Whole-genome sequencing reveals distinct genetic bases for insulinomas and non-functional pancreatic neuroendocrine tumours: leading to a new classification system

Abstract

Objective: Insulinomas and non-functional pancreatic neuroendocrine tumours (NF-PanNETs) have distinctive clinical presentations but share similar pathological features. Their genetic bases have not been comprehensively compared. Herein, we used whole-genome/whole-exome sequencing (WGS/WES) to identify genetic differences between insulinomas and NF-PanNETs.

Design: The mutational profiles and copy-number variation (CNV) patterns of 211 PanNETs, including 84 insulinomas and 127 NF-PanNETs, were obtained from WGS/WES data provided by Peking Union Medical College Hospital and the International Cancer Genome Consortium. Insulinoma RNA sequencing and immunohistochemistry data were assayed.

Results: PanNETs were categorised based on CNV patterns: amplification, copy neutral and deletion. Insulinomas had CNV amplifications and copy neutral and lacked CNV deletions. CNV-neutral insulinomas exhibited an elevated rate of YY1 mutations. In contrast, NF-PanNETs had all three CNV patterns, and NF-PanNETs with CNV deletions had a high rate of loss-of-function mutations of tumour suppressor genes. NF-PanNETs with CNV alterations (amplification and deletion) had an elevated risk of relapse, and additional DAXX/ATRX mutations could predict an increased relapse risk in the first 2-year period.

Conclusion: These WGS/WES data allowed a comprehensive assessment of genetic differences between insulinomas and NF-PanNETs, reclassifying these tumours into novel molecular subtypes. We also proposed a novel relapse risk stratification system using CNV patterns and DAXX/ATRX mutations.

Keywords: gene mutation; neuroendocrine tumours; pancreas.

Figure 1

Differences in SMGs and CNVs between insulinomas and NF-PanNETs. SMGs and mutation rates in (A) 84 insulinomas and (B) 127 NF-PanNETs. NF-PanNET. Landscapes of somatic CNVs in (C) insulinomas (D) and NF-PanNETs; chromosomes and samples are shown on the y-axis and x-axis, respectively. Cluster classification indicated the presence of two subtypes with distinct CNV patterns within the insulinomas and three subtypes within the NF-PanNETs; the subtypes are highlighted with different colours. CNV, copy-number variation; Ins-Amp, insulinoma amplification subtype; Ins-Neutral, insulinoma neutral subtype; NF-Amp, NF-PanNET amplification subtype; NF-Del, NF-PanNET deletion subtype; NF-Neutral, NF-PanNET neutral subtype; NF-PanNET, non-functional pancreatic neuroendocrine tumour; SMG, significantly mutated gene.

Figure 2

Distinctive SNV characteristics within the same CNV patterns. Comparison of frequent mutations between (A) Ins-Neutral and NF-Neutral and between (B) Ins-Amp and NF-Amp. (C) Comparison of the molecular timing of chromosome amplifications between Ins-Amp and NF-Amp. (D) Fractional distributions of chromosomes that were involved in early amplification events (%pmt <20%) in Ins-Amp and NF-Amp tumours. ***P<0.001. CNV, copy-number variation; Ins-Amp, insulinoma amplification subtype; Ins-Neutral, insulinoma neutral subtype; NF-Amp, NF-PanNET amplification subtype; NF-Neutral, NF-PanNET neutral subtype; NF-PanNET, non-functional pancreatic neuroendocrine tumour; pmt, point mutation time; SNV, single-nucleotide variant.

Figure 3

Five molecular subtypes and corresponding RFS for insulinomas and NF-PanNETs. (A) The x-axis represents the chromosomal alterations. The y-axis represents the percentage of patients with insulinomas or NF-PanNETs in the current study. (B) RFS among CNV subtypes. CNV, copy-number variation; Ins-Amp, insulinoma amplification subtype; Ins-Neutral, insulinoma neutral subtype; NF-Amp, NF-PanNET amplification subtype; NF-Del, NF-PanNET deletion subtype; NF-Neutral, NF-PanNET neutral subtype; NF-PanNET, non-functional pancreatic neuroendocrine tumour; RFS, relapse-free survival.

Figure 4

Molecular subtypes within insulinomas. (A) Comparison of tumour diameters between Ins-Neutral and Ins-Amp. (B) Comparison of histological grades between Ins-Neutral and Ins-Amp. (C) Comparison of YY1 mutation rates between Ins-Neutral and Ins-Amp. (D) Rates of amplification events on early amplified chromosomes involved in mTOR-related genes. (E) IHC p-S6 scores in Ins-Neutral, Ins-Amp tumours and NF-PanNETs. (F) Demonstration of IHC p-S6 staining, with scores of 80, 40 and 0. *P<0.05. IHC, immunohistochemistry; Ins-Amp, insulinoma amplification subtype; Ins-Neutral, insulinoma neutral subtype; NF-PanNET, non-functional pancreatic neuroendocrine tumour; NS, not significant.

Figure 5

Molecular subtypes within NF-PanNETs. (A) Tumour diameters of the NF-PanNET. (B) The distribution of histological grades in the NF-PanNETs. (C) The presence of lymph node metastases in the NF-PanNETs. (D) Comparison of TMB among NF-PanNET CNV groups. (E) TSG two-hit inactivation in NF-PanNETs. *P<0.05, **P<0.01, ***P<0.001. CNV, copy-number variation; NF-Amp, NF-PanNET amplification subtype; NF-Del, NF-PanNET deletion subtype; NF-Neutral, NF-PanNET neutral subtype; NF-PanNET, non-functional pancreatic neuroendocrine tumour; NS, not significant; TMB, tumour mutation burden; TSG, tumour suppressor gene.

Figure 6

Clinical significance of CNV pattern and DAXX/ATRX mutation status. (A) RFS among three prognostic subtypes. (B) RFS between NF-Amp/Del with or without DAXX/ATRX mutations. (C) The presence of synchronous and metachronous distant metastases among NF-Neutral and NF-Amp/Del with or without DAXX/ATRX mutations. (D) Clinically oriented risk stratification diagram. *P<0.05. CNV, copy-number variation; NF-Amp/Del, NF-PanNET amplification and deletion subtypes; NF-Neutral, NF-PanNET neutral subtype; NF-PanNET, non-functional pancreatic neuroendocrine tumour; NS, not significant; RFS, relapse-free survival.