DNA methylation is a crucial epigenetic mechanism for gene expression regulation and cell differentiation. Furthermore, it was found to play a major role in multiple pathological processes, including cancer. In pancreatic neuroendocrine neoplasms (PNENs), epigenetic deregulation is also considered to be of significance, as the most frequently mutated genes have an important function in epigenetic regulation. However, the exact changes in DNA methylation between PNENs and the endocrine cells of the pancreas, their likely cell-of-origin, remain largely unknown. Recently, two subtypes of PNENs have been described which were linked to cell-of-origin and have a different prognosis. A difference in the expression of the transcription factor PDX1 was one of the key molecular differences. In this study, we performed an exploratory genome-wide DNA methylation analysis using Infinium Methylation EPIC arrays (Illumina) on 26 PNENs and pancreatic islets of five healthy donors. In addition, the methylation profile of the PDX1 region was used to perform subtyping in a global cohort of 83 PNEN, 2 healthy alpha cell and 3 healthy beta cell samples. In our exploratory analysis, we identified 26,759 differentially methylated CpGs and 79 differentially methylated regions. The gene set enrichment analysis highlighted several interesting pathways targeted by altered DNA methylation, including MAPK, platelet-related and immune system-related pathways. Using the PDX1 methylation in 83 PNEN, 2 healthy alpha cell and 3 healthy beta cell samples, two subtypes were identified, subtypes A and B, which were similar to alpha and beta cells, respectively. These subtypes had different clinicopathological characteristics, a different pattern of chromosomal alterations and a different prognosis, with subtype A having a significantly worse prognosis compared with subtype B (HR 0.22 [95% CI: 0.051–0.95], p = 0.043). Hence, this study demonstrates that several cancer-related pathways are differently methylated between PNENs and normal islet cells. In addition, we validated the use of the PDX1 methylation status for the subtyping of PNENs and its prognostic importance.

Background: Detection of tumor-specific alterations in cell-free DNA (cfDNA) has proven valuable as a liquid biopsy for several types of cancer. So far, use of cfDNA remains unexplored for pancreatic neuroendocrine tumor (PNET) patients.Methods: From 10 PNET patients, fresh frozen tumor tissue, buffy coat and plasma samples were collected. Whole-exome sequencing of primary tumor and germline DNA was performed to identify tumor-specific variants and copy number variations (CNVs). Subsequently, tumor-specific variants were quantified in plasma cfDNA with droplet digital PCR. In addition, CNV analysis of cfDNA was performed using shallow whole-genome sequencing.Results: Tumor-specific variants were detected in perioperative plasma samples of two PNET patients, at variant allele fractions (VAFs) of respectively 19 and 21%. Both patients had metastatic disease at time of surgery, while the other patients presented with localized disease. In the metastatic patients, CNV profiles of tumor tissue and cfDNA were significantly correlated. A follow-up plasma sample of a metastatic patient demonstrated an increased VAF (57%) and an increased chromosomal instability, in parallel with an increase in tumor burden.Conclusions: We are the first to report the presence of tumor-specific genetic alterations in cfDNA of metastatic PNET patients and their evolution during disease progression. Additionally, CNV analysis in cfDNA shows potential as a liquid biopsy.

Mutations in DAXX/ATRX, MEN1 and genes involved in the phosphoinositide-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway have been implicated in pancreatic neuroendocrine neoplasms (pNENs). However, mainly mutations present in the majority of tumor cells have been identified, while proliferation-driving mutations could be present only in small fractions of the tumor. This study aims to identify high- and low-abundance mutations in pNENs using ultra-deep targeted resequencing. Formalin-fixed paraffin-embedded matched tumor-normal tissue of 38 well-differentiated pNENs was sequenced using a HaloPlex targeted resequencing panel. Novel amplicon-based algorithms were used to identify both single nucleotide variants (SNVs) and insertion-deletions (indels) present in >10% of reads (high abundance) and in <10% of reads (low abundance). Found variants were validated by Sanger sequencing. Sequencing resulted in 416,711,794 reads with an average target base coverage of 2663 ± 1476. Across all samples, 32 high-abundance somatic, 3 germline and 30 low-abundance mutations were withheld after filtering and validation. Overall, 92% of high-abundance and 84% of low-abundance mutations were predicted to be protein damaging. Frequently, mutated genes were MEN1, DAXX, ATRX, TSC2, PI3K/Akt/mTOR and MAPK-ERK pathway-related genes. Additionally, recurrent alterations on the same genomic position, so-called hotspot mutations, were found in DAXX, PTCH2 and CYFIP2. This first ultra-deep sequencing study highlighted genetic intra-tumor heterogeneity in pNEN, by the presence of low-abundance mutations. The importance of the ATRX/DAXX pathway was confirmed by the first-ever pNEN-specific protein-damaging hotspot mutation in DAXX. In this study, both novel genes, including the pro-apoptotic CYFIP2 gene and hedgehog signaling PTCH2, and novel pathways, such as the MAPK-ERK pathway, were implicated in pNEN.