Lung neuroendocrine tumours: deep sequencing of the four World Health Organization histotypes reveals chromatin-remodelling genes as major players and a prognostic role for TERT, RB1, MEN1 and KMT2D

Abstract

Next-generation sequencing (NGS) was applied to 148 lung neuroendocrine tumours (LNETs) comprising the four World Health Organization classification categories: 53 typical carcinoid (TCs), 35 atypical carcinoid (ACs), 27 large-cell neuroendocrine carcinomas, and 33 small-cell lung carcinomas. A discovery screen was conducted on 46 samples by the use of whole-exome sequencing and high-coverage targeted sequencing of 418 genes. Eighty-eight recurrently mutated genes from both the discovery screen and current literature were verified in the 46 cases of the discovery screen, and validated on additional 102 LNETs by targeted NGS; their prevalence was then evaluated on the whole series. Thirteen of these 88 genes were also evaluated for copy number alterations (CNAs). Carcinoids and carcinomas shared most of the altered genes but with different prevalence rates. When mutations and copy number changes were combined, MEN1 alterations were almost exclusive to carcinoids, whereas alterations of TP53 and RB1 cell cycle regulation genes and PI3K/AKT/mTOR pathway genes were significantly enriched in carcinomas. Conversely, mutations in chromatin-remodelling genes, including those encoding histone modifiers and members of SWI-SNF complexes, were found at similar rates in carcinoids (45.5%) and carcinomas (55.0%), suggesting a major role in LNET pathogenesis. One AC and one TC showed a hypermutated profile associated with a POLQ damaging mutation. There were fewer CNAs in carcinoids than in carcinomas; however ACs showed a hybrid pattern, whereby gains of TERT, SDHA, RICTOR, PIK3CA, MYCL and SRC were found at rates similar to those in carcinomas, whereas the MEN1 loss rate mirrored that of TCs. Multivariate survival analysis revealed RB1 mutation (p = 0.0005) and TERT copy gain (p = 0.016) as independent predictors of poorer prognosis. MEN1 mutation was associated with poor prognosis in AC (p = 0.0045), whereas KMT2D mutation correlated with longer survival in SCLC (p = 0.0022). In conclusion, molecular profiling may complement histology for better diagnostic definition and prognostic stratification of LNETs. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Keywords: KMT2D; MEN1; RB1; TERT; exome sequencing; lung tumours; neuroendocrine; targeted sequencing.

Figure 1

Flow chart of the experiments performed on 148 LNETs. The chart shows the workflow of sequencing analysis, including discovery screen on 46 fresh‐frozen cases and validation on 102 FFPE cases.

Figure 2

Recurrent alterations in 148 LNETs. Cases are grouped according to the four histotypes defined by the WHO classification. The histograms on the left report the alteration frequency of each gene expressed as a percentage. Alterations are annotated by different colours, according to their impact on the gene product. (A) The upper histogram shows the number of mutations in recurrently altered genes for each sample. Asterisks indicate the two hypermutated cases that emerged from WES. The matrix shows 26 genes that were mutated in at least three cases. (B) The matrix shows the copy number status of 13 genes in the whole cohort.

Figure 3

Somatic copy number alterations detected in 20 LNETs by WES. Cases are grouped according to the four histotypes defined by the WHO classification, and arranged from the most to the least altered within each category. Asterisks indicate the two hypermutated cases that emerged from WES. (A) Copy gain (red/yellow) or loss (cyan/blue) of large chromosomal regions. (B) Overview of genes affected by either somatic mutation or copy number alteration. For each case, the upper panel shows somatic mutations, and the lower panel shows copy number alterations, both annotated according to the colour panels at the bottom.

Figure 4

Somatic mutations in chromatin‐remodelling and cell cycle checkpoint genes for 148 LNETs. Cases are grouped according to the four histotypes defined by the WHO classification. Micrographs illustrate a representative case for each histotype. Genes are grouped as per molecular pathway. The histograms on the left show the mutation frequency of each gene expressed as a percentage. Mutations are annotated according to the colour panels at the bottom.

Figure 5

Disease‐specific survival according to pathological and molecular features. (A) Disease‐specific survival of patients with LNET (n = 117) is significantly affected by histology (p < 0.0001). (B, C) The presence of MEN1 mutation in ACs correlates with poor prognosis (p = 0.0045) (B), whereas KMT2D mutation is linked to better prognosis in patients with SCLC (p = 0.0022) (C). Survival time is expressed in months. Kaplan–Meier and log‐rank statistics were used to determine levels of significance. x‐Axes are trimmed for visualization purposes.