Gene expression analysis of TIL rich HPV-driven head and neck tumors reveals a distinct B-cell signature when compared to HPV independent tumors

Abstract

Human papilloma virus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) has a better prognosis than it's HPV negative (HPV(−)) counterpart. This may be due to the higher numbers of tumor-infiltrating lymphocytes (TILs) in HPV positive (HPV(+)) tumors. RNA-Sequencing (RNA-Seq) was used to evaluate whether the differences in clinical behaviour simply reflect a numerical difference in TILs or whether there is a fundamental behavioural difference between TILs in these two settings. Thirty-nine HNSCC tumors were scored for TIL density by immunohistochemistry. After the removal of 16 TILlow tumors, RNA-Seq analysis was performed on 23 TILhigh/med tumors (HPV(+) n=10 and HPV(−) n=13). Using EdgeR, differentially expressed genes (DEG) were identified. Immune subset analysis was performed using Functional Analysis of Individual RNA-Seq/ Microarray Expression (FAIME) and immune gene RNA transcript count analysis. In total, 1,634 DEGs were identified, with a dominant immune signature observed in HPV(+) tumors. After normalizing the expression profiles to account for differences in B- and T-cell number, 437 significantly DEGs remained. A B-cell associated signature distinguished HPV(+) from HPV(−) tumors, and included the DEGs CD200, GGA2, ADAM28, STAG3, SPIB, VCAM1, BCL2 and ICOSLG; the immune signal relative to T-cells was qualitatively similar between TILs of both tumor cohorts. Our findings were validated and confirmed in two independent cohorts using TCGA data and tumor-infiltrating B-cells from additional HPV(+) HNSCC patients. A B-cell associated signal segregated tumors relative to HPV status. Our data suggests that the role of B-cells in the adaptive immune response to HPV(+) HNSCC requires re-assessment.

Validation of Immune Cell Modules in Multicellular Transcriptomic Data

“…B cell depletion after rituximab treatment [21] and NK cell depletion in the uteri of women treated with the progesterone receptor modulator asoprisinil [22] were best identified by B and NK cell modules with the greatest MDI scores (Fig 4E & 4F). These findings were observed in both Agilent and Affymetrix microarray data, but in order to extend our MDI validation further, we identified RNA-Seq expression data from human head and neck cancer samples where tumour infiltrating lymphocyte (TIL) numbers were classified as high or moderate [23]. The differences in T cell module expression between high and moderate TIL tumours closely correlated with T cell module MDI score (Fig 4G).…”

A prognostic mRNA expression signature of four 16q24.3 genes in radio(chemo)therapy‐treated head and neck squamous cell carcinoma (HNSCC)

“…Due to the complexity and heterogeneity of tumors, it is likely that instead of a single gene, the prognostic value of a gene signature with regard to patient outcome would be more powerful (Ginos et al ., ; Lohavanichbutr et al ., ; Pavon et al ., ). Up to now, mRNA expression signatures associated with metastasis (Lian et al ., ; Roepman et al ., ), hypoxia (Eustace et al ., ; Toustrup et al ., ), HPV status (Cancer Genome Atlas, ; Slebos et al ., ), and immune response (Chung et al ., ; Wood et al ., ) have been reported in HNSCC. Although, for example, the immune response signature of Chung et al .…”

“…Due to the complexity and heterogeneity of tumors, it is likely that instead of a single gene, the prognostic value of a gene signature with regard to patient outcome would be more powerful (Ginos et al, 2004;Lohavanichbutr et al, 2013;Pavon et al, 2012). Up to now, mRNA expression signatures associated with metastasis (Lian et al, 2013;Roepman et al, 2006), hypoxia (Eustace et al, 2013;Toustrup et al, 2012), HPV status (Cancer Genome Atlas, 2015; Slebos et al, 2006), and immune response (Chung et al, 2004;Wood et al, 2016) have been reported in HNSCC. Although, for example, the immune response signature of Chung et al and the hypoxia signatures developed by Eustace et al and Toustrup et al could be confirmed in subsequent publications (Keck et al, 2015;Tawk et al, 2016), many molecular signatures, some of them very complex, fail independent validation and therefore to change practice in a clinical setting.…”

Assessing Tumor-Infiltrating Lymphocytes in Solid Tumors: A Practical Review for Pathologists and Proposal for a Standardized Method from the International Immuno-Oncology Biomarkers Working Group: Part 2: TILs in Melanoma, Gastrointestinal Tract Carcinomas, Non–Small Cell Lung Carcinoma and Mesothelioma, Endometrial and Ovarian Carcinomas, Squamous Cell Carcinoma of the Head and Neck, Genitourinary Carcinomas, and Primary Brain Tumors

“…Increased numbers of FOXP3 + T regs , PD-1 + T cells and CD20 + B cells within immune infiltrates in HPV + tumors have also been described [234,235]. Using gene expression analysis, Wood et al found increased expression of genes encoding PD-1, CTLA-4, and TIM3 in HPV + tumors, indicating an exhausted immune response [236]. Interestingly, a B cell signature distinguished HPV + from HPV − tumors, suggesting B cells rather than T cells account for the increased TILs seen in HPV + tumors [236].…”

“…Using gene expression analysis, Wood et al found increased expression of genes encoding PD-1, CTLA-4, and TIM3 in HPV + tumors, indicating an exhausted immune response [236]. Interestingly, a B cell signature distinguished HPV + from HPV − tumors, suggesting B cells rather than T cells account for the increased TILs seen in HPV + tumors [236]. Immunohistochemical studies have shown CD8 + and FOXP3 + TILs to significantly correlate with improved prognosis in oropharyngeal HPV + tumors [229–236] and probably also HPV − tumors [233,236].…”