Abstract 6678: Sensitive HLA loss of heterozygosity detection reveals allele-specific neoantigen expansion as resistance mechanism to anti-PD-1 therapy
Authors: Pyke Rachel Marty , Abbott Charles , Mellacheruvu Dattatreya , Zhang Simo V. , Bedi Nikita , Colevas A. Dimitrios , Sunwoo John , West John , Chen Richard , Boyle Sean Michael 
Published: September 2020

Abstract
Loss of heterozygosity (LOH) in the HLA locus is increasingly being recognized as an important mechanism of immune escape and a proposed biomarker for immunotherapy response. Neoantigens that bind to a deleted HLA allele will no longer be presented to the immune system, potentially allowing subclones with these deletions to escape immune surveillance. Despite interest in the field, few methods exist to detect HLA LOH, and their sensitivity is not well understood. Moreover, the mechanistic impact of HLA LOH in response to immune checkpoint inhibitors (ICI) remains unexplored. Here, using a novel tool to detect HLA LOH, DASH (Deletion of Allele-Specific HLAs), we reveal allele-specific neoantigen expansion in response to ICIs in a head and neck carcinoma cohort and the widespread occurrence of HLA LOH across several tumor types.
We performed exome sequencing with ImmunoID NeXT, which enhances coverage of the HLA locus, on tumor and normal samples from 260 patients to create a training dataset for our model. For each patient, we mapped reads to each of their allele-specific HLAs and manually annotated LOH. Then, using purity, ploidy and two novel features (normalized b-allele frequency and allele-specific coverage ratios), we trained an XGBoost model.
To evaluate our tool, we compared DASH predictions on held out tumor samples to deletion calls from a standard copy number tool in the regions flanking each HLA gene (91% concordance, 0.73 F1-score), ascertained our sensitivity by diluting cell line data with known HLA LOH to imitate variable purity (100% accuracy in samples above 17% purity) and confirmed the functional impact of HLA LOH using immunopeptidomics data of tumor samples (average of 47% fewer unique peptides binding to lost alleles than kept). To explore the mechanistic impact of HLA LOH in response to ICIs, we studied a cohort of nine head and neck carcinoma patients who received a single dose of nivolumab, and sequenced pre- and posttreatment tumor biopsies for each patient. With DASH, we detected HLA LOH in four of the patients, pretreatment. For these patients, we found a significant posttreatment expansion of neoantigens predicted to bind to the deleted HLA alleles in comparison to the pretreatment biopsy (p=0.046, Wilcoxon signed-rank), revealing the evolutionary force of HLA LOH as a resistance mechanism during ICI therapy. To assess the pervasiveness of HLA LOH across tumor types, we applied DASH to over 500 pretreatment tumors across 13 tumor types and found highly variable frequencies of HLA LOH across the tumor types.
In summary, we developed a sensitive method to detect HLA LOH and exposed neoantigen expansion to deleted HLA alleles in response to ICI therapy, emphasizing the limitations of deleted alleles to ignite an immune response. Moreover, we found widespread occurrences of HLA LOH across tumor types, highlighting the importance of accurate HLA LOH detection as a pan-cancer biomarker.
Citation Format: Rachel Marty Pyke, Charles Abbott, Dattatreya Mellacheruvu, Simo V. Zhang, Nikita Bedi, A. Dimitrios Colevas, John Sunwoo, John West, Richard Chen, Sean Michael Boyle. Sensitive HLA loss of heterozygosity detection reveals allele-specific neoantigen expansion as resistance mechanism to anti-PD-1 therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6678.