Next-Generation Sequencing of Pulmonary Large Cell Neuroendocrine … · 2016. 5. 3. · Biology of Human Tumors Next-Generation Sequencing of Pulmonary Large Cell Neuroendocrine

Biology of Human Tumors

Next-Generation Sequencing of Pulmonary LargeCell Neuroendocrine Carcinoma Reveals SmallCell Carcinoma–like and Non–Small CellCarcinoma–like SubsetsNatasha Rekhtman1, Maria C. Pietanza2, Matthew D. Hellmann2, Jarushka Naidoo2,Arshi Arora3, Helen Won4, Darragh F. Halpenny5, Hangjun Wang1, Shaozhou K. Tian1,Anya M. Litvak2, Paul K. Paik2, Alexander E. Drilon2, Nicholas Socci6, John T. Poirier2,Ronglai Shen3, Michael F. Berger1,4, Andre L. Moreira1,WilliamD.Travis1, Charles M. Rudin2,and Marc Ladanyi1,7

Abstract

Purpose: Pulmonary large cell neuroendocrine carcinoma(LCNEC) is a highly aggressive neoplasm, whose biologic rela-tionship to small cell lung carcinoma (SCLC) versus non-SCLC(NSCLC) remains unclear, contributing to uncertainty regardingoptimal clinical management. To clarify these relationships, weanalyzed genomic alterations in LCNEC compared with othermajor lung carcinoma types.

Experimental Design: LCNEC (n ¼ 45) tumor/normal pairsunderwent targeted next-generation sequencing of 241 cancergenes by Memorial Sloan Kettering-Integrated Mutation Profilingof Actionable Cancer Targets (MSK-IMPACT) platform and com-prehensive histologic, immunohistochemical, and clinical anal-ysis. Genomic data were compared withMSK-IMPACT analysis ofother lung carcinoma histologies (n ¼ 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 and1 minor subsets: SCLC-like (n¼ 18), characterized by TP53þRB1co-mutation/loss and other SCLC-type alterations, including

MYCL amplification; NSCLC-like (n ¼ 25), characterized by thelack of coaltered TP53þRB1 and nearly universal occurrence ofNSCLC-type mutations (STK11, KRAS, and KEAP1); and carci-noid-like (n ¼ 2), characterized by MEN1 mutations and lowmutation burden. SCLC-like and NSCLC-like subsets revealedseveral clinicopathologic differences, including higher prolifer-ative activity in SCLC-like tumors (P < 0.0001) and exclusiveadenocarcinoma-type differentiation marker expression inNSCLC-like tumors (P ¼ 0.005). While exhibiting predominantsimilarity with lung adenocarcinoma, NSCLC-like LCNEC har-bored several distinctive genomic alterations, including morefrequent mutations in NOTCH family genes (28%), implicatedas key regulators of neuroendocrine differentiation.

Conclusions: LCNEC is a biologically heterogeneous group oftumors, comprising distinct subsets with genomic signatures ofSCLC, NSCLC (predominantly adenocarcinoma), and rarely,highly proliferative carcinoids. Recognition of these subsets mayinform the classification andmanagement of LCNECpatients.ClinCancer Res; 1–12. �2016 AACR.

IntroductionPulmonary large cell neuroendocrine carcinoma (LCNEC) is

defined pathologically as a neoplasm that shares with small celllung carcinoma (SCLC) a neuroendocrine (NE) phenotype andan extremely high proliferation rate but which lacks the classiccytomorphology of SCLC. Diagnostic criteria for LCNEC were

first introduced in 1991 by Travis and colleagues (1), and thiscategory became adopted as a distinct entity by the WorldHealth Organization (WHO) classification of lung tumors in1999 and subsequent editions (2). Prior to this, these uncon-ventional neuroendocrine tumors were classified under a vari-ety of diagnostic terms and criteria (3). While the LCNEC

1Department of Pathology, Memorial Sloan Kettering Cancer Center,New York, New York. 2Thoracic Oncology Service, Division of SolidTumor Oncology, Department of Medicine, Memorial Sloan KetteringCancer Center, New York, New York. 3Department of Epidemiologyand Biostatistics, Memorial Sloan Kettering Cancer Center, New York,NewYork. 4SloanKettering Institute,Memorial SloanKetteringCancerCenter, New York, New York. 5Department of Radiology, MemorialSloan Kettering Cancer Center, New York, New York. 6BioinformaticsCore, Memorial Sloan Kettering Cancer Center, New York, New York.7Human Oncology and Pathogenesis Program, Memorial Sloan Ket-tering Cancer Center, New York, New York.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Current affiliation for J. Naidoo: Upper Aerodigestive Division, Sidney KimmelComprehensive Cancer Center at Johns Hopkins, Baltimore, MD; current affil-iation for H. Wang: Department of Pathology, Jewish General Hospital, McGillUniversity, Montr�eal, Qu�ebec, Canada; and current affiliation for A.L. Moreira:Department of Pathology, New York University Langone Medical Center, NewYork, NY.

Corresponding Author: Natasha Rekhtman, Department of Pathology, Memo-rial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065.Phone: 212-639-5900, Fax: 646-422-2070; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-2946

�2016 American Association for Cancer Research.

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category captured in a single group highly proliferative non–small cell neuroendocrine neoplasms, it has long been recog-nized that tumors falling under this umbrella term are histo-logically heterogeneous (4, 5). In particular, although theprototypical LCNECs are morphologically sharply distinct fromSCLC, some LCNECs enter in a close morphologic differentialdiagnosis with SCLC (6). Clinically, LCNEC are generallyassociated with high rate of metastases and poor patient sur-vival, but remarkably broad survival ranges have been reported(7). In addition, highly variable results have been reportedregarding the chemosensitivity of LCNEC to platinum/etopo-side–based regimens utilized for SCLC (8–10), resulting in thelack of consensus on whether LCNEC should be clinicallymanaged as SCLC versus non-SCLC (NSCLC).

At that crux of the problem is the unresolved biologic relation-ship between LCNEC and SCLC, which is compounded by thepaucity of clinical studies and relative rarity of these tumors (�3%of all lung carcinomas; ref. 11). Because LCNEC shares severalfundamental clinicopathologic features with SCLC, includingaggressive clinical behavior, strong link to smoking, exceptionallyhigh proliferation rates, neuroendocrine gene expression, andsome morphologic features (in at least a subset of cases), com-bined with the evidence from experimental models of a closerelationship between SCLC and LCNEC (12), it has been postu-lated that LCNECs may represent a variant of SCLC. To date, geneexpression and limited genomic analyses have yielded conflictingresults regarding the biologic relationship between LCNEC andSCLC. Both entirely overlapping (13) and distinct (14) geneexpression profiles have been reported. Genomically, LCNEC isknown to share with SCLC frequent alterations in RB1 and TP53,albeit with variable reported frequencies (15–17). Currently, onlylimited published data are available on comprehensive genomicprofiles of LCNEC by next-generation sequencing (NGS) meth-ods. Notably, in a study that included whole-exome sequencingof 15 LCNECs, it was concluded that LCNEC was overall similarto SCLC but harbored occasional alterations characteristic of

other tumor types (17). On the other hand, there have beenseveral reports of molecular alterations typical of adenocarci-noma in histologically pure LCNEC with no overt adenocarci-noma component, including EGFR mutations (18, 19), ALKrearrangements (20), and KRAS mutations (21), drawing asharp contradistinction with classic SCLC, which in the purede novo form is consistently devoid of adenocarcinoma-typedriver mutations (22–24).

The goal of this study was therefore to perform detailedgenomic characterization of LCNEC to clarify its biologic rela-tionship with SCLC and other major lung cancer types, aswell as to describe the landscape of potentially targetablemolecular alterations in these tumors. Here, we report theresults of targeted NGS of 45 LCNECs, in conjunction withdetailed morphologic, immunohistochemical, and clinicopath-ologic analysis. Furthermore, we compared mutation profiles inLCNEC with those of other lung cancer histologies analyzed bythe same NGS platform.

Materials and MethodsPatients and samples

The study was performed with the approval of the InstitutionalReview Board of the Memorial Sloan Kettering Cancer Center(MSKCC, New York, NY). Surgically resected LCNECs wereincluded in the study after central pathology review by 3 thoracicpathologists (N. Rekhtman, A.L. Moreira, and W.D. Travis). Onlyhistologically pure LCNECs were included [i.e., tumors lackingmorphologically identifiable adenocarcinoma, squamous cellcarcinoma (SqCC), or SCLC component]. All cases met theWHOcriteria for LCNEC, including (i) neuroendocrine morphology,including organoid nesting, palisading, trabeculae, and/orrosettes; (ii) high grade, defined as >10mitoses per 10 high-powerfields (HPF) and typically necrosis; and (iii) IHC expression of atleast oneneuroendocrinemarker (synaptophysin, chromogranin-A, CD56/NCAM) in �10% of tumor cells (2). LCNEC wasdistinguished from SCLC based on a constellation of cytomor-phologic features, including the presence of nucleoli (contrastingwith the sine qua non nuclear appearance of SCLC characterized bydispersed granular chromatin lacking prominent nucleoli) and/orabundant cytoplasm, and typically but not invariably largernuclear size (2).

Molecular analysisDNA was extracted from approximately 80 mm of formalin-

fixed, paraffin-embedded tumor sections and matched benigntissue. Each tumor samplewasmacrodissected to enrich for tumorcells. Tissue was deparaffinized and DNA extracted using DNeasyBlood & Tissue Kit (Qiagen).

NGS of paired tumor/normal tissue samples was performedusing Memorial Sloan Kettering-Integrated Mutation Profiling ofActionable Cancer Targets (MSK-IMPACT) platform, a customhybrid capture-based assay for targeted deep sequencing of allexons and selected introns of key cancer genes. The selected genes(n ¼ 241) encompass the majority of established oncogenes andtumor suppressors, including actionable targets of approvedtherapies and agents under clinical investigation at MSKCC (fulllist in Supplementary Table S1). Briefly, barcoded DNA librariesfrom tumor and normal samples were captured using customoligonucleotide probes, massively parallel sequenced on an Illu-mina HiSeq 2500 platform, and subjected to a custom analysis

Translational Relevance

Currently, the data on comprehensive genomic profiles ofpulmonary large cell neuroendocrine carcinoma (LCNEC) arelimited, contributing to uncertainty regarding its biologicrelationship with other major lung cancer types and optimalclinical management. Here, we report on targeted next-gener-ation sequencing of 45 LCNECs with detailed clinicopatho-logic and immunohistochemical correlation. This analysisrevealed distinct subgroups within LCNEC, exhibiting geno-mic signatures of SCLC, non-SCLC (NSCLC; predominantlyadenocarcinoma), and, in a minority of cases, carcinoidtumors. These subsets were associated with several distinctclinicopathologic features, includinghigher proliferative activ-ity of SCLC-like tumors and exclusive exocrine differentiationmarker expression inNSCLC-like tumors.We also describe thelandscape of potentially targetable genomic alterations inLCNEC. Our data yield novel insight into the biology ofLCNEC andmay provide a useful framework for future clinicalinvestigations of cytotoxic and targeted therapies in patientswith these tumors.

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pipeline to identify single-nucleotide variants, small indels (<30bp), copy number alterations (CNA), and selected structuralrearrangements (including ALK fusions), as described in detailelsewhere (25). All candidate mutations and indels were manu-ally reviewed using Integrative Genomics Viewer.

Mutations predicted to be deleterious, defined as beingreported in the Catalogue of Somatic Mutation in Cancer orhaving medium/high functional impact score by Mutation Asses-sor [accessed via cBioPortal for Cancer Genomics (26)], wereexamined and manually curated for potential actionability usingapproved or investigational compounds. For comparison withother lung tumor histologies (N ¼ 242), MSK-IMPACT resultswere reviewed for lung adenocarcinomas (n ¼ 151), SqCC (n ¼36), SCLC (n ¼ 42), and carcinoids (n ¼ 13) that were consec-utively sequenced as part of routine clinical care under an insti-tutional initiative (27), or as part of clinical protocols or retro-spective studies, with detailed results reported in part elsewhere(24, 28).

Histologic, immunohistochemical, and clinical analysisHematoxylin and eosin–stained tissue sections were reviewed

to quantify the mitotic rate by manually counting the mitoticfigures in 10 HPFs, calculated as an average of at least 5 sets of 10HPF counts. Prominence of nucleoli and abundance of cyto-plasm, the key histologic features distinguishing LCNEC fromSCLC (2), were recorded. Tumors featuring prominent nucleoli(defined as nucleoli that were readily identifiable using a 10�microscope objective) and/or abundant cytoplasm (defined ascytoplasm of sufficient volume to render cytoplasmicmembranesreadily visible) were classified as "NSCLC-spectrum" morpholo-gy, whereas tumors with identifiable yet not prominent nucleoliand cytoplasm were classified as "SCLC-spectrum" morphology(i.e., themorphology that enters in the differential diagnosis withSCLC, yet is sufficiently distinct to qualify for the diagnosis ofLCNEC). Tumors with intermediate or geographically mixedmorphologic features were classified as "mixed." IHC was per-formed utilizing the following primary antibodies: synaptophy-sin (clone Snp88), chromogranin-A (LK2H10), CD56 (MRQ-42),Ki67 (MIB1), ASCL1 (24B72D11.1), pRb (13A100), STK11(Ley37D/G6), Napsin-A (IP64), and p40 (BC28; see Supplemen-tary Table S2 for detailed protocols and scoring criteria).

Electronic medical records were reviewed to collect demo-graphic and clinical information. Staging was performed accord-ing to the American Joint Committee on Cancer 7th Edition.Overall and recurrence-free survival were estimated using theKaplan–Meier method, with the mean follow-up period of 4.1years (range 0.02–14.5 years). For patients with localized diseasetreated with curative intent, recurrence-free survival was deter-mined as time to recurrence or death, whichever came first. Toassess treatment response to first-line chemotherapy, CT scanswere reviewed up to 7 months (mean 3 months) from a baselinescan using the standard RECIST guidelines (version 1.1) if scanswere available for review.

Statistical analysisStatistical analysis was performed using R software (R version

3.2.2). P values were computed using Fisher exact test and non-parametric Mann–Whitney U test for categorical and continuousvariables, respectively. Survival analysis was performed usingsurvival package in CRAN, version 2.38.

ResultsClinicopathologic characteristics

Tumor and patient characteristics of 45 LCNECs in this seriesare summarized in Supplementary Table S3. All but one patientwere current or former smokers, with a mean smoking history of50 pack-years (range 0–150). The mean age at diagnosis was 64(range 45–79) and 51% were male. Stage distribution was asfollows: stage I, 40%; stage II–IIIA, 44%; and stage IIIB/IV, 15%.The mean mitotic rate was 68/10 HPF (range 14–110), and themean Ki67 proliferation index was 75% (range 30%–100%). Alltumors expressed at least one conventional neuroendocrinemark-er (synaptophysin, chromogranin-A, CD56), 80% of tumorsexpressed 2 to 3 conventional markers, and 71% expressed theinvestigational neuroendocrine marker ASCL1.

Overall landscape of somatic mutationTargeted NGS of 45 LCNEC tumor/normal tissue pairs by

MSK-IMPACT platform identified a total of 535 nonsynon-ymous somatic mutations (392 missense, 66 nonsense, 37splice site, 34 frameshift, 4 deletion, 2 insertion), 61 gains,and 15 losses (Supplementary Table S4). Given the total pro-tein-coding territory of MSK-IMPACTof 1.2 Mb, we estimatethat the mean rate of nonsynonymous mutations in LCNEC is10.5/Mb. Of the single-nucleotide variants, 47.5% were G>T(C>A) transversions, typical of tobacco-induced carcinogenesis.The mean number of nonsynonymous mutations, gains, andlosses per case was 11.8 (range 1–30), 1.3 (range 0–7), and 0.3(0–2), respectively. The mean tumor coverage depth was 486-fold (range 60–1,086 fold).

Recurrently altered genesCommonly altered genes in LCNEC included TP53 (n ¼ 35;

78%), RB1 (n¼ 17; 38%), STK11 (n¼ 15; 33%), KEAP1 (n¼ 14;31%), and KRAS (n ¼ 10; 22%; Fig. 1A). Analogous to lungadenocarcinoma, KRAS, STK11, and KEAP1 mutations were notmutually exclusive (30, 31). Ninety-five percent of TP53 muta-tions affected the functionally critical DNA-binding domain. Themajority (81%) of RB1 mutations and many STK11 and KEAP1mutations were protein truncating (frameshift and nonsense),consistent with their tumor suppressor role. KRAS mutationscomprised the canonical missense mutations: G12C (n ¼ 6),G12D (n ¼ 1), G13D (n ¼ 2), and Q61L (n ¼ 1; example ofKRAS-mutant LCNEC shown in Supplementary Fig. S1).

Given the prior observations that the loss of pRB (29) andSTK11 (30, 31) expression may occur by nonmutationalmechanisms in lung carcinomas, LCNECs with sufficient tissuewere further evaluated by anti-pRb (n ¼ 42) and anti-STK11(n ¼ 40) IHC. As shown in Fig. 1B and C, all tested LCNECswith RB1 (n ¼ 16) and STK11 (n ¼ 15) gene alterations showedcomplete loss of the respective protein expression. In addition,complete loss of protein expression was identified in 2 of26 cases lacking RB1 and 3 of 25 cases lacking STK11 genealterations.

Molecular subsetsOn the basis of the recent NGS studies showing that joint

inactivation of RB1þTP53 is a virtually invariable signature eventin SCLC (22), we classified LCNEC with coaltered RB1þTP53 as"SCLC-like" (n¼ 18; 40%), whereas tumors lacking this coaltera-tionwere classified as "NSCLC-like" (n¼ 25; 68%) after exclusion

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of 2 outlier cases with carcinoid-like molecular and cytomorpho-logic features (see below).

Cooccurring genomic alterations in these 3 subgroups areshown in Fig. 2A. The most striking finding was that nearly allLCNECs lacking RB1þTP53 alterations (NSCLC-like subset) har-bored additional NSCLC-typemutations: STK11 and KRASmuta-tions, typical of adenocarcinoma, occurred in 15 (60%) and 10(40%) cases, respectively, with a combined rate of STK11 muta-tions/protein loss in NSCLC-like tumors of 72% (18/25 cases).Either STK11 mutation/protein loss or KRAS mutation werepresent in 21 of 25 (84%) cases. KEAP1-NFE2L2 mutations,typical of either adenocarcinoma or SqCC, were present in 9(36%) of cases. Overall, STK11, KRAS, or KEAP1-NFE2L2 muta-tions were present in 24 of 25 NSCLC-like LCNECs. Notably, allSTK11 and KRAS alterations were entirely restricted to thissubset, and none occurred in tumors bearing RB1þTP53 coaltera-tions (P < 0.0001 and P ¼ 0.0024, respectively; Fig. 2B). Con-versely, KEAP1–NFE2L2 mutations were not mutually exclusivewith RB1þTP53 (see below). Only a single NSCLC-like tumor(KRAS mutated) harbored RB1 mutation/protein loss, but thiscase lacked TP53 mutations. Several other alterations character-istic of NSCLC were identified in isolated cases this subset,including CDKN2A losses (n¼ 2),MAP2K1 (n¼ 1), and PIK3CA(n ¼ 1) mutations. NKX2.1 amplification, an alteration charac-teristic of both lung adenocarcinoma and SCLC, was identified inboth NSCLC-like (24%) and SCLC-like (17%) subsets. Interest-ingly, NSCLC-like tumors occasionally harbored alterations char-acteristic of SCLC and other neuroendocrine/neuronal tumors,including isolated cases with highMYCN amplification (8.5-fold)and IRS2 amplification.

Conversely, tumor with RB1þTP53 comutation/loss (SCLC-like subset) featured exclusive or preferential coalterations ofseveral genes typical of SCLC, including MYCL amplification(17%), SOX2 amplification (22%), PTEN mutation/loss(17%), and FGFR1 amplification (5%). In contrast, STK11 and

KRAS mutations were entirely absent, whereas KEAP1–NFE2L2alterations occurred in 39% of these cases (see Discussion).

The third, minor, subset of LCNEC, carcinoid-like, included 2outlier cases, characterized by a low total number of detectedalterations (two and one per case) compared with the mean of 13(range 4–32) genomic alterations per case for other LCNECs (P¼0.007; Supplementary Fig. S2). In addition, in contrast to all otherLCNEC in our dataset, both of these tumors harbored MEN1mutations - the hallmark of carcinoid tumors. BothMEN1muta-tions were protein truncating and were associated with highmutant allele frequencies (79% and 89%), suggesting associatedloss-of-heterozygosity.

Other frequently altered genes and functional groups,including NOTCH family genes

We identified the following 4 gene families/functional groupsto be frequently altered in LCNECoverall, with nopredilection foreither the SCLC-like or NSCLC-like subset. This included (i)chromatin modifiers (78% of LCNEC), with particularly highmutation frequency in MLL histone methyltransferases (33%)and SWI/SNF chromatin–remodeling genes (40%); (ii) genesimplicated in neurogenesis, including NOTCH1–4 (33%) andNTRK2/3 (19%); (iii) DNA replication/repair genes (52%); and(iv) PI3K–AKT–mTOR pathway genes (49%; Supplementary Fig.S3). Themajority (17/19) ofNOTCH familymutationswere overtloss-of-function (protein truncating)mutations or were predictedto be functionally deleterious by PROVEAN or SIFT (Supplemen-tary Table S5). Many mutations were located in the extracellularEGF-like domain (Supplementary Fig. S4), analogous to the loss-of-function NOTCH mutations in SCLC (22) and SqCC (32).

Molecular alterations in LCNEC subsets versus conventionallung carcinomas

Figure 3A illustratesmutation frequencies in LCNEC versus othermajor lung cancer histologies analyzed by the same NGS platform

RB1 gene Wild typeMut/lost

pRB

Prot

ein Lo

st 216

Expr

esse

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240

0%

10%

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40%

50%

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80%GainLossMut + Loss Muta�on

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95% CI 63%–

TP53

RB1

STK11

KEAP

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KRAS

PTPRT

NKX2-1

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NOTCH1

NOTCH4

EPHA

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89% 24%–54%

20%–49%

18%–47%

11%–37%

11%–37%

10%–35%

8%–32%

8%–32%

7%–30%

7%–30%

78%

38%33% 31%

22% 22% 20% 18%18% 16%16% STK11 gene Wild type Mutated

STK1

1 Pr

otei

n

Lost 315

Expr

esse

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220

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 genealterations 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.

Rekhtman et al.





(MSK-IMPACT), which highlighted the overall similarity betweenthe SCLC-like subset and SCLC and the NSCLC-like subset andadenocarcinoma. This comparison also revealed that despite theoverall similarity, therewere several distinctive alterations in LCNECsubsets comparedwith their corresponding conventional carcinomacounterparts. Significant differences between NSCLC-like LCNECand conventional adenocarcinoma included more frequent muta-tions in STK11 (60% vs. 16%; P ¼ 0.0001), NOTCH1–4 (28% vs.7%; P ¼ 0.005), chromatin modifiers (80% vs. 49%, P ¼ 0.005),fewer sensitizing EGFRmutations (0% vs. 25%, P¼ 0.003), as wellas significantly elevated comutation rate in STK11þTP53 (40% vs.8%; P ¼ 0.0002), respectively. Conversely, SCLC-like LCNEC dif-fered from SCLC by significantly higher rate of KEAP1 alterations(33% vs. 5%, respectively; P ¼ 0.008).

We further noted that the profile of major mutations inNSCLC-like LCNEC was reminiscent of the "proximal-prolif-erative" transcriptional subset of lung adenocarcinoma inThe Cancer Genome Atlas (TCGA) series (33), which is

similarly characterized by frequent mutations in STK11,KEAP1, and KRAS. To test the hypothesis that NSCLC-likeLCNEC may be related specifically to this subset of lungadenocarcinoma, we analyzed publicly available TCGA data-set for the expression of neuroendocrine genes, ASCL1 anddopa decarboxylase, previously found to be overexpressed ina fraction of lung adenocarcinomas (34–36). This analysisrevealed that, indeed, proximal-proliferative subset was sig-nificantly enriched in tumors with high expression of ASCL1(P ¼ 6.92e�10) and dopa decarboxylase (P ¼ 3.86e�14;Supplementary Fig. S5).

Finally, we compared overall mutation burden detected byMSK-IMPACT across LCNEC and other lung cancer types. Thisanalysis revealed that both NSCLC-like and, to a lesser extent,SCLC-like LCNEC subsets harbored a higher mutation load thantheir conventional counterparts: NSCLC-like subset exhibitedgreater number of mutations than both lung adenocarcinoma(P < 0.0001) and SqCC (P ¼ 0.0002), and the SCLC-like subset

05

101520253035

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gene

sper

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Carcinoid-like (n = 2)

Loss

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SCLC

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SCLC/SqCC

SCLC-like (n = 18) NSCLC-like (n = 25)

TP53_78%RB1_40%

MYCL_7%MYCN#_2%

IRS2#_4%

SOX2_11%FGFR1#_4%

PTEN_4%

KRAS_22%STK11_40%

MAP2K1_2%

KEAP1_31%NFE2L2_4%

CDKN2A#_4%

MYC¥_13%NKX2-1_20%

MEN1_4%

Case IDKi67

Napsin-Ap40 (ΔNp63)Morphology

Chemosensi�vity

RB1+TP53 Coaltera�onPresent(n = 18)

Absent(n = 25) P

STK11 mut_ 150 <0.0001(60%)STK11 mut/lossT 180 <0.0001(72%)

KRAS mut_ 100 0.0024(40%)STK11 mut/loss or KRAS mut_ 210 <0.0001(84%)

KEAP1-NFE2L2 mut/loss 7 9(39%) ns(36%)

SCLC/Adeno

Figure 2.Molecular subsets of LCNEC. A, OncoPrint depicting coalterations in selected genes in LCNEC with presence or absence of RB1þTP53 coalteration defining themajor 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 forwhich 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 ofexpression 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: þþþ >80%; þþ 60%–80%; þ 40%–50%; Napsin-A and p40: f, focally expressed byIHC; 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 RB1þTP53 and STK11/KRAS alterations. ns, not significant.






had marginally elevated mutation burden compared with con-ventional SCLC (P ¼ 0.03; Fig. 3B).

Potentially targetable genomic alterationsNo sensitizing EGFR mutations or ALK rearrangements were

identified. Nevertheless, at least one alteration potentially target-able by investigational agents was present in 30 of 45 (67%)LCNEC, more commonly in NSCLC-like than SCLC-like subset(84% vs. 50%, respectively; P¼ 0.02). Individual drug targets arelisted in Supplementary Table S6.

Molecular subsets of LCNEC and histomorphologic featuresWe next asked whether SCLC-like and NSCLC-like molecular

subsets of LCNEC were distinguishable morphologically. Indeed,we found that at least focal SCLC-spectrum morphology (seeMaterials and Methods) was over-represented in SCLC-likemolecular subset (72%), whereas NSCLC-spectrum morphologypredominated in tumors with NSCLC-like molecular features(76%), P ¼ 0.006 (Table 1; Fig. 4A and B). Despite this associ-ation, there was a substantial overlap in morphologic featuresbetween these subsets, with a total of 35% of cases exhibitingdiscordant or mixed morphology, particularly in SCLC-likemolecular group.

Two other histologic/immunophenotypic features differed inSCLC-like and NSCLC-like molecular subsets (Table 1). First, as agroup, tumors with SCLC-like molecular features displayed sig-nificantly higher proliferative activity thanNSCLC-like tumors, asrevealedbyKi67proliferationmarker expression (P<0.0001) andmitotic figure count (P¼ 0.017), although both subsets showed awide range of proliferation rates with substantial overlap inindividual cases (Fig. 5). Second, 9 (37%) NSCLC-like LCNECsexhibited low-level (weak and/or focal, 5%–30% tumor cells)reactivity for Napsin-A, a well-established and highly specificmarker of exocrine differentiation, whereas none of the SCLC-like tumors expressed this marker (P¼ 0.005). Conversely, a low-

level expression (�10% tumor cells) of squamous differentiationmarker, p40, was identified only in SCLC-like LCNECs (5 cases).Napsin-A and p40 expression was seen in areas of classic LCNECmorphology, lacking overt glandular or squamous differentiation(Fig. 4A and B). Neuroendocrine marker expression (synapto-physin, chromogranin-A, CD56, ASCL1) was equivalent in theSCLC-like and NSCLC-like subsets.

The 2 tumors with carcinoid-like molecular features met WHOcriteria for LCNEC based on mitotic rate exceeding 10 mitosis/10HPFs (featuring 30 and 14 mitoses per 10 HPF and Ki67 rate of40% and 30%, respectively), but these tumors did have distinctcarcinoid-type cytomorphology (Fig. 4C). Notably, althoughhigher than typical of carcinoid tumors, these proliferation ratesfell in the lowest range of proliferative activity seen in otherLCNECs (Fig. 5).

Molecular subsets of LCNEC and clinical features, includingresponse to cytotoxic therapy

Comparison of patient characteristics in the SCLC-like andNSCLC-like molecular subsets did not reveal significant differ-ences in the distribution of patient age, gender, stage, smokinghistory, or survival, although NSCLC-like subset was numericallyenriched in females compared with SCLC-like subset and includ-ed the sole never-smoker (Table 1). In addition, SCLC-like subsetexhibited a nonsignificant trend toward shorter recurrence-freesurvival (Supplementary Fig. S6).

In this series, only 11 patients received cytotoxic chemother-apy (almost all platinum based) for the de novo or recurrentstage IV LCNEC (Supplementary Table S7). Only 3 of 11patients had radiologic response to treatment (all partialresponses). Although the small number of evaluable patientsprecludes statistical analysis, we did note that all 3 responderswere in the SCLC-like molecular subset (3/4 treated patients),whereas none of the treated patients in NSCLC-like subsetshowed objective responses (0/6; Fig. 2), although 4 of 6

40 60 32 4 16 24 4 4 4 28 24 80

33 6 89 17 11 17 22 6 17 44 17 72

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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-likeand NSCLC-like LCNEC subsets versus conventional SCLC (n ¼ 42), adenocarcinoma (n ¼ 151), SqCC (n ¼ 36), and carcinoids (n ¼ 13), with bubble areaindicating frequency of an alteration. Significant differences (P < 0.01; Fisher exact test) are shown for SCLC-like LCNEC versus SCLC and NSCLC-like LCNECversus 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-IMPACTgene 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.

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NSCLC-like patients did show stable disease on therapy, sug-gesting some treatment benefit. One patient with carcinoid-likeLCNEC received cytotoxic chemotherapy and showed stabledisease.

DiscussionNSCLC-like subset

The first major finding in this study is that 56% of LCNECdisplayed NSCLC-like molecular features, characterized by thelack of RB1þTP53 coalteration and virtually invariable presenceof NSCLC-type mutations (STK11, KRAS, KEAP1, NFE2L2), withSTK11/KRAS mutations occurring in 80% of these tumors. Inconventional lung carcinomas, both STK11 and KRASmutationsoccur almost exclusively in adenocarcinomas (33), and theyare either entirely absent or exceptionally rare in SCLC, as previ-ously reported (22–24) and as seen in this study. This findingdraws a sharp contrast between NSCLC-like LCNEC and SCLCand highlights a predominant genomic similarity between thesetumors and lung adenocarcinoma. A link to adenocarcinoma isfurther supported by our finding that 37% of NSCLC-like LCNECexhibited low-level expression of pulmonary exocrine cell markerNapsin-A - an aspartic proteinase involved in surfactant protein Bprocessing, which is highly specific for lung adenocarcinoma andis consistently negative in SCLC (37).We suggest that NSCLC-likesubset of LCNEC may represent the extreme end of the spectrum

with tumors designated "NSCLC with NE differentiation,"the tumors in which neuroendocrine gene expression occur inthe absence of overt neuroendocrine morphology (34–36).The known phenomenon that neuroendocrine gene expressionhas a predilection for lung adenocarcinomas and is rare inSqCC (35, 36) parallels our finding of a predominant relation-ship with adenocarcinoma in NSCLC-like LCNEC. Our obser-vation that the profile of major mutations in NSCLC-likeLCNEC resembles the "proximal-proliferative" transcriptionalsubgroup of lung adenocarcinoma in TCGA series (33), whichis similarly dominated by STK11, KRAS, and KEAP1 mutations,and which we found to be significantly enriched in tumors withhigh neuroendocrine gene expression, further suggests a poten-tial link between NSCLC-like LCNEC and this specific subset oflung adenocarcinoma.

Despite the overall genomic similarity with adenocarcinoma,we found several molecular differences between NSCLC-likeLCNEC and conventional lung adenocarcinoma, which supportsthe concept that these tumors represent related but separateentities. The most striking difference was an unusually highrate of STK11 mutations (60%) in NSCLC-like LCNEC (with acombined rate of STK11 mutations and protein loss of 72%),compared with 16% to 17% STK11 mutation rate in lung ade-nocarcinoma in prior (33) and current series, respectively. STK11(also known as LKB1) is a tumor suppressor, which affects diversecell pathways, including cellular metabolism, pluripotency and

Table 1. Clinicopathologic features of SCLC-like versus NSCLC-like molecular subsets of LCNEC

SCLC-like (n ¼ 18) NSCLC-like (n ¼ 25) P

Age - mean (range) 63 (50–78) 64 (45–79) 0.803

Gender (female) 6 (33%) 16 (64%) 0.067

Smoking (pack-years) - mean (range) 50 (25–80) 51 (0–150) 0.830Never smoker 0 1Current/former smoker 18 24

StageI 7 (39%) 11 (44%) 0.922II–IIIA 8 (44%) 10 (40%)IIIB/IV 3 (17%) 4 (16%)

Syn, Chr, CD56 expressiona

þþ 14 (78%) 21 (84%) 0.701þ 4 (22%) 4 (16%)

ASCL1 Expression 10/16 (62%) 19/24 (79%) 0.295

Ki67 (MIB1) Proliferation rateb - mean (range) 88 (60–100) 68 (40–90) <0.0001>80% 12 (67%) 2 (8%)60–80% 6 (33%) 17 (68%)40–50% 0 6 (24%)

Mitotic count per 10 HPFb - mean (range) 83 (40–110) 60 (14–110) 0.017

Napsin-A expression 0/17 9/24 (38%)c 0.005

p40 (DNp63) expression 5/16 (31%)d 0/23 0.006

CytomorphologyLCNEC, SCLC-spectrum 9 (50%) 4 (16%) 0.006LCNEC, NSCLC-spectrum 5 (28%) 19 (76%)LCNEC, mixed 4 (22%) 2 (8%)

Survival (log-rank test)e

Overall survival: 5-yr (median) 59.5% (5.1 y) 53.8% (5.3 y) 0.883Recurrence-free survival: 5-yr (median) 27.5% (0.9 y) 51.4% (5.3 y) 0.475

Abbreviations: Chr, chromogranin-A; Syn, synaptophysin,aSyn, Chr, CD56 scoring: (þþ) at least one marker staining diffusely; (þ) all markers focal (staining <20% tumor cells).bSee Fig. 5 for scatter plots of Ki67 rates and mitotic figure counts.cDetected Napsin-A expression was weak and/or focal (5%–30% tumor cells labeling) in all cases and was seen in tumor areas with usual LCNEC morphology.dDetected p40 expression was focal (�10% tumor cells labeling) in all cases and was seen in tumor areas with usual LCNEC morphology.eSee Supplementary Fig. S6 for Kaplan–Meier survival curves.






SCLC

-like

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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 cytoplasmon hematoxylin and eosin (H&E)]. IHC illustrates complete loss of pRb in tumor cells (arrowhead indicates intact pRB expression in stromal cells, serving asinternal positive control), intact STK11 expression, high Ki67 index (100%), negative Napsin-A (arrowhead indicates Napsin-A–positive peritumoralpneumocytes, 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, completeloss of STK11 expression, Ki67 proliferation index of 70%, weak/focal Napsin-A expression, and negative p40. C, LCNEC with carcinoid-like molecularprofile (case ID LCNEC-36). H&E illustrates morphology characteristic of carcinoid tumors (bland uniform nuclei, abundant rosettes), but with unusually-highmitotic rate (arrowheads) and elevated Ki67 rate (40%). SYN synaptophysin.

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phenotypic plasticity (38), and whose inactivation in lungadenocarcinoma is associated with accelerated tumor growthandmetastases, particularly in cooperation with KRASmutations(39, 40). These functional roles of STK11 may be contributingto dysregulated differentiation and aggressive clinical phenotypeof LCNEC.

Other notable differences in NSCLC-like LCNEC from lungadenocarcinoma included frequent alterations in key neuro-genesis regulators, NOTCH family and NTRK2/3 and isolatedalterations typical of SCLC and other neuroendocrine/neuronaltumors,MYCN, and IRS2 amplifications. In agreement with ourfindings, frequent mutations in the NOTCH family genes (41)and NTRK2/3 (42) were identified in LCNEC by other inves-tigators. NOTCH family alterations are of particular interestgiven the strong evidence for their crucial role in neuroendo-crine fate specification in normal development (43) and inSCLC (22). Also of potential relevance is our finding of aremarkably high rate of mutations in chromatin modifiers inLCNEC, as this feature has recently emerged as a hallmark ofvarious neuroendocrine tumors (22, 44). Collectively, wehypothesize that alterations in genes regulating neurogenesisand the epigenetic landscape may contribute to the genesis ofneuroendocrine phenotype in NSCLC-like LCNEC.

SCLC-like subsetThe second major finding in this study is that 40% of

LCNEC exhibited SCLC-like genomic profile, characterized byRB1þTP53 coalteration, complete absence of STK11 and KRASmutations, and exclusive or enriched occurrence of MYCL,SOX2, and FGFR1 amplifications and PTEN mutation/loss,analogous to conventional SCLC (22–24). Importantly, theSCLC-like LCNECs displayed several clinicopathologic featuresthat paralleled those seen in conventional SCLC, includinghigher proliferative activity, a trend toward shorter recur-rence-free survival, and, in a subset analysis, a suggestion ofgreater platinum-based chemosensitivity (see below) thanNSCLC-like tumors. Furthermore, SCLC-like molecular subsetwas enriched in tumors with morphology that approached themorphologic spectrum of SCLC, yet met the criteria for LCNEC.Although these data support a close relationship of SCLC-likeLCNEC and conventional SCLC, whether these tumors may beregarded as a single group for management purposes willrequire further clinical studies.

Despite the overall similarity, a notable difference in SCLC-likeLCNEC from conventional SCLC identified in this study was an

elevated rate of KEAP1–NFE2L2 alterations (39%). These altera-tions only rarely occur in conventional SCLC, and in the absenceof concurrent STK11/KRASmutations, they are a common featureof SqCC. In fact, a close histogenetic relationship of some con-ventional SCLC and SqCC has been previously suggested, giventhat these tumors share several key genomic aberrations, includ-ing SOX2 and FGFR1 amplification and PTEN inactivation (45). Itis thus tempting to speculate that at least some SCLC-like LCNECmay share an even stronger histogenetic link with SqCC thanconventional SCLC, which may account for their atypical mor-phology and higher rate of KEAP1–NFE2L2 alterations. Thishypothesis is supported by our finding of focal expression ofsquamous marker p40 in this group of tumors.

Although the joint loss of RB1þTP53 has emerged as avirtually universal feature of SCLC (22), this coalteration isnot entirely specific to SCLC, as it also occurs in a minority ofconventional NSCLC (�6% in our series). Nevertheless, the useof RB1þTP53 coalteration as a classifier for SCLC-like subtypeof LCNEC is supported by the evidence from experimentalmodels showing that neuroendocrine cells are selectively vul-nerable to pRB loss, contrasting with the ability of non-neu-roendocrine cells to compensate for pRb inactivation (46),which suggests that pRB inactivation may expert selectivegrowth-promoting effects specifically in the context of neuro-endocrine carcinomas.

Carcinoid-like subsetThe finding that among LCNECs in this series 2 cases had

carcinoid-like molecular profiles, characterized by inactivatingMEN1 mutations and low overall mutation burden, analogousto conventional carcinoids (44), should not be interpreted asevidence that some bona fide LCNEC represent transformed car-cinoid tumors. It is important to emphasize that both carcinoid-like LCNECs were clear morphologic outliers in that they dis-played the overt carcinoid-type morphology, unlike the otherLCNECs in this series. Their classification as LCNEC was basedentirely on their proliferation rate exceeding the maximal prolif-erative activity currently accepted for lung carcinoids (i.e., 10mitoses/10 HPF, with typical Ki67 rate under 20%), whichqualifies them for the diagnosis of LCNECunder theWHOcriteria(2). This special issue will require further study, but overall, ourdata support carcinoid family kinship of these unusual tumorsand expand the spectrum of proliferative capacity currently rec-ognized for lung carcinoids, analogous to the recent findings forgastroenteropancreatic neuroendocrine tumors (47).

SCLC-like NSCLC-like Carcinoid-like

%Ki

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ells

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Figure 5.Scatter plots for Ki67 proliferationindex (A) and mitotic figure count inLCNEC subsets (B). Lines indicate themean.






Therapeutic implicationsA possible implication of our findings is that biologic sub-

groups of LCNEC could underlie the reported heterogeneity ofresponses to platinum-based chemotherapy in patients withLCNEC. Given the small number of patients with evaluabletreatment responses in this study, we could not definitely assessthis hypothesis. However, in a small subgroup analysis, therewas a suggestion that SCLC-like LCNECs exhibited greaterplatinum-based chemosensitivity than other LCNECs. Prospec-tive genomic analysis of patients with advanced disease, whichis currently ongoing at our institution, should enable furthercorrelation of molecular profiles and clinical behavior inLCNEC patients.

With respect to targeted therapy implications, the relativepaucity of potentially actionable alterations in SCLC-like sub-set, which included rare PTEN and FGFR1 alterations, parallelsthe observations in conventional SCLC (22–24). For NSCLC-like subset, the profile of potential targets resembles those seenin smoking-related lung adenocarcinomas. Although in thisstudy, we did not identify sensitizing EGFR mutations or ALKrearrangements, which are inversely associated with smokinghistory, these alterations have been reported in isolated cases ofLCNEC (18–20). Despite the apparent low incidence, it isimportant to include LCNEC in screening for EGFR and ALKalterations because impressive clinical responses to tyrosinekinase inhibitors have been reported in several EGFR-mutatedLCNECs (18, 19).

Our findings suggest several other special therapeutic consid-erations for LCNEC. First, the finding that both NSCLC-like and,to a lesser extent, SCLC-like LCNEC harbor extremely high muta-tion burden, exceeding that of conventional NSCLC and SCLC,suggests that these tumors may be particularly sensitive to PD-1/PD-L1 immune checkpoint inhibitors, whose activity has beenshown to correlate with high nonsynonymous mutational load(48). In addition, frequently altered chromatin modifiers inLCNEC may serve as potential targets for emerging epigenetictherapies (49).

PathologyAn important practical question raised by our findings is

whether SCLC-like and NSCLC-like subsets of LCNEC can beidentified by routine pathologic examination in the absence ofmolecular analysis. Although we did find that genomicallydefined subgroups were enriched in tumors with SCLC-likeand NSCLC-like morphologic features, respectively, and that, asa group, tumors with SCLC-like molecular features were moreproliferative, we also found a substantial histologic overlap,emphasizing that these molecular subtypes cannot be accurate-ly predicted by routine morphologic examination in individualcases. On the other hand, we found that RB1 alteration, the keydefining feature of SCLC-like group, can be accurately identifiedby IHC, which also detected few instances of pRB protein lossoccurring in the absence of gene mutations/CNAs, which maybe mediated by mechanisms like promoter hypermethylation,as described in SCLC (29). Thus, in a case with LCNEC mor-phology, intact pRb expression by IHC could serve as a markerof NSCLC-like subtype, even in the absence of moleculartesting. Conversely, the loss of pRb expression, although favor-ing SCLC-like subset, would require molecular confirmation ofconcurrent TP53 mutation given the rare possibility of pRb lossin the absence of TP53 mutation, as illustrated in a single case

of KRAS-mutated LCNEC with isolated RB1 mutation in thisstudy. Unlike pRb, we found p53 IHC to be an imperfectsurrogate for molecular testing in LCNEC (data not shown).In addition, our data suggest that the loss of STK11 expressionand/or presence of focal Napsin-A expression may serve asspecific albeit incompletely sensitive IHC-based markers ofNSCLC-like subset. Overall, pending further validation, ourdata suggest that IHC could serve to predict molecular subtypeof LCNEC in at least a subset of cases at the time of histopath-ologic evaluation.

Finally, it is worth commenting on a potential limitation ofour targeted NGS platform compared with more comprehen-sive NGS methodologies. Although 241 genes represented inour gene panel encompass the majority of the known func-tionally important cancer genes (27), we cannot exclude thepossibility that our analysis may have failed to identify relevantalterations in untested genes or non-exonic regions. Therefore,studies utilizing whole-genome/exome sequencing technolo-gies will be needed for more comprehensive characterization ofLCNEC, including more accurate determination of overallmutation burden in these tumors, which can only be estimatedon the basis of targeted NGS methods. Finally, analysis of alarger cohort of cases will be needed to capture a full spectrumof genomic profiles in LCNEC.

In conclusion, by performing targetedNGS in conjunctionwithdetailed histopathologic and clinical analysis, we gained insightthat LCNEC is composed of biologically distinct subgroups. Thesefindings have potential implications for classification, clinicalmanagement, and future investigations of LCNEC. Notably, thesedata parallel the recent general trend of genomics providingessential insights into the biologic relationships of tumors thathave been notoriously difficult to categorize by traditional meth-ods (17, 50).

Disclosure of Potential Conflicts of InterestM.C. Pietanza is an employee of Merck. M. Hellmann reports receiving

a commercial research grant from Bristol-Myers Squibb and is a consul-tant/advisory board member for AstraZeneca, Bristol-Meyers Squibb,Genentech, Inovio, Merck, and Neon. C.M. Rudin is a consultant/advisoryboard member for Bristol-Meyers Squibb, Celgene, ImaginAb, and Med-ivation. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: N. Rekhtman, M.C. Pietanza, J. Naidoo, A.L. Moreira,W.D. Travis, M. LadanyiDevelopment ofmethodology:N. Rekhtman, J. Naidoo, S.K. Tian, M.F. Berger,A.L. Moreira, W.D. TravisAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): N. Rekhtman, M.C. Pietanza, M.D. Hellmann,J. Naidoo, D.F. Halpenny, S.K. Tian, A.M. Litvak, P.K. Paik, M.F. Berger,A.L. Moreira, W.D. Travis, C.M. RudinAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): N. Rekhtman, M.C. Pietanza, M.D. Hellmann,J. Naidoo, A. Arora, H. Won, D.F. Halpenny, S.K. Tian, P.K. Paik, A.E. Drilon,N. Socci, J.T. Poirier, R. Shen, M.F. Berger, A.L. Moreira, W.D. Travis, M. LadanyiWriting, review, and/or revision of the manuscript: N. Rekhtman,M.C. Pietanza, M.D. Hellmann, J. Naidoo, H. Won, D.F. Halpenny, S.K. Tian,P.K. Paik, A.E. Drilon, J.T. Poirier, A.L. Moreira, W.D. Travis, C.M. Rudin,M. LadanyiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): N. Rekhtman, J. Naidoo, H. Wang, S.K. Tian,W.D. TravisStudy supervision: N. Rekhtman, J. Naidoo, M. Ladanyi


Rekhtman et al.




Grant SupportThis study was supported in part by the grant from the Fiona and Stanley

Druckenmiller Center for Lung Cancer Research (to N. Rekhtman), MSKCCDepartment of Pathology Research and Development grant (to N.Rekhtman), and P01CA129243 (to M. Ladanyi, N. Rekhtman, and M.F.Berger). The MSK-IMPACT program is supported in part by the Marie-Jos�eeand Henry R. Kravis Center for Molecular Oncology at Memorial SloanKettering Cancer Center and Cycle for Survival. This research was made

possible by infrastructure support provided by the NIH/NCI Cancer CenterSupport Grant P30 CA008748.

The costs of publication of this article were defrayed in part by the pay-ment of page charges. This article must therefore be hereby marked advertise-ment in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ReceivedDecember 4, 2015; revised February 11, 2016; accepted February 28,2016; published OnlineFirst March 9, 2016.

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Rekhtman et al.




Published OnlineFirst March 9, 2016.Clin Cancer Res Natasha Rekhtman, Maria C. Pietanza, Matthew D. Hellmann, et al.

like Subsets−Small Cell Carcinoma−like and Non−Neuroendocrine Carcinoma Reveals Small Cell Carcinoma

Next-Generation Sequencing of Pulmonary Large Cell

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Next-Generation Sequencing of Pulmonary Large Cell Neuroendocrine … · 2016. 5. 3. · Biology of Human Tumors Next-Generation Sequencing of Pulmonary Large Cell Neuroendocrine