Comprehensive Genomic Profiling of Gastroenteropancreatic Neuroendocrine Neoplasms (GEP-NENs)

Abstract

Purpose: GEP-NENs are rare malignancies with increasing incidence. Their molecular characteristics are still undefined. We explored the underlying biology of GEP-NENs and the differences between gastrointestinal (GI) and pancreatic (PNEN), high-grade (HG), and low-grade (LG) tumors.

Experimental design: GEP-NENs were analyzed using next-generation sequencing (NGS; MiSeq on 47 genes, NextSeq on 592 genes), IHC, and in situ hybridization. Tumor mutational burden (TMB) was calculated on the basis of somatic nonsynonymous missense mutations, and microsatellite instability (MSI) was evaluated by NGS of known MSI loci.

Results: In total, 724 GEP-NENs were examined: GI (N = 469), PNEN (N = 255), HG (N = 135), and LG (N = 335). Forty-nine percent were female, and median age was 59. Among LG tumors, the most frequently mutated genes were ATRX (13%), ARID1A (10%), and MEN1 (10%). HG tumors showed TP53 (51%), KRAS (30%), APC (27%), and ARID1A (23%). Immune-related biomarkers yielded a lower prevalence in LG tumors compared with HG [MSI-H 0% vs. 4% (P = 0.04), PD-L1 overexpression 1% vs. 6% (P = 0.03), TMB-high 1% vs. 7% (P = 0.05)]. Compared with LG, HG NENs showed a higher mutation rate in BRAF (5.4% vs. 0%, P < 0.0001), KRAS (29.4% vs. 2.6%, P < 0.0001), and PI3KCA (7% vs. 0.3%, P < 0.0001). When compared with GI, PNEN carried higher frequency of MEN1 (25.9% vs. 0.0%, P < 0.0001), FOXO3 (8.6% vs. 0.8%, P = 0.005), ATRX (20.6% vs. 2.0%, P = 0.007), and TSC2 (6.3% vs. 0.0%, P = 0.007), but lower frequency of mutations in APC (1.0% vs. 13.8%, P < 0.0001).

Conclusions: Significant molecular differences were observed in GEP-NENs by tumor location and grade, indicating differences in carcinogenic pathways and biology.

Figure 1 –

Patient Demographics. NEN from GI (N = 469), PNEN (N = 255), HG (N = 135), and LG (N = 335). Female to male ratio was 49%/51% and the median age was 59.

Figure 2.. Oncoprint

Comprehensive molecular profile of 724 GEP-NENs. Overall, the most frequently mutated genes were TP53 (18%), ATRX (12%), KRAS (11%), MEN1 (11%), ARID1A (11%), and APC (10%). Gene amplification events, as determined by NGS, were rare across the cohort. The most frequently amplified genes are MYC (2%), FGF6 (2%), CCND2 (2%), and FOXA1 (2%). Each column is a single patient; gray boxes are those in which no alteration was detected. Samples were arranged by those that harbored a mutation in the genes listed as rows top to bottom (those with TP53 mutation show up on left, then by KRAS mt, then by APC, etc). The stacked graph at the top is a representation of the number of alterations that case had (the higher the line the more alterations). Mutation frequencies were determined on a per gene level excluding cases from that analysis where a particular gene was determined to be indeterminate. The variation of sample size per gene comes from the fact that not all genes within a single case are evaluable.

Figure 3. Molecular differences between PNEN and GI-NEN.

Compared to GI, PNEN carried significantly higher frequency of MEN1 (25.9% vs 0.0%), FOXO3 (8.6% vs 0.8%), ATRX (20.6% vs 2.0%), and TSC2 (6.3% vs 0.0%), but lower frequency of mutations in APC (1.0% vs 13.8%). PNEN had a significantly higher expression of PR (38.9% vs 3.7%), but interestingly, GI-NEN had higher expression levels of ER (12.5% vs 2.4%).

Figure 4.. Differences in LG PNET and LG GI-NET

Molecular differences between LG PNET and LG GI-NET. Compared to LG GI-NET, LG PNET carried significantly higher frequency of MEN1 (24.3% vs 0.0%), ATRX (33.3% vs 0.0%), and FOXO3 (12.2% vs 0.0%). LG GI-NET had a higher mutation rate in APC (1.6% vs 0.0%) and CDKNB1 (4.9% vs 2.5%), although neither was significant. Molecular profiles of LG tumors were similar to those of the entire cohort for each primary location.

Figure 5

Figure 5a – Molecular differences between HG and LG GEP-NEN. Among LG tumors, the most frequently mutated genes were ATRX (13%), ARID1A (10%) and MEN1 (10%). Among HG, TP53 (51%), KRAS (30%), APC (27%), ARID1A (23%) and RB1 (11%). Compared to LG, HG NENs showed a higher mutation rate in BRAF (5.4% vs 0%), KRAS (29.4% v 2.6%) and PI3KCA (7% vs 0.3%), among others. 5b – Differences in immune markers between HG vs LG GEP-NEN. Immune-related biomarkers showed lower prevalence in LG tumors compared to HG: lower mean TML (5.1 mut/MB vs 9.5, P < .0001), MSI-H 0% vs 4% (P = .04), PD-L1 expression 1% vs 6% (P = .03).

Figure 5

Figure 5a – Molecular differences between HG and LG GEP-NEN. Among LG tumors, the most frequently mutated genes were ATRX (13%), ARID1A (10%) and MEN1 (10%). Among HG, TP53 (51%), KRAS (30%), APC (27%), ARID1A (23%) and RB1 (11%). Compared to LG, HG NENs showed a higher mutation rate in BRAF (5.4% vs 0%), KRAS (29.4% v 2.6%) and PI3KCA (7% vs 0.3%), among others. 5b – Differences in immune markers between HG vs LG GEP-NEN. Immune-related biomarkers showed lower prevalence in LG tumors compared to HG: lower mean TML (5.1 mut/MB vs 9.5, P < .0001), MSI-H 0% vs 4% (P = .04), PD-L1 expression 1% vs 6% (P = .03).