Genomic Profiling of Lung Adenocarcinoma in Never-Smokers

Abstract

Purpose: Approximately 10%-40% of patients with lung cancer report no history of tobacco smoking (never-smokers). We analyzed whole-exome and RNA-sequencing data of 160 tumor and normal lung adenocarcinoma (LUAD) samples from never-smokers to identify clinically actionable alterations and gain insight into the environmental and hereditary risk factors for LUAD among never-smokers.

Methods: We performed whole-exome and RNA-sequencing of 88 and 69 never-smoker LUADs. We analyzed these data in conjunction with data from 76 never-smoker and 299 smoker LUAD samples sequenced by The Cancer Genome Atlas and Clinical Proteomic Tumor Analysis Consortium.

Results: We observed a high prevalence of clinically actionable driver alterations in never-smoker LUADs compared with smoker LUADs (78%-92% v 49.5%; P < .0001). Although a subset of never-smoker samples demonstrated germline alterations in DNA repair genes, the frequency of samples showing germline variants in cancer predisposing genes was comparable between smokers and never-smokers (6.4% v 6.9%; P = .82). A subset of never-smoker samples (5.9%) showed mutation signatures that were suggestive of passive exposure to cigarette smoke. Finally, analysis of RNA-sequencing data showed distinct immune transcriptional subtypes of never-smoker LUADs that varied in their expression of clinically relevant immune checkpoint molecules and immune cell composition.

Conclusion: In this comprehensive genomic and transcriptome analysis of never-smoker LUADs, we observed a potential role for germline variants in DNA repair genes and passive exposure to cigarette smoke in the pathogenesis of a subset of never-smoker LUADs. Our findings also show that clinically actionable driver alterations are highly prevalent in never-smoker LUADs, highlighting the need for obtaining biopsies with adequate cellularity for clinical genomic testing in these patients.

FIG 1.

The data-driven smoking classification in the LUAD cohorts (Institutional, CPTAC, and TCGA). (A) The flowchart presents the analysis steps included in deriving the scoring model (continuous smoking score range from 0 to 1). The bar charts show the smoking score distribution in each of the three cohorts. The smoking score (0.3 as the lower-bound cutoff and 0.7 as the upper-bound cutoff) along with the self-reported smoking status, and mutagen signature validation was used to infer smoking status. The pie charts represent the NS and S composition of each of the three cohorts on the basis of the inferred smoking status. In total, the NS group consisted of 160 samples and the S group contained 299 samples. (B) The violin plot shows the comparisons of log₂-scaled total mutation counts and mutation fractions that contributed to the SS between NS and S samples (as inferred from steps described in Fig 1A). CPTAC, Clinical Proteomic Tumor Analysis Consortium; DNP, dinucleotide polymorphism; LUAD, lung adenocarcinoma; NS, never-smokers; S, smokers; SS, smoking signature; TCGA, The Cancer Genome Atlas.

FIG 2.

Driver alterations in NS LUAD samples. (A) Distribution of driver alterations in NS samples across the three LUAD cohorts (Institutional, CPTAC, and TCGA). (B) The mutually exclusive or co-occurring set of somatic alterations in LUAD-related genes (somatic mutations + gene fusions + CNVs) and cancer predisposition genes with pathogenic and likely pathogenic germline variants (genes altered with germline variants in at least two samples in the Data Supplement) detected by performing pairwise Fisher's exact test. CNV, copy number variation; CPTAC, Clinical Proteomic Tumor Analysis Consortium; LUAD, lung adenocarcinoma; NS, never-smokers; TCGA, The Cancer Genome Atlas.

FIG 3.

Discovery of rare germline predisposition variants in NS. (A) Number of manually reviewed rare germline pathogenic, likely pathogenic, and prioritized VUS identified by using the CharGer tool for each data set. Variants were considered pathogenic, on the basis of their classification in ClinVar or other curated databases; likely pathogenic if their CharGer score was ≥ 9; and prioritized VUS if their CharGer score was ≥ 5. Only variants affecting one of 152 cancer predisposition genes are included in these counts. (B and C) Percentage of S and NS carrying rare pathogenic and likely pathogenic germline variants in cancer predisposition genes in our combined cohort (B) and in each individual data set (C). (D and E) Distribution of rare pathogenic and likely pathogenic germline events in cancer predisposition genes in S and NS. (D) The variant counts for each gene. (E) The variant types. (F) Burden test results for the affected genes in S and NS against the gnomAD noncancer data set. The numbers in each box indicate the percentage of carriers of pathogenic and likely pathogenic variants in each gene in the specified cohort. Gray outlines indicate suggestive enrichment (FDR ≤ 0.15) for pathogenic and likely pathogenic variants in that gene. No significant (FDR ≤ 0.05) enrichment was observed. (G) Pathogenic, likely pathogenic, and prioritized VUSs undergoing LOH in the NS subset. Dots represent individual variants, and the diagonal line indicates neutral selection of the germline variant, where the normal and tumor VAFs are the same. Only genes carrying suggestive and/or significant LOH events in cancer predisposition genes are labeled. (H) All variants undergoing significant or suggestive LOH (depicted in G) were checked for WT allele loss using somatic copy number results from GISTIC2. LOH events were classified as deletion of WT allele if somatic copy number results showed lower ploidy below threshold in the gene region (shown in red), amplification of alternate allele when CNV results showed higher ploidy above threshold in the gene region (no events in the three genes shown in H), or unclassified LOH if the event was not classifiable as either of these two (shown in blue). Events in other samples, apart from those significant or suggestive for LOH, are represented as well and labeled as none (shown in gray). CNV, copy number variation; CPTAC, Clinical Proteomic Tumor Analysis Consortium; FDR, false discovery rate; LOH, loss of heterozygosity; NS, never-smokers; S, smokers; TCGA, The Cancer Genome Atlas; VAF, variant allele frequency; VUS, variants of undetermined significance; WT, wild-type.

FIG 4.

The contributions of smoking-related mutagens to the phenotypes. The heatmap shows the correlation coefficient between the mutational signatures identified 160 NS and 299 S (x-axis) samples and smoking-related mutagen signatures (y-axis) described by Kucab et al. AFR, African; AMR, Admixed American; ASN, Asian; CPTAC, Clinical Proteomic Tumor Analysis Consortium; EUR, European; FDR, false discovery rate; NS, never-smoker; PAH, polycyclic aromatic hydrocarbon; S, smoker; TCGA, The Cancer Genome Atlas.

FIG 5.

Identification of immune subtypes in NS by RNA-sequencing–based cell-type enrichment. (A) Consensus clustering of cell-type enrichment data and gene-level expression patterns of immune checkpoints and potential immunotherapy target genes in NS samples from the Institutional LUAD cohort (discovery cohort) shows three immune clusters—IM-1, IM-2, and IM-3. Each sample and the attributes (summarized in legend on top right), immune cell composition, and checkpoint molecule expression level (shown as Z-scores) are represented in individual columns on the x-axis. (B) Individual box plots showing differences in immune cell composition with the associated level of statistical significance between Institutional cohort samples from the three immune clusters. AFR, African; AMR, Admixed American; EUR, European; IM, immune subtype; LUAD, lung adenocarcinoma; NS, never-smoker; SAS, South Asian.