Epigenetic Dysregulation of 5-hydroxymethylcytosine in Well-Differentiated Pancreatic Neuroendocrine Tumors

Abstract

Dysregulation of epigenetic mechanisms, reflected by loss of expression of 5-hydroxymethylcytosine (5-hmC) is being increasingly recognized as a marker of aggressive behavior in several neoplasms; however, the role of such epigenetic modifiers in pancreatic neuroendocrine tumors (PanNETs) has not been studied. Annotated cohort of 60 PanNETs was evaluated for 5-hmC expression using immunohistochemistry. Univariable and multivariable analyses were performed. To determine intratumor heterogeneity of 5-hmC expression, 26 additional synchronous metastatic deposits of PanNETs from 8 patients were evaluated for 5-hmC expression. 5-hmC level showed significant association with the presence of distant metastases (P = 0.02), female sex (P = 0.04), and Ki-67 proliferation index (P = 0.002). A multivariate model created using the stepwise logistic regression analysis showed the presence of nodal metastases (odds ratio = 6.15), lymphovascular invasion (odds ratio = 4.07) and lack of 5-hmC expression (odds ratio = 5.34) were predictive of the risk of distant metastasis in PanNETs with a c-statistic of 0.845. Epigenetic intratumoral heterogeneity of 5-hmC expression was seen in 37.5% cases (3/8). Our work provides evidence that epigenetic regulators are involved in the pathobiology of PanNETs and immunohistochemical analysis of 5-hmC may be able to refine prognostic evaluation of these tumors.

The role of DNA methylation in human pancreatic neuroendocrine tumours

“…(Sengelaub et al, 2016, Shen et al, 2015 Of note, most studies that have reported on the DNA methylome in PNETs, have focused on 5mC, with only one study reporting loss of global 5hmC to be associated with tumourigenesis. (Sharma et al, 2022)…”

“…(Stricker et al, 2012, Choi et al, 2007 No study has reported a difference in PNET methylation between sexes, (Choi et al, 2007, Campana et al, 2018, Ban et al, 2022, Boons et al, 2020, although loss of global 5hmC was reported to be associated with tumourigenesis and to correlate with distant metastasis and female gender in a multivariate analysis of 60 well-differentiated PNETs. (Sharma et al, 2022) The DNA methylome has also been used to cluster PNETs into different prognostic categories. (Di Domenico et al, 2020, Chan et al, 2018, Simon et al, 2022, Boons et al, 2020 Studies clustering PNETs into two categories (A-D-M/ATRX-DAXX-MEN1) mutant vs wild-type, have reported that the A-D-M mutant category (ARX positive, PDX1 negative) had an overall worse prognosis, when compared to A-D-M wild-type (Chan et al, 2018) and PNETs with a beta-like cell methylation signature.…”

“…There have been 32 studies reporting DNA methylation in human PNETs, and one study which has looked at global 5hmC ( Tables 2 and 3 ). The PNET methylome has been profiled in 24 studies in a gene-specific manner ( Muscarella et al 1998 , Bartsch et al 2000 , Serrano et al 2000 , Chan et al 2003 , Dammann et al 2003 , House et al 2003 , Wild et al 2003 , Liu et al 2005 , Arnold et al 2007 , Choi et al 2007 , Dejeux et al 2009 , Malpeli et al 2011 , Stricker et al 2012 , Ohki et al 2014 , Schmitt et al 2014 , Stefanoli et al 2014 , Liu et al 2014 , Cros et al 2016 , Ushiku et al 2016 , Campana et al 2018 , Conemans et al 2018 , Zhang et al 2020 , Ban et al 2022 , Evans et al 2022 a ) and subsequently by both global methylation ( Marinoni et al 2017 ) and hydroxymethylation ( Sharma et al 2022 ) and by specific CpG site assessment with array-based technologies ( Chan et al 2003 , Tirosh et al 2019 , Boons et al 2020 , Di Domenico et al 2020 , Lakis et al 2021 , Simon et al 2022 , Yachida et al 2022 ). Out of the 24 studies looking at a specific subset of genes, 58% (14/24) have used methylation-specific polymerase chain reaction (MSP) to investigate the percentage of methylation present at specific gene promoters.…”

“…PNETs clustered into (i) MEN1 alterations with RASSF1A , PDX1, and CDX2 promoter hypermethylation and (ii) hypomethylation group including: HNF4A , MGMT, and TERT . Hydroxymethylation assessment Sharma et al (2022) 60 PNETs IHC 5hmC staining of formalin-fixed paraffin-embedded slides Loss of 5hmC was associated with metastatic disease 450K, Infinium HumanMethylation450 BeadChip; ARX, Aristaless-related homeobox; A-D-M, ATRX-DAXX-MEN1; ATAC-seq, assay for transposase-accessible chromatin with high-throughput sequencing; ATRX, ATRX chromatin remodeler; CDKN2A(p14, p16); cyclin-dependent kinase inhibitor 2A; CDX2, caudal-type homeobox protein 2; DAXX, death domain-associated protein; EPIC, Infinium MethylationEPIC BeadChip; HNF4A, hepatocyte nuclear factor 4 alpha; IHC, immunohistochemistry; LINE-1, long interspersed nuclear element 1; MEN1, multiple endocrine neoplasia type 1; MGMT, O6-methyl–guanine methyltransferase; mRNA, messenger ribonucleic acid; MSP, methylation-specific PCR, NF, non-functioning; PNEC, pancreatic neuroendocrine carcinoma; PNEN, pancreatic neuroendocrine neoplasm; PNET, pancreatic neuroendocrine tumour; PSQ, pyrosequencing; RASSF1A, Ras association domain family member 1; TERT, telomerase reverse transcriptase; VHL, Von Hippel–Lindau tumour suppressor; WES, whole-exome sequencing; WGS, whole genome sequencing. …”

“…PTPRN2 is highly expressed in islet cells and is upregulated in other cancers, including breast and hepatocellular cancer ( Shen et al 2015 , Sengelaub et al 2016 ). Of note, most studies that have reported on the DNA methylome in PNETs have focused on 5mC, with only one study reporting loss of global 5hmC to be associated with tumourigenesis ( Sharma et al 2022 ).…”