e15540 Background: Evolution of tumor immunogenicity during immune checkpoint blockade therapy affects tumor response. This study is to investigate the potential mechanisms of the evolution by identifying the differences in immunogenetics between primary tumor and various metastaticlesions after immunotherapy. Methods: This study was approved by IRB. Three types of tissues from a patient with advanced squamous esophageal cancer were obtained three months after anti-PD-1 therapy. The primary tumor (E) was obtained from surgery, the metastatic tumor tissues were obtained from biopsy of the testicular (T)and skin (S) lesions. The presence of immune cell infiltrates and the availability of target antigens defined by mutated neoantigens in these 3 different tissues were examined. The genetic changes from these lesions were analyzed by whole-exome sequencing and RNA sequencing.The inter-lesional immunogenetic heterogeneity between responsive and non-responsive tumor lesions is interpreted. Results: The patient was evaluated at 1 and 3 months after anti-PD-1 therapy with image examinations. The skin metastatic lesion showed significantly decrease in size, but the testicular metastatic lesion failed to respond to the therapy. To correlate tumor response with immunogenetic heterogeneity, 3 specific genotypic changes were examined and compared. 1. Phylogenetic analysis of multi-site mutational profiles showed that metastases were more closely related to each other than to the primary tumor. Truncal and shared mutations were generally clonal with high cellular prevalence, whereas private mutations had medium to low cellular prevalence indicating subclonal status. 2. Although human leukocyte antigen (HLA) loss occurred in S, the numbers of high expression mutation that can be recognized and presented by HLA were the highest. Compared to E,S and T, an overall higher expression of HLA genes was observed in the S. 3. The number and state of different kinds of immune cell were evaluated. The analysesrevealed an increase in distinct immune cell subsets and activation of specific transcriptional networks that were more pronounced in S than T.s and activation of specific transcriptional networks that were more pronounced in S than T. Conclusions: Recent work has shown that HLA-LOH may result in reduced antigen presentation and thus facilitate immune evasion. This study analysis of inter-lesional heterogeneity during anti-PD-1 therapy demonstrated differential clonal evolution within tumors and potential impact of HLA-LOH on neoantigen presentation and expression.