Publications by authors named "Maria Scardoni"

12 Publications

  • Page 1 of 1

Whole-genome landscape of pancreatic neuroendocrine tumours.

Nature 2017 03 15;543(7643):65-71. Epub 2017 Feb 15.

QIMR Berghofer Medical Research Institute, Herston Road, Brisbane 4006, Australia.

The diagnosis of pancreatic neuroendocrine tumours (PanNETs) is increasing owing to more sensitive detection methods, and this increase is creating challenges for clinical management. We performed whole-genome sequencing of 102 primary PanNETs and defined the genomic events that characterize their pathogenesis. Here we describe the mutational signatures they harbour, including a deficiency in G:C > T:A base excision repair due to inactivation of MUTYH, which encodes a DNA glycosylase. Clinically sporadic PanNETs contain a larger-than-expected proportion of germline mutations, including previously unreported mutations in the DNA repair genes MUTYH, CHEK2 and BRCA2. Together with mutations in MEN1 and VHL, these mutations occur in 17% of patients. Somatic mutations, including point mutations and gene fusions, were commonly found in genes involved in four main pathways: chromatin remodelling, DNA damage repair, activation of mTOR signalling (including previously undescribed EWSR1 gene fusions), and telomere maintenance. In addition, our gene expression analyses identified a subgroup of tumours associated with hypoxia and HIF signalling.
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http://dx.doi.org/10.1038/nature21063DOI Listing
March 2017

Next-generation sequencing for genetic testing of familial colorectal cancer syndromes.

Hered Cancer Clin Pract 2015 21;13(1):18. Epub 2015 Aug 21.

ARC-Net Research Centre, University and Hospital Trust of Verona, Verona, Italy ; Department of Pathology and Diagnostics, University and Hospital Trust of Verona, Verona, Italy ; ARC-Net Research Centre, Department of Pathology & Diagnostics, University of Verona, Policlinico GB Rossi, Piazzale L.A. Scuro, 10, Verona, Italy.

Background: Genetic screening in families with high risk to develop colorectal cancer (CRC) prevents incurable disease and permits personalized therapeutic and follow-up strategies. The advancement of next-generation sequencing (NGS) technologies has revolutionized the throughput of DNA sequencing.

Methods: A series of 16 probands for either familial adenomatous polyposis (FAP; 8 cases) or hereditary nonpolyposis colorectal cancer (HNPCC; 8 cases) were investigated for intragenic mutations in five CRC familial syndromes-associated genes (APC, MUTYH, MLH1, MSH2, MSH6) applying both a custom multigene Ion AmpliSeq NGS panel and conventional Sanger sequencing.

Results: Fourteen pathogenic variants were detected in 13/16 FAP/HNPCC probands (81.3 %); one FAP proband presented two co-existing pathogenic variants, one in APC and one in MUTYH. Thirteen of these 14 pathogenic variants were detected by both NGS and Sanger, while one MSH2 mutation (L280FfsX3) was identified only by Sanger sequencing. This is due to a limitation of the NGS approach in resolving sequences close or within homopolymeric stretches of DNA. To evaluate the performance of our NGS custom panel we assessed its capability to resolve the DNA sequences corresponding to 2225 pathogenic variants reported in the COSMIC database for APC, MUTYH, MLH1, MSH2, MSH6. Our NGS custom panel resolves the sequences where 2108 (94.7 %) of these variants occur. The remaining 117 mutations reside inside or in close proximity to homopolymer stretches; of these 27 (1.2 %) are imprecisely identified by the software but can be resolved by visual inspection of the region, while the remaining 90 variants (4.0 %) are blind spots. In summary, our custom panel would miss 4 % (90/2225) of pathogenic variants that would need a small set of Sanger sequencing reactions to be solved.

Conclusions: The multiplex NGS approach has the advantage of analyzing multiple genes in multiple samples simultaneously, requiring only a reduced number of Sanger sequences to resolve homopolymeric DNA regions not adequately assessed by NGS. The implementation of NGS approaches in routine diagnostics of familial CRC is cost-effective and significantly reduces diagnostic turnaround times.
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http://dx.doi.org/10.1186/s13053-015-0039-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4546256PMC
August 2015

Mixed adenoneuroendocrine carcinomas of the gastrointestinal tract: targeted next-generation sequencing suggests a monoclonal origin of the two components.

Neuroendocrinology 2014 20;100(4):310-6. Epub 2014 Oct 20.

ARC-Net Research Centre, University and Hospital Trust of Verona, Verona, Italy.

Background: Mixed adenoneuroendocrine carcinomas (MANECs) of the gastrointestinal tract are rare neoplasms characterized by coexisting exocrine and neuroendocrine neoplastic components. MANECs' histogenetic classification and molecular characterization remain unclear, significantly affecting the identification of innovative therapeutic options for these tumors.

Methods: The exocrine and neuroendocrine components of 6 gastrointestinal MANECs were microdissected and subjected to the simultaneous mutation assessment in selected regions of 54 cancer-associated genes using Ion Torrent semiconductor-based next-generation sequencing. Sanger sequencing and immunohistochemistry were used as validation of the mutational status.

Results: A total of 20 driver gene somatic mutations were observed among the 12 neoplastic components investigated. In 11 of 12 (91.7%) samples, at least one mutation was detected; 7 samples (58.3%) were found to have multiple mutations. TP53 gene mutations were the most frequent genetic alterations observed in the series, occurring in 11/12 samples (91.7%). Somatic mutations in other genes were detected at lower frequencies: ATM, CTNNB1, ERBB4, JAK3, KDR, KRAS, RB1.

Conclusions: Five of the 6 MANECs presented an overlapping mutational profile in both components, suggesting a monoclonal origin of the two MANEC components.
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http://dx.doi.org/10.1159/000369071DOI Listing
October 2015

Reporting tumor molecular heterogeneity in histopathological diagnosis.

PLoS One 2014 15;9(8):e104979. Epub 2014 Aug 15.

Applied Research on Cancer Network (ARC-NET) and Department of Pathology and Diagnostics, University and Hospital Trust of Verona, Verona, Italy.

Background: Detection of molecular tumor heterogeneity has become of paramount importance with the advent of targeted therapies. Analysis for detection should be comprehensive, timely and based on routinely available tumor samples.

Aim: To evaluate the diagnostic potential of targeted multigene next-generation sequencing (TM-NGS) in characterizing gastrointestinal cancer molecular heterogeneity.

Methods: 35 gastrointestinal tract tumors, five of each intestinal type gastric carcinomas, pancreatic ductal adenocarcinomas, pancreatic intraductal papillary mucinous neoplasms, ampulla of Vater carcinomas, hepatocellular carcinomas, cholangiocarcinomas, pancreatic solid pseudopapillary tumors were assessed for mutations in 46 cancer-associated genes, using Ion Torrent semiconductor-based TM-NGS. One ampulla of Vater carcinoma cell line and one hepatic carcinosarcoma served to assess assay sensitivity. TP53, PIK3CA, KRAS, and BRAF mutations were validated by conventional Sanger sequencing.

Results: TM-NGS yielded overlapping results on matched fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissues, with a mutation detection limit of 1% for fresh-frozen high molecular weight DNA and 2% for FFPE partially degraded DNA. At least one somatic mutation was observed in all tumors tested; multiple alterations were detected in 20/35 (57%) tumors. Seven cancers displayed significant differences in allelic frequencies for distinct mutations, indicating the presence of intratumor molecular heterogeneity; this was confirmed on selected samples by immunohistochemistry of p53 and Smad4, showing concordance with mutational analysis.

Conclusions: TM-NGS is able to detect and quantitate multiple gene alterations from limited amounts of DNA, moving one step closer to a next-generation histopathologic diagnosis that integrates morphologic, immunophenotypic, and multigene mutational analysis on routinely processed tissues, essential for personalized cancer therapy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0104979PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4134249PMC
December 2015

High-throughput mutation profiling improves diagnostic stratification of sporadic medullary thyroid carcinomas.

Virchows Arch 2014 Jul 15;465(1):73-8. Epub 2014 May 15.

ARC-Net Research Centre, University and Hospital Trust of Verona, 37134, Verona, Italy.

Sporadic medullary thyroid carcinoma (MTC) harbors RET gene somatic mutations in up to 50 % of cases, and RAS family gene mutations occur in about 10 %. A timely and comprehensive characterization of molecular alterations is needed to improve MTC diagnostic stratification and design-tailored therapeutic approaches. Twenty surgically resected sporadic MTCs, previously analyzed for RET mutations by Sanger sequencing using DNA from formalin-fixed paraffin-embedded samples, were investigated for intragenic mutations in 50 cancer-associated genes applying a multigene Ion AmpliSeq next-generation sequencing (NGS) technology. Thirteen (65 %) MTCs harbored a RET mutation; 10 were detected at both Sanger and NGS sequencing, while 3 undetected by Sanger were revealed by NGS. One of the 13 RET-mutated cases also showed an F354L germline mutation in STK11. Of the seven RET wild-type MTCs, four cases (57.1 %) harbored a RAS mutation: three in HRAS (all Q61R) and one in KRAS (G12R). The three remaining MTCs (15 %) resulted as wild-type for all the 50 cancer-related genes. Follow-up was available in all but one RET-mutated case. At the end of follow-up, 7 of 12 (58 %) RET-mutated patients had relapsed, while the 4 RAS-mutated MTC patients were disease-free. Two of the three patients with MTC wild-type for all 50 genes relapsed during the follow-up period. Detection of mutations by NGS has the potential to improve the diagnostic stratification of sporadic MTC.
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http://dx.doi.org/10.1007/s00428-014-1589-3DOI Listing
July 2014

Molecular typing of lung adenocarcinoma on cytological samples using a multigene next generation sequencing panel.

PLoS One 2013 13;8(11):e80478. Epub 2013 Nov 13.

ARC-NET Research Centre, University of Verona, Verona, Italy ; Department of Pathology and Diagnostics, University of Verona, Verona, Italy.

Identification of driver mutations in lung adenocarcinoma has led to development of targeted agents that are already approved for clinical use or are in clinical trials. Therefore, the number of biomarkers that will be needed to assess is expected to rapidly increase. This calls for the implementation of methods probing the mutational status of multiple genes for inoperable cases, for which limited cytological or bioptic material is available. Cytology specimens from 38 lung adenocarcinomas were subjected to the simultaneous assessment of 504 mutational hotspots of 22 lung cancer-associated genes using 10 nanograms of DNA and Ion Torrent PGM next-generation sequencing. Thirty-six cases were successfully sequenced (95%). In 24/36 cases (67%) at least one mutated gene was observed, including EGFR, KRAS, PIK3CA, BRAF, TP53, PTEN, MET, SMAD4, FGFR3, STK11, MAP2K1. EGFR and KRAS mutations, respectively found in 6/36 (16%) and 10/36 (28%) cases, were mutually exclusive. Nine samples (25%) showed concurrent alterations in different genes. The next-generation sequencing test used is superior to current standard methodologies, as it interrogates multiple genes and requires limited amounts of DNA. Its applicability to routine cytology samples might allow a significant increase in the fraction of lung cancer patients eligible for personalized therapy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0080478PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3827450PMC
July 2014

ICAT is a novel Ptf1a interactor that regulates pancreatic acinar differentiation and displays altered expression in tumours.

Biochem J 2013 May;451(3):395-405

Grupo de Carcinogénesis Epitelial, Programa de Patología Molecular, CNIO-Spanish National Cancer Research Center, 28029 Madrid, Spain.

The PTF1 (pancreas transcription factor 1) complex is a master regulator of differentiation of acinar cells, responsible for the production of digestive enzymes. In the adult pancreas, PTF1 contains two pancreas-restricted transcription factors: Ptf1a and Rbpjl. PTF1 recruits P/CAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor] which acetylates Ptf1a and enhances its transcriptional activity. Using yeast two-hybrid screening, we identified ICAT (inhibitor of β-catenin and Tcf4) as a novel Ptf1a interactor. ICAT regulates the Wnt pathway and cell proliferation. We validated and mapped the ICAT-Ptf1a interaction in vitro and in vivo. We demonstrated that, following its overexpression in acinar tumour cells, ICAT regulates negatively PTF1 activity in vitro and in vivo. This effect was independent of β-catenin and was mediated by direct binding to Ptf1a and displacement of P/CAF. ICAT also modulated the expression of Pdx1 and Sox9 in acinar tumour cells. ICAT overexpression reduced the interaction of Ptf1a with Rbpjl and P/CAF and impaired Ptf1a acetylation by P/CAF. ICAT did not affect the subcellular localization of Ptf1a. In human pancreas, ICAT displayed a cell-type-specific distribution; in acinar and endocrine cells, it was nuclear, whereas in ductal cells, it was cytoplasmic. In ductal adenocarcinomas, ICAT displayed mainly a nuclear or mixed distribution and the former was an independent marker of survival. ICAT regulates acinar differentiation and it does so through a novel Wnt pathway-independent mechanism that may contribute to pancreatic disease.
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http://dx.doi.org/10.1042/BJ20120873DOI Listing
May 2013

Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes.

Nature 2012 Nov 24;491(7424):399-405. Epub 2012 Oct 24.

The Kinghorn Cancer Centre, 370 Victoria Street, Darlinghurst, Sydney, New South Wales 2010, Australia.

Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.
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http://dx.doi.org/10.1038/nature11547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3530898PMC
November 2012

Application of microfluidic technology to the BIOMED-2 protocol for detection of B-cell clonality.

J Mol Diagn 2012 Jan 24;14(1):30-7. Epub 2011 Oct 24.

Department of Pathology and Diagnostics, Section of Anatomic Pathology, University of Verona, Verona, Italy.

The BIOMED-2 protocol is widely used for detecting clonality in lymphoproliferative disorders. The protocol requires multiple PCR reactions, which are analyzed by either capillary electrophoresis (GeneScan analysis) or heteroduplex PAGE analysis. We tested a microfluidic chip-based electrophoresis device (Agilent 2100 Bioanalyzer) for the analysis of B-cell clonality using PCR for the three framework subregions (FR) of the Ig heavy chain gene (IGH) and PCR for two rearrangements occurring in the Ig κ chain gene (IGK-VJ and IGK-DE). We analyzed 62 B-cell lymphomas (33 follicular and 29 nonfollicular) and 16 reactive lymph nodes. Chip-based electrophoresis was conclusive for monoclonality in 59/62 samples; for 20 samples, it was compared with GeneScan analysis. Concordant results were obtained in 45/55 IGH (FR1, FR2, and FR3) gene rearrangements, and in 34/37 IGK gene rearrangements. However, when the chip device was used to analyze selected IGK gene rearrangements (biallelic IGK rearrangements or IGK rearrangements in a polyclonal background), its performance was not completely accurate. We conclude, therefore, that this microfluidic chip-based electrophoresis device is reliable for testing cases with dominant PCR products but is less sensitive than GeneScan in detecting clonal peaks in a polyclonal background for IGH PCR, or with complex IGK rearrangement patterns.
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http://dx.doi.org/10.1016/j.jmoldx.2011.07.007DOI Listing
January 2012

MEN1 in pancreatic endocrine tumors: analysis of gene and protein status in 169 sporadic neoplasms reveals alterations in the vast majority of cases.

Endocr Relat Cancer 2010 Sep 16;17(3):771-83. Epub 2010 Aug 16.

ARC-NET Research Center Department of Pathology, University of Verona, Policlinico G.B. Rossi c/o Piastra Odontoiatrica, Piazzale L.A. Scuro 10, Verona, Italy.

Pancreatic endocrine tumors (PETs) may be part of hereditary multiple endocrine neoplasia type 1 (MEN1) syndrome. While MEN1 gene mutation is the only ascertained genetic anomaly described in PETs, no data exist on the cellular localization of MEN1-encoded protein, menin, in normal pancreas and PETs. A total of 169 PETs were used to assess the i) MEN1 gene mutational status in 100 clinically sporadic PETs by direct DNA sequencing, ii) immunohistochemical expression of menin in normal pancreas and 140 PETs, including 71 cases screened for gene mutations, and iii) correlation of these findings with clinical-pathological parameters. Twenty-seven PETs showed mutations that were somatic in 25 patients and revealed to be germline in 2 patients. Menin immunostaining showed strong nuclear and very faint cytoplasmic signal in normal islet cells, whereas it displayed abnormal location and expression levels in 80% of tumors. PETs harboring MEN1 truncating mutations lacked nuclear protein, and most PETs with MEN1 missense mutations showed a strong cytoplasmic positivity for menin. Menin was also misplaced in a significant number of cases lacking MEN1 mutations. In conclusion, the vast majority of PETs showed qualitative and/or quantitative alterations in menin localization. In 30% of cases, this was associated with MEN1 mutations affecting sequences involved in nuclear localization or protein-protein interaction. In cases lacking MEN1 mutations, the alteration of one of the menin interactors may have prevented its proper localization, as suggested by recent data showing that menin protein shuttles between the nucleus and cytoplasm and also affects the subcellular localization of its interactors.
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http://dx.doi.org/10.1677/ERC-10-0028DOI Listing
September 2010

Primary mediastinal B-cell lymphoma: hypermutation of the BCL6 gene targets motifs different from those in diffuse large B-cell and follicular lymphomas.

Haematologica 2004 Sep;89(9):1091-9

Dipartimento di Patologia, Sezione di Anatomia Patologica Università di Verona, Strada Le Grazie I-37134 Verona, Italy.

Background And Objectives: Somatic hypermutation of the BCL6 gene and its expression in lymphoma represent specific markers for B-cell transit through the germinal center. Thus, analysis of BCL6 may aid in clarifying the relationship between primary mediastinal B-cell lymphoma (PMBL) and other non-thymic diffuse large cell lymphomas (DLCL).

Design And Methods: Twenty-four PMBL were analyzed for BCL6 status, including first intron mutations, by quantitative reverse transcription polymerase chain reaction (RT-PCR), and immunohistochemistry. We also performed a meta-analysis of reported BCL6 mutations in PMBL (n=141), DLCL (n=233), and follicular lymphoma (n=120).

Results: Thirteen PMBL (54%) showed hypermutation of BCL6. All cases showed bcl6 mRNA and immunohistochemical expression. Meta-analysis demonstrated that the preferentially altered sequence motifs of BCL6 in PMBL were TA (p=0.002) and AT (p=0.0008) dinucleotides and TAT trinucleotides (p=0.001). GC and RGYW/WRCY motifs were a target in DLCL and FL but not in PMBL. Moreover, the DNA stretch spanning nucleotides 150-270 was highly targeted only in PMBL.

Interpretation And Conclusions: The consistent expression of bcl6 protein and occurrence of hypermutation indicate that PMBL should be considered of germinal center origin. The fact that the hypermutation sites and mutational spectrum of BCL6 in PMBL differ from those found in FL and DLCL might suggest that the maturation block of the transforming cells differs among these tumor types, and that the characteristic mutational pattern is present before neoplastic transformation. Thus, our findings strengthen the hypothesis that PMBL originate from an already defined sub-population of B-cells, which are different from those leading to either DLCL or FL.
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September 2004