Whole-exome sequencing and immune profiling of early-stage lung adenocarcinoma with fully annotated clinical follow-up

Abstract

Background: Lung adenocarcinomas (LUADs) lead to the majority of deaths attributable to lung cancer. We performed whole-exome sequencing (WES) and immune profiling analyses of a unique set of clinically annotated early-stage LUADs to better understand the pathogenesis of this disease and identify clinically relevant molecular markers.

Methods: We performed WES of 108 paired stage I-III LUADs and normal lung tissues using the Illumina HiSeq 2000 platform. Ten immune markers (PD-L1, PD-1, CD3, CD4, CD8, CD45ro, CD57, CD68, FOXP3 and Granzyme B) were profiled by imaging-based immunohistochemistry (IHC) in a subset of LUADs (n = 92). Associations among mutations, immune markers and clinicopathological variables were analyzed using ANOVA and Fisher's exact test. Cox proportional hazards regression models were used for multivariate analysis of clinical outcome.

Results: LUADs in this cohort exhibited an average of 243 coding mutations. We identified 28 genes with significant enrichment for mutation. SETD2-mutated LUADs exhibited relatively poor recurrence- free survival (RFS) and mutations in STK11 and ATM were associated with poor RFS among KRAS-mutant tumors. EGFR, KEAP1 and PIK3CA mutations were predictive of poor response to adjuvant therapy. Immune marker analysis revealed that LUADs in smokers and with relatively high mutation burdens exhibited increased levels of immune markers. Analysis of immunophenotypes revealed that LUADs with STK11 mutations exhibited relatively low levels of infiltrating CD4+/CD8+ T-cells indicative of a muted immune response. Tumoral PD-L1 was significantly elevated in TP53 mutant LUADs whereas PIK3CA mutant LUADs exhibited markedly down-regulated PD-L1 expression. LUADs with TP53 or KEAP1 mutations displayed relatively increased CD57 and Granzyme B levels indicative of augmented natural killer (NK) cell infiltration.

Conclusion(s): Our study highlights molecular and immune phenotypes that warrant further analysis for their roles in clinical outcomes and personalized immune-based therapy of LUAD.

Keywords: immune profiles; lung adenocarcinoma; whole-exome sequencing.

Figure 1.

Prognostic and predictive significantly mutated genes and CNVs in early-stage LUAD. (A) RFS of early-stage LUADs who did not receive adjuvant therapy (nontreated) based on mutational status of SETD2. (B) Multivariate analysis demonstrating association of mutations in either STK11, ATM or LRRIQ3 with relatively poor RFS in nontreated KRAS mutant early-stage LUADs. (C) Identification by multivariate analysis of mutated genes (C) or a combination of mutated genes and CNVs (D) that were associated with relatively poor RFS in early-stage LUADs who received adjuvant therapy. P-values were obtained using the log-rank test and the Kaplan–Meier method for estimation of survival probability. CNV, copy number variation; LUAD, lung adenocarcinoma; RFS, recurrence-free survival.

Figure 2.

Mutation signatures and immune marker expression in 108 early-stage LUADs. Exonic somatic mutations were identified as described in supplementary Methods, available at Annals of Oncology online. Columns represent LUADs, arranged from left to right by decreasing proportion of C > A transversions (top panel). Middle rows indicate pathological stage, mutation burden categorized into four groups, pack years and smoking status. Rows represent significantly mutated genes that are ordered vertically in decreasing order of NS mutation frequency. Corresponding (bottom heatmap) expression levels of immune markers were log-transformed and analyzed by hierarchical clustering using Gower’s distance and ward’s linkage method (yellow, relatively up-regulated; blue, relatively down-regulated). LUADs, lung adenocarcinomas; NS, non-silent.

Figure 3.

Differential expression of immunophenotypes and immune markers based on gene mutation status. (A) Score indicative of T-cell exclusion was computed as described in the supplementary Methods, available at Annals of Oncology online and statistically analyzed by STK11 mutation status using Fisher’s exact test (A). Significantly increased levels of expression of tumor core CD4 (B, left) and CD8 (B, right) in STK11 mutant LUADs compared with STK11-WT LUADs. A score indicative NK cell infiltration (C and E) was computed as described in supplementary Methods, available at Annals of Oncology online and statistically compared and contrasted between the indicated subgroups using Fisher’s exact test. Significantly elevated tumor core and peritumoral Granzyme B levels in TP53 mutant and KEAP1 mutant LUADs, respectively (D and F, right panels). Significantly elevated peritumoral CD57 in KEAP1 mutant relative to KEAP1 WT LUADs (F, left panel). Tumor core CD57 followed the same trend in TP53 mutant relative to TP53 WT LUADs albeit not reaching statistically significance (D, left panel). Upper panels in (B, D and F) represent graphical summaries (box plots) of the log₂-transformed expression of the indicated immune markers. Lower panels comprise representative photomicrographs of the immunohistochemical expression of the immune markers. P-values in panels B, D and F were obtained by t-tests; values < 0.05 were considered statistically significant. LUADs, lung adenocarcinomas; WT, wild type; NK, natural killer.