We estimated TICs by CIBERSORT again. homologous recombination (HR)-deficient tumors. However, new treatment strategies, such as immune checkpoint inhibition, are required for patients with HR-proficient tumors. Methods A total of 80 cases of HGSC were analyzed in this study. Whole exome and RNA sequencing was performed for these tumors. Methylation arrays were also carried out to examine and promoter methylation status. Mutations, neoantigen load, antigen presentation machinery, and local immune profile were investigated, and the relationships of these factors with clinical outcome were also analyzed. Results As expected, the numbers of predicted neoAgs were lower in HR-proficient (n=46) than HR-deficient tumors (n=34). However, 40% of the patients with HR-proficient tumors still had higher than median numbers of neoAgs and better survival than patients with lower numbers of neoAgs. Incorporation of human leukocyte antigen (HLA)-class I expression status into the survival analysis revealed that patients with both high neoAg numbers and high HLA-class I expression (neoAghiHLAhi) had the best progression-free survival (PFS) in HR-proficient HGSC (p=0.0087). Gene set enrichment analysis demonstrated that the genes for effector memory CD8 T cells, TH1 T cells, the interferon- response, and other immune-related genes, were enriched in these patients. Interestingly, this subset of patients also had better PFS (p=0.0015) and a more T-cell-inflamed tumor phenotype than patients with the same phenotype (neoAghiHLAhi) in HR-deficient HGSC. Conclusions Our results suggest that immune checkpoint inhibitors might be an alternative to explore in HR-proficient cases which currently do not benefit from PARP inhibition. mutation-associated advanced ovarian cancer after treatment with multiple chemotherapies.3 Subgroup analyses from the phase III Nova (niraparib) and ARIEL3 (rucaparib) trials demonstrated the dramatic efficacy of PARP inhibitors in HGSC patients with HR-deficient tumors. In contrast, the efficacy was rather limited for HR-proficient tumors.3 4 Therefore, there is a need to improve outcomes of HGSC patients with HR-proficient tumors. New treatment modalities, such as immunotherapy, are urgently required. Tumors exhibit multiple somatic mutations in the course of development. Mutational burden varies across different types of human cancers.5 Neoantigens derived from such tumor-specific mutations are good potential targets for effective antitumor immune responses because they are foreign to the immune system.6C8 Recent reports document that a clinical benefit of immune checkpoint inhibitors (ICI) was more likely to be achieved in melanoma and lung cancer patients with tumors harboring abundant neoantigens,9C12 although it is becoming increasingly clear that patients with high mutation burden do not always have clinical benefits by ICI possibly due to many mechanisms dampening antitumor immune responses in the tumor microenvironment. In contrast, the efficacy of ICI has been limited in cancers such as HGSC with a lower tumor mutation burden (TMB) and thus fewer potential neoantigens. A phase II trial of pembrolizumab for ovarian cancer yielded a response rate for HGSC of only 8.0%.13 Nonetheless, a small number of patients obviously do benefit from ICI and experience durable responses.14 Therefore, in those types of cancers, stricter criteria for patient selection would be desirable. Other than the TMB, antigen presentation machinery, interferon (IFN)- signatures and combinations of those factors might be employed for this purpose. In the present study, we investigated the status of neoantigen load and immunologic characteristics of HGSCs, especially focusing on HR-proficient cancer using integrated molecular analysis to determine which tumors would be the best candidates for immunotherapy. Methods Sample description and preparation Genomic DNA and total RNA were extracted from frozen tumor samples after cryostat sectioning, using DNA and AllPrep DNA/RNA Mini Kits (Qiagen, Hilden, Germany). Genomic DNA was isolated from matched peripheral blood samples using QIAamp DNA Mini Kits (Qiagen). Eighty HGSC samples were analyzed in this study. Whole-exome sequencing, read mapping and detection of somatic mutations Paired tumor and blood genomic DNA libraries were constructed according to the protocol provided with the Delavirdine mesylate KAPA Hyper Prep Kit (Kapa Biosystems). Whole-exome capture was performed with the SureSelect Human All Exon kit V.4 and V.5 (Agilent Technologies) following the manufacturer’s protocols. We sequenced exome capture libraries on the HiSeq 2000 platform according to the manufacturer’s instructions, and 2100?bp paired-end reads were generated. Image analysis and base calling were performed using the Illumina pipeline with default settings.15 Exome reads were independently mapped to the human genome (GRCh37/hg19) using Burrows-Wheeler Aligner and Novoalign software. Reads with a minimal editing distance to the reference genome were taken to represent optimal alignments. Bam files were then locally realigned with short-read micro re-aligner (SRMA). Normal-tumor pair bam files were processed using.Methylation arrays were also carried out to examine and promoter methylation status. with homologous recombination (HR)-deficient tumors. However, new treatment strategies, such as immune checkpoint inhibition, are required for patients with HR-proficient tumors. Methods A total of 80 cases of HGSC were analyzed in this study. Whole exome and RNA sequencing was performed for these tumors. Methylation arrays were also carried out to examine and promoter methylation status. Mutations, neoantigen load, antigen presentation machinery, and local immune profile were investigated, and the relationships of these factors with Delavirdine mesylate clinical outcome were also analyzed. Results As expected, the numbers of predicted neoAgs were lower in HR-proficient (n=46) than HR-deficient tumors (n=34). However, 40% of the patients with HR-proficient tumors still had higher than median numbers of neoAgs and better survival than patients with lower numbers of neoAgs. Incorporation of human leukocyte antigen (HLA)-class I expression status into the survival analysis revealed that patients with both high neoAg numbers and high HLA-class I expression (neoAghiHLAhi) had the best progression-free survival (PFS) in HR-proficient HGSC (p=0.0087). Gene set enrichment analysis demonstrated that the genes for effector memory CD8 T cells, TH1 T cells, the interferon- response, and other immune-related genes, were enriched in these patients. Interestingly, this subset of patients also had better PFS (p=0.0015) and a more T-cell-inflamed tumor phenotype than patients with the same phenotype (neoAghiHLAhi) in HR-deficient HGSC. Conclusions Our results suggest that immune checkpoint inhibitors might be an alternative to explore in HR-proficient cases which currently do not benefit from PARP inhibition. mutation-associated advanced ovarian cancer after treatment with multiple chemotherapies.3 Subgroup analyses from the phase III Nova (niraparib) and ARIEL3 (rucaparib) trials demonstrated the dramatic efficacy of PARP inhibitors in HGSC patients with HR-deficient tumors. In contrast, the efficacy was rather limited for HR-proficient tumors.3 4 Therefore, there is a need to improve outcomes of HGSC patients Delavirdine mesylate with HR-proficient tumors. New treatment modalities, such as immunotherapy, are urgently required. Tumors exhibit multiple somatic mutations in the course of development. Mutational burden varies across different types of human cancers.5 Neoantigens derived from such tumor-specific mutations are good potential targets for effective antitumor immune responses because they are foreign to the immune system.6C8 Recent reports document that a clinical benefit of immune checkpoint inhibitors (ICI) was more likely to be achieved in melanoma Delavirdine mesylate and lung cancer patients with tumors harboring abundant neoantigens,9C12 although it is becoming increasingly clear that patients with high mutation burden do not always have clinical benefits by ICI possibly due to many mechanisms dampening antitumor immune responses in the tumor microenvironment. In contrast, the efficacy of ICI has been limited in cancers such as HGSC with a lower tumor mutation burden (TMB) and thus fewer potential neoantigens. A phase II trial of pembrolizumab for ovarian cancer yielded a response rate for HGSC of only 8.0%.13 Nonetheless, a small number of patients obviously do benefit from ICI and experience durable responses.14 Therefore, in those types of cancers, stricter criteria for patient selection would be desirable. Other than the TMB, antigen presentation machinery, interferon (IFN)- signatures and combinations of those CCR1 factors might be employed for this purpose. In the present study, we investigated the status of neoantigen load and immunologic characteristics of HGSCs, especially focusing on HR-proficient cancer using integrated molecular analysis to determine which tumors would be the best candidates for immunotherapy. Methods Sample description and preparation Genomic DNA and total RNA were extracted from frozen tumor samples after cryostat sectioning, using DNA and AllPrep DNA/RNA Mini Kits (Qiagen, Hilden, Germany). Genomic DNA was isolated from matched peripheral blood samples using QIAamp DNA Mini Kits (Qiagen). Eighty HGSC samples were analyzed in this study. Whole-exome sequencing, read mapping and detection of.