Mutation profiles in early-stage lung squamous cell carcinoma with clinical follow-up and correlation with markers of immune function

Abstract

Background: Lung squamous cell carcinoma (LUSC) accounts for 20–30% of non-small cell lung cancers (NSCLCs). There are limited treatment strategies for LUSC in part due to our inadequate understanding of the molecular underpinnings of the disease. We performed whole-exome sequencing (WES) and comprehensive immune profiling of a unique set of clinically annotated early-stage LUSCs to increase our understanding of the pathobiology of this malignancy.

Methods: Matched pairs of surgically resected stage I-III LUSCs and normal lung tissues (n = 108) were analyzed by WES. Immunohistochemistry and image analysis-based profiling of 10 immune markers were done on a subset of LUSCs (n = 91). Associations among mutations, immune markers and clinicopathological variables were statistically examined using analysis of variance and Fisher’s exact test. Cox proportional hazards regression models were used for statistical analysis of clinical outcome.

Results: This early-stage LUSC cohort displayed an average of 209 exonic mutations per tumor. Fourteen genes exhibited significant enrichment for somatic mutation: TP53, MLL2, PIK3CA, NFE2L2, CDH8, KEAP1, PTEN, ADCY8, PTPRT, CALCR, GRM8, FBXW7, RB1 and CDKN2A. Among mutated genes associated with poor recurrence-free survival, MLL2 mutations predicted poor prognosis in both TP53 mutant and wild-type LUSCs. We also found that in treated patients, FBXW7 and KEAP1 mutations were associated with poor response to adjuvant therapy, particularly in TP53-mutant tumors. Analysis of mutations with immune markers revealed that ADCY8 and PIK3CA mutations were associated with markedly decreased tumoral PD-L1 expression, LUSCs with PIK3CA mutations exhibited elevated CD45ro levels and CDKN2A-mutant tumors displayed an up-regulated immune response.

Conclusion(s): Our findings pinpoint mutated genes that may impact clinical outcome as well as personalized strategies for targeted immunotherapies in early-stage LUSC.

Figure 1.

Genes with significant enrichment for mutations in the 108 early-stage LUSCs. Early-stage LUSCs are denoted by columns and are arranged from left to right by the number of exonic non-silent somatic mutations (top panel). Rows represent genes with statistical enrichment for mutations. The significantly mutated genes are ordered vertically in decreasing order of non-silent mutation frequency. Middle rows indicate smoking status (former and current), gender and pathological stage of the LUSC patients. Colored rectangles indicate mutation categories.

Figure 2.

Genome-wide copy number gains and losses in the 108 early-stage LUSCs. Gains and losses within 1 Mb chromosomal intervals were identified as described in supplementary Methods, available at Annals of Oncology online. Frequency of chromosomal interval copy number gain (red) and loss (blue) were plotted along the genome. Bona fide driver genes within recurrent chromosomal interval gains and losses and in significant CNV peak regions are labeled.

Figure 3.

Prognostic and predictive significantly mutated genes and CNVs in early-stage LUSC. (A) Mutations in MLL2, CDH8, NFE2L2 or RB1 were associated with significantly poorer RFS. (B) Among TP53 mutant LUSCs, mutations in MLL2, CALCR, NFE2L2 or RB1 were associated with poor RFS whereas mutations in PIK3CA predicted better RFS. Among those who received adjuvant chemotherapy, mutations in KEAP1 or FBXW7 exhibited a trend for predicting poor response to therapy (C) which reached statistical significance when assessed in TP53 mutant LUSCs (D). P-values were obtained using the log-rank test and the Kaplan–Meier method for estimation of survival probability.

Figure 4.

Differential expression of immune markers in early-stage LUSCs with significantly mutated genes. Significantly decreased expression of tumoral PD-L1 expression in ADCY8 (A) and PIK3CA (B) mutant LUSCs compared with wild-type tumors. (C) Significantly attenuated peritumoral PD-L1 expression in PIK3CA mutant relative to wild type LUSCs. (D) Significantly elevated CD45ro expression in immune cells infiltrating TP53 mutant relative to wild type LUSCs. Upper panels represent graphical summaries (box plots) of the log (base 2) transformed expression of the indicated immune markers. Lower panels comprise representative photomicrographs (200 × magnification) of the immunohistochemical expression of the immune markers. P-values were obtained by t-tests; values < 0.05 were considered statistically significant.