Homologous Recombination Genes Research Articles

571P BRCA and beyond: Wider genetic testing of women with epithelial ovarian cancer

Epithelial ovarian cancer (EOC) is one of the most heritable cancers. It is associated with pathogenic variants (PVs) in mismatch repair (MMR) and homologous recombination (HR) genes, including moderate susceptibility gene BRIP1.Background: Epithelial ovarian cancer (EOC) is one of the most heritable cancers. It is associated with pathogenic variants (PVs) in mismatch repair (MMR) and homologous recombination (HR) genes, including moderate susceptibility gene BRIP1.

Activation of recombinational repair in Ewing sarcoma cells carrying EWS-FLI1 fusion gene by chromosome translocation

Chromosome translocation (TL) is an important mode of genomic changes underlying human tumorigenesis, the detailed mechanisms of which are, however, still not well understood. The two major modalities of DNA double strand break repair, i.e. homologous recombination (HR) and non-homologous end-joining (NHEJ), have been hypothesized. In a typical TL+ human neoplasm, Ewing sarcoma, which is frequently associated with t(11;22) TL encoding the EWS-FLI1 fusion gene, NHEJ has been regarded as a model to explain the disease-specific TL. Using comprehensive microarray approaches, we observed that expression of the HR genes, particularly of RAD51, is upregulated in TL+ Ewing sarcoma cell lines, WE-68 and SK-N-MC, as in the other TL+ tumor cell lines and one defective in DNA mismatch repair (MMR). The upregulated RAD51 expression indeed lead to frequent focus formation, which may suggest an activation of the HR pathway in these cells. Furthermore, sister chromatid exchange was frequently observed in the TL+ and MMR-defective cells. Intriguingly, ionizing irradiation revealed that the decrease of 53BP1 foci was significantly retarded in the Ewing sarcoma cell lines, suggesting that the NHEJ pathway may be less active in the cells. These observations may support an HR involvement, at least in part, to explain TL in Ewing sarcoma.

Reduction Sensitive Polymers Delivering Cationic Platinum Drugs as STING Agonists for Enhanced Chemo‐Immunotherapy

AbstractSTING agonists have made great progress in tumor immunotherapy. However, the inherent instability and low bioavailability have limited their wide applications. Herein, a reduction sensitive polymer with pair‐wised carboxyl groups that further encapsulate a cationic phenanthriplatin drug (PhenPt) as STING agonists into nanoparticles (PhenPt NPs) via electrostatic interactions is designed. PhenPt NP can release PhenPt in cancer cells, which then induce DNA damage, activate the STING signaling pathway, stimulate innate and adaptive immune responses, and improve the chemo‐immunotherapy efficacy. In vitro, for the first time it is found that PhenPt NP can activate cGAS‐STING pathway. Further genome‐wide RNA‐sequencing reveals that DNA replication, mismatch repair, homologous recombination, and other gene repair‐related pathways are involved. In vivo, PhenPt NP are found to completely inhibit the tumor growth, thereby shifting the tumor microenvironment from immunosuppressive to immunostimulatory phenotype, and boosting antitumor immune responses for long‐term immunity. In addition, PhenPt NP combined with checkpoint blockade therapy (a‐PD‐L1) can elicit long‐term immune response on both primary and distant tumors by activating the cGAS‐STING pathway. Overall, this nano‐delivery system with cationic chemotherapeutic drugs can greatly enhance DNA damage and activate immunity, hence providing a promising strategy for enhanced chemo‐immunotherapy.

RAD54B mutations enhance the sensitivity of ovarian cancer cells to poly(ADP-ribose) polymerase (PARP) inhibitors

Synthetic lethal targeting of homologous recombination (HR)–deficient ovarian cancers (OvCas) with poly(ADP-ribose) polymerase inhibitors (PARPis) has attracted considerable attention. Olaparib was the first PARPi approved by the Food and Drug Administration, offering significant clinical benefits in BRCA1/2-deficient OvCas. However, only approximately 20% of OvCa patients harbor BRCA1/2 mutations. Given the shared roles that BRCA1/2 have with other HR regulators, alterations in HR genes may also contribute to “BRCAness profiles” in OvCas. RAD54B has been considered a key player in HR repair, although its roles and therapeutic potential in cancers need further investigation. Here, we identified 22 frequently mutated HR genes by whole-exome sequencing of OvCa tissues from 82 patients. To our surprise, 7.3% of patients were found to harbor mutations of RAD54B, the third-highest mutated gene among patients. We determined that RAD54B-mutated tumor tissues harbored more DNA double-strand breaks than normal tissues. Additionally, we found that RAD54B knockdown inhibited HR repair, enhanced sensitivities of OvCa cells with increased DNA double-strand breaks to olaparib, and induced apoptosis. Enhanced inhibitory effects of olaparib on the growth of ES2 xenograft tumors were further demonstrated by RAD54B knockdown. Finally, we show that restoration with wildtype RAD54B rather than RAD54BN593S and RAD54BH219Y, identified in patients, abolished the effects of RAD54B knockdown, indicating these RAD54B mutants probably malfunctioned in HR repair. Our investigations may offer insight into the contributions of RAD54B mutations to synthetic lethality with olaparib treatment in OvCas, enrich the gene list for “HR deficiency scoring,” and expand the applications of PARPis.

Examining the Diagnostic Yield of Tumour Testing and Qualifying Germline Concordance for Hereditary Cancer Variants in Patients with High-Grade Serous Carcinoma.

Despite advances in treatment, prognosis for most patients with high-grade serous carcinoma (HGSC) remains poor. Genomic alterations in the homologous recombination (HR) pathway are used for cancer risk assessment and render tumours sensitive to platinum-based chemotherapy and poly (ADP-ribose) polymerase inhibitors (PARPi), which can be associated with more favourable outcomes. In addition to patients with tumours containing BRCA1 or BRCA2 pathologic variants, there is emerging evidence that patients with tumours harbouring pathologic variants in other HR genes may also benefit from PARPi therapy. The objective of this study is to assess the feasibility of primary-tumour testing by examining the concordance of variant detection between germline and tumour-variant status using a custom hereditary cancer gene panel (HCP). From April 2019 to November 2020, HCP variant testing was performed on 146 HGSC formalin-fixed, paraffin-embedded tissue samples using next-generation sequencing. Of those, 78 patients also underwent HCP germline testing using blood samples. A pathogenic variant was detected in 41.1% (60/146) of tumours tested, with 68.3% (41/60) having either a BRCA1 or BRCA2 variant (n = 36), or BRCA1/2 plus a second variant (n = 5), and 31.2% (19/60) carrying a pathogenic variant in another HCP gene. The overall variant rate among the paired germline and tumour samples was 43.6% (34/78), with the remaining 56% (44/78) having no pathogenic variant detected in the germline or tumour. The overall BRCA1/2 variant rate for paired samples was 33.3% (26/78), with germline variants detected in 11.5% (9/78). A non-BRCA1/2 germline variant in another HCP gene was detected in 9.0% (7/78). All germline variants were detected in the tumour, demonstrating 100% concordance. These data provide evidence supporting the feasibility of primary-tumour testing for detecting germline and somatic variants in HCP genes in patients with HGSC, which can be used to guide clinical decision-making, and may provide opportunity for improving patient triage and clinical genetic referral practices.

Correlation of CCNE1 copy number levels with molecular profiles and outcomes for high grade serous ovarian and endometrial cancer (121)

<b>Objectives:</b> This study aimed to characterize <i>CCNE1</i> copy number (CN) in relation to other molecular characteristics of tumors, along with patient outcomes, to help guide future clinical trial design and potential novel targeted therapeutics development for these aggressive molecular subtypes of high-grade serous ovarian (HGSOC) and endometrial cancers (EMCA). <b>Methods:</b> Tumor samples were analyzed using Next-Gen sequencing (NGS) of the DNA (NextSeq, 592 genes or NovaSeq, whole-exome sequencing) and RNA (NovaSeq, whole-transcriptome sequencing) and immunohistochemistry (IHC) (Caris Life Sciences, Phoenix, AZ). PD-L1 IHC used SP-142 (cut-off ≥1%). Microsatellite instability (MSI) was tested by fragment analysis, IHC, and NGS. Tumor mutation burden (TMB) was measured by totaling somatic mutations per tumor (high ≥10 mutations/MB). Relative abundance of immune-cell infiltrates was calculated by MCPCounter. <i>CCNE1</i> amplification (<i>CCNE1</i>^Amp) was defined as CN ≥6 and calculated by NGS/WES. Real-world overall survival (OS) was obtained from insurance claims data, and Kaplan-Meier estimates were calculated for molecularly defined patient cohorts. Statistical significance was determined using Chi-square and Wilcoxon rank-sum test, and p-values adjusted for multiple comparisons (q) to be <0.05. <b>Results:</b> <i>CCNE1</i> was amplified in 608/6306 (9.6%) of HGSOC, 214/2098 (10.2%) of serous EMCA, 191/749 (15.3%) of uterine carcinosarcomas (UCS), and 17/2053 (0.80%) of endometrioid EMCA. <i>TP53</i> mutations positively correlated with <i>CCNE1</i>^Amp. <i>CCNE1</i>^Amp HGSOC had fewer mutations in <i>BRCA1/2</i>, <i>ARID1A</i>, and <i>NF1</i> but increased CN amplification in other genes (Table 1). <i>PTEN</i> mutations were negatively correlated with <i>CCNE1</i>^Amp in UCS and endometrioid EMCA. There was no difference in immuno-oncology biomarkers (TMB, dMMR/MSI, PD-L1). However, increased <i>CCNE1</i> levels were associated with decreased fibroblasts in HGSOC and serous EMCA, decreased B cells in serous EMCA, and decreased CD3+ T cells in UCS. In HGSOC, <i>CCNE1</i>^Amp was associated with increased expression of cell-cycle checkpoint regulators, Fanconi anemia, homologous recombination, mismatch repair, and single-strand break repair pathway genes, but decreased expression of these genes in endometrioid EMCA. In tumors with >1 specimen serially submitted, there was a significant increase in <i>CCNE1</i> CN over time in 29% (49/169) of HGSOC and 23% of EMCA (36/158). There was no difference in <i>CCNE1</i> CN in tumors harboring <i>BRCA</i> reversion mutations (<i>n</i>=8) compared to wild-type (<i>n</i>=5110). <i>CCNE1</i>^Amp correlated with mRNA expression in HGSOC and EMCA. <i>CCNE1</i>^Amp was associated with worse OS after carboplatin-based treatment in HGSOC as well as in serous EMCA and UCS. <b>Conclusions:</b> <i>CCNE1</i>^Amp in HGSOC and EMCA is less common than previously identified. Both HGSOC and endometrioid EMCAs have distinct molecular characteristics of <i>CCNE1</i>^Amp tumors. <i>CCNE1</i> CN may increase with the emergence of treatment resistance. Finally, <i>CCNE1</i>^Amp tumors appear overall non-immunogenic.

A Systematic Review and Meta-Analysis on the Prognostic Value of BRCA Mutations, Homologous Recombination Gene Mutations, and Homologous Recombination Deficiencies in Cancer.

Patients with BRCA1/2 mutations (BRCAm), loss-of-function mutations in other homologous recombination repair (HRRm) genes, or tumors that are homologous recombination deficiency positivity (HRD+) demonstrate a robust response to PARPi therapy. We conducted a systematic literature review and meta-analysis to evaluate the prognostic value of BRCAm, HRRm, and HRD+ on overall survival (OS) among those treated by chemotherapy or targeted therapy other than PARPi across tumor types. A total of 135 eligible studies were included. Breast cancer (BC) patients with BRCA1/2m had a similar overall survival (OS) to those with wild-type BRCA1/2 (BRCA1/2 wt) across 18 studies. Ovarian cancer (OC) patients with BRCA1/2m had a significantly longer OS than those with BRCA1/2 wt across 24 studies reporting BRCA1m and BRCA2m, with an HR of 0.7 (0.6–0.8). Less OS data were reported for other tumors: 6 studies for BRCA2m compared with BRCA2 wt in prostate cancer with an HR of 1.9 (1.1–3.2) and 2 studies for BRCA1/2m compared with BRCA1/2 wt in pancreatic cancer with an HR of 1.5 (0.8–3.1). Only 4 studies reported HRD+ by either BRCA m or genomic instability score (GIS) ≥ 42 and OS by HRD status. The HR was 0.67 (0.43–1.02) for OS with HRD+ vs. HRD−. A total of 15 studies reported the association between HRRm and OS of cancers in which one or more HRR genes were examined. The HR was 1.0 (0.7–1.4) comparing patients with HRRm to those with HRR wild-type across tumors. Our findings are useful in improving the precision and efficacy of treatment selection in clinical oncology.

Rare germline variants in PALB2 and BRCA2 in familial and sporadic chordoma.

Chordoma is a rare bone tumor with genetic risk factors largely unknown. We conducted a whole-exome sequencing (WES) analysis of germline DNA from 19 familial chordoma cases in five pedigrees and 137 sporadic chordoma patients and identified 17 rare germline variants in PALB2 and BRCA2, whose products play essential roles in homologous recombination (HR) and tumor suppression. One PALB2 variant showed disease cosegregation in a family with four affected people or obligate gene carrier. Chordoma cases had a significantly increased burden of rare variants in both genes when compared to population-based controls. Four of the six PALB2 variants identified from chordoma patients modestly affected HR function and three of the 11 BRCA2 variants caused loss of function in experimental assays. These results, together with previous reports of abnormal morphology and Brachyury expression of the notochord in Palb2 knockout mouse embryos and genomic signatures associated with HR defect and HR gene mutations in advanced chordomas, suggest that germline mutations in PALB2 and BRCA2 may increase chordoma susceptibility. Our data shed light on the etiology of chordoma and support the previous finding that PARP-1 inhibitors may be a potential therapy for some chordoma patients.

Neoadjuvant afatinib with paclitaxel for triple-negative breast cancer and the molecular characteristics in responders and non-responders

The prognosis of triple-negative breast cancer (TNBC) is worse and a major proportion of TNBC expresses epidermal growth factor receptor (EGFR). Afatinib can inhibit EGFR signal pathway; however, its treatment effect for TNBC is unknown. Thus, we aimed to assess the efficacy and biomarkers of afatinib in combination with paclitaxel in a neoadjuvant setting. Patients with stage II to III TNBC were enrolled. They received 40mg of afatinib daily for 14 days, followed by daily afatinib and weekly paclitaxel (80mg/m2) every 21 days for four to six cycles. To explore the mechanisms of responsiveness and non-responsiveness, 409 cancer-associated genes were sequenced. Twenty-one patients were enrolled and one patient achieved a complete clinical response; however, a 2mm residual tumor was noted in the surgical specimen. Overall, 33.0% patients were responders. Fifteen patients received molecular testing. No activated mutation of EGFR or Her2 were found. Activated PI3K or JAK2 pathway were trended to associate with non-responder (p=0.057). Mutation of homologous recombination (HR) genes were correlated with non-responsiveness (p=0.005). Seven patients did not have altered PI3K, JAK2 or HR pathway; six (85.7%) of them were responder. Patients with the amplified DAXX gene was associated with a favorable trend of response (p=0.109). Adding afatinib to neoadjuvant paclitaxel generated a modest effect in TNBC. Exploratory molecular analysis suggested that activated PI3K, JAK2 pathways and mutation of HR genes were associated with therapeutic non-responsiveness, and amplification of DAXX genes was associated with responsiveness to afatinib in combination with paclitaxel.

Targeting Therapeutic Resistance and Multinucleate Giant Cells in CCNE1-Amplified HR-Proficient Ovarian Cancer.

Approximately 20% of high-grade serous ovarian cancers (HGSOC) have CCNE1 amplification. CCNE1-amplified tumors are homologous recombination (HR) proficient and resistant to standard therapies. Therapy resistance is associated with increased numbers of polyploid giant cancer cells (PGCC). We sought to identify new therapeutic approaches for patients with CCNE1-amplified tumors. Using TCGA data, we find that the mTOR, HR, and DNA checkpoint pathways are enriched in CCNE1-amplified ovarian cancers. Furthermore, Interactome Mapping Analysis linked the mTOR activity with upregulation of HR and DNA checkpoint pathways. Indeed, we find that mTOR inhibitors (mTORi) downregulate HR/checkpoint genes in CCNE1-amplified tumors. As CCNE1-amplified tumors are dependent on the HR pathway for viability, mTORi proved selectively effective in CCNE1-amplified tumors. Similarly, via downregulation of HR genes, mTORi increased CCNE1-amplifed HGSOC response to PARPi. In contrast, overexpression of HR/checkpoint proteins (RAD51 or ATR), induced resistance to mTORi. In vivo, mTORi alone potently reduced CCNE1-amplified tumor growth and the combination of mTORi and PARPi increased response and tumor eradication. Tumors treated with mTORi demonstrated a significant reduction in ALDH+ PGCCs. Finally, as a proof of principle, we identified three patients with CCNE1 amplified tumors who were treated with an mTORi. All three obtained clinical benefits from the therapy. Our studies and clinical experience indicate mTORi are a potential therapeutic approach for patients with CCNE1-amplified tumors.

Homologous Recombination Deficiency in Ovarian, Breast, Colorectal, Pancreatic, Non-Small Cell Lung and Prostate Cancers, and the Mechanisms of Resistance to PARP Inhibitors.

Homologous recombination (HR) is a highly conserved DNA repair mechanism that protects cells from exogenous and endogenous DNA damage. Breast cancer 1 (BRCA1) and breast cancer 2 (BRCA2) play an important role in the HR repair pathway by interacting with other DNA repair proteins such as Fanconi anemia (FA) proteins, ATM, RAD51, PALB2, MRE11A, RAD50, and NBN. These pathways are frequently aberrant in cancer, leading to the accumulation of DNA damage and genomic instability known as homologous recombination deficiency (HRD). HRD can be caused by chromosomal and subchromosomal aberrations, as well as by epigenetic inactivation of tumor suppressor gene promoters. Deficiency in one or more HR genes increases the risk of many malignancies. Another conserved mechanism involved in the repair of DNA single-strand breaks (SSBs) is base excision repair, in which poly (ADP-ribose) polymerase (PARP) enzymes play an important role. PARP inhibitors (PARPIs) convert SSBs to more cytotoxic double-strand breaks, which are repaired in HR-proficient cells, but remain unrepaired in HRD. The blockade of both HR and base excision repair pathways is the basis of PARPI therapy. The use of PARPIs can be expanded to sporadic cancers displaying the “BRCAness” phenotype. Although PARPIs are effective in many cancers, their efficacy is limited by the development of resistance. In this review, we summarize the prevalence of HRD due to mutation, loss of heterozygosity, and promoter hypermethylation of 35 DNA repair genes in ovarian, breast, colorectal, pancreatic, non-small cell lung cancer, and prostate cancer. The underlying mechanisms and strategies to overcome PARPI resistance are also discussed.

Abstract 1542: MALAT1 ablation dismantles homologous recombination repair machinery and sensitizes castrate resistant prostate cancer cells to PARP inhibitor

Abstract Poly (ADP-ribose) polymerase (PARP) inhibitors have emerged as a promising target of intervention for metastatic castration-resistant prostate cancer (mCRPC), as approximately one-third of mCRPC patients harbor mutations in the genes associated with the homologous recombination (HR) pathway. Nonetheless, most patients inevitably develop resistance to PARP inhibition (PARPi), due to the induction of reversion mutations in the HR pathway. These mutations restore the function of HR genes and revert to the HR-proficient stage, which in turn decreases the vulnerability of cancer cells to PARP inhibitors. Here, we report that the clinical utility of PARPi could be efficaciously extended by targeting Metastasis associated lung adenocarcinoma transcript (MALAT1), a long non-coding RNA (lncRNA) often elevated in advanced-stage prostate cancer (PCa). In support of this, we show that mCRPC patients exhibit higher expression of genes involved in HR, which positively correlate with MALAT1 levels. Furthermore, RNA interference (RNAi)-mediated depletion of MALAT1 in CRPC cells perturb the expression of key HR genes, namely BRCA1/2, RAD51, EXO1, CHEK1/2, subsequently resulting in HR deficiency. This, in turn, escalates accumulation of DNA lesions in the MALAT1 ablated cells as noticed by abundance in γH2AX, a marker for DNA damage. As a consequence, MALAT1 ablated cells instigates G1/S phase arrest to fetch additional time to repair the damaged DNA or to induce apoptosis. Furthermore, we also provide evidence that the HR deficiency induced by MALAT1 depletion phenocopies “BRCAness” and exhibits synergy with clinically approved DNA repair inhibitors such as Olaparib. In particular, co-inhibition of MALAT1 and PARP1 exhibits a significant anti-proliferative effect reduced clonogenic survival and delays the resolution of γH2AX foci in CRPC cell lines. Taken together, our results establish that MALAT1 plays a central role in regulating the DNA damage response and provides a mechanistic rationale for dual targeting of MALAT1 and PARP in mCRPC patients. Citation Format: Anjali Yadav, Tanay Biswas, Ayush Praveen, Bushra Ateeq. MALAT1 ablation dismantles homologous recombination repair machinery and sensitizes castrate resistant prostate cancer cells to PARP inhibitor [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 1542.

Abstract 3763: Detection of tumor-associated gene inactivation in clinical blood draws via cell-free DNA methylation profiling

Abstract Introduction: The ability to detect genome-wide epigenetic changes, such as DNA methylation, has expanded translational applications in oncology settings. Because these changes occur early in carcinogenesis, they can be used for early cancer detection when genomic technologies fall short due to lower sensitivity, and in the early and late-stage cancer setting for minimal residual disease detection, disease monitoring and therapy selection. In this analysis, we demonstrated our detection of differential methylations that classify cancer from healthy normals, as well as the quantification of promoter methylation, using a highly sensitive targeted assay that simultaneously captures both genomic alterations and methylation signatures in cell-free DNA (cfDNA). Methods: Methylation signals were profiled with a broad genomic panel (15.2 Mb) targeting regions that are unmethylated in plasma cfDNA from cancer-free donors. The panel covers the promoter regions of 925 out of 1,217 known tumor suppressor genes (TSGs) (e.g. TP53, APC, RB1, PTEN), homologous recombination and repair (HRR) genes (e.g. ATM, BRCA1/2, CDK12, RAD51C/D). We applied our genomic and epigenomic assay on cfDNA from 1,968 colorectal cancer (CRC) patients, 480 patients with other 6 common cancers, and 2,037 cancer-free donors. To test the sensitivity of our epigenomic assay, we generated an in-silico dataset by computationally mixing reads from the cancer patients with those from cancer-free donors at a low tumor fraction (TF) of 0.1%. Results: Among 62 clinically relevant TSG and HRR genes, 56 (90%) were differentially methylated (Wilcoxon p&amp;lt;0.05) between the 2,448 cancer vs. 2,037 cancer-free donor samples. In the in-silico dataset, 51 of the 56 differentially methylated genes remained statistically significant (Wilcoxon p&amp;lt;0.05) at 0.1% TF. In the 1,968 CRC patients, we observed a significant association (Fisher’s p&amp;lt;1e-05) between MLH1 promoter methylation and microsatellite instability (MSI-H): 70% of MSI-H samples has MLH1 promoter methylation above a predefined “high methylation” threshold, while only 7% of microsatellite-stable samples has MLH1 promoter methylation above this threshold. Our result is consistent with previous studies that 54-100% of CRC patients with MSI-H tumors harbor MLH1 promoter methylation. Conclusion: We demonstrate that our assay can accurately detect cancer-driven DNA methylation across the genome in clinical plasma samples. Detection of differential methylation in cancerous versus non-cancerous tissues could allow for early cancer detection, leading to better survival and prediction of recurrence prior to imaging. The highly sensitive detection of promoter methylation shown with TSG, HRR, and MLH1 genes may provide orthogonal information to oncologists in therapeutic selection with high confidence. Citation Format: Sai Chen, Shile Zhang, Tingting Jiang, Jennifer Yen, Yupeng He, Ariel Jaimovich, Yvonne Kim, Dustin Ma, Giao Tran, Daniel P. Gaile, Rebecca J. Nagy, Elena Helman, Han-Yu Chuang. Detection of tumor-associated gene inactivation in clinical blood draws via cell-free DNA methylation profiling [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 3763.

Abstract 5777: Germline variants of cancer predisposition genes in a large cohort of Chinese sarcoma patients

Abstract Background: Sarcomas represent a rare and heterogeneous group of soft tissue and bone cancers, including more than 100 histological subtypes. Although genomic profiling has identified oncogenic fusion genes as the major molecular drivers of many sarcoma subtypes, direct targeting of these fusion genes is clinically challenging and novel treatment approaches are needed. Methods: To explore new therapeutic targets in sarcoma, we performed germline testing of 148-cancer predisposition genes (OncoPanscan࣪, Genetronhealth) on genomic DNA from a cohort of 822 unselected Chinese sarcoma patients. Results: The median age of this cohort was 45 (1-88) years, and 84% were soft tissue sarcoma (STS), 16% were osteosarcoma. 9.3% (n =82) of patients harbored at least one predicted pathogenic germline variant in 37 cancer disposition genes including APC, BRCA1, BRCA2, CDKN2A, CHEK2, DICER1, MSH6, PMS2, SBDS, SMARCA4, and TP53. Twenty-four (2.9%) patients had a variant that mapped to a known sarcoma-associated cancer predisposition syndrome gene (CHEK2, DICER1, NF1, RECQL4, SMARCA4, TP53, WRN). The most frequently mutated genes are involved in the DNA damage repair pathway, with a prevalence of 6.3% (n =52). Twelve patients carried pathogenic mutations in TP53, which was associated with cancer-predisposing Li-Fraumeni syndrome (LFS). Interestingly, we also observed 12 patients harboring the same SBDS c.258+2T&amp;gt;C variant. Biallelic SBDS mutations were linked to Shwachman-Diamond Syndrome (SDS) associated with leukemia predisposition. Additionally, four patients carried pathogenic mutations in SMARCA4 (n =2) and SMARCE1 (n =2), two genes encoding key components of the BAF chromatin-remodeling complex. Patients with pathogenic SMARCA4 germline variants could participate in clinical trials of EZH2 inhibitor tazemetostat. Four patients harbored pathogenic germline variants in core homologous recombination genes (BRCA1/2 and PALB2), which may be targeted with platinum-based chemotherapy or PARP inhibitor olaparib. Conclusions: In summary, our genomic profiling approach revealed novel predisposition variants associated with sarcoma, and some of them can serve as therapeutic targets for future clinical investigation. Our work provided proof of principle that sarcoma patients may benefit from genetic counseling and participate in genomically-matched clinical trials. Citation Format: Jingnan Shen, Xiaomo Li, Xianbiao Xie, Si Liu, Tonghui Ma. Germline variants of cancer predisposition genes in a large cohort of Chinese sarcoma patients [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 5777.

Abstract 798: Comprehensive genomic profiling of tissue and liquid biopsies reveals the landscape of reversion mutations

Abstract Loss of function mutations in homologous recombination (HR) repair genes predispose individuals to cancer due to the genomic instability brought about by the inability to properly repair DNA. These alterations may also identify patients eligible for treatment with poly (ADP-ribose) polymerase (PARP) inhibitors and platinum drugs that exploit synthetic lethality; however, resistance eventually occurs. This resistance is largely the result of reversion mutations, which restore the DNA repair function of mutated HR genes. We employed a computational approach to identify patients with breast, ovarian, pancreas, or prostate cancer who underwent comprehensive genomic profiling (CGP; Foundation Medicine, Inc.) and had reversion mutations in the BRCA1, BRCA2, PALB2, RAD51C, or RAD51D genes. Reversion mutations were identified in six different categories: 1) missense mutations that occur at the same amino acid position as a deleterious alteration, 2) non-frameshift deletion mutations that encompass a deleterious alteration, 3) frameshift mutations that restore the open reading frame of another frameshift mutation, 4) splice-site mutations that occur at the same exon as a deleterious alteration, 5) copy number losses that remove a deleterious alteration, and 6) intragenic deletions that lead to loss of deleterious mutation. Using our computational approach, we identified 483 patients who 1,112 reversion mutation pairs in both solid tissue- and liquid biopsy-derived samples. The most frequently observed category was a non-frameshift deletion of a deleterious mutation. Overall, a greater number of reversion mutations were found in liquid biopsy samples. BRCA2 was the most frequently reverted gene followed by BRCA1, PALB2, RAD51D, and RAD51C. BRCA1-mutated samples had up to six reversion mutations, and for BRCA2, we observed as many as 228 putative reversion pairs in one sample. PALB2, RAD51C, and RAD51D had no more than two reversion mutations. We further examined co-occurring alterations and found correlations with genes in multiple signaling pathways including the MYC signaling pathway, which contained more mutations in reversion-positive samples. Lastly, we explored real-world longitudinal data from reversion-positive samples and found that the presence of a reversion mutation was significantly associated with lower overall survival for breast, ovarian, and prostate cancer patients. In summary, we characterized the landscape of reversion mutations in patients who underwent tissue or liquid biopsy-based CGP during clinical cancer treatment. Our data provide a better perspective for the identification and treatment of cancers involving a reversion mechanism. Citation Format: Shannon T. Bailey, Meijuan Li, James Thornton, Lei Yang, Chenming Cui, Mark Hartman, Garrett Frampton, Brennan Decker, Christine Vietz. Comprehensive genomic profiling of tissue and liquid biopsies reveals the landscape of reversion mutations [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 798.

Current Methods for Recombination Detection in Bacteria

The role of genetic exchanges, i.e., homologous recombination (HR) and horizontal gene transfer (HGT), in bacteria cannot be overestimated for it is a pivotal mechanism leading to their evolution and adaptation, thus, tracking the signs of recombination and HGT events is importance both for fundamental and applied science. To date, dozens of bioinformatics tools for revealing recombination signals are available, however, their pros and cons as well as the spectra of solvable tasks have not yet been systematically reviewed. Moreover, there are two major groups of software. One aims to infer evidence of HR, while the other only deals with horizontal gene transfer (HGT). However, despite seemingly different goals, all the methods use similar algorithmic approaches, and the processes are interconnected in terms of genomic evolution influencing each other. In this review, we propose a classification of novel instruments for both HR and HGT detection based on the genomic consequences of recombination. In this context, we summarize available methodologies paying particular attention to the type of traceable events for which a certain program has been designed.

Detection of homologous recombination deficiency (HRD) in cell-free DNA (cfDNA) using an amplicon-based next-generation sequencing (NGS) assay.

3042 Background: Homologous recombination (HR) deficiency is characterized by tumor genomic instability, often due to alterations in BRCA1/2 and other HR-related genes. HRD predicts sensitivity to PARP inhibitors (PARPi) in prostate, ovarian and breast cancers. For the subset of cancers that have genomic instability without detectable alterations in HR genes, profiling biomarkers of HRD such as loss of heterozygosity (LOH) could identify additional HRD positive (HRD+) patients that may benefit from PARPi. Here, we present a novel cfDNA NGS assay that can detect tumor LOH non-invasively to improve assessment of HRD+ status. Methods: An amplicon-based HRD NGS assay covering &gt; 1000 SNPs was developed to detect LOH both globally and on the gene level in cfDNA. Analytical validation was done using LOH+ BRCA-mutant cell lines in limiting dilution admixtures. Clinical performance was assessed by benchmarking findings from 46 tumor tissue DNA samples against results from an orthogonal NGS-based genomic scarring assay. Clinical utility of the HRD assay was evaluated in 75 cfDNA samples, including 72% (54/75) from BRCA-associated cancers (36 breast, 12 prostate, 4 ovarian, 2 pancreatic) and 28% from other cancer types. All cfDNA samples were previously clinically tested by an NGS assay that included BRCA1/2 (LiquidHALLMARK). A subset of cfDNA samples identified as HRD+ were further assessed for alterations in 26 key HR genes ( ARID1A, ATM, ATR, ATRX, BAP1, BARD1, BRCA1, BRCA2, BRIP1, CDK12, CHEK1, CHEK2, FANCA, FANCC, FANCG, FANCL, MRE11, NBN, PALB2, PTEN, RAD51, RAD51B, RAD51C, RAD51D, RAD54L, and XRCC2). Results: In analytical validation, the assay could detect HRD in admixtures with as low as 18% tumor fraction. In tissue samples, overall concordance of HRD status with the orthogonal test was 91.3% (42/46), with a positive percent agreement of 94.4% (34/36) and negative percent agreement of 80.0% (8/10). In cfDNA, 26.7% (20/75) of samples were HRD+, including 29.6% of BRCA-associated cancers (16/54; 12 breast, 2 prostate, 1 ovarian, and 1 pancreatic) and 19.1% (4/21) of other cancer types. In the three HRD+ breast cancers with pathogenic BRCA mutations, the assay also identified BRCA-specific LOH. Nine HRD+ breast cancers (two BRCA+ and seven BRCA-) were further analyzed for alterations in 26 HR genes. Of the two BRCA+ HRD+ breast cancers, one harbored an additional PTEN loss-of-function (LOF) mutation. Of the seven BRCA- HRD+ breast cancers, one harbored biallelic PALB2 and CDK12 LOF mutations, while another harbored a CDK12 LOF mutation. Conclusions: LOH detection in cfDNA provides additional diagnostic yield of HRD+ status in multiple cancer types, even in the absence of pathogenic HR gene alterations. Further clinical studies to evaluate the utility of HRD detection in cfDNA using LOH and to determine concordance with tumor tissue are ongoing.

Landscape of tumor mutation burden and correlation to clinical outcomes in 1,744 solid cancers.

2667 Background: Tumor mutation burden (TMB) has proven clinical utility and efficacy related to immune checkpoint inhibitor (ICI) treatment in different tumor types. However, limited data were available to understand the landscape and matched clinical outcomes to a pan-cancer extent. Methods: Target sequencing results, using TruSight Oncology 500, from 1,744 solid cancer between 2019 and 2021 as a part of real-world clinical practice were collected. Matched clinical and histological outcomes, including PD-L1 expression ( n= 798) and immunotherapy treatment ( n= 400), were analyzed. Results: Among the 33 different cancer types, high TMB was observed in 257 patients (14.7%) with a significantly higher incidence observed in carcinoma of unknown origin (33.3%), bladder cancer (32.3%), and small bowel cancer (30.0%). TMB was positively correlated with the degree of mutation in homologous recombination genes (HRD, 0.76) and microsatellite instability (MSI, 0.51). The predictive value of high TMB to ICI treatment seemed to be enhanced when it was combined with PD-L1 expression, showing higher response rates in the PD-L1 high group (63.3%) than in the low group (22.2%). In the patients with high TMB, both longer overall survival ( p = 0.015) and progression-free survival ( p = 0.020) were observed compared to the patients with low TMB. Conclusions: We examined the landscape of TMB in a different type of cancer using clinical-level target sequencing outcomes. High TMB seems to be a promising predictive biomarker of ICI, showing a positive correlation with alteration in other DNA damage response and repair-related genes and additive value to PD-L1, which needs to be validated through further prospective trials.

Characterization of mutational signatures of homologous recombination deficiency (HRD) in Chinese breast cancer.

e15129 Background: Homologous recombination deficiency (HRD) is a common molecular characteristic of genomic instability and has been proved to be an important biomarker for personalized therapy decisions and stratified management in breast cancer. In this study, we explored the mutational features of HRD tumors identified by HRD score in Chinese breast cancer. Methods: Tumor tissue DNA from 68 breast cancer patients was isolated and sequenced by next-generation sequencing (NGS) technologies with AcornMed 808-genes panel. HRD score was calculated as an unweighted sum of loss of heterozygosity (LOH) score, telomeric allelic imbalance (TAI) score and large-scale state transition (LST) score by AcornHRD algorithm, and mutations in 39 homologous recombination (HR) related genes including BRCA1/2 were identified for each case. Results: Based on the mutation alternation profiles from our cohort, somatic BRCA1/2 mutations were present in 15 patients (22.1%) while 29 patients (42.6%) harbored one or more mutations in HR-related genes excluding BRCA1/2. Germline mutations of BRCA1/2 and other HR-related genes occurred in 11.8% and 4.4% of cases detected, respectively.In our cohort, the median HRD score was 40 (range from 3 to 92). Sixty-eight cases were divided into HRD-high or HRD-low group based on a cut-off value of ≥ 42 or &lt; 42, with HRD-high group accounting for approximately 47% of our cohort, indicating the combination with HRD score yielded an increment of 26.5% in coverage of HRD within BRCA1/2 wildtype cases. The HRD-high group covered 82.4% of the BRCA-mutated cases and also showed higher mutation level of BRCA1/2-excluded HR genes than HRD-low group (p = 0.001). Prevalence of mutations in a total of 12 genes, including BRCA1(31.3% vs 5.6%, p = 0.009) and NBN (25% vs 5.6%, p = 0.038), were significant difference between HRD-high and HRD-low group. Besides, Cosmic Signature 1 (spontaneous deamination of 5-methylcytosine) and 5 (unknown) were existed in both groups, whereas Signature 6 (defective DNA mismatch repair) was only discovered in HRD-high group. Compared with HRD-low ones, copy number variations ratio was higher in HRD-high group (p = 0.031). Meanwhile, the patients with ERBB2 amplification (HER2+) showed a lower level of HRD score than HER2- ones. Conclusions: This study comprehensively delineated the genetic characteristics of HRD tumors, which suggests that HRD status marked by HRD score is strongly associated with BRCA1/2 and HR-related genes mutation as well as other genomic instability signatures, thus indicating its predictive value as a promising biomarker for DNA repair deficiency related drugs or therapies in Chinese breast cancer.

Landscape of homologous recombination reversion mutations in gynecologic malignancies.

5576 Background: Homologous recombination (HR) reversion mutations (REV) are biomarkers for predicting resistance to platinum and PARP inhibitor therapies. The biologic diversity of REV represents a diagnostic challenge. An automated computational approach was used to detect REV for analysis of genomic features of REV-positive ovarian epithelial, fallopian tube, and peritoneal cancers. Methods: Retrospective study of tissue (n = 23,612) and liquid biopsy (n = 869) samples from patients undergoing hybrid-capture comprehensive genomic profiling during routine clinical care 11/2012-03/2021. A proprietary algorithm tested for seven distinct REV mechanisms in BRCA1, BRCA2, PALB2, RAD51C, or RAD51D. For subjects with multiple samples, the earliest REV-positive sample was used for downstream analyses. Results: Among 23,866 ovarian epithelial, fallopian tube, and peritoneal cancers, 16.4% (n = 3,920) had at least one pathogenic variant (PV) in BRCA1 (10%, n = 2,383), BRCA2 (5.5%, n = 1,320), PALB2 (0.67%, n = 160), RAD51C (0.64%, n = 152), or RAD51D (0.47%, n = 113). 3.9% (154/3,920) of patients with one or more PV had REV. REV were found in tumors with PV in BRCA2 at twice the frequency of BRCA1 (6.0%, [79/1,320] vs 3.0% [71/2,383]; p &lt; 0.001). REV involving RAD51D (1.8%, 2/113), RAD51C (0.7%, 1/152), or PALB2 (0.6%, 1/160) were rarer. A total of 193 REV pairs were identified. The most frequent REV mechanism was an exonic non-frameshift deletion completely encompassing a PV (45%, 87/193). Other recurrent mechanisms included restoration of the reading frame of a frameshift PV (21%, 42/193), replacement of a PV with a benign missense substitution at the same codon (16%, 32/193), and deletion with intronic breakpoints encompassing a PV (15%, 29/193). REV were significantly more prevalent in liquid biopsies than in tissue samples with PV (16% [20/124] vs. 3.5% [134/3,796]; p &lt; 0.001). A range of 1-6 REV pairs were found per sample (liquid: 1-6, tissue: 1-3). Multiple REV per sample was more common in liquid biopsies (35% [7/20] vs. 11% [15/134]; p = 0.011). These differences likely reflect stage of disease and sampling of multiple subclones. Conclusions: BRCA2 PV are most frequently reverted in gynecologic tumors. REV are also common in tumors with BRCA1 PV and can more rarely occur in other HR genes. Liquid biopsy is enriched for detection of polyclonal resistance. Diverse REV mechanisms highlight a need for robust detection to incorporate REV in identifying treatment resistance and guiding downstream therapy selection.