ATRX, DAXX or MEN1 mutant pancreatic neuroendocrine tumors are a distinct alpha-cell signature subgroup

Abstract

The commonly mutated genes in pancreatic neuroendocrine tumors (PanNETs) are ATRX, DAXX, and MEN1. We genotyped 64 PanNETs and found 58% carry ATRX, DAXX, and MEN1 mutations (A-D-M mutant PanNETs) and this correlates with a worse clinical outcome than tumors carrying the wild-type alleles of all three genes (A-D-M WT PanNETs). We performed RNA sequencing and DNA-methylation analysis to reveal two distinct subgroups with one consisting entirely of A-D-M mutant PanNETs. Two genes differentiating A-D-M mutant from A-D-M WT PanNETs were high ARX and low PDX1 gene expression with PDX1 promoter hyper-methylation in the A-D-M mutant PanNETs. Moreover, A-D-M mutant PanNETs had a gene expression signature related to that of alpha-cells (FDR q-value < 0.009) of pancreatic islets including increased expression of HNF1A and its transcriptional target genes. This gene expression profile suggests that A-D-M mutant PanNETs originate from or transdifferentiate into a distinct cell type similar to alpha cells.

Fig. 1

Mutational landscape of ATRX, DAXX, and MEN1 in PanNETs. a Oncoprint mutational profile for PanNETs samples. ATRX/DAXX/MEN1 mutations were identified in 37/64 (58 %) of PanNETs using Sanger sequencing. b Among 44 patients who initially presented with localized PanNETs (without distant metastasis), those with A-D-M mutant genotype had a worse recurrence free survival outcome than those A-D-M WT genotype in their primary tumors. A-D-M mutated samples are annotated as any mutation (n = 20) and A-D-M WT samples annotated as WT (n = 24)

Fig. 2

A-D-M mutant and WT PanNETs as two distinct gene expression groups. a Unsupervised clustering of PanNETs using top 3000 variant genes across all samples revealed two distinct robust clusters. The two clusters almost perfectly separate A-D-M WT panNETs from A-D-M mutant panNETs. b Principal component analysis using top 3000 variant genes separated the A-D-M mutant from A-D-M WT PanNETs along the first principal component (PC1). A-D-M mutant panNETs were more homogeneous in gene expression than A-D-M WT as shown by smaller variation along PC1. c Heatmap of pair-wise Pearson correlation of panNETs using top 3000 variant genes across all samples revealed a higher correlation among A-D-M mutants as compared to A-D-M WT panNETs. Red color represents higher correlation and blue represents lower correlation. d Heatmap of top variants genes showing liver, complement, and coagulation genes highly expressed in A-D-M mutant panNETs. Star (*) below sample names represent liver metastatic samples (except for A_mk12 which is a lymph node)

Fig. 3

Distinct DNA methylation pattern between A-D-M mutant and A-D-M WT PanNETs. a Unsupervised clustering of PanNETs using top 2000 variant CpG sites across all samples revealed two clusters. The two clusters separate A-D-M mutant from A-D-M WT PanNETs. b Principal component analysis using top 2000 variant CpG sites separated A-D-M mutant from A-D-M WT PanNETs along PC1

Fig. 4

A-D-M mutant PanNETs with alpha-cell signature. a Heatmap of gene expression for top 20 alpha and beta cell-specific genes from Muraro et al revealed alpha cell specific genes are highly expressed in A-D-M mutant panNETs. A-D-M WT panNETs are more heterogeneous in gene expression but some show high beta cell specific gene expression. Red color represents higher correlation with alpha cell specific genes. b Gene set enrichment analysis show A-D-M mutant PanNETs to be enriched for expression of alpha cell specific genes. Pancreas cell type (alpha, beta, delta, PP, acinar, ductal) gene signatures were obtained from three different published dataset to access enrichment of cell type signatures in A-D-M mutant vs A-D-M WT PanNETs. Table represents GSEA results where size is the number of genes in gene set. All alpha cell gene sets (from three different sources) are significantly enriched in A-D-M mutant panNETs (highlighted in red). No other cell types were enriched in A-D-M mutant or A-D-M WT panNETs. c GSEA plots of significant alpha cell signatures from Bramswig et al., Wang et al., and Muraro et al.

Fig. 5

Validation of A-D-M mutant PanNET and alpha cell signatures. a Pearson correlation boxplot for two independent PanNET datasets show significant positive and negative correlations of A-D-M mutant and WT PanNETs with our A-D-M mutant PanNETs signature respectively (red represent A-D-M mutant and blue represent A-D-M WT with Wilcox p-value; center line is median, bounds of box are first and third quartile, and whiskers are min and max). b GSEA analysis shows A-D-M mutant PanNETs from ICGC PAEN and Sadanandam et al. are enriched for A-D-M mutant and alpha cell gene signatures. c GSEA enrichment plot for significant gene set for A-D-M mutant and alpha cell gene signatures from Sadanandam et al. dataset

Fig. 6

HNF1A pathway with transcriptionally up-regulation in A-D-M mutant panNETs and alpha cells. a Boxplot of HNF1A gene expression for A-D-M mutant and A-D-M WT PanNETs. HNF1A was homogeneously expressed 2.93 fold higher in A-D-M mutants PanNETs (corrected p-val < 0.004, Benjamini–Hochberg). Center line, median; bounds of box, the 1st and 3rd quartiles; and upper and lower whisker is defined to be 1.5 × IQR more than the third and first quartile. b Table represents significant KEGG pathways where genes were differentially expressed between A-D-M mutants and A-D-M WT panNETs. c Table represents transcription factor motifs significantly enriched in promoters of genes differentially expressed in A-D-M mutants and A-D-M WT panNETs. Three HNF1 related motif gene sets from GSEA showed significant enrichment in genes over-expressed in A-D-M mutant panNETs. GSEA was used to find pathway enrichment from genes differentially expressed between A-D-M mutant and A-D-M WT PanNETs

Fig. 7

PDX1 has promoter hypermethylated and lower gene expression in A-D-M mutant panNETs. a Four PDX1 promoter CpG sites show strong hypermethylation in A-D-M mutant PanNETs (corrected p-val < 0.05, Benjamini–Hochberg). The range of beta values is from 0 to 1 and represented as blue (hypo-methylation) to red (hyper-methylation). b PDX1 expression and promoter methylation (TSS1500 cg27033418 CpG site) across all samples showing separation of A-D-M mutant and A-D-M WT PanNETs