Loss Of Heterozygosity Score Research Articles

METB-14. INTEGRATED GERMLINE AND SOMATIC PROFILING OF PEDIATRIC BRAIN TUMORS REVEALS INCIDENTAL FINDINGS AND NOVEL TUMOR BIOLOGY

Abstract BACKGROUND The contribution of rare pathogenic germline variation to central nervous system (CNS) tumor formation in pediatric patients without a family history of cancer remains unclear. METHODS We characterized the prevalence of pathogenic germline variants in 214 cancer predisposition genes (CPGs) in 837 patients profiled in the Pediatric Brain Tumor Atlas by whole genome or exome sequencing (n=820 and n=17, respectively). Rare SNVs and small INDELs were annotated as pathogenic (P) or likely pathogenic (LP) consistent with American College of Medical Genetics criteria using AutoGVP, our open-source automated pathogenicity assessment tool. We classified pathogenicity of germline structural variants (SVs) using ClassifyCNV and AnnotSV. Somatic alterations and mutational signatures from matched tumor sequencing were integrated to identify functional consequences associated with germline pathogenic variation. RESULTS We observed 206 germline P/LP SNVs/small INDELs and 18 SVs (16 deletions, 2 duplications) within 78 unique CPGs in 195 patients (23.3%). We detected syndrome-associated P/LP variants in 45/58 patients with a clinically-reported cancer predisposition syndrome and incidentally in 41 patients. Ninety-five (42%) germline P/LP variants co-occurred with at least one somatic alteration in the same gene in matched tumors: n=6 oncogenic SNVs, n=28 CNVs, n=54 loss of heterozygosity (LOH), n=26 differential gene or protein expression, and n=18 alternative splicing. NF1, TSC1, and TSC2 germline P/LP carriers exhibited significantly higher tumor LOH scores and lower gene expression in tumors relative to non-P/LP carriers. Patients with germline splice region P/LP variants exhibited significantly increased skipping of most the proximal exon in matched tumor relative to patients with non-splice region P/LP variants (W=401, p=1.1E-04). CONCLUSION We have identified rare germline P/LP variants associated with cancer predisposition syndromes and diverse functional consequences in pediatric CNS tumor patients. Efforts are underway to extend this work to include 1,801 additional probands and 1,105 parents to further characterize the prevalence and heritability of germline pathogenic variation in children diagnosed with CNS tumors.

The characteristics and clinical relevance of tumor fusion burden in non-EBV (+) gastric cancer with MSS

BackgroundNext-generation sequencing (NGS) is maturely applied for gene fusion detection. Although tumor fusion burden (TFB) has been identified as an immune marker for cancer, the relationship between these fusions and the immunogenicity and molecular characteristics of gastric cancer (GC) patients remains unclear. GCs have different clinical significance depending on their subtypes, and thus, this study aimed to investigate the characteristics and clinical relevance of TFB in non-Epstein–Barr-virus-positive (EBV+) GC with microsatellite stability (MSS).MethodsA total of 319 GC patients from The Cancer Genome Atlas stomach adenocarcinoma (TCGA-STAD) and a cohort of 45-case from ENA (PRJEB25780) were included. The cohort characteristics and distribution of TFB among the patients were analyzed. Additionally, the correlations of TFB with mutation characteristics, pathway differences, relative abundance of immune cells, and prognosis were examined in the TCGA-STAD cohort of MSS and non-EBV (+) patients.ResultsWe observed that in the MSS and non-EBV (+) cohort, the TFB-low group exhibited significantly lower gene mutation frequency, gene copy number, loss of heterozygosity score, and tumor mutation burden than in the TFB-high group. Additionally, the TFB-low group exhibited a higher abundance of immune cells. Furthermore, the immune gene signatures were significantly upregulated in the TFB-low group, 2-year disease-specific survival was markedly increased in the TFB-low group compared with to the TFB-high group. The rates of TFB-low cases were significantly higher TFB-than high cases in durable clinical benefit (DCB) and response groups with pembrolizumab treatment. Low TFB may serve as a predictor of GC prognosis, and the TFB-low group exhibits higher immunogenicity.ConclusionIn conclusion, this study reveals that the TFB-based classification of GC patient may be instructive for individualized immunotherapy regimens.

Diagnosis of Ovarian Carcinoma Homologous Recombination DNA Repair Deficiency From Targeted Gene Capture Oncology Assays.

Homologous recombination DNA repair deficiency (HRD) is a therapeutic biomarker for sensitivity to platinum and poly(ADP-ribose) polymerase inhibitor therapies in breast and ovarian cancers. Several molecular phenotypes and diagnostic strategies have been developed to assess HRD; however, their clinical implementation remains both technically challenging and methodologically unstandardized. We developed and validated an efficient and cost-effective strategy for HRD determination on the basis of calculation of a genome-wide loss of heterozygosity (LOH) score through targeted, hybridization capture and next-generation DNA sequencing augmented with 3,000 common, polymorphic single-nucleotide polymorphism (SNP) sites distributed genome-wide. This approach requires minimal sequence reads and can be readily integrated into targeted gene capture workflows already in use for molecular oncology. We interrogated 99 ovarian neoplasm-normal pairs using this method and compared results with patient mutational genotypes and orthologous predictors of HRD derived from whole-genome mutational signatures. LOH scores of ≥11% had >86% sensitivity for identifying tumors with HRD-causing mutations in an independent validation set (90.6% sensitivity for all specimens). We found strong agreement of our analytic approach with genome-wide mutational signature assays for determining HRD, yielding an estimated 96.7% sensitivity and 50% specificity. We observed poor concordance with mutational signatures inferred using only mutations detected by the targeted gene capture panel, suggesting inadequacy of the latter approach. LOH score did not significantly correlate with treatment outcomes. Targeted sequencing of genome-wide polymorphic SNP sites can be used to infer LOH events and subsequently diagnose HRD in ovarian tumors. The methods presented here are readily generalizable to other targeted gene oncology assays and could be adapted for HRD diagnosis in other tumor types.

114. Comparison of optical genome mapping, CMA, and 523-gene NGS panel for Homologous Recombination Deficiency calculation

Homologous Recombination Deficiency (HRD) is characterized by the inability of a cell to repair the double-stranded breaks using the homologous recombination repair (HRR) pathway. The deficiency of the HRR pathway results in defective DNA repair, leading to genomic instability and tumorigenesis. The presence of HRD has been found to make tumors sensitive to ICL-inducing platinum-based therapies and poly(adenosine diphosphate [ADP]?ribose) polymerase (PARP) inhibitors (PARPi). However, there are no standardized methods to define, measure, and report HRD in diagnostic laboratories. Herein, we compare optical genome mapping (OGM), chromosomal microarray (CMA), and 523-gene NGS panel for HRD scar calculations. Traditionally, CMA has been used to calculate either loss of heterozygosity (LOH), or a combination of LOH, telomeric allelic imbalance (TAI), and large scale transitions (LST), where a combination is more accurate than LOH score alone for HRD calculation. We demonstrate the use of a 523-gene NGS panel to detect HRD using NxClinical software with automated HRD calculation using LOH, TAI, and LST. More interestingly, we compare OGM with CMA and NGS panel and highlight the potential advantages of using OGM, which reveals the genomic instability with a much higher resolution compared to other methodologies. For example, in a case of glioblastoma, the HRD score with CMA was 7 (TAI:1, HRD-LOH: 2, LST: 4) compared to 29 with OGM (TAI:1, HRD-LOH:2, LST:25). The HRD definition, measurement, and reporting criteria need to be standardized for each platform, while OGM might be the most sensitive platform for HRD calculation.

PRECISION ONCOLOGY- BASIC TO BEDSIDE

Basic science technologies and platforms have unraveled the molecular basis of carcinogenesis, both genetic and epigenetic events. Few of the events which are repeatedly observed and are actionable, gave birth to the science “Precision Oncology”. The evolution of molecular testing has made a major stride in the recent times, and has empowered personalized cancer medicine over the conventional blanket treatment. Among the important diagnostic technologies, Next Generation Sequencing (NGS) based in-vitro diagnostics have contributed substantially to comprehensive genomic profiling (CGP) in clinical practice that is meant for the detection of substitutions, insertions and deletions (indels), copy number alterations, and gene rearrangements, as well as genomic signatures including Microsatellite Instability (MSI), Loss of Heterozygosity (LOH), Homologous Recombination Defect (HRD Score), Tumor Mutation Burden (TMB). CGP has come into clinical practice with various guidelines endorsing its clinical utility in various indications, both tumor specific and tumor agnostic. Lung cancer is an excellent example where the role of NGS based comprehensive genomic profiling (CGP) has been recommended upfront either on tissue or blood (Liquid Biopsy). In ovarian cancer, calculation like LOH or HRD score, based on the tissue NGS, has widen the eligibility of PARP inhibitor drugs. Further, NTRK fusions, MSI and TMB biomarkers are universal supporting tumor agnostic approach. CGP is a tremendous diagnostic application but brings challenges especially interpretation which are affected by intra/inter tumor heterogeneity, identification of multiple driver mutations, multiple targets and treatment options, cross talk of various mutations and genomic signatures, and various signaling pathways, variable variant allele frequency (VAF), cut-offs for various scores like TMB, LOH, HRD. All these challenges are currently overcome by Molecular Tumor Boards, where basic science understanding in conjunct with clinical experience, has made a huge difference. Thus, offering true precision oncology with unique treatment options for individual patient. 

Frequency of homologous recombination deficiency in a large community-based cohort of epithelial ovarian cancer cases (597)

Frequency of homologous recombination deficiency in a large community-based cohort of epithelial ovarian cancer cases (597)

Chemotherapy response score (CRS) as a surrogate marker for homologous recombination deficiency (HRD) based on loss of heterozygosity (LOH) in high grade serous ovarian cancer (HGSOC) (165)

Chemotherapy response score (CRS) as a surrogate marker for homologous recombination deficiency (HRD) based on loss of heterozygosity (LOH) in high grade serous ovarian cancer (HGSOC) (165)

Abstract 80: Genomic characterization of PDX models from rare cancer patients in the NCI Patient-Derived Models Repository

Abstract Background: The National Cancer Institute’s Patient-Derived Models Repository (NCI PDMR; https://pdmr.cancer.gov) has developed a large number of patient-derived xenograft (PDX) models from a diverse set of rare cancers. These models have been genomically characterized using whole-exome sequencing (WES) and RNAseq. The resource provides a unique opportunity to explore the genomic features of rare tumor models in NCI PDMR and to understand the oncogenic processes in pre-clinical models to identify biomarkers associated with therapeutic responses. Methods: Genomic characterization was done in 4-6 PDX samples across multiple passages and lineages from each model. As the samples exhibited a high level of genomic stability within each model, consensus mutation and copy number variation (CNV), microsatellite instability (MSI), genomic loss of heterozygosity (LOH), homologous recombination deficiency score (scarHRD), and mutational signature data were generated from WES. Fusions were identified from RNASeq data using Star-Fusion and FusionInspector. Gene set enrichment analysis was conducted from the gene expression data obtained from RNAseq. Results: 1) 233 PDX models have been developed and characterized from more than 45 different rare malignancies. Most frequent cancer types are different sarcomas (n=63), head & neck squamous cell carcinoma (n=61), and malignant fibrous histiocytoma (MFH) (n=11); 2) TP53 was the most frequently altered gene, mutated in 51% of models, followed by NOTCH1 (16%) and PIK3CA (11%). In terms of CNVs, ovarian epithelial cancer (OVT) showed relatively high chromosomal instability, while uterine endometrioid carcinoma (UEC) and synovial sarcoma (SYNS) had low instability; 3) MSI-H was observed in only 7 models. Esophageal adenocarcinoma (ESCA), OVT, and cervical squamous cell carcinoma (CESC) had high scarHRD and genomic LOH scores, while both scores were low in UEC and anal squamous cell carcinoma (ANSC). COSMIC v2 mutational signature 3 is significantly associated with a high scarHRD score (p-value < 0.01, Wilcoxon rank-sum test); 4) Characteristic fusions were observed in certain sarcoma models: SS18-SSX1 and ASPSCR1-TFE3 fusions were observed in SYNS and alveolar soft part sarcoma (ASPS) models respectively. EWSR1-FLI1 fusion was present in 2 out of 3 Ewing sarcoma (ES) models. 5) Gene set enrichment analysis from RNASeq data showed that epithelial-mesenchymal transition score could accurately distinguish carcinoma from sarcoma models, confirming the divergent gene expression programs. Conclusion: Comprehensive genomic characterization of NCI PDMR models generated from rare cancers solves an unmet need in the community. It will serve as a valuable resource for translational researchers interested in pre-clinical drug development and discovery. Citation Format: Li Chen, Rini Pauly, Ting-Chia Chang, Biswajit Das, Yvonne A. Evrard, Chris A. Karlovich, Tomas Vilimas, Alyssa Chapman, Nikitha Nair, Luis Romero, Anna Lee Fong, Amanda Peach, Shahanawaz Jiwani, Nastaran Neishaboori, Lindsay Dutko, Kelly Benauer, Gloryvee Rivera, Erin Cantu, Corinne Camalier, Thomas Forbes, Michelle Gottholm-Ahalt, John Carter, Suzanne Borgel, Chelsea McGlynn, Candace Mallow, Emily Delaney, Tiffanie Miner, Michelle A. Eugeni, Dianne Newton, Melinda G. Hollingshead, P. Mickey Williams, James H. Doroshow. Genomic characterization of PDX models from rare cancer patients in the NCI Patient-Derived Models Repository [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 80.

Abstract P1-08-12: The status of homologous recombination deficiency is a potential biomarker for platinum-based chemotherapy in triple-negative breast cancer

Abstract BackgroundTriple-negative breast cancer (TNBC) is a subtype of breast cancer that is highly susceptible to recurrence and metastasis. The therapeutic drugs are limited due to the lack of effective biomarkers predicting the therapeutic efficacy and prognosis of disease. Previous studies have shown that platinum-containing regimens are effective for both early and advanced TNBC, therefore, it is particularly important to explore biomarkers for predicting the efficacy of platinum drugs and to screen the population sensitive to platinum drugs. Methods All patients with available tumor specimens from National Cancer Center in China were eligible for this prospective-retrospective study (n=189), including 149 patients with early TNBC (NCT01150513, CH-BC-007) and 40 patients with advanced TNBC (12-123/657, CH-BC-018) who are treated with or without platinum. The primary endpoint is disease-free survival (DFS) for early TNBC and progression free survival (PFS) for advanced TNBC. For HRD status and genomic signatures, indexed libraries were subjected to probe-based hybridization with a customized NGS panel targeting 733 cancer-related genes. 3DMed-HRD algorithm was evaluated based on loss of heterozygosity score (LOH), telomeric allelic imbalance score (TAI) and large-scale state transition score (LST) to characterize genomic instability using over 10,000 single-nucleotide polymorphisms distributed across human genome, adjusted by tumor ploidy and purity. HRD positive is defined by HRD score above the threshold (cut-off ≥30) and/or deleterious mutation in BRCA1/2. ResultsDeleterious BRCA1/2 mutations were detected in 21.2% (40/189) of TNBC patients and 48.1% (91/189) were defined as HRD positive. In advanced TNBC cohort, 21 patients received platinum-containing and 19 received platinum-free chemotherapy regimens for first-line chemotherapy. The progression-free survival (PFS) of the platinum-containing group was longer than that of the platinum-free group (median PFS 9.13 vs 2.97 months, HR 0.39, 95%CI, 0.19-0.81, P=0.011), and the PFS of patients with HRD positive was significantly longer than HRD negative patients (median PFS 13.6 vs 6.80 months, HR 0.38, 95%CI, 0.15-0.99, P=0.048) in platinum-containing group. Interestingly, HRD-positive patients have a significantly shorter PFS than HRD-negative in platinum-free group (median PFS 1.97 vs 4.52 months, HR 3.67, 95%CI, 1.20-11.22, P=0.023). For the HRD-positive patients, median PFS was significantly better in platinum-based group than platinum-free group (median PFS 13.6 vs 1.97 months, HR 0.12, 95%CI, 0.03-0.43, P=0.001). In early-stage TNBC cohort (n=149), 74 patients received platinum-containing and 75 received platinum-free chemotherapy regimens for adjuvant chemotherapy. In platinum-containing group, no statistically significant difference was found in DFS between HRD-positive and HRD-negative patients (P=0.118). High-risk TNBC group (Ki-67 ≥ 15%) analysis revealed that HRD-positive patients had a numerical better DFS than HRD-negative patients (HR 0.43, 95%CI, 0.12-1.50, P=0.180). Among patients with HRD-positive, patients in platinum-containing group had a tendency to benefit more than those in platinum-free group (HR 0.35, 95%CI, 0.11-1.10, P=0.062). Conclusions: We developed a novel algorithm to evaluate 3DMed-HRD status and highlights the potential utility of HRD in guiding chemotherapy for TNBC patients in this retrospective analysis. In comparison to platinum-free chemotherapy, the advanced TNBC HRD positive patients with advanced TNBC receiving platinum-based chemotherapy is a preferable regimen, and the HRD negative patients might be on the contrary. Prospective validation with larger sample size is needed. Citation Format: Yimeng Chen, Xue Wang, Feng Du, Jian Yue, Yiran Si, Lina Cui, Bei Zhang, Binghe Xu, Peng Yuan. The status of homologous recombination deficiency is a potential biomarker for platinum-based chemotherapy in triple-negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-12.

Landscape of homologous recombination deficiencies in solid tumours: analyses of two independent genomic datasets

BackgroundDNA repair deficiencies are characteristic of cancer and homologous recombination deficiency (HRD) is the most common. HRD sensitizes tumour cells to PARP inhibitors so it is important to understand the landscape of HRD across different solid tumour types.MethodsGermline and somatic BRCA mutations in breast and ovarian cancers were evaluated using sequencing data from The Cancer Genome Atlas (TCGA) database. Secondly, a larger independent genomic dataset was analysed to validate the TCGA results and determine the frequency of germline and somatic mutations across 15 different candidate homologous recombination repair (HRR) genes, and their relationship with the genetic events of bi-allelic loss, loss of heterozygosity (LOH) and tumour mutation burden (TMB).ResultsApproximately one-third of breast and ovarian cancer BRCA mutations were somatic. These showed a similar degree of bi-allelic loss and clinical outcomes to germline mutations, identifying potentially 50% more patients that may benefit from precision treatments. HRR mutations were present in sizable proportions in all tumour types analysed and were associated with high TMB and LOH scores. We also identified numerous BRCA reversion mutations across all tumour types.ConclusionsOur results will facilitate future research into the efficacy of precision oncology treatments, including PARP and immune checkpoint inhibitors.

Rucaparib in patients presenting a metastatic breast cancer with homologous recombination deficiency, without germline BRCA1/2 mutation

BackgroundBreast cancer may present genomic alterations leading to homologous recombination deficiency (HRD). PARP inhibitors have proven their efficacy in patients with HER2-negative (HER2-) metastatic breast cancer (mBC) harbouring germline (g) BRCA1/2 mutations in 3 phases III trials. The single-arm phase II RUBY trial included 42 patients, 40 of whom received at least one dose of rucaparib. RUBY study assessed the efficacy of rucaparib in HER2-mBC with either high genomic loss of heterozygosity (LOH) score or non-germline BRCA1/2 mutation. Patients and methodsThe primary objective was the clinical benefit rate (CBR), and the study was powered to see 20% CBR using a 2-stage Simon design. ResultsThe primary-end point was not reached with a CBR of 13.5%. Two LOH-high patients, without somatic BRCA1/2 mutation, presented a complete and durable response (12 and 28.5 months). Whole-genome analysis was performed on 24 samples, including 5 patients who presented a clinical benefit from rucaparib. HRDetect tended to be associated with response to rucaparib, without reaching statistical significance (median HRDetect responders versus non-responders: 0.465 versus 0.040; p = 0.2135). Finally, 220 of 711 patients with mBC screened for LOH upstream from RUBY presented a high LOH score associated with a higher likelihood of death (hazard ratio = 1.39; 95% CI: 1.11–1.75; p = 0.005). ConclusionOur data suggest that a small subset of patients with high LOH scores without germline BRCA1/2 mutation could derive benefit from PARP inhibitors. However, the RUBY study underlines the need to develop additional biomarkers to identify selectively potential responders.

PTCH1 mutation promotes antitumor immunity and the response to immune checkpoint inhibitors in colorectal cancer patients

Immunotherapy has emerged as an effective therapeutic strategy for various cancers, including colorectal cancer (CRC), but only a subset of MSI-H patients can benefit from such therapy. Patched1 (PTCH1) is a frequently altered gene in CRCs and its mutations contribute to unregulated Hedgehog (Hh) signaling. In the study, we evaluated the association of PTCH1 mutations with CRC immunity based on our single-center cohort and multiple cancer genomic datasets. Among 21 enrolled patients, six (28.6%) harbored a PTCH1 mutation based on WES analyses. In CRC patients, the PTCH1 mutation subgroup experienced a higher durable clinical benefit rate than the PTCH1 wild-type subgroup (100% vs. 40%, P = 0.017). In addition, patients with the PTCH1 mutation experienced greater progression-free survival (PFS, P = 0.037; HR, 0.208) and overall survival (OS, P = 0.045; HR, 0.185). A validation cohort from the MSKCC also confirmed the correlation between PTCH1 mutation and better prognosis (P = 0.022; HR, 0.290). Mechanically, diverse antitumor immune signatures were more highly enriched in PTCH1-mutated tumors than in PTCH1 wild-type tumors. Furthermore, PTCH1-mutated tumors had higher proportions of CD8 + T cells, activated NK cells, and M1 type macrophage infiltration, as well as elevated gene signatures of several steps in the cancer-immunity cycle. Notably, the PTCH1 mutation was correlated with tumor mutational burden (TMB), loss of heterozygosity score, and copy number variation burden. Our results show that the mutation of PTCH1 is a potential biomarker for predicting the response of CRC patients to immunotherapy.

Efficacy of platinum-based chemotherapy in advanced triple-negative breast cancer in association with homologous recombination deficiency.

e13051 Background: Previous studies have identified that at least 50% of triple negative breast cancer (TNBC) harbor mutation characteristics of homologous recombination deficiency (HRD). Thus, more sophisticated research into comprehensive genomic profiling of HRD is urgently needed. Whereas BRCA1/2-deficient advanced TNBC patients are sensitive to treatment with platinum, it is not yet clear whether HRD status could predict platinum response. Methods: 3DMed-HRD algorithm was developed based on loss of heterozygosity score (LOH), telomeric allelic imbalance score (TAI) and large-scale state transition score (LST) as previously described. HRD status was defined as HRD positive (deleterious mutation in BRCA1/2 or HRD score ≥30) or HRD negative (no deleterious mutation in BRCA1/2 and HRD score < 30). Tumor samples from 207 TNBC patients were analyzed by next-generation sequencing. Deleterious or suspected deleterious mutations were included for analysis. Among the overall cohort, 34 patients with advanced TNBC treated with chemotherapy were analyzed. Cox regression model was applied to evaluate the relationship between HRD status and clinical outcomes. Results: Deleterious BRCA1/2 mutations were detected in 22.2% (46/207) of TNBC patients as well as 54.6% (113/207) were defined as HRD positive. The most frequent mutations in HRD-positive patients were TP53 (93.5%), MYC (29.0%), PIK3R1 (22.6%), PTEN (22.6%) and MCL1 (19.4%), while TP53 (77.8%), MYC (29.6%), PIK3CA (18.5%), KMT2C (14.8%) and RIT1 (14.8%) enriched in HRD-negative patients. Mutations in DNA Damage Response (DDR), P53, Checkpoint and Receptor Tyrosine Kinase (RTK) pathways were most involved in HRD positive patients. In advanced TNBC cohort, 19 patients received platinum-based and 15 received platinum-free chemotherapy in the first-line treatment. The progression-free survival (PFS) of the platinum-based group was longer than that of the platinum-free group (media PFS 9.1 vs 2.2 months, HR 0.44, 95%CI, 0.20-0.94, P = 0.009). In HRD-positive patients, median PFS was significantly longer in platinum-based group (N = 6) than platinum-free group (N = 8) (media PFS 13.6 vs 1.9 months, HR 0.30, 95%CI, 0.09-0.95, P = 0.008). No significant difference in PFS between platinum-based and platinum-free group (p = 0.332) in patients with HRD-negative tumors. Patients with mutations in homologous recombination (HR) pathway had a worse PFS compared to wild-type in platinum-free group (1.4 vs 3.0 months, p = 0.050). Conclusions: Our findings illustrate the potential of HRD status as a marker to guide chemotherapy in advanced TNBC. In HRD positive patients, platinum-based chemotherapy might be a preferable regimen. Patients with mutations in HR pathway had a shorter PFS in platinum-free group. Prospective study with a larger sample-size is needed for further validation of our findings.

Genomic characteristics of endometrial cancer in China.

e17569 Background: Molecular classification of endometrial cancer has been applied in patient stratification and treatment strategy. Although the Cancer Genome Atlas project and other genomic studies have established comprehensive molecular classification, its genomic characteristics in Chinese endometrial cancer remain unclear. Methods: Tumor tissue and matched white blood cells from 316 patients with endometrial cancer underwent next-generation sequencing (NGS), which targeted the whole coding region of 733 or 381 genes. Endometrial cancer was classified into four subtypes according to genomic characteristics: polymerase (POLE) ultramutated, microsatellite instability (MSI), TP53 wild type (TP53wt), and TP53 mutant (TP53mut). Tumor mutation burden (TMB) was calculated based on the number of somatic single nucleotide variants and indels in examined coding regions. Deleterious or suspected deleterious genes were included for analysis. Genomic instability was also evaluated with Homologous Recombination Deficiency (HRD) score by over 10, 000 single-nucleotide polymorphisms distributed across human genome. HRD algorithm combined loss of heterozygosity score (LOH), telomeric allelic imbalance score (TAI) and large-scale state transition score (LST). Results: The 316 endometrial cancer patients were divided into four subtypes: POLE ultramutated (16, 5.06%), MSI tumors (70, 22.2%), TP53wt (131, 41.5%), and TP53mut (99, 31.3%). Median TMB was 26.5 mut/Mb in overall cohort and POLE ultramutated subtype possessed the highest TMB (181 mut/Mb), followed by MSI (54.1 mut/Mb), TP53mut (7.11 mut/Mb) and TP53wt (6.61 mut/Mb) subtypes. POLE, PTEN, LRP1B, BRCA2 and ATRX were the top frequently mutated genes in POLE ultramutated subtype, which mutation rate are more that 80%. The most frequent mutations were detected in ARID1A, PTEN, PIK3CA in MSI subtype, TP53, PIK3CA, PTEN in TP53mut subtype, and PTEN, PIK3CA, ARID1A in TP53wt subtype. HRD score was significantly higher in TP53mut subtype, followed by TP53wt, MSI and POLE ultramutated subtype (median HRD score, 21.7, 6.15, 3.65 and 0; P < 0.001). Conclusions: Our study revealed the heterogeneous genomic characteristics of four molecular subtypes in Chinese endometrial cancer. POLE ultramutated and MSI subtypes display higher TMB. TP53mut subtype showed genome features of HRD and would likely benefit from HRD-targeted therapy.

Abstract PS4-46: Homologous recombination deficiency score predicts patient characteristics and outcomes of triple negative breast cancer in China

Abstract BackgroundPrevious studies found homologous recombination deficiency (HRD) score could predict response to platinum-containing neoadjuvant chemotherapy in patients with triple negative breast cancer (TNBC), but its predictive effects of adjuvant chemotherapy is unexplored. Methods We developed the 3DMed-HRD algorithm, which combines loss of heterozygosity score (LOH), telomeric allelic imbalance score (TAI) and large-scale state transition score (LST) to characterize genomic instability using over 10000 SNPs, adjusted by tumor ploidy and purity. HRD positive is defined by deleterious mutation in BRCA1/2 or HRD score above the threshold (cut-off ≥30). Tumor samples were retrospectively obtained from 149 TNBC patients who received platinum-based (paclitaxel and cisplatin, TP) or platinum-free (cyclophosphamide and adriamycin, EC) adjuvant chemotherapy after surgery. All patients underwent the 3DMed-HRD testing. ResultsIn 25.5% (38/149) of TNBC patients deleterious germline or somatic mutation in BRCA 1/2 was detected. HRD results were available for all patients and 81of the 149 patients (54.4%) were defined as HRD positive. HRD-positive patients had younger age at diagnosis (median 46.7 years vs 51.5 years, P=0.002), higher differentiated (59.8% vs 39.5%; p=0.037), lower N-stage (55.7% vs 33.3%; p=0.006) and more common at high expression of Ki67 (64.7% vs 39.0%; p=0.015). In TP treatment group, patients with HRD positive tumors had a lower 5-year recurrence rate although the difference was not statistically significant (8.82% vs 16.1%; p=0.463). Patients in EC treatment cohort, 5-year recurrence rate was comparable between HRD positive and negative subgroups (15.4% vs 13.3%; p=1.0). In the 81 patients in HRD-positive cohort, patients in TP treatment group showed a numerical lower 5-year recurrence rates compared with patients in EC treatment group (8.82% vs 15.4%; p=0.489). Conclusions54.5% patients were characterized as HRD positive in TNBC patients in our cohort. HRD was associated with clinicopathologic characteristics. Our results suggested that HRD status mightguide chemotherapy treatment decisions. Prospective validation with larger sample size is needed. Citation Format: Xue Wang, Feng Du, Jian Yue, Yiran Si, Lina Cui, Peng Yuan. Homologous recombination deficiency score predicts patient characteristics and outcomes of triple negative breast cancer in China [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-46.

Abstract CT269: A phase 2 study of olaparib in combination with pembrolizumab in patients with previously treated advanced solid tumors with homologous recombination repair mutation (HRRm) and/or homologous recombination repair deficiency (HRD): KEYLYNK-007

Abstract Background: Treatment with the anti-PD-1 antibody pembrolizumab has improved clinical outcomes in multiple previously treated advanced solid tumors. The poly (ADP-ribose) polymerase (PARP) inhibitor olaparib has shown antitumor activity as monotherapy in patients with previously treated advanced ovarian, breast, pancreatic, and prostate cancers with BRCA1/BRCA2 mutations (BRCAm). Activity was also seen in patients with previously treated advanced solid tumors with other HRRm and in those with ovarian cancer with HRD phenotype. PARP inhibitors have been found to increase interferon signaling and tumor infiltrating lymphocytes, enhancing tumor susceptibility to immune checkpoint blockade. Antitumor activity of PD-(L)1 plus PARP inhibition was found to be higher than expected with either agent alone in patients with recurrent ovarian cancer regardless of BRCAm or HRD status and in patients with BRCAm breast cancer. KEYLYNK-007 (NCT04123366) evaluates the antitumor activity and safety of olaparib in combination with pembrolizumab in patients with previously treated advanced solid tumors with HRRm and/or HRD. Methods: This phase 2, nonrandomized, multicenter, open-label study plans to enroll approximately 300 patients aged ≥18 years with histologically/cytologically confirmed, previously treated, advanced solid tumors with HRRm and/or HRD per Lynparza HRR-HRD assay (Foundation Medicine, Inc., Cambridge, MA, USA), with an ECOG PS of 0-1. Patients will be grouped by biomarker status: subgroup 1: BRCAm; subgroup 2: HRRm without BRCAm; and subgroup 3: HRD positive without HRRm (loss of heterozygosity score ≥16 per Lynparza HRR-HRD assay). Patients will receive olaparib 300 mg twice daily + pembrolizumab 200 mg intravenously Q3W (35 cycles) until PD, unacceptable AEs, intercurrent illness, investigator decision, withdrawal of consent, or pregnancy. Tumor imaging assessment by blinded independent central review (BICR) per RECIST version 1.1 or Prostate Cancer Working Group (PCWG)-modified RECIST version 1.1 for prostate cancer will occur Q9W for 12 months, then Q12W until PD, start of new anticancer treatment, withdrawal of consent, pregnancy, or death. AEs will be monitored throughout the study and for 30 days after final dose (90 days for serious AEs) and are graded by National Cancer Institute Common Terminology Criteria for Adverse Events version 5. The primary endpoint is ORR (RECIST version 1.1 or PCWG-modified RECIST version 1.1 by BICR). Secondary endpoints include duration of response (DOR) and PFS (RECIST version 1.1 or PCWG-modified RECIST version 1.1 by BICR), OS, and safety. Point estimate and exact Clopper-Pearson CI for ORR, and Kaplan-Meier estimates for DOR, PFS, and OS will be calculated. Citation Format: Timothy A. Yap, Mallika Dhawan, Andrew E. Hendifar, Michele Maio, Taofeek K. Owonikoko, Miguel Quintela-Fandino, Ronnie Shapira-Frommer, Sanatan Saraf, Ping Qiu, Fan Jin, Alexander Gozman, Douglas A. Levine. A phase 2 study of olaparib in combination with pembrolizumab in patients with previously treated advanced solid tumors with homologous recombination repair mutation (HRRm) and/or homologous recombination repair deficiency (HRD): KEYLYNK-007 [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 CT269.

Abstract 3548: Loss of heterozygosity (LOH) as a candidate biomarker of PARP inhibitor sensitivity in Chinese solid tumor patients

Abstract Background: Poly (ADP-ribose) polymerase (PARP) plays a vital role in the repair of single-strand DNA breaks through the base excision repair pathway. Several PARP inhibitor approved for advanced BRCA-mutated (BRCAm) ovarian cancer. PARP inhibitors may benefit patients whose tumors are dysfunctional in DNA repair mechanisms unrelated to BRCA1/2. Here we descript the clinical application of LOH score as a candidate biomarker to predicate therapeutic response of PARP inhibitor in Chinese solid tumor patients. Methods: Formalin fixed paraffin embedded (FFPE) samples of 16 Chinese solid tumor patients were collected for next-generation sequencing (NGS) based 450 genes panel assay. Genomic alterations including single base substitution, short and long insertions/deletions, copy number variations, gene fusions and rearrangement were assessed. Loss of heterozygosity (LOH) status, Microsatellite stable (MSS) status and TMB (tumor mutational burden) were also acquired by an NGS algorithm. Results: There are 16 patients were analyzed in this retrospective study that most patients having advanced disease stage, Including 9 males and 7 females. The median age is 64 (range, 40-72) years old. The tumor type of these patients including Hepatocellular carcinoma (N=3), Cholangiocarcinoma (N=7), Breast cancer (N=2), Prostate cancer (N=2), Lymphoepithelioma like carcinoma (N=1) and Gallbladder adenocarcinoma (N=1). All 16 patients were treated with PARP inhibitor (Olaparib) including 14 patients harboring BRCA1/2 mutation. 15 patients were evaluable, OS (overall survival) time was calculated until abstract publish. 1 patients achieved complete response (CR), 4 patients achieved partial response (PR), 9 had stable disease (SD) and 1 had progressive disease (PD). Two patients with BRCA wild type achieved SD and the OS time was 13 and 37 months, respectively. LOH status was evaluated all tumors, 25% (4/16) of patients would be considered as having high LOH (LOH-H), including 2 patients without BRCA mutation. 75% (12/16) patients considered as having low LOH (LOH-L). LOH-high group has a longer OS time (P=0.056), median OS time of LOH-H group (N=4) is 25 month (range, 4-42) and LOH-L group (N=11) is 6 month (range, 2-23). Also we explored the association between TMB and OS, identified TMB-H patients (N=6) morel likely has a longer OS (median OS, 11.5 VS 5 month, P=0.017). Conclusion: In patients with advanced solid cancer, High LOH status was associated with good therapeutic response of PARP inhibitor. LOH status may become a potential indicator for the treatment of PARP inhibitors. Due to the limitations of the number and clinical baseline of patients, the results may require further research and validation. Citation Format: Jinwei Hu, Shuirong Zhang, Kai You, Lijuan Chen, Peng Zhang, Junping Shi, Ming Yao, Mo Wang, Kai Wang. Loss of heterozygosity (LOH) as a candidate biomarker of PARP inhibitor sensitivity in Chinese solid tumor patients [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 3548.

Abstract 3551: Simple vs. comprehensive prediction models of homologous recombination deficiency based on mutational and clinical features in three independent breast cancer datasets

Abstract Homologous recombination deficiency (HRD) phenotype often observed in tumors with BRCA1/2 deficiency is considered to be associated with efficacy of PARP inhibitors and platinum-based therapies. However, analysis on the degree of HRD requires comprehensive “genomic scar” analyses like Myriad MyChoice, therefore, we sought to develop a simple HRD prediction model in breast cancer patients without compromising its predictive value. HRD score as a consequence of genomic scars was calculated counting loss of heterozygosity score (LOH), telomeric allelic imbalance score (TAI), and large-scale state transition score (LST) for three independent breast cancer cohorts; TCGA (n= 744), ICGA (International Cancer Genome Consortium, n= 477), and Japanese AYA cohorts (n= 46). The HRD-high phenotype was defined as HRD scores ≥ 42. Genetic and pathological factors in HRD-high cases were determined by analyzing the sequencing data and DNA methylation chip analysis data as well as clinico-pathological information of the cohorts. The sequencing data comprised somatic mutation status of 82 significantly mutated genes (SMGs), germline/somatic mutations of 25 cancer susceptibility genes and three other HR-related genes. First, we adopted an Elastic Net regularized regression approach that controls for co-varying features within high-dimensional data in a TCGA breast cancer (Area under the curve [AUC] = 0.876), then applied the model onto two validation datasets. Among 16 selected features in the model, features with top-10 coefficient values were hypermethylation status, germline mutations of BRCA1, BRCA2, and PALB2, somatic mutations of BRCA1, BRCA2, PALB2 and TP53, triple negative subtype, and higher nuclear grade. The AUC values in ICGC and Japanese AYA were 0.899 and 0.941, respectively. Second, a simple predictive model for the HRD-high phenotype was developed using a TCGA data set (Area under the curve [AUC] = 0.838) based on only 5 features which were statistically significant (P < 0.01) in logistic regression analysis; germline BRCA1, BRCA2 and somatic TP53 mutations, triple negative subtype and higher nuclear grades. Its prediction power was validated in ICGC (AUC = 0.873) and Japanese AYA cohorts (AUC = 0.936). After excluding most features in the comprehensive Elastic Net model, the simple prediction model derived from five features still showed high AUC values comparable to those from the comprehensive models in the validation sets. These five features can be assessed by gene panel tests and daily pathological analyses. Thus, this study clarifies genomic and pathological factors associated with HRD phenotype of breast cancer and provides a simple predictive model of HRD to identify breast cancer patients who would benefit from PARP and/or platinum therapies in the clinical setting. Citation Format: Maki Tanioka, Tomoko Watanabe, Takayuki Honda, Hirohiko Totsuka, Eri Arai, Yae Kanai, Kouya Shiraishi, Kenji Tamura, Takashi Kohno. Simple vs. comprehensive prediction models of homologous recombination deficiency based on mutational and clinical features in three independent breast cancer datasets [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 3551.

Abstract A28: Intratumor heterogeneity and homologous recombination deficiency of high-grade serous ovarian cancer are associated with prognosis and molecular subtypes and change in treatment course

Abstract Background: High-grade serous ovarian cancers (HGSOC) are genomically characterized by homologous recombination deficiency (HRD) and TP53 mutations, which lead to intratumor heterogeneity (ITH). High degree of loss of heterozygosity (LOH) indicates HRD, a characteristic associated with sensitivity to platinum agents and poly-(ADP ribose) polymerase (PARP) inhibitors. This study aimed to reveal the relationship between HRD, ITH, and prognosis and analyze their changes during treatment. Methods: We obtained 573 single-nucleotide polymorphism (SNP) array and gene expression array data from The Cancer Genome Atlas. SNP array data were processed to calculate the Clonality Index (CI) and LOH scores. Gene expression array data were used for classifying molecular subtypes. Pathway analysis was performed by Ingenuity Pathway Analysis (IPA). Additionally, we obtained 33 samples from 20 HGSOC patients, including 4 samples from interval debulking surgery (IDS) and 9 samples from recurrent surgery. DNA extracted from FFPE specimens was analyzed with an OncoScan FFPE Assay Kit and the number of clones and the LOH score was calculated. GISTIC 2.0 was used to identify copy number aberrations in tumors. Results: We divided HGSOC samples into 2 groups by the CI and LOH scores, respectively. The high CI group (CI≧3) showed statistically shorter disease-free survival (DFS) and progression-free survival (PFS), but there was no statistically significant difference about overall survival (OS) (p<0.001, 0.01, p=0.419, respectively). The high LOH group (LOH score≧16) showed statistically longer DFS, PFS, and OS (p<0.001, 0.01, 0.001, respectively). Combining the two factors, the high LOH/low CI group showed a statistically good prognosis. In terms of molecular subtypes, the mesenchymal subtype, which had a poor prognosis, showed a high CI with statistically significant difference (p=0.0403) and the immunoreactive subtype, which had a good prognosis, showed a tendency to have a high LOH score (p=0.0762). With IPA analysis, it was predicted that the activation, migration, and adhesion of immune cells were activated in the high LOH group and high LOH/low CI group. Throughout treatment, the CI at primary surgery was 3 in 3 cases and 2 in 1 case, and at IDS, the CI decreased to 1 in all 4 cases, and then the CI increased at the secondary surgery in all 3 recurrent cases. The LOH score did not change in 2 cases and greatly decreased in 2 cases from primary surgery to IDS and then increased at secondary surgery. With GISTIC analysis, a pattern of the copy number variant compared between primary and recurrent surgery had few differences, but amplification of 8q24 was found at IDS with statistical significance. Conclusions: ITH and HRD were associated with prognosis in HGSOC. ITH of remaining tumors at IDS decreased compared with that of primary tumors. This study indicated that it is important to analyze tumors that remain after chemotherapy for investigating the mechanism of the development of chemoresistance. Citation Format: Hisamitsu Takaya, Hidekatsu Nakai, Kosuke Murakami, Noriomi Matsumura. Intratumor heterogeneity and homologous recombination deficiency of high-grade serous ovarian cancer are associated with prognosis and molecular subtypes and change in treatment course [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A28.

Abstract A49: Homologous recombination deficiency status-based classification of high-grade serous ovarian carcinoma

Abstract Background: Homologous recombination repair (HRR) pathway deficiency (HRD) is deeply involved in the tumorigenesis and progression of high-grade serous ovarian carcinoma (HGSOC) as well as in the sensitivity to platinum chemotherapy drugs. Until now, the proposed cutoff value for the HRD score, determined by calculating genomic scar signature scores associated with HRD, namely, the loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale state transitions (LST), has been ≥ 42 based on a combined dataset of HGSOC and breast cancer. In this study, we propose a rational method to classify HGSOC based on HRD status and investigated its clinical significance. Methods: We obtained data from The Cancer Genome Atlas (TCGA) on HGSOC and determined the HRD score as the sum of LOH, TAI, and LST scores. We then investigated the relationships among the score, genetic alterations in HRR-related genes, and the clinical data. Results: BRCA1/2 mutations were enriched in the group with HRD scores ≥ 63. BRCA1/2 mutations represented 38% of the cases with HRD scores ≥ 63 (49/128), 10% with HRD scores between 42 and 62 (12/118), and 10% with HRD scores 41 and below (5/50); no enrichment of BRCA1/2 mutation cases with HRD scores from 42 to 62 was observed compared with those with scores ≤ 41. Compared with the groups with scores ≤ 62, this group had a good prognosis (p < 0.0001); we thus considered HRD scores ≥ 63 to be the best cutoff point for identifying HRD cases in HGSOC. Among the other HRR pathway gene mutations, only CHEK1 homozygous deletions and PTEN homozygous deletions were associated with high HRD scores (p = 0.0038 and p = 0.035, respectively). In terms of promoter methylation, BRCA1 methylations and RAD51C methylations were associated with high HRD scores (p < 0.0001 and p = 0.029, respectively). Classification of HGSOC cases by the HRD status revealed a better prognosis for HRD cases caused by genetic alterations (HRD-G) than those caused by epigenetic changes (HRD-E), those caused by undetermined reasons (HRD-U), and non-HRD cases (p < 0.0001). A combined analysis of HRD status and residual tumor after primary debulking surgery revealed that, among cases without macroscopic residual tumors, 11 of 12 HRD-G cases survived after the median observation period of 6.6 years, showing remarkably higher survival rates than HRD-E, HRD-U, and non-HRD cases (p = 0.0059). Conclusion: HRD score ≥ 63 is an optimal cut-off to define HRD in HGSOC. HGSOC can be classified into subtypes with different prognoses according to HRD status. This classification could be useful for personalized HGSOC treatment. Citation Format: Noriomi Matsumura, Hisamitsu Takaya, Shiro Takamatsu, Hidekatsu Nakai. Homologous recombination deficiency status-based classification of high-grade serous ovarian carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A49.