Lymphocyte-activation gene 3 in non-small-cell lung carcinomas: correlations with clinicopathologic features and prognostic significance

Abstract

Lymphocyte-activation gene 3 (LAG-3) modulates the tumor microenvironment through immunosuppressive effects. Its associations with clinicopathologic parameters and prognostic significance in non-small-cell lung carcinomas remain unclear. We examined LAG-3 expression in 368 resected non-small-cell lung carcinomas (including 218 adenocarcinomas and 150 squamous-cell carcinomas) using tissue microarrays, with normalization to CD8⁺ T-cell count (LAG-3/CD8 index), and correlated LAG-3, CD8, and LAG-3/CD8 index with clinicopathologic features, molecular status, and survival. LAG-3 expression in the immune cells (ranged 0.35-540.1 cells/mm²) was identified in 92% of non-small-cell lung carcinomas. In adenocarcinomas and squamous-cell carcinomas, LAG-3 expression correlated with CD8⁺ T-cell count and PD-L1 expression. In adenocarcinomas, high LAG-3 expression (defined as >median) was additionally associated with smoking history, high T stage, aggressive pathologic features (solid-predominant histologic pattern, lymphovascular invasion, and nodal metastasis), and lack of EGFR mutation. In the entire resected tumor cohort and in adenocarcinomas, high LAG-3 and LAG-3/CD8 index were each associated with worse overall survival. In squamous-cell carcinomas, high CD8 was associated with better overall survival. In an exploratory analysis of pretreatment samples from advanced non-small-cell lung carcinoma patients treated with pembrolizumab, high CD8 was predictive of improved overall and progression-free survival, while high LAG-3, but not high LAG-3/CD8 index, was associated with improved progression-free survival. In conclusion, the clinicopathologic correlations and prognostic impact of LAG-3 in non-small-cell lung carcinoma are histotype-dependent, highlighting differences in the immune microenvironment between adenocarcinomas and squamous-cell carcinomas. The predictive impact of LAG-3 warrants further investigation.

Figure 1.. Associations of LAG-3 expression with clinicopathologic and molecular features in primary lung adenocarcinomas.

A. Representative images of LAG-3 and CD8 immunohistochemical staining in acinar- (top row) and solid- (bottom row) pattern adenocarcinomas. H&E: hematoxylin and eosin. B. LAG-3 (left), CD8 (middle), and LAG-3/CD8 index (right) stratified by clinicopathologic and molecular features. Diamonds and bars represent medians and interquartile ranges, respectively. Vertical dotted lines indicate medians of the cohort. Open diamonds indicate p<0.05 (Wilcoxon rank-sum tests). C. Multivariate logistic regression analysis. Diamonds and bars represent odds ratios and 95% confidence intervals, respectively. Open diamonds indicate p<0.05 (logistic regression analysis). pN1+ encompasses pN1 and pN2; pT2+ encompasses pT2, pT3, and pT4. Pleural inv.: pleural invasion. LVI: lymphovascular invasion. WT: wild type; mut: mutated.

Figure 2.. Associations of LAG-3 expression with clinicopathologic and molecular features in primary lung squamous cell carcinomas.

A. Representative images of LAG-3 and CD8 immunohistochemical staining in well-differentiated (top row) and poorly-differentiated (bottom row) squamous cell carcinomas. H&E: hematoxylin and eosin. B. LAG-3 (left), CD8 (middle), and LAG-3/CD8 index (right) stratified by clinicopathologic and molecular features. Diamonds and bars represent medians and interquartile ranges, respectively. Vertical dotted lines indicate medians of the cohort. Open diamonds indicate p<0.05 (Wilcoxon rank-sum tests). C. Multivariate logistic regression analysis. Diamonds and bars represent odds ratios and 95% confidence intervals, respectively. Open diamonds indicate p<0.05 (logistic regression analysis). W: well-differentiated; M: moderately-differentiated; P: poorly-differentiated. Pleural inv.: pleural invasion. LVI: lymphovascular invasion.

Figure 3.. Overall survival and multivariate analysis in patients with resected non-small cell lung carcinomas stratified by LAG-3, CD8, and LAG-3/CD8 index.

A-E. Overall survival (OS) in the combined adenocarcinoma and squamous cell carcinoma cohort stratified by LAG-3 (A), CD8 (B), and LAG-3/CD8 index (D). Forest plots of hazard ratios (HR), including LAG-3 and CD8 separately (C) and LAG-3/CD8 index (E) as covariates. F-J: OS in adenocarcinomas stratified by LAG-3 (F), CD8 (G), and LAG-3/CD8 ratio (I). Forest plots of HR, including LAG-3 and CD8 separately (H) and LAG-3/CD8 index (J) as covariates. K-O: OS in squamous cell carcinomas stratified by LAG-3 (K), CD8 (L), and LAG-3/CD8 index (N). Forest plots of HR includng LAG-3 and CD8 separately (M) and LAG-3/CD8 index (O) as covariates. “Low” and “high” refer to ≤ and > median, respectively. Sample sizes represent the number at risk at time 0. Open diamonds indicate p<0.05 (Cox proportional hazards).

Figure 4.. LAG-3 expression in pretreatment samples from pembrolizumab-treated advanced non-small cell lung carcinoma patients.

A. Distribution of primary and metastatic pretreatment samples in the pembrolizumab-treated patient cohort (n=38 with evaluable LAG-3). Met: metastasis; LN: lymph node. B. LAG-3 (left), CD8 (middle), and LAG-3/CD8 index (right) in the pembrolizumab-treated (IO) and non-IO cohorts. Bars represent medians and 95% confidence intervals. C. Progression-free (top) and overall (bottom) survival in the IO cohort stratified by LAG-3, CD8, and LAG-3/CD8. “Low” and “high” refer to ≤ and > median, respectively. Sample sizes represent the number at risk at time 0.