We analyzed all cases that underwent CGP testing at Foundation Medicine between August 2014 and June 2020. Available clinical information for the patient samples were extracted from accompanying test requisition form and pathology reports.

All specimens were assigned a diagnosis by a board-certified pathologist based on microscopic examination of a H&E-stained slide from the formalin-fixed-paraffin-embedded (FFPE) tissue, pathology report, and clinical information provided by the ordering physician.

CGP was performed on hybridization-captured, adaptor ligation-based libraries using DNA extracted from FFPE tumor tissue in a Clinical Laboratory Improvement Amendments (CLIA)-certified and College of American Pathologists (CAP)-accredited laboratory (Foundation Medicine, Cambridge, Massachusetts, USA). The samples were sequenced for up to 324 cancer related genes and/or select gene rearrangements.14

CN alterations were detected using a comparative genomic hybridization-like method applied to next generation sequencing data.14 15 In the laboratory, each specimen was analyzed alongside a process-matched normal control (an internally validated mixture of 10 heterozygous diploid samples from the HapMap project), with custom algorithms to normalize the sequence coverage distribution across captured DNA regions. Log-ratios of normalized coverage data for exonic, intronic, and SNP targets accounting for stromal admixture, as well as genome-wide SNP frequencies, were used to generate the profiles. Using circular binary segmentation, custom algorithms further clustered groups of targets and SNP frequencies to define upper and lower bounds of genomic segments. Empirical Bayesian algorithms employed a distribution of parameters including purity and base ploidy and probability matrices were derived using different statistical sampling methodologies to fit these data. Specimen level ploidy was estimated as described by Sun et al15. Computational models were reviewed by expert analysts for each sample.14 CD274 CN gain was defined as any CN >ploidy of the specimen, CD274 CN neutral was any CN that equaled the ploidy of the specimen, and CD274 CN loss was any CN <ploidy of the specimen. We also explored prevalence rates of different CN cut offs (ploidy +1 (CN 3), ploidy +2 (CN 4), ploidy +3 (CN 5), ploidy +4 (CN 6), and ploidy +8 (CN 10)) to define CD274 CN positivity.

TMB was determined on 0.8–1.1 Mb of sequenced DNA and calculated as the number of non-driver somatic coding mut/Mb of genome sequenced.16 For TMB, we considered a TMB cut-off of at least 10 mut/Mb as TMB-H in our analysis based on the FDA pan-solid tumor CDx approval for pembrolizumab.17 MSI status was determined by analyzing 114 intronic homopolymer repeat loci for length variability, as previously described.18 MSI positivity was defined as MSI-H as per the pan-tumor approval for pembrolizumab.19

As an exploratory analysis, we hypothesized that the high rates of CD274 CN gains in cervix squamous cell carcinoma (SCC) and Head and Neck (HN) SCC could be due to human papillomavirus (HPV) infection, and therefore explored the HPV status of these patients. To determine the HPV status of the samples, we performed de novo assembly of non-human sequencing reads and BLASTn comparison against all viral nucleotide sequences in the NCBI RefSeq database were used to detect the presence of HPV genome sequences (Research Use Only).

PD-L1 IHC testing was run and interpreted by board-certified pathologists according to the manufacturer instructions in a CLIA-certified and CAP-accredited laboratory (Foundation Medicine, Morrisville, North Carolina) for a subset of specimens in this cohort.20 21 Specially, we examined the tumor types with a PD-L1 CDx approval: DAKO 22C3 PD-L1 assay for NSCLC (tumor proportion score cut-off ≥1), cervical carcinoma (combined positive score (CPS) cut-off ≥1), head and neck SCC (CPS cut-off ≥1), gastric/gastroesophageal adenocarcinoma (CPS cut-off ≥1), urothelial carcinoma (CPS cut-off ≥10), and esophageal SCC (CPS cut-off ≥10); and VENTANA SP142 PD-L1 assay for breast carcinoma at tumor infiltrating immune cell cut-off of 1%.22–24

Statistical analyses were performed using R V.3.6.0 (R Foundation for Statistical Computing, Vienna, Austria) and Python V.2.7.16.25 26 Differences among categorical variables were assessed using the Fisher’s exact test. Statistical tests were two sided and multiple hypothesis testing correction was performed using the Benjamini-Hochberg procedure.

Currently, PD-L1 IHC is the gold standard for the tumor types with a PD-L1 CDx claim. We further sought to investigate the sensitivity, specificity, positive predictive value and negative predictive value of CD274 CN positivity (based on different CN cut-offs) when compared with PD-L1 IHC positivity.

We analyzed CD274 CN in 244 584 solid tumor samples representing 290 solid tumor types. Overall, 17.6% (42,983/244,584) had CD274 CN gains (CN >ploidy), 44.6% (108,970/244,584) were CD274 CN neutral (CN=ploidy), and 37.9% (92,631/244,584) had CD274 CN loss (CN <ploidy) (online supplemental table 1).

Among tumor types with ≥1000 samples, cervical SCC (31.3%), lung small-cell undifferentiated carcinoma (27.0%), and head and neck SCC (HNSCC) (25.7%) had the highest frequencies of CD274 CN gain (figure 1). Conversely, skin melanoma (8.1%), pancreatic ductal adenocarcinoma (8.5%), and glioblastoma (9.3%) had the lowest prevalence of CD274 gain. In addition, in the cohort with CD274 CN gains, we observed the highest magnitude of CN gains in tumor types with SCC morphology (figure 2). A positive correlation of CD274 CN gains with HPV infection in cervical SCC and HNSCC was observed (OR=1.4, p=0.17; OR 3.8, p=8.8×10⁻³¹). Pancreatic ductal adenocarcinoma (56.3%), gallbladder adenocarcinoma (55.5%), and cutaneous melanoma (55.4%) had the highest frequencies of CD274 CN loss. This contrasted with cervical SCC (14.4%), appendiceal adenocarcinoma (16.4%), and uterine endometrial adenocarcinoma (16.6%), which had the lowest frequencies of CD274 CN loss (figure 1).

In addition, we explored the prevalence of CD274 CN positivity based on different CN cut offs. When using ploidy +1 (CN 3) as the cut-off, the overall positivity was 17.4% (42,636/244,584); when using ploidy +2 (CN 4) as the cut-off, the overall positivity was 6.2% (15,183/244,584); when using ploidy +3 (CN 5) as the cut-off, the overall positivity was 2.2% (5375/244 584); when using ploidy +4 (CN 6) as the cut-off, the overall positivity was 1.1% (2712/244 584); and when using ploidy +8 (CN 10) as the cut-off, the overall positivity was 0.2% (434/244 584). CD274 CN positivity in different tumor types also varied using these different cut-offs (figure 3 and online supplemental table 2).

CD274 CN gains were highly correlated with PD-L1 IHC positive status in almost all tumor types where a CDx assay was available (figure 4A). Interestingly, HNSCC had the highest OR in this comparison and gastric/esophageal adenocarcinoma which had the lowest OR (8.44, p=3.1×10⁻²; 1.41, p=8.7×10⁻²; respectively). NSCLC, urothelial carcinoma, breast carcinoma, cervical carcinoma, and esophageal SCC all had a positive and significant OR (3.29, p=3.2×10⁻¹⁷³; 2.97, p=3.2×10⁻¹⁵; 1.96, p=1.6×10⁻¹³; 4.51, p=2.1×10⁻⁵; and 3.81, p=8.7×10⁻², respectively).

While CD274 CN changes were highly correlated with PD-L1 IHC status across multiple tumor types, at a population level, there was still a subset of patients in which they were not. Specifically, 4.6% (1378/29887) of the overall cohort with CD274 CN and PD-L1 IHC data had CD274 gain but were PD-L1 negative (table 1). Conversely, 23.3% (6953/29 887) had CD274 loss but were PD-L1 IHC positive (table 1).

When compared with PD-L1 IHC, CD274 CN positivity (at different CN cut-offs) is highly specific and has high positive predictive value (figure 4B and online supplemental table 3). On the other hand, sensitivity and the negative predictive value is lower. Importantly, the sensitivity, specificity, positive predictive value, and negative predictive value varied depending on which cut-off we used to define CD274 CN positivity and varied depending on tumor type/PD-L1 CDx assay and scoring algorithm used (figure 4B and online supplemental table 3).

CD274 CN gains were not significantly correlated with TMB-H in almost all (98.6%, 286/290) tumor types. CD274 CN gain were significantly correlated with TMB in only four tumor types: lung adenocarcinoma, gastric adenocarcinoma, uterine endometrial adenocarcinoma, and bladder urothelial carcinoma (OR: 1.2, p=9.3×10⁻⁶; 2.3, p=1.6×10⁻⁵; 2.3, p=4.7×10⁻⁵; and 1.4, p=4.6×10⁻², respectively) (figure 5A).

In the tumor types in which MSI is most clinically relevant (gastric adenocarcinoma, colorectal adenocarcinoma, uterine endometrial adenocarcinoma, esophageal adenocarcinoma, and gastroesophageal junction adenocarcinoma), we observed a significant correlation between CD274 CN gains and MSI-H status (OR: 5.2, p=4.9×10⁻¹⁰; 1.9, p=6.7×10⁻⁸; 3.2, p=2.1×10⁻⁶; 3.7, p=5.5×10⁻³; and 6.5, p=2.3×10⁻², respectively) (figure 5B). Most of the remaining tumor types did not have significant correlation between CD274 CN gains and MSI-H.