Genomic Amplification of CD274 (PD-L1) in Small-Cell Lung Cancer

This article is featured in Highlights of This Issue, p. 1121

Small-cell lung cancer (SCLC) is the most aggressive type of lung cancer and accounts for about 15% of all lung cancer cases (1). Unfortunately, most SCLC cases are diagnosed at advanced, metastatic stage, when treatment is only palliative. Furthermore, although most SCLC tumors respond initially to chemotherapy, disease recurs quickly in all patients and is universally fatal within only few months. Beyond chemotherapy, no effective drugs have been approved over the last decades, thus necessitating novel and effective therapeutic approaches (2).

Blockade of immune checkpoints increases antitumor immunity (3, 4). Clinical trials have shown that inhibiting cytotoxic T-lymphocyte antigen 4 (CTLA-4) or the interaction between programmed death-1 (PD-1) and the PD-1 ligand, PD-L1, can overcome the intrinsic resistance to immune surveillance in various types of cancers, including lung cancer, thus leading to significant and durable clinical responses (5–8). More recently, anti–PD-1/PD-L1–based immunotherapies have also shown early signs of clinical efficacy in SCLC (9–11).

Expression of PD-L1 protein in tumor cells has been proposed as a predictive biomarker of response to anti-PD-1/ PD-L1 treatment (7, 8, 12). However, the mechanisms governing levels of PD-L1 expression in tumor cells remain poorly understood and several patients respond to checkpoint blockade, whose tumors lacked expression of PD-L1. Thus, efficacy might not primarily be related to basic levels of PD-L1 expression. In addition, efficacy of PD-1 blockade segregates with mutational burden in the tumor in melanoma and lung cancer and with mutations in DNA repair genes in colorectal cancer (13).

We have recently shown that PD-L1 expression is almost universally absent in SCLC tumor cells (14). However, approximately 50% of cases exhibited PD-L1 expression in tumor-infiltrating immune cells. Although two other studies reported high expression of PD-L1 in SCLC tumor cells in the majority of the cases (15, 16), both studies had involved an antibody that has been discontinued due to lack of specificity for PD-L1. When examining PD-L1 expression by RNA sequencing, which is less prone to these methodological distortions, PD-L1 expression in SCLC is generally low to absent (14). We therefore sought to determine in detail genomic alterations that might affect the levels of PD-L1 expression in SCLC.

Between 1985 and 2013, tumor and corresponding noncancerous tissues were obtained from SCLC patients at surgery, autopsy, or biopsy with the approval of each institutional review board. Two independent cohorts were examined. Cohort 1 comprised 88 tumors from 72 patients recruited from National Cancer Center Hospital, Tokyo, Japan, Saitama Medical University, Saitama, Japan, the University of Tsukuba, Ibaraki, Japan, and hospitals in the Metropolitan Baltimore area. Sixty-two of these samples had been previously subjected to genome scanning (17). Cohort 2 comprised 138 tumors from 138 SCLC patients diagnosed with stage I-IV SCLC. These tumor samples are enriched for Caucasian ethnicity and were collected by multiple collaborating centers. Cohort 2 cases were previously analyzed in genomic studies for SCLC (18). Information regarding age, sex, ethnicity, pathologic TNM stage (the 7^th classification), and smoking status was collected retrospectively (Supplementary Tables S1 and S4).

Copy-number alterations in cohort 1 were examined by quantitative real-time PCR. Genomic DNA was extracted using a QIAamp DNA mini kit (QIAGEN) in accordance with the manufacturer's instructions. Quantitative real-time PCR (qRT-PCR) was performed using a TaqMan copy number assay (Thermo Fisher Scientific) and the 7900 HT Fast Real-Time PCR System (Thermo Fisher Scientific). DNA (10 ng) was added to each 10 μL PCR reaction, which contained TaqMan Universal Genotyping Master Mix. All assays were performed in quadruplicate and investigators were blinded to the clinical outcome. The following TaqMan probes were purchased from Thermo Fisher Scientific: CD274 (Assay ID: Hs00268713_cn), PDCD1LG2 (Hs01115641_cn), JAK2 (Hs02831068_cn), PLGRKT (Hs03723314_cn), KIAA1432 (Hs03076906_cn), FLJ41200 (Hs03718447_cn), and NFIB (Hs01324052_cn). RNase P (#4403326) was used as a reference control. The predicted copy number was calculated using CopyCaller v2.0 (Thermo Fisher Scientific) and normalized to the mean of noncancerous tissues from ten Japanese individuals in this study. Samples from cohort 1 were further analyzed with human 250K Nsp SNP arrays, followed by data processing using Copy Number Analyzer for Affymetrix GeneChip Mapping Array (CNAG) software, ver.2.0 (17). Copy number was determined with respect to the copy number of noncancerous lung tissues of the same patient. In addition, cases from cohort 1 (n = 10) were analyzed by SNP 6.0 array following the procedure as previously described (18).

Copy number alterations of samples from cohort 2 (n = 138) were calculated from Affymetrix Genome-Wide Human SNP array 6.0 data (n = 132) or by referring to copy-number data calculated from whole-genome sequencing (n = 98). The data were analyzed as previously described (18). Genomic regions are determined according to the human reference genome NCBI37/hg19.

GISTIC 2.0 was used to determine significant copy number alterations for the SNP 6.0 Array data from samples of cohort 1 and 2 (19). Genomic regions with a log₂ ratio of above 0.5 were considered as amplified. The significance threshold was set at Q = 0.25.

Whole-genome sequencing was performed as previously described (18). Genomic copy numbers were deduced from the number of sequence reads and described as integral copy number (iCN) values. Transcriptome sequencing data of cohort 1 (n = 7; ref. 17) and cohort 2 (n = 74; ref. 18) were analyzed following the computational approach as described previously (18). The transcript levels of each gene were analyzed as FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values. The transcriptome sequencing data were used to analyze for transcripts specific for B cells (CD19) and T cells (CD3E), and to further define T-cell subsets for CD8 T cells (average of CD8A and CD8B transcripts), CD4 T cells (CD4) and regulatory T cells (Tregs, average of FOXP3 and IL2RA transcripts).

Tissue block sections (4-μmthick) were deparaffinized, incubated in xylene and ethanol, and then washed with water. For antigen retrieval, sections were boiled in 10 mmol/L sodium citrate buffer for 10 minutes at 121°C, rinsed with water, incubated in 3% peroxidase, and then washed with water followed by buffer. For PD-L1 staining, sections were incubated with anti-PD-L1 (E1L3N; Cell Signaling Technology) diluted 1:800 with antibody diluent (Signal Stain Antibody Diluent #8112, Cell Signaling Technology), followed by a peroxidase-labeled secondary antibody (EnVision/HRP System; DAKO), rinsed in buffer, and immersed in DAB substrate. IHC of a Cohort 2 sample showing focal CD274 amplification was performed as previously described (14). FFPE sections were stained for SCLC marker: CD56 (clone 123C3, dilution: 1:500, Zytomed), synaptophysin (clone SP11, dilution: 1:50, Thermo Fisher Scientific), chromogranin A (clone DAK-A3, dilution 1:500; Dako Cytomation), and Ki-67 (clone SP6, dilution: 1:100, Cellmarque). The following immune cell markers were used for IHC: CD3 (clone SP7, dilution: 1:50, Thermo Fisher Scientific), CD4 (clone 4B12, dilution: 1:100, Thermo Fisher Scientific), CD8A (clone C8/144B, dilution: 1:200, Dako) and CD20 (clone L26, dilution: 1:1250, Dako).

Immunostaining for PD-L1 was evaluated by at least two independent observers (M. Saito, K. Tsuta, and A.H. Scheel) using the H-score method, as previously described (20). Briefly, staining percentages (0%–100%) and the intensity (0, negative; 1, very weak expression; 2, moderate expression; 3, strong expression) in tumor cells were evaluated and then immunostained slides were scored ranging from 0 to 300 by multiplying the percentage of tumor area. For PD-L1 expression, a score of 3 was used as the cut-off score due to the antibody specificity.

We first sought for mutations affecting the CD274 gene in four SCLC sequencing studies; however, no recurrent mutations were found (18, 21–23). We then examined in detail changes in the chromosomal copy number of the CD274 (PD-L1) locus in two cohorts of SCLC tumors. We had previously observed copy number gains of 9p24, in which CD274 resides (17) and thus performed genomic quantitative PCR in a larger cohort (54 primary SCLC tumors and 34 metastases) obtained from 72 patients (Table 1 and Supplementary Table S2). In addition to CD274, we also examined the copy number states of representative cancer-related genes that mapped telomeric and centromeric to CD274, such as NFIB and FLJ41200 at 9p23 and KIAA1432, PDCD1LG2, PLGRKT, and JAK2 at 9p24.1(Fig. 1A). NFIB and JAK2 have been suggested to play a role in lung cancer and were therefore included in this analysis (24, 25). Three tumors showed an increase in somatic CD274 copy number (≥4; Fig. 1A, Supplementary Fig. S1, Supplementary Table S3), and two of these (9-P/SM09-010T and 38-P/R-513T) exhibited focal and high-level amplification (copy numbers, 39.9 and 5.7, respectively) of the segment containing the CD274 gene and of the neighboring genes, KIAA1432 and PDCD1LG2, the latter of which encodes the PD-L2 protein. Tumor specimen 6-P/SM09-006T exhibited polysomy of chromosome 9p (Fig. 1A, Supplementary Fig. S1). These findings were confirmed by analyzing SNP array data that revealed focal, high-level amplification of the locus comprising CD274 (Fig. 1B) in case 9-P and 38-P, but not in the polysomic case, 6-P (Supplementary Fig. S1). In the cases, 9-P and 38-P, amplification occurred on one allele within the chromosome 9p24 segment (17), as evidenced by analyses of allele-specific copy number (Supplementary Fig. S1).

Tumor specimens from cohort 2 (135 primary lung tumors and three metastatic tumors; Table 1 and Supplementary Table S2) were analyzed by SNP 6.0 arrays and whole genome sequencing (18), which revealed focal amplification of CD274 in four cases (Fig. 1C), of which two tumors had copy numbers greater than four (case S00213 and S02404, Fig. 1D and Supplementary Table S3). Detailed analyses of the copy number gain affecting the 9p24 locus revealed that the minimally amplified region included CD274 and the neighboring genes, PDCD1LG2 and KIAA1432 (Fig. 1B and D). SNP 6.0 array data that was available for samples from both cohorts (refs. 17, 18; Supplementary Table S3), and the combined analysis further pointed to a significant and focal amplification of the 9p24 locus harboring CD274 (Q-value = 0.11, Supplementary Fig. S2A). SCLC tumor cases with significant CD274 amplifications showed the same level of genomic amplification across the genome as observed in other SCLC cases with amplified genomic regions (P = 0.21, Supplementary Fig. S2B). In summary, four SCLC tumors from two cohorts (4/210, 1.9%) exhibited focal and high-level amplification of CD274 and the neighboring PDCD1LG2 and KIAA1432 genes. Of note, three of these four cases were of Asian ethnicity. However, these four SCLC patients had no clinical or pathological features discriminating them from other cases (Supplementary Tables S1 and S4).

By analyzing whole-genome sequencing data, we observed multiple intra- and interchromosomal rearrangements in case 9-P (cohort 1) and case S02404 (cohort 2), which affected the 9p24.1 segment (Fig. 2A, Supplementary Fig. S3, and Supplementary Table S5). Of note, a genomic rearrangement occurred in the upstream promoter and 5′-UTR region of CD274. This led to a tandem duplication of the region upstream of CD274 in case 9-P, compatible with amplification of CD274 enhancer elements (26). Case S02404, on the contrary, revealed intra- and interchromosomal rearrangements, which involved a series of amplification patterns causing the observed CD274 amplification. However, the copy number state within the genomic segment encoding the CD274 open reading frame was not altered (Fig. 2A). This finding raises the possibility that genomic rearrangements in the 9p24 locus upstream of CD274 may result in deregulation of CD274 expression, without altering the coding region.

We therefore analyzed transcriptome sequencing data from these tumors, which revealed high expression of the CD274 transcript in cases with high-level focal amplification of CD274 (Supplementary Table S3, Supplementary Table S6). Of note, amplification of 9p24 also resulted in higher expression of other genes contained in the amplified locus (Supplementary Fig. S4A, Supplementary Table S6). However, of the genes in the minimally amplified region, CD274 was the most highly expressed in case 9-P and one of the two most highly expressed in case S00213 (Fig. 2B). Thus, CD274 appears to be the target of the 9p24 amplification.

We next examined whether high-level focal CD274 amplification and high transcript levels were associated with PD-L1 protein expression. We therefore performed immunohistochemical analyses of PD-L1 protein expression using the PD-L1 antibody E1L3N. The specificity of this antibody has been validated in knockdown studies and has been used in several studies examining PD-L1 expression (20, 27, 28), including SCLC (14). We studied tumors bearing focal CD274 amplification and 15 additional tumors from cohort 1, including the 9p-polysomic case. Only the four focally amplified cases contained tumor cells that stained positive for PD-L1 while confirming the classic SCLC morphology (Supplementary Fig. S4B); all other tumors, including 9p-polysomic case, were PD-L1-negative (Fig. 2C, Supplementary Fig. S4C, and Supplementary Table S7). We additionally quantified the level of immune cell infiltrates based on transcriptome sequencing data. PD-L1 expression positively correlated with infiltration of immune cells, as determined by expression of markers specific for B cells and T-cell subsets (Supplementary Fig. S5A). Tumors bearing CD274 amplification ranged among those SCLC tumor cases which showed elevated transcript levels for immune cell markers, which was further confirmed by IHC (Supplementary Fig. S5B). These results suggest that focal CD274 amplification is associated with high PD-L1 antigen expression.

Here, we report that a subset (2%) of SCLC bear focal and high-level amplification of CD274 resulting in high expression of PD-L1. By contrast, tumors with nonfocal, low-amplitude copy number gain of 9p24 (where CD274 resides), or tumors lacking gain or amplification of 9p24, did not exhibit high expression of PD-L1. These findings suggest that genomic upregulation of the CD274 gene is possibly a mechanism for immune evasion in SCLC and thus such tumors might be particularly vulnerable to immune checkpoint inhibition.

Recent studies emphasized the role of focal gene amplifications in activation of proto-oncogenes in various cancers (29–34), and a genomic study across 22 major cancer types, which does not include SCLC, found that CD274 was amplified in 12% of the cases (35). Focal amplification of the MYCL1, MYCN, and MYC oncogenes are known genomic events in SCLC (36). Our findings suggest that amplification of CD274 may be yet another mechanism during oncogenic transformation providing a specific route to immune evasion. Of the other genes that are contained in the focal 9p24 amplicon, only CD274, PDCD1LG2, and KIAA1432 genes are in the minimally amplified region. Of note, co-amplification of the neighboring gene PDCD1LG2, encoding another PD-1 ligand, PD-L2, did not lead to strong upregulation of corresponding PD-L2 transcript levels as observed for CD274 (Fig. 2D). Therefore, PD-L1, rather than PD-L2, seems to play a major role in suppressing immune responses in 9p24 amplified SCLC tumors.

In summary, our results provide evidence for genomic upregulation of PD-L1 expression in a subset of SCLC. We speculate that SCLC tumors with amplification of CD274 may be particularly sensitive to therapeutic PD-1/ PD-L1 inhibition.

R. Nishikawa reports receiving speakers bureau honoraria from Asteras, Chugai, Eisai, MSD, Nobel Pharma, Novocure, Ohtsuka, and Ohno. M. Peifer is a consultant/advisory board member for NEO New Oncology GmbH. I. Petersen is a consultant/advisory board member for Bristol-Myers Squibb, MSD, and Roche. R.K. Thomas is a consultant/advisory board member for Clovis, Johnson & Johnson, New Oncology AG, and Roche. No potential conflicts of interest were disclosed by the other authors.

Conception and design: J. George, M. Saito, R. Büttner, J. Yokota, R.K. Thomas, T. Kohno

Development of methodology: J. George, K. Tsuta, M. Peifer, R.K. Thomas

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J. George, K. Tsuta, K. Shiraishi, A.H. Scheel, S. Uchida, S. Watanabe, R. Nishikawa, M. Noguchi, S.J. Jang, R. Büttner, C.C. Harris, J. Yokota

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. George, M. Saito, R. Iwakawa, A.H. Scheel, R.K. Thomas

Writing, review, and/or revision of the manuscript: J. George, M. Saito, K. Tsuta, R. Iwakawa, A.H. Scheel, M. Peifer, S.J. Jang, C.C. Harris, R.K. Thomas, T. Kohno

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S.J. Jang, I. Petersen, R. Büttner, J. Yokota

Study supervision: J. George, R.K. Thomas

We thank Drs. Akiteru Goto and Masayuki Tsuneki for support and comments.

This work was supported by Grants-in-Aid from the Japan Agency for Medical Research and Development (AMED: 16ck0106012h0003) and a Grant-in-Aid from the Japanese Society for the Promotion of Science (JSPS) and Scientific Research (B: 26293200). NCC Biobank was supported by the National Cancer Center Research and Development Fund (26A-1). C C Harris was supported by intermural funding from the National Cancer Institute, NIH. This work was also supported by the German Cancer Aid (Deutsche Krebshilfe) as part of the small-cell lung cancer genome sequencing consortium (grant ID: 109679; to R.K. Thomas, R. Büttner, and M. Peifer), by the German Ministry of Science and Education (BMBF) as part of the e:Med program (grant no. 01ZX1303A; to R.K. Thomas, R. Büttner, and M. Peifer), by the Deutsche Forschungsgemeinschaft (DFG; through TH1386/3-1 to R.K. Thomas), by the Deutsche Krebshilfe as part of the Oncology Centers of Excellence funding program (to R.K. Thomas) and by the German Cancer Consortium (DKTK) Joint Funding program.

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