Array Comparative Genomic Hybridization-Based Characterization of Genetic Alterations in Pulmonary Neuroendocrine Tumors

Array comparative genomic hybridization-based characterization of genetic alterations in pulmonary neuroendocrine tumors

Johannes Voortmana,1,2, Jih-Hsiang Leea,1, Jonathan Keith Killianb, Miia Suuriniemib, Yonghong Wangb, Marco Lucchic, William I. Smith, Jr.d, Paul Meltzerb, Yisong Wanga, and Giuseppe Giacconea,3

aMedical Oncology Branch and bGenetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; cDivision of Thoracic Surgery, Cardiac and Thoracic Department, University of Pisa, 56120 Pisa, Italy; and dDepartment of Pathology, Suburban Hospital, Bethesda, MD 20892

Communicated by Ira Pastan, National Cancer Institute, National Institutes of Health, Bethesda, MD, June 11, 2010 (received for review April 12, 2010) The goal of this study was to characterize and classify pulmonary indicated that SCLC and bronchial carcinoids display distinct neuroendocrine tumors based on array comparative genomic hybrid- chromosomal imbalances and that, in general, bronchial carci- ization (aCGH). Using aCGH, we performed karyotype analysis of 33 noids tend to have fewer chromosomal imbalances (15). Further small cell lung cancer (SCLC) tumors, 13 SCLC cell lines, 19 bronchial advances of these studies, however, are hampered by small case carcinoids, and 9 gastrointestinal carcinoids. In contrast to the numbers as well as by low resolution attained by conventional relatively conserved karyotypes of carcinoid tumors, the karyotypes CGH analysis. Array-based CGH (aCGH) analysis has the ad- of SCLC tumors and cell lines were highly aberrant. High copy number vantages of high resolution and high-throughput genome-wide (CN) gains were detected in SCLC tumors and cell lines in cytogenetic screening of genomic imbalances (16–18) and has been used to bands encoding JAK2, FGFR1, and MYC family members. In some of successfully characterize several malignant disorders (19, 20). those samples, the CN of these genes exceeded 100, suggesting that Many lung-cancer cell lines have been developed and exten- they could represent driver alterations and potential drug targets in sively used as research tools to study cancer biology and to aid in subgroups of SCLC patients. In SCLC tumors, as well as bronchial drug development (21–23). Whether cancer cell lines are reliable carcinoids and carcinoids of gastrointestinal origin, recurrent CN representatives of in vivo cancer biology is, however, still a mat-

alterations were observed in 203 genes, including the RB1 gene and ter of debate. Cancer cell lines are developed under selection MEDICAL SCIENCES 59 microRNAs of which 51 locate in the DLK1-DIO3 domain. These pressure and, after many passages in vitro, may no longer be findings suggest the existence of partially shared CN alterations in representative of the original primary culture from which the cell thesetumor types. In contrast, CN alterationsofthe TP53geneandthe line was derived (21, 24). In line with this, changes in gene ex- MYC family members were predominantly observed in SCLC. Further- pression were observed during the establishment of SCLC cell more, we demonstrated that the aCGH profile of SCLC cell lines highly lines (25). Thus far, little is known about gene copy number resembles that of clinical SCLC specimens. Finally, by analyzing alterations (CNA) between primary SCLC and cell lines derived potential drug targets, we provide a genomics-based rationale for from SCLC. targeting the AKT-mTOR and apoptosis pathways in SCLC. Using aCGH analysis, we explored genomic alterations in SCLC, bronchial carcinoids, and carcinoids of GI origin. We also com- carcinoid | cell line | gene dosage | small cell lung carcinoma | therapy pared to what degree SCLC cell lines are similar to clinical SCLC specimens. We were able to demonstrate that SCLC cell lines and euroendocrine tumors of the lung compose a spectrum of SCLC tumors displayed very similar aCGH patterns. Furthermore, Ntumors ranging from low-grade typical carcinoids to small we identified genetic alterations that could define potential targets cell lung cancer (SCLC) (1). SCLC and bronchial carcinoids are for treatment of SCLC and bronchial carcinoids. characterized by neuroendocrine features and are thought to be derived from the Kultschitzky cells of the bronchial tree (2). Results These tumors have distinct histological morphologies and very Recurrent Copy Number Alterations in Pulmonary Neuroendocrine different clinical behaviors (1, 3). The 10-y overall survival rate Tumors. Fig. 1 depicts the karyotypes of SCLC tumors, SCLC cell of patients with a typical bronchial carcinoid has been reported lines (Fig. 1A), bronchial carcinoids (Fig. 1B), and carcinoids of GI at 87%, whereas the median overall survival rate of SCLC is only origin (Fig. 1C). As expected, the karyotype of bronchial carci- 2–3 mo if left untreated and approximately 1 y with treatment. noids was more conserved than that of SCLC tumors or SCLC cell Whereas SCLC is typically caused by cigarette smoke, the etio- lines. Whereas recurrent CNA tended to involve the whole arm of pathogenesis of carcinoids is obscure, and no clear correlation some chromosomes in SCLC tumors or cell lines, the alterations were more often located in narrower gene regions in bronchial with smoking has been found (1). Although both bronchial car- carcinoids. Fewer cytogenetic bands and fewer genes in these cinoids and carcinoids of gastrointestinal (GI) origin are well- bands with recurrent CNA were observed in bronchial carcinoids differentiated neuroendocrine tumors, there are site-specific compared with SCLC tumors or cell lines (Table 1). In SCLC differences between the two tumors in terms of clinicopatho- logical features, disease course, and prognosis (4, 5). It is pre- sumed that bronchial carcinoids are embryologically derived Author contributions: P.M. and G.G. designed research; J.V., J.-H.L., J.K.K., and M.S. per- from the foregut, whereas most carcinoids of GI origin are de- formed research; J.K.K., M.L., W.I.S., and P.M. contributed new reagents/analytic tools; J.V., rived from the midgut or hindgut. The cells of origin of these J.-H.L., Y.W., P.M., and Y.W. analyzed data; and J.V., J.-H.L., and G.G. wrote the paper. three tumors are possibly different, and the carcinogenetic The authors declare no conflict of interest. events are likely to be distinct (6). It is not clear whether a similar Data deposition: The data reported in this paper have been deposited in the Gene Ex- sequence of early oncogenic events may occur at the origin of pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE 21468). bronchial carcinoids, carcinoids of GI origin, and SCLC. 1J.V. and J.-H.L. contributed equally to this work. Traditional cytogenetic studies and conventional comparative 2Present address: VU University Medical Center, Amsterdam, The Netherlands. genomic hybridization (CGH) analyses have been performed in 3To whom correspondence should be addressed. E-mail: [email protected]. – both SCLC [7 10; reviewed by Balsara et al. (11)] and bronchial This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. carcinoids [12, 13; reviewed by Leotlela et al. (14)]. These studies 1073/pnas.1008132107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1008132107 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 the MPDZ protein (also known as MUPP-1) is a component of tight-junctions (30). Another high CN gain in one sample was detected in the chromosomal region 19q13.12, which contains 10 genes encoding factors involved in transcriptional regulation (SI Appendix, Table S3). Eight (61.5%) SCLC cell lines carried high CN gains of genes of the MYC family (SI Appendix, Table S5). CN gains (log2 ratio >0.2) of at least one MYC family member were detected in 27 of 33 SCLC tumors (81.8%) and in 1 of 19 bronchial carcinoids (4.3%, P < 0.001), which is in agreement with the notion that alteration of MYC family genes correlates with tumor aggressiveness.

Genetic Alterations Shared by SCLC and Carcinoid Tumors. Because both SCLC and carcinoids share neuroendocrine features (2, 31), we hypothesized that they may share common genetic alterations during the process of carcinogenesis. The number of genes and microRNAs affected by CNA in SCLC tumors, bronchial carcinoids, and carcinoids of GI origin is depicted in Fig. 2. In total, 203 genes and 59 microRNAs were found of which CNA were common for SCLC tumors, bronchial carcinoids, and carcinoids of GI origin (Fig. 2; Table 2; and SI Appendix,TableS6). None of these genes or microRNAs maps to a known chromosome fragile site (32). The retinoblastoma 1 (RB1) tumor suppressor gene was among the genes encoded on chromosome 13q that was commonly lost (SI Appendix, Table S6). Notably, the whole cluster of imprinted genes delineated by the delta-like homolog 1 gene and the type III iodothyronine deiodinase gene (DLK1-DIO3), located on chro- Fig. 1. Frequency of genomic alterations in neuroendocrine tumors and cell mosome 14q32 where many small nucleolar RNAs (snoRNAs) lines. Genome-wide frequency of copy number alterations in (A)SCLCtumor and 51 microRNAs are encoded, was on the list of shared genes samples (n = 33) and SCLC cell lines (n = 13), (B) bronchial carcinoids (n = 19), and (details of the snoRNAs are listed in the SI Appendix,TableS6). (C) carcinoids of GI origin (n = 9). (D) Comparison of the frequency of copy The delta-like homolog 1 (Dlk1) protein is expressed in neuro- number alterations in SCLC tumors obtained from lung (n = 19) or from meta- endocrine-lineage tumors, such as adrenal gland tumors, neuro- static sites (n = 14). Gains and losses are shown in green and red, respectively. blastoma, pheochromcytoma, and some SCLC cell lines (33, 34). Eight microRNAs in the DLK1-DIO3 domain are potential tumor suppressors (35). This suggests that genomic alterations of the tumors, there were recurrent copy number (CN) gains on chro- DLK1-DIO3 domain may play a role in the development of mosomes 1, 3q, 5p, 6p, 12, 14, 17q, 18, 19, and 20 and recurrent tumors with neuroendocrine features. CN losses on chromosomes 3p, 4, 5q, 10, 13, 16q, and 17p. This pattern is comparable to what was found in prior cytogenetic studies or by conventional CGH analysis (9–11). In bronchial Similarity of Copy Number Alterations Between SCLC Tumors and Cell carcinoids, recurrent CN gains were observed on chromosomes 5, Lines. SCLC cell lines shared similar karyotype patterns with SCLC 7, and 14, and recurrent CN losses were observed on chromo- tumors, with the exception of more CN losses observed on chromosome 2 in cell lines (Fig. 1A). Among ∼20,000 genes analyzed, somes 3, 11, and 22q. In carcinoids of GI origin, regions of re- ∼ fi current CNA were disseminated across autosomes with the only 771 genes ( 4%) showed signi cantly different frequencies of CNA (P < 0.01) between SCLC tumors and cell lines. In contrast, exception of CN gain of the chromosome 20. Three of the 9 ∼ fi carcinoids of GI origin harbored CN gain of chromosome 5, which 7,868 genes ( 39%) were observed to have signi cantly different was observed recurrently in bronchial carcinoids. frequencies of CNA between SCLC tumors and bronchial carcinoids and 4,189 genes (∼21%) between SCLC tumors and carci- High Copy Number Gains Are Associated with SCLCs but Not with noids of GI origin. It should be noted that most SCLC cell lines were derived from malignant pleural effusions or bone marrow Carcinoids. Considering that gene amplification is common in cancer and often related to activation of specific genes and pathways with cultures rather than from the primary tumors (Table 1). However, oncogenic properties (26), we studied cytogenetic bands or genes among 33 SCLC tumors, samples obtained from the primary lung > (n = 19) and metastatic sites (n = 14) demonstrated similar kar- with high CN gain (log2 ratio 3). The nonprotein coding plasma- D cytoma variant translocation (PVT1) gene was of interest because it yotype patterns (Fig. 1 ), and CN losses on chromosome 2 were is immediately downstream of the MYC gene and thought to be not observed in SCLC tumors obtained from metastatic sites. oncogenic (27, 28), and PVT1-CHD7 fusions were found in the We further selected cytogenetic bands in which the difference of NCI-H2171 and LU-135 SCLC cell lines (29). In our aCGH study, frequencies of CNA between SCLC tumors and cell lines was greater SI Appendix than 50% and statistically significant (Table 3). In total, only 74 genes PVT1 intragenic CN gain was observed ( ,Fig.S1). By fi SI Appendix using real-time PCR to study the CN of the PVT1 gene in SCLC cell were identi ed in these cytogenetic bands ( ,TableS7). lines, we found that the CN could be higher than 100 for specimens Cytogenetic band 7q34 was the only band for which a CN gain was with high CN gain in the PVT1 gene, and intragenic CNA in the observed more frequently in cell lines. This band contains two genes, NCI-H82 and NCI-H620 cells was confirmed (SI Appendix,Table PRSS1 and PRSS2, encoding serine protease trypsin 1 and trypsin S1). High CN gains were not observed in bronchial carcinoids or 2, respectively. carcinoids of GI origin (Table 1). High CN gains were found in four distinct cytogenetic bands in SCLC tumors (SI Appendix,TableS2), Potential Drug Targets of Bronchial Carcinoids and SCLC Identified by encoding 41 genes (SI Appendix,TableS3). Nine SCLC cell lines aCGH. To identify genes that may serve as predictive biomarkers for were observed to have high CN gains of 11 cytogenetic bands, anticancer therapies and/or constitute potential targets of ap- encoding a total of 39 genes (SI Appendix,TableS4). proved drugs and drugs under development, we performed real- High CN gains were observed for the fibroblast growth factor time PCR to confirm the accuracy of CNA of 10 selected genes receptor 1 (FGFR1) gene in one SCLC tumor sample (SI Ap- detected by aCGH assay (SI Appendix,Fig.S3). We observed pendix, Fig. S2A) and for the Janus kinase 2 (JAK2) gene in a highly significant correlation (P < 0.001) between the ratios, another sample (SI Appendix, Fig. S2B), suggesting that ampli- obtained by real-time PCR and by aCGH, of the CN of selected fication of these tyrosine kinases could have been driving the genes to the endogenous control RPPH1 gene (SI Appendix,Table malignant behavior of these individual tumors. A high CN gain S8 and Fig. S4). We demonstrated a trend for higher mRNA ex- of the multiple PDZ (MPDZ) gene was detected in one sample; pression of JAK2 or FGFR1 in SCLC cells with CN gains (SI Ap-

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1008132107 Voortman et al. Downloaded by guest on September 26, 2021 Table 1. Baseline characteristics of all samples and cell lines SCLC tumors SCLC cell lines Bronchial carcinoids Carcinoids of GI origin

Number 33 13 19 9 Age (y) Median 69 56 63 Range 52–94 30–83 30–85 Gender Male 15 12 3 4 Female 18 1 16 5 Origin of samples Lung 19 19 Mediastinum 6 Lymph node 2 Liver 3 1 Soft tissue 2 1 Brain 1 Pleural effusion 8 Bone marrow 4 Small intestine 6 Rectum 2 Cytogenetic bands with recurrent CNA Gains 122 98 86 92 Losses 48 71 45 89 Genes in cytogenetic bands with recurrent CNA Gains 8459 6851 536 3406 Losses 5085 7232 1021 1178 MicroRNAs in cytogenetic bands with recurrent CN aberrancy Gains 320 201 69 196 Losses 164 255 37 34 Cytogenetic bands with high CN gain 4 11 0 0 Genes in cytogenetic bands with high CN gain 41 39 0 0 MEDICAL SCIENCES MicroRNAs in cytogenetic bands with high CN gain 1 6 0 0

CN, copy number; CNA, copy number alternation, referring to cytogenetic band with log2 ratio > 0.2 (gains) or < −0.2 (losses); high CN gains are cytogenetic bands with log2 ratio >3.

pendix, Fig. S5). A total of 31 druggable genes were identified on the cently developed array-based CGH assay. To perform traditional basis of our aCGH and real-time PCR analyses (Table 4). The cytogenetic studies, metaphase chromosomes of cancer cells are frequencies of the observed CNA in our study were comparable to used for evaluation (7, 8). Whereas conventional CGH studies in data obtained from the Tumorscape (http://www.broadinstitute. SCLC detect genomic alterations involving extensive regions of org/tumorscape/pages/portalHome.jsf) (36). the genome, i.e., at the megabase level as shown by Levin et al. In bronchial carcinoids, genes encoding epidermal growth factor (9), aCGH analysis identifies genomic alterations at the kilobase receptor (EGFR), hepatocyte growth factor receptor (MET), platelet- level, readily defining the boundaries and thus the genes carried derived growth factor receptor 2 (PDGFRB), protein kinase B with regions of CNA. [reviewed by Feuk et al. (37)]. Molecular (AKT1/PKB), mammalian target of rapamycin (FRAP1), and Kirsten studies of SCLC are often hampered by the difficulty to obtain rat sarcoma viral oncogene homolog (KRAS) exhibited the most tumor samples because surgical interventions are relatively rare frequent CNA; however, the frequencies of CNA of candidate genes in the treatment course of this tumor type. Because of this, were below 30%. In contrast, the frequencies of CNA of selected drug formalin fixed, paraffin-embedded (FFPE) samples are often the target genes reached more than 75%. Frequent CNA were observed only resource available for further molecular analysis. Our for genes encoding proteins involved in the PI3K-AKT (PIK3CA, analysis is the largest array-based CGH study performed on AKT1, and PTEN) and apoptosis (BCL2, MCL1, and PMAIP1) pulmonary neuroendocrine tumors, focusing on FFPE SCLC pathways for SCLC, indicating that both pathways may be important specimens obtained up to two decades ago (38), and is the most for SCLC carcinogenesis or tumor maintenance and are potential comprehensive screening of CNA in the SCLC genome. The drug targets. karyotype of SCLC samples in our analysis is comparable to what was found in prior cytogenetic studies or by CGH analysis (9–11, Discussion 39), which may help open the door for a wider application of Genomic CN imbalances can be studied by cytogenetic analysis, aCGH analysis using FFPE SCLC samples. We observed that, in conventional chromosome-based CGH techniques, or the re- SCLC tumors, recurrent CNA were observed in more than half of all autosomal genes. The intricate and abundant CNA are important hallmarks of SCLC as they may be associated with the higher proliferation rate of SCLC (40). Using conventional CGH techniques, comparative analyses of the genomic differences among SCLC, bronchial carcinoids, and GI carcinoids have been conducted (41–43). Through comparison of bronchial carcinoids and SCLCs, Ullmann et al. (41) found that carcinoids carried fewer chromosomal aberrancies than SCLC tumors and that SCLC tumors were characterized by many gains and losses in their genome. Zhao et al. (42) reported on marked genomic imbalance differences between bronchial Fig. 2. Common copy number alterations across three types of neuroen- carcinoids and carcinoids of GI origin. The chromosome 11q13 docrine tumors. Venn diagram of (A) genes and (B) microRNAs with re- deletion is a hallmark of multiple endocrine neoplasia type 1 current CNA shared by SCLC tumors, bronchial carcinoids, and carcinoids of (MEN-1)-related foregut carcinoids and was observed in 5 of 19 GI origin. G: gains; L: losses. bronchial carcinoids in our analysis (13, 14). Because carcinoids

Voortman et al. PNAS Early Edition | 3of6 Downloaded by guest on September 26, 2021 Table 2. Cytogenetic bands with recurrent CNA in SCLC tumors, bronchial carcinoids, and carcinoids of GI origin Cytogenetic band No. of genes Region length (bp) Selected genes*

Gains 1 q25.1 2 237,106 2 p11.1 1 213,248 5 p15.33-p15.31 16 5,580,620 IRX2, IRX1, ADAMTS16, MED10, SRD5A1, POLS, FASTKD3 5 p15.2 7 3,704,524 DAP, CTNND2 5 p15.1 5 1,188,763 7 q11.22 2 29,091 7 q11.21 3 144,657 PMS2L3 7 q11.21–11.22 60 3,531,411 HIP1, ELN, FZD9, LIMK1, BAZ1B, GTF2IRD1, GTF2I, GTF2IRD2, GTF2IRD2B 12 q24.22-q24.23 1 622,164 14 q32.2-q32.31 49 1,099,667 RTL1 17 q24.3 1 1,594,088 SOX9 17 q25.1 1 63,059 17 q25.1 3 77,123 17 q25.3 2 765,774 CBX2 Losses 11 q21 8 698,251 MAML2 13 q13.3-q14.11 18 2,882,639 FOXO1, ELF1 13 q14.2 4 300,043 ITM2B, RB1 13 q32.3 6 673,267 15 q22.31 2 350,716 DAPK2 22 q13.1 12 1,185,129 SGSM3, EP300, RBX1

*Genes classiﬁed by Gene Ontology (GO) as related to cell proliferation, cell differentiation, cell cycle regulation, apoptosis, or DNA damage and repair are presented.

of GI origin may originate from midgut or hindgut embryologi- genes in other cancer types (36). In contrast, recurrent copy cally (5), the chromosome 11q13 deletion was observed in only number gain of the MYC family members and recurrent copy one of nine carcinoids of GI origin in our analysis. Our analysis number loss of the TP53 gene were observed in SCLC tumors as was in agreement with the hypothesis of site-specific difference well as in other cancer types (36) but were seldom observed in in the carcinogenesis of bronchial carcinoids and carcinoids of carcinoids. Amplification of the DLK1-DIO3 domain was ob- GI origin (14). served rarely in cancers other than SCLC and esophageal By comparing somatic CNA shared by several cancer types squamous cell carcinoma (36). We assumed that deletion of the from a large collection of cancer samples, Beroukhim et al. (36) RB1 gene may be an early event of all benign and malignant explored key genes with causal roles in oncogenesis. Considering neoplasms; deletion of the TP53 gene and amplification of the that the three tumor types in our analysis share neuroendocrine MYC genes may be related to malignant neoplasms only; fre- characteristics, we hypothesized that certain common molecular quent DLK1-DIO3 domain amplification tends to be a pattern of events may be related to the potential common origin of these neuroendocrine tumors. tumor types. Our analysis addressed what these three tumor Cancer cell lines are widely used in research, but their ability types genetically have in common. For example, the RB1 tumor to represent primary tumors has been questioned, and only a few suppressor gene was found to display a recurrent copy number studies have addressed this issue. Jones et al. (44) reported that loss in all three tumor types and is one of the leading deleted few new mutations were observed during the development of

Table 3. Cytogenetic bands in which the difference of CNA frequencies between SCLC tumors and cell lines were more than 50% Cytogenetic band No. of genes Region length (bp) Frequency in tumors (%) Frequency in cell lines (%) Selected genes*

Gains 1 p36.32 6 172,645 69.7 15.4 TNFRSF14, HES5, MMEL1 1 p36.32 1 417,368 60.6 7.7 PRDM16 1 p36.31 10 404,420 69.7 15.4 HES2, HES3, ZBTB48, CAMTA1 1 p36.31-p36.23 6 1,066,347 66.7 15.4 7 q34 3 228,766 18.2 69.2 PRSS1, PRSS2 18 p11.21-q11.1 1 1,143,946 54.5 0.0 Losses 2 q13 1 166,560 15.2 69.2 2 q13 2 172,569 18.2 69.2 2 q14.1-q14.2 4 1,229,775 18.2 69.2 2 q14.2 2 125,682 15.2 69.2 STEAP3, EN1 2 q14.2 1 52,720 9.1 61.5 CLASP1 2 q21.1 24 1,490,881 12.1 69.2 2 q21.3 5 645,481 18.2 69.2 ACMSD, CCNT2 2 q22.1 1 618,210 18.2 69.2 2 q22.1 1 396,539 24.2 76.9 2 q22.1 1 229,309 24.2 76.9 2 q22.3 1 285,232 15.2 69.2 2 q31.1 1 146,828 15.2 69.2 2 q33.3 3 232,140 9.1 61.5

P < 0.001 for all cytogenetic bands. *Genes classiﬁed by Gene Ontology (GO) as related to cell proliferation, cell differentiation, cell cycle regulation, apoptosis, or DNA damage and repair are presented.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1008132107 Voortman et al. Downloaded by guest on September 26, 2021 Table 4. Frequency of CNA among genes encoding drug target proteins in bronchial carcinoids and SCLC tumors Tumorscape: Bronchial Genes Protein Drug Event all cancers(%) Tumorscape: SCLC (%) SCLC tumors (%) SCLC cells (%) carcinoids (%) IGF1R igf1r MK0646 Gain 16.4 20 21.2 15.4 10.5 EGFR egfr Geﬁtinib Gain 31.6 37.5 30.3 15.4 26.3 ERBB2 her2 BIBW-2992 Gain 25.7 40 39.4 30.8 10.5 ERBB3 her3 Gain 19 27.5 36.4 15.4 0 ERBB4 her4 Lapatinib Gain 13.1 15 21.2 0 21.1 KDR vegfr2 Sorafenib Gain 10.3 10 24.2 15.4 0 KIT c-kit Imatinib Gain 10.5 10 36.4 15.4 0 MET met XL-184, MK8033 Gain 27.6 52.5 18.2 53.8 26.3 FGFR1 fgfr PD-173074 Gain 19.9 27.5 33.3 30.8 15.8 RET ret XL-184 Gain 11.2 7.5 3.0 0 5.3 PDGFRB pdgfr2 Imatinib Gain 16.9 12.5 6.1 7.7 26.3 JAK2 jak2 INCB018424 Gain 11.4 42.5 27.3 30.8 15.8 PIK3CA PI3K BGT-226 Gain 21.6 57.5 75.8 53.8 21.1 AKT1 akt GSK690693 Gain 16.3 47.5 63.6 38.5 26.3 PTEN pten Loss 24.2 62.5 75.8 69.2 21.1 FRAP1 mtor Everolimus Gain 13.4 45 54.5 15.4 26.3 SRC src Dasartinib Gain 29.4 37.5 60.6 38.5 15.8 ALK alk PF-02341066 Gain 16.5 27.5 30.3 23.1 10.5 KRAS k-ras PD-325901 Gain 24.8 45 21.2 30.8 26.3 PTCH1 patched GDC-0449 Loss 21.8 27.5 30.3 38.5 0 VHL vhl Sunitinib Loss 23.6 80 75.8 61.5 21.1 CDKN1A p21 Flavopiridol Loss 9.7 15 30.3 30.8 21.1 CDKN2A p16 Flavopiridol Loss 40 22.5 30.3 30.8 5.3 BRCA1 brca1 PARP inhibitor Loss 11.8 10 6.1 15.4 5.3 BRCA2 brca2 PARP inhibitor Loss 27.2 57.5 63.6 69.2 15.8 BCL2 bcl-2 Obatoclax Gain 11.9 47.5 51.5 61.5 10.5 MCL1 Mcl1 Obatoclax Gain 36.7 57.5 75.8 61.5 5.3 PMAIP1 noxa ABT-263 Gain 11.7 45 66.5 61.5 5.3 MEDICAL SCIENCES TP53 p53 Flavopiridol Loss 34.7 7.5 51.5 61.5 10.5 ERCC1 ercc1 Cisplatin Gain 13.1 40 51.5 30.8 15.8 RRM1 rrm1 Gemcitabine Gain 9.4 10 15.2 7.6 0

Also included in this table are data downloaded from Tumorscape (http://www.broadinstitute.org/tumorscape/pages/portalHome.jsf) (36).

colon cancer cell lines from primary tumors. Others suggested and the JAK2 gene in SCLC tumors were only 33.3% and 27.3%, that the genomes of a panel of 84 human non-small cell lung respectively, it is possible that subgroups of SCLC may respond cancer (NSCLC) cell lines are highly representative of the to FGFR1 inhibitors or JAK2 inhibitors, given the very high level original primary NSCLC tumors from which they were derived of amplification of this gene in individual samples. The fre- (45). By comparing the results of previously reported preclinical quencies of CNA in the drug target list in Table 4 in bronchial studies and clinical trials, Voskoglou-Nomikos et al. (46) sug- carcinoids were less than 30%, and high CN gain was not ob- gested that cancer cell lines can be useful in predicting the phase served; this is consistent with the low response rate of targeted II clinical trial performance of anticancer drugs. Our analysis therapies, including everolimus and sunitinib, in the treatment of demonstrated that copy number aberrancies of most genes, some bronchial carcinoids (1). of which encoded potential drug targets, were common in SCLC In conclusion, our analyses suggest that CNA of some genes, tumors and SCLC cell lines. including the RB1 tumor suppressor gene, were common in By comparing the CNA of SCLC tumors and cell lines, we SCLC, bronchial carcinoids, and carcinoids of GI origin. We found 74 genes locating in cytogenetic bands for which the dif- confirmed that SCLC cell lines represent SCLC tumors well ference of CNA frequencies between the two groups was greater karyotypically, but found that they do not contain the full spec- than 50%. Among the 74 genes, CN gains of the PRSS1 and trum of high copy number amplifications found in tumors. On PRSS2 genes, which encode trypsin 1 and trypsin 2, respectively, the basis of our analysis, we provide a genomics-based rationale are of interest. Trypsin expression is increased in several cancer for targeting the AKT-mTOR and apoptosis pathways in SCLC. types and may mediate cancer cell proliferation, metastasis, and invasion through the interaction with matrix metalloproteinases Materials and Methods and the proteinase-activated receptor 2 (47). It is possible that Sample Acquisition. Thirteen SCLC cell lines were selected for this study. The trypsin 1 and trypsin 2 may account for the aggressiveness of SCLC cells were GLC4 and GLC4-CDDP (kindly provided by S. de Jong, De- SCLC in patients and for rapid expansion in culture. partment of Medical Oncology, University Medical Center Groningen, 9713 GZ There has been little progress in the medical treatment of Groningen, The Netherlands), NCI-H69, NCI-H82, NCI-H128, NCI-H146, NCI- SCLC in the past two decades (48). We identified several po- H187, NCI-H526, NCI-N592, NCI-H620, NCI-H678, NCI-H792, NCI-H1173. Sixty- tential drug target genes on the basis of our aCGH data (Table one FFPE or cytology specimens of bronchial carcinoids, carcinoids of GI ori- 4). It is notable that genes encoding members of the PI3K-AKT- gin, or SCLC cases were included (SI Appendix, Table S9). Samples originated mTOR pathway and apoptotic regulating proteins had relatively from the VU University Medical Center, Amsterdam, The Netherlands (38); high frequencies of CNA in SCLC tumors, and codeletion and/or Suburban Hospital; the National Cancer Institute, National Institutes of coamplification of different members in the same pathway were Health (NIH); Johns Hopkins Hospital; and the University of Pisa. Use of human observed. Our study lays a strong foundation for further func- samples was approved by institutional review boards according to the legal tional characterization of the affected genes in the PI3K-AKT- regulations of the participating countries. All tissue samples were reviewed at mTOR and apoptotic pathways in SCLCs, which may help to the National Cancer Institute, NIH. Diagnosis was verified, and tissue sections define multitarget-specific strategies for the treatment of SCLC hosting more than 50% tumor materials were selected for aCGH. patients in the future. In addition, we observed high CN gain of the FGFR1 gene and the JAK2 gene in two individual SCLC DNA and RNA Extraction. Total genomic DNA was isolated from cell lines, tumors. Although the frequencies of CN gains of the FGFR gene cytology specimens, or deparaffinated FFPE specimens using reagents of the

Voortman et al. PNAS Early Edition | 5of6 Downloaded by guest on September 26, 2021 DNeasy Blood and Tissue Kit, QIAquick PCR Puriﬁcation Kit (Qiagen) as well as transcribed using High Capacity cDNA Reverse Transcription Kit, and then the Dako Target Retrieval Solution in protocols optimized for maximum yield mRNA expression was determined using TaqMan Gene Expression Assays from the various types of samples. Total RNA from cell lines was extracted by (Applied Biosystems). The CN of genes of interest were studied by TaqMan TRIzol (Invitrogen) in accordance with the manufacturer’s instructions. Fur- copy number assay (Applied Biosystems), in which the probes for the gene of ther details are available upon request. interest and the endogenous control gene were labeled by FAM and VIC reporters, respectively, and were measured in the same well. Primers for Array CGH Analysis. aCGH was performed using the SurePrint G3 Human CGH mRNA expression study and for gene CN study are available upon request. Microarray Kit 180K or 105K (Agilent Technologies) as well as reference The GAPDH gene and the RPPH1 gene were used as endogenous reference genomic male DNA (Promega). For Cy3/Cy5 labeling, the Genomic DNA ULS controls for mRNA expression study and gene CN study, respectively. Real- labeling kit was used (Agilent Technologies). Slides were scanned on an time PCR were operated on theABI 7900HT fast real-time PCR system (Applied −ΔΔ Agilent Microarray Scanner, followed by data extraction and normalization Biosystems). mRNA expression was analyzed by the 2 Ct method, gene CN by Feature Extraction v10.5 software (Agilent Technologies). Data analysis was analyzed by CopyCaller software v1.0 (Applied Biosystems), and the CN was carried out using Nexus 4.0 software (Biodiscovery). Sex chromosomes of a gene in a sample was calibrated to the CN of reference genomic DNA

were excluded from analysis. The thresholds of log2 ratio values for gain and (Promega), which was supposed to be two. loss were 0.2 and −0.2, respectively; the thresholds for high CN gain and homozygous deletion were 3.0 and −1.0, respectively. CNA was recurrent if Statistical Analysis. Comparisons of CGH patterns between cancer cell lines more than 35% of the samples of a speciﬁc tumor type carried the same CNA and clinical samples and between different histotypes were analyzed by with a P value less than 0.05. Fisher’s exact test. P < 0.01 was regarded as signiﬁcant.

Real-Time PCR. Total RNA and genomic DNA from SCLC cell lines were used for ACKNOWLEDGMENTS. We thank Dr. Hye-Seung Lee for a review of the mRNA expression and CN determination, respectively. Total RNA was reverse pathology discussed in this article.

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