The immunologic landscape of HRAS-mutant head and neck squamous-cell carcinoma

Abstract

Background: HRAS mutations define a distinct biologic subset of head and neck squamous-cell carcinoma (HNSCC). There are limited data regarding HRAS-mutant (mut) tumors' sensitivity to immunotherapy. We sought to evaluate the mutational landscape and transcriptional profile, as well as analyze the tumor microenvironment (TME) of HRAS-mut tumors to provide the conceptual framework for combinatorial treatment approaches.

Materials and methods: We analyzed mutational and transcriptome data from The Cancer Genome Atlas (TCGA). In addition, genomic DNA from baseline tumor biopsies was targeted for sequencing. Our study included 10 patients with HRAS-mut and 40 with HRAS-wild-type (WT) HNSCC. Programmed death-ligand 1 (PD-L1) expression in formalin-fixed paraffin-embedded tumor samples was assessed using the PD-L1 IHC 22C3 pharmDx assay. We characterized subpopulations of exhausted CD8(+) T cells by measuring the expression of T-cell factor-1 (TCF1) and programmed cell death protein 1 (PD-1) in both the center and the periphery of the tumors using multiplex immunohistochemistry, followed by analysis using a manually trained algorithm in QuPath software.

Results: The analysis of TCGA HNSCC mutation and mRNA expression data demonstrated that 6% of HNSCCs harbor mutant HRAS. Transcriptome analysis showed that HRAS-mut HNSCCs are infiltrated by immune cells (CD8A, CD8B, CD2) and have higher expression levels of CXCL11, CXCL10, CXCL9 and CCL4 chemokines. Moreover, the percentage of HRAS-mut samples increased in higher PD-L1 score groups (11% versus 20% versus 100% in tumor positive scores <1%, 1%-49% and ≥50%, respectively, P = 0.006). The analysis of TME showed that HRAS-mut tumors have a statistically significant higher number of total immune cells (5123.17/mm² versus 3527.93/mm², P = 0.002) and a higher percentage of pre-exhausted CD8(+) PD-1(+) TCF1(+) T cells in the periphery (384.67/mm² versus 51.18/mm², P = 0.040) than HRAS-WT tumors.

Conclusions: HRAS-mut HNSCCs are characterized by a significantly increased number of pre-exhausted PD-1(+) TCF1(+) T cells and PD-L1 expression, suggesting a potential sensitivity to immunotherapy.

Keywords: HRAS mutations; PD-L1 expression; TCF1; head and neck squamous-cell carcinoma; pre-exhausted CD8 (+) T cells.

Figure 1

HRAS alterations in head and neck cancer. (A) TCGA HRAS mutation and transcriptomic data. Tumors with HRAS expression that corresponds to z-score ≥1.5 were assigned to the HRAS-overexpressing group (HRAS-WT^OV). (B) Chart showing the relative size of HRAS-mut and HRAS-WT^OV groups in TCGA HNSCC cohort (PanCancer Atlas). (C) GO analysis of differentially up-regulated genes in the HRAS-mut group. (D) Heatmap indicating the mRNA expression levels of HRAS and immune genes based on the TCGA HNSCC mutation and transcriptomic data (PanCancer Atlas). (E) Fold change expression of specific immune genes in the HRAS-mut group of tumors compared with the non-altered HRAS group of tumors based on TCGA transcriptomic data (PanCancer Atlas). GO, gene ontology; HNSCC, head and neck squamous-cell carcinoma; mut, mutant; NF-κB, nuclear factor-κB; NK, natural killer; TCGA, The Cancer Genome Atlas; WT, wild type.

Figure 2

Immune profile of tumors with HRAS alterations in head and neck cancer. (A) Heatmap indicating the mRNA expression levels of HRAS and immune genes based on the TCGA HNSCC mutation and transcriptomic data (PanCancer Atlas). (B, C) Boxplot indicating the CD8A and CD274 mRNA expression levels among the HRAS-mut, HRAS-WT^OV and non-altered HRAS group of head and neck tumors. ANOVA was used for the statistical analysis of expression levels among the three groups. ANOVA, analysis of variance; HNSCC, head and neck squamous-cell carcinoma; mut, mutant; TCGA, The Cancer Genome Atlas; WT, wild type.

Figure 3

Gene expression profile of the HRAS-WT^OV group of tumors. (A) Gene ontology analysis of differentially up-regulated genes in the HRAS-WT^OV group. (B) Heatmap indicating the mRNA expression levels of HRAS and mitochondrial DNA genes based on the TCGA HNSCC transcriptomic data (PanCancer Atlas). (C) Fold change expression of mitochondrial DNA genes in the HRAS-WT^OV group of tumors compared with the non-altered HRAS group of tumors based on TCGA transcriptomic data (PanCancer Atlas). HNSCC, head and neck squamous-cell carcinoma; TCGA, The Cancer Genome Atlas; WT, wild type.

Figure 4

Bar graph showing that the percentage of pre-exhausted CD8+ T cells, defined as PD-1+ TCF1+, is elevated in the P ofHRAS-mut tumors (384.67/mm²versus 51.18/mm²,P= 0.040). CI, confidence interval; mut, mutant; P, periphery; PD-1, programmed cell death protein 1; TCF1, T-cell factor-1; WT, wild type.

Figure 5

Bar graph showing that exhausted T cells, defined asPD-1+TCF1− are more abundant in the C ofHRAS-mut tumors compared with WT (13.77% versus 2.67% of total CD8+ cells,P= 0.022). C, center; CI, confidence interval; mut, mutant; P, periphery; PD-1, programmed cell death protein 1; TCF1, T-cell factor-1; WT, wild type.