Intraductal neoplasms are important precursors to invasive pancreatic cancer and provide an opportunity to detect and treat pancreatic neoplasia before an invasive carcinoma develops. The diagnostic evaluation of these lesions is challenging, as diagnostic imaging and cytological sampling do not provide accurate information on lesion classification, the grade of dysplasia or the presence of invasion. Moreover, the molecular driver gene mutations of these precursor lesions have yet to be fully characterized. Fifty‐two intraductal papillary neoplasms, including 48 intraductal papillary mucinous neoplasms (IPMNs) and four intraductal tubulopapillary neoplasms (ITPNs), were subjected to the mutation assessment in 51 cancer‐associated genes, using ion torrent semiconductor‐based next‐generation sequencing. P16 and Smad4 immunohistochemistry was performed on 34 IPMNs and 17 IPMN‐associated carcinomas. At least one somatic mutation was observed in 46/48 (96%) IPMNs; 29 (60%) had multiple gene alterations. GNAS and/or KRAS mutations were found in 44/48 (92%) of IPMNs. GNAS was mutated in 38/48 (79%) IPMNs, KRAS in 24/48 (50%) and these mutations coexisted in 18/48 (37.5%) of IPMNs. RNF43 was the third most commonly mutated gene and was always associated with GNAS and/or KRAS mutations, as were virtually all the low‐frequency mutations found in other genes. Mutations in TP53 and BRAF genes (10% and 6%) were only observed in high‐grade IPMNs. P16 was lost in 7/34 IPMNs and 9/17 IPMN‐associated carcinomas; Smad4 was lost in 1/34 IPMNs and 5/17 IPMN‐associated carcinomas. In contrast to IPMNs, only one of four ITPNs had detectable driver gene (GNAS and NRAS) mutations. Deep sequencing DNA from seven cyst fluid aspirates identified 10 of the 13 mutations detected in their associated IPMN. Using next‐generation sequencing to detect cyst fluid mutations has the potential to improve the diagnostic and prognostic stratification of pancreatic cystic neoplasms. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA) and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR) was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF) tissues, 6 formalin-fixed paraffin-embedded (FFPE) tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard workflow for qualification of DNA preparations should include the sequential combination of NanoDrop and Qubit to assess the purity and quantity of dsDNA, respectively.

Gastroenteropancreatic (GEP) neuroendocrine neoplasms (NENs) are heterogeneous regarding site of origin, biological behavior, and malignant potential. There has been a rapid increase in data publication during the last 10 years, mainly driven by high-throughput studies on pancreatic and small intestinal neuroendocrine tumors (NETs). This review summarizes the present knowledge on genetic and epigenetic alterations. We integrated the available information from each compartment to give a pathway-based overview. This provided a summary of the critical alterations sustaining neoplastic cells. It also highlighted similarities and differences across anatomical locations and points that need further investigation. GEP-NENs include well-differentiated NETs and poorly differentiated neuroendocrine carcinomas (NECs). NENs are graded as G1, G2, or G3 based on mitotic count and/or Ki-67 labeling index, NECs are G3 by definition. The distinction between NETs and NECs is also linked to their genetic background, as TP53 and RB1 inactivation in NECs set them apart from NETs. A large number of genetic and epigenetic alterations have been reported. Recurrent changes have been traced back to a reduced number of core pathways, including DNA damage repair, cell cycle regulation, and phosphatidylinositol 3-kinase/mammalian target of rapamycin signaling. In pancreatic tumors, chromatin remodeling/histone methylation and telomere alteration are also affected. However, also owing to the paucity of disease models, further research is necessary to fully integrate and functionalize data on deregulated pathways to recapitulate the large heterogeneity of behaviors displayed by these tumors. This is expected to impact diagnostics, prognostic stratification, and planning of personalized therapy.

One-hundred-fifty-three biliary cancers, including 70 intrahepatic cholangiocarcinomas (ICC), 57 extrahepatic cholangiocarcinomas (ECC) and 26 gallbladder carcinomas (GBC) were assessed for mutations in 56 genes using multigene next-generation sequencing. Expression of EGFR and mTOR pathway genes was investigated by immunohistochemistry. At least one mutated gene was observed in 118/153 (77%) cancers. The genes most frequently involved were KRAS (28%), TP53 (18%), ARID1A (12%), IDH1/2 (9%), PBRM1 (9%), BAP1 (7%), and PIK3CA (7%). IDH1/2 (p=0.0005) and BAP1 (p=0.0097) mutations were characteristic of ICC, while KRAS (p=0.0019) and TP53 (p=0.0019) were more frequent in ECC and GBC. Multivariate analysis identified tumour stage and TP53 mutations as independent predictors of survival. Alterations in chromatin remodeling genes (ARID1A, BAP1, PBRM1, SMARCB1) were seen in 31% of cases. Potentially actionable mutations were seen in 104/153 (68%) cancers: i) KRAS/NRAS/BRAF mutations were found in 34% of cancers; ii) mTOR pathway activation was documented by immunohistochemistry in 51% of cases and by mutations in mTOR pathway genes in 19% of cancers; iii) TGF-ß/Smad signaling was altered in 10.5% cancers; iv) mutations in tyrosine kinase receptors were found in 9% cases. Our study identified molecular subgroups of cholangiocarcinomas that can be explored for specific drug targeting in clinical trials.

Tumor mutational burden (TMB) is a numeric index that expresses the number of mutations per megabase (muts/Mb) harbored by tumor cells in a neoplasm. TMB can be determined using different approaches based on next-generation sequencing. In the case of high values, it indicates a potential response to immunotherapy. In this systematic review, we assessed the potential predictive role of high-TMB in pancreatic ductal adenocarcinoma (PDAC), as well as the histo-molecular features of high-TMB PDAC. High-TMB appeared as a rare but not-negligible molecular feature in PDAC, being present in about 1.1% of cases. This genetic condition was closely associated with mucinous/colloid and medullary histology (p < 0.01). PDAC with high-TMB frequently harbored other actionable alterations, with microsatellite instability/defective mismatch repair as the most common. Immunotherapy has shown promising results in high-TMB PDAC, but the sample size of high-TMB PDAC treated so far is quite small. This study highlights interesting peculiarities of PDAC harboring high-TMB and may represent a reliable starting point for the assessment of TMB in the clinical management of patients affected by pancreatic cancer.