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Massimo DOMINICI

Professore Ordinario
Dipartimento di Scienze Mediche e Chirurgiche Materno-Infantili e dell'Adulto

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Pubblicazioni

- Device for cell culture [Brevetto]
Dominici, Massimo; G., Bellini; M., Brogli; Conte, Pierfranco
abstract

The device for cell culture comprises: a container body (2) which has an internal compartment (5) to contain a quantity of cells to be cultured and a culture surface (3A; 120) for said cells placed in said internal compartment (5); an introduction opening (6; 106) for introducing said cells to be cultured in said compartment (5); said container body (2) comprising: first half shell means (3; 103) and second half shell means (4; 104) which can be fixed in mutually facing relation by removable fixing means (8; 108, 108') and which together define said internal compartment (5) and said culture surface (3A; 120); at least one out feed opening (7; 107) for a transport and/or culture fluid for said cells.

- Method For Obtaining A Population Of Stromal Progenitor Cells [Brevetto]
Dominici, Massimo; Cafarelli, Luigi; Veronesi, Elena; Piccinno, MARIA SERENA; Paolucci, Paolo; DE SANTIS, Giorgio; Conte, P. F.
abstract

E' STATO ELABORATO UN NUOVO E ORIGINALE SISTEMA DI ISOLAMENTO DI POPOLAZIONI CELLULARI.

- METHOD FOR PRODUCTION OF ANTI-TUMOR TRAIL PROTEIN [Brevetto]
Dominici, Massimo; Bussolari, Rita; Grisendi, Giulia; Conte, Pierfranco
abstract

The method for production of anti-tumor TRAIL comprises: inserting a TRAIL molecule, encoded by a viral vector irreversibly derived from a cell line, into a carrier cell, thereby obtaining a stably TRAIL-producing carrier cell, said TRAIL molecule comprising a soluble molecule.

- Targeting the homing of stem cells via suppression of cell adhesion in the peripheral vasculature [Brevetto]
R., Schaefer; L., Danielyan; R., Kehlbach; R., Bantleon; G., Siegel; A., Ameln Mayerhofer; H. P., Wendel; T., Kluba; Dominici, Massimo; C. D., Claussen; H., Northoff
abstract

The general approach of the so far developed technologies is the attempt to increase the homing rate of transplanted stem cells by modifying the molecules/receptors that interact directly with the chemokines/ligands in the damaged tissue. Our unique technology does not interact with the natural repertoire of specific molecules/receptors mediating homing of stem cells to the damaged tissue (SPECIFIC HOMING) but impairs the function of molecules/receptors that are responsible for the adhesion of stem cells throughout the non-damaged vasculature (NON-SPECIFIC CELL ADHESION). Moreover and most importantly, our highly effective technology is non-toxic, does not affect stem cell function and does not require genetic modifications thus making it a most promising candidate for clinical use

2024 - Proteomics Analysis of Formalin-Fixed Paraffine-Embedded Tissue Reveals Key Proteins Related to Lung Dysfunction in Idiopathic Pulmonary Fibrosis. [Articolo su rivista]
Samarelli, ANNA VALERIA; Tonelli, Roberto; Raineri, Giulia; Bruzzi, Giulia; Andrisani, Dario; Gozzi, Filippo; Marchioni, Alessandro; Costantini, Matteo; Fabbiani, Luca; Genovese, Filippo; Pinetti, Diego; Manicardi, Linda; Castaniere, Ivana; Masciale, Valentina; Aramini, Beatrice; Tabbi', Luca; Rizzato, Simone; Bettelli, Stefania; Manfredini, Samantha; Dominici, Massimo; Clini, Enrico; Cerri, Stefania
abstract

Idiopathic pulmonary fibrosis (IPF) severely affects the lung leading to aberrant deposition of extracellular matrix and parenchymal stiffness with progressive functional derangement. The limited availability of fresh tissues represents one of the major limitations to study the molecular profiling of IPF lung tissue. The primary aim of this study was to explore the proteomic profiling yield of archived formalin-fixed paraffin-embedded (FFPE) specimens of IPF lung tissues. We further determined the protein expression according to respiratory functional decline at the time of biopsy. The total proteins isolated from 11 FFPE samples of IPF patients compared to 3 FFPE samples from a non-fibrotic lung defined as controls, were subjected to label-free quantitative proteomic analysis by liquid chromatography-mass spectrometry (LC-MS/MS) and resulted in the detection of about 400 proteins. After the pairwise comparison between controls and IPF, functional enrichment analysis identified differentially expressed proteins that were involved in extracellular matrix signaling pathways, focal adhesion and transforming growth factor β (TGF‐β) signaling pathways strongly associated with IPF onset and progression. Five proteins were significantly over-expressed in the lung of IPF patients with either advanced disease stage (Stage II) or impaired pulmonary function (FVC<75, DLco<55) compared to controls; these were lymphocyte cytosolic protein 1 (LCP1), peroxiredoxin-2 (PRDX2), transgelin 2 (TAGLN2), lumican (LUM) and mimecan (OGN) that might play a key role in the fibrogenic processes. Our work showed that the analysis of FFPE samples was able to identify key proteins that might be crucial for the IPF pathogenesis. These proteins are correlated with lung carcinogenesis or involved in the immune landscape of lung cancer, thus making possible common mechanisms between lung carcinogenesis and fibrosis progression, two pathological conditions at risk for each other in the real life.

2024 - Real-World Data and Clinical Implications of Next-Generation Sequencing (NGS)-Based Analysis in Metastatic Breast Cancer Patients [Articolo su rivista]
Canino, F; Tornincasa, A; Bettelli, S; Manfredini, S; Barbolini, M; Moscetti, L; Omarini, C; Toss, A; Tamburrano, F; Antonelli, G; Baglio, F; Belluzzi, L; Martinelli, G; Natalizio, S; Ponzoni, O; Dominici, M; Piacentini, F.
abstract

Over the last two decades, the use of Next-Generation Sequencing (NGS) in medical oncology has increased the likelihood of identifying druggable mutations that may be potentially susceptible to targeted treatments. The European Society for Medical Oncology (ESMO) currently does not recommend the use of the NGS test to determine the therapeutic course of patients with metastatic breast cancer (mBC) in daily clinical practice. However, the aim of this work is to evaluate the potential contribution of the NGS test in selecting targeted therapies for patients with mBC. Data were retrospectively collected from 101 patients diagnosed with metastatic breast cancer and treated at the Modena Cancer Center between January 2015 and April 2022. A NGS test was performed on the tumor tissue of each patient at the Laboratory of Molecular Pathology of the University Hospital of Modena. This study analyzed the clinical-pathological characteristics and mutational profile of the population using NGS tests, with a focus on actionable mutations that could be targeted in advanced stages of clinical development. The indicator of this study was to quantify the actionable mutations that resulted in a change of cancer treatment. In total, 101 patients with metastatic breast cancer were analyzed, including 86 with luminal phenotype, 10 who were HER2-positive and 5 who were triple-negative. Median age was 52 years. NGS analysis was conducted on 47 samples of primary breast cancer, 52 on metastatic sites of disease and 2 on liquid biopsies. A total of 85 gene mutations were found. The most common mutations were identified in the PIK3CA (47%), FGFR (19%) and ERBB2 genes (12%), and to a lesser extent in other genes. Of the 61 patients with pathogenic mutations, 46 (75%) had at least one actionable mutation. Of these, nine received treatment with a molecular target drug: eight patients with a mutation of the PIK3CA gene were treated with alpelisib and fulvestrant; one patient with FGFR1/2 amplifications received TAS120. Median PFS for these patients was 3.8 months. The study results show that using the NGS test on cancer tissue of metastatic breast cancer could influence the therapeutic choices, considering the small sample size and limited follow-up. About 9% of the study population had their therapy modified based on the results of NGS. The growing number of detectable mutations and increased accessibility of the test may lead to a greater number of potential therapeutic implications for the NGS assay. Perspectives suggest that NGS analysis can be implemented in daily clinical practice, particularly in contexts where a Molecular Tumor Board (MTB) is active.

2023 - A new strategy to prevent biofilm and clot formation in medical devices: the use of atmospheric non-thermal plasma assisted deposition of silver-based nanostructured coatings [Articolo su rivista]
Gallingani, T; Resca, E; Dominici, M; Gavioli, G; Laurita, R; Liguori, A; Mari, G; Ortolani, L; Pericolini, E; Sala, A; Laghi, G; Petrachi, T; Arnauld, Gf; Accorsi, L; Rizzoli, R; Colombo, V; Gherardi, M; Veronesi, E
abstract

In industrialized countries, health care associated infections, the fourth leading cause of dis- ease, are a major health issue. At least half of all cases of nosocomial infections are associ- ated with medical devices. Antibacterial coatings arise as an important approach to restrict the nosocomial infection rate without side effects and the development of antibiotic resis- tance. Beside nosocomial infections, clot formation affects cardiovascular medical devices and central venous catheters implants. In order to reduce and prevent such infection, we develop a plasma-assisted process for the deposition of nanostructured functional coatings on flat substrates and mini catheters. Silver nanoparticles (Ag NPs) are synthesized exploit- ing in-flight plasma-droplet reactions and are embedded in an organic coating deposited through hexamethyldisiloxane (HMDSO) plasma assisted polymerization. Coating stability upon liquid immersion and ethylene oxide (EtO) sterilization is assessed through chemical and morphological analysis carried out by means of Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). In the perspective of future clinical appli- cation, an in vitro analysis of anti-biofilm effect has been done. Moreover, we employed a murine model of catheter-associated infection which further highlighted the performance of Ag nanostructured films in counteract biofilm formation. The anti-clot performances coupled by haemo- and cytocompatibility assays have also been performed.

2023 - Adjuvant Endocrine Therapy in Premenopausal Women With Hormone Receptor-Positive Early-Stage Breast Cancer: Risk Stratification in a Real-World Setting [Articolo su rivista]
D'Onofrio, Raffaella; Omarini, Claudia; Toss, Angela; Sperduti, Isabella; Piacentini, Federico; Barbolini, Monica; Cortesi, Laura; Barbieri, Elena; Pettorelli, Elisa; Chiavelli, Chiara; Dominici, Massimo; Moscetti, Luca
abstract

Background: Ovarian function suppression (OFS) and hormone therapy (HT) represent an adjuvant option in premenopausal hormone receptor-positive early breast cancer (HR+EBC). The SOFT-TEXT trials showed improved outcomes upon receiving aromatase inhibitors (AIs)/OFS. Methods: In order to estimate the magnitude of absolute improvements, we conducted a retrospective study applying composite risk (CR) to 237 premenopausal HR+EBC patients. Results: Overall, 119 of these received tamoxifen (T)/OFS and 118 received AIs/OFS. The median age was 45 years (ys). After a median follow up of 65 months, recurrence was 6.7% in T patients and 10.2% in AI ones. CR (cutoff: 2.67) and ET duration (five-year cutoff) was found to have a significant impact on DFS. Invasive disease-free survival (IDFS) at 5 ys amounted to 82.9% for a CR>2.67 and 95% with CR

2023 - Clinical Significance of Molecular Subtypes in Western Advanced Gastric Cancer: A Real-World Multicenter Experience [Articolo su rivista]
Salati, Massimiliano; Ghidini, Michele; Paccagnella, Matteo; Reggiani Bonetti, Luca; Bocconi, Alessandro; Spallanzani, Andrea; Gelsomino, Fabio; Barbin, Francesca; Garrone, Ornella; Daniele, Bruno; Dominici, Massimo; Facciorusso, Antonio; Petrillo, Angelica
abstract

: In recent years, the molecular subtyping of gastric cancer has led to the identification of novel clinically relevant biomarkers as well as promising therapeutic targets. In parallel, the advent of checkpoint inhibitors has expanded treatment options beyond conventional chemotherapy. Compelling evidence has shown unprecedented efficacy results for anti-PD1-based therapies in the molecular subgroups of dMMR/MSI-h, EBV+ and PD-L1 CPS+ patients, to the point that these are granted approval for gastric cancer adenocarcinoma (AGC) in several countries. Despite this, cytotoxic chemotherapy remains the only treatment choice for the considerable proportion of biomarkers-negative patients. In this context, little is known about the association between subtypes-defining biomarkers (HER2, MMR/MSI, PD-L1, and EBV) and the efficacy of standard chemotherapy in non-Asian AGC. Here, we aimed to investigate the prevalence, the clinic-pathologic features, and the impact on treatment outcome of clinical molecular subtypes in a new-diagnosed Western cohort of AGC.

2023 - Impact of soluble tumor necrosis factor-related apoptosis-inducing ligand released by engineered adipose mesenchymal stromal cells on white blood cells [Articolo su rivista]
Casari, Giulia; Dall'Ora, Massimiliano; Melandri, Aurora; Masciale, Valentina; Chiavelli, Chiara; Prapa, Malvina; Neri, Giovanni; Spano, Maria Carlotta; Murgia, Alba; D'Esposito, Angela; Baschieri, Maria Cristina; Ceccherelli, Giovanni Battista; Dominici, Massimo; Grisendi, Giulia
abstract

Background aims: The proapoptotic protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is physiologically expressed by immune cells and performs regulatory functions in infections, autoimmune diseases and cancer, where it acts as a tumor suppressor. Adipose-derived mesenchymal stromal cells (AD-MSCs) also may play immunomodulatory roles in both primary and acquired immune responses. We have previously demonstrated the efficacy of an anticancer gene therapy based on AD-MSC engineered to secrete a soluble TRAIL variant (sTRAIL) against pancreatic cancer. However, the impact of AD-MSC sTRAIL on leukocyte subsets has been not yet considered also to predict a possible immunotoxicity profile in the clinical translation of this cell-based anticancer strategy. Methods: Monocytes, polymorphonuclear cells and T lymphocytes were freshly isolated from the peripheral blood of healthy donors. Immunophenotype and functional (DR4 and DR5) and decoy (DcR1 and DcR2) TRAIL receptors were tested by flow cytometry. The viability of white blood cells treated with sTRAIL released by gene-modified AD-MSC or co-cultured with AD-MSC sTRAIL was then evaluated by both metabolic assays and flow cytometry. In addition, cytokine profile in co-cultures was analyzed by multiplex enzyme-linked immunosorbent assay.Results: Monocytes and polymorphonuclear cells showed high positivity for DR5 and DcR2, respectively, whereas T cells revealed negligible expression of all TRAIL receptors. Irrespective of TRAIL receptors' pres-ence on the cell membrane, white blood cells were refractory to the proapoptotic effect displayed by sTRAIL secreted by gene-modified AD-MSC, and direct cell-to-cell contact with AD-MSC sTRAIL had negligible impact on T-cell and monocyte viability. Cytokine crosstalk involving interleukin 10, tumor necrosis factor alpha, and interferon gamma secreted by T lymphocytes and vascular endothelial growth factor A and inter-leukin 6 released by AD-MSC was highlighted in T-cell and AD-MSC sTRAIL co-cultures. Conclusions: In summary, this study demonstrates the immunological safety and thus the clinical feasibility of an anticancer approach based on AD-MSC expressing the proapoptotic molecule sTRAIL.(c) 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

2023 - Inhibition of ERK1/2 signaling prevents bone marrow fibrosis by reducing osteopontin plasma levels in a myelofibrosis mouse model [Articolo su rivista]
Bianchi, Elisa; Rontauroli, Sebastiano; Tavernari, Lara; Mirabile, Margherita; Pedrazzi, Francesca; Genovese, Elena; Sartini, Stefano; Dall'Ora, Massimiliano; Grisendi, Giulia; Fabbiani, Luca; Maccaferri, Monica; Carretta, Chiara; Parenti, Sandra; Fantini, Sebastian; Bartalucci, Niccolò; Calabresi, Laura; Balliu, Manjola; Guglielmelli, Paola; Potenza, Leonardo; Tagliafico, Enrico; Losi, Lorena; Dominici, Massimo; Luppi, Mario; Vannucchi, Alessandro Maria; Manfredini, Rossella
abstract

Clonal myeloproliferation and development of bone marrow (BM) fibrosis are the major pathogenetic events in myelofibrosis (MF). The identification of novel antifibrotic strategies is of utmost importance since the effectiveness of current therapies in reverting BM fibrosis is debated. We previously demonstrated that osteopontin (OPN) has a profibrotic role in MF by promoting mesenchymal stromal cells proliferation and collagen production. Moreover, increased plasma OPN correlated with higher BM fibrosis grade and inferior overall survival in MF patients. To understand whether OPN is a druggable target in MF, we assessed putative inhibitors of OPN expression in vitro and identified ERK1/2 as a major regulator of OPN production. Increased OPN plasma levels were associated with BM fibrosis development in the Romiplostim-induced MF mouse model. Moreover, ERK1/2 inhibition led to a remarkable reduction of OPN production and BM fibrosis in Romiplostim-treated mice. Strikingly, the antifibrotic effect of ERK1/2 inhibition can be mainly ascribed to the reduced OPN production since it could be recapitulated through the administration of anti-OPN neutralizing antibody. Our results demonstrate that OPN is a novel druggable target in MF and pave the way to antifibrotic therapies based on the inhibition of ERK1/2-driven OPN production or the neutralization of OPN activity.

2023 - Label-Free Optical Sensing and Medical Grade Resins: An Advanced Approach to Investigate Cell–Material Interaction and Biocompatibility [Articolo su rivista]
Bergamini, V.; Resca, E.; Portone, A.; Petrachi, T.; Ganzerli, F.; Truzzi, S.; Mari, G.; Rovati, L.; Dominici, M.; Veronesi, E.
abstract

: The Corning Epic® label-free (ELF) system is an innovative technology widely used in drug discovery, immunotherapy, G-protein-associated studies, and biocompatibility tests. Here, we challenge the use of ELF to further investigate the biocompatibility of resins used in manufacturing of blood filters, a category of medical devices representing life-saving therapies for the increasing number of patients with kidney failure. The biocompatibility assays were carried out by developing a cell model aimed at mimicking the clinical use of the blood filters and complementing the existing cytotoxicity assay requested by ISO10993-5. Experiments were performed by putting fibroblasts in both direct contact with two types of selected resins, and indirect contact by means of homemade customized well inserts that were precisely designed and developed for this technology. For both types of contact, fibroblasts were cultured in medium and human plasma. ELF tests confirmed the biocompatibility of both resins, highlighting a statistically significant different biological behavior of a polyaromatic resin compared to control and ion-exchanged resin, when materials were in indirect contact and soaking with plasma. Overall, the ELF test is able to mimic clinical scenarios and represents a promising approach to investigate biocompatibility, showing peculiar biological behaviors and suggesting the activation of specific intracellular pathways.

2023 - LOW-FREQUENCY ALLELE VARIANTS IN NGS MULTI-GENE PANELS FOR HEREDITARY CANCER TESTING: ARTIFACTS, CHIP OR MOSAICS? MANAGING THE RESULTS IN THE LABORATORY ROUTINE [Abstract in Rivista]
Tenedini, E.; Bonamici, L.; Artuso, L.; Marino, M.; Botticelli, L.; Barbieri, E.; Toss, A.; Venturelli, M.; Trenti, T.; Dominici, M.; Cortesi, L.; Tagliafico, E.
abstract

2023 - Management of PALB2-associated breast cancer: A literature review and case report [Articolo su rivista]
Toss, Angela; Ponzoni, Ornella; Riccò, Beatrice; Piombino, Claudia; Moscetti, Luca; Combi, Francesca; Palma, Enza; Papi, Simona; Tenedini, Elena; Tazzioli, Giovanni; Dominici, Massimo; Cortesi, Laura
abstract

Key Clinical Message Germline pathogenic variants (PV) of the PALB2 tumor suppressor gene are associated with an increased risk of breast, pancreatic, and ovarian cancer. In previous research, PALB2-associated breast cancer showed aggressive clinicopathological phenotypes, particularly triple-negative subtype, and higher mortality regardless of tumor stage, type of chemotherapy nor hormone receptor status. The identification of this germline alteration may have an impact on clinical management of breast cancer (BC) from the surgical approach to the systemic treatment choice. We herein report the case of a patient with a germline PV of PALB2, diagnosed with locally advanced PD-L1 positive triple-negative BC, who progressed after an immune checkpoint inhibitor (ICI)-containing regimen and then experienced a pathologic complete response after platinum-based chemotherapy. This case report hints a major role of the germline PALB2 alteration compared to the PD-L1 expression as cancer driver and gives us the opportunity to extensively review and discuss the available literature on the optimal management of PALB2-associated BC. Overall, our case report and review of the literature provide additional evidence that the germline analysis of PALB2 gene should be included in routine genetic testing for predictive purposes and to refine treatment algorithms.

2023 - Molecular portraits of patients with intrahepatic cholangiocarcinoma who diverge as rapid progressors or long survivors on chemotherapy [Articolo su rivista]
O'Rourke, Colm J; Salati, Massimiliano; Rae, Colin; Carpino, Guido; Leslie, Holly; Pea, Antonio; Prete, Maria G; Bonetti, Luca R; Amato, Francesco; Montal, Robert; Upstill-Goddard, Rosie; Nixon, Colin; Sanchon-Sanchez, Paula; Kunderfranco, Paolo; Sia, Daniela; Gaudio, Eugenio; Overi, Diletta; Cascinu, Stefano; Hogdall, Dan; Pugh, Sian; Domingo, Enric; Primrose, John N; Bridgewater, John; Spallanzani, Andrea; Gelsomino, Fabio; Llovet, Josep M; Calvisi, Diego F; Boulter, Luke; Caputo, Francesco; Lleo, Ana; Jamieson, Nigel B; Luppi, Gabriele; Dominici, Massimo; Andersen, Jesper B; Braconi, Chiara
abstract

Objective: Cytotoxic agents are the cornerstone of treatment for patients with advanced intrahepatic cholangiocarcinoma (iCCA), despite heterogeneous benefit. We hypothesised that the pretreatment molecular profiles of diagnostic biopsies can predict patient benefit from chemotherapy and define molecular bases of innate chemoresistance. Design: We identified a cohort of advanced iCCA patients with comparable baseline characteristics who diverged as extreme outliers on chemotherapy (survival <6 m in rapid progressors, RP; survival >23 m in long survivors, LS). Diagnostic biopsies were characterised by digital pathology, then subjected to whole-transcriptome profiling of bulk and geospatially macrodissected tissue regions. Spatial transcriptomics of tumour-infiltrating myeloid cells was performed using targeted digital spatial profiling (GeoMx). Transcriptome signatures were evaluated in multiple cohorts of resected cancers. Signatures were also characterised using in vitro cell lines, in vivo mouse models and single cell RNA-sequencing data. Results: Pretreatment transcriptome profiles differentiated patients who would become RPs or LSs on chemotherapy. Biologically, this signature originated from altered tumour-myeloid dynamics, implicating tumour-induced immune tolerogenicity with poor response to chemotherapy. The central role of the liver microenviroment was confrmed by the association of the RPLS transcriptome signature with clinical outcome in iCCA but not extrahepatic CCA, and in liver metastasis from colorectal cancer, but not in the matched primary bowel tumours. Conclusions: The RPLS signature could be a novel metric of chemotherapy outcome in iCCA. Further development and validation of this transcriptomic signature is warranted to develop precision chemotherapy strategies in these settings.

2023 - Multiparametric analysis of tumor infiltrating lymphocytes in solid tumors [Capitolo/Saggio]
Borella, R.; Paolini, A.; Aramini, B.; Gibellini, L.; Masciale, V.; Lo Tartaro, D.; Dominici, M.; De Biasi, S.; Cossarizza, A.
abstract

The use of single-cell technologies in characterizing the interactions between immune and cancer cells is in continuous expansion. Indeed, the combination of different single-cell approaches enables the definition of novel phenotypic and functional aspects of the immune cells infiltrating the tumor microenvironment (TME). This approach is promoting the discovery of relevant and reliable predictive biomarkers, along with the development of new promising treatments. In this chapter, we describe the main subsets of tumor-infiltrating lymphocytes from a phenotypical and functional point of view and discuss the use of single-cell technologies used to characterize these cell populations within TME.

2023 - Novel bioprinted 3D model to human fibrosis investigation [Articolo su rivista]
Petrachi, T.; Portone, A.; Arnaud, G. F.; Ganzerli, F.; Bergamini, V.; Resca, E.; Accorsi, L.; Ferrari, A.; Delnevo, A.; Rovati, L.; Marra, C.; Chiavelli, C.; Dominici, M.; Veronesi, E.
abstract

Fibrosis is shared in multiple diseases with progressive tissue stiffening, organ failure and limited therapeutic options. This unmet need is also due to the lack of adequate pre-clinical models to mimic fibrosis and to be challenged novel by anti-fibrotic therapeutic venues. Here using bioprinting, we designed a novel 3D model where normal human healthy fibroblasts have been encapsulated in type I collagen. After stimulation by Transforming Growth factor beta (TGFβ), embedded cells differentiated into myofibroblasts and enhanced the contractile activity, as confirmed by the high level of α − smooth muscle actin (αSMA) and F-actin expression. As functional assays, SEM analysis revealed that after TGFβ stimulus the 3D microarchitecture of the scaffold was dramatically remolded with an increased fibronectin deposition with an abnormal collagen fibrillar pattern. Picrius Sirius Red staining additionally revealed that TGFβ stimulation enhanced of two logarithm the collagen fibrils neoformation in comparison with control. These data indicate that by bioprinting technology, it is possible to generate a reproducible and functional 3D platform to mimic fibrosis as key tool for drug discovery and impacting on animal experimentation and reducing costs and time in addressing fibrosis.

2023 - Oligorecurrent Non-Small-Cell Lung Cancer Treated by Chemo-Radiation Followed by Immunotherapy and Intracranial Radiosurgery: A Case Report and Mini Review of Literature [Articolo su rivista]
Bruni, Alessio; Bertolini, Federica; D'Angelo, Elisa; Guaitoli, Giorgia; Imbrescia, Jessica; Cappelli, Anna; Guidi, Gabriele; Stefani, Alessandro; Dominici, Massimo; Lohr, Frank
abstract

Locally advanced non-small-cell lung cancer still represents a "grey zone" in terms of the best treatment choice and optimal clinical outcomes. Indeed, most patients may be suitable to receive different treatments with similar outcomes such as chemo-radiotherapy (CHT-RT) followed by immunotherapy (IO) or surgery followed by adjuvant local/systemic therapies. We report a clinical case of a patient submitted to primary thoracic surgery who developed a mediastinal nodal recurrence successfully treated by CHT-RT-IO. Subsequently, a single brain lesion was found to have been successfully treated by single fraction stereotactic ablative radiotherapy. The patient is still on follow-up and she is free from disease having a good quality of life. In this report, we also perform a mini review about the role of CHT-RT followed by IO in treating loco-regional relapse after surgery. The role of SABR after IO is also evaluated, finding that it is safe and well tolerated. More robust and larger clinical data are needed in this particular setting to better define the role of the combination of systemic and local treatments in the management of intrathoracic and intracranial relapse for patients already submitted to CHT-RT followed by immunotherapy.

2023 - Personalized Systemic Therapies in Hereditary Cancer Syndromes [Articolo su rivista]
Mastrodomenico, L.; Piombino, C.; Ricco, B.; Barbieri, E.; Venturelli, M.; Piacentini, F.; Dominici, M.; Cortesi, L.; Toss, A.
abstract

Hereditary cancer syndromes are inherited disorders caused by germline pathogenic variants (PVs) that lead to an increased risk of developing certain types of cancer, frequently at an earlier age than in the rest of the population. The germline PVs promote cancer development, growth and survival, and may represent an ideal target for the personalized treatment of hereditary tumors. PARP inhibitors for the treatment of BRCA and PALB2-associated tumors, immune checkpoint inhibitors for tumors associated with the Lynch Syndrome, HIF-2α inhibitor in the VHL-related cancers and, finally, selective RET inhibitors for the treatment of MEN2-associated medullary thyroid cancer are the most successful examples of how a germline PVs can be exploited to develop effective personalized therapies and improve the outcome of these patients. The present review aims to describe and discuss the personalized systemic therapies for inherited cancer syndromes that have been developed and investigated in clinical trials in recent decades.

2023 - Targeting PI3K/AKT/mTOR Pathway in Breast Cancer: From Biology to Clinical Challenges [Articolo su rivista]
Cerma, K.; Piacentini, F.; Moscetti, L.; Barbolini, M.; Canino, F.; Tornincasa, A.; Caggia, F.; Cerri, S.; Molinaro, A.; Dominici, M.; Omarini, C.
abstract

Breast cancer (BC) is the most common women cancer and cause of cancer death. Despite decades of scientific progress in BC treatments, the clinical benefit of new drugs is modest in several cases. The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway mutations are frequent in BC (20-40%) and are significant causes of aggressive tumor behavior, as well as treatment resistance. Improving knowledge of the PI3K/AKT/mTOR pathway is an urgent need. This review aims to highlight the central role of PI3K-mTORC1/C2 mutations in the different BC subtypes, in terms of clinical outcomes and treatment efficacy. The broad base of knowledge in tumor biology is a key point for personalized BC therapy in the precision medicine era.

2023 - Thromboembolism and Adjuvant Endocrine Therapy (AET) in Hormone Receptor-Positive Early Breast Cancer (EBC): Did Treatment Evolution Change Incidence of the Adverse Event? A Meta-Analysis [Articolo su rivista]
D'Onofrio, R.; Sperduti, I.; Piacentini, F.; Barbolini, M.; Omarini, C.; Toss, A.; Cortesi, L.; Barbieri, E.; Canino, F.; Dominici, M.; Moscetti, L.
abstract

: The adjuvant endocrine therapy (AET) of HR+ EBC has been changing in recent years. Aromatase inhibitors (AIs) as an upfront strategy (or as part of a switch strategy) have been added to the choice of Tamoxifen (T) alone. Increased TE risk is well known in T-treated patients, while AIs have shown a reduced TE rate. By adding the cyclin dependent kinase 4/6 inhibitors (CDK4/6) to AIs, an increase in TE rate has been shown. We conducted this meta-analysis to evaluate the impact of the AETs on TE incidence. Twelve randomized phase III trials were included. Four trials evaluated the upfront strategy, 6 assessed the switch and 2 the combination with a CDK4/6 inhibitor. The new AETs did not significantly modify or affect the rate of TE events (OR 0.847, 95% CI, 0.528-1.366, P = .489). The OR for CDK4/6 inhibitor plus ET vs. ET was 3.635 (P = .002). Excluding the CDK4/6 inhibitors, the overall OR for AIs vs. T was 0.628 (P < .001), while it was 0.781 (P = .151) for switching T vs. continuing T for 5 years, and 0.52 (P < .0001) for the upfront strategies with AIs. The AIs alone or plus CDK4/6 inhibitors did not affect the rate of TE events. AIs as an upfront strategy is the safest AET, associated with the lowest TE incidence. The switch strategy increases TE rate, whereas the addition of CDK4/6 to the standard AET was shown to significantly increase TE events. The results of the currently ongoing trials with CDK4/6 inhibitors will help obtain additional data to evaluate any differences among the different CDK4/6 inhibitors and clarify the weight of TE adverse events in the benefit/risk balance of this new adjuvant strategy.

2022 - A Roadmap for the Production of a GMP-Compatible Cell Bank of Allogeneic Bone Marrow-Derived Clonal Mesenchymal Stromal Cells for Cell Therapy Applications [Articolo su rivista]
Pakzad, M.; Hassani, S. N.; Abbasi, F.; Hajizadeh-Saffar, E.; Taghiyar, L.; Fallah, N.; Haghparast, N.; Samadian, A.; Ganjibakhsh, M.; Dominici, M.; Baharvand, H.
abstract

Background: Allogeneic mesenchymal stromal cells (MSCs) have been used extensively in various clinical trials. Nevertheless, there are concerns about their efficacy, attributed mainly to the heterogeneity of the applied populations. Therefore, producing a consistent population of MSCs is crucial to improve their therapeutic efficacy. This study presents a good manufacturing practice (GMP)-compatible and cost-effective protocol for manufacturing, banking, and lot-release of a homogeneous population of human bone marrow-derived clonal MSCs (cMSCs). Methods: Here, cMSCs were isolated based on the subfractionation culturing method. Afterward, isolated clones that could reproduce up to passage three were stored as the seed stock. To select proliferative clones, we used an innovative, cost-effective screening strategy based on lengthy serial passaging. Finally, the selected clones re-cultured from the seed stock to establish the following four-tired cell banking system: initial, master, working, and end of product cell banks (ICB, MCB, WCB, and EoPCB). Results: Through a rigorous screening strategy, three clones were selected from a total of 21 clones that were stored during the clonal isolation process. The selected clones met the identity, quality, and safety assessments criteria. The validated clones were stored in the four-tiered cell bank system under GMP conditions, and certificates of analysis were provided for the three-individual ready-to-release batches. Finally, a stability study validated the EoPCB, release, and transport process of the frozen final products. Conclusion: Collectively, this study presents a technical and translational overview of a GMP-compatible cMSCs manufacturing technology that could lead to the development of similar products for potential therapeutic applications. Graphical Abstract: [Figure not available: see fulltext.]

2022 - A 3D Platform to Investigate Dynamic Cell-to-Cell Interactions Between Tumor Cells and Mesenchymal Progenitors [Articolo su rivista]
Golinelli, G.; Talami, R.; Frabetti, S.; Candini, O.; Grisendi, G.; Spano, C.; Chiavelli, C.; Arnaud, G. F.; Mari, G.; Dominici, M.
abstract

We here investigated the dynamic cell-to-cell interactions between tumor and mesenchymal stromal/stem cells (MSCs) by the novel VITVOⓇ 3D bioreactor that was customized to develop in vivo-like metastatic nodules of Ewing’s sarcoma (ES). MSCs are known to contribute to tumor microenvironment as cancer associated fibroblast (CAF) precursors and, for this reason, they have also been used as anti-cancer tools. Using dynamic conditions, the process of tissue colonization and formation of metastatic niches was recreated through tumor cell migration aiming to mimic ES development in patients. ES is an aggressive tumor representing the second most common malignant bone cancer in children and young adults. An urgent and unmet need exists for the development of novel treatment strategies to improve the outcomes of metastatic ES. The tumor-tropic ability of MSCs offers an alternative approach, in which these cells can be used as vehicles for the delivery of antitumor molecules, such as the proapoptotic TNF-related apoptosis inducing ligand (TRAIL). However, the therapeutic targeting of metastases remains challenging and the interaction occurring between tumor cells and MSCs has not yet been deeply investigated. Setting up in vitro and in vivo models to study this interaction is a prerequisite for novel approaches where MSCs affinity for tumor is optimized to ultimately increase their therapeutic efficacy. Here, VITVOⓇ integrating a customized scaffold with an increased inter-fiber distance (VITVO50) was used to develop a dynamic model where MSCs and tumor nodules were evaluated under flow conditions. Colonization and interaction between cell populations were explored by droplet digital PCR (ddPCR). VITVO50 findings were then applied in vivo. An ES metastatic model was established in NSG mice and biodistribution of TRAIL-expressing MSCs in mice organs affected by metastases was investigated using a 4-plex ddPCR assay. VITVOⓇ proved to be an easy handling and versatile bioreactor to develop in vivo-like tumor nodules and investigate dynamic cell-to-cell interactions with MSCs. The proposed fluidic system promises to facilitate the understanding of tumor-stroma interaction for the development of novel tumor targeting strategies, simplifying the analysis of in vivo data, and ultimately accelerating the progress towards the early clinical phase.

2022 - Anti-GD2 CAR MSCs against metastatic Ewing's sarcoma [Articolo su rivista]
Golinelli, G.; Grisendi, G.; Dall'Ora, M.; Casari, G.; Spano, C.; Talami, R.; Banchelli, F.; Prapa, M.; Chiavelli, C.; Rossignoli, F.; Candini, O.; D'Amico, R.; Nasi, M.; Cossarizza, A.; Casarini, L.; Dominici, M.
abstract

Background: Ewing's sarcoma (ES) is an aggressive cancer affecting children and young adults. We pre-clinically demonstrated that mesenchymal stromal/stem cells (MSCs) can deliver tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) against primary ES after local injection. However, ES is often metastatic calling for approaches able to support MSC targeting to the ES multiple remote sites. Considering that the disialoganglioside GD2 is expressed by ES and to optimise MSC tumour affinity, bi-functional (BF) MSCs expressing both TRAIL and a truncated anti-GD2 chimeric antigen receptor (GD2 tCAR) were generated and challenged against ES. Methods: The anti-GD2 BF MSCs delivering a soluble variant of TRAIL (sTRAIL) were tested in several in vitro ES models. Tumour targeting and killing by BF MSCs was further investigated by a novel immunodeficient ES metastatic model characterized by different metastatic sites, including lungs, liver and bone, mimicking the deadly clinical scenario. Findings: In vitro data revealed both tumour affinity and killing of BF MSCs. In vivo, GD2 tCAR molecule ameliorated the tumour targeting and persistence of BF MSCs counteracting ES in lungs but not in liver. Interpretation: We here generated data on the potential effects of BF MSCs within a complex ES metastatic in vivo model, exploring also the biodistribution of MSCs. Our BF MSC-based strategy promises to pave the way for potential improvements in the therapeutic delivery of TRAIL for the treatment of metastatic ES and other deadly GD2-positive malignancies.

2022 - Autologous Marrow Mesenchymal Stem Cell Driving Bone Regeneration in a Rabbit Model of Femoral Head Osteonecrosis [Articolo su rivista]
Mastrolia, I.; Giorgini, A.; Murgia, A.; Loschi, P.; Petrachi, T.; Rasini, V.; Pinelli, M.; Pinto, V.; Lolli, F.; Chiavelli, C.; Grisendi, G.; Baschieri, M. C.; Santis, G. D.; Catani, F.; Dominici, M.; Veronesi, E.
abstract

Osteonecrosis of the femoral head (ONFH) is a progressive degenerative disease that ultimately requires a total hip replacement. Mesenchymal stromal/stem cells (MSCs), particularly the ones isolated from bone marrow (BM), could be promising tools to restore bone tissue in ONFH. Here, we established a rabbit model to mimic the pathogenic features of human ONFH and to challenge an autologous MSC-based treatment. ON has been originally induced by the synergic combination of surgery and steroid administration. Autologous BM-MSCs were then implanted in the FH, aiming to restore the damaged tissue. Histological analyses confirmed bone formation in the BM-MSC treated rabbit femurs but not in the controls. In addition, the model also allowed investigations on BM-MSCs isolated before (ON-BM-MSCs) and after (ON+BM-MSCs) ON induction to dissect the impact of ON damage on MSC behavior in an affected microenvironment, accounting for those clinical approaches foreseeing MSCs generally isolated from affected patients. BM-MSCs, isolated before and after ON induction, revealed similar growth rates, immunophenotypic profiles, and differentiation abilities regardless of the ON. Our data support the use of ON+BM-MSCs as a promising autologous therapeutic tool to treat ON, paving the way for a more consolidated use into the clinical settings.

2022 - Biological effects of COVID-19 on lung cancer: can we drive our decisions? [Articolo su rivista]
Aramini, Beatrice; Masciale, Valentina; Samarelli, Anna V.; Tonelli, Roberto; Cerri, Stefania; Clini, Enrico; Stella, Franco; Dominici, Massimo
abstract

COVID-19 infection caused by SARS-CoV-2 is considered catastrophic because it affects multiple organs, particularly those of the respiratory tract. Although the consequences of this infection are not fully clear, it causes damage to the lungs, the cardiovascular and nervous systems, and other organs, subsequently inducing organ failure. In particular, the effects of SARS-CoV-2-induced inflammation on cancer cells and the tumor microenvironment need to be investigated. COVID-19 may alter the tumor microenvironment, promoting cancer cell proliferation and dormant cancer cell (DCC) reawakening. DCCs reawakened upon infection with SARS-CoV-2 can populate the premetastatic niche in the lungs and other organs, leading to tumor dissemination. DCC reawakening and consequent neutrophil and monocyte/macrophage activation with an uncontrolled cascade of pro-inflammatory cytokines are the most severe clinical effects of COVID-19. Moreover, neutrophil extracellular traps have been demonstrated to activate the dissemination of premetastatic cells into the lungs. Further studies are warranted to better define the roles of COVID-19 in inflammation as well as in tumor development and tumor cell metastasis; the results of these studies will aid in the development of further targeted therapies, both for cancer prevention and the treatment of patients with COVID-19.

2022 - Cancer Stem Cells and Cell Cycle Genes as Independent Predictors of Relapse in Non-small Cell Lung Cancer: Secondary Analysis of a Prospective Study [Articolo su rivista]
Masciale, V.; Banchelli, F.; Grisendi, G.; D'Amico, R.; Maiorana, A.; Stefani, A.; Morandi, U.; Stella, F.; Dominici, M.; Aramini, B.
abstract

PURPOSE: Cancer stem cells (CSCs) are described as resistant to chemotherapy and radiotherapy. It has been shown that CSCs influence disease-free survival in patients undergoing surgery for lung cancer (NCT04634630). We recently described an overexpression of CSCs recurrence-related genes (RG) in lung cancer. This study aims to investigate CSC frequency and RG expression as predictors of disease-free survival in lung cancer. EXPERIMENTAL DESIGN: This secondary analysis of a prospective cohort study involved 22 surgical tumor specimens from 22 patients harboring early (I-II) and locally advanced (IIIA) stages ACL and SCCL. Cell population frequency analysis of ALDHhigh (CSCs) and ALDHlow (cancer cells) was performed on each tumor specimen. In addition, RG expression was assessed for 31 target genes separately in ALDHhigh and ALDHlow populations. CSCs frequency and RG expression were assessed as predictors of disease-free survival by Cox analysis. RESULTS: CSCs frequency and RG expression were independent predictors of disease-free survival. CSC frequency was not related to disease-free survival in early-stage patients (HR = 0.84, 95%CI = 0.53-1.33, P = .454), whereas it was a risk factor for locally advanced-stage patients (HR = 1.22, 95%CI = 1.09-1.35, P = .000). RG expression-if measured in CSCs-was related to a higher risk of recurrence (HR = 1.19, 95%CI = 1.03-1.39, P = .021). The effect of RG expression measured in cancer cells on disease-free survival was lower and was not statistically significant (HR = 1.12, 95%CI = 0.94-1.33, P = .196). CONCLUSIONS: CSCs frequency and RG expression are independent predictors of relapse in lung cancer. Considering these results, CSCs and RG may be considered for both target therapy and prognosis.

2022 - Cancer Stem Cells (CSCs), Circulating Tumor Cells (CTCs) and Their Interplay with Cancer Associated Fibroblasts (CAFs): A New World of Targets and Treatments [Articolo su rivista]
Aramini, B.; Masciale, V.; Arienti, C.; Dominici, M.; Stella, F.; Martinelli, G.; Fabbri, F.
abstract

The importance of defining new molecules to fight cancer is of significant interest to the scientific community. In particular, it has been shown that cancer stem cells (CSCs) are a small sub-population of cells within tumors with capabilities of self-renewal, differentiation, and tumorigenicity; on the other side, circulating tumor cells (CTCs) seem to split away from the primary tumor and appear in the circulatory system as singular units or clusters. It is becoming more and more important to discover new biomarkers related to these populations of cells in combination to define the network among them and the tumor microenvironment. In particular, cancer-associated fibroblasts (CAFs) are a key component of the tumor microenvironment with different functions, including matrix deposition and remodeling, extensive reciprocal signaling interactions with cancer cells and crosstalk with immunity. The settings of new markers and the definition of the molecular connections may present new avenues, not only for fighting cancer but also for the definition of more tailored therapies.

2022 - Carcinoid Crisis: A Misunderstood and Unrecognized Oncological Emergency [Articolo su rivista]
Bardasi, C.; Benatti, S.; Luppi, G.; Garajova, I.; Piacentini, F.; Dominici, M.; Gelsomino, F.
abstract

Carcinoid Crisis represents a rare and extremely dangerous manifestation that can occur in patients with Neuroendocrine Tumors (NETs). It is characterized by a sudden onset of hemodynamic instability, sometimes associated with the classical symptoms of carcinoid syndrome, such as bronchospasm and flushing. Carcinoid Crisis seems to be caused by a massive release of vasoactive substances, typically produced by neuroendocrine cells, and can emerge after abdominal procedures, but also spontaneously in rare instances. To date, there are no empirically derived guidelines for the management of this cancer-related medical emergency, and the available evidence essentially comes from single-case reports or dated small retrospective series. A transfer to the Intensive Care Unit may be necessary during the acute setting, when the severe hypotension becomes unresponsive to standard practices, such as volemic filling and the infusion of vasopressor therapy. The only effective strategy is represented by prevention. The administration of octreotide, anxiolytic and antihistaminic agents represents the current treatment approach to avoid hormone release and prevent major complications. However, no standard protocols are available, resulting in great variability in terms of schedules, doses, ways of administration and timing of prophylactic treatments.

2022 - CRISPR-mediated T cell engineering against Non-Small Cell Lung Cancer [Abstract in Atti di Convegno]
Benati, Daniela; Ferrari, Tommaso; Masciale, Valentina; Grisendi, Giulia; Aramini, Beatrice; Dominici, Massimo; Recchia, Alessandra
abstract

2022 - CtDNA-guided rechallenge with anti-EGFR therapy in RASwt metastatic colorectal cancer: Evidence from clinical practice [Articolo su rivista]
D’Onofrio, Raffaella; Caputo, Francesco; Prampolini, Francesco; Spallanzani, Andrea; Gelsomino, Fabio; Bettelli, Stefania; Manfredini, Samantha; Reggiani Bonetti, Luca; Carotenuto, Pietro; Bocconi, Alessandro; Dominici, Massimo; Luppi, Gabriele; Salati, Massimiliano
abstract

Aim: To apply extended ctDNA-based RAS genotyping to clinical criteria for improving the selection of patients eligible for anti-EGFR-based rechallenge in a real-world setting. Methods: ctDNA testing was prospectively applied to RASwt mCRC progressed after a first-line anti-EGFR-containing regimen and at least one other line. The primary endpoint was the objective response rate. Results: Among ten enrolled patients, the anti-EGFR rechallenge resulted in an objective response rate and disease control rate of 70% and 90%. The median progression-free survival was 11.3 months and overall survival was not reached. Compared with a historical cohort retreated with anti-EGFR agents based on clinical criteria, the ctDNA-driven approach resulted in a higher chance of achieving an objective response and longer survival. Conclusions: Blood-based RASwt status may enrich metastatic colorectal cancer more likely to benefit from anti-EGFR-based rechallenge. RAS genotyping in ctDNA represents a feasible, fast, and cost-effective tool to be implemented in the clinic for advancing precision medicine.

2022 - Current Status of Mesenchymal Stem/Stromal Cells for Treatment of Neurological Diseases [Articolo su rivista]
Soares, M. B. P.; Goncalves, R. G. J.; Vasques, J. F.; da Silva-Junior, A. J.; Gubert, F.; Santos, G. C.; de Santana, T. A.; Almeida Sampaio, G. L.; Silva, D. N.; Dominici, M.; Mendez-Otero, R.
abstract

Neurological disorders include a wide spectrum of clinical conditions affecting the central and peripheral nervous systems. For these conditions, which affect hundreds of millions of people worldwide, generally limited or no treatments are available, and cell-based therapies have been intensively investigated in preclinical and clinical studies. Among the available cell types, mesenchymal stem/stromal cells (MSCs) have been widely studied but as yet no cell-based treatment exists for neurological disease. We review current knowledge of the therapeutic potential of MSC-based therapies for neurological diseases, as well as possible mechanisms of action that may be explored to hasten the development of new and effective treatments. We also discuss the challenges for culture conditions, quality control, and the development of potency tests, aiming to generate more efficient cell therapy products for neurological disorders.

2022 - Delayed Effect of Dendritic Cells Vaccination on Survival in Glioblastoma: A Systematic Review and Meta‐Analysis [Articolo su rivista]
Cozzi, S.; Najafi, M.; Gomar, M.; Ciammella, P.; Iotti, C.; Iaccarino, C.; Dominici, M.; Pavesi, G.; Chiavelli, C.; Kazemian, A.; Jahanbakhshi, A.
abstract

Background: Dendritic cell vaccination (DCV) strategies, thanks to a complex immune response, may flare tumor regression and improve patients’ long‐term survival. This meta‐analysis aims to assess the efficacy of DCV for newly diagnosed glioblastoma patients in clinical trials. Meth-ods: The study databases, including PubMed, Web of Knowledge, Google Scholar, Scopus, and Cochrane, were searched by two blinded investigators considering eligible studies based on the following keywords: “glioblastoma multiforme”, “dendritic cell”, “vaccination”, “immunother-apy”, “immune system”, “immune response”, “chemotherapy”, “recurrence”, and “te-mozolomide”. Among the 157 screened, only 15 articles were eligible for the final analysis. Results: Regimens including DCV showed no effect on 6‐month progression‐free survival (PFS, HR = 1.385, 95% CI: 0.822–2.335, p = 0.673) or on 6‐month overall survival (OS, HR = 1.408, 95% CI: 0.882–2.248, p = 0.754). In contrast, DCV led to significantly longer 1‐year OS (HR = 1.936, 95% CI: 1.396–2.85, p = 0.001) and longer 2‐year OS (HR = 3.670, 95% CI: 2.291–5.879, p = 0.001) versus control groups. Hence, introducing DCV could lead to increased 1 and 2‐year survival of patients by 1.9 and 3.6 times, respectively. Conclusion: Antitumor regimens including DCV can effectively improve mid-term survival in patients suffering glioblastoma multiforme (GBM), but its impact emerges only after one year from vaccination. These data indicate the need for more time to achieve an anti‐GBM immune response and suggest additional therapeutics, such as checkpoint inhibitors, to empower an earlier DCV action in patients affected by a very poor prognosis.

2022 - Development and Multicenter Validation of a Novel Immune-Inflammation-Based Nomogram to Predict Survival in Western Resectable Gastric and Gastroesophageal Junction Adenocarcinoma (GEA): The NOMOGAST [Articolo su rivista]
Salati, Massimiliano; De Ruvo, Nicola; Giglio, Mariano Cesare; Sorrentino, Lorena; Esposito, Giuseppe; Fenocchi, Sara; Cucciarrè, Giovanni; Serra, Francesco; Rossi, Elena Giulia; Vittimberga, Giovanni; Radi, Giorgia; Solaini, Leonardo; Morgagni, Paolo; Grizzi, Giulia; Ratti, Margherita; Gelsomino, Fabio; Spallanzani, Andrea; Ghidini, Michele; Ercolani, Giorgio; Dominici, Massimo; Gelmini, Roberta
abstract

2022 - Development and Multicentre Validation of the Modena Score to Predict Survival in Advanced Biliary Cancers Undergoing Second-Line Chemotherapy [Articolo su rivista]
Salati, M.; Marcheselli, L.; Messina, C.; Merz, V.; Messina, M.; Carotenuto, P.; Caputo, F.; Gelsomino, F.; Spallanzani, A.; Bonetti, L. R.; Caramaschi, S.; Luppi, G.; Dominici, M.; Ghidini, M.
abstract

Background: The role of second-line chemotherapy in advanced biliary cancers (ABCs) has only recently been established in phase III randomized trial and the optimal selection of patients most likely to benefit from it remains challenging. Methods: A cohort of 98 ABC treated second-line chemotherapy was used as a developmental dataset to identify covariates independently associated with overall survival (OS). Kaplan–Meier analysis was used to investigate the association between variables and OS and those retaining statistically significance were combined in a multiplexed score. Results: The following pretreatment variables were independently associated with OS: ECOG PS > 0, peritoneal disease, LDH > 430 UI/L, albumin <3.5 gr/dL, gamma-GT >100 UI/L, sodium <140 mEq/L, absolute lymphocyte count <1000/mmc, and PFS to first-line <6 months. Based on these results, a scoring system was developed that identified three subgroups with statistically different OS: low-risk (mOS 18 months), intermediate-risk (mOS 9.4 months) and high-risk (mOS 2.9 months) (p < 0.001). The prognostic model was both internally and externally validated in a multicentre cohort of 120 ABCs. Conclusion: The Modena score is a multiplexed scoring system capable of accurately risk-stratified ABCs treated with second-line chemotherapy. Based on its reproducibility, usability and generalizability, it has the potential for assisting therapeutic decision-making in the clinic and risk-stratification in future trials.

2022 - Dissecting Immunotherapy Strategies for Small Cell Lung Cancer: Antibodies, Ionizing Radiation and CAR-T [Articolo su rivista]
Guaitoli, G.; Neri, G.; Cabitza, E.; Natalizio, S.; Mastrodomenico, L.; Talerico, S.; Trudu, L.; Lauro, C.; Chiavelli, C.; Baschieri, M. C.; Bruni, A.; Dominici, M.; Bertolini, F.
abstract

Small cell lung cancer (SCLC) is a highly aggressive malignancy that accounts for about 14% of all lung cancers. Platinum-based chemotherapy has been the only available treatment for a long time, until the introduction of immune checkpoint inhibitors (ICIs) recently changed first-line standard of care and shed light on the pivotal role of the immune system. Despite improved survival in a subset of patients, a lot of them still do not benefit from first-line chemo-immunotherapy, and several studies are investigating whether different combination strategies (with both systemic and local treatments, such as radiotherapy) may improve patient outcomes. Moreover, research of biomarkers that may be used to predict patients’ outcomes is ongoing. In addition to ICIs, immunotherapy offers other different strategies, including naked monoclonal antibodies targeting tumor associated antigens, conjugated antibody, bispecific antibodies and cellular therapies. In this review, we summarize the main evidence available about the use of immunotherapy in SCLC, the rationale behind combination strategies and the studies that are currently ongoing in this setting, in order to give the reader a clear and complete view of this rapidly expanding topic.

2022 - Dissecting Tumor Growth: The Role of Cancer Stem Cells in Drug Resistance and Recurrence [Articolo su rivista]
Aramini, B.; Masciale, V.; Grisendi, G.; Bertolini, F.; Mauer, M.; Guaitoli, G.; Chrystel, I.; Morandi, U.; Stella, F.; Dominici, M.; Haider, K. H.
abstract

Emerging evidence suggests that a small subpopulation of cancer stem cells (CSCs) is responsible for initiation, progression, and metastasis cascade in tumors. CSCs share characteristics with normal stem cells, i.e., self-renewal and differentiation potential, suggesting that they can drive cancer progression. Consequently, targeting CSCs to prevent tumor growth or regrowth might offer a chance to lead the fight against cancer. CSCs create their niche, a specific area within tissue with a unique microenvironment that sustains their vital functions. Interactions between CSCs and their niches play a critical role in regulating CSCs’ self-renewal and tumorigenesis. Differences observed in the frequency of CSCs, due to the phenotypic plasticity of many cancer cells, remain a challenge in cancer therapeutics, since CSCs can modulate their transcriptional activities into a more stem-like state to protect themselves from destruction. This plasticity represents an essential step for future therapeutic approaches. Regarding self-renewal, CSCs are modulated by the same molecular pathways found in normal stem cells, such as Wnt/β-catenin signaling, Notch signaling, and Hedgehog signaling. Another key characteristic of CSCs is their resistance to standard chemotherapy and radiotherapy treatments, due to their capacity to rest in a quiescent state. This review will analyze the primary mechanisms involved in CSC tumorigenesis, with particular attention to the roles of CSCs in tumor progression in benign and malignant diseases; and will examine future perspectives on the identification of new markers to better control tumorigenesis, as well as dissecting the metastasis process.

2022 - Exceptional response to lurbinectedin and irinotecan in BRCA-mutated platinum-resistant ovarian cancer patient: a case report [Articolo su rivista]
Cortesi, L.; Venturelli, M.; Barbieri, E.; Baldessari, C.; Bardasi, C.; Coccia, E.; Baglio, F.; Rimini, M.; Greco, S.; Napolitano, M.; Pipitone, S.; Dominici, M.
abstract

Lurbinectedin is responsible for DNA recognition and binding, producing double-strand DNA (dsDNA) breaks thus resulting in apoptosis. Sensitivity to lurbinectedin is linked to the nucleotide excision repair (NER) system. Furthermore, irinotecan, a topoisomerase I inhibitor, provokes dsDNA breaks that could be reinforced abrogating the NER system using lurbinectedin. BRCA-mutated patients, already treated with platinum-derived drugs, who suffered DNA damage, cannot repair the breaks due to lurbinectedin interaction, whereas irinotecan provokes a dsDNA break that promotes synthetic lethality. This article describes an exceptional response to lurbinectedin alone followed by the association with irinotecan in a BRCA-mutated platinum-resistant ovarian cancer patient. A 44-year-old BRCA1-mutated ovarian cancer patient was treated in sixth line with lurbinectedin and irinotecan with a time to further progression (TTFP) equal to 8 months. In our case, the association with irinotecan overcame the resistance to lurbinectedin alone. In conclusion, lurbinectedin and irinotecan demonstrated a promising response in platinum-resistant patients. However, further studies should be conducted to validate our findings and future trials will be important to further define the clinical utility of lurbinectedin.

2022 - Human Adipose Mesenchymal Stromal/Stem Cells Improve Fat Transplantation Performance [Articolo su rivista]
Piccinno, M. S.; Petrachi, T.; Pignatti, M.; Murgia, A.; Grisendi, G.; Candini, O.; Resca, E.; Bergamini, V.; Ganzerli, F.; Portone, A.; Mastrolia, I.; Chiavelli, C.; Castelli, I.; Bernabei, D.; Tagliazucchi, M.; Bonetti, E.; Lolli, F.; De Santis, G.; Dominici, M.; Veronesi, E.
abstract

The resorption rate of autologous fat transfer (AFT) is 40–60% of the implanted tissue, requiring new surgical strategies for tissue reconstruction. We previously demonstrated in a rabbit model that AFT may be empowered by adipose-derived mesenchymal stromal/stem cells (AD-MSCs), which improve graft persistence by exerting proangiogenic/anti-inflammatory effects. However, their fate after implantation requires more investigation. We report a xenograft model of adipose tissue engineering in which NOD/SCID mice underwent AFT with/without human autologous AD-MSCs and were monitored for 180 days (d). The effect of AD-MSCs on AFT grafting was also monitored by evaluating the expression of CD31 and F4/80 markers. Green fluorescent protein-positive AD-MSCs (AD-MSC-GFP) were detected in fibroblastoid cells 7 days after transplantation and in mature adipocytes at 60 days, indicating both persistence and differentiation of the implanted cells. This evidence also correlated with the persistence of a higher graft weight in AFT-AD-MSC compared to AFT alone treated mice. An observation up to 180 d revealed a lower resorption rate and reduced lipidic cyst formation in the AFT-AD-MSC group, suggesting a long-term action of AD-MSCs in support of AFT performance and an anti-inflammatory/proangiogenic activity. Together, these data indicate the protective role of adipose progenitors in autologous AFT tissue resorption.

2022 - Immune-Checkpoint-Inhibitor-Related Lung Toxicity: A Multicentre Real-Life Retrospective Portrait from Six Italian Centres [Articolo su rivista]
Cameli, P.; Faverio, P.; Ferrari, K.; Bonti, V.; Marsili, S.; Mazzei, M. A.; Mazzoni, F.; Bartolucci, M.; Scotti, V.; Bertolini, F.; Barbieri, F.; Baldessari, C.; Veronese, C.; Boffi, R.; Brighenti, M.; Cortinovis, D.; Dominici, M.; Pesci, A.; Bargagli, E.; Luppi, F.
abstract

Background: Immune checkpoint inhibitors (ICIs) have revolutionized the therapeutic horizons of various cancers. However, immune-related adverse events have been reported, including interstitial lung diseases. Our aim was to describe the clinical and radiological features and survival of a multicentre cohort of patients who developed ICI-related lung toxicity. Methods: Six Italian centres were involved in the study. Patients who were treated with anti-PD-1/PD-L1 and CTLA-4 mAbs and developed ICI-related lung toxicity were recruited retrospectively to study clinical, radiological, immunological and survival data. Results: A total of 41 patients (25 males, 66.8 ± 9.9 years) were enrolled. Lung toxicity occurred after 204.3 ± 208.3 days of therapy, with ground glass opacities being the most common HRCT pattern (23 cases). Male sex, lung cancer and acute respiratory failure were associated with a shorter latency of toxicity (p = 0.0030, p = 0.0245 and p = 0.0390, respectively). Patients who required high-flow oxygen therapy showed significantly worse survival (p = 0.0028). Conclusions: Our cohort showed heterogeneous clinical and radiological aspects of ICI-related lung toxicity, with a latency not limited to the first year of treatment. Severity was mainly mild to moderate, although life-threatening events did occur. Our data indicate that strict long-term follow-up is needed to enable early diagnosis and appropriate management.

2022 - Multicentre match-paired analysis of advanced biliary cancer long-term survivors: The BILONG study [Articolo su rivista]
Caputo, F.; Gelsomino, F.; Spallanzani, A.; Pettorelli, E.; Benatti, S.; Ghidini, M.; Grizzi, G.; Ratti, M.; Merz, V.; Messina, C.; Tonelli, R.; Luppi, G.; Melisi, D.; Dominici, M.; Salati, M.
abstract

Background: Advanced biliary cancers (ABC) are aggressive malignancies with a median overall survival (mOS) <12 months when treated with first-line chemotherapy. Nevertheless, a subset of patients experiencing longer survival has been described in the updated analysis of ABC-02 trial. We aimed to provide a real-world description of ABC long-term survivors (LS), identifying which factors impact on survival. Methods: Patients diagnosed with ABC at three Institutions between 2002 and 2019, and who survived ≥18 months, were retrospectively identified. We compared them with a control cohort (C) with a mOS <18 months, matched on age, gender, ECOG PS, disease status, primary tumor site, prior surgery, and treatment modality. Their clinical features, treatments, and outcome were analyzed. Results: A total of 78 patients was included, 39 in each group. Both LS and C cohorts had superimposable baseline characteristics, without significant differences. mOS was 29 (95%CI 24.6–33.5) and 9 months (95%CI 6.6–12.9) in the two groups, respectively. After performing a logistic regression analysis, three factors were significantly associated with long-term outcome: low neutrophil-to-lymphocyte ratio (NLR < 3) (Odds Ratio [OR] 0.38), achievement of objective response to treatment (OR 0.16), and the number of lines received (OR 0.29). Conclusions: We described a considerable subset of ABC experiencing long-term survival with conventional chemotherapy in a real-world scenario. Beyond clinical factors, we identified low NLR as a prognostic determinant that may allow for a more accurate selection of long survivors. While waiting for a deeper molecular characterization of this subgroup, we propose NLR as a stratification factor for daily practice and clinical trials.

2022 - Myocarditis and diaphragmatic rhabdomyolysis with respiratory failure in a patient with metastatic melanoma treated with Nivolumab [Articolo su rivista]
Baldessari, C.; Pugliese, G.; Venturelli, M.; Greco, S.; Ferrara, L.; Longo, G.; Dominici, M.; Depenni, R.
abstract

Introduction: Immunotherapy dramatically changed history of melanoma patients with a clinical benefit never seen before. Nevertheless, severe and unexpected adverse effects can occur, fortunately rarely. Case presentation: We reported the case of a 75-year-old male patient affected by metastatic melanoma who developed myocarditis and acute rhabdomyolysis with secondary diaphragmatic dysfunction and consequent pulmonary restrictive syndrome after Nivolumab monotherapy. Blood tests and ultrasonography of the diaphragm revealing left hypokinesis suggested a Nivolumab-related rhabdomyolysis, as an immune-mediated adverse event. The rhabdomylolysis involved the diaphragm with consequent diaphragmatic weakness and respiratory distress. Mangement & outcome: The patient had a slow but slight and progressive improvement of symptoms and vital signs post-treatment with high-dose corticosteroids. Discussion: With this case report, we want to highlight the importance of rapid recognition and treatment of rare and unexpected, but potential serious immune-related adverse events. These events might happen despite the remarkable clinical benefits of immune checkpoint inhibitors. We do not know which patients will benefit from these therapies and why, when and in which cases adverse event will occur: we must not lower our attention.

2022 - Ocular Toxicity in Breast Cancer Management: Manual for The Oncologist [Articolo su rivista]
Canino, F.; Omarini, C.; Cerma, K.; Moscetti, L.; Tornincasa, A.; Trudu, L.; Dominici, M.; Piacentini, F.
abstract

Ocular adverse events are common to many antineoplastic agents, although often misunderstood. In most cases, they are easily manageable, but sometimes they require instrumental diagnostics and specific treatments. There are currently no international guidelines for the management of these toxicities. In this review we summarized the main ocular adverse events related to the antineoplastic agents used in the treatment of breast cancer, analyzing their clinical presentation and management, trying to provide a useful tool to be used in clinical practice.

2022 - Phenotypic, functional, and metabolic heterogeneity of immune cells infiltrating non–small cell lung cancer [Articolo su rivista]
Aramini, B.; Masciale, V.; Samarelli, A. V.; Dubini, A.; Gaudio, M.; Stella, F.; Morandi, U.; Dominici, M.; De Biasi, S.; Gibellini, L.; Cossarizza, A.
abstract

Lung cancer is the leading cancer in the world, accounting for 1.2 million of new cases annually, being responsible for 17.8% of all cancer deaths. In particular, non-small cell lung cancer (NSCLC) is involved in approximately 85% of all lung cancers with a high lethality probably due to the asymptomatic evolution, leading patients to be diagnosed when the tumor has already spread to other organs. Despite the introduction of new therapies, which have improved the long-term survival of these patients, this disease is still not well cured and under controlled. Over the past two decades, single-cell technologies allowed to deeply profile both the phenotypic and metabolic aspects of the immune cells infiltrating the TME, thus fostering the identification of predictive biomarkers of prognosis and supporting the development of new therapeutic strategies. In this review, we discuss phenotypic and functional characteristics of the main subsets of tumor-infiltrating lymphocytes (TILs) and tumor-infiltrating myeloid cells (TIMs) that contribute to promote or suppress NSCLC development and progression. We also address two emerging aspects of TIL and TIM biology, i.e., their metabolism, which affects their effector functions, proliferation, and differentiation, and their capacity to interact with cancer stem cells.

2022 - Predictive factors for relapse in triple-negative breast cancer patients without pathological complete response after neoadjuvant chemotherapy [Articolo su rivista]
Toss, A.; Venturelli, M.; Civallero, M.; Piombino, C.; Domati, F.; Ficarra, G.; Combi, F.; Cabitza, E.; Caggia, F.; Barbieri, E.; Barbolini, M.; Moscetti, L.; Omarini, C.; Piacentini, F.; Tazzioli, G.; Dominici, M.; Cortesi, L.
abstract

IntroductionTriple-negative breast cancer (TNBC) patients who do not obtain pathological complete response (pCR) after neoadjuvant chemotherapy (NACT) present higher rate of relapse and worse overall survival. Risk factors for relapse in this subset of patients are poorly characterized. This study aimed to identify the predictive factors for relapse in TNBC patients without pCR after NACT. MethodsWomen with TNBC treated with NACT from January 2008 to May 2020 at the Modena Cancer Center were included in the analysis. In patients without pCR, univariate and multivariable Cox analyses were used to determine factors predictive of relapse. ResultsWe identified 142 patients with a median follow-up of 55 months. After NACT, 62 patients obtained pCR (43.9%). Young age at diagnosis (<50 years) and high Ki-67 (20%) were signi!cantly associated with pCR. Lack of pCR after NACT resulted in worse 5-year event-free survival (EFS) and overall survival (OS). Factors independently predicting EFS in patients without pCR were the presence of multifocal disease [hazard ratio (HR), 3.77; 95% CI, 1.45-9.61; p=0.005] and residual cancer burden (RCB) III (HR, 3.04; 95% CI, 1.09-9.9; p=0.04). Neither germline BRCA status nor HER2-low expression were associated with relapse. DiscussionThese data can be used to stratify patients and potentially guide treatment decision-making, identifying appropriate candidates for treatment intensi!cation especially in neo-/adjuvant setting.

2022 - Redistribution of CD8+ T cell subsets in metastatic renal cell carcinoma patients treated with anti-PD-1 therapy [Articolo su rivista]
De Biasi, S.; Guida, A.; Lo Tartaro, D.; Fanelli, M.; Depenni, R.; Dominici, M.; Finak, G.; Porta, C.; Paolini, A.; Borella, R.; Bertoldi, C.; Cossarizza, A.; Sabbatini, R.; Gibellini, L.
abstract

Renal-cell carcinoma (RCC) is responsible for the majority of tumors arising from the kidney parenchyma. Although a progressive improvement in median overall survival has been observed after the introduction of anti-PD-1 therapy, many patients do not benefit from this treatment. Therefore, we have investigated T cell dynamics to find immune modification induced by anti-PD-1 therapy. Here, we show that, after therapy, RCC patients (5 responders and 14 nonresponders) are characterized by a redistribution of different subsets across the memory T cell compartment.

2022 - Role of Intrinsic Subtype Analysis with PAM50 in Hormone Receptors Positive HER2 Negative Metastatic Breast Cancer: A Systematic Review. [Articolo su rivista]
Canino, F; Piacentini, F; Omarini, C; Toss, A; Barbolini, M; Vici, P; Dominici, M; Moscetti, L.
abstract

Endocrine therapy (ET), associated with CDK 4/6 inhibitors, represents the first choice of treatment for HR+/HER2- metastatic breast cancer (mBC). Primary or secondary endocrine resistance could develop; however validated biomarkers capable of predicting such a conditions are not available. Several studies have shown that HR+/HER2- mBC comprises five intrinsic subtypes. The purpose of this systematic review was to analyze the potential correlations between intrinsic subtype, efficacy of treatment, and patient outcome. Five papers that analyzed the intrinsic subtype with PAM50 assay in patients (pts) with HR+/HER2- mBC treated with ET (alone or in combination) within seven phase III clinical trials (EGF30008, BOLERO-2, PALOMA-2,3, MONALEESA-2,3,7) were identified. Non-luminal subtypes are more frequent in endocrine-resistant pts and in metastatic sites (vs. primary tumors), have less benefit from ET, and worse prognosis. Among these, HER2-enriched subtypes are similar to HER2+ tumors and benefit from the addition of anti-HER2 agents (lapatinib) and, for less clear reasons, of ribociclib (unconfirmed data for palbociclib and everolimus). Basal-like subtypes are similar to triple-negative tumors, making them more sensitive to chemotherapy. The intrinsic subtype is also not static but can vary over time with the evolution of the disease. Currently, the intrinsic subtype does not play a decisive role in the choice of treatment in clinical practice, but has potential prognostic and predictive value that should be further investigated.

2022 - Statins increase pathological response in locally advanced rectal cancer treated with chemoradiation: a multicenter experience. [Articolo su rivista]
Caputo, F; Santini, C; Casadei-Gardini, A; Cerma, K; Bardasi, C; Garajovà, I; Lattanzi, E; Passardi, A; Rapposelli, Ig; Spallanzani, A; Salati, M; Bonetti, Lr; Gelmini, R; Meduri, B; Piccoli, M; Pecchi, A; Benatti, S; Piacentini, F; Dominici, M; Luppi, G; Gelsomino, F.
abstract

Aims: To investigate the influence of various concomitant medications on outcomes in patients with locally advanced rectal cancer undergoing neoadjuvant chemoradiation. Materials & methods: The authors retrospectively identified 246 patients from 2003 to 2018, collecting demographic and clinicopathological data of interest. Odds ratio (OR) was used to assess the association between concomitant drugs and outcomes. Results: The authors found an association between statins and a Dworak regression grade of 3-4 (OR = 8.78; p = 0.01). Furthermore, statins were significantly associated with more frequent chemoradiation-related toxicity (OR = 2.39; p = 0.0098) and chemotherapy dose reduction or discontinuation (OR = 2.26; p = 0.03). Conclusion: Despite higher frequency of radiotherapy and chemotherapy interruption or dose reduction, the concomitant use of statins during neoadjuvant chemoradiation proved to be associated with better tumor regression.

2022 - Successes and failures of angiogenesis blockade in gastric and gastro-esophageal junction adenocarcinoma [Articolo su rivista]
Salati, M.; Caputo, F.; Bocconi, A.; Cerri, S.; Baldessari, C.; Piacentini, F.; Dominici, M.; Gelsomino, F.
abstract

Gastric and gastro-esophageal junction adenocarcinoma (GEA) remains a considerable major public health problem worldwide, being the fifth most common cancer with a fatality-to-case ratio that stands still at 70%. Angiogenesis, which is a well-established cancer hallmark, exerts a fundamental role in cancer initiation and progression and its targeting has been actively pursued as a promising therapeutic strategy in GEA. A wealth of clinical trials has been conducted, investigating anti-angiogenic agents including VEGF-directed monoclonal antibodies, small molecules tyrosine kinase inhibitors and VEGF-Trap agents both in the resectable and advanced setting, reporting controversial results. While phase III randomized trials testing the anti-VEGFR-2 antibody Ramucirumab and the selective VEGFR-2 tyrosine kinase inhibitor Apatinib demonstrated a significant survival benefit in later lines, the shift of angiogenesis inhibitors in the perioperative and first-line setting failed to improve patients’ outcome in GEAs. The molecular landscape of disease, together with novel combinatorial strategies and biomarker-selected approaches are under investigation as key elements to the success of angiogenesis blockade in GEA. In this article, we critically review the existing literature on the biological rationale and clinical development of antiangiogenic agents in GEA, discussing major achievements, limitations and future developments, aiming at fully realizing the potential of this therapeutic approach.

2022 - Survey on the effectiveness of telephone-based communication with relatives of hospitalized cancer patients in COVID-19 era in Italy [Articolo su rivista]
Ricco, B.; Fiorani, C.; Ferrara, L.; Potenza, L.; Saviola, A.; Malavasi, N.; Acquaviva, G.; Carboni, C.; Scarabelli, L.; Dominici, M.; Luppi, M.; Longo, G.
abstract

Objective: No-visitor policies adopted to prevent coronavirus disease-19 (COVID-19) spread in hospital wards have deeply impacted communication with patients and their relatives. Whereas in pre-COVID-19 era family-clinician meetings were held in person, during the pandemic interactions often took place over the phone, frequently causing feelings of uncertainty and distress to the close ones at home. The goal of this study was to assess and improve the effectiveness of structured telephone-based communication with hospitalized onco-hematological patients’ relatives in COVID-19 era. Methods: After no-visitor policy was adopted in the Onco-Hematological Unit of Modena, inpatients’ relatives were contacted daily for clinical updates. After discharge, a telephone satisfaction survey was administered to all contact people of patients consecutive admitted between December 2020 and January 2021 (n = 97). Mean score of response and potential statistically significative differences depending on respondents’ characteristics were assessed. Results: Most relatives were satisfied with the communication received with a mean total score of 4.69 on a 5-point Likert scale (standard deviation: 0.60). Results showed high satisfaction rate with both the informative (mean ± SD: 4.66 ± 0.64) and emotional (mean ± SD: 4.66 ± 0.58) content, with no significant difference depending on respondents’ demographic characteristics (p > 0.05). Conclusion: A structured telephone-based communication may be a reasonable substitute for face-to-face meetings; especially if regular in time, conducted by the same doctor and integrated with video calls. Our findings might assist health workers in implementing measures to minimize the psychological effects of no-visitor policies during hospitalization. Clinical updates delivery through structured phone calls and video calls could become an opportunity also in post-COVID era.

2022 - T-DM1 efficacy in trastuzumab-pertuzumab pre-treated HER2 positive metastatic breast cancer patients: a meta-analysis. [Articolo su rivista]
Omarini, C; Piacentini, F; Sperduti, I; Cerma, K; Barbolini, M; Canino, F; Nasso, C; Isca, C; Caggia, F; Dominici, M; Moscetti, L.
abstract

Background: Current guidelines consider T-DM1 the standard 2nd line therapy for HER2 positive metastatic breast cancer (MBC) patients following trastuzumab (T) + pertuzumab (P) and taxane 1st line treatment. Despite this, there are no prospective studies supporting this sequence. Methods: We performed a meta-analysis using real world data to determine the efficacy of T-DM1 after 1st line TP in HER2 positive MBC patients. We used a random-effect model to find differences in the rate of 1-year progression free survival (PFS) between TP pre-treated population and the EMILIA phase III pivotal trial. Results: Seven studies were eligible. The meta-analysis showed a combined 1-year PFS risk difference for T-DM1 efficacy after TP in 2nd or more lines of -0.122, with lower and upper limits of -0.253 and 0.010, respectively (p = 0.07), with low heterogeneity among studies (I2 0.01%, p = 0.836). Considering the four studies on T-DM1 in 2nd line setting, 1-year PFS risk was -0.034 (95% CI -0.207 - 0,139; p = 0.701) (I2 0.01%, p = 0.91). Conclusion: Overall, the efficacy of T-DM1 after TP seems to be similar to that previously reported in the EMILIA trial. In the second line setting, data are not mature enough to confirm T-DM1 efficacy in TP pre-treated population.

2022 - The impact of UEFA Euro 2020 football championship on Takotsubo Syndrome: Results of a multicenter national registry [Articolo su rivista]
Polimeni, A.; Spaccarotella, C.; Ielapi, J.; Esposito, G.; Ravera, A.; Martuscelli, E.; Ciconte, V.; Menichelli, M.; Varbella, F.; Imazio, M.; Navazio, A.; Sinagra, G.; Oberhollenzer, R.; Sibilio, G.; Cacciavillani, L.; Meloni, L.; Dominici, M.; Tomai, F.; Amico, F.; Corda, M.; Musumeci, G.; Lupi, A.; Zezza, L.; De Caterina, R.; Cernetti, C.; Metra, M.; Rossi, L.; Calabro, P.; Murrone, A.; Volpe, M.; Caldarola, P.; Carugo, S.; Cortese, B.; Valenti, R.; Boriani, G.; Fedele, F.; Ventura, G.; Manes, M. T.; Colavita, A. R.; Feola, M.; Versaci, F.; Assennato, P.; Arena, G.; Ceravolo, R.; Amodeo, V.; Tortorici, G.; Nassiacos, D.; Antonicelli, R.; Esposito, N.; Favale, S.; Licciardello, G.; Tedesco, L.; Indolfi, C.
abstract

Objectives: The UEFA 2020 European Football Championship held in multiple cities across Europe from June 11 to July 11, 2021, was won by Italy, providing an opportunity to examine the relationship between emotional stress and the incidence of acute cardiovascular events (ACE). Methods and results: Cardiovascular hospitalizations in the Cardiac Care Units of 49 hospital networks in Italy were assessed by emergency physicians during the UEFA Euro 2020 Football Championship. We compared the events that occurred during matches involving Italy with events that occurred during the remaining days of the championship as the control period. ACE was assessed in 1,235 patients. ACE during the UEFA Euro 2020 Football Championship semifinal and final, the most stressful matches ended with penalties and victory of the Italian team, were assessed. A significant increase in the incidence of Takotsubo Syndrome (TTS) by a factor of 11.41 (1.6–495.1, P < 0.003), as compared with the control period, was demonstrated during the semifinal and final, whereas no differences were found in the incidence of ACS [IRR 0.93(0.74–1.18), P = 0.57]. No differences in the incidence of ACS [IRR 0.98 (0.87–1.11; P = 0.80)] or TTS [IRR 1.66(0.80–3.4), P = 0.14] were found in the entire period including all matches of the UEFA Euro 2020 compared to the control period. Conclusions: The data of this national registry demonstrated an association between the semifinal and final of UEFA Euro 2020 and TTS suggesting that it can be triggered by also positive emotions such as the victory in the European Football Championship finals.

2022 - The quality of palliative sedation in end-stage disease: audit from a department of oncology and haematology [Articolo su rivista]
Alessia, S.; Matilde, S. F.; Chrystel, I.; Massimiliano, S.; Daniele, D.; Claudia, F.; Raffaella, P.; Flavia, C.; Leonardo, F.; Chiara, C.; Leonardo, P.; Dominici, Massimo; Luppi, Mario; Giuseppe, L.
abstract

Purpose: Palliative sedation (PS) plays a critical role to give suffering relief from refractory symptoms at the end of life. Our audit aimed to assess and improve quality of PS at the Department of Oncology and Hematology of University Hospital of Modena, to verify the adherence to international guidelines, the cooperation among members of care team, focusing with attention on family’s perception of this delicate situation. Methods: From December 2016 to June 2019, data of patients undergoing PS in the Department were collected by an electronic folder tool, “Sedation Tool” (ST), that recorded clinical and PS informations, D-PaP, Rudkin score, and family’s perception. Results: In total, 245 patients were enrolled. Eighty-two percent had a Karnofsky Performance Status 10–20%. The most common cancer types were lung and gastro-intestinal carcinomas (27% and 21% respectively). Refractory symptoms observed were confusion and agitation (76%), dyspnea (39%), pain (15%), delirium (10%), and psychological distress (5%). Midazolam was the drug of choice for PS. Most of patients had Rudkin score 5 after 24 h and 33% had terminal event within a period of 24 h from the beginning of PS. During PS, most of patient’s relatives reported peacefulness (65%), agitation/impatience in 6% of cases, and concern for suffering (16%). Conclusion: PS is used in case of worsening general conditions at the end-stage disease to relieve refractory symptoms with dignity. The ST can become a simple instrument to evaluate and improve PS quality, providing more attention on the impact of PS on relatives to then possibly develop new supportive procedures for patients and their families.

2022 - Third-line chemotherapy in advanced biliary cancers (ABC): pattern of care, treatment outcome and prognostic factors from a multicenter study [Articolo su rivista]
Salati, M.; Rizzo, A.; Merz, V.; Messina, C.; Francesco, C.; Gelsomino, F.; Spallanzani, A.; Ricci, A. D.; Palloni, A.; Frega, G.; De Lorenzo, S.; Carotenuto, P.; Pettorelli, E.; Benatti, S.; Luppi, G.; Melisi, D.; Brandi, G.; Dominici, M.
abstract

Objectives: Here, we aim at describing the pattern of care, survival outcome and prognostic factors of ABC patients (pts) receiving third-line chemotherapy. Methods: Institutional registries across three academic medical centers were retrospectively reviewed. Kaplan–Meier estimators were used to calculate survival, the log-rank test to make comparisons, and the Cox proportional hazard models to assess the progostic impact of variables. Results: Among 101 pts included in the analysis. 68 (67.3%), 19 (18.8%) and 14 (13.8%) had intrahepatic and extrahepatic cholangiocarcinoma and gallbladder cancer, respectively. Atotal of 63 (62.3%) pts received monochemotherapy, while 38 (37.6%) were treated with adoublet. The median OS and PFS were 5 and 3 months, respectively. Disease control rate was achieved in 23 (22.7%) pts, with 2 (2%) partial responses. Grade 3–4 treatment-related adverse events were reported in 22 (21.7%) pts. At multivariate analysis, ECOG PS (p < 0.001), tumor burden (p = 0.01) and lymphocyte-to-monocyte ratio (p =0.02) were independent predictors of survival. Conclusions: Third-line chemotherapy displayed limited activity in this real-world cohort, although prognostic factors have been identified that may assist in treatment decision. The results of this multicenter experience, highlight the need for more effective therapies and provide a benchmark for future trials in this setting.

2022 - Thyroid function impairment after chemo-immunotherapy for advanced NSCLC: A single institutional retrospective report [Articolo su rivista]
Trudu, L.; Guaitoli, G.; Bertolini, F.; Maur, M.; Santini, C.; Papapietro, V. R.; Talerico, S.; Natalizio, S.; Isca, C.; Dominici, M.; Barbieri, F.
abstract

Introduction: Aims of the study were to explore outcomes and toxicities of chemotherapy-immunotherapy (CT-IT) for patients (pts) with metastatic nonsquamous non-small-cell lung cancer (mNSCLC) in a real-world population. Materials & methods: Clinical data of 26 pts with mNSCLC treated with CT-IT at our institution from January 2020 to January 2021 were collected retrospectively. Results: Median follow-up time was 7.7 months. Median progression-free survival was 9.5 months. The most frequent immune-related adverse event was thyroid dysfunction (ThD): 30.7%. Conclusion: There was a higher rate of ThD in this study population compared with the literature, with a possible correlation with clinical outcomes. Plain language summary Aims of the study were to explore outcomes and toxicities of chemotherapy-immunotherapy (CT-IT) for patients (pts) with metastatic nonsquamous NSCLC (mNSCLC) in a real practice experience, because practice experience not always could be the same of experimental experience. We collected clinical data of 26 pts with mNSCLC, treated with CT-IT at our Institution from January 2020 to January 2021. We observed efficacy and tolerability of treatment were similar to known data, except for thyroid disfunctions (ThD) that was more frequent in our experience. This collateral effect was not cause of treatment interruption, indeed the pts with this manifestation would seem responder better to this therapy. However, we need more time and kind of studies for confirm this observation.

2021 - A Novel Three-Dimensional Culture Device Favors a Myelinating Morphology of Neural Stem Cell-Derived Oligodendrocytes [Articolo su rivista]
Flagelli, A.; Candini, O.; Frabetti, S.; Dominici, M.; Giardino, L.; Calza, L.; Baldassarro, V. A.
abstract

The complexity of the central nervous system (CNS) requires researchers to consider all the variables linked to the interaction between the different cell inhabitants. On this basis, any in vitro study of the physiological and pathological processes regarding the CNS should consider the balance between the standardization of the assay and the complexity of the cellular system which mimics the in vivo microenvironment. One of the main structural and functional components of the CNS is the oligodendrocyte precursor cell (OPC), responsible for developmental myelination and myelin turnover and repair during adulthood following differentiation into mature oligodendrocytes. In the present brief research report, we describe a 3D culture tool (VITVO) based on an inert and biocompatible synthetic polymer material scaffold, functionalized with laminin coating, and tested as a new culture microenvironment for neural stem/precursor cell (NSPC) differentiation compared to standard 2D cultures. NSPCs spontaneously differentiate in the three neural lineages (neurons, astrocytes and OPCs), identified by specific markers, along the fibers in the 3D structure. Analysis of the mRNA levels for lineage differentiation markers reveals a higher expression compared to those seeded on a 2D surface, suggesting an acceleration of the differentiation process. We then focused on the oligodendroglial lineage, showing that in VITVO, mature oligodendrocytes exhibit a myelinating morphology, proven by 3D image elaboration, linked to a higher expression of mature oligodendrocyte markers. This preliminary study on an innovative 3D culture system is the first robust step in producing new microenvironment-based strategies to investigate in vitro OPC and oligodendrocyte biology.

2021 - Adjuvant exemestane or tamoxifen plus ovarian suppression in premenopausal women: single institution analysis [Abstract in Rivista]
D'Onofrio, R.; Piacentini, F.; Barbolini, M.; Isca, C.; Nasso, C.; Caggia, F.; Dominici, M.; Moscetti, L.; Omarini, C.
abstract

Background: The combined analysis of data from TEXT and SOFT trials shows that among premenopausal women with hormone receptor-positive (HR+) breast cancer (BC), adjuvant endocrine therapy (AET) with exemestane (EXE) plus ovarian function suppression (OFS) improved disease-free survival compared to tamoxifen (TAM) plus OFS. We conducted a single institution analysis to compare the activity and safety of both treatment strategies. Patients And Methods: The data on tumor and patient’s characteristics of premenopausal women treated with AET from January 2014 to December 2018 in our institution were retrospectively collected. Treatment toxicities were graded according to CTCAE v5. Survival data were analyzed by Kaplan Meier curves and log rank test. Results: 237 patients were included in the study: 120 on TAM / OFS and 117 on EXE/OFS. Notably, 43 patients (18%) started AET in 2014 (before TEXT/SOFT data): 93% of these were treated with TAM/OFS versus only 7% with EXE/OFS. Women on EXE/OFS had more high-risk early BC compared to those on TAM/OFS (STAGE III 23,9% vs 6,6%; luminal B-like 34,2% vs 21,6%; T> 2 cm 68,4% vs 32,5%; nodal status positive 66,6% vs 36,6% - all p value <0,01). According with risk of relapse, the number of patients pre-treated with chemotherapy was higher in EXE/OFS group (79,5% versus 37,5%, p value <0,001) than TAM/OFS one. Extended therapy was accepted by 50% of patients in the TAM/OFS group and 47% in the EXE/OFS group. Any grade adverse events (AE) were observed in 77 (64%) and 101 (86%) patients in TAM/OFS and EXE/OFS group, respectively. In particular, the incidence of G3 AEs was significantly higher in the EXE/OFS group and mainly represented by muscoloskeletal symptoms, osteoporosis and hypertension. Eighteen (15,4%) women discontinued EXE and switched to an alternate ET (TAM or NSAI) due to treatment toxicity. No statistically significant difference in terms of relapse freesurvival was observed between the two groups. A – Breast Cancer 27 Conclusions: In our analysis, the choice of the AET is driven mainly from the risk of relapse. EXE/OFS represents the main choice in the high-risk patients as per SOFT/ TEXT trials results. TAM/OFS represent the main chose antecedent to the SOFT/TEXT results (2014/2015).The frequency and the grade of AEs were higher in EXE group than TAM one. The AET should be proposed based on both, risk of relapse and treatment toxicity profile. An update analysis will be presented at the meeting.

2021 - Alternative splicing of NF-YA promotes prostate cancer aggressiveness and represents a new molecular marker for clinical stratification of patients [Poster]
Belluti, Silvia; Semeghini, Valentina; Rigillo, Giovanna; Ronzio, Mirko; Benati, Daniela; Torricelli, Federica; REGGIANI BONETTI, Luca; Carnevale, Gianluca; Grisendi, Giulia; Ciarrocchi, Alessia; Dominici, Massimo; Recchia, Alessandra; Dolfini, Diletta; Imbriano, Carol
abstract

2021 - Alternative splicing of NF-YA promotes prostate cancer aggressiveness and represents a new molecular marker for clinical stratification of patients [Articolo su rivista]
Belluti, Silvia; Semeghini, Valentina; Rigillo, Giovanna; Ronzio, Mirko; Benati, Daniela; Torricelli, Federica; Reggiani Bonetti, Luca; Carnevale, Gianluca; Grisendi, Giulia; Ciarrocchi, Alessia; Dominici, Massimo; Recchia, Alessandra; Dolfini, Diletta; Imbriano, Carol
abstract

Approaches based on expression signatures of prostate cancer (PCa) have been proposed to predict patient outcomes and response to treatments. The transcription factor NF-Y participates to the progression from benign epithelium to both localized and metastatic PCa and is associated with aggressive transcriptional profile. The gene encoding for NF-YA, the DNA-binding subunit of NF-Y, produces two alternatively spliced transcripts, NF-YAs and NF-YAl. Bioinformatic analyses pointed at NF-YA splicing as a key transcriptional signature to discriminate between different tumor molecular subtypes. In this study, we aimed to determine the pathophysiological role of NF-YA splice variants in PCa and their association with aggressive subtypes.

2021 - Assessing biocompatibility of face mask materials during covid-19 pandemic by a rapid multi-assays strategy [Articolo su rivista]
Petrachi, T.; Ganzerli, F.; Cuoghi, A.; Ferrari, A.; Resca, E.; Bergamini, V.; Accorsi, L.; Burini, F.; Pasini, D.; Arnaud, G. F.; Piccini, M.; Aldrovandi, L.; Mari, G.; Tomasi, A.; Rovati, L.; Dominici, M.; Veronesi, E.
abstract

During the coronavirus disease 2019 (COVID-19) pandemic, scientific authorities strongly suggested the use of face masks (FMs). FM materials (FMMs) have to satisfy the medical device biocompatibility requirements as indicated in the technical standard EN ISO 10993-1:2018. The biologic evaluation must be confirmed by in vivo tests to verify cytotoxicity, sensitisation, and skin irritation. Some of these tests require an extensive period of time for their execution, which is incompatible with an emergency situation. In this study, we propose to verify the safety of FMMs combining the assessment of 3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide (MTT) with quantification of nitric oxide (NO) and interleukin-6 (IL-6), as predictive markers of skin sensitisation or irritation based on human primary fibroblasts. Two hundred and forty-two FMMs were collected and classified according to spectrometer IR in polypropylene, paper, cotton, polyester, polyethylene terephthalate, 3-dimensional printing, and viscose. Of all FMMs tested, 50.8% passed all the assays, 48% failed at least one, and only 1.2% failed all. By a low cost, rapid and highly sensitive multi assays strategy tested on human skin fibroblasts against a large variety of FMMs, we propose a strategy to promptly evaluate biocompatibility in wearable materials.

2021 - Body composition and inflammation impact in non-small cell lung cancer patients treated by first-line immunotherapy [Articolo su rivista]
Baldessari, Cinzia; Pecchi, Annarita; Marcheselli, Raffaella; Guaitoli, Giorgia; Bonacini, Riccardo; Valoriani, Filippo; Torricelli, Pietro; Reverberi, Linda; Menozzi, Renata; Pugliese, Giuseppe; Vitale, Maria Giuseppa; Sabbatini, Roberto; Bertolini, Federica; Barbieri, Fausto; Dominici, Massimo
abstract

Lay abstract Inflammation and malnutrition in cancer patients may affect the immune system and response to therapy. We noticed an increase in inflammation and visceral fat and a decrease in muscle and subcutaneous fat during therapy. No variation showed a significant correlation with survival. Muscle mass, adipose tissue and body mass index do not confirm any prognostic impact or relationship with response to therapy. More interesting results were observed with parameters related to inflammation. Probably, for the best treatment choice, a combination of clinical and biological factors will be necessary. Further studies with a multidimensional approach are needed to propose the best treatment and the best support to everyone.Tweetable abstract Body composition, nutritional and inflammatory status changed during first-line immunotherapy on NSCLC patients. Inflammation has interesting prognostic implications. Combined with other factors, these clinical characteristics may be important to optimize the care of patients.Background: Immunotherapy changed the landscape of non-small cell lung cancer (NSCLC). Efforts were made to implement its action. This study aims to describe body composition, nutritional and inflammatory status in NSCLC patients treated by first-line immunotherapy, their correlation, variation and impact. Patients and methods: We retrospectively analyzed 44 consecutive patients who received pembrolizumab treatment. Results: During the therapy, inflammation and visceral fat increased, whereas muscle and subcutaneous fat decreased. Parameters related to inflammation had an interesting prognostic impact. High numbers of white blood cells remained significantly correlated with a high risk of death in multivariate model. Conclusion: For the best treatment choice, a combination of clinical and biological factors will be most likely be necessary. Prospective and larger studies with a multidimensional approach are needed.

2021 - Cabozantinib and nivolumab as first-line treatment in advanced renal cell carcinoma [Articolo su rivista]
Vitale, M. G.; Nasso, C.; Oltrecolli, M.; Baldessari, C.; Fanelli, M.; Dominici, M.; Sabbatini, R.
abstract

Introduction: In the last decade, there have been substantial changes in the management of metastatic renal cell carcinoma (mRCC) with combined regimens with immune checkpoint inhibitors (ICI) replacing targeted therapies. These combined regimens include the combination of cabozantinib plus nivolumab. Areas covered: Here, we provide an overview of clinical trials evaluating the combination of cabozantinib and nivolumab and the current clinical data on mechanism of action, pharmacokinetics, efficacy, and safety profile. Expert opinion: Dual immune checkpoint inhibition with nivolumab and ipilimumab as well as the combination of a vascular endothelial growth factor (VEGF) inhibitor and an immune checkpoint inhibitor have shown to improve outcomes in phase III trials in comparison to sunitinib (axitinib plus pembrolizumab, axitinib plus avelumab, bevacizumab plus atezolizumab, cabozantinib plus nivolumab, lenvatinib plus pembrolizumab). However, to date, there are no head-to-head trials comparing these new combination therapies and no biomarkers are available to guide the optimal choice of first line therapy.

2021 - Cancer stem cells and macrophages: molecular connections and future perspectives against cancer. [Articolo su rivista]
Aramini, Beatrice; Masciale, Valentina; Grisendi, Giulia; Banchelli, Federico; D'Amico, Roberto; Maiorana, Antonino; Morandi, Uliano; Dominici, Massimo; Husnain Haider, Khawaja
abstract

Cancer stem cells (CSCs) have been considered the key drivers of cancer initiation and progression due to their unlimited self-renewal capacity and their ability to induce tumor formation. Macrophages, particularly tumor-associated macrophages (TAMs), establish a tumor microenvironment to protect and induce CSCs development and dissemination. Many studies in the past decade have been performed to understand the molecular mediators of CSCs and TAMs, and several studies have elucidated the complex crosstalk that occurs between these two cell types. The aim of this review is to define the complex crosstalk between these two cell types and to highlight potential future anti-cancer strategies.

2021 - Cemiplimab- and nivolumab-induced myasthenia gravis: two clinical cases [Articolo su rivista]
Canino, F.; Pugliese, G.; Baldessari, C.; Greco, S.; Depenni, R.; Dominici, M.
abstract

Immune-related myasthenia gravis is a rare, disabling, and potentially fatal adverse event of immune checkpoint inhibitor treatment. It is important to identify and manage it promptly. We present two cases of immune-related de novo myasthenia gravis observed at the Modena Cancer Center in two elderly patients treated with two anti-PD-1 monoclonal antibodies: cemiplimab and nivolumab.

2021 - Circulating and Intracellular miRNAs as Prognostic and Predictive Factors in HER2-Positive Early Breast Cancer Treated with Neoadjuvant Chemotherapy: A Review of the Literature [Articolo su rivista]
Isca, Chrystel; Piacentini, Federico; Mastrolia, Ilenia; Masciale, Valentina; Caggia, Federica; Toss, Angela; Piombino, Claudia; Moscetti, Luca; Barbolini, Monica; Maur, Michela; Dominici, Massimo; Omarini, Claudia
abstract

2021 - Circulating mucosal-associated invariant T cells identify patients responding to anti-PD-1 therapy [Articolo su rivista]
De Biasi, S.; Gibellini, L.; Lo Tartaro, D.; Puccio, S.; Rabacchi, C.; Mazza, E. M. C.; Brummelman, J.; Williams, B.; Kaihara, K.; Forcato, M.; Bicciato, S.; Pinti, M.; Depenni, R.; Sabbatini, R.; Longo, C.; Dominici, M.; Pellacani, G.; Lugli, E.; Cossarizza, A.
abstract

Immune checkpoint inhibitors are used for treating patients with metastatic melanoma. Since the response to treatment is variable, biomarkers are urgently needed to identify patients who may benefit from such therapy. Here, we combine single-cell RNA-sequencing and multiparameter flow cytometry to assess changes in circulating CD8+ T cells in 28 patients with metastatic melanoma starting anti-PD-1 therapy, followed for 6 months: 17 responded to therapy, whilst 11 did not. Proportions of activated and proliferating CD8+ T cells and of mucosal-associated invariant T (MAIT) cells are significantly higher in responders, prior to and throughout therapy duration. MAIT cells from responders express higher level of CXCR4 and produce more granzyme B. In silico analysis support MAIT presence in the tumor microenvironment. Finally, patients with >1.7% of MAIT among peripheral CD8+ population show a better response to treatment. Our results thus suggest that MAIT cells may be considered a biomarker for patients responding to anti-PD-1 therapy.

2021 - CLINICAL AND PATHOLOGICAL FEATURES OF BREAST CANCER IN PATIENTS WITH SYSTEMIC SCLEROSIS: PRELIMINARY DATA FROM THE SCLERO-BREAST STUDY [Abstract in Rivista]
Toss, Angela; Spinella, Amelia; Isca, Chrystel; Vacchi, Caterina; Ficarra, Guido; Fabbiani, Luca; Iannone, Anna; Magnani, Luca; Castrignanò, Paola; Macripo', Pierluca; Gasparini, Elisa; Piana, Simonetta; Cortesi, Laura; Maiorana, Antonino; Salvarani, Carlo; Dominici, Massimo; Giuggioli, Dilia
abstract

Systemic Sclerosis (SSc) is a chronic disease associated with a 1.5-fold increase in cancer risk, including lung cancer, hematological malignancies, and breast cancer (BC). This is a retrospective study aiming to explore the clinical and pathological features of BC developed by SSc patients. A total of 54.5% of patients developed BC before SSc (median interval: 5 years), whereas 45.5% of patients developed BC after SSc (median delay: 8 years). A total of 93.1% of patients were diagnosed with an early stage tumor. Among invasive carcinomas, 70.8% presented with a low Mib1, 8.3% with a tubular histotype, and 42.8% with a Luminal A-like phenotype. A total of 66.6% of patients underwent breast-conserving surgery and 55.5% RT. A total of 40% of patients developed interstitial lung disease after RT and 20% diffuse cutaneous SSc. The cause of death of the six deceased patients was PAH. A significant association was observed between the use of immunosuppressive therapy and diffuse skin extension, negative ACA, positive Anti-Scl-70, and interstitial lung disease, but not BC status. SSc patients developed BC at a good prognosis, suggesting a de-escalation strategy of cancer therapies. In particular, ionizing radiation and chemotherapeuticals should be limited to higher-risk cases. Finally, proper screening is mandatory in order to allow for early cancer detection in SSc patients.

2021 - Clinical and Pathological Features of Breast Cancer in Systemic Sclerosis: Results from the Sclero-Breast Study [Articolo su rivista]
Toss, Angela; Spinella, Amelia; Isca, Chrystel; Vacchi, Caterina; Ficarra, Guido; Fabbiani, Luca; Iannone, Anna; Magnani, Luca; Castrignanò, Paola; Macripò, Pierluca; Gasparini, Elisa; Piana, Simonetta; Cortesi, Laura; Maiorana, Antonino; Salvarani, Carlo; Dominici, Massimo; Giuggioli, Dilia
abstract

Systemic Sclerosis (SSc) is a chronic disease associated with a 1.5-fold increase in cancer risk, including lung cancer, hematological malignancies, and breast cancer (BC). This is a retrospective study aiming to explore the clinical and pathological features of BC developed by SSc patients. A total of 54.5% of patients developed BC before SSc (median interval: 5 years), whereas 45.5% of patients developed BC after SSc (median delay: 8 years). A total of 93.1% of patients were diagnosed with an early stage tumor. Among invasive carcinomas, 70.8% presented with a low Mib1, 8.3% with a tubular histotype, and 42.8% with a Luminal A-like phenotype. A total of 66.6% of patients underwent breast-conserving surgery and 55.5% RT. A total of 40% of patients developed interstitial lung disease after RT and 20% diffuse cutaneous SSc. The cause of death of the six deceased patients was PAH. A significant association was observed between the use of immunosuppressive therapy and diffuse skin extension, negative ACA, positive Anti-Scl-70, and interstitial lung disease, but not BC status. SSc patients developed BC at a good prognosis, suggesting a de-escalation strategy of cancer therapies. In particular, ionizing radiation and chemotherapeuticals should be limited to higher-risk cases. Finally, proper screening is mandatory in order to allow for early cancer detection in SSc patients.

2021 - Clinical implications of malnutrition in the management of patients with pancreatic cancer: Introducing the concept of the nutritional oncology board [Articolo su rivista]
Rovesti, G.; Valoriani, F.; Rimini, M.; Bardasi, C.; Ballarin, R.; Di Benedetto, F.; Menozzi, R.; Dominici, M.; Spallanzani, A.
abstract

Pancreatic cancer represents a very challenging disease, with an increasing incidence and an extremely poor prognosis. Peculiar features of this tumor entity are represented by pancreatic exocrine insufficiency and an early and intense nutritional imbalance, leading to the highly prevalent and multifactorial syndrome known as cancer cachexia. Recently, also the concept of sarcopenic obesity has emerged, making the concept of pancreatic cancer malnutrition even more multifaceted and complex. Overall, these nutritional derangements play a pivotal role in contributing to the dismal course of this malignancy. However, their relevance is often underrated and their assessment is rarely applied in clinical daily practice with relevant negative impact for patients’ outcome in neoadjuvant, surgical, and metastatic settings. The proper detection and management of pancreatic cancer-related malnutrition syndromes are of primary importance and deserve a specific and multidisciplinary (clinical nutrition, oncology, etc.) approach to improve survival, but also the quality of life. In this context, the introduction of a “Nutritional Oncology Board” in routine daily practice, aimed at assessing an early systematic screening of patients and at implementing nutritional support from the time of disease diagnosis onward seems to be the right path to take.

2021 - Clinicopathologic Profile of Breast Cancer in Germline ATM and CHEK2 Mutation Carriers [Articolo su rivista]
Toss, Angela; Tenedini, Elena; Piombino, Claudia; Venturelli, Marta; Marchi, Isabella; Gasparini, Elisa; Barbieri, Elena; Razzaboni, Elisabetta; Domati, Federica; Caggia, Federica; Grandi, Giovanni; Combi, Francesca; Tazzioli, Giovanni; Dominici, Massimo; Tagliafico, Enrico; Cortesi, Laura
abstract

The most common breast cancer (BC) susceptibility genes beyond BRCA1/2 are ATM and CHEK2. For the purpose of exploring the clinicopathologic characteristics of BC developed by ATM or CHEK2 mutation carriers, we reviewed the archive of our Family Cancer Clinic. Since 2018, 1185 multi-gene panel tests have been performed. Nineteen ATM and 17 CHEK2 mutation carriers affected by 46 different BCs were identified. A high rate of bilateral tumors was observed in ATM (26.3%) and CHEK2 mutation carriers (41.2%). While 64.3% of CHEK2 tumors were luminal A-like, 56.2% of ATM tumors were luminal B-like/HER2-negative. Moreover, 21.4% of CHEK2-related invasive tumors showed a lobular histotype. About a quarter of all ATM-related BCs and a third of CHEK2 BCs were in situ carcinomas and more than half of ATM and CHEK2-related BCs were diagnosed at stage I-II. Finally, 63.2% of ATM mutation carriers and 64.7% of CHEK2 mutation carriers presented a positive BC family history. The biological and clinical characteristics of ATM and CHEK2-related tumors may help improve diagnosis, prognostication and targeted therapeutic approaches. Contralateral mastectomy should be considered and discussed with ATM and CHEK2 mutation carriers at the first diagnosis of BC.

2021 - CRISPR-Mediated Genome Editing to Redirect T Cells against Non-Small Cell Lung Cancer [Abstract in Atti di Convegno]
Benati, Daniela; Masciale, Valentina; Grisendi, Giulia; Marchionni, Matteo; Ferrari, Tommaso; Aramini, Beatrice; Dominici, Massimo; Recchia, Alessandra
abstract

2021 - CRISPR-mediated genome editing to redirect T cells against Non-Small Cell Lung Cancer [Abstract in Atti di Convegno]
Benati, Daniela; Masciale, Valentina; Grisendi, Giulia; Ferrari, Tommaso; Cattin, Eleonora; Marchionni, Matteo; Aramini, Beatrice; Dominici, Massimo; Recchia, Alessandra
abstract

2021 - CRISPR-mediated genome editing to redirect T cells against Non-Small Cell Lung Cancer [Abstract in Atti di Convegno]
Benati, Daniela; Masciale, Valentina; Grisendi, Giulia; Ferrari, Tommaso; Cattin, Eleonora; Aramini, Beatrice; Dominici, Massimo; Recchia, Alessandra
abstract

2021 - Critical considerations for the development of potency tests for therapeutic applications of mesenchymal stromal cell-derived small extracellular vesicles [Articolo su rivista]
Gimona, M.; Brizzi, M. F.; Choo, A. B. H.; Dominici, M.; Davidson, S. M.; Grillari, J.; Hermann, D. M.; Hill, A. F.; de Kleijn, D.; Lai, R. C.; Lai, C. P.; Lim, R.; Monguio-Tortajada, M.; Muraca, M.; Ochiya, T.; Ortiz, L. A.; Toh, W. S.; Yi, Y. W.; Witwer, K. W.; Giebel, B.; Lim, S. K.
abstract

Mesenchymal stromal/stem cells (MSCs) have been widely tested against many diseases, with more than 1000 registered clinical trials worldwide. Despite many setbacks, MSCs have been approved for the treatment of graft-versus-host disease and Crohn disease. However, it is increasingly clear that MSCs exert their therapeutic functions in a paracrine manner through the secretion of small extracellular vesicles (sEVs) of 50–200 nm in diameter. Unlike living cells that can persist long-term, sEVs are non-living and non-replicative and have a transient presence in the body. Their small size also renders sEV preparations highly amenable to sterilization by filtration. Together, acellular MSC-sEV preparations are potentially safer and easier to translate into the clinic than cellular MSC products. Nevertheless, there are inherent challenges in the development of MSC-sEV drug products. MSC-sEVs are products of living cells, and living cells are sensitive to changes in the external microenvironment. Consequently, quality control metrics to measure key identity and potency features of MSC-sEV preparations have to be specified during development of MSC-sEV therapeutics. The authors have previously described quantifiable assays to define the identity of MSC-sEVs. Here the authors discuss requirements for prospective potency assays to predict the therapeutic effectiveness of the drug substance in accordance with International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. Although potency assays should ideally reflect the mechanism of action (MoA), this is challenging because the MoA for the reported efficacy of MSC-sEV preparations against multiple diseases of diverse underlying pathology is likely to be complex and different for each disease and difficult to fully elucidate. Nevertheless, robust potency assays could be developed by identifying the EV attribute most relevant to the intended biological activity in EV-mediated therapy and quantifying the EV attribute. Specifically, the authors highlight challenges and mitigation measures to enhance the manufacture of consistent and reproducibly potent sEV preparations, to identify and select the appropriate EV attribute for potency assays despite a complex “work-in-progress” MoA and to develop assays likely to be compliant with regulatory guidance for assay validation.

2021 - Deepening the knowledge of ros1 rearrangements in non-small cell lung cancer: Diagnosis, treatment, resistance and concomitant alterations [Articolo su rivista]
Guaitoli, G.; Bertolini, F.; Bettelli, S.; Manfredini, S.; Maur, M.; Trudu, L.; Aramini, B.; Masciale, V.; Grisendi, G.; Dominici, M.; Barbieri, F.
abstract

ROS proto-oncogene 1 (ROS1) rearrangements are reported in about 1–2% of non-squamous non-small-cell lung cancer (NSCLC). After efficacy of crizotinib was demonstrated, identification of ROS1 translocations in advanced disease became fundamental to give patients the chance of specific and effective treatment. Different methods are available for detection of rearrangements, and probably the real prevalence of ROS1 rearrangements is higher than that reported in literature, as our capacity to detect gene rearrangements is improving. In particular, with next generation sequencing (NGS) techniques, we are currently able to assess multiple genes simultaneously with increasing sensitivity. This is leading to overcome the “single oncogenic driver” paradigm, and in the very near future, the co-existence of multiple drivers will probably emerge more frequently and represent a therapeutic issue. Since recently, crizotinib has been the only available therapy, but today, many other tyrosine kinase inhibitors (TKI) are emerging and seem promising both in first and subsequent lines of treatment. Indeed, novel inhibitors are also able to overcome resistance mutations to crizotinib, hypothesizing a possible sequential strategy also in ROS1-rearranged disease. In this review, we will focus on ROS1 rearrangements, dealing with diagnostic aspects, new therapeutic options, resistance issues and the coexistence of ROS1 translocations with other molecular alterations.

2021 - Developing cell therapies as drug products [Articolo su rivista]
Ciccocioppo, R.; Comoli, P.; Astori, G.; del Bufalo, F.; Prapa, M.; Dominici, M.; Locatelli, F.
abstract

In the last 20 years, the global regulatory frameworks for drug assessment have been managing the challenges posed by using cellular products as new therapeutic tools. Currently, they are defined as “Advanced Therapy Medicinal Products”, comprising a large group of cellular types that either alone or in combination with gene and tissue engineering technology. They have the potential to change the natural course of still lethal or highly debilitating diseases, including cancers, opportunistic infections and chronic inflammatory conditions. Globally, more than 50 cell-based products have obtained market authorization. This overview describes the advantages and unsolved challenges on developing cells as innovative therapeutic vehicles. The main cell therapy players and the legal framework are discussed, starting from chimeric antigen receptor T-cells for leukaemia and solid tumours, dealing then with lymphocytes as potent anti-microbiological tools and then focusing on mesenchymal stem/stromal cells whose role covers regenerative medicine, immunology and anti-tumour therapy.

2021 - Development and validation of a new storage procedure to extend the in-use stability of azacitidine in pharmaceutical formulations [Articolo su rivista]
Iudicello, A.; Genovese, F.; Strusi, V.; Dominici, M.; Ruozi, B.
abstract

Stability studies performed by the pharmaceutical industry are principally designed to fulfill licensing requirements. Thus, post-dilution or post-reconstitution stability data are frequently limited to 24 h only for bacteriological reasons, regardless of the true physicochemical stability which could, in many cases, be longer. In practice, the pharmacy-based centralized preparation may require preparation in advance for administration, for example, on weekends, holidays, or in general when pharmacies may be closed. We report an innovative strategy for storing resuspended solutions of azacitidine, a well-known chemotherapic agent, for which the manufacturer lists maximum stability of 22 h. By placing the syringe with the azacitidine reconstituted suspension between two refrigerant gel packs and storing it at 4 °C, we found that the concentration of azacitidine remained above 98% of the initial concentration for 48 h, and no change in color nor the physicochemical properties of the suspension were observed throughout the study period. The physicochemical and microbiological properties were evaluated by HPLC–UV and UHPLC-HRMS analysis, FTIR spectroscopy, pH determination, visual and subvisual examination, and sterility assay. The HPLC-UV method used for evaluating the chemical stability of azacitidine was validated according to ICH. Precise control of storage temperature was obtained by a digital data logger. Our study indicates that by changing the storage procedure of azacitidine reconstituted suspension, the usage window of the drug can be significantly extended to a time frame that better copes with its use in the clinical environment.

2021 - Dissecting the role of mesenchymal stem cells in idiopathic pulmonary fibrosis: cause or solution? [Articolo su rivista]
Samarelli, Av; Tonelli, R; Heijink, I; Martin Medina, A; Marchioni, A; Bruzzi, G; Castaniere, I; Andrisani, D; Gozzi, F; Manicardi, L; Moretti, A; Cerri, S; Fantini, R; Tabbì, L; Nani, C; Mastrolia, I; Weiss, Dj; Dominici, M; Clini, E.
abstract

Idiopathic pulmonary fibrosis (IPF) is one of the most aggressive forms of idiopathic interstitial pneumonias, characterized by chronic and progressive fibrosis subverting the lung’s architecture, pulmonary functional decline, progressive respiratory failure, and high mortality (median survival 3 years after diagnosis). Among the mechanisms associated with disease onset and progression, it has been hypothesized that IPF lungs might be affected either by a regenerative deficit of the alveolar epithelium or by a dysregulation of repair mechanisms in response to alveolar and vascular damage. This latter might be related to the progressive dysfunction and exhaustion of the resident stem cells together with a process of cellular and tissue senescence. The role of endogenous mesenchymal stromal/stem cells (MSCs) resident in the lung in the homeostasis of these mechanisms is still a matter of debate. Although endogenous MSCs may play a critical role in lung repair, they are also involved in cellular senescence and tissue ageing processes with loss of lung regenerative potential. In addition, MSCs have immunomodulatory properties and can secrete anti-fibroticfactors. Thus, MSCs obtained from other sources administered systemically or directly into the lung have been investigated for lung epithelial repair and have been explored as a potential therapy for the treatment of lung diseases including IPF. Given these multiple potential roles of MSCs, this review aims both at elucidating the role of resident lung MSCs in IPF pathogenesis and the role of administered MSCs from other sources for potential IPF therapies.

2021 - Fibrotic idiopathic interstitial lung disease: the molecular and cellular key players. [Articolo su rivista]
Samarelli, A; Tonelli, R; Marchioni, A; Bruzzi, G; Gozzi, F; Andrisani, D; Castaniere, I; Manicardi, L; Moretti, A; Tabbì, L; Cerri, S; Beghe', B; Dominici, M; Clini, E.
abstract

Interstitial lung disease (ILDs) that are known as diffuse parenchymal lung diseases (DPLDs) lead to the damage of alveolar epithelium and lung parenchyma culminating into inflammation and widespread fibrosis. ILDs that account for more than 200 different pathologies, can be di-vided into two groups: ILDs that have a known cause and those where the cause is unknown clas-sified as Idiopathic Interstitial Pneumonia (IIPs). IIPs include idiopathic pulmonary fibrosis (IPF), non-specific interstitial pneumonia (NSIP), cryptogenic organizing pneumonia (COP) known also as bronchiolitis obliterans organizing pneumonia (BOOP), Acute interstitial pneumonia (AIP), Desquamative Interstitial Pneumonia (DIP), Respiratory bronchiolitis-associated interstitial lung disease (RB-ILD), and lymphocytic interstitial pneumonia (LIP). In this review our aim is to de-scribe the pathogenic mechanisms that lead to the onset and progression of the different IIPs, starting from IPF as the most studied, in order to find both common and standalone molecular and cellular key players among them. Finally, a deeper molecular and cellular characterization of different interstitial lung disease without known cause, would contribute to give a more accurate diagnosis to the patients, that would translate in a more effective treatment decision.

2021 - GD2 CAR T cells against human glioblastoma [Articolo su rivista]
Prapa, M.; Chiavelli, C.; Golinelli, G.; Grisendi, G.; Bestagno, M.; Di Tinco, R.; Dall'Ora, M.; Neri, G.; Candini, O.; Spano, C.; Petrachi, T.; Bertoni, L.; Carnevale, G.; Pugliese, G.; Depenni, R.; Feletti, A.; Iaccarino, C.; Pavesi, G.; Dominici, M.
abstract

Glioblastoma is the most malignant primary brain tumor and is still in need of effective medical treatment. We isolated patient-derived glioblastoma cells showing high GD2 antigen expression representing a potential target for CAR T strategy. Data highlighted a robust GD2 CAR antitumor potential in 2D and 3D glioblastoma models associated with a significant and CAR T-restricted increase of selected cytokines. Interestingly, immunosuppressant TGF β1, expressed in all co-cultures, did not influence antitumor activity. The orthotopic NOD/SCID models using primary glioblastoma cells reproduced human histopathological features. Considering still-conflicting data on the delivery route for targeting brain tumors, we compared intracerebral versus intravenous CAR T injections. We report that the intracerebral route significantly increased the length of survival time in a dose-dependent manner, without any side effects. Collectively, the proposed anti-GD2 CAR can counteract human glioblastoma potentially opening a new therapeutic option for a still incurable cancer.

2021 - Impact of body composition, nutritional and inflammatory status on outcome of non-small cell lung cancer patients treated with immunotherapy [Articolo su rivista]
Baldessari, C.; Guaitoli, G.; Valoriani, F.; Bonacini, R.; Marcheselli, R.; Reverberi, L.; Pecchi, A.; Menozzi, R.; Torricelli, P.; Bertolini, F.; Barbieri, F.; Dominici, M.
abstract

Background and aims: Body composition and balance of nutritional and inflammatory status are important for the immune system. Alterations of these aspects may impact on response, outcome and toxicities of immunotherapy. In this review we try to clarify some definitions and tools used for the assessment of the different aspects of nutritional disorders, body composition and inflammatory status with a focus on lung cancer. Methods: We primary investigate the definitions of malnutrition, cachexia, sarcopenia and overweight. Secondary, tools used to measure body composition, nutritional and inflammatory status, mainly in lung cancer are reviewed. Results: All these features, in the time of precision medicine may improve assessment and selection of patients, incorporating also early palliative care in standard therapy. Conclusions: A multimodal approach based on nutrition assessment and physical exercise should be evaluated to improve aspects of the immune response against cancer and to propose the best treatment to every patient.

2021 - Invited Response on: Comments on "Autologous Fat Grafting for the Oral and Digital Complications of Systemic Sclerosis: Results of a Prospective Study" [Articolo su rivista]
Pignatti, Marco; Spinella, Amelia; Cocchiara, Emanuele; Boscaini, Giulia; Lusetti, Irene Laura; Citriniti, Giorgia; Lumetti, Federica; Setti, Giacomo; Dominici, Massimo; Salvarani, Carlo; De Santis, Giorgio; Giuggioli, Dilia
abstract

2021 - Irinotecan-based chemotherapy in extrapulmonary neuroendocrine carcinomas: survival and safety data from a multicentric Italian experience [Articolo su rivista]
Bardasi, C.; Spallanzani, A.; Benatti, S.; Spada, F.; Laffi, A.; Antonuzzo, L.; Lavacchi, D.; Marconcini, R.; Ferrari, M.; Rimini, M.; Caputo, F.; Santini, C.; Cerma, K.; Casadei-Gardini, A.; Andrikou, K.; Salati, M.; Bertolini, F.; Fontana, A.; Dominici, M.; Luppi, G.; Gelsomino, F.
abstract

Purpose: Neuroendocrine carcinomas (NECs) are a rare subgroup of neuroendocrine neoplasms that occasionally originate from gastro-entero-pancreatic (GEP) tract. Evidence of the effectiveness of chemotherapy is scarce. Platinum plus Etoposide regimens are currently the standard treatment in first-line, while little data are available on second-line treatments. The aim of this study is to evaluate the efficacy and safety of irinotecan (IRI)-based chemotherapy in a series of extrapulmonary NECs. Methods: Patients with NEC diagnosis treated at University Hospitals of Modena, Florence, Pisa, and European Institute of Oncology of Milan with an IRI-based regimen (FOLFIRI or XELIRI) after progression to a first-line platinum-based therapy were enrolled. Objective responses were assessed according to RECIST criteria. Progression-free survival (PFS) and overall survival (OS) were calculated. Results: Thirty-four patients, 16 males, and 18 females, median age of 59 years (range 32–77), with metastatic NEC were included. Twenty-seven patients had Ki-67 ≥ 55% and four patients Ki-67 of <55% (for three patients data were not available). The median number of treatment cycles of the IRI-based regimen was 7.5 (range 1–16). Six partial responses (17.6%) and 9 stable diseases (26.5%) were observed, with a disease control rate of 44.1%. Median PFS and OS were 4.4 and 5.9 months, respectively. Neutropenia, anemia, and nausea were the only G3–G4 toxicities reported. Conclusions: Despite the relatively small sample size, IRI-based therapy demonstrated to be a valid option for patients with pretreated extrapulmonary NEC.

2021 - Long progression-free survival with cabozantinib in a heavily pretreated patient with metastatic renal cell carcinoma: a case report [Articolo su rivista]
Nasso, C.; Sabbatini, R.; Baldessari, C.; Dominici, M.; Vitale, M. G.
abstract

Renal cell carcinoma accounts for 3% of all tumors. Over the last decades, the prognosis of metastatic renal cell carcinoma (mRCC) has improved owing to the approval of several drugs such as tyrosine kinase inhibitors and immunotherapy. The median progression-free survival (PFS) does not exceed 8 months with the available drugs in pretreated patients with mRCC. We present a case of a patient with a long-term response to fourth-line treatment with cabozantinib. Our patient obtained a PFS of 33 months, which is much higher than that reported in literature.

2021 - Long survival of a young patient with Xp11.2 translocation metastatic clear cell renal carcinoma: case report [Articolo su rivista]
Pipitone, S.; Vitale, M. G.; Baldessari, C.; Dominici, M.; Sabbatini, R.
abstract

Introduction: Xp11.2 translocation is a rare subtype of renal cell carcinoma (RCC), identified as a single entity only from 2004 by World Health Organization (WHO). These tumors involve pediatric age group and rarely patients over 40 years old. Children show indolent disease; adult population has invasive tumor at diagnosis with rapid progression. Case report: We describe a case report of a young woman affected by metastatic clear cell renal carcinoma with Xp11.2 translocation. She achieved a longer stable disease (SD) to first line treatment with atezolizumab plus bevacizumab, obtaining a progression free survival (PFS) of 21 months. After she received cabozantinib, sunitinib and then sorafenib. Conclusions: The patient had an overall survival (OS) of 51 months, which is much higher than that reported in literature data. Unfortunately, the biology of Xp11.2 translocation RCC and its therapeutic management are still unclear.

2021 - Mesenchymal stem cell immunomodulation: In pursuit of controlling COVID-19 related cytokine storm [Articolo su rivista]
Song, N.; Wakimoto, H.; Rossignoli, F.; Bhere, D.; Ciccocioppo, R.; Chen, K. -S.; Khalsa, J. K.; Mastrolia, I.; Samarelli, A. V.; Dominici, M.; Shah, K.
abstract

The Coronavirus disease 2019 (COVID-19) pandemic has grown to be a global public health crisis with no safe and effective treatments available yet. Recent findings suggest that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the coronavirus pathogen that causes COVID-19, could elicit a cytokine storm that drives edema, dysfunction of the airway exchange, and acute respiratory distress syndrome in the lung, followed by acute cardiac injury and thromboembolic events leading to multiorgan failure and death. Mesenchymal stem cells (MSCs), owing to their powerful immunomodulatory abilities, have the potential to attenuate the cytokine storm and have therefore been proposed as a potential therapeutic approach for which several clinical trials are underway. Given that intravenous infusion of MSCs results in a significant trapping in the lung, MSC therapy could directly mitigate inflammation, protect alveolar epithelial cells, and reverse lung dysfunction by normalizing the pulmonary microenvironment and preventing pulmonary fibrosis. In this review, we present an overview and perspectives of the SARS-CoV-2 induced inflammatory dysfunction and the potential of MSC immunomodulation for the prevention and treatment of COVID-19 related pulmonary disease.

2021 - Microfragmented adipose tissue is associated with improved ex vivo performance linked to HOXB7 and b-FGF expression [Articolo su rivista]
Casari, G.; Resca, E.; Giorgini, A.; Candini, O.; Petrachi, T.; Piccinno, M. S.; Foppiani, E. M.; Pacchioni, L.; Starnoni, M.; Pinelli, M.; De Santis, G.; Selleri, F.; Catani, F.; Dominici, M.; Veronesi, E.
abstract

Introduction: Adipose tissue (AT) has become a source of mesenchymal stromal/stem cells (MSC) for regenerative medicine applications, in particular skeletal disorders. Several enzymatic or mechanical procedures have been proposed to process AT with the aim to isolate cells that can be locally implanted. How AT is processed may impact its properties. Thus, we compared AT processed by centrifugation (C-AT) to microfragmentation (MF-AT). Focusing on MF-AT, we subsequently assessed the impact of synovial fluid (SF) alone on both MF-AT and isolated AT-MSC to better understand their cartilage repair mechanisms. Materials and methods: MF-AT and C-AT from the same donors were compared by histology and qRT-PCR immediately after isolation or as ex vivo cultures using a micro-tissue pellet system. The in vitro impact of SF on MF-AT and AT-MSC was assessed by histological staining and molecular analysis. Results: The main AT histological features (i.e., increased extracellular matrix and cellularity) of the freshly isolated or ex vivo-cultured MF-AT persisted compared to C-AT, which rapidly deteriorated during culture. Based on our previous studies of HOX genes in MSC, we investigated the involvement of Homeobox Protein HOX-B7 (HOXB7) and its target basic Fibroblast Growth Factor (bFGF) in the molecular mechanism underlying the improved performance of MF-AT. Indeed, both these biomarkers were more prominent in freshly isolated MF-AT compared to C-AT. SF alone preserved the AT histological features of MF-AT, together with HOXB7 and bFGF expression. Increased cell performance was also observed in isolated AT-MSC after SF treatment concomitant with enhanced HOXB7 expression, although there was no apparent association with bFGF. Conclusions: Our findings show that MF has a positive effect on the maintenance of AT histology and may trigger the expression of trophic factors that improve tissue repair by processed AT.

2021 - Microscopic and chemical characterization of PVC tube used for dialysis lines: A new approach [Articolo su rivista]
Petrachi, T.; Arnaud, G. F.; Roncioni, S.; Resca, E.; Veronesi, E.; Dominici, M.; Tomasi, A.; Cuoghi, A.
abstract

Polyvinylchloride is universally agreed upon to be the material of choice for tubings and for containers for medical application. Many alterations of the chemical/physical surface conditions, mainly due to an altered extrusion process, could influence its biocompatibility by promoting platelet aggregation. Biocompatibility and safety of the medical device must be preserved, also monitoring the migration of additives within polyvinylchloride during the diffusion process. A large variety of methods are used to verify the correct composition and extrusion of polyvinylchloride but, generally, they need long experimental time and are expensive. The aim of the study is to propose a simple, economic and rapid approach based on Fourier transform-infrared spectroscopy and Coomassie Blue staining. The method has been used to detect chemical and morphological defects caused by an altered extrusion process on 20/75 polyvinylchloride tubings in a blind test. This approach positively identified altered samples in 80% of the cases. The suggested approach represents a reliable and versatile method to detect and monitor surface defects by an easy, inexpensive and reproducible method.

2021 - Molecular mechanisms and cellular contribution from lung fibrosis to lung cancer development. [Articolo su rivista]
Samarelli, ANNA VALERIA; Masciale, Valentina; Aramini, Beatrice; Pamela Colò, Georgina; Tonelli, Roberto; Marchioni, Alessandro; Bruzzi, Giulia; Gozzi, Filippo; Andrisani, Dario; Castaniere, Ivana; Manicardi, Linda; Moretti, Antonio; Tabbì, Luca; Guaitoli, Giorgia; Cerri, Stefania; Dominici, Massimo; Clini, Enrico
abstract

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrosing interstitial lung 28 disease (ILD) of unknown etiology, with a median survival of 2-4 years from the time of diagnosis. 29 Although IPF has unknown etiology by definition, there have been identified several risks factors 30 increasing the probability of the onset and progression of the disease in IPF patients such as cigarette 31 smoking and environmental risks factors associated to domestic and occupational exposure. Among 32 them, cigarette smoking together with concomitant emphysema might predispose IPF patients to 33 lung cancer (LC), mostly to non-small cell lung cancer (NSCLC), increasing the risk of lung cancer 34 development. To this purpose, IPF and LC share several cellular and molecular processes driving 35 the progression of both pathologies such as fibroblast transition proliferation and activation, endo- 36 plasmic reticulum stress, oxidative stress, and many genetic and epigenetic markers that predispose 37 the IPF patients to LC development. Nintedanib, a tyrosine-kinase inhibitor, was firstly developed 38 as an anticancer drug and then recognized as an anti-fibrotic agent based on the common target 39 molecular pathway. In this review our aim is to describe the updated studies on common cellular 40 and molecular mechanisms between IPF and lung cancer, whose knowledge might help to find 41 novel therapeutic targets for this disease combination.

2021 - New Perspectives in Different Gene Expression Profiles for Early and Locally Advanced Non-Small Cell Lung Cancer Stem Cells. [Articolo su rivista]
Masciale, Valentina; Banchelli, Federico; Grisendi, Giulia; D'Amico, Roberto; Maiorana, Antonino; Stefani, Alessandro; Morandi, Uliano; Dominici, Massimo; Aramini, Beatrice
abstract

Introduction: Lung cancer is one of the most common cancers in the world, causing over 1.7 million deaths in 2018. Thus far, no effective treatments against lung cancer for advanced stages have been found. For early stages, although surgery is considered the gold standard treatment, 30–55% of patients develop recurrence within the first 5 years of surgery. Our aim is to assess whether cancer stem cells (CSC) display overexpression of a pool of genes that were previously identified for adenocarcinoma recurrence in patients with early and locally advanced stages of non-small cell lung cancer (NSCLC). Methods: This cross-sectional study was carried out by harvesting surgical tumor specimens obtained from patients harboring early (I-II) and locally advanced (IIIA) stages of NSCLC. For each patient, cell sorting was performed to identify and isolate the ALDHhigh (CSC) and ALDHlow (cancer cells) populations. The mRNA expressions of 31 recurrence-related genes (target genes) in both ALDHhigh and ALDHlow populations were then assessed and compared. Results: Surgical specimens were obtained from 22 patients harboring NSCLC. Sixteen (51.6%) out of 31 recurrence-related genes were significantly overexpressed in ALDHhigh cells in the early stages and 9 (29.0%) were overexpressed in the locally advanced stages of NSCLC. Overall, the relative mRNA expressions for these recurrence-related genes were higher in early-stage patients. The average fold change, considering all 31 recurrence-related genes together, was 4.5 (95% CI = 3.1-6.3) in early-stage patients and 1.6 (95% CI = 1.2-2.2) in locally advanced-stage patients. Conclusions: Our study represents the first attempt toward identifying genes associated with recurrence that are overexpressed in cancer stem cells in patients with early and ocally advanced stages of NSCLC. This finding may contribute to the identification of new target therapies tailored for NSCLC stages.

2021 - Next Generation Sequencing (NGS): a possible game changer in metastatic breast cancer [Abstract in Rivista]
Barbolini, M.; Omarini, C.; Moscetti, L.; Canino, F.; Trudu, L.; Tornincasa, A.; Caggia, F.; Bettelli, S.; Manfredini, S.; Isca, C.; Molinaro, A.; Dominici, M.; Piacentini, F.
abstract

Background: NGS has been introduced into the clinics with the aim of sequencing long and complex genes for tumor sample, in order to identify driver and/or targetable alterations. Several companies and academic centers have implemented NGS assays to guide treatment decisions, even though there are no clear recommendations from scientific societies about their use in daily clinical practice. Patients and methods: Since 2019 NGS analysis was performed in 32 MBC patients’ tissues at Modena Cancer Center, as for clinical judgement during the course of MBC. Oncomine™ was mainly used for the assay. The aim was to define the PI3K mutational status, since Alpelisib - an a-subunit selective PI3K inhibitor - had shown to improve PFS in PI3K mutated HR+/HER2- MBC patients in SOLAR-1 and BYLIEVE trials. Results: Twenty (62%) NGS analysis were performed on MBC samples, the other (13) on primary breast cancer. Table1 summarize the clinical-pathological characteristics of patients. At least 1 mutation was found in 25 (78%) samples. A PI3K mutation was detected in 14 (44%) cases, with E542K as the most frequent. In 10 out of 14 cases, PI3K mutation was associated with other gene mutations. FGFR3, FGFR4 mutations and FGFR2 amplification were described in 4, 2 and one patients respectively. Two patients showed AKT1 mutation, in one case was associated with PTEN mutation. Two of the patients with PI3K mutation were treated with Alpelisib + Fulvestrant. The patient with FGFR1 amplification was eligible for a phase II clinical trial. Conclusions: NGS performed in this cohort of MBC patients allowed therapeutic decisions in about 10% of cases. Although PI3K mutational status for eligibility to Alpelisib can be cheaply studied by RT-PCR, NGS assay can provide wider information about other gene mutations, useful for patients’ selection for clinical trials. In the era of precision medicine, knowing the mutational status of MBC early in patient history can change the therapeutic algorithm.

2021 - Osteonecrosis of the femoral head safely healed with autologous, expanded, bone marrow-derived mesenchymal stromal cells in a multicentric trial with minimum 5 years follow-up [Articolo su rivista]
Gomez-Barrena, E.; Padilla-Eguiluz, N. G.; Rosset, P.; Hernigou, P.; Baldini, N.; Ciapetti, G.; Gonzalo-Daganzo, R. M.; Avendano-Sola, C.; Rouard, H.; Giordano, R.; Dominici, M.; Schrezenmeier, H.; Layrolle, P.
abstract

10.3390/jcm10030508.

2021 - Palbociclib in a patient with HR+/HER2- advanced breast cancer and HIV1 infection: A case report [Articolo su rivista]
Canino, F.; Moscetti, L.; Borghi, V.; Dominici, M.; Piacentini, F.
abstract

The use of drugs that affect the cell cycle represents one of the common strategies for the control of some unrelated pathologies, such as chronic viral HIV infections or cancer. The authors report the case of a patient followed for a hormone receptor-positive (HR+)/HER2 negative (HER2-) advanced breast cancer, treated with hormone therapy and CDK 4/6 inhibitors, and a concomitant HIV infection under antiretroviral treatment. The authors consider the function of the sterile alpha motif and HD domain-containing protein-1 (SAMHD1) enzyme, its implications in the control of viral replication and the correlation between its activity and the mechanism of action of the CDK 4/6 inhibitor palbociclib.

2021 - Pazopanib-related secondary polycythemia in metastatic myxofibrosarcoma: A case report and review of the literature [Articolo su rivista]
Fanelli, M.; Caputo, F.; Cerma, K.; Gelsomino, F.; Bari, A.; Dominici, M.; Pozzi, S.
abstract

Introduction: Pazopanib, a tyrosine kinase inhibitor (TKI), is a standard treatment for various tumours, including metastatic non-adipocytic soft-tissue sarcomas. In literature, erythrocytosis has been described as a TKI-related condition. Case report: A 59-year-old man underwent surgical removal of a sub-scapular mass consistent with myxofibrosarcoma. After distant relapse, he first started chemotherapy, and then Pazopanib. He was found to have increased levels of hemoglobin (Hb) and hematocrit (Hct). He was asymphtomatic, with no history of pulmonary disease nor smoking habit. Erythropoietin (EPO) level was higher than normal. A polycythemia vera was ruled out. Management & outcome: The patient started a prophylactic therapy with lysine acetylsalicylate, and we observed a reduction of Hb, but not Hct. Due to disease progression, we interrupted Pazopanib. After a week from drug discontinuation, Hb levels got back to the normal range, Hct was lowering. We decided not to perform phlebotomy, considering the declining trend in Hb and Hct values and the absence of symptoms. Discussion: We postulated a Pazopanib-related secondary erythrocytosis, since Hb and Hct levels increased from baseline during treatment, then normalized when Pazopanib was discontinued. We used the Naranjo Nomogram to assess the correlation between the adverse effect and Pazopanib, the correlation was “Probable”, a score of 5. To the best of our knowledge, this is the first case report of Pazopanib-related secondary polycythemia in a patient with sarcoma. It is important to pay attention to blood count and to any symptoms potentially related to erythrocytosis in patients treated with TKIs.

2021 - Pembrolizumab rechallenge in squamous non-small-cell lung cancer and HIV-positivity: A case report [Articolo su rivista]
Guaitoli, G.; Barbieri, F.; Barbolini, M.; Molinaro, E.; Emidio, K. D.; Borghi, V.; Dominici, M.; Bertolini, F.
abstract

Immune checkpoint inhibitors (ICIs) changed management of non-small-cell lung cancer, but resistance usually develops. Today, at ICIs failure, chemotherapy is the treatment of choice, but the chance of immunotherapy rechallenge is appealing. Another challenging issue is whether it is safe to treat HIV-positive patients with ICIs: safety and efficacy of immunotherapy have been marginally considered in this subgroup. We report the case of a non-small-cell lung cancer patient treated by PD-1 inhibitors rechallenge despite his HIV-positivity, achieving good partial response with significant clinical benefit and without toxicities. Our experience underlines that HIV-positive patients can be treated similarly to HIV-negative individuals. HIV-positivity should be considered similar to other comorbidities, and not as a sufficient reason to preclude them the best available treatments.

2021 - Persistency of Mesenchymal Stromal/Stem Cells in Lungs [Articolo su rivista]
Ferrini, E.; Stellari, F. F.; Franceschi, V.; Macchi, F.; Russo, L.; Murgia, A.; Grisendi, G.; Villetti, G.; Dominici, M.; Donofrio, G.
abstract

Mesenchymal stromal/stem cells (MSCs) are a fibroblast-like cell population with high regenerative potential that can be isolated from many different tissues. Several data suggest MSCs as a therapeutic tool capable of migrating to a site of injury and guide tissue regeneration mainly through their secretome. Pulmonary first-pass effect occurs during intravenous administration of MSCs, where 50 to 80% of the cells tend to localize in the lungs. This phenomenon has been exploited to study MSC potential therapeutic effects in several preclinical models of lung diseases. Data demonstrated that, regardless of the lung disease severity and the delivery route, MSCs were not able to survive longer than 24 h in the respiratory tract but still surprisingly determined a therapeutic effect. In this work, two different mouse bone marrow-derived mesenchymal stromal/stem cell (mBM-MSC) lines, stably transduced with a third-generation lentiviral vector expressing luciferase and green fluorescent protein reporter genes tracking MSCs in vivo biodistribution and persistency, have been generated. Cells within the engrafted lung were in vivo traced using the high-throughput bioluminescence imaging (BLI) technique, with no invasiveness on animal, minimizing biological variations and costs. In vivo BLI analysis allowed the detection and monitoring of the mBM-MSC clones up to 28 days after implantation independently from the delivery route. This longer persistency than previously observed (24 h) could have a strong impact in terms of pharmacokinetics and pharmacodynamics of MSCs as a therapeutic tool.

2021 - Quality of life of therapies for hormone receptor positive advanced/metastatic breast cancer: Regulatory aspects and clinical impact in Europe [Articolo su rivista]
Moscetti, L.; Sperduti, I.; Frassoldati, A.; Musolino, A.; Nasso, C.; Toss, A.; Omarini, C.; Dominici, M.; Piacentini, F.
abstract

In recent years, the number of trials incorporating health-related quality of life (HRQoL) data has increased. The impact of HRQoL on regulatory decision making in the European context and on clinical practice is not well established. We conducted an analysis of the role of QoL data extracted from the clinical trials of the drugs approved for hormone receptor positive/HER2-negative advanced/metastatic breast cancer (mBC). The results from the HRQoL were collected and a meta-analysis was performed to evaluate the impact of experimental drugs compared to standard treatments. The results showed a non-detrimental effect in HRQoL from the new treatments. As regards the approval process, from an examination of the European Medicine Agency (EMA) documents, HRQoL was reported nonextensively and contained and discussed in the European assessment reports (EPARs) for eleven trials in the approval process and cited in three cases in the EPARs and summary of medicinal product characteristics (SmPC). An effort should be made by all the stakeholders to increase the visibility of the HRQoL results in order to allow increased consideration in the approval process to make QoL data more easily and visibly available for the clinician and the patients. The evaluation should be reflected in the SmPC in order to increase the amount of information provided to the physician.

2021 - Quality of life of therapies for hormone receptor positive advanced/metastatic breast cancer (HR+/HER2- mBC): regulatory aspects and clinical impact in Europe [Abstract in Rivista]
Moscetti, ; Sperduti, I.; Frassoldati, A.; Musolino, A.; Nasso, C.; Toss, A.; Omarini, C.; Dominici, M.; Piacentini, F.
abstract

Background: In recent years the number of trials that incorporated health related quality of life (HRQoL) data has increased. The impact of HRQoL on the regulatory decision making in the European regulatory context and on clinical practice is not well established. We conduct an analysis of the role of QoL data extracted from the pivotal trials of the drugs approved for HR+/HER2- mBC, to discuss their impact on the regulatory decision making in the European regulatory context and the possible impact on clinical practice. Methods: We identified all products approved for mBC by the European Medicines Agency (EMA) based on the European public assessment reports (EPARs) that are publicly available on the agency’s website.The following substances has been evaluated: letrozole, anastrozole, exemestane, fulvestrant, ribociclib, palbociclib, abemaciclib, alpelisib. The results of the HRQoL analysis form the pivotal trials have been collected and a metanalysis has been performed to evaluate the impact of experimental drugs if compared to the standard treatments. All the EPARs available from the EMA website have been checked to verify the presence of the HRQoL in the discussion and in the benefit risk assessment. The related summary of medicine products characteristics (SmPCs) have been verified to evaluate the presence of the HRQoL data in the section 5.1 Results: 7 out of the 9 active substances taken in account in the current analysis incorporated the HRQoL data in the description of the result of the pivotal trials. Seventeen trial has been identified, in fourteen the QoL was included as a secondary endpoint. A global improvement in the global QoL, considering the Time To deterioration >10, was observed, pointing out the consistency of the efficacy of the new substances if compared to the standard treatment. As regards the approval process from the analysis of the EMA documents, the HRQoL were reported quite shortly and contained and discussed in the EPARs of eleven trials in the approval process and cited in three cases in the EPARs and summary of medicine products characteristics (SmPC). Conclusions: An effort should be done from all the stakeholders to increase the visibility of the HRQoL results in order to allow an increasing consideration in the approval process to make QoL data more easily and visibly available for the clinician and the patients. The evaluation should be reflected in the SmPC in order to increase the amount of information provided to the physician.

2021 - Splenic macrophage phagocytosis of intravenously infused mesenchymal stromal cells attenuates tumor localization [Articolo su rivista]
Hasgur, S.; Desbourdes, L.; Relation, T.; Overholt, K. M.; Stanek, J. R.; Guess, A. J.; Yu, M.; Patel, P.; Roback, L.; Dominici, M.; Otsuru, S.; Horwitz, E. M.
abstract

Mesenchymal stromal cells (MSCs) possess remarkable tumor tropism, making them ideal vehicles to deliver tumor-targeted therapeutic agents; however, their value in clinical medicine has yet to be realized. A barrier to clinical utilization is that only a small fraction of infused MSCs ultimately localize to the tumor. In an effort to overcome this obstacle, we sought to enhance MSC trafficking by focusing on the factors that govern MSC arrival within the tumor microenvironment. Our findings show that MSC chemoattraction is only present in select tumors, including osteosarcoma, and that the chemotactic potency among similar tumors varies substantially. Using an osteosarcoma xenograft model, we show that human MSCs traffic to the tumor within several hours of infusion. After arrival, MSCs are observed to localize in clusters near blood vessels and MSC-associated bioluminescence signal intensity is increased, suggesting that the seeded cells expand after engraftment. However, our studies reveal that a significant portion of MSCs are eliminated en route by splenic macrophage phagocytosis, effectively limiting the number of cells available for tumor engraftment. To increase MSC survival, we transiently depleted macrophages with liposomal clodronate, which resulted in increased tumor localization without substantial reduction in tumor-associated macrophages. Our data suggest that transient macrophage depletion will significantly increase the number of MSCs in the spleen and thus improve MSC localization within a tumor, theoretically increasing the effective dose of an anti-cancer agent. This strategy may subsequently improve the clinical efficacy of MSCs as vehicles for the tumor-directed delivery of therapeutic agents.

2021 - TDM-1 efficacy in trastuzumab-pertuzumab pre-treated HER2 positive metastatic breast cancer patients: A meta-analysis [Abstract in Atti di Convegno]
Omarini, Claudia; Piacentini, Federico; Sperduti, Isabella; Cerma, Krisida; Barbolini, Monica; Canino, Fabio; Nasso, Cecilia; Dominici, Massimo; Moscetti, Luca
abstract

Background: Based on the results reported in Emilia trial population, current guidelines consider TDM-1 the standard second-line therapy for HER2 positive metastatic breast cancer (MBC) patients. Despite that, there are no prospective studies supporting the efficacy of TDM-1 following trastuzumab (T) + pertuzumab (P) and taxane first-line treatment. Currently, only real-world data have investigated this sequence with controversial results Methods: We performed a meta-analysis of the available real world data to determine the efficacy of TDM-1 after first-line TP in HER2 positive MBC patients. We used a random-effect model to find differences in the rate of 1-year progression free survival (PFS) between TP pre-treated population and the phase III Emilia trial (T pre-treated population). Results: Seven studies were eligible, in three of them data were from sub-group population analysis. The meta-analysis showed a combined 1-years PFS risk difference for TDM-1 efficacy after TP in second or more lines of -0.122, with lower and upper limits of -0.253 and 0.010, respectively (p=0.07) , with low heterogeneity among studies (I2 < 0.0001, p =0.836). Considering the four studies on TDM-1 in second-line setting, 1-years PFS risk was -0.034 (95% CI -0.207 – 0,139; p=0.701) (I2 < 0.0001, p =0.91). Conclusions: Results from the meta-analysis show that the efficacy of TDM-1 after TP double-block seems to be similar to the previously reported in Emilia trial. In the second line setting, available data are not mature enough to confirm TDM-1 efficacy in TP pre-treated population. Currently, TP pretreated patients should receive TDM-1 as indicated in the guidelines.

2021 - TDM-1 efficacy in trastuzumabpertuzumab pre-treated HER2 positive metastatic breast cancer patients: a meta-analysis [Abstract in Rivista]
Omarini, C.; Piacentini, F.; Sperduti, I.; Cerma, K.; Barbolini, M.; Canino, F.; Nasso, C.; Isca, C.; Caggia, F.; Dominici, M.; Moscetti, L.
abstract

Background: Based on the results reported in Emilia trial population, current guidelines consider TDM-1 the standard 2nd line therapy for HER2 positive metastatic breast cancer (MBC) patients. Despite that, there are no prospective studies supporting the efficacy of TDM-1 following trastuzumab (T) + pertuzumab (P) and taxane 1st line treatment. Currently, only real-world data have investigated this sequence with controversial results. Methods: We performed a meta-analysis of the available real world data to determine the efficacy of T-DM1 after 1st line TP in HER2 positive MBC patients. We used a random- effect model to find differences in the rate of 1-year progression free survival (PFS) between TP pre-treated population and the phase III Emilia trial (T pre-treated population). Results: Seven studies were eligible, in three of them data were from sub-group population analysis. The meta-analysis showed a combined 1-years PFS risk difference for TDM-1 efficacy after TP in 2nd or more lines of -0.122, with lower and upper limits of -0.253 and 0.010, respectively (p=0.07), with low heterogeneity among studies (I2 < 0.0001, p =0.836). Considering the four studies on TDM-1 in 2nd line setting, 1-years PFS risk was -0.034 (95% CI -0.207 – 0,139; p=0.701) (I2 < 0.0001, p =0.91). Conclusions: Results from the meta-analysis show that the efficacy of TDM-1 after TP double-block seems to be similar to the previously reported in Emilia trial. In the second line setting, available data are not mature enough to confirm TDM-1 efficacy in TP pre-treated population. Currently, TP pretreated patients should receive T-DM1 as indicated in the guidelines.

2021 - The Coronavirus Pandemic: A Pitfall or a Fast Track for Validating Cell Therapy Products? [Articolo su rivista]
Khoury, M.; Ikonomou, L.; Dominici, M.; Leblanc, K.; Levine, B. L.; Weiss, D. J.
abstract

The global COVID-19 pandemic has prompted urgent need for potential therapies for severe respiratory consequences resulting from coronavirus infection. New therapeutic agents that will attenuate ongoing inflammation and at the same time promote regeneration of injured lung epithelial cells are urgently needed. Cell-based therapies, primarily involving mesenchymal stromal cells (MSCs) and their derivatives, are currently investigated worldwide for SARS-CoV-2-induced lung diseases. A significant number of academic centers and companies globally have already initiated such trials. However, at a time of unprecedented need, it is also foreseen that families and caregivers will seek all available options, including access to cell-based and other investigational products, even before proven safety and efficacy as well as regulatory approval. This should not be an excuse for opportunists to sell or advertise unproven therapies of any kind. "Compassionate use" should be conducted in the context of a clinical investigation framed by strict ethical and regulatory permissions, with the goal of obtaining mechanistic information wherever possible.

2021 - The evolving role of fgfr2 inhibitors in intrahepatic cholangiocarcinoma: From molecular biology to clinical targeting [Articolo su rivista]
Salati, M.; Caputo, F.; Baldessari, C.; Carotenuto, P.; Messina, M.; Caramaschi, S.; Dominici, M.; Bonetti, L. R.
abstract

Intrahepatic cholangiocarcinoma (iCCA) is an anatomically and biologically distinct entity with a rising incidence and a poor prognosis on conventional treatments. Surgery followed by adjuvant chemotherapy is a potentially curative option in resectable cases, while palliative-intent chemotherapy is the standard-of-care in the advanced setting. Technological advances through massive parallel sequencing have enabled a deeper under-standing of disease biology with the identification of several druggable molecular vulnerabilities in nearly 50% of cases. Among them, gene fusions involving the fibroblast growth factor receptor 2 (FGFR2) are the most therapeutically exploited so far with a number of Phase II clinical trials investigating FGFR2 inhibitors showing unprecedented efficacy results in this molecular subgroup. Over the last year, these efforts have culminated in the US FDA-approval of pemigatinib and infigratinib, the first two oral selective FGFR2 targeted agents for previously treated, locally advanced or metastatic iCCA driven by FGFR2 fusion or rearrangements. While first-line Phase III trials are currently underway to test these targeted approach against standard-of-care chemotherapy, translational studies are trying to better understand primary and secondary resistance mechanisms in order to optimize FGFR2 blockade in iCCA. In this article, we extensively reviewed the current evidence on the biological rationale, as well as preclinical and clinical development of FGFR inhibitors in iCCA.

2021 - The growing skyline of advanced hepatocellular carcinoma treatment: A review. [Articolo su rivista]
Schipilliti, F. M.; Garajova, I.; Rovesti, G.; Balsano, R.; Piacentini, F.; Dominici, M.; Gelsomino, F.
abstract

Hepatocellular carcinoma (HCC) is the main type of liver cancer. In the majority of cases, HCC is diagnosed at the advanced stage, leading to poor prognosis. In recent years, many efforts have been devoted to investigating potential new and more effective drugs and, indeed, the treatment armamentarium for advanced HCC has broadened tremendously, with targeted- and immune-therapies, and probably the combination of both, playing pivotal roles. Together with new established knowledge, many issues are emerging, with the role of neoadjuvant/adjuvant settings, the definition of the best transitioning time from loco-regional treatments to systemic therapy, the identification of potential predictive biomarkers, and radiomics being just some of the topics that will have to be further explored in the next future. Clearly, the current COVID-19 pandemic has influenced the management of HCC patients and some considerations about this topic will be elucidated.

2021 - The harmonization of World Health Organization International Nonproprietary Names definitions for cell and cell-based gene therapy substances: when a name is not enough [Articolo su rivista]
Loizides, U.; Dominici, M.; Manderson, T.; Rizzi, M.; Robertson, J. S.; de Sousa Guimaraes Koch, S.; Timon, M.; Balocco, R.
abstract

The World Health Organization (WHO) assigns International Nonproprietary Names (INN) to pharmaceutical substances, including advanced therapy medicinal products, to ensure that each substance is globally recognized by a unique name. The majority of INN are published in the WHO Drug Information in accordance with the nomenclature rules of the International Union of Pure and Applied Chemistry. However, advanced therapy medicinal products, and in particular cell therapy and cell-based gene therapy substances, cannot be defined by such chemical nomenclature. Instead, they are published together with a textual definition paragraph to unambiguously describe their characteristics. These definitions are an integral part of the INN nomenclature system, and their presence contributes to pharmacovigilance and patient safety, as they help to distinguish regulated substances from cell-based interventions that have no INN and are marketed without regulatory oversight. Particular attention is therefore allocated to these descriptive paragraphs, as they form the basis for defining the uniqueness of a particular cell substance. This review describes the INN nomenclature system for cell-based substances and focuses on the progress made by the WHO INN Programme to develop and harmonize these definition paragraphs, which is reflected in a newly revised INN application form for cell therapy substances.

2021 - The Prognostic Role of Early Skeletal Muscle Mass Depletion in Multimodality Management of Patients with Advanced Gastric Cancer Treated with First Line Chemotherapy: A Pilot Experience from Modena Cancer Center [Articolo su rivista]
Rimini, M; Pecchi, A; Prampolini, F; Bussei, C; Salati, M; Forni, D; Martelli, F; Valoriani, F; Canino, F; Bocconi, A; Gelsomino, F; Reverberi, L; Benatti, S; Piacentini, F; Menozzi, R; Dominici, M; Luppi, G; Spallanzani, A
abstract

Background: Few data about the link between nutritional status and survival are available in the metastatic gastric cancer (GC) setting. The aim of this work was to evaluate the prognostic role of tissue modifications during treatment and the benefit of a scheduled nutritional assessment in this setting. Methods: Clinical and laboratory variables of 40 metastatic GC patients treated at Modena Cancer Center were retrieved: 20 received a nutritional assessment on the oncology’s discretion, the other 20 received a scheduled nutritional assessment at baseline and every 2–4 weeks. Anthropometric parameters were calculated on Computed Tomography (CT) images at the baseline and after 3 months of chemotherapy. Results: A correlation between baseline Eastern Cooperative Oncology Group Performance Status (ECOG PS), Lymphocyte to Monocyte Ratio (LMR), C-reactive protein (PCR), Prognostic Nutritional Index (PNI) and Overall survival (OS) was highlighted. Among the anthropometric parameters, early skeletal muscle mass depletion (ESMMD) >10% in the first months of treatment significantly impacted on mOS (p = 0.0023). A link between ESMMD and baseline LDH > 460 U/L, baseline CRP > 2.2 mg/dL and weight decrease during treatment emerged. Patients evaluated with a nutritional scheduled support experienced a mean gain in subcutaneous and visceral fat of 11.4% and 10.21%, respectively. Conclusion: We confirm the prognostic impact of ESMMD > 10% during chemotherapy in metastatic GC. The prognostic role of a scheduled nutritional assessment deserves further confirmation in large prospective trials.

2021 - The release of inflammatory mediators from acid-stimulated mesenchymal stromal cells favours tumour invasiveness and metastasis in osteosarcoma [Articolo su rivista]
Avnet, S.; Lemma, S.; Cortini, M.; Di Pompo, G.; Perut, F.; Lipreri, M. V.; Roncuzzi, L.; Columbaro, M.; Errani, C.; Longhi, A.; Zini, N.; Heymann, D.; Dominici, M.; Grisendi, G.; Golinelli, G.; Consolino, L.; Longo, D. L.; Nanni, C.; Righi, A.; Baldini, N.
abstract

Osteosarcoma is the most frequent primary malignant bone tumour with an impressive tendency to metastasise. Highly proliferative tumour cells release a remarkable amount of protons into the extracellular space that activates the NF-kB inflammatory pathway in adjacent stromal cells. In this study, we further validated the correlation between tumour glycolysis/acidosis and its role in metastases. In patients, at diagnosis, we found high circulating levels of inflammatory mediators (IL6, IL8 and miR-136-5p-containing extracellular vesicles). IL6 serum levels significantly correlated with disease-free survival and18F-FDG PET/CT uptake, an indirect measurement of tumour glycolysis and, hence, of acidosis. In vivo subcutaneous and orthotopic models, co-injected with mesenchymal stromal (MSC) and osteosarcoma cells, formed an acidic tumour microenvironment (mean pH 6.86, as assessed by in vivo MRI-CEST pH imaging). In these xenografts, we enlightened the expression of both IL6 and the NF-kB complex subunit in stromal cells infiltrating the tumour acidic area. The co-injection with MSC also significantly increased lung metastases. Finally, by using 3D microfluidic models, we directly showed the promotion of osteosarcoma invasiveness by acidosis via IL6 and MSC. In conclusion, osteosarcoma-associated MSC react to intratumoural acidosis by triggering an inflammatory response that, in turn, promotes tumour invasiveness at the primary site toward metastasis development.

2021 - The Role of Exosomes in Breast Cancer Diagnosis. [Articolo su rivista]
Piombino, C; Mastrolia, I; Omarini, C; Candini, O; Dominici, M; Piacentini, F; Toss, A.
abstract

The importance of molecular re-characterization of metastatic disease with the purpose of monitoring tumor evolution has been acknowledged in numerous clinical guidelines for the management of advanced malignancies. In this context, an attractive alternative to overcome the limitations of repeated tissue sampling is represented by the analysis of peripheral blood samples as a 'liquid biopsy'. In recent years, liquid biopsies have been studied for the early diagnosis of cancer, the monitoring of tumor burden, tumor heterogeneity and the emergence of molecular resistance, along with the detection of minimal residual disease. Interestingly, liquid biopsy consents the analysis of circulating tumor cells, circulating tumor DNA and extracellular vesicles (EVs). In particular, EVs play a crucial role in cell communication, carrying transmembrane and nonmembrane proteins, as well as metabolites, lipids and nucleic acids. Of all EVs, exosomes mirror the biological fingerprints of the parental cells from which they originate, and therefore, are considered one of the most promising predictors of early cancer diagnosis and treatment response. The present review discusses current knowledge on the possible applications of exosomes in breast cancer (BC) diagnosis, with a focus on patients at higher risk.

2021 - Thromboembolism (TE) and adjuvant endocrine therapy (AET) in hormone receptor positive (HR+) early breast cancer (EBC): Did the evolution of treatment change the incidence of the adverse event? A metanalysis [Abstract in Atti di Convegno]
Moscetti, Luca; Omarini, Claudia; Sperduti, Isabella; Canino, Fabio; Barbolini, Monica; Nasso, Cecilia; Toss, Angela; Dominici, Massimo; Piacentini, Federico
abstract

Background: The AET of HR+ EBC has been changing in the recent years. Aromatase inhibitors (AI) as upfront, or in a planned switch strategy after Tamoxifen (T), have been added to the choice of T alone. An increased risk of TE is well known in the T treated patients while AIs have showed a reduced rate of TE. Recently, adding the cyclin dependent kinase 4/6 inhibitors (CDK4/6) abemaciclib to AIs, has showed a positive impact in the high risk HR+ EBC subgroups, but we are seeing an increase of the TE rate. We conducted this meta-analysis to evaluate the impact of the new AETs on the incidence of TE if compared to the standard monotherapy. Methods: We performed a meta-analysis of the randomized phase III trials comparing the experimental AETs and the endocrine standard therapy. A random-effect model to find differences in the rate of TE events between the experimental treatments and the standard therapy has been used. Results: Twelve phase III trials were included. Five trials evaluated the upfront strategy, 6 studies the switch and one the combination with a CDK4/6inhibitor (i.e. abemaciclib). Overall, the new AETs did not significantly modify or affect the rate of TE events (OR 0.708, 0.444-1.130, p = 0.148) with high heterogeneity among studies (I2 87, p < 0.0001). Excluding the abemaciclib trial, the incidence of TE is reduced (OR 0.609, 0.462-0.802, p < 0.0001) with a moderate heterogeneity among the studies (I2 59, p < 0.006). Considering the upfront strategies with AIs, the TE events are reduced (OR 0.507, 0.394-0.651, p < 0.0001) but they are not if we consider the trials in which T is used upfront before AIs (OR 0.762, 0.546-1.065, p = 0.112). Conclusions: Overall, the new treatments (AIs alone or plus CDK4/6 inhibitors) did not affect the rate in TE events. AIs as upfront strategy is the safest AETs of HR+ EBC, being associated to the lowest incidence of TE. The switch strategy increases the TE rate whereas the addition of abemaciclib to the standard AET showed to significantly increase the TE events. The results of the currently ongoing trials with the CDK4/6 inhibitors will help to obtain additional data to evaluate any differences among the different CDK4/6 inhibitors and to clarify the weight of the TE adverse events in the balance of benefit/risk of this new adjuvant strategy.

2021 - TRAIL receptors are expressed in both malignant and stromal cells in pancreatic ductal adenocarcinoma [Articolo su rivista]
Dall'Ora, Massimiliano; Rovesti, Giulia; Reggiani Bonetti, Luca; Casari, Giulia; Banchelli, Federico; Fabbiani, Luca; Veronesi, Elena; Petrachi, Tiziana; Magistri, Paolo; Di Benedetto, Fabrizio; Spallanzani, Andrea; Chiavelli, Chiara; Spano, Maria Carlotta; Maiorana, Antonino; Dominici, Massimo; Grisendi, Giulia
abstract

: This study assesses the expression of all TNF-related apoptosis-inducing ligand (TRAIL) receptors in pancreatic ductal adenocarcinoma (PDAC) tumor tissue. We aimed to include TRAIL receptor expression as an inclusion parameter in a future clinical study using a TRAIL-based therapy approach for PDAC patients. Considering the emerging influence of PDAC desmoplastic stroma on the efficacy of anti-PDAC therapies, this analysis was extended to tumor stromal cells. Additionally, we performed PDAC stroma characterization. Our retrospective cohort study (N=50) included patients with histologically confirmed PDAC who underwent surgery. The expression of TRAIL receptors (DR4, DR5, DcR1, DcR2, and OPG) in tumor and stromal cells was evaluated by immunohistochemistry (IHC). The amount of tumor stroma was assessed by anti-vimentin IHC and Mallory's trichrome staining. The prognostic impact was determined by the univariate Cox proportional hazards regression model. An extensive expression of functional receptors DR4 and DR5 and a variable expression of decoy receptors were detected in PDAC tumor and stromal cells. Functional receptors were detected also in metastatic tumor and stromal cells. A poor prognosis was associated with low or absent expression of decoy receptors in tumor cells of primary PDAC. After assessing that almost 80% of tumor mass was composed of stroma, we correlated a cellular-dense stroma in primary PDAC with reduced relapse-free survival. We demonstrated that TRAIL functional receptors are widely expressed in PDAC, representing a promising target for TRAIL-based therapies. Further, we demonstrated that a low expression of DcR1 and the absence of OPG in tumor cells, as well as a cellular-dense tumor stroma, could negatively impact the prognosis of PDAC patients.

2021 - Two-month stop in mammographic screening significantly impacts on breast cancer stage at diagnosis and upfront treatment in the COVID era [Articolo su rivista]
Toss, A; Isca, C; Venturelli, M; Nasso, C; Ficarra, G; Bellelli, V; Armocida, C; Barbieri, E; Cortesi, L; Moscetti, L; Piacentini, F; Omarini, C; Andreotti, A; Gambini, A; Battista, R; Dominici, M; Tazzioli, G
abstract

The present analysis aims to evaluate the consequences of a 2-month interruption of mammographic screening on breast cancer (BC) stage at diagnosis and upfront treatments in a region of Northern Italy highly affected by the severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) virus.

2020 - A new bioactive glass with extremely high crystallization temperature and outstanding biological performance [Articolo su rivista]
Bellucci, D.; Veronesi, E.; Dominici, M.; Cannillo, V.
abstract

In this work, a new bioactive glass was designed, prepared by means of a melt-quenching route and characterized in terms of both thermal properties and biological performance. The main objective was to obtain a novel product with high temperature of crystallization in view of possible thermal treatments, as well as remarkable biological responsiveness. Thermal behavior was investigated by heating microscopy, differential thermal analysis (DTA) and sintering tests. The glass displayed a very high crystallization temperature and the samples remained completely amorphous after sintering. Bioactivity was evaluated by means of Simulated Body Fluid (SBF) assay, which is a widely used method to preliminary investigate samples' reactivity in vitro; the glass showed a strong apatite forming ability. Additionally, in order to exclude cytotoxic effects, biocompatibility was verified according to ISO standard 10993. Finally, the biological potential of the new glass was tested by using an innovative 3D cellular model, that mimicked the potential clinical application of a given biomaterial. Human bone marrow mesenchymal stem cells (BM-MSCs) were employed to study the performance of bioactive glass granules in such 3D cellular model. The results showed that the bioactive glass supported human BM-MSCs adhesion, colonization and bone differentiation. Thus, this new bioactive glass looks particularly promising for orthopedic applications, bone tissue engineering and regenerative medicine, especially when a thermal treatment is necessary for the production of specific devices.

2020 - ALDH Expression in Angiosarcoma of the Lung: A Potential Marker of Aggressiveness? [Articolo su rivista]
Aramini, Beatrice; Masciale, Valentina; Bianchi, Daniel; Manfredini, Beatrice; Banchelli, Federico; D'Amico, Roberto; Bertolini, Federica; Dominici, Massimo; Morandi, Uliano; Maiorana, Antonino
abstract

Background: Primary angiosarcoma of the lung is a very aggressive rare malignant disease resulting in a severe prognosis (1). This type of cancer represents about 2% of all soft tissue sarcomas and has a high rate of metastasis through the hematogenous route. For the rarity of this malignant vascular tumor it is still challenging to set a diagnosis (1). The diagnostic features that have thus far been considered include primarily clinical and radiological findings. In some cases, immunohistochemical characteristics based on the most common markers used in pathology have been described. The aim of this report is to present two cases of angiosarcoma of the lung in which the aldehyde dehydrogenase (ALDH) marker was analyzed by immunohistochemistry. Methods: We report two cases of angiosarcoma of the lung in patients underwent lung surgery at our Unit. In addition to the standard histopathological analysis for this disease, immunohistochemistry using an ALDH1A1 antibody was performed in both of the cases. For ALDH quantification, a semi-quantitative method based on the positivity of the tumor cells was used: 0 (<5%), 1 (5–25%), 2 (>25–50%), 3 (>50–75%), 4 (>75%). Results: One patient with recurrent lung disease survived, achieving complete remission after chemo- and radiotherapy. The second patient died of recurrent disease within 5 years of diagnosis. ALDH1A1 was evaluated in both of these cases using an immunohistochemistry scoring system based on the positivity for this marker. The scores were consistent with the patients’ clinical outcomes, as the lower (score 1) was observed in the patient with the better clinical outcome, while the higher (score 3) was seen in the patient with the worse outcome. Conclusion: Our data suggest that ALDH may be an important clinical marker in angiosarcoma of the lung. Although further studies need to be performed in a larger cohort of patients, we believe that, if the results will be confirmed, ALDH1A1 may be used to stratify patients in terms of prognosis and for targeted therapy.

2020 - Arming Mesenchymal Stromal/Stem Cells Against Cancer: Has the Time Come? [Articolo su rivista]
Golinelli, Giulia; Mastrolia, Ilenia; Aramini, Beatrice; Masciale, Valentina; Pinelli, Massimo; Pacchioni, Lucrezia; Casari, Giulia; Dall’Ora, Massimiliano; Botelho Pereira Soares, Milena; Kauanna Fonseca Damasceno, Patrícia; Nascimento Silva, Daniela; Dominici, Massimo; Grisendi, Giulia
abstract

Since mesenchymal stromal/stem cells (MSCs) were discovered, researchers have been drawn to study their peculiar biological features, including their immune privileged status and their capacity to selectively migrate into inflammatory areas, including tumors. These properties make MSCs promising cellular vehicles for the delivery of therapeutic molecules in the clinical setting. In recent decades, the engineering of MSCs into biological vehicles carrying anticancer compounds has been achieved in different ways, including the loadingof MSCs with chemotherapeutics or drug functionalized nanoparticles (NPs), genetic modifications to force the production of anticancer proteins, and the use of oncolytic viruses. Recently, it has been demonstrated that wild-type and engineered MSCs can release extracellular vesicles (EVs) that contain therapeutic agents. Despite the enthusiasm for MSCs as cyto-pharmaceutical agents, many challenges, including controlling the fate of MSCs after administration, must still be considered. Preclinical results demonstrated that MSCs accumulate in lung, liver, and spleen, which could prevent their engraftment into tumor sites. For this reason, physical, physiological, and biological methods have been implemented to increase MSC concentration in the target tumors. Currently, there are more than 900 registered clinical trials using MSCs. Only a small fraction of these are investigating MSC-based therapies for cancer, but the number of these clinical trials is expected to increase as technology and our understanding of MSCs improve. This review will summarize MSC-based antitumor therapies to generate an increasing awareness of their potential and limits to accelerate their clinical translation.

2020 - Author Correction: A Novel 3D In Vitro Platform for Pre-Clinical Investigations in Drug Testing, Gene Therapy, and Immuno-oncology (Scientific Reports, (2019), 9, 1, (7154), 10.1038/s41598-019-43613-9) [Articolo su rivista]
Candini, O.; Grisendi, G.; Foppiani, E. M.; Brogli, M.; Aramini, B.; Masciale, V.; Spano, C.; Petrachi, T.; Veronesi, E.; Conte, P.; Mari, G.; Dominici, M.
abstract

The original version of this Article contained an error in Affiliation 5, which was incorrectly given as ‘Department of Surgery, Oncology and Gastroenterology, University of Padova, Istituto Oncologico Veneto IRCCS, Padova, Italy’. The correct affiliation is listed below: Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy In addition, the original version of this Article omitted an affiliation for Pierfranco Conte. The correct affiliations for Pierfranco Conte are listed below: Medical Oncology 2, Veneto Institute of Oncology IOV, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Padova, Italy Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy These errors have now been corrected in the HTML and PDF versions of this Article, and in the accompanying Supplementary Information.

2020 - Autologous Fat Grafting for the Oral and Digital Complications of Systemic Sclerosis: Results of a Prospective Study [Articolo su rivista]
Pignatti, Marco; Spinella, Amelia; Cocchiara, Emanuele; Boscaini, Giulia; Lusetti, Irene Laura; Citriniti, Giorgia; Lumetti, Federica; Setti, Giacomo; Dominici, Massimo; Salvarani, Carlo; De Santis, Giorgio; Giuggioli, Dilia
abstract

Background: Systemic sclerosis is a connective tissue disease. Skin involvement of the mouth and hand may compromise function and quality of life. Autologous fat grafting has been described as a specific treatment of these clinical features. We report the results of our prospective study designed to treat and prevent skin complications in systemic sclerosis. Materials and methods: We treated 25 patients with mouth and/or hand involvement (microstomia, xerostomia, skin sclerosis, Raynaud's phenomenon and long-lasting digital ulcers) with autologous fat grafting, according to the Coleman's technique, around the mouth and/or at the base of each finger. The surgical procedures were repeated in each patient every 6 months for a total of two or three times. Clinical data were collected before the first surgery and again 6 months after each surgical procedure. Pain, skin thickness, saliva production and disability were assessed with validated tests. Results: Overall we performed 63 autologous fat grafting sessions (either on the mouth, on the hands or on both anatomical areas). Results at 6 moths after the last session included improvement of xerostomia evaluated with a sialogram, reduction of the skin tension around the mouth and, in the hands, reduction of the Raynaud phenomenon as well as skin thickness. Pain was reduced while the perception of disability improved. Digital ulcers healed completely in 8/9 patients. Conclusions: Our results confirm the efficacy and safety of autologous fat grafting for the treatment of skin complications and digital ulcers due to systemic sclerosis. In addition, the patients' subjective well-being improved. Level of evidence V This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 . Keywords: Autologous fat grafting; Digital ulcers; Microstomia; Stem cell transplantation; Systemic sclerosis; Xerostomia.

2020 - Cancer Stem-Like Cells in a Case of an Inflammatory Myofibroblastic Tumor of the Lung. [Articolo su rivista]
Masciale, Valentina; Grisendi, Giulia; Banchelli, Federico; D'Amico, Roberto; Maiorana, Antonino; Sighinolfi, Pamela; Brugioni, Lucio; Stefani, Alessandro; Morandi, Uliano; Dominici, Massimo; Aramini, Beatrice
abstract

Background: Inflammatory myofibroblast tumor (IMT) is a rare tumor with obscure etiopathogenesis in which different inflammatory cells and myofibroblastic spindle cells are seen histologically. Although the majority of these neoplasms have a benign clinical course, the malignant form has also been reported. The gold standard is surgical treatment for complete removal. Our report describes a 50-year-old woman who underwent surgery for IMT of the lung. The aim is to determine whether cancer stem cells may be present in IMT of the lung. Methods: In April 2018, the patient underwent surgery for tumor mass asportation through lateral thoracotomy. The histology of the tumor was consistent with IMT of the lung. The ALDEFLUOR assay, after tissue digestion, was used to identify and sort human lung cancer cells expressing high and low aldehyde dehydrogenase (ALDH) activity. SOX2, NANOG, OCT-4, and c-MYC positivity were additionally determined by immunohistochemistry. Results: The specimen contained 1.10% ALDHhigh cells among all viable lung cancer cells, which indicates the population of cancer stem cells is not negligible. Immunohistochemically assessed cell positivity for ALDH1A1, SOX2, NANOG, OCT-4, and c-MYC, which are considered as lung cancer stem-like cells markers. Conclusion: For the first time, we demonstrated the presence of cancer stem cells in a case of IMT of the lung. This finding may provide a base for considering new pathological and molecular aspects of this tumor. This perspective suggests further studies to understand the possibility of developing recurrence depending on the presence of cancer stem cells.

2020 - Cancer treatment during the coronavirus disease 2019 pandemic: Do not postpone, do it! [Articolo su rivista]
Omarini, C.; Maur, M.; Luppi, G.; Narni, F.; Luppi, M.; Dominici, M.; Longo, G.; Piacentini, F.
abstract

At the end of January 2020, a novel betacoronavirus, known as severe acute respiratory syndrome coronavirus 2, progressively spread in Italy. Patients with cancer are considered more prone to infections because of the immunosuppressive status due to both malignancy and anticancer treatments. From the first Italian government restrictions (23rd February), Modena Cancer Center adopted practical health vigilance recommendations to minimise the risk of exposure to the virus without overlooking cancer management. From 23rd February to 31st March 2020, 1257 patients on active anticancer treatment for oncological or haematological malignancies attended our institution. All the staff activities were rescheduled following our practical coronavirus disease 2019 (COVID-19) guideline. During this period, we have tallied 9 cases of COVID-19 infection (0.71%) in patients with cancer and 3 cases (1.66%) in health workers. The mortality rate of our patients with cancer was 22%, consistent with the data reported in the literature. In conclusion, following our practical health vigilance recommendations, physicians should be confident in maintaining life-saving anticancer treatment without exceedingly increasing the risk of nosocomial COVID-19 infection. The high rate of mortality suggested that all patients on active anticancer treatment with flu-like symptoms have to be carefully screened for COVID-19 infection.

2020 - CD44+/EPCAM+ cells detect a subpopulation of ALDHhigh cells in human non-small cell lung cancer: A chance for targeting cancer stem cells? [Articolo su rivista]
Masciale, Valentina; Grisendi, Giulia; Banchelli, Federico; D'Amico, Roberto; Maiorana, Antonino; Sighinolfi, Pamela; Stefani, Alessandro; Morandi, Uliano; Dominici, Massimo; Aramini, Beatrice
abstract

Objectives: Several studies demonstrated that aldehyde dehydrogenase (ALDH) and CD44 are the most considered cancer stem cells (CSC) markers. However, a comparison between ALDH high cells and CD44+ cells have been previously described with no significant correlation. Indeed, the aim of the present research is to identify a superficial marker able to match with ALDH high cells population in freshly isolated human lung cancer cells. Materials and Methods: This cross-sectional study analyzed the expression of ALDHhigh/low cells and the positivity for CD44 and epithelium cell adhesion molecule (EPCAM) antigens in surgical lung cancer tissues. The main approach was a cytofluorimetric analysis of ALDH expression and positivity for CD44/EPCAM on primary cell population obtained from 23 patients harboring NSCLC. Results: There was a highly positive correlation between the expressions of ALDHhigh and CD44+/EPCAM+ cells, with a Pearson’s correlation coefficient equal to 0.69 (95% CI 0.39–0.86; P = 0.0002), and Spearman’s correlation coefficient equal to 0.52 (P = 0.0124). The average paired difference between the expression of ALDHhigh and CD44+/EPCAM+ cells was very close to 0, being 0.1% (SD 2.5%); there was no difference between these subpopulations in terms of means (95% CI = –1.0; 1.2%, P = 0.8464). These results highlight a strong similarity between ALDHhigh and CD44+/EPCAM+ cells. Conclusions: Our study is the first attempt which identifies a high correlation between the ALDHhigh and the CD44+/EPCAM+ cells, thus suggesting the possibility to use this superficial marker for future target treatments against lung cancer stem cells.

2020 - Clinical Prognosticators in Patients Treated with CDK 4/6 Inhibitors for Hormone Receptors Positive Advanced Breast Cancer [Articolo su rivista]
Isca, Chrystel; Omarini, Claudia; Cortesi, Giulia; Moscetti, Luca; Barbolini, Monica; Dominici, Massimo; Piacentini, Federico
abstract

Background: CDK4/6 inhibitors are the new standard of care in hormonal receptors positive (HR+) advanced breast cancer (ABC). Phase III trials demonstrated an improvement in survival outcomes in patients with combined endocrine approach compared to endocrine therapy (ET) alone. The aim of this retrospective study was to assess prognostic factors for clinical response to CDK4/6 inhibitors. Methods: All patients receiving CDK4/6 inhibitors from September 2016 to July 2019 were registered in a database. Data on tumor and patient’s characteristics as well as concomitant medications were collected. Survival data were analyzed by Kaplan Meier curves and log rank test. Treatment toxicities were graded according to CTCAE v5. A drug-drug interactions analysis among CDK 4/6 inhibitors and co-administered medications was performed too. Results: 121 patients were included in the study: 49% of patients treated in 1st -line, 25% in 2nd -line and 26% in 3rd –or further lines. 1st-line objective response rate (ORR) and clinical benefit rate (CBR) was 56% and 68%, compared to 40% and 50% in 2nd-line and 31% and 47% in heavily pre-treated patients, respectively. Median PFS according to line setting was: not reached in 1st-line, 17 months (95% CI 13-21) in 2nd-line and 7 months (95% CI 4-12) in 3rd or further lines. Negative prognostic factors in term of PFS were: previous chemotherapy for metastatic disease (p=0.0001), visceral metastatic sites (p=0.002) and endocrine sensitivity (p=0.001). No association among concomitant drugs administered and survival outcome was found. 94% of patients experienced neutropenia (G3-G4 60%) with 3% of febrile neutropenia. 71% of patients treated with Abemaciclib had diarrhea. Management of AE included 63% of treatment delay, 44% of 1st dose reduction and 15% of 2nd dose reduction, all due to neutropenia. No treatment discontinuation due to any toxicity was observed. Conclusion: Data on efficacy and safety profile of CDK 4/6 inhibitors administered outside the context of a clinical trial are consistent with those reported in Phase III trials. Previous chemotherapy for metastatic disease, visceral metastatic site as well as previous endocrine sensitivity negatively affect CDK 4/6 inhibitors efficacy. Concomitant medications did not affect survival outcome or safety profile.

2020 - Combined endocrine approaches vs endocrine therapy alone as first line treatment in elderly patients with hormone receptor-positive, HER2 negative, advanced breast cancer: To prescribe for the patient or the physician? A meta-analysis of phase II and III randomized clinical trials [Articolo su rivista]
Omarini, C.; Piacentini, F.; Sperduti, I.; Barbolini, M.; Isca, C.; Toss, A.; Cortesi, L.; Barbieri, E.; Dominici, M.; Moscetti, L.
abstract

Background: Elderly patients are underrepresented in clinical study where combined endocrine strategies were compared to endocrine therapy (ET) in hormone receptors positive, HER2 negative, metastatic breast cancer. The role of the new endocrine approaches in elderly women is still unclear. Methods: We performed a meta-analysis of first line phase II/III randomized trials on ET versus combined strategies considering clinical benefit and safety profile. Trials with hazard ratio (HR) for PFS in elderly patients were included. Results: Overall, the meta-analysis showed a PFS advantage for the experimental arms [HR 0.77, p 0.016] with a significant high/moderate heterogeneity [I2 65.46%, p 0.005]. For patients on CDK 4/6 inhibitors and ET, HR was 0.57 (p < 0.0001), with low heterogeneity [I2 0.0001%, p 0.96]. Hematological adverse events, as well as diarrhea with Abemaciclib, were significantly higher in elderly population. Conclusions: The magnitude of PFS benefit due to the combined strategies in elderly patients is similar to those reported in the overall clinical trial population. Adding CDK4/6 inhibitors to ET significantly prolongs PFS, even if toxicity profile have to be carefully considered. Future trials should be designed taking into account patients' age, geriatric assessment and comorbidity.

2020 - Early efficacy evaluation of mesenchymal stromal cells (MSC) combined to biomaterials to treat long bone non-unions [Articolo su rivista]
Gomez-Barrena, E.; Padilla-Eguiluz, N.; Rosset, P.; Gebhard, F.; Hernigou, P.; Baldini, N.; Rouard, H.; Sensebe, L.; Gonzalo-Daganzo, R. -M.; Giordano, R.; Garcia-Rey, E.; Cordero-Ampuero, J.; Rubio-Suarez, J. C.; Garcia-Simon, M. D.; Stanovici, J.; Ehrnthaller, C.; Huber-Lang, M.; Flouzat-Lachaniette, C. H.; Chevallier, N.; Donati, D. M.; Spazzoli, B.; Ciapetti, G.; Fleury, S.; Fernandez, M. -N.; Cabrera, J. -R.; Avendano-Sola, C.; Montemurro, T.; Panaitescu, C.; Veronesi, E.; Rojewski, M. T.; Lotfi, R.; Dominici, M.; Schrezenmeier, H.; Layrolle, P.
abstract

Background and study aim: Advanced therapy medicinal products (ATMP) frequently lack of clinical data on efficacy to substantiate a future clinical use. This study aims to evaluate the efficacy to heal long bone delayed unions and non-unions, as secondary objective of the EudraCT 2011-005441-13 clinical trial, through clinical and radiological bone consolidation at 3, 6 and 12 months of follow-up, with subgroup analysis of affected bone, gender, tobacco use, and time since the original fracture. Patients and methods: Twenty-eight patients were recruited and surgically treated with autologous bone marrow derived mesenchymal stromal cells expanded under Good Manufacturing Practices, combined to bioceramics in the surgical room before implantation. Mean age was 39 ± 13 years, 57% were males, and mean Body Mass Index 27 ± 7. Thirteen (46%) were active smokers. There were 11 femoral, 4 humeral, and 13 tibial non-unions. Initial fracture occurred at a mean ± SD of 27.9 ± 31.2 months before recruitment. Efficacy results were expressed by clinical consolidation (no or mild pain if values under 30 in VAS scale), and by radiological consolidation with a REBORNE score over 11/16 points (value of or above 0.6875). Means were statistically compared and mixed models for repeated measurements estimated the mean and confidence intervals (95%) of the REBORNE Bone Healing scale. Clinical and radiological consolidation were analyzed in the subgroups with Spearman correlation tests (adjusted by Bonferroni). Results: Clinical consolidation was earlier confirmed, while radiological consolidation at 3 months was 25.0% (7/28 cases), at 6 months 67.8% (19/28 cases), and at 12 months, 92.8% (26/28 cases including the drop-out extrapolation of two failures). Bone biopsies confirmed bone formation surrounding the bioceramic granules. All locations showed similar consolidation, although this was delayed in tibial non-unions. No significant gender difference was found in 12-month consolidation (95% confidence). Higher consolidation scale values were seen in non-smoking patients at 6 (p = 0.012, t-test) and 12 months (p = 0.011, t-test). Longer time elapsed after the initial fracture did not preclude the occurrence of consolidation. Conclusion: Bone consolidation was efficaciously obtained with the studied expanded hBM-MSCs combined to biomaterials, by clinical and radiological evaluation, and confirmed by bone biopsies, with lower consolidation scores in smokers.

2020 - Emerging Neuroblastoma 3D In Vitro Models for Pre-Clinical Assessments [Articolo su rivista]
Corallo, D.; Frabetti, S.; Candini, O.; Gregianin, E.; Dominici, M.; Fischer, H.; Aveic, S.
abstract

The potential of tumor three-dimensional (3D) in vitro models for the validation of existing or novel anti-cancer therapies has been largely recognized. During the last decade, diverse in vitro 3D cell systems have been proposed as a bridging link between two-dimensional (2D) cell cultures and in vivo animal models, both considered gold standards in pre-clinical settings. The latest awareness about the power of tailored therapies and cell-based therapies in eradicating tumor cells raises the need for versatile 3D cell culture systems through which we might rapidly understand the specificity of promising anti-cancer approaches. Yet, a faithful reproduction of the complex tumor microenvironment is demanding as it implies a suitable organization of several cell types and extracellular matrix components. The proposed 3D tumor models discussed here are expected to offer the required structural complexity while also assuring cost-effectiveness during pre-selection of the most promising therapies. As neuroblastoma is an extremely heterogenous extracranial solid tumor, translation from 2D cultures into innovative 3D in vitro systems is particularly challenging. In recent years, the number of 3D in vitro models mimicking native neuroblastoma tumors has been rapidly increasing. However, in vitro platforms that efficiently sustain patient-derived tumor cell growth, thus allowing comprehensive drug discovery studies on tailored therapies, are still lacking. In this review, the latest neuroblastoma 3D in vitro models are presented and their applicability for a more accurate prediction of therapy outcomes is discussed.

2020 - Expression of ALDH and SOX-2 in Pulmonary Sclerosing Pnemocytoma (PSP) of the Lung: Is There a Meaning Behind? [Articolo su rivista]
Aramini, Beatrice; Masciale, Valentina; Manfredini, Beatrice; Bianchi, Daniel; Banchelli, Federico; D'Amico, Roberto; Bertolini, Federica; Dominici, Massimo; Morandi, Uliano; Maiorana, Antonino
abstract

Background: Pulmonary sclerosing pneumocytoma (PSP) is a rare benign pulmonary tumor that derives from primitive respiratory epithelium of the pulmonary alveolus. The etiology and pathogenesis are still unclear. Histopathological diagnosis focuses on cells that are positive for TTF1, EMA, cytokeratin-7, and CAM 5.2. The aim of our study is to highlight the elevated expression of ALDH and the presence of SOX-2 in pulmonary sclerosing pneumocytoma. Methods: We report five cases of pulmonary sclerosing pneumocytoma undergone surgery at our Division of Thoracic Surgery, during a period between 1994 and 2011. ALDH and SOX-2 markers were also tested for positivity in all the patients. Results: Patients showed elevated expression of ALDH during immunohistochemistry and mild expression of SOX-2, although in two cases in which SOX-2 was highly expressed. Among these two patients, one presented with lymph node recurrence while the other had no recurrence with a PET-positive nodule. In particular, the patient who had developed recurrence had an ALDH score of 4 and a SOX-2 score of 3, whereas the patient with the PET-positive nodule showed an ALDH score of 4 with a mild SOX-2expression of score 1. Conclusions: This is the first attempt demonstrating the elevated expression of ALDH in this disease. SOX-2 expression was noted in both the patient who developed recurrence and the patient with a PET-positive nodule. We believe that further investigation may be highly useful to better characterize these two markers as well as understandtheir function.

2020 - Genetic Engineering as a Strategy to Improve the Therapeutic Efficacy of Mesenchymal Stem/Stromal Cells in Regenerative Medicine [Articolo su rivista]
Damasceno, P. K. F.; de Santana, T. A.; Santos, G. C.; Orge, I. D.; Silva, D. N.; Albuquerque, J. F.; Golinelli, G.; Grisendi, G.; Pinelli, M.; Ribeiro dos Santos, R.; Dominici, M.; Soares, M. B. P.
abstract

Mesenchymal stem/stromal cells (MSCs) have been widely studied in the field of regenerative medicine for applications in the treatment of several disease settings. The therapeutic potential of MSCs has been evaluated in studies in vitro and in vivo, especially based on their anti-inflammatory and pro-regenerative action, through the secretion of soluble mediators. In many cases, however, insufficient engraftment and limited beneficial effects of MSCs indicate the need of approaches to enhance their survival, migration and therapeutic potential. Genetic engineering emerges as a means to induce the expression of different proteins and soluble factors with a wide range of applications, such as growth factors, cytokines, chemokines, transcription factors, enzymes and microRNAs. Distinct strategies have been applied to induce genetic modifications with the goal to enhance the potential of MCSs. This review aims to contribute to the update of the different genetically engineered tools employed for MSCs modification, as well as the factors investigated in different fields in which genetically engineered MSCs have been tested.

2020 - IDH signalling pathway in cholangiocarcinoma: From biological rationale to therapeutic targeting [Articolo su rivista]
Salati, M.; Caputo, F.; Baldessari, C.; Galassi, B.; Grossi, F.; Dominici, M.; Ghidini, M.
abstract

Biliary tract cancers are anatomically distinct and genetically diverse tumors, evenly characterized by poor response to standard treatments and a bleak outlook. The advent of comprehensive genomic profiling using next-generation sequencing has unveiled a plethora of potentially actionable aberrations, changing the view of biliary tract cancers from an “orphan” to a “target-rich” disease. Recently, mutations in isocitrate dehydrogenase genes (IDH1/2) and fusions of the fibroblast growth factor receptor have emerged as the most amenable to molecularly targeted inhibition, with several compounds actively investigated in advanced-phase clinical trials. Specifically, the IDH1 inhibitor ivosidenib has been the first targeted agent to show a survival benefit in a randomized phase III trial of cholangiocarcinoma patients harboring IDH1 mutations. In this review article, we will focus on the IDH1/IDH2 pathway, discussing the preclinical rationale of its targeting as well as the promises and challenges of the clinical development of IDH inhibitors in biliary tract cancers.

2020 - Integrated intErventional bronchoscopy in the treatment of locally adVanced non-small lung cancER with central Malignant airway Obstructions: a multicentric REtrospective study (EVERMORE). [Articolo su rivista]
Marchioni, A; Andrisani, D; Tonelli, R; Piro, R; Andreani, A; Cappiello, Gf; Meschiari, E; Dominici, M; Bavieri, M; Barbieri, F; Taddei, S; Casalini, E; Falco, F; Gozzi, F; Bruzzi, G; Fantini, R; Tabbì, L; Castaniere, I; Facciolongo, N; Clini, E.
abstract

Objectives- Despite new therapeutic perspectives, the presence of central airways occlusion (CAO) in patients with locally advanced non-small cell lung cancer (NSCLC) is associated with poor survival. There is no clear evidence on the clinical impact of interventional bronchoscopy as a part of an integrated treatment to cure these patients. Materials and methods- This retrospective cohort study was conducted in two teaching hospitals over a 10 years period (January 2010-January 2020) comparing patients with NSCLC at stage IIIB and CAO at disease onset treated with chemotherapy/radiotherapy (standard therapy-ST) with those receiving interventional bronchoscopy plus ST (integrated treatment-IT). Primary outcome was 1-year survival. The onset of respiratory events, symptoms-free interval, hospitalization, need for palliation, and overall mortality served as secondary outcomes. Results- A total of 100 patients were included, 60 in the IT and 40 in the ST group. Unadjusted Kaplan-Meier estimates showed greater effect of IT compared to ST on 1-year survival (HR=2.1 95%CI[1.1-4.8], p=0.003). IT showed a significantly higher survival gain over ST in those patients showing KRAS mutation (7.6 VS 0.8 months,<0.0001), a lumen occlusion >65% (6.6 VS 2.9 months,<0.001), and lacking the involvement of left bronchus (7 VS 2.3 months,<0.0001). Compared to ST, IT also showed a favorable difference in terms of new hospitalizations (p=0.03), symptom-free interval (p=0.02), and onset of atelectasis (p=0.01). Conclusions- In patients with NSCLC stage IIIB and CAO, additional interventional bronchoscopy might impact on 1-year survival. Genetic and anatomic phenotyping might allow identifying those patients who may gain life expectancy from the endoscopic intervention.

2020 - International Society for Extracellular Vesicles and International Society for Cell and Gene Therapy statement on extracellular vesicles from mesenchymal stromal cells and other cells: considerations for potential therapeutic agents to suppress coronavirus disease-19 [Articolo su rivista]
Borger, V.; Weiss, D. J.; Anderson, J. D.; Borras, F. E.; Bussolati, B.; Carter, D. R. F.; Dominici, M.; Falcon-Perez, J. M.; Gimona, M.; Hill, A. F.; Hoffman, A. M.; de Kleijn, D.; Levine, B. L.; Lim, R.; Lotvall, J.; Mitsialis, S. A.; Monguio-Tortajada, M.; Muraca, M.; Nieuwland, R.; Nowocin, A.; O'Driscoll, L.; Ortiz, L. A.; Phinney, D. G.; Reischl, I.; Rohde, E.; Sanzenbacher, R.; Thery, C.; Toh, W. S.; Witwer, K. W.; Lim, S. K.; Giebel, B.
abstract

STATEMENT: The International Society for Cellular and Gene Therapies (ISCT) and the International Society for Extracellular Vesicles (ISEV) recognize the potential of extracellular vesicles (EVs, including exosomes) from mesenchymal stromal cells (MSCs) and possibly other cell sources as treatments for COVID-19. Research and trials in this area are encouraged. However, ISEV and ISCT do not currently endorse the use of EVs or exosomes for any purpose in COVID-19, including but not limited to reducing cytokine storm, exerting regenerative effects or delivering drugs, pending the generation of appropriate manufacturing and quality control provisions, pre-clinical safety and efficacy data, rational clinical trial design and proper regulatory oversight.

2020 - Interstitial Lung Disease in Abemaciclib-treated Patients during SARS-CoV-2 Pandemic: A Case Series. [Articolo su rivista]
Barbolini, Monica; Omarini, Claudia; Toss, Angela; Cortesi, Giulia; Fiorani, Claudia; Piombino, Claudia; Dominici, Massimo; Piacentini, Federico
abstract

Abemaciclib is a cyclin-dependent 4/6 inhibitor approved by FDA in 2017 and by EMA in 2018 for treatment of hormone receptors positive, HER2 negative metastatic breast cancer in association with endocrine therapy. In 2019 FDA warned for a rare but severe lung inflammation possibly related to CDK 4/6 inhibitors. At the end of 2019 the world comes to know to a new corona virus causing potential fatal lung injury. Here we report three different cases of interstitial lung disease in patients treated with Abemaciclib during SARS-CoV-2 pandemic at Modena Cancer Center.

2020 - LETTER TO THE EDITOR: Henoch-schönlein Purpura (HSP) in a Patient on Abemaciclib [Articolo su rivista]
Omarini, Caudia; Molinaro, Eleonora; Barbolini, Monica; Dominici, Massimo; Piacentini, Federico
abstract

No abstract available.

2020 - Mesenchymal stromal cells and their secreted extracellular vesicles as therapeutic tools for COVID-19 pneumonia? [Articolo su rivista]
Muraca, M.; Pessina, A.; Pozzobon, M.; Dominici, M.; Galderisi, U.; Lazzari, L.; Parolini, O.; Lucarelli, E.; Perilongo, G.; Baraldi, E.
abstract

The COVID-19 epidemic represents an unprecedented global health emergency, further aggravated by the lack of effective therapies. For this reason, several clinical trials are testing different off-label drugs, already approved for other pathologies. Mesenchymal stem/stromal cells (MSCs) have been tested during the last two decades for the treatment of various pathologic conditions, including acute and chronic lung diseases, both in animal models and in patients. In particular, promising results have been obtained in the experimental therapy of acute respiratory distress syndrome, which represents the most threatening complication of COVID-19 infection. Furthermore, more recently, great interest has been devoted to the possible clinical applications of extracellular vesicles secreted by MSCs, nanoparticles that convey much of the biological effects and of the therapeutic efficacy of their cells of origin. This review summarizes the experimental evidence underlying the possible use of MSCs and of MSC-EVs in severe COVID-19 infection and underlines the need to evaluate the possible efficacy of these therapeutic approaches through controlled studies under the supervision of the Regulatory Authorities.

2020 - Modulating endothelial adhesion and migration impacts stem cell therapies efficacy [Articolo su rivista]
Schafer, R.; Schwab, M.; Siegel, G.; von Ameln-Mayerhofer, A.; Buadze, M.; Lourhmati, A.; Wendel, H. -P.; Kluba, T.; Krueger, M. A.; Calaminus, C.; Scheer, E.; Dominici, M.; Grisendi, G.; Doeppner, T. R.; Schlechter, J.; Finzel, A. K.; Gross, D.; Klaffschenkel, R.; Gehring, F. K.; Spohn, G.; Kretschmer, A.; Bieback, K.; Kramer-Albers, E. -M.; Barth, K.; Eckert, A.; Elser, S.; Schmehl, J.; Claussen, C. D.; Seifried, E.; Hermann, D. M.; Northoff, H.; Danielyan, L.
abstract

Background: Limited knowledge of stem cell therapies‘ mechanisms of action hampers their sustainable implementation into the clinic. Specifically, the interactions of transplanted stem cells with the host vasculature and its implications for their therapeutic efficacy are not elucidated. We tested whether adhesion receptors and chemokine receptors on stem cells can be functionally modulated, and consequently if such modulation may substantially affect therapeutically relevant stem cell interactions with the host endothelium. Methods: We investigated the effects of cationic molecule polyethylenimine (PEI) treatment with or without nanoparticles on the functions of adhesion receptors and chemokine receptors of human bone marrow-derived Mesenchymal Stem Cells (MSC). Analyses included MSC functions in vitro, as well as homing and therapeutic efficacy in rodent models of central nervous system´s pathologies in vivo. Findings: PEI treatment did not affect viability, immunomodulation or differentiation potential of MSC, but increased the CCR4 expression and functionally blocked their adhesion receptors, thus decreasing their adhesion capacity in vitro. Intravenously applied in a rat model of brain injury, the homing rate of PEI-MSC in the brain was highly increased with decreased numbers of adherent PEI-MSC in the lung vasculature. Moreover, in comparison to untreated MSC, PEI-MSC featured increased tumour directed migration in a mouse glioblastoma model, and superior therapeutic efficacy in a murine model of stroke. Interpretation: Balanced stem cell adhesion and migration in different parts of the vasculature and tissues together with the local microenvironment impacts their therapeutic efficacy. Funding: Robert Bosch Stiftung, IZEPHA grant, EU grant 7 FP Health

2020 - Modulation of Mutational Landscape in HER2-Positive Breast Cancer after Neoadjuvant Chemotherapy [Articolo su rivista]
Omarini, Claudia; Bettelli, Stefania Raffaella; Manfredini, Samantha; Barbolini, Monica; Isca, Chrystel; Cortesi, Giulia; Maiorana, Antonino; Tazzioli, Giovanni; Dominici, Massimo; Piacentini, Federico
abstract

2020 - Multicentre validation of an immune-inflammation-based nomogram to predict survival in western resectable gastroesophageal adenocarcinoma: The NOMOGAST [Abstract in Rivista]
Salati, M; Marcheselli, L; De Ruvo, N; Esposito, G; Fenocchi, S; Cucciarre, G; Serra, F; Cautero, N; Cabry, F; Gelmini, R; Vittimberga, G; Radi, G; Solaini, L; Morgagni, P; Ercolani, G; Ghidini, M; Grizzi, G; Ratti, M; Gelsomino, F; Luppi, G; Dominici, M; Spallanzani, A
abstract

2020 - On the in Vitro Biocompatibility Testing of Bioactive Glasses [Articolo su rivista]
Bellucci, Devis; Veronesi, Elena; Dominici, Massimo; Cannillo, Valeria
abstract

2020 - Overall survival in patients with lung adenocarcinoma harboring "niche" mutations: an observational study [Articolo su rivista]
Aramini, Beatrice; Banchelli, Federico; Bettelli, Stefania Raffaella; Manfredini, Samantha; D'Amico, Roberto; Masciale, Valentina; Pinelli, Massimo; MORETTI FANTERA, Margherita; Stefani, Alessandro; Bertolini, Federica; Dominici, Massimo; Morandi, Uliano; Maiorana, Antonino
abstract

Objective: In addition to the most common somatic lung cancer mutations (i. e., KRAS and EGFR mutations), other genes may harbor mutations that could be relevant for lung cancer. We defined BRAF, c-MET, DDR2, HER2, MAP2K1, NRAS, PIK3CA, and RET mutations as “niche” mutations and analyzed. The aim of this retrospective cohort study was to assess the differences in the overall survival (OS) of patients with lung adenocarcinoma harboring niche somatic mutations. Results: Data were gathered for 252 patients. Mutations were observed in all genes studied, except c-MET, DDR2, MAP2K1, and RET. The multivariable analysis showed that 1) niche mutations had a higher mortality than EGFR mutations (HR = 2.3; 95% CI = 1.2–4.4; p = 0.009); 2) KRAS mutations had a higher mortality than EGFR mutations (HR = 2.5; 95% CI = 1.4–4.5; p = 0.003); 3) niche mutations presented a similar mortality to KRAS mutations (HR = 0.9; 95% CI = 0.6–1.5; p = 0.797). Methods: Three cohorts of mutations were selected from patients with lung adenocarcinoma and their OS was compared. Mutations that were searched for, were 1) BRAF, c-MET, DDR2, HER2, MAP2K1, NRAS, PIK3CA, and RET; 2) K-RAS; and 3) EGFR. Differences in OS between these three cohorts were assessed by means of a multivariable Cox model that adjusted for age, sex, smoking habits, clinical stages, and treatments. Conclusions: Niche mutations exhibited an increased risk of death when compared with EGFR mutations and a similar risk of death when compared with KRAS mutations.

2020 - PD-1 blockade in deficient mismatch repair mixed adenoneuroendocrine carcinoma of the stomach: new hope for an orphan disease [Articolo su rivista]
Ricco, B.; Salati, M.; Reggiani Bonetti, L.; Dominici, M.; Luppi, G.
abstract

Introduction: Mixed adenoneuroendocrine carcinoma (MANEC) is an uncommon and aggressive tumor arising throughout the entire gastrointestinal tract. Treatment options are limited, and survival is dismal with conventional therapies. Case description: We present the case of a 66-year-old man who was diagnosed with a locally advanced MANEC of the gastroesophageal junction. He was treated with perioperative chemotherapy and total gastrectomy. After anastomotic tumor recurrence was detected, he underwent three systemic regimens, including chemoradiotherapy. The patient was then tested for mismatch repair (MMR) protein status, revealing defective expression of MLH1 and PMS2 proteins. Treatment with anti–programmed death 1 (PD-1) receptor monoclonal antibody pembrolizumab was started and radiologic response is ongoing after 11 months. Clinical benefit was evident, and no immune therapy–related side effect was detected. Conclusions: To our knowledge, this is the first report of a heavily pretreated and rapidly progressing deficient MMR gastroesophageal MANEC experiencing a durable benefit from anti-PD1 treatment.

2020 - Precision Medicine in Lung Cancer: Challenges and Opportunities in Diagnostic and Therapeutic Purposes. [Capitolo/Saggio]
Aramini, Beatrice; Masciale, Valentina; Banchelli, Federico; D'Amico, Roberto; Dominici, Massimo; Husnain Haider, Khawaja
abstract

Lung cancer is one of the leading causes of cancer death among both men and women, making up almost 25% of all cancer deaths. Precision medicine shows promise for improving many aspects of health and healthcare, including tests, drugs, and other technologies that support innovation, with the possibility of new partnerships with scientists in a wide range of specialties. Non–small-cell lung cancer (NSCLC) has become a prominent example of the success of precision medicine in treating solid tumor malignancies. The first step in this process involves new bloodbased diagnostics, which can now noninvasively provide clinically useful information. However, the identification of novel biomarkers that could be used in early diagnosis is urgently needed, especially for guiding initial therapy and predicting relapse or drug resistance following the administration of novel targeted therapies.

2020 - Predictors of HER2 gene amplification in immunohistochemistry score 2+ Early Breast Cancer according to 2018 ASCO/CAP guidelines: a single institution analysis. [Poster]
Barbolini, M.; Omarini, C.; Bettelli, S.; Manfredini, S.; Dominici, M.; Piacentini, F.
abstract

Background: HER2 overexpression occurs in approximately 15-20% of invasive breast cancers (BC). From a pathological point of view HER2 positivity is defined by intense circumferential membrane complete staining in more than 10% of tumour cells in immunohistochemistry (IHC score 3+). When complete circumferential staining is weak to moderate (IHC score 2+) double probe in situ ibridation (ISH) is mandatory to define HER2 status. In 2018 ASCO/CAP guidelines were updated to provide additional guidance in HER2 equivocal cases to allow a greater discrimination between positive and negative cases. Our aim is to find predictors of HER2 positivity among IHC score 2+ early breast cancer specimens analysed according to 2018 ASCO/CAP guidelines. Patients and methods: 253 cases of early BC diagnosed at Modena Cancer Center between November 2013 and August 2017 were identified. Stage, ISH result, hormonal receptor status (HR), proliferation index (MIB1), and histological grade were captured; menopausal status was available too. All IHC score 2+ cases were reclassified according to 2018 ASCO/CAP guidelines. The association between pathological tumour features, clinical characteristics and ISH positivity was assessed using Fisher test. Results: Overall, 25.7% IHC score 2+ BC resulted HER2 amplified in double probe ISH. High tumour grade (G3 vs G1-2) and MIB1 > 20% significantly predict HER2 ISH amplification (p=0,0001). No correlation was found according to HR, stage, or menopausal status. The majority (185; 98.4%) of HER2-ve BC were reclassified as group 5 (HER2/ CEP17 ratio <2 and HER2 copy number <4 signals/cell) except for 3 specimens classified as group 4 (HER2/CEP17 RATIO <2 and HER2 copy number ³4 but <6 signals/cell). In HER2+ve group the majority (62; 95.3%) specimens were group 1 (HER2/CEP17 RATIO >2 and HER2 copy number =4 signals/cell), no specimen was group 2, and only 3 cases were classified as group 3 (HER2/CEP17 RATIO <2 and HER2 copy number >6 signals/cell). Conclusions: In this IHC score 2+ BC series, reclassification according to 2018 ASCO/CAP guidelines identified only 4.6% group 3 and 1.6% group 4 cases. The routinely assessment of grading and proliferation index could help to predict HER2 amplification in IHC score 2+ samples even if it must not substitute ISH assay in determining eligibility for HER2 targeted therapies.

2020 - Primary treatment strategy in elderly patients with hormone receptor positive early breast cancer: is breast cancer surgery. [Poster]
Nasso, C.; Barbolini, M.; Isca, C.; D'Onofrio, R.; Cortesi, G.; Dominici, M.; Piacentini, F.; Omarini, C.
abstract

Background: Older age, ECOG performance status, major comorbidities and concomitant medications influence the primary treatment strategy of hormone receptor positive (HR+) early breast cancer (EBC) patients. In case of frail patients, oncologist may choose primary endocrine therapy (ET) instead of breast cancer surgery (BCS) even if its clinical impact is still unknown. Methods: We performed a retrospective study on women aged 75 years and older with HR+ EBC diagnosed at the Modena Cancer Center from 2010 to 2016. According to primary treatment strategy patients were divided into two groups: patients who underwent BCS (BCS group) versus patients treated with only ET (ET group). Patients’ clinical data and tumor characteristics were collected. Disease Free Survival (DFS) and Breast cancer-specific survival (BCSS) were estimated by long rank test and Kaplan-Meier curves. Results: 143 patients were involved in the study: 105 in BCS group and 38 in ET one. Patients who underwent surgery had significantly better ECOG (p=0.000001), low tumour grade (p=0.04) and early clinical stage (p=0.0001) compared to those in the ET group. In patients with negative lymph-nodes at the diagnosis, tumor stage I-II and low ki67 (<20%), BCS did not improve 5-years DFS in univariate and multivariate analysis (p = 0.099, 95%CI 0.152-1.175). No differences were found in terms of 5-years BCSS between the two groups (p = 0.195). Conclusions: Stage I-II at the diagnosis, negative axillary lymph nodes and low ki67 identified a subgroup of HR+ EBC patients aged > 75 with low risk of disease progression who may not benefit from primary BCS. In elderly frail patients, primary ET instead of BCS could be a valid treatment strategy that should be considered on a case-bycase basis.

2020 - PROGNOSIS AND RESPONSE TO NEOADJUVANT CHEMOTHERAPY ACCORDING TO HER2 EXPRESSION IN EARLY BREAST CANCER: a retrospective single institution analysis [Poster]
D'Onofrio, R.; Omarini, C.; Barbolini, M.; Nasso, C.; Isca, C.; Dominici, M.; Piacentini, F.
abstract

Background: The assessment of HER2 status plays a key role in the treatment decision making process of early breast cancer (BC).HER2 status is routinely assessed by immunohistochemistry and/or in situ hybridization (ISH), according to ASCO/CAP 2018 guidelines. The dichotomous definition of HER2+ versus HER2- disease does not reflect the spectrum of variable levels of HER2 protein expression. We have analyzed the clinical outcomes of a cohort of BC patients treated with neoadjuvant chemotherapy (NACT) according to HER2 score. Patients and methods: We performed a retrospective analysis of 483 women with early BC treated with NACT from March 2002 to November 2018 at Modena Cancer Center. Clinical and pathological characteristics of patients and disease were collected and compared to HER2 score by Pearson’s chi square test. The impact of HER2 status on pathological complete response (pCR) was set according to logistic regression model. Results: According to HER2 score, patients were divided in 5 groups: 79 (16,3%) of them in HER2 score0 group, 153 (31,7%) in HER2 score1+, 53 (11%) HER2 score2+/ ISH-, 43 (9%) score2+/ISH+ and 155 (32%) score3+. Of note, 59% of women had hormone receptor positive BC. Overall, the HER2 status significant correlated to histotype (p=0,003), grading (p=0,026) and pCR (p=0,0001). The pCR was achieved in 132 (27%) patients: 46 (9,5%) in HER2 score0/1+ group, 9 (1,9%) in HER2 score2+/ISH-, 8 (1,7%) in HER2 score2+/ISH+ and 69 (14,3%) in HER2 score3+. The pCR rate was not statistically different when both HER2 score0/1+ and HER2 score2+/ISHgroups were compared to HER2 score2+/ISH+ one. Considering HER2+ BCs, the pCR rate was significantly higher in HER2 score3+ compared to HER2 score2+/ ISH+ (p=0,002). No statistically significant differences in terms of RFS and OS inter-subgroups were observed. Conclusions: The HER2 protein expression levels better correlated to pCR compare to HER2 gene amplification. In particular, the lack of difference in pCR rate between HER2 score2+/ISH+ and HER2 score2+/ISH– groups may suggests a paradigm shift in the current classification of HER2 status, consistently with emerging data on “HER2 low” BC landscape.

2020 - Targeting GD2-positive glioblastoma by chimeric antigen receptor empowered mesenchymal progenitors [Articolo su rivista]
Golinelli, Giulia; Grisendi, Giulia; Prapa, Malvina; Bestagno, Marco; Spano, Carlotta; Rossignoli, Filippo; Bambi, Franco; Sardi, Iacopo; Cellini, Monica; Horwitz, Edwin M.; Feletti, Alberto; Pavesi, Giacomo; Dominici, Massimo
abstract

Tumor targeting by genetically modified mesenchymal stromal/stem cells (MSCs) carrying anti-cancer molecules represents a promising cell-based strategy. We previously showed that the pro-apoptotic agent tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can be successfully delivered by MSCs to cancer sites. While the interaction between TRAIL and its receptors is clear, more obscure is the way in which MSCs can selectively target tumors and their antigens. Several neuroectoderm-derived neoplasms, including glioblastoma (GBM), sarcomas, and neuroblastoma, express high levels of the tumor-associated antigen GD2. We have already challenged this cell surface disialoganglioside by a chimeric antigen receptor (CAR)-T cell approach against neuroblastoma. With the intent to maximize the therapeutic profile of MSCs delivering TRAIL, we here originally developed a bi-functional strategy where TRAIL is delivered by MSCs that are also gene modified with the truncated form of the anti-GD2 CAR (GD2 tCAR) to mediate an immunoselective recognition of GD2-positive tumors. These bi-functional MSCs expressed high levels of TRAIL and GD2 tCAR associated with a robust anti-tumor activity against GD2-positive GBM cells. Most importantly, the anti-cancer action was reinforced by the enhanced targeting potential of such bi-functional cells. Collectively, our results suggest that a truncated anti-GD2 CAR might be a powerful new tool to redirect MSCs carrying TRAIL against GD2-expressing tumors. This affinity-based dual targeting holds the promise to combine site-specific and prolonged retention of MSCs in GD2-expressing tumors, thereby providing a more effective delivery of TRAIL for still incurable cancers.

2020 - Two Decades of Global Progress in Authorized Advanced Therapy Medicinal Products: An Emerging Revolution in Therapeutic Strategies [Articolo su rivista]
Ramezankhani, R.; Torabi, S.; Minaei, N.; Madani, H.; Rezaeiani, S.; Hassani, S. N.; Gee, A. P.; Dominici, M.; Silva, D. N.; Baharvand, H.; Hajizadeh-Saffar, E.
abstract

The introduction of advanced therapy medicinal products (ATMPs) to the global pharma market has been revolutionizing the pharmaceutical industry and has opened new routes for treating various types of cancers and incurable diseases. In the past two decades, a noticeable part of clinical practices has been devoting progressively to these products. The first step to develop such an ATMP product is to be familiar with other approved products to obtain a general view about this industry trend. The present paper depicts an overall perspective of approved ATMPs in different countries, while reflecting the degree of their success in a clinical point of view and highlighting their main safety issues and also related market size as a whole. In this regard, published articles regarding safety, efficacy, and market size of approved ATMPs were reviewed using the search engines PubMed, Scopus, and Google Scholar. For some products which the related papers were not available, data on the relevant company website were referenced. In this descriptive study, we have introduced and classified approved cell, gene, and tissue engineering-based products by different regulatory agencies, along with their characteristics, manufacturer, indication, approval date, related regulatory agency, dosage, product description, price and published data about their safety and efficacy. In addition, to gain insights about the commercial situation of each product, we have gathered accessible sale reports and market size information that pertain to some of these products.

2019 - A Novel 3D In Vitro Platform for Pre-Clinical Investigations in Drug Testing, Gene Therapy, and Immuno-oncology. [Articolo su rivista]
Candini, O; Grisendi, G; Foppiani, Em; Brogli, M; Aramini, B; Masciale, V; Spano, C; Petrachi, T; Veronesi, E; Conte, P; Mari, G; Dominici, M.
abstract

Tumors develop within complex cell-to-cell interactions, with accessory cells playing a relevant role starting in the early phases of cancer progression. This event occurs in a three-dimensional (3D) environment, which to date, has been difficult to reproduce in vitro due to its complexity. While bi-dimensional cultures have generated substantial data, there is a progressive awareness that 3D culture strategies may rapidly increase the understanding of tumor development and be used in anti-cancer compound screening and for predicting response to new drugs utilizing personalized approaches. However, simple systems capable of rapidly rebuilding cancer tissues ex-vivo in 3D are needed and could be used for a variety of applications. Therefore, we developed a flat, handheld and versatile 3D cell culture bioreactor that can be loaded with tumor and/or normal cells in combination which can be monitored using a variety of read-outs. This biocompatible device sustained 3D growth of tumor cell lines representative of various cancers, such as pancreatic and breast adenocarcinoma, sarcoma, and glioblastoma. The cells repopulated the thin matrix which was completely separated from the outer space by two gas-permeable membranes and was monitored in real-time using both microscopy and luminometry, even after transportation. The device was tested in 3D cytotoxicity assays to investigate the anti-cancer potential of chemotherapy, biologic agents, and cell-based therapy in co-cultures. The addition of luciferase in target cancer cells is suitable for comparative studies that may also involve parallel in vivo investigations. Notably, the system was challenged using primary tumor cells harvested from lung cancer patients as an innovative predictive functional assay for cancer responsiveness to checkpoint inhibitors, such as nivolumab. This bioreactor has several novel features in the 3D-culture field of research, representing a valid tool useful for cancer investigations, drug screenings, and other toxicology approaches.

2019 - Acid microenvironment promotes cell survival of human bone sarcoma through the activation of cIAP proteins and NF-κB pathway [Articolo su rivista]
Avnet, Sofia; Chano, Tokuhiro; Massa, Annamaria; Bonuccelli, Gloria; Lemma, Silvia; Falzetti, Luigi; Grisendi, Giulia; Dominici, Massimo; Baldini, Nicola
abstract

Extracellular acidification is a very common cause of stress in tumor microenvironment and of Darwinian pressure. In acid areas of the tumor, most cancer cells are-albeit slowly proliferating-more resistant to cell death than those in well-perfused regions. Tumor acidosis can directly regulate the expression of pro-survival proteins since a low extracellular pH activates the caspase-dependent cell death machinery. This mechanism has never been explored in bone sarcomas. We cultured osteosarcoma and Ewing sarcoma cells under low pH (pH 6.5), and we performed deep-sequencing and protein analysis. Both in in vitro and in vivo models, acidification activity enhanced tumor cells survival. However, we did not observe any change in ERK1 phosphorylation. On the contrary, both at the mRNA and protein level, we found a significant induction of TRAF adaptor proteins and of cIAP proteins (BIRC2 and/or BIRC3). As a consequence, the downstream nuclear transcription factor kappa B (NF-κB) survival pathway was increased. Furthermore, the treatment with the cIAP inhibitor LCL161 reverted the protection from apoptosis under low pH. In vitro results were confirmed both in Ewing sarcoma xenograft and in osteosarcoma patients, since the analysis of tumor tissues demonstrated that the levels of expression of TRAF1 or NF-κB1 significantly correlate with the level of expression of the vacuolar ATPase (V-ATPase), the most important proton pump in eukaryotes. Moreover, in the tissue sections of xenograft model, the nuclear translocation of RelB, a key subunit of the NF-κB transcriptional complex, localized in the tumor region that also corresponded to the acid microenvironment associated with the highest levels of expression of LAMP2 and V-ATPase, in the internal area of the tumor, as revealed by immunohistochemistry. Our data confirm that tumor acid microenvironment activates a stress-regulated switch to promote cell survival of bone sarcoma, and support the hypothesis that this mechanism is mediated by the recruitment of TRAF/cIAP complexes. Altogether, these results suggest that TRAF/cIAP can be considered as a target for anti-cancer therapies.

2019 - Breast cancer follow-up: a national survey of current clinical practice by the centers of Italian Oncological Group of Clinical Research (GOIRC). [Poster]
Omarini, Claudia; Piacentini, Federico; Frassoldati, Antonio; Musolino, Antonino; Dominici, Massimo; Moscetti on behalf of GOIRC, Luca
abstract

Background: The number of breast cancer (BC) survivors is increasing due to the aging of the population and the improvement of survival rates. Survivors have health care needs including detection of early recurrences, treatment of therapy-related complications and psychological support. No randomized data exist to support any individual follow-up (FU) sequence or protocol. Pphysicians’ adherence to international guidelines is unknown. The aim of this study is to investigate the survivorship care plan in Cancer Centers affiliated to Italian Oncological Group of Clinical Research (GOIRC). Methods: A questionnaire survey with 12 questions was e-mailed to the members of GOIRC in March 2019. Respondents were asked how they follow-up BC survivors. We have collected the survey data and compared them to national/international guidelines. Results: 20 out of 30 GOIRC centers completed the survey. The majority of the oncologists (75%) reported to follow AIOM guideline in FU management. Although, 14 respondents (70%) are used to perform routinely tumor markers and imaging tests (chest X-ray and liver ultrasound) as screening tools for early detection of recurrence. Advanced imaging studies (bone scan, CT scan, PET/FDG CT) are routinely recommended in high-risk patients by 4 interviewed. Considering patients on aromatase inhibitors, all the respondents recommend lipid profile and bone density evaluation every two years. Moreover, nutritional counselling is offered in 7 centers (35%). Frequency of checkup is scheduled according with BC risk of relapse in 11 centers (55%), while visits are conducted six-monthly in the other 9 cases. Duration of FU is variable: 60% of interviewed monitor the patients until the end of the adjuvant endocrine therapy while in the other cases checkup is carried on over 10 years. At the end of oncology FU, all the interviewed recommended yearly mammography, in four cases annual tumor markers check is suggested too. Conclusion A majority of respondents in Italian Cancer Centers perform more intensive follow-up compared to guidelines recommendations. FU of BC survivors is still an unmet clinical need. Randomized national trial on survivorship care plan should be considered.

2019 - Cancer stem-neuroendocrine cells in an atypical carcinoid case report. [Articolo su rivista]
Masciale, Valentina; Grisendi, Giulia; Banchelli, Federico; D'Amico, Roberto; Maiorana, Antonino; Morandi, Uliano; Dominici, Massimo; Aramini, Beatrice
abstract

Lung neuroendocrine cells tumor (NET) classification and diagnosis, particularly for typical and atypical carcinoids, are complicated by a variable natural history and nonspecific symptoms. Mechanisms for the development and progression of well-differentiated lung NETs are still unclear. An accurate and timely diagnosis can ensure the implementation of appropriate treatment and impact on prognosis. One of the main unclear point is the definition of these cells’ composition. In fact, it is known that carcinoids are mainly constituted by neuroendocrine cells. Aim of our report is to show for the first time the presence of a high percentage of cancer stem cells (CSCs) in an atypical carcinoid. The ALDEFLUOR assay was used to identify and sort ALDHhigh and ALDHlow human lung cancer cells following tissue digestion. SOX2 was additionally determined by immunohistochemistry. All specimens contained the 53.10% of ALDHhigh cells among all viable lung cancer cells, which indicates that more than half of the entire tumor cell population was composed by CSCs. As expected also in immunohistochemistry, about a half of the nuclei of the cells were positive for SOX2. We strongly support the hypothesis of the presence of cancer stem-neuroendocrine cells (CSCs-NETs) as subpopulation in these types of tumors.

2019 - Challenges in Clinical Development of Mesenchymal Stromal/Stem Cells: Concise Review [Articolo su rivista]
Mastrolia, I.; Foppiani, E. M.; Murgia, A.; Candini, O.; Samarelli, A. V.; Grisendi, G.; Veronesi, E.; Horwitz, E. M.; Dominici, M.
abstract

Identified 50 years ago, mesenchymal stromal/stem cells (MSCs) immediately generated a substantial interest among the scientific community because of their differentiation plasticity and hematopoietic supportive function. Early investigations provided evidence of a relatively low engraftment rate and a transient benefit for challenging congenital and acquired diseases. The reasons for these poor therapeutic benefits forced the entire field to reconsider MSC mechanisms of action together with their ex vivo manipulation procedures. This phase resulted in advances in MSCs processing and the hypothesis that MSC-tissue supportive functions may be prevailing their differentiation plasticity, broadening the spectrum of MSCs therapeutic potential far beyond their lineage-restricted commitments. Consequently, an increasing number of studies have been conducted for a variety of clinical indications, revealing additional challenges and suggesting that MSCs are still lagging behind for a solid clinical translation. For this reason, our aim was to dissect the current challenges in the development of still promising cell types that, after more than half a century, still need to reach their maturity. Stem Cells Translational Medicine 2019;8:1135–1148.

2019 - Clinical-Pathological Characteristics of HER2+ Breast Cancers patients among BRCA1/2+ carriers tested in Modena Cancer Center. [Poster]
Barbolini, M; Omarini, C; Viola, L; Isca, C; Marchi, I; Caggia, F; Barbieri, E; Toss, A; Cortesi, L; Dominici, M; Piacentini, F.
abstract

Background and Objectives Prevalence of HER2 positive breast cancer (BC) in BRCA1/2 carriers is still underestimated. Clinical behaviour of HER2+/BRCA+ BC could be in some ways different from sporadic HER2+ disease. The aim of this research is to describe clinical-pathological characteristics of this rare entity in patients treated at Modena Cancer Center. Methods Between January 2005 and July 2019, 2911 BC patients have been tested for BRCA1/2. 231 (7.9%) and 173 (5.9%) were found positive for BRCA1 and BRCA2 pathogenetic mutations respectively. We considered the HER2+ subgroup (14 patients), focusing on hormone receptor status, IGF-1R expression (semi-quantitative expression by immunohistochemistry with Anti-IGF-1R rabbit monoclonal), PI3K mutation (NGS Sequenom analysis) and clinical characteristics. Results All but one patients received trastuzumab as part of their treatment. Six patients were treated with neoadjuvant chemotherapy (NACT), comprehensive of trastuzumab +/- pertuzumab, achieving a pathological complete response in 33% of cases. Two patients experienced disease recurrence after NACT (median EFS = 5.7 years). From a pathological point of view, the majority of cancers were ductal infiltrating, grading 3 and hormone receptor positive. 3 cases a PI3K mutation was found: in one of them a double mutation (p.R38H + p.E545K) was detected. IGF-1R showed high expression in 2 cases (IHC score 3+), intermediate expression in 2 cases (score 2+), low or absent expression in 3 cases. Interestingly one HER2+/BRCA1 mutated patient shows both PI3K mutation and IGF-1R overexpression, achieving a partial response after NACT and still NED.  Conclusions Here we report a low frequency of HER2+ BC in BRCA1/2 germline carriers. This rate is higher in patients with BRCA2 mutation, accordingly with previous literature. It is not clear, in this peculiar population that displays different possible pathogenetic drivers, which could be the main druggable

2019 - Correlating tumor-infiltrating lymphocytes and lung cancer stem cells: a cross-sectional study. [Articolo su rivista]
Masciale, Valentina; Grisendi, Giulia; Banchelli, Federico; D'Amico, Roberto; Maiorana, Antonino; Sighinolfi, Pamela; Pinelli, Massimo; Lovati, Eleonora; Stefani, Alessandro; Morandi, Uliano; Dominici, Massimo; Aramini, Beatrice
abstract

Background: Lung cancer stem cells (LCSCs) are endowed with high aldehyde dehydrogenase (ALDH) expression and play roles in tumor proliferation, metastasis, and drug resistance. Their elusive nature may allow them to escape the immune response by tumor-infiltrating lymphocytes (TILs), which can positively affect the outcome in non-small cell lung cancer (NSCLC) patients. Despite independent investigations on both LCSCs and TILs, the relationship between the two has been very marginally considered. We analyzed whether these two cell types may be related as a prerequisite for novel diagnostic and therapeutic approaches. Methods: In this cross-sectional study, NSCLC human surgical specimens from 12 patients were tested by ALDEFLUOR assay to identify ALDHhigh cells. Fluorescence-activated cell sorting (FACS) analyses for CD3+, CD4+, and CD8+ TILs were performed in combination with immunohistochemistry evaluation. Results: Statistically positive correlations were found between ALDH+ and CD8+, and between ALDH+ and CD3+ cells populations; no correlation was found between ALDH+ and CD4+ cells. The expression of CD3+ and CD8+ by cells accounted for 40.1% and 58.7%, respectively, of the variability of ALDH+ cell expression by an R-squared index, which highlights the strong correlation between TILs and LCSCs. Immunohistochemistry revealed 6–25% positive cells. Conclusions: We report a correlation between cytotoxic TILs and LCSCs, which may contribute to the future development of targeted therapies focusing on the different roles of lymphocytes against lung cancer.

2019 - Defining mesenchymal stromal cell (MSC)-derived small extracellular vesicles for therapeutic applications [Articolo su rivista]
Dominici, Massimo
abstract

Small extracellular vesicles (sEVs) from mesenchymal stromal/stem cells (MSCs) are transiting rapidly towards clinical applications. However, discrepancies and controversies about the biology, functions, and potency of MSC-sEVs have arisen due to several factors: the diversity of MSCs and their preparation; various methods of sEV production and separation; a lack of standardized quality assurance assays; and limited reproducibility of in vitro and in vivo functional assays. To address these issues, members of four societies (SOCRATES, ISEV, ISCT and ISBT) propose specific harmonization criteria for MSC-sEVs to facilitate data sharing and comparison, which should help to advance the field towards clinical applications. Specifically, MSC-sEVs should be defined by quantifiable metrics to identify the cellular origin of the sEVs in a preparation, presence of lipid-membrane vesicles, and the degree of physical and biochemical integrity of the vesicles. For practical purposes, new MSC-sEV preparations might also be measured against a well-characterized MSC-sEV biological reference. The ultimate goal of developing these metrics is to map aspects of MSC-sEV biology and therapeutic potency onto quantifiable features of each preparation.

2019 - Dissecting the Pharmacodynamics and Pharmacokinetics of MSCs to Overcome Limitations in Their Clinical Translation [Articolo su rivista]
Salvadori, M.; Cesari, N.; Murgia, A.; Puccini, P.; Riccardi, B.; Dominici, M.
abstract

Recently, mesenchymal stromal stem cells (MSCs) have been proposed as therapeutic agents because of their promising preclinical features and good safety profile. However, their introduction into clinical practice has been associated with a suboptimal therapeutic profile. In this review, we address the biodistribution of MSCs in preclinical studies with a focus on the current understanding of the pharmacodynamics (PD) and pharmacokinetics (PK) of MSCs as key aspects to overcome unsatisfactory clinical benefits of MSC application. Beginning with evidence of MSC biodistribution and highlighting PK and PD factors, a new PK-PD model is also proposed. According to this theory, MSCs and their released factors are key players in PK, and the efficacy biomarkers are considered relevant for PD in more predictive preclinical investigations. Accounting for the PK-PD relationship in MSC translational research and proposing new models combined with better biodistribution studies could allow realization of the promise of more robust MSC clinical translation. The number of clinical trials based on MSCs that are publicly available exceeds 800; however, data regarding MSC pharmacodynamics (PD), pharmacokinetics (PK), and biodistribution are still scarce. For this reason, we dissected the PD and PK properties of MSCs, presenting factors that may influence MSC-based PK studies to then conceive a new PK-PD model that would support better and more robust MSC clinical translation.

2019 - Feasibility and safety of treating non-unions in tibia, femur and humerus with autologous, expanded, bone marrow-derived mesenchymal stromal cells associated with biphasic calcium phosphate biomaterials in a multicentric, non-comparative trial [Articolo su rivista]
Gómez-Barrena, Enrique; Rosset, Philippe; Gebhard, Florian; Hernigou, Philippe; Baldini, Nicola; Rouard, Helène; Sensebé, Luc; Gonzalo-Daganzo, Rosa M.; Giordano, Rosaria; Padilla-Eguiluz, Norma; García-Rey, Eduardo; Cordero-Ampuero, José; Rubio-Suárez, Juan Carlos; Stanovici, Julien; Ehrnthaller, Christian; Huber-Lang, Markus; Flouzat-Lachaniette, Charles Henri; Chevallier, Nathalie; MariaDonati, Davide; Ciapetti, Gabriela; Fleury, Sandrine; Fernandez, Manuel-Nicolás; Cabrera, José-Rafael; Avendaño-Solá, Cristina; Montemurro, Tiziana; Panaitescu, Carmen; Veronesi, Elena; Rojewski, Markus Thomas; Lofti, Ramin; Dominici, Massimo; Schrezenmeier, Hubert; Layrolle, Pierre
abstract

ORTHO-1 is a European, multicentric, first in human clinical trial to prove safety and feasibility after surgical implantation of commercially available biphasic calcium phosphate bioceramic granules associated during surgery with autologous mesenchymal stromal cells expanded from bone marrow (BM-hMSC) under good manufacturing practices, in patients with long bone pseudarthrosis. Methods: Twenty-eight patients with femur, tibia or humerus diaphyseal or metaphyso-diaphyseal non-unions were recruited and surgically treated in France, Germany, Italy and Spain with 100 or 200 million BM-hMSC/mL associated with 5–10 cc of bioceramic granules. Patients were followed up during one year. The investigational advanced therapy medicinal product (ATMP) was expanded under the same protocol in all four countries, and approved by each National Competent Authority. Findings: With safety as primary end-point, no severe adverse event was reported as related to the BM-hMSC. With feasibility as secondary end-point, the participating production centres manufactured the BM-hMSC as planned. The ATMP combined to the bioceramic was surgically delivered to the non-unions, and 26/28 treated patients were found radiologically healed at one year (3 out of 4 cortices with bone bridging). Interpretation: Safety and feasibility were clinically proven for surgical implantation of expanded autologous BM-hMSC with bioceramic. Funding: EU-FP7-HEALTH-2009, REBORNE Project (GA: 241876)

2019 - Human mesenchymal stem cell combined with a new strontium-enriched bioactive glass: An ex-vivo model for Bone Regeneration [Articolo su rivista]
Bellucci, D.; Veronesi, E.; Strusi, V.; Petrachi, T.; Murgia, A.; Mastrolia, I.; Dominici, M.; Cannillo, V.
abstract

A 3D cellular model that mimics the potential clinical application of a biomaterial is here applied for the first time to a bioactive glass, in order to assess its biological potential. A recently developed bioactive glass (BGMS10), whose composition contained strontium and magnesium, was produced in the form of granules and fully investigated in terms of biocompatibility in vitro. Apart from standard biological characterization (Simulated Body Fluid (SBF) testing and biocompatibility as per ISO10993), human bone marrow mesenchymal stromal/stem cells (BM-MSCs) were used to investigate the performance of the bioactive glass granules in an innovative 3D cellular model. The results showed that BGMS10 supported human BM-MSCs adhesion, colonization, and bone differentiation. Thus, bioactive glass granules seem to drive osteogenic differentiation and thus look particularly promising for orthopedic applications, bone tissue engineering and regenerative medicine.

2019 - Impact of HOXB7 overexpression on human adipose-derived mesenchymal progenitors [Articolo su rivista]
Foppiani, E. M.; Candini, O.; Mastrolia, I.; Murgia, A.; Grisendi, G.; Samarelli, A. V.; Boscaini, G.; Pacchioni, L.; Pinelli, M.; De Santis, G.; Horwitz, E. M.; Veronesi, E.; Dominici, M.
abstract

Background: The ex vivo expansion potential of mesenchymal stromal/stem cells (MSC) together with their differentiation and secretion properties makes these cells an attractive tool for transplantation and tissue engineering. Although the use of MSC is currently being tested in a growing number of clinical trials, it is still desirable to identify molecular markers that may help improve their performance both in vitro and after transplantation. Methods: Recently, HOXB7 was identified as a master player driving the proliferation and differentiation of bone marrow mesenchymal progenitors. In this study, we investigated the effect of HOXB7 overexpression on the ex vivo features of adipose mesenchymal progenitors (AD-MSC). Results: HOXB7 increased AD-MSC proliferation potential, reduced senescence, and improved chondrogenesis together with a significant increase of basic fibroblast growth factor (bFGF) secretion. Conclusion: While further investigations and in vivo models shall be applied for better understanding, these data suggest that modulation of HOXB7 may be a strategy for innovative tissue regeneration applications.

2019 - Inducible Caspase9-mediated suicide gene for MSC-based cancer gene therapy [Articolo su rivista]
Rossignoli, Filippo; Grisendi, Giulia; Spano, Carlotta; Golinelli, Giulia; Recchia, Alessandra; Rovesti, Giulia; Orsi, Giulia; Veronesi, Elena; Horwitz, Edwin M.; Dominici, Massimo
abstract

Cellular therapies based on mesenchymal stromal/stem cells (MSC) are promising strategies in regenerative medicine and oncology. Despite encouraging results, there is still some level of concerns on inoculating MSC in cancer patients. To face this issue, one possibility resides in engineering MSC by incorporating a suicide gene in order to control their fate once infused. Strategies based on Herpes Simplex Virus Thymidine Kinase (HSV-TK) and the Cytosine Deaminase genes have been developed and more recently a novel suicide gene, namely, iCasp9, has been proposed. This approach is based on a variant of human Caspase9 that binds with high affinity to a synthetic, bioinert small molecule (AP20187) leading to cell death. Based on this technology so far marginally applied to MSC, we tested the suitability of iCasp9 suicide strategy in MSC to further increase their safety. MSC have been transfected by a lentiviral vector carrying iCasp9 gene and then tested for viability after AP20187 treatment in comparison with mock-transfected cells. Moreover, accounting our anti-tumor approaches based on MSC expressing potent anti-cancer ligand TNF-Related Apoptosis-Inducing Ligand (TRAIL), we generated adipose MSC co-expressing iCasp9 and TRAIL successfully targeting an aggressive sarcoma type. These data show that anti-cancer and suicide mechanisms can coexist without affecting cells performance and hampering the tumoricidal activity mediated by TRAIL. In conclusion, this study originally indicates the suitability of combining a MSC-based anti-cancer gene approach with iCasp9 demonstrating efficiency and specificity.

2019 - Isolation and Identification of Cancer Stem-Like Cells in Adenocarcinoma and Squamous Cell Carcinoma of the Lung: A Pilot Study [Articolo su rivista]
Masciale, Valentina; Grisendi, Giulia; Banchelli, Federico; D'Amico, Roberto; Maiorana, Antonino; Sighinolfi, Pamela; Stefani, Alessandro; Morandi, Uliano; Dominici, Massimo; Aramini, Beatrice
abstract

Background: Lung cancer stem cells (CSCs) share many characteristics with normal stem cells, such as self-renewal and multipotentiality. High expression of aldehyde dehydrogenase (ALDH) has been detected in many tumors, particularly in the CSC compartment, and it plays an important role in tumor proliferation, metastasis, and drug resistance. CD44 is commonly used as a cell surface marker of cancer stem-like cells in epithelial tumors. The aim of this study was to isolate and analyze cancer stem-like cells from surgically removed specimens to compare lung adenocarcinoma (ADENO) and squamous (SQUAMO) cell carcinoma. Methods: The ALDEFLUOR assay was used to identify and sort ALDHhigh and ALDHlow human lung cancer cells following tissue digestion. Fluorescence-activated cell sorting analysis for CD44 was performed with tumor cells. Quantitative real-time PCR was performed to assess the expression of SOX2 and NANOG as stemness markers. ALDH1A1 expression was additionally determined by immunohistochemistry. Anchorage-independent ALDHhigh cell growth was also evaluated. ALDHhigh ADENO and SQUAMO cells were cultured to analyze spheroid formation. Results: All specimens contained 0.5–12.5% ALDHhigh cells with 3.8–18.9% CD44-positive cells. SOX2 and NANOG relative expression in ALDHhigh compared to ALDHlow cells in ADENO and SQUAMO was analyzed and compared between the histotypes. Immunohistochemistry confirmed the presence of ALDH1A1 in the sections. SOX2 and NANOG were expressed at higher levels in the ALDHhigh subpopulation than in the ALDHlow subpopulation only in ADENO cells, and the opposite result was seen in SQUAMO cells. In vitro functional assays demonstrated that ALDHhigh cells exhibited migration capacity with distinct behaviors between ALDHhigh spheres in ADENO vs. SQUAMO samples. Conclusions: Our results highlight the importance of a better characterization of cancer stem-like cells in ADENO and SQUAMO histotypes. This may suggest new differential approaches for prognostic and therapeutic purposes in patients with non-small-cell lung cancer.

2019 - MSC-delivered soluble TRAIl and paclitaxel as novel combinatory treatment for pancreatic adenocarcinoma [Articolo su rivista]
Rossignoli, Filippo; Spano, Carlotta; Grisendi, Giulia; Foppiani, Elisabetta Manuela; Golinelli, Giulia; Mastrolia, Ilenia; Bestagno, Marco; Candini, Olivia; Petrachi, Tiziana; Recchia, Alessandra; Miselli, Francesca; Rovesti, Giulia; Orsi, Giulia; Veronesi, Elena; Medici, Gregorio; Petocchi, Benedetta; Pinelli, Massimo; Horwitz, Edwin M.; Conte, Pierfranco; Dominici, Massimo
abstract

Pancreatic cancer is the fourth leading cause of cancer death in western countries with more than 100,000 new cases per year in Europe and a mortality rate higher than 90%. In this scenario, advanced therapies based on gene therapies are emerging, thanks to a better understanding of tumour architecture and cancer cell alterations. We have demonstrated the efficacy of an innovative approach for pancreatic cancer based on mesenchymal stromal cells (MSC) genetically engineered to produce TNF-related Apoptosis Inducing Ligand (TRAIL). Here we investigated the combination of this MSC-based approach with the administration of a paclitaxel (PTX)-based chemotherapy to improve the potential of the treatment, also accounting for a possible resistance onset. Methods: Starting from the BXPC3 cell line, we generated and profiled a TRAIL-resistant model of pancreatic cancer, testing the impact of the combined treatment in vitro with specific cytotoxicity and metabolic assays. We then challenged the rationale in a subcutaneous mouse model of pancreatic cancer, assessing its effect on tumour size accounting stromal and parenchymal organization. Results: PTX was able to restore pancreatic cancer sensitivity to MSC-delivered TRAIL by reverting its pro-survival gene expression profile. The two compounds cooperate both in vitro and in vivo and the combined treatment resulted in an improved cytotoxicity on tumour cells. Conclusion: In summary, this study uncovers the potential of a combinatory approach between MSC-delivered TRAIL and PTX, supporting the combination of cell-based products and conventional chemotherapeutics as a tool to improve the efficacy of the treatments, also addressing possible mechanisms of resistance.

2019 - Response to Nature commentary “Clear up this stem-cell mess” [Articolo su rivista]
Galipeau, Jacques; Weiss, Daniel J.; Dominici, Massimo
abstract

n/a

2019 - Soluble TRAIL Armed Human MSC As Gene Therapy For Pancreatic Cancer [Articolo su rivista]
Spano, Carlotta; Grisendi, Giulia; Golinelli, Giulia; Rossignoli, Filippo; Prapa, Malvina; Bestagno, Marco; Candini, Olivia; Petrachi, Tiziana; Recchia, Alessandra; Miselli, Francesca; Rovesti, Giulia; Orsi, Giulia; Maiorana, Antonino; Manni, Paola; Veronesi, Elena; Piccinno, Maria Serena; Murgia, Alba; Pinelli, Massimo; Horwitz, Edwin M.; Cascinu, Stefano; Conte, Pierfranco; Dominici, Massimo
abstract

Pancreatic ductal adenocarcinoma (PDAC) is still one of the most aggressive adult cancers with an unacceptable prognosis. For this reason novel therapies accounting for PDAC peculiarities, such as the relevant stromal reaction, are urgently needed. Here adipose mesenchymal stromal/stem cells (AD-MSC) have been armed to constantly release a soluble trimeric and multimeric variant of the known anti-cancer TNF-related apoptosis-inducing ligand (sTRAIL). This cancer gene therapy strategy was in vitro challenged demonstrating that sTRAIL was thermally stable and able to induce apoptosis in the PDAC lines BxPC-3, MIA PaCa-2 and against primary PDAC cells. sTRAIL released by AD-MSC relocated into the tumor stroma was able to significantly counteract tumor growth in vivo with a significant reduction in tumor size, in cytokeratin-7+ cells and by an anti-angiogenic effect. In parallel, histology on PDAC specimens form patients (n = 19) was performed to investigate the levels of TRAIL DR4, DR5 and OPG receptors generating promising insights on the possible clinical translation of our approach. These results indicate that adipose MSC can very efficiently vehicle a novel TRAIL variant opening unexplored opportunities for PDAC treatment.

2019 - Stem cells and lung cancer : between advanced diagnostics and new therapeutics. [Capitolo/Saggio]
Masciale, V; Grisendi, G; Morandi, U; Dominici, M; Aramini, B.
abstract

Lung cancers (LCs) remain a significant and devastating cause of morbidity and mortality worldwide. Despite the very recent success of immunotherapy, the diagnosis and treatment of LC remain one of the greatest challenges in chest surgery, clinical oncology, and molecular medicine. A growing number of investigations on normal/cancer stem cells and cellular therapies are offering exciting new avenues to advance knowledge on LC. Here, we will be focusing on the multiple relationships between LC and stem cells accounting for cancer stem cell (CSC) diagnostics and progenitor-based therapeutics for LC. Cancer cell repopulation after chemotherapy and/or radiotherapy still represents a major factor limiting the efficacy of treatment since CSCs play critical roles during this process by reciprocal connections between CSCs and tumor microenvironment. This calls for new opportunities to integrate advanced CSC diagnostics and targeted approaches also based on immunotherapy. In addition, recent discoveries on malignant pleural and other LC highlight that mesenchymal stromal/stem cells may be a novel platform for drug delivery within still unexplored gene therapy strategies. This chapter will dissect these two apparently distant technologies within a unified stem-cell-based vision aimed at providing better diagnostics and therapeutics for LC at the forefront of modern clinical oncology.

2018 - Cancer stem cells and their microenvironment. [Capitolo/Saggio]
Masciale, Valentina; Grisendi, Giulia; Banchelli, Federico; D'Amico, Roberto; Morandi, Uliano; Dominici, Massimo; Husnain Haider, Khawaja; Aramini, Beatrice
abstract

2018 - Cell, tissue and gene products with marketing authorization in 2018 worldwide [Articolo su rivista]
Cuende, N.; Rasko, JOHN E. J.; Koh, MICKEY B. C.; Dominici, M.; Ikonomou, L.
abstract

Cell and gene therapies (CGTs) are progressively entering into clinical practice in different parts of the world. The International Society for Cell & Gene Therapy (ISCT), a global scientific society, has been committed since 1992 to supporting and developing knowledge on clinical applications of CGTs. Considering the number of products that have been progressively approved and, in some cases, withdrawn in recent years, the ISCT would like to present a brief annual report on CGTs with marketing authorization (MA) in different regions. This article reflects the dynamic momentum around authorized CGTs coinciding with the parallel increase of unproven approaches where cells are delivered without appropriate and rigorous scientific and regulatory assessment and authorization. This is intended to be a living document with a yearly update linked to a dedicated section of the ISCT website for faster adjustments. The aim is to ultimately inform, by periodic snapshots, the scientific community, healthcare stakeholders and patient associations on authorized CGT products as a way to increase communication around the approved therapeutic approaches charged with heightened expectations.

2018 - Challenging the efficacy of checkpoint inhibitors: a new 3D model for a precision medicine approach. [Abstract in Atti di Convegno]
Foppiani, ELISABETTA MANUELA; Candini, Olivia; Aramini, Beatrice; Masciale, Valentina; Grisendi, Giulia; Matteo, Brogli; Giorgio, Mari; Dominici, Massimo
abstract

Challenging the efficacy of checkpoint inhibitors: a new 3D model for a precision medicine approach.

2018 - Dynamic cultivation of mesenchymal stem cell aggregates [Articolo su rivista]
Egger, Dominik; Tripisciano, Carla; Weber, Viktoria; Dominici, Massimo; Kasper, Cornelia
abstract

Mesenchymal stem cells (MSCs) are considered as primary candidates for cell-based therapies due to their multiple effects in regenerative medicine. Pre-conditioning of MSCs under physiological conditions—such as hypoxia, three-dimensional environments, and dynamic cultivation—prior to transplantation proved to optimize their therapeutic efficiency. When cultivated as three-dimensional aggregates or spheroids, MSCs display increased angiogenic, anti-inflammatory, and immunomodulatory effects as well as improved stemness and survival rates after transplantation, and cultivation under dynamic conditions can increase their viability, proliferation, and paracrine effects, alike. Only few studies reported to date, however, have utilized dynamic conditions for three-dimensional aggregate cultivation of MSCs. Still, the integration of dynamic bioreactor systems, such as spinner flasks or stirred tank reactors might pave the way for a robust, scalable bulk expansion of MSC aggregates or MSC-derived extracellular vesicles. This review summarizes recent insights into the therapeutic potential of MSC aggregate cultivation and focuses on dynamic generation and cultivation techniques of MSC aggregates.

2018 - Extracellular vesicles derived from mesenchymal cells: Perspective treatment for cutaneous wound healing in pediatrics [Articolo su rivista]
Pelizzo, Gloria; Avanzini, Maria Antonietta; Icaro Cornaglia, Antonia; De Silvestri, Annalisa; Mantelli, Melissa; Travaglino, Paola; Croce, Stefania; Romano, Piero; Avolio, Luigi; Iacob, Giulio; Dominici, Massimo; Calcaterra, Valeria
abstract

Aim: We evaluated the effects of the intradermal injection of extracellular vesicles (EVs) derived from adipose stem cells (ASC-EVs) and bone marrow cells (BM-EVs) in an experimental cutaneous wound repair model. Methods: Mesenchymal stem cells (MSCs) were in vitro expanded from adipose (ASC) or BM tissues (BM-MSC) of rabbits. EVs were separated from the supernatants of confluent ASC and BM-MSCs. Two skin wounds were induced in each animal and treated with MSC or EV injections. Histological examination was performed postinoculation. Results: EV-treated wounds exhibited a better restoration compared with the counterpart MSC treatment. ASC-EV-treated wounds were significantly better than BM-EVs (p = 0.036). Conclusion: EV topical inoculation provides restored architecture during cutaneous wound healing and represents a promising solution for regenerative medicine in children.

2018 - Extracellular vesicles released from mesenchymal stromal cells stimulate bone growth in osteogenesis imperfecta [Articolo su rivista]
Otsuru, Satoru; Desbourdes, Laura; Guess, Adam J.; Hofmann, Ted J.; Relation, Theresa; Kaito, Takashi; Dominici, Massimo; Iwamoto, Masahiro; Horwitz, Edwin M.
abstract

Background Systemic infusion of mesenchymal stromal cells (MSCs) has been shown to induce acute acceleration of growth velocity in children with osteogenesis imperfecta (OI) despite minimal engraftment of infused MSCs in bones. Using an animal model of OI we have previously shown that MSC infusion stimulates chondrocyte proliferation in the growth plate and that this enhanced proliferation is also observed with infusion of MSC conditioned medium in lieu of MSCs, suggesting that bone growth is due to trophic effects of MSCs. Here we sought to identify the trophic factor secreted by MSCs that mediates this therapeutic activity. Methods To examine whether extracellular vesicles (EVs) released from MSCs have therapeutic activity, EVs were isolated from MSC conditioned medium by ultracentrifugation. To further characterize the trophic factor, RNA or microRNA (miRNA) within EVs was depleted by either ribonuclease (RNase) treatment or suppressing miRNA biogenesis in MSCs. The functional activity of these modified EVs was evaluated using an in vitro chondrocyte proliferation assay. Finally, bone growth was evaluated in an animal model of OI treated with EVs. Results We found that infusion of MSC-derived EVs stimulated chondrocyte proliferation in the growth plate, resulting in improved bone growth in a mouse model of OI. However, infusion of neither RNase-treated EVs nor miRNA-depleted EVs enhanced chondrocyte proliferation. Conclusion MSCs exert therapeutic effects in OI by secreting EVs containing miRNA, and EV therapy has the potential to become a novel cell-free therapy for OI that will overcome some of the current limitations in MSC therapy.

2018 - Human Herpes simplex 1 virus infection of endometrial decidual tissue-derived MSC alters HLA-G expression and immunosuppressive functions [Articolo su rivista]
Bortolotti, Daria; Rossignoli, Filippo; Rotola, Antonella; Campioni, Diana; Cultrera, Rosario; Grisendi, Giulia; Dominici, Massimo; Rizzo, Roberta
abstract

Objectives: Mesenchymal stromal/stem cells have immunosuppressive functions. Our previous results demonstrated that one of the players of this immunomodulation can be ascribed to the Human Leukocyte Antigen-G. HLA-G, a non classical HLA class I antigen, is involved in immune tolerance during pregnancy, organ transplantation, autoimmune and inflammatory diseases. In this study we wanted to verify whether human endometrial decidual tissue derived (EDT)-MSC could express HLA-G. Additionally we assessed the permissivity to Human Herpesvirus infections, using HSV-1 as a model, and the possible effect on EDT-MSC immunosuppressive functions towards peripheral blood mononuclear cell (PBMC) proliferation. Methods: We analyzed immune-inhibitory functions and HLA-G expression in human EDT-MSC before and after HSV-1 infection. Results: We observed that EDT-MSC express HLA-G molecules, that partly are responsible for the immune-inhibitory functions of EDT-MSC towards PBMC proliferation. EDT-MSC are permissive for a productive infection by HSV-1, that decreases HLA-G expression and affects EDT-MSC immune-inhibitory functions. Conclusions: We demonstrate that EDT-MSC are susceptible to HSV-1 infection, that reduces HLA-G expression and their immune-inhibitory function. These data could have a clinical implication in the use of EDT-MSC as an immunosuppressant, in particular in steroid-refractory GvHD after allogeneic hematopoietic stem cell transplantation and in autoimmune diseases.

2018 - In vitro and in vivo discrepancy in inducing apoptosis by mesenchymal stromal cells delivering membrane-bound tumor necrosis factor–related apoptosis inducing ligand in osteosarcoma pre-clinical models [Articolo su rivista]
Guiho, Romain; Biteau, Kevin; Grisendi, Giulia; Chatelais, Mathias; Brion, Regis; Taurelle, Julien; Renault, Sarah; Heymann, Dominique; Dominici, Massimo; Redini, Françoise
abstract

Background: Osteosarcoma (OS) is the most frequent pediatric malignant bone tumor. OS patients have not seen any major therapeutic progress in the last 30 years, in particular in the case of metastatic disease, which requires new therapeutic strategies. The pro-apoptotic cytokine Tumor necrosis factor (TNF)–Related Apoptosis Inducing Ligand (TRAIL) can selectively kill tumor cells while sparing normal cells, making it a promising therapeutic tool in several types of cancer. However, many OS cell lines appear resistant to recombinant human (rh)TRAIL-induced apoptosis. We, therefore, hypothesized that TRAIL presentation at the membrane level of carrier cells might overcome this resistance and trigger apoptosis. Methods: To address this, human adipose mesenchymal stromal cells (MSCs) transfected in a stable manner to express membrane-bound full-length human TRAIL (mbTRAIL) were co-cultured with several human OS cell lines. Results: This induced apoptosis by cell-to-cell contact even in cell lines initially resistant to rhTRAIL. In contrast, mbTRAIL delivered by MSCs was not able to counteract tumor progression in this OS orthotopic model. Discussion: This was partly due to the fact that MSCs showed a potential to support tumor development. Moreover, the expression of mbTRAIL did not show caspase activation in adjacent tumor cells.

2018 - Intratumoral Delivery of Interferonγ-Secreting Mesenchymal Stromal Cells Repolarizes Tumor-Associated Macrophages and Suppresses Neuroblastoma Proliferation In Vivo [Articolo su rivista]
Relation, T; Yi, T; Guess, Aj; La Perle, K; Otsuru, S; Hasgur, S; Dominici, M; Breuer, C; Horwitz, Em
abstract

Neuroblastoma, the most common extracranial solid tumor in childhood, remains a therapeutic challenge. However, one promising patient treatment strategy is the delivery of anti-tumor therapeutic agents via mesenchymal stromal cell (MSC) therapy. MSCs have been safely used to treat genetic bone diseases such as osteogenesis imperfecta, cardiovascular diseases, autoimmune diseases, and cancer. The pro-inflammatory cytokine interferon-gamma (IFNγ) has been shown to decrease tumor proliferation by altering the tumor microenvironment (TME). Despite this, clinical trials of systemic IFNγ therapy have failed due to the high blood concentration required and associated systemic toxicities. Here, we developed an intra-adrenal model of neuroblastoma, characterized by liver and lung metastases. We then engineered MSCs to deliver IFNγ directly to the TME. In vitro, these MSCs polarized murine macrophages to the M1 phenotype. In vivo, we attained a therapeutically active TME concentration of IFNγ without increased systemic concentration or toxicity. The TME-specific IFNγ reduced tumor growth rate and increased survival in two models of T cell deficient athymic nude mice. Absence of this benefit in NOD SCID gamma (NSG) immunodeficient mouse model indicates a mechanism dependent on the innate immune system. IL-17 and IL-23p19, both uniquely M1 polarization markers, transiently increased in the tumor interstitial fluid. Finally, the MSC vehicle did not promote tumor growth. These findings reveal that MSCs can deliver effective cytokine therapy directly to the tumor while avoiding systemic toxicity. This method transiently induces inflammatory M1 macrophage polarization, which reduces tumor burden in our novel neuroblastoma murine model.

2018 - Label-free toxicology screening of primary human mesenchymal cells and iPS-derived neurons [Articolo su rivista]
Piccinno, Maria Serena; Petrachi, Tiziana; Resca, Elisa; Strusi, Valentina; Bergamini, Valentina; Mulas, Giuseppe Antonio; Mari, Giorgio; Dominici, Massimo; Veronesi, Elena
abstract

The high-throughput, label-free Corning Epic assay has applications in drug discovery, pharmacogenomics, cell receptor signaling, cell migration, and viral titration. The utility of Epic technology for biocompatibility testing has not been well established. In manufacturing of medical devices, in vitro and in vivo biocompatibility assessments are mandatory, according to ISO 10993. The new medical device regulation MDR 745/2017 specifies that ex vivo assays that can closely recapitulate in vivo scenarios are needed to better evaluate biomedical devices. We propose herein that Epic technology—which enables detection of variations in cell mass distribution—is suitable for biocompatibility screening of compounds. In this study, we challenged primary human osteoblasts, endothelial cells, and neurons derived from induced pluripotent stem cells with specific concentrations of methyl methacrylate (MMA). Polymeric MMA has long been applied in cranioplasty, where it makes contact with multiple cell types. Application of Epic technology yielded real-time cytotoxicity profiles for all considered cell types. The results were compared with those from microscopic observation of the same culture plate used in the Epic analyses. The Epic assay should be further examined for its utility for cell biology, genomics, and proteomics companion assays. Our results suggest that Epic technology can be applied to biocompatibility evaluation of human cells in medical device development.

2018 - Mineralization by mesenchymal stromal cells is variously modulated depending on commercial platelet lysate preparations [Articolo su rivista]
Boraldi, Federica; Burns, Jorge S.; Bartolomeo, Angelica; Dominici, Massimo; Quaglino, Daniela
abstract

Background aims: Numerous cellular models have been developed to investigate calcification for regenerative medicine applications and for the identification of therapeutic targets in various complications associated with age-related diseases. However, results have often been contradictory due to specific culture conditions, cell type ontogeny and aging status. Human platelet lysate (hPL) has been recently investigated as valuable alternative to fetal bovine serum (FBS) in cell culture and bone regeneration. A parallel comparison of how all these multiple factors may converge to influence mineralization has yet to be reported. Methods: To compare mineralization of human mesenchymal cell types known to differ in extracellular matrix calcification potency, bone marrow–derived mesenchymal stromal cells and dermal fibroblasts from neonatal and adult donors, at both low and high passages, were investigated in an ex vivo experimental model by supplementing the osteogenic induction medium with FBS or with hPL. Four commercial hPL preparations were profiled by liquid chromatography/electrospray ionization quadrupole time-of-flight spectrometry, and mineralization was visualized by von Kossa staining and quantified by morphometric evaluations after 9, 14 and 21 days of culture. Results: Data demonstrate that (i) commercial hPL preparations differ according to mass spectra profiles, (ii) hPL variously influences mineral deposition depending on cell line and possibly on platelet product preparation methods, (iii) donor age modifies mineral deposition in the presence of the same hPL and (iv) reduced in vitro proliferative capacity affects osteogenic induction and response to hPL. Conclusion: Despite the standardized procedures applied to obtain commercial hPL, this study highlights the divergent effects of different preparations and emphasizes the importance of cellular ontology, donor age and cell proliferative capacity to optimize the osteogenic induction capabilities of mesenchymal stromal cells and design more effective cell-based therapeutic protocols.

2018 - Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines [Articolo su rivista]
Théry, Clotilde; Witwer, Kenneth W; Aikawa, Elena; Alcaraz, Maria Jose; Anderson, Johnathon D; Andriantsitohaina, Ramaroson; Antoniou, Anna; Arab, Tanina; Archer, Fabienne; Atkin-Smith, Georgia K; Ayre, D Craig; Bach, Jean-Marie; Bachurski, Daniel; Baharvand, Hossein; Balaj, Leonora; Baldacchino, Shawn; Bauer, Natalie N; Baxter, Amy A; Bebawy, Mary; Beckham, Carla; Bedina Zavec, Apolonija; Benmoussa, Abderrahim; Berardi, Anna C; Bergese, Paolo; Bielska, Ewa; Blenkiron, Cherie; Bobis-Wozowicz, Sylwia; Boilard, Eric; Boireau, Wilfrid; Bongiovanni, Antonella; Borràs, Francesc E; Bosch, Steffi; Boulanger, Chantal M; Breakefield, Xandra; Breglio, Andrew M; Brennan, Meadhbh Á; Brigstock, David R; Brisson, Alain; Broekman, Marike LD; Bromberg, Jacqueline F; Bryl-Górecka, Paulina; Buch, Shilpa; Buck, Amy H; Burger, Dylan; Busatto, Sara; Buschmann, Dominik; Bussolati, Benedetta; Buzás, Edit I; Byrd, James Bryan; Camussi, Giovanni; Carter, David RF; Caruso, Sarah; Chamley, Lawrence W; Chang, Yu-Ting; Chaudhuri, Amrita Datta; Chen, Chihchen; Chen, Shuai; Cheng, Lesley; Chin, Andrew R; Clayton, Aled; Clerici, Stefano P; Cocks, Alex; Cocucci, Emanuele; Coffey, Robert J; Cordeiro-da-Silva, Anabela; Couch, Yvonne; Coumans, Frank AW; Coyle, Beth; Crescitelli, Rossella; Criado, Miria Ferreira; D’Souza-Schorey, Crislyn; Das, Saumya; de Candia, Paola; De Santana, Eliezer F; De Wever, Olivier; del Portillo, Hernando A; Demaret, Tanguy; Deville, Sarah; Devitt, Andrew; Dhondt, Bert; Di Vizio, Dolores; Dieterich, Lothar C; Dolo, Vincenza; Dominguez Rubio, Ana Paula; Dominici, Massimo; Dourado, Mauricio R; Driedonks, Tom AP; Duarte, Filipe V; Duncan, Heather M; Eichenberger, Ramon M; Ekström, Karin; EL Andaloussi, Samir; Elie-Caille, Celine; Erdbrügger, Uta; Falcón-Pérez, Juan M; Fatima, Farah; Fish, Jason E; Flores-Bellver, Miguel; Försönits, András; Frelet-Barrand, Annie; Fricke, Fabia; Fuhrmann, Gregor; Gabrielsson, Susanne; Gámez-Valero, Ana; Gardiner, Chris; Gärtner, Kathrin; Gaudin, Raphael; Gho, Yong Song; Giebel, Bernd; Gilbert, Caroline; Gimona, Mario; Giusti, Ilaria; Goberdhan, Deborah CI; Görgens, André; Gorski, Sharon M; Greening, David W; Gross, Julia Christina; Gualerzi, Alice; Gupta, Gopal N; Gustafson, Dakota; Handberg, Aase; Haraszti, Reka A; Harrison, Paul; Hegyesi, Hargita; Hendrix, An; Hill, Andrew F; Hochberg, Fred H; Hoffmann, Karl F; Holder, Beth; Holthofer, Harry; Hosseinkhani, Baharak; Hu, Guoku; Huang, Yiyao; Huber, Veronica; Hunt, Stuart; Ibrahim, Ahmed Gamal-Eldin; Ikezu, Tsuneya; Inal, Jameel M; Isin, Mustafa; Ivanova, Alena; Jackson, Hannah K; Jacobsen, Soren; Jay, Steven M; Jayachandran, Muthuvel; Jenster, Guido; Jiang, Lanzhou; Johnson, Suzanne M; Jones, Jennifer C; Jong, Ambrose; Jovanovic-Talisman, Tijana; Jung, Stephanie; Kalluri, Raghu; Kano, Shin-ichi; Kaur, Sukhbir; Kawamura, Yumi; Keller, Evan T; Khamari, Delaram; Khomyakova, Elena; Khvorova, Anastasia; Kierulf, Peter; Kim, Kwang Pyo; Kislinger, Thomas; Klingeborn, Mikael; Klinke, David J; Kornek, Miroslaw; Kosanović, Maja M; Kovács, Árpád Ferenc; Krämer-Albers, Eva-Maria; Krasemann, Susanne; Krause, Mirja; Kurochkin, Igor V; Kusuma, Gina D; Kuypers, Sören; Laitinen, Saara; Langevin, Scott M; Languino, Lucia R; Lannigan, Joanne; Lässer, Cecilia; Laurent, Louise C; Lavieu, Gregory; Lázaro-Ibáñez, Elisa; Le Lay, Soazig; Lee, Myung-Shin; Lee, Yi Xin Fiona; Lemos, Debora S; Lenassi, Metka; Leszczynska, Aleksandra; Li, Isaac TS; Liao, Ke; Libregts, Sten F; Ligeti, Erzsebet; Lim, Rebecca; Lim, Sai Kiang; Linē, Aija; Linnemannstöns, Karen; Llorente, Alicia; Lombard, Catherine A; Lorenowicz, Magdalena J; Lörincz, Ákos M; Lötvall, Jan; Lovett, Jason; Lowry, Michelle C; Loyer, Xavier; Lu, Quan; Lukomska, Barbara; Lunavat, Taral R; Maas, Sybren LN; Malhi, Harmeet; Marcilla, Antonio; Mariani, Jacopo; Mariscal, Javier; Martens-Uzunova, Elena S; Martin-Jaular, Lorena; Martinez, M Carmen; Martins, Vilma Regina; Mathieu, Mathild
abstract

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

2018 - Nivolumab-Induced Impressive Response of Refractory Pulmonary Sarcomatoid Carcinoma with Brain Metastasis [Articolo su rivista]
Salati, Massimiliano; Baldessari, Cinzia; Calabrese, Fiorella; Rossi, Giulio; Pettorelli, Elisa; Grizzi, Giulia; Dominici, Massimo; Barbieri, Fausto
abstract

Background: Pulmonary sarcomatoid carcinoma is a rare, poorly differentiated, and highly aggressive type of non-small cell lung cancer. High tumor mutational burden and PD-L1 overexpression make it an excellent candidate for immunotherapy. Objectives and Method: We presented the case of a patient who underwent left inferior lobectomy with concurrent right paravertebral muscular metastasectomy for an infiltrative neoplastic mass, whose final diagnosis was consistent with stage IV pulmonary sarcomatoid carcinoma. He received first- and second-line chemotherapy without any benefit. Since March 2016, he has been treated with the anti-PD1 agent nivolumab with a dramatic improvement of symptoms, disappearance of a brain lesion, and partial response on other metastatic sites. He tolerated the treatment well and is still responding after 22 months from the beginning. Results and Conclusions: In very lethal non-small cell lung cancer subtypes such as the sarcomatoid variants, high tumor burden and deteriorated general conditions should not preclude, at least in some cases, the use of immunotherapy. Anti-PD1 may also have a reliable role in disease control in the brain. Lastly, the strong rationale behind sarcomatoid histology should further prompt trials exploring immunotherapeutic approaches in this subset of non-small cell lung cancer.

2018 - Proceedings of the signature series symposium “cellular therapies for orthopaedics and musculoskeletal disease proven and unproven therapies—promise, facts and fantasy,” international society for cellular therapies, montreal, canada, May 2, 2018 [Articolo su rivista]
Piuzzi, NICOLAS S.; Dominici, M.; Long, M. A. R. C.; PASCUAL-GARRIDO, C.; Rodeo, S.; Huard, J.; Guicheux, Jérome; Mcfarland, R.; Goodrich, LAURIE R.; Maddens, Stéphane; Robey, PAMELA G.; Bauer, THOMAS W; Barrett, J. O. H. N.; Barry, F.; Karli, D.; Chu, CONSTANCE R.; Weiss, DANIEL J.; Martin, I. V. A. N.; Jorgensen, C.; Muschler, GEORGE F.
abstract

The Signature Series Symposium “Cellular Therapies for Orthopaedics and Musculoskeletal Disease Proven and Unproven Therapies—Promise, Facts and Fantasy” was held as a pre-meeting of the 26th International Society for Cellular Therapy (ISCT) annual congress in Montreal, Canada, May 2, 2018. This was the first ISCT program that was entirely dedicated to the advancement of cell-based therapies for musculoskeletal diseases. Cellular therapies in musculoskeletal medicine are a source of great promise and opportunity. They are also the source of public controversy, confusion and misinformation. Patients, clinicians, scientists, industry and government share a commitment to clear communication and responsible development of the field. Therefore, this symposium convened thought leaders from around the world in a forum designed to catalyze communication and collaboration to bring the greatest possible innovation and value to patients with musculoskeletal conditions.

2017 - A new Approach to Investigate Biofilm Formation in Medical Devices [Abstract in Rivista]
Resca, E; Petrachi, T; Piccinno, Ms; Gavioli, G; Dominici, M; Veronesi, E
abstract

Abstracts from the 10th Congress of the Vascular Access Society, 5-8 April 2017, Ljubljana, Slovenia

2017 - An alternative approach to investigate biofilm in medical devices: A feasibility study [Articolo su rivista]
Petrachi, Tiziana; Resca, Elisa; Piccinno, Maria Serena; Biagi, Francesco; Strusi, Valentina; Dominici, Massimo; Veronesi, Elena
abstract

Biofilms are assemblages of bacterial cells irreversibly associated with a surface where moisture is present. In particular, they retain a relevant impact on public health since through biofilms bacteria are able to survive and populate biomedical devices causing severe nosocomial infections that are generally resistant to antimicrobial agents. Therefore, controlling biofilm formation is a mandatory feature during medical device manufacturing and during their use. In this study, combining a crystal violet staining together with advanced stereomicroscopy, we report an alternative rapid protocol for both qualitative and semi-quantitative biofilm determination having high specificity, high repeatability, and low variability. The suggested approach represents a reliable and versatile method to detect, monitor, and measure biofilm colonization by an easy, more affordable, and reproducible method.

2017 - Autologous Porcine Bone Marrow Mesenchymal Cells for Reconstruction of a Resorbed Alveolar Bone: A Preclinical Model in Mini-Pigs [Articolo su rivista]
Gudveig Gjerde, Cecilie; De Santis, Daniele; Dominici, Massimo; Zanotti, Guglielmo; Hellem, Sølve; Piccinno, MARIA SERENA; Burns, Jorge Phillip Joaquin Sans; Murgia, Alba; Candini, Olivia; Krampera, Mauro; Nocini, Pierfrancesco; Addis, Alessandro; Amiaud, Jérôme; Layrolle, Pierre; Mustafa, Kamal; Veronesi, Elena
abstract

Regeneration of atrophied alveolar bone prior to insertion of dental implants is a major challenge for oral and maxillofacial surgery. It has been reported that Bone Marrow (BM) derived Mesenchymal Stromal Cells (MSC) retain therapeutic potential for bone regeneration. In the present study, a preclinical mini-pig model simulating the clinical setting was established in order to evaluate the efficacy of autologous MSC for mandible regeneration. Under general anaesthesia, BM aspirates were collected from tibia of mini-pigs (n = 5) and MSC were isolated, characterized and expanded. At the same time, a narrow alveolar ridge was simultaneously created by bilateral extraction of two premolar teeth and removal of the buccal bone in order to simulate the pathological situation in humans. After ex vivo expansion, cells were delivered fresh to the surgical operating room and seeded on Biphasic Calcium Phosphate (BCP) granules for 1 hour followed by implantation into the simulated alveolar defects in one pig. The surgical defects were closed with sutures and left to heal for eight weeks. A bone biopsy was taken and dental implants were placed in the newly formed bone. The bone biopsy taken during the procedure showed mineralized bone containing substantial amount of new bone with BCP granules embedded in osteoid tissues and dispersed throughout the newly formed bone matrix. The data demonstrate the osteogenic potential of autologous MSC combined with BCP, providing crucial pre-clinical information in a large animal aimed at the reconstruction of resorbed alveolar bone.

2017 - Blocking tumor-educated MSC paracrine activity halts osteosarcoma progression [Articolo su rivista]
Baglio, Sr; Lagerweij, T; Pérez Lanzón, M; Xuan Ho, D; Léveillé, N; Melo, Sa; Cleton-Jansen, Am; Jordanova, Es; Roncuzzi, L; Greco, M; van Eijndhoven, Ma; Grisendi, G; Dominici, M; Bonafede, R; Lougheed, S; de Gruijl, Td; Zini, N; Cervo, S; Steffan, A; Canzonieri, V; Martson, A; Maasalu, K; Koks, S; Wurdinger, T; Baldini, N; Pegtel, Dm.
abstract

Purpose: Human osteosarcoma is a genetically heterogeneous bone malignancy with poor prognosis despite the employment of aggressive chemotherapy regimens. Because druggable driver mutations have not been established, dissecting the interactions between osteosarcoma cells and supporting stroma may provide insights into novel therapeutic targets.Experimental Design: By using a bioluminescent orthotopic xenograft mouse model of osteosarcoma, we evaluated the effect of tumor extracellular vesicle (EV)-educated mesenchymal stem cells (TEMSC) on osteosarcoma progression. Characterization and functional studies were designed to assess the mechanisms underlying MSC education. Independent series of tissue specimens were analyzed to corroborate the preclinical findings, and the composition of patient serum EVs was analyzed after isolation with size-exclusion chromatography.Results: We show that EVs secreted by highly malignant osteosarcoma cells selectively incorporate a membrane-associated form of TGFβ, which induces proinflammatory IL6 production by MSCs. TEMSCs promote tumor growth, accompanied with intratumor STAT3 activation and lung metastasis formation, which was not observed with control MSCs. Importantly, intravenous administration of the anti-IL6 receptor antibody tocilizumab abrogated the tumor-promoting effects of TEMSCs. RNA-seq analysis of human osteosarcoma tissues revealed a distinct TGFβ-induced prometastatic gene signature. Tissue microarray immunostaining indicated active STAT3 signaling in human osteosarcoma, consistent with the observations in TEMSC-treated mice. Finally, we isolated pure populations of EVs from serum and demonstrated that circulating levels of EV-associated TGFβ are increased in osteosarcoma patients.Conclusions: Collectively, our findings suggest that TEMSCs promote osteosarcoma progression and provide the basis for testing IL6- and TGFβ-blocking agents as new therapeutic options for osteosarcoma patients.

2017 - Concise review: An (Im)Penetrable Shield- How the Tumor Microenvironment Protects Cancer Stem Cells [Articolo su rivista]
Relation, T; Dominici, M; Horwitz, Em
abstract

Cancer stem cells (CSCs) are defined by their unlimited self-renewal ability and their capacity to initiate and maintain malignancy, traits that are not found in most cells that comprise the tumor. Although current cancer treatments successfully reduce tumor burden, the tumor will likely recur unless CSCs are effectively eradicated. This challenge is made greater by the protective impact of the tumor microenvironment (TME), consisting of infiltrating immune cells, endothelial cells, extracellular matrix, and signaling molecules. The TME acts as a therapeutic barrier through immunosuppressive, and thereby tumor-promoting, actions. These factors, outside of the cancer cell lineage, work in concert to shelter CSCs from both the body's intrinsic anticancer immunity and pharmaceutical interventions to maintain cancer growth. Emerging therapies aimed at the TME offer a promising new tool in breaking through this shield to target the CSCs, yet definitive treatments remain unrealized. In this review, we summarize the mechanisms by which CSCs are protected by the TME and current efforts to overcome these barriers

2017 - GD2 expression in breast cancer. [Articolo su rivista]
Orsi, Giulia; Barbolini, Monica; Ficarra, G; Tazzioli, Giovanni; Manni, Paola; Petrachi, Tiziana; Mastrolia, Ilenia; Orvieto, E; Spano, Maria Carlotta; Prapa, Malvina; Kaleci, Shaniko; D'Amico, Roberto; Guarneri, V; Dieci, Mv; Cascinu, Stefano; Conte, P; Piacentini, Federico; Dominici, Massimo
abstract

Breast cancer (BC) is a heterogeneous disease, including different subtypes having diverse incidence, drug-sensitivity and survival rates. In particular, claudin-low and basal-like BC have mesenchymal features with a dismal prognosis. Disialoganglioside GD2 is a typical neuroectodermal antigen expressed in a variety of cancers. Despite its potential relevance in cancer diagnostics and therapeutics, the presence and role of GD2 require further investigation, especially in BC. Therefore, we evaluated GD2 expression in a cohort of BC patients and its correlation with clinical-pathological features.Sixty-three patients with BC who underwent surgery without prior chemo- and/or radiotherapy between 2001 and 2014 were considered. Cancer specimens were analyzed by immunohistochemistry and GD2-staining was expressed according to the percentage of positive cells and by a semi-quantitative scoring system.Patient characteristics were heterogeneous by age at diagnosis, histotype, grading, tumor size, Ki-67 and receptor-status. GD2 staining revealed positive cancer cells in 59% of patients. Among them, 26 cases (41%) were labeled with score 1+ and 11 (18%) with score 2+. Notably, the majority of metaplastic carcinoma specimens stained positive for GD2. The univariate regression logistic analysis revealed a significant association of GD2 with triple-receptor negative phenotype and older age (> 78) at diagnosis.We demonstrate for the first time that GD2 is highly prevalent in a cohort of BC patients clustering on very aggressive BC subtypes, such as triple-negative and metaplastic variants.

2017 - Hematopoietic derived cells do not contribute to osteogenesis as osteoblasts [Articolo su rivista]
Otsuru, Satoru; Overholt, Kathleen M.; Olson, Timothy S.; Hofmann, Ted J.; Guess, Adam J.; Velazquez, Victoria M.; Kaito, Takashi; Dominici, Massimo; Horwitz, Edwin M.
abstract

Despite years of extensive investigation, the cellular origin of heterotopic ossification (HO) has not been fully elucidated. We have previously shown that circulating bone marrow-derived osteoblast progenitor cells, characterized by the immunophenotype CD45 −/CD44 +/CXCR4 +, contributed to the formation of heterotopic bone induced by bone morphogenetic protein (BMP)-2. In contrast, other reports have demonstrated the contribution of CD45 + hematopoietic derived cells to HO. Therefore, in this study, we developed a novel triple transgenic mouse strain that allows us to visualize CD45 + cells with red fluorescence and mature osteoblasts with green fluorescence. These mice were generated by crossing CD45-Cre mice with Z/RED mice that express DsRed, a variant of red fluorescent protein, after Cre-mediated recombination, and then crossing with Col2.3GFP mice that express green fluorescent protein (GFP) in mature osteoblasts. Utilizing this model, we were able to investigate if hematopoietic derived cells have the potential to give rise to mature osteoblasts. Analyses of this triple transgenic mouse model demonstrated that DsRed and GFP did not co-localize in either normal skeletogenesis, bone regeneration after fracture, or HO. This indicates that in these conditions hematopoietic derived cells do not differentiate into mature osteoblasts. Interestingly, we observed the presence of previously unidentified DsRed positive bone lining cells (red BLCs) which are derived from hematopoietic cells but lack CD45 expression. These red BLCs fail to produce GFP even under in vitro osteogenic conditions. These findings indicate that, even though both osteoblasts and hematopoietic cells are developmentally derived from mesoderm, hematopoietic derived cells do not contribute to osteogenesis in fracture healing or HO.

2017 - Identification of a murine CD45-F4/80lo HSC-derived marrow endosteal cell associated with donor stem cell engraftment [Articolo su rivista]
Overholt, Kathleen M; Otsuru, Satoru; Olson, Timothy S; Guess, Adam J; Velazquez, Victoria M; Desbourdes, Laura; Dominici, Massimo; Horwitz, Edwin M
abstract

Hematopoietic stem cells (HSCs) reside in specialized microenvironments within the marrow designated as stem cell niches, which function to support HSCs at homeostasis and promote HSC engraftment after radioablation. We previously identified marrow space remodeling after hematopoietic ablation, including osteoblast thickening, osteoblast proliferation, and megakaryocyte migration to the endosteum, which is critical for effective engraftment of donor HSCs. To further evaluate the impact of hematopoietic cells on marrow remodeling, we used a transgenic mouse model (CD45Cre/iDTR) to selectively deplete hematopoietic cells in situ. Depletion of hematopoietic cells immediately before radioablation and hematopoietic stem cell transplantation abrogated donor HSC engraftment and was associated with strikingly flattened endosteal osteoblasts with preserved osteoblast proliferation and megakaryocyte migration. Depletion of monocytes, macrophages, or megakaryocytes (the predominant hematopoietic cell populations that survive short-term after irradiation) did not lead to an alteration of osteoblast morphology, suggesting that a hematopoietic-derived cell outside these lineages regulates osteoblast morphologic adaptation after irradiation. Using 2 lineage-tracing strategies, we identified a novel CD45-F4/80lo HSC-derived cell that resides among osteoblasts along the endosteal marrow surface and, at least transiently, survives radioablation. This newly identified marrow cell may be an important regulator of HSC engraftment, possibly by influencing the shape and function of endosteal osteoblasts.

2017 - Impact of time to surgery after neoadjuvant chemotherapy in operable breast cancer patients [Articolo su rivista]
Omarini, Claudia; Guaitoli, Giorgia; Noventa, Silvia; Andreotti, Alessia; Gambini, Anna; Palma, E; Papi, Simona; Tazzioli, Giovanni; Balduzzi, Sara; Dominici, Massimo; Cascinu, Stefano; Piacentini, Federico
abstract

The optimal time interval between the end of neoadjuvant systemic therapy (NST) and breast surgery is still unclear. It is not known if a delay in surgery might influence the benefit of primary chemotherapy. The aim of this study is to evaluate the relationship between time to surgery (TTS) and survival outcomes.

2017 - Introduction and overview [Articolo su rivista]
Dominici, Massimo; Zhang, Nan; Barrett, A. John
abstract

n/A

2017 - Safety Profile of Good Manufacturing Practice Manufactured Interferon γ-Primed Mesenchymal Stem/Stromal Cells for Clinical Trials [Articolo su rivista]
Guess, Aj; Daneault, B; Wang, R; Bradbury, H; La Perle, Kmd; Fitch, J; Hedrick, Sl; Hamelberg, E; Astbury, C; White, P; Overolt, K; Rangarajan, H; Abu-Arja, R; Devine, Sm; Otsuru, S; Dominici, M; O'Donnell, L; Horwitz, Em
abstract

Mesenchymal stem/stromal cells (MSCs) are widely studied by both academia and industry for a broad array of clinical indications. The collective body of data provides compelling evidence of the clinical safety of MSC therapy. However, generally accepted proof of therapeutic efficacy has not yet been reported. In an effort to generate a more effective therapeutic cell product, investigators are focused on modifying MSC processing protocols to enhance the intrinsic biologic activity. Here, we report a Good Manufacturing Practice-compliant two-step MSC manufacturing protocol to generate MSCs or interferon γ (IFNγ) primed MSCs which allows freshly expanded cells to be infused in patients on a predetermined schedule. This protocol eliminates the need to infuse cryopreserved, just thawed cells which may reduce the immune modulatory activity. Moreover, using (IFNγ) as a prototypic cytokine, we demonstrate the feasibility of priming the cells with any biologic agent. We then characterized MSCs and IFNγ primed MSCs prepared with our protocol, by karyotype, in vitro potential for malignant transformation, biodistribution, effect on engraftment of transplanted hematopoietic cells, and in vivo toxicity in immune deficient mice including a complete post-mortem examination. We found no evidence of toxicity attributable to the MSC or IFNγ primed MSCs. Our data suggest that the clinical risk of infusing MSCs or IFNγ primed MSCs produced by our two-step protocol is not greater than MSCs currently in practice. While actual proof of safety requires phase I clinical trials, our data support the use of either cell product in new clinical studies.

2017 - Therapeutic potential of the metabolic modulator phenformin in targeting the stem cell compartment in melanoma [Articolo su rivista]
Petrachi, Tiziana; Romagnani, Alessandra; Albini, Adriana; Longo, Caterina; Argenziano, Giuseppe; Grisendi, Giulia; Dominici, Massimo; Ciarrocchi, Alessia; Dallaglio, Katiuscia
abstract

Melanoma is the most dangerous and treatment-resistant skin cancer. Tumor resistance and recurrence are due to the persistence in the patient of aggressive cells with stem cell features, the cancer stem cells (CSC). Recent evidences have shown that CSC display a distinct metabolic profile as compared to tumor bulk population: a promising anti-tumor strategy is therefore to target specific metabolic pathways driving CSC behavior. Biguanides (metformin and phenformin) are anti-diabetic drugs able to perturb cellular metabolism and displaying anti-cancer activity. However, their ability to target the CSC compartment in melanoma is not known. Here we show that phenformin, but not metformin, strongly reduces melanoma cell viability, growth and invasion in both 2D and 3D (spheroids) models. While phenformin decreases melanoma CSC markers expression and the levels of the pro-survival factor MITF, MITF overexpression fails to prevent phenformin effects. Phenformin significantly reduces cell viability in melanoma by targeting both CSC (ALDHhigh) and non-CSC cells and by significantly reducing the number of viable cells in ALDHhighand ALDHlowderived spheroids. Consistently, phenformin reduces melanoma cell viability and growth independently from SOX2 levels. Our results show that phenformin is able to affect both CSC and non-CSC melanoma cell viability and growth and suggests its potential use as anti-cancer therapy in melanoma.

2016 - Altered pH gradient at the plasma membrane of osteosarcoma cells is a key mechanism of drug resistance [Articolo su rivista]
Avnet, Sofia; Lemma, Silvia; Cortini, Margherita; Pellegrini, Paola; Perut, Francesca; Zini, Nicoletta; Kusuzaki, Katsuyuki; Chano, Tokuhiro; Grisendi, Giulia; Dominici, Massimo; De Milito, Angelo; Baldini, Nicola
abstract

Current therapy of osteosarcoma (OS), the most common primary bone malignancy, is based on a combination of surgery and chemotherapy. Multidrug resistance mediated by P-glycoprotein (P-gp) overexpression has been previously associated with treatment failure and progression of OS, although other mechanisms may also play a role. We considered the typical acidic extracellular pH (pHe) of sarcomas, and found that doxorubicin (DXR) cytotoxicity is reduced in P-gp negative OS cells cultured at pHe 6.5 compared to standard 7.4. Short-time (24-48 hours) exposure to low pHe significantly increased the number and acidity of lysosomes, and the combination of DXR with omeprazole, a proton pump inhibitor targeting lysosomal acidity, significantly enhanced DXR cytotoxicity. In OS xenografts, the combination treatment of DXR and omeprazole significantly reduced tumor volume and body weight loss. The impaired toxicity of DXR at low pHe was not associated with increased autophagy or lysosomal acidification, but rather, as shown by SNARF staining, with a reversal of the pH gradient at the plasma membrane (ΔpHcm), eventually leading to a reduced DXR intracellular accumulation. Finally, the reversal of ΔpHcm in OS cells promoted resistance not only to DXR, but also to cisplatin and methotrexate, and, to a lesser extent, to vincristine. Altogether, our findings show that, in OS cells, shortterm acidosis induces resistance to different chemotherapeutic drugs by a reversal of ΔpHcm, suggesting that buffer therapies or regimens including proton pump inhibitors in combination to low concentrations of conventional anticancer agents may offer novel solutions to overcome drug resistance.

2016 - Cell therapies for pancreatic beta-cell replenishment. [Articolo su rivista]
Okere, Bernard; Lucaccioni, Laura; Dominici, Massimo; Iughetti, Lorenzo
abstract

The current treatment approach for type 1 diabetes is based on daily insulin injections, combined with blood glucose monitoring. However, administration of exogenous insulin fails to mimic the physiological activity of the islet, therefore diabetes often progresses with the development of serious complications such as kidney failure, retinopathy and vascular disease. Whole pancreas transplantation is associated with risks of major invasive surgery along with side effects of immunosuppressive therapy to avoid organ rejection. Replacement of pancreatic beta-cells would represent an ideal treatment that could overcome the above mentioned therapeutic hurdles. In this context, transplantation of islets of Langerhans is considered a less invasive procedure although long-term outcomes showed that only 10 % of the patients remained insulin independent five years after the transplant. Moreover, due to shortage of organs and the inability of islet to be expanded ex vivo, this therapy can be offered to a very limited number of patients. Over the past decade, cellular therapies have emerged as the new frontier of treatment of several diseases. Furthermore the advent of stem cells as renewable source of cell-substitutes to replenish the beta cell population, has blurred the hype on islet transplantation. Breakthrough cellular approaches aim to generate stem-cell-derived insulin producing cells, which could make diabetes cellular therapy available to millions. However, to date, stem cell therapy for diabetes is still in its early experimental stages. This review describes the most reliable sources of stem cells that have been developed to produce insulin and their most relevant experimental applications for the cure of diabetes.

2016 - International society for cellular therapy perspective on immune functional assays for mesenchymal stromal cells as potency release criterion for advanced phase clinical trials [Articolo su rivista]
Galipeau, Jacques; Krampera, Mauro; Barrett, John; Dazzi, Francesco; Deans, Robert J.; Debruijn, Joost; Dominici, Massimo; Fibbe, Willem E.; Gee, Adrian P.; Gimble, Jeffery M.; Hematti, Peiman; Koh, Mickey B. C.; Leblanc, Katarina; Martin, Ivan; Mcniece, Ian K.; Mendicino, Michael; Oh, Steve; Ortiz, Luis; Phinney, Donald G.; Planat, Valerie; Prockop, Darwin J.; Shi, Yufang; Stroncek, David F.; Viswanathan, Sowmya; Weiss, Daniel J.; Sensebe, Luc
abstract

Mesenchymal stromal cells (MSCs) as a pharmaceutical for ailments characterized by pathogenic autoimmune, alloimmune and inflammatory processes now cover the spectrum of early- to late-phase clinical trials in both industry and academic sponsored studies. There is a broad consensus that despite different tissue sourcing and varied culture expansion protocols, human MSC-like cell products likely share fundamental mechanisms of action mediating their anti-inflammatory and tissue repair functionalities. Identification of functional markers of potency and reduction to practice of standardized, easily deployable methods of measurements of such would benefit the field. This would satisfy both mechanistic research as well as development of release potency assays to meet Regulatory Authority requirements for conduct of advanced clinical studies and their eventual registration. In response to this unmet need, the International Society for Cellular Therapy (ISCT) addressed the issue at an international workshop in May 2015 as part of the 21st ISCT annual meeting in Las Vegas. The scope of the workshop was focused on discussing potency assays germane to immunomodulation by MSC-like products in clinical indications targeting immune disorders. We here provide consensus perspective arising from this forum. We propose that focused analysis of selected MSC markers robustly deployed by in vitro licensing and metricized with a matrix of assays should be responsive to requirements from Regulatory Authorities. Workshop participants identified three preferred analytic methods that could inform a matrix assay approach: quantitative RNA analysis of selected gene products; flow cytometry analysis of functionally relevant surface markers and protein-based assay of secretome. We also advocate that potency assays acceptable to the Regulatory Authorities be rendered publicly accessible in an "open-access" manner, such as through publication or database collection.

2016 - Microglia are less pro-inflammatory than myeloid infiltrates in the hippocampus of mice exposed to status epilepticus [Articolo su rivista]
Vinet, Jonathan; Vainchtein, Ilia D.; Spano, Maria Carlotta; Giordano, Carmela; Bordini, Domenico; Curia, Giulia; Dominici, Massimo; Boddeke, Hendrikus W. G. M.; Eggen, Bart J. L.; Biagini, Giuseppe
abstract

Activated microglia, astrogliosis, expression of pro-inflammatory cytokines, blood brain barrier (BBB) leakage and peripheral immune cell infiltration are features of mesial temporal lobe epilepsy. Numerous studies correlated the expression of pro-inflammatory cytokines with the activated morphology of microglia, attributing them a pro-epileptogenic role. However, microglia and myeloid cells such as macrophages have always been difficult to distinguish due to an overlap in expressed cell surface molecules. Thus, the detrimental role in epilepsy that is attributed to microglia might be shared with myeloid infiltrates. Here, we used a FACS-based approach to discriminate between microglia and myeloid infiltrates isolated from the hippocampus 24 h and 96 h after status epilepticus (SE) in pilocarpine-treated CD1 mice. We observed that microglia do not express MHCII whereas myeloid infiltrates express high levels of MHCII and CD40 96 h after SE. This antigen-presenting cell phenotype correlated with the presence of CD4pos T cells. Moreover, microglia only expressed TNFα 24 h after SE while myeloid infiltrates expressed high levels of IL-1β and TNFα. Immunofluorescence showed that astrocytes but not microglia expressed IL-1β. Myeloid infiltrates also expressed matrix metalloproteinase (MMP)-9 and 12 while microglia only expressed MMP-12, suggesting the involvement of both cell types in the BBB leakage that follows SE. Finally, both cell types expressed the phagocytosis receptor Axl, pointing to phagocytosis of apoptotic cells as one of the main functions of microglia. Our data suggests that, during early epileptogenesis, microglia from the hippocampus remain rather immune supressed whereas myeloid infiltrates display a strong inflammatory profile.

2016 - Part 1: Defining unproven cellular therapies [Articolo su rivista]
Srivastava, Alok; Mason, Chris; Wagena, Edwin; Cuende, Natividad; Weiss, Daniel J.; Horwitz, Edwin M.; Dominici, Massimo
abstract

Given the potential of cell-based products, including stem/progenitor cells and immune cells, there is a global effort to introduce these therapies into the clinic to correct organ dysfunctions, to treat cancer and to abrogate autoimmune diseases and a wide variety of pathological conditions [1–3]. Relatively easy access to these cells, obtained from marrow, adipose, cord blood and other human tissues, provides tremendous opportunity for translational research, particularly for indications with no satisfactory medical solution for patients with “unmet medical needs.” Prenatal and adult stem cells (including induced pluripotent stem cells have significant potential to rebuild tissues and correct dysfunctional organs in human diseases.

2016 - Part 2: Making the "unproven" "proven" [Articolo su rivista]
Weiss, Daniel J; Rasko, John E. J.; Cuende, Natividad; Ruiz, Milton A.; Ho, Hong Nerng; Nordon, Robert; Wilton, Steve; Dominici, Massimo; Srivastava, Alok
abstract

n/a

2016 - Part 5: Unproven cell therapies and the commercialization of cell-based products [Articolo su rivista]
Deans, Robert J; Gunter, Kurt C.; Dominici, Massimo; Forte, Miguel
abstract

n/a

2016 - Potency biomarker signature genes from multiparametric osteogenesis assays: Will cGMP human bone marrow mesenchymal stromal cells make bone? [Articolo su rivista]
Murgia, Alba; Veronesi, Elena; Candini, Olivia; Caselli, Anna; D'Souza, Naomi; Rasini, Valeria; Giorgini, Andrea; Catani, Fabio; Iughetti, Lorenzo; Dominici, Massimo; Burns, Jorge S.
abstract

In skeletal regeneration approaches using human bone marrow derived mesenchymal stromal cells (hBM-MSC), functional evaluation before implantation has traditionally used biomarkers identified using fetal bovine serum-based osteogenic induction media and time courses of at least two weeks. However, emerging pre-clinical evidence indicates donordependent discrepancies between these ex vivo measurements and the ability to form bone, calling for improved tests. Therefore, we adopted a multiparametric approach aiming to generate an osteogenic potency assay with improved correlation. hBM-MSC populations from six donors, each expanded under clinical-grade (cGMP) conditions, showed heterogeneity for ex vivo growth response, mineralization and bone-forming ability in a murine xenograft assay. A subset of literature-based biomarker genes was reproducibly upregulated to a significant extent across all populations as cells responded to two different osteogenic induction media. These 12 biomarkers were also measurable in a one-week assay, befitting clinical cell expansion time frames and cGMP growth conditions. They were selected for further challenge using a combinatorial approach aimed at determining ex vivo and in vivo consistency. We identified five globally relevant osteogenic signature genes, notably TGF-β1 pathway interactors; ALPL, COL1A2, DCN, ELN and RUNX2. Used in agglomerative cluster analysis, they correctly grouped the bone-forming cell populations as distinct. Although donor #6 cells were correlation slope outliers, they contrastingly formed bone without showing ex vivo mineralization. Mathematical expression level normalization of the most discrepantly upregulated signature gene COL1A2, sufficed to cluster donor #6 with the bone-forming classification. Moreover, attenuating factors causing genuine COL1A2 gene down-regulation, restored ex vivo mineralization. This suggested that the signature gene had an osteogenically influential role; nonetheless no single biomarker was fully deterministic whereas all five signature genes together led to accurate cluster analysis. We show proof of principle for an osteogenic potency assay providing early characterization of primary cGMP-hBM-MSC cultures according to their donor-specific bone-forming potential.

2016 - President's letter on unproven cellular therapy [Articolo su rivista]
Dominici, Massimo
abstract

The International Society for CellularTherapy (ISCT; www.celltherapysociety.org), with a strategy adopted more than 5 year ago (Gunter KC et al. Cytotherapy 2010), wishes to continue raising awareness about unproven cellular therapies. All over the world, there is a legitimate effort to introduce cellular therapies into the clinic; however, at the same time a large number of clinics have begun offering cell-based intervention for a variety of diseases with questionable safety or efficacy data or an unclear scientific rationale.This has led to many vulnerable patients subjecting themselves to unproven cellular therapies with no adequate regulatory oversight or reliable information about risks or benefit. For this reason, ISCT feels the need to provide a new source of information: an open document that represents a sharing of knowledge by leaders in the field.

2016 - Resistance to neoplastic transformation of ex-vivo expanded human mesenchymal stromal cells after exposure to supramaximal physical and chemical stress [Articolo su rivista]
Conforti, Antonella; Starc, Nadia; Biagini, Simone; Tomao, Luigi; Pitisci, Angela; Algeri, Mattia; Sirleto, Pietro; Novelli, Antonio; Grisendi, Giulia; Candini, Olivia; Carella, Cintia; Dominici, Massimo; Locatelli, Franco; Bernardo, Maria Ester
abstract

The risk of malignant transformation of ex-vivo expanded human mesenchymal stromal cells (huMSCs) has been debated in the last years; however, the biosafety of these cells after exposure to supramaximal physical and chemical stress has never been systematically investigated.We established an experimental in vitro model to induce supramaximal physical (ionizing radiation, IR) and chemical (starvation) stress on ex-vivo expanded bone marrow (BM)-derived huMSCs and investigated their propensity to undergo malignant transformation. To this aim, we examined MSC morphology, proliferative capacity, immune-phenotype, differentiation potential, immunomodulatory properties and genetic profile before and after stressor exposure. Furthermore, we investigated the cellular mechanisms underlying MSC response to stress. MSCs were isolated from 20 healthy BM donors and expanded in culture medium supplemented with 5% platelet lysate (PL) up to passage 2 (P2). At this stage, MSCs were exposed first to escalating doses of IR (30, 100, 200 Gy) and then to starvation culture conditions (1% PL).With escalating doses of radiation, MSCs lost their typical spindle-shaped morphology, their growth rate markedly decreased and eventually stopped (at P4-P6) by reaching early senescence. Irradiated and starved MSCs maintained their typical immune-phenotype, ability to differentiate into adipocytes/osteoblasts and to inhibit mitogen-induced T-cell proliferation. The study of the genetic profile of irradiated/starved MSCs did not show any alteration. While the induction of supramaximal stress triggered production of ROS and activation of DNA damage response pathway via multiple mechanisms, our data indicate that irradiated/starved MSCs, although presenting altered morphology/growth rate, do not display increased propensity for malignant transformation.

2016 - SOLUBLE TRAIL-ARMED HUMAN AD-MSC AS NOVEL CELL THERAPY APPROACH FOR PANCREATIC DUCTAL ADENOCARCINOMA [Abstract in Atti di Convegno]
Candini, C. Spano. G. Grisendi. O.; Petrachi, G. Golinelli. T.; Rossignoli, F.; Prapa, M.; Orsi, G.; Barbolini, M.; Rovesti, G.; Maiorana, A.; Marmi, P.; Piacentini, F.; Conte, P.; Dominici, M.
abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive adult tumors and its prognosis is still poor since the number of deaths almost equal the number of new cases. New therapeutic approaches are therefore urgently needed. In our model human adipose mesenchy.mal stromallstem cells (ADMSC) have been armed with a novel soluble TRAIL variant that is constantly released by modified progenitors (sTRAIL AD-MSC). The wild type TRAIL form is known to act as a trimer stabilized by a zinc-binding site. In this srudy, by gene engineering, we allow AD-MSC to secrete a trimeric zincindependent soluble TRAIL variant. The molecule has been then challenged in vitro and in vivo, either using sTRAIL AD-MSC supernatant or injecting sTRAIL AD-MSC cells in a PDAC xenotransplant rnodel, We demonstrated that sTRAIL was stable at 37°C far at least 24 hours and was able to induce apoptosis in the PDAC lines BxPC-3 and MIA PaCa-2 and, more interestingly, against primary PDAC cells. Moreover, sTRAIL released by AD-MSC was able to significantly counteract tumor growth with a reduction of the cytokeratin-7 positive cells and by an anti-angiogenic effect. In parallel, a retrospective study on PDAC specimens form patients (n = 19) has been conducted in order to investigate TRAIL DR4, DR5 and OPG receptor expression in "real" PDAC tissue and generate insights on the possible clinical translation of our approach. Our results suggest that MSC can be vehicles for novel TRAIL variants opening novel opportunities for PDAC treatrnent by multiple mechanisms.

2016 - The Survey on Cellular and Engineered Tissue Therapies in Europe in 2013 [Articolo su rivista]
Martin, Ivan; Ireland, Hilary; Baldomero, Helen; Dominici, Massimo; Saris, Daniël B.; Passweg, Jakob
abstract

Following the coordinated efforts of five established scientific organizations, this report, the sixth of its kind, describes activity in Europe for the year 2013 in the area of cellular and engineered tissue therapies, excluding hematopoietic stem cell (HSC) treatments for the reconstitution of hematopoiesis. Three hundred eighteen teams from 31 countries responded to the cellular and engineered tissue therapy survey; 145 teams from 25 countries reported treating 2187 patients, while a further 173 teams reported no activity. Indications were musculoskeletal/rheumatological disorders (45%; 89% autologous), cardiovascular disorders (20%; 99% autologous), hematology/oncology, predominantly prevention or treatment of graft versus host disease (GvHD) and HSC graft enhancement, (19%; <1% autologous), neurological disorders (3%; 100% autologous), gastrointestinal disorders (2%; 32% autologous), and other indications (11%; 67% autologous). The majority of autologous cells (88%) were used to treat musculoskeletal/rheumatological (57%) and cardiovascular (27%) disorders, whereas allogeneic cells were used mainly for hematology/oncology (64%). The reported cell types were mesenchymal stem/stromal cells (MSC) (49%), HSC (28%), chondrocytes (11%), dendritic cells (2%), keratinocytes (1%), and others (9%). In 46% of the grafts, cells were delivered following ex vivo expansion, sorted in 17% of the reported cases and transduced in only 3%. Thirty three percent of treatments were delivered intravenously or intra-arterially, and of the remaining 67%, 37% used a membrane/scaffold, 28% a suspension, and 2% a gel. The data are compared to those previously collected to identify trends in a still unpredictably evolving field.

2016 - TRAIL delivered by mesenchymal stromal/stem cells counteracts tumor development in orthotopic Ewing sarcoma models [Articolo su rivista]
Guiho, Romain; Biteau, Kevin; Grisendi, Giulia; Taurelle, Julien; Chatelais, Mathias; Gantier, Malika; Heymann, Dominique; Dominici, Massimo; Redini, Françoise
abstract

Ewing sarcoma (EWS) is the second most frequent pediatric malignant bone tumor. EWS patients have not seen any major therapeutic progress in the last 30 years, in particular in the case of metastatic disease, which requires new therapeutic strategies. The pro-apoptotic cytokine TNF-Related Apoptosis Inducing Ligand (TRAIL) can selectively kill tumor cells while sparing normal cells, making it a promising therapeutic tool in several types of cancer. However, certain EWS cell lines appear resistant to recombinant human (rh) TRAIL-induced apoptosis. We therefore hypothesized that a TRAIL presentation at the surface of the carrier cells might overcome this resistance and trigger apoptosis. For this purpose, human adipose mesenchymal stromal/stem cells (MSC) transfected in a stable manner to express full-length human TRAIL were co-cultured with several human EWS cell lines, inducing apoptosis by cell-to-cell contact even in cell lines initially resistant to rhTRAIL or AMG655, an antibody agonist to the death receptor, DR5. In vivo, TRAIL delivered by MSCs was able to counteract tumor progression in two orthotopic models of Ewing sarcoma, associated with caspase activation, indicating that a cell-based delivery of a potent apoptosis-inducing factor could be relevant in EWS.

2016 - Tumor Stroma Manipulation By MSC [Articolo su rivista]
Grisendi, Giulia; Spano, Carlotta; Rossignoli, Filippo; D. Souza, Naomi; Golinelli, Giulia; Fiori, Agnese; Horwitz, Edwin M; Guarneri, Valentina; Piacentini, Federico; Paolucci, Paolo; Dominici, Massimo
abstract

Tumor stroma (TS) plays relevant roles in all steps of cancer development. We here address several fundamental aspects related with the interaction between cancer cells and their stromal counterparts. Dissecting these players is of pivotal importance to understand oncogenesis, immunoescape and drug resistance. In addition, this better comprehension will allow the introduction of novel and more effective therapeutic approaches where manipulated stromal elements may become detrimental for tumor growth. Our group and others rely on the use of multipotent mesenchymal stromal/stem cells (MSC) as anti-cancer tools, since these putative TS cell precursors can deliver potent apoptosis-inducing agents. Multimodal-armed MSC can target a variety of cancers in vitro and, when injected in vivo, they localize into tumors mediating cell death without evident toxicities to normal tissues. While several aspects of these strategies shall require further investigations, these approaches collectively indicate how TS manipulation by MSC represents a tool to influence the fate of cancer cells, creating a new generation of anti-cancer strategies.

2015 - A novel anti-GD2/4-1BB chimeric antigen receptor triggers neuroblastoma cell killing [Articolo su rivista]
Prapa, Malvina; Caldrer, Sara; Spano, Maria Carlotta; Bestagno, Marco; Golinelli, Giulia; Grisendi, Giulia; Petrachi, Tiziana; Conte, Pierfranco; Horwitz, EDWIN MARK; Campana, Dario; Paolucci, Paolo; Dominici, Massimo
abstract

Chimeric antigen receptor (CAR)-expressing T cells are a promising therapeutic option for patients with cancer. We developed a new CAR directed against the disialoganglioside GD2, a surface molecule expressed in neuroblastoma and in other neuroectoderm-derived neoplasms. The anti-GD2 single-chain variable fragment (scFv) derived from a murine antibody of IgM class was linked, via a human CD8α hinge-transmembrane domain, to the signaling domains of the costimulatory molecules 4-1BB (CD137) and CD3-ζ. The receptor was expressed in T lymphocytes by retroviral transduction and anti-tumor activities were assessed by targeting GD2-positive neuroblastoma cells using in vitro cytotoxicity assays and a xenograft model. Transduced T cells expressed high levels of anti-GD2 CAR and exerted a robust and specific anti-tumor activity in 4- and 48-hour cultures with neuroblastoma cells. Cytotoxicity was associated with the release of pro-apoptotic molecules such as TRAIL and IFN-γ. These results were confirmed in a xenograft model, where anti-GD2 CAR T cells infiltrating tumors and persisting into blood circulation induced massive apoptosis of neuroblastoma cells and completely abrogated tumor growth. This anti-GD2 CAR represents a powerful new tool to redirect T cells against GD2. The preclinical results of this study warrant clinical testing of this approach in neuroblastoma and other GD2-positive malignancies.

2015 - Carbonic anhydrase IX inhibition is an effective strategy for osteosarcoma treatment [Articolo su rivista]
Perut, Francesca; Carta, Fabrizio; Bonuccelli, Gloria; Grisendi, Giulia; Di, Pompo Gemma; Avnet, Sofia; Sbrana, Francesca Vittoria; Hosogi, Shigekuni; Dominici, Massimo; Kusuzaki, Katsuyuki; Supuran, Claudiu T.; Baldini, Nicola
abstract

Objective: Hypoxia-inducible factor 1, a regulator of CA IX activity, is often overexpressed in human osteosarcoma (OS) but not in normal tissues, and its expression levels correlate with prognosis. In this study, we investigated the therapeutic potential of newly synthesized CA IX sulfonamide inhibitors in OS. Methods: CA IX expression was evaluated in OS cell lines and bone marrow stromal cells (BMSC). After treatment with CA IX inhibitors, cell proliferation, apoptosis, cell cycle, extracellular and cytosolic pH changes were evaluated both in vitro and in mouse OS xenografts. Results: CA IX expression levels were significantly higher in OS than in BMSC. Accordingly, CA IX inhibitor 3 induced remarkable cytotoxicity on OS cells without affecting BMSC proliferation. This activity was increased under hypoxia, and was mediated by cell cycle arrest and by the modulation of cytosolic and extracellular pH. In vivo, CA IX inhibitor 3 reduced tumor growth by inducing significant necrosis. Conclusions: Our results provide a strong rationale for the clinical use of the newly synthesized CA IX inhibitor 3 in human OS.

2015 - CD271 Mediates Stem Cells to Early Progeny Transition in Human Epidermis [Articolo su rivista]
Truzzi, Francesca; Saltari, Annalisa; Palazzo, Elisabetta; Lotti, Roberta; Petrachi, Tiziana; Dallaglio, Katiuscia; Gemelli, Claudia; Grisendi, Giulia; Dominici, Massimo; Pincelli, Carlo; Marconi, Alessandra
abstract

CD271 is the low-affinity neurotrophin (p75NTR) receptor that belongs to the tumor necrosis factor receptor superfamily. Because in human epidermis, CD271 is predominantly expressed in transit-amplifying (TA) cells, we evaluated the role of this receptor in keratinocyte differentiation and in the transition from keratinocyte stem cells (KSCs) to progeny. Calcium induced an upregulation of CD271 in subconfluent keratinocytes, which was prevented by CD271 small interfering RNA. Furthermore, CD271 overexpression provoked the switch of KSCs to TA cells, whereas silencing CD271 induced TA cells to revert to a KSC phenotype, as shown by the expression of β1-integrin and by the increased clonogenic ability. CD271(+) keratinocytes sorted from freshly isolated TA cells expressed more survivin and keratin 15 (K15) compared with CD271(-) cells and displayed a higher proliferative capacity. Early differentiation markers and K15 were more expressed in the skin equivalent generated from CD271(+) TA than from those derived from CD271(-) TA cells. By contrast, late differentiation markers were more expressed in skin equivalents from CD271(-) than in reconstructs from CD271(+) TA cells. Finally, skin equivalents originated from CD271(-) TA cells displayed a psoriatic phenotype. These results indicate that CD271 is critical for keratinocyte differentiation and regulates the transition from KSCs to TA cells.

2015 - cGMP-compliant transportation conditions for a prompt therapeutic use of marrow mesenchymal stromal/stem cells [Capitolo/Saggio]
Veronesi, Elena; Burns, Jorge Phillip Joaquin Sans; Murgia, Alba; Candini, Olivia; Rasini, Valeria; Mastrolia, Ilenia; Catani, Fabio; Paolucci, Paolo; Dominici, Massimo
abstract

We recently described conditions for safe 18-h manufacturer-to-patient transportation of freshly harvested hBM-MSC expanded under cGMP protocols using human platelet lysate (hPL), that allowed prompt use as an advanced therapeutic medicinal product. Here we outline important considerations when comparing different transportation conditions, highlighting that although cell transportation may involve a reduction in viability, this did not undermine the ultimate bone-forming regenerative potential of the cGMP-hBM-MSC population.

2015 - Detection of microparticles from human red blood cells by multiparametric flow cytometry [Articolo su rivista]
Grisendi, Giulia; Finetti, Elena; Manganaro, Daniele; Cordova, Nicoletta; Montagnani, Giuliano; Spano, Maria Carlotta; Prapa, Malvina; Guarneri, Valentina; Otsuru, Satoru; Horwitz, Edwin M.; Mari, Giorgio; Dominici, Massimo
abstract

Background: During storage, red blood cells (RBC) undergo chemical and biochemical changes referred to as "storage lesions". These events determine the loss of RBC integrity, resulting in lysis and release of microparticles. There is growing evidence of the clinical importance of microparticles and their role in blood transfusion-related side effects and pathogen transmission. Flow cytometry is currently one of the most common techniques used to quantify and characterise microparticles. Here we propose multiparametric staining to monitor and quantify the dynamic release of microparticles by stored human RBC. Material and methods: RBC units (n=10) were stored under blood bank conditions for up to 42 days. Samples were tested at different time points to detect microparticles and determine the haemolysis rate (HR%). Microparticles were identified by flow cytometry combining carboxyfluorescein diacetate succinimidyl ester (CFSE) dye, annexin V and anti-glycophorin A antibody. Results: We demonstrated that CFSE can be successfully used to label closed vesicles with an intact membrane. The combination of CFSE and glycophorin A antibody was effective for monitoring and quantifying the dynamic release of microparticles from RBC during storage. Double staining with CFSE/glycophorin A was a more precise approach, increasing vesicle detection up to 4.7-fold vs the use of glycophorin A/annexin V alone. Moreover, at all the time points tested, we found a robust correlation (R=0.625; p=0.0001) between HR% and number of microparticles detected. Discussion: Multiparametric staining, based on a combination of CFSE, glycophorin A antibody and annexin V, was able to detect, characterise and monitor the release of microparticles from RBC units during storage, providing a sensitive approach to labelling and identifying microparticles for transfusion medicine and, more broadly, for cell-based therapies.

2015 - Effects of a ceramic biomaterial on immune modulatory properties and differentiation potential of human mesenchymal stromal cells of different origin [Articolo su rivista]
Bassi, Giulio; Guilloton, Fabien; Menard, Cedric; Di Trapani, Mariano; Deschaseaux, Frederic; Sensebé, Luc; Schrezenmeier, Hubert; Giordano, Rosaria; Bourin, Philippe; Dominici, Massimo; Tarte, Karin; Krampera, Mauro
abstract

The aim of this study was to assess the immune modulatory properties of human mesenchymal stromal cells obtained from bone marrow (BM-MSCs), fat (ASCs), and cord blood (CB-MSCs) in the presence of a hydroxyapatite and tricalcium-phosphate (HA/TCP) biomaterial as a scaffold for MSC delivery. In resting conditions, a short-term culture with HA/TCP did not modulate the anti-apoptotic and suppressive features of the various MSC types toward T, B, and NK cells; in addition, when primed with inflammatory cytokines, MSCs similarly increased their suppressive capacities in the presence or absence of HA/TCP. The long-term culture of BM-MSCs with HA/TCP induced an osteoblast-like phenotype with upregulation of OSTERIX and OSTEOCALCIN, similar to what was obtained with dexamethasone and, to a higher extent, with bone morphogenetic protein 4 (BMP-4) treatment. MSC-derived osteoblasts did not trigger immune cell activation, but were less efficient than undifferentiated MSCs in inhibiting stimulated T and NK cells. Interestingly, their suppressive machinery included not only the activation of indoleamine-2,3 dioxygenase (IDO), which plays a central role in T-cell inhibition, but also cyclooxygenase-2 (COX-2) that was not significantly involved in the immune modulatory effect of human undifferentiated MSCs. Since COX-2 is significantly involved in bone healing, its induction by HA/TCP could also contribute to the therapeutic activity of MSCs for bone tissue engineering.

2015 - Foreword [Guide tocell therapy GxP] [Breve Introduzione]
Dominici, Massimo
abstract

n/a

2015 - Genomic and functional comparison of mesenchymal stromal cells prepared using two isolation methods [Articolo su rivista]
Otsuru, Satoru; Hofmann, Ted J.; Raman, Pichai; Olson, Timothy S.; Guess, Adam J.; Dominici, Massimo; Horwitz, Edwin M.
abstract

Background aims: Mesenchymal stromal cells (MSCs) have been applied to patients in cell therapy for various diseases. Recently, we introduced a novel MSC separation filter device which could yield approximately 2.5-fold more MSCs from bone marrow in a closed system compared with the conventional open density gradient centrifugation method. MSCs isolated with these two methods were phenotypically similar and met the criteria defining human MSC proposed by the International Society for Cellular Therapy. However, these criteria do not reflect the functional capacity of MSCs. It has been shown that the donor, source, isolation method, culture condition and cryopreservation of MSCs have potential to alter their therapeutic efficacy. To determine the equivalency of MSCs isolated by these two methods, we compared their genomic profiles as an index of their biologic potential and evaluated their growth promoting potential as an index of function. Methods: The gene expression profiles of human MSCs isolated from 5 healthy donors with two distinct methods were obtained from microarray analyses. The functional activity of freshly expanded/cryopreserved MSCs from these two isolation methods was evaluated using an invitro chondrocyte proliferation assay. Results: Freshly expanded MSCs isolated by these two methods were found to exhibit similar gene expression profiles and equivalent therapeutic effects, while freshly thawed, cryopreserved MSCs lacked all measureable therapeutic activity. Conclusions: The MSC separation device generates genomically and functionally equivalent MSCs compared with the conventionally isolated MSCs, although freshly thawed, cryopreserved MSCs, isolated by either method, are devoid of activity in our bioassay.

2015 - Impressive response to dose-dense chemotherapy in a patient with NUT midline carcinoma [Articolo su rivista]
Maur, Michela; Toss, Angela; Dominici, Massimo; Frassoldati, Antonio; Corradini, Paolo; Maiorana, Antonio; Fontana, Annalisa; Conte, Pierfranco
abstract

Objective: Rare disease Background: NUT midline carcinoma (NMC) is a rare, highly lethal malignancy that results from a chromosome translocation and mostly arises in the midline organs. To date, no treatment has been established. Most patients receive combinations of chemotherapy regimens and radiation, and occasionally subsequent resection; nevertheless, patients have an average survival hardly exceeding 7 months. Case Report: A 21-year-old patient was admitted to our division with a large mediastinal mass with lung nodules, multiple vertebral metastases, and massive nodal involvement. In a few days, the patient developed a superior vena cava syndrome and an acute respiratory failure. Due to the rapid course of the disease, based on preliminary histology of poorly differentiated carcinoma, a dose-dense biweekly chemotherapy with paclitaxel, ifosfamide, and cisplatin was started. In the meantime, the diagnosis of NMC was confirmed. A surprising clinical benefit was obtained after the first cycle of chemotherapy, and after 6 cycles a PET-CT scan showed a very good response. At this point, radiotherapy was started but the disease progressed outside of the radiation field. The patient entered into a compassionate use protocol with Romidepsin, but a PET/CT scan after the first course showed disease progression with peritoneal and retroperitoneal carcinosis. A treatment with Pemetrexed was then started but the patient eventually died with rapid progressive disease. Conclusions: Our case history adds some interesting findings to available knowledge: NMC can be chemosensitive and radiosensitive. This opens the possibility to study more aggressive treatments, including high-dose consolidation chemotherapy and to evaluate the role of biological agents as maintenance treatments.

2015 - In vitro differentiation of human amniotic epithelial cells into insulin-producing 3D spheroids [Articolo su rivista]
Okere, Bernard; Alviano, Francesco; Costa, Roberta; Quaglino, Daniela; Ricci, Francesca; Dominici, Massimo; Paolucci, Paolo; Bonsi, Laura; Iughetti, Lorenzo
abstract

Regenerative medicine and stem cell therapy may represent the solution for the treatment of non-curable human diseases such as type 1 diabetes. In this context of growing demand for functional and safe stem cells, human amniotic epithelial cells (hAECs) from term placenta have attracted increasing interest for their wide availability, stem cell properties, and differentiation plasticity, which make them a promising tool for stem cell-based therapeutic applications. We initially assayed the stemness characteristics of hAECs in serum-free conditions. Subsequently we developed a culture procedure on extracellular matrix for the formation of three-dimensional (3D) spheroids. Finally, we tested the immunomodulation and differentiation potential of hAEC spheroids: the presence of pancreatic endocrine hormones was revealed with transmission electron microscopy and immunofluorescence analyses; the release of C-peptide in hyperglycemic conditions was assayed with ELISA.The serum-free culture conditions we applied proved to maintain the basic stemness characteristics of hAECs. We also demonstrated that 3D spheroids formed by hAECs in extracellular matrix can be induced to differentiate into insulin-producing cells. Finally, we proved that control and induced cells equally inhibit the proliferation of activated mononuclear cells.The results of this study highlight the properties of amnion derived epithelial cells as promising and abundant source for cell-based therapies. In particular we are the first group to show the in vitro pancreatic induction of hAECs cultured on extracellular matrix in a 3D fashion. We accordingly propose the outcomes of this study as a novel contribution to the development of future cell replacement therapies involving placenta-derived cells.

2015 - Mesenchymal progenitors aging highlights a mir-196 switch targeting HOXB7 as master regulator of proliferation and osteogenesis [Articolo su rivista]
Candini, Olivia; Spano, Maria Carlotta; Murgia, Alba; Grisendi, Giulia; Veronesi, Elena; Piccinno, MARIA SERENA; Ferracin, Manuela; Negrini, Massimo; Giacobbi, Francesca; Bambi, Franco; Horwitz, Edwin Mark; Conte, Pierfranco; Paolucci, Paolo; Dominici, Massimo
abstract

Human aging is associated with a decrease in tissue functions combined with a decline in stem cells frequency and activity followed by a loss of regenerative capacity. The molecular mechanisms behind this senescence remain largely obscure, precluding targeted approaches to counteract aging. Focusing on mesenchymal stromal/stem cells (MSC) as known adult progenitors, we identified a specific switch in miRNA expression during aging, revealing a miR-196a up-regulation which was inversely correlated with MSC proliferation through HOXB7 targeting. A forced HOXB7 expression was associated with an improved cell growth, a reduction of senescence and an improved osteogenesis linked to a dramatic increase of autocrine bFGF secretion. These findings, along with the progressive decrease of HOXB7 levels observed during skeletal aging in mice, indicate HOXB7 as a master factor driving progenitors behavior lifetime, providing a better understanding of bone senescence and leading to an optimization of MSC performance.

2015 - Mesenchymal progenitors expressing TRAIL induce apoptosis in sarcomas [Articolo su rivista]
Grisendi, Giulia; Spano, Maria Carlotta; D'Souza, Naomi; Rasini, Valeria; Veronesi, Elena; Prapa, Malvina; Petrachi, Tiziana; Piccinno, MARIA SERENA; Rossignoli, Filippo; Burns, Jorge Phillip Joaquin Sans; Fiorcari, Stefania; Granchi, Donatella; Baldini, Nicola; Horwitz, EDWIN MARK; Guarneri, Valentina; Conte, Pierfranco; Paolucci, Paolo; Dominici, Massimo
abstract

Sarcomas are frequent tumors in children and young adults that, despite a relative chemo-sensitivity, show high relapse rates with up to 80% of metastatic patients dying in 5 years from diagnosis. The real ontogeny of sarcomas is still debated and evidences suggest they may derive from precursors identified within mesenchymal stromal/stem cells (MSC) fractions. Recent studies on sarcoma microenvironment additionally indicated that MSC could take active part in generation of a supportive stroma. Based on this knowledge, we conceived to use modified MSC to deliver tumor necrosis factor related apoptosis inducing ligand (TRAIL) targeting different sarcoma histotypes. Gene modified MSC expressing TRAIL were co-cultured with different osteosarcoma, rhabdomyosarcoma and Ewing's Sarcoma (ES) cell lines assessing viability and caspase-8 activation. An in vivo model focused on ES was then implemented considering the impact of MSC-TRAIL on tumor size, apoptosis and angiogenesis. MSC expressing TRAIL induced significantly high apoptosis in all tested lines. Sarcoma death was specifically associated with caspase-8 activation starting from 8 hours of co-culture with MSC-TRAIL. When injected into pre-established ES xenotransplants, MSC-TRAIL persisted within its stroma, causing significant tumor apoptosis versus control groups. Additional histological and in vitro studies reveal that MSC-TRAIL could also exert potent anti-angiogenic functions. Our results suggest that MSC as TRAIL vehicles could open novel therapeutic opportunities for sarcoma by multiple mechanisms.

2015 - Mesenchymal stem/stromal cells as a delivery platform in cell and gene therapies [Articolo su rivista]
D’Souza, Naomi; Rossignoli, Filippo; Golinelli, Giulia; Grisendi, Giulia; Spano, Maria Carlotta; Candini, Olivia; Osturu, Satoru; Catani, Fabio; Paolucci, Paolo; Horwitz, Edwin M.; Dominici, Massimo
abstract

Regenerative medicine relying on cell and gene therapies is one of the most promising approaches to repair tissues. Multipotent mesenchymal stem/stromal cells (MSC), a population of progenitors committing into mesoderm lineages, are progressively demonstrating therapeutic capabilities far beyond their differentiation capacities. The mechanisms by which MSC exert these actions include the release of biomolecules with anti-inflammatory, immunomodulating, anti-fibrogenic, and trophic functions. While we expect the spectra of these molecules with a therapeutic profile to progressively expand, several human pathological conditions have begun to benefit from these biomolecule-delivering properties. In addition, MSC have also been proposed to vehicle genes capable of further empowering these functions. This review deals with the therapeutic properties of MSC, focusing on their ability to secrete naturally produced or gene-induced factors that can be used in the treatment of kidney, lung, heart, liver, pancreas, nervous system, and skeletal diseases. We specifically focus on the different modalities by which MSC can exert these functions. We aim to provide an updated understanding of these paracrine mechanisms as a prerequisite to broadening the therapeutic potential and clinical impact of MSC.

2015 - Mesenchymal stromal cells for cutaneous wound healing in a rabbit model: pre-clinical study applicable in the pediatric surgical setting [Articolo su rivista]
Pelizzo, Gloria; Avanzini, Maria Antonietta; Icaro Cornaglia, Antonia; Osti, Monica; Romano, Piero; Avolio, Luigi; Maccario, Rita; Dominici, Massimo; De Silvestri, Annalisa; Andreatta, Erika; Costanzo, Federico; Mantelli, Melissa; Ingo, Daniela; Piccinno, MARIA SERENA; Calcaterra, Valeria
abstract

Objective: Mesenchymal stromal cells (MSCs) expanded in vitro have been proposed as a potential therapy for congenital or acquired skin defects in pediatrics. The aim of this pre-clinical study was to investigate the effects of intradermal injections of MSC in experimental cutaneous wound repair comparing allogeneic and autologous adipose stem cells (ASCs) and autologous bone marrow-mesenchymal stromal cells (BM-MSCs). Methods: Mesenchymal stromal cells were in vitro expanded from adipose and BM tissues of young female New Zealand rabbits. MSCs were characterized for plastic adhesion, surface markers, proliferation and differentiation capacity. When an adequate number of cells (ASCs 10 × 10<sup>6</sup> and BM-MSCs 3 × 10<sup>6</sup>, because of their low rate of proliferation) was reached, two skin wounds were surgically induced in each animal. The first was topically treated with cell infusions, the second was used as a control. The intradermal inoculation included autologous or allogeneic ASCs or autologous BM-MSCs. For histological examination, animals were sacrificed and wounds were harvested after 11 and 21 days of treatment. Results: Rabbit ASCs were isolated and expanded in vitro with relative abundance, cells expressed typical surface markers (CD49e, CD90 and CD29). Topically, ASC inoculation provided more rapid wound healing than BM-MSCs and controls. Improved re-epithelization, reduced inflammatory infiltration and increased collagen deposition were observed in biopsies from wounds treated with ASCs, with the best result in the autologous setting. ASCs also improved restoration of skin architecture during wound healing. Conclusion: The use of ASCs may offer a promising solution to treat extended wounds. Pre-clinical studies are however necessary to validate the best skin regeneration technique, which could be used in pediatric surgical translational research.

2015 - Positioning a Scientific Community on Unproven Cellular Therapies: The 2015 International Society for Cellular Therapy Perspective [Articolo su rivista]
Dominici, Massimo; Nichols, Karen; Srivastava, Alok; Weiss, Daniel J.; Eldridge, Paul; Cuende, Natividad; Deans, Robert J.; Rasko, John E. J.; Levine, Aaron D.; Turner, Leigh; Griffin, Deborah L.; O'Donnell, Lynn; Forte, Miguel; Mason, Chris; Wagena, Edwin; Janssen, William; Nordon, Robert; Wall, Dominic; Ho, Hong Nerng; Ruiz, Milton A.; Wilton, Steve; Horwitz, Edwin M.; Gunter, Kurt C.
abstract

"Currently, there are many unproven or insufficiently" "proven cell-based treatments commercially available for hopeful individuals seeking cures for a variety of conditions. Typically, these so-called “therapies” are currently being advertised, sold and administered to patients, although they fail to achieve recognized" "biological/medical standards of proof for safety or ef-" "ficacy. In addition, they are often expensive and offered outside the cover of routine clinical care for treat- ments, outside the realm of conventional clinical trials supervised and monitored by regulatory agencies.This paper summarizes a position document to be pub- lished by the International Society for Cellular Therapy (ISCT) as an open manuscript intended for professionals and patient associations. Avoiding a systematic overview of the relevant peer-reviewed literature and investigations, its purpose is to examine multiple aspects of unproven cell therapy interventions including definitions, manufacturing issues, regulations, economic factors and communication.With this document, the ISCT intends to promote a cooperative approach to facilitate the development of safe and effective therapies while minimizing and balancing risks for patients to ultimately establish a coalition of stakeholders that fulfill the vision of a broad, pro-patient cell therapy alliance.

2015 - The phenotypes of microglia and macrophages during epileptogenesis [Abstract in Rivista]
Vinet, Jonathan; Vainchtein, I. D.; Spano, Maria Carlotta; Giordano, Carmela; Bordini, D.; Dominici, Massimo; Eggen, B. J. L.; Biagini, Giuseppe
abstract

Question: A growing body of evidence is now supporting a relationship between inflammation and epilepsy. Indeed, activated microglia, reactive astrocytes, local expression of pro-inflammatory cytokines, blood brain barrier leakage and peripheral immune cell infiltration have all been observed in temporal lobe epilepsy (TLE) animal models as well as in humans. Accordingly, inflammatory mechanisms are thought to play a central role in the initiation and maintenance of seizures, starting in the acute phase represented by status epilepticus (SE) induction. Microglia activation has been correlated with the expression of several pro-inflammatory cytokines which are thought to contribute to the neuronal cell death occurring after SE. Data point towards a pro-inflammatory phenotype of microglia that precedes neuronal injury and cell death. Because of this, microglia are generally considered to play a pro-epileptogenic role. However, infiltration of peripheral immune cells during epileptogenesis such as leukocytes, granulocytes and monocytes/macrophages might also contribute to the development of chronic epilepsy and recurrent seizures. Uncertainty on the role of these POSTER ABSTRACTS E355 GLIA different inflammatory cells depended on technical limitations in the discrimination of microglia from macrophages. For this reason, it is possible that the detrimental function that is currently attributed to microglia might be incorrect and should be ascribed to infiltrating macrophages. Methods: Both microglia and macrophages were acutely isolated from the hippocampi of control and pilocarpine-treated CD1 mice (24h and 96h after SE) and FACS sorted. Microglia were defined as CD11b+ CD45int Ly-6Cneg and infiltrated macrophages as CD11b+ CD45hi Ly-6Cpos. After sorting, qPCR and flow cytometry analysis were performed. Results: During epileptogenesis, microglia displayed a weakly immune-activated phenotype, based on the expression of MHCII, co-stimulatory molecule CD40 and pro-inflammatory gene IL-1. In contrast, infiltrated macrophages were strongly immune activated. Both cell types expressed high levels of the phagocytosis marker AXL. Conclusions: These data suggest that macrophages might be more detrimental than microglia during epileptogenesis.

2014 - A novel function for amniotic fluid: original or authentic? [Articolo su rivista]
Burns, Jorge Phillip Joaquin Sans; Dominici, Massimo
abstract

N/A

2014 - Bimodal Action of MSC in Regenerative Medicine [Monografia/Trattato scientifico]
Veronesi, Elena; Dominici, Massimo
abstract

Worldwide investigations on mesenchymal stromal/stem cells (MSC) have highlighted their regenerative potentials. Subsequent ex vivo & in vivo studies, with or without biomaterial combination, have explored MSC efficiency to repair damaged tissues and organs. This book investigates some of key factors on MSC biology & performance, such as the cell transportation procedures and the vasculogenesis after transplantation. One of the prerequisites for MSC introduction into clinic is their manipulation under good manufacturing practice (GMP) facilities that are often far from the operating theatre. Appropriate cell shipment conditions are poorly defined, therefore in this book we describe the impact of transportation on functional outcome focusing on bone regeneration. We then focused on an innovative approach of adipose tissue regeneration that reduced necrosis and significantly increased vasculogenesis compared with common surgical procedure for adipose tissue reconstruction based on autologous fat transfer. These data may be useful for anyone who studies MSC looking for their introduction into regenerative medicine approaches based on both their differentiation and secretory potentials

2014 - Bimodal Action of MSC in Rigenerative Medicine [Monografia/Trattato scientifico]
Veronesi, Elena; Dominici, Massimo
abstract

Worldwide investigations on mesenchymal stromal/stem cells (MSC) have highlighted their regenerative potentials. Subsequent ex vivo & in vivo studies, with or without biomaterial combination, have explored MSC efficiency to repair damaged tissues and organs. This book investigates some of key factors on MSC biology & performance, such as the cell transportation procedures and the vasculogenesis after transplantation. One of the prerequisites for MSC introduction into clinic is their manipulation under good manufacturing practice (GMP) facilities that are often far from the operating theatre. Appropriate cell shipment conditions are poorly defined, therefore in this book we describe the impact of transportation on functional outcome focusing on bone regeneration. We then focused on an innovative approach of adipose tissue regeneration that reduced necrosis and significantly increased vasculogenesis compared with common surgical procedure for adipose tissue reconstruction based on autologous fat transfer. These data may be useful for anyone who studies MSC looking for their introduction into regenerative medicine approaches based on both their differentiation and secretory potentials

2014 - In Vitro derivation of insulin-producing cells from 3D spheroids of human amniotic epithelial cells [Abstract in Rivista]
Okere, B; Alviano, F; Patianna, V; Costa, R; Predieri, Barbara; Quaglino, Daniela; Ricci, F; Dominici, Massimo; Paolucci, P; Bonsi, L; Iughetti, Lorenzo
abstract

Objective: Regenerative medicine and stem cell therapy represent a promising tool for the treatment of non-curable human diseases such as type 1 diabetes. Human amniotic epithelial cells (hAECs) from term placenta have attracted growing interest for their immunological properties, plasticity and availability which make them a promising tool for stem cell-based therapeutic applications. The aim of our study was to culture hAECs in serum-free condition preserving their phenotypic and genetic traits, evaluating their pancreatic differentiative potential in a 3D fashion. Methods: hAECs were isolated and cultured in standard serum-rich medium and serum-free optimized media. Flow Cytometry analysis was performed to evaluate stemness and specific epithelial cells markers. qPCR assessed stem cell and proliferation markers. We established a 3D culture procedure on basement membrane extracts to obtain spheroids mimicking the in vivo morphology and spatial organization of pancreatic islets. Results: The serum free protocol we developed proved to maintain hAECs stemness characteristics and confirmed their immunomodulatory activity on PHA stimulated PBMCs as revealed by proliferation assays. Immunofluorescence revealed the presence of pancreatic endocrine hormones and transmission electron microscopy (TEM) analysis showed a clear membrane-associated organization of secretory granules, consistent with beta cell ultrastructure in vivo. Conclusion: We accordingly propose the outcomes of this study as a novel contribution to the development of a future cell replacement therapy for type 1 diabetes.

2014 - Mesenchymal stem cell biodistribution, migration, and homing in vivo [Articolo su rivista]
Zhao, Weian; Phinney, Donald G.; Bonnet, Dominique; Dominici, Massimo; Krampera, Mauro
abstract

n/a

2014 - Pazopanib-Induced Heart Failure In A Metastastic Sarcoma Patient: Between Reversible Side Effect and Efficacy [Articolo su rivista]
Lucarini, Valaria; Madrigali, Stefano; Lugli, Roberto; Maur, Michela; Fontana, Annalisa; Masini, Cristina; Guarneri, Valentina; Conte, Piefranco; Dominici, Massimo
abstract

Introduction: Pazopanib, a multi-target tyrosine-kinase inhibitor (TKI), is a relatively novel anticancer agent registered for advanced renal cell carcinoma recently emerged in the setting of advanced soft-tissue sarcoma (STS). In the early clinical trials pazopanib has been very marginally linked to left ventricular ejection fraction (LVEF) dysfunction as, on contrary, reported for other anti-angiogenesis TKIs, such as Sunitinib and Sorafenib. Presentation of Case: We here present a case of severe, but reversible, congestive cardiac failure in a 37-year old Caucasian man affected by soft-tissue sarcoma during an efficacious treatment with pazopanib. Conclusion: Cardiac damage from novel TKI treatments is still an underestimated phenomenon. In our patient, pazopanib was the only treatment ensuring stability of disease and its discontinuation meant disease progression. Post-approval monitoring of novel TKIs should be taken into account by clinicians including a careful monitoring of LVEF and all symptoms suggestive of cardiac dysfunction, in particular for drugs potentially capable to change the natural history of still uncurable cancer.

2014 - Pazopanib-Induced Heart Failure in a Metastatic Sarcoma Patient: between Reversible Side Effect and Efficacy [Articolo su rivista]
Lucarini, Valeria; Madrigali, Stefano; Lugli, Roberta; Maur, Michela; Bertolini, Federica; Fontana, Annalisa; Masini, Cristina; Guarneri, Valentina; Conte, Pierfranco; Dominici, Massimo
abstract

Introduction: Pazopanib, a multi-target tyrosine-kinase inhibitor (TKI), is a relatively novel anticancer agent registered for advanced renal cell carcinoma recently emerged in the setting of advanced soft-tissue sarcoma (STS). In the early clinical trials pazopanib has been very marginally linked to left ventricular ejection fraction (LVEF) dysfunction as, on contrary, reported for other anti-angiogenesis TKIs, such as Sunitinib and Sorafenib. Presentation of Case: We here present a case of severe, but reversible, congestive cardiac failure in a 37-year old Caucasian man affected by soft-tissue sarcoma during an efficacious treatment with pazopanib. Conclusion: Cardiac damage from novel TKI treatments is still an underestimated phenomenon. In our patient, pazopanib was the only treatment ensuring stability of disease and its discontinuation meant disease progression. Post-approval monitoring of novel TKIs should be taken into account by clinicians including a careful monitoring of LVEF and all symptoms suggestive of cardiac dysfunction, in particular for drugs potentially capable to change the natural history of still uncurable cancer.

2014 - Quantitative expression of estrogen receptor on relapse biopsy for ER-positive breast cancer: prognostic impact [Articolo su rivista]
Dieci, Maria Vittoria; Piacentini, Federico; Dominici, Massimo; Omarini, Claudia; Goubar, Aicha; Ficarra, Guido; Conte, Pierfranco; Guarneri, Valentina
abstract

BACKGROUND: The aim of this study was to evaluate the prognostic impact of quantitative estrogen receptor (ER) expression at relapse for ER-positive breast cancer with ER-positive recurrence. PATIENTS AND METHODS: A total of 81 patients with ER-positive primary breast cancer and ER-positive paired recurrence were included. ER expression was evaluated as the percentage of tumor cells staining for ER under immunohistochemistry. Samples were defined as ER-high (ER>50%) or ER-low (ER≥10% and ≤50%). RESULTS: Quantitative ER expression on relapse biopsy was an independent prognostic factor for overall survival in multivariate analysis, both as a continuous (hazard ratio=0.8; 95% confidence interval=0.7-0.92, p=0.001) and as a categorical (ER-high vs. ER-low; hazard ratio=0.26; 95% confidence interval=0.11-0.59, p=0.001) variable. Patients whose status changed from ER-high (primary BC) to ER-low (relapse) had the poorest outcome, with a 10-year overall survival rate of 14%. CONCLUSION: Even in the case of maintenance of ER-positivity on primary and relapse of breast cancer, recurrence biopsy provides prognostic information.

2014 - Rare breast cancer subtypes: Histological, molecular, and clinical peculiarities [Articolo su rivista]
Dieci, Maria Vittoria; Orvieto, Enrico; Dominici, Massimo; Conte, Pierfranco; Guarneri, Valentina
abstract

Breast cancer encompasses a collection of different diseases characterized by different biological and pathological features, clinical presentation, response to treatments, clinical behavior, and outcome. On the basis of cell morphology, growth, and architecture patterns, breast cancer can be classified in up to 21 distinct histological types. Breast cancer special types, including the classic lobular invasive carcinoma, represent 25% of all breast cancers. The histological diversity of breast carcinomas has relevant prognostic implications. Indeed, the rare breast cancer group includes subtypes with very different prognoses, ranging from the tubular carcinoma, associated with an indolent clinical course, to metaplastic cancer, whose outcome is generally unfavorable. New approaches based on gene expression profiling allow the identification of molecularly defined breast cancer classes, with distinct biological features and clinical behavior. In clinical practice, immunohistochemical classification based on the expression of human epidermal growth factor receptor 2 and Ki67 is applied as a surrogate of the intrinsic molecular subtypes. However, the identification of intrinsic molecular subtypes were almost completely limited to the study of ductal invasive breast cancer. Moreover, some good-prognosis triple-negative histotypes, on the basis of gene expression profiling, can be classified among the poor-prognosis group. Therefore, histopathological classification remains a crucial component of breast cancer diagnosis. Special histologies can be very rare, and the majority of information on outcome and treatments derives from small series and case reports. As a consequence, clear recommendations about clinical management are still lacking. In this review, we summarize current knowledge about rare breast cancer histologies.

2014 - Role of mesenchymal stem cells in osteosarcoma and metabolic reprogramming of tumor cells [Articolo su rivista]
Bonuccelli, Gloria; Avnet, Sofia; Grisendi, Giulia; Salerno, Manuela; Granchi, Donatella; Dominici, Massimo; Kusuzaki, Katsuyuki; Baldini, Nicola
abstract

The tumor microenvironment plays an important role in cancer progression. Here, we focused on the role of reactive mesenchymal stem cells (MSC) in osteosarcoma (OS), and used human adipose MSC and a panel of OS cell lines (Saos-2, HOS, and 143B) to investigate the mutual effect of normal-cancer cell metabolic programming. Our results showed that MSC are driven by oxidative stress induced by OS cells to undergo Warburg metabolism, with increased lactate production. Therefore, we analyzed the expression of lactate monocarboxylate transporters. By real time PCR and immunofluorescence, in MSC we detected the expression of MCT-4, the transporter for lactate efflux, whereas MCT-1, responsible for lactate uptake, was expressed in OS cells. In agreement, silencing of MCT-1 by siRNA significantly affected the ATP production in OS cancer cells. Thus, cancer cells directly increase their mitochondrial biogenesis using this energy-rich metabolite that is abundantly provided by MSC as an effect of the altered microenvironmental conditions induced by OS cells. We also showed that lactate produced by MSC promotes the migratory ability of OS cells. These data provide novel information to be exploited for cancer therapies targeting the mutual metabolic reprogramming of cancer cells and their stroma.

2014 - Staminali per la cura del tumore del pancreas: tra evidenze scienti che e possibile trasferimento clinico [Articolo su rivista]
Golinelli, Giulia; Grisendi, Giulia; Spano, Carlotta; Rossignoli, Filippo; Dominici, Massimo
abstract

Una nuova promessa terapeutica arriva dall'utilizzo di cellule umane ca- paci di indurre la morte delle cellule tumorali tramite il rilascio di composti tossici.

2014 - Suppression of invasion and metastasis of triple-negative breast cancer lines by pharmacological or genetic inhibition of slug activity [Articolo su rivista]
Ferrari Amorotti, Giovanna; Chiodoni, Claudia; Shen, Fei; Cattelani, Sara; Soliera, Angela Rachele; Manzotti, Gloria; Grisendi, Giulia; Dominici, Massimo; Rivasi, Francesco; Colombo, Mario Paolo; Fatatis, Alessandro; Calabretta, Bruno
abstract

Most triple-negative breast cancers (TNBCs) exhibit gene expression patterns associated with epithelial-to-mesenchymal transition (EMT), a feature that correlates with a propensity for metastatic spread. Overexpression of the EMT regulator Slug is detected in basal and mesenchymal-type TNBCs and is associated with reduced E-cadherin expression and aggressive disease. The effects of Slug depend, in part, on the interaction of its N-terminal SNAG repressor domain with the chromatin-modifying protein lysine demethylase 1 (LSD1); thus, we investigated whether tranylcypromine [also known as trans-2-phenylcyclopropylamine hydrochloride (PCPA) or Parnate], an inhibitor of LSD1 that blocks its interaction with Slug, suppresses the migration, invasion, and metastatic spread of TNBC cell lines. We show here that PCPA treatment induces the expression of E-cadherin and other epithelial markers and markedly suppresses migration and invasion of TNBC cell lines MDA-MB-231 and BT-549. These effects were phenocopied by Slug or LSD1 silencing. In two models of orthotopic breast cancer, PCPA treatment reduced local tumor growth and the number of lung metastases. In mice injected directly in the blood circulation with MDA-MB-231 cells, PCPA treatment or Slug silencing markedly inhibited bone metastases but had no effect on lung infiltration. Thus, blocking Slug activity may suppress the metastatic spread of TNBC and, perhaps, specifically inhibit homing/colonization to the bone.

2014 - Surrounding pancreatic adenocarcinoma by killer mesenchymal stromal/stem cells [Articolo su rivista]
Golinelli, Giulia; Grisendi, Giulia; Spano, Maria Carlotta; Dominici, Massimo
abstract

N/A

2014 - The puzzling situation of hospital exemption for advanced therapy medicinal products in Europe and stakeholders' concerns [Articolo su rivista]
Cuende, Natividad; Boniface, Christelle; Bravery, Christopher; Forte, Miguel; Giordano, Rosaria; Hildebrandt, Martin; Izeta, Ander; Dominici, Massimo
abstract

N/A

2014 - Transportation conditions for prompt use of Ex Vivo expanded and freshly harvested clinical-grade bone marrow mesenchymal stromal/stem cells for bone regeneration [Articolo su rivista]
Veronesi, Elena; Murgia, Alba; Caselli, Anna; Grisendi, Giulia; Piccinno, MARIA SERENA; Rasini, Valeria; Giordano, Rosaria; Montemurro, Tiziana; Bourin, Philippe; Sensebé, Luc; Rojewski, Markus T.; Schrezenmeier, Hubert; Layrolle, Pierre; Ginebra, Maria Pau; Panaitescu, Carmen Bunu; Gómez Barrena, Enrique; Catani, Fabio; Paolucci, Paolo; Burns, Jorge Phillip Joaquin Sans; Dominici, Massimo
abstract

Successful preliminary studies have encouraged a more translational phase for stem cell research. Nevertheless, advances in the culture of human bone marrow-derived mesenchymal stromal/stem cells (hBM-MSC) and osteoconductive qualities of combined biomaterials can be undermined if necessary cell transportation procedures prove unviable. We aimed at evaluating the effect of transportation conditions on cell function, including the ability to form bone in vivo, using procedures suited to clinical application. hBM-MSC expanded in current Good Manufacturing Practice (cGMP) facilities (cGMP-hBM-MSC) to numbers suitable for therapy were transported overnight within syringes and subsequently tested for viability. Scaled-down experiments mimicking shipment for 18 h at 4°C tested the influence of three different clinical-grade transportation buffers (0.9\% saline alone or with 4\% human serum albumin [HSA] from two independent sources) compared with cell maintenance medium. Cell viability after shipment was &gt;80\% in all cases, enabling evaluation of (1) adhesion to plastic flasks and hydroxyapatite tricalcium phosphate osteoconductive biomaterial (HA/β-TCP 3D scaffold); (2) proliferation rate; (3) ex vivo osteogenic differentiation in contexts of 2D monolayers on plastic and 3D HA/β-TCP scaffolds; and (4) in vivo ectopic bone formation after subcutaneous implantation of cells with HA/β-TCP scaffold into NOD/SCID mice. Von Kossa staining was used to assess ex vivo osteogenic differentiation in 3D cultures, providing a quantifiable test of 3D biomineralization ex vivo as a rapid, cost-effective potency assay. Near-equivalent capacities for cell survival, proliferation, and osteogenic differentiation were found for all transportation buffers. Moreover, cGMP-hBM-MSC transported from a production facility under clinical-grade conditions of 4\% HSA in 0.9\% saline to a destination 18 h away showed prompt adhesion to HA/β-TCP 3D scaffold and subsequent in vivo bone formation. A successfully validated transportation protocol extends the applicability of fresh stem cells involving multicentric trials for regenerative medicine.

2013 - Adipose stromal/stem cells assist fat transplantation reducing necrosis and increasing graft performance. [Articolo su rivista]
PICCINNO, MARIA SERENA; VERONESI, Elena; LOSCHI, Pietro; M., Pignatti; MURGIA, ALBA; GRISENDI, Giulia; I., Castelli; D., Bernabei; CANDINI, Olivia; P., Conte; PAOLUCCI, Paolo; E. M., Horwitz; DE SANTIS, Giorgio; IUGHETTI, Lorenzo; DOMINICI, Massimo
abstract

Autologous fat transfer (AFT) is a procedure for adipose tissue (AT) repair after trauma, burns, post-tumor resections and lipodystrophies still negatively impacted by the lack of graft persistence. The reasons behind this poor outcome are unclear and seem to involve damages in either harvested/transplanted mature adipocytes or on their mesenchymal progenitors, namely adipose stromal/stem cells (ASC), and due to post-transplant AT apoptosis and involution. A rabbit subcutaneous AT regeneration model was here developed to first evaluate graft quality at different times after implant focusing on related parameters, such as necrosis and vasculogenesis. Standard AFT was compared with a strategy where purified autologous ASC, combined with hyaluronic acid (HA), assisted AFT. Five million of autologous ex vivo isolated CD29+, CD90+, CD49e+ ASC, loaded into HA, enriched 1&nbsp;ml of AT generating an early significant protective effect in reducing AFT necrosis and increasing vasculogenesis with a preservation of transplanted AT architecture. This beneficial impact of ASC assisted AFT was then confirmed at three months with a robust lipopreservation and no signs of cellular transformation. By a novel ASC assisted AFT approach we ensure a reduction in early cell death favoring an enduring graft performance possibly for a more stable benefit in patients.

2013 - Delayed marrow infusion in mice enhances hematopoietic and osteopoietic engraftment by facilitating transient expansion of the osteoblastic niche [Articolo su rivista]
Marino, Roberta; Otsuru, Satoru; Hofmann, Ted J.; Olson, Timothy S.; Rasini, Valeria; Veronesi, Elena; Boyd, Kelli; Gaber, Mostafa Waleed; Martinez, Caridad; Paolucci, Paolo; Dominici, Massimo; Horwitz, Edwin Mark
abstract

Transplantation of bone marrow cells leads to engraftment of osteopoietic and hematopoietic progenitors. We sought to determine whether the recently described transient expansion of the host osteoblastic niche after marrow radioablation promotes engraftment of both osteopoietic and hematopoietic progenitor cells. Mice infused with marrow cells 24&nbsp;hours after total body irradiation (TBI) demonstrated significantly greater osteopoietic and hematopoietic progenitor chimerism than did mice infused at 30&nbsp;minutes or 6&nbsp;hours. Irradiated mice with a lead shield over 1 hind limb showed greater hematopoietic chimerism in the irradiated limb than in the shielded limb at both the 6- and 24-hour intervals. By contrast, the osteopoietic chimerism was essentially equal in the 2 limbs at each of these intervals, although it significantly increased when cells were infused 24&nbsp;hours compared with 6&nbsp;hours after TBI. Similarly, the number of donor phenotypic long-term hematopoietic stem cells was equivalent in the irradiated and shielded limbs after each irradiation-to-infusion interval but was significantly increased at the 24-hour interval. Our findings indicate that a 24-hour delay in marrow cell infusion after TBI facilitates expansion of the endosteal osteoblastic niche, leading to enhanced osteopoietic and hematopoietic engraftment.

2013 - IGF-1-mediated osteoblastic niche expansion enhances long-term hematopoietic stem cell engraftment after murine bone marrow transplantation [Articolo su rivista]
Caselli, Anna; Olson, Timothy S.; Otsuru, Satoru; Chen, Xiaohua; Hofmann, Ted J.; Nah, Hyun Duck; Grisendi, Giulia; Paolucci, Paolo; Dominici, Massimo; Horwitz, Edwin Mark
abstract

The efficiency of hematopoietic stem cell (HSC) engraftment after bone marrow (BM) transplantation depends largely on the capacity of the marrow microenvironment to accept the transplanted cells. While radioablation of BM damages osteoblastic stem cell niches, little is known about their restoration and mechanisms governing their receptivity to engraft transplanted HSCs. We previously reported rapid restoration and profound expansion of the marrow endosteal microenvironment in response to marrow radioablation. Here, we show that this reorganization represents proliferation of mature endosteal osteoblasts which seem to arise from a small subset of high-proliferative, relatively radio-resistant endosteal cells. Multiple layers of osteoblasts form along the endosteal surface within 48 hours after total-body irradiation, concomitant with a peak in marrow cytokine expression. This niche reorganization fosters homing of the transplanted hematopoietic cells to the host marrow space and engraftment of long-term (LT)-HSC. Inhibition of insulin-like growth factor (IGF)-1-receptor tyrosine kinase signaling abrogates endosteal osteoblast proliferation and donor HSC engraftment, suggesting that the cytokine IGF-1 is a crucial mediator of endosteal niche reorganization and consequently donor HSC engraftment. Further understanding of this novel mechanism of IGF-1-dependent osteoblastic niche expansion and HSC engraftment may yield clinical applications for improving engraftment efficiency after clinical HSC transplantation.

2013 - Improved isolation and expansion of bone marrow mesenchymal stromal cells using a novel marrow filter device [Articolo su rivista]
Otsuru, Satoru; Hofmann, Ted J.; Olson, Timothy S.; Dominici, Massimo; Horwitz, Edwin Mark
abstract

Background aims. Mesenchymal stromal cells (MSCs) have been studied as cell therapy to treat a vast array of diseases. In clinical MSC production, the isolated cells must undergo extensive ex vivo expansion to obtain a sufficient dose of MSCs for the investigational treatment. However, extended tissue culture is fraught with potential hazards, including contamination and most worrisome, malignant transformation. Moreover, changes of gene expression with prolonged culture may alter the therapeutic potential of the cells. Therefore, increasing the recovery of MSCs from the freshly harvested bone marrow allowing for less ex vivo expansion would represent a major advance in MSC therapy. Methods. Human bone marrow cells from 8 healthy donors were processed using a marrow filter device and, in parallel, using buoyant density centrifugation by two independent investigators. The initial nucleated cell recovery as well as the final yield, immunophenotype, and trilineage differentiation potential of passage 2 MSCs were examined. Results. The marrow filter device generated significantly greater initial cell recovery requiring less investigator time and resulted in approximately 2.5-fold more MSCs after passage 2. The immunophenotype and differentiation potential of MSCs isolated using the two methods was equivalent and consistent the defining criteria. The two independent investigators generated comparable results. Conclusions. This novel filter device is a fast, efficient, and reliable system to isolate MSCs and should greatly expedite preclinical and clinical investigations of MSC therapy.

2013 - Isolation, characterization, and transduction of endometrial decidual tissue multipotent mesenchymal stromal/stem cells from menstrual blood [Articolo su rivista]
Rossignoli, Filippo; Caselli, Anna; Grisendi, Giulia; Piccinno, MARIA SERENA; Burns, Jorge Phillip Joaquin Sans; Murgia, Alba; Veronesi, Elena; Loschi, Pietro; Masini, Cristina; Conte, Pierfranco; Paolucci, Paolo; Horwiz, Edwin M.; Dominici, Massimo
abstract

Mesenchymal stromal/stem cells (MSCs) reveal progenitor cells-like features including proliferation and differentiation capacities. One of the most historically recognized sources of MSC has been the bone marrow, while other sources recently include adipose tissue, teeth, bone, muscle, placenta, liver, pancreas, umbilical cord, and cord blood. Frequently, progenitor isolation requires traumatic procedures that are poorly feasible and associated with patient discomfort. In the attempt to identify a more approachable MSC source, we focused on endometrial decidual tissue (EDT) found within menstrual blood. Based also on recent literature findings, we hypothesized that EDT may contain heterogeneous populations including some having MSC-like features. Thus, we here sought to isolate EDT-MSC processing menstrual samples from multiple donors. Cytofluorimetric analyses revealed that resulting adherent cells were expressing mesenchymal surface markers, including CD56, CD73, CD90, CD105 and CD146, and pluripotency markers such as SSEA-4. Moreover, EDT-MSC showed a robust clonogenic potential and could be largely expanded in vitro as fibroblastoid elements. In addition, differentiation assays drove these cells towards osteogenic, adipogenic, and chondrogenic lineages. Finally, for the first time, we were able to gene modify these progenitors by a retroviral vector carrying the green fluorescent protein. From these data, we suggest that EDT-MSC could represent a new promising tool having potential within cell and gene therapy applications.

2013 - Megakaryocytes promote murine osteoblastic HSC niche expansion and stem cell engraftment after radioablative conditioning [Articolo su rivista]
Olson, Timothy S; Caselli, Anna; Otsuru, Satoru; Hofmann, Ted J; Williams, Richard; Paolucci, Paolo; Dominici, Massimo; Horwitz, EDWIN MARK
abstract

Successful hematopoietic stem cell (HSC) transplantation requires donor HSC engraftment within specialized bone marrow microenvironments known as HSC niches. We have previously reported a profound remodeling of the endosteal osteoblastic HSC niche after total body irradiation (TBI), defined as relocalization of surviving megakaryocytes to the niche site and marked expansion of endosteal osteoblasts. We now demonstrate that host megakaryocytes function critically in expansion of the endosteal niche after preparative radioablation and in the engraftment of donor HSC. We show that TBI-induced migration of megakaryocytes to the endosteal niche depends on thrombopoietin signaling through the c-MPL receptor on megakaryocytes, as well as CD41 integrin-mediated adhesion. Moreover, niche osteoblast proliferation post-TBI required megakaryocyte-secreted platelet-derived growth factor-BB. Furthermore, blockade of c-MPL-dependent megakaryocyte migration and function after TBI resulted in a significant decrease in donor HSC engraftment in primary and competitive secondary transplantation assays. Finally, we administered thrombopoietin to mice beginning 5 days before marrow radioablation and ending 24 hours before transplant to enhance megakaryocyte function post-TBI, and found that this strategy significantly enhanced donor HSC engraftment, providing a rationale for improving hematopoietic recovery and perhaps overall outcome after clinical HSC transplantation.

2013 - Mesenchymal Stem Cells - Basics and Clinical Application II [Monografia/Trattato scientifico]
Weyand, Birgit; Dominici, Massimo; Hass, Ralf; Jacobs, Roland; Kasper, Cornelia
abstract

Mesenchymal stem cells (MSC) represent one of the most interesting progenitors to date due to their biodiverse functionalities. Among the fascinating multiple properties of MSC are their supportive roles in wound healing and in the regeneration of damaged tissues and organs. This implies the capacity of MSC to migrate towards injured tissue, to undergo differentiation, to modulate the activation of immune cells and to activate endothelial cells contributing to both angiogenesis and neo-vascularisation. Together with their self-renewal capability, the maintenance of stem cell homeostasis, the release of several bioactive compounds like chemokines, cytokines, micro RNAs and exosomes, MSC can be certainly considered as cellular all-round supporters. These multi-functional MSC properties are highlighted in the present book. While some chapters are focused on differentiation capacities of MSC, even beyond the more consolidated mesodermal lineages, others provide novel insights on the stimulatory signals involved in MSC survival and trafficking. Moreover, MSC role in regulating cancer progression for novel therapeutics is assessed. In-depth molecular analyses of MSC functions are also covered additionally including initial characterizations of distinct proteomic patterns that are specific for discrete MSC populations. Technical aspects for the isolation and enrichment of selected MSC populations are here additionally addressed in relationship to new cell sources and in the attempt to open new therapeutic platforms for potential clinical applications. Although MSC research is progressively bridging to more consolidated clinical applications, it still represents a dynamically developing field,where a variety of intriguing aspects remain to be addressed. We feel this present book represents a comprehensive summary gathering a panel of up-to-date articles which combine the diverse MSC biological functionalities and their potential in translational cell therapy as highlighted from different angles with a wide interdisciplinary view.

2013 - Mesenchymal stem cells: Basics and clinical application I [Curatela]
Weyand, Birgit; Dominici, Massimo; Hass, Ralf; Jacobs, Roland; Kasper, Cornelia
abstract

Mesenchymal stem cells (MSC) represent one of the most interesting progenitors to date, due to their biodiverse functionalities. Among the fascinating multiple properties of MSC are their supportive roles in wound healing and in the regeneration of damaged tissues and organs. This implies the capacity of MSC to migrate towards injured tissue, to undergo differentiation, to modulate the activation of immune cells and to activate endothelial cells contributing to both angiogenesis and neo-vascularisation. Together with their self-renewal capability, the maintenance of stem cell homeostasis, the release of several bioactive compounds like chemokines, cytokines, micro RNAs and exosomes, MSC can be certainly considered as cellular all-round supporters. These multi-functional MSC properties are highlighted in the present volume. While some chapters are focused on differentiation capacities of MSC, even beyond the more consolidated mesodermal lineages, others provide novel insights into the stimulatory signals involved in MSC survival and trafficking. Moreover, the MSC role in regulating cancer progression for novel therapeutics is assessed. In-depth molecular analyses of MSC functions are also covered, additionally including initial characterisations of distinct proteomic patterns that are specific for discrete MSC populations. Technical aspects for the isolation and enrichment of selected MSC populations are here additionally addressed in relationship to new cell sources and in the attempt to open new therapeutic platforms for potential clinical applications. Although MSC research is progressively bridging to more consolidated clinical applications, it still represents a dynamically developing field, where a variety of intriguing aspects remain to be addressed. We feel this volume represents a comprehensive summary gathering a panel of up-to-date articles which combine the diverse MSC biological functionalities and their potential in translational cell therapy, as highlighted from different angles with a broad interdisciplinary perspective.

2013 - Mesenchymal stromal/stem cells markers in the human bone marrow [Articolo su rivista]
Rasini, Valeria; Dominici, Massimo; Kluba, Torsten; Siegel, Georg; Lusenti, Giulia; Northoff, Hinnak; Horwitz, EDWIN MARK; Schäfer, Richard
abstract

Mesenchymal stromal/stem cells (MSCs) can be isolated from human bone marrow (BM), expanded ex&nbsp;vivo and identified via numerous surface antigens. Despite the importance of these cells in regenerative therapy programs, it is unclear whether the cell membrane signature defining MSC preparations ex vivo is determined during culture or may reflect an in vivo counterpart. BM-MSC phenotype in vivo requires further investigation.To characterize cells in their natural BM environment, we performed multi-parametric immunohistochemistry on trabecular bone biopsy specimens from multiple donors and described cells by different morphology and micro-anatomic localization in relationship to a precise pattern of MSC antigen expression.Microscopically examined high-power field marrow sections revealed an overlapping in vivo expression of antigens characterizing ex vivo expanded BM-MSCs, including CD10, CD73, CD140b, CD146, GD2 and CD271. Expanding this panel to proteins associated with pluripotency, such as Oct4, Nanog and SSEA-4, we were able to identify different cellular populations in the human trabecular bone and BM expressing different progenitor cell markers.Targeting several multipotency and pluripotency markers, we found that the BM contains identifiable and distinct progenitor cells further justifying their introduction for a wide range of applications in regenerative medicine.

2013 - MSC and Tumors: Homing, Differentiation and Secretion Influence The Therapeutic Potentials [Capitolo/Saggio]
D'Souza, Naomi; Burns, Jorge Sans; Grisendi, Giulia; Candini, Olivia; Veronesi, Elena; Piccinno, Serena; Horwitz, EDWIN MARK; Paolucci, Paolo; Conte, Pierfranco; Dominici, Massimo
abstract

Mesenchymal stromal/stem cells (MSC) are adult multipotent progenitors with fibroblast-like morphology able to differentiate into adipocytic, osteogenic, chondrogenic, and myogenic lineages. Due to these properties, MSC have been studied and introduced as therapeutics in regenerative medicine. Preliminary studies have also shown a possible involvement of MSC as precursors of cellular elements within tumor microenvironments, in particular tumor-associated fibroblasts (TAF). Among a number of different possible origins, TAF may originate from a pool of circulating progenitors from bone marrow or adipose tissue-derived MSC. There is growing evidence to corroborate that cells immunophenotypically defined as MSC are able to reside as TAF influencing the tumor microenvironment in a potentially bi-phasic and obscure manner: either promoting or inhibiting growth depending on tumor context and MSC sources. Here we focus on relationships between the tumor microenvironment, cancer cells, and MSC, analyzing their diverse ability to influence neoplastic development. Associated activities include MSC homing driven by the secretion of various mediators, differentiation towards TAF phenotypes, and reciprocal interactions with the tumor cells. These are reviewed here with the aim of understanding the biological functions of MSC that can be exploited for innovative cancer therapy.

2013 - Proinflammatory stimuli induce galectin-9 in human mesenchymal stromal cells to suppress T-cell proliferation [Articolo su rivista]
Gieseke, Friederike; Kruchen, Anne; Tzaribachev, Nikolay; Bentzien, Frank; Dominici, Massimo; Müller, Ingo
abstract

Human multipotent mesenchymal stromal cells (MSCs) are clinically applied to treat autoimmune diseases and graft-versus-host disease due to their immunomodulatory properties. Several molecules have been identified to mediate these effects, including constitutively expressed galectin-1. However, there are indications in the literature that MSCs exert enhanced immunosuppressive functions after interaction with an inflammatory environment. Therefore, we analyzed how inflammatory stimuli influence the expression of the galectin network in MSCs and functionally tested the relevance for the immunomodulatory effects of MSCs. We found that galectin-9 was strongly induced in MSCs upon interaction with activated PBMCs. Proinflammatory cytokines, such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α), and also ligands of the Toll-like receptors (TLRs) TLR2, TLR3, and TLR4 elicited similar induction of galectin-9 in activated PBMCs. Galectin-9 was not only upregulated intracellularly, but also released by MSCs in significant amounts into the supernatant after exposure to proinflammatory stimuli. In proliferation assays, MSCs with a galectin-9 knockdown lost a significant portion of their antiproliferative effects on T cells. In conclusion, we found that unlike constitutively expressed galectin-1, galectin-9 is induced by several proinflammatory stimuli and released by MSCs. Thus, galectin-9 contributes to the inducible immunomodulatory functions of MSCs.

2013 - Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: A joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT) [Articolo su rivista]
Bourin, Philippe; Bunnell, Bruce A.; Casteilla, Louis; Dominici, Massimo; Katz, Adam J.; March, Keith L.; Redl, Heinz; Rubin, J. Peter; Yoshimura, Kotaro; Gimble, Jeffrey M.
abstract

Adipose tissue is a rich and very convenient source of cells for regenerative medicine therapeutic approaches. However, a characterization of the population of adipose-derived stromal and stem cells (ASCs) with the greatest therapeutic potential remains unclear. Under the authority of International Federation of Adipose Therapeutics and International Society for Cellular Therapy, this paper sets out to establish minimal definitions of stromal cells both as uncultured stromal vascular fraction (SVF) and as an adherent stromal/stem cells population.Phenotypic and functional criteria for the identification of adipose-derived cells were drawn from the literature.In the SVF, cells are identified phenotypically by the following markers: CD45-CD235a-CD31-CD34+. Added value may be provided by both a viability marker and the following surface antigens: CD13, CD73, CD90 and CD105. The fibroblastoid colony-forming unit assay permits the evaluation of progenitor frequency in the SVF population. In culture, ASCs retain markers in common with other mesenchymal stromal/stem cells (MSCs), including CD90, CD73, CD105, and CD44 and remain negative for CD45 and CD31. They can be distinguished from bone-marrow-derived MSCs by their positivity for CD36 and negativity for CD106. The CFU-F assay is recommended to calculate population doublings capacity of ASCs. The adipocytic, chondroblastic and osteoblastic differentiation assays serve to complete the cell identification and potency assessment in conjunction with a quantitative evaluation of the differentiation either biochemically or by reverse transcription polymerase chain reaction.The goal of this paper is to provide initial guidance for the scientific community working with adipose-derived cells and to facilitate development of international standards based on reproducible parameters.

2013 - Transplanted murine long-term repopulating hematopoietic cells can differentiate to osteoblasts in the marrow stem cell niche [Articolo su rivista]
Hofmann, Ted J.; Otsuru, Satoru; Marino, Roberta; Rasini, Valeria; Veronesi, Elena; Murgia, Alba; Lahti, Jill; Boyd, Kelli; Dominici, Massimo; Horwitz, Edwin Mark
abstract

Bone marrow transplantation (BMT) can give rise to donor-derived osteopoiesis in mice and humans; however, the source of this activity, whether a primitive osteoprogenitor or a transplantable marrow cell with dual hematopoietic and osteogenic potential, has eluded detection. To address this issue, we fractionated whole BM from mice according to cell surface immunophenotype and assayed the hematopoietic and osteopoietic potentials of the transplanted cells. Here, we show that a donor marrow cell capable of robust osteopoiesis possesses a surface phenotype of c-Kit(+) Lin(-) Sca-1(+) CD34(-/lo), identical to that of the long-term repopulating hematopoietic stem cell (LTR-HSC). Secondary BMT studies demonstrated that a single marrow cell able to contribute to hematopoietic reconstitution in primary recipients also drives robust osteopoiesis and LT hematopoiesis in secondary recipients. These findings indicate that LTR-HSC can give rise to progeny that differentiate to osteoblasts after BMT, suggesting a mechanism for prompt restoration of the osteoblastic HSC niche following BM injury, such as that induced by clinical BMT preparative regimens. An understanding of the mechanisms that regulate this differentiation potential may lead to novel treatments for disorders of bone as well as methods for preserving the integrity of endosteal hematopoietic niches.

2012 - Cardiorenal Syndrome Type 1 May Be Immunologically Mediated: A Pilot Evaluation of Monocyte Apoptosis. [Articolo su rivista]
G. M., Virzì; R., Torregrossa; D. N., Cruz; C. Y., Chionh; M., de Cal; S., S: Soni; Dominici, Massimo; G., Vescovo; M. H., Rosner; C., Ronco
abstract

Background: Cardiorenal syndrome (CRS) type 1 is characterized by a rapid worsening of cardiac function leading to acute kidney injury (AKI). An immune-mediated damage and alteration of immune response have been postulated as potential mechanisms involved in CRS type 1. In this pilot study, we examined the possible role of the immune-mediated mechanisms in the pathogenesis of this syndrome. The main objective was to analyze in vitro that plasma of CRS type 1 patients was able to trigger a response in monocytes resulting in apoptosis. The secondary aim was to evaluate TNF-α and IL-6 plasma levels of CRS type 1 patients. Methods: Fifteen patients with acute heart failure (AHF) and CRS type 1 were enrolled and 20 healthy volunteers without AHF or AKI were recruited as control group. Plasma from these two groups was incubated with monocytes and, subsequently, cell apoptosis was evaluated. In addition, the activity of caspase-8 was assessed after 24 h incubation. Quantitative determination of TNF-α and IL-6 levels was performed. Results: Plasma-induced apoptosis was significantly higher in CRS type 1 patients compared with healthy controls at 72 h (78 vs. 11%) and 96 h (81 vs. 11%). At 24 h, the activity of caspase-8 was significantly higher in monocytes incubated with plasma from the CRS type 1 group. TNF-α (2.39 vs. 28.49 pg/ml) and IL-6 (4.8 vs. 16.5 pg/ml) levels were significantly elevated in the CRS type 1 group (p &lt; 0.01). Conclusions: In conclusion, there is a defective regulation of monocyte apoptosis in CRS type 1 patients, and inflammatory pathways may have a central role in the pathogenesis of CRS type 1 and may be fundamental in damage to distant organs

2012 - Clinical perspectives of mesenchymal stem cells [Articolo su rivista]
Kramer, Jan; Dazzi, Francesco; Dominici, Massimo; Schlenke, Peter; Wagner, Wolfgang
abstract

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2012 - Discordance in receptor status between primary and recurrent breast cancer has a prognostic impact: a single Institution analysis [Articolo su rivista]
Dieci, Maria Vittoria; Barbieri, Elena; Piacentini, Federico; Ficarra, G; Bettelli, Stefania Raffaella; Dominici, Massimo; Conte, Pierfranco; Guarneri, Valentina
abstract

Background: Tumor phenotype may change during breast cancer progression. This study evaluates the prognostic impact of receptor discordance between paired primaries and recurrences.Patients and Methods: 139 patients underwent histological sampling of suspected breast cancer recurrence. All the pathology assessments (ER, PgR and HER2) on both primaries and confirmed recurrences were performed at the same laboratory. Results: A breast cancer recurrence was confirmed in 119 cases. Rates of discordance were 13.4%, 39% and 11.8% for ER, PgR and HER2, respectively. Ninety-two patients maintained the same tumor phenotype (i.e., the same hormone receptors and HER2 status) whereas 27 (22.7%) changed during progression. The loss of hormone receptor-positivity and the loss of HER2-positivity resulted in a worse post-recurrence survival (p=0.01 and p=0.008, respectively) and overall survival (p=0.06 and p=0.0002, respectively), compared to the corresponding concordant-positive cases. Tumor phenotype discordance was associated with worse post-recurrence and overall survival (p=0.006 and p=0.002, respectively); those cases who turned into triple-negative experienced the poorest outcome, respect to the concordant group (p=0.001, overall survival).Conclusions: We demonstrated for the first time an impact on overall survival of phenotype discordance between primary breast cancer and relapse. Among discordant cases, receptor-loss resulted the main determinant of poorer outcome.

2012 - ESF Exploratory Workshop on Mesenchymal Stem Cells. Around Mesenchymal Stem Cells: Dissection And Exploitation Of Secretory Activity Of MSC For Regenerative Medicine And Anticancer Therapies; Bologna (Italy) [Esposizione]
N., Baldini; Dominici, Massimo
abstract

Goal of the workshop is to establish collaborative research in the emerging field of cell-free therapy based on the paracrine activity of MSC. Participants will present state of the art of and new insights on specific fields. Workshop will define: 1) potential clinical application of cell-free therapies, 2) major advantages/limitations 3) gaps in current knowledge. Future activities include: 1) Identification of research issues for clinical translation, 2) Strategy development for joint collaborative research, 3) Education/research training networks, and 4) Preparation of a consensus paper as a preliminary step for grant application.

2012 - In vitro anti-myeloma activity of TRAIL-expressing adipose-derived mesenchymal stem cells. [Articolo su rivista]
S., Ciavarella; Grisendi, Giulia; Dominici, Massimo; M., Tucci; O., Brunetti; F., Dammacco; F., Silvesitris
abstract

Recently, genetically modified mesenchymal stem cells (MSCs) have beenexploited to deliver anti-cancer bio-drugs directly within the tumour mass.Here, we explored whether adipose-derived MSCs (AD-MSCs), engineeredto express the pro-apoptotic ligand TRAIL (also known as TNFSF10), killmultiple myeloma (MM) cells and migrate toward MM cells in vitro. DifferentMM cell lines were assessed for their sensitivity to recombinanthuman (rh) TRAIL alone and in combination with the proteasome inhibitorbortezomib, which was shown to enhance the effect of rhTRAIL.TRAIL+-AD-MSCs were co-cultured with bortezomib-pretreated MM cellsand their killing activity was evaluated in presence or absence of caspaseinhibition, whereas AD-MSC migration toward media conditioned by bothmyeloma cells and myeloma bone fragments was also investigated. Despitemoderate MM cell sensitivity to rhTRAIL, TRAIL+-AD-MSCs in combinationwith bortezomib significantly induced myeloma cell death. This effectwas associated with caspase-8 activation and abrogated by capsase inhibition.On the other hand, co-culture experiments were performed to evaluatewhether unmodified AD-MSCs affect myeloma cell growth in vitro.AD-MSCs appeared ineffective on myeloma cell growth and showed powerfulmigratory capacity toward MM cells in vitro. These data emphasize theanti-myeloma activity of TRAIL-engineered AD-MSCs and provide supportfor a future model of a cell-based approach against MM.

2012 - Inhibiting interactions of lysine demethylase LSD1 with Snail/Slug blocks cancer cell invasion. [Articolo su rivista]
Ferrari, Giovanna; Fragliasso, Valentina; Esteki, R; Prudente, Zelia; Soliera, Angela Rachele; Cattelani, Sara; Manzotti, Gloria; Grisendi, Giulia; Dominici, Massimo; Pieraccioli, M; Raschellà, G; Claudia, C; Colombo, Mp; Calabretta, Bruno
abstract

The process of epithelial-mesenchymal transition (EMT) which is required for cancer cell invasion is regulated by a family of E-box binding transcription repressors which include Snail (SNAI) and Slug (SNAI2). Snail appears to repress the expression of the EMT marker E-cadherin by epigenetic mechanisms dependent on the interaction of its N-terminal SNAG domain with chromatin modifying proteins including lysine specific demethylase 1 (LSD1/KDM1A). We assessed whether blocking Snail/Slug-LSD1 interaction by treatment with Parnate, an enzymatic inhibitor of LSD1, or TAT-SNAG, a cell-permeable peptide corresponding to the SNAG domain of Slug, suppresses the motility and invasiveness of cancer cells of different origin and genetic background. We show here that either treatment blocked Slug-dependent repression of the E-cadherin promoter and inhibited the motility and invasion of tumor cell lines without any effect on their proliferation. These effects correlated with induction of epithelial and repression of mesenchymal markers and were phenocopied by LSD1 or Slug down-regulation. Parnate treatment also inhibited bone marrow homing/engraftment of Slug-expressing K562 cells. Together, these studies support the concept that targeting Snail/Slug-dependent transcription repression complexes may lead to the development of novel drugs selectively inhibiting the invasive potential of cancer cells.

2012 - Predictors of human epidermal growth factor receptor 2 fluorescence in-situ hybridisation amplification in immunohistochemistry score 2+ infiltrating breast cancer: a single institution analysis. [Articolo su rivista]
Dieci, Maria Vittoria; Barbieri, Elena; Bettelli, Stefania Raffaella; Piacentini, Federico; Omarini, Claudia; Ficarra, G; Balduzzi, Sara; Dominici, Massimo; Conte, Pierfranco; Guarneri, Valentina
abstract

Eligibility for anti-human epidermal growth factor receptor 2 (HER2) treatments in breast cancer requires a correct HER2 status assessment. Testing guidelines recommend fluorescence in-situ hybridisation (FISH) for samples scored as 2+ by immunohistochemistry. This study investigates the correlation between pathological features and FISH amplification in HER2 2+ breast cancer cases.Methods: 480 HER2 2+ breast cancer samples were included. The association between tumour grade, hormone receptor status, proliferation index (Ki67) and FISH amplification, using both US Food and Drug Administration (ratio ≥2) and American Society of Clinical Oncologists/College of American Pathologists cut-offs (ratio >2.2) was evaluated.Results: 90.2% of the samples were hormone receptor positive. The median Ki67 value was 23.5%; 311 (64.8%) samples showed a Ki67 value of 15% or greater. Tumour grade was evaluable in 421 cases (87.7%), 268 (55.8%) being grade 3. FISH amplification rates were 27.5% (ratio ≥2.0) and 20.8% (ratio >2.2). Grade 3 tumours were more frequently amplified than grades 1-2 tumours: 34% versus 18% (ratio ≥2.0, p<0.001) and 27% versus 9% (ratio >2.2, p<0.001). Samples with Ki67 of 15% or greater showed higher amplification ratesthan low Ki67 samples: 31% versus 21% (ratio ≥2.0, p=0.022) and 25% versus 12% (ratio >2.2, p=0.003). The OR for FISH amplification was significant in the case of grade 3 and high Ki67 with both cut-offs.Conclusions: In this study, high tumour grade and high Ki67 significantly predicted FISH amplification in 480 HER2 2+breast cancer samples.

2012 - Sarcomas as a mise en abyme of Mesenchymal Stem Cells; exploiting interrelationships for cell mediated anticancer therapy [Articolo su rivista]
J. S., Burns; A., Safwat; Grisendi, Giulia; M., Kassem; Dominici, Massimo
abstract

Mise en abyme meaning "placed into abyss or infinite recurrence" is an apt paradigm for the relentless growth of sarcoma cells. Its alternative meaning, "self-reflexive embedding" fits the central role attributed to cancer stem cells (CSCs). Diversely sourced and defined, mesenchymal stem cells (MSCs) may be the cells of sarcoma origin, evolve a CSC phenotype and/or contribute to tumor growth through inherent qualities for homing, neovascularization, paracrine cross-feeding, microvesicle secretion, cell fusion, entosis and immune modulation. Exploiting these qualities, MSC expressing modified forms of the TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) are being developed to complement more conventional radiation and chemotherapy.

2012 - Transplanted bone marrow mononuclear cells and MSCs impart clinical benefit to children with osteogenesis imperfecta through different mechanisms [Articolo su rivista]
S., Otsuru; P. L., Gordon; K., Shimono; R., Jethva; R., Marino; C. L., Phillips; T. J., Hofmann; Veronesi, Elena; Dominici, Massimo; M., Iwamoto; E. M., Horwitz
abstract

Transplantation of whole bone marrow (BMT) as well as ex vivo-expanded mesenchymal stromal cells (MSCs) leads to striking clinical benefits in children with osteogenesis imperfecta (OI); however, the underlying mechanism of these cell therapies has not been elucidated. Here, we show that non-(plastic)-adherent bone marrow cells (NABMCs) are more potent osteoprogenitors than MSCs in mice. Translating these findings to the clinic, a T cell-depleted marrow mononuclear cell boost (&gt; 99.99% NABMC) given to children with OI who had previously undergone BMT resulted in marked growth acceleration in a subset of patients, unambiguously indicating the therapeutic potential of bone marrow cells for these patients. Then, in a murine model of OI, we demonstrated that as the donor NABMCs differentiate to osteoblasts, they contribute normal collagen to the bone matrix. In contrast, MSCs do not substantially engraft in bone, but secrete a soluble mediator that indirectly stimulates growth, data which provide the underlying mechanism of our prior clinical trial of MSC therapy for children with OI. Collectively, our data indicate that both NABMCs and MSCs constitute effective cell therapy for OI, but exert their clinical impact by different, complementary mechanisms. The study is registered at www.clinicaltrials.gov as NCT00187018.

2012 - 4th Annual Meeting of The Forum of Italian Researchers on Mesenchymal and Stromal cells (FIRST) Pavia (Italy). [Esposizione]
M., Gnecchi; Dominici, Massimo; R., Giordano; L., Lazzari
abstract

This year the historical frame of Collegio Borromeo in Pavia hosted the participants of the 4th FIRST meeting, 122 delegates from 3 European Countries, who had the occasion to debate the most intriguing and recent developments in mesenchymal stem cell with some of the principal experts in the field. Surrounded by the fresco paintings of the main episodes of Saint Charles Borromeo life and warmly welcomed by the rector of the Collegio, the speakers discussed the futuristic aspects of mesenchymal stem cell research. Special attention has been given to the strategies to better exploit mesenchymal stem cell potential against the well-known killers of our era, namely heart diseases and cancer. A further step in heart disease treatment has been presented by Paolo Madeddu. He showed how a population of CD34+ cells, located around the aorta adventitia with typical pericyte, mesenchymal and stemness markers, and a robust regenerative potential in a mouse model of hind limb ischemia, can be also successfully isolated from saphenous vein leftovers of patients undergoing coronary artery bypass graft surgery and sustained long-term improvements in a mouse model of myocardial infarction. Carlo Ventura illustrated, in a multidisciplinary context, important chemical and physical strategies to empower mesenchymal stem cell pluripotentiality. Endorphin peptides, hyaluronan esters of butyric and retinoic acids, extremely low frequency magnetic fields (ELF-MF) and RF energy from Wi-Fi technologies are some of the new tools that can be applied in order to enhance the regenerative potential of mesenchymal stem cells, especially towards cardiomyocyte differentiation, as shown by his numerous in vitro and in vivo experiments. The dark side of mesenchymal stem cells has been explored by Paola Chiarugi who clearly summarized her experiments addressed to explore the role of cancer-associated fibroblasts (CAFs) in epithelial mesenchymal transition (EMT) of prostate carcinoma cells. Interleukine-6, metalloproteases (MMP)-2 and 9, cycloxygenase-2, nuclear factor-?B and hypoxia inducible factor-1 have been identified as the key-players in the reciprocal interplay between prostate carcinoma cells and CAFs. What happens to mesenchymal stem cells and tumor microenvironment when a patient receives chemotherapy? Which is the role of mesenchymal stem cells in establishing and maintaining chemoresistence? Emile Voest gave an important contribution to answer these questions. He showed how, after exposure to commonly used chemotherapeutics such as cisplatin, oxaliplatin and carboplatin, MSC become activated to secrete very specific fatty acids, the so-called platinum induced fatty acids (PIFA), with important in vivo effect on chemoresistance. Inhibitors of the enzymes involved in PIFA synthesis could therefore prevent PIFA-mediated chemoresistance but also enhance the anti-tumor activity of chemotherapy in vivo. A nano-scale view on the possibility to bioengineer mesenchymal stem cells: the lecture by Jeffrey M. Karp opened a new perspective on the methods to enhance the homing of systematically infused mesenchymal stem cells through cell surface engineering and to orchestrate mesenchymal stem cell-mediated drug delivery with an intracellular depot of phenotype altering agents. Moreover, he showed how cell surface sensors can be used to detect signals within the cellular nano-environment with unprecedented spatial and temporal resolution thus providing a useful tool for monitoring the cell secretome after transplantation. From the future to the past and back to the future: the lecture from Bruno Péault beautifully summarized the fascinating ancestry of mesenchymal stem cells from the potential of perivascular stem cells through their mechanisms of control in situ and towards clinical application of adipose tissue derived autologous perivascular cells in bone repair. And then, biomaterials: Nicol

2011 - An adoptive immuno-gene therapy approach targeting neuroblastoma. [Abstract in Rivista]
Caldrer, Sara; Spano, Maria Carlotta; Grisendi, Giulia; Dominici, Massimo; Paolucci, Paolo
abstract

Although dose intensification of chemotherapy has increased initial response rates in neuroblastoma (NB), this effect has not translated into durable remissions in patients with disseminated disease. Immunotherapy may be an alternative approach following cytoreductive chemotherapy providing a long-term disease control (I). We have engineered human cytotoxic T lymphocytes (CTL) to express a chimeric receptor (CR) for the specific recognition of GD2 that is expressed in NB (II). Chimeric lymphocytes (CL) have been previously introduced towards several tumors (III). Thus, we have generated a novel CR consisting of a single-chain variable domain of an anti-GD2 antibody in conjunction with a signal domain of 4-1BB molecule (CD137). Retroviral particles encoding for the flag gene only (GFP), for the whole CR and for a truncated receptor were generated. Peripheral blood cells from buffy-coat were specifically stimulated and transuced obtaining a cytotoxic population of T cells having high level of transduction (50±25%). Co-culture experiments were then performed against NB cell lines. In short (4h) co-culture experiments (T:E=1:20) by Cr51 release assay, CLs exerted a powerful and specific cytotoxicity against GD2-positive NB cells, compared with truncated controls (35±5% and 10,8±4% ; p<0,05). Moreover, in a longer co-culture assay (5 days) in vitro at higher ratio (T:E =1:5), using as read-out FACS analyses targeting GD2 expressing cells, we observed a significant decrease (from 92±3% to 19±5% ; p<0.05) of the NB cells compared with controls (from 92±3% to 76,5±4% %; p<0.05). These preliminary data in vitro suggest that CL against GD2-positive NB cells may represent a powerful new tool for T-cell therapy in patients with GD2-positive NB or other GD2-positive malignancies.

2011 - Bone marrow derived mesenchymal stem/stromal cells transduced with full length human TRAIL repress the growth of rhabdomyosarcoma cells in vitro [Articolo su rivista]
Barti-Juhasz, H.; Mihalik, R.; Nagy, K.; Grisendi, G.; Dominici, M.; Petak, I.
abstract

Our results with BM-MSCs together with the results ofKuci et al. with CIK cells1 emphasize that cell mediateddelivery of TRAIL to metastatic RMS tumor sites can bea useful approach in RMS therapy. Targeted delivery ofTRAIL to tumors may allow systemic exposure ofpatients to drugs (e.g. the proteasome inhibitor bortezomib)that may overcome resistance for TRAIL-inducedapoptosis in RMS cells.

2011 - Cytokine-Induced Osteopoietic Differentiation of Transplanted Marrow Cells [Articolo su rivista]
S., Otsuru; Rasini, Valeria; Bussolari, Rita; T. J., Hofmann; Dominici, Massimo; E. M., Horwitz
abstract

Transplantation of whole bone marrow (BMT) leads to engraftment of both osteoprogenitor cells and hematopoietic cells; however, the robust osteopoietic chimerism seen early after BMT decreases with time. Using our established murine model, we demonstrate that a post-BMT regimen of either granulocyte-colony stimulating factor, growth hormone, parathyroid hormone, or stem cell factor each stimulates greater donor osteoblast chimerism at 4 months posttransplantation than saline-treated controls and approximates the robust osteopoietic chimerism seen early after BMT; however, only growth hormone led to significantly more donor-derived osteocytes than controls. Importantly, there were no adverse hematologic consequences of the different treatments. Our data demonstrate that these cytokines can stimulate the differentiation of transplanted donor marrow cells into the osteopoietic lineage after BMT. Post-BMT cytokine therapy may generate durable osteopoietic engraftment, which should lead to sustained clinical benefit and render BMT more applicable to bone disorders.

2011 - Membro Board Editoriale [Direzione o Responsabilità Riviste]
Dominici, Massimo
abstract

2011 - Mesenchymal stem cells: a new promise in anti-cancer therapy [Articolo su rivista]
Ciavarella, Sabino; Dominici, Massimo; Dammacco, Franco; Silvestris, Franco
abstract

Novel cell-based and gene therapies represent promising approaches for the treatment of incurable diseases,including cancer. Following the success of the hematopoietic stem cell-based transplantation, other populationsof adult progenitor cells, including mesenchymal stem cells (MSCs), have been identified as powerful therapeutictools in humans. The intrinsic capability of MSCs to migrate toward injured tissues emphasizes theirsuitability to deliver anticancer agents for new clinical applications in addition to the tissue repairing capacity.Here, we revisit the experimental history of MSCs, the most exciting features of their biology in keeping withtheir promising applications in cell-based therapeutic strategies for cancer treatment.

2011 - Mesenchymal Stromal/Stem Cells from Tissue Repair to Destruction of Tumor Cells [Capitolo/Saggio]
Bussolari, Rita; Grisendi, Giulia; L., Cafarelli; Loschi, Pietro; L., Scarabelli; A., Frassoldati; M., Maur; DE SANTIS, Giorgio; Paolucci, Paolo; P. F., Conte; Dominici, Massimo
abstract

A tumor is a complex framework composed of tumor cells (TC) and stroma where the extracellular matrix (ECM) and cellular components such as immune cells, vessels cells and fibroblasts interact closely together. Here these complex interactions are addressed in the light of the possible use of mesenchymal stomal cells to target tumors.

2011 - Una cellula effettrice modificata per il trattamento di neoplasie esprimenti il disialonganglioside GD2 [Brevetto]
Dominici, Massimo; S., Caldrer; Spano, Maria Carlotta; M., Bestagno; D., Campana; Paolucci, Paolo
abstract

Sviluppo di cellule ingenierizzate da utilizzare nel trattamento di tumori GD2-positivi. Tali cellule, costituite da un citoplasma, nel quale è presente un nucleo, il tutto racchiuso da una membrana plasmatica, vengono dotate di uno specifico recettore chimerico. Tale Recettore Chimerico (CR) è la molecola da noi ingegnerizzata, e comprende una porzione intra-citoplasmatica, e una porzione extra-citoplasmatica che è fusa con la porzione intra-citoplasmatica

2011 - Understanding Tumor-Stroma Interplays for Targeted Therapies by Armed Mesenchymal Stromal Progenitors: The Mesenkillers. [Articolo su rivista]
Grisendi, Giulia; Bussolari, Rita; Veronesi, Elena; Piccinno, MARIA SERENA; Burns, J. S.; DE SANTIS, Giorgio; Loschi, Pietro; Pignatti, M.; DI BENEDETTO, Fabrizio; Ballarin, Roberto; C., Di Gregorio; V., Guarneri; Piccinini, Lino; Em, Horwitz; Paolucci, Paolo; P., Conte; Dominici, Massimo
abstract

Tumor represents a complex structure containing malignant cells strictly coupled with a large variety of surroundingcells constituting the tumor stroma (TS). In recent years, the importance of TS for cancer initiation, development,local invasion and metastases became increasingly clear allowing the identification of TS as one of the possibleways to indirectly target tumors. Inside the heterogeneous stromal cell population, tumor associated fibroblasts(TAF) play a crucial role providing both functional and supportive environments. During both tumor and stroma development,several findings suggest that TAF could be recruited from different sources such as locally derived host fibroblasts,via epithelial/endothelial mesenchymal transitions or from circulating pools of fibroblasts deriving form mesenchymalprogenitors, namely mesenchymal stem/stromal cells (MSC). These insights prompted scientists to identifymultimodal approaches to target TS by biomolecules, monoclonal antibodies and, more recently, via cell basedstrategies. These latter appear extremely promising, although associated with still debated and unclear findings. Thisreview discusses on crosstalk between cancers and their stroma, dissecting specific tumor types, such as sarcoma,pancreatic and breast carcinoma where stroma plays distinct paradigmatic roles. The recognition of these distinctstromal functions may help in planning effective and safer approaches aimed either to eradicate or to substitute TSby novel compounds and/or MSC having specific killing activities

2011 - 17th Annual ISCT Meeting. May 18-21, 2011. Rotterdam [Esposizione]
Dominici, Massimo; I., Slaper Cortenbach; F., Falkenburg
abstract

Cell therapy continues to grow at rapid pace and ISCT continues to be the premier global society promoting the principles and practice of all areas within the vast arena of cell therapy. Every year, we gather at a wonderful destination around the world to talk about our work, rekindle old friendships, and make new ones. These yearly events are the forum for us to learn about new breakthroughs, and exchange our thoughts and ideas. This year Ineke, Massimo and Fred have done an outstanding job in producing what promises to be the best meeting ever! We truly have something for everyone! The plenary sessions will present the most outstanding science in cell therapy covering a wide array of topics from stem cells to immunotherapy. You will hear about everything from the basic research in cell therapy to the translation this knowledge into the clinics and, most importantly, how cell therapy is impacting the practice of medicine. The oral abstract sessions highlight the very best “cutting-edge” science at the meeting and the poster sessions generate the informal exchanges that stimulate the ideas which ultimately move the field forward. Our workshops provide the opportunity to discuss and debate the controversies in the field leaving all participants with a broader view than when they arrived. We continue to have a special track at the meeting focused on the cell processing technologists, the unsung heroes of cell therapy. We pay special attention to the legal and regulatory issues that are required to move cell therapy into the clinics…after all, we are the translational cell therapy society.

2011 - 3rd Annual Meeting of The Forum of Italian Researchers on Mesenchymal and Stromal cells (FIRST); Milano (Italy) [Esposizione]
Dominici, Massimo; R., Giordano; L., Lazzari; E., Montelatici; N., Baldini
abstract

On April 18th 2011, FIRST third meeting was held in Milan, Italy, at Palazzo delle Stelline with the participation of more than 100 attendees from 5 European Countries and particular emphasis was put on the contribution of young researchers who were also awarded for the best abstracts. Presentations included studies on neuroregenerative properties of MSC and kidney repair, on MSC senescence, on mechanisms underlying MSC multipotency and their immunomodulatory properties. Moreover, the limits and bottlenecks of induced pluripotent stem cells (iPS) have been reviewed, with particular attention to cardiovascular applications.

2010 - Adipose-derived mesenchymal stem cells as stable source of tumor necrosis factor-related apoptosis-inducing ligand delivery for cancer therapy [Articolo su rivista]
Grisendi, Giulia; Bussolari, Rita; Cafarelli, Luigi; Petak, I.; Rasini, Valeria; Veronesi, Elena; DE SANTIS, Giorgio; Spano, Maria Carlotta; Tagliazzucchi, M.; Barti Juhasz, H.; Scarabelli, Laura; Bambi, F.; Frassoldati, A.; Rossi, G.; Casali, Christian; Morandi, Uliano; Horwitz, E. M.; Paolucci, Paolo; Conte, P.; Dominici, Massimo
abstract

Adipose-derived mesenchymal stromal/stem cells (AD-MSC) may offer efficient tools for cell-based gene therapy approaches. In this study, we evaluated whether AD-MSC could deliver proapoptotic molecules for cancer treatment. Human AD-MSCs were isolated and transduced with a retroviral vector encoding full-length human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a proapoptotic ligand that induces apoptosis in a variety of human cancers but not normal tissues. Although several studies have documented the antitumor activity of recombinant human TRAIL, its use in vivo is limited by a short half-life in plasma due to a rapid clearance by the kidney. We found that these limitations can be overcome using stably transduced AD-MSC, which could serve as a constant source of TRAIL production. AD-MSC armed with TRAIL targeted a variety of tumor cell lines in vitro, including human cervical carcinoma, pancreatic cancer, colon cancer, and, in combination with bortezomib, TRAIL-resistant breast cancer cells. Killing activity was associated with activation of caspase-8 as expected. When injected i.v. or s.c. into mice, AD-MSC armed with TRAIL localized into tumors and mediated apoptosis without significant apparent toxicities to normal tissues. Collectively, our results provide preclinical support for a model of TRAIL-based cancer therapy relying on the use of adipose-derived mesenchymal progenitors as cellular vectors.

2010 - Co-Editore Cytotherapy [Direzione o Responsabilità Riviste]
J., Barrett; C., Bollard; Dominici, Massimo; E. M., Horwitz; J., Rasko
abstract

2010 - Getting beneath the skin to understand MSC complexity [Articolo su rivista]
Dominici, Massimo; E. M., Horwitz
abstract

Since their initial description, mesenchymal progenitors have exhibited two general functions: to directly generate tissue by differentiation and to support the survival and proliferation of other cells/tissues by secreting cytokines or growth factors. This dual-function paradigm was represented in the original description of mesenchymal progenitors, now designated as marrow stromal cells (MSC), by their capacity to give rise to bone cells and to support hematopoiesis in vitro.

2010 - GMP-manufactured density gradient media for optimized mesenchymal stromal/stem cell isolation and expansion. [Articolo su rivista]
Grisendi, Giulia; Annerén, C; Sternieri, R; Veronesi, Elena; Cervo, Gl; Luminari, Stefano; Maur, M; Frassoldati, A; Palazzi, G; Otsuru, S; Bambi, F; Paolucci, Paolo; Conte, Pierfranco; Horwitz, E; Dominici, Massimo; Cafarelli, Luigi
abstract

BACKGROUND AIMS: Bone marrow (BM) mesenchymal stromal/stem cells (MSC) are therapeutic tools in regenerative medicine and oncology. MSC isolation is often performed starting from a separation step based on research-grade 1.077 g/mL density gradient media (DGM). However, MSC clinical application should require the introduction of good manufacturing practice (GMP) reagents. We took advantage of two novel GMP DGM with densities of 1.077 and 1.073 g/mL (Ficoll-Paque PREMIUM and Ficoll-Paque PREMIUM 1.073, respectively) to test whether these reagents could isolate MSC efficiently while simultaneously comparing their performance.METHODS: BM samples were processed using either 1.077 or 1.073 g/mL GMP DGM. BM mononucleated cell (MNC) fractions were analyzed for viability, immunophenotype, clonogenic potential, ex vivo expansion and differentiation potential.RESULTS: No differences were noticed in cell recovery and viability between the groups. Fluorescence-activated cell-sorting (FACS) analyzes on freshly isolated cells indicated that the 1.073 g/mL GMP DGM more efficiently depleted the CD45(+) fraction in comparison with 1.077 GMP DGM. Moreover, in the 1.073 group, fibroblastic colony-forming units (CFU-F) were 1.5 times higher and the final MSC yield 1.8 times increased after four passages. Both reagents isolated MSC with the expected phenotype; however, 1.073-isolated MSC showed a higher expression of CD90, CD146 and GD2. Additionally, MSC from both groups were capable of fully differentiating into bone, adipose cells and cartilage.CONCLUSIONS: Both GMP DGM enriched MSC from BM samples, suggesting that these reagents would be suitable for clinical-grade expansions. In addition, the density of 1.073 g/mL provides a significant advantage over 1.077 g/mL GMP DGM, impacting the quantity of MSC obtained and reducing the ex vivo expansion time for optimized cell-based clinical applications.

2010 - Human multipotent mesenchymal stromal cells use galectin-1 to inhibit immune effector cells [Articolo su rivista]
F., Gieseke; J., Böhringer; Bussolari, Rita; Dominici, Massimo; R., Handgretinger; I., Müller
abstract

Human multipotent mesenchymal stromal cells (MSC) suppress proliferation and alloreactivity of T cells. Several signalling molecules and enzymes contribute to this effect. We focused on carbohydrate-protein interactions and investigated if lectins are involved in immune modulation by MSC. Gene expression profiling of MSC revealed that one of the most important lectins in this setting, galectin-1, was highly expressed. Galectin-1 protein was detected intracellularly and on the cell surface of MSC. In addition, galectin-1 was released into the cell culture supernatant by MSC. In order to analyze the functional role of galectin-1, a stable knockdown of galectin-1 in MSC using a retroviral transfection system was established. Galectin-1 knockdown in MSC resulted in a significant loss of their immunomodulatory properties, when compared to MSC infected with non-targeting control sequences. The galectin-1 knockdown partially restored the proliferation of CD4(+) and CD8(+) T cells. By contrast, the effect of MSC on non-alloreactive NK cells was unaffected by downregulation of galectin-1 expression. Furthermore, MSC-derived galectin-1 significantly modulated the release of cytokines involved in GvHD and autoimmunity, e. g. TNFalpha, IFNgamma, IL-2 and IL-10. These results identify galectin-1 as the first lectin mediating the immunomodulatory effect of MSC on allogeneic T cells.

2010 - IGF1-mediated osteoblastic niche expansion after marrow ablation enhance lon-term hematopoietic stem cell engraftment and hematopoietic reconstitution after bone marrow transplantation [Abstract in Rivista]
Caselli, Anna; T. S., Olson; S., Otsuru; Paolucci, Paolo; Dominici, Massimo; E., Horwitz
abstract

The capacity for hematopoietic stem cells (HSCs) to engraft in bone marrow (BM) and reconstitute long-term multilineage hematopoiesis after transplantation depends on their interaction with a specific microenvironment, the stem cell niche. We previously demonstrated a profound reorganization of the marrow endosteal microenvironment after lethal total body irradiation (TBI) of mice that leads to a rapid restoration of the osteoblastic HSC niche, followed by a transient, reversible expansion of this niche. Here, we show that the niche restoration and expansion is driven by a small number of highly proliferative osteoprogenitors via an IGF1 dependent mechanism, that directly facilities long-term hematopoietic stem cell homing and engraftment. At homeostasis, BM osteoblasts (OB) are organized as a single layer of flattened cells at the endosteal surface. Within 3 h of TBI administered to C57BL6 mice, few OB were identified at the endosteal surface. However, by 6 h post-TBI, a new single layer of OB was evident, followed by a marked increase in OB proliferation resulting in multiple layers of cells at the endosteum. The OB proliferation showed half maximal expansion at 24 h post-TBI and maximal expansion at 48 h, at which time the expanded OB constituted significantly more cell tiers than in unirradiated controls (p<0.005). BrdU incorporation/Ki67 expression dual marker studies, conducted over 48 h post-TBI, revealed clusters of highly proliferative cells that expand to repopulate the endosteum and to drive the osteoblastic niche expansion, suggesting a pool of radioresistant osteoprogenitors as the source of the niche restoration and expansion. Real-time qPCR of BM cells harvested after TBI showed an increased gene expression of Sdf1, Pdgfβ, TGFβ, Fgf2, and notably, Igf1, that peaks at 48 h. Using an ELISA, we, similarly, found an increase in protein expression, relative to unirradiated controls, that also peaks at 48 h (SDF1, 2.3-fold increase, p<0.005; PDGFβ, 6.1-fold, p<0.005; TGFβ, 4.4-fold, p<0.005; FGF2, 3.2-fold, p=0.05; IGF1, 5.3-fold, p<0.005). We propose that this extensive niche remodeling, closely correlated with increased expression of highly relevant cytokines, serves to optimize the osteoblastic niche for homing and engraftment of HSCs. To test the hypothesis, we used competitive repopulation secondary transplantation assays to assess the capacity of the osteoblastic niche to foster HSC engraftment. Primary recipients were transplanted with GFP-transgenic BM (3x106 cells/mouse) at 0.5 h, 24 h, or 48 h (maximal niche expansion) after TBI. At 24 h after transplantation, BM cells (representing 12.5% of the total marrow population) harvested from the primary recipients were transplanted into lethally irradiated secondary recipients with 2x105 competitor (unlabeled) BM cells. Secondary recipients, receiving marrow from primary recipients transplanted at 48 h post TBI, showed greater engraftment at 3 (p<0.05), 6 (p<0.001), and 18 (p<0.05) weeks after BMT compared with the 0.5 h and 24 h groups, indicating that 48 h of niche expansion facilitates homing/early engraftment of short- and long-term hematopoietic repopulating cells. To further define the importance of niche expansion and to determine the contribution of IGF1 signaling, we performed a competitive repopulation secondary transplantation assay with and without picropodophyllin (PPP), a specific IGF1 receptor tyrosine kinase inhibitor. Treated mice were either sacrificed for evaluation of niche expansion or transplanted with GFP-transgenic BM cells at 48 h post TBI. The short half-life of PPP (3 h) indicates that the drug will be eliminated prior to transplantation, 36 h after the last PPP dose. Histological analysis of irradiated, PPP-treated mice revealed that the osteoblastic niche was restored but did not expand to multiple layers at 48 h. Secondary recipients, receiving marrow from PPP-treated primary recipients

2010 - International Society for Cellular Therapy - 2nd European Meeting; Belgirate, Italy 11 - 14 September 2010 [Esposizione]
F., Lanza; I., Slaper Cortenbach; J., Gratama; Dominici, Massimo
abstract

not available

2010 - Membro Board Editoriale [Direzione o Responsabilità Riviste]
Dominici, Massimo
abstract

2010 - Osteopoietic engraftment after bone marrow transplantation: Effect of inbred strain of mice [Articolo su rivista]
S., Otsuru; Rasini, Valeria; T. J., Hofmann; Veronesi, Elena; Dominici, Massimo; Horwitz, EDWIN MARK
abstract

OBJECTIVE: Transplantable osteoprogenitors, as well as hematopoietic progenitors, reside in bone marrow. We previously reported the first clinical trial of bone marrow transplantation (BMT) for a genetic disorder of bone, osteogenesis imperfecta. Although the patients demonstrated striking clinical benefits after transplantation, measured osteopoietic engraftment was low and did not seem to be durable. Therefore, we sought an animal model, which closely reflects the clinical experience, to facilitate development of strategies to improve the efficiency of osteoprogenitor engraftment after BMT.MATERIALS AND METHODS: We transplanted unfractionated bone marrow cells from green fluorescent protein-transgenic mice into lethally irradiated recipients in four combinations of inbred mouse strains: from C57BL/6 into C57BL/6 (C-C), from C57BL/6 into FVB/N (C-F), from FVB/N into C57BL/6 (F-C), and from FVB/N into FVB/N (F-F). At 2 weeks after transplantation, we assessed donor hematopoietic and osteopoietic engraftment by flow cytometry, using a novel mean fluorescence assay, and by immunohistochemical staining for green fluorescent protein.RESULTS: Hematopoietic reconstitution by donor cells was complete in all four combinations. Although osteopoietic engraftment of the transplanted cells was also documented in all the four groups, the magnitude of osteopoietic engraftment differed markedly among the strains where F-F &gt; C-F &gt; F-C &gt; C-C.CONCLUSION: Our findings indicate that the genetic background of inbred mouse strains affects efficiency of osteopoietic engraftment after BMT. Thus, the murine strain must be considered when comparing experimental outcomes. Moreover, comparing the genetic variation among murine strains may lend insight into the factors governing osteopoietic differentiation of transplanted marrow cells.

2010 - Phase I study of intraperitoneal MHC unrestricted adoptive cell therapy with TALL-104 cells in patients with peritoneal carcinosis [Abstract in Rivista]
C., Bengala; Rasini, Valeria; Sternieri, Rita; Dominici, Massimo; Andreotti, Alessia; Gelmini, Roberta; Cafarelli, Luca; P., Bevini; A., Berti; Conte, Pierfranco
abstract

Background: TALL-104 is an irradiated human leukemic T cell line (CD3 + , CD4– CD8 + , CD56 + , CD16–) grown in IL-2-containing medium, that has the ability to kill tumor cells in preclinical models in a MHC unrestricted way. A phase I trial in metastatic breast cancer patients, has shown that multiple i.v. infusions (infs) of TALL-104 cells can be given safely. In order to optimise the tumor:effector cell ratio, we have designed a phase I study of intraperitoneal administrations of g-irradiated TALL-104 cells. Methods: Patients (pts) with peritoneal carcinosis from solid tumors resistant to standard treatment were eligible for the study. The treatment included 5 i.p. infs (day 1, 3, 5, 15, 30) and the study was designed to test three cell dose levels: 1 × 108, 5 × 108, 2.5 × 109. End points were: safety, kinetic of TALL-104 cells on ascites (if present) and peripheral blood (PB), levels of cytokines (TGF-b, GM-CSF, IL-2, IL-4, IL-10, IFN-g, TNF-a and -b, HGF, sIL-2R, sICAM-1) on ascites and serum, and immunological monitoring. Results: Fifteen pts have been treated: 8 with ovarian and 7 with GI cancer. Five pts have been treated at each dose level. No treatment-related adverse events were observed. TALL-104 cells were detected in ascites (100% of the pts) and PB (3.3% of the pts) up to 48 hrs after the infs. Cytotoxicity of autologous MNC showed an increase up to 5% at day 3 through 7 and it was still evident at day 30. HLA-DR + /CD14 + cells increased up to 21% and 30% in the serum and ascites respectively at day 7 through 15. GM-CSF, IL-2, IL-4, IL-10, IFN-g, TNF were not detectable neither in serum nor in ascitic fl uid. sIL-2R showed an increase up to 22% and 109% in serum and ascites respectively. IL-10 showed an increase up to 34% at 1st dose level and a decrease down to 20% and 40% in ascites and serum respectively at 2nd dose level. TGF-b1 and -b2 showed an increase both in serum and ascites at 1st and 2nd dose level. sICAM-1 showed an increase up to 22% at 1st dose level and a decrease down to 41% at 2nd dose level. Six patients had a confi rmed stable disease with a median duration of response of 5 months (3–7). Conclusion: These results show that the i.p. infusion of TALL- 104 is safe. Moreover, the increased autologous cell-mediated cytotoxicity and the levels of soluble cytokines after i.p. infs indicate that TALL-104 cells may elicit potential antitumor activity and deserve further evaluation in patients with a less severe stage of disease.

2010 - Toward cell therapy using placenta-derived cells: Disease mechanisms, cell biology, preclinical studies, and regulatory aspects at the round table [Articolo su rivista]
Parolini, Ornella; Alviano, Francesco; Bergwerf, Irene; Boraschi, Diana; De Bari, Cosimo; De Waele, Peter; Dominici, Massimo; Evangelista, Marco; Falk, Werner; Hennerbichler, Simone; Hess, David C.; Lanzoni, Giacomo; Liu, Bing; Marongiu, Fabio; Mcguckin, Colin; Mohr, Stefan; Nolli, Maria Luisa; Ofir, Racheli; Ponsaerts, Peter; Romagnoli, Luca; Solomon, Abraham; Soncini, Maddalena; Strom, Stephen; Surbek, Daniel; Venkatachalam, Sankar; Wolbank, Susanne; Zeisberger, Steffen; Zeitlin, Andy; Zisch, Andreas; Borlongan, Cesar V.
abstract

Among the many cell types that may prove useful to regenerative medicine, mounting evidence suggests that human term placenta-derived cells will join the list of significant contributors. In making new cell therapy-based strategies a clinical reality, it is fundamental that no a priori claims are made regarding which cell source is preferable for a particular therapeutic application. Rather, ongoing comparisons of the potentiality and characteristics of cells from different sources should be made to promote constant improvement in cell therapies, and such comparisons will likely show that individually tailored cells can address disease-specific clinical needs. The principle underlying such an approach is resistance to the notion that comprehensive characterization of any cell type has been achieved, neither in terms of phenotype nor risks-to-benefits ratio. Tailoring cell therapy approaches to specific conditions also requires an understanding of basic disease mechanisms and close collaboration between translational researchers and clinicians, to identify current needs and shortcomings in existing treatments. To this end, the international workshop entitled "Placenta-derived stem cells for treatment of inflammatory diseases: moving toward clinical application" was held in Brescia, Italy, in March 2009, and aimed to harness an understanding of basic inflammatory mechanisms inherent in human diseases with updated findings regarding biological and therapeutic properties of human placenta-derived cells, with particular emphasis on their potential for treating inflammatory diseases. Finally, steps required to allow their future clinical application according to regulatory aspects including good manufacturing practice (GMP) were also considered. In September 2009, the International Placenta Stem Cell Society (IPLASS) was founded to help strengthen the research network in this field

2010 - 2nd Annual Meeting of The Forum of Italian Researchers on Mesenchymal and Stromal cells (FIRST); Modena (Italy) [Esposizione]
Dominici, Massimo; R., Giordano; L., Lazzari
abstract

Questa seconda edizione del meeting italiano sulle cellule mesenchimali staminali (MSC) è stato caratterizzato dalla partecipazione di relatori italiani e stranieri di grande capacità ed esperienza e di un numero elevato di partecipanti (più di 100). Il meeting è stato molto intenso e sono stati presi in esame vari aspetti, sia di ricerca di base che traslazionali. Sono state discusse le varie origini e proprietà delle MSC , le loro utilizzazioni ed effetti terapeutici, specialmente in modelli animali.

2009 - Basic research and clinical applications of non-hematopoietic stem cells, 4-5 April 2008, Tubingen, Germany. [Articolo su rivista]
Schafer, R; Dominici, Massimo; Muller, I; Horwitz, E; Asahara, T; Bulte, J; Bieback, K; LE BLANC, K; Buhring, H; Capogrossi, M; Dazzi, F; Gorodetsky, R; Henschler, R; Handgretinger, R; Kajstura, J; Kluger, P; Lange, C; VON LUETTICHAU, I; Mertsching, H; Schrezenmeier, H; Sievert, K; Strunk, D; Verfaillie, C; Northoff, H.
abstract

From 4 to 5 April 2008, international experts met for the second time in Tubingen, Germany, to present and discuss the latest proceedings in research on non-hematopoietic stem cells (NHSC). This report presents issues of basic research including characterization, isolation, good manufacturing practice (GMP)-like production and imaging as well as clinical applications focusing on the regenerative and immunomodulatory capacities of NHSC.

2009 - Cell therapy for disorders of bone [Articolo su rivista]
R., Jethva; S., Otsuru; Dominici, Massimo; E. M., Horwitz
abstract

Bone marrow transplantation (BMT) has changed the course of treatment for an array of diseases, including disorders of bone. Hematopoietic stem cells (HSC) within the marrow are known to be the precursors of osteoclastic bone cells, and trials of BMT in osteopetrosis, a disorder characterized by a deficiency of osteoclasts, have resulted in significant clinical improvement in patients. The origin of the other major bone cell, the osteoblast, remains uncertain, although studies have identified osteoprogenitor cells within the marrow, leading to further investigation of both mesenchymal stromal cells (MSC) and HSC as candidates for this role. A better understanding of the source of osteoblasts and normal bone metabolism is crucial to efforts to develop effective cell therapy for bone disorders characterized by deficient or abnormal osteoblast function. This review focuses on systemic and local cell therapy in the treatment of several genetic bone disorders and osteoporosis, an acquired disorder caused by abnormal bone metabolism, with the intent of presenting both the progress and challenges associated with this emerging form of therapy. Although the risks of systemic transplantation must be carefully considered, cell therapy for disorders of bone carries the potential for long-term and potentially curative benefits, justifying further intensive research on this important treatment option.

2009 - Heterogeneity of multipotent mesenchimal stromal cells: from stromal cells to stem cells and viceversa [Articolo su rivista]
Dominici, Massimo; Paolucci, Paolo; Conte, Pierfranco; E. M., Horwitz
abstract

Discovered more than 40 years ago, the biological features of multipotent mesenchymal stromal cells (MSC) were progressively compared first with hematopoietic stem cells (HSC) and, more recently, with embryonic stem cells (ESC). Although these comparisons have been crucial in helping to clarify their nature, there is now a robust amount of data indicating that MSC in vitro represent an independent and heterogeneous group of progenitors with distinct self-renewal properties and established differentiation potentials. However, research developments both in humans and animals have progressively revealed the limits that MSC may face in vivo. To recognize these issues and challenge MSC stemness may seem to be a step backward. Nevertheless, it might also represent the beginning of a phase in which the introduction of novel preclinical approaches could provide better characterization and standardization of the in vivo factors influencing cell engraftment and survival, allowing a more successful impact of mesenchymal progenitors in several clinical settings.

2009 - Heterogeneity of multipotent mesenchymal stromal cells: from stromal cells to stem cells and vice versa [Articolo su rivista]
Dominici, M.; Paolucci, P.; Conte, P.; Horwitz, E. M.
abstract

Discovered more than 40 years ago, the biological features of multipotent mesenchymal stromal cells (MSC) were progressively compared first with hematopoietic stem cells (HSC) and, more recently, with embryonic stem cells (ESC). Although these comparisons have been crucial in helping to clarify their nature, there is now a robust amount of data indicating that MSC in vitro represent an independent and heterogeneous group of progenitors with distinct self-renewal properties and established differentiation potentials. However, research developments both in humans and animals have progressively revealed the limits that MSC may face in vivo. To recognize these issues and challenge MSC stemness may seem to be a step backward. Nevertheless, it might also represent the beginning of a phase in which the introduction of novel preclinical approaches could provide better characterization and standardization of the in vivo factors influencing cell engraftment and survival, allowing a more successful impact of mesenchymal progenitors in several clinical settings.

2009 - Optimizing the niche conditions for maximal stem cell engraftment: human and animal model data [Articolo su rivista]
Dominici, Massimo; E. M., Horwitz
abstract

While animal models are ideal to uncover the biologic basis of pathologic observations and therapeutic interventions, whether such scientific findings, in reality, apply to patients is always an open question. The exceptional human data of Zweegman et al corroborate our murine findings and suggest that the novel mechanisms we describe may, in fact, apply to clinical hematopoietic cell transplantation.

2009 - Refining GMP-manufactured density gradient media for optimised mesenchymal stromal/stem cell isolation and expansion. [Abstract in Rivista]
Grisendi, Giulia; C., Anneren; L., Cafarelli; Veronesi, Elena; R., Sternieri; G. L., Cervo; Luminari, Stefano; A., Frassoldati; M., Maur; G., Palazzi; Paolucci, Paolo; Conte, Pierfranco; Dominici, Massimo
abstract

not available

2009 - Restoration and reversible expansion of the osteoblastic hematopoietic stem cell niche after marrow radioablation [Articolo su rivista]
Dominici, Massimo; Rasini, Valeria; Bussolari, Rita; X., Chen; T. J., Hofmann; Spano, Maria Carlotta; D., Bernabei; Veronesi, Elena; F., Bertoni; Paolucci, Paolo; Conte, Pierfranco; E. M., Edwin
abstract

Adequate recovery of hematopoietic stem cell (HSC) niches after cytotoxic conditioning regimens is essential to successful bone marrow transplantation. Yet, very little is known about the mechanisms that drive the restoration of these niches after bone marrow injury. Here we describe a profound disruption of the marrow microenvironment after lethal total body irradiation of mice that leads to the generation of osteoblasts restoring the HSC niche, followed by a transient, reversible expansion of this niche. Within 48 hours after irradiation, surviving host megakaryocytes were observed close to the endosteal surface of trabecular bone rather than in their normal parasinusoidal site concomitant with an increased stromal-derived factor-1 level. A subsequent increase in 2 megakaryocyte-derived growth factors, platelet-derived growth factor-beta and basic fibroblast growth factor, induces a 2-fold expansion of the population of N-cadherin-/osteopontin-positive osteoblasts, relative to the homeostatic osteoblast population, and hence, increases the number of potential niches for HSC engraftment. After donor cell engraftment, this expanded microenvironment reverts to its homeostatic state. Our results demonstrate the rapid recovery of osteoblastic stem cell niches after marrow radioablation, provide critical insights into the associated mechanisms, and suggest novel means to manipulate the bone marrow microenvironment to promote HSC engraftment.

2009 - Rigenerand srl - Spin off Università di Modena e Reggio Emilia [Spin Off]
Dominici, Massimo; Conte, Pierfranco; Paolucci, Paolo; G., Bellini
abstract

2009 - 1st Annual Meeting of The Forum of Italian Researchers on Mesenchymal and Stromal cells (FIRST); Milano (Italy). [Esposizione]
L., Lazzari; Dominici, Massimo; R., Giordano
abstract

A questo Meeting scientifico, alla sua prima edizione, hanno partecipato i più rappresentativi ricercatori italiani che lavorano sulle cellule staminali mesenchimali da diverse sorgenti tissutali. Inoltre, ricercatori stranieri hanno apportato il loro contributo. L’argomento è stato estremamente innovativo e interessante ed i docenti sono risulatati di assoluto riferimento in materia della ricerca sulle cellule staminali adulte.

2008 - Application of multipotent mesenchymal stromal cells in pediatric patients following allogeneic stem cell transplantation [Articolo su rivista]
Müller, Ingo; Kordowich, Sandra; Holzwarth, Christina; Isensee, Gesa; Lang, Peter; Neunhoeffer, Felix; Dominici, Massimo; Greil, Johann; Handgretinger, Rupert
abstract

Multipotent mesenchymal stromal cells (MSC) have immunomodulatory effects. The aim of this study was to demonstrate safety and feasibility of MSC transfusion in pediatric patients who had undergone allogeneic stem cell transplantation from MMFD, MUD, MMUD and MSD. Patients with posttransplant complications based on deregulated immune effector cells who may benefit from an immunomodulatory effect of MSC had been selected. MSC were isolated from the hematopoietic stem cell donors in five cases and from a third party parental donor in two cases. We transfused ex vivo-expanded MSC in 11 doses into seven pediatric patients. Cell doses were escalated based on availability from 0.4x10(6) to 3.0x10(6) per kg bodyweight No adverse effects were detected with a maximum follow-up of 29 months. One out of three patients showed slight improvement of chronic GVHD. Two patients with severe acute GvHD did not progress to cGvHD. One patient received MSC to stabilize graft function after secondary haploidentical transplantation. One patient recovered from trilineage failure due to severe hemophagocytosis. This is the first case of a pediatric patient treated with MSC for trilineage failure after haploidentical stem cell transplantation from her father. We report the first series of 11 transfusions of expanded MSC in pediatric patients with immunological complications after allogeneic transplantation. Transfusion of MSC was safe and encouraging improvements in some patients were observed

2008 - Donor Cell-Derived Osteopoiesis Originates from a Self-Renewing Stem Cell with a Limited Regenerative Contribution after Transplantation [Articolo su rivista]
Dominici, Massimo; R., Marino; Rasini, Valeria; Spano, Maria Carlotta; Paolucci, Paolo; Conte, Pierfranco; T. J., Hofmann; E. M., Horwitz
abstract

n principle,bone marrow transplantation should offer effective treatment for disorders originating from defects in mesenchymal stem cells. Results with the bone disease osteogenesis imperfecta support this hypothesis,although the rate of clinical improvement seen early after transplantation does not persist long term,raising questions as to the regenerative capacity of the donor-derived mesenchymal progenitors. We therefore studied the kinetics and histologic/anatomic pattern of osteopoietic engraftment after transplantation of GFP-expressing nonadherent marrow cells in mice. Serial tracking of donor-derived GFP(+) cells over 52 weeks showed abundant clusters of donor-derived osteoblasts/osteocytes in the epiphysis and metaphysis but not the diaphysis,a distribution that paralleled the sites of initial hematopoietic engraftment. Osteopoietic chimerism decreased from approximately 30% to 10% by 24 weeks after transplantation,declining to negligible levels thereafter. Secondary transplantation studies provided evidence for a self-renewing osteopoietic stem cell in the marrow graft. We conclude that a transplantable,primitive,self-renewing osteopoietic cell within the nonadherent marrow cell population engrafts in an endosteal niche,like hematopoietic stem cells,and regenerates a significant fraction of all bone cells. The lack of durable donor-derived osteopoiesis may reflect an intrinsic genetic program or exogenous environmental signaling that suppresses the differentiation capacity of the donor stem cells.

2008 - Epidermal growth factor receptor (EGFR) high gene copy number and activating mutations in lung adenocarcinomas are not consistently accompanied by positivity for EGFR protein by standard immunohistochemistry [Articolo su rivista]
F., Pinter; J., Papay; A., Almasi; Z., Sapi; E., Szabo; M., Kanya; A., Tamasi; B., Jori; E., Varkondi; J., Moldvay; K., Szondy; G., Keri; Dominici, Massimo; Conte, Pierfranco; S., Eckhardt; L., Kopper; R., Schwab; I., Petak
abstract

The purpose of this study was to investigate whether detectable protein biomarker overexpression is a prerequisite for the presence of increased gene copy number or activating mutations and responsiveness to the epidermal growth factor receptor (EGFR) inhibitors gefitinib and erlotinib in patients with lung adenocarcinomas. EGFR status was prospectively analyzed in tumor biopsy samples by three methods: protein expression (n = 117) by standardized immunohistochemistry (IHC), gene copy number (n = 97) by fluorescent in situ hybridization (FISH), and mutation analysis by sequencing (n = 126). Fifty-nine percent of the samples were positive by IHC, 40% were positive by FISH, and 13.5% contained activating kinase domain mutations. Thirty-four percent of the FISH-positive and 27% of the mutant samples were also IHC-negative. All EGFR mutant patients had major clinical responses (five complete response and five partial response) to gefitinib or erlotinib treatment, although three of these tumors were IHC-negative and four were FISH-negative. In a retrospective analysis of samples from nine patients with excellent therapeutic responses (three complete response, five partial response, one stable disease) to erlotinib or gefitinib, mutations were identified in eight cases, but IHC was negative in four of these tumors. These results indicate that molecular diagnostic methods appear to be most important for the identification of lung adenocarcinoma patients who may benefit from EGFR inhibitor treatments.

2008 - How do mesenchymal stromal cells exert their therapeutic benefit? [Articolo su rivista]
E. M., Horwitz; Dominici, Massimo
abstract

n recent years mesenchymal stromal cells (MSC) have emerged as a major new form of cell therapy. While the original perception was that MSC were stem/progenitor cells with the potential to contribute to the regeneration of tissue, more recent data suggest that the principal mechanism of MSC activity is through the release of soluble mediators that elicit the observed biologic response. Future studies are needed to identify more completely the spectrum of therapeutic applications and delineate better the associated molecular and cellular mechanisms.

2008 - Transplantable marrow osteoprogenitors engraft in discrete saturable sites in the marrow microenvironment [Articolo su rivista]
R., Marino; C., Martinez; K., Boyd; Dominici, Massimo; T. J., Hofmann; E. M., Horwitz
abstract

OBJECTIVE: Based on the recognition that marrow contains progenitors for bone as well as blood, we undertook the first trial of bone marrow transplantation (BMT) for a genetic disorder of bone, osteogenesis imperfecta. While we documented striking clinical benefit soon after transplantation, the measured level of osteopoietic engraftment was low. To improve the efficacy of BMT for bone disorders, we sought to gain insight into the cellular mechanism of engraftment of transplantable marrow osteoprogenitors.MATERIALS AND METHODS:We transplanted unfractionated bone marrow harvested from green fluorescent protein-transgenic FVB/N mice into lethally irradiated FVB/N recipients. At 3 weeks posttransplantation, we assessed hematopoietic engraftment by flow cytometry and osteopoietic engraftment by immunohistochemical staining for the green fluorescent protein.RESULTS:We show that engraftment of transplantable marrow osteoprogenitors is saturable with a maximal engraftment of about 15% of all bone cells in the epiphysis and metaphysis of the femur at 3 weeks after transplantation. The number of engrafting sites is not up- or downregulated in response to initial progenitor cell engraftment, and there is no evidence for clonal succession of osteopoietic differentiation of engrafted progenitors.CONCLUSIONS:Our findings indicate that the capacity for initial osteopoietic engraftment after BMT is limited and "megadose" stem cell transplantation is unlikely to enhance engraftment. Thus, novel strategies to foster osteopoietic chimerism must be developed.

2007 - CELLULE STAMINALI: REALTA’ E PROSPETTIVE [Capitolo/Saggio]
Paolucci, Paolo; Dominici, Massimo; M. C., Cano; A. L., Bonetti; C., China; Spano, Maria Carlotta; Rasini, Valeria; Conte, Pierfranco
abstract

non disponibile

2007 - Focusing on the borders between bone and blood to better understand stem cells based regeneration [Abstract in Rivista]
Dominici, Massimo; Rasini, Valeria; Veronesi, Elena; Paolucci, Paolo
abstract

It has been proposed that areas which border both bone and marrow contain specific niches which are crucial for hematopoietic stem cell (HSC) engraftment (Calvi LM, 2003). Similarly, we have shown that bone regeneration takes place after BM transplantation in discrete districts close to bone within the so called bone lining cells compartment where early osteopoietic engraftment originates as well (Dominici et al. 2004 and 2008). Very little is known about the mechanisms that drive the restoration of these compartments following a bone marrow (BM) injury. Researchers have been focused almost entirely on how HSCs are regulated by their microenvironmental niches (Zhang J 2003), with little attention paid to the recovery of cellular constituents of these niches after use of cytotoxic conditioning regimens. Moreover, while several models have been proposed, the precise anatomic location of niches within the BM microenvironment is not well understood. Thus, focusing on ionizing radiation (IR) exposure, we asked how the niches may respond to specific stress and affect mesenchymal engraftment as well. Based on this animal model, we report a profound disruption of the BM microenvironment after IR exposure that leads to a survival and generation of osteoblast constituting the niche (Dominici et al. 2009). These complex events are associated with a megakaryocytes re-location close to the bone lining cell compartment capable to stimulate osteoblasts which are then keen to host transplanted donor HSC. Thus, following irradiation, a hematopoietic cell lineage contributes to both skeletal turnover and to HSC osteoblast niche homeostasis. These results provide critical insights into the mechanisms of recovery of stem cell niches after BM radioablation, and suggest novel means to manipulate the BM microenvironment to promote HSC and bone engraftment.

2007 - Human bone marrow mesenchymal stromal cells express the neural ganglioside GD2: A novel surface marker for the identification of MSCs [Articolo su rivista]
C., Martinez; T. J., Hofmann; R., Marino; Dominici, Massimo; E. M., Horwitz
abstract

esenchymal stromal cells (MSCs) have enormous potential for the regeneration of bone, cartilage, and other tissues derived from primitive mesoderm. Despite extensive research, there is still no single marker that reliably identifies MSCs within the bone marrow. Using immunocytochemistry and flow cytometry, we demonstrate here that the neural ganglioside GD2 is expressed by MSCs either newly isolated from bone marrow or expanded in tissue culture; this finding was supported by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showing expression of the mRNA for GD2 synthase, an essential enzyme for GD2 biosynthesis. GD2 was also expressed on MSCs isolated from adipose tissue, but not on foreskin fibroblasts. Importantly, MSCs were the only cells within normal marrow that expressed this marker. Thus, GD2 appears to be the first reported single surface marker that uniquely distinguishes MSCs from other marrow elements. GD2 may prove valuable to study MSC biology and for the preparation of MSCs for clinical applications.

2007 - La cardiopatia iatrogena in oncologia [Articolo su rivista]
V., Silingardi; R., Sternieri; Dominici, Massimo; A., Frassoldati
abstract

The iatrogenic cardiopathies are late complications in oncology patients and represent a relevant cause of morbidity in the long term survivors. The large majority of the chemotherapeutic agents may potentially damage the cardiovascular structures, however the related clinical consequences deserve particular cautions only for some. Although the anthracyclines are considered as very active anti-tumor molecules, they are associated with a robust and, sometimes, deathly cardiotoxicity. Therefore, their uses require either a careful evaluation of pre-existing risk factors before their administration or an adequate follow-up during and at the end of the therapeutic programs. In addition, cardiac damages are reported in relationships with other chemotherapy agents (such as cyclophosphamide, cisplatinum, 5-fluorouracile and taxanes), with hormonal therapy and, more recently with the introduction of the so called “targeted therapies”. Between them, the trastuzumab (a monoclonal antibody anti-Erb-B2) has been related with the onset of different heart problems especially in association with anthracyclines and/or taxanes. The bevacizumab (a monoclonal antibody directed against the vascular endothelial growth factor) may cause ipertension in the treated patients, while the imatinib (a tyrosine kinase inhibitor) has been recently related with various ventricular disfunctions. Next to the molecules with anti-tumor activity, the radiotherapy has been associated in the past with severe damages to the cardiac tissue, in particular regarding the coronary arteries. Typically, these complications appear very late after the exposure, showing a poor prognosis. The availability of modern diagnostic tools and the comprehension of risk factors may identify the patients which are more keen in developing heart problems inducing the adoption of specific therapeutic options, such as the drug withdrawal. Hopefully, the constant progresses in the biotechnology will permit the development of less cardiotoxic therapeutic options with the reduction of such a terrible complication in oncology.

2007 - Linee mesenchimali multipotenti da tessuto connettivale orale; studio pilota. [Abstract in Rivista]
Bertoldi, Carlo; Cafarelli, Luigi; Veronesi, Elena; Consolo, Ugo; Dominici, Massimo
abstract

Vedi allegato

2007 - Progress in characterization, preparation and clinical applications of non-hematopoietic stem cells, 29-30 September 2006, Tübingen, Germany [Articolo su rivista]
Schafer, R; Dominici, Massimo; Muller, I; Dazzi, F; Bieback, K; Godthardt, K; Blanc, Kl; Meisel, R; Pochampally, R; Richter, R; Skutella, T; Steinhoff, G; Mitterberger, M; Wendel, H; Wiskirchen, J; Handgretinger, R; Northoff, H.
abstract

The first „Congress on Characterization, Preparation and Clinical Applications of non Hematopoietic Stem Cells” was held 29-30 September 2006 in Tuebingen/Germany. The meeting has been focused on characterization and isolation of non hematopoietic stem cells (NHSC) as well as on immunological aspects and clinical applications of NHSC. Further topics have been labeling and homing of NHSC. This meeting report will present the key informations of the talks as a contribution to parts of the current research of this field.

2007 - 0.1 grams of sub-cutaneous adipose tissue (SCAT) as source of adult multipotent mesenchymal stromal cells (MSC) for cell-based therapies [Abstract in Rivista]
Dominici, Massimo; Sternieri, R.; De Fazio, D.; Cafarelli, L.; Guaraldi, Giovanni; Spano, C.; Rasini, V.; Paolucci, P.; Cilli, M.; Piccardi, F.; Astori, G.; Hofmann, T. J.; Horwitz, E.; Conte, P. F.
abstract

The purification of multipotent mesenchymal stromal cell (MSC) from sub-cutaneous adipose tissue (SCAT) represents a promising approach for several clinical applications. We here report a novel and safe approach to isolate multipotent cells from small SCAT specimens to be used as platform for cell based therapies with minimal donor's discomfort.

2006 - Animal serum-free culture conditions for isolation and expansion of multipotent mesenchymal stromal cells from human BM [Articolo su rivista]
I., Muller; S., Kordowich; C., Holzwarth; Spano, Maria Carlotta; G., Isensee; A., Staiber; S., Viebahn; F., Gieseke; H., Langer; Mp, Gawaz; Em, Horwitz; Conte, Pierfranco; R., Handgretinger; Dominici, Massimo
abstract

Background Multipotent mesenchymal stromal cells ( MSC) have become important tools in regenerative and transplantation medicine. Rapidly increasing numbers of patients are receiving in vitro-expanded MSC. Culture conditions typically include FSC because human serum does not fully support growth of human MSC in vitro (MSCFCS). Concerns regarding BSE, other infectious complications and host immune reactions have fueled investigation of alternative culture supplements. Methods As PDGF has long been identified as a growth factor for MSC, we tested media supplementation with platelet lysate for support of MSC proliferation. Results We found that primary cultures of BM-derived MSC can be established with animal serum-free media containing fresh frozen plasma and platelets (MSCFFPP). Moreover, MSCFFPP showed vigorous proliferation that was superior to classical culture conditions containing FCS. MSCFFPP morphology was equivalent to MSC FCS, and MSCFFPP expressed CD73, CD90, CD105, CD106, CD146 and HLA-ABC while being negative for CD34, CD45 and surface HLA-DR, as expected. In addition to being phenotypically identical, MSCFFPP could efficiently differentiate into adipocytes and osteoblasts. In terms of immune regulatory properties, MSCFFPP were indistinguishable from MSC FCS. Proliferation of PBMC induced by IL-2 in combination with OKT-3 or by PHA was inhibited in the presence of MSCFFPP. Discussion Taken together, FCS can be replaced safely by FFPP in cultures of MSC for clinical purposes.

2006 - Characterization of human MSC propagated in animal serum-free cultures and clinical application of MSC in paediatric patients. [Abstract in Rivista]
Holzwarth, C; Kordowich, S; Isensee, G; Viebahn, S; Gieseke, F; Lang, P; Greil, J; Dominici, Massimo; Conte, Pierfranco; Handgretinger, R; Mueller, I.
abstract

Multipotent mesenchymal stromal cells (MSC) have become important tools in regenerative and transplantation medicine. Rapidly increasing numbers of patients are receiving in vitro-expanded MSC. Culture conditions typically include FSC because human serum does not fully support growth of human MSC in vitro (MSC(FCS)). Concerns regarding BSE, other infectious complications and host immune reactions have fueled investigation of alternative culture supplements. As PDGF has long been identified as a growth factor for MSC, we tested media supplementation with platelet lysate for support of MSC proliferation. We found that primary cultures of BM-derived MSC can be established with animal serum-free media containing fresh frozen plasma and platelets (MSC(FFPP)). Moreover, MSC(FFPP) showed vigorous proliferation that was superior to classical culture conditions containing FCS. MSC(FFPP) morphology was equivalent to MSC(FCS), and MSC(FFPP) expressed CD73, CD90, CD105, CD106, CD146 and HLA-ABC while being negative for CD34, CD45 and surface HLA-DR, as expected. In addition to being phenotypically identical, MSC(FFPP) could efficiently differentiate into adipocytes and osteoblasts. In terms of immune regulatory properties, MSC(FFPP) were indistinguishable from MSC(FCS). Proliferation of PBMC induced by IL-2 in combination with OKT-3 or by PHA was inhibited in the presence of MSC(FFPP). Taken together, FCS can be replaced safely by FFPP in cultures of MSC for clinical purposes.

2006 - Human multipotent mesenchymal stromal cells (MSCs) induce proliferation of allogeneic peripheral blood mononuclear cells. [Abstract in Rivista]
Dominici, Massimo; Rasini, Valeria; Sternieri, R; Cafarelli, L; Spano, C; Cervo, Gl; Bonetti, A; China, C; Paolucci, Paolo; Frassoldati, A; Bambi, F; Campana, D; Conte, Pierfranco; Horwitz, Em
abstract

not available

2006 - Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement [Articolo su rivista]
Dominici, Massimo; K., Le Blanc; I., Mueller; I., Slaper Cortenbach; Fc, Marini; Ds, Krause; Rj, Deans; A., Keating; Dj, Prockop; Em, Horwitz
abstract

The considerable therapeutic potential of human multipotent mesenchymal stromal cells ( MSC) has generated markedly increasing interest in a wide variety of biomedical disciplines. However, investigators report studies of MSC using different methods of isolation and expansion, and different approaches to characterizing the cells. Thus it is increasingly difficult to compare and contrast study outcomes, which hinders progress in the field. To begin to address this issue, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposes minimal criteria to define human MSC. First, MSC must be plastic-adherent when maintained in standard culture conditions. Second, MSC must express CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79 alpha or CD19 and HLA-DR surface molecules. Third, MSC must differentiate to osteoblasts, adipocytes and chondroblasts in vitro. While these criteria will probably require modification as new knowledge unfolds, we believe this minimal set of standard criteria will foster a more uniform characterization of MSC and facilitate the exchange of data among investigators.

2006 - Phase I study of intraperitoneal MHC un resctrected adoptive cell therapy with TALL-104 cells in patients with peritoneal carcinosis: preliminary results. [Abstract in Atti di Convegno]
Bengala, Carmelo; Rasini, Valeria; Sternieri, Rita; Dominici, Massimo; Gelmini, Roberta; Cafarelli, Luigi; Bevini, Paola; Berti, Andrea; Conte, Pierfranco
abstract

Phase I study of intraperitoneal MHC un resctrected adoptive cell therapy with TALL-104 cells in patients with peritoneal carcinosis: preliminary results.

2006 - Proteasome inhibitors sensitize colon carcinoma cells to TRAIL-induced apoptosis via enhanced release of Smac/DIABLO from the mitochondria [Articolo su rivista]
K., Nagy; K., SZEKELY SZUTS; K., Izeradjene; L., Douglas; M., Tillman; H., BARTI JUHASZ; Dominici, Massimo; Spano, Maria Carlotta; G. L., Cervo; Conte, Pierfranco; J. A., Houghton; R., Mihalik; L., Kopper; I., Petak
abstract

The synergistic interaction between proteasome inhibitors and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising approach to induce cell death in tumor cells. However, the molecular and biochemical mechanisms of this synergism have been proven to be cell type specific. We therefore focused our investigation on TRAIL-resistant colon carcinoma cells in this study. DNA fragmentation, mitochondrial membrane depolarization and increased caspase-3-like enzyme activity was exclusively induced only by combined treatment with proteasome inhibitors (epoxomicin, MG132, bortezomib/PS-341) and TRAIL. The expression level of anti-apoptotic proteins (XIAP, survivin, Bcl-2, Bcl-XL), regulated by NF-kappaB transcription factor, was not effected by any of these treatments. TRAIL alone induced only partial activation of caspase-3 (p20), while the combination of TRAIL and proteasome inhibition led to the full proteolytic activation of caspase-3 (p17). Only the combination treatment induced marked membrane depolarization and the release of cytochrome c, HtrA2/Omi and Smac/DIABLO. Apoptosis-inducing factor (AIF) was not released in any of these conditions. These results are consistent with a model where the full activation of caspase-3 by caspase-8 is dependent on the release of Smac/DIABLO in response to the combined treatment. This molecular mechanism, independent of the inhibition NF-kappaB activity, may provide rationale for the combination treatment of colon carcinomas with proteasome inhibitors and recombinant TRAIL or agonistic antibody of TRAIL receptors.

2006 - Transcriptional link between blood and bone: the stem cell leukemia gene and its+19 stem cell enhancer are active in bone cells [Articolo su rivista]
Je, Pimanda; L., Silberstein; Dominici, Massimo; B., Dekel; M., Bowen; S., Oldham; A., Kallianpur; Sj, Brandt; D., Tannahill; B., Gottgens; Ar, Green
abstract

Blood and vascular cells are generated during early embryogenesis from a common precursor, the hemangioblast. The stem cell leukemia gene (SCL/tal 1) encodes a basic helix-loop-helix transcription factor that is essential for the normal development of blood progenitors and blood vessels. We have previously characterized a panel of SCL enhancers including the +19 element, which directs expression to hematopoietic stem cells and endothelium. Here we demonstrate that SCL is expressed in bone primordia during embryonic development and in adult osteoblasts. Despite consistent expression in cells of the osteogenic lineage, SCL protein is not required for bone specification of embryonic stem cells. In transgenic mice, the SCL +19 core enhancer directed reporter gene expression to vascular smooth muscle and bone in addition to blood and endothelium. A 644-bp fragment containing the SCL +19 core enhancer was active in both blood and bone cell lines and was bound in vivo by a common array of Ets and GATA transcription factors. Taken together with the recent observation that a common progenitor can give rise to blood and bone cells, our results suggest that the SCL +19 enhancer targets a mesodermal progenitor capable of generating hematopoietic, vascular, and osteoblastic progeny.

2006 - Trophoblast stem cells rescue placental defect in SOCS3-deficient mice [Articolo su rivista]
Y., Takahashi; Dominici, Massimo; J., Swift; C., CRISTIE NAGY; J. N., Ihle
abstract

Stem cells have important clinical and experimental potentials. Trophoblast stem (TS) cells possess the ability to differentiate into trophoblast subtypes in vitro and contribute to the trophoblast lineage in vivo. Suppressor of cytokine signaling 3 (SOCS3) is a negative regulator of cytokine signaling. Targeted disruption of SOCS3 revealed embryonic lethality on E12.5; it was caused by placental defect with enhanced leukemia inhibitory factor receptor signaling. A complementation of the wild-type (WT) placenta by using tetraploid rescue technique showed that the embryonic lethality in SOCS3-deficient embryo was due to the placental defect. Here we demonstrate that TS cells supplementation rescues placental defect in SOCS3-deficient embryos. In the rescued placenta, TS cells were integrated into the placental structure, and a substantial structural improvement was observed in the labyrinthine layer that was disrupted in the SOCS3-deficient placenta. Importantly, by supplying TS cells, living SOCS3-deficient embryos were detected at term. These results indicate a functional contribution of TS cells in the placenta and their potential application.

2005 - A NOVEL SERUM DEPRIVED MEDIUM FOR ISOLATION AND EXPANSION OF FUNCTIONAL DIFFERENTIATING MESENCHYMAL STEM CELLS [Abstract in Atti di Convegno]
I., Mariotti; S., Spaggiari; A., Bonetti; Dominici, Massimo; I., Mueller; L., Cafarelli; R., Sternieri; C., Spano; V., Rasini; P., Conte; Paolucci, Paolo
abstract

non disp

2005 - Clarification of the nomenclature for MSC: the international society for cellular therapy position statement [Articolo su rivista]
E. M., Horwitz; K., Le Blanc; Dominici, Massimo; I., Mueller; I., Slaper Cortenbach; F. C., Marini; R. J., Deans; D. S., Krause; A., Keating
abstract

The plastic-adherent cells isolated from BM and other sources have come to be widely known as mesenchymal stem cells ( MSC). However, the recognized biologic properties of the unfractionated population of cells do not seem to meet generally accepted criteria for stem cell activity, rendering the name scientifically inaccurate and potentially misleading to the lay public. Nonetheless, a bona fide MSC most certainly exists. To address this inconsistency between nomenclature and biologic properties, and to clarify the terminology, we suggest that the fibroblast-like plastic-adherent cells, regardless of the tissue from which they are isolated, be termed multipotent mesenchymal stromal cells, while the term mesenchymal stem cells is used only for cells that meet specified stem cell criteria. The widely recognized acronym, MSC, may be used for both cell populations, as is the current practice; thus, investigators must clearly define the more scientifically correct designation in their reports. The International Society for Cellular Therapy (ISCT) encourages the scientific community to adopt this uniform nomenclature in all written and oral communications.

2005 - Immunoterapia genica anti-tumorale con linfociti chimerici: prospettive in oncologia pediatrica [Articolo su rivista]
I., Mariotti; S., Spaggiari; S., Bonetti; C., China; C., Spano; D., Campana; V., Rasini; Dominici, Massimo; Pf, Conte; Paolucci, Paolo
abstract

NON DISPONIBILE

2005 - SMALL VOLUMES OF SUBCUTANEOUS ADIPOSE TISSUE AS SOURCE OF ADULT MESENCHIMAL STEM CELLS FOR REGENERATIVE MEDICINE AND CANCER CELL-THERAPIES [Abstract in Rivista]
Dominici, Massimo; L., Caffarelli; Guaraldi, Giovanni; S., Kordowich; R., Sternieri; D., Campioni; Paolucci, Paolo; G., Cervo; C., Spano; I., Mariotti; T., Hofman; E., Horwitz; P., Conte
abstract

THE POSSIBILITY OF GENERATING MESENCHIMAL STEM CELLS FROM SMALL VOLUMES OF ADIPOSE TISSUE OPENS NEW PERSPECTIVES TO CLINICAL APPLICATIONS OF REGENERATIVE MEDICINE AND CANCER CELL-THERAPIES

2005 - Transgenic Mice with Pancellular Enhanced Green Fluorescent Protein Expression in Primitive Hematopoietic Cells and All Blood Cell Progeny [Articolo su rivista]
Dominici, Massimo; M., Tadjali; E. R., Allay; S., Kepes; K., Boyd; P. A., Ney; E. M., Horwitz; D. A., Persons
abstract

Transgenic mice homogeneously expressing enhanced green fluorescence protein (EGFP) in primitive hematopoietic cells and all blood cell progeny, including erythrocytes and platelets, have not been reported. Given previous data indicating H2Kb promoter activity in murine hematopoietic stem cells (HSCs), bone marrow (BM), and lymphocytes, an H2Kb enhancer/promoter EGFP construct was used to generate transgenic mice. These mice demonstrated pancellular EGFP expression in both primitive BM Sca-1+Lin-Kit+ cells and side population (SP) cells. Additionally, all peripheral blood leukocytes subsets, erythrocytes, and platelets uniformly expressed EGFP strongly. Competitive BM transplantation assays established that transgenic H2Kb-EGFP HSCs had activity equivalent to wildtype HSCs in their ability to reconstitute hematopoiesis in lethally irradiated mice. In addition, immunohistochemistry revealed EGFP transgene expression in all tissues examined. This transgenic strain should be a useful reagent for both murine hematopoiesis studies and functional studies of specific cell types from particular tissues.

2005 - Translational Research Using Marrow Mesenchymal Cells in Bone Repair/Osteogenesis Imperfecta [Abstract in Rivista]
E. M., Horwitz; Dominici, Massimo; T. J., Hofmann
abstract

not available

2005 - Varicella zoster virus reactivation in patients with breast cancer and multiple myeloma after autologous stem cell transplantation [Abstract in Rivista]
Maur, M; Sabbatini, R; Cuoghi, A; Cafarelli, L; Dominici, Massimo; Piacentini, Federico; Giovannelli, S; Conte, Pierfranco
abstract

not available

2004 - A role for SCL in developing bone? the SCL+19 core enhancer targets expression to cells of developing bone, osteocytes, chondrocytes and bone lining cells [Abstract in Rivista]
Silberstein, L; Pimanda, Je; Piltz, S; Delaney, L; Oldham, S; Kallianpur, A; Dominici, Massimo; Brandt, S; Gottgens, B; Green, A.
abstract

The transcription factor SCL/TAL1 is essential for haematopoiesis and vascular development in the embryo. It is also expressed in the developing skeletal and central nervous systems, but lethality of SCL null mice has precluded detailed analysis of the role of this gene in these tissues.We have previously demonstrated that the SCL +18/19 enhancer directs reporter gene expression to haemangioblasts, endothelium and haematopoietic progenitors. Using human placental alkaline phosphatase as a reporter gene, we now report that the +19 core enhancer also directs expression to osteocytes, articular chondrocytes and bone lining cells in adult mice. In E14.5 embryos, the transgene is expressed in cells within the cartilaginous template of future bone and in the perichondrium. The pattern of expression of the transgene resembles that of endogenous SCL RNA and protein expression in age matched embryos. At E16.5, transgene-positive cells are seen within ossification centres. Activity of the enhancer during osteogenesis was observed both in bones formed through intra-membranous (e.g. cranial vault) and endochondral ossification (limbs and axial skeleton). We found that the murine pre-osteoblast cell line, MC3T3, expresses endogenous SCL. Stable transfection of these cells with luciferase reporter constructs that include the +19 core enhancer produce a 5-fold increase in luciferase activity. There is a progressive reduction in SCL RNA expression in these cells during osteogenic differentiation. This is consistent with a previous report that expression of SCL protein is down-regulated in skeletal tissues of more advanced stage embryos, and with our finding that the MLO-Y4 cell line, which phenotypically and functionally resembles mature osteocytes, does not express SCL. We addressed the issue of SCL function in bone formation using in-vitro differentiation of embryonic stem (ES) cells. Wild type and SCL–/– J1 ES cells did not differ in their ability to form bone nodules. These data demonstrate that SCL is not required for bone specification, but do not exclude a role for SCL in regulating bone formation in vivo. The SCL +19 core enhancer directs expression to haematopoietic progenitors and endothelium together with their putative precursors, haematopoietic endothelium and haemangioblasts. Our demonstration that the +19 core enhancer also targets expression to osteogenic cells is consistent with recent data suggesting that haematopoietic and osteogenic cells share a common precursor (Olmsted-Davis, EA et al PNAS 2003, Dominici M et al PNAS 2004) and that osteoblastic bone lining cells are intimately associated with haematopoietic stem cells (Calvi LM et al Nature 2003; Zhang J et al Nature 2003).

2004 - Hematopoietic cells and osteoblasts are derived from a common marrow progenitor after bone marrow transplantation [Articolo su rivista]
Dominici, Massimo; C., Pritchard; J. E., Garlits; T. J., Hofmann; D. A., Persons; E. M., Horwitz
abstract

Bone and bone marrow are closely aligned physiologic compartments, suggesting that these tissues may represent a single functional unit with a common bone marrow progenitor that gives rise to both osteoblasts and hematopoietic cells. Although reports of multilineage engraftment by a single marrow-derived stem cell support this idea, more recent evidence has challenged claims of stem cell transdifferentiation and therefore the existence of a multipotent hematopoietic/osteogenic progenitor cell. Using a repopulation assay in mice, we show here that gene-marked, transplantable marrow cells from the plastic-nonadherent population can generate both functional osteoblasts/osteocytes and hematopoietic cells. Fluorescent in situ hybridization for the X and Y chromosomes and karyotype analysis of cultured osteoblasts confirmed the donor origin of these cells and excluded their generation by a fusion process. Molecular analysis demonstrated a common retroviral integration site in clonogenic hematopoietic cells and osteoprogenitors from each of seven animals studied, establishing a shared clonal origin for these ostensibly independent cell types. Our findings indicate that the bone marrow contains a primitive cell able to generate both the hematopoietic and osteocytic lineages. Its isolation and characterization may suggest novel treatments for genetic bone diseases and bone injuries.

2004 - Isolation and expansion of functional differentiating mesenchymal stem cells with a serum deprived medium. [Abstract in Rivista]
Dominici, Massimo; Mueller, I; Cafarelli, L; Kordowich, S; Sternieri, R; Spano, C; Chiodino, C; Mariotti, I; Petak, I; Horwitz, E; Paolucci, Paolo; Conte, Pierfranco
abstract

The infusion of mesenchymal stem cells (MSC) is an increasingly utilized therapeutic approach for congenital and acquired diseases in regenerative medicine. Recently, MSC have been used for the treatment of graft-versus-host disease after allogeneic stem cell transplantation[1]. These strategies require ex vivo MSC expansion with media containing fetal bovine serum (FBS). Since the use of FBS may be associated with immunological responses against the cultured MSC[2] and may unknowingly transmit prions[4], the development of a serum free medium (SFM) for MSC isolation and expansion is essential for the widely applicable MSC therapy. We evaluated a novel serum deprived medium (Quantum 333R) for the isolation and expansion of MSC as compared to the standard medium (SM), DMEM + 10% FBS. Fibroblastoid colony forming unit (CFU-F), as MSC progenitors, and MSC expansion potential were considered as measurable parameters. The number of CFU-F found at 14 days of culture in the SFM (average: 1 CFU-F/ 1.78 x 105 bone marrow mononuclear cells, BMMNC) and the SM (1 CFU-F/ 2.3 x 105 BMMNC) were similar (p=0.18 t-test), but the trend suggested that the SFM may be superior. Passage 5 MSC in both culture conditions showed similar percentages of proliferating, i. e. BrdU+ cells: 7,5% (+/– 1,3%) and 10% (+/– 2,1%) in MSF and in SM, respectively. Flow cytometric analysis of MSC for side and forward scatter properties showed that MSCSFM were smaller in the side scatter than MSC SM. Further immunophenotypical characterization showed, in the MSCSFM, the absence of CD45, CD34, CD31 and the presence of CD90, CD105, CD73 confirming the phenotype of the "classical" MSCSM. CD73 and CD105 were upregulated in MSCSFM in comparison to MSCSM. Still, MSCSFM retained the capacity to inhibit the cytokine-induced proliferation of PBMC as reported for MSCSM. In addition, to test the differentiation potential of MSCSFM, the cells were successfully driven into osteogenic, adipogenic, myogenic and chondrogenic phenotypes. Our data support that Quantum 333R is able to isolate and expand MSC clonogenic precursors without affecting their proliferation, immunophenotype, differentiation and immunomodulatory potentials, thus suggesting a superior technique to isolate clinical grade cell population for safer clinical applications.

2004 - Le Cellule Staminali Mesenchimali: Aspetti Biologici ed Approcci Terapeutici [Articolo su rivista]
Dominici, Massimo; Guarneri, Valentina; Cafarelli, Luigi; Conte, Pierfranco
abstract

The pioneeristic studies, started thirty years ago, were able to uncover an interesting bone marrow derived cell population named as mesenchymal stem cells (MSC). This cell type, used in the last decade in both pre-clinical and clinical phases in several fields of biomedical sciences, opened up innovative branches of translational research. In this review we analyze the biological background of the MSC with the purpose to identify their actual therapeutical applications with a special focus on their possible future role in oncology.

2003 - Development and functional characterization of human bone marrow mesenchymal cell immortalized by enforced expression of telomerase [Articolo su rivista]
K., Mihara; C., Imai; Coustan, Smith; J. S., DOME JS; Dominici, Massimo; E., Vanin; D., Campana
abstract

To create immortal mesenchymal cell lines, we transduced primary human bone marrow mesenchymal cells with telomerase reverse transcriptase (TERT). TERT+ mesenchymal cells continued to grow for > 2 years; parallel TERT- cultures underwent senescence after 15 weeks. TERT+ mesenchymal cells did not form foci in soft agar, had a normal karyotype and could differentiate into osteoblasts and chondrocytes. Their capacity to support leukaemic lymphoblasts and normal CD34+ haematopoietic cells was equal to or greater than that of primary cells; 42 TERT+ mesenchymal cell clones varied in their supporting capacity. Immortalized mesenchymal cells offer a promising tool for identifying molecules that regulate human haematopoiesis

2003 - Enrichment of CD105+ and fibroblast stromal cells from healthy donors and AML patients [Articolo su rivista]
D., Campioni; Dominici, Massimo; F., Lanza
abstract

Fractions isolated with CD105 MicroBeads and Anti-Fibroblast MicroBeads represent sources of bonemarrow stromal cells useful for in vitro investigations of hematological disorders.

2003 - Functional and immunophenotypic characteristics of isolated CD105(+) and fibroblast(+) stromal cells from AML: implications for their plasticity along endothelial lineage [Articolo su rivista]
D., Campioni; F., Lanza; S., Moretti; Dominici, Massimo; M., Punturieri; S., Pauli; T. J., Hofmann; E. M., Horwitz; G., Castoldi
abstract

BACKGROUND: In vitro cultures of BM cells from newly diagnosed patients with AML displayed a defective BM stromal compartment, with a reduced number of fibroblast-colony-forming unit (CFU-F: 1 +/- 1.25 SD) and a decreased proliferative ability. The purposes of our study were: 1). to select BM mesenchymal stem cells (MSC) and BM-derived stromal cells (BMDSCs) from AML patients at diagnosis and from healthy subjects, using an immunomagnetic system and either anti-CD105 or anti-fibroblast MAbs; 2). to study the immunophenotypic and functional properties of freshly isolated and cultured mesenchymal cells; 3). to test the in vitro plasticity of the selected cells to differentiate towards an endothelial phenotype.METHODS: Fresh mononuclear cells obtained from BM of 20 patients newly diagnosed with AML and from eight healthy subjects were selected by using anti-fibroblast and anti-CD105 MAbs. Freshly isolated cells were analyzed, characterized by flow cytometry using a wide panel of MAbs and seeded in long-term culture medium to assess CFU-F formation. The level of confluence after 30 days and functional capacity in a long-term colony-forming cell culture (LTC-CFC) were tested. Furthermore, the cultured selected cell populations were assayed for their ability to differentiate into an endothelial-like cell phenotype with the addition of vascular endothelial growth factor (VEFG) and endothelial cell growth supplement (ECGS).RESULTS: In normal subjects the selection produced an increase of the CFU-F number of 2.6-fold with anti-fibroblast MAb and 2.7-fold with the anti-CD105 MAb. Anti-fibroblast and anti-CD105 MAb selection from AML BM cells resulted in a statistically significant greater count of CFU-F that was respectively 10.6-fold (P = 0.04) and 14.4-fold (P = 0.00001) higher in comparison with the unselected AML samples. Interestingly, in 80% of AML samples immunoselection was also able to restore the capacity of the CFU-F to proliferate and form confluent stromal layers. The isolation of those layers sustained the proliferation and differentiation of hematopoietic stem cells in the LTC-CFC. The phenotypic profile of cultured BMDSCs was different from that of the freshly isolated cells, and changed in relation to the culture conditions: CD105+ selected cells cultured with VEGF and ECGS expressed endothelial markers, a finding that suggests that this cell subpopulation may have the potential to differentiate toward an endothelial-like phenotype.DISCUSSION: We report that immunomagnetic selection represents a valid tool for the selection of BM mesenchymal cells in samples obtained from both healthy subjects and patients with AML. This technique was able to rescue two functional and immunophenotypic compartments related to two different selected populations. In particular, the CD105+ cells isolated in AML displayed, after stimulation with VEGF and ECGS, the ability to change towards an endothelial-like cell phenotype, thus revealing an unexpected plasticity. Both CD105+ and fibroblast+ cells once successfully isolated might represent sources of mesenchymal cells populations useful for in vitro investigations and, above all, as therapeutic devices.

2002 - Acute colon diverticulitis in multiple myeloma patient: an unusual presentation of a colonic perforation. Case report. [Articolo su rivista]
G., Soliani; Dominici, Massimo; L., Bergossi; E., Basaglia; S., Pauli; P., Carcoforo
abstract

This case report describes an acute colonic diverticular perforation occurred to a multiple myeloma patient, taking corticosteroid and morphine therapy, revealed by a subcutaneous emphysema of upper chest and right abdomen as initial presentation. Sigmoid diverticulitis with perforation and generalized peritonitis is a severe complication of the diverticular disease and it is due to diverticular microperforation. This condition occurs more frequently in patients with widespread diverticolosis and usually after 50 years of age, and the frequency of related complications increases with age (and with the use of corticosteroids). Extraperitoneal air from the sigmoid-rectum perforation can escape diffusing superiorly though paravertebral retroperitoneal tissues and via the diaphragmatic iatus into the mediastinum, producing pneumomediastinum and it diffuses to yield superior thoracic emphysema. This report suggests that the diagnosis of retroperitoneal perforation is usually difficult because of the lack of signs of peritoneal irritation and the paucity of symptoms, particularly in patients treated with corticosteroids.

2002 - Comparison of single and dual platform methodologies for the estimation of CD34+ hematopoietic progenitor cells: correlation with colony assay [Articolo su rivista]
S., Moretti; M., Dabusti; B., Castagnari; A., Tieghi; L., Ferrari; D., Campioni; M., Punturieri; Dominici, Massimo; G. L., Castoldi; F., Lanza
abstract

In this study three assays for the enumeration of CD34+ progenitors were compared: 1) a modified version of the Milan protocol, used in the standard dual-platform format; 2) a dual-platform version of the ISHAGE protocol; 3) the ProCOUNT software version 2.0/ProCOUNT kit. The assays were compared to validate the accuracy of CD34+ cell counts in mobilized peripheral blood (PB), apheresis products (AP), and cord blood (CB). The ProCOUNT protocol uses reference beads for absolute CD34+ cell counting, whereas CD34 counts by other techniques are derived from a separate leukocyte count performed by a hematology analyzer. A good correlation between the ISHAGE and ProCOUNT methods was obtained for estimation of CD34+ counts in PB (n=42 samples analyzed) and AP (n=35)--except for samples having a leukocyte count >25 x 10(9)/L or a CD34 count <0.0025 x 10(9)/L)--while a suboptimal correlation between the methods was observed for CB (n=30). The ProCOUNT system proved to be effective in reducing the variability in CD34+ cell counting and appeared to be useful for intralaboratory methodology standardization. The main disadvantage of the ProCOUNT assay was its inability to calculate CD34 counts in leukopenic samples and in CB samples showing a high erythroblast count. As far as the correlation with hematopoietic colonies is concerned, data collected from apheresis samples showed a good correlation between the three flow cytometry methods and colony-forming unit granulocyte-macrophage (CFU-GM) counts, confirming the value of the flow cytometric test as a real-time, truly predictive test to measure the hematopoietic potential of the graft. In summary, all methods are suitable for enumeration of most PB samples, while the single-platform methodology should be preferred for the analysis of AP and CB. We also found the dual-platform format of the ISHAGE method precise and accurate for the estimation of CD34+ cells from CB samples. Based on these data it can be concluded that the single-platform flow cytometry assay format should be the preferred approach for CD34+ stem cell enumeration in different types of samples.

2002 - On the development of cell therapy for genetic disorders [Articolo su rivista]
E. M., HORWITZ; T. J., HOFMANN; J. E., GARLITS; D., CAMPIONI; DOMINICI, Massimo
abstract

Cell therapy is the process of using intact cells to treat adisease or other unwanted condition. For genetic disorders,cell therapy is based on the premise that allogeneic, geneticallynormal, stem or progenitor cells may be used torepopulate the cells of a genetically defective tissue, effectivelycorrecting the underlying genetic disorder. Theclinical potential of cell therapy is enormous. To begin toappreciate the possible uses of cell therapy in medicine, it isimportant to understand the relationship of cell therapy toother treatment strategies, such as drugs and gene therapy.

2001 - Angiogenesis in multiple myeloma: Correlation between in vitro endothelial colony growth (CFU-En) and clinical biological features [Articolo su rivista]
Dominici, Massimo; Luppi, Mario; F., Lanza; Campioni, D.; Barozzi, Patrizia; S., Pauli; R., Milani; F., Cavazzini; M., Punturieri; R., Trovato; G., Torelli; G., Castoldi
abstract

Mouse models and studies performed on fixed bone marrow (BM) specimens obtained from patients with multiple myeloma (MM) suggest that plasma cell growth is dependent on endothelial cell (EC) proliferation within the BM microenvironment. In order to assess whether EC overgrowth in MM reflects a spontaneous in vitro angiogenesis, BM mononucleated cells from 13 untreated (UT) MM, 20 treated (11 with melphalan and nine with DAV schedule) MM, eight patients with monoclonal gammopathy of uncertain significance (MGUS) and eight controls were seeded in an unselective medium to assess EC proliferation. Furthermore, the influence of IL6 on the EC growth was investigated. Endothelial colonies (CFU-En) appeared as small clusters, formed by at least 100 slightly elongated and sometimes bi-nucleated cells expressing factor VIII, CD31 and CD105 (endoglin). The CFU-En mean number/10(6) BM mononucleated cells in untreated MM samples (2.07 s.d. +/- 1.3) was significantly higher than in normal BM (0.28 +/- 0.48), while no difference was seen between normal BM and MGUS (0.28 +/- 0.54). Interestingly, the mean number of CFU-En in the DAV group (1.88 +/- 1.6) did not differ from the UT, while it was found to be lower in the melphalan group (0.31 +/- 0.63). The addition of anti-IL6 monoclonal antibody induced a reduction of both the plasma cells in the supernatant and the CFU-En number. This study describes a rapid and feasible assay providing support for the association between EC and plasma cells further suggesting that the in vitro angiogenesis process may parallel that observed in vivo.

2001 - Bone marrow mesenchymal cells: biological properties and clinical applications. [Articolo su rivista]
Dominici, Massimo; T., Hofmann; E. M., Horwitz
abstract

In postnatal mammals, the primary site of hematopoiesis is the bone marrow (BM) extravascular compartment. As cells mature, they are released into the vascular compartment through the endothelium and the adventitial layer of mature blood elements. In the BM extravascular space, a dense network of cells constitutes the bone marrow microenvironment (BMM). The BMM provides structural and functional support for the self-renewal and differentiation of hematopoietic progenitor cells as cytokines and cell-to-cell interactions within proliferation niches of the BMM regulate these processes. In this paper, recent advances in understanding the nature of BMM cells are described with a specific focus on mesenchymal progenitors describing their main biological properties and potential clinical applications.

2001 - CD 123 (Interleukin 3 receptor alpha chain) [Articolo su rivista]
S., Moretti; F., Lanza; M., Dabusti; A., Tieghi; D., Campioni; Dominici, Massimo; G. L., Castoldi
abstract

not available

2001 - CD34 (+) cell subsets and long-term culture colony-forming cells evaluated on both autologous and normal bone marrow stroma predict long-term hematopoietic engraftment in patients undergoing autologous peripheral blood stem cell transplantation. [Articolo su rivista]
F., Lanza; D., Campioni; S., Moretti; Dominici, Massimo; M., Punturieri; E., Focarile; S., Pauli; M., Dabusti; A., Tieghi; M., Bacilieri; G. L., Scapoli; C., DE ANGELI; L., Galluccio; G. L., Castoldi
abstract

The aim of this study was to evaluate which CD34(+) cell subset contained in leukapheresis products could be regarded as the most predictive of long-term hematopoietic recovery after autologous peripheral blood stem cell transplantation (auto-PBSCT). MATERIALS AND METHODS: Based on data from 34 patients with hematologic malignancies, doses of CD34(+) cells and CD34(+) cell subsets, defined by the expression of HLA-DR, CD38, CD117 (c-kit/R), CD123 (alpha subunit of IL-3/R), CD133 (AC133), and CD90 (Thy-1) antigens, were correlated with the number of short-term (i.e., colony-forming cells [CFC]) and long-term culture CFC (LTC-CFC) (generated at week 5 of culture) and with the kinetics of hematopoietic engraftment following auto-PBSCT. The capacity of autologous stroma (AS), normal human bone marrow stroma, and M2-10B4 murine cell line to sustain CD34(+) cell growth was comparatively evaluated in the LTC assay. RESULTS: Our data demonstrated that some of the most primitive progenitor subsets (CD34(+)CD117(-)HLA-DR(-), and CD34(+)CD38(+)HLA-DR(-)) showed the strongest correlation with LTC-CFC numbers generated within the AS, whereas no significant correlation was noted using normal bone marrow stroma. Multivariate analysis showed that the only CD34 cell subset independently associated with long-term (3 to 6 months) platelet engraftment after auto-bone marrow transplantation was the CD34(+)CD117(-)HLA-DR(-) phenotype; long-term erythrocyte engraftment was correlated with CD34(+)CD38(+)HLA-DR(-) cell content. The latter further influenced platelet engraftment in the first 3 months after auto-PBSCT. The most predictive parameters for neutrophil engraftment were CD34(+)CD38(+)HLA-DR(-) cell subtype and the total LTC-CFC quantity infused. CONCLUSIONS: These data further support the hypothesis that the type of stromal feeders influences the frequency of LTC-CFC, possibly because they differ in their ability to interact with distinct subsets of hematopoietic stem cells. Furthermore, as the use of AS in LTC assay can mimic in vitro the human bone marrow microenvironment, it can be speculated that this culture system could be a useful means to study the kinetics of recovery of bone marrow stroma following chemotherapy and PBSCT. From these results, it can be concluded that some CD34(+) cell subsets appear to be more reliable predictors of long-term hematopoietic recovery rates than total CD34(+) cell quantity

2000 - Bone marrow stromal cells in acute promyelocytic leukemia: comparison with other acute leukemia subtypes [Abstract in Rivista]
Dominici, Massimo; D., Campioni; F., Lanza; Pauli, S; Punturieri, M; Castagnari, B; Moretti, S; Capoli, G; Castoldi, G.
abstract

Acute promyelocytic leukemia (APL) with t(15;17), significantly differs from other acute myelogenous leukemias (AML) by the prognosis which has been significantly improved after introduction of retinoic acid, as a differentiating agent. In an attempt to clarify whether APL differs from AML with regards to the stromal cell compartment too, bone marrow mononucleated cells (BM/MNC) from 21 untreated AML patients (M0: 1; M1: 4; M2: 4; APL: 6; M4: 2; M5:4) were plated in long-term culture. The fibroblastoid clonogenic precursors (CFU-F/ 106 BM/MNC; Collagen I/II/III+, CD68+) and the endothelial colonies (CFUEn CD31+, factor VIII+) content were assessed after 15 days by an immunohistochemical technique. Fibroblast confluence , in the T25 flask, was evaluated after 40 days by inverted microscope. CFU-F % conference p (Wilcoxon) AML (all cases) (mean±s.d) 4.4±4.7 50±30 0.01 non-APL AML 3.4±2.9 38.1±31.2 0.01 APL 7.6±2.1 71±11 n.s Controls 12.3±4.4 90±10 Considering CFU-En, the non-APL group displayed a 3.8-fold higher value than the control group (1.1±1 vs 0.28±0.4; p=0.05), while this difference was not present in APL samples (0.5±1.1). In conclusion, the biological characteristc of bone marrow stromal cells was found to be different in APL, in comparison with other AML subvarieties. This may be due to the different nature of the diseases. Moreover, we give evidence of an high angiogenesis rate in AML which appeared more prominent in non-APL samples.

2000 - Bone marrow stromal cells in acute promyelocytic leukemia: comparison with other acute myelogenous leukemia subtypes [Articolo su rivista]
Dominici, M.; Campioni, D.; Lanza, F.; Moretti, S.; Horwitz, E. M.; Spanedda, R.; Castoldi, G.
abstract

Acute promyelocytic leukemia (APL) with t(15;17), significantly differs from other acute myelogenous leukemias (AML) by the prognosis which has been significantly improved after introduction of retinoic acid, as differentiating agent. In the attempt to clarify if APL differs from AML regarding stromal cell compartment too, bone marrow mononucleated cells (bmmnc) from 21 untreated AML patients (MO: 1 ; Ml : 4; M2:4; APL: 6; M4: 2; M5:4) were plated in long-term culture. The fibroblastoid clonogenic precursors (CFU-F/ 106 bmmnc; Collagen I/II/1II+, CD68+) and the endothelial colonies (CFU-En CD31+, Factor VIII+) content, as expression of angiogenic potential of the AML samples, were assessed after 15 days by immunohistochemical technique. Fibroblast confluence (see table), in T25 flask, was evaluated after 40 days by inverted microscope. Results are in detail described in the table below. Considering CFU-En. no-APL group display 3.8-fold value than control (1.1+/-1 vs 0.28+/-0.4; p=0.05), while this difference is not present in APL samples (0.5+/-1.1). In conclusion, the biologic characteristics of bm stromal cell was found to be different in APL, in comparison with other AML subvarieties. This may be due to the different nature of the diseases. Moreover, we give evidence of an high angiogenesis rate in AML which appeared more prominent in no-APL samples. CFU-F %Confluence p(wilcoxon) AMLfall cases) 4.4+/-4.T 50+/-30 0.01 AML 3.4+/-2.9 38+/-31 0.01 APL 7.6+/-2.1 71+/-11 not signif. Controls 12.3+/-4.4 90+/-10.

2000 - IMPAIRMENT OF THE COMPARTMENT OF STROMAL CELLS IN MYELODYSPLASTIC SYNDROMES [Abstract in Rivista]
D., Campioni; Dominici, Massimo; Lanza, F; Punturieri, M; Pauli, S; Tieghi, A; Dabusti, M; Ferrari, L; Spanedda, R; Castoldi, G.
abstract

We assessed the in vitro behavior of bone marrow stromal cells (BM-SC) in myelodysplastic syndromes (MDS) with a particular focus on hypoplastic MDS (hMDS). H-MDS is an entity characterized by cytopenia, BM hypoplasia sharing similarities with severe aplastic anemia (SAA), except for the presence of a normal number of CD34+ cells, occurrence of abnormal localized immune precursors, dysmegakaryocytopoiesis and involvement of -7/7q- chromosome. Thirty-two cases were considered: 10 refractory anemia (RA), 3 RA with ring sideroblasts (RARS), 8 RA with excess of blasts (RAEB), 5 RAEB-t and 6 cases of hMDS. BM cells were cultured with long-term culture medium to evaluate the mesenchymal clonogenic precursors (CFU-F) and the stromal layer confluence capacity in a T25 flask. MDS stromal cells gave evidence of defective cell growth capacity vs controls (see Figures 1 & 2). In hMDS, CFU-F significantly (Wilcoxon test) differed from that in controls, RA/RARS (p=0.02) and RAEB (p=0.01) groups; the same difference was maintained considering the stromal confluence (p=0.01). An increased apoptosis process involving both the hemopoietic compartment and BM-SC has been reported in MDS, with the maximum rate occurring in the RAEB-t samples (Raza et al., Blood, 1995). Our in vitro findings confirm an impairment of BM-SC in MDS, suggesting how hMDS differs from both MDS or SAA, in which the stromal cell compartment seems to be spared, adding a further clue that differentiates these two similar forms of BM failure.

2000 - PCR with degenerate primers for highly conserved DNA polymerase gene of the herpesvirus family shows neither human herpesvirus 8 nor a related variant in bone marrow stromal cells from multiple myeloma patinets. [Articolo su rivista]
Dominici, Massimo; Luppi, Mario; D., Campioni; F., Lanza; Barozzi, Patrizia; R., Milani; S., Moretti; G., Nadali; R., Spanedda; R., Trovato; Torelli, Giuseppe; G., Castoldi
abstract

The possibility has been raised that either a human herpesvirus-8 (HHV-8) variant or a novel, unidentified, gamma-herpesvirus related to HHV-8 is frequently associated with multiple myeloma (MM), which could explain the lack of antibodies to HHV-8 antigens and the discordant results from polymerase chain reaction (PCR) studies of HHV-8-specific sequences in MM patients. Thus, we used a sensitive PCR assay with degenerate primers targeting the highly conserved DNA polymerase gene of the herpesvirus family to examine the long-term cultures of bone marrow stromal cells (BMSCs) from 19 MM, 3 monoclonal gammopathies of undetermined significance and 6 control patients. Both the culture supernatant and the adherent stromal layer were examined from the 2nd until the 8th week of culture to assess the immunophenotype of the various cell types harvested for the molecular analysis. BMSCs consisted of a mixed population of fibroblast, macrophage, dendritic and endothelial cells. An amplified product of the expected size was obtained only in 3 MM cases, both in the adherent and nonadherent fractions. Direct sequencing and alignment of the nucleotide and amino acid sequences showed that the DNA sequences were 100% identical to Epstein-Barr virus (EBV) DNA. The PCR positivity was due to the presence of EBV-infected lymphoblastoid cells with plasmacytoid features, expressing the EBV-encoded latent membrane protein-1 and detectable either in the stromal cells or in the culture supernatant. Our data do not support a causal role of either HHV-8 or a novel herpesviral variant related to HHV-8 in MM.

2000 - Prolonged remission state of refractory adult onset Still's disease following CD34-selected autologous peripheral blood stem cell transplantation [Articolo su rivista]
F., Lanza; Dominici, Massimo; M., Govoni; S., Moretti; D., Campioni; R. L., Corte; A., Latorraca; A., Tieghi; B., Castagnari; F., Trotta; G. L., Castoldi
abstract

We report a 38-year-old patient affected by refractory adult onset Still's disease who achieved a prolonged remission following CD34-selected ABMT. The conditioning regimen was based on the use of CY and anti-thymocyte globulin. A 3.0 and 2.0 log reduction of T (CD3+) and B (CD19+) lymphocytes, respectively, was obtained using a Ceprate device to select CD34+ cells from PBSC. In the pre-transplant period (1994-1998) the patient had a chronic persistent disease course with frequent and recurrent systemic articular flares and loss of some functional abilities, despite daily prednisone, pulses of CY and immunosuppressive therapy (CYA or MTX). At the time of ABMT the patient had become non-ambulatory. Within 3 weeks of ABMT the patient showed a marked decrease in joint swelling, and morning stiffness. Joint pain and systemic symptoms disappeared, the patient was able to walk and run and gained general well being. ESR, C-reactive protein and WBC count were significantly decreased, while Hb level increased. This partial remission persisted for at least 1 year after ABMT, although at 15 months of follow-up a reappearance of moderate synovitis in the knees and wrists was noted. Our data further showed that both patient BM microenvironment and stem-progenitor cell function (as assessed by LTC-IC assay) were damaged even 1 year after CD34-selected ABMT, suggesting that the persistence of these alterations could have facilitated the favorable outcome of the disease following ABMT

1999 - Assessment of distribution of CD34 epitope classes in fresh and cryopreserved peripheral blood progenitor cells and acute myeloid leukemic blasts. [Articolo su rivista]
F., Lanza; S., Moretti; B., Castagnari; F., Montanelli; A., Latorraca; L., Ferrari; A., Bardi; Dominici, Massimo; D., Campioni; M., Dabusti; N., Piva; G., LODI G; R., Reverberi; G. L., Castoldi
abstract

So far several reports have described changes in the expression of surface antigens in progenitor cells and blasts following cryopreservation. However, there are no data on the effects of cryopreservation on the expression of the three CD34 epitope classes, and on their relationship with the clonogenic capacity of PBPC collected by leukapheresis. DESIGN AND METHODS: In order to analyze the effects of freezing/thawing procedures (Eth 80C storage for 3 months) and use of dimethylsulfoxide (DMSO) on the immunophenotype profile and colony production of peripheral blood progenitor cells (PBPC) in apheresis products derived from 20 patients with stage 0-III non-Hodgkin's lymphoma (nHL), a flow cytometry study was undertaken using different CD34 monoclonal antibodies (MoAbs) capable of recognizing the 3 epitope classes of CD34 molecule (class III: HPCA-2/FITC, HPCA-2/PE, 581/FITC, 581/PE; class II: Q-Bend 10/PE; class I: ICH3/PE, BI3C5-PE, Immu-133-PE). CD34 epitope expression was also analyzed in thawed CD34+ blasts obtained from 14 patients with acute myeloid leukemia (AML), who were analyzed using a larger number (#17) of CD34 epitope class I, II, and III reactive MoAbs. RESULTS: Under our experimental conditions it was found that class III and class II CD34 epitopes (differentially resistant to enzymatic cleavage with neuraminidase, chymopapain and glycoprotease) are better preserved than class I epitope Eth sensitive to degradation Eth after cell exposure to cryoprotectant DMSO and the freezing- thawing procedures. Results further showed a concomitant decrease in class I CD34+ counts and in BFU-E colony production. A significant increase in CD34 antigen expression levels (i.e. antibody binding capacity, ABC) by cryopreserved cells stained with CD34 epitope class III, and class II reactive MoAbs was also documented, while no changes after cryopreservation were noted using class I-reactive MoAbs. The slight increase in the percentage of CD34+ cells detected after frozen storage was correlated to a concomitant decrease in the number of more mature myeloid cells (CD15+, CD13+, CD33+). Compared to pre-cryopreservation values, a slight reduction in class I CD34 epitope expression was also found in thawed CD34+ AML blasts. INTERPRETATION AND CONCLUSIONS: As far as the reduction of class I CD34 epitope is concerned, it may be hypothesized that the freezing procedure, use of DMSO, and/or lysis methodology may either damage a CD34 subset, or induce distinct alterations of the CD34 glycoprotein, possibly determining a reduction in their immunoreactivity with some CD34 MoAbs. In conclusion, this study has shown that exposure to the cryoprotectant DMSO and the freezing/thawing procedures modifies the distribution of CD34 epitopes as well as the clonogenic capacity of PBPCs from nHL patients, and CD34+ blasts from AML. These findings need to considered when selecting CD34 MoAbs for enumeration and positive selection of stem/progenitor cells for research and clinical purposes.

1999 - Caratterizzazione degli epitopi del CD34 e analisi dei recettori per fattori di crescita nelle cellule staminali ematopoietiche [Articolo su rivista]
F., Lanza; S., Moretti; A., Latorraca; B., Castagnari; F., Montanelli; R., MILANI R; Dominici, Massimo; M., Dabusti; L., Ferrari; G. L., Castoldi
abstract

non disponibile

1999 - Lack of confirmation of an association between HTLV-I infection and myelodysplastic syndrome [Articolo su rivista]
M., Morselli; Luppi, Mario; Barozzi, Patrizia; Dominici, Massimo; Temperani, Paola; D., Campione; F., Lanza; R., Trovato; Marasca, Roberto; G., Longo; G., Emilia; Torelli, Giuseppe
abstract

No abstract available

1999 - Neither Human Herpesvirus 8 nor a related variant could be detected in bone marrow stromal cells from multiple myeloma patients. [Abstract in Rivista]
Dominici, Massimo; Luppi, Mario; D., Campioni; F., Lanza; Barozzi, Patrizia; R., Milani; A., Latorraca; S., Moretti; Torelli, Giuseppe; G. L., Castoldi
abstract

The possibility has been raised that either a human herpesvirus- 8 (HHV-8) variant or a novel, unidentified, g-herpesvirus related to HHV-8 is frequently associated with multiple myeloma (MM), which could explain the lack of antibodies to HHV-8 antigens and the discordant results from polymerase chain reaction (PCR) studies of HHV-8-specific sequences in MM patients. Thus, we used a sensitive PCR assay with degenerate primers targeting the highly conserved DNA polymerase gene of the herpesvirus family to examine the longterm cultures of bone marrow stromal cells (BMSCs) from 19 MM, 3 monoclonal gammopathies of undetermined significance and 6 control patients. Both the culture supernatant and the adherent stromal layer were examined from the 2nd until the 8th week of culture to assess the immunophenotype of the various cell types harvested for the molecular analysis. BMSCs consisted of a mixed population of fibroblast, macrophage, dendritic and endothelial cells. An amplified product of the expected size was obtained only in 3 MM cases, both in the adherent and nonadherent fractions. Direct sequencing and alignment of the nucleotide and amino acid sequences showed that the DNA sequences were 100% identical to Epstein-Barr virus (EBV) DNA. The PCR positivity was due to the presence of EBV-infected lymphoblastoid cells with plasmacytoid features, expressing the EBV-encoded latent membrane protein-1 and detectable either in the stromal cells or in the culture supernatant. Our data do not support a causal role of either HHV-8 or a novel herpesviral variant related to HHV-8 in MM.

1999 - PCR con primer degenerati non evidenzia sequenze virali di HHV-8 ne di una sua variante in cellule stromali midollari di pazienti affetti da gammopatie monoclonali [Abstract in Rivista]
Dominici, Massimo; Luppi, Mario; D., Campioni; F., Lanza; Barozzi, Patrizia; R., Milani; S., Moretti; R., Spanedda; Torelli, Giuseppe; G. L., Castoldi
abstract

The association of human herpesvirus-8 (HHV-8) infection with multiple myeloma (MM) is still controversial. The possibility has been raised that either a HHV-8 variant or a novel, as yet unidentified, γ-herpesvirus related to HHV-8, are frequently associated with MM, which could explain the lack of antibodies to HHV-8 antigens and the discordant results from polymerase chain reaction studies (PCR) of HHV-8 specific genomic sequences in MM patients. Thus, we used a sensitive PCR assay with degenerate primers targeting the highly conserved DNA polymerase gene of the herpesvirus family to examine the long term-cultures (LTC) of bone marrow stromal cells (BMSCs) from 21 MM, 3 monoclonal gammopathy of undetermined significance (MGUS) and 6 control patients. Both the culture supernatant and the adherent stromal layer were examined from the 2nd until the 8th week of culture to assess the immunophenotype of the various cell types harvested for the molecular analysis. BMSCs consisted of a mixed population of fibroblast, macrophage, dendritic and endothelial cells. An amplified product of the expected size was obtained only in 3 MM cases, both in the adherent and non adherent fractions. Direct sequencing and alignment of the nucleotide and amino acidic sequences showed that the DNA sequences were 100% identical to Epstein-Barr virus DNA. PCR with specific primers targeting the latent membrane protein-1 (LMP-1) gene confirmed the presence of EBV. The PCR positivity was due to the presence of EBV infected lymphoblastoid cells (LCLs) with plasmacytoid features, expressing the LMP-1 protein and detectable either in the stromal cells or in the culture supernatant. Our data do not support a causal role of either HHV-8 or a novel herpesviral variant related to HHV-8 in MM, not even in the presence of EBV co-infection.