Abstract 5725: Systematic identification of tumour-specific neoantigens(by whole-genome sequencing) and correlation between tumour neoantigen burden, PD-L1 expression and immune infiltrates in 158Asian colorectal cancers
Authors: Koo Si-Lin , Yeong Joe Poh Sheng , Nguyen Andy , Chua Clarinda Wei Ling , Sanborn J Zachary , Benz Steve , Tan Wah Siew , Tang Choong Leong , Yan Su , Chew Min Hoe , Goh Brian , Chan Chung Yip , Koh Xiao Qing , Lezhava Alexander , Lim Tony Kiat Hon , Rabizadeh Shahrooz , Skanderup Anders , Tan Iain Beehuat 
Published: August 2018

Background: Somatic mutations are attractive therapeutic targets for “individualized neoantigen vaccines” because of lack of host central tolerance and reduced risk of autoimmunity. Here, we perform large-scale-omic analyses to assess the neoantigen landscape of colorectal cancer (CRC), a cancer largely refractory to immune-checkpoint inhibition.

Methods: We performed whole genome sequencing (WGS) (60x tumor, 30x normal) and deep whole transcriptomic sequencing (RNA-Seq) (∼200x106 reads per tumor) on 158 colorectal cancers of which 32 are microsatellite instability high (MSI-H) tumours and 126 are microsatellite stable (MSS). Whole exome sequencing (200x tumor, 100x normal) was also performed on 120 tumours. HLA typing, somatic mutations, gene expression and neoepitope predictions were computationally evaluated. Inferred HLA-A alleles were orthogonally validated with Pacbio long-read sequencing. Tissue microarrays (TMAs) with tumour core, tumour edge and normal adjacent tissue of these 158 CRCs were constructed. Histopathological analyses using multiplex immunohistochemistry (mIHC) to simultaneously evaluate 7 markers, i.e. cytokeratin (CK), CD3, CD8, FOXP3, CD68, PD-L1, DAPI, have been performed.

Results: The most common HLAs were, by allele count: A*11:01: 56; A*33:03: 38; B*58:01: 33; B*46:01: 29; B*40:01: 26; C*01:02: 41; C*07:02: 33. Inferred HLA-A alleles from WGS data was largely concordant (&gt;90%) with Pacbio long-read sequencing. There were a median of 2,850 (1229-6909) [MSI] & 213 (27-13,835) [MSS] coding variants, from which 10,487 (4,307-27,365) [MSI] & 726.5 (50-59,096) [MSS] possible neoepitopes were derived, after accounting for epitope processing, the normal proteome and general population variome based on dbSNP, Of these, 5,707 (2,608-15,218) [MSI] & 320 (14-25,243) [MSS] neoepitopes are expressed (based on RNA-Seq). Epitope prediction algorithms revealed a median of 423 (17-1,056) [MSI] & 26 (0-1,102) [MSS] bound & expressed neoepitopes. 5 MSS tumors did not have any predicted bound nor expressed neoepitopes, 112 of 126 (89%) of MSS tumors had at least 5 predicted bound, expressed neoepitopes. Histopathological correlations between extent of immune infiltrates in fixed tissues, tumour PD-L1 expression and neoantigen burden is ongoing.

Conclusions: There is substantial variability in the neoantigen landscape amongst MSI & MSS CRCs. MSI contains multiple-fold higher neo-antigens. Amongst MSS tumours, 89% of patients have at least 5 predicted bound and expressed neo-epitopes that could be targeted in neoantigen-based vaccines for personalized immunotherapy.

Citation Format: Si-Lin Koo, Joe Poh Sheng Yeong, Andy Nguyen, Clarinda Wei Ling Chua, J Zachary Sanborn, Steve Benz, Wah Siew Tan, Choong Leong Tang, Su Yan, Min Hoe Chew, Brian Goh, Chung Yip Chan, Xiao Qing Koh, Alexander Lezhava, Tony Kiat Hon Lim, Shahrooz Rabizadeh, Anders Skanderup, Iain Beehuat Tan. Systematic identification of tumour-specific neoantigens(by whole-genome sequencing) and correlation between tumour neoantigen burden, PD-L1 expression and immune infiltrates in 158Asian colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5725.