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

Abstract

Purpose: Programmed death ligand-1 (PD-L1), encoded by the CD274 gene, is a target for immune checkpoint blockade; however, little is known about genomic CD274 alterations. A subset of small-cell lung cancer (SCLC) exhibits increased copy number of chromosome 9p24, on which CD274 resides; however, most SCLCs show low expression of PD-L1. We therefore examined whether CD274 is a target of recurrent genomic alterations.Experimental Design: We examined somatic copy number alterations in two patient cohorts by quantitative real-time PCR in 72 human SCLC cases (cohort 1) and SNP array analysis in 138 human SCLC cases (cohort 2). Whole-genome sequencing revealed the detailed genomic structure underlying focal amplification. PD-L1 expression in amplified cases from cohorts 1 and 2 was further examined by transcriptome sequencing and immunohistochemical (IHC) staining.Results: By examining somatic copy number alterations in two cohorts of primary human SCLC specimens, we observed 9p24 copy number gains (where CD274 resides) and focal, high-level amplification of CD274 We found evidence for genomic targeting of CD274, suggesting selection during oncogenic transformation. CD274 amplification was caused by genomic rearrangements not affecting the open reading frame, thus leading to massively increased CD274 transcripts and high level expression of PD-L1.Conclusions: A subset (4/210, 1.9%) of human SCLC patient cases exhibits massive expression of PD-L1 caused by focal amplification of CD274 Such tumors may be particularly susceptible to immune checkpoint blockade. Clin Cancer Res; 23(5); 1220-6. ©2016 AACR.

Figure 1.

Focal amplification of CD274 in small-cell lung carcinoma (SCLC). A, Schematic representation of the 9p locus encompassing CD274 (top). The highlighted genes were subjected to quantitative real-time genomic PCR analysis in cohort 1 samples (bottom). B, Raw copy numbers of the 9p24 locus derived from 250 K Affymetrix SNP array data were plotted for tumors 9-P and 38-P. Dashed black lines indicate the minimally amplified region for cohort 1 samples. Dashed blue lines point to the CD274 locus. C, Copy number analysis of cohort 2 samples based on Affymetrix SNP 6.0 and whole-genome sequencing (WGS) data. Shown are normalized and segmented copy number data of the tumors with the highest 9p24 copy number. Tumors are sorted according to copy number. D, Detailed representation of the copy number states in cohort 2 samples with 9p24 amplification. Dashed black lines indicate the minimally amplified region for cohort 2 samples. Dashed blue lines point to the CD274 locus. E, Bar plot displaying the copy number state for CD274 in cohort 1 and 2.*, cases with focal 9p24 amplifications.

Figure 2.

High-level PD-L1 expression in SCLC tumors with high-level focal amplification of CD274. A, Copy number states and genomic rearrangements determined by whole-genome sequencing for cases 9-P and S02404. The 9p24 locus encompassing the genes PLGRKT, CD274, PDCD1LG2, and KIAA1432 is displayed. Copy numbers are plotted as integral copy numbers (iCN), and genomic breaks leading to genomic rearrangements are annotated below the graph as noninverted rearrangements (blue) or inversions (5^′-to-5^′ as green, 3^′-to-3^′ as orange). Dashed black lines highlight the chromosomal locus of CD274. Exon regions in CD274 are represented as boxes (small boxes: noncoding exons, large boxes: coding exons). B, Expression of genes located in the minimally amplified region of the 9p24.1 amplicon, expressed as FPKM values. Case 9-P and S00213 were compared with 75 nonamplified cases (meanþSD). C, Immunohistochemistry for PD-L1 protein expression in 9p24-amplified SCLC tumors. Scale bar as indicated.