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Elena TENEDINI
Professore Associato
Dipartimento di Scienze Mediche e Chirurgiche Materno-Infantili e dell'Adulto
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Pubblicazioni
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
- 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
- LOW-FREQUENCY ALLELE VARIANTS IN NGS MULTI-GENE PANELS FOR HEREDITARY CANCER TESTING: ARTIFACTS,
CHIP OR MOSAICS? MANAGING THE RESULTS IN THE LABORATORY ROUTINE
[Abstract in Rivista]
Tenedini, E.; Bonamici, L.; Artuso, L.; Marino, M.; Botticelli, L.; Barbieri, E.; Toss, A.; Venturelli, M.; Trenti, T.; Dominici, M.; Cortesi, L.; Tagliafico, E.
abstract
2023
- Management of PALB2-associated breast cancer: A literature review and case report
[Articolo su rivista]
Toss, Angela; Ponzoni, Ornella; Riccò, Beatrice; Piombino, Claudia; Moscetti, Luca; Combi, Francesca; Palma, Enza; Papi, Simona; Tenedini, Elena; Tazzioli, Giovanni; Dominici, Massimo; Cortesi, Laura
abstract
Key Clinical Message
Germline pathogenic variants (PV) of the PALB2 tumor suppressor gene are associated with an increased risk of breast, pancreatic, and ovarian cancer. In previous research, PALB2-associated breast cancer showed aggressive clinicopathological phenotypes, particularly triple-negative subtype, and higher mortality regardless
of tumor stage, type of chemotherapy nor hormone receptor status. The identification of this germline alteration may have an impact on clinical management of breast cancer (BC) from the surgical approach to the systemic treatment choice. We herein report the case of a patient with a germline PV of PALB2, diagnosed with locally advanced PD-L1 positive triple-negative BC, who progressed after an immune checkpoint inhibitor (ICI)-containing regimen and then experienced a pathologic complete response after platinum-based chemotherapy. This case report hints a major role of the germline PALB2 alteration compared to the PD-L1 expression as cancer driver and gives us the opportunity to extensively review and discuss the available literature on the optimal management of PALB2-associated
BC. Overall, our case report and review of the literature provide additional evidence that the germline analysis of PALB2 gene should be included in routine genetic testing for predictive purposes and to refine treatment algorithms.
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
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
- 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
- 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
- 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
- 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
- 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.
2020
- WISP-2 expression induced by Teriparatide treatment affects in vitro osteoblast differentiation and improves in vivo osteogenesis
[Articolo su rivista]
Smargiassi, A.; Bertacchini, J.; Checchi, M.; Poti, F.; Tenedini, E.; Montosi, G.; Magaro, M. S.; Amore, E.; Cavani, F.; Ferretti, M.; Grisendi, G.; Maurel, D. B.; Palumbo, C.
abstract
The Osteocyte, recognized as a major orchestrator of osteoblast and osteoclast activity, is the most important key player during bone remodeling processes. Imbalances occurring during bone remodeling, caused by hormone perturbations or by mechanical loading alterations, can induce bone pathologies such as osteoporosis. Recently, the active fraction of parathormone, PTH (1-34) or Teriparatide (TPTD), was chosen as election treatment for osteoporosis. The effect of such therapy is dependent on the temporal manner of administration. The molecular reasons why the type of administration regimen is so critical for the fate of bone remodeling are numerous and not yet well known. Our study attempts to analyze diverse signaling pathways directly activated in osteocytes upon TPTD treatment. By means of gene array analysis, we found many molecules upregulated or downregulated in osteocytes. Later, we paid attention to Wisp-2, a protein involved in the Wnt pathway, that is secreted by MLO-Y4 cells and increases upon TPTD treatment and that is able to positively influence the early phases of osteogenic differentiation. We also confirmed the pro osteogenic property of Wisp-2 during mesenchymal stem cell differentiation into the preliminary osteoblast phenotype. The same results were confirmed with an in vivo approach confirming a remarkable Wisp-2 expression in metaphyseal trabecular bone. These results highlighted the anabolic roles unrolled by osteocytes in controlling the action of neighboring cells, suggesting that the perturbation of certain signaling cascades, such as the Wnt pathway, is crucial for the positive regulation of bone formation.
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
- 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.
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
- 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
- Wisp2 overexpression induced by short Teriparatide treatment affects IDG-SW3 osteogenic differentiation.
[Abstract in Rivista]
Bertacchini, Jessika; Smargiassi, Alberto; Checchi, Marta; Magaro', MARIA SARA; Poti', Francesco; Tenedini, Elena; Montosi, Giuliana; Magaro', MARIA SARA; Vinet, Jonathan; Maurel, Delphine; Palumbo, Carla
abstract
The study supports the importance of osteocytes in controlling the action of the other bone cells and suggests that the perturbation of certain signaling cascades, such as the Wnt pathway, is crucial for the positive regulation of bone formation.
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
- 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
- Osteocytes signaling events induced by intermittent vs continuous Teriparatide treatment affect in vitro osteoblast differentiation and mineralization
[Abstract in Rivista]
Bertacchini, Jessika; Smargiassi, Alberto; Checchi, Marta; Tenedini, Elena; Montosi, Giuliana; Vinet, Jonathan; Ferretti, Marzia; Palumbo, Carla
abstract
PTH(1-34), also known as Teriparatide, is an active anabolic drug used in the treatment of some forms of osteoporosis and occasionally exploited to speed fracture healing. The effect of such therapies are dependent on the type of administration, in fact it has been largely demonstrated that a short administration of Teriparatide (also called intermittent) increases the bone mass, meanwhile a long administration of the same agent (known as continuous) leads to an increased resorption. The molecular reason why the type of administration is so critical for the fate of the bone remodeling is still largely unknown but it is probably due to the fact that it affects several signaling pathways and alters the biological activity of a cohort of cells: osteoblasts, lining cells, osteoclasts, and osteocytes. In the present work, we firstly focused the attention on molecular events induced by intermittent vs continuous Teriparatide treatment in a well-known osteocytes in vitro model, the MLO-Y4 cells. By the use of a gene array platform, we found many molecules upregulated or downregulated depending on the the temporal administration modes, suggesting that the drug affects in diverse manner the osteocytes related signaling pathways. In particular, we paid attention to Wisp-2, a protein of the Wnt pathway that has been demonstrated to be able to interact and influence the differentiation of osteoblasts into osteocytes and their mineralization. Secondly, through the mineralization assay, we analyzed the functional effects, involving the differentiation of osteoblast IDG-SW3 cell line, upon the conditioning culture with MLO-Y4 medium, that were pre-treated with short and long time administration of Teriparatide. These findings, consistent with the crucial role performed by osteocytes on osteoblast differentiation, clarify the molecular events downstream the short treatment with Teriparatide, suggesting that the perturbation of certain signaling patwhays, such as the Wnt pathway, is crucial for the positive regulation of bone formation.
2017
- The chaperone activity of 4PBA ameliorates the skeletal phenotype of Chihuahua, a zebrafish model for dominant osteogenesis imperfecta
[Articolo su rivista]
Gioia, Roberta; Tonelli, Francesca; Ceppi, Ilaria; Biggiogera, Marco; Leikin, Sergey; Fisher, Shannon; Tenedini, Elena; Yorgan, Timur A.; Schinke, Thorsten; Tian, Kun; Schwartz, Jean-Marc; Forte, Fabiana; Wagener, Raimund; Villani, Simona; Rossi, Antonio; Forlino, Antonella
abstract
Classical osteogenesis imperfecta (OI) is a bone disease caused by type I collagen mutations and characterized by bone fragility, frequent fractures in absence of trauma and growth deficiency. No definitive cure is available for OI and to develop novel drug therapies, taking advantage of a repositioning strategy, the small teleost zebrafish (Danio rerio) is a particularly appealing model. Its small size, high proliferative rate, embryo transparency and small amount of drug required make zebrafish the model of choice for drug screening studies, when a valid disease model is available. We performed a deep characterization of the zebrafish mutant Chihuahua, that carries a G574D (p.G736D) substitution in the α1 chain of type I collagen. We successfully validated it as a model for classical OI. Growth of mutants was delayed compared with WT. X-ray, mCT, alizarin red/alcian blue and calcein staining revealed severe skeletal deformity, presence of fractures and delayed mineralization. Type I collagen extracted from different tissues showed abnormal electrophoretic migration and low melting temperature. The presence of endoplasmic reticulum (ER) enlargement due to mutant collagen retention in osteoblasts and fibroblasts of mutant fish was shown by electron and confocal microscopy. Two chemical chaperones, 4PBA and TUDCA, were used to ameliorate the cellular stress and indeed 4PBA ameliorated bone mineralization in larvae and skeletal deformities in adult, mainly acting on reducing ER cisternae size and favoring collagen secretion. In conclusion, our data demonstrated that ER stress is a novel target to ameliorate OI phenotype; chemical chaperones such as 4PBA may be, alone or in combination, a new class of molecules to be further investigated for OI treatment.
2017
- uPA/uPAR system activation drives a glycolytic phenotype in melanoma cells
[Articolo su rivista]
Laurenzana, Anna; Chillà, Anastasia; Luciani, Cristina; Peppicelli, Silvia; Biagioni, Alessio; Bianchini, Francesca; Tenedini, Elena; Torre, Eugenio; Mocali, Alessandra; Calorini, Lido; Margheri, Francesca; Fibbi, Gabriella; Del Rosso, Mario
abstract
In this manuscript, we show the involvement of the uPA/uPAR system in the regulation of aerobic glycolysis of melanoma cells. uPAR over-expression in human melanoma cells controls an invasive and glycolytic phenotype in normoxic conditions. uPAR down-regulation by siRNA or its uncoupling from integrins, and hence from integrin-linked tyrosine kinase receptors (IL-TKRs), by an antagonist peptide induced a striking inhibition of the PI3K/AKT/mTOR/HIF1α pathway, resulting into impairment of glucose uptake, decrease of several glycolytic enzymes and of PKM2, a checkpoint that controls metabolism of cancer cells. Further, binding of uPA to uPAR regulates expression of molecules that govern cell invasion, including extracellular matrix metallo-proteinases inducer (EMPPRIN) and enolase, a glycolytyc enzyme that also serves as a plasminogen receptor, thus providing a common denominator between tumor metabolism and phenotypic invasive features. Such effects depend on the α5β1-integrin-mediated uPAR connection with EGFR in melanoma cells with engagement of the PI3K-mTOR-HIFα pathway. HIF-1α trans-activates genes whose products mediate tumor invasion and glycolysis, thus providing the common denominator between melanoma metabolism and its invasive features. These findings unveil a unrecognized interaction between the invasion-related uPAR and IL-TKRs in the control of glycolysis and disclose a new pharmacological target (i.e., uPAR/IL-TKRs axis) for the therapy of melanoma.
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
- 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 (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
- 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
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. 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.
The main aim of this study is to
find prognostic and predictive
molecular biomarkers for the
management of metastatic BC
patients in clinical practice.
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
- 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
- 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
- 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
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
- New modulated genes in psoriasis-derived keratinocyte subpopulations.
[Abstract in Rivista]
Lotti, Roberta; Tenedini, Elena; Fabiano, A; Truzzi, Francesca; Saltari, Annalisa; Morandi, Paolo; Marconi, Alessandra; Pincelli, Carlo
abstract
T cells play a crucial role in the pathogenesis of psoriasis, recent data emphasize the key role of keratinocytes that, by carrying intrinsic alterations, could determine the formation of skin lesions resembling psoriasis, without the participation of T-cell derived cytokines. In particular, transit amplifying (TA) cells, the stem cell progeny, seems to be responsible for the psoriatic phenotype. The aim of this study was to analyze the role of keratinocytes sub-populations in the pathogenesis of psoriasis. We analyzed the gene expression profile (GEP) of human keratinocyte sub-populations (stem, “early” TA (ETA) and “late” TA (LTA) cells), derived from healthy skin, lesional and non-lesional psoriatic skin. The total RNA samples, extracted from keratinocyte subpopulations immediately after separation, were hybridized onto the Affymetrix human U133 plus 2.0 GeneChip Array. We identified a small number of up-regulated genes (12 probe sets, corresponding to 8 genes) in keratinocyte subpopulations derived from lesional psoriasis vs. healthy and non-lesional psoriasis. We confirmed the increased expression levels of TCN1, S100A7A, KYNU, SERPINB13, FOXE1, but solely due to the keratinocyte component. We identified for the first time the up-regulation of TMEM171, CLEC7A and NDRG4, which seems to correlate with the pathophysiology of psoriasis. Moreover, GEP analysis of lesional psoriasis sub-populations, as compared to the non-lesional psoriatic counterpart revealed the modulation of 17 probe sets, corresponding to 13 genes. Among these genes, we recognized for the first time the up-regulation of IL13RA1, CCDC109B and CD47. These results indicate the importance of keratinocyte compartment in psoriasis, opening the way to the study of new genes potentially critical in the pathogenesis of psoriasis.
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
- 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
- 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.
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.
2011
- Expression profiling of FSHD-1 and FSHD-2 cells during myogenic differentiation evidences common and distinctive gene dysregulation patterns
[Articolo su rivista]
S., Cheli; S., François; B., Bodega; F., Ferrari; Tenedini, Elena; Roncaglia, Enrica; Ferrari, Sergio; E., Ginelli; R., Meneveri
abstract
BackgroundDetermine global gene dysregulation affecting 4q-linked (FSHD-1) and non 4q-linked (FSHD-2) cells during early stages of myogenic differentiation. This approach has been never applied to FSHD pathogenesis.Methodology/Principal FindingsBy in vitro differentiation of FSHD-1 and FSHD-2 myoblasts and gene chip analysis we derived that gene expression profile is altered only in FSHD-1 myoblasts and FSHD-2 myotubes. The changes seen in FSHD-1 regarded a general defect in cell cycle progression, probably due to the upregulation of myogenic markers PAX3 and MYOD1, and a deficit of factors (SUV39H1 and HMGB2) involved in D4Z4 chromatin conformation. On the other hand, FSHD-2 mytubes were characterized by a general defect in RNA metabolism, protein synthesis and degradation and, to a lesser extent, in cell cycle. Common dysregulations regarded genes involved in response to oxidative stress and in sterol biosynthetic process. Interestingly, our results also suggest that miRNAs might be implied in both FSHD-1 and FSHD-2 gene dysregulation. Finally, in both cell differentiation systems, we did not observe a gradient of altered gene expression throughout the 4q35 chromosome.Conclusions/SignificanceFSHD-1 and FSHD-2 cells showed, in different steps of myogenic differentiation, a global deregulation of gene expression rather than an alteration of expression of 4q35 specific genes. In general, FSHD-1 and FSHD-2 global gene deregulation interested common and distinctive biological processes. In this regard, defects of cell cycle progression (FSHD-1 and to a lesser extent FSHD-2), protein synthesis and degradation (FSHD-2), response to oxidative stress (FSHD-1 and FSHD-2), and cholesterol homeostasis (FSHD-1 and FSHD-2) may in general impair a correct myogenesis. Taken together our results recapitulate previously reported defects of FSHD-1, and add new insights into the gene deregulation characterizing both FSHD-1 and FSHD-2, in which miRNAs may play a role.
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
- ANALISYS OF GENE EXPRESSION PROFILE DURING MYOGENESIS EVIDENCES DIFFERENT MOLECULAR DEFECTS AT THE BASIS OF FSHD-1 AND FSHD-2 PATHOGENESIS
[Abstract in Atti di Convegno]
Cheli, Stefania; Francois, Stéphanie; Bodega, Beatrice; Ferrari, Francesco; Tenedini, Elena; Roncaglia, Enrica; Ferrari, Sergio; Ginelli, Enrico; Meneveri, Raffaella
abstract
Germ line cell-derived pluripotent stem cells (GPSCs) are derived from spermatogonial stem cells and are similar to
embryonic stem (ES) cells in that they can proliferate intensively and differentiate into a variety of cell types. Previous
studies have revealed some inherent differences in gene expression between undifferentiated mouse ES cells and
GPSCs. Our aims were to generate functional hepatocytes from mouse GPSCs in vitro and to investigate whether the
differences in gene expression may impact on the hepatocyte differentiation capacity of the GPSCs compared with ES
cells. Mouse GPSCs and ES cells were induced to differentiate into hepatocytes through embryoid body formation, with
very high efficiency. These hepatocytes were characterized at cellular, molecular, and functional levels. The GPSCderived
hepatocytes expressed hepatic markers and were metabolically active as shown by albumin and haptoglobin
secretion, urea synthesis, glycogen storage, and indocyanine green uptake. We also performed an unprecedented DNA
microarray analysis comparing different stages of hepatocyte differentiation. Gene expression profiling demonstrated a
strong similarity between GPSC and ES cells at different stages of induced hepatic differentiation. Moreover, Pearson
correlation analysis of the microarray datasets suggested that, at late hepatic differentiation stages, the in vitro-derived
cells were closer to fetal mouse primary hepatocytes than to those obtained from neonates. We have shown for the first
time that adult GPSCs can be induced to differentiate into functional hepatocytes in vitro. Moreover, our ongoing in
vivo work shows that GPSC-derived hepatocytes can colonize the liver of monocrotaline-treated, partially
hepatectomised mice. These GPSC-derived hepatocytes thus offer great potential for cell replacement therapy for a
wide variety of liver diseases.
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, F; Orlandi, C; 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 myelopoieis: role of hsa-mir- 299-5p in CD34+ progenitor cells commitment
[Relazione in Atti di Convegno]
Tenedini, Elena
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 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
- c-Myb supports erythropoiesis by transactivating KLF1 and LMO2 expression
[Abstract in Atti di Convegno]
Bianchi, Elisa; Zini, Roberta; Salati, Simona; Tenedini, Elena; Norfo, Ruggiero; Ferrari, Sergio; Manfredini, Rossella
abstract
The c-Myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during
differentiation. c-myb is essential for the hematopoietic development, as c-myb-/- mice die at E15 due to failure of fetal
hepatic erythropoiesis. To gain further insights into the role of c-myb during the hematopoietic lineage commitment, we
studied the effects of c-Myb silencing in human CD34+ hematopoietic stem/progenitor cells. c-Myb silencing in CD34+
cells was performed by transfection of siRNAs using the Amaxa Nucleofector® Technology. In order to keep c-Myb
expression silenced for all the commitment phase of CD34+ cells, each sample was nucleofected 3 times, once a day.
Moreover, to exclude non-specific effects of siRNA nucleofection, for each experiment, together with the sample
transfected with the siRNAs targeting c-Myb, one sample electroporated without siRNAs and one transfected with a
non-targeting siRNA were performed. c-Myb silencing effects on CD34+ cells differentiation ability were studied by
methylcellulose and collagen-based clonogenic assays and by morphological and immunophenotypic analyses after
liquid culture. Furthermore, we investigated by microarray analysis the changes in gene expression induced by c-Myb
silencing. Methylcellulose assay revealed a remarkable increase of the percentage of monocyte (CFU-M) colonies and a
decrease of the erythroid ones (BFU-E) in c-Myb-silenced CD34+ cells. Moreover, collagen-based clonogenic assay
demonstrated that c-Myb silencing strongly enhances the megakaryocyte commitment of CD34+ cells. In agreement
with these data, flow cytometric analysis showed an increase in mono-macrophage and megakaryocyte fractions in cmyb-silenced
cells, while the erythroid population was strongly decreased. Morphological evaluation of May
Grunwald-Giemsa stained cytospins further supported the conclusion that c-myb silencing forces the CD34+ cells
commitment towards the macrophage and megakaryocyte lineages at the expense of the erythroid one. Gene expression
profiling of c-Myb silenced CD34+ cells enabled us to identify new 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. Functional rescue experiments showed that the
retroviral vector-mediated overexpression of KLF1 and LMO2 transcription factors in c-Myb silenced cells is able to
rescue, at least in part, the impaired erythroid differentiation. 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. In particular, we identified c-Myb-driven KLF1 and LMO2 transactivation
as the molecular mechanism through which c-Myb regulates erythroid versus megakaryocyte lineage fate decision.
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.
2007
- Eosinophils, but not neutrophils, exibit an efficient DNA repair machinary and high nucleolar activity
[Articolo su rivista]
Salati, Simona; Bianchi, Elisa; Zini, Roberta; Tenedini, Elena; Quaglino, Daniela; Manfredini, Rossella; Ferrari, Sergio
abstract
BACKGROUND AND OBJECTIVES: Traditionally eosinophils have been considered terminally differentiated cells that play a role in host protection against parasites. However, there is some evidence showing that eosinophils are, in fact, multifunctional leukocytes involved in inflammatory responses, as well as in tissue homeostasis. We characterized the transcriptome profile of human eosinophils, and, for the purpose of comparison, the transcriptome profile of neutrophils, monocytes and hematopoietic progenitor cells. Moreover, we studied the activation of selected cellular processes for which a significant differential expression was demonstrated. DESIGN AND METHODS: We profiled gene expression using Affymetrix GeneChips. DNA repair capacity was tested using the comet assay. Nucleoli and their activity were characterized by transmission electron microscopy analysis, silver staining of nucleolus regions (AgNOR) and RNA staining. RESULTS: Gene expression profiling showed that eosinophils appear hierarchically closer to monocytes than to neutrophils. Gene ontology mapping of differentially expressed genes revealed that eosinophils express categories very similar to those expressed by monocytes, related to DNA repair and nucleolar functions. Moreover, our data show that eosinophils and monocytes maintain the ability to repair both double and single strand DNA breaks, whereas neutrophils lack this capacity. Furthermore, eosinophils exhibit nucleolar activity, which is lacking in neutrophils, but resembles that in monocytes. INTERPRETATION AND CONCLUSIONS: The presence of large, active nucleoli in eosinophils, coupled to marked activity of DNA repair systems, suggests that eosinophils are not terminally differentiated cells. Indeed, their transcriptome profile and functional properties are more similar to those of non-terminally differentiated cells such as monocytes, rather than to neutrophils.
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
- 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
- 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 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 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
- Tfe3 expression is closely associated to macrophage terminal differentiation of human hematopoietic myeloid precursors.
[Articolo su rivista]
ZANOCCO MARANI, Tommaso; Vignudelli, Tatiana; Gemelli, Claudia; Pirondi, Sara; Testa, Anna; Montanari, Monica; Parenti, Sandra; Tenedini, Elena; Grande, Alexis; Ferrari, Sergio
abstract
The MItf-Tfe family of basic helix–loop–helix leucine zipper (bHLH-Zip) transcription factors encodes four family members: MItf, Tfe3, TfeB and TfeC. In vitro, each protein of the family binds DNA in a homo- or heterodimeric form with other family members. Tfe3 is involved in chromosomal translocations recurrent in different tumors and it has been demonstrated, by in vivo studies, that it plays, redundantly with MItf, an important role in the process of osteoclast formation, in particular during the transition from mono-nucleated to multi-nucleated osteoclasts. Since mono-nucleated osteoclasts derive from macrophages we investigated whether Tfe3 might play a role upstream during hematopoietic differentiation. Here we show that Tfe3 is able to induce mono-macrophagic differentiation of U937 cells, in association with a decrease of cell proliferation and an increase of apoptosis. We also show that Tfe3 does not act physiologically during commitment of CD34+ hematopoietic stem cells (HSCs), since it is not able to direct HSCs toward a specific lineage as observed by clonogenic assay, but is a strong actor of terminal differentiation since it allows human primary myeloblasts' maturation toward the macrophage lineage.
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.
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 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 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 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.