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. 2018 Dec 1;110(12):1386-1392.
doi: 10.1093/jnci/djy060.

Tobacco Smoking-Associated Alterations in the Immune Microenvironment of Squamous Cell Carcinomas

Alexis Desrichard  1   2 , Fengshen Kuo  1 , Diego Chowell  1   2 , Ken-Wing Lee  1   2 , Nadeem Riaz  1   3 , Richard J Wong  4 , Timothy A Chan  1   2   3 , Luc G T Morris  1   2   4
Affiliations

Tobacco Smoking-Associated Alterations in the Immune Microenvironment of Squamous Cell Carcinomas

Alexis Desrichard et al. J Natl Cancer Inst. .

Abstract

Background: Tobacco smoking creates DNA damage, inducing mutations and potentially altering the tumor immune microenvironment. These types of genetic and immune microenvironment alterations are critical factors known to affect tumor response to immunotherapy. Here we analyze the association between the mutational signature of tobacco smoking, tumor mutational load, and metrics of immune activity in squamous cell carcinomas arising in the head and neck and lung.

Methods: Using RNA and DNA sequencing data from The Cancer Genome Atlas head and neck (HNSC; n = 287) and lung (LUSC; n = 130) squamous cell carcinoma data sets and two independent gene expression data sets (HNSC, n = 136; LUSC, n = 75), we examined associations between the mutational smoking signature, mutation count, immune cell infiltration, cytolytic activity, and interferon-γ signaling.

Results: An increasing mutational smoking signature was associated with statistically significantly increased overall mutational load in both HNSC (ρ = .33, P = 1.01 × 10-7) and LUSC (ρ = .49, P = 2.80 × 10-9). In HNSC, a higher mutational smoking signature was associated with lower levels of immune infiltration (ρ = -.37, P = 1.29 × 10-10), cytolytic activity (ρ = -.28, P = 4.07 × 10-6), and interferon-γ pathway signaling (ρ = .39, P = 3.20 × 10-11). In LUSC, these associations were reversed (ρ = .19, P = .03; ρ = .20, P = .02; and ρ = .18, P = .047, respectively). Differentially expressed genes between smoking-high and smoking-low tumors revealed broad tobacco-induced immunosuppression in HNSC, in contrast to a tumor-inflamed microenvironment in smokers with LUSC.

Conclusions: In squamous cell carcinomas, the genetic smoking signature is associated with higher mutational load, but variable effects on tumor immunity can occur, depending on anatomic site. In HNSC, smoking is predominantly immunosuppressive; in LUSC, more pro-inflammatory. Both tumor mutation load and immune microenvironment affect clinical response to immunotherapy. Thus, the mutational smoking signature is likely to have relevance for immunotherapeutic investigation in smoking-associated cancers.

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Figures

Figure 1.
Figure 1.
Overall survival in (A) head and neck squamous cell carcinoma (HNSC; median follow-up = 536 days), (B) human papillomavirus–negative HNSC (499 days), and (C) lung squamous cell carcinoma (686 days), by genetic smoking signature status. Smoking signature was defined as highest or lowest quartile, and hazard ratios determined using Cox regression. CI = confidence interval; HNSC = head and neck squamous cell carcinoma; HPV = human papillomavirus; HR = hazard ratio; LUSC = lung squamous cell carcinoma.
Figure 2.
Figure 2.
Scatterplots of associations between genetic smoking signature and (A) tumor mutational load (log-transformed), (B) cytolytic score (log-transformed), (C) enrichment in interferon-γ signaling, and (D) overall immune infiltration, as measured by ESTIMATE Immune Score. Correlations for head and neck squamous cell carcinoma (HNSC; all sites), HNSC (larynx only), and lung squamous cell carcinoma were determined using Spearman correlation (ρ with P value) and curve fitting with LOESS regression. CYT = cytolytic; H&N = head and neck; SCC = squamous cell carcinoma.
Figure 3.
Figure 3.
A) Heatmap of Ingenuity Pathway Analysis–defined canonical pathways altered in either head and neck squamous cell carcinoma (HNSC) or lung squamous cell carcinoma (LUSC), showing an immunosuppressive phenotype in smoking-high HNSC, with the color legend representing directional z-score (Supplementary Tables 8 and 9, available online). B) Dot plot of results from Gene Set Enrichment Analysis (GSEA) of pathways enriched in smoking-high tumors, showing a pro-inflammatory phenotype in smoking-high LUSC, but an immunosuppressive phenotype in smoking-high HNSC. Complete expression data of smoking-high and smoking-low LUSC and HNSC (human papillomavirus–negative only) were analyzed for enrichment of GSEA hallmark gene sets, and results are plotted by color (normalized enrichment score; legend on the upper right) and size (–log false discovery rate [FDR]–adjusted P value; legend on the lower right, with * indicating FDR-adjusted P < .05). HNSC = head and neck squamous cell carcinoma; LUSC = lung squamous cell carcinoma; NES = normalized enrichment score.
Figure 4.
Figure 4.
Scatter box plots of differentially expressed immunologic genes in smoking-high vs smoking-low tumors, where dots represent samples; horizontal lines represent median values; boxes represent interquartile range; whiskers represent minimum/maximum values; outlier values beyond 1.5× the interquartile range. CD8A = cluster of differentiation 8a (encoding the CD8 alpha chain); GZMA = granzyme A; HNSC = head and neck squamous cell carcinoma; IDO1 = indoleamine 2,3-dioxygenase 1; IFNG = interferon gamma; IL2RA = interleukin 2 receptor subunit alpha; LUSC = lung squamous cell carcinoma; PDCD1LG2 = programmed cell death 1 ligand 2 (PD-L2).
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