Next-Generation Sequencing of Pulmonary Large Cell Neuroendocrine Carcinoma Reveals Small Cell Carcinoma-like and Non-Small Cell Carcinoma-like Subsets

Abstract

Purpose: Pulmonary large cell neuroendocrine carcinoma (LCNEC) is a highly aggressive neoplasm, whose biologic relationship to small cell lung carcinoma (SCLC) versus non-SCLC (NSCLC) remains unclear, contributing to uncertainty regarding optimal clinical management. To clarify these relationships, we analyzed genomic alterations in LCNEC compared with other major lung carcinoma types.

Experimental design: LCNEC (n = 45) tumor/normal pairs underwent targeted next-generation sequencing of 241 cancer genes by Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) platform and comprehensive histologic, immunohistochemical, and clinical analysis. Genomic data were compared with MSK-IMPACT analysis of other lung carcinoma histologies (n = 242).

Results: Commonly altered genes in LCNEC included TP53 (78%), RB1 (38%), STK11 (33%), KEAP1 (31%), and KRAS (22%). Genomic profiles segregated LCNEC into 2 major and 1 minor subsets: SCLC-like (n = 18), characterized by TP53+RB1 co-mutation/loss and other SCLC-type alterations, including MYCL amplification; NSCLC-like (n = 25), characterized by the lack of coaltered TP53+RB1 and nearly universal occurrence of NSCLC-type mutations (STK11, KRAS, and KEAP1); and carcinoid-like (n = 2), characterized by MEN1 mutations and low mutation burden. SCLC-like and NSCLC-like subsets revealed several clinicopathologic differences, including higher proliferative activity in SCLC-like tumors (P < 0.0001) and exclusive adenocarcinoma-type differentiation marker expression in NSCLC-like tumors (P = 0.005). While exhibiting predominant similarity with lung adenocarcinoma, NSCLC-like LCNEC harbored several distinctive genomic alterations, including more frequent mutations in NOTCH family genes (28%), implicated as key regulators of neuroendocrine differentiation.

Conclusions: LCNEC is a biologically heterogeneous group of tumors, comprising distinct subsets with genomic signatures of SCLC, NSCLC (predominantly adenocarcinoma), and rarely, highly proliferative carcinoids. Recognition of these subsets may inform the classification and management of LCNEC patients. Clin Cancer Res; 22(14); 3618-29. ©2016 AACR.

Figure 1

Commonly altered genes in LCNEC. A, histogram of commonly altered genes detected by NGS (MSK-IMPACT) in LCNEC. CI, confidence intervals. B, RB1 gene alterations versus pRb expression by IHC. C, STK11 mutations versus STK11 expression by IHC. IHC not available for pRB and STK11 IHC in 3 and 5 cases, respectively. Mut, mutated.

Figure 2

Molecular subsets of LCNEC. A, OncoPrint depicting coalterations in selected genes in LCNEC with presence or absence of RB1+TP53 coalteration defining the major SCLC-like and NSCLC-like subsets, respectively, and MEN1 mutations (mut) and low total mutation burden defining carcinoid-like subset. Left, conventional lung cancer types characteristically associated with indicated gene alterations are shown. ^¥, MYC amplifications also occur in SqCC. ^#, genes for which only CNAs are shown. Loss of pRB and STK11 expression by IHC (↓) is only shown for cases without gene mutations/losses. For all other cases, loss of expression and molecular results were concordant. No pRB IHC available for cases 27, 897, 299; and no STK11 IHC available for cases 27, 55, 637, 299, and 913. Selected clinicopathologic features are designated as follows: Ki67: +++ >80%; ++ 60%–80%; + 40%–50%; Napsin-A and p40: f, focally expressed by IHC; 0, no expression; na, not available; morphology: S, SCLC spectrum; N, NSCLC spectrum; M, mixed; C, carcinoid-like; chemosensitivity: PR, partial response; SD, stable disease; PD, progressive disease. B, mutual exclusivity of RB1+TP53 and STK11/KRAS alterations. ns, not significant.

Figure 3

Genomic alteration in LCNEC versus other lung cancer types analyzed by MSK-IMPACT. A, bubble graph illustrating frequency of selected alterations in SCLC-like and NSCLC-like LCNEC subsets versus conventional SCLC (n = 42), adenocarcinoma (n = 151), SqCC (n = 36), and carcinoids (n = 13), with bubble area indicating frequency of an alteration. Significant differences (P < 0.01; Fisher exact test) are shown for SCLC-like LCNEC versus SCLC and NSCLC-like LCNEC versus adenocarcinoma. EGFR and ALK alterations (not shown) were absent in LCNEC, compared with 25% and 7% rate, respectively, in adenocarcinoma. See Supplementary Fig. S3 for individual genes in “chromatin modifiers” group. B, dot plot graph illustrating total number of nonsynonymous mutations (mut) per case in LCNEC versus other lung tumors. Lines indicate the mean. This comparison was performed for 222 genes shared across several MSK-IMPACT gene panel versions used to sequence different tumor types. Three data points (2 adenocarcinomas, 1 SqCC) are outside the axis limit. amp, amplification; co-alt, coalteration. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Figure 4

Morphologic and immunohistochemical features of genomically defined LCNEC subsets. Representative examples from each molecular subset are illustrated. A, LCNEC with SCLC-like molecular profile (case ID LCNEC-47) and SCLC-spectrum morphology [identifiable but not prominent nucleoli and cytoplasm on hematoxylin and eosin (H&E)]. IHC illustrates complete loss of pRb in tumor cells (arrowhead indicates intact pRB expression in stromal cells, serving as internal positive control), intact STK11 expression, high Ki67 index (100%), negative Napsin-A (arrowhead indicates Napsin-A–positive peritumoral pneumocytes, serving as internal positive control), and focal (<5%) tumor cell labeling for p40. B, LCNEC with NSCLC-like molecular profile (case ID LCNEC-40, STK11-mutated) and NSCLC-spectrum morphology (prominent nucleoli and abundant cytoplasm on H&E). IHC illustrates intact pRB expression, complete loss of STK11 expression, Ki67 proliferation index of 70%, weak/focal Napsin-A expression, and negative p40. C, LCNEC with carcinoid-like molecular profile (case ID LCNEC-36). H&E illustrates morphology characteristic of carcinoid tumors (bland uniform nuclei, abundant rosettes), but with unusually-high mitotic rate (arrowheads) and elevated Ki67 rate (40%). SYN synaptophysin.

Figure 5

Scatter plots for Ki67 proliferation index (A) and mitotic figure count in LCNEC subsets (B). Lines indicate the mean.