Enrico TAGLIAFICO - personale UniMoRe

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Enrico TAGLIAFICO

Professore Associato
Dipartimento di Scienze Mediche e Chirurgiche materno infantili e dell'adulto - Sede ex Scienze Biomediche

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Pubblicazioni

2024 - Metastatic site patterns by intrinsic subtype and HER2DX in early HER2-positive breast cancer [Articolo su rivista]
Dieci, M. V.; Conte, P.; Bisagni, G.; Bartolini, S.; Frassoldati, A.; Generali, D.; Piacentini, F.; Griguolo, G.; Tagliafico, E.; Braso Maristany, F.; Chic, N.; Pare, L.; Miglietta, F.; Vicini, R.; D'Amico, R.; Balduzzi, S.; Prat, A.; Guarneri, V.
abstract

Background: Even with contemporary treatment strategies, more than 10% of HER2-positive early stage breast cancer patients may experience distant metastasis as first event during follow-up. Tools for predicting unique patterns of metastatic spread are needed to plan personalized surveillance. We evaluated how molecular heterogeneity affects the pattern of distant relapse in HER2-positive breast cancer. Methods: A total of 677 HER2-positive stage I-III breast cancer patients from ShortHER trial, Cher-LOB trial, and 2 institutional cohorts were included. PAM50 molecular subtypes and research-based HER2DX scores were evaluated. The cumulative incidence of distant relapse as the first event (any site and site specific) was evaluated using competing risk analysis. Median follow-up was 8.4 years. Tests of statistical significance are 2-sided. Results: Stage III and high HER2DX risk score identified patients at the highest risk of distant relapse as first event (10-year incidence 24.5% and 19.7%, respectively). Intrinsic molecular subtypes were associated with specific patterns of metastatic spread: compared with other subtypes, HER2-enriched tumors were more prone to develop brain metastases (10-year incidence 3.8% vs 0.6%, P =. 005), basal-like tumors were associated with an increased risk of lung metastases (10-year incidence 11.1% vs 2.6%, P =. 001), and luminal tumors developed more frequently bone-only metastases (10-year incidence 5.1% vs 2.0%, P =. 042). When added to stage or HER2DX risk score in competing risk regression models, intrinsic subtype maintained an independent association with site-specific metastases. Conclusions: The integration of intrinsic molecular subtypes with stage or HER2DX risk score predicts site-specific metastatic risk in HER2-positive breast cancer, with potential implications for personalized surveillance and clinical trials aimed at preventing site-specific recurrence.

2023 - Abstracts from the 55th European Society of Human Genetics (ESHG) Conference: Hybrid Posters [Abstract in Rivista]
Tenedini, Elena; Piana, Simonetta; Toss, Angela; Marino, Marco; Barbieri, Elena; Artuso, Lucia; Venturelli, Marta; Gasparini, Elisa; Dario Mandato, Vincenzo; Marchi, Isabella; Castellano, Sara; Luppi, Mario; Trenti, Tommaso; Cortesi, Laura; Tagliafico, Enrico
abstract

2023 - Allele specific CRISPR/Cas9 editing of dominant Epidermolysis Bullosa Simplex in human epidermal stem cells [Articolo su rivista]
Cattaneo, C; Enzo, E; De Rosa, L; Sercia, L; Consiglio, F; Forcato, M; Bicciato, S; Paiardini, A; Basso, G; Tagliafico, E; Paganelli, A; Fiorentini, C; Magnoni, C; Latella, M C; De Luca, M
abstract

: Epidermolysis Bullosa Simplex (EBS) is a rare skin disease inherited mostly in an autosomal dominant manner. Patients display a skin fragility that leads to blisters and erosions caused by minor mechanical trauma. EBS phenotypic and genotypic variants are caused by genetic defects in intracellular proteins whose function is to provide the attachment of basal keratinocytes to the basement membrane zone and most of EBS cases display mutations in keratin 5 (KRT5) and keratin 14 (KRT14) genes. Besides palliative treatments, there is still no long-lasting effective cure to correct the mutant gene and abolish dominant negative effect of the pathogenic protein over its wild-type counterpart. Here, we propose a molecular strategy for EBS01 patient's keratinocytes carrying a monoallelic c.475/495del21 mutation in KRT14 exon1. Through the CRISPR/Cas9 system we performed a specific cleavage only on the mutant allele and restore a normal cellular phenotype and a correct intermediate filament network, without affecting the epidermal stem cell, referred to as holoclones, which play a crucial role in epidermal regeneration.

2023 - Characteristics and clinical behavior of acute myeloid leukemia harboring rare non-A/B/D nucleophosmin (NPM1) gene mutation subtypes: a single-center experience and review of the literature [Articolo su rivista]
Mutti, M.; Cordella, S.; Parisotto, A.; Bettelli, F.; Morselli, M.; Cuoghi, A.; Bresciani, P.; Messerotti, A.; Gilioli, A.; Pioli, V.; Giusti, D.; Colaci, E.; Cassanelli, L.; Paolini, A.; Martinelli, S.; Maffei, R.; Riva, G.; Nasillo, V.; Sarti, M.; Trenti, T.; Comoli, P.; Tagliafico, E.; Manfredini, R.; Eccher, A.; Lagreca, I.; Barozzi, P.; Potenza, L.; Marasca, R.; Candoni, A.; Luppi, M.; Forghieri, F.
abstract

2023 - Chromosome 9p Duplication Promotes T-Cell Exhaustion and Enhances Stem Cell Clonogenic Potential in JAK2-Mutant Myeloproliferative Neoplasms [Abstract in Rivista]
Norfo, Ruggiero; Carretta, Chiara; Parenti, Sandra; Badii, Filippo; Bertesi, Matteo; Rontauroli, Sebastiano; Tavernari, Lara; Genovese, Elena; Sperduti, Samantha; Enzo, Elena; Mirabile, Margherita; Pedrazzi, Francesca; Pessina, Chiara; Colugnat, Ilaria; Mora, Barbara; Maccaferri, Monica; Tenedini, Elena; Martinelli, Silvia; Bianchi, Elisa; Casarini, Livio; Potenza, Leonardo; Luppi, Mario; Tagliafico, Enrico; Guglielmelli, Paola; Simoni, Manuela; Passamonti, Francesco; Vannucchi, Alessandro Maria; Manfredini, Rossella
abstract

2023 - Clinical application of NGS in the diagnosis of iron overload disorders or hyperferritinemia of genetic origin [Abstract in Rivista]
Ricci, A.; Bergamini, E.; Scarlini, S.; Buzzetti, E.; Caleffi, A.; Rabacchi, C.; Ventura, P.; Artuso, L.; Tenedini, E.; Tagliafico, E.; Pietrangelo, A.; Corradini, E.
abstract

2023 - Clinically relevant low-frequency Next Generation Sequencing variants in hereditary cancer patients: an operational multi-step algorithm for laboratory managing [Articolo su rivista]
Tenedini, E.; Bonamici, L.; Marino, M.; Artuso, L.; Luppi, M.; Trenti, T.; Tagliafico, E.
abstract

2023 - De novo variants and recombination at 4q35: hints for preimplantation genetic testing in facioscapulohumeral muscular dystrophy [Articolo su rivista]
Pini, Sara; Napoli, Floriana Maria; Tagliafico, Enrico; Marca, Antonio La; Bertucci, Emma; Salsi, Valentina; Tupler, Rossella
abstract

2023 - From gating to computational flow cytometry: Exploiting artificial intelligence for MRD diagnostics [Articolo su rivista]
Riva, Giovanni; Luppi, Mario; Tagliafico, Enrico
abstract

The era of AI-based methods to improve flow cytometry diagnostics in haematology is now at the beginning. The study by Nguyen and colleagues explored an emerging machine learning approach to assess phenotypic MRD in chronic lymphocytic leukaemia patients, showing that such AI-driven computational analysis may represent a robust and feasible tool for advanced diagnostics of haematological malignancies.Commentary on: Nguyen et al. Computational flow cytometry provides accurate assessment of measurable residual disease in chronic lymphocytic leukaemia. Br J Haematol 2023 (Online ahead of print). doi:

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 - 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 - PAX2/Renal Coloboma Syndrome Expresses Extreme Intrafamilial Phenotypic Variability [Articolo su rivista]
Giovanella, Silvia; Pasini, Andrea; Ligabue, Giulia; Testa, Francesca; Mori, Giacomo; Tagliafico, Enrico; Magistroni, Riccardo
abstract

Renal coloboma syndrome (RCS) is a disease characterized by kidney and ocular anomalies (kidney hypodysplasia and coloboma). RCS is caused, in half of the cases, by mutations in the paired box 2 (PAX2) gene, a critical organogenesis transcriptional factor. We report the case of a newborn with kidney hypodysplasia in a negative parental context where mother and father were phenotypically unaffected at the initial evaluation. The maternal family presented an important history of kidney disease with undefined diagnosis. Molecular characterization identified a PAX2 variant, classified as likely pathogenic. This variant segregates with the disease, and it was also found in the newborn, explaining his severe symptoms. It is noteworthy that the mother shows the same PAX2 variant, with an apparently negative kidney phenotype, displaying the possibility of an extreme variable expressivity of the disease. This feature suggests extreme caution in segregation analysis and family counseling of PAX2 pedigrees.

2023 - Prognostic Relevance of Multi-Antigenic Myeloma-Specific T-Cell Assay in Patients with Monoclonal Gammopathies [Articolo su rivista]
Lagreca, Ivana; Nasillo, Vincenzo; Barozzi, Patrizia; Castelli, Ilaria; Basso, Sabrina; Castellano, Sara; Paolini, Ambra; Maccaferri, Monica; Colaci, Elisabetta; Vallerini, Daniela; Natali, Patrizia; Debbia, Daria; Pirotti, Tommaso; Ottomano, Anna Maria; Maffei, Rossana; Bettelli, Francesca; Giusti, Davide; Messerotti, Andrea; Gilioli, Andrea; Pioli, Valeria; Leonardi, Giovanna; Forghieri, Fabio; Bresciani, Paola; Cuoghi, Angela; Morselli, Monica; Manfredini, Rossella; Longo, Giuseppe; Candoni, Anna; Marasca, Roberto; Potenza, Leonardo; Tagliafico, Enrico; Trenti, Tommaso; Comoli, Patrizia; Luppi, Mario; Riva, Giovanni
abstract

: Multiple Myeloma (MM) typically originates from underlying precursor conditions, known as Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM). Validated risk factors, related to the main features of the clonal plasma cells, are employed in the current prognostic models to assess long-term probabilities of progression to MM. In addition, new prognostic immunologic parameters, measuring protective MM-specific T-cell responses, could help to identify patients with shorter time-to-progression. In this report, we described a novel Multi-antigenic Myeloma-specific (MaMs) T-cell assay, based on ELISpot technology, providing simultaneous evaluation of T-cell responses towards ten different MM-associated antigens. When performed during long-term follow-up (mean 28 months) of 33 patients with either MGUS or SMM, such deca-antigenic myeloma-specific immunoassay allowed to significantly distinguish between stable vs. progressive disease (p < 0.001), independently from the Mayo Clinic risk category. Here, we report the first clinical experience showing that a wide (multi-antigen), standardized (irrespective to patients' HLA), MM-specific T-cell assay may routinely be applied, as a promising prognostic tool, during the follow-up of MGUS/SMM patients. Larger studies are needed to improve the antigenic panel and further explore the prognostic value of MaMs test in the risk assessment of patients with monoclonal gammopathies.

2022 - BTK Inhibitors Impair Platelet-Mediated Antifungal Activity [Articolo su rivista]
Nasillo, V.; Lagreca, I.; Vallerini, D.; Barozzi, P.; Riva, G.; Maccaferri, M.; Paolini, A.; Forghieri, F.; Fiorcari, S.; Maffei, R.; Martinelli, S.; Atene, C. G.; Castelli, I.; Marasca, R.; Potenza, L.; Comoli, P.; Manfredini, R.; Tagliafico, E.; Trenti, T.; Luppi, M.
abstract

In recent years, the introduction of new drugs targeting Bruton’s tyrosine kinase (BTK) has allowed dramatic improvement in the prognosis of patients with chronic lymphocytic leukemia (CLL) and other B-cell neoplasms. Although these small molecules were initially considered less immunosuppressive than chemoimmunotherapy, an increasing number of reports have described the occurrence of unexpected opportunistic fungal infections, in particular invasive aspergillosis (IA). BTK represents a crucial molecule in several signaling pathways depending on different immune receptors. Based on a variety of specific off-target effects on innate immunity, namely on neutrophils, monocytes, pulmonary macrophages, and nurse-like cells, ibrutinib has been proposed as a new host factor for the definition of probable invasive pulmonary mold disease. The role of platelets in the control of fungal growth, through granule-dependent mechanisms, was described in vitro almost two decades ago and is, so far, neglected by experts in the field of clinical management of IA. In the present study, we confirm the antifungal role of platelets, and we show, for the first time, that the exposure to BTK inhibitors impairs several immune functions of platelets in response to Aspergillus fumigatus, i.e., the ability to adhere to conidia, activation (as indicated by reduced expression of P-selectin), and direct killing activity. In conclusion, our experimental data suggest that antiplatelet effects of BTK inhibitors may contribute to an increased risk for IA in CLL patients.

2022 - Constitutional Mosaicism: A Critical Issue in the Definition of BRCA-Inherited Cancer Risk [Articolo su rivista]
Tenedini, Elena; Piana, Simonetta; Toss, Angela; Marino, Marco; Barbieri, Elena; Artuso, Lucia; Venturelli, Marta; Gasparini, Elisa; Mandato, Vincenzo Dario; Marchi, Isabella; Castellano, Sara; Luppi, Mario; Trenti, Tommaso; Cortesi, Laura; Tagliafico, Enrico
abstract

2022 - Homologous recombination deficiency, RB-loss gene signatures, intrinsic subtype and response to neoadjuvant treatment in HR+/HER2- early breast cancer: a correlative analysis of two phase II trials [Poster]
Griguolo, Gaia; Miglietta, Federica; Paré, Laia; Generali, Daniele G.; Frassoldati, Antonio; Musolino, Antonino; Spazzapan, Simon; Vernaci, Grazia; Giarratano, Tommaso; Lo Mele, Marcello; Bisagni, Giancarlo; Piacentini, Federico; Tagliafico, Enrico; Cagossi, Katia; Schiavi, Francesca; Pinato, Claudia; Prat, Aleix; Guarneri, Valentina; Dieci, Maria Vittoria
abstract

Background: Hormone-receptor (HR)+/HER2- breast cancer (BC) is a biologically heterogeneous disease. Homologous recombination deficiency (HRD) and BRCA mutations have been previously reported to be associated with worse outcomes in HR+/HER2- metastatic BC patients receiving CDK4/6 inhibitors and endocrine therapy. Here, we assess the relation between HRD and RB-loss signatures, intrinsic subtyping, the PAM50-based chemo-endocrine score, and response to chemotherapy-based therapy and endocrine treatment in HR+/HER2- early BC. Methods: GIADA is a multicentric neoadjuvant phase II trial that treated premenopausal patients with Luminal B (LumB)-like BC (HR-positive, HER2-negative, with Ki67>20% and/or histologic Grade 3) with a combination of chemotherapy, immunotherapy and endocrine treatment. Expression of 758 genes on baseline tumor samples from all 43 patients was quantified by nCounter platform. The LETLOB phase II trial randomized postmenopausal women with clinical stage II-IIIA HR+/HER2- BC to neoadjuvant letrozole + lapatinib or letrozole + placebo for 6 months (Guarneri, JCO 2014). Gene-expression data (Affymetrix platform) from pre-treatment frozen core-biopsies was available from 66 out of 92 pts enrolled. Intrinsic subtype was assigned using a research-based PAM50 subtype predictor. A published HRD signature (Peng, Nat Commun 2014) and a signature of RB loss (RBsig), previously reported to potentially predict resistance to CDK4/6 inhibitors in HR+/HER2- BC (Malorni, Oncotarget 2016) were computed. The PAM50 based chemo-endocrine score (CES) was calculated using published definition (Prat, CCR 2017). Higher values of CES indicate increased endocrine sensitivity, while lower values indicate chemosensitivity. Association between genomic signatures was assessed through Pearson’s correlation coefficient. Association of genomic signatures with pCR was assessed through logistic regression and association with PEPI scores was assessed through Kruskal-Wallis test. Results: HRD signature levels were significantly higher in non-luminal (Basal-like and HER2-enriched) tumors as compared to Luminal (A or B) tumors (p>0.001 in the GIADA trial, p=0.021 in the LETLOB trial). Moreover, higher levels of HRD signature were associated with higher levels of RB-loss signature (Pearson correlation 0.355, p=0.020 in the GIADA trial; Pearson correlation 0.942, p< 0.001 in the LETLOB trial), higher levels of Basal-like signature (Pearson correlation 0.502, p< 0.001 in the GIADA trial; Pearson correlation 0.373, p=0.002 in the LETLOB trial) and lower levels of CES (Pearson correlation -0.422, p=0.005 in the GIADA trial; Pearson correlation -0.763, p< 0.001 in the LETLOB trial), indicative of higher chemosensitivity. In the GIADA trial, higher levels of HRD signature (p=0.018) and RBloss signature (p=0.073) and lower levels of CES (p=0.007) were associated with higher pCR rates after chemo, endocrine and immunotherapy. In the LETLOB trial, lower levels of HRD signature (p=0.068) and RBloss signature (p=0.042) and higher levels of CES (p=0.050) were associated with higher sensitivity to endocrine treatment (lower PEPI scores, 0 vs 1-3 vs 4 or more, after neoadjuvant letrozole). Conclusions: In HR+/HER2- early BC, HRD gene signatures, RB-loss gene signatures and non-luminal (especially Basal-like) intrinsic subtyping are associated with each other and associated with higher sensitivity to chemotherapy-based therapy and lower sensitivity to endocrine treatment. These observations might help correctly tailor systemic therapy, including biologic agents, in patients with HR+/HER2- early and advanced BC.

2022 - Implementation of preventive and predictive BRCA testing in patients with breast, ovarian, pancreatic, and prostate cancer: a position paper of Italian Scientific Societies [Articolo su rivista]
Russo, A.; Incorvaia, L.; Capoluongo, E.; Tagliaferri, P.; Gori, S.; Cortesi, L.; Genuardi, M.; Turchetti, D.; De Giorgi, U.; Di Maio, M.; Barberis, M.; Dessena, M.; Del Re, M.; Lapini, A.; Luchini, C.; Jereczek-Fossa, B. A.; Sapino, A.; Cinieri, S.; Beretta, G.; Bella, M. A.; Bracarda, S.; Colombo, N.; Conteduca, V.; Del Mastro, L.; Galvano, A.; Gristina, V.; Guarneri, V.; La Verde, N.; Lorusso, D.; Marchetti, P.; Normanno, N.; Ottini, L.; Pensabene, M.; Pignata, S.; Procopio, G.; Ricevuto, E.; Silvestris, N.; Tassone, P.; Tucci, M.; Donato, V.; Carrara, S.; Paiella, S.; Gentilini, O.; Gunelli, R.; Nicolis, F.; Buttitta, F.; Colecchia, M.; Fassan, M.; Malapelle, U.; Marchetti, A.; Marchio, C.; Scarpa, A.; Truini, M.; Zamboni, G.; Gion, M.; Trevisiol, C.; Gronchi, A.; Danesi, R.; Di Marco, V.; Carrera, P.; Ghiorzo, P.; Pasini, B.; Varesco, L.; Artibani, W.; Ludovico, G.; Campanella, O.; Vatrano, S.; Tagliafico, E.
abstract

2022 - Linking COVID-19, monocyte activation and sepsis: MDW, a novel biomarker from cytometry [Articolo su rivista]
Riva, G.; Nasillo, V.; Luppi, M.; Tagliafico, E.; Trenti, T.
abstract

2022 - Pattern of distant relapse according to intrinsic molecular subtype in patients with HER2-positive breast cancer: a combined analysis of ShortHER, CherLOB, and two institutional cohorts [Poster]
Dieci, Maria Vittoria; Bisagni, Giancarlo; Bartolini, Stefania; Frassoldati, Antonio; Giulio Generali, Daniele; Piacentini, Federico; Griguolo, Gaia; Tagliafico, Enrico; Brasó-Maristany, Fara; Chic, Nuria; Porra, Francesca; Vicini, Roberto; D’Amico, Roberto; Balduzzi, Sara; Prat, Aleix; Conte, Pierfranco; Guarneri, Valentina
abstract

Background: All intrinsic molecular subtypes are represented among HER2-positive breast cancer, with implications on clinical outcome and treatment sensitivity. The impact of molecular subtypes on the pattern and site of relapse is largely unexplored. Methods: 677 patients with HER2-positive early breast cancer from the Shorther trial (n=437), the CherLOB trial (n=84) and two Institutional cohorts (Istituto Oncologico Veneto IRCCS Padova n=39 and Hospital Clinic Barcelona n=117) were included. Only patients with available PAM50 intrinsic molecular subtyping were considered. We analyzed the incidence of distant relapse (at any site and at specific sites) as the first event. Cumulative incidence was estimated according to competing risk analysis (Fine and Gray’s method). Competing risk regression was used to calculate the subdistribution Hazard Ratios (subHR) and their 95% Confidence Interval (CI). Results: The distribution of molecular subtypes was: 130 LumA (19%), 75 LumB (11%), 347 HER2-e (51%), 46 Basal (7%), 79 Normal (12%). Median follow up was 8.4 years (95%CI 8.2-8.6). The 10-yr cumulative incidence rates of distant relapse as first event were: LumA 7.9%, LumB 14.8%, HER2-e 14.7%, Basal 15.5%, Normal 10.4% (HER2-e vs LumA: SubHR 2.21, 95%CI 1.05-4.64, p=0.037). Table 1 shows the 5-yr and 10-yr cumulative incidence rates of distant metastases at specific sites (as first event) according to intrinsic subtype. HER2-e enriched and Basal tumors were more prone as compared to other subtypes to develop brain and lung metastasis as first event, respectively. Isolated brain metastases without extracranial disease occurred only in patients with HER2-e tumors. All brain metastases as first event occurred within 5 years from diagnosis. Bone-only disease as first event was less frequent in HER2-e and Basal subtype (subHR HER2-e vs LumA: 0.32, 95%CI 0.10-10.4. p=0.058). Next, we analyzed the frequency of site-specific first metastasis among patients who experienced a distant metastasis as first event (n=77). Lung metastases were more frequent in Basal tumors (LumA 25.0%, LumB 20.0%, HER2-e 24.4%, Basal 71.4%, Normal 0.0%, p=0.037) and bone metastases were more frequent in Luminal tumors (LumA 100.0%, LumB 60.0%, HER2-e 31.1%, Basal 42.9%, Normal 57.1%, p=0.006). Among 45 HER2-e patients with a first distant relapse, 25.6% were diagnosed with a brain metastasis and 15.6% had brain-only disease. Conclusions: Molecular subtypes influence the metastatic behaviour of clinically HER2-positive breast cancer. These results, if further validated, may have implication in planning personalized monitoring strategies.

2022 - The Response to Oxidative Damage Correlates with Driver Mutations and Clinical Outcome in Patients with Myelofibrosis [Articolo su rivista]
Genovese, E.; Mirabile, M.; Rontauroli, S.; Sartini, S.; Fantini, S.; Tavernari, L.; Maccaferri, M.; Guglielmelli, P.; Bianchi, E.; Parenti, S.; Carretta, C.; Mallia, S.; Castellano, S.; Colasante, C.; Balliu, M.; Bartalucci, N.; Palmieri, R.; Ottone, T.; Mora, B.; Potenza, L.; Passamonti, F.; Voso, M. T.; Luppi, M.; Vannucchi, A. M.; Tagliafico, E.; Manfredini, R.
abstract

Myelofibrosis (MF) is the Philadelphia-negative myeloproliferative neoplasm characterized by the worst prognosis and no response to conventional therapy. Driver mutations in JAK2 and CALR impact on JAK-STAT pathway activation but also on the production of reactive oxygen species (ROS). ROS play a pivotal role in inflammation-induced oxidative damage to cellular components including DNA, therefore leading to greater genomic instability and promoting cell transformation. In order to unveil the role of driver mutations in oxidative stress, we assessed ROS levels in CD34+ hematopoietic stem/progenitor cells of MF patients. Our results demonstrated that ROS production in CD34+ cells from CALR-mutated MF patients is far greater compared with patients harboring JAK2 mutation, and this leads to increased oxidative DNA damage. Moreover, CALR-mutant cells show less superoxide dismutase (SOD) antioxidant activity than JAK2-mutated ones. Here, we show that high plasma levels of total antioxidant capacity (TAC) correlate with detrimental clinical features, such as high levels of lactate dehydrogenase (LDH) and circulating CD34+ cells. Moreover, in JAK2-mutated patients, high plasma level of TAC is also associated with a poor overall survival (OS), and multivariate analysis demonstrated that high TAC classification is an independent prognostic factor allowing the identification of patients with inferior OS in both DIPSS lowest and highest categories. Altogether, our data suggest that a different capability to respond to oxidative stress can be one of the mechanisms underlying disease progression of myelofibrosis.

2022 - The Role of T Cell Immunity in Monoclonal Gammopathy and Multiple Myeloma: From Immunopathogenesis to Novel Therapeutic Approaches [Articolo su rivista]
Lagreca, I.; Riva, G.; Nasillo, V.; Barozzi, P.; Castelli, I.; Basso, S.; Bettelli, F.; Giusti, D.; Cuoghi, A.; Bresciani, P.; Messerotti, A.; Gilioli, A.; Pioli, V.; Colasante, C.; Vallerini, D.; Paolini, A.; Maccaferri, M.; Donatelli, F.; Forghieri, F.; Morselli, M.; Colaci, E.; Leonardi, G.; Marasca, R.; Potenza, L.; Manfredini, R.; Tagliafico, E.; Trenti, T.; Comoli, P.; Luppi, M.
abstract

Multiple Myeloma (MM) is a malignant growth of clonal plasma cells, typically arising from asymptomatic precursor conditions, namely monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM). Profound immunological dysfunctions and cyto-kine deregulation are known to characterize the evolution of the disease, allowing immune escape and proliferation of neoplastic plasma cells. In the past decades, several studies have shown that the immune system can recognize MGUS and MM clonal cells, suggesting that anti-myeloma T cell immunity could be harnessed for therapeutic purposes. In line with this notion, chimeric antigen receptor T cell (CAR-T) therapy is emerging as a novel treatment in MM, especially in the re-lapsed/refractory disease setting. In this review, we focus on the pivotal contribution of T cell im-pairment in the immunopathogenesis of plasma cell dyscrasias and, in particular, in the disease progression from MGUS to SMM and MM, highlighting the potentials of T cell-based immunother-apeutic approaches in these settings.

2021 - A single-tube multiplex method for monitoring mutations in cysteine 481 of Bruton Tyrosine Kinase (BTK) gene in chronic lymphocytic leukemia patients treated with ibrutinib [Articolo su rivista]
Maffei, R.; Fiorcari, S.; Atene, C. G.; Martinelli, S.; Scarfo, L.; Bonfiglio, S.; Maccaferri, M.; Ljungstrom, V.; Zucchini, P.; Forghieri, F.; Potenza, L.; Ghia, P.; Marasca, R.; Trenti, T.; Tagliafico, E.; Luppi, M.
abstract

2021 - Arresto di crescita nei primi mesi di vita: quando l’ipolipidemia e la steatorrea fanno diagnosi. [Abstract in Atti di Convegno]
Poluzzi, Silvia; Bruzzi, Patrizia; Trevisani, Viola; Lucaccioni, Laura; Predieri, Barbara; Madeo, Simona Filomena; Tagliafico, Enrico; Iughetti, Lorenzo
abstract

2021 - Automated capture-based NGS workflow: one thousand patients experience in a clinical routine framework [Articolo su rivista]
Tenedini, E; Celestini, F; Iapicca, P; Marino, M; Castellano, S; Artuso, L; Biagiarelli, F; Cortesi, L; Toss, A; Barbieri, E; Roncucci, L; Pedroni, M; Manfredini, R; Luppi, M; Trenti, T; Tagliafico, E
abstract

Objectives: The Next Generation Sequencing (NGS) based mutational study of hereditary cancer genes is crucial to design tailored prevention strategies in subjects with different hereditary cancer risk. The ease of amplicon-based NGS library construction protocols contrasts with the greater uniformity of enrichment provided by capture-based protocols and so with greater chances for detecting larger genomic rearrangements and copy-number variations. Capture-based protocols, however, are characterized by a higher level of complexity of sample handling, extremely susceptible to human bias. Robotics platforms may definitely help dealing with these limits, reducing hands-on time, limiting random errors and guaranteeing process standardization.Methods: We implemented the automation of the CE-IVD SOPHiA Hereditary Cancer Solution (TM) (HCS) libraries preparation workflow by SOPHiA GENETICS on the Hamilton's STARlet platform. We present the comparison of results between this automated approach, used for more than 1,000 DNA patients' samples, and the performances of the manual protocol evaluated by SOPHiA GENETICS onto 240 samples summarized in their HCS evaluation study.Results: We demonstrate that this automated workflow achieved the same expected goals of manual setup in terms of coverages and reads uniformity, with extremely lower standard deviations among samples considering the sequencing reads mapped onto the regions of interest.Conclusions: This automated solution offers same reliable and affordable NGS data, but with the essential advantages of a flexible, automated and integrated framework, minimizing possible human errors and depicting a laboratory's walk-away scenario.

2021 - Automation of a capture-based NGS workflow: one thousand patients experience in a diagnostic clinical routine framework [Abstract in Rivista]
Tenedini, E.; Celestini, F.; Iapicca, P.; Marino, M.; Castellano, S.; Artuso, L.; Biagiarelli, F.; Cortesi, L.; Toss, A.; Barbieri, E.; Roncucci, L.; Pedroni, M.; Manfredini, R.; Luppi, M.; Trenti, T.; Tagliafico, E.
abstract

2021 - Ceruloplasmin gene variants are associated with hyperferritinemia and increased liver iron in patients with {NAFLD} [Articolo su rivista]
Corradini, Elena; Buzzetti, Elena; Dongiovanni, Paola; Scarlini, Stefania; Caleffi, Angela; Pelusi, Serena; Bernardis, Isabella; Ventura, Paolo; Rametta, Raffaela; Tenedini, Elena; Tagliafico, Enrico; Ludovica Fracanzani, Anna; Fargion, Silvia; Pietrangelo, Antonello; Vittorio Valenti, Luca
abstract

Non-alcoholic fatty liver disease (NAFLD) is a multifactorial disorder resulting from genetic and environmental factors. Hyperferritinemia has been associated with increased hepatic iron stores and worse outcomes in patients with NAFLD. The aim of this study was to evaluate the prevalence of variants of iron-related genes and their association with hyperferritinemia, hepatic iron stores and liver disease severity in patients with NAFLD.

2021 - Characterization of new ATM deletion associated with hereditary breast cancer [Articolo su rivista]
Parenti, S.; Rabacchi, C.; Marino, M.; Tenedini, E.; Artuso, L.; Castellano, S.; Carretta, C.; Mallia, S.; Cortesi, L.; Toss, A.; Barbieri, E.; Manfredini, R.; Luppi, M.; Trenti, T.; Tagliafico, E.
abstract

Next-generation sequencing (NGS)-based cancer risk screening with multigene panels has become the most successful method for programming cancer prevention strategies. ATM germ-line heterozygosity has been described to increase tumor susceptibility. In particular, families carrying heterozygous germ-line variants of ATM gene have a 5-to 9-fold risk of developing breast cancer. Recent studies identified ATM as the second most mutated gene after CHEK2 in BRCA-negative patients. Nowadays, more than 170 missense variants and several truncating mutations have been identified in ATM gene. Here, we present the molecular characterization of a new ATM deletion, identified thanks to the CNV algorithm implemented in the NGS analysis pipeline. An automated workflow implementing the SOPHiA Genetics’ Hereditary Cancer Solution (HCS) protocol was used to generate NGS libraries that were sequenced on Illumina MiSeq Platform. NGS data analysis allowed us to identify a new inactivating deletion of exons 19–27 of ATM gene. The deletion was characterized both at the DNA and RNA level.

2021 - Chromosome 16 changes do not always come for good [Articolo su rivista]
Paolini, A.; Nasillo, V.; Lusenti, B.; Roncati, L.; Tagliafico, E.; Luppi, M.
abstract

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 - Detection of germline variants in 450 breast/ovarian cancer families with a multi‐gene panel including coding and regulatory regions [Articolo su rivista]
Guglielmi, C.; Scarpitta, R.; Gambino, G.; Conti, E.; Belle, F.; Tancredi, M.; Cervelli, T.; Falaschi, E.; Cosini, C.; Aretini, P.; Congregati, C.; Marino, M.; Patruno, M.; Pilato, B.; Spina, F.; Balestrino, L.; Tenedini, E.; Carnevali, I.; Cortesi, L.; Tagliafico, E.; Tibiletti, M. G.; Tommasi, S.; Ghilli, M.; Vivanet, C.; Galli, A.; Caligo, M. A.
abstract

With the progress of sequencing technologies, an ever‐increasing number of variants of unknown functional and clinical significance (VUS) have been identified in both coding and noncoding regions of the main Breast Cancer (BC) predisposition genes. The aim of this study is to identify a mutational profile of coding and intron‐exon junction regions of 12 moderate penetrance genes (ATM, BRIP1, CDH1, CHEK2, NBN, PALB2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53) in a cohort of 450 Italian patients with Hereditary Breast/Ovarian Cancer Syndrome, wild type for germline mutation in BRCA1/2 genes. The analysis was extended to 5′UTR and 3′UTR of all the genes listed above and to the BRCA1 and BRCA2 known regulatory regions in a subset of 120 patients. The screening was performed through NGS target resequencing on the Illumina platform MiSeq. 8.7% of the patients analyzed is carriers of class 5/4 coding variants in the ATM (3.6%), BRIP1 (1.6%), CHEK2 (1.8%), PALB2 (0.7%), RAD51C (0.4%), RAD51D (0.4%), and TP53 (0.2%) genes, while variants of uncertain pathological significance (VUSs)/class 3 were identified in 9.1% of the samples. In intron‐exon junctions and in regulatory regions, variants were detected respectively in 5.1% and in 32.5% of the cases analyzed. The average age of disease onset of 44.4 in non‐coding variant carriers is absolutely similar to the average age of disease onset in coding variant carriers for each pro-band’s group with the same cancer type. Furthermore, there is not a statistically significant difference in the proportion of cases with a tumor onset under age of 40 between the two groups, but the presence of multiple non‐coding variants in the same patient may affect the aggressiveness of the tumor and it is worth underlining that 25% of patients with an aggressive tumor are carriers of a PTEN 3′UTR‐variant. This data provides initial information on how important it might be to extend mutational screening to the regulatory regions in clinical practice.

2021 - Gene expression profile correlates with molecular and clinical features in patients with myelofibrosis [Articolo su rivista]
Rontauroli, S.; Castellano, S.; Guglielmelli, P.; Zini, R.; Bianchi, E.; Genovese, E.; Carretta, C.; Parenti, S.; Fantini, S.; Mallia, S.; Tavernari, L.; Sartini, S.; Mirabile, M.; Mannarelli, C.; Gesullo, F.; Pacilli, A.; Pietra, D.; Rumi, E.; Salmoiraghi, S.; Mora, B.; Villani, L.; Grilli, A.; Rosti, V.; Barosi, G.; Passamonti, F.; Rambaldi, A.; Malcovati, L.; Cazzola, M.; Bicciato, S.; Tagliafico, E.; Vannucchi, A. M.; Manfredini, R.
abstract

Myelofibrosis (MF) belongs to the family of classic Philadelphia-negative myeloproliferative neoplasms (MPNs). It can be primary myelofibrosis (PMF) or secondary myelofibrosis (SMF) evolving from polycythemia vera (PV) or essential thrombocythemia (ET). Despite the differences, PMF and SMF patients are currently managed in the same way, and prediction of survival is based on the same clinical and genetic features. In the last few years, interest has grown concerning the ability of gene expression profiles (GEPs) to provide valuable prognostic information. Here, we studied the GEPs of granulocytes from 114 patients with MF, using a microarray platform to identify correlations with patient characteristics and outcomes. Cox regression analysis led to the identification of 201 survival-related transcripts characterizing patients who are at high risk for death. High-risk patients identified by this gene signature displayed an inferior overall survival and leukemia-free survival, together with clinical and molecular detrimental features included in contemporary prognostic models, such as the presence of high molecular risk mutations. The high-risk group was enriched in post-PV and post-ET MF and JAK2V617F homozygous patients, whereas pre-PMF was more frequent in the low-risk group. These results demonstrate that GEPs in MF patients correlate with their molecular and clinical features, particularly their survival, and represent the proof of concept that GEPs might provide complementary prognostic information to be applied in clinical decision making.

2021 - How to improve prognostication in acute myeloid leukemia with cbfb‐myh11 fusion transcript: Focus on the role of molecular measurable residual disease (mrd) monitoring [Articolo su rivista]
Talami, A.; Bettelli, F.; Pioli, V.; Giusti, D.; Gilioli, A.; Colasante, C.; Galassi, L.; Giubbolini, R.; Catellani, H.; Donatelli, F.; Maffei, R.; Martinelli, S.; Barozzi, P.; Potenza, L.; Marasca, R.; Trenti, T.; Tagliafico, E.; Comoli, P.; Luppi, M.; Forghieri, F.
abstract

Acute myeloid leukemia (AML) carrying inv(16)/t(16;16), resulting in fusion transcript CBFB‐MYH11, belongs to the favorable‐risk category. However, even if most patients obtain morphological complete remission after induction, approximately 30% of cases eventually relapse. While well‐established clinical features and concomitant cytogenetic/molecular lesions have been recognized to be relevant to predict prognosis at disease onset, the independent prognostic impact of measurable residual disease (MRD) monitoring by quantitative real‐time reverse transcriptase polymerase chain reaction (qRT‐PCR), mainly in predicting relapse, actually supersedes other prognostic factors. Although the ELN Working Party recently indicated that patients affected with CBFB‐MYH11 AML should have MRD assessment at informative clinical timepoints, at least after two cycles of intensive chemotherapy and after the end of treatment, several controversies could be raised, especially on the frequency of subsequent serial monitoring, the most significant MRD thresholds (most commonly 0.1%) and on the best source to be analyzed, namely, bone marrow or peripheral blood samples. Moreover, persisting low‐level MRD positivity at the end of treatment is relatively common and not predictive of relapse, provided that transcript levels remain stably below specific thresholds. Rising MRD levels suggestive of molecular relapse/progression should thus be confirmed in subsequent samples. Further prospective studies would be required to optimize postremission monitoring and to define effective MRD‐based therapeutic strategies.

2021 - Immune microenvironment and intrinsic subtyping in hormone receptor-positive/HER2-negative breast cancer. [Articolo su rivista]
Griguolo, G; Dieci, Mv; Paré, L; Miglietta, F; Generali, Dg; Frassoldati, A; Cavanna, L; Bisagni, G; Piacentini, F; Tagliafico, E; Cagossi, K; Ficarra, G; Prat, A; Conte, P; Guarneri, V.
abstract

Little is known regarding the interaction between immune microenvironment and tumor biology in hormone receptor (HR)+/HER2- breast cancer (BC). We here assess pretreatment gene-expression data from 66 HR+/HER2- early BCs from the LETLOB trial and show that non-luminal tumors (HER2-enriched, Basal-like) present higher tumor-infiltrating lymphocyte levels than luminal tumors. Moreover, significant differences in immune infiltrate composition, assessed by CIBERSORT, were observed: non-luminal tumors showed a more proinflammatory antitumor immune infiltrate composition than luminal ones.

2021 - Increased Plasma Levels of lncRNAs LINC01268, GAS5 and MALAT1 Correlate with Negative Prognostic Factors in Myelofibrosis [Articolo su rivista]
Fantini, Sebastian; Rontauroli, Sebastiano; Sartini, Stefano; Mirabile, Margherita; Bianchi, Elisa; Badii, Filippo; Maccaferri, Monica; Guglielmelli, Paola; Ottone, Tiziana; Palmieri, Raffaele; Genovese, Elena; Carretta, Chiara; Parenti, Sandra; Mallia, Selene; Tavernari, Lara; Salvadori, Costanza; Gesullo, Francesca; Maccari, Chiara; Zizza, Michela; Grande, Alexis; Salmoiraghi, Silvia; Mora, Barbara; Potenza, Leonardo; Rosti, Vittorio; Passamonti, Francesco; Rambaldi, Alessandro; Voso, Maria Teresa; Mecucci, Cristina; Tagliafico, Enrico; Luppi, Mario; Vannucchi, Alessandro Maria; Manfredini, Rossella
abstract

: Long non-coding RNAs (lncRNAs) have been recently described as key mediators in the development of hematological malignancies. In the last years, circulating lncRNAs have been proposed as a new class of non-invasive biomarkers for cancer diagnosis and prognosis and to predict treatment response. The present study is aimed to investigate the potential of circulating lncRNAs as non-invasive prognostic biomarkers in myelofibrosis (MF), the most severe among Philadelphia-negative myeloproliferative neoplasms. We detected increased levels of seven circulating lncRNAs in plasma samples of MF patients (n = 143), compared to healthy controls (n = 65). Among these, high levels of LINC01268, MALAT1 or GAS5 correlate with detrimental clinical variables, such as high count of leukocytes and CD34+ cells, severe grade of bone marrow fibrosis and presence of splenomegaly. Strikingly, high plasma levels of LINC01268 (p = 0.0018), GAS5 (p = 0.0008) or MALAT1 (p = 0.0348) are also associated with a poor overall-survival while high levels of LINC01268 correlate with a shorter leukemia-free-survival. Finally, multivariate analysis demonstrated that the plasma level of LINC01268 is an independent prognostic variable, suggesting that, if confirmed in future in an independent patients' cohort, it could be used for further studies to design an updated classification model for MF patients.

2021 - Inflammatory microenvironment and specific t cells in myeloproliferative neoplasms: Immunopathogenesis and novel immunotherapies [Articolo su rivista]
Nasillo, V.; Riva, G.; Paolini, A.; Forghieri, F.; Roncati, L.; Lusenti, B.; Maccaferri, M.; Messerotti, A.; Pioli, V.; Gilioli, A.; Bettelli, F.; Giusti, D.; Barozzi, P.; Lagreca, I.; Maffei, R.; Marasca, R.; Potenza, L.; Comoli, P.; Manfredini, R.; Maiorana, A.; Tagliafico, E.; Luppi, M.; Trenti, T.
abstract

The Philadelphia-negative myeloproliferative neoplasms (MPNs) are malignancies of the hematopoietic stem cell (HSC) arising as a consequence of clonal proliferation driven by somatically acquired driver mutations in discrete genes (JAK2, CALR, MPL). In recent years, along with the advances in molecular characterization, the role of immune dysregulation has been achieving increasing relevance in the pathogenesis and evolution of MPNs. In particular, a growing number of studies have shown that MPNs are often associated with detrimental cytokine milieu, expansion of the monocyte/macrophage compartment and myeloid-derived suppressor cells, as well as altered functions of T cells, dendritic cells and NK cells. Moreover, akin to solid tumors and other hemato-logical malignancies, MPNs are able to evade T cell immune surveillance by engaging the PD-1/PD-L1 axis, whose pharmacological blockade with checkpoint inhibitors can successfully restore effective antitumor responses. A further interesting cue is provided by the recent discovery of the high immunogenic potential of JAK2V617F and CALR exon 9 mutations, that could be harnessed as in-triguing targets for innovative adoptive immunotherapies. This review focuses on the recent insights in the immunological dysfunctions contributing to the pathogenesis of MPNs and outlines the potential impact of related immunotherapeutic approaches.

2021 - Ivar, an interpretation‐oriented tool to manage the update and revision of variant annotation and classification [Articolo su rivista]
Castellano, S.; Cestari, F.; Faglioni, G.; Tenedini, E.; Marino, M.; Artuso, L.; Manfredini, R.; Luppi, M.; Trenti, T.; Tagliafico, E.
abstract

The rapid evolution of Next Generation Sequencing in clinical settings, and the resulting challenge of variant reinterpretation given the constantly updated information, require robust data management systems and organized approaches. In this paper, we present iVar: a freely available and highly customizable tool with a user‐friendly web interface. It represents a platform for the unified management of variants identified by different sequencing technologies. iVar accepts variant call format (VCF) files and text annotation files and elaborates them, optimizing data organization and avoiding redundancies. Updated annotations can be periodically re‐uploaded and associated with variants as historically tracked attributes, i.e., modifications can be recorded whenever an updated value is imported, thus keeping track of all changes. Data can be visualized through variant‐centered and sample‐centered interfaces. A customizable search function can be exploited to periodically check if pathogenicity‐related data of a variant has changed over time. Patient recontacting ensuing from variant reinterpretation is made easier by iVar through the effective identification of all patients present in the database carrying a specific variant. We tested iVar by uploading 4171 VCF files and 1463 annotation files, obtaining a database of 4166 samples and 22,569 unique variants. iVar has proven to be a useful tool with good performance in terms of collecting and managing data from a medium‐throughput laboratory.

2021 - Monocyte Distribution Width (MDW) as novel inflammatory marker with prognostic significance in COVID-19 patients [Articolo su rivista]
Riva, G.; Castellano, S.; Nasillo, V.; Ottomano, A. M.; Bergonzini, G.; Paolini, A.; Lusenti, B.; Milic, J.; De Biasi, S.; Gibellini, L.; Cossarizza, A.; Busani, S.; Girardis, M.; Guaraldi, G.; Mussini, C.; Manfredini, R.; Luppi, M.; Tagliafico, E.; Trenti, T.
abstract

Monocyte Distribution Width (MDW), a new cytometric parameter correlating with cytomorphologic changes occurring upon massive monocyte activation, has recently emerged as promising early biomarker of sepsis. Similar to sepsis, monocyte/macrophage subsets are considered key mediators of the life-threatening hyper-inflammatory disorder characterizing severe COVID-19. In this study, we longitudinally analyzed MDW values in a cohort of 87 COVID-19 patients consecutively admitted to our hospital, showing significant correlations between MDW and common inflammatory markers, namely CRP (p < 0.001), fibrinogen (p < 0.001) and ferritin (p < 0.01). Moreover, high MDW values resulted to be prognostically associated with fatal outcome in COVID-19 patients (AUC = 0.76, 95% CI: 0.66–0.87, sensitivity 0.75, specificity 0.70, MDW threshold 26.4; RR = 4.91, 95% CI: 1.73–13.96; OR = 7.14, 95% CI: 2.06–24.71). This pilot study shows that MDW can be useful in the monitoring of COVID-19 patients, as this innovative hematologic biomarker is: (1) easy to obtain, (2) directly related to the activation state of a fundamental inflammatory cell subset (i.e. monocytes, pivotal in both cytokine storm and sepsis immunopathogenesis), (3) well correlated with clinical severity of COVID-19-associated inflammatory disorder, and, in turn, (4) endowed with relevant prognostic significance. Additional studies are needed to define further the clinical impact of MDW testing in the management of COVID-19 patients.

2021 - Multiparametric flow cytometry for MRD monitoring in hematologic malignancies: Clinical applications and new challenges [Articolo su rivista]
Riva, G.; Nasillo, V.; Ottomano, A. M.; Bergonzini, G.; Paolini, A.; Forghieri, F.; Lusenti, B.; Barozzi, P.; Lagreca, I.; Fiorcari, S.; Martinelli, S.; Maffei, R.; Marasca, R.; Potenza, L.; Comoli, P.; Manfredini, R.; Tagliafico, E.; Trenti, T.; Luppi, M.
abstract

In hematologic cancers, Minimal Residual Disease (MRD) monitoring, using either molecular (PCR) or immunophenotypic (MFC) diagnostics, allows the identification of rare cancer cells, readily detectable either in the bone marrow or in the peripheral blood at very low levels, far below the limit of classic microscopy. In this paper, we outlined the state-of-the-art of MFC-based MRD detection in different hematologic settings, highlighting main recommendations and new challenges for using such a method in patients with acute leukemias or chronic hematologic neoplasms. The combination of new molecular technologies with advanced flow cytometry is progressively allowing clinicians to design a personalized therapeutic path, proportionate to the biological aggressiveness of the disease, in particular by using novel immunotherapies, in view of a modern decision-making process, based on precision medicine. Along with the evolution of immunophenotypic and molecular diagnostics, the assessment of Minimal Residual Disease (MRD) has progressively become a keystone in the clinical management of hematologic malignancies, enabling valuable post-therapy risk stratifications and guiding risk-adapted therapeutic approaches. However, specific prognostic values of MRD in different hematological settings, as well as its appropriate clinical uses (basically, when to measure it and how to deal with different MRD levels), still need further investigations, aiming to improve standardization and harmonization of MRD monitoring protocols and MRD-driven therapeutic strategies. Currently, MRD measurement in hematological neoplasms with bone marrow involvement is based on advanced highly sensitive methods, able to detect either specific genetic abnormalities (by PCRbased techniques and next-generation sequencing) or tumor-associated immunophenotypic profiles (by multiparametric flow cytometry, MFC). In this review, we focus on the growing clinical role for MFC-MRD diagnostics in hematological malignancies-from acute myeloid and lymphoblastic leukemias (AML, B-ALL and T-ALL), to chronic lymphocytic leukemia (CLL) and multiple myeloma (MM)-providing a comparative overview on technical aspects, clinical implications, advantages and pitfalls of MFC-MRD monitoring in different clinical settings.

2021 - Mutated clones driving leukemic transformation are already detectable at the single-cell level in CD34-positive cells in the chronic phase of primary myelofibrosis [Articolo su rivista]
Parenti, Sandra; Rontauroli, Sebastiano; Carretta, Chiara; Mallia, Selene; Genovese, Elena; Chiereghin, Chiara; Peano, Clelia; Tavernari, Lara; Bianchi, Elisa; Fantini, Sebastian; Sartini, Stefano; Romano, Oriana; Bicciato, Silvio; Tagliafico, Enrico; Della Porta, Matteo; Manfredini, Rossella
abstract

Disease progression of myeloproliferative neoplasms is the result of increased genomic complexity. Since the ability to predict disease evolution is crucial for clinical decisions, we studied single-cell genomics and transcriptomics of CD34-positive cells from a primary myelofibrosis (PMF) patient who progressed to acute myeloid leukemia (AML) while receiving Ruxolitinib. Single-cell genomics allowed the reconstruction of clonal hierarchy and demonstrated that TET2 was the first mutated gene while FLT3 was the last one. Disease evolution was accompanied by increased clonal heterogeneity and mutational rate, but clones carrying TP53 and FLT3 mutations were already present in the chronic phase. Single-cell transcriptomics unraveled repression of interferon signaling suggesting an immunosuppressive effect exerted by Ruxolitinib. Moreover, AML transformation was associated with a differentiative block and immune escape. These results suggest that single-cell analysis can unmask tumor heterogeneity and provide meaningful insights about PMF progression that might guide personalized therapy.

2021 - Neoantigen-specific T-cell immune responses: The paradigm of NPM1-mutated acute myeloid leukemia [Articolo su rivista]
Forghieri, F.; Riva, G.; Lagreca, I.; Barozzi, P.; Bettelli, F.; Paolini, A.; Nasillo, V.; Lusenti, B.; Pioli, V.; Giusti, D.; Gilioli, A.; Colasante, C.; Galassi, L.; Catellani, H.; Donatelli, F.; Talami, A.; Maffei, R.; Martinelli, S.; Potenza, L.; Marasca, R.; Tagliafico, E.; Manfredini, R.; Trenti, T.; Comoli, P.; Luppi, M.
abstract

The C-terminal aminoacidic sequence from NPM1-mutated protein, absent in normal human tissues, may serve as a leukemia-specific antigen and can be considered an ideal target for NPM1-mutated acute myeloid leukemia (AML) immunotherapy. Different in silico instruments and in vitro/ex vivo immunological platforms have identified the most immunogenic epitopes from NPM1-mutated protein. Spontaneous development of endogenous NPM1-mutated-specific cytotoxic T cells has been observed in patients, potentially contributing to remission maintenance and prolonged survival. Genetically engineered T cells, namely CAR-T or TCR-transduced T cells, directed against NPM1-mutated peptides bound to HLA could prospectively represent a promising therapeutic approach. Although either adoptive or vaccine-based immunotherapies are unlikely to be highly effective in patients with full-blown leukemia, these strategies, potentially in combination with immune-checkpoint inhibitors, could be promising in maintaining remission or preemptively eradicat-ing persistent measurable residual disease, mainly in patients ineligible for allogeneic hematopoietic stem cell transplant (HSCT). Alternatively, neoantigen-specific donor lymphocyte infusion derived from healthy donors and targeting NPM1-mutated protein to selectively elicit graft-versus-leukemia effect may represent an attractive option in subjects experiencing post-HSCT relapse. Future studies are warranted to further investigate dynamics of NPM1-mutated-specific immunity and explore whether novel individualized immunotherapies may have potential clinical utility in NPM1-mutated AML patients.

2021 - Pre-existing cytopenia heralding de novo acute myeloid leukemia: Uncommon presentation of NPM1-mutated AML in a single-center study [Articolo su rivista]
Galassi, L.; Colasante, C.; Bettelli, F.; Gilioli, A.; Pioli, V.; Giusti, D.; Morselli, M.; Paolini, A.; Nasillo, V.; Lusenti, B.; Colaci, E.; Donatelli, F.; Catellani, H.; Pozzi, S.; Barbieri, E.; del Rosso, M. N.; Barozzi, P.; Lagreca, I.; Martinelli, S.; Maffei, R.; Riva, G.; Tenedini, E.; Roncati, L.; Marasca, R.; Potenza, L.; Comoli, P.; Trenti, T.; Manfredini, R.; Tagliafico, E.; Luppi, M.; Forghieri, F.
abstract

2021 - Single-keratinocyte transcriptomic analyses identify different clonal types and proliferative potential mediated by FOXM1 in human epidermal stem cells [Articolo su rivista]
Enzo, E.; Secone Seconetti, A.; Forcato, M.; Tenedini, E.; Polito, M. P.; Sala, I.; Carulli, S.; Contin, R.; Peano, C.; Tagliafico, E.; Bicciato, S.; Bondanza, S.; De Luca, M.
abstract

Autologous epidermal cultures restore a functional epidermis on burned patients. Transgenic epidermal grafts do so also in genetic skin diseases such as Junctional Epidermolysis Bullosa. Clinical success strictly requires an adequate number of epidermal stem cells, detected as holoclone-forming cells, which can be only partially distinguished from the other clonogenic keratinocytes and cannot be prospectively isolated. Here we report that single-cell transcriptome analysis of primary human epidermal cultures identifies categories of genes clearly distinguishing the different keratinocyte clonal types, which are hierarchically organized along a continuous, mainly linear trajectory showing that stem cells sequentially generate progenitors producing terminally differentiated cells. Holoclone-forming cells display stem cell hallmarks as genes regulating DNA repair, chromosome segregation, spindle organization and telomerase activity. Finally, we identify FOXM1 as a YAP-dependent key regulator of epidermal stem cells. These findings improve criteria for measuring stem cells in epidermal cultures, which is an essential feature of the graft.

2021 - The prognostic and predictive role of somatic brca mutations in ovarian cancer: Results from a multicenter cohort study [Articolo su rivista]
Toss, A.; Piombino, C.; Tenedini, E.; Bologna, A.; Gasparini, E.; Tarantino, V.; Filieri, M. E.; Cottafavi, L.; Giovanardi, F.; Madrigali, S.; Civallero, M.; Marcheselli, L.; Marchi, I.; Domati, F.; Venturelli, M.; Barbieri, E.; Grandi, G.; Tagliafico, E.; Cortesi, L.
abstract

Previous research involving epithelial ovarian cancer patients showed that, compared to germline BRCA (gBRCA) mutations, somatic BRCA (sBRCA) mutations present a similar positive impact with regard to overall survival (OS) and platinum and PARP (poly (ADP-ribose) polymerase) inhibitor sensitivity. Nevertheless, molecular testing in these studies did not include copy number variation (CNV) analyses of BRCA genes. The aim of this study was to explore the prognostic and predictive role of sBRCA mutations as compared to gBRCA mutations in patients who were also tested for CNVs. Among the 158 patients included in the study, 17.09% of patients carried a pathogenic or likely pathogenic gBRCA variant and 15.19% of patients presented pathogenetic or likely pathogenic sBRCA variants and/or CNVs. Overall, 81.6% of the patients included in this study were diagnosed with a serous histotype, and 77.2% were in advanced stages. Among women diagnosed in advanced stages, gBRCA patients showed better progression-free survival and OS as compared to sBRCA and wild-type patients, whereas sBRCA patients did not show any advantage in outcome as compared to wild-type patients. In this study, the introduction of CNV analyses increased the detection rate of sBRCA mutations, and the resulting classification among gBRCA, sBRCA and wild-type patients was able to properly stratify the prognosis of OC patients. Particularly, sBRCA mutation patients failed to show any outcome advantage as compared to wild-type patients.

2020 - A multivariable prognostic score to guide systemic therapy in early-stage HER2-positive breast cancer: a retrospective study with an external evaluation [Articolo su rivista]
Prat, A; Guarneri, V; Paré, L; Griguolo, G; Pascual, T; Dieci, Mv; Chic, N; González-Farré, B; Frassoldati, A; Sanfeliu, E; Cejalvo, Jm; Muñoz, M; Bisagni, G; Brasó-Maristany, F; Urso, L; Vidal, M; Brandes, Aa; Adamo, B; Musolino, A; Miglietta, F; Conte, B; Oliveira, M; Saura, C; Pernas, S; Alarcón, J; Llombart-Cussac, A; Cortés, J; Manso, L; López, R; Ciruelos, E; Schettini, F; Villagrasa, P; Carey, La; Perou, Cm; Piacentini, F; D'Amico, R; Tagliafico, E; Parker, Js; Conte, P
abstract

Background: In early-stage HER2-positive breast cancer, escalation or de-escalation of systemic therapy is a controversial topic. As an aid to treatment decisions, we aimed to develop a prognostic assay that integrates multiple data types for predicting survival outcome in patients with newly diagnosed HER2-positive breast cancer. Methods: We derived a combined prognostic model using retrospective clinical-pathological data on stromal tumour-infiltrating lymphocytes, PAM50 subtypes, and expression of 55 genes obtained from patients who participated in the Short-HER phase 3 trial. The trial enrolled patients with newly diagnosed, node-positive, HER2-positive breast cancer or, if node negative, with at least one risk factor (ie, tumour size >2 cm, histological grade 3, lymphovascular invasion, Ki67 >20%, age ≤35 years, or hormone receptor negativity), and randomly assigned them to adjuvant anthracycline plus taxane-based combinations with either 9 weeks or 1 year of trastuzumab. Trastuzumab was administered intravenously every 3 weeks (8 mg/kg loading dose at first cycle, and 6 mg/kg thereafter) for 18 doses or weekly (4 mg/kg loading dose in the first week, and 2 mg/kg thereafter) for 9 weeks, starting concomitantly with the first taxane dose. Median follow-up was 91·4 months (IQR 75·1-105·6). The primary objective of our study was to derive and evaluate a combined prognostic score associated with distant metastasis-free survival (the time between randomisation and distant recurrence or death before recurrence), an exploratory endpoint in Short-HER. Patient samples in the training dataset were split into a training set (n=290) and a testing set (n=145), balancing for event and treatment group. The training set was further stratified into 100 iterations of Monte-Carlo cross validation (MCCV). Cox proportional hazard models were fit to MCCV training samples using Elastic-Net. A maximum of 92 features were assessed. The final prognostic model was evaluated in an independent combined dataset of 267 patients with early-stage HER2-positive breast cancer treated with different neoadjuvant and adjuvant anti-HER2-based combinations and from four other studies (PAMELA, CHER-LOB, Hospital Clinic, and Padova) with disease-free survival outcome data. Findings: From Short-HER, data from 435 (35%) of 1254 patients for tumour size (T1 vs rest), nodal status (N0 vs rest), number of tumour-infiltrating lymphocytes (continuous variable), subtype (HER2-enriched and basal-like vs rest), and 13 genes composed the final model (named HER2DX). HER2DX was significantly associated with distant metastasis-free survival as a continuous variable (p<0·0001). HER2DX median score for quartiles 1-2 was identified as the cutoff to identify low-risk patients; and the score that distinguished quartile 3 from quartile 4 was the cutoff to distinguish medium-risk and high-risk populations. The 5-year distant metastasis-free survival of the low-risk, medium-risk, and high-risk populations were 98·1% (95% CI 96·3-99·9), 88·9% (83·2-95·0), and 73·9% (66·0-82·7), respectively (low-risk vs high-risk hazard ratio [HR] 0·04, 95% CI 0·0-0·1, p<0·0001). In the evaluation cohort, HER2DX was significantly associated with disease-free survival as a continuous variable (HR 2·77, 95% CI 1·4-5·6, p=0·0040) and as group categories (low-risk vs high-risk HR 0·27, 0·1-0·7, p=0·005). 5-year disease-free survival in the HER2DX low-risk group was 93·5% (89·0-98·3%) and in the high-risk group was 81·1% (71·5-92·1). Interpretation: The HER2DX combined prognostic score identifies patients with early-stage, HER2-positive breast cancer who might be candidates for escalated or de-escalated systemic treatment. Future clinical validation of HER2DX seems warranted to establish its use in different scenarios, especially in the neoadjuvant setting.

2020 - Acute myeloid leukemia in patients living with HIV infection: Several questions, fewer answers [Articolo su rivista]
Forghieri, F.; Nasillo, V.; Bettelli, F.; Pioli, V.; Giusti, D.; Gilioli, A.; Mussini, C.; Tagliafico, E.; Trenti, T.; Cossarizza, A.; Maffei, R.; Barozzi, P.; Potenza, L.; Marasca, R.; Narni, F.; Luppi, M.
abstract

Both human immunodeficiency virus (HIV) infection and acute myeloid leukemia (AML) may be considered relatively uncommon disorders in the general population, but the precise incidence of AML in people living with HIV infection (PLWH) is uncertain. However, life expectancy of newly infected HIV-positive patients receiving anti-retroviral therapy (ART) is gradually increasing, rivaling that of age-matched HIV-negative individuals, so that the occurrence of AML is also expected to progressively increase. Even if HIV is not reported to be directly mutagenic, several indirect leukemogenic mechanisms, mainly based on bone marrow microenvironment disruption, have been proposed. Despite a well-controlled HIV infection under ART should no longer be considered per se a contraindication to intensive chemotherapeutic approaches, including allogeneic hematopoietic stem cell transplantation, in selected fit patients with AML, survival outcomes are still generally unsatisfactory. We discussed several controversial issues about pathogenesis and clinical management of AML in PLWH, but few evidence-based answers may currently be provided, due to the limited number of cases reported in the literature, mainly as case reports or small retrospective case series. Prospective multicenter clinical trials are warranted to more precisely investigate epidemiology and cytogenetic/molecular features of AML in PLWH, but also to standardize and further improve its therapeutic management.

2020 - Brca detection rate in an italian cohort of luminal early-onset and triple-negative breast cancer patients without family history: When biology overcomes genealogy [Articolo su rivista]
Toss, A.; Molinaro, E.; Venturelli, M.; Domati, F.; Marcheselli, L.; Piana, S.; Barbieri, E.; Grandi, G.; Piombino, C.; Marchi, I.; Tenedini, E.; Tagliafico, E.; Tazzioli, G.; Cortesi, L.
abstract

NCCN Guidelines recommend BRCA genetic testing in individuals with a probability >5% of being a carrier. Nonetheless, the cost-effectiveness of testing individuals with no tumor family history is still debated, especially when BRCA testing is offered by the national health service. Our analysis evaluated the rate of BRCA pathogenic or likely-pathogenic variants in 159 triplenegative breast cancer (TNBC) patients diagnosed ≤60 years, and 109 luminal-like breast cancer (BC) patients diagnosed ≤35 without breast and/or ovarian family histories. In TNBC patients, BRCA mutation prevalence was 22.6% (21.4% BRCA1). Mutation prevalence was 64.2% ≤30 years, 31.8% in patients aged 31–40, 16.1% for those aged 41–50 and 7.9% in 51–60s. A total of 40% of patients with estrogen receptors (ER) 1–9% were BRCA1 carriers. BRCA detection rate in early-onset BCs was 6.4% (4.6% BRCA2). Mutation prevalence was 0% between 0–25 years, 9% between 26–30 years and 6% between 31–35 years. In conclusion, BRCA testing is recommended in TNBC patients diagnosed ≤60 years, regardless of family cancer history or histotype, and by using immunohistochemical staining <10% for both ER and/PR. In luminal-like early-onset BC, a lower BRCA detection rate was observed, suggesting a role for other predisposing genes along with BRCA genetic testing.

2020 - Changes in gene expression in human skeletal stem cells transduced with constitutively active Gsα correlates with hallmark histopathological changes seen in fibrous dysplastic bone [Articolo su rivista]
Raimondo, D.; Remoli, C.; Astrologo, L.; Burla, R.; Torre, M. L.; Verni, F.; Tagliafico, E.; Corsi, A.; Giudice, S. D.; Persichetti, A.; Giannicola, G.; Robey, P. G.; Riminucci, M.; Saggio, I.
abstract

Fibrous dysplasia (FD) of bone is a complex disease of the skeleton caused by dominant activating mutations of the GNAS locus encoding for the α subunit of the G protein-coupled receptor complex (Gsα). The mutation involves a substitution of arginine at position 201 by histidine or cysteine (GsαR201H or R201C), which leads to overproduction of cAMP. Several signaling pathways are implicated downstream of excess cAMP in the manifestation of disease. However, the pathogenesis of FD remains largely unknown. The overall FD phenotype can be attributed to alterations of skeletal stem/progenitor cells which normally develop into osteogenic or adipogenic cells (in cis), and are also known to provide support to angiogenesis, hematopoiesis, and osteoclastogenesis (in trans). In order to dissect the molecular pathways rooted in skeletal stem/progenitor cells by FD mutations, we engineered human skeletal stem/progenitor cells with the GsαR201C mutation and performed transcriptomic analysis. Our data suggest that this FD mutation profoundly alters the properties of skeletal stem/progenitor cells by pushing them towards formation of disorganized bone with a concomitant alteration of adipogenic differentiation. In addition, the mutation creates an altered in trans environment that induces neovascularization, cytokine/chemokine changes and osteoclastogenesis. In silico comparison of our data with the signature of FD craniofacial samples highlighted common traits, such as the upregulation of ADAM (A Disintegrin and Metalloprotease) proteins and other matrix-related factors, and of PDE7B (Phosphodiesterase 7B), which can be considered as a buffering process, activated to compensate for excess cAMP. We also observed high levels of CEBPs (CCAAT-Enhancer Binding Proteins) in both data sets, factors related to browning of white fat. This is the first analysis of the reaction of human skeletal stem/progenitor cells to the introduction of the FD mutation and we believe it provides a useful background for further studies on the molecular basis of the disease and for the identification of novel potential therapeutic targets.

2020 - COVID-19: More than a cytokine storm [Articolo su rivista]
Riva, G.; Nasillo, V.; Tagliafico, E.; Trenti, T.; Comoli, P.; Luppi, M.
abstract

2020 - COVID-19: Room for treating T cell exhaustion? [Articolo su rivista]
Riva, G.; Nasillo, V.; Tagliafico, E.; Trenti, T.; Luppi, M.
abstract

2020 - Genomic analysis of hematopoietic stem cell at the single-cell level: Optimization of cell fixation and whole genome amplification (WGA) protocol [Articolo su rivista]
Carretta, C.; Mallia, S.; Genovese, E.; Parenti, S.; Rontauroli, S.; Bianchi, E.; Fantini, S.; Sartini, S.; Tavernari, L.; Tagliafico, E.; Manfredini, R.
abstract

Single-cell genomics has become the method of choice for the study of heterogeneous cell populations and represents an elective application in defining the architecture and clonal evolution in hematological neoplasms. Reconstructing the clonal evolution of a neoplastic population therefore represents the main way to understand more deeply the pathogenesis of the neoplasm, but it is also a potential tool to understand the evolution of the tumor population with respect to its response to therapy. Pre-analytical phase for single-cell genomics analysis is crucial to obtain a cell population suitable for single-cell sorting, and whole genome amplification is required to obtain the necessary amount of DNA from a single cell in order to proceed with sequencing. Here, we evaluated the impact of different methods of cellular immunostaining, fixation and whole genome amplification on the efficiency and yield of single-cell sequencing.

2020 - Npm1‐mutated myeloid neoplasms with <20% blasts: A really distinct clinico‐pathologic entity? [Articolo su rivista]
Forghieri, F.; Nasillo, V.; Paolini, A.; Bettelli, F.; Pioli, V.; Giusti, D.; Gilioli, A.; Colasante, C.; Acquaviva, G.; Riva, G.; Barozzi, P.; Maffei, R.; Potenza, L.; Marasca, R.; Fozza, C.; Tagliafico, E.; Trenti, T.; Comoli, P.; Longo, G.; Luppi, M.
abstract

Nucleophosmin (NPM1) gene mutations rarely occur in non‐acute myeloid neoplasms (MNs) with <20% blasts. Among nearly 10,000 patients investigated so far, molecular analyses documented NPM1 mutations in around 2% of myelodysplastic syndrome (MDS) cases, mainly belonging to MDS with excess of blasts, and 3% of myelodysplastic/myeloproliferative neoplasm (MDS/MPN) cases, prevalently classified as chronic myelomonocytic leukemia. These uncommon malignancies are associated with an aggressive clinical course, relatively rapid progression to overt acute myeloid leukemia (AML) and poor survival outcomes, raising controversies on their classification as distinct clinico‐pathologic entities. Furthermore, fit patients with NPM1‐mutated MNs with <20% blasts could benefit most from upfront intensive chemotherapy for AML rather than from moderate intensity MDS‐directed therapies, although no firm conclusion can currently be drawn on best therapeutic approaches, due to the limited available data, obtained from small and mainly retrospective series. Caution is also suggested in definitely diagnosing NPM1‐mutated MNs with blast count <20%, since NPM1‐mutated AML cases frequently present dysplastic features and multilineage bone marrow cells showing abnormal cytoplasmic NPM1 protein delocalization by immunohistochemical staining, therefore belonging to NPM1‐mutated clone regardless of blast morphology. Further prospective studies are warranted to definitely assess whether NPM1 mutations may become sufficient to diagnose AML, irrespective of blast percentage.

2020 - P2X7 receptor activity limits accumulation of T cells within tumors [Articolo su rivista]
Romagnani, Andrea; Rottoli, Elsa; Mazza, Emilia Maria Cristina; Rezzonico-Jost, Tanja; De Ponte Conti, Benedetta; Proietti, Michele; Perotti, Michela; Civanelli, Elisa; Perruzza, Lisa; Catapano, Alberico L; Baragetti, Andrea; Tenedini, Elena; Tagliafico, Enrico; Falzoni, Simonetta; Di Virgilio, Francesco; Norata, Giuseppe Danilo; Bicciato, Silvio; Grassi, Fabio
abstract

Extracellular adenosine triphosphate (eATP) is a signaling molecule which variably affects all cells of the immune system either directly or after hydrolysis to adenosine. Although eATP is virtually absent in the interstitium of normal tissues, it can be present in the hundreds of micromolar range in tumors, a concentration compatible with activation of the ATP-gated ionotropic P2X7 receptor. Here we show that P2X7 activity in tumor-infiltrating T cells (TILs) induces cellular senescence and limits tumor suppression. P2X7 stimulation affected cell cycling of effector T cells and resulted in generation of mitochondrial reactive oxygen species (ROS) and p38 MAPK-dependent upregulation of cyclin-dependent kinase inhibitor 1A (Cdkn1a, encoding for p21Waf1/Cip1). Lack of P2X7 promoted a transcriptional signature that correlated with enhanced cytotoxic T cell response in human solid tumors. In mice, transfer of tumor specific T cells with deletion of P2rx7 significantly reduced tumor growth and extended survival. Collectively, these findings uncover a purinergic checkpoint that can be targeted to improve the efficacy of cancer immunotherapy strategies.

2019 - BRCA mutations among triple negative breast cancer without family history of breast and ovarian cancer: The Modena family cancer clinic experience [Abstract in Rivista]
Molinaro, E.; Venturelli, M.; Toss, A.; Piombino, C.; Barbieri, E.; Marcheselli, L.; Marchi, I.; Tagliafico, E.; Cascinu, S.; Cortesi, L.
abstract

2019 - Calreticulin Ins5 and Del52 mutations impair unfolded protein and oxidative stress responses in K562 cells expressing CALR mutants [Articolo su rivista]
Salati, Simona; Genovese, Elena; Carretta, Chiara; Zini, Roberta; Bartalucci, Niccolò; Prudente, Zelia; Pennucci, Valentina; Ruberti, Samantha; Rossi, Chiara; Rontauroli, Sebastiano; Enzo, Elena; Calabresi, Laura; Balliu, Manjola; Mannarelli, Carmela; Bianchi, Elisa; Guglielmelli, Paola; Tagliafico, Enrico; Vannucchi, Alessandro M; Manfredini, Rossella
abstract

Somatic mutations of calreticulin (CALR) have been described in approximately 60-80% of JAK2 and MPL unmutated Essential Thrombocythemia and Primary Myelofibrosis patients. CALR is an endoplasmic reticulum (ER) chaperone responsible for proper protein folding and calcium retention. Recent data demonstrated that the TPO receptor (MPL) is essential for the development of CALR mutant-driven Myeloproliferative Neoplasms (MPNs). However, the precise mechanism of action of CALR mutants haven't been fully unraveled. In this study, we showed that CALR mutants impair the ability to respond to the ER stress and reduce the activation of the pro-apoptotic pathway of the unfolded protein response (UPR). Moreover, our data demonstrated that CALR mutations induce increased sensitivity to oxidative stress, leading to increase oxidative DNA damage. We finally demonstrated that the downmodulation of OXR1 in CALR-mutated cells could be one of the molecular mechanisms responsible for the increased sensitivity to oxidative stress mediated by mutant CALR. Altogether, our data identify novel mechanisms collaborating with MPL activation in CALR-mediated cellular transformation. CALR mutants negatively impact on the capability of cells to respond to oxidative stress leading to genomic instability and on the ability to react to ER stress, causing resistance to UPR-induced apoptosis.

2019 - CXCR3 identifies human naive CD8+ T cells with enhanced effector differentiation potential [Articolo su rivista]
De Simone, G.; Mazza, E. M. C.; Cassotta, A.; Davydov, A. N.; Kuka, M.; Zanon, V.; De Paoli, F.; Scamardella, E.; Metsger, M.; Roberto, A.; Pilipow, K.; Colombo, F. S.; Tenedini, E.; Tagliafico, E.; Gattinoni, L.; Mavilio, D.; Peano, C.; Price, D. A.; Singh, S. P.; Farber, J. M.; Serra, V.; Cucca, F.; Ferrari, F.; Orru, V.; Fiorillo, E.; Iannacone, M.; Chudakov, D. M.; Sallusto, F.; Lugli, E.
abstract

In mice, the ability of naive T (TN) cells to mount an effector response correlates with TCR sensitivity for self-derived Ags, which can be quantified indirectly by measuring surface expression levels of CD5. Equivalent findings have not been reported previously in humans. We identified two discrete subsets of human CD8+ TN cells, defined by the absence or presence of the chemokine receptor CXCR3. The more abundant CXCR3+ TN cell subset displayed an effector-like transcriptional profile and expressed TCRs with physicochemical characteristics indicative of enhanced interactions with peptide-HLA class I Ags.Moreover, CXCR3+ TN cells frequently produced IL-2 and TNF in response to nonspecific activation directly ex vivo and differentiated readily into Ag-specific effector cells in vitro. Comparative analyses further revealed that human CXCR3+ TN cells were transcriptionally equivalent to murine CXCR3+ TN cells, which expressed high levels of CD5. These findings provide support for the notion that effector differentiation is shaped by heterogeneity in the preimmune repertoire of human CD8+ T cells.

2019 - Gene expression profiles of human granulosa cells treated with bioequivalent doses of corifollitropin alfa (CFA) or recombinant human follicle-stimulating hormone (recFSH) [Articolo su rivista]
Sacchi, Sandro; Tenedini, Elena; Tondelli, Debora; Parenti, Sandra; Tagliasacchi, Daniela; Xella, Susanna; Marsella, Tiziana; Tagliafico, Enrico; La Marca, Antonio
abstract

Using recombinant DNA technologies, a chimeric gene containing the coding sequences of follicle stimulating hormone (FSH) β-subunit and C-terminal peptide of the human chorionic gonadotrophin (hCG) β-subunit have been designed to generate a new gonadotrophin named corifollitropin alfa (CFA). CFA has longer elimination half-life and slower rate of absorption compared with FSH, which makes CFA a long-acting hormone employed as a substitute of the recombinant FSH (recFSH) in the controlled ovarian stimulation (COS). The purpose of this study is to compare the gene expression profiles elicited by bioequivalent doses of CFA or recFSH in primary cultures of human granulosa cells (hGCs). Gonadotrophins exert their functions by binding FSH receptors (FSHRs), activating signaling pathways that increase the cyclic adenosine monophosphate (cAMP) intracellular content. Bioequivalence has been defined as the dose/duration of gonadotrophin treatment able to promote the same amount of intracellular cAMP. hGCs were treated with different doses of either gonadotrophin and the cAMP was measured after different incubation times to establish the bioequivalence. Results obtained by comparing the bioequivalent treatments, showed that CFA is more effective than recFSH in inducing aromatase gene expression after 6 and 24 h from the initial stimulation in agreement with its long-acting characteristic.

2019 - Hereditary pancreatic cancer: A retrospective single-center study of 5143 Italian families with history of BRCA-related malignancies [Articolo su rivista]
Toss, Angela; Venturelli, Marta; Molinaro, Eleonora; Pipitone, Stefania; Barbieri, Elena; Marchi, Isabella; Tenedini, Elena; Artuso, Lucia; Castellano, Sara; Marino, Marco; Tagliafico, Enrico; Razzaboni, Elisabetta; De Matteis, Elisabetta; Cascinu, Stefano; Cortesi, Laura
abstract

The identification of BRCA mutations plays a crucial role in the management of hereditary cancer prevention and treatment. Nonetheless, BRCA-testing in pancreatic cancer (PC) patients is not universally introduced in clinical practice. A retrospective analysis was conducted, firstly, to evaluate the rate of BRCA-positive families among those presenting a family history of PC besides breast and/or ovarian cancer. Secondly, the relationship between BRCA pathogenic variants and PC risk was evaluated. Finally, the characteristics of PC developed in BRCA families were described. Among 5143 family trees reporting breast and/or ovarian cancer cases, 392 showed a family history of PC. A total of 35 families (24.5% selected by the Modena Criteria and 21.3% by the NCCN Criteria) were positive to BRCA testing. Among the BRCA1 mutations, 36.8% were found within a region defined by c.3239–c.3917, whilst 43.7% of BRCA2 mutations were located within c.7180–c.8248. This study confirmed that an increase in the rate of positive tests in families with PC when associated to breast and/or ovarian tumors. Moreover, this analysis indicated two possible Pancreatic Cancer Cluster Regions that should be verified in future research. Finally, PC in families with breast and/or ovarian cancer history, particularly in BRCA families, were diagnosed at younger age and showed better one-year overall survival.

2019 - Immune infiltrate composition across intrinsic subtypes in hormone receptor (HR)+/HER2- early breast cancer (BC) enrolled in the prospective LETLOB trial. [Poster]
Griguolo, G; V Dieci, M; Paré, L; Miglietta, F; G Generali, D; Frassoldati, A; Bisagni, G; Piacentini, F; Tagliafico, E; Cagossi, K; Ficarra, G; Prat, A; F Conte, P; Guarneri, V
abstract

Background In HR+/HER2- early BC, high tumour infiltrating lymphocytes (TIL) levels predict higher pathological complete response to neoadjuvant chemotherapy, but are associated with shorter overall survival (Denkert, Lancet Oncol 2018). HR+/HER2- BC is a biologically heterogeneous disease, encompassing all BC molecular intrinsic subtypes, with different clinical behaviour (Cejalvo, CTR 2018). Little is known concerning the distribution of TIL levels and immune infiltrate composition across intrinsic subtypes in HR+/HER2- BC. Methods Gene-expression data (Affymetrix platform) from pre-treatment frozen core-biopsies was available from 66 postmenopausal patients with HR+/HER2- early BC from the LETLOB trial (neoadjuvant letrozole+/-lapatinib) (Guarneri, JCO 2014). Intrinsic subtype was assigned using a research-based PAM50 subtype predictor. Relative leukocyte fractions were calculated using CIBERSORT (Newman, Nature Methods 2015), a deconvolution method based on RNA gene-expression signatures. Pre-treatment stromal TILs were assessed on centralized HES slides according to recommendations (Salgado, Ann Oncol 2015). Results Intrinsic subtype distribution was as follows: basal 18% (N = 12), HER2-enriched 8% (N = 5), Luminal A 39% (N = 25), Luminal B 36% (N = 24). Non-luminal subtypes (HER2-enriched and Basal) had significantly higher baseline TIL levels than luminal subtypes (median (range): 7 (0-100) and 2 (0-35), respectively; p = 0.038). Non-luminal subtypes also presented higher fractions of CD4 memory activated T-cells (p = 0.018), γδ T-cells (p = 0.010) and M1 macrophages (p = 0.001) and lower fractions of T-regulatory cells (p = 0.002) than luminal subtypes. Conclusions In HR+/HER2- early BC, non-luminal subtypes show higher TIL levels and a more pro-inflammatory anti-tumour immune infiltrate composition. This immune heterogeneity across intrinsic subtypes should be considered when analysing the complex prognostic role of TILs in HR+/HER2- early BC.

2019 - MICAL2 is expressed in cancer associated neo-angiogenic capillary endothelia and it is required for endothelial cell viability, motility and VEGF response [Articolo su rivista]
Barravecchia, Ivana; Mariotti, Sara; Pucci, Maria Angela; Scebba, Francesca; De Cesari, Chiara; Bicciato, Silvio; Tagliafico, Enrico; Tenedini, Elena; Vindigni, Carla; Cecchini, Marco; Berti, Gabriele; Vitiello, Marianna; Poliseno, Laura; Mazzanti, Chiara Maria; Angeloni, Debora
abstract

The capacity of inducing angiogenesis is a recognized hallmark of cancer cells. The cancer microenvironment, characterized by hypoxia and inflammatory signals, promotes proliferation, migration and activation of quiescent endothelial cells (EC) from surrounding vascular network. Current anti-angiogenic drugs present side effects, temporary efficacy, and issues of primary resistance, thereby calling for the identification of new therapeutic targets. MICALs are a unique family of redox enzymes that destabilize F-actin in cytoskeletal dynamics. MICAL2 mediates Semaphorin3A-NRP2 response to VEGFR1 in rat ECs. MICAL2 also enters the p130Cas interactome in response to VEGF in HUVEC. Previously, we showed that MICAL2 is overexpressed in metastatic cancer. A small-molecule inhibitor of MICAL2 exists (CCG-1423). Here we report that 1) MICAL2 is expressed in neo-angiogenic ECs in human solid tumors (kidney and breast carcinoma, glioblastoma and cardiac myxoma, n = 67, were analyzed with immunohistochemistry) and in animal models of ischemia/inflammation neo-angiogenesis, but not in normal capillary bed; 2) MICAL2 protein pharmacological inhibition (CCG-1423) or gene KD reduce EC viability and functional performance; 3) MICAL2 KD disables ECs response to VEGF in vitro. Whole-genome gene expression profiling reveals MICAL2 involvement in angiogenesis and vascular development pathways. Based on these results, we propose that MICAL2 expression in ECs participates to inflammation-induced neo-angiogenesis and that MICAL2 inhibition should be tested in cancer- and noncancer-associated neo-angiogenesis, where chronic inflammation represents a relevant pathophysiological mechanism.

2019 - Usefulness and limitations of comprehensive characterization of mRNA splicing profiles in the definition of the clinical relevance of BRCA1/2 variants of uncertain significance [Articolo su rivista]
Gelli, E.; Colombo, M.; Pinto, A. M.; De Vecchi, G.; Foglia, C.; Amitrano, S.; Morbidoni, V.; Imperatore, V.; Manoukian, S.; Baldassarri, M.; Lo Rizzo, C.; Catania, L.; Frullanti, E.; Tagliafico, E.; Cortesi, L.; Spaggiari, F.; Mencarelli, M. A.; Trevisson, E.; Radice, P.; Renieri, A.; Ariani, F.
abstract

Highly penetrant variants of BRCA1/2 genes are involved in hereditary predisposition to breast and ovarian cancer. The detection of pathogenic BRCA variants has a considerable clinical impact, allowing appropriate cancer-risk management. However, a major drawback is represented by the identification of variants of uncertain significance (VUS). Many VUS potentially affect mRNA splicing, making transcript analysis an essential step for the definition of their pathogenicity. Here, we characterize the impact on splicing of ten BRCA1/2 variants. Aberrant splicing patterns were demonstrated for eight variants whose alternative transcripts were fully characterized. Different events were observed, including exon skipping, intron retention, and usage of de novo and cryptic splice sites. Transcripts with premature stop codons or in-frame loss of functionally important residues were generated. Partial/complete splicing effect and quantitative contribution of different isoforms were assessed, leading to variant classification according to Evidence-based Network for the Interpretation of Mutant Alleles (ENIGMA) consortium guidelines. Two variants could be classified as pathogenic and two as likely benign, while due to a partial splicing effect, six variants remained of uncertain significance. The association with an undefined tumor risk justifies caution in recommending aggressive risk-reduction treatments, but prevents the possibility of receiving personalized therapies with potential beneficial effect. This indicates the need for applying additional approaches for the analysis of variants resistant to classification by gene transcript analyses.

2018 - Calreticulin affects hematopoietic stem/progenitor cell fate by impacting erythroid and megakaryocytic differentiation [Articolo su rivista]
Salati, Simona; Prudente, Zelia; Genovese, Elena; Pennucci, Valentina; Rontauroli, Sebastiano; Bartalucci, Niccolo'; Mannarelli, Carmela; Ruberti, Samantha; Zini, Roberta; Rossi, Chiara; Bianchi, Elisa; Guglielmelli, Paola; Tagliafico, Enrico; Vannucchi, Alessandro M; Manfredini, Rossella
abstract

Calreticulin (CALR) is a chaperone protein that localizes primarily to the endoplasmic reticulum (ER) lumen where it is responsible for the control of proper folding of neo-synthesized glycoproteins and for the retention of calcium. Recently, mutations affecting exon 9 of the CALR gene have been described in approximately 40% of patients with myeloproliferative neoplasms (MPNs). Although the role of mutated CALR in the development of MPNs has begun to be clarified, there are still no data available on the function of wild-type (WT) CALR during physiological hematopoiesis. In order to shed light on the role of WT CALR during normal hematopoiesis, we performed gene silencing and overexpression experiments in Hematopoietic Stem Progenitor Cells (HSPCs). Our results showed that CALR overexpression is able to affect physiological hematopoiesis by enhancing both erythroid and megakaryocytic (MK) differentiation. In agreement with overexpression data, CALR silencing caused a significant decrease in both erythroid and MK differentiation of human HSPCs. Gene expression profiling (GEP) analysis showed that CALR is able to affect the expression of several genes involved in HSPCs differentiation towards both the erythroid and MK lineages. Moreover, GEP data also highlighted the modulation of several genes involved in ER stress response, unfolded protein response (UPR), DNA repair and of several genes already described to play a role in MPN development, such as pro-inflammatory cytokines and hematological neoplasms-related markers. Altogether, our data unraveled a new and unexpected role for CALR in the regulation of normal hematopoietic differentiation. Moreover, by showing the impact of CALR on the expression of genes involved in several biological processes already described in cellular transformation, our data strongly suggest a more complex role for CALR in MPN development that goes beyond the activation of the THPO receptor and involves ER stress response, UPR and DNA repair.

2018 - ERBB2 mutations in hormone receptor positive primary breast cancers samples and in their matched endocrine-resistant recurrences. [Poster]
Venturelli, M.; Toss, A.; Piacentini, F.; Artuso, L.; Bernardis, I.; Parenti, S.; Tenedini, E.; Omarini, C.; Moscetti., 1; Cascinu, S.; Tagliafico, E.; Cortesi, L.
abstract

Previous preclinical studies showed that mutations in ERBB2 might represent an alternative mechanism for HER2 activation and the rate of mutations in BC is around 2%. They occur more frequently in HER2-negative (HER2-) BC and are associated with poor survival. On these bases, HER2- pts with mutation are potentially candidates for HER2-targeted therapy, as already showed by Neratinib. We evaluated the incidence of ERBB2 mutations in 14 hormone receptor (HR) positive BC and in their matched endocrine-resistant recurrences. Using an NGS technology, we evaluated a panel of genes including ERBB2, in FFPE tissues. We analysed 14 HR positive BCs and their matched recurrences. All the relapses have been developed during an endocrine treatment. 29% of pts were diagnosed with HER2+ BC, while 71% of pts developed HER2- BC. 3 pts were diagnosed at stage I, 6 pts at stage II, 5 pts at stage III. We found 8 different mutations in 9 samples: A356D, Q1206X, Q396X, Q393X, P523L, I654V, G1220C, 135+3G>T. Only I654V was previously described in literature. All but one (135+3G>T) of these mutations are exonic variants. 5 mutations were in the extracellular domain, 1 in the tyrosine kinase domain and 2 in the carboxy tail. 28.6% of pts had ERBB2 mutations in the primary BCs and 35.7% in the relapsed site. 66.6% of HER2+ primary BCs showed an ERBB2 mutation, while only 21% of HER2- samples brought a mutation. 2 patients acquired a new mutation in the relapsed site, while 1 patient lost the mutation in the relapsed tissue. The mDFS was 35.3 months. mDFS in HER2+ and/or mutated pts was 46.4 months, while mDFS in HER2- wild type pts was 28.5. The mOS was 104 months (6 pts still alive). mOS in HER2+ and/or mutated pts was 115.6 months while mOS in HER2- wild type pts was 97.5. We found an overall detection rate of mutations higher than that described in literature (ERBB2 mutations were present in 32.1% of our samples), meaning that our pts have been highly selected. In fact, only tumors relapsing 26 Tumori Journal 104(4S) under an endocrine treatment, and thus with proved endocrine resistance, have been included in our analyses. The identification of an ERBB2 mutation in primary BCs might justify a more targeted neo/adjuvant approach and, might guide the subsequent treatment choices when the mutation is identified in the relapsed tissue. Contrary to previous literature, in our study the majority of mutations occurred in HER2+ samples and HER2+ and/or mutated samples did not show worse outcomes.

2018 - Genomic alterations at the basis of treatment resistance in metastatic breast cancer: Clinical applications [Articolo su rivista]
Toss, Angela; Piacentini, Federico; Cortesi, Laura; Artuso, Lucia; Bernardis, Isabella; Parenti, Sandra; Tenedini, Elena; Ficarra, Guido; Maiorana, Antonino; Iannone, Anna; Omarini, Claudia; Moscetti, Luca; Cristofanilli, Massimo; Federico, Massimo; Tagliafico, Enrico
abstract

The standard of care for breast cancer has gradually evolved from empirical treatments based on clinical-pathological characteristics to the use of targeted approaches based on the molecular profile of the tumor. Consequently, an increasing number of molecularly targeted drugs have been developed. These drugs target specific alterations, called driver mutations, which confer a survival advantage to cancer cells. To date, the main challenge remains the identification of predictive biomarkers for the selection of the optimal treatment. On this basis, we evaluated a panel of 25 genes involved in the mechanisms of targeted treatment resistance, in 16 primary breast cancers and their matched recurrences, developed during treatment. Overall, we found a detection rate of mutations higher than that described in the literature. In particular, the most frequently mutated genes were ERBB2 and those involved in the PI3K/AKT/mTOR and the MAPK signaling pathways. The study revealed substantial discordances between primary tumors and metastases, stressing the need for analysis of metastatic tissues at recurrence. We observed that 85.7% of patients with an early-stage or locally advanced primary tumor showed at least one mutation in the primary tumor. This finding could explain the subsequent relapse and might therefore justify more targeted adjuvant treatments. Finally, the mutations detected in 50% of relapsed tissues could have guided subsequent treatment choices in a different way. This study demonstrates that mutation events may be present at diagnosis or arise during cancer treatment. As a result, profiling primary and metastatic tumor tissues may be a major step in defining optimal treatments.

2018 - Involvement of MAF/SPP1 axis in the development of bone marrow fibrosis in PMF patients [Articolo su rivista]
Ruberti, S; Bianchi, E; Guglielmelli, P; Rontauroli, S; Barbieri, G; Tavernari, L; Fanelli, T; Norfo, R; Pennucci, V; Fattori, G. Corbizi; Mannarelli, C; Bartalucci, N; Mora, B; Elli, L; Avanzini, M. A; Rossi, C; Salmoiraghi, S; Zini, R; Salati, S; Prudente, Z; Rosti, V; Passamonti, F; Rambaldi, A; Ferrari, S; Tagliafico, E; Vannucchi, A. M; Manfredini, R.
abstract

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by hyperplastic megakaryopoiesis and myelofibrosis. We recently described the upregulation of MAF (v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog) in PMF CD34+ hematopoietic progenitor cells (HPCs) compared to healthy donor. Here we demonstrated that MAF is also upregulated in PMF compared with the essential thrombocytemia (ET) and polycytemia vera (PV) HPCs. MAF overexpression and knockdown experiments shed some light into the role of MAF in PMF pathogenesis, by demonstrating that MAF favors the megakaryocyte and monocyte/macrophage commitment of HPCs and leads to the increased expression of proinflammatory and profibrotic mediators. Among them, we focused our further studies on SPP1 and LGALS3. We assessed SPP1 and LGALS3 protein levels in 115 PMF, 47 ET and 24 PV patients plasma samples and we found that SPP1 plasma levels are significantly higher in PMF compared with ET and PV patients. Furthermore, in vitro assays demonstrated that SPP1 promotes fibroblasts and mesenchymal stromal cells proliferation and collagen production. Strikingly, clinical correlation analyses uncovered that higher SPP1 plasma levels in PMF patients correlate with a more severe fibrosis degree and a shorter overall survival. Collectively our data unveil that MAF overexpression contributes to PMF pathogenesis by driving the deranged production of the profibrotic mediator SPP1.

2018 - KLF4 mediates the effect of 5-ASA on the b-catenin pathway in colon cancer cells [Articolo su rivista]
Parenti, Sandra; Montorsi, Lucia; Fantini, Sebastian; Mammoli, Fabiana; Gemelli, Claudia; Atene, Claudio Giacinto; Losi, Lorena; Frassineti, Chiara; Calabretta, Bruno; Tagliafico, Enrico; Ferrari, Sergio; Zanocco-Marani, Tommaso; Grande, Alexis
abstract

Mesalazine (5-ASA) is an aminosalicylate anti-inflammatory drug capable of inducing m-protocadherin, a protein expressed by colorectal epithelial cells that is downregulated upon malignant transformation. Treatment with 5-ASA restores m-protocadherin expression and promotes the sequestration of b-catenin to the plasma membrane. Here, we show that 5-ASA–induced m-protocadherin expression is directly regulated by the KLF4 transcription factor. In addition, we suggest the existence of a dual mechanism whereby 5-ASA–mediated b-catenin inhibition is caused by m-protocadherin–dependent sequestration of b-catenin to the plasma membrane and by the direct binding of KLF4 to b-catenin. In addition, we found that 5-ASA treatment suppresses the expression of miR-130a and miR-135b, which target KLF4 mRNA, raising the possibility that this mechanism is involved in the increased expression of KLF4 induced by 5-ASA.

2018 - Role of TGF-β1/miR-382-5p/SOD2 axis in the induction of oxidative stress in CD34+ cells from primary myelofibrosis [Articolo su rivista]
Rossi, Chiara; Zini, Roberta; Rontauroli, Sebastiano; Ruberti, Samantha; Prudente, Zelia; Barbieri, Greta; Bianchi, Elisa; Salati, Simona; Genovese, Elena; Bartalucci, Niccolò; Guglielmelli, Paola; Tagliafico, Enrico; Rosti, Vittorio; Barosi, Giovanni; Vannucchi, Alessandro M.; Manfredini, Rossella
abstract

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by an excessive production of pro-inflammatory cytokines resulting in chronic inflammation and genomic instability. Besides the driver mutations in JAK2, MPL, and CALR genes, the deregulation of miRNA expression may also contribute to the pathogenesis of PMF. To this end, we recently reported the upregulation of miR-382-5p in PMF CD34+ cells. In order to unveil the mechanistic details of the role of miR-382-5p in pathogenesis of PMF, we performed gene expression profiling of CD34+ cells overexpressing miR-382-5p. Among the downregulated genes, we identified superoxide dismutase 2 (SOD2), which is a predicted target of miR-382-5p. Subsequently, we confirmed miR-382-5p/SOD2 interaction by luciferase assay and we showed that miR-382-5p overexpression in CD34+ cells causes the decrease in SOD2 activity leading to reactive oxygen species (ROS) accumulation and oxidative DNA damage. In addition, our data indicate that inhibition of miR-382-5p in PMF CD34+ cells restores SOD2 function, induces ROS disposal, and reduces DNA oxidation. Since the pro-inflammatory cytokine transforming growth factor-β1 (TGF-β1) is a key player in PMF pathogenesis, we further investigated the effect of TGF-β1 on ROS and miR-382-5p levels. Our data showed that TGF-β1 treatment enhances miR-382-5p expression and reduces SOD2 activity leading to ROS accumulation. Finally, inhibition of TGF-β1 signaling in PMF CD34+ cells by galunisertib significantly reduced miR-382-5p expression and ROS accumulation and restored SOD2 activity. As a whole, this study reports that TGF-β1/miR-382-5p/SOD2 axis deregulation in PMF cells is linked to ROS overproduction that may contribute to enhanced oxidative stress and inflammation. Our results suggest that galunisertib may represent an effective drug reducing abnormal oxidative stress induced by TGF-β1 in PMF patients. Database linking: GEO: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE103464.

2018 - The early expansion of anergic NKG2Apos/CD56dim/CD16neg natural killer represents a therapeutic target in haploidentical hematopoietic stem cell transplantation [Articolo su rivista]
Roberto, Alessandra; Di Vito, Clara; Zaghi, Elisa; Mazza, Emilia Maria Cristina; Capucetti, Arianna; Calvi, Michela; Tentorio, Paolo; Zanon, Veronica; Sarina, Barbara; Mariotti, Jacopo; Bramanti, Stefania; Tenedini, Elena; Tagliafico, Enrico; Bicciato, Silvio; Santoro, Armando; Roederer, Mario; Marcenaro, Emanuela; Castagna, Luca; Lugli, Enrico; Mavilio, Domenico
abstract

Natural killer cells are the first lymphocyte population to reconsti-tute early after non-myeloablative and T cell-replete haploidenti-cal hematopoietic stem cell transplantation with post-transplant infusion of cyclophosphamide. The study herein characterizes the transient and predominant expansion starting from the second week following haploidentical hematopoietic stem cell transplantation of a donor-derived unconventional subset of NKp46neg-low/CD56dim/CD16neg natural killer cells expressing remarkably high levels of CD94/NKG2A. Both transcription and phenotypic profiles indicated that unconventional NKp46neg-low/CD56dim/CD16neg cells are a distinct natural killer cell subpopulation with features of late stage differentiation, yet retaining prolifera-tive capability and functional plasticity to generate conventional NKp46pos/CD56bright/CD16neg-low cells in response to interleukin-15 plus interleukin-18. While present at low frequency in healthy donors, unconventional NKp46neg-low/CD56dim/CD16neg cells are greatly expanded in the seven weeks following haploidentical hematopoietic stem cell transplantation, and express high levels of the activating receptors NKG2D and NKp30 as well as of the lytic granules Granzyme-B and Perforin. Nonetheless, NKp46neg-low/CD56dim/CD16neg cells displayed a markedly defective cytotoxicity that could be reversed by blocking the inhibitory receptor CD94/NKG2A. These data open new and important perspectives to better understand the ontogenesis/homeostasis of human natural killer cells and to develop a novel immune-therapeutic approach that targets the inhibitory NKG2A check-point, thus unleashing natural killer cell alloreactivity early after haploidentical hematopoietic stem cell transplantation.

2018 - Workload measurement for molecular genetics laboratory: A survey study [Articolo su rivista]
Tagliafico, Enrico; Bernardis, Isabella; Grasso, Marina; D’Apice, Maria Rosaria; Lapucci, Cristina; Botta, Annalisa; Giachino, Daniela Francesca; Marinelli, Maria; Primignani, Paola; Russo, Silvia; Sani, Ilaria; Seia, Manuela; Fini, Sergio; Rimessi, Paola; Tenedini, Elena; Ravani, Anna; Genuardi, Maurizio; Ferlini, Alessandra
abstract

Genetic testing availability in the health care system is rapidly increasing, along with the diffusion of next-generation sequencing (NGS) into diagnostics. These issues make imperative the knowledge-drive optimization of testing in the clinical setting. Time estimations of wet laboratory procedure in Italian molecular laboratories offering genetic diagnosis were evaluated to provide data suitable to adjust efficiency and optimize health policies and costs. A survey was undertaken by the Italian Society of Human Genetics (SIGU). Forty-two laboratories participated. For most molecular techniques, the most time-consuming steps are those requiring an intensive manual intervention or in which the human bias can affect the global process time-performances. For NGS, for which the study surveyed also the interpretation time, the latter represented the step that requiring longer times. We report the first survey describing the hands-on times requested for different molecular diagnostics procedures, including NGS. The analysis of this survey suggests the need of some improvements to optimize some analytical processes, such as the implementation of laboratory information management systems to minimize manual procedures in pre-analytical steps which may affect accuracy that represents the major challenge to be faced in the future setting of molecular genetics laboratory.

2017 - CALR mutational status identifies different disease subtypes of essential thrombocythemia showing distinct expression profiles [Articolo su rivista]
Zini, Roberta; Guglielmelli, Paola; Pietra, Daniela; Rumi, Elisa; Rossi, Chiara; Rontauroli, Sebastiano; Genovese, Elena; Fanelli, Tiziana; Calabresi, Laura; Bianchi, Elisa; Salati, Simona; Cazzola, Mario; Tagliafico, Enrico; Vannucchi, Alessandro M.; Manfredini, Rossella
abstract

Polycythemia vera (PV) and essential thrombocythemia (ET) are Philadelphia-negative myeloproliferative neoplasms (MPNs) characterized by erythrocytosis and thrombocytosis, respectively. Approximately 95% of PV and 50-70% of ET patients harbor the V617F mutation in the exon 14 of JAK2 gene, while about 20-30% of ET patients carry CALRins5 or CALRdel52 mutations. These ET CALR-mutated subjects show higher platelet count and lower thrombotic risk compared to JAK2-mutated patients. Here, we showed that CALR-mutated and JAK2V617F-positive CD34+ cells display different gene and miRNA expression profiles. Indeed, we highlighted several pathways differentially activated between JAK2V617F- and CALR-mutated progenitors, i.e., mTOR, MAPK/PI3K, and MYC pathways. Furthermore, we unveiled that the expression of several genes involved in DNA repair, chromatin remodeling, splicing, and chromatid cohesion are decreased in CALR-mutated cells. According to the low risk of thrombosis in CALR-mutated patients, we also found the downregulation of several genes involved in thrombin signaling and platelet activation. As a whole, these data support the model that CALR-mutated ET could be considered as a distinct disease entity from JAK2V617F-positive MPNs and may provide the molecular basis supporting the different clinical features of these patients.

2017 - Deregulated expression of miR-29a-3p, miR-494-3p and miR-660-5p affects sensitivity to tyrosine kinase inhibitors in CML leukemic stem cells [Articolo su rivista]
Salati, Simona; Salvestrini, Valentina; Carretta, Chiara; Genovese, Elena; Rontauroli, Sebastiano; Zini, Roberta; Rossi, Chiara; Ruberti, Samantha; Bianchi, Elisa; Barbieri, Greta; Curti, Antonio; Castagnetti, Fausto; Gugliotta, Gabriele; Rosti, Gianantonio; Bergamaschi, Micaela; Tafuri, Agostino; Tagliafico, Enrico; Lemoli, Roberto; Manfredini, Rossella
abstract

The development of Imatinib mesylate (IM), which targets the oncogenic BCRABL fusion protein, has greatly improved the outcome of Chronic Myeloid Leukemia (CML) patients. However, BCR-ABL-positive progenitors can be detected in CML patients in complete cytogenetic response. Several evidence suggests that CML stem cells are intrinsically resistant to Tyrosine Kinase Inhibitors (TKI), and therefore they represent the most likely candidate responsible for disease relapse. In this work, we investigated the microRNA (miRNA) expression profile of different subpopulations of CML Leukemic Stem Cells (LSCs): Lin-CD34+CD38-and Lin-CD34-CD38-cells. These cell fractions have been previously shown to be endowed with TKI intrinsic resistance. Our analysis identified 33 common deregulated miRNAs in CML LSCs. Among those, 8 miRNAs were deregulated in CML independently from BCR-ABL kinase activity and therefore are likely to be involved in the BCR-ABL-independent resistance to TKI that characterizes CML LSCs. In particular, the up-regulation of miR-29a-3p and miR-660-5p observed in CML LSCs, led to the down-regulation of their respective targets TET2 and EPAS1 and conferred TKI-resistance to CML LSCs in vitro. On the other hand, miR-494-3p down-regulation in CML LSCs, leading to c-MYC up-regulation, was able to decrease TKI-induced apoptosis. These results demonstrate that aberrant miRNA expression in CML LSCs could contribute to the intrinsic TKIresistance observed in these cell populations, and support the development of novel therapies aimed at targeting aberrantly regulated miRNAs or their targets in order to effectively eradicate CML LSCs.

2017 - Identification of miR-31-5p, miR-141-3p, miR-200c-3p, and GLT1 as human liver aging markers sensitive to donor-recipient age-mismatch in transplants [Articolo su rivista]
Capri, Miriam; Olivieri, Fabiola; Lanzarini, Catia; Remondini, Daniel; Borelli, Vincenzo; Lazzarini, Raffaella; Graciotti, Laura; Albertini, Maria Cristina; Bellavista, Elena; Santoro, Aurelia; Biondi, Fiammetta; Tagliafico, Enrico; Tenedini, Elena; Morsiani, Cristina; Pizza, Grazia; Vasuri, Francesco; D'Errico, Antonietta; Dazzi, Alessandro; Pellegrini, Sara; Magenta, Alessandra; D'Agostino, Marco; Capogrossi, Maurizio C.; Cescon, Matteo; Rippo, Maria Rita; Procopio, Antonio Domenico; Franceschi, Claudio; Grazi, Gian Luca
abstract

To understand why livers from aged donors are successfully used for transplants, we looked for markers of liver aging in 71 biopsies from donors aged 12-92 years before transplants and in 11 biopsies after transplants with high donor-recipient age-mismatch. We also assessed liver function in 36 age-mismatched recipients. The major findings were the following: (i) miR-31-5p, miR-141-3p, and miR-200c-3p increased with age, as assessed by microRNAs (miRs) and mRNA transcript profiling in 12 biopsies and results were validated by RT-qPCR in a total of 58 biopsies; (ii) telomere length measured by qPCR in 45 samples showed a significant age-dependent shortage; (iii) a bioinformatic approach combining transcriptome and miRs data identified putative miRs targets, the most informative being GLT1, a glutamate transporter expressed in hepatocytes. GLT1 was demonstrated by luciferase assay to be a target of miR-31-5p and miR-200c-3p, and both its mRNA (RT-qPCR) and protein (immunohistochemistry) significantly decreased with age in liver biopsies and in hepatic centrilobular zone, respectively; (iv) miR-31-5p, miR-141-3p and miR-200c-3p expression was significantly affected by recipient age (older environment) as assessed in eleven cases of donor-recipient extreme age-mismatch; (v) the analysis of recipients plasma by N-glycans profiling, capable of assessing liver functions and biological age, showed that liver function recovered after transplants, independently of age-mismatch, and recipients apparently 'rejuvenated' according to their glycomic age. In conclusion, we identified new markers of aging in human liver, their relevance in donor-recipient age-mismatches in transplantation, and offered positive evidence for the use of organs from old donors.

2017 - Mesenchymal stromal cells (MSCs) induce ex vivo proliferation and erythroid commitment of cord blood haematopoietic stem cells (CB-CD34+ cells) [Articolo su rivista]
Perucca, Simone; Di Palma, Andrea; Piccaluga, Pier Paolo; Gemelli, Claudia; Zoratti, Elisa; Bassi, Giulio; Giacopuzzi, Edoardo; Lojacono, Andrea; Borsani, Giuseppe; Tagliafico, Enrico; Scupoli, Maria Teresa; Bernardi, Simona; Zanaglio, Camilla; Cattina, Federica; Cancelli, Valeria; Malagola, Michele; Krampera, Mauro; Marini, Mirella; Almici, Camillo; Ferrari, Sergio; Russo, Domenico
abstract

A human bone marrow-derived mesenchymal stromal cell (MSCs) and cord blood-derived CD34+ stem cell co-culture system was set up in order to evaluate the proliferative and differentiative effects induced by MSCs on CD34+ stem cells, and the reciprocal influences on gene expression profiles. After 10 days of co-culture, non-adherent (SN-fraction) and adherent (AD-fraction) CD34+ stem cells were collected and analysed separately. In the presence of MSCs, a significant increase in CD34+ cell number was observed (fold increase = 14.68), mostly in the SN-fraction (fold increase = 13.20). This was combined with a significant increase in CD34+ cell differentiation towards the BFU-E colonies and with a decrease in the CFU-GM. These observations were confirmed by microarray analysis. Through gene set enrichment analysis (GSEA), we noted a significant enrichment in genes involved in heme metabolism (e.g. LAMP2, CLCN3, BMP2K), mitotic spindle formation and proliferation (e.g. PALLD, SOS1, CCNA1) and TGF-beta signalling (e.g. ID1) and a down-modulation of genes participating in myeloid and lymphoid differentiation (e.g. PCGF2) in the co-cultured CD34+ stem cells. On the other hand, a significant enrichment in genes involved in oxygenlevel response (e.g. TNFAIP3, SLC2A3, KLF6) and angiogenesis (e.g. VEGFA, IGF1, ID1) was found in the co-cultured MSCs. Taken together, our results suggest that MSCs can exert a priming effect on CD34+ stem cells, regulating their proliferation and erythroid differentiation. In turn, CD34+ stem cells seem to be able to polarise the BM-niche towards the vascular compartment by modulating molecular pathways related to hypoxia and angiogenesis.

2017 - miR-494-3p overexpression promotes megakaryocytopoiesis in primary myelofibrosis hematopoietic stem/progenitor cells by targeting SOCS6 [Articolo su rivista]
Rontauroli, Sebastiano; Norfo, Ruggiero; Pennucci, Valentina; Zini, Roberta; Ruberti, Samantha; Bianchi, Elisa; Salati, Simona; Prudente, Zelia; Rossi, Chiara; Rosti, Vittorio; Guglielmelli, Paola; Barosi, Giovanni; Vannucchi, Alessandro; Tagliafico, Enrico; Manfredini, Rossella
abstract

Primary myelofibrosis (PMF) is a chronic Philadelphia-negative myeloproliferative neoplasm characterized by hematopoietic stem cell-derived clonal myeloproliferation, involving especially the megakaryocyte lineage. To better characterize how the altered expression of microRNAs might contribute to PMF pathogenesis, we have previously performed the integrative analysis of gene and microRNA expression profiles of PMF hematopoietic stem/progenitor cells (HSPCs), which allowed us to identify miR- 494-3p as the upregulated microRNA predicted to target the highest number of downregulated mRNAs. To elucidate the role of miR-494-3p in hematopoietic differentiation, in the present study we demonstrated that miR-494-3p enforced expression in normal HSPCs promotes megakaryocytopoiesis. Gene expression profiling upon miR-494-3p overexpression allowed the identification of genes commonly downregulated both after microRNA overexpression and in PMF CD34+ cells. Among them, suppressor of cytokine signaling 6 (SOCS6) was confirmed to be a miR-494-3p target by luciferase assay. Western blot analysis showed reduced level of SOCS6 protein as well as STAT3 activation in miR-494-3p overexpressing cells. Furthermore, transient inhibition of SOCS6 expression in HSPCs demonstrated that SOCS6 silencing stimulates megakaryocytopoiesis, mimicking the phenotypic effects observed upon miR-494-3p overexpression. Finally, to disclose the contribution of miR-494-3p upregulation to PMF pathogenesis, we performed inhibition experiments in PMF HSPCs, which showed that miR-494-3p silencing led to SOCS6 upregulation and impaired megakaryocyte differentiation. Taken together, our results describe for the first time the role of miR-494- 3p during normal HSPC differentiation and suggest that its increased expression, and the subsequent downregulation of its target SOCS6, might contribute to the megakaryocyte hyperplasia commonly observed in PMF patients.

2017 - Role of miR-34a-5p in Hematopoietic Progenitor Cells Proliferation and Fate Decision: Novel Insights into the Pathogenesis of Primary Myelofibrosis [Articolo su rivista]
Bianchi, Elisa; Ruberti, Samantha; Rontauroli, Sebastiano; Guglielmelli, Paola; Salati, Simona; Rossi, Chiara; Zini, Roberta; Tagliafico, Enrico; Vannucchi, Alessandro Maria; Manfredini, Rossella
abstract

Primary Myelofibrosis (PMF) is a chronic Philadelphia-negative myeloproliferative neoplasm characterized by a skewed megakaryopoiesis and an overproduction of proinflammatory and profibrotic mediators that lead to the development of bone marrow (BM) fibrosis. Since we recently uncovered the upregulation of miR-34a-5p in PMF CD34+ hematopoietic progenitor cells (HPCs), in order to elucidate its role in PMF pathogenesis here we unravelled the effects of miR-34a-5p overexpression in HPCs. We showed that enforced expression of miR-34a-5p partially constrains proliferation and favours the megakaryocyte and monocyte/macrophage commitment of HPCs. Interestingly, we identified lymphoid enhancer-binding factor 1 (LEF1) and nuclear receptor subfamily 4, group A, member 2 (NR4A2) transcripts as miR-34a-5p-targets downregulated after miR-34a-5p overexpression in HPCs as well as in PMF CD34+ cells. Remarkably, the knockdown of NR4A2 in HPCs mimicked the antiproliferative effects of miR-34a-5p overexpression, while the silencing of LEF1 phenocopied the effects of miR-34a-5p overexpression on HPCs lineage choice, by favouring the megakaryocyte and monocyte/macrophage commitment. Collectively our data unravel the role of miR-34a-5p in HPCs fate decision and suggest that the increased expression of miR-34a-5p in PMF HPCs could be important for the skewing of megakaryopoiesis and the production of monocytes, that are key players in BM fibrosis in PMF patients.

2016 - DIAGNOSI MOLECOLARE DELLE IPERTRIGLICERIDEMIE PRIMITIVE ATTRAVERSO “NGS” (NEXT GENERATION SEQUENCING) [Abstract in Atti di Convegno]
Rabacchi, Claudio; Tenedini, Elena; Bernardis, Isabella; Simone, Maria Luisa; Tagliafico, Enrico; Tarugi, Patrizia Maria
abstract

L’ipertrigliceridemia severa è una condizione caratterizzata da elevati livelli di trigliceridi (TG) superiori a 1000 mg/dl ed accumulo di chilomicroni a digiuno. Questa condizione è rivelata dalla presenza di plasma lattescente e può essere secondaria (es. in corso di diabete scompensato, sindrome nefrosica grave etc.) o primitiva su base genetica. La forma primitiva prende il nome di Chilomicronemia Familiare (CF). Il quadro clinico della CF può comprendere: coliche addominali, pancreatiti ricorrenti, xantomi eruttivi, lipemia retinalis, ed epatomegalia. Questo disordine ha una modalità di trasmissione autosomica recessiva ed è dovuto a mutazioni in uno dei geni coinvolti nella cascata lipolitica intravascolare, il processo attraverso il quale i trigliceridi trasportati dai chilomicroni e dalle VLDL sono idrolizzati nel plasma. I principali geni candidati tradizionalmente considerati sono cinque: il gene LPL che codifica per l’enzima lipasi lipoproteica; il gene APOC2 ed il gene APOA5 che codificano per due apolipoproteine che svolgono il ruolo di attivatori della LPL; il gene GPIHBP1 che codifica la piattaforma molecolare per la LPL, ed infine il gene LMF1 che codifica per una proteina coinvolta nella maturazione intracellulare della LPL

2016 - Epidemiology and clinical relevance of mutations in postpolycythemia vera and postessential thrombocythemia myelofibrosis: A study on 359 patients of the AGIMM group [Articolo su rivista]
Rotunno, Giada; Pacilli, Annalisa; Artusi, Valentina; Rumi, Elisa; Maffioli, Margherita; Delaini, Federica; Brogi, Giada; Fanelli, Tiziana; Pancrazzi, Alessandro; Pietra, Daniela; Bernardis, Isabella; Belotti, Clara; Pieri, Lisa; Sant'Antonio, Emanuela; Salmoiraghi, Silvia; Cilloni, Daniela; Rambaldi, Alessandro; Passamonti, Francesco; Barbui, Tiziano; Manfredini, Rossella; Cazzola, Mario; Tagliafico, Enrico; Vannucchi, Alessandro M; Guglielmelli, Paola
abstract

Transformation to secondary myelofibrosis (MF) occurs as part of the natural history of polycythemia vera (PPV-MF) and essential thrombocythemia (PET-MF). Although primary (PMF) and secondary MF are considered similar diseases and managed similarly, there are few studies specifically focused on the latter. The aim of this study was to characterize the mutation landscape, and describe the main clinical correlates and prognostic implications of mutations, in a series of 359 patients with PPV-MF and PET-MF. Compared with PV and ET, the JAK2V617F and CALR mutated allele burden was significantly higher in PPV-MF and/or PET-MF, indicating a role for accumulation of mutated alleles in the process of transformation to MF. However, neither the allele burden nor the type of driver mutation influenced overall survival (OS), while absence of any driver mutation (triple negativity) was associated with significant reduction of OS in PET-MF, similar to PMF. Of the five interrogated subclonal mutations (ASXL1, EZH2, SRSF2, IDH1, and IDH2), that comprise a prognostically detrimental high molecular risk (HMR) category in PMF, only SRSF2 mutations were associated with reduced survival in PET-MF, and no additional mutation profile with prognostic relevance was highlighted. Overall, these data indicate that the molecular landscape of secondary forms of MF is different from PMF, suggesting that unknown mutational events might contribute to the progression from chronic phase disease to myelofibrosis. These findings also support more extended genotyping approaches aimed at identifying novel molecular abnormalities with prognostic relevance for patients with PPV-MF and PET-MF. Am. J. Hematol. 91:681–686, 2016. © 2016 Wiley Periodicals, Inc.

2016 - Expression of μ-protocadherin is negatively regulated by the activation of the β-catenin signaling pathway in normal and cancer colorectal enterocytes [Articolo su rivista]
Montorsi, Lucia; Parenti, Sandra; Losi, Lorena; Ferrarini, F; Gemelli, Claudia; Rossi, A; Manco, Gianrocco; Ferrari, Sergio; Calabretta, Bruno; Tagliafico, Enrico; ZANOCCO MARANI, Tommaso; Grande, Alexis
abstract

Mu-protocadherin (MUCDHL) is an adhesion molecule predominantly expressed by colorectal epithelial cells which is markedly downregulated upon malignant transformation. Notably, treatment of colorectal cancer (CRC) cells with mesalazine lead to increased expression of MUCDHL, and is associated with sequestration of β-catenin on the plasma membrane and inhibition of its transcriptional activity. To better characterize the causal relationship between β-catenin and MUCDHL expression, we performed various experiments in which CRC cell lines and normal colonic organoids were subjected to culture conditions inhibiting (FH535 treatment, transcription factor 7-like 2 siRNA inactivation, Wnt withdrawal) or stimulating (LiCl treatment) β-catenin activity. We show here that expression of MUCDHL is negatively regulated by functional activation of the β-catenin signaling pathway. This finding was observed in cell culture systems representing conditions of physiological stimulation and upon constitutive activation of β-catenin in CRC. The ability of MUCDHL to sequester and inhibit β-catenin appears to provide a positive feedback enforcing the effect of β-catenin inhibitors rather than serving as the primary mechanism responsible for β-catenin inhibition. Moreover, MUCDHL might have a role as biomarker in the development of CRC chemoprevention drugs endowed with β-catenin inhibitory activity.

2016 - Integrated evaluation of PAM50 subtypes and immune modulation of pCR in HER2-positive breast cancer patients treated with chemotherapy and HER2-targeted agents in the CherLOB trial [Articolo su rivista]
Dieci, M. V; Prat, A; Tagliafico, Enrico; Paré, L; Ficarra, G; Bisagni, G; Piacentini, Federico; Generali, D. G; Conte, P; Guarneri, V.
abstract

In the CherLOB study, intrinsic subtypes, TILs and immune signatures all contribute to the chance of pCR after neoadjuvant chemotherapy plus anti-HER2 agents for HER2-positive breast cancer. However, immune gene signatures rather than TILs provide significant independent prediction of pCR beyond intrinsic subtypes.The aim of this work was to evaluate the impact of (and relative contribution of) tumor-related and immune-related diversity of HER2-positive disease on the response to neoadjuvant chemotherapy plus anti-HER2 agents.The CherLOB phase II study randomized 121 HER2-positive breast cancer patients to neoadjuvant chemotherapy plus trastuzumab, lapatinib or both. Tumor samples from diagnostic core biopsy were centralized. Tumor-infiltrating lymphocytes (TILs) were evaluated on H&E slides. Intrinsic subtyping was carried out using the research-based 50-gene prediction analysis of a microarray (PAM50) subtype predictor. Immune-related gene signatures were also evaluated.Continuous Str-TILs and It-TILs were significantly associated with pCR [OR 1.03, 95% CI 1.02-1.05 (P < 0.001) and OR 1.09, 95% CI 1.04-1.15 (P < 0.001) for Str-TILs and It-TILs, respectively]. According to PAM50, the subtype distribution was as follows: HER2-enriched 26.7%, Luminal A 25.6%, Luminal B 16.3%, Basal-like 14% and Normal-like 17.4%. The highest rate of pCR was observed for the HER2-enriched subtype (50%), followed by Basal-like, Luminal B and Luminal A (chi(2) test, P = 0.026). Immune gene signatures significantly associated with pCR in univariate analyses were identified: most of them maintained a significant association with pCR in multivariate analyses corrected for PAM50 subtypes, whereas TILs did not.In this study, both tumor-related and immune-related features contribute to the modulation of pCR after neoadjuvant chemotherapy plus anti-HER2 agents. Immune signatures rather than TILs added significant prediction of pCR beyond PAM50 intrinsic subtypes.

2016 - Integrative analysis of copy number and gene expression data suggests novel pathogenetic mechanisms in primary myelofibrosis [Articolo su rivista]
Salati, Simona; Zini, Roberta; Nuzzo, Simona; Guglielmelli, Paola; Pennucci, Valentina; Prudente, Zelia; Ruberti, Samantha; Rontauroli, Sebastiano; Norfo, Ruggiero; Bianchi, Elisa; Bogani, Costanza; Rotunno, Giada; Fanelli, Tiziana; Mannarelli, Carmela; Rosti, Vittorio; Salmoiraghi, Silvia; Pietra, Daniela; Ferrari, Sergio; Barosi, Giovanni; Rambaldi, Alessandro; Cazzola, Mario; Bicciato, Silvio; Tagliafico, Enrico; Vannucchi, Alessandro M; Manfredini, Rossella
abstract

Primary myelofibrosis (PMF) is a Myeloproliferative Neoplasm (MPN) characterized by megakaryocyte hyperplasia, progressive bone marrow fibrosis, extramedullary hematopoiesis and transformation to Acute Myeloid Leukemia (AML). A number of phenotypic driver (JAK2, CALR, MPL) and additional subclonal mutations have been described in PMF, pointing to a complex genomic landscape. To discover novel genomic lesions that can contribute to disease phenotype and/or development, gene expression and copy number signals were integrated and several genomic abnormalities leading to a concordant alteration in gene expression levels were identified. In particular, copy number gain in the polyamine oxidase (PAOX) gene locus was accompanied by a coordinated transcriptional up-regulation in PMF patients. PAOX inhibition resulted in rapid cell death of PMF progenitor cells, while sparing normal cells, suggesting that PAOX inhibition could represent a therapeutic strategy to selectively target PMF cells without affecting normal hematopoietic cells' survival. Moreover, copy number loss in the chromatin modifier HMGXB4 gene correlates with a concomitant transcriptional down-regulation in PMF patients. Interestingly, silencing of HMGXB4 induces megakaryocyte differentiation, while inhibiting erythroid development, in human hematopoietic stem/progenitor cells. These results highlight a previously un-reported, yet potentially interesting role of HMGXB4 in the hematopoietic system and suggest that genomic and transcriptional imbalances of HMGXB4 could contribute to the aberrant expansion of the megakaryocytic lineage that characterizes PMF patients.

2016 - NF-YA splice variants have different roles on muscle differentiation [Articolo su rivista]
Basile, Valentina; Baruffaldi, Fiorenza; Dolfini, Diletta; Belluti, Silvia; Benatti, Paolo; Ricci, Laura; Artusi, Valentina; Tagliafico, Enrico; Mantovani, Roberto; Molinari, Susanna; Imbriano, Carol
abstract

The heterotrimeric CCAAT-binding factor NF-Y controls the expression of a multitude of genes involved in cell cycle progression. NF-YA is present in two alternatively spliced isoforms, NF-YAs and NF-YAl, differing in 28 aminoacids in the N-terminal Q-rich activation domain. NF-YAs has been identified as a regulator of stemness and proliferation in mouse embryonic cells (mESCs) and human hematopoietic stem cells (hHSCs), whereas the role of NF-YAl is not clear. In the muscle system, NF-YA expression is observed in proliferating cells, but barely detectable in terminally differentiated cells in vitro and adult skeletal muscle in vivo. Here, we show that NF-YA inactivation in mouse myoblasts impairs both proliferation and differentiation. The overexpression of the two NF-YA isoforms differentially affects myoblasts fate: NF-YAs enhance cell proliferation, while NF-YAl boosts differentiation. The molecular mechanisms were investigated by expression profilings, detailing the opposite programs of the two isoforms. Bioinformatic analysis of the regulated promoters failed to detect a significant presence of CCAAT boxes in the regulated genes. NF-YAl activates directly Mef2D, Six genes, and p57kip2 (Cdkn1c), and indirectly the myogenic regulatory factors (MRFs). Specifically, Cdkn1c activation is induced by NF-Y binding to its CCAAT promoter and by reducing the expression of the lncRNA Kcnq1ot1, a negative regulator of Cdkn1c transcription. Overall, our results indicate that NF-YA alternative splicing is an influential muscle cell determinant, through direct regulation of selected cell cycle blocking genes, and, directly and indirectly, of muscle-specific transcription factors.

2016 - No Identical ‘‘Mesenchymal Stem Cells’’ at Different Times and Sites: Human Committed Progenitors of Distinct Origin and Differentiation Potential Are Incorporated as Adventitial Cells in Microvessels [Articolo su rivista]
Sacchetti, Benedetto; Funari, Alessia; Remoli, Cristina; Giannicola, Giuseppe; Kogler, Gesine; Liedtke, Stefanie; Cossu, Giulio; Serafini, Marta; Sampaolesi, Maurilio; Tagliafico, Enrico; Tenedini, Elena; Saggio, Isabella; Robey, Pamela G.; Riminucci, Mara; Bianco, Paolo
abstract

A widely shared view reads that mesenchymal stem/stromal cells ("MSCs") are ubiquitous in human connective tissues, can be defined by a common in vitro phenotype, share a skeletogenic potential as assessed by in vitro differentiation assays, and coincide with ubiquitous pericytes. Using stringent in vivo differentiation assays and transcriptome analysis, we show that human cell populations from different anatomical sources, regarded as "MSCs" based on these criteria and assumptions, actually differ widely in their transcriptomic signature and in vivo differentiation potential. In contrast, they share the capacity to guide the assembly of functional microvessels in vivo, regardless of their anatomical source, or in situ identity as perivascular or circulating cells. This analysis reveals that muscle pericytes, which are not spontaneously osteochondrogenic as previously claimed, may indeed coincide with an ectopic perivascular subset of committed myogenic cells similar to satellite cells. Cord blood-derived stromal cells, on the other hand, display the unique capacity to form cartilage in vivo spontaneously, in addition to an assayable osteogenic capacity. These data suggest the need to revise current misconceptions on the origin and function of so-called "MSCs," with important applicative implications. The data also support the view that rather than a uniform class of "MSCs," different mesoderm derivatives include distinct classes of tissue-specific committed progenitors, possibly of different developmental origin.

2016 - STRATEGIES TO PREDICT TREATMENT RESPONSE AND SELECT THERAPIES IN METASTATIC BREAST CANCER PATIENTS USING A NEXT GENERATION SEQUENCING MULTI-GENE PANEL [Abstract in Atti di Convegno]
Toss, Angela; Cortesi, Laura; Artuso, Lucia; Tenedini, Elena; Bernardis, Isabella; Ficarra, G; Piacentini, Federico; Federico, Massimo; Tagliafico, Enrico
abstract

2016 - STRATEGIES TO PREDICT TREATMENT RESPONSE AND SELECT THERAPIES IN METASTATIC BREAST CANCER PATIENTS USING A NEXT GENERATION SEQUENCING (NGS) MULTI-GENE PANEL [Poster]
Toss, Angela; Cortesi, Laura; Artuso, Lucia; Tenedini, Elena; Bernardis, Isabella; Parenti, Sandra; Ficarra, Guido; Piacentini, Federico; Federico, Massimo; Tagliafico, Enrico
abstract

The standard of care for many patients with advanced breast cancer (BC )is gradually evolving from empirical treatment based on clinicalpathological characteristics to the use of targeted approaches based on the molecular profile of the tumor. In the last decade, an increasing number of molecularly targeted drugs have been developed for the treatment of metastatic BC. These drugs target specific molecular abnormalities that confer to cancer cells a survival advantage [1]. Interestingly, the ability to perform multigene testing for a range of molecular alterations may provide an opportunity to clarify the mechanisms of treatment response, to find the strategies to overcome treatment resistance and thus, to identify patients who are more likely to develop relapse and who may be candidates for matched targeted therapies [2-3]. The main aim of this study is to find prognostic and predictive molecular biomarkers for the management of metastatic BC patients in clinical practice. MATERIALS AND METHODS The amplicon-sequencing analyses took advantage of the Ion AmpliSeq™ technology (Thermo Fisher, Waltham, MA, USA). A custom panel was designed with the help of the Designer online tool (www.ampliseq.com), which was employed to generate optimized primers encompassing the coding DNA sequences (with 100bp of exon padding and the UTRs regions) of 25 genes in the Human Reference Genome (hg19); these genes were selected searching and screening scientific literature for treatments resistance in BC and are reported in Table 1. Primer pairs were divided into two pools to optimize multiplex PCR conditions and the coverage, that assessed to 89.02%. The customized Ion AmpliSeq panel was employed on samples from 7 primary BC samples and matched metastatic sites (3 skin, 3 lymph node and 1 lung metastases). They were all processed using the Ion AmpliSeq Library Kit 2.0, starting from 15 nanograms of FFPE extracted DNA/pool. Samples were barcoded with the Ion Express Kit to optimize matched patients pooling on the same 318 Chip v2 sequencing chip. The template-positive Ion Sphere Particles were sequenced on a Personal Genome Machine (Thermo Fisher, Waltham, MA, USA). RESULTS The mutation profiles of paired primary and secondary tumors of the seven patients enrolled in this study are presented in Table 2. Ten different genes (PTEN, PIK3CA, mTOR, ERBB2, ERBB3, MET, INPP4B, MAP2K1, CDK6, KRAS) in 6 different patients showed possible damaging variants as shown in Table 2. • Four patients (number 1, 3, 5 and 6) showed no additional or different mutations in secondary tumors if compared to primary samples. • In patient number 2, the metastatic site presented new mutations if compared to the primary tumor. • Finally in patient number 4 and 7 we did not detect in metastases some of the mutations found in the primary tumor. DISCUSSION In 5 patients (71,4%) the mutational status of primary tumor could explain treatment resistance and thus predict relapse, in one patient the mutational status of the new subclones could be relevant for guiding differently the subsequent treatment choices. In 2 patients (28,5%) we were not able to detect in metastases some of the mutations found in the primary tumor. This could be explained by considering the clonal evolution of metastases. These preliminary data suggest that the multi-gene panel analysis of primary and secondary tumors may help clinicians: • in discriminating BC patients HR+ and/or HER2+ with mutations predicting an increased risk of adjuvant treatment resistance and thus relapse • in guiding treatment selection strategies in the metastatic setting. The study is still open and we are currently recruiting other patients.

2016 - Tie2 expressing monocytes in the spleen of patients with primary myelofibrosis [Articolo su rivista]
Campanelli, R.; Fois, G.; Catarsi, P.; Poletto, V.; Villani, L.; Erba, B. G.; Maddaluno, L.; Jemos, B.; Salmoiraghi, S.; Guglielmelli, P.; Abbonante, V.; Di Buduo, C. A.; Balduini, A.; Iurlo, A.; Barosi, G.; Rosti, V.; Massa, M.; Vannucchi, A. M.; Balliu, M.; Bartalucci, N.; Bogani, C.; Bosi, A.; Calabresi, L.; Corbizzi Fattori, G.; Fanelli, T.; Fjerza, R.; Gesullo, F.; Mannarelli, C.; Merli, L.; Pacilli, A.; Pancrazzi, A.; Paoli, C.; Pieri, L.; Rotunno, G.; Sant'Antonio, E.; Bonetti, E.; Cazzola, M.; Ambaglio, I.; Bernasconi, P.; Casetti, C. I.; Catricala, S.; Elena, C.; Fugazza, E.; Galli, A.; Malcovati, L.; Milanesi, C.; Pascutto, C.; Pietra, D.; Ripamonti, F.; Rossi, M.; Rumi, E.; Dejana, E.; Breviario, F.; Corada, M.; Malinverno, M.; Rambaldi, A.; Chioda, G.; Ferrari, M. L.; Finazzi, G.; Finazzi, M. C.; Belotti, C.; Boroni, C.; Amaru, A.; Golay, J.; Bortoluzzi, S.; Bisognin, A.; Coppe, A.; Saccoman, C.; Manfredini, R.; Artuso, L.; Bernardis, I.; Bianchi, E.; Montanari, M.; Pennucci, V.; Prudente, Z.; Rontauroli, S.; Rossi, C.; Ruberti, S.; Salati, S.; Tagliafico, E.; Tenedini, E.; Zini, R.
abstract

Primary myelofibrosis (PMF) is a Philadelphia-negative (Ph-) myeloproliferative disorder, showing abnormal CD34 + progenitor cell trafficking, splenomegaly, marrow fibrosis leading to extensive extramedullary haematopoiesis, and abnormal neoangiogenesis in either the bone marrow or the spleen. Monocytes expressing the angiopoietin-2 receptor (Tie2) have been shown to support abnormal angiogenic processes in solid tumors through a paracrine action that takes place in proximity to the vessels. In this study we investigated the frequency of Tie2 expressing monocytes in the spleen tissue samples of patients with PMF, and healthy subjects (CTRLs), and evaluated their possible role in favouring spleen angiogenesis. We show by confocal microscopy that in the spleen tissue of patients with PMF, but not of CTRLs, the most of the CD14 + cells are Tie2 + and are close to vessels; by flow cytometry, we found that Tie2 expressing monocytes were Tie2 + CD14 low CD16 bright CDL62 - CCR2 - (TEMs) and their frequency was higher (p = 0.008) in spleen tissue-derived mononuclear cells (MNCs) of patients with PMF than in spleen tissue-derived MNCs from CTRLs undergoing splenectomy for abdominal trauma. By in vitro angiogenesis assay we evidenced that conditioned medium of immunomagnetically selected spleen tissue derived CD14 + cells of patients with PMF induced a denser tube like net than that of CTRLs; in addition, CD14 + Tie2 + cells sorted from spleen tissue derived single cell suspension of patients with PMF show a higher expression of genes involved in angiogenesis than that found in CTRLs. Our results document the enrichment of Tie2 + monocytes expressing angiogenic genes in the spleen of patients with PMF, suggesting a role for these cells in starting/maintaining the pathological angiogenesis in this organ.

2016 - Unravelling the Complexity of Inherited Retinal Dystrophies Molecular Testing: Added Value of Targeted Next-Generation Sequencing [Articolo su rivista]
Bernardis, Isabella; Chiesi, Laura; Tenedini, Elena; Artuso, Lucia; Percesepe, Antonio; Artusi, Valentina; Simone, Maria Luisa; Manfredini, Rossella; Camparini, Monica; Rinaldi, Chiara; Ciardella, Antonio; Graziano, Claudio; Balducci, Nicole; Tranchina, Antonia; Cavallini, Gian Maria; Pietrangelo, Antonello; Marigo, Valeria; Tagliafico, Enrico
abstract

To assess the clinical utility of targeted Next-Generation Sequencing (NGS) for the diagnosis of Inherited Retinal Dystrophies (IRDs), a total of 109 subjects were enrolled in the study, including 88 IRD affected probands and 21 healthy relatives. Clinical diagnoses included Retinitis Pigmentosa (RP), Leber Congenital Amaurosis (LCA), Stargardt Disease (STGD), Best Macular Dystrophy (BMD), Usher Syndrome (USH), and other IRDs with undefined clinical diagnosis. Participants underwent a complete ophthalmologic examination followed by genetic counseling. A custom AmpliSeq� panel of 72 IRD-related genes was designed for the analysis and tested using Ion semiconductor Next-Generation Sequencing (NGS). Potential disease-causing mutations were identified in 59.1% of probands, comprising mutations in 16 genes. The highest diagnostic yields were achieved for BMD, LCA, USH, and STGD patients, whereas RP confirmed its high genetic heterogeneity. Causative mutations were identified in 17.6% of probands with undefined diagnosis. Revision of the initial diagnosis was performed for 9.6% of genetically diagnosed patients. This study demonstrates that NGS represents a comprehensive cost-effective approach for IRDs molecular diagnosis. The identification of the genetic alterations underlying the phenotype enabled the clinicians to achieve a more accurate diagnosis. The results emphasize the importance of molecular diagnosis coupled with clinic information to unravel the extensive phenotypic heterogeneity of these diseases.

2015 - A Next Generation Sequencing amplicon-based strategy to explore Inherited Retinal Degeneration complexity [Abstract in Atti di Convegno]
Artusi, Valentina; Chiesi, Laura; Bernardis, Isabella; Tenedini, Elena; Artuso, Lucia; Cavallini, Gian Maria; Percesepe, Antonio; Marigo, Valeria; Tagliafico, Enrico
abstract

Inherited Retinal Degeneration (IRD) are a group of eye disorders, characterized by photoreceptors degeneration which include: Retinitis Pigmentosa, Stargardt disease, Usher Syndrome and Leber congenital amaurosis. The high genetic heterogeneity, the incompleteness of disease specific databases and the elevated number of genes involved in IRD, often hamper the correct molecular diagnosis and patients stratification. To clarify IRD molecular profile, we used a next Generation Sequencing (NGS) strategy and designed a custom AmpliSeq panel (Life Technologies), containing the coding sequences of 72 disease related genes, for a total of 1649 amplicons. An in-house bioinformatic pipeline was optimized to filter synonymous variants and polymorphism and to annotate variants with prediction algorithm (dbNSFP) and disease specific databases (LOVD eye diseases, Retina International, RPGR database). A cohort of 40 samples was selected (29 patients, 11 healthy relatives). They underwent a complete ophthalmologic examination (visual acuity, anterior segment and fundus examination, ERG and/or EOG, OCT), as well as a genetic counselling. Possibly causative mutations were detected in 62% patients (n=18). We found mutations in 8 genes. The most recurrent gene was mutated in 38% (n=7) of patients. The remaining seven genes harboured lower frequencies with just one or two patients mutated. Overall, seven genes were inherited with an autosomal recessive pattern and one gene was X-linked. Of note, less than 21% of variants have been already described in specific databases. These preliminary results highlight the need to further explore the molecular complexity and heterogeneity of RD in order to translate these analyses into clinical practice.

2015 - Abnormal expression patterns of WT1-as, MEG3 and ANRIL long non-coding RNAs in CD34+ cells from patients with primary myelofibrosis and their clinical correlations [Articolo su rivista]
Pennucci, Valentina; Zini, Roberta; Norfo, Ruggiero; Guglielmelli, Paola; Bianchi, Elisa; Salati, Simona; Sacchi, Giorgia; Prudente, Zelia; Tenedini, Elena; Ruberti, Samantha; Paoli, Chiara; Fanelli, Tiziana; Mannarelli, Carmela; Tagliafico, Enrico; Ferrari, Sergio; Vannucchi, ALESSANDRO MARIA; Manfredini, Rossella; Associazione Italiana per la Ricerca sul Cancro Gruppo Italiano Malattie Mieloproliferative, Investigators
abstract

Abnormal expression patterns of WT1-as, MEG3 and ANRIL long non-coding RNAs in CD34+ cells from patients with primary myelofibrosis and their clinical correlations.

2015 - Amplicon-based Next Generation Sequencing: an effective approach to molecular diagnosis of Epidermolysis Bullosa [Abstract in Atti di Convegno]
Tenedini, Elena; Artuso, Lucia; Bernardis, Isabella; Artusi, Valentina; Percesepe, Antonio; Manfredini, Rossella; DE ROSA, Laura; Contin, Roberta; Pellacani, Giovanni; Giannetti, Alberto; Pagani, Jacopo; DE LUCA, Michele; Tagliafico, Enrico
abstract

Epidermolysis Bullosa (EB) is caused by mutations in genes encoding for proteins of the epidermal–dermal junction assembly. Due to the extreme clinical/genetic heterogeneity of the disease, current methods in EB diagno- stics comprise immunohistochemistry on bioptic samples and transmission electron microscopy followed by single candidate gene Sanger Sequencing (SS) that therefore represents the final phase of a labour intensive and ex- pensive clinical pathway. Methods: Participants in a cross sectional study included individuals with Muenke syndrome (P250R mutation in FGFR3) and their mutation negative siblings. Participants completed validated assessments of executive functio- ning (Behavior Rating Inventory of Executive Function; BRIEF) and adaptive behavior skills (Adaptive Behavior Assessment System; ABAS-II). According to the recently published recommendations for diagnosis and treatment in EB, the assessment of mutational landscape is instead a fun- damental step to a comprehensive diagnosis path; Next Generation Sequen- cing (NGS), throughout parallel ultra-deep sequencing of many genes, would represent a proper method for reducing timing and costs in EB diagnostics. We developed an EB disease-comprehensive amplicon panel (AmpliSeq pa- nel), to accomplish NGS onto Ion Torrent PGM platform. The panel was dealt on ten patients with known genetic diagnosis, and then employed in eight family trios with unknown molecular footprinting. Results: Forty-four FGFR3 mutation positive individuals, median age 9, range 6 months to 52 years were evaluated with the BRIEF and ABAS-II. Additio- nally, 10 unaffected siblings were used as controls. For the General Executive Composite scale of the BRIEF, 32.1% of the cohort had scores greater than +1.5 SD, signifying “Potential Clinical Significance.” For the General Adaptive Composite of the ABAS-II, 28.2% of affected individuals scored in the “Ex- tremely Low” category” (3rd -8th percentile of normative population) and 53.9% were below the “Average” category (less than the 25th percentile). Multiple regression analysis showed that the presence of craniosynostosis was not a predictor (P = 0.7) of BRIEF and ABAS-II scores. The AmpliSeq panel, obtaining a proof of concept of the sensitivity, specificity, and accuracy of this kind of procedure, showed successful in finding the causative mutations in all the ten patients with known mutations, fully confirming SS data. Besides, showing consistent with the clinical diagnosis, it was effective in trios, identifying all the variants, even the ones SS missed or in case of de novo mutations. NGS

2015 - Amplicon-based next-generation sequencing: an effective approach for the molecular diagnosis of epidermolysis bullosa [Articolo su rivista]
Tenedini, Elena; Artuso, Lucia; Bernardis, Isabella; Artusi, Valentina; Percesepe, Antonio; De Rosa, Laura; Contin, Roberta; Manfredini, Rossella; Pellacani, Giovanni; Giannetti, Alberto; Pagani, J.; De Luca, Michele; Tagliafico, Enrico
abstract

Epidermolysis bullosa (EB) is caused by mutations in genes that encode proteins belonging to the epidermal-dermal junction assembly. Due to the extreme clinical/genetic heterogeneity of the disease, the current methods available for diagnosing EB involve immunohistochemistry of biopsy samples and transmission electron microscopy followed by single-candidate gene Sanger sequencing (SS), which are labour-intensive and expensive clinical pathways.

2015 - AMPLICON-BASED NGS: AN EFFECTIVE APPROACH FOR THE MOLECULAR DIAGNOSIS OF EPIDERMOLYSIS BULLOSA [Abstract in Atti di Convegno]
Tenedini, Elena; Artuso, Lucia; Bernardis, Isabella; Artusi, Valentina; Percesepe, A; DE ROSA, Laura; Contin, Roberta; Manfredini, Rossella; Pellacani, Giovanni; Giannetti, A; DE LUCA, Michele; Tagliafico, Enrico
abstract

Background: Epidermolysis Bullosa (EB) is caused by mutations in genes that encode proteins belonging to the epidermal-dermal junction assembly. Due to the extreme clinical/genetic heterogeneity of the disease, the current methods available for diagnosing EB involve immunohistochemistry of bioptic samples and transmission electron microscopy followed by single candidate gene Sanger Sequencing (SS), which are labour intensive and expensive clinical pathways. Objectives: According to the recently published recommendations for the EB diagnosis and treatment, the assessment of the mutational landscape is now a fundamental step for developing a comprehensive diagnostic path. Next-Generation Sequencing (NGS) via the parallel ultra-deep sequencing of many genes represents a proper method for reducing the processing time and costs of EB diagnostics. Methods: We developed an EB disease-comprehensive AmpliSeq panel to accomplish the NGS on the Ion Torrent PGM platform. The panel was performed on ten patients with known genetic diagnoses and was then employed in eight family trios with unknown molecular footprints. Results: The panel was successful in finding the causative mutations in all ten of the patients with known mutations, fully confirming the SS data and providing proof of concept of the sensitivity, specificity, and accuracy of this procedure. In addition to being consistent with the clinical diagnosis, it was also effective in the trios, identifying all of the variants, including ones that the SS missed or de novo mutations. Conclusions: The NGS and AmpliSeq were shown to be an effective approach for the diagnosis of EB, resulting in a costand time-effective 72-hour procedure.

2015 - Implementation of an NGS-based workflow for BRCA1 and BRCA2 mutation screening [Abstract in Atti di Convegno]
Artuso, Lucia; Medici, Veronica; Bernardis, Isabella; Tenedini, Elena; Artusi, Valentina; Simone, Maria Luisa; Tarugi, Patrizia Maria; Manfredini, Rossella; Cortesi, Laura; Tagliafico, Enrico
abstract

probability to develop familiar breast cancer. To detect BRCA1/2 germline mutations we developed a next-generation sequencing (NGS) routine dia- gnostic workflow, based on the Ion Torrent PGMTM System platform. The Ion AmpliSeqTM BRCA1 and BRCA2 Community Panel was handled with a semi- automatized procedure for multiplex PCR-based library preparation and se- quencing. Data analysis required the implementation of a custom designed bioinformatic pipeline for sequences alignment and for the identification, annotation and filtration of genetic variants. Sanger sequencing was perfor- med to validate candidate mutations, and to re-sequence amplicons having low NGS coverage (<50 reads per amplicon). Negative samples were ana- lyzed using the BRCA HP Kit (Multiplicom) for an effective homopolymeric stretches detection. This workflow together with the potentiality of our bio- informatic pipeline was blindly tested and validated onto a small cohort of patients previously Sanger sequenced, fine-tuning the parameter settings and resulting in a sensitivity of 100% in variant detection. Subsequently, 244 patients were analyzed thus confirming the need of a double check for the homopolymeric stretches with both NGS sequencing and BRCA HP Kit. The NGS-based workflow here proposed was able to decrease the overall

2015 - Prospective biomarker analysis of the randomized CHER-LOB study evaluating the dual anti-HER2 treatment with trastuzumab and lapatinib plus chemotherapy as neoadjuvant therapy for HER2-positive breast cancer [Articolo su rivista]
Guarneri, Valentina; Dieci, Maria Vittoria; Frassoldati, Antonio; Maiorana, Antonino; Ficarra, Guido; Bettelli, Stefania Raffaella; Tagliafico, Enrico; Bicciato, Silvio; Generali, Daniele Giulio; Cagossi, Katia; Bisagni, Giancarlo; Sarti, Samanta; Musolino, Antonino; Ellis, Catherine; Crescenzo, Rocco; Conte, Pierfranco
abstract

Background. The CHER-LOB randomized phase II study showed that the combination of lapatinib and trastuzumab plus chemotherapy increases the pathologic complete re- mission (pCR) rate compared with chemotherapy plus either trastuzumab or lapatinib. A biomarker program was prospectively planned to identify potential predictors of sensitivity to different treatments and to evaluate treatment effect on tumor biomarkers. Materials and Methods. Overall, 121 breast cancer patients positive for human epidermal growth factor 2 (HER2) were randomly assigned to neoadjuvant chemotherapy plus trastu- zumab, lapatinib, or both trastuzumab and lapatinib. Pre-and post-treatment samples were centrally evaluated for HER2, p95- HER2, phosphorylated AKT (pAKT), phosphatase and tensin homolog, Ki67, apoptosis, and PIK3CA mutations. Fresh-frozen tissue samples were collected for genomic analyses. Results. A mutation in PIK3CA exon 20 or 9 was documented in 20% of cases. Overall, the pCR rates were similar in PIK3CA wild- type and PIK3CA-mutated patients (33.3% vs. 22.7%; p 5.323). For patients receiving trastuzumab plus lapatinib, the probabil- ity of pCR was higher in PIK3CA wild-type tumors (48.4% vs. 12.5%; p 5.06). Ki67, pAKT, and apoptosis measured on the residual disease were significantly reduced from baseline. The degree of Ki67 inhibition was significantly higher in patients receiving the dual anti-HER2 blockade.The integrated analysis of gene expression and copy number data demonstrated that a 50- gene signature specifically predicted the lapatinib-induced pCR. Conclusion. PIK3CA mutations seem to identify patients who are less likely to benefit from dual anti-HER2 inhibition. p95-HER2 and markers of phosphoinositide 3-kinase pathway deregulation are not confirmed as markers of different sensitivity to trastuzumab or lapatinib.

2015 - Transcriptional Response of Human Neurospheres to helper-dependent CAV-2 vectors involves the modulation of DNA damage response, microtubule and centromere gene groups [Articolo su rivista]
Piersanti, Stefania; Burla, Romina; Licursi, Valerio; Brito, Catarina; La Torre, Mattia; Alves, Paula M.; Simao, Daniel; Mottini, Carla; Salinas, Sara; Negri, Rodolfo; Tagliafico, Enrico; Kremer, Eric J.; Saggio, Isabella
abstract

Brain gene transfer using viral vectors will likely become a therapeutic option for several disorders. Helper-dependent (HD) canine adenovirus type 2 vectors (CAV-2) are well suited for this goal. These vectors are poorly immunogenic, efficiently transduce neurons, are retrogradely transported to afferent structures in the brain and lead to long-term transgene expression. CAV-2 vectors are being exploited to unravel behavior, cognition, neural networks, axonal transport and therapy for orphan diseases. With the goal of better understanding and characterizing HD-CAV-2 for brain therapy, we analyzed the transcriptomic modulation induced by HD-CAV-2 in human differentiated neurospheres derived from midbrain progenitors. This 3D model system mimics several aspects of the dynamic nature of human brain. We found that differentiated neurospheres are readily transduced by HDCAV- 2 and that transduction generates two main transcriptional responses: a DNA damage response and alteration of centromeric and microtubule probes. Future investigations on the biochemistry of processes highlighted by probe modulations will help defining the implication of HD-CAV-2 and CAR receptor binding in enchaining these functional pathways. We suggest here that the modulation of DNA damage genes is related to viral DNA, while the alteration of centromeric and microtubule probes is possibly enchained by the interaction of the HD-CAV-2 fibre with CAR.

2014 - DNA microarray to analyze adenovirus-host interactions [Capitolo/Saggio]
Piersanti, Stefania; Tagliafico, Enrico; Saggio, Isabella
abstract

Defining the molecular toxicity of viral vectors that are or will be in use for clinical trials is a prerequisite for their safe application in humans. DNA chips allow high-throughput evaluation of the profile of transduced cells and have contributed to underlining specific aspects of vector toxicity both in in vitro and in vivo assets. With gene chips we have been able to identify vector-specific properties, such as the cell cycle alteration induced by vector genomic DNA, along with the activation of specific innate immune pathways that can be ascribed to viral particles. We herein describe a detailed protocol for the use of gene chips to dissect the toxicogenomic signature of human and canine helper-dependent adenoviral vectors. We suggest specific procedures suited for the study of these viral vectors, but we also give indications that can be applied to different experimental contexts. In addition, we discuss the in silico elaboration of gene chip raw data which is a crucial step to extrapolate biological information from gene chip studies. © 2014 Springer Science+Business Media, LLC.

2014 - Double-blind, placebo-controlled, multicenter, randomized, phase IIb neoadjuvant study of letrozole-lapatinib in postmenopausal hormone receptor-positive, human epidermal growth factor receptor 2-negative, operable breast cancer [Articolo su rivista]
Guarneri, Valentina; Generali, Daniele Giulio; Frassoldati, Antonio; Artioli, Fabrizio; Boni, Corrado; Cavanna, Luigi; Tagliafico, Enrico; Maiorana, Antonino; Bottini, Alberto; Cagossi, Katia; Bisagni, Giancarlo; Piacentini, Federico; Ficarra, Guido; Bettelli, Stefania Raffaella; Roncaglia, Enrica; Nuzzo, Simona; Swaby, Ramona; Ellis, Catherine; Holford, Clare; Conte, Pierfranco
abstract

Purpose This is a randomized, double-blind, placebo-controlled study aimed to evaluate the clinical and biologic effects of letrozole plus lapatinib or placebo as neoadjuvant therapy in hormone receptor (HR) –positive/human epidermal growth factor receptor 2 (HER2) –negative operable breast cancer. Methods Ninety-two postmenopausal women with stage II to IIIA primary breast cancer were randomly assigned to preoperative therapy consisting of 6 months of letrozole 2.5 mg orally daily plus lapatinib 1,500 mg orally daily or placebo. Surgery was performed within 2 weeks from the last study medication. Clinical response was assessed by ultrasonography. Pre- and post-treatment samples were evaluated for selected biomarkers. Fresh-frozen tissue samples were collected for genomic analyses. Results Numerically similar clinical response rates (partial complete response) were observed (70% for letrozole-lapatinib and 63% for letrozole-placebo). Toxicities were generally mild and manageable. A significant decrease in Ki-67 and pAKT expression from baseline to surgery was observed in both arms. Overall, 34 patients (37%) had a mutation in PIK3CA exon 9 or 20. In the letrozole-lapatinib arm, the probability of achieving a clinical response was significantly higher in the presence of PIK3CA mutation (objective response rate, 93% v 63% in PIK3CA wild type; P .040). Conclusion The combination of letrozole-lapatinib in early breast cancer was feasible, with expected and manageable toxicities. In unselected estrogen receptor–positive/HER2-negative patients, letrozolelapatinib and letrozole-placebo resulted in a similar overall clinical response rate and similar effect on Ki-67 and pAKT. Our secondary end point findings of a significant correlation between PIK3CA mutation and response to letrozole-lapatinib in HR-positive/HER2-negative early breast cancer must now be independently confirmed.

2014 - FOXP1 and TP63 involvement in the progression of myelodysplastic syndrome with 5q- and additional cytogenetic abnormalities [Articolo su rivista]
L'Abbate, Alberto; Lo Cunsolo, Crocifissa; Macrì, Ettore; Iuzzolino, Paolo; Mecucci, Cristina; Doglioni, Claudio; Coco, Michelina; Muscarella, Lucia Anna; Salati, Simona; Tagliafico, Enrico; Minoia, Carla; De Tullio, Giacoma; Guarini, Attilio; Testoni, Nicoletta; Agostinelli, Claudio; Storlazzi, Clelia Tiziana
abstract

The progression of low-risk del(5q) myelodysplastic syndrome to acute myeloid leukemia is increased when associated with mutations of TP53, or with additional chromosomal abnormalities. However, to date the prognostic impact and molecular consequences of these rearrangements were poorly investigated. Single additional alterations to del(5q) by balanced chromosome rearrangements were rarely found in myelodysplasia. In particular, balanced alterations involving TP63 and FOXP1 genes were never reported in the literature.

2014 - Impact of mutational status on outcomes in myelofibrosis patients treated with ruxolitinib in the COMFORT-II study [Articolo su rivista]
Guglielmelli, Paola; Biamonte, Flavia; Rotunno, Giada; Artusi, Valentina; Artuso, Lucia; Bernardis, Isabella; Tenedini, Elena; Pieri, Lisa; Paoli, Chiara; Mannarelli, Carmela; Fjerza, Rajmonda; Rumi, Elisa; Stalbovskaya, Viktoriya; Squires, Matthew; Cazzola, Mario; Manfredini, Rossella; Harrison, Claire; Tagliafico, Enrico; Vannucchi, ALESSANDRO MARIA
abstract

The JAK1/JAK2 inhibitor ruxolitinib produced significant reductions in splenomegaly and symptomatic burden and improved survival in patients with myelofibrosis (MF), irrespective of their JAK2 mutation status, in 2 phase III studies against placebo (COMFORT-I) and best available therapy (COMFORT-II). We performed a comprehensive mutation analysis to evaluate the impact of 14 MF-associated mutations on clinical outcomes in 166 patients included in COMFORT-II. We found that responses in splenomegaly and symptoms, as well as the risk of developing ruxolitinib-associated anemia and thrombocytopenia, occurred at similar frequencies across different mutation profiles. Ruxolitinib improved survival independent of mutation profile and reduced the risk of death in patients harboring a set of prognostically detrimental mutations (ASXL1, EZH2, SRSF2, IDH1/2) with an hazard ratio of 0.57 (95% confidence interval: 0.30-1.08) vs best available therapy. These data indicate that clinical efficacy and survival improvement may occur across different molecular subsets of patients with MF treated with ruxolitinib.

2014 - MafB is a downstream target of the IL-10/STAT3 signaling pathway, involved in the regulation of macrophage de-activation [Articolo su rivista]
Gemelli, C.; Zanocco Marani, T.; Bicciato, S.; Mazza, E. M. C.; Boraschi, D.; Salsi, V.; Zappavigna, V.; Parenti, S.; Selmi, T.; Tagliafico, E.; Ferrari, S.; Grande, A.
abstract

In spite of the numerous reports implicating MafB transcription factor in the molecular control of monocyte-macrophage differentiation, the precise genetic program underlying this activity has been, to date, poorly understood. To clarify this issue, we planned a number of experiments that were mainly conducted on human primary macrophages. In this regard, a preliminary gene function study, based on MafB inactivation and over-expression, indicated MMP9 and IL-. 7R genes as possible targets of the investigated transcription factor. Bioinformatics analysis of their promoter regions disclosed the presence of several putative MARE elements and a combined approach of EMSA and luciferase assay subsequently demonstrated that expression of both genes is indeed activated by MafB through a direct transcription mechanism. Additional investigation, performed with similar procedures to elucidate the biological relevance of our observation, revealed that MafB is a downstream target of the IL-10/STAT3 signaling pathway, normally inducing the macrophage de-activation process. Taken together our data support the existence of a signaling cascade by which stimulation of macrophages with the IL-10 cytokine determines a sequential activation of STAT3 and MafB transcription factors, in turn leading to an up-regulated expression of MMP9 and IL-. 7R genes. © 2014 Elsevier B.V.

2014 - miRNA-mRNA integrative analysis in primary myelofibrosis CD34+ cells: role of miR-155/JARID2 axis in abnormal megakaryopoiesis [Articolo su rivista]
Norfo, Ruggiero; Zini, Roberta; Pennucci, Valentina; Bianchi, Elisa; Salati, Simona; Guglielmelli, Paola; Bogani, Costanza; Fanelli, Tiziana; Mannarelli, Carmela; Rosti, Vittorio; Pietra, Daniela; Salmoiraghi, Silvia; Bisognin, Andrea; Ruberti, Samantha; Rontauroli, Sebastiano; Sacchi, Giorgia; Prudente, Zelia; Barosi, Giovanni; Cazzola, Mario; Rambaldi, Alessandro; Bortoluzzi, Stefania; Ferrari, Sergio; Tagliafico, Enrico; Vannucchi, Alessandro M; Manfredini, Rossella; Associazione Italiana per la Ricerca sul Cancro Gruppo Italiano Malattie Mieloproliferative, Investigators
abstract

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by megakaryocyte (MK) hyperplasia, bone marrow fibrosis, and abnormal stem cell trafficking. PMF may be associated with somatic mutations in JAK2, MPL, or CALR. Previous studies have shown that abnormal MKs play a central role in the pathophysiology of PMF. In this work, we studied both gene and microRNA (miRNA) expression profiles in CD34(+) cells from PMF patients. We identified several biomarkers and putative molecular targets such as FGR, LCN2, and OLFM4. By means of miRNA-gene expression integrative analysis, we found different regulatory networks involved in the dysregulation of transcriptional control and chromatin remodeling. In particular, we identified a network gathering several miRNAs with oncogenic potential (eg, miR-155-5p) and targeted genes whose abnormal function has been previously associated with myeloid neoplasms, including JARID2, NR4A3, CDC42, and HMGB3. Because the validation of miRNA-target interactions unveiled JARID2/miR-155-5p as the strongest relationship in the network, we studied the function of this axis in normal and PMF CD34(+) cells. We showed that JARID2 downregulation mediated by miR-155-5p overexpression leads to increased in vitro formation of CD41(+) MK precursors. These findings suggest that overexpression of miR-155-5p and the resulting downregulation of JARID2 may contribute to MK hyperplasia in PMF.

2014 - Pre-mir146a e FSHR sono marker di mosaicismo tiroideo nel carcinoma follicolare della tiroide [Abstract in Atti di Convegno]
Vighi, Eleonora; Pignatti, Elisa; Magnani, Elisa; Kara, Elda; Artuso, Lucia; Bernardis, Isabella; V., Cirello; Tagliafico, Enrico; Maiorana, Antonino; L., Fugazzola; Rochira, Vincenzo; Carani, Cesare; Simoni, Manuela
abstract

This study investigates the pre-mir146a within follicular thyroid cancer tissue and normal thyroid

2014 - Targeted cancer exome sequencing reveals recurrent mutations in myeloproliferative neoplasms [Articolo su rivista]
Tenedini, Elena; Bernardis, Isabella; Artusi, Valentina; Artuso, Lucia; Roncaglia, E.; Guglielmelli, P.; Pieri, L.; Bogani, C.; Biamonte, F.; Rotunno, G.; Mannarelli, C.; Bianchi, Elisa; Pancrazzi, A.; Fanelli, T.; MALAGOLI TAGLIAZUCCHI, Guidantonio; Ferrari, Sergio; Manfredini, Rossella; Vannucchi, A. M.; Tagliafico, Enrico
abstract

With the intent of dissecting the molecular complexity of Philadelphia-negative myeloproliferative neoplasms (MPN), we designed a target enrichment panel to explore, using next-generation sequencing (NGS), the mutational status of an extensive list of 2,000 cancer-associated genes and microRNAs. The genomic DNA of granulocytes and in-vitro-expanded CD3+ T-lymphocytes, as a germline control, was target-enriched and sequenced in a learning cohort of 20 MPN patients using Roche 454 technology. We identified 141 genuine somatic mutations, most of which were not previously described. To test the frequency of the identified variants, a larger validation cohort of 189 MPN patients was additionally screened for these mutations using Ion Torrent AmpliSeq NGS. Excluding the genes already described in MPN, for 8 genes (SCRIB, MIR662, BARD1, TCF12, FAT4, DAP3, POLG, and NRAS), we demonstrated a mutation frequency between 3 and 8%. We also found that mutations at codon 12 of NRAS (NRASG12V and NRASG12D) were significantly associated, for primary myelofibrosis (PMF), with highest DIPSS-plus score categories. This association was then confirmed in 66 additional PMF patients composing a final dataset of 168 PMF showing an NRAS mutation frequency of 4.7%, which was associated with a worse outcome, as defined by the DIPSS plus score.

2014 - The barley Frost resistance-H2 locus [Articolo su rivista]
Pasquariello, Marianna; Delfina, Barabaschi; Axel, Himmelbach; Burkhard, Steuernagel; Ruvini, Ariyadasa; Nils, Stein; Gandolfi, Francesco; Tenedini, Elena; Bernardis, Isabella; Tagliafico, Enrico; Pecchioni, Nicola; Francia, Enrico
abstract

Frost resistance-H2 (Fr-H2) is a major QTL affecting freezing tolerance in barley, yet its molecular basis is still not clearly understood. To gain a better insight into the structural characterization of the locus, a high-resolution linkage map developed from the Nure x Tremois cross was initially implemented to map 13 loci which divided the 0.602 cM total genetic distance into ten recombination segments. A PCR-based screening was then applied to identify positive bacterial artificial chromosome (BAC) clones from two genomic libraries of the reference genotype Morex. Twenty-six overlapping BACs from the integrated physical-genetic map were 454 sequenced. Reads assembled in contigs were subsequently ordered, aligned and manually curated in 42 scaffolds. In a total of 1.47 Mbp, 58 protein-coding sequences were identified, 33 of which classified according to similarity with sequences in public databases. As three complete barley C-repeat Binding Factors (HvCBF) genes were newly identified, the locus contained13 full-length HvCBFs, four Related to AP2 Triticeae (RAPT) genes, and at least five CBF pseudogenes. The final overall assembly of Fr-H2 includes more than 90 % of target region: all genes were identified along the locus, and a general survey of Repetitive Elements obtained. We believe that this gold-standard sequence for the Morex Fr-H2 will be a useful genomic tool for structural and evolutionary comparisons with Fr-H2 in winter-hardy cultivars along with Fr-2 of other Triticeae crops.

2013 - Abnormal expression of WT1-as, MEG3 and ANRIL long non-coding RNAs in primary myelofibrosis and their clinical correlates [Abstract in Atti di Convegno]
Pennucci, Valentina; Zini, Roberta; Norfo, Ruggiero; Guglielmelli, P.; Bianchi, Elisa; Salati, Simona; Sacchi, G.; Prudente, Z.; Tenedini, Elena; Ruberti, S.; Rontauroli, S.; Paoli, C.; Fanelli, T.; Mannarelli, C.; Tagliafico, E.; Vannucchi, A. M.; Ferrari, S.; Manfredini, R.
abstract

Long non-coding RNAs (lncRNAs) are emerging as key regulators of gene expression in normal and cancer cells by recruiting chromatin remodeling complexes. Despite their characterization in several tumor types, little is known about the role of lncRNAs in malignant hematopoiesis. In particular, lncRNAs expression has never been investigated in cells from primary myelofibrosis (PMF) patients. PMF is a Philadelphia negative chronic Myeloproliferative Neoplasm (MPN) that originates from deregulated clonal proliferation of hematopoietic stem cell associated with overproduction of mature blood cells. Molecular basis underlying MPN pathogenesis were partially unraveled in 2005-2006 with the identification of somatic mutations of JAK2 and MPL, after which many other mutations were identified. Recently, several new molecular pathogenetic mechanisms were proposed, such as the aberrant expression of coding and non-coding RNAs. In order to identify other molecular abnormalities harbored by PMF patients, we investigated the expression of CDKN2B-antisense (ANRIL), MEG3 and WT1-antisense lncRNAs, previously described as potentially involved in hematological malignancies, in CD34+ cells from PMF patients. The results evidence that the majority of PMF samples displays a co-upregulation of WT1 and its antisense RNA compared to controls. These samples also show an increased MEG3 expression. In these patients, we found a correlation with high Dynamic International Prognostic Scoring System (DIPPS) plus score and elevated number of circulating CD34+ cells. Moreover, the expression pattern of CDKN2B/ANRIL distinguishes a group of patients characterized by an upregulation of CDKN2B, and, among these, a subgroup with downregulated ANRIL. Of note, this group of patients was characterized by a higher grade of bone marrow fibrosis and by the presence of JAK2V617F mutation. Our results suggest that a deregulated expression of these lncRNAs could play a role in PMF pathogenesis and progression.

2013 - Correlation between eight-gene expression profiling and response to therapy of newly diagnosed multiple myeloma patients treated with thalidomide-dexamethasone incorporated into double autologous transplantation. [Articolo su rivista]
Terragna, C; Renzulli, M; Remondini, D; Tagliafico, Enrico; Di Raimondo, F; Patriarca, F; Martinelli, G; Roncaglia, E; Masini, L; Tosi, P; Zamagni, E; Tacchetti, P; Ledda, A; Brioli, A; Angelucci, E; Testoni, N; Marzocchi, G; Galieni, P; Gozzetti, A; Martello, M; Dico, F; Mancuso, K; Cavo, M.
abstract

We performed a molecular study aimed at identifying a gene expression profile (GEP) signature predictive of attainment of at least near complete response (CR) to thalidomide-dexamethasone (TD) as induction regimen in preparation for double autologous stem cell transplantation in 112 younger patients with newly diagnosed multiple myeloma. A GEP supervised analysis was performed on a training set of 32 patients, allowing to identify 157 probe sets differentially expressed in patients with CR versus those failing CR to TD. We then generated an eight-gene GEP signature whose performance was subsequently validated in a training set of 80 patients. A correct prediction of response to TD was found in 71 % of the cases analyzed. The eight genes were downregulated in patients who achieved CR to TD. Comparisons between post-autotransplantation outcomes of the 44 non-CR-predicted patients and of the 36 CR-predicted patients showed that this latter subgroup had a statistically significant benefit in terms of higher rate of CR after autotransplant(s) and longer time to progression, event-free survival, and overall survival. These results can be an important first step to identify at diagnosis those patients who will respond more favourably to a particular treatment strategy.

2013 - Differentiated Neuroprogenitor Cells Incubated with Human or Canine Adenovirus, or Lentiviral Vectors Have Distinct Transcriptome Profiles [Articolo su rivista]
Piersanti, Stefania; Astrologo, Letizia; Licursi, Valerio; Costa, Rossella; Roncaglia, Enrica; Gennetier, Aurelie; Ibanes, Sandy; Chillon, Miguel; Negri, Rodolfo; Tagliafico, Enrico; Kremer, Eric J.; Saggio, Isabella
abstract

Several studies have demonstrated the potential for vector-mediated gene transfer to the brain. Helper-dependent (HD) human (HAd) and canine (CAV-2) adenovirus, and VSV-G-pseudotyped self-inactivating HIV-1 vectors (LV) effectively transduce human brain cells and their toxicity has been partly analysed. However, their effect on the brain homeostasis is far from fully defined, especially because of the complexity of the central nervous system (CNS). With the goal of dissecting the toxicogenomic signatures of the three vectors for human neurons, we transduced a bona fide human neuronal system with HD-HAd, HD-CAV-2 and LV. We analysed the transcriptional response of more than 47,000 transcripts using gene chips. Chip data showed that HD-CAV-2 and LV vectors activated the innate arm of the immune response, including Toll-like receptors and hyaluronan circuits. LV vector also induced an IFN response. Moreover, HD-CAV-2 and LV vectors affected DNA damage pathways - but in opposite directions - suggesting a differential response of the p53 and ATM pathways to the vector genomes. As a general response to the vectors, human neurons activated pro-survival genes and neuron morphogenesis, presumably with the goal of re-establishing homeostasis. These data are complementary to in vivo studies on brain vector toxicity and allow a better understanding of the impact of viral vectors on human neurons, and mechanistic approaches to improve the therapeutic impact of brain-directed gene transfer. © 2013 Piersanti et al.

2013 - Impact Of Prognostically Detrimental Mutations (ASXL1, EZH2, SRSF2, IDH1/2) On Outcomes In Patients With Myelofibrosis Treated With Ruxolitinib In COMFORT-II [Abstract in Rivista]
Guglielmelli, Paola; Biamonte, Flavia; Pieri, Lisa; Rotunno, Giada; Paoli, Chiara; Fjerza, Rajmonda; Tagliafico, Enrico; Manfredini, Rossella; Artusi, Valentina; Tenedini, Elena; Artuso, Lucia; Bernardis, Isabella; Stalbovskaya, Viktoriya; Squires, Matthew; Harrison, Claire N; Vannucchi, Alessandro M.
abstract

Abstract Background Ruxolitinib (RUX) is a JAK1 & JAK2 inhibitor that resulted in rapid and durable reductions in splenomegaly and improved disease-related symptoms and quality of life in patients (pts) with myelofibrosis (MF) compared with either placebo (COMFORT-I) or best available therapy (BAT; COMFORT-II). In addition, RUX-treated pts had longer overall survival (OS) compared with placebo and BAT. We recently reported that, among 879 primary MF pts receiving conventional BAT, those harboring a mutation in any one of EZH2, ASXL1, IDH1/2 and SRSF2 constituted an IPSS- and DIPSS-plus prognostic score–independent “high molecular risk” (HMR+) category associated with shorter OS and greater risk of leukemia compared with pts with no mutations (“low molecular risk”; LMR) (Vannucchi AM, et al. Leukemia. 2013). The aim of this study was to analyze the impact of mutational status on spleen volume reduction, anemia development, and OS in pts receiving RUX in the COMFORT II trial. Patients and methods In COMFORT-II, pts with primary or post–polycythemia vera/–essential thrombocythemia MF were randomized to receive RUX (n=146) or BAT (n=73). Mutations in 12 genes (JAK2, MPL, EZH2, ASXL1, TET2, IDH1/2, CBL, SRSF2, SOCS1, SOCS2, SOCS3, and SH2B3) were genotyped, in DNA derived from whole blood, at baseline in 166 pts (RUX n=120, BAT n=46). Analysis was performed by next-generation sequencing with the Ion Torrent PGM or Roche 454 platform. Sequencing data were analyzed with Nextgene software or Roche 454 Analysis software v2.6 with variant frequency cutoff adjusted to 5%. All mutations were confirmed at least twice. Missense, nonsense, and frameshift mutations only were considered; in the case of novel mutations, SNPs were excluded by database searching and by germline DNA genotyping when available. Development of anemia was defined as a drop of hemoglobin level by more than 1 g/dL from baseline to a value <10 g/dL within the first 48 weeks of treatment. Survival estimates were obtained with Kaplan-Meier method. The treatment effect and the prognostic value of the molecular variables with regard to OS were analyzed by Cox regression and adjusted for the IPSS category. Results The frequency of mutations was: JAK2V617F 75.47%; MPLW515 7.74%; ASXL1 32.53%; TET2 10.69%; EZH2 7.24%; CBL 4.4%; SRSF2 3.01%; SH2B3 1.3%; IDH1-2 0.7%; SOCS1 0.65%; SOCS2 0.65%; SOCS3 0.0%, with no difference between RUX and BAT. Forty-six (38.3%) and 20 (43.5%) pts in the RUX and BAT groups, respectively, were classified as HMR+. We first determined whether an HMR+ status impacted the achievement of a ≥35% spleen volume reduction (primary study endpoint). The percentage of RUX treated pts achieving ≥35% spleen volume reduction was 36.3% (16/44) and 33.8% (21/62) at 24 wk and 30.7% (12/39) and 36.3% (20/55) at 48 wk in the HMR+ and LMR categories, respectively. Mean spleen volume reduction was also similar: -29.0% and -23.5% in HMR+ vs -29.9% and -30.6% in LMR pts at 24 and 48 wk. None of the other mutations analyzed correlated with spleen volume reduction in pts receiving RUX. We also found that an HMR+ status did not predict for the development of anemia associated with RUX administration: the percentage of anemic pts was 74% in the HMR+ group vs 72% in the LMR group. This was also independent of the presence of mutation in any one of the genes associated with JAK2/STAT signaling (JAK2, MPL, SH3B2, CBL, and SOCSs): anemic pts were 74% in mutated vs 72% in wild-type ones. The survival estimate at 114 wk of follow-up in BAT pts was 0.58 and 0.71 in HMR+ and LMR pts, confirming the negative impact of the mutational risk category. In the RUX arm, the survival estimate was 0.79 and 0.85 for HMR+ and LMR pts, indicating a benefit of RUX treatment in both groups. In the multivariate Cox model, a risk of death with RUX compared with BAT was reduced by 43% (HR=0.57, 95% CI: 0.30-1.08) and LMR patients had

2013 - Integrative Analysis Of mRNA/miRNA Expression Profiles Identified JARID2 As a Shared Target Of Deregulated Mirnas In Primary Myelofibrosis [Abstract in Rivista]
Zini, Roberta; Norfo, Ruggiero; Pennucci, Valentina; Bianchi, Elisa; Salati, Simona; Paola, Guglielmelli; Andrea, Bisognin; Vittorio, Rosti; Daniela, Pietra; Silvia, Salmoiraghi; Costanza, Bogani; Tiziana, Fanelli; Ruberti, Samantha; Sacchi, Giorgia; Prudente, Zelia; Giovanni, Barosi; Mario, Cazzola; Alessandro, Rambaldi; Stefania, Bortoluzzi; Ferrari, Sergio; Tagliafico, Enrico; Alessandro M., Vannucchi; Manfredini, Rossella
abstract

Ph-negative myeloproliferative neoplasms (MPNs) are characterized by many somatic mutations which have already been shown useful in the prognostic assessment of MPN patients [A.M. Vannucchi et al., Leukemia, 2013]. Moreover, aberrant microRNA (miRNA) expression seems to add to the molecular complexity of MPNs, as specific miRNA signatures capable of discriminating MPN cells from those of normal donors were previously reported [P. Guglielmelli et al., Exp Hematol, 2007]. In order to have a comprehensive picture of miRNA deregulation and its relationship with differential gene expression in primary myelofibrosis (PMF) cells, we obtained gene- (GEP) and miRNA expression profiles (miEP) of CD34+ cells from 31 healthy donors and 42 PMF patients using Affymetrix technology (HG-U219 and miRNA 2.0 arrays). Among 726 differentially expressed genes (DEG) we found that several putative cancer markers (WT1, ANGPT1) and several genes related to PMF progression, i.e. involved in megakaryocyte (MK) differentiation (NFE2, CD9), and fibrosis development (DLK1, LEPR1), were significantly more expressed in PMF samples than in the normal counterpart. Similarly, as regards the miEP, among 74 human differentially expressed miRNAs (DEM) in PMF compared to controls we found the upregulation of several miRNAs associated with hematological malignancies or known as oncomiRs (i.e. hsa-miR-155-5p [S. Jiang et al., Cancer Res, 2010], miRNAs belonging to the miR-17-92 cluster [L. Venturini et al., Blood, 2007]), and other aberrantly expressed miRNAs never described in hematopoiesis (i.e. hsa-miR-335-5p). Then, in order to construct regulatory networks of the functional human miRNA-target interactions, we performed an integrative analysis (IA) with Ingenuity Pathway analysis software, which combines the miRNA expression profile with computational predicted targets and with the gene expression data. IA between DEG and DEM disclosed a high number of predicted targets with anti-correlated expression to the trend of their targeting miRNAs. Of note, IA identified an interaction network (see Figure) in which the upregulated oncomirs miR-155-5p [R.M. O'Connel et al., J Exp Med, 2008], miR29a-3p [Y.C. Han et al., J Exp Med, 2010] and miR-19b-3p [K.J. Mavrakis et al., Nat Cell Biol, 2010] could explain the downregulation of targets whose lower expression was already described as involved in myeloproliferative phenotypes, such as NR4A3, CDC42, HMGB3. Additionally, IA disclosed the chromatin remodeler JARID2, which is frequently deleted in leukemic transformation of chronic myeloid malignancies, as a shared target of several upregulated miRNAs in PMF samples (i.e. miR-155-5p, miR-152-3p). Noteworthy, these miRNA-mRNA interactions were functionally confirmed by 3' UTR luciferase reporter assays. Next, in order to characterize the role of JARID2 in PMF pathogenesis, we performed RNAi-mediated gene silencing experiments on CD34+ cells of healthy donor. Interestingly, inhibition of JARID2 expression produces in silenced cells a significant increase of CD41 expression when compared with control (28.6±3.1% vs 15.3±1.8% at day 8, 52.6±7.6% vs 35.4±4.9% at day 12 of serum free liquid culture) and a remarkable increase in CFU-MK colonies (59.6±6.5% vs 39.8±5.9%). The values are reported as mean ± 2S.E.M from five independent experiments. Moreover, morphological analysis after May-Grunwald-Giemsa staining showed that JARID2 silencing induces in normal CD34+ cells a considerable enrichment in MK precursors at different stages of maturation. This study allowed the identification of different networks possibly involved in PMF onset, highlighting the potential contribution of miRNAs to PMF pathogenesis. Furthermore, for the first time, we demonstrated that the JARID2 downregulation in CD34+ cells might contribute to the abnormal megakaryopoiesis typical of PMF.

2013 - Isolation of human keratinocyte stem cells and high-throughput screening approach for their characterization [Abstract in Atti di Convegno]
Di Rocco, Antonio; Carulli, Sonia; Tenedini, Elena; Bianchi, Elisa; Tagliafico, Enrico; Manfredini, Rossella; Pellegrini, Graziella; DE LUCA, Michele
abstract

In the last three decades, regenerative medicine has opened new horizons for the in vitro reconstruction of epithelial tissues and gene therapy treatment of skin disorders involving the use of adult keratinocyte stem cells (KSCs). Although the ability to identify and isolate these cells represents an important prerequisite for the development of these approaches, molecular markers and their precise in vivo localization are still lacking. In order to define genes involved in the control of stemness and commitment of KSCs, we developed a non-invasive, stem cell-preserving magnetic micro beads based method in order to obtain a KSCs enriched population for high throughput screening experiments. After 3T3 murine fibroblast feeder layer depletion from our keratinocyte cultures, we isolated a subpopulation of basal epithelial cells on the basis of the different expression levels of the a6β4 integrin. By using different approaches, including clonal analysis and p63 bright cells quantification, we clearly showed that a6β4 integrin bright cells have greater growth potential and clonogenic capacity compared to the remaining cell fraction and they include the KSCs population. Comparing gene expression profile of a KSCs-enriched and a terminally differentiated cell population coming from the same original primary cell culture we defined a set of genes most probably involved in stemness maintenance. Ongoing gene profiling on single clone type will allow us to validate this gene signature and to start functional studies on selected genes. Extending this approach to different ectodermal derived tissues will provide a genome wide signature of the molecular pathways underlying self-renewal, commitment and differentiation of KSCs.

2013 - Mutations and prognosis in primary myelofibrosis [Articolo su rivista]
Vannucchi, Am; Lasho, Tl; Guglielmelli, P; Biamonte, F; Pardanani, A; Pereira, A; Finke, C; Score, J; Gangat, N; Mannarelli, C; Ketterling, Rp; Rotunno, G; Knudson, Ra; Susini, Mc; Laborde, Rr; Spolverini, A; Pancrazzi, A; Pieri, L; Manfredini, Rossella; Tagliafico, Enrico; Zini, Roberta; Jones, A; Zoi, K; Reiter, A; Duncombe, A; Pietra, D; Rumi, E; Cervantes, F; Barosi, G; Cazzola, M; Cross, Nc; Tefferi, A.
abstract

Patient outcome in primary myelofibrosis (PMF) is significantly influenced by karyotype. We studied 879 PMF patients to determine the individual and combinatorial prognostic relevance of somatic mutations. Analysis was performed in 483 European patients and the seminal observations were validated in 396 Mayo Clinic patients. Samples from the European cohort, collected at time of diagnosis, were analyzed for mutations in ASXL1, SRSF2, EZH2, TET2, DNMT3A, CBL, IDH1, IDH2, MPL and JAK2. Of these, ASXL1, SRSF2 and EZH2 mutations inter-independently predicted shortened survival. However, only ASXL1 mutations (HR: 2.02; P<0.001) remained significant in the context of the International Prognostic Scoring System (IPSS). These observations were validated in the Mayo Clinic cohort where mutation and survival analyses were performed from time of referral. ASXL1, SRSF2 and EZH2 mutations were independently associated with poor survival, but only ASXL1 mutations held their prognostic relevance (HR: 1.4; P=0.04) independent of the Dynamic IPSS (DIPSS)-plus model, which incorporates cytogenetic risk. In the European cohort, leukemia-free survival was negatively affected by IDH1/2, SRSF2 and ASXL1 mutations and in the Mayo cohort by IDH1 and SRSF2 mutations. Mutational profiling for ASXL1, EZH2, SRSF2 and IDH identifies PMF patients who are at risk for premature death or leukemic transformation.

2013 - Regulatory mRNA/microRNA networks in CD34+ cells from Primary Myelofibrosis [Abstract in Atti di Convegno]
Norfo, Ruggiero; Zini, Roberta; Pennucci, Valentina; Ruberti, S.; Bianchi, Elisa; Salati, Simona; Gugliemelli, P.; Bisognin, A.; Rosti, V.; Pietra, D.; Ricci, C.; Fanelli, T.; Salmoiraghi, S.; Sacchi, G.; Prudente, Z.; Rontauroli, S.; Barosi, G.; Cazzola, M.; Bortoluzzi, S.; Tagliafico, E.; Vannucchi, A. M.; Ferrari, S.; Manfredini, R.
abstract

Primary myelofibrosis (PMF) is a clonal disorder of a hematopoietic stem cell included in the Philadelphia chromosome-negative chronic myeloproliferative neoplasms (MPNs), together with polycythemia vera and essential thrombocythemia. The molecular mechanisms of these diseases were partially unravelled in 2005 with the identification of somatic gain-of-function of Janus kinase 2 (JAK2) and Thrombopoietin Receptor (MPL), after which many other mutated genes were found. Moreover, aberrant microRNA (miRNA) expression seems to add up to the molecular complexity of MPNs, as specific miRNA signatures discriminates MPN cells from those of normal donors. In order to have a comprehensive picture of miRNA deregulation and its relationship with differential gene expression in PMF cells, we obtained mRNA and miRNA profiles in the same CD34+ cells from 31 healthy donors and 42 PMF patients by means of Affymetrix technology. Several miRNAs involved in hematological malignancies or known as oncomirs resulted upregulated in PMF samples (hsa-miR-155-5p, miRNAs belonging to the miR-17-92 cluster), whereas other aberrantly expressed miRNAs have never been described in the hematological context (hsa-miR-335). Next, we carried out an in silico integrative analysis (IA) with Ingenuity Pathway Analysis software, which combines the computational predicted targets with the gene expression data to construct regulatory networks of the functional miRNA-mRNA interactions. Of note, IA identified a network potentially involved in PMF pathogenesis, in which the upregulated oncomirs miR-155-5p and miR29a-3p could explain the downregulation of targets whose lower expression was already described in myeloproliferative phenotypes (NR4A3, CDC42, HMGB3), and of the chromatin remodeler JARID2, which is frequently deleted in leukemic transformation of MPNs. Finally, we demonstrated the JARID2 downregulation in CD34+ cells plays a role in the abnormal megakaryopoiesis, and contributes to PMF pathogenesis.

2013 - Regulatory mRNA/microRNA networks in CD34+ cells from Primary Myelofibrosis [Abstract in Atti di Convegno]
Pennucci, Valentina; Zini, Roberta; Norfo, Ruggiero; Ruberti, S.; Bianchi, Elisa; Salati, Simona; Gugliemelli, P.; Bisognin, A.; Rosti, V.; Pietra, D.; Ricci, C.; Fanelli, T.; Salmoiraghi, S.; Sacchi, G.; Prudente, Z.; Rontauroli, S.; Barosi, G.; Cazzola, M.; Bortoluzzi, S.; Tagliafico, E.; Vannucchi, A. M.; Ferrari, S.; Manfredini, R.
abstract

Primary myelofibrosis (PMF) is a clonal disorder of a hematopoietic stem cell included in the Philadelphia chromosome-negative chronic myeloproliferative disorders (MPD), together with polycythemia vera and essential thrombocythemia. The molecular mechanisms of these diseases were partially unravelled in 2005 with the identification of somatic gain-of-function of Janus kinase 2 (JAK2) and Thrombopoietin Receptor (MPL), after which many other mutated genes were found. Moreover, aberrant microRNA (miRNA) expression especially seems to add up to the molecular complexity of MPNs, as specific miRNA signatures discriminates MPN from normal donors. In order to have a comprehensive picture of miRNA deregulation and its relationship with differential gene expression in PMF cells, we obtained mRNA and miRNA profiles in the same CD34+ cells from 31 healthy donors and 42 PMF patients by means of Affymetrix technology. Several miRNAs involved in hematological malignancies or known as oncomirs were upregulated in PMF samples (hsa-miR-155-5p, miRNAs belonging to the miR-17-92 cluster), whereas other aberrantly expressed miRNAs have never been described in the hematological context (has-miR-335). Next, we carried out an in silico integrative analysis (IA) with Ingenuity Pathway Analysis software, which combines the computational predicted targets with the gene expression data to construct regulatory networks of the functional miRNA-mRNA interactions. Of note, IA identified a significant network in which the upregulated oncomirs miR-155-5p and miR29a-3p could explain the downregulation of targets whose lower expression was already described in myeloproliferative phenotypes (NR4A3, CDC42, HMGB3), and of the chromatin remodeler JARID2, which is frequently deleted in leukemic transformation of MPNs. This approach allowed the identification of different networks potentially involved in PMF onset and progression, highlighting the potential contribution of miRNAs to PMF pathogenesis.

2013 - TRANSCRIPTOME ANALYSIS IN THE INTERACTION BRACHYPODIUM – PUCCINIA BRACHYPODII [Abstract in Atti di Convegno]
Mazzamurro, Valentina; Laviano, Luca; Thierri, Marcel; Milc, Justyna Anna; Francia, Enrico; Rients, Niks; T., Vozabova; David, Garvin; Enrica, Roncaglia; MALAGOLI TAGLIAZUCCHI, Guidantonio; Bicciato, Silvio; Tagliafico, Enrico; Pecchioni, Nicola
abstract

The model grass Brachypodium distachyon L (Brachypodium) has recently revealed its potential for studying grass-pathogen interactions. In particular, the identification of genomic regions associated with resistance to the false brome rust fungus Puccinia brachypodii offered perspectives to elucidate the genetic and molecular basis of this trait. In this study, we aimed to: 1) provide an initial whole-genome expression dataset for Brachypodium-P. brachypodii interaction in the two inbred lines Bd3-1 (resistant) and Bd1-1 (susceptible), and 2) fine mapping and cloning Rpbq2 and Rpbq3: to increase the resolution of QTL mapping and to reduce the number of candidate genes underlying QTL LOD curves. For the first, aim the two inbred lines have been characterized macroscopically and by confocal microscopy to follow the development of the fungus and the formation of rust infection structures. The expression of six brachypodium genes, homologous to known wheat and barley defence-related genes, was monitored by qRT-PCR analysis in Bd3-1 and Bd1-1 at three time points (18, 24 and 72 hours post infection, hpi). The 18 hpi time point was selected for transcriptome profiling on the basis of the expression profiles of the defence genes. The Affymetrix Brachypodium Tiling Array (BradiAR1b520742) revealed that expression levels of a set of genes (more than 100 in total) were altered in infected plants, mainly in the resistant line Bd3-1. At 18 hpi a significant re-programming of host metabolism occurred in infected leaves, with a modulation of genes involved in different metabolic networks such as defence, glycolysis, aminoacid and nitrogen metabolism. This study represents the first characterization of the functional genomic basis of resistance to a rust species in the model plant Brachypodium, and could be useful for translational genomics to ‘complex’ cereals. For the second aim, fine mapping Rpbq2 and Rpbq3, a new large segregating RIL population has been developed for each QTL separately. Selection of Bd3-1 x Bd1-1 RILs heterozygous for the QTLs has been completed based on flanking marker haplotypes, with the target QTL in a heterozygous state, while the other two QTLs were selected to be homozygous for the susceptible allele. These marker-selected heterozygous RILs have been selfed to obtain large segregating populations for each QTL. These results represent the first steps of a genetic approach towards the cloning of Rpbq2 and Rpbq3 determinants, and for their possible exploitation in cereals.

2012 - Effects of bile duct ligation and cholic acid treatment on fatty liver in two rat models of non-alcoholic fatty liver disease [Articolo su rivista]
Gabbi, Chiara; Bertolotti, Marco; Anzivino, Claudia; Macchioni, Daria; Del Puppo, Marina; Ricchi, Matteo; Carubbi, Francesca; Tagliafico, Enrico; Romagnoli, Dante; Odoardi, Maria Rosaria; Loria, Paola; Losi, Luisa; Carulli, Nicola
abstract

Non-alcoholic fatty liver disease, one of the most prevalent liver disorders in Western countries, is characterized by hepatic accumulation of triglycerides. Bile acids have long been known to affect triglyceride homeostasis through a not completely understood mechanism.

2012 - Regulatory Mrna/Microrna Networks in CD34+ Cells From Primary Myelofibrosis [Abstract in Rivista]
Norfo, Ruggiero; Zini, Roberta; Pennucci, Valentina; Bianchi, Elisa; Salati, Simona; Guglielmelli, P.; Bisognin, A; Rosti, V.; Pietra, D.; Ricci, C.; Fanelli, T.; Salmoiraghi, S.; Sacchi, G.; Ruberti, S.; Barosi, G.; Cazzola, M.; Bortoluzzi, S.; Ferrari, S.; Tagliafico, E.; Vannucchi, A. M.; Manfredini, R.
abstract

Molecular mechanisms underlying Philadephia-negative myeloproliferative neoplasm (MPN) pathogenesis were partially unraveled in 2005–2006 with the identification of somatic gain-of-function of JAK2 and MPL, after which many other mutated genes were found. Recently, several new molecular pathogenetic mechanisms were identified. Among them, aberrant microRNA (miRNA) expression especially seems to add to the molecular complexity of MPNs, as specific miRNA signatures capable of discriminating MPN cells from those of normal donors were previously reported (P. Guglielmelli et al., Exp Hematol, 2007). In order to have a comprehensive picture of miRNA deregulation and its relationship with differential gene expression in primary myelofibrosis (PMF) cells, we obtained coding gene- (GEP) and miRNA expression profiles (miEP) in the same CD34+ sample from 31 healthy donors and 42 PMF patients by means of Affymetrix technology (HG-U219 and miRNA 2.0 arrays). 726 genes were found as differentially expressed (DEG) (fold change contrast â{per thousand}¥2, false discovery rate â{per thousand}¤0.05) (FIG. 1) and further analysis pointed out that several DEG are related to processes involved in PMF progression as megakaryocyte (MK) differentiation, fibrosis and migration. Of interest, we found the upregulation of some putative cancer markers, such as WT1 (K. Inoue et al., Blood, 1994) and ANGPT1 (C.L. Cheng, Br J Cancer, 2011) whose expression has already been associated with poor prognosis in hematological neoplasms and in other malignancies. Among the deregulated transcription factors, we detected several genes involved in CD34+ commitment, and potentially in their transformation, such as NFE-2 (C. LAbbaye et al., J Clin Invest, 1995) and KLF3 (A.P. Funnell, Mol Cell Biol, 2012). As regards miEP, we achieved a list of 74 human miRNAs modulated in PMF (DEM) (fold change contrast â{per thousand}¥1.5, false discovery rate â{per thousand}¤0.05), some of which associated with hematological malignancies or known as oncomirs are upregulated, i.e. hsa-miR-155-5p (S. Jiang, Cancer Res, 2010), miRNAs belonging to the miR-17–92 cluster (L. Venturini et al., Blood, 2007), whereas other aberrantly expressed miRNAs have never been described in any hematological context. Next, we performed an in silico integrative analysis (IA) with Ingenuity Pathway analysis software, which combines the computational predicted targets with the gene expression data, in order to construct regulatory networks of the functional human miRNA-target interactions. IA between DEG and DEM disclosed a high number of predicted targets with anti-correlated expression to the trend of their targeting miRNAs. This approach allowed the identification of different networks potentially involved in PMF onset and progression, such as MK differentiation and chromatin remodeling, highlighting the potential contribution of miRNAs to PMF pathogenesis. In particular, the integrative analysis has identified an interaction network involving the oncomirs miR-155-5p and miR-29a-3p (R. M. O'Connel et al, J Exp Med, 2008, Y.C. Han et al, j Exp Med, 2010) and their targets (FIG. 2). In this network the upregulation of miR-155-5p and mir-29a-3p could explain the negative regulation of two tumor suppressor genes, HBP1 and TP53INP1, and of SPTB1, CDC42 and KLF3, whose downregulation is involved in malignant hematopoiesis (L.Yang et al, Blood 2007). This network also shows the upregulation of some miRNAs whose function is unknown in the hematopoietic context as miR-335-5p, with the negative regulation of its predicted targets, NR4A3 and PRDM2, which are described as implicated in myeloproliferation (AM Ramirez-Herrick et al, Blood 2001). The present findings lay the groundwork for functional in vitro validation of selected networks in normal and PMF CD34+ cells by means of DEG/DEM overexpression and silencing experiments; furthermore, expression data will b

2012 - Survival features of EBV-stabilized cells from centenarians: morpho-functional and transcriptomic analyses. [Articolo su rivista]
Matarrese, P; Tinari, A; Ascione, B; Gambardella, L; Remondini, D; Salvioli, S; Tenedini, Elena; Tagliafico, Enrico; Franceschi, C; Malorni, W.
abstract

In the present work, we analyzed the survival features of six different Epstein–Barr virus (EBV)-stabilized lymphoid cell lines obtained from adult subjects and from subjects of more than 95 years. For the first, we found that lymphoid B cells from centenarians were more resistant to apoptosis induction and displayed a more developed lysosomal compartment, the most critical component of phagic machinery, in comparison with lymphoid B cells from adult subjects. In addition, cells from centenarians were capable of engulfing and digesting other cells, i.e., their siblings (even entire cells), whereas lymphoid cells from “control samples”, i.e., from adults, did not. This behavior was improved by nutrient deprivation but, strikingly, it was unaffected by the autophagy-modulating drug, rapamycin, an autophagy inducer, and 3-methyladenine, an autophagy inhibitor. Transcriptomic analyses indicated that: (1) aspartyl proteases, (2) cell surface molecules such as integrins and cadherins, and (3) some components of cytoskeletal network could contribute to establish this survival phenotype. Also, Kyoto Encyclopedia of Genes and Genomes pathways such as Wnt signaling pathway, an essential contributor to cell migration and actin cytoskeleton remodeling, appeared as prominent. Although we cannot rule out the possibility that EBV-immortalization could play a role, since we observed this phagic behavior in cells from centenarians but not in those from adults, we hypothesize that it may represent an important survival determinant in cells from centenarians.

2012 - Transcriptional profiles underlying vulnerability and resilience in rats exposed to an acute unavoidable stress [Articolo su rivista]
Benatti, Cristina; Valensisi, Cristina; Blom, Johanna Maria Catharina; Alboni, Silvia; Montanari, Claudia; Ferrari, Francesco; Tagliafico, Enrico; Julien, Mendlewicz; Brunello, Nicoletta; Tascedda, Fabio
abstract

A complex interplay between gene and environment influences the vulnerability or the resilience to stressful events. In the acute escape deficit (AED) paradigm, rats exposed to an acute unavoidable stress (AUS) develop impaired reactivity to noxious stimuli. Here we assessed the behavioral and molecular changes in rats exposed to AUS. A genome-wide microarray experiment generated a comprehensive picture of changes in gene expression in the hippocampus and the frontal cortex of animals exposed or not to AUS. Exposure to AUS resulted in two distinct groups of rats with opposite behavioral profiles: one developing an AED, called “stress vulnerable,” and one that did not develop an AED, called “stress resilient.” Genome-wide profiling revealed a low percentage of overlapping mechanisms in the two areas, suggesting that, in the presence of stress, resilience or vulnerability to AUS is sustained by specific changes in gene expression that can either buffer or promote the behavioral and molecular adverse consequences of stress. Specifically, we observed in the frontal cortex a downregulation of the transcript coding for interferon-β and leukemia inhibitory factor in resilient rats and an upregulation of neuroendocrine related genes, growth hormone and prolactin, in vulnerable rats. In the hippocampus, the muscarinic M2 receptor was downregulated in vulnerable but upregulated in resilient rats. Our findings demonstrate that opposite behavioral responses did not correspond to opposite regulatory changes of the same genes, but resilience rather than vulnerability to stress was associated with specific changes, with little overlap, in the expression of patterns of genes.

2012 - Transplantation of genetically corrected human iPSC-derived progenitors in mice with limb-girdle muscular dystrophy [Articolo su rivista]
Tedesco, F. S.; Gerli, M. F.; L., Perani; S., Benedetti; F., Ungaro; M., Cassano; S., Antonini; Tagliafico, Enrico; Artusi, Valentina; E., Longa; R., Tonlorenzi; M., Ragazzi; G., Calderazzi; H., Hoshiya; O., Cappellari; M., Mora; B., Schoser; P., Schneiderat; M., Oshimura; R., Bottinelli; M., Sampaolesi; Y., Torrente; V., Broccoli; G., Cossu
abstract

Mesoangioblasts are stem/progenitor cells derived from a subset of pericytes found in muscle that express alkaline phosphatase. They have been shown to ameliorate the disease phenotypes of different animal models of muscular dystrophy and are now undergoing clinical testing in children affected by Duchenne's muscular dystrophy. Here, we show that patients with a related disease, limb-girdle muscular dystrophy 2D (LGMD2D), which is caused by mutations in the gene encoding α-sarcoglycan, have reduced numbers of this pericyte subset and thus produce too few mesoangioblasts for use in autologous cell therapy. Hence, we reprogrammed fibroblasts and myoblasts from LGMD2D patients to generate human induced pluripotent stem cells (iPSCs) and developed a protocol for the derivation of mesoangioblast-like cells from these iPSCs. The iPSC-derived mesoangioblasts were expanded and genetically corrected in vitro with a lentiviral vector carrying the gene encoding human α-sarcoglycan and a promoter that would ensure expression only in striated muscle. When these genetically corrected human iPSC-derived mesoangioblasts were transplanted into α-sarcoglycan-null immunodeficient mice, they generated muscle fibers that expressed α-sarcoglycan. Finally, transplantation of mouse iPSC-derived mesoangioblasts into α-sarcoglycan-null immunodeficient mice resulted in functional amelioration of the dystrophic phenotype and restoration of the depleted progenitors. These findings suggest that transplantation of genetically corrected mesoangioblast-like cells generated from iPSCs from LGMD2D patients may be useful for treating this type of muscular dystrophy and perhaps other forms of muscular dystrophy as well.

2012 - Valproic acid triggers erythro/megakaryocyte lineage decision through induction of GFI1B and MLLT3 expression [Articolo su rivista]
Zini, Roberta; Norfo, Ruggiero; Ferrari, Francesco; Bianchi, Elisa; Salati, Simona; Pennucci, Valentina; Sacchi, Giorgia; Carboni, Chiara; Ceccherelli, Gb; Tagliafico, Enrico; Ferrari, Sergio; Manfredini, Rossella
abstract

Histone deacetylase inhibitors represent a family of targeted anticancer compounds that are widely used against hematological malignancies. So far little is known about their effects on normal myelopoiesis. Therefore, in order to investigate the effect of histone deacetylase inhibitors on the myeloid commitment of hematopoietic stem/progenitor cells, we treated CD34(+) cells with valproic acid (VPA). Our results demonstrate that VPA treatment induces H4 histone acetylation and hampers cell cycle progression in CD34(+) cells sustaining high levels of CD34 protein expression. In addition, our data show that VPA treatment promotes erythrocyte and megakaryocyte differentiation. In fact, we demonstrate that VPA treatment is able to induce the expression of growth factor-independent protein 1B (GFI1B) and of mixed-lineage leukemia translocated to chromosome 3 protein (MLLT3), which are crucial regulators of erythrocyte and megakaryocyte differentiation, and that the up-regulation of these genes is mediated by the histone hyperacetylation at their promoter sites. Finally, we show that GFI1B inhibition impairs erythroid and megakaryocyte differentiation induced by VPA, while MLLT3 silencing inhibits megakaryocyte commitment only. As a whole, our data suggest that VPA sustains the expression of stemness-related markers in hematopoietic stem/progenitor cells and is able to interfere with hematopoietic lineage commitment by enhancing erythrocyte and megakaryocyte differentiation and by inhibiting the granulocyte and mono-macrophage maturation.

2011 - Double-blind, placebo-controlled, multicentric randomized phase IIb neoadjuvant study of letrozole-lapatinib in postmenopausal HER2-negative, hormone receptor-positive operable breast cancer [Abstract in Rivista]
Conte, Pierfranco; Guarneri, Valentina; D. G., Generali; A., Bottini; L., Bazzola; Piacentini, Federico; F., Artioli; K., Cagossi; G., Bisagni; M., Bagnalasta; Tagliafico, Enrico; Barbieri, Elena; L., Cavanna; A., Ravaioli; D'Amico, Roberto; Vicini, Roberto; A., Frassoldati
abstract

Background: The crosstalk between the ER pathway and erbB receptor family is emerging as a mechanism of resistance to hormonal therapy. The combination of lapatinib-letrozole might prevent or delay the development of endocrine resistance. On these premises we have designed a multicentric phase IIb randomized, double-blind, placebo controlled study to evaluate the clinical and biological effects of combined letrozole+lapatinib/placebo as neoadjuvant therapy in previously untreated ER+ve/HER2-ve breast cancer patients. Primary aim is the percentage of breast clinical response, as measured by ultrasonography (US). Secondary aims include pathologic response, percentage of breast conserving surgery, modulation of Ki67 and HER2/EGFR pathways, and gene expression analysis. Methods: After diagnostic core biopsy, patients were randomized to letrozole 2.5 mg continuous daily dosing (CDD) + lapatinib 1500mg CDD or to letrozole- placebo, given for 24 weeks before surgery. Results: As of October 2010, the planned accrual has been completed. Ninety-two postmenopausal women have been randomized. Patient characteristics were as follows: median age 68 yrs (range 48-89 yrs); stage at diagnosis: IIA 49%; IIB 41%, IIIA 10%. Median ER expression 95% (range 30-100%); median PgR expression 68% (range 0-100%). At diagnosis, mean US tumor size was 3 cm (range 1.2-8 cm). Seventy-one patients are evaluable so far. The ORR (PR + CR) by US at the completion of therapy was 61%; SD was observed in 27% of the patients. Four patients experienced PD. Five patients prematurely discontinued therapy due to toxicity (n=1), consent withdrawal (n=3) or lost to follow up (n=1). No change in mean LVEF was observed at the 3- and 6-month evaluations. The data will be unblinded by April 2011. Conclusions: Preliminary blinded results suggest that the combination of letrozole+lapatinib/placebo is associated with clear tumor downstaging. Final unblinded results per treatment arm, including pathologic response, clinical response by centralized review and biomarker analyses will be presented at the meeting.

2011 - Final results of a phase II randomized trial of neoadjuvant anthracycline-taxane chemotherapy plus lapatinib, trastuzumab, or both in HER2-positive breast cancer (CHER-LOB trial). [Abstract in Rivista]
Guarneri, Valentina; A., Frassoldati; A., Bottini; D. G., Generali; K., Cagossi; F., Artioli; G., Bisagni; C., Boni; A., Ravaioli; D., Amadori; A., Musolino; L., Cavanna; M., Untch; L., Orlando; G., Giardina; Piacentini, Federico; Tagliafico, Enrico; M., Bagnalasta; D'Amico, Roberto; Conte, Pierfranco
abstract

Background: This is a randomized phase II trial of preoperative taxane-anthracycline in combination with trastuzumab, lapatinib, or combined trastuzumab and lapatinib in HER2 positive, stage II-IIIA breast cancer patients. Primary aim of the study is the percentage pathological complete response (pCR), defined as complete disappearance of invasive tumor in breast and axillary nodes. Methods: chemotherapy (CT) consists of weekly paclitaxel x 12 followed by FE75C x 4. Pts randomized to arm A receive CT plus weekly trastuzumab; in arm B pts receive CT plus lapatinib 1250 mg po daily; in arm C pts receive CT plus weekly trastuzumab and lapatinib 750 mg po daily. The study sample size has been calculated according to the two-step Simon’s design. The overall planned accrual was 120 pts. P95HER2 expression will be measured by bioTheranostics, Inc (San Diego) to explore if there is a clinically relevant difference in the pCR rate according to p-95 status. Gene expression profile analysis to identify a predictive signature is ongoing. Results: 121 pts have been randomized as of November 2010. Pts characteristics are the following: median age 49 yrs (26-68 yrs); stage IIA 32%, IIB 50%; IIIA 18%; ER and or PgR positivity: 59%. Eighty pts have completed surgery and are evaluable for response: 50 pts (62.5%) received breast conservation (BCS). A conversion from mastectomy to BCS was observed in 23/44 pts initially candidate to mastectomy (conversion rate: 52%). The pCR rate is 36.2% (28% in arm A, 32% in arm B, and 48% in arm C). By using a 30% cutoff for p95 positivity, in a preliminary analysis on 48 cases, 57% resulted as p-95 positive. In this preliminary analysis, the pCR rate in 15 trastuzumab treated pts is 86% in p-95-negative and 13% in p-95-positive cases. Mean Left ventricular ejection fraction (range) at baseline was 62% (52%-77%), 61% (44%-78%) after 12-13 weeks, and 61% (44%-74%) at the end of therapy. No patient had symptomatic cardiac events. Conclusions: Preliminary activity data are promising, and cardiac safety data are reassuring. Final results per treatment arm, along with definitive biomarker correlations will be presented at the meeting.

2011 - Gene expression profiling in MDS and AML: potential and future avenues [Articolo su rivista]
K., Theilgaard Mönch; J., Boultwood; Ferrari, Sergio; K., Giannopoulos; J. M., Hernandez Rivas; A., Kohlmann; M., Morgan; B., Porse; Tagliafico, Enrico; C. M., Zwaan; J., Wainscoat; M. M., Van den Heuvel Eibrink; K., Mills; L., Bullinger
abstract

Today, the classification systems for myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) already incorporate cytogenetic and molecular genetic aberrations in an attempt to better reflect disease biology. However, in many MDS/AML patients no genetic aberrations have been identified yet, and even within some cytogenetically well-defined subclasses there is considerable clinical heterogeneity. Recent advances in genomics technologies such as gene expression profiling (GEP) provide powerful tools to further characterize myeloid malignancies at the molecular level, with the goal to refine the MDS/AML classification system, incorporating as yet unknown molecular genetic and epigenetic pathomechanisms, which are likely reflected by aberrant gene expression patterns. In this study, we provide a comprehensive review on how GEP has contributed to a refined molecular taxonomy of MDS and AML with regard to diagnosis, prediction of clinical outcome, discovery of novel subclasses and identification of novel therapeutic targets and novel drugs. As many challenges remain ahead, we discuss the pitfalls of this technology and its potential including future integrative studies with other genomics technologies, which will continue to improve our understanding of malignant transformation in myeloid malignancies and thereby contribute to individualized risk-adapted treatment strategies for MDS and AML patients.

2011 - Liver Aging in Transplantation: Future Perspective on Donor-Recipient Age-Mismatch [Abstract in Rivista]
Grazi, Gl; Cescon, M; Olivieri, F; Capri, M; Lanzarini, C; Bellavista, E; Santoro, A; Martucci, M; Tenedini, Elena; Tagliafico, Enrico; Lazzarini, R; Remondini, D; Castellani, G; Ferrari, Sergio; Vasuri, F; D'Errico Grigioni, A; Procopio, A; Franceschi, C.
abstract

Research Areas:Gastroenterology & Hepatology; Surgery; Transplantation Web of Science Categories:Gastroenterology & Hepatology; Surgery; Transplantation

2010 - c-Myb supports erythropoiesis through the transactivation of KLF1 and LMO2 expression. [Articolo su rivista]
Bianchi, Elisa; Zini, Roberta; Salati, Simona; Tenedini, Elena; Norfo, Ruggiero; Tagliafico, Enrico; Manfredini, Rossella; Ferrari, Sergio
abstract

The c-Myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during differentiation. To define its role during the hematopoietic lineage commitment, we silenced c-Myb in human CD34+ hematopoietic stem/progenitor cells. Noteworthy, c-myb silencing increased the commitment capacity towards the macrophage and megakaryocyte lineages, while erythroid differentiation was impaired, as demonstrated by clonogenic assay, morphological and immunophenotypic data. Gene expression profiling and computational analysis of promoter regions of genes modulated in c-Myb-silenced CD34+ cells identified the transcription factors KLF1 and LMO2 as putative targets which can account for c-Myb knockdown effects. Indeed, Chromatin Immunoprecipitation and Luciferase reporter assay demonstrated that c-Myb binds to KLF1 and LMO2 promoters and transactivates their expression. Consistently, the retroviral vector-mediated overexpression of either KLF1 or LMO2 partially rescued the defect in erythropoiesis caused by c-Myb silencing, while only KLF1 was also able to repress the megakaryocyte differentiation enhanced in Myb-silenced CD34+ cells. Our data collectively demonstrate that c-Myb plays a pivotal role in human primary hematopoietic stem/progenitor cells lineage commitment, by enhancing erythropoiesis at the expense of megakaryocyte diffentiation. Indeed, we identified KLF1 and LMO2 transactivation as the molecular mechanism underlying Myb-driven erythroid versus megakaryocyte cell fate decision.

2010 - Cytogenetic abnormalities and clinical features in a patient cohort affected by three or more synchronous or metachronous primitive malignancies. [Articolo su rivista]
Ponti, Giovanni; Luppi, G; Giacobbi, F; Corradini, G; Temperani, Paola; Losi, Lorena; Ferrara, L; Pagano, M; Seidenari, Stefania; Tagliafico, Enrico; Torelli, Giuseppe; Conte, Pierfranco
abstract

The multiple cancers (MC) phenotype represents an intriguing entity from both the clinical and the biomolecular points of view. Multiple cancers can arise in a patient either synchronously or metachronously and are frequently detected in hereditary disorders. Here we report the clinical and cytogenetic characterization of 48 patients developing at least three malignancies outside the context of a known genetic condition and 30 control individuals. Medical and pathology reports were registered, blood was collected for cytogenetic studies, and the standard G-banding technique was used for chromosomal analysis of the lymphocyte cultures. Chromosomal analysis of the peripheral blood cultures revealed high cytogenetic instability in 83% of patients' karyotypes that displayed structural rearrangements most often involving chromosomes X, 1, 6, and 7. Peculiar telomeric associations and marker chromosomes were detected in patients with a suspected cancer family history. The MC condition can be observed over a wide clinical range, which includes either apparently sporadic cases or families with a strong history of tumors. These findings indicate that Xq, 6p, and 7q are likely to harbor genes of importance in cancer development, and the present cytogenetic mapping may be crucial for further molecular genetic investigations to recognize a predictive cytogenetic signature useful to detect patients with a high risk of multiple malignancies.

2010 - HGM 2010 Programme / Abstract [Abstract in Rivista]
Tenedini, Elena; Roncaglia, Enrica; Ferrari, Francesco; Orlandi, Claudia; Bianchi, Elisa; Bicciato, Silvio; Tagliafico, Enrico; Ferrari, Sergio
abstract

Hematopoiesis entails a series of hierarchically organized events that proceed throughout cell specification and terminates with cell differentiation. Commitment needs the transcription factors effort that, in concert with microRNAs, drives cell fate and responds to promiscuous patterns of gene expression by turning-on lineage-specific genes and repressing alternate lineage transcripts. We obtained microRNAs profiles from human CD34+ hematopoietic progenitor cells and in-vitro differentiated erythroblasts, megakaryoblasts, monoblasts and myeloblasts precursors, that we analyzed together with their gene expression profiles. The integrated analysis of microRNA-mRNA expression levels highlighted an inverse correlation between microRNAs specifically up-regulated in one single cell progeny and their putative target genes, which resulted down-regulated. Among the up-regulated lineage-enriched microRNAs, hsa-miR-299-5p emerged as having a role in controlling CD34+ progenitors fate, grown in multilineage culture conditions. Gain- and loss-of-function experiments revealed that hsa-miR-299-5p participates the regulation of hematopoietic progenitors fate, modulating megakaryocytic-granulocytic versus erythroid-monocytic differentiation.

2010 - Integrated analysis of microRNA and mRNA expression profiles in physiological myelopoieis: role of hsa-miR-299-5p in CD34+ progenitor cells commitment [Abstract in Atti di Convegno]
Tenedini, Elena; Roncaglia, Enrica; Ferrari, Francesco; Orlandi, Claudia; Bianchi, Elisa; Bicciato, Silvio; Tagliafico, Enrico; Ferrari, Sergio
abstract

Cell fate decisions in the hematopoietic system appear to be directed by an antagonistic or synergistic interplay of transcription factors that pivot immature blood progenitors for cell specification. Multipotent progenitors initially trigger a promiscuous transcriptional program and, as soon as they commit to a restricted fate, they reinforce unilineage gene expression and withdraw transcripts affiliated with alternative blood cell types. MicroRNAs appear to be especially pertinent in driving this particular behavior representing a new component of the hematopoietic gene regulatory network. In fact, the archetypal microRNA can potentially regulate hundreds of genes even if most targets contain isolated microRNA recognition sites that may be inadequate for complete gene silencing. According to Bartel’s theory, microRNAs mediated post-transcriptional control offers a more flexible and rapid way of tuning genes compared to transcriptional control (Bartel DP and Chen CZ, Nat Rev Genet 2004). These issues encouraged some investigators to explore the association of microRNAs and genes expression profiles obtained from the same cell type and advocated that microRNAs evolved to regulate gene expression programs and remove gene products unnecessary or potentially dangerous more rapidly than might occur by natural decay. Although many studies addressed the role of microRNAs during the normal myeloid differentiation process, only Georgantas and co-workers focused onto the impact of microRNAs on mRNA expression levels but limited the analyses to data obtained from human CD34+ stem/progenitor cells (Georgantas RW 3rd et al, PNAS 2007). In order to shed light onto the interplay of mRNAs and microRNAs during the normal myeloid commitment and verify that increased expression of a microRNA is skillful to modulate the levels of corresponding target mRNAs, we obtained microRNAs profiles from CD34+ hematopoietic progenitor cells (CD34 HPCs) and in-vitro differentiated precursors: erythroblasts, megakaryoblasts, monoblasts and myeloblasts (ERY, MKC, MONO and MYELO). We therefore analyzed these microRNA expression profiles together with the gene expression profiles of the same populations and observed that for the most part of the microRNAs specifically up-regulated in one single progeny an inverse correlation between microRNAs and down-regulated putative targets expression levels occurs, i.e. down-regulated genes showed an enrichment for the conserved putative targets of up-regulated microRNA. Among these microRNAs, hsa-miR-299-5p emerged as an interesting candidate to demonstrate how the integrated analysis of microRNA and mRNA expression data can help shedding light on the regulatory mechanisms governing cell differentiation. In particular, we used hsa-miR-299-5p to prove that the forced expression of a single lineage-specific microRNA is able to control the cell fate of CD34 HPCs grown in multilineage culture conditions. Clonogenic and liquid culture differentiation assays after gain- and loss-of-function experiments revealed that indeed hsa-miR-299-5p regulates hematopoietic progenitors fate modulating megakaryocytic-granulocytic versus erythroid-monocytic development.

2010 - Integrated analysis of microRNA and mRNA expression profiles in physiological myelopoiesis: role of hsa-mir-299-5p in CD34+ progenitor cells commitment [Articolo su rivista]
Tenedini, Elena; Roncaglia, Enrica; Orlandi, Claudia; Bianchi, Elisa; Bicciato, Silvio; Tagliafico, Enrico; Ferrari, Sergio; Ferrari, Francesco
abstract

Hematopoiesis entails a series of hierarchically organized events that proceed throughout cell specification and terminates with cell differentiation. Commitment needs the transcription factors' effort, which, in concert with microRNAs, drives cell fate and responds to promiscuous patterns of gene expression by turning on lineage-specific genes and repressing alternate lineage transcripts. We obtained microRNA profiles from human CD34+ hematopoietic progenitor cells and in vitro differentiated erythroblasts, megakaryoblasts, monoblasts and myeloblast precursors that we analyzed together with their gene expression profiles. The integrated analysis of microRNA-mRNA expression levels highlighted an inverse correlation between microRNAs specifically upregulated in one single-cell progeny and their putative target genes, which resulted in downregulation. Among the upregulated lineage-enriched microRNAs, hsa-miR-299-5p emerged as having a role in controlling CD34+ progenitor fate, grown in multilineage culture conditions. Gain- and loss-of-function experiments revealed that hsa-miR-299-5p participates in the regulation of hematopoietic progenitor fate, modulating megakaryocytic-granulocytic versus erythroid-monocytic differentiation

2010 - Integrated analysis of microRNA and mRNA expression profiles in physiological myelopoieis: role of hsa-mir-299-5p in CD34+ progenitor cells commitment [Abstract in Atti di Convegno]
Tenedini, Elena; Roncaglia, Enrica; Ferrari, F; Orlandi, C; Bianchi, Elisa; Bicciato, Silvio; Tagliafico, Enrico; Ferrari, Sergio
abstract

2010 - Nfix Regulates Fetal-Specific Transcription in Developing Skeletal Muscle [Articolo su rivista]
Messina, G; Biressi, S; Monteverde, S; Magli, A; Cassano, M; Perani, L; Roncaglia, Enrica; Tagliafico, Enrico; Starnes, L; Campbell, Ce; Grossi, M; Goldhamer, Dj; Gronostajski, Rm; Cossu, G.
abstract

Skeletal myogenesis, like hematopoiesis, occurs in successive developmental stages that involve dif- ferent cell populations and expression of different genes. We show here that the transcription factor nuclear factor one X (Nfix), whose expression is acti- vated by Pax7 in fetal muscle, in turn activates the transcription of fetal specific genes such as MCK and b-enolase while repressing embryonic genes such as slow myosin. In the case of the MCK promoter, Nfix forms a complex with PKC theta that binds, phosphorylates, and activates MEF2A. Pre- mature expression of Nfix activates fetal and sup- presses embryonic genes in embryonic muscle, whereas muscle-specific ablation of Nfix prevents fetal and maintains embryonic gene expression in the fetus. Therefore, Nfix acts as a transcriptional switch from embryonic to fetal myogenesis.

2010 - Polarization dictates iron handling by inflammatory and alternatively activated macrophages [Articolo su rivista]
Corna, G; Campana, L; Pignatti, E; Castiglioni, A; Tagliafico, Enrico; Bosurgi, L; Campanella, A; Brunelli, S; Manfredi, Aa; Apostoli, P; Silvestri, L; Camaschella, C; Rovere Querini, P.
abstract

BackgroundMacrophages play a key role in iron homeostasis. In peripheral tissues, they are known to polarize into classically activated (or M1) macrophages and alternatively activated (or M2) macrophages. Little is known on whether the polarization program influences the ability of macrophages to store or recycle iron and the molecular machinery involved in the processes.Design and MethodsInflammatory/M1 and alternatively activated/M2 macrophages were propagated in vitro from mouse bone-marrow precursors and polarized in the presence of recombinant interferon-γ or interleukin-4. We characterized and compared their ability to handle radioactive iron, the characteristics of the intracellular iron pools and the expression of molecules involved in internalization, storage and export of the metal. Moreover we verified the influence of iron on the relative ability of polarized macrophages to activate antigen-specific T cells.ResultsM1 macrophages have low iron regulatory protein 1 and 2 binding activity, express high levels of ferritin H, low levels of transferrin receptor 1 and internalize – albeit with low efficiency -iron only when its extracellular concentration is high. In contrast, M2 macrophages have high iron regulatory protein binding activity, express low levels of ferritin H and high levels of transferrin receptor 1. M2 macrophages have a larger intracellular labile iron pool, effectively take up and spontaneously release iron at low concentrations and have limited storage ability. Iron export correlates with the expression of ferroportin, which is higher in M2 macrophages. M1 and M2 cells activate antigen-specific, MHC class II-restricted T cells. In the absence of the metal, only M1 macrophages are effective.ConclusionsCytokines that drive macrophage polarization ultimately control iron handling, leading to the differentiation of macrophages into a subset which has a relatively sealed intracellular iron content (M1) or into a subset endowed with the ability to recycle the metal (M2).

2009 - Gene expression in grapevine cultivars in response to BOIS NOIR phytoplasma infection [Articolo su rivista]
Albertazzi, Giorgia; Milc, Justyna Anna; Caffagni, Alessandra; Francia, Enrico; Roncaglia, Enrica; F., Ferrari; Tagliafico, Enrico; Stefani, Emilio; Pecchioni, Nicola
abstract

Bois Noir phytoplasma is an emerging disease of Vitis vinifera in several regions of the world. No completely resistant grapevine cultivars are known and the physiology of disease remains still poorly understood so far. Affymetrix GeneChip® oligonucleotide arrays have been used to identify differentially expressed genes between infected and recovered samples from cv. Chardonnay and between infected and healthy samples from cv. Manzoni Bianco. In the field, cv. Manzoni showed reduced symptoms,while cv. Chardonnay was highly susceptible to the disease. Results showed that expression levels of few hundreds genes were altered in infected plants, both common and specific for each cultivar, with effects on various metabolic pathways. In cv. Chardonnay a serious inhibition of whole photosynthetic chain and photosystem I activity, Calvin-cycle enzymes transcription, lipid metabolism and phenylpropanoid biosynthesis was observed. Increasing physical barriers to limit phytoplsma spread in the plant was observed in both Chardonnay and Manzoni infected plants, with the repression of genes responsible for cell wall degradation and the induction of genes involved in cell wall reinforcement. Interestingly, specifically in cv. Manzoni the expression of a Myb transcription factor, belonging to a gene family that has a role in defense response, was induced.This is the first analysis of gene expression profiling in a grapevine-phytoplasma interaction using Affymetrix GeneChip® array. Presented data provide an interesting picture of the transcriptional response of grapevine to Bois Noir and allowed the selection of several candidate genes for future functional analysis.

2009 - Gene expression profiling of monocytes displaying herpes simplex virus 1 induced dysregulation of antifungal defences [Articolo su rivista]
Cermelli, Claudio; Orsi, Carlotta Francesca; A., Cuoghi; Ardizzoni, Andrea; Tagliafico, Enrico; Neglia, Rachele Giovanna; Peppoloni, Samuele; Blasi, Elisabetta
abstract

Recently, we showed that herpes simplex virus 1 (HSV-1)-infected monocytes have altered antifungal defences, in particular they show augmented phagocytosis of Candida albicans followed by a failure of the intracellular killing of the ingested fungi. On the basis of these functional data, comparative studies were carried out on the gene expression profile of cells infected with HSV-1 and/or C. albicans in order to investigate the molecular mechanisms underlying such virus-induced dysfunction. Affymetrix GeneChip technology was used to evaluate the cell transcription pattern, focusing on genes involved in phagocytosis, fungal adhesion, antimicrobial activity and apoptosis. The results indicated there was: (a) prevalent inhibition of opsonin-mediated phagocytosis, (b) upregulation of several pathways of antibody- and complement-independent phagocytosis, (c) inhibition of macrophage activation, (d) marked dysregulation of oxidative burst, (e) induction of apoptosis.

2009 - Microarray analysis in hippocampus of rats treated with escitalopram in the chronic escape deficit model of depression [Abstract in Rivista]
Caggia, Federica; Valensisi, Cristina; Alboni, Silvia; Benatti, Cristina; Corsini, Daniela; F., Ferrari; Tagliafico, Enrico; J., Mendlewicz; Tascedda, Fabio; Brunello, Nicoletta
abstract

Currently, the biological bases of depression and the molecular mechanisms underlying antidepressant action are not completely understood. Valuable tools to better understand the pathophysiology of this disease are behavioural models of depression eventually combined with genome-wide gene expression analysis. The Chronic Escape Deficit (CED) is a validated behavioural model of depression, based on the induction of an escape deficit after exposure of rats to an unavoidable stress. This model allows to evaluate the capacity of a treatment to revert the escape deficit. The antidepressant drugs tested in CED model need to be administered for at least 3−4 weeks in order to revert the escape deficit [1,2]. In this study, we demonstrated that already after one week of treatment with Escitalopram, a widely used SSRI, 50% of the animals responded reverting the escape deficit induced. Moreover, the other 50% of treated animals did not respond also after 3−4 weeks of treatment. Since in the CED model the behavioural alteration is induced by stress application and reverted by escitalopram treatment in only half of animals, the aims of our study were two fold: (i) to investigate transcriptional changes activated by stress; (ii) to study the different gene expression pattern involved into mechanisms of the response and not response to the pharmacological treatment. To address these issues we performed a microarray experiment in the rat hippocampus using Affymetrix GeneChip Rat Exon 1.0 ST evaluating both gene-level and exon-level expression profiling on the whole genome. Total RNA extracted from hippocampus of each animal was utilized to chip a single array using the Affymetrix protocols. 20 single arrays were utilized for data analysis and divided into five replicates for each experimental group (control, stress, stress-escitalopram responders and stress escitalopram-not responders). Using two parallel analyses (gene level and exon level) of raw data files carried out in Expression Console software using iterPLIER algorithms, we identified genes and exons that were differentially regulated in each pairwise comparison considered. The exons identified in this study were examinated for their biological association to gene ontology (GO) categories using eGOn software. Moreover, all exons differentially expressed were also uploaded into Ingenuity Pathways Analysis (Ingenuity® Systems, www.ingenuity.com) in order to identify molecular pathways and functions related to stress and escitalopram response. Our results suggest that stress may exert a negative effect on gene transcription since the largest number of genes was downregulated. Moreover from our data it seems that a different pattern of gene expression exhibits between animals that respond and that did not respond to escitalopram treatment. Functional analysis of exon dataset, arising from stress protocol and escitalopram treatment, reflects interesting different biological features. More specifically, the biological functions regard both molecular and cellular functions, such as cellular growth and proliferation, gene expression and signal transduction, as well as involvement of central neurotransmission and immune response. We believe that this pharmacogenomic approach will be helpful to understand the molecular mechanisms involved in the pathogenesis of depression as well as in the response to antidepressant drugs.

2008 - A balance between NF-Y and p53 governs the pro- and anti-apoptotic transcriptional response [Articolo su rivista]
Benatti, Paolo; Basile, Valentina; Merico, D; Fantoni, Luca Isaia; Tagliafico, Enrico; Imbriano, Carol
abstract

The transcription factor NF-Y is a trimer with histone-likesubunits that binds and activates CCAAT-containingpromoters. NF-Y controls the expressionof several key regulators of the cell cycle. In thisstudy, we examined the functional and moleculareffects of NF-YB knockdown. Cell cycle progressionis affected with a G2/M-specific depletion. This isdue to the inability of activation of G2/M-specificgenes, as evidenced by expression profiling, RT-PCRand ChIP data. Surprisingly, apoptosis is alsoobserved, with Caspase 3/7/8 cleavage. A role ofp53 and Bcl-2 family members is important. NF-YBinactivation is sufficient to functionally activate p53,in the absence of DNA damage. Failure to maintain aphysiologic level of CCAAT-dependent transcriptionof anti-apoptotic genes contributes to impairment ofBax/Bcl-2 and Bax/Bcl-XL ratios. Our data highlightthe importance of fine balancing the NF-Y-p53 duofor cell survival by (i) maintaining transcription ofanti-apoptotic genes and (ii) preventing p53 activationthat triggers the apoptotic cascade.

2008 - Microarray analysis of the chronic escape deficit model of depression: Effects of escitalopram treatment in hippocampus [Abstract in Rivista]
Caggia, Federica; Valensisi, Cristina; Alboni, Silvia; Benatti, Cristina; Corsini, Daniela; Ferrari, F; Tagliafico, Enrico; Mendlewicz, J; Brunello, Nicoletta; Tascedda, Fabio
abstract

Objective: Currently, the biological bases of depression and the molecular mechanisms underlying antidepressant action are not completely understood. Behavioural models of depression and genome-wide gene expression analysis can be relevant to better understand the pathophysiology of this disease. Chronic escape deficit is a valid and useful model of depression and is based on the induction of an escape deficit after exposure of rats to unavoidable stress. This behavioural model allows to evaluate the capacity of a treatment to revert the escape deficit. The majority of antidepressant drugs need to be administered for at least 3−4 weeks in order to revert the escape deficit. In this study, we demonstrated that only one week of treatment with Escitalopram, a widely used SSRI, is effective in the chronic escape deficit model of depression. Also, our study demonstrated that only 50% of the animals receiving ESC responded to the treatment. The mechanisms underlying the action of escitalopram are still poorly understood and the molecular targets and pathways involved remain to be identified. In order to identify the biological target involved in the response to escitalopram, we performed a microarray experiment using the chronic escape deficit model of depression after a 7 day treatment with escitalopram. Methods: Gene expression patterns in the rat hippocampus were analyzed using Affymetrix GeneChip Rat Exon 1.0 ST evaluating both gene-level and exon-level expression profiling on the whole genome. Total RNA extracted from hippocampus of each treated animal was utilized to chipping a single array using the Affymetrix protocols. 20 single arrays were utilized for data analysis and divided into five replicates for each experimental group (naive, stress, escitalopram responders and not responders). With two parallel analyses (gene level and exon level) of raw data files carried out in Expression Console software using iterPLIER algorithms, we identified various transcripts that were differentially regulated in each pairwise comparison. In order to identify biological processes and signalling networks regulated by escitalopram response, we performed a functional analysis using Ingenuity web tool. Results: Functional annotation of selected genes reflected interesting different biological features between escitalopram responders and not responders. More specifically, the biological functions regard cellular growth and proliferation, gene expression and signal transduction. Conclusion: We believe that this pharmacogenomic approach will be helpful to understand the molecular mechanisms involved in the pathogenesis of depression as well as in the response to antidepressant drugs.

2008 - Role of the transcriprion factor NF-Y in cell cycle regulation [Poster]
Benatti, Paolo; Basile, Valentina; Merico, Daniele; Fantoni, Luca Isaia; Tagliafico, Enrico; Imbriano, Carol
abstract

The CCAAT-binding factor NF-Y plays an important role in controlling the transcription of cell cycle regulated genes. NF-Y binding sites belong to the regulatory module NF-Y-CDE-CHR, which controls cell cycle-dependent transcription of G2/M genes. NF-Y functions as an heterotrimer composed by NF-YA, NF-YB and NF-YC subunits. NF-YB knock-down impairs cell cycle progression by reducing G2/M cells and inducing p53-dependent apoptosis. Failure to maintain a physiological level of anti-apoptotic genes by NF-Y transcriptional activity, contributes to the triggering of the apoptotic cascade. Increasing the levels of NF-Y expression protects cells from entering a p53-mediated apoptosis: NF-Y reverts cytochrome c release into the cytoplasm following Adriamycin treatment, preventing p53 transcriptional activation. To investigate the role of the different NF-Y subunits in controlling cell cycle progression, we have separately knocked-down the three subunits by lentiviral shRNAs. NF-YA silencing shows a more severe impairment in cell cycle progression with respect to NF-YB knock-down. p53 is a common player of the cell cycle block observed following both NF-YA and NF-YB silencing. The identification of the signaling pathways through which p53 is activated will shed light on the molecular mechanism controlling the cross-talk between NF-Y and p53.

2008 - Self-Renewing Osteoprogenitors in Bone Marrow Sinusoids Can Organize a Hematopoietic Microenvironment (DOI:10.1016/j.cell.2007.08.025) [Articolo su rivista]
Sacchetti, B.; Funari, A.; Michienzi, S.; Di Cesare, S.; Piersanti, S.; Saggio, I.; Tagliafico, E.; Ferrari, S.; Robey, P. G.; Riminucci, M.; Bianco, P.
abstract

2008 - The homeobox gene Arx is a novel positive regulator of embryonic myogenesis [Articolo su rivista]
Biressi, S; Messina, G; Collombat, P; Tagliafico, Enrico; Monteverde, S; Benedetti, L; CUSELLA DE ANGELIS, Mg; Mansouri, A; Ferrari, Stefano; Tajbakhsh, S; Broccoli, V; Cossu, G.
abstract

Skeletal muscle fibers form in overlapping, but distinct phases that depend on the generation of temporally different lineages of myogenic cells. During primary myogenesis (E10.5–E12.5 in the mouse), embryonic myoblasts fuse homotypically to generate primary fibers, whereas during later development (E14.5–E17.5), fetal myoblasts differentiate into secondary fibers. How these myogenic waves are regulated remains largely unknown. Studies have been hampered by the lack of markers which would distinguish embryonic from fetal myoblast populations. We show here that the homeobox gene Arx is strongly expressed in differentiating embryonic muscle, downstream of myogenic basic helix–loop–helix (bHLH) genes. Its expression progressively decreases during development. When overexpressed in the C2C12 myogenic cell line, Arx enhances differentiation. Accordingly, it stimulates the transcriptional activity from the Myogenin promoter and from multimerized E-boxes when co-expressed with MyoD and Mef2C in CH310T1/2. Furthermore, Arx co-immunoprecipitates with Mef2C, suggesting that it participates in the transcriptional regulatory network acting in embryonic muscle. Finally, embryonic myoblasts isolated from Arx-deficient embryos show a delayed differentiation in vivo together with an enhanced clonogenic capacity in vitro. We propose here that Arx acts as a novel positive regulator of embryonic myogenesis by synergizing with Mef2C and MyoD and by establishing an activating loop with Myogenin.

2008 - Transcriptional changes in grapevine in response to Bois Noir infection [Abstract in Atti di Convegno]
Albertazzi, Giorgia; Caffagni, Alessandra; Milc, Justyna Anna; Francia, Enrico; Roncaglia, Enrica; Ferrari, Francesco; Tagliafico, Enrico; Pecchioni, Nicola
abstract

2007 - ALTERATA REATTIVITA’ MACROFAGICA IN CORSO DI INFEZIONE MISTA DA VIRUS E FUNGHI:VALUTAZIONI FUNZIONALI, CITOFLUORIMETRICHE E DI ESPRESSIONE GENICA [Abstract in Atti di Convegno]
Cermelli, Claudio; Peppoloni, Samuele; Ardizzoni, Andrea; Tagliafico, Enrico; Blasi, Elisabetta
abstract

Base: I casi clinici di infezioni miste da funghi e virus sono in aumento, soprattutto negli ospiti immunocompromessi.Ciononostante, gli eventi biomolecolari che caratterizzano l’andamento di infezioni polimicrobiche sono tuttora poco conosciuti:scarse sono le conoscenze sulle interazioni che si verificano tra i patogeni e sui derivanti effetti, sinergistici o antagonistici.Nell’ambito del presente lavoro, abbiamo indagato sulla reattività macrofagica nel corso di infezioni miste, sostenute davirus HSV-1 e funghi opportunisti patogeni.Metodi: Sulla base di recenti studi (Cermelli C. et al., 2006), cellule THP-1 infettate per 18 ore con HSV-1 venivano esposte aCandida albicans o Cryptococcus neoformans e quindi saggiate per fagitosi, killing (CFU), marker fenotipici (citofluorimetria)ed espressione genica (microarray di RNA).Risultati: La fagocitosi di entrambi i miceti risulta significativamente aumentata nei monociti infettati da HSV-1 mentre l’attivitàantifungina è diminuita (significativa sopravvivenza e replicazione intracellulare dei due miceti). Al citofluorimetro, celluleTHP-1 infettate mostrano a) significativa downregolazione di TLR2 e TLR4, importanti molecole coinvolte nel riconoscimentodei miceti; b) ridotta espressione di CD38 e CD69, marker di attivazione cellulare; 3) aumento dei marker di apoptosi enecrosi. Il profilo di espressione genica indica un drastico calo (circa il 50%) nella quantità di geni espressi e una modulazionedell’espressione dei geni che comunque restano accesi nelle cellule infettate da HSV-1 rispetto ai controlli (> 7.500 genisovra- o sotto-espressi di almeno 3 volte). In particolare, l’analisi genica per cluster mostra uno spegnimento dei geni coinvoltinella fagocitosi opsonizzata e un aumento dell’espressione di quelli associati alla fagocitosi non opsonizzata. I geni di TLR2and TLR4 risultano downregolati così come molti geni coinvolti nel killing intracellulare.Conclusioni: Questi dati dimostrano che HSV-1 è in grado di alterare la funzione del macrofago fino a renderlo inerme o addiritturapromotore della sopravvivenza e della replicazione del fungo, sottolineando la possibilità di effetti sinergici in vivo nelcorso di infezioni miste.

2007 - Gene expression analysis of angioimmunoblastic lymphoma indicates derivation from T follicular helper cells and vascular endothelial growth factor deregulation [Articolo su rivista]
PICCALUGA P., P; Agostinelli, C; Califano, A; Carbone, A; Fantoni, L; Ferrari, Sergio; Gazzola, A; Gloghini, A; Righi, S; Rossi, M; Tagliafico, Enrico; ZINZANI P., G; Zupo, S; Baccarani, M; Pileri, S. A.
abstract

Angioimmunoblastic lymphoma (AILT) is the second most common subtype of peripheral T-cell lymphoma (PTCL) and is characterized by dismal prognosis. Thus far, only a few studies have dealt with its molecular pathogenesis. We performed gene expression profile (GEP) analysis of six AILT, six anaplastic large cell lymphomas (ALCL), 28 PTCL-unspecified (PTCL/U), and 20 samples of normal T lymphocytes (including CD4+, CD8+, and activated and resting subpopulations), aiming to (a) assess the relationship of AILT with other PTCLs, (b) establish the relationship between AILT and normal T-cell subsets, and (c) recognize the cellular programs deregulated in AILT possibly looking for novel potential therapeutic targets. First, we found that AILT and other PTCLs have rather similar GEP, possibly sharing common oncogenic pathways. Second, we found that AILTs are closer to activated CD4+, rather than to resting or CD8+ lymphocytes. Furthermore, we found that the molecular signature of follicular T helper cells was significantly overexpressed in AILT, reinforcing the idea that AILT may arise from such cellular counterpart. Finally, we identified several genes deregulated in AILT, including PDGFRA, REL, and VEGF. The expression of several molecules was then studied by immunohistochemistry on tissue microarrays containing 45 independent AILT cases. Notably, we found that the vascular endothelial growth factor (VEGF) was expressed not only by reactive cells, but also by neoplastic cells, and that nuclear factor-B (NF-B) activation is uncommon in AILT, as suggested by frequent exclusively cytoplasmic c-REL localization. Our study provides new relevant information on AILT biology and new candidates for possible therapeutic targets such as PDGFRA (platelet-derived growth factor ) and VEGF. [Cancer Res 2007;67(22):10703–10]

2007 - HEPATIC EXPRESSION OF NUCLEAR RECEPTORS AND BILIARY TRANSPOTERS IN HUMAN CHOLESTEROL GALLSTONE DISEASE. [Abstract in Rivista]
Bertolotti, Marco; Gabbi, C; Anzivino, Claudia; Tagliafico, Enrico; Carulli, Lucia; Ricchi, M; Rossi, A; Loria, Paola; Carulli, Nicola
abstract

Little is known on the molecular mechanisms underlying cholesterol cholelithiasis even if previous evidence has suggested that reduced production of bile acids might play a role. AIM of the present study was to analyze the hepatic expression of a number of genes involved in bile acid metabolism in human cholelithiasis. METHODS. Surgical liver biopsies were obtained in 11 patients with untreated cholesterol cholelithiasis and 9 gallstone-free subjects; mRNA levels of CYP7A1 and related nuclear receptors and coactivators were assayed by real-time quantitative RT-PCR. RESULTS. No differences were detected the expression of any of the genes studied, with the exception of PPAR-gamma coactivator 1 (PGC-1), a transcriptional coactivator of CYP7A1 involved in insulin sensitivity and energy balance, which was significantly (p < 0.01) less expressed in gallstone subjects. Expression of PGC-1 was linearly correlated with the bile acid receptor FXR in the population of gallstone patients (r = 0.87 on a log scale, p < 0.01). CONCLUSIONS. PGC-1 appears to play a role in the prevention of cholesterol gallstone disease in humans; the finding might suggest a link with insulin resistance conditions. This effect might take place via interaction with the bile acid receptor FXR, whose protective role in cholelithiasis has been suggested by recent evidence in animal models. PGC-1 and related genes might therefore represent molecular targets for the prevention and/or treatment of gallstone disease.

2007 - HEPATIC NUCLEAR RECEPTOR EXPRESSION AND REGULATION OF BILE ACID SYNTHESIS IN HUMANS [Abstract in Rivista]
Bertolotti, Marco; C., Gabbi; Anzivino, Claudia; M., Crestani; E., Defabiani; Tagliafico, Enrico; Carulli, Lucia; M., Ricchi; Loria, Paola; Carulli, Nicola
abstract

Bile acid synthesis plays a crucial role in cholesterol homeostasis. Recent evidence has highlighted the role of nuclear receptors in the regulation of the expression and activity of cholesterol 7alpha-hydroxylase (CYP7A1), the limiting enzyme, in cellular and animal models. Understanding the regulatory role of nuclear receptors in humans might help to define molecular targets for pharmacological intervention aimed to enhance hepatic cholesterol degradation. AIM of the present study was to analyze the expression of CYP7A1 and a number of related nuclear receptors in human liver. METHODS. Surgical liver biopsies were obtained in 40 patients; 30 of them were untreated, presenting gallbladder stones (12), non-metastatic abdominal cancer (10) or obstructive cholestasis (8); 10 subjects were receiving standard dose of CDCA (3), UDCA (5) or cholestyramine (2). mRNA levels of CYP7A1 and of the main nuclear receptors involved in its regulation (FXR, SHP, LRH-CPF-1, HNF-4, PGC-1) were assayed by real-time RT-PCR, using custom-designed primers and with sybr-green as an intercalator of double-stranded DNA. RESULTS. CYP7A1 mRNA showed a high degree of variability. No difference was detected between untreated gallstone and gallstone-free subjects regarding the expression of CYP7A1 and other genes, with the exception of PGC-1 which was significantly (p < 0.05 on a log scale) less expressed in gallstone subjects. In untreated, non-cholestatic subjects no correlation could be detected between CYP7A1 or other genes and age. Stepwise regression analysis of data from all non-cholestatic subjects, with CYP7A1 mRNA levels as the dependent variable, showed the strongest correlation with HNF-4 as the independent variable (r = 0.471 on a log scale, p < 0.05), all other genes (including SHP) bringing non-significant further contribution to the correlation. A very strong direct correlation (r = 0.880 on a log scale, p< 0.05) was detected between HNF-4 and LRH-CPF-1 expression. Finally, no difference was observed between cholestatic and non-cholestatic patients.CONCLUSIONS. Our data suggest that HNF-4 might play a relevant role in the regulation of CYP7A1 transcription in humans; on the other hand no evidence for a suppressive role of SHP, which was well documented in cellular models, was observed. As a whole, the interrelationships between the different nuclear receptors, and their physiological role, have still to be defined. Data on CYP7A1 expression support the view that post-transcriptional, and possibly post-translational levels of regulation may also play a critical role in the control of bile acid synthesis.

2007 - Intrinsic phenotypic diversity of embryonic and fetal myoblasts is revealed by genome-wide gene expression analysis on purified cells [Articolo su rivista]
S., Biressi; Tagliafico, Enrico; G., Lamorte; S., Monteverde; Tenedini, Elena; Roncaglia, Enrica; Ferrari, Stefano; Ferrari, Sergio; MG CUSELLA DE, Angelis; S., Tajbakhsh; G., Cossu
abstract

Skeletal muscle development occurs asynchronously and it has been proposed to be dependent upon the generation of temporally distinct populations of myogenic cells. This long-held hypothesis has not been tested directly due to the inability to isolate and analyze purified populations of myoblasts derived from specific stages of prenatal development. Using a mouse strain with the GFP reporter gene targeted into the Myf5 locus, a cell-sorting method was developed for isolating embryonic and fetal myoblasts. The two types of myoblasts show an intrinsic difference in fusion ability, proliferation, differentiation and response to TGFβ, TPA and BMP-4 in vitro. Microarray and quantitative PCR were used to identify differentially expressed genes both before and after differentiation, thus allowing a precise phenotypic analysis of the two populations. Embryonic and fetal myoblasts differ in the expression of a number of transcription factors and surface molecules, which may control different developmental programs. For example, only embryonic myoblasts express a Hox code along the antero-posterior axis, indicating that they possess direct positional information. Taken together, the data presented here demonstrate that embryonic and fetal myoblasts represent intrinsically different myogenic lineages and provide important information for the understanding of the molecular mechanisms governing skeletal muscle development.

2007 - Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells [Articolo su rivista]
Dellavalle, A; Sampaolesi, M; Tonlorenzi, R; Tagliafico, Enrico; Sacchetti, B; Perani, L; Innocenzi, A; Galvez, Bg; Messina, G; Morosetti, R; Li, S; Belicchi, M; Peretti, G; Chamberlain, Js; Wright, We; Torrente, Y; Ferrari, Stefano; Bianco, P; Cossu, G.
abstract

Cells derived from blood vessels of human skeletal muscle can regenerate skeletal muscle, similarly to embryonic mesoangioblasts. However, adult cells do not express endothelial markers, but instead express markers of pericytes, such as NG2 proteoglycan and alkaline phosphatase ( ALP), and can be prospectively isolated from freshly dissociated ALP(+) cells. Unlike canonical myogenic precursors ( satellite cells), pericyte-derived cells express myogenic markers only in differentiated myotubes, which they form spontaneously with high efficiency. When transplanted into severe combined immune deficient-X-linked, mouse muscular dystrophy (scid-mdx) mice, pericyte-derived cells colonize host muscle and generate numerous fibres expressing human dystrophin. Similar cells isolated from Duchenne patients, and engineered to express human mini-dystrophin, also give rise to many dystrophin-positive fibres in vivo. These data show that myogenic precursors, distinct from satellite cells, are associated with microvascular walls in the human skeletal muscle, may represent a correlate of embryonic 'mesoangioblasts' present after birth and may be a promising candidate for future cell-therapy protocols in patients.

2007 - Self-Renewing Osteoprogenitors in Bone Marrow Sinusoids Can Organize a Hematopoietic microenvironment [Articolo su rivista]
Sacchetti, B; Funari, A; Michienzi, S; DI CESARE, S; Piersanti, S; Saggio, I; Tagliafico, Enrico; Ferrari, Stefano; GEHRON ROBEY, P; Riminucci, M; Bianco, P.
abstract

The identity of cells that establish the hematopoietic microenvironment (HME) in human bone marrow (BM), and of clonogenic skeletal progenitors found in BM stroma, has long remained elusive. We show that MCAM/CD146-expressing, subendothelial cells in human BM stroma are capable of transferring, upon transplantation, the HME to heterotopic sites, coincident with the establishment of identical subendothelial cells within a miniature bone organ. Establishment of subendothelial stromal cells in developing heterotopic BM in vivo occurs via specific, dynamic interactions with developing sinusoids. Subendothelial stromal cells residing on the sinusoidal wall are major producers of Angiopoietin-1 (a pivotal molecule of the HSC “niche” involved in vascular remodeling). Our data reveal the functional relationships between establishment of the HME in vivo, establishment of skeletal progenitors in BM sinusoids, and angiogenesis.

2007 - Transcriptional profiles in melanocytes from clinically unaffected skin distinguish the neoplastic growth pattern in patients with melanoma [Articolo su rivista]
Magnoni, Cristina; Tenedini, Elena; Ferrari, Francesco; Benassi, Luisa; Bernardi, Chiara; Gualdi, Giulio; Bertazzoni, Giorgia; Roncaglia, Enrica; Fantoni, Luca Isaia; Manfredini, Rossella; Bicciato, Silvio; Ferrari, Sergio; Giannetti, Alberto; Tagliafico, Enrico
abstract

Background It is generally accepted that sunlight may contribute to the development of melanoma. Objectives To analyse gene expression of melanocytes obtained from clinically unaffected skin of patients with melanoma and healthy controls before and after exposure to ultraviolet B radiation. Methods Using GeneChip array technology, the gene expression of melanocytes obtained from the two donor groups was profiled, in order to identify transcriptional differences affecting susceptibility to melanoma. Results The data collected did not show any difference between the expression profiles of melanocytes purified from normal donors and from patients with melanoma that was able to give a statistically significant class separation. However, by means of unsupervised clustering our data could be divided into two main classes. The first class included the transcriptome profiles of melanocytes obtained from skin samples of patients with a vertical growth phase (VGP) melanoma, while the second class included the transcriptome profiles of melanocytes obtained from skin samples of patients with a radial growth phase (RGP) melanoma. Conclusions These data suggest that melanocytes in patients with VGP and RGP melanomas show significant differences in gene expression profiles, which allow us to classify patients with melanoma also from clinically unaffected skin.

2006 - Embryonic stem-derived versus somatic neural stem cells: A comparative analysis of their developmental potential and molecular phenotype [Articolo su rivista]
Colombo, Elena; Giannelli, Serena G.; Galli, Rossella; Tagliafico, Enrico; Foroni, Chiara; Tenedini, Elena; Ferrari, Sergio; Ferrari, Stefano; Corte, Giorgio; Vescovi, Angelo; Cossu, Giulio; Broccoli, Vania
abstract

Reliable procedures to induce neural commitment of totipotent undifferentiated embryonic stem (ES) cells have provided new tools for investigating the molecular mechanisms underlying cell fate choices. We extensively characterized the developmental potential of ES-induced neural cells obtained using an adaptation of the multistep induction protocol. We provided evidence that ES-derived neural proliferating cells are endowed with stem cell properties such as extensive self-renewal capacity and single-cell multipotency. In differentiating conditions, cells matured exclusively into neurons, astrocytes, and oligodendrocytes. All these features have been previously described in only somatic neural stem cells (NSCs). Therefore, we consider it more appropriate to rename our cells ES-derived NSCs. These similarities between the two NSC populations induced us to carefully compare their proliferation ability and differentiation potential. Although they were very similar in overall behavior, we scored specific differences. For instance, ES-derived NSCs proliferated at higher rate and consistently generated a higher number of neurons compared with somatic NSCs. To further investigate their relationships, we carried out a molecular analysis comparing their transcriptional profiles during proliferation. We observed a large fraction of shared expressed transcripts, including genes previously described to be critical in defining somatic NSC traits. Among the genes differently expressed, candidate genes possibly responsible for divergences between the two cell types were selected and further investigated. In particular, we showed that an enhanced MAPK (mitogen-activated protein kinase) signaling is acting in ES-induced NSCs, probably triggered by insulin-like growth factor-H. This may contribute to the high proliferation rate exhibited by these cells in culture.

2006 - Ex vivo treatment with nitric oxide increases mesoangioblast therapeutic efficacy in muscular dystrophy [Articolo su rivista]
C., Sciorati; Bg, Galvez; S., Brunelli; Tagliafico, Enrico; Ferrari, Stefano; G., Cossu; E., Clementi
abstract

Muscular dystrophies are characterized by primary wasting of skeletal muscle for which no satisfactory therapy is available. Studies in animal models have shown that stem cell-based therapies may improve the outcome of the disease, and that mesoangioblasts are promising stem cells in this respect. The efficacy of mesoangioblasts in yielding extensive muscle repair is, however, still limited. We found that mesoangioblasts treated with nitric oxide ( NO) donors and injected intra-arterially in alpha-sarcoglycan-null dystrophic mice have a significantly enhanced ability to migrate to dystrophic muscles, to resist their apoptogenic environment and engraft into them, yielding a significant recovery of alpha-sarcolgycan expression. In vitro NO-treated mesoangioblasts displayed an enhanced chemotactic response to myotubes, cytokines and growth factors generated by the dystrophic muscle. In addition, they displayed an increased ability to fuse with myotubes and differentiating myoblasts and to survive when exposed to cytotoxic stimuli similar to those present in the dystrophic muscle. All the effects of NO were cyclic GMP-dependent since they were mimicked by treatment with the membrane permeant cyclic-GMP analogue 8-bromo-cGMP and prevented by inhibiting guanylate cyclase. We conclude that NO donors exert multiple beneficial effects on mesoangioblasts that may be used to increase their efficacy in cell therapy of muscular dystrophies.

2006 - Identification of a molecular signature for leukemic promyelocytes and their normal counterparts: focus on DNA repair genes [Articolo su rivista]
I., Casorelli; Tenedini, Elena; Tagliafico, Enrico; Mf, Blasi; A., Giuliani; M., Crescenzi; E., Pelosi; U., Testa; C., Peschle; L., Mele; D., Diverio; M., Breccia; F., Lo Coco; Ferrari, Sergio; M., Bignami
abstract

Acute promyelocytic leukemia (APL) is a clonal expansion of hematopoietic precursors blocked at the promyelocytic stage. Gene expression profiles of APL cells obtained from 16 patients were compared to eight samples of CD34(+)-derived normal promyelocytes. Malignant promyelocytes showed widespread changes in transcription in comparison to their normal counterpart and 1020 differentially expressed genes were identified. Discriminating genes include transcriptional regulators (FOS, JUN and HOX genes) and genes involved in cell cycle and DNA repair. The strong upregulation in APL of some transcripts (FLT3, CD33, CD44 and HGF) was also confirmed at protein level. Interestingly, a trend toward a transcriptional repression of genes involved in different DNA repair pathways was found in APL and confirmed by real-time polymerase chain reactor (PCR) in a new set of nine APLs. Our results suggest that both inefficient base excision repair and recombinational repair might play a role in APLs development. To investigate the expression pathways underlying the development of APL occurring as a second malignancy (sAPL), we included in our study eight cases of sAPL. Although both secondary and de novo APL were characterized by a strong homogeneity in expression profiling, we identified a small set of differentially expressed genes that discriminate sAPL from de novo cases.

2006 - IDENTIFICATION OF A MOLECULAR SIGNATURE PREDICTIVE OF REFRACTORINESS IN ACUTE MYELOID LEUKEMIA [Abstract in Atti di Convegno]
Tenedini, Elena; Tagliafico, Enrico; Manfredini, Rossella; Ferrari, Francesco; Roncaglia, Enrica; Fantoni, Luca; Grande, Alexis; Parenti, Sandra; ZANOCCO MARANI, Tommaso; Gemelli, Claudia; Tatiana Vignudelli, Tatiana; Montanari, Monica; Zini, Roberta; Salati, Simona; Bianchi, Elisa; Bicciato, Silvio; Ferrari, Sergio
abstract

Acute Myeloid Leukemia (AML) blast cells are immature committed myeloid cells unable to spontaneously undergo terminal maturation, characterized by heterogeneous sensitivity to natural differentiation inducers. No data are available so far by which infer the AML’s response to differentiating therapy. Thus, we have initially profiled by GeneChip arrays the gene expression of several AML cell lines: they derived by the original blast cell populations and are still characterized by the same immunophenotype, retain a different sensitivity or resistance to All-Trans Retinoic-Acid (ATRA) and Vitamin-D3 (VD) and never undergo spontaneously terminal maturation. Here we show that differences exist by which predict the cell line differentiation fate. Next we constructed a signature able to predict resistance or sensitivity to the differentiation induction and tested it, using a TaqMan platform, for its capability to predict the in-vitro response of 28 VD or ATRA treated AML blast cell populations. Finally, by a meta-analysis of public available microarray data we demonstrated that our signature of 11 genes, among them is particularly intriguing the presence of Meis1 and ID3, that was formerly designed to identify differentiation therapy resistant populations, turned out to be a good classifier for clusters of patients known to have poor prognostic significance.

2006 - Identification of a molecular signature predictive of sensitivity to differentiation induction in acute myeloid leukemia [Articolo su rivista]
Tagliafico, Enrico; Tenedini, Elena; Manfredini, Rossella; Grande, Alexis; Ferrari, F.; Roncaglia, Enrica; Bicciato, Silvio; Zini, Roberta; Salati, Simona; Bianchi, Elisa; Gemelli, Claudia; Montanari, Monica; Vignudelli, Tatiana; ZANOCCO MARANI, Tommaso; Parenti, Sandra; Paolucci, Paolo; Martinelli, G.; Piccaluga, P. P.; Baccarani, M.; Specchia, G.; Torelli, U.; Ferrari, Sergio
abstract

Acute myeloid leukemia (AML) blasts are immature committed myeloid cells unable to spontaneously undergo terminal maturation, and characterized by heterogeneous sensitivity to natural differentiation inducers. Here, we show a molecular signature predicting the resistance or sensitivity of six myeloid cell lines to differentiation induced in vitro with retinoic acid or vitamin D. The identified signature was further validated by TaqMan assay for the prediction of response to an in vitro differentiation assay performed on 28 freshly isolated AML blast populations. The TaqMan assay successfully predicts the in vitro resistance or responsiveness of AML blasts to differentiation inducers. Furthermore, performing a meta-analysis of publicly available microarray data sets, we also show the accuracy of our prediction on known phenotypes and suggest that our signature could become useful for the identification of patients eligible for new therapeutic strategies.

2006 - Identification of new p63 targets in human keratinocytes [Articolo su rivista]
B., Testoni; S., Borrelli; Tenedini, Elena; D., Alotto; C., Castagnoli; S., Piccolo; Tagliafico, Enrico; Ferrari, Sergio; M. A., Vigano; Mantovani, Roberto
abstract

P63 is a transcription factor involved in the development of ectodermal tissues, including limb, skin and, in general, multilayered epithelia. We identified both activated and repressed genes in human keratinocytes via gene expression profiling of p63 depleted cells and validated 21 new primary targets by RT-PCR and ChIP location analysis. The p63 isoforms differentially activate or repress selected promoters. ChIPs in primary keratinocytes indicate that p63 targets are generally shared with p53, but some are p63-specific. Several growth suppressors are among repressed genes. The newly identified genes belong to pathways of growth and differentiation and are regulated in HaCaT differentiation and in stratification of human skin.

2006 - MOLECULAR REGULATION OF STEROL METABOLISM BY BILE ACIDS IN CULTURED HUMAN HEPATOCYTES [Abstract in Rivista]
Anzivino, Claudia; Bertolotti, Marco; C., Gabbi; M., Ricchi; Tagliafico, Enrico; Tenedini, Elena; Carulli, Lucia; Carubbi, Francesca; Loria, Paola; Carulli, Nicola
abstract

Disruption of hepatic cholesterol homeostasis may predispose to important clinical conditions such as cholelithiasis and atherosclerosis. The regulatory role of nuclear receptors has recently been underlined but the integration of the different metabolic pathways is largely unknown. AIM of the present study is to analyze the expression of a number of genes involved in cholesterol and bile acid metabolism in cultured human hepatocytes. METHODS. HepG2 cells were incubated with 100 micromol concentrations of different bile acids (DCA, CDCA, UDCA) for 24 hr. mRNA levels of cholesterol 7alpha-hydroxylase (CYP7A1), LDL-receptor, HMG-CoA reductase and a number of nuclear receptors and coactivators involved in sterol metabolism were assayed by real-time RT-PCR. RESULTS. A significant effect of bile acid treatment, detected by ANOVA, was shown on the expression of CYP7A1 and SHP, which were respectively reduced and increased by treatment with the hydrophobic bile acids DCA and CDCA, but not with UDCA; expression of SREBP-2 and LDL-receptor were also significantly increased by hydrophobic bile acids. CONCLUSIONS. Hydrophobic, but not hydrophilic bile acids suppress CYP7A1 expression, possibly via increased expression of SHP. Surprisingly, the same bile acids seem to enhance the expression of SREBP-2 and of genes involved in LDL uptake, mimicking a condition of cholesterol depletion. Knowledge of the subtle relationships linking bile acid and cholesterol metabolism might provide useful information for the management of cholesterol accumulation conditions.

2006 - MyoD expression restores defective myogenic differentiation of human mesoangioblasts from inclusion-body myositis muscle [Articolo su rivista]
R., Morosetti; M., Mirabella; C., Gliubizzi; A., Broccolini; L., De Angelis; Tagliafico, Enrico; M., Sampaolesi; T., Gidaro; M., Papacci; Roncaglia, Enrica; S., Rutella; Ferrari, Stefano; Pa, Tonali; E., Ricci; G., Cossu
abstract

Inflammatory myopathies (IM) are acquired diseases of skeletal muscle comprising dermatomyositis (DM), polymyositis (PM), and inclusion-body myositis (IBM). Immunosuppressive therapies, usually beneficial for DM and PM, are poorly effective in IBM. We report the isolation and characterization of mesoangioblasts, vessel-associated stem cells, from diagnostic muscle biopsies of IM. The number of cells isolated, proliferation rate and lifespan, markers expression, and ability to differentiate into smooth muscle do not differ among normal and IM mesoangioblasts. At variance with normal, DM and PM mesoangioblasts, cells isolated from IBM, fail to differentiate into skeletal myotubes. These data correlate with lack in connective tissue of IBM muscle of alkaline phosphatase (ALP)-positive cells, conversely dramatically increased in PM and DM. A myogenic inhibitory basic helix-loop-helix factor B3 is highly expressed in IBM mesoangioblasts. Indeed, silencing this gene or overexpressing MyoD rescues the myogenic defect of IBM mesoangioblasts, opening novel cell-based therapeutic strategies for this crippling disorder.

2006 - Retroviral vector integration deregulates gene expression but has no consequence on the biology and function of transplanted T cells [Articolo su rivista]
Recchia, Alessandra; C., Bonini; Z., Magnani; Urbinati, Fabrizia; D., Sartori; S., Muraro; Tagliafico, Enrico; A., Bondanza; Mtl, Stanghellini; M., Bernardi; A., Pescarollo; F., Ciceri; C., Bordignon; Mavilio, Fulvio
abstract

The use of retroviral vectors in gene therapy has raised safety concerns for the genotoxic risk associated with their uncontrolled insertion into the human genome. We have analyzed the consequences of retroviral transduction in T cells from leukemic patients treated with allogeneic stem cell transplantation and donor lymphocytes genetically modified with a suicide gene (HSV-TK). Retroviral vectors integrate preferentially within or near transcribed regions of the genome, with a preference for sequences around promoters and for genes active in T cells at the time of transduction. Quantitative transcript analysis shows that one fifth of these integrations affect the expression of nearby genes. However, transduced T cell populations maintain remarkably stable gene expression profiles, phenotype, biological functions, and immune repertoire in vivo, with no evidence of clonal selection up to 9 yr after administration. Analysis of integrated proviruses in transduced cells before and after transplantation indicates that integrations interfering with normal T cell function are more likely to lead to clonal ablation than expansion in vivo. Despite the potentially dangerous interactions with the T cell genome, retroviral integration has therefore little consequence on the safety and efficacy of T cell transplantation.

2006 - Virally mediated MafB transduction induces the monocyte commitment of human CD34+ hematopoietic stem/progenitor cells [Articolo su rivista]
Gemelli, Claudia; Montanari, Monica; Tenedini, Elena; ZANOCCO MARANI, Tommaso; Vignudelli, Tatiana; Siena, Michela; Zini, Roberta; Salati, Simona; Tagliafico, Enrico; Manfredini, Rossella; Specchia, G; Grande, Alexis; Ferrari, Sergio
abstract

Upregulation of specific transcription factors is a generally accepted mechanism to explain the commitment of hematopoietic stem cells along precise maturation lineages. Based on this premise, transduction of primary hematopoietic stem/progenitor cells with viral vectors containing the investigated transcription factors appears as a suitable experimental model to identify such regulators. Although MafB transcription factor is believed to play a role in the regulation of monocytic commitment, no demonstration is, to date, available supporting this function in normal human hematopoiesis. To address this issue, we retrovirally transduced cord blood CD34+ hematopoietic progenitors with a MafB cDNA. Immunophenotypic and morphological analysis of transduced cells demonstrated the induction of a remarkable monomacrophage differentiation. Microarray analysis confirmed these findings and disclosed the upregulation of macrophage-related transcription factors belonging to the AP-1, MAF, PPAR and MiT families. Altogether our data allow to conclude that MafB is a key regulator of human monocytopoiesis.

2005 - Correlation between differentiation plasticity and mRNA expression profiling of CD34+-derived CD14- and CD14+ human normal myeloid precursors [Articolo su rivista]
Montanari, Monica; Gemelli, Claudia; Tenedini, Elena; ZANOCCO MARANI, Tommaso; Vignudelli, T; Siena, M; Zini, Roberta; Salati, Simona; Chiossi, G; Tagliafico, Enrico; Manfredini, Rossella; Grande, Alexis; Ferrari, Sergio
abstract

In spite of their apparently restricted differentiation potentiality, hematopoietic precursors are plastic cells able to trans-differentiate from a maturation lineage to another. To better characterize this differentiation plasticity, we purified CD14- and CD14+ myeloid precursors generated by 'in vitro' culture of human CD34+ hematopoietic progenitors. Morphological analysis of the investigated cell populations indicated that, as expected, they consisted of granulocyte and monocyte precursors, respectively. Treatment with differentiation inducers revealed that CD14- cells were bipotent granulo-monocyte precursors, while CD14+ cells appeared univocally committed to a terminal macrophage maturation. Flow cytometry analysis demonstrated that the conversion of granulocyte precursors to the mono-macrophage maturation lineage occurs through a differentiation transition in which the granulocyte-related myeloperoxidase enzyme and the monocyte-specific CD14 antigen are co-expressed. Expression profiling evidenced that the observed trans-differentiation process was accompanied by a remarkable upregulation of the monocyte-related MafB transcription factor.

2005 - IDENTIFICATION OF A MOLECULAR SIGNATURE PREDICTIVE OF REFRACTORINESS IN ACUTE MYELOID LEUKEMIA [Abstract in Atti di Convegno]
Tagliafico, Enrico; Tenedini, Elena; Manfredini, Rossella; Ferrari, Sergio; Roncaglia, Enrica; Fantoni, Luca; Grande, Alexis; Parenti, Sandra; ZANOCCO MARANI, Tommaso; Gemelli, Claudia; Vignudelli, Tatiana; Montanari, Monica; Zini, Roberta; Salati, Simona; Bianchi, Elisa; Bicciato, Silvio; Specchia, Giorgina; Martinelli, Giovanni; Baccarani, Michele; Piccaluga, Pier Paolo; Torelli, Umberto; Ferrari, Sergio
abstract

Acute Myeloid Leukemia (AML) blast cells are immature committed myeloid cells unable to spontaneously undergo terminal maturation, characterized by heterogeneous sensitivity to natural differentiation inducers. No data are available so far by which infer the AML’s response to differentiating therapy. Thus, we have initially profiled by GeneChip arrays the gene expression of several AML cell lines: they derived by the original blast cell populations and are still characterized by the same immunophenotype, retain a different sensitivity or resistance to ATRA and VD and never undergo spontaneously terminal maturation. Here we show that differences exist by which predict the cell line differentiation fate. Next we constructed a signature able to predict resistance or sensitivity to the differentiation induction and tested it, using a TaqMan platform, for its capability to predict the in-vitro response of 28 VD or ATRA treated AML blast cell populations. Finally, by a meta-analysis of public available microarray data we demonstrated that our signature, that was formerly designed to identify differentiation therapy resistant populations, turned out to be a good classifier for clusters of patients with citogenetically and molecularly defined lesions that are known to have poor prognostic significance.

2005 - The kinetic status of hematopoietic stem cell subpopulations underlies a differential expression of genes involved in self-renewal, commitment, and engraftment. [Articolo su rivista]
Manfredini, Rossella; Zini, Roberta; Salati, Simona; Siena, M; Tenedini, Elena; Tagliafico, Enrico; Montanari, Monica; ZANOCCO MARANI, Tommaso; Gemelli, Claudia; Vignudelli, T; Grande, A; Fogli, M; Rossi, L; Fagioli, Me; Catani, L; Lemoli, Rm; Ferrari, Sergio
abstract

The gene expression profile of CD34(-) hematopoietic stem cells (HSCs) and the correlations with their biological properties are still poorly understood. To address this issue, we used the DNA microarray technology to compare the expression profiles of different peripheral blood hemopoietic stem/progenitor cell subsets, lineage-negative (Lin(-)) CD34(-), Lin(-)CD34(+), and Lin(+)CD34(+) cells. The analysis of gene categories differentially expressed shows that the expression of CD34 is associated with cell cycle entry and metabolic activation, such as DNA, RNA, and protein synthesis. Moreover, the significant upregulation in CD34(-) cells of pathways inhibiting HSC proliferation induces a strong differential expression of cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, and growth-arrest genes. According to the expression of their receptors and transducers, interleukin (IL)-10 and IL-17 showed an inhibitory effect on the clonogenic activity of CD34(-) cells. Conversely, CD34(+) cells were sensitive to the mitogenic stimulus of thrombopoietin. Furthermore, CD34(-) cells express preferentially genes related to neural, epithelial, and muscle differentiation. The analysis of transcription factor expression shows that the CD34 induction results in the upregulation of genes related to self-renewal and lineage commitment. The preferential expression in CD34(+) cells of genes supporting the HSC mobilization and homing to the bone marrow, such as chemokine receptors and integrins, gives the molecular basis for the higher engraftment capacity of CD34(+) cells. Thus, the different kinetic status of CD34(-) and CD34(+) cells, detailed by molecular and functional analysis, significantly influences their biological behavior

2004 - Gene expression profiling of normal and malignant CD34-derived megakaryocytic cells [Articolo su rivista]
Tenedini, Elena; Fagioli, Me; Vianelli, N; Tazzari, Pl; Ricci, F; Tagliafico, Enrico; Ricci, P; Gugliotta, L; Martinelli, G; Tura, S; Baccarani, M; Ferrari, Sergio; Catani, L.
abstract

Gene expression profiles of bone marrow (BM) CD34-derived megakaryocytic cells (MKs) were compared in patients with essential thrombocythemia (ET) and healthy subjects using oligonucleotide microarray analysis to identify differentially expressed genes and disease-specific transcripts. We found that proapoptotic genes such as BAX, BNIP3, and BNIP3L were down-regulated in ET MKs together with genes that are components of the mitochondrial permeability transition pore complex, a system with a pivotal role in apoptosis. Conversely, antiapoptotic genes such as IGF1-R and CFLAR were up-regulated in the malignant cells, as was the SDF1 gene, which favors cell survival. On the basis of the array results, we characterized apoptosis of normal and ET MKs by time-course evaluation of annexin-V and sub-G1 peak DNA stainings of immature and mature MKs after culture in serum-free medium with an optimal thrombopoietin concentration, and annexin-V-positive MKs only, with decreasing thrombopoietin concentrations. ET MKs were more resistant to apoptosis than their normal counterparts. We conclude that imbalance between proliferation and apoptosis seems to be an important step in malignant ET megakaryocytopoiesis.

2004 - Msx2 and Necdin regulate smooth muscle determination in mesoangioblasts. [Articolo su rivista]
Brunelli, S.; Tagliafico, Enrico; De Angelis, F. G.; Tonlorenzi, R.; Baesso, S.; Ferrari, Sergio; Niinobe, M.; Yoshikawa, K.; Schwartz, R. J.; Bozzoni, I.; Ferrari, Stefano; Cossu, G.
abstract

Little is known about the molecular mechanism underlying specification and differentiation of smooth muscle (SM), and this is, at least in part, because of the few cellular systems available to study the acquisition of a SM phenotype in vitro. Mesoangioblasts are vessel-derived stem cells that can be induced to differentiate into different cell types of the mesoderm, including SM. We performed a DNA microarray analysis of a mesoangioblast clone that spontaneously expresses an immature SM phenotype and compared it with a sister clone mainly composed of undifferentiated progenitor cells. This study allowed us to define a gene expression profile for “stem” cells versus smooth muscle cells (SMCs) in the absence of differentiation inducers such as transforming growth factor . Two transcription factors, msx2 and necdin, are expressed at least 100 times more in SMCs than in stem cells, are coexpressed in all SMCs and tissues, are induced by transforming growth factor , and, when coexpressed, induce a number of SM markers in mesoangioblast, fibroblast, and endothelial cell lines. Conversely, their downregulation through RNA interference results in a decreased expression of SM markers. These data support the hypothesis that Msx2 and necdin act as master genes regulating SM differentiation in at least a subset of SMCs.

2004 - TGFβ/BMP activate the smooth muscle/bone differentiation programs in mesoangioblasts [Articolo su rivista]
Tagliafico, Enrico; S., Brunelli; A., Bergamaschi; L., De Angelis; R., Scardigli; D., Galli; Battini, Renata; P., Bianco; Ferrari, Sergio; G., Cossu; Ferrari, Stefano
abstract

Mesoangioblasts are vessel-derived stem cells that can be induced to differentiate into different cell types of the mesoderm such as muscle and bone. The gene expression profile of four clonal derived lines of mesoangioblasts was determined by DNA micro-array analysis: it was similar in the four lines but different from 10T1/2 embryonic fibroblasts, used as comparison. Many known genes expressed by mesoangioblasts belong to response pathways to developmental signalling molecules, such as Writ or TGFbeta/BMP Interestingly, mesoangioblasts express receptors of the TGFbeta/BMP family and several Smads and, accordingly, differentiate very efficiently into smooth muscle cells in response to TGFbeta and into osteoblasts in response to BMP In addition, insulin signalling promotes adipogenic differentiation, possibly through the activation of IGF-R. Several Writs and Frizzled, Dishevelled and Tcfs are expressed, suggesting the existence of an autocrine loop for proliferation and indeed, forced expression of Frzb-1 inhibits cell division. Mesoangioblasts also express many neuro-ectodermal genes and yet undergo only abortive neurogenesis, evens after forced expression of neurogenin 1 or 2, MASH or NeuroD. Finally, mesoangioblasts express several pro-inflammatory genes, cytokines and cytokine receptors, which may explain their ability to be recruited by tissue inflammation. Our data define a unique phenotype for mesoangioblasts, explain several of their biological features and set the basis for future functional studies on the role of these cells in tissue histogenesis and repair.

2003 - Development of an IL-6 antagonist peptide that induces apoptosis in IL-6 dependent 7TD1 cells. [Articolo su rivista]
Manfredini, Rossella; Tenedini, Elena; M., Siena; Tagliafico, Enrico; Montanari, Monica; Grande, Alexis; ZANOCCO MARANI, Tommaso; C., Poligani; Zini, Roberta; A., Bergamaschi; DE RIENZO, Francesca; DE BENEDETTI, Pier Giuseppe; Menziani, Maria Cristina; Ferrari, Sergio
abstract

Interleukin-6 (IL-6) is a pleiotropic cytokine involved in the regulation of proliferation and differentiation of hematopoietic cells and in the pathogenesis of many diseases, including multiple myeloma. This study pursues a way to interfere with IL-6 pathway in an attempt to modulate its biological activity. Here we describe the rational design and biological evaluation of peptides able to antagonize the murine IL-6 activity by interfering with IL-6 Receptor alpha in 7TD1 cells, a IL-6-dependent B-cell line. Of the peptide tested, only Guess 4a is capable of interfering with IL-6 transducing pathway, therefore inducing growth arrest and apoptosis of 7TD1 cells.

2003 - Requirement of the coiled-coil domains of p92(c-Fes) for nuclear localization in myeloid cells upon induction of differentiation [Articolo su rivista]
Tagliafico, Enrico; M., Siena; ZANOCCO MARANI, Tommaso; Manfredini, Rossella; Tenedini, Elena; M., Montanari; Grande, Alexis; Ferrari, Sergio
abstract

The nonreceptor tyrosine kinase Fes is implicated in myeloid cells differentiation. It has been observed that its localization can be cytoplasmic, perinuclear, or nuclear. To further characterize this point, we studied Fes subcellular localization in myeloid cell lines (HL60 and K562) and in COS1 cells. Fes was observed in both the nucleus and the cytoplasm of HL60, K562 cells over-expressing Fes and only in the cytoplasm of COS1 cells, suggesting that nuclear localization is cell context dependent. Moreover, in myeloid cells, the treatment with differentiation-inducing agents such as retinoic acid, phorbol esters and vitamin D, is followed by an increase of the oligomeric form of Fes in the nucleus. In fact, oligomerization seems to be necessary for translocation to occur, since Fes mutants missing the coiled-coil domains are not able to form oligomers and fail to localize in the nucleus. The active form of Fes is tyrosine phosphorylated; however, phosphorylation is not required for Fes to localize in the nucleus, since tyrosine kinase inhibitors do not block the translocation process.

2002 - Gene expression Profile of Human Myeloid Cells [Abstract in Atti di Convegno]
Siena, M; Manfredini, R; Bergamaschi, A; Tenedini, Elena; Tagliafico, Enrico; ZANOCCO MARANI, Tommaso; Montanari, Monica; Gemelli, Claudia; Grande, Alexis; Ferrari, Sergio
abstract

Array technologies have made it possible to monitor simultaneously the expression pattern of thousands of genes. Working on normal human hempoietic stem cells it is possible to evaluate their gene expression profile, changes in gene expression occurring in their early commitment phase and to compare the gene expression profiling with normallly differentiated myeloid cells, i.e. granulocytes and monocytes.

2002 - Gene Expression profile of Vitamin D3 treated HL60 cells shows a phenotypic but not a complete functional conversion to monocytes [Abstract in Atti di Convegno]
Tenedini, Elena; Bergamaschi, A; Manfredini, Rossella; Percudani, R; Siena, M; ZANOCCO MARANI, Tommaso; Grande, Alexis; Montanari, Monica; Gemelli, Claudia; Torelli, U; Ferrari, Sergio; Tagliafico, Enrico
abstract

Acute Myeloid leukemia blast cells are characterized by their inability to proceed spontaneously toward terminal differentiation. To tackle this problem we have studied the changes occurring in the gene expression profile during the differentiation of HL60 cells treated with VD using the Affymetrix GeneChip technology and we have compared the molecular phenotype of VD induced cells to that of CD14+ pheripheral monocytes.

2002 - Gene expression profile of vitamin D3 treated HL60 cells shows an incomplete molecular phenotypic conversion to monocytes [Articolo su rivista]
Tagliafico, Enrico; Tenedini, Elena; A., Bergamaschi; ZANOCCO MARANI, Tommaso; R., Percudani; M., Siena; Manfredini, Rossella; Grande, Alexis; M., Montanari; C., Gemelli; U., Torelli; Ferrari, Sergio
abstract

By high density oligonucleotide microarrays we have studied the expression profile of proliferating and VD treated HL60 cells and the molecular phenotype of VD monocytes and that of CD14+ peripheral monocytes has been compared. The results indicate that important changes in functional categories of the differentially expressed genes underlie the differentiation transition from myeloblasts to monocytes. This differential gene expression pattern leads to an increased expression of mRNAs involved in surface and external activities since many of the VD induced genes belong to ligand binding, receptors, cell surface antigens, defense/immunity and adhesion molecules functional categories. results also indicate that the molecular phenotypes of monocytes and VD induced cells diverge for a small but significant set of defense related genes. Particularly, class II MHC genes are not expressed in these cells. Furthermore, the high levels of expression of these genes induced by serum treatment of monocytes are decreased by VD.

2002 - Physiological levels of 1 alpha, 25 di-hydroxyvitamin D3 induce the monocytic commitment of CD34+ hematopoietic progenitors [Articolo su rivista]
Grande, Alexis; M., Montanari; Tagliafico, Enrico; Manfredini, Rossella; ZANOCCO MARANI, Tommaso; M., Siena; Tenedini, Elena; A., Gallinelli; Ferrari, Sergio
abstract

Although supraphysiological levels of 1alpha, 25 dihydroxyvitamin D3 (VD) have been demonstrated extensively to induce the monomacrophagic differentiation of leukemic myelo- and monoblasts, little is known about the role that physiological levels of this vitamin could play in the regulation of normal hematopoiesis. To clarify this issue, we adopted a liquid-culture model in which cord blood CD34+ hematopoietic progenitors, induced to differentiate in the presence of different combinations of cytokines, were exposed to VD at various concentrations and stimulation modalities. The data obtained show that physiological levels of VD promote a differentiation of CD34+ hematopoietic progenitors characterized by the induction of all the monomacrophagic immunophenotypic and morphological markers. This effect is not only exerted at the terminal maturation but also at the commitment level, as demonstrated by the decrease of highly undifferentiated CD34+CD38-hematopoietic stem cells, the down-regulation of CD34 antigen, and the increase of monocyte-committed progenitors. Molecular analysis suggests that the VD genomic signaling pathway underlies the described differentiation effects.

2002 - Requirement of the coiled coil domains of p92c-Fes for nuclear translocation in myeloid cells upon induction of differentiation [Abstract in Atti di Convegno]
ZANOCCO MARANI, Tommaso; Siena, M; Tagliafico, Enrico; Manfredini, Rossella; Tenedini, Elena; Montanari, Monica; Grande, Alexis; Gemelli, Claudia; Ferrari, Sergio
abstract

The non-receptor tyrosine kinase Fes is expressed in hematopoietic progenitors, differentiated myeloid cells and other cell types, such as vascular endothelial cells and neuroblastoma cell lines. To further clarify this point we performed confocal microscopy and western blot experiments on myeloid cell lines and COS1 cells. In myeloid cells the treatment with differentiation inducing agents such as ATRA, PMA and VD is followed by an increase of Fes abundance in the nuclear compartment. The active form of Fes is phosphorylated on residue 713 and is present into the nucleus while treated cells with the tyrosine kinase inhibitor Genistein clearly showed that phosphorylation is not a required event in order to Fes to translocate to the nucleus.

2001 - A functionally active RAR alpha nuclear receptor is expressed in retinoic acid non responsive early myeloblastic cell lines [Articolo su rivista]
Grande, Alexis; M., Montanari; Manfredini, Rossella; Tagliafico, Enrico; ZANOCCO MARANI, Tommaso; F., Trevisan; Ligabue, Giulia; M., Siena; Ferrari, Stefano; Ferrari, Sergio
abstract

Although ail-trans retinoic acid (ATRA) can restore the differentiation capacity of leukemic promyelocytes, early leukemic myeloblasts are conversely not responsive to ATRA induced granulocytic differentiation. To assess whether this resistance to ATRA is related to an impaired function of the Retinoic Acid Receptor alpha (RAR alpha), we performed an analysis of RAR alpha expression and transactivation activity, in several myeloid leukemic cell lines, representative of different types of spontaneous acute myeloid leukemias. Our results indicate that a functionally active RAR alpha nuclear receptor is expressed in all the analyzed cell lines, regardless of their differentiation capacity following exposure to ATRA. The observation that ATRA treatment is able to induce the expression of retinoic acid target genes, in late- but not in early-myetoblastic leukemic cells, raises the possibility that the differentiation block of these cells is achieved through a chromatin mediated mechanism. Acetylation is apparently not involved in this process, since the histone deacetylase inhibitor trichostatin A, is not able to restore the differentiation capacity of early leukemic myeloblasts. Further investigation is needed to clarify whether myeloid transcription factors, distinct to RAR alpha, play a role in the resistance of these cells to ATRA treatment.

2001 - Suppression of bile acid synthesis, but not of hepatic cholesterol 7alpha-hydroxylase expression, by obstructive cholestasis in humans [Articolo su rivista]
Bertolotti, Marco; Carulli, Lucia; Concari, M; Martella, P; Loria, Paola; Tagliafico, Enrico; Ferrari, S; Del Puppo, M; Amati, B; De Fabiani, E; Crestani, M; Amorotti, Claudio; Manenti, A; Carubbi, Francesca; Pinetti, A; Carulli, N.
abstract

Regulation of bile acid synthesis, a key determinant of cholesterol homeostasis, is still incompletely understood. To elucidate the feedback control exerted on bile acid biosynthesis in humans with obstructive cholestasis, 16 patients with bile duct obstruction were studied. In vivo 7alpha-hydroxylation, reflecting bile acid synthesis, was assayed in 13 of them by tritium release analysis. Serum 27-hydroxycholesterol was determined by gas chromatography-mass spectrometry. In a subgroup, hepatic cholesterol 7alpha-hydroxylase mRNA was assayed by real-time polymerase chain reaction (PCR), enzyme activity was determined by isotope incorporation, and microsomal cholesterol content was assayed by gas chromatography-mass spectrometry. Age-matched control subjects were studied in parallel. Hydroxylation rates were lower in cholestatic patients (108 +/- 33 mg of cholesterol per day, mean +/- SEM; controls: 297 +/- 40 mg/d; P <.01). The reduction was proportional to the severity of cholestasis, and synthetic rates were normalized in 4 subjects restudied after resolution of biliary obstruction. Consistent findings were obtained by analysis of serum 7alpha-hydroxycholesterol levels. On the other hand, hepatic cholesterol 7alpha-hydroxylase mRNA, microsomal enzyme activity, and cholesterol content tended to be increased in cholestasis. Finally, serum 27-hydroxycholesterol levels were slightly reduced in cholestatic subjects and were not related with the severity of the disease. Suppression of in vivo bile acid synthesis with no corresponding reduction in tissue 7alpha-hydroxylase expression and activity is consistent with nontranscriptional, posttranslational levels of regulation; these may play a role in the feedback control of bile acid synthesis in particular conditions. Alteration of the alternate biosynthetic pathway seems unlikely according to the present data.

2000 - Suprression of in vivo bile acid synthesis, but not of in vitro cholesterol 7 alpha-hydroxylase expression, by biliary obstruction in humans [Articolo su rivista]
Bertolotti, Marco; Carulli, Lucia; Concari, M; Martella, P; Loria, Paola; Tagliafico, Enrico; De Fabiani, E; Amorotti, Claudio; Carulli, Nicola
abstract

1999 - Induction of a functional vitamin D receptor in all-trans-retinoic acid- induced monocytic differentiation of M2-type leukemic blast cells [Articolo su rivista]
Manfredini, R.; Trevisan, F.; Grande, A.; Tagliafico, E.; Montanari, M.; Lemoli, R.; Visani, G.; Tura, S.; Ferrari, S.; Ferrari, S.
abstract

Different types of acute myeloid leukemia blast cells were induced to differentiate in vitro with all-trans-retinoic acid (ATRA) and vitamin D3 (VD). M0/M1 leukemic cells are not sensitive to differentiating agents, whereas M3 leukemic cells are induced to undergo granulocytic differentiation after ATRA treatment but are not sensitive to VD. M2 leukemic blast cells behave differently because they undergo monocytic differentiation with both the differentiation inducers. To gain some insight into the maturation of M2- type leukemic cells, we studied the molecular mechanisms underlying monocytic differentiation induced by ATRA and VD in spontaneous M2 blast cells as well as in Kasumi-1 cells (an acute myeloid leukemia M2-type cell line). Our results indicate that ATRA as well as VD efficiently increases the nuclear abundance of VD receptor (VDR) and promotes monocytic differentiation. VDR is functionally active in ATRA-treated Kasumi-1 cells because it efficiently heterodimerizes with retinoid X receptor, binds to a DR3-type vitamin D- responsive element, and activates the transcription of a vitamin D-responsive element-regulated reporter gene. Consistent with these findings, VD- responsive genes are induced by ATRA treatment of Kasumi-1 cells, suggesting that the genetic program underlying monocytic differentiation is activated. The molecular mechanism by which ATRA increases the nuclear abundance of a functional VDR is still unknown, but our data clearly indicate that the M2 leukemic cell context is only permissive of monocytic differentiation.

1999 - Regulation of ob gene expression: evidence for epinephrine-induced suppression in human obesity [Articolo su rivista]
Carulli, Lucia; Ferrari, S; Bertolini, M; Tagliafico, Enrico; DEL RIO, Graziano
abstract

Leptin acts as satiety factor and increases energy expenditure. Studies conducted on animals and in vitro on adipocytes culture have shown that infusion of catecholamines leads to a significant reduction of ob gene expression; it appears of interest to evaluate the in vivo effects of adrenergic activation on the expression of the ob gene in humans. We studied ob gene expression in adipose tissue samples from 13 obese subjects before and after epinephrine (25 ng/min x kg ideal body weight for 3 h) and 6 obese patients during saline infusion. Hormonal infusion led to a significant increase in epinephrine plasma levels (from 27 +/- 4 to 339 +/- 75 pg/mL; P < 0.001), plasma free fatty acids (from 0.73 +/- 0.05 to 0.98 +/- 0.07; P < 0.05), heart rate (13.5 +/- 3.1 beats/min; F = 2.9; P < 0.03), and systolic blood pressure (F = 2.7; P < 0.05), whereas diastolic blood pressure did not show significant variation. Plasma leptin levels decreased by the end of the infusion (from 63 +/- 13 to 49 +/- 11 ng/mL; P < 0.05), and ob messenger ribonucleic acid levels were significantly reduced (decrease amounting to 47 +/- 5% of basal values). Our study shows that adrenergic activation contributes to regulate ob messenger ribonucleic acid levels in humans. The interaction between epinephrine and leptin may operate during metabolic and psychological stress to regulate energy expenditure and food intake.

1998 - Antisense inhibition of Bax mRNA increases survival of terminally differentiated HL60 cells [Articolo su rivista]
Manfredini, R.; Capobianco, M. L.; Trevisan, F.; Rauzi, F.; Barbieri, D.; Citro, G.; Tagliafico, E.; Ferrari, S.
abstract

Cell sensitivity to programmed cell death is primarily modulated by members of the Bcl-2 family, as the balance of homodimer or heterodimer formation between proapoptotic and antiapoptotic members defines apoptosis susceptibility in the great majority of cellular contexts. It is, therefore, important to clarify if the Bax protein is limiting for activation of the genetic program of programmed cell death or can be complemented by different Bcl-2 family members, such as Bah or Bad. To gain some insight into the role of Bax in the molecular mechanisms of apoptosis of myeloid cells, we inhibited this gene in all-trans-retinoic acid (ATRA)-treated HL60 cells using the methodology of antisense oligodeoxynucleotides (AS-ODN). Our results indicate that Bax inhibition has no effect on the proliferation and differentiation capacity of HL60 cells. Instead, the survival rate of terminally differentiated Bax-inactivated HL60 (Bax((-)) HL60) cells is almost three times higher in respect to control cultures, indicating that in mature granulocytes Bax is not efficiently complemented by others members of the Bcl-2 family proteins.

1997 - Antisense inhibition of c-fes proto-oncogene blocks PMA-induced macrophage differentiation in HL60 and in FDC-P1/MAC-11 cells [Articolo su rivista]
Manfredini, Rossella; R., Balestri; Tagliafico, Enrico; F., Trevisan; Grande, Alexis; M., Pizzanelli; D., Barbieri; P., Zucchini; G., Citro; C., Franceschi; Ferrari, Sergio
abstract

To gain some insight into the role of c-fes in macrophage differentiation, we have analyzed the ability of HL60 leukemic promyelocytic cells and FDC-P1/MAC-11 murine myeloid precursor cells to differentiate in response to phorbol esters after inhibition of c-fes function. Fes inactivation has been obtained by using oligodeoxynucleotides (ODN) complementary to the 5´ region of c-fes mRNA and to 5´ splice junctions of c-fes primary transcript. After 5 days (d) in culture, in several separate experiments performed with different ODN preparations, a complete inhibition of c-fes expression was observed in HL60 and in FDC-P1/MAC-11 cells. No perturbation of cell growth was evident in our experimental conditions in both cell lines after c-fes inhibition. Furthermore, in HL60 cells lacking c-fes product, an almost complete downregulation of the alpha 4 beta 1 fibronectin receptor occurred. However, in both cell lines, the induction of macrophage differentiation by phorbol esters resulted in an almost complete maturation arrest as evaluated by morphological, cytochemical, immunological criteria, and by the cytofluorimetric cell cycle analysis. A loss of the adhesion capacity of both myeloid cell lines, when compared to terminally differentated macrophages, was also observed. These results suggest that HL60 and FDC-P1/MAC-11 cells, when treated with phorbol 12-myristate 13-acetate, require c-fes protein expression to activate the genetic program underlying macrophage differentiation.

1997 - Correlations between the FAB phenotype and differentiation potential in leukemic myelopoiesis [Relazione in Atti di Convegno]
Manfredini, R.; Trevisan, F.; Grande, A.; Tagliafico, E.; Montanari, M.; Pignatti, E.; Ligabue, G.; Barbieri, D.; Ferrari, Se.
abstract

1997 - Presence of a functional vitamin D receptor does not correlate with vitamin D3 phenotypic effects in myeloid differentiation [Articolo su rivista]
Grande, Alexis; Manfredini, Rossella; M., Pizzanelli; Tagliafico, Enrico; R., Balestri; F., Trevisan; D., Barbieri; C., Franceschi; Battini, Renata; Ferrari, Stefano; Ferrari, Sergio
abstract

Although VDR is expressed in all the acute myeloid leukemia cell populations studied, most of these leukemias do not exibit any phenotypic response when exposed to VD. To determine whether VD resistance is related to an altered VDR function,we performed an analysis of VDR expression, phosphorylation, DNA binding capacity and transactivation activity in several leukemic myeloid cell lines arrested at different levels of maturation. Our results indicate that VD induces a clear phenotypic effect, i.e. terminal monocytic differentiation, only in leukemic cells of M2/M3 (intermediate myeloblasts) and M5 (monoblasts) types but not in erythroid precursor cells, early leukemic myeloblasts (M0/M1 type) and promyelocytes (M3 type). VDR expression and function are evident in all the nuclear extracts obtained from the different myeloid cell lines after 12 h of VD treatment, but VD activation of monocytic differentiation is limited to a narrow differentiation window characterized by the M2 type myeloid cellular context.

1996 - Bile acid structure and regulation of biliary protein secretion and composition in man [Articolo su rivista]
M., Bozzoli; Loria, Paola; Carubbi, Francesca; M., Concari; M. E., Guicciardi; Bertolotti, Marco; Tagliafico, Enrico; Carulli, Nicola
abstract

To evaluate the effect of bile acid structure on total protein secretion and composition, 4 different bile acids, deoxycholic acid, chenodeoxycholic acid, cholic acid and ursodeoxycholic acid, in order of decreasing hydrophobicity, were infused intraduodenally in 5 cholecystectomized T-tube patients with interrupted enterohepatic circulation, Concentration and composition of biliary proteins were evaluated before and after acute substitution of the endogenous bile acid pool with each bile acid, Total biliary protein concentration and secretion increased progressively with increasing hydrophobicity of the infused bile acid and was highest for deoxycholic acid, followed by chenodeoxycholic acid, cholic acid and ursodeoxycholic acid, A significant increase in the 120 and 150 kDa protein bands on gel-electrophoresis was found after infusion with the more hydrophobic bile acids (deoxycholic acid and chenodeoxycholic acid), Quantitative and qualitative changes in biliary proteins, associated with the administration of bile acids that have different physico-chemical structures, may be relevant to the process of cholesterol crystal nucleation and the:pathogenesis of gallstone formation.

1995 - All-trans-retinoic acid induces simultaneously granulocytic differentiation and expression of inflammatory cytokines in HL-60 cells [Articolo su rivista]
Grande, Alexis; Manfredini, Rossella; Tagliafico, Enrico; R., Balestri; M., Pizzanelli; S., Papa; P., Zucchini; L., Bonsi; G., Bagnara; U., Torelli; Ferrari, Sergio
abstract

All-trans-retinoic acid (ATRA) can induce granulocytic differentiation both in vitro and in vivo, and its activity is mediated by the retinoic acid receptor-alpha (RAR-alpha). In the present study, we evaluated the ability of this inducer in HL-60 cells, to stimulate simultaneously granulocytic differentiation and the expression of the cytokines interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-3, IL-6, tumor necrosis factor-a: (TNF-a), and stem cell factor (SCF). The level of expression of these cytokines in ATRA-treated HL-60 cells was compared with that observed in normal and lipopolysaccharide (LPS)-heated peripheral granulocytes. The results indicate that the expression of these cytokines is enhanced during differentiation so that the pattern observed in ATRA-treated HL-60 cells is close to that of LPS-stimulated normal granulocytes. In addition, tetra phorbol acetate (TPA)-treated HL-60 cells express several of the above listed cytokines. It is concluded that ATRA not only induces granulocytic differentiation of HL-60 cells, but also activation of these terminally differentiated cells. The activating cytokine expression in these cells appears related to the progress of the differentiation program induced by ATRA since normal granulocytes do not respond to this inducer by activation of the expression of these genes. Furthermore, the cytokine activation is a specific effect of ATRA, since DMSO does not have any stimulatory effect.

1995 - ROLE OF C-FES PROTOONCOGENE IN MYELOID DIFFERENTIATION [Articolo su rivista]
FERRARI, Sergio; GRANDE, Alexis; MANFREDINI, Rossella; TAGLIAFICO, Enrico
abstract

The main purpose of this report is to provide a review of the present knowledge on the structure, function, and possible regulatory role of c-fes in the genetic programs underlying the proliferation and differentiation of hematopoietic myeloid cells. Fes encodes a non-receptor tyrosine kinase that is highly expressed in immature and differentiated cells of the granulocytic and mono-macrophagic lineages. It is therefore possible that c-fes is involved in the signal transduction of myeloid cell differentiation, even if the specific substrates phosphorylated by this protooncogene are only poorly characterised. Several experimental models have been established to evaluate the role of c-fes in myeloid differentiation, in particular: the differentiation capacity of HL60 cells lacking the p92(c-fes) protein, the transfection of c-fes gene into K562 cells and transgenic animals overexpressing c-fes, The results obtained point to the importance of c-fes in myeloid cells, since it appears to be involved in granulocytic maturation as an antiapoptotic gene, and in macrophagic maturation as a regulatory gene.

1994 - Antiapoptotic effect of c-fes protooncogene during granulocytic differentiation [Articolo su rivista]
Ferrari, Sergio; Manfredini, Rossella; Tagliafico, Enrico; Grande, Alexis; D., Barbieri; R., Balestri; M., Pizzanelli; Zucchini, Patrizia; G., Citro; G., Zupi
abstract

The c-fes protooncogene is expressed at high levels in the terminal stages of granulocytic differentiation. Its product, p92c-fes, exhibits a tyrosine-kinase activity and is involved in the cellular response to GM-CSF, but its role is not yet clarified. To study this problem, the c-fes protooncogene expression has been inhibited in HL60 cells and in fresh leukemic blast cells of Acute Promyelocytic Leukemia (APL) induced to differentiate with All-Trans-Retinoic Acid (ATRA). Inhibition of c-fes function was obtained by treatment of the cells with a specific antisense oligomer complementary to the 5' region of the c-fes mRNA. It was observed that the cells, rather then differentiate to granulocytes, underwent premature cell death showing the morphological and molecular characteristics of apoptosis. Superimposable results are obtained on blast cells from APL. It is possible to conclude that the loss of cell viability that occurs during the in vitro differentiation of myeloid cells, after the complete inhibition of c-fes expression and treatment with ATRA, is due to activation of programmed cell death rather than an accelerated differentiation. Our data suggest that the c-fes product is essential for the survival of myeloid cells during differentiation.

1994 - Patogenesi della colelitiasi [Articolo su rivista]
Loria, Paola; Bozzoli, M; Concari, M; Giucciardi, Me; Dilengite, Ma; Carubbi, Francesca; Carulli, Nicola; Tagliafico, Enrico
abstract

No abstract

1993 - Expression of interleukins 1, 3, 6, stem cell factor and their receptors in acute leukemia blast cells and in normal peripheral lymphocytes and monocytes [Articolo su rivista]
Ferrari, S.; Grande, A.; Manfredini, R.; Tagliafico, E.; Zucchini, P.; Torelli, G.; Torelli, U.
abstract

Abstract: Reverse transcriptase‐polymerase chain reaction amplification (RT‐PCR) and Southern blot analysis were used to evaluate ligand and receptor expression of interleukin 1α (IL‐1α), interleukin 3 (IL‐3), interleukin 6 (IL‐6) and stem cell factor (SCF) in peripheral blood lymphocytes and monocytes and in several acute leukemia blast cell populations. Resting peripheral lymphocytes and monocytes expressed both ligand and receptor of the four cytokines at considerable levels. The leukemic blast cells of the M1‐M4 phenotypes are characterized by almost complete lack of expression of IL‐1α, IL‐3 and IL‐6 and the constant and usually high expression of SCF. On the other hand, these myeloid blast cells express generally high levels of the four cytokine receptors. The data suggest that the regulation of the expression of IL‐1α, IL‐3 and IL‐6, at least in our limited number of leukemic cell populations studied, is independent of that of SCF. The results indicate that, at least in most of the leukemic myeloid blasts cells, the expression of SCF and its receptor, the c‐kit oncogene, may permit an autocrine regulation of cell cycling. © Munksgaard 1993

1993 - Inhibition of c-fes expression by an antisense oligomer causes apoptosis oh HL60 cells induced to granulocytic differentiation [Articolo su rivista]
Manfredini, Rossella; Grande, Alexis; Tagliafico, Enrico; D., Barbieri; P., Zucchini; G., Citro; G., Zupi; C., Franceschi; U., Torelli; Ferrari, Sergio
abstract

The c-fes protooncogene is expressed at high levels in the terminal stages of granulocytic differentiation, but so far no definite function has been attributed to the product of this oncogene. To tackle this problem, the c-fes protooncogene expression has been inhibited in HL60 cells, and fresh leukemic promyelocytes of acute promyelocytic leukemia have been induced to differentiate with retinoic acid (RA) and dimethylsulfoxide (DMSO). Inhibition was obtained by incubating the cells with a specific c-fes antisense oligodeoxynucleotide. It was observed that the cells, rather than differentiating, underwent premature cell death showing the morphological and molecular characteristics of apoptosis. This process was inhibited by granulocyte and granulocyte/macrophage colony-stimulating factor, but not by interleukin 3 (IL-3), IL-6, or stem cell factor. Our present results demonstrate that the loss of cell viability that occurs during the in vitro differentiation of myeloid cells, after the complete inhibition of the c-fes gene product and treatment with RA-DMSO, is due to activation of programmed cell death. It is concluded that a possible role of the c-fes gene product is to exert an antiapoptotic effect during granulocytic differentiation.

1993 - Overexpression of c-kit in a leukemic cell population carrying a trisomy 4 and its relationship with the proliferative capacity [Articolo su rivista]
Ferrari, S.; Grande, A.; Zucchini, P.; Manfredini, R.; Tagliafico, E.; Rossi, E.; Temperani, P.; Torelli, G.; Emilia, G.; Torelli, U.
abstract

The expression of c-kit and its ligand, the stem cell factor (SCF), was studied in five cases of acute myeloid leukemia. One of these had a trisomy of chromosome 4, where the c-kit oncogene is located. In this case, the c-kit oncogene was overexpressed, but matched by a low expression of its ligand, SCF. The molecular evaluation of the growth rate by c-myc and the histone H3 expression indicated that the growth fraction of this cell population was very low. In one of the other leukemic cell populations studied, characterized by a low expression of c-kit and an elevated expression of the SCF, the growth fraction was also very low. Our results suggest that at least for some receptor oncogenes, the simple overexpression cannot be taken as an indication that the oncogene is involved in the deregulation of cell proliferation. © 1993 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.

1992 - Abundance of the Primary Transcript and Its Processed Product of Growth-related Genes in Normal and Leukemic Cells during Proliferation and Differentiation [Articolo su rivista]
Ferrari, S.; Tagliafico, E.; Manfredini, R.; Grande, A.; Rossi, E.; Zucchini, P.; Torelli, G.; Torelli, U.
abstract

The relative abundance of primary transcript and mature mRNA of the c-myc, calcyclin, S14 ribosomal protein, and rRNA genes was deter-mined densitometrically after reverse transcriptase-polymerase chain reaction and Northern blotting analysis in resting and mitogen-stimulated lymphocytes, proliferating and terminally differentiated HL-60 cells, and leukemic blast cells. Transcription and processing of c-myc and rRNA gene transcripts increased proportionally after mitogen stimulation, whereas these processes were independent of cell cycling status in the case of the SI 4 gene. Normal lymphocytes showed an unexpectedly large amount of primary transcript of the calcyclin gene, whereas the corresponding mRNA was undetectable. The abundance of c-myc, calcyclin, and S14 mRNA in terminally differentiated HL-60 cells decreased sharply, compared to their proliferating counterparts. This decrease reflected post-transcriptional modulation, since the abundance of precursor remained essentially unchanged. After HL-60 differentiation, the 32S rRNA levels remained relatively high, whereas the 45S primary transcript almost disappeared. Leukemic blast cells displayed highly variable precursor/mRNA ratios of c-myc, calcyclin, and S14 genes but consistently high ratios of 32S to 45S RNA, suggesting that the cleavage rate of the 32S rRNA was sharply reduced in these cells, resulting in an accumulation of this molecule. These results suggest the importance of efficient processing of primary transcript to generate adequate functional mRNA, thus regulating gene expression. Furthermore, in terminally differentiated cells and leukemic blast cells a stabilization of the primary transcript without efficient processing can be observed. The role of the stabilization of the primary transcript in terminal differentiation is further supported by the results of run-off transcription, indicating a sharp decrease of c-myc and calcyclin transcription rate in retinoic acid/dimethyl sulfoxide-treated HL-60 cells. © 1992, American Association for Cancer Research. All rights reserved.

1990 - Chronic myelogenous leukemia with typical clinical and morphological features can be Philadelphia chromosome negative and "bcr negative". [Articolo su rivista]
Selleri, L; Emilia, Giovanni; Luppi, Mario; Temperani, Paola; Zucchini, Patrizia; Tagliafico, Enrico; Artusi, Tullio; Sarti, M; Donelli, A; Castoldi, Gl
abstract

The Philadelphia (Ph1) chromosome is found in the majority of patients affected by chronic myelogenous leukemia (CML), being considered the hallmark of the disease, but around 5-8% of patients diagnosed as CML lack the Ph1 chromosome-negative (Ph-) CML has been discussed extensively in the literature because of its heterogeneity. However, it is now accepted that some of the Ph1-CML patients have a disease indistinguishable from Ph1-positive (Ph+) CML. It was investigated whether Ph- CML with clinical and morphological features indicating true CML would always have bcr rearrangements, as the relocation of c-abl from 9q34 into the breakpoint cluster region on 22q11 is considered a crucial event in the pathogenesis of CML. From molecular studies, it seemed that Ph- CML with features of true CML always have the bcr rearrangement, while Ph- patients, lacking such rearrangement, have atypical forms of CML. Here we describe 8 Ph- CML and myeloproliferative syndrome (MPS) patients of whom 6 were by all respects true CML cases. Nevertheless, bcr rearrangement and expression of the classic bcr/abl chimeric mRNA was found in only 1 of the 6 patients. More advanced molecular techniques will be needed to understand which molecular mechanisms underlie Ph-, bcr- CML, resulting in phenotypes sometimes indistinguishable from Ph+, bcr+ CML.

1990 - Detection of low abundance mRNA of myeloid specific genes in cells of acute and chronic lymphoid leukemias by cRNA hybridization [Articolo su rivista]
Ferrari, S.; Ceccherelli, G.; Tagliafico, E.; Zucchini, P.; Manfredini, R.; Torelli, G.; Emilia, G.; Torelli, U.
abstract

The hybridization to a complementary RNA (cRNA) probe both in situ and in solution was used to assay tiny amounts of mRNA of the lactoferrin (LF) and myeloperoxidase (MPO) genes in normal bone marrow cells and in acute and chronic lymphoid leukemias. Evidence is reported that this technique is much more sensitive than the standard Northern blot technique. The LF mRNA was detectable in three of seven cases of acute lymphoblastic leukemia (ALL) and in three of seven cases of chronic lymphocytic leukemia (CLL). Four cases of ALL were also positive when tested with the MPO cRNA. It is apparent from these results that myeloid specific mRNA, different from MPO, may be detected in leukemic cells with lymphoid phenotype using a method more sensitive than the Northern blot technique. Whether or not the molecular events observed in these cell populations reflect events physiologically occurring rather than a deregulation of gene expression associated to leukemogenesis remains to be established.

1990 - Differential effects of c-myb and c-fes antisense oligodeoxynucleotides on granulocytic differentiation of human myeloid leukemia HL60 cells [Articolo su rivista]
Ferrari, S.; Donelli, A.; Manfredini, R.; Sarti, M.; Roncaglia, R.; Tagliafico, E.; Rossi, E.; Torelli, G.; Torelli, U.
abstract

To gain some insight into the role of c-myb and c-fes in myeloid differentiation, the authors have analyzed the ability of HL60 cells to differentiate in response to several different inducers after inhibition of c-myb and c-fes function. This function has been inhibited almost completely by using deoxynucleotides complementary to two 18-nucleotide sequences of c-myb and c-fes encoding mRNA. After 5 days in culture, in several separate experiments with different oligomer preparations, more than 90% growth inhibition was observed in c-myb antisense-treated HL60 cells. At this time, independent of the differentiation inducer used, c-myb antisense-treated HL60 cells differentiate only along the monocytic pathway, whereas in sense oligomer-treated cultures, retinoic acid and dimethyl sulfoxide induced granulocytic differentiation. No perturbation of the HL60 cell growth was observed after 5 days of treatment with antisense c-fes oligomer. However, induction to granulocytic differentiation by retinoic acid and dimethyl sulfoxide resulted in progressive cell death, whereas monocytic differentiation by other differentiation inducers was only marginally affected. These results suggest that granulocytic, unlike monocytic, differentiation requires c-myb-conditioned proliferation and the activity of the protein encoded by c-fes.

1990 - Noncoordinated Expression of S6, S11, and S14 Ribosomal Protein Genes in Leukemic Blast Cells [Articolo su rivista]
Ferrari, S.; Manfredini, R.; Tagliafico, E.; Rossi, E.; Donelli, A.; Torelli, G.; Torelli, U.
abstract

The steady state levels of mRNAs codying for the ribosomal proteins S6, Sil, and SI4 have been evaluated in quiescent and proliferating human fibroblasts and in resting and proliferating human peripheral blood mononuclear cells. It was found that the amounts of ribosomal protein mRN A are very similar and are not increased by serum or mitogen stimulation. The constitutive expression of these genes appears to be coordinately regulated and it is not modified after protein synthesis inhibition by cycloheximide. The ribosomal protein mRNA was also assayed in 15 different populations of human leukemic blast cells. In these populations the abundance of each ribosomal protein mRNA is remarkably different from the other. The results of our present experiments indicate that the expression of the three ribosomal protein genes undergoes independent noncoordinated changes in the large majority of the leukemic populations studied. © 1990, American Association for Cancer Research. All rights reserved.

1990 - Overexpression of the MPO gene occurring in a case of APL without unusual genotypic characteristics [Articolo su rivista]
Ferrari, S.; Tagliafico, E.; Temperani, P.; Manfredini, R.; Ceccherelli, G.; Zucchini, P.; Tabilio, A.; Donelli, A.; Torelli, G.; Emilia, G.; Torelli, U.
abstract

Northern blot analysis of four typical cases of acute promyelocytic leukemia showed that one of the cell population examined was characterized by a very high level of expression of the myeloperoxidase (MPO) gene. Western blot analysis confirms that the protein content of the cells corresponded to the levels of the MPO mRNA. Southern blot studies of the DNA of this cell population ruled out the presence of any genome amplification or rearrangement. Chromosome hybridization studies in situ confirmed that the MPO gene was translocated on the long arm of chromosome 15. The observation that a typical genomic pattern may or may not be associated with the MPO overexpression leads us to believe that so far it is impossible to reach any conclusion about the significance of the translocation in the genesis of MPO overexpression. © 1990.

1990 - Ratios between the abundance of messenger RNA and the corresponding protein of two growth-related genes, c-myc and vimentin, in leukemia blast cells.. [Articolo su rivista]
Ferrari, S; Tagliafico, Enrico; D'Incá, M; Ceccherelli, G; Manfredini, R; Selleri, L; Donelli, A; Sacchi, Stefano; Torelli, Giuseppe; Torelli, U.
abstract

The abundance of the mRNAs of two growth-related genes, vimentin and c-myc, and that of the corresponding proteins have been studied in unstimulated and phytohemagglutinin-stimulated lymphocytes as well as in 18 populations of leukemic blast cells. The quantitative assay was carried out by densitometric scanning of Northern and Western blots. In normal lymphocytes the mRNA and the protein of both genes were almost undetectable. The phytohemagglutinin stimulation led to a sharp increase of the mRNA and the proteins of vimentin and c-myc. The increase was followed by a progressive fall of the gene products. The rate of decrease of the two mRNAs was similar to that of the corresponding proteins. In some leukemic populations very similar amounts of the vimentin protein were accompanied by amounts of the mRNA differing at least 25 times. Not unlikely, very similar amounts of p62c-myc corresponded to mRNA abundances differing at least 16 times. The coordinated biogenesis of both messenger RNAs and proteins, which occurs in mitogen-stimulated lymphocytes, is substituted, in approximately 30% of the leukemic blast cell populations, by molecular events leading to the accumulation of an excess of mRNA.

1989 - Cytogenetic and molecular studies in primary myelofibrosis [Articolo su rivista]
Emilia, G.; Temperani, P.; Ferrari, S.; Zucchini, P.; Tagliafico, E.; Selleri, L.; Torelli, G.; Artusi, T.; Torelli, U.
abstract

Cytogenetic and molecular data of three patients affected by primary myelofibrosis with myeloid metaplasia (PMMM) evolving to blastic crisis are reported. The cytogenetic findings were uncommon. The first patient (female) showed an idic(X)(q13) as the sole alteration in chronic phase, with an additional r(7) in 67% of the cells of the blast crisis; the other two patients showed, in blast crisis, a partial trisomy of the long arm of chromosome 1, without translocation, as a unique structural abnormality. These findings confirm the presence of nonrandom, although nonspecific, alterations in PMMM that, in our cases, seem to be related to the multistep progression of the neoplastic process. Molecular investigations have been applied to study the genomic organization and the level of expression of genes such as bcr and calcyclin and c-fms protooncogene possibly involved in the molecular mechanisms underlying cell proliferation in hematopoietic cells. The data obtained are discussed with respect to the myeloproliferative disorder. © 1989.

1989 - Differential relationship between the abundance of vimentin c-myc and p53 mRNAs and the corresponding proteins in acute leukemia blast cells [Abstract in Rivista]
Ferrari, S; Tagliafico, E; D'Incà, M; Ceccherelli, G; Manfredini, R; Selleri, L; Sacchi, Stefano; Torelli, Umberto; Torelli, U.
abstract

no abstract

1989 - Expression of myeloperoxidase and lactoferrin genes in blast cells of acute lymphoblastic leukemia [Articolo su rivista]
Manfredini, R.; Tagliafico, E.; Rossi, E.; Ceccherelli, G.
abstract

The myeloperoxidase (MPO) and lactoferrin genes are strictly associated to the granulocytic differentiation pathway. Many reports show that immunologic and molecular cell markers, though to be lineage specific, are in fact expressed in cells of different lineages. We have identified lymphoid blast cell populations expressing the MPO gene at the mRNA level, but lacking any detectable MPO protein. We addressed this issue studying the expression of the MPO and lactoferrin genes in extremely homogenous Acute Lymphoblastic Leukemia (ALL) blast cell populations. The study was carried out by solution hybridization of total cellular mRNA extracted from the leukemic populations with an antisense RNA transcribed by riboprobes. Either MPO or lactoferrin mRNAs were detectable in some ALL populations. We suggest that the expression of these two genes could be a physiologic event in the very early stages of lymphoid cell differentiation.

1989 - Expression of the myeloperoxidase gene in acute and chronic myeloid leukemias: relationship to the expression of cell cycle-related genes. [Articolo su rivista]
Ferrari, Sergio; Tagliafico, Enrico; Ceccherelli, G; Selleri, L; Calabretta, Bruno; Donelli, A; Temperani, Paola; Sarti, M; Sacchi, Stefano; Emilia, Giovanni; Torelli, Giuseppe; Torelli, Umberto
abstract

The expression of the myeloperoxidase (MPO) gene was studied, by means of Northern blot analysis in 14 cases of acute myeloid leukemia (AML), 11 cases of chronic myeloid leukemia (CML), and 6 cases of CML blast crisis, and in HL60 cells before and after induction of terminal differentiation with retinoic acid (RA), phorbol esters (TPA), or vitamin D. The expression of a panel of cell cycle-related genes, namely C-MYC, histone H3, ornithine decarboxylase, P53, vimentin, and calcyclin, was also studied in the same cell populations. Our results indicate that: (a) MPO gene expression (steady state mRNA levels) is strictly confined to the first stages of myeloid differentiation, reaching its peak at the promyelocyte stage and becoming undetectable in mature granulocytes and monocytes; (b) cells devoid of any detectable MPO enzymatic activity such as leukemic basophils have a high content of MPO mRNA; and (c) MPO gene expression is not related to the growth activity of the cell population. Finally, our results show that the pattern of expression of growth-regulated genes in the neoplastic myeloid disorders AML, CML, and CML blast crisis is remarkably different.

1989 - Livelli di espressione degli RNA e delle corrispondenti proteine dei geni vimentina e c-myc in blasti di leucemia acuta. [Abstract in Rivista]
Tagliafico, E; D'Inca, M; Ceccherelli, G; Manfredini, R; Selleri, L; Sacchi, Stefano; Torelli, G; Torelli, U; Ferrari, S.
abstract

1989 - Philadelphia-positive chronic myelogenous leukemia with typical bcr/abl molecular features and atypical, prolonged survival [Articolo su rivista]
Selleri, L.; Emilia, G.; Temperani, P.; Grassilli, E.; Zucchini, P.; Tagliafico, E.; Bonati, A.; Venezia, L.; Ferrari, S.; Torelli, U.; Torelli, G.
abstract

Despite the major breakthrough in the knowledge of the molecular events underlying the t(9;22) translocation, still no consistent data have been found on the evolution of Ph1 positive CML from the chronic to the accelerated or blastic phase of the disease. In most patients in fact the bcr/abl rearrangements are identical both in chronic phase and in blast crisis, and overall differences in chronic phase duration, related to different location of breakpoints inside the bcr region, were found to be marginal. We approached this problem by studying the molecular features of the bcr/abl abnormality in rare CML patients with very long, atypical chronic phase. The three patients studied, whose chronic phase duration is 17, 19, and 21 years, respectively, have typical genomic bcr rearrangements, and two of them show, hybridizing Northern blots to c-abl, the 8.5 kb mRNA, as that typically present in CML. It seems that genomic alterations within bcr and abl cannot account, alone, for the duration of the chronic phase of Ph1 positive CML and those quantitative and/or qualitative alterations of the p210 bcr/abl protein, unluckily awkward to prove, might be responsible for the atypical clinical features of these CML long survivors.

1989 - Polymerase chain reaction for the diagnostic identification of HIVinfection [Articolo su rivista]
Selleri, L; Grassilli, E; Tagliafico, Enrico; Corradi, M; Luppi, Mario; Ceccherelli, G; Borghi, V.
abstract

The Acquired Immune deficiency Syndrome (AIDS), caused by the Human Immunodeficiency Virus (HIV) is now a worldwide social problem. Routine diagnostic procedures to identify infected individuals are based on the presence of antibodies against viral epitopes in the serum. There is nevertheless impelling need to detect directly the virus in people infected by HIV, independently of a serological response. In this study we describe the procedure which allows amplification of a specific segment of the HIV genome, through the Polymerase Chain Reaction (PCR), in infected individuals. This new approach represents a precious tool towards the diagnosis of HIV infection, it can be easily and quickly carried out on a large scale and will be capable of identifying HIV infected subjects before the development of antibodies.

1988 - Myeloperoxidase gene expression in blast cells with a lymphoid phenotype in cases of acute lymphoblastic leukemia. [Articolo su rivista]
Ferrari, Sergio; M. T., Mariano; Tagliafico, Enrico; M., Sarti; G., Ceccherelli; L., Selleri; F., Merli; Narni, Franco; A., Donelli; Torelli, Giuseppe
abstract

By using a cDNA clone of the myeloperoxidase (MPO) gene, we have studied, by Northern blot analysis, the level of MPO mRNA in eight cases of acute lymphoblastic leukemia (ALL). The blast cell populations studied were characterized by morphologic, cytochemical, immunochemical, and molecular criteria. With all the methods used the populations were found to be highly homogeneous and showed a typical lymphoid phenotype. In particular, the Ig heavy-chain gene rearrangement was largely prevalent, and the germ line configuration was almost absent. However, in three of eight cases, high levels of MPO mRNA were detected. The remarkable homogeneity of the cell populations examined suggests that the MPO mRNA observed was present in cellular elements certainly identified as lymphoid. The absence of contamination by myeloid cells was confirmed by the results of Western blot analysis of the proteins of the cell population studied: no MPO protein was detectable. The levels of mRNA observed were high enough to be comparable to those observed in a promyelocytic cell population.

1988 - Possible correlations between proliferation and differentiation in blast cells of acute promyelocytic leukemia: A molecular study [Articolo su rivista]
Tagliafico, E.; Manfredini, R.; Selleri, L.; Zucchini, P.; Ceccherelli, G.
abstract

The level of expression of the myeloperoxidase gene (MPO), strictly associated with myeloid differentiation as well as of some cell cycle related genes, such as c-myc and Histone H3, was evaluated by northern blot analysis in blast cells of 4 cases of Acute Promyelocytic Leukemia. MPO gene expression was found to be not related to the growth activity of the cell population. It appears that the differentiation arrest which occurs in the majority of the blast cells is not strictly associated with the proliferation impairment.

1988 - Studio citogenetico e molecolare in un caso di mesotelioma maligno [Abstract in Rivista]
Zucchini, P; Temperani, P; Tagliafico, E; Artusi, T; Ferrari, S; Narni, F; Sacchi, Stefano; Emilia, G.
abstract

Università degli studi di Modena e Reggio Emilia

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