Investigating the immunogenomic profile of anal HPV driven disease for novel therapeutic discovery.

3522 Background: Anal squamous cell carcinoma (ASCC) is driven by Human Papilloma Virus and arises from high-grade squamous intraepithelial lesions (HSIL) which, when treated, reduces the risk of cancer progression. However, current treatment options for anal HSIL are limited with considerable morbidity. Although a proportion of patients with localised ASCC can be successfully treated with pelvic chemoradiotherapy, there are significant associated side effects. Given the stepwise evolution of disease, there is an opportunity for the identification of novel therapeutic approaches for cancer prevention. Methods: This study investigates the mutational and immune landscape of matched fresh frozen anal cancer and HSIL samples as well as blood samples from 8 patients using multi-omic analysis (WES, RNAseq and FUME-TCRseq). We compared candidate driver mutations and pathways (including known immune escape mechanisms), copy number alterations (CNAs), differential gene expression, predicted neoantigen profiles (using NeoPredPipe) and TCR repertoire composition and diversity. In addition, digital cell classification of H&E stained sections was used to characterise the distribution of 4 immune cell types. Results: There was considerable copy number profile overlap between anal HSIL and cancer with 79.2% CNA concordance. Samples clustered by patient rather than pathology on gene expression and few differentially expressed genes were identified. There was no difference in predicted neoantigen burden (p = 0.11) nor the proportion of unique or common neoantigens (p = 0.64), illustrating shared immunogenicity between anal cancers with corresponding pre-cancerous lesions. However, we observed a shift in TCR repertoire composition between HSIL and cancer in all patients, with HSIL regions containing larger clusters of related TCR clonotypes (p = 0.02). Driver mutations in PIK3CA , KMT2C , PBRM1 , KLF5, STK11 and CUL1 were shared between matched samples. Enriched GO terms, Kegg pathways and Reactome pathways shared by 2 or more samples included ubiquitination, lipid metabolism and glycosylation. There was higher PD-L1 and CTLA4 expression in anal cancer compared with HSIL, suggestive of immune escape at the transition to invasive cancer. Pathogenic mutations in the Endoplasmic reticulum aminopeptidase 1 ( ERAP1 ) gene, responsible for modulating the peptide repertoire presented by MHC class I molecules, were found in one HSIL sample and two cancer samples. Conclusions: For the first time, this study demonstrates compelling overlap in the immunogenomic profiles of advanced anal HSIL and neighbouring invasive cancer. The shared neoantigen burden and overall immunogenicity supports future vaccine development in the treatment of anal HSIL and subsequent anal cancer prevention. Furthermore, the evidence for immune escape at the transition to invasion could motivate the use of immunotherapy in this setting.