Publications by authors named "Caterina Vicentini"

35 Publications

Alternative Lengthening of Telomeres (ALT) in Pancreatic Neuroendocrine Tumors: Ready for Prime-Time in Clinical Practice?

Curr Oncol Rep 2021 Jul 16;23(9):106. Epub 2021 Jul 16.

Department of Diagnostics and Public Health, Section of Pathology, University and Hospital Trust of Verona, 37134, Verona, Italy.

Purpose Of Review: Alternative lengthening of telomeres (ALT) is a telomerase-independent mechanism used by some types of malignancies, including pancreatic neuroendocrine tumors, to overcome the issue of telomere shortening, thus supporting tumor growth and cell proliferation. This review is focused on the most important achievements and opportunities deriving from ALT assessment in PanNET onco-pathology, highlighting the most promising fields in which such biomarker could be implemented in clinical practice.

Recent Findings: In pancreatic neuroendocrine tumors (PanNET), ALT is strongly correlated with the mutational status of two chromatin remodeling genes, DAXX and ATRX. Recent advances in tumor biology permitted to uncover important roles of ALT in the landscape of PanNET, potentially relevant for introducing this biomarker into clinical practice. Indeed, ALT emerged as a reliable indicator of worse prognosis for PanNET, helping in clinical stratification and identification of "high-risk" patients. Furthermore, it is a very specific marker supporting the pancreatic origin of neuroendocrine neoplasms and can be used for improving the diagnostic workflow of patients presenting with neuroendocrine metastasis from unknown primary. The activation of this process can be determined by specific FISH analysis. ALT should be introduced in clinical practice for identifying "high-risk" PanNET patients and improving their clinical management, and as a marker of pancreatic origin among neuroendocrine tumors.
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http://dx.doi.org/10.1007/s11912-021-01096-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8285324PMC
July 2021

Exosomal miRNA signatures of pancreatic lesions.

BMC Gastroenterol 2020 May 6;20(1):137. Epub 2020 May 6.

ARC-NET Research Centre, University of Verona, Verona, Italy.

Background: Pancreatic and peri-pancreatic neoplasms encompass a variety of histotypes characterized by a heterogeneous prognostic impact. miRNAs are considered efficient candidate biomarkers due to their high stability in tissues and body fluids. We applied Nanostring profiling of circulating exosomal miRNAs to distinct pancreatic lesions in order to establish a source for biomarker development.

Methods: A series of 140 plasma samples obtained from patients affected by pancreatic ductal adenocarcinoma (PDAC, n = 58), pancreatic neuroendocrine tumors (PanNET, n = 42), intraductal papillary mucinous neoplasms (IPMN, n = 20), and ampulla of Vater carcinomas (AVC, n = 20) were analyzed. Comprehensive miRNA profiling was performed on plasma-derived exosomes. Relevant miRNAs were validated by qRT-PCR and in situ hybridization (ISH).

Results: Lesion specific miRNAs were identified through multiple disease comparisons. Selected miRNAs were validated in the plasma by qRT-PCR and at tissue level by ISH. We leveraged the presence of clinical subtypes with each disease cohort to identify miRNAs that are differentially enriched in aggressive phenotypes.

Conclusions: This study shows that pancreatic lesions are characterized by specific exosomal-miRNA signatures. We also provide the basis for further explorations in order to better understand the relevance of these signatures in pancreatic neoplasms.
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http://dx.doi.org/10.1186/s12876-020-01287-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7204029PMC
May 2020

Targeted next-generation sequencing identifies genomic abnormalities potentially driving the prognosis of early-stage invasive lobular breast carcinoma patients stratified according to a validated clinico-pathological model.

Breast 2020 Apr 27;50:56-63. Epub 2020 Jan 27.

Oncologia Medica, Fondazione Policlinico Universitario A. Gemelli IRCCS, Università Cattolica Del Sacro Cuore, Roma, Italy. Electronic address:

Introduction: The clinico-pathological and molecular factors that drive the prognosis of invasive lobular breast carcinoma (ILC) are not entirely explored. In this regard, the development and validation of a prognostic model for ILC and the investigation of the distribution of molecular abnormalities (focusing on CDK4/6 alterations) according to prognosis were the aims of this study.

Patients And Methods: Two clinico-pathological multi-center data-sets of early-stage ILC patients (Training/Validation Set, TS/VS) were gathered. A 3-class model was developed according to the multivariate analysis for disease-free-survival (DFS) and externally validated. Mutational, copy number variation and transcriptomic analyses by targeted next generation sequencing (NGS) were performed (and validated with quantitative PCR) in an explorative cohort of patients with poor and good prognosis.

Results: Data from overall 773 patients (TS/VS: 491/282) were gathered. The developed model significantly discriminated low/intermediate/high risk in the TS (10-years DFS: 76.3%/67.6%/39.8%, respectively, p<0.0001) and in the VS (p<0.0001). In the explorative cohort for molecular analysis (34 patients), CDK4 gain was present exclusively in the poor prognosis group (35.0%, p = 0.03; OR 7.98, 95%CI 1.51-42.1, p = 0.014). Moreover, CDK4 and 6 overexpression showed a trend toward an association with poor prognosis (OR 2.7, 95%CI 0.4-18.1, p = 0.3; OR 3.29, 95%CI 0.56-19.25, p = 0.18).

Conclusions: A risk stratification model, able to accurately separate early-stage ILC patients' prognosis into different risk classes according to clinico-pathological variables, allowed to investigate potential biomarkers of prognosis with targeted NGS. CDK4 gain is suggested for future validation as a prognostic biomarker and a potential therapeutic opportunity in ILC patients.
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http://dx.doi.org/10.1016/j.breast.2020.01.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7375560PMC
April 2020

Current role of non-coding RNAs in the clinical setting.

Noncoding RNA Res 2019 Sep 17;4(3):82-85. Epub 2019 Sep 17.

Department of Medicine (DIMED), University of Padua, Padua, PD, Italy.

Non-coding RNAs (ncRNAs) have long been considered as "junk" material of the human genome until functional studies have exposed them as critical regulators of gene expression in both physiological and pathological conditions. Mounting evidences have also shown that ncRNAs may serve as diagnostic markers for several disorders, predictor for drugs response, and targets for new therapeutic approaches. In this mini-review, we discuss the state of the art of non-coding RNAs in drug development and their involvement in conventional treatments response.
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http://dx.doi.org/10.1016/j.ncrna.2019.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926199PMC
September 2019

Modulation of Biliary Cancer Chemo-Resistance Through MicroRNA-Mediated Rewiring of the Expansion of CD133+ Cells.

Hepatology 2020 09 10;72(3):982-996. Epub 2020 Sep 10.

Medical Oncology and Hematology Unit, Humanitas Cancer Center, Humanitas Clinical and Research Center-IRCCS, Rozzano, Italy.

Background And Aims: Changes in single microRNA (miRNA) expression have been associated with chemo-resistance in biliary tract cancers (BTCs). However, a global assessment of the dynamic role of the microRNome has never been performed to identify potential therapeutic targets that are functionally relevant in the BTC cell response to chemotherapy.

Approach And Results: High-throughput screening (HTS) of 997 locked nucleic acid miRNA inhibitors was performed in six cholangiocarcinoma cell lines treated with cisplatin and gemcitabine (CG) seeking changes in cell viability. Validation experiments were performed with mirVana probes. MicroRNA and gene expression was assessed by TaqMan assay, RNA-sequencing, and in situ hybridization in four independent cohorts of human BTCs. Knockout of microRNA was achieved by CRISPR-CAS9 in CCLP cells (MIR1249KO) and tested for effects on chemotherapy sensitivity in vitro and in vivo. HTS revealed that MIR1249 inhibition enhanced chemotherapy sensitivity across all cell lines. MIR1249 expression was increased in 41% of cases in human BTCs. In validation experiments, MIR1249 inhibition did not alter cell viability in untreated or dimethyl sulfoxide-treated cells; however, it did increase the CG effect. MIR1249 expression was increased in CD133+ biliary cancer cells freshly isolated from the stem cell niche of human BTCs as well as in CD133+ chemo-resistant CCLP cells. MIR1249 modulated the chemotherapy-induced enrichment of CD133+ cells by controlling their clonal expansion through the Wnt-regulator FZD8. MIR1249KO cells had impaired expansion of the CD133+ subclone and its enrichment after chemotherapy, reduced expression of cancer stem cell markers, and increased chemosensitivity. MIR1249KO xenograft BTC models showed tumor shrinkage after exposure to weekly CG, whereas wild-type models showed only stable disease over treatment.

Conclusions: MIR1249 mediates resistance to CG in BTCs and may be tested as a target for therapeutics.
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http://dx.doi.org/10.1002/hep.31094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7590111PMC
September 2020

Comparative Lesions Analysis Through a Targeted Sequencing Approach.

J Vis Exp 2019 11 5(153). Epub 2019 Nov 5.

ARC-Net Research Centre, University and Hospital Trust of Verona; Department of Diagnostics and Public Health, Section of Pathology, University and Hospital Trust of Verona;

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective anti-tumor strategies. Although concerns are frequently raised due to differences in sample processing and depth of coverage, next-generation sequencing of solid tumors have unraveled a highly variable degree of ITH across tumor types. Capturing the genetic relatedness between primary and metastatic lesions through the identification of clonal and subclonal populations is critical to the design of therapies for advance-stage diseases. Here, we report a method for comparative lesions analysis that allows for the identification of clonal and subclonal populations among different specimens from the same patient. The experimental approach described here integrates three well-established approaches: histological analysis, high-coverage multi-lesion sequencing, and immunophenotypic analyses. In order to minimize the effects on the detection of subclonal events by inappropriate sample processing, we subjected tissues to careful pathological examination and neoplastic cell enrichment. Quality controlled DNA from neoplastic lesions and normal tissues was then subjected to high coverage sequencing, targeting the coding regions of 409 relevant cancer genes. While only looking at a limited genomic space, our approach enables evaluating the extent of heterogeneity among somatic alterations (single-nucleotide mutations and copy-number variations) in distinct lesions from a given patient. Through comparative analysis of sequencing data, we were able to distinguish clonal vs. subclonal alterations. The majority of ITH is often ascribed to passenger mutations; therefore, we also used immunohistochemistry to predict functional consequences of mutations. While this protocol has been applied to a specific tumor type, we anticipate that the methodology described here is broadly applicable to other solid tumor types.
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http://dx.doi.org/10.3791/59844DOI Listing
November 2019

Gene Expression Profiling of Lung Atypical Carcinoids and Large Cell Neuroendocrine Carcinomas Identifies Three Transcriptomic Subtypes with Specific Genomic Alterations.

J Thorac Oncol 2019 09 11;14(9):1651-1661. Epub 2019 May 11.

Comprehensive Cancer Center, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy; Sacred Hearth Catholic University, Rome, Italy.

Introduction: DNA mutational profiling showed that atypical carcinoids (ACs) share alterations with large cell neuroendocrine carcinomas (LCNECs). Transcriptomic studies suggested that LCNECs are composed of two subtypes, one of which shares molecular anomalies with SCLC. The missing piece of information is the transcriptomic relationship between ACs and LCNECs, as a direct comparison is lacking in the literature.

Methods: Transcriptomic and genomic alterations were investigated by next-generation sequencing in a discovery set of 14 ACs and 14 LCNECs and validated on 21 ACs and 18 LCNECs by using custom gene panels and immunohistochemistry for Men1 and Rb1.

Results: A 58-gene signature distinguished three transcriptional clusters. Cluster 1 comprised 20 LCNECs and one AC harboring concurrent inactivation of tumor protein p53 gene (TP53) and retinoblastoma 1 gene (RB1) in the absence of menin 1 gene (MEN1) mutations; all cases lacked Rb1 nuclear immunostaining. Cluster 3 included 20 ACs and four LCNECs lacking RB1 alterations and having frequent MEN1 (37.5%) and TP53 mutations (16.7%); menin nuclear immunostaining was lost in 75% of cases. Cluster 2 included 14 ACs and eight LCNECs showing intermediate features: TP53, 40.9%; MEN1, 22.7%; and RB1, 18.2%. Patients in cluster C1 had a shorter cancer-specific survival than did patients in C2 or C3.

Conclusions: ACs and LCNECs comprise three different and clinically relevant molecular diseases, one AC-enriched group in which MEN1 inactivation plays a major role, one LCNEC-enriched group whose hallmark is RB1 inactivation, and one mixed group with intermediate molecular features. These data support a progression of malignancy that may be traced by using combined molecular and immunohistochemical analysis.
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http://dx.doi.org/10.1016/j.jtho.2019.05.003DOI Listing
September 2019

miR-224 Is Significantly Upregulated and Targets Caspase-3 and Caspase-7 During Colorectal Carcinogenesis.

Transl Oncol 2019 Feb 16;12(2):282-291. Epub 2018 Nov 16.

Department of Medicine (DIMED), University of Padua, Padua, Italy.

miR-224 has recently emerged as a driver oncomiR in sporadic colorectal carcinogenesis, but its pathogenetic role is still controversial. A large phenotypical and molecularly characterized series of preinvasive and invasive colorectal lesions was investigated for miR-224 expression by qRT-PCR and in situ hybridization. The caspase-3 and caspase-7 status was also assessed and correlated to miR-224 dysregulation. miR-224 was significantly upregulated during the adenoma-carcinoma sequence and in the context of inflammatory bowel disease dysplastic lesions, whereas its expression was significantly downregulated among BRAF-mutated tumors and in the presence of a DNA mismatch repair deficiency. miR-224 targets caspase-3 and caspase-7 in colorectal cancer, and this inverse relation was already evident from the earliest phases of transformation in intestinal mucosa. The miR-224/caspases axis may represent an interesting field of study for innovative biomarkers/therapeutics for BRAF-mutated/DNA mismatch repair-deficient tumors.
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http://dx.doi.org/10.1016/j.tranon.2018.10.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6240712PMC
February 2019

Molecular alterations associated with metastases of solid pseudopapillary neoplasms of the pancreas.

J Pathol 2019 01 27;247(1):123-134. Epub 2018 Nov 27.

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

Solid pseudopapillary neoplasms (SPN) of the pancreas are rare, low-grade malignant neoplasms that metastasise to the liver or peritoneum in 10-15% of cases. They almost invariably present somatic activating mutations of CTNNB1. No comprehensive molecular characterisation of metastatic disease has been conducted to date. We performed whole-exome sequencing and copy-number variation (CNV) analysis of 10 primary SPN and comparative sequencing of five matched primary/metastatic tumour specimens by high-coverage targeted sequencing of 409 genes. In addition to CTNNB1-activating mutations, we found inactivating mutations of epigenetic regulators (KDM6A, TET1, BAP1) associated with metastatic disease. Most of these alterations were shared between primary and metastatic lesions, suggesting that they occurred before dissemination. Differently from mutations, the majority of CNVs were not shared among lesions from the same patients and affected genes involved in metabolic and pro-proliferative pathways. Immunostaining of 27 SPNs showed that loss or reduction of KDM6A and BAP1 expression was significantly enriched in metastatic SPNs. Consistent with an increased transcriptional response to hypoxia in pancreatic adenocarcinomas bearing KDM6A inactivation, we showed that mutation or reduced KDM6A expression in SPNs is associated with increased expression of the HIF1α-regulated protein GLUT1 at both primary and metastatic sites. Our results suggest that BAP1 and KDM6A function is a barrier to the development of metastasis in a subset of SPNs, which might open novel avenues for the treatment of this disease. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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http://dx.doi.org/10.1002/path.5180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588017PMC
January 2019

Mutational and copy number asset of primary sporadic neuroendocrine tumors of the small intestine.

Virchows Arch 2018 Dec 16;473(6):709-717. Epub 2018 Sep 16.

ARC-Net Research Centre, University and Hospital Trust of Verona, Policlinico GB Rossi, Piazzale L.A. Scuro, 10, Piastra Odontoiatrica (II floor), Verona, Italy.

Small intestine neuroendocrine tumors (SI-NETs) represent the most common histotype among small intestine neoplasms, and metastatic disease is usually present at diagnosis. A retrospective series of 52 sporadic primary surgically resected SI-NETs, which were metastatic at diagnosis, was analyzed by high-coverage target sequencing (HCTS) for the mutational status of 57 genes and copy number status of 40 genes selected from recently published genome sequencing data. Seven genes were found to be recurrently mutated: CDKN1B (9.6%), APC and CDKN2C (each 7.7%), BRAF, KRAS, PIK3CA, and TP53 (each 3.8%). Copy number analysis showed frequent allelic loss of 4 genes located on chromosome 18 (BCL2, CDH19, DCC, and SMAD4) in 23/52 (44.2%) and losses on chromosomes 11 (38%) and 16 (15%). Other recurrent copy number variations were gains for genes located on chromosomes 4 (31%), 5 (27%), 14 (36%), and 20 (20%). Univariate survival analysis showed that SRC gene copy number gains were associated with a poorer prognosis (p = 0.047). Recurrent copy number variations are important events in SI-NET and SRC may represent a novel prognostic biomarker for this tumor type.
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http://dx.doi.org/10.1007/s00428-018-2450-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6267237PMC
December 2018

ERG alterations and mTOR pathway activation in primary prostate carcinomas developing castration-resistance.

Pathol Res Pract 2018 Oct 29;214(10):1675-1680. Epub 2018 Aug 29.

ARC-NET Research Centre, University of Verona, Verona, Italy.

Introduction: One of the most common sites of distant metastasization of prostate cancer is bone, but to date reliable biomarkers able to predict the risk and timing of bone metastasization are still lacking.

Patients And Methods: Surgically resected paraffin embedded samples from 12 primary prostate cancers that developed metachronous bone metastasis at different time points were studied (six cases within 2 years, six cases after 5 years from surgery). A targeted next-generation DNA and RNA sequencing able to assess simultaneously mutations, copy number alterations and fusion events of multiple genes was used. Immunohistochemistry was used to assess mTOR pathway activation.

Results: Rearrangements of ETS family genes, molecular alterations in PTEN and TP53 genes were detected in 10, 6 and 5 cancers, respectively. Nine samples showed TMPRSS2-ERG fusions, which were associated with increased ERG expression at immunohistochemistry. mTOR pathway activation was documented in 6 patients, with a clear trend of prevalence in late-metastatic patients (p = 0.08).

Conclusions: A simultaneous next-generation targeted DNA and RNA sequencing is applicable on routine formalin-fixed paraffin-embedded tissues to assess the multigene molecular asset of individual prostate cancers. This approach, coupled with immunohistochemistry for ERG and mTOR pathway proteins, may help to better characterize prostate cancer molecular features with a potential impact on clinical decisions.
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http://dx.doi.org/10.1016/j.prp.2018.08.031DOI Listing
October 2018

Unmasking the impact of Rictor in cancer: novel insights of mTORC2 complex.

Carcinogenesis 2018 07;39(8):971-980

Section of Pathology, Department of Diagnostics and Public Health, University of Verona, Verona, Italy.

Genomic alterations affecting components of the mechanistic target of rapamycin (mTOR) pathway are found rather frequently in cancers, suggesting that aberrant pathway activity is implicated in oncogenesis of different tumor types. mTOR functions as the core catalytic kinase of two distinct complexes, mTOR complex 1 (mTORC1) and 2 (mTORC2), which control numerous vital cellular processes. There is growing evidence indicating that Rictor, an essential subunit of the mTORC2 complex, is inappropriately overexpressed across numerous cancer types and this is associated with poor survival. To date, the candidate mechanisms responsible for aberrant Rictor expression described in cancer are two: (i) gene amplification and (ii) epigenetic regulation, mainly by microRNAs. Moreover, different mTOR-independent Rictor-containing complexes with oncogenic role have been documented, revealing alternative routes of Rictor-driven tumorigenesis, but simultaneously, paving the way for identifying novel biomarkers and therapeutic targets. Here, we review the main preclinical and clinical data regarding the role of Rictor in carcinogenesis and metastatic behavior as well as the potentiality of its alteration as a target.
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http://dx.doi.org/10.1093/carcin/bgy086DOI Listing
July 2018

Genetic alterations analysis in prognostic stratified groups identified TP53 and ARID1A as poor clinical performance markers in intrahepatic cholangiocarcinoma.

Sci Rep 2018 05 8;8(1):7119. Epub 2018 May 8.

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

The incidence and mortality rates of intrahepatic cholangiocarcinoma have been rising worldwide. Few patients present an early-stage disease that is amenable to curative surgery and after resection, high recurrence rates persist. To identify new independent marker related to aggressive behaviour, two prognostic groups of patient were selected and divided according to prognostic performance. All patients alive at 36 months were included in good prognostic performers, while all patients died due to disease within 36 months in poor prognostic performers. Using high-coverage target sequencing we analysed principal genetic alterations in two groups and compared results to clinical data. In the 33 cases included in poor prognosis group, TP53 was most mutated gene (p = 0.011) and exclusively present in these cases. Similarly, ARID1A was exclusive of this group (p = 0.024). TP53 and ARID1A are mutually exclusive in this study. Statistical analysis showed mutations in TP53 and ARID1A genes and amplification of MET gene as independent predictors of poor prognosis (TP53, p = 0.0031, ARID1A, p = 0.0007, MET, p = 0.0003 in Cox analysis). LOH in PTEN was also identified as marker of disease recurrence (p = 0.04) in univariate analysis. This work improves our understanding of aggressiveness related to this tumour type and has identified novel prognostic markers of clinical outcome.
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http://dx.doi.org/10.1038/s41598-018-25669-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5940669PMC
May 2018

Fhit down-regulation is an early event in pancreatic carcinogenesis.

Virchows Arch 2017 Jun 13;470(6):647-653. Epub 2017 Mar 13.

Comprehensive Cancer Center, Department of Cancer Biology and Genetics, The Ohio State University, Columbus, OH, USA.

Aberrant Fhit expression characterizes a large proportion of primary pancreatic ductal adenocarcinomas (PDACs), but fragmentary information is available on Fhit expression during the phenotypic changes of pancreatic ductal epithelium during multistep transformation. We assessed Fhit expression by immunohistochemistry in two different multistep pancreatic carcinogenic processes: pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasia (IPMN). We considered 105 surgically treated PDACs/IPMNs and selected 30 samples of non-neoplastic pancreatic parenchyma, 50 PanIN lesions, 30 IPMNs, 15 IPMNs with associated invasive carcinoma, and 60 adenocarcinomas. Normal pancreatic ducts and surrounding acinar cells consistently showed moderate to strong Fhit immunoreactivity. Significant down-regulation of Fhit expression was observed in association with increasing severity of dysplastia/neoplastia in both carcinogenic processes. This was further confirmed by studying multiple lesions obtained from the same surgical specimen. Of 60 PDACs, only 14 showed Fhit expression comparable to normal pancreatic ductal epithelium, while the remainder (77%) showed clearly negative or reduced Fhit expression. This study demonstrates that Fhit down-regulation is an early event in both multistep carcinogenic processes leading to PDAC.
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http://dx.doi.org/10.1007/s00428-017-2105-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5568551PMC
June 2017

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

Identification of microRNAs implicated in the late differentiation stages of normal B cells suggests a central role for miRNA targets ZEB1 and TP53.

Oncotarget 2017 Feb;8(7):11809-11826

Department of Diagnostics and Public Health, Section of Pathological Anatomy, University of Verona, Verona, Italy.

In the late B cell differentiation stages, miRNAs expression modifications promoting or inhibiting key pathways are only partially defined. We isolated 29 CD19+ human B cell samples at different stages of differentiation: B cells from peripheral blood; naïve, germinal center (GC) and subepithelial (SE) B cells from tonsils. SE cells were further split in activated and resting B cell. The miRNA expression profile of these B cells was assessed by microarray analysis and selected miRNAs were validated by quantitative RT-PCR and in situ hybridization on normal tonsils. The comparison of all samples showed changes in 107 miRNAs in total. Among 48 miRNAs differentially expressed in naïve, GC and SE cells, we identified 8 miRNAs: mir-323, mir-138, mir-9*, mir-211, mir-149, mir-373, mir-135a and mir-184, strictly specific to follicular cells that had never been implicated before in late stages of B cell development. Moreover, we unveiled 34 miRNAs able to discriminate between CD5- activated B cells and resting B cells. The miRNAs profile of CD5- resting B cells showed a higher similarity to naïve CD5+ than CD5- activated B cells. Finally, network analysis on shortest paths connecting gene targets suggested ZEB1 and TP53 as key miRNA targets during the follicular differentiation pathway. These data confirm and extend our knowledge on the miRNAs-related regulatory pathways involved in the late B cell maturation.
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http://dx.doi.org/10.18632/oncotarget.14683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5355306PMC
February 2017

Lung neuroendocrine tumours: deep sequencing of the four World Health Organization histotypes reveals chromatin-remodelling genes as major players and a prognostic role for TERT, RB1, MEN1 and KMT2D.

J Pathol 2017 03 29;241(4):488-500. Epub 2016 Dec 29.

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

Next-generation sequencing (NGS) was applied to 148 lung neuroendocrine tumours (LNETs) comprising the four World Health Organization classification categories: 53 typical carcinoid (TCs), 35 atypical carcinoid (ACs), 27 large-cell neuroendocrine carcinomas, and 33 small-cell lung carcinomas. A discovery screen was conducted on 46 samples by the use of whole-exome sequencing and high-coverage targeted sequencing of 418 genes. Eighty-eight recurrently mutated genes from both the discovery screen and current literature were verified in the 46 cases of the discovery screen, and validated on additional 102 LNETs by targeted NGS; their prevalence was then evaluated on the whole series. Thirteen of these 88 genes were also evaluated for copy number alterations (CNAs). Carcinoids and carcinomas shared most of the altered genes but with different prevalence rates. When mutations and copy number changes were combined, MEN1 alterations were almost exclusive to carcinoids, whereas alterations of TP53 and RB1 cell cycle regulation genes and PI3K/AKT/mTOR pathway genes were significantly enriched in carcinomas. Conversely, mutations in chromatin-remodelling genes, including those encoding histone modifiers and members of SWI-SNF complexes, were found at similar rates in carcinoids (45.5%) and carcinomas (55.0%), suggesting a major role in LNET pathogenesis. One AC and one TC showed a hypermutated profile associated with a POLQ damaging mutation. There were fewer CNAs in carcinoids than in carcinomas; however ACs showed a hybrid pattern, whereby gains of TERT, SDHA, RICTOR, PIK3CA, MYCL and SRC were found at rates similar to those in carcinomas, whereas the MEN1 loss rate mirrored that of TCs. Multivariate survival analysis revealed RB1 mutation (p = 0.0005) and TERT copy gain (p = 0.016) as independent predictors of poorer prognosis. MEN1 mutation was associated with poor prognosis in AC (p = 0.0045), whereas KMT2D mutation correlated with longer survival in SCLC (p = 0.0022). In conclusion, molecular profiling may complement histology for better diagnostic definition and prognostic stratification of LNETs. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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http://dx.doi.org/10.1002/path.4853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5324596PMC
March 2017

ERK Activation Globally Downregulates miRNAs through Phosphorylating Exportin-5.

Cancer Cell 2016 Nov;30(5):723-736

Department of Cancer Biology and Genetics, Ohio State University, Columbus, OH 43210, USA. Electronic address:

MicroRNAs (miRNA) are mostly downregulated in cancer. However, the mechanism underlying this phenomenon and the precise consequence in tumorigenesis remain obscure. Here we show that ERK suppresses pre-miRNA export from the nucleus through phosphorylation of exportin-5 (XPO5) at T345/S416/S497. After phosphorylation by ERK, conformation of XPO5 is altered by prolyl isomerase Pin1, resulting in reduction of pre-miRNA loading. In liver cancer, the ERK-mediated XPO5 suppression reduces miR-122, increases microtubule dynamics, and results in tumor development and drug resistance. Analysis of clinical specimens further showed that XPO5 phosphorylation is associated with poor prognosis for liver cancer patients. Our study reveals a function of ERK in miRNA biogenesis and suggests that modulation of miRNA export has potential clinical implications.
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http://dx.doi.org/10.1016/j.ccell.2016.10.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127275PMC
November 2016

Wnt signalling modulates transcribed-ultraconserved regions in hepatobiliary cancers.

Gut 2017 07 12;66(7):1268-1277. Epub 2016 Sep 12.

The Institute of Cancer Research, London, UK.

Objective: Transcribed-ultraconserved regions (T-UCR) are long non-coding RNAs which are conserved across species and are involved in carcinogenesis. We studied T-UCRs downstream of the Wnt/β-catenin pathway in liver cancer.

Design: Hypomorphic Apc mice () and thiocetamide (TAA)-treated rats developed Wnt/β-catenin dependent hepatocarcinoma (HCC) and cholangiocarcinoma (CCA), respectively. T-UCR expression was assessed by microarray, real-time PCR and in situ hybridisation.

Results: Overexpression of the T-UCR uc.158- could differentiate Wnt/β-catenin dependent HCC from normal liver and from β-catenin negative diethylnitrosamine (DEN)-induced HCC. uc.158- was overexpressed in human HepG2 versus Huh7 cells in line with activation of the Wnt pathway. In vitro modulation of β-catenin altered uc.158- expression in human malignant hepatocytes. uc.158- expression was increased in -mutated human HCCs compared with non-mutated human HCCs, and in human HCC with nuclear localisation of β-catenin. uc.158- was increased in TAA rat CCA and reduced after treatment with Wnt/β-catenin inhibitors. uc.158- expression was negative in human normal liver and biliary epithelia, while it was increased in human CCA in two different cohorts. Locked nucleic acid-mediated inhibition of uc.158- reduced anchorage cell growth, 3D-spheroid formation and spheroid-based cell migration, and increased apoptosis in HepG2 and SW1 cells. miR-193b was predicted to have binding sites within the uc.158- sequence. Modulation of uc.158- changed miR-193b expression in human malignant hepatocytes. Co-transfection of uc.158- inhibitor and anti-miR-193b rescued the effect of uc.158- inhibition on cell viability.

Conclusions: We showed that uc.158- is activated by the Wnt pathway in liver cancers and drives their growth. Thus, it may represent a promising target for the development of novel therapeutics.
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http://dx.doi.org/10.1136/gutjnl-2016-312278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5530482PMC
July 2017

RASSF1 tumor suppressor gene in pancreatic ductal adenocarcinoma: correlation of expression, chromosomal status and epigenetic changes.

BMC Cancer 2016 Jan 12;16:11. Epub 2016 Jan 12.

ARC-NET Centre for Applied Research on Cancer, Department of Pathology and Diagnostics, The Hospital and University of Verona, Verona, Italy.

Background: The Ras Association Domain Family Member 1 (RASSF1) is one of the most frequently reported methylation-inactivated tumor suppressor genes in primary pancreatic ductal adenocarcinomas (PDAC). Limited information is still available about the impact of RASSF1 gene silencing on the expression of its different isoforms in neoplastic cells.

Methods: A series of 96 primary PDAC, with known clinico-pathological parameters, was tested for RASSF1 methylation status by methylation-specific PCR, RASSF1 locus copy number alterations by fluorescence in situ hybridization, and Rassf1a protein expression by immunohistochemistry. A further series of 14 xenografted primary PDAC and 8 PDAC-derived cell lines were tested to obtain a detailed methylation mapping of CpG islands A and C of the RASSF1 locus by pyrosequencing and to evaluate the expression of Rassf1 variants by qRT-PCR.

Results: Methylation of CpG island A of the RASSF1 gene was observed in 35% of the tumors and allelic loss of RASSF1 locus was seen in 30 disomic and in 20 polysomic cases (52%). Rassf1a immunohistochemical expression was downregulated in half of primary PDAC, and this downregulation was neither correlated with methylation of RASSF1 promoter nor with RASSF1 copy number alterations. RASSF1 status did not influence patients' prognosis. The expression of the seven RASSF1 isoforms in xenografts and cell lines showed that RASSF1A, RASSF1B, and RASSF1C isoforms were present in all xenografts and cell lines, whereas RASSF1D, RASSF1E, and RASSF1F isoforms were variably expressed among samples. RASSF1G was never expressed in either xenografts or cell lines. The variable expression of RASSF1 isoforms in PDAC xenografts and cell lines was not dependent on RASSF1 methylation status of CpG islands A and C.

Conclusions: RASSF1 alterations occurring in PDAC mainly consist in variations of expression of the different isoforms. Different genetic mechanisms seem to contribute to RASSF1 deregulation in this setting, but RASSF1 methylation does not seem to substantially affect RASSF1 isoforms expression.
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http://dx.doi.org/10.1186/s12885-016-2048-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4710004PMC
January 2016

Monosomy of chromosome 17 in breast cancer during interpretation of HER2 gene amplification.

Am J Cancer Res 2015 15;5(7):2212-21. Epub 2015 Jun 15.

Department of Pathology and Diagnostic, Anatomic Pathology, University and Hospital Trust Verona, Italy.

Monosomy of chromosome 17 may affect the assessment of HER2 amplification. Notably, the prevalence ranges from 1% up to 49% due to lack of consensus in recognition. We sought to investigate the impact of monosomy of chromosome 17 to interpretation of HER2 gene status. 201 breast carcinoma were reviewed for HER2 gene amplification and chromosome 17 status. FISH analysis was performed by using double probes (LSI/CEP). Absolute gene copy number was also scored per each probe. HER2 FISH test was repeated on serial tissue sections, ranging in thickness from 3 to 20 µm. Ratio was scored and subsequently corrected by monosomy after gold control test using the aCGH method to overcome false interpretation due to artefactual nuclear truncation. HER2 immunotests was performed on all cases. 26/201 cases were amplified (13%). Single signals per CEP17 were revealed in 7/201 (3.5%) cases. Five out of 7 cases appeared monosomic with aCGH (overall, 5/201, 2.5%) and evidenced single signals in >60% of nuclei after second-look on FISH when matching both techniques. Among 5, one case showed amplification with a pattern 7/1 (HER2/CEP17>2) of copies (3+ at immunotest); three cases revealed single signals per both probes (LSI/CEP=1) and one case revealed a 3:1 ratio; all last 4 cases showed 0/1+ immunoscore. We concluded that: 1) monosomy of chromosome 17 may be observed in 2.5% of breast carcinoma; 2) monosomy of chromosome 17 due to biological reasons rather than nuclear truncation was observed when using the cut-off of 60% of nuclei harboring single signals; 3) the skewing of the ratio due to single centromeric 17 probe may lead to false positive evaluation; 4) breast carcinomas showing a 3:1 ratio (HER2/CEP17) usually show negative 0/1+ immunoscore and <6 gene copy number at FISH.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4548332PMC
September 2015

miR-15b/16-2 deletion promotes B-cell malignancies.

Proc Natl Acad Sci U S A 2015 Sep 31;112(37):11636-41. Epub 2015 Aug 31.

Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, OH 43210;

The central role of the microRNA (miR) 15a/16-1 cluster in B-cell oncogenesis has been extensively demonstrated, with over two-thirds of B-cell chronic lymphocytic leukemia characterized by the deletion of the miR-15a/16-1 locus at 13q14. Despite the well-established understanding of the molecular mechanisms occurring during miR-15a/16-1 dysregulation, the oncogenic role of other miR-15/16 family members, such as the miR-15b/16-2 cluster (3q25), is still far from being elucidated. Whereas miR-15a is highly similar to miR-15b, miR-16-1 is identical to miR-16-2; thus, it could be speculated that both clusters control a similar set of target genes and may have overlapping functions. However, the biological role of miR-15b/16-2 is still controversial. We generated miR-15b/16-2 knockout mice to better understand the cluster's role in vivo. These mice developed B-cell malignancy by age 15-18 mo with a penetrance of 60%. At this stage, mice showed significantly enlarged spleens with abnormal B cell-derived white pulp enlargement. Flow cytometric analysis demonstrated an expanded CD19+ CD5+ population in the spleen of 40% knockout mice, a characteristic of the chronic lymphocytic leukemia-associated phenotype found in humans. Of note, miR-15b/16-2 modulates the CCND2 (Cyclin D2), CCND1 (Cyclin D1), and IGF1R (insulin-like growth factor 1 receptor) genes involved in proliferation and antiapoptotic pathways in mouse B cells. These results are the first, to our knowledge, to suggest an important role of miR-15b/16-2 loss in the pathogenesis of B-cell chronic lymphocytic leukemia.
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http://dx.doi.org/10.1073/pnas.1514954112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4577143PMC
September 2015

MicroRNA-224 is implicated in lung cancer pathogenesis through targeting caspase-3 and caspase-7.

Oncotarget 2015 Sep;6(26):21802-15

Department of Molecular Virology, Immunology and Medical Genetics and Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA.

We recently reported that miR-224 was significantly up-regulated in non-small cell lung cancer (NSCLC) tissues, in particular in resected NSCLC metastasis. We further demonstrated that miR-224 functions as an oncogene in NSCLC by directly targeting TNFAIP1 and SMAD4. However, the biological functions of miR-224 in NSCLC are controversial and underlying mechanisms of miR-224 in the progression and metastasis of lung cancer remain to be further explored. Here we report that caspase3 (CASP3) and caspase7 (CASP7) are previously unidentified targets of miR-224 in NSCLC, and that miR-224 promotes lung cancer cells proliferation and migration in part by directly targeting CASP7 and down-regulating its expression. In addition, miR-224 attenuated TNF-α induced apoptosis by direct targeting of CASP3 resulting in reduction of cleaved PARP1 expression in lung cancer cells. Furthermore, the expression of miR-224 negatively correlates with the expression of CASP7 and CASP3 in tissue samples from patients with lung cancer. Finally, we found that activated NF-κB signaling is involved in the regulation of miR-224 expression in lung cancer. Our study provides new insight in understanding of oncogenic role of miR-224 in the lung cancer pathogenesis and suggests that NF-κB/miR-224/CASP3, 7 pathway could be a putative therapeutic target in lung cancer.
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http://dx.doi.org/10.18632/oncotarget.5224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4673127PMC
September 2015

A differentially expressed set of microRNAs in cerebro-spinal fluid (CSF) can diagnose CNS malignancies.

Oncotarget 2015 Aug;6(25):20829-39

MVIMG, The Ohio State University, Columbus, OH, USA.

Central Nervous System malignancies often require stereotactic biopsy or biopsy for differential diagnosis, and for tumor staging and grading. Furthermore, stereotactic biopsy can be non-diagnostic or underestimate grading. Hence, there is a compelling need of new diagnostic biomarkers to avoid such invasive procedures. Several biological markers have been proposed, but they can only identify specific prognostic subtype of Central Nervous System tumors, and none of them has found a standardized clinical application.The aim of the study was to identify a Cerebro-Spinal Fluid microRNA signature that could differentiate among Central Nervous System malignancies.CSF total RNA of 34 neoplastic and of 14 non-diseased patients was processed by NanoString. Comparison among groups (Normal, Benign, Glioblastoma, Medulloblastoma, Metastasis and Lymphoma) lead to the identification of a microRNA profile that was further confirmed by RT-PCR and in situ hybridization.Hsa-miR-451, -711, 935, -223 and -125b were significantly differentially expressed among the above mentioned groups, allowing us to draw an hypothetical diagnostic chart for Central Nervous System malignancies.This is the first study to employ the NanoString technique for Cerebro-Spinal Fluid microRNA profiling. In this article, we demonstrated that Cerebro-Spinal Fluid microRNA profiling mirrors Central Nervous System physiologic or pathologic conditions. Although more cases need to be tested, we identified a diagnostic Cerebro-Spinal Fluid microRNA signature with good perspectives for future diagnostic clinical applications.
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http://dx.doi.org/10.18632/oncotarget.4096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4673232PMC
August 2015

MicroRNA-224 promotes tumor progression in nonsmall cell lung cancer.

Proc Natl Acad Sci U S A 2015 Aug 17;112(31):E4288-97. Epub 2015 Jul 17.

Department of Molecular Virology, Immunology and Medical Genetics and Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210;

Lung cancer is the leading cause of cancer-related deaths worldwide. Despite advancements and improvements in surgical and medical treatments, the survival rate of lung cancer patients remains frustratingly poor. Local control for early-stage nonsmall cell lung cancer (NSCLC) has dramatically improved over the last decades for both operable and inoperable patients. However, the molecular mechanisms of NSCLC invasion leading to regional and distant disease spread remain poorly understood. Here, we identify microRNA-224 (miR-224) to be significantly up-regulated in NSCLC tissues, particularly in resected NSCLC metastasis. Increased miR-224 expression promotes cell migration, invasion, and proliferation by directly targeting the tumor suppressors TNFα-induced protein 1 (TNFAIP1) and SMAD4. In concordance with in vitro studies, mouse xenograft studies validated that miR-224 functions as a potent oncogenic miRNA in NSCLC in vivo. Moreover, we found promoter hypomethylation and activated ERK signaling to be involved in the regulation of miR-224 expression in NSCLC. Up-regulated miR-224, thus, facilitates tumor progression by shifting the equilibrium of the partially antagonist functions of SMAD4 and TNFAIP1 toward enhanced invasion and growth in NSCLC. Our findings indicate that targeting miR-224 could be effective in the treatment of certain lung cancer patients.
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http://dx.doi.org/10.1073/pnas.1502068112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4534291PMC
August 2015

Break-apart interphase fluorescence in situ hybridization assay in papillary thyroid carcinoma: on the road to optimizing the cut-off level for RET/PTC rearrangements.

Eur J Endocrinol 2015 May 19;172(5):571-82. Epub 2015 Feb 19.

Department of Pathology and DiagnosticsARC-NET Research CentreUniversity of Verona, Policlinico GB Rossi, Piazzale LA Scuro, 10, Piastra Odontoiatrica (II floor), 37134 Verona, ItalyDepartment of Internal MedicineEndocrinology, and Metabolism and Biochemistry, University of Siena, Siena, ItalyDepartment of Surgery and OncologyUniversity of Verona, Verona, ItalyDivision of SurgicalMolecular and Ultrastructural, Section of Cytopathology, University Hospital of Pisa, Pisa, ItalyNuclear Medicine UnitUniversity Hospital of Verona, Verona, Italy Department of Pathology and DiagnosticsARC-NET Research CentreUniversity of Verona, Policlinico GB Rossi, Piazzale LA Scuro, 10, Piastra Odontoiatrica (II floor), 37134 Verona, ItalyDepartment of Internal MedicineEndocrinology, and Metabolism and Biochemistry, University of Siena, Siena, ItalyDepartment of Surgery and OncologyUniversity of Verona, Verona, ItalyDivision of SurgicalMolecular and Ultrastructural, Section of Cytopathology, University Hospital of Pisa, Pisa, ItalyNuclear Medicine UnitUniversity Hospital of Verona, Verona, Italy.

Objective: Chromosomal rearrangements of the RET proto-oncogene is one of the most common molecular events in papillary thyroid carcinoma (PTC). However, their pathogenic role and clinical significance are still debated. This study aimed to investigate the prevalence of RET/PTC rearrangement in a cohort of BRAF WT PTCs by fluorescence in situ hybridization (FISH) and to search a reliable cut-off level in order to distinguish clonal or non-clonal RET changes.

Design: Forty BRAF WT PTCs were analyzed by FISH for RET rearrangements. As controls, six BRAFV600E mutated PTCs, 13 follicular adenomas (FA), and ten normal thyroid parenchyma were also analyzed.

Methods: We performed FISH analysis on formalin-fixed, paraffin-embedded tissue using a commercially available RET break-apart probe. A cut-off level equivalent to 10.2% of aberrant cells was accepted as significant. To validate FISH results, we analyzed the study cohort by qRT-PCR.

Results: Split RET signals above the cut-off level were observed in 25% (10/40) of PTCs, harboring a percentage of positive cells ranging from 12 to 50%, and in one spontaneous FA (1/13, 7.7%). Overall, the data obtained by FISH matched well with qRT-PCR results. Challenging findings were observed in five cases showing a frequency of rearrangement very close to the cut-off.

Conclusions: FISH approach represents a powerful tool to estimate the ratio between broken and non-broken RET tumor cells. Establishing a precise FISH cut-off may be useful in the interpretation of the presence of RET rearrangement, primarily when this strategy is used for cytological evaluation or for targeted therapy.
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http://dx.doi.org/10.1530/EJE-14-0930DOI Listing
May 2015

MicroRNAs as tools and effectors for patient treatment in gastrointestinal carcinogenesis.

Curr Drug Targets 2015 ;16(4):383-92

University of Padua, Department of Medicine, Surgical Pathology and Cytopathology Unit, Via Aristide Gabelli, 61, 35100 - Padua, Italy.

In the last 20 years, microRNAs (miRNAs) have become the most promising class of diagnostic and prognostic biomarkers for human cancer. From a therapeutic perspective, advances in the understanding of the molecular role of miRNAs in the pathological processes have significantly influenced the selection of new therapeutic modalities. Moreover, the intrinsic characteristics that confer stability to miRNAs in vitro, allow a longer molecular/structural resistance and activity in vivo. Preclinical models have consistently underlined the feasibility and efficacy of miRNA-based therapies, either alone or in combination with current targeted therapies. The appealing strength of such therapeutic option dwells in miRNAs' ability to concurrently target multiple genes, frequently in the context of a specific network/pathway. This property allows miRNA-based therapy to be extremely efficient in regulating distinct biological processes relevant to normal and pathological cell homeostasis. The purpose of this review is to summarize the role of miRNAs in gastrointestinal carcinogenesis and their potential use as novel biomarkers and therapeutics.
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http://dx.doi.org/10.2174/1389450116666141210091454DOI Listing
January 2016

Transcribed ultraconserved noncoding RNAs (T-UCR) are involved in Barrett's esophagus carcinogenesis.

Oncotarget 2014 Aug;5(16):7162-71

Department of Medicine (DIMED), Surgical Pathology & Cytopathology Unit, University of Padua, Padua, Italy. Istituto Oncologico Veneto - IOV-IRCCS, Padua, Italy.

Barrett's esophagus (BE) involves a metaplastic replacement of native esophageal squamous epithelium (Sq) by columnar-intestinalized mucosa, and it is the main risk factor for Barrett-related adenocarcinoma (BAc). Ultra-conserved regions (UCRs) are a class non-coding sequences that are conserved in humans, mice and rats. More than 90% of UCRs are transcribed (T-UCRs) in normal tissues, and are altered at transcriptional level in tumorigenesis. To identify the T-UCR profiles that are dysregulated in Barrett's mucosa transformation, microarray analysis was performed on a discovery set of 51 macro-dissected samples obtained from 14 long-segment BE patients. Results were validated in an independent series of esophageal biopsy/surgery specimens and in two murine models of Barrett's esophagus (i.e. esophagogastric-duodenal anastomosis). Progression from normal to BE to adenocarcinoma was each associated with specific and mutually exclusive T-UCR signatures that included up-regulation of uc.58-, uc.202-, uc.207-, and uc.223- and down-regulation of uc.214+. A 9 T-UCR signature characterized BE versus Sq (with the down-regulation of uc.161-, uc.165-, and uc.327-, and the up-regulation of uc.153-, uc.158-, uc.206-, uc.274-, uc.472-, and uc.473-). Analogous BE-specific T-UCR profiles were shared by human and murine lesions. This study is the first demonstration of a role for T-UCRs in the transformation of Barrett's mucosa.
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http://dx.doi.org/10.18632/oncotarget.2249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4196192PMC
August 2014

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
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