Comparative analysis of the tumor microbiome, molecular profiles, and immune cell abundances by HPV status in mucosal head and neck cancers and their impact on survival

Abstract

Head and Neck Squamous Cell Carcinoma (HNSCC) comprises a diverse group of tumors with variable treatment response and prognosis. The tumor microenvironment (TME), which includes microbiome and immune cells, can impact outcomes. Here, we sought to relate the presence of specific microbes, gene expression, and tumor immune infiltration using tumor transcriptomics from The Cancer Genome Atlas (TCGA) and associate these with overall survival (OS). RNA sequencing (RNAseq) from HNSCC tumors in TCGA was processed through the exogenous sequences in tumors and immune cells (exotic) pipeline to identify and quantify low-abundance microbes. The detection of the Papillomaviridae family of viruses assessed HPV status. All statistical analyses were performed using R. A total of 499 RNAseq samples from TCGA were analyzed. HPV was detected in 111 samples (22%), most commonly Alphapapillomavirus 9 (90.1%). The presence of Alphapapillomavirus 9 was associated with improved OS [HR = 0.60 (95%CI: 0.40-0.89, p = .01)]. Among other microbes, Yersinia pseudotuberculosis was associated with the worst survival (HR = 3.88; p = .008), while Pseudomonas viridiflava had the best survival (HR = 0.05; p = .036). Microbial species found more abundant in HPV- tumors included several gram-negative anaerobes. HPV- tumors had a significantly higher abundance of M0 (p < .001) and M2 macrophages (p = .035), while HPV+ tumors had more T regulatory cells (p < .001) and CD8+ T-cells (p < .001). We identified microbes in HNSCC tumor samples significantly associated with survival. A greater abundance of certain anaerobic microbes was seen in HPV tumors and pro-tumorigenic macrophages. These findings suggest that TME can be used to predict patient outcomes and may help identify mechanisms of resistance to systemic therapies.

Keywords: HNSCC; HPV; Tumor microbiome; immune cell abundances; microbiome; molecular profiles.

Figure 1.

Kaplan-Meier curve showing the difference in survival between the HPV-positive and HPV-negative tumors. HPV-positive tumors aggregate several papilloma virus strains. Patients with HPV-positive HNSCC have better overall survival. This curve does not control for covariates.

Figure 2.

Hazard ratio (x-axis) and p-values (y-axis) generated from the cox-hazard proportional model that analyzed the overall survival with the presence of each microbe, controlling for stage, smoking status, and age. The diamond represents the alphapapillomavirus 9, a strain of HPV.

Figure 3.

Hazard ratio (x-axis) and p-values (y-axis) from the cox-hazard proportional model analyzing the overall survival with the presence of each microbe, after excluding microbes present in less than 10 samples.

Figure 4.

Differentially expressed microbes by HPV-status. The x-axis shows HPV-negative (left, negative -log2 fold change) and HPV-positive (right, positive -log2fold change), and the y-axis is the negative log of p-value for the microbes associated with each type of tumor.

Figure 5.

Relative abundance of pertinent immune cell types found in HPV-positive and negative tumors. CD8+ T cells, helper T cells, and naïve B cells were found to be more abundant in HPV-positive tumors. M0 macrophages were more abundant in the HPV-negative tumors.