Progressive epigenetic dysregulation in neuroendocrine tumour liver metastases.

The incidence of small intestinal neuroendocrine tumours (SINETs) is increasing and distant metastases are present at diagnosis in 70% of cases, the liver being the commonest site of metastasis (Yao et al. 2008). Despite this, our understanding of the mechanisms underlying metastatic progression of SINETs is currently limited, and prior studies of the molecular biology of SINET liver metastases (LM) have been performed predominantly in small cohorts utilising candidate-based techniques. SINETs have a low rate of mutations compared to most cancers. The most frequently mutated gene is CDKN1B (encoding p27, a cell cycle regulator); however, mutations in this gene occur in only 8% of tumours, and there is no characteristic mutational hotspot (Francis et al. 2013). Furthermore, mutation of CDKN1B does not correlate with the expression of p27 (Crona et al. 2015). We previously identified that SINETs are epigenetically dysregulated, and a panel of candidate driver epimutation genes has been identified (Karpathakis et al. 2016). Therefore, we postulated that metastatic progression in SINETs may also be epigenetically regulated. Here, we present the findings from the largest molecular profiling study of SINET LM performed to date, integrating copy number variance (CNV), DNA methylation, and RNA expression profiling to characterise the mechanisms underlying metastatic progression. Experimental details of DNA methylation, CNV and RNA expression profiling are as previously published (Karpathakis et al. 2016). Patients provided informed consent for their tissue to be analysed in this study, which was Research Ethics Committee approved (Ref: 09/H0722/27). All cases were reviewed by two expert NET histopathologists (TVL/MN). Nucleic acids were extracted using standard methods (Qiagen, QIAamp DNA Mini kit; Roche, High Pure RNA Paraffin kit). H&E-stained sections were evaluated to ensure >80% purity of tumour specimens. Methylation profiling was performed on the HumanMethylation450 BeadChip (HM450) (Illumina). Methylation data were analysed using ChAMP pipeline (https://www.bioconductor.org/packages/release/bioc/ html/ChAMP.html). Whole-genome methylation profiling using methylated DNA immunoprecipitation sequencing (MeDIP) was performed as previously described. MeDIP data were analysed using the custom pipeline MeDUSA, v2.0 (https://www.ucl.ac.uk/cancer/research/departmentcancer-biology/medical-genomics-group/past-projects/ medusa-project). Gene expression levels were quantified using the whole genome cDNA-mediated annealing, selection and ligation (DASL) (Illumina) assay. Expression data were analysed using the ‘LIMMA’ package in R (https://bioconductor.org/packages/release/bioc/html/ limma.html). Raw data from this study has previously been deposited in GEO (Accession number: GSE73832) (Karpathakis et al. 2016). In summary, n = 90 samples underwent array-based DNA methylation analysis, n = 26 samples underwent methylation-specific immunoprecipitation followed by DNA sequencing and n = 49 underwent array-based RNA expression analysis. Of cases with relevant clinical data, 93% had received no systemic treatment prior to specimen collection (27/29 cases). The CNV profile of SINET LM (n = 20) mirrors that of primary tumours with the most frequent alteration of chr18 LOH seen in 79% of cases. A greater proportion of LM demonstrates amplification of chr20 (42%) and deletion of chr19 (35%), whereas gain of 17q is found only in LM (21%). A trend of increased incidence of CNVs was seen in LM compared to that of SINET primary tumours (SINET primary: median 78 mbp; LM: median 114 mbp, P = 0.08). Comparison of methylation profiles of SINET LM to those of primary SINETs identified 29,263 methylation