`Analysis of neo-antigens and immune profiles of Asian breast cancer patients, focusing on common germline deletion of APOBEC3B cytosine deaminase

Breast cancer is the most common cancer among women, and in Asia, breast cancer incidence is increasing at 3% per annum because of increasing "westernization" and urbanization, along with changes in lifestyle factors. Much effort to catalogue the genomic alterations in common types of cancers including breast cancer, but this has mainly been done in women of European descent. Molecular profiling of breast cancer in multi-ethnic Asian populations has been limited in part because of cost and unavailability of large collections of good quality, clinically annotated tumour samples. The goal of understanding the genomic basis of Asian breast cancers can now be realized because newer DNA sequencing technologies provide high-throughput and accurate data at a significantly reduced cost.
We have decided to focus on breast cancers arising in germline carriers of APOBEC3B (A3B) deletion, which is three times more common in Asians compared to Caucasians. The APOBEC ("apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like") family of proteins are evolutionarily conserved cytosine deaminases associated with intrinsic mutations in response to viral and bacterial infection. Recently, germline A3B deletions were reported to be associated with increased risk to breast cancer, and breast cancers that have a hypermutator phenotype and activated immune signature. However, in part because germline mutations are less common in Caucasian populations and the majority of large sequencing projects have thus far been conducted in Caucasian sample collections, there remains limited information about A3B in breast cancers, particularly in Asian breast cancer samples.
Tumour-infiltrating immune cells play a role in immunesurveillance and immune checkpoint inhibitors such as anti-PD-1 and anti-CTLA-4 antibodies have been shown to elicit remarkable response in treatment of melanoma, non-small-cell lung cancer, renal-cell carcinoma, and other cancers. However, only a small fraction of patients benefit and it appears that cancers with higher mutation load are more likely benefit due to immune activation and consequently increased tumour-infiltrating immune cells. Indeed, colorectal patients with high mutation burden because of mismatch repair deficiency showed better immune-related objective response rate and progression-free survival, suggesting that high somatic mutation burden may be a predictive potential biomarker to immune checkpoint inhibitors.
To date, there has been limited data of tumour response to immune checkpoint therapy in breast cancer. The majority of breast cancers in women of European descent are post menopausal breast cancer which is more likely to be positive for estrogen receptor and have a lower mutational load. However, for a subset of breast cancers, for example, those with triple negative breast cancers, with BRCA1 or BRCA2 germline alterations, and with A3B germline mutations, there is higher mutational load. Notably, the mutational load in Asian breast cancer samples, where A3B germline mutations are 3 times more common, has not yet been systematically tested in Asian women.
Since 2012, we have established hospital-based cohort of breast cancer samples collected in fresh frozen format and all samples are associated with in depth risk factor information, sequencing for a comprehensive panel of 30 breast cancer predisposition genes, and A3B germline status analysis, thus providing a unique opportunity for this collaboration. In this project, we propose to conduct a systematic analysis of the mutational profile and immune response in a cohort of women with either wild-type, 1 copy deletion or 2 copy deletion of A3B and determine germline A3B status is associated with anti-tumour response. We believe that this work will lead to a better understanding of a common germline alteration in Asian women and may lay the grounds for future studies on immunotherapy in Asian breast cancer patients.

GOALS OF THE PROJECT: The goal of this proposed study is to focus on the impact of common A3B germline alterations on the generation of neo-antigens and an immune signature in Asian breast cancer samples. This will be accomplished through the following methodology:
Specific Aim 1: Paired-end whole exome sequencing of 400 fresh frozen tumour and corresponding normal (blood) samples will be performed at 100x and 50x depth coverage respectively using Illumina's TruSeq Rapid Exome on Illumina's HiSeq 4000 platform. For variant analysis, SNVs will be called using CaVEMAN while indels will be called using Pindel whereas mutational signatures will be called using NMF. Risk of developing hypermutator profile will be ascertained. Lastly, bioinformatics tools POLYSOLVER, neoantigen prediction pipeline by Rajasagi et al., and netMHCpan will be used to infer neoepitope load from initial sequencing data. Association between genetic alteration, hypermutation profile, and neoantigen burden will be assessed.
Specific Aim 2: Transcriptomic sequencing of mRNA (~30M reads) will be performed on HiSeq 2500 platform. Sequencing reads will be aligned using MapSlice and quantitation will be performed using RSEA. GSEA and DAVID will be used to identify gene sets and pathways significantly enriched in A3B-del group with focus on immune response-related gene sets and pathways. Overall abundance of TILs will be inferred using ESTIMATE while levels of mRNA transcripts corresponding to specific T cell markers and that of corresponding to specific immune checkpoint molecules will be compared across the different genotypes. IHC validation will be performed by staining for CD4, CD8, A3B and PD-1 and CTLA-4 in a representative set of samples. Association between immune signature, specific T cell markers, and immune checkpoint molecules will be assessed.

Breast cancer is the most common cancer among women, and in Asia, breast cancer incidence is increasing at 3% per annum because of increasing "westernization" and urbanization, and changes in lifestyle factors. Although sequencing approaches to understand the genomic basis of breast cancer subtypes are increasingly common and has led to more accurate selection of treatment and estimation of prognosis, thus far, genome sequencing analysis of breast cancer in the setting of a multi-ethnic Asian populations has been limited in part because of cost and lack of availability of large collections of good quality, clinically annotated tumour samples. The goal of understanding the genomic basis of Asian breast cancers can now be realized mainly because DNA sequencing technologies provide high-throughput and accurate data at a significantly reduced cost. In this project, we plan to apply technological advances in next generation sequencing to uncover the mechanisms of breast cancer development in Asian women, with a focus on whether a common genetic alteration in Asian women is associated with the development of breast cancers which may be amenable to treatment using immunotherapy. We hope that this research will lay the foundations for determining which breast cancer patients may benefit from immunotherapy.