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

// Oliver Wood 1, * , Jeongmin Woo 1, * , Gregory Seumois 3, * , Natalia Savelyeva 1 , Katy J. McCann 1 , Divya Singh 3 , Terry Jones 2 , Lailah Peel 1 , Michael S. Breen 1 , Matthew Ward 1 , Eva Garrido Martin 1 , Tilman Sanchez-Elsner 1, # , Gareth Thomas 1, # , Pandurangan Vijayanand 1, 3, # , Christopher H. Woelk 1, # , Emma King 1, # , Christian Ottensmeier 1, # , for the SPARC Consortium 1 Faculty of Medicine, University of Southampton & University Hospital Southampton, Southampton, UK 2 Department of Molecular and Clinical Cancer Medicine, University of Liverpool, Liverpool, UK 3 La Jolla Institute for Allergy & Immunology, La Jolla, CA, USA * These first authors have contributed equally to this work # These senior authors have contributed equally to this work Correspondence to: Emma King, email: e.king@soton.ac.uk Christopher H. Woelk, email: C.H.Woelk@soton.ac.uk Keywords: head and neck squamous cell carcinoma, human papilloma virus, tumor-infiltrating lymphocyte, RNA-Sequencing, transcriptome Received: February 15, 2016     Accepted: June 30, 2016     Published: July 22, 2016 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 TIL low tumors, RNA-Seq analysis was performed on 23 TIL high/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.