Exploratory genomic analysis of high-grade neuroendocrine neoplasms across diverse primary sites

Abstract

High-grade (grade 3) neuroendocrine neoplasms (G3 NENs) have poor survival outcomes. From a clinical standpoint, G3 NENs are usually grouped regardless of primary site and treated similarly. Little is known regarding the underlying genomics of these rare tumors, especially when compared across different primary sites. We performed whole transcriptome (n = 46), whole exome (n = 40), and gene copy number (n = 43) sequencing on G3 NEN formalin-fixed, paraffin-embedded samples from diverse organs (in total, 17 were lung, 16 were gastroenteropancreatic, and 13 other). G3 NENs despite arising from diverse primary sites did not have gene expression profiles that were easily segregated by organ of origin. Across all G3 NENs, TP53, APC, RB1, and CDKN2A were significantly mutated. The CDK4/6 cell cycling pathway was mutated in 95% of cases, with upregulation of oncogenes within this pathway. G3 NENs had high tumor mutation burden (mean 7.09 mutations/MB), with 20% having >10 mutations/MB. Two somatic copy number alterations were significantly associated with worse prognosis across tissue types: focal deletion 22q13.31 (HR, 7.82; P = 0.034) and arm amplification 19q (HR, 4.82; P = 0.032). This study is among the most diverse genomic study of high-grade neuroendocrine neoplasms. We uncovered genomic features previously unrecognized for this rapidly fatal and rare cancer type that could have potential prognostic and therapeutic implications.

Keywords: APC; CDK4; CDKN2A; RB1; TP53; cell cycling; genomics; grade 3; high grade; neuroendocrine carcinoma; neuroendocrine neoplasm; neuroendocrine tumor; sequencing; tumor mutation burden.

Figure 1.. Clinical characteristics

(a) Forest plot of Cox multivariate analysis of 46 patients. (b) Kaplan-Meier survival curve of patients separated by tissue origin. Table lists the numbers at risk. (c) Dot plot of systemic therapies received per patient. In the No Systemic Treatment group, surgery, radiation and/or hospice services were administered. (d) Number of treatments of each chemotherapy received. Some patients received the same chemotherapy more than once. Only the most frequent chemotherapies were listed. Abbreviations: GEP, gastroenteropancreatic. Misc, miscellaneous. AJCC, American Joint Committee on Cancer.

Figure 2.. Transcriptomic profiles

(a) Principal Component Analysis (PCA) of adjacent normal tissue (n = 21), grade 3 neuroendocrine neoplasms (NENs) from gastroenteropancreatic (GEP) (n = 16), lung (n = 17) and miscellaneous primary sites (n = 13; gynecologic, head and neck, breast, bladder and unknown primary). Divided line marks separation between normal and tumor samples. (b) Unsupervised clustering heatmap of above samples based on the top 30,000 differentially expressed genes. Clustering method was complete linkage with Pearson correlation. (c) Schematic of machine learning and network analysis algorithm to integrate 4 layers of data. (d) Bar graph showing subtypes from integrated genomic and transcriptomic analysis (n = 40). Gap statistic determined the optimal number of subtypes was three. Abbreviations: GEP, gastroenteropancreatic. WGS, whole-genome sequencing. WES, whole-exome sequencing. PC1, principal component 1. PC2, principal component 2.

Figure 3.. Significantly mutated genes and CDK4/6 cell cycling pathway dysregulation

(a) Oncoplot showing significantly mutated genes marked by asterisk(s) per dNdScv method. Low-pass WGS n = 43; WES n = 40. (b) TP53 mutations were enriched in lung, APC in gastroenteropancreatic, and RB1 in non-lung/non-GEP neoplasms (p < 0.05, pairwise and groupwise Fisher exact test). N = 40. (c) EXPANDS analysis showing the clone size of each significantly mutated gene. Dotted line shows the 70% threshold often used to define a founder clone. (d) Schematic of CDK4/6 cell cycling pathway. (e) Boxplot showing gene expression levels of the CDK4/6 pathway oncogenes (CCND1, CCDN2, CCND3, CCNE1, CDK2, CDK4, CDK6, E2F1, E2F3) between neoplasms and normal tissue. Mean ± SD (colorectal normal: 4.26 ± 0.15, n = 7; colorectal tumor: 4.77 ± 0.35, n = 8; lung normal: 4.34 ± 0.11, n = 9; lung tumor: 4.71 ± 0.48, n = 17). Statistical test: Holm-Bonferroni method. (f) Frequency of mutations in canonical oncogenic pathways upstream of CDK4/6 pathway. (g) Unsupervised clustering heatmap of gene pathways. Gene pathways universally upregulated by all neoplasms compared to normal tissue (n = 42) comprised mostly of cell cycling pathways (n = 23). Abbreviations: WGS, whole genome sequencing. WES, whole exome sequencing. GEP, gastroenteropancreatic. NEN, neuroendocrine neoplasm. Misc, miscellaneous.

Figure 4.. Tumor mutation burden

(a) Grade 3 neuroendocrine neoplasms (NEN) rank 4^th in order of highest tumor mutation burden (TMB) when compared to 33 tumor types in The Cancer Genome Atlas project. Red line indicates median TMB. N = 40. (b) Mean TMB of grade 3 NENs (7.08, SD = 8.5, n = 40) compared to lower grade tumors previously reported (pancreas 0.82, small intestine 0.77, lung 0.4). (Fernandez-Cuesta, et al. 2014; Mafficini and Scarpa 2019; Yao, et al. 2019) (c) TMB among grade 3 NENs separated by tissue origin. Dotted line marks 10 mutations/MB, a known predictor of response to immunotherapy as validated in other cancers. The miscellaneous group has the highest TMB (vs GEP: p = 0.04), with head and neck primaries in this group showing the highest TMB (21.5 mutations/MB). Mean ± SD: GEP (4.28 ± 8.17, n = 15), Lung (6.01 ± 3.82, n = 14), Misc (12.28 ± 11.24, n = 11). One-way ANOVA/Tukey’s multiple comparison test. (d) Patients with high TMB had better overall survival than patients with lower TMB. Multivariate analysis accounted for age, stage, primary site, resected tumor and number of treatment lines, p = 0.03. Abbreviations: GEP, gastroenteropancreatic. NEN, neuroendocrine neoplasm. TMB, tumor mutation burden. SD, standard deviation.

Figure 5.. Two somatic copy number alterations confer increased risk of death.

(a) Focal somatic copy number alterations per GISTIC analysis, q > 0.25. N = 43. (b) Oncoplot of focal somatic copy number alterations. Frequency of arm 19q amplification and focal 22q13.31 deletion are mapped (gray = affected). (c) Arm level somatic copy number alterations. (d) Kaplan-Meier survival curves for differing burdens of copy number alteration. Focal deletion in 22q13.31 was associated with worse survival (HR 7.82, p = 0.034 by multivariate Cox analysis). Arm amplification of 19q was associated with worse survival (HR 4.82, p = 0.032 by multivariate Cox). Abbreviations: GEP, gastroenteropancreatic. NEN, neuroendocrine neoplasm; SCNA, somatic copy number alteration.