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. 2024 Oct 2;12(10):e009617.
doi: 10.1136/jitc-2024-009617.

Cancer cell-specific PD-L1 expression is a predictor of poor outcome in patients with locally advanced oral cavity squamous cell carcinoma

Minyu Wang  1   2   3 , Lei Qin  2   4 , Kevin Thia  5   2   3 , Thu Nguyen  5   2 , Sean MacDonald  5   2   3 , Simone Belobrov  6 , Sevastjan Kranz  7 , David Goode  2   4 , Joseph A Trapani  5   2   3 , David Wiesenfeld  6   8   9 , Paul Joseph Neeson  1   2   3
Affiliations

Cancer cell-specific PD-L1 expression is a predictor of poor outcome in patients with locally advanced oral cavity squamous cell carcinoma

Minyu Wang et al. J Immunother Cancer. .

Abstract

Background: Locally advanced oral cavity squamous cell carcinoma (OCSCC) presents a significant clinical challenge despite being partially responsive to standard treatment modalities. This study investigates the prognostic implications of programmed death-ligand 1 (PD-L1) expression in these tumors, focusing on its association with treatment outcomes and the immune microenvironment.

Methods: We assessed tumor-infiltrating lymphocytes (TILs) in 132 patients with OCSCC to evaluate their impact on survival. Multiplex immunohistochemistry staining for CD3, CD68, CD11c, PD-L1, and P40 was used to explore correlations with clinical outcomes in patients with early-stage (n=22) and locally advanced (n=36) OCSCC. These initial findings were validated through differential gene expression analysis, gene set enrichment, and immune cell deconvolution in a The Cancer Genome Atlas cohort of 163 locally advanced OCSCC tumors. Additionally, single-cell RNA sequencing (scRNA-seq) on a smaller cohort (n=10) further characterized the PD-L1hi or PD-L1lo cancer cells in these tumors.

Results: Elevated PD-L1 expression was associated with poor outcomes in patients with locally advanced OCSCC undergoing standard adjuvant therapy, irrespective of "hot" or "cold" classification based on TILs assessment. PD-L1hi tumors exhibited an active immune response phenotype, enriched with M1 macrophages, CD8+ T cells and T regulatory cells in the tumor microenvironment. Notably, the negative impact of PD-L1 expression on outcomes was primarily attributed to its expression by cancer cells, rather than immune cells. Furthermore, scRNA-seq revealed that immune interactions were not essential for PD-L1 upregulation in cancer cells, instead, complex regulatory networks were involved. Additionally, PD-L1lo locally advanced tumors exhibited more complex pathway enrichment and diverse T-cell populations compared with those in the early-stage.

Conclusion: Our findings underscore the prognostic significance of PD-L1 expression in locally advanced OCSCC, and unveil the complex interplay between PD-L1 expression, immune responses, and molecular pathways in the tumor microenvironment. This study provides insights that may inform future therapeutic strategies, including the possibility of tailored immunotherapeutic approaches for patients with PD-L1hi locally advanced OCSCC.

Keywords: Adjuvant; Head and Neck Cancer; Immune Checkpoint Inhibitor; Radiotherapy/Radioimmunotherapy; Tumor Microenvironment.

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Conflict of interest statement

Competing interests: No, there are no competing interests.

Figures

Figure 1
Figure 1. Elevated PD-L1 expression in locally advanced OCSCC indicates poor outcome. (A) Survival stratification based on stromal TILs (sTILs) assessment on H&E slides from patients with OCSCC with all stages (n=132), early-stage (n=78) or locally advanced stage (n=45). (B) Survival stratification based on CD3+, CD68+, CD11c+ or PD-L1+ cell densities (per mm2 in early-stage OCSCC (n=22). (C) Survival stratification based on CD3+, CD68+, CD11c+ or PD-L1+ cell densities (per mm2 in locally advanced OCSCC (n=36). (D) Differences of CD3+, CD68+, CD11c+ or PD-L1+ cell densities (per mm2 between patients with good or poor outcomes in locally advanced OCSCC (n=36). (E) Survival differences between PD-L1hi (top 25% n=41) and PD-L1lo (bottom 25% n=41) in the TCGA—locally advanced OCSCC cohort. (F) Differences in PD-L1 messenger RNA expression between patients with good or poor outcomes in the TCGA—locally advanced OCSCC cohort (n=163). Data are expressed as mean±SEM. OCSCC, oral cavity squamous cell carcinoma; PD-L1, programmed death ligand 1; TCGA, The Cancer Genome Atlas; TILs, tumor-infiltrating lymphocytes.
Figure 2
Figure 2. PD-L1hi tumors demonstrate an active immune response in the tumor microenvironment in locally advanced OCSCC. (A) Differences in CD3+, CD68+ and CD11c+ cells densities (per mm2 between PD-L1hi (n=18) and PD-L1lo (n=18) locally advanced OCSCC. (B) Differentially expressed genes between PD-L1hi (n=41) and PD-L1lo (n=41) samples from the TCGA—locally advanced OCSCC cohort. (C) Spearman’s correlation was performed to assess the correlations between individual hallmark gene sets and PD-L1 mRNA expression. Gene sets with rho values above 0.40 were illustrated. (D) Differences in immune cell composition using quanTIseq deconvolution analysis between PD-L1hi (n=41, left) and PD-L1lo (n=41, right) samples. (E) Differences of B cell, M1, M2, monocyte, neutrophil, NK cell, CD4+ non-regulatory cell, CD8+ T cell, Tregs, and myeloid dendritic cell populations between PD-L1hi (n=41) and PD-L1lo (n=41) samples using quanTIseq method. (F) Differences of total TCR, TCR-α, and TCR-β reads, Shannon’s entropy index, number of clones and neoantigens between PD-L1hi (n=41) and PD-L1lo (n=41) samples. Data are expressed as mean±SEM. DEGs, differentially expressed genes; FDR, false discovery rate; NK cell, natural killer cell; OCSCC, oral cavity squamous cell carcinoma; PD-L1, programmed death ligand 1; TCGA, The Cancer Genome Atlas; TCR, T-cell receptor; Tregs, T regulatory cells.
Figure 3
Figure 3. PD-L1 expression on the cancer cells, not immune cells, is associated with poor outcomes in the locally advanced OCSCC. (A) Differences in CD3+PD-L1+, CD68+PD-L1+, CD11c+PD-L1+ and P40+PD-L1+ cell densities (per mm2 between patients with good and poor outcomes (n=36). (B) Survival stratification based on CD3+PD-L1+, CD68+PD-L1+, CD11c+PD-L1+ and P40+PD-L1+ cell densities (per mm2 in locally advanced OCSCC (n=36). (C) Examples of multiplex staining for CD11c (magenta), CD68 (yellow), PD-L1 (green), CD3 (red) and P40 (white) in patients with good (n=2) and poor (n=2) outcomes. Scale bar, 50 µm. (D) Survival stratification based on stromal TILs (sTILs) and PD-L1 densities in locally advanced OCSCC tumors (n=36). Data are expressed as mean±SEM. OCSCC, oral cavity squamous cell carcinoma; PD-L1, programmed death ligand 1; TILs, tumor-infiltrating lymphocytes.
Figure 4
Figure 4. Complex regulatory networks other than immune interactions are involved in PD-L1 expression on cancer cells in locally advanced tumors. (A) Cancer cell clusters based on sample IDs from the single cell RNA sequencing cohort. (B) PD-L1 expression on individual cancer cells from different tumor samples. (C) Violin plot of PD-L1 expression on individual cancer cells from different tumor samples. (D) Single-cell compositions in the samples (n=10) with evaluable cancer cells (cell number >10). (E) Top 50 differentially expressed genes (DEGs) in cancer cells between samples 6 and 20 (PD-L1hi locally advanced tumors) and samples 17,18, 25, 26 and 28 (PD-L1lo locally advanced tumors). (F) Volcano plot of 6,475 variables from the comparison in panel E with immune-related genes labeled. (G) Enriched hallmark gene set analysis of DEGs from the comparison in panel E. NES, normalized enrichment score; PD-L1, programmed death ligand 1; UMAP, uniform manifold approximation and projection.
Figure 5
Figure 5. More complex pathway enrichment and diverse T cells in PD-L1lo locally advanced tumors compared with early-stage ones. (A) Top 50 differentially expressed genes (DEGs) in cancer cells between samples 5, 16 and 22 (PD-L1 lo early-stage tumors) and samples 17,18, 25, 26 and 28 (PD-L1lo locally advanced tumors). (B) Volcano plot of 1,572 variables from the comparison in panel A with immune-related genes labeled. (C) Enriched hallmark gene set analysis of DEGs from the comparison in panel A. (D) Composition of cell phenotypes of T cells from samples 17, 18 and 25 (PD-L1lo locally advanced tumors) using the Monaco method. (E) Composition of cell phenotypes of T cells from samples 5 and 16 (PD-L1lo early-stage tumors) using the Monaco method. (F) Differences in T-cell composition between PD-L1lo locally advanced tumors (samples 17, 18 and 25) and PD-L1lo early-stage tumors (samples 5 and 16). NES, normalized enrichment score; PD-L1, programmed death ligand 1; Th cells: T helper cells; UMAP, uniform manifold approximation and projection; Vd2 gd T cells: V-delta 2 gamma delta T cell.
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