No abstract

Many genes undergo aberrant methylation in human cancers, and microarray platforms enable more comprehensive profiling of aberrant DNA methylation patterns.Results: 1,010 of 87,922 probes on the 88 K promoter array (606 genes) had a higher signal (log 2 > 2) in the pancreatic cancer line, Panc-1 compared to the non-neoplastic pancreatic duct line, HPDE. Using this cut-off, bisulfite sequencing and/ or MSP confirmed differential methylation of all 27 genes (66 probes) predicted to be methylated by the MCA array. More than half of the genes aberrantly hypermethylated in Panc-1 were not expressed in the pancreatic duct (HPDE) by expression array analysis. Using the 244 K CpG island array, 1,968 CpG islands were differentially methylated in MiaPaca2 compared to normal pancreas. The MCA method was more likely to identify hypermethylation within CpG islands than a cocktail of methylation sensitive restriction enzymes. DNA methylation profiles using 10 ng of DNA were highly correlated with those obtained using 5 mg of DNA (R2 = 0.98). Analysis of 57 pancreatic cancers and 34 normal pancreata using MSP identified MDFI, hsa-miR-9-1, ZNF415, CNTNAP2 and ELOVL4 as methylated in 96%, 89%, 86%, 82% and 68% of the cancers vs. 9%, 15%, 6%, 3% and 9% of normal pancreata, respectively. Methods: We used methylated CpG island amplification (MCA) and Agilent promoter and CpG island microarrays to identify differential DNA methylation patterns in pancreatic cancer vs. normal pancreas. We examined MCA array reproducibility, compared it to methylation profiles obtained using a cocktail of methylation-sensitive restriction enzymes and examined gene expression of methylated genes.Conclusion: Promoter and CpG island array analysis finds aberrant methylation of hundreds of promoters and CpG islands in pancreatic cancer cells.

Pancreatic cysts are common and often pose a management dilemma, because some cysts are precancerous, whereas others have little risk of developing into invasive cancers. We used supervised machine learning techniques to develop a comprehensive test, CompCyst, to guide the management of patients with pancreatic cysts. The test is based on selected clinical features, imaging characteristics, and cyst fluid genetic and biochemical markers. Using data from 436 patients with pancreatic cysts, we trained CompCyst to classify patients as those who required surgery, those who should be routinely monitored, and those who did not require further surveillance. We then tested CompCyst in an independent cohort of 426 patients, with histopathology used as the gold standard. We found that clinical management informed by the CompCyst test was more accurate than the management dictated by conventional clinical and imaging criteria alone. Application of the CompCyst test would have spared surgery in more than half of the patients who underwent unnecessary resection of their cysts. CompCyst therefore has the potential to reduce the patient morbidity and economic costs associated with current standard-of-care pancreatic cyst management practices.

No abstract

The remarkable aggressiveness of pancreatic cancer has never been fully explained. Although clearly multifactorial, we postulate that venous invasion, a finding seen in most pancreatic cancers but not in most cancers of other organs, may be a significant, underappreciated contributor to the aggressiveness of this disease. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

ObjectiveRecent studies have found aristaless-related homeobox gene (ARX)/pancreatic and duodenal homeobox 1 (PDX1), alpha-thalassemia/mental retardation X-linked (ATRX)/death domain-associated protein (DAXX) and alternative lengthening of telomeres (ALT) to be promising prognostic biomarkers for non-functional pancreatic neuroendocrine tumours (NF-PanNETs). However, they have not been comprehensively evaluated, especially among small NF-PanNETs (≤2.0 cm). Moreover, their status in neuroendocrine tumours (NETs) from other sites remains unknown.DesignAn international cohort of 1322 NETs was evaluated by immunolabelling for ARX/PDX1 and ATRX/DAXX, and telomere-specific fluorescence in situ hybridisation for ALT. This cohort included 561 primary NF-PanNETs, 107 NF-PanNET metastases and 654 primary, non-pancreatic non-functional NETs and NET metastases. The results were correlated with numerous clinicopathological features including relapse-free survival (RFS).ResultsATRX/DAXX loss and ALT were associated with several adverse prognostic findings and distant metastasis/recurrence (p<0.001). The 5-year RFS rates for patients with ATRX/DAXX-negative and ALT-positive NF-PanNETs were 40% and 42% as compared with 85% and 86% for wild-type NF-PanNETs (p<0.001 and p<0.001). Shorter 5-year RFS rates for ≤2.0 cm NF-PanNETs patients were also seen with ATRX/DAXX loss (65% vs 92%, p=0.003) and ALT (60% vs 93%, p<0.001). By multivariate analysis, ATRX/DAXX and ALT status were independent prognostic factors for RFS. Conversely, classifying NF-PanNETs by ARX/PDX1 expression did not independently correlate with RFS. Except for 4% of pulmonary carcinoids, ATRX/DAXX loss and ALT were only identified in primary (25% and 29%) and NF-PanNET metastases (62% and 71%).ConclusionsATRX/DAXX and ALT should be considered in the prognostic evaluation of NF-PanNETs including ≤2.0 cm tumours, and are highly specific for pancreatic origin among NET metastases of unknown primary.

Background and Aims Targeting costimulatory receptors with agonistic antibodies is a promising cancer immunotherapy option. We aimed to investigate costimulatory receptor expression, particularly 4‐1BB (CD137 or tumor necrosis factor receptor superfamily member 9), on tumor‐infiltrating CD8+ T cells (CD8+ tumor‐infiltrating lymphocytes [TILs]) and its association with distinct T‐cell activation features among exhausted CD8+ TILs in hepatocellular carcinoma (HCC). Approach and Results Tumor tissues, adjacent nontumor tissues, and peripheral blood were collected from HCC patients undergoing surgical resection (n = 79). Lymphocytes were isolated and used for multicolor flow cytometry, RNA‐sequencing, and in vitro functional restoration assays. Among the examined costimulatory receptors, 4‐1BB was most prominently expressed on CD8+ TILs. 4‐1BB expression was almost exclusively detected on CD8+ T cells in the tumor—especially on programmed death 1 (PD‐1)high cells and not PD‐1int and PD‐1neg cells. Compared to PD‐1int and 4‐1BBnegPD‐1high CD8+ TILs, 4‐1BBposPD‐1high CD8+ TILs exhibited higher levels of tumor reactivity and T‐cell activation markers and significant enrichment for T‐cell activation gene signatures. Per‐patient analysis revealed positive correlations between percentages of 4‐1BBpos cells among CD8+ TILs and levels of parameters of tumor reactivity and T‐cell activation. Among highly exhausted PD‐1high CD8+ TILs, 4‐1BBpos cells harbored higher proportions of cells with proliferative and reinvigoration potential. Our 4‐1BB–related gene signature predicted survival outcomes of HCC patients in the The Cancer Genome Atlas cohort. 4‐1BB agonistic antibodies enhanced the function of CD8+ TILs and further enhanced the anti‐PD‐1–mediated reinvigoration of CD8+ TILs, especially in cases showing high levels of T‐cell activation. Conclusion 4‐1BB expression on CD8+ TILs represents a distinct activation state among highly exhausted CD8+ T cells in HCC. 4‐1BB costimulation with agonistic antibodies may be a promising strategy for treating HCCs exhibiting prominent T‐cell activation.

Intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs) are non-invasive neoplasms that are often observed in association with invasive pancreatic cancers, but their origins and evolutionary relationships are poorly understood. In this study, we analyze 148 samples from IPMNs, MCNs, and small associated invasive carcinomas from 18 patients using whole exome or targeted sequencing. Using evolutionary analyses, we establish that both IPMNs and MCNs are direct precursors to pancreatic cancer. Mutations in SMAD4 and TGFBR2 are frequently restricted to invasive carcinoma, while RNF43 alterations are largely in non-invasive lesions. Genomic analyses suggest an average window of over three years between the development of high-grade dysplasia and pancreatic cancer. Taken together, these data establish non-invasive IPMNs and MCNs as origins of invasive pancreatic cancer, identifying potential drivers of invasion, highlighting the complex clonal dynamics prior to malignant transformation, and providing opportunities for early detection and intervention.