Mutations In RAD50 Research Articles

RAD50 Deficiency and Its Effects on Zebrafish Embryonic Development and DNA Repair Mechanisms

The MRE11-RAD50-NBS1 (MRN) complex is essential in detecting, signaling, and repairing DNA double-strand breaks (DSBs), thus maintaining genomic integrity. Mutations in RAD50 are linked to severe conditions such as microcephaly, mental retardation, and growth retardation in humans. This study investigates the developmental impact of RAD50 protein disruption in zebrafish embryos. Zebrafish embryos were treated with MIRIN (35 µM) to inhibit RAD50 and subsequently exposed to gamma-ray irradiation (15 Gy) to analyze the role of RAD50 in managing DNA damage during embryogenesis. Time-point analysis indicated that inhibiting RAD50 and ATM proteins during early embryonic stages (at 1 hpf) leads to increased embryonic mortality and abnormalities. These adverse effects were exacerbated by irradiation, underscoring the critical role of RAD50 in DNA DSB repair. The study concludes that RAD50 deficiencies can lead to embryonic lethality and human deformities due to the inability of tissues to repair DNA DSBs effectively.

Sae2 controls Mre11 endo- and exonuclease activities by different mechanisms

DNA double-strand breaks (DSBs) must be repaired to ensure cell survival and genomic integrity. In yeast, the Mre11-Rad50-Xrs2 complex (MRX) collaborates with Sae2 to initiate DSB repair. Sae2 stimulates two MRX nuclease activities, endonuclease and 3’−5’ exonuclease. However, how Sae2 controls the two nuclease activities remains enigmatic. Using a combined genetic and biochemical approach, we identified a separation-of-function rad50 mutation, rad50-C47, that causes a defect in Sae2-dependent MRX 3’−5’ exonuclease activity, but not endonuclease activity. We found that both the endo- and 3’−5’ exonuclease activities are essential to release Spo11 from DNA ends, whereas only the endonuclease activity is required for hairpin removal. We also uncovered that MRX-Sae2 endonuclease introduces a cleavage at defined distances from the Spo11-blocked end with gradually decreasing efficiency. Our findings demonstrate that Sae2 stimulates the MRX endo- and exonuclease activities via Rad50 by different mechanisms, ensuring diverse actions of MRX-Sae2 nuclease at DNA ends.

The RAD51 S181P mutation shortens lifespan of female mice

RAD51 is critical to the homologous recombination (HR) pathway that repairs DNA double strand breaks (DSBs) and protects replication forks (RFs). Previously, we showed that the S181P (SP) mutation in RAD51 causes defective RF maintenance but is proficient for DSB repair. Here we report that SP/SP female mice exhibit a shortened lifespan compared to +/+ females but not males. Histological analysis found that most mice in this study died from lymphoma, independent of genotype and sex. We propose that a potential cause for shortened lifespan in SP/SP females is due to the RF defect.

Expression patterns of mismatch repair proteins in cervical cancer uncover independent prognostic value of MSH-2

ObjectiveAlthough early-detected cervical cancer is associated with good survival, the prognosis for late-stage disease is poor and treatment options are sparse. Mismatch repair deficiency (MMR-D) has surfaced as a predictor...

A Rad50-null mutation in mouse germ cells causes reduced DSB formation, abnormal DSB end resection and complete loss of germ cells.

The conserved MRE11-RAD50-NBS1/Xrs2 complex is crucial for DNA break metabolism and genome maintenance. Although hypomorphic Rad50 mutation mice showed normal meiosis, both null and hypomorphic rad50 mutation yeast displayed impaired meiosis recombination. However, the in vivo function of Rad50 in mammalian germ cells, particularly its in vivo role in the resection of meiotic double strand break (DSB) ends at the molecular level remains elusive. Here, we have established germ cell-specific Rad50 knockout mouse models to determine the role of Rad50 in mitosis and meiosis of mammalian germ cells. We find that Rad50-deficient spermatocytes exhibit defective meiotic recombination and abnormal synapsis. Mechanistically, using END-seq, we demonstrate reduced DSB formation and abnormal DSB end resection occurs in mutant spermatocytes. We further identify that deletion of Rad50 in gonocytes leads to complete loss of spermatogonial stem cells due to genotoxic stress. Taken together, our results reveal the essential role of Rad50 in mammalian germ cell meiosis and mitosis, and provide in vivo views of RAD50 function in meiotic DSB formation and end resection at the molecular level.

Abstract 946: Landscape of HRD aberration in EGFR mutated lung cancer and the role of PARP-inhibitor in EGFR mutated lung cancer with HRD aberration

Abstract Introduction: Olaparib, an oral poly polymerase (PARP) inhibitor has received approval for treating malignancies displaying homologous recombination deficiency (HRD). Current data is insufficient in establishing the role of PARP inhibitors in non-small cell lung cancer (NSCLC) in the setting of third generation EGFR tyrosine kinase inhibitor (TKI) use. Preclinical investigation suggests heightened PARP inhibitor sensitivity of TKI-resistant EGFR mutant NSCLC cells compared to TKI-sensitive cells. Methods: We utilized The Cancer Genome Atlas (TCGA) to investigate the prevalence of HRD gene mutations in patients with NSCLC harboring EGFR mutations. We also present a case demonstrating a favorable response to the dual therapy of olaparib and osimertinib in NSCLC harboring EGFR, RAD50, and ARID1A mutations that progressed on osimertinib. Results: There were 2298 patients in the NSCLC cohort (MSKCC database). Among 687 patients with EGFR mutations, 24 patients (3.5%) exhibited ARID1A mutations, 8 patients (1.2%) had BRCA1 mutations, 11 patients (1.6%) had BRCA2 mutations, and 5 (0.7%) had RAD50 mutations. A 71-year-old female presented with stage IV adenocarcinoma of the lung. She had recurrent malignant pericardial effusion requiring pericardiocentesis for previous cardiac tamponade. Computed tomography (CT) of the chest revealed a 3cm irregular left hilar mass with bilateral mediastinal and hilar lymph nodes. Tissue next generation sequencing (NGS) at baseline identified EGFR exon19 deletion along with RAD50 R726H which is a HRD pathway gene. Circulating tumor DNA (ct-DNA) NGS (Guardant360) showed EGFR E746_A750del 4.8%. The patient achieved partial response to osimertinib for 28 months. Follow-up CT showed disease progression. Olaparib 200mg twice daily was added to osimertinib based on repeat ct-DNA NGS indicating a new ARID1A alteration (splice site SNV) along with the known BRCA1 mutation. Follow up scans currently have consistently demonstrated ongoing partial response for 19 months. Combining olaparib with osimertinib can be effective in an osimertinib-resistant NSCLC patient with EGFR mutation and HRD aberration. The favorable response could be attributed not only to pre-existing RAD50 mutation but also to acquired mutations in ARID1A HRD genes. Conclusion: HRD aberrations are uncommon in EGFR mutated lung cancer patients. Further investigation on the role of PARP inhibitor in EGFR mutated lung cancer is warranted. Citation Format: Jinah Kim, Horyun Choi, Liam Il-Young Chung, Young Kwang Chae. Landscape of HRD aberration in EGFR mutated lung cancer and the role of PARP-inhibitor in EGFR mutated lung cancer with HRD aberration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 946.

Abstract 5603: Defining the functions of the BRCA2 BRC repeats in modulating RAD51 binding and activity

Abstract The BRCA2 (Breast Cancer Susceptibility 2) gene is critical for preserving genome integrity by regulating homology-directed repair (HDR) of DNA double-strand breaks (DSBs). Germline mutations in BRCA2 predispose individuals to a high risk for ovarian, breast, prostate, and pancreatic cancer. BRCA2 contains eight BRC repeats that mediate binding to RAD51. It remains unclear how exactly the different BRC repeats regulate RAD51 functions. In our study, we explore the importance of each BRC repeat, their interconnections, and their impact on RAD51 binding and filament stability. We engineered amino acid substitutions into the BRC FxTAS motif required for specific contacts within a hydrophobic binding pocket of RAD51 to disrupt BRC binding to RAD51. We further created mutations in RAD51 (F86E/A89E) that either prevent self-association but retain binding to the BRC repeats or a mutation (K133R) that results in a hyper-stable RAD51 nucleoprotein filament. Using both cell-based models and biochemical analyses, we have begun studies to parse out the specific functions of each BRC repeat. Our ultimate goal is to incorporate single amino acid changes into each BRC repeat within the context of the full-length BRCA2 protein to comprehensively characterize the contribution of each BRC repeat to HDR and response to chemotherapeutics such as PARP inhibitors. Citation Format: Julia R. Jensen, Ryan B. Jensen. Defining the functions of the BRCA2 BRC repeats in modulating RAD51 binding and activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5603.

Survival impact of homologous recombination deficiency in veterans with cholangiocarcinoma including mutual exclusivity with pathogenic KRAS and TP53.

546 Background: Cholangiocarcinoma (CCA) has poor prognosis and limited treatment options. The US Veteran Health Administration’s (VHA) National Precision Oncology Program (NPOP) was established to characterize pathogenic drivers across the integrated VA network. Here, we summarize an important subgroup of CCA with homologous recombination deficiency (HRD) and its impact on overall survival (OS). Methods: Next-generation sequencing (NGS) was performed using FoundationOne CDx Analysis included clinicopathological, molecular alterations including, single nucleotide variants, gene amplification/loss, and gene fusion/rearrangements obtained from centralized data of the VHA’s Corporate Data Warehouse. Homologous recombination deficiency was defined by pathogenic variant mutations (MT) in ARID1A, ATM, ATR, BAP1, BRCA1, BRCA2, CDK12, CHEK2, FANC, NBN, PALB2, and RAD51. Statistical analysis was performed using Chi-squared test for concurrent gene enrichment and log-rank Kaplan-Meier analysis. Results: 483 CCA samples underwent tissue NGS as standard of care. Pathogenic variants in HRD genes included ARID1A (15.5%, n=75), BAP1 (9.5%, n=46), ATM (2.7%, n=13), PALB2 (2.3%, n=11) and BRCA (2.0%, n=10), representing 31.7% (n=153) of the population. HRD mutations were found in similar proportions of localized (31.8%) and metastatic (31.5%) clinical presentations at diagnosis. OS was not significantly different between CCA with HRD mutations (12.3 mo WT vs. 13.8 months with HRD MT, p=64). KRAS MT (n=77, 15.9% of population) did not impact OS (WT 12.3 mo v. MT 13.1 mo) including subgroup analysis stratified by HRD status. There was increased frequency in KRAS MT with HRD WT 18.5% versus HRD MT10.4% (p<0.03). Additionally, there was increased frequency in TP53 MT with HRD WT 54.8% versus HRD MT 28.1% (p<0.00001). TP53 MT (n=224, 46.3% of population) conferred inferior OS (WT 12.3 mo v. MT 13.1 mo) including subgroup analysis stratified by HRD status. Subgroup analysis of TP53 MT had worsened OS with concurrent HRD MT (n=39, 4.1 mo) v. HRD WT (n=158, 9.0 mo; p<0.04). Conclusions: Across a diverse integrated healthcare system of Veterans, mutations associated with HRD were found to be common, however, not prognostic in OS across a diverse patient population of CCA. Further work is needed to clarify exposure to platinum containing chemotherapy. TP53 MT CCA remains a critical unmet need that drives worsened outcomes including here in patients with mutations in HRD. Future analysis should consider mutation signatures of HRD, loss in heterozygosity of HRD, and platinum exposure to further define the subgroups of CCA with mutations in HRD. [Table: see text]

Abstract C062: Defining the interplay between PARP and the RB/E2F axis in prostate cancer

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related death in American men. Non-Hispanic Black men are 1.8 times more likely to develop PCa and 2.3 times more likely to die from PCa. Tumor suppressor retinoblastoma (RB1) is frequently deleted in PCa. RB represses the transcriptional activity of E2Fs and the phosphorylation of RB by CDK4/6 results in dissociation of the complex and a subsequent increase in E2F transcriptional activity, leading to cell cycle progression. RB loss is associated with tumor aggressiveness occurring in 10-15% of castration-resistant prostate cancer (CRPC) tumors and corresponds to a decrease in overall survival. RB depletion leads to increased expression of DNA repair proteins due to elevated transcriptional activity of E2F1. PARP-1 is an enzyme that plays a role in DNA repair. Elevated PARP-1 enzymatic activity is associated with PCa progression and correlates with poor clinical outcomes in CRPC. E2F1 transcriptional activity is supported by PARP enzymatic activity, however the consequences of the functional interaction between the RB/E2F axis and PARP remains an open line of inquiry. Defining the interplay between PARP and RB/E2F is likely to contribute to the identification of novel treatment strategies and/or biomarker response. Canonically, E2F1-3 are transcription factors that positively regulate gene expression programs associated with pro-proliferative signals. Prior research by other groups has shown that E2F target genes expression is elevated in tumors excised from Black men compared to Asian and White men. There was an increase in germline RAD51 mutations observed. RAD51 is an E2F target gene involved in DDR, therefore attenuating PARP enzymatic activity may be a potential mode of targeting this axis. These data suggest that there may be differential DNA damage response (DDR) in African American men driven by aberrations in the RB/E2F signaling axis, however the mechanism is yet to be fully explored. Analysis of publicly available patient datasets were performed and demonstrate that RB1 deletions is more frequent than RB1/BRCA2 co-deletion in metastatic PCa. Isogenic models of RB1 knockdown were used to evaluate the impact of RB loss on PARP enzymatic activity. PARP-1 mRNA and PARP-1 protein are elevated upon RB1 depletion with a concomitant increase in PARP activity. Growth curve assays were conducted to examine the biological response to PARP inhibition in the isogenic models. In hormone-depleted conditions, results suggest a differential response between RB depleted models compared to the control when challenged with PARP inhibition. Furthermore, an IC50 dose of Olaparib elicits differential PARP enzymatic response in the RB1 depleted model. RB1 loss is associated with an enrichment in E2F1 binding at the PARP-1 locus. Future studies have been designed to determine the impact of the RB/E2F pathway alterations on downstream consequences PARP enzymatic function and establish the contribution of PARP and the RB/E2F axis on racial disparity in PCa. Citation Format: Latese Evans. Defining the interplay between PARP and the RB/E2F axis in prostate cancer [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C062.

Fanconi anemia-associated mutation in RAD51 compromises the coordinated action of DNA-binding and ATPase activities

Fanconi anemia (FA) is a rare genetic disease caused by a defect in DNA repair pathway for DNA interstrand crosslinks. These crosslinks can potentially impede the progression of the DNA replication fork, consequently leading to DNA double-strand breaks. Heterozygous RAD51-Q242R mutation has been reported to cause FA-like symptoms. However, the molecular defect of RAD51 underlying the disease is largely unknown. In this study, we conducted a biochemical analysis of RAD51-Q242R protein, revealing notable deficiencies in its DNA-dependent ATPase activity and its ATP-dependent regulation of DNA-binding activity. Interestingly, although RAD51-Q242R exhibited the filament instability and lacked the ability to form D-loops, it efficiently stimulated the formation of D-loops mediated by wild-type RAD51. These findings facilitate understanding of the biochemical properties of the mutant protein and how RAD51 works in the FA patient cells.

Genomic analysis of plasma circulating tumor DNA in patients with heavily pretreated HER2 + metastatic breast cancer

We explored accumulated genomic alterations in patients with heavily treated HER2 + metastatic breast cancer enrolled in the KCSG BR18-14/KM10B trial. Targeted sequencing was performed with circulating tumor DNAs (ctDNAs) collected before the treatment of 92 patients. ctDNAs collected at the time of disease progression from seven patients who had a durable response for > 12 months were also analyzed. Sixty-five genes were identified as pathogenic alterations in 99 samples. The most frequently altered genes were TP53 (n = 48), PIKCA (n = 21) and ERBB3 (n = 19). TP53 and PIK3CA mutations were significantly related with shorter progression free survival (PFS), and patients with a higher ctDNA fraction showed a worse PFS. The frequency of homologous recombination deficiency (HRD)-related gene mutations was higher than that in matched tumor tissues, and these mutations tended to be associated with shorter PFS. New pathogenic variants were found at the end of treatment in all seven patients, including BRCA2, VHL, RAD50, RB1, BRIP1, ATM, FANCA, and PIK3CA mutations. In conclusion, TP53 and PIK3CA mutations, as well as a higher ctDNA fraction, were associated with worse PFS with trastuzumab and cytotoxic chemotherapy. The enrichment of HRD-related gene mutations and newly detected variants in ctDNA may be related to resistance to treatment.

Identification of New Potential Prognostic and Predictive Markers in High-Grade Osteosarcoma Using Whole Exome Sequencing.

Conventional high-grade osteosarcoma (OS) is the most common primary cancer of bone and it typically affects the extremities of adolescents. OS has a complex karyotype, and molecular mechanisms related to carcinogenesis, progression and resistance to therapy are still largely unknown. For this reason, the current standard of care is associated with considerable adverse effects. In this study, our aim was to identify gene alterations in OS patients using whole exome sequencing (WES) to find new potential prognostic biomarkers and therapeutic targets. We performed WES on formalin-fixed paraffin-embedded (FFPE) biopsy materials collected from 19 patients affected by conventional high-grade OS. The clinical and genetic data were analyzed according to response to therapy, presence of metastasis and disease status. By comparing good and poor responders to neoadjuvant therapy, we detected a clear prevalence of mutations in the ARID1A, CREBBP, BRCA2 and RAD50 genes in poor responders that negatively influence the progression-free survival time. Moreover, higher tumor mutational burden values correlated with worse prognosis. The identification of mutations in ARID1A, CREBBP, BRCA2 and RAD50 may support the use of a more specific therapy for tumors harboring these alterations. In particular, BRCA2 and RAD50 are involved in homologous recombination repair, and could thus be used as specific therapy targets of inhibitors of the enzyme Poly ADP Ribose Polymerase (PARP). Finally, tumor mutational burden is found to be a potential prognostic marker for OS.

Abstract B054: Inherited mutations of DNA damage repair genes in a racially diverse cohort of men with prostate cancer

Abstract Introduction: DNA damage repair genes (DDRGs) play an important role in genomic stability and mutations in DDRGs contribute to clinically significant prostate cancer (PCa). However, the association of germline mutations in DDRGs with PCa progression and therapeutic stratification remain to be defined in African American (AA) men. Our objective was to genomically profile all known DDRGs and provide ancestry specific findings, focusing on PCa patients with African ancestry. Methods: Germline mutations in all DDRGs (N=276) was evaluated by whole genome sequence (WGS) analysis of archived blood DNA samples from 600 PCa patients (300 AA and 300 CA) who underwent primary treatment at Walter Reed National Military Medical Center. The WGS mean coverage exceeded 37x. Principal Component Analysis using the Peddy program was conducted, and ancestry prediction was based on a machine learning approach using 1000G as reference. Variant frequencies in CPDR PCa patients were compared to variant frequencies available from the Exome Aggregation Consortium (ExAC) controls with no PCa. Data analysis approaches included single variant association, gene-based total frequency test and clinico-pathological associations for germline mutations. All statistical tests were two-sided, and FDR adjusted. FDR < 0.05 was set as significance threshold. Results: Comprehensive analysis of 276 DDRGs revealed that germline mutation (Pathogenic/Likely Pathogenic; P/LP) carrier rate was 23.5%, which is three times higher than reported before in both AA and CA men, due to our unbiased WGS approach. We found that only 13 of the 46 DDRGs identified with P/LP mutations were common to AA and CA men, demonstrating clear disparity along ancestry lines. Importantly, RAD family genes (RAD54L, RAD54B, RAD51), which are potentially targetable, as well as PMS2 and BRCA1, were among the most frequently mutated DDRGs in AA, but not in CA men. Germline mutations in RAD51 and PMS2 were enriched in AA cohort as compared to CA men (P 0.01 and 0.03 respectively), while germline mutations in FANCA were significantly enriched in CA cohort (P < 0.01). Pathway analysis showed that the HR pathway (with 15 mutations) was associated with faster disease progression to biochemical recurrence (BCR) (P <0.05), while the NHEJ pathway (with three mutations) was found to be marginally associated with metastasis (p <0.05). Additionally, we found that germline mutations in DDRGs was associated with shorter time to BCR (P <0.05) in AA patients, but not in CA men. Conclusion: Our findings highlight distinct DDRG germline mutation profile across AA and CA prostate cancer men. This may help refine patient stratification for specific targeted therapeutic options especially for under-studied AA men. Future replication studies in an independent patient cohort are warranted to validate our findings. Citation Format: Indu Kohaar, Xijun Zhang, Gregory Chesnut, Clifton Dalgard, Matthew D. Wilkerson, Gyorgy Petrovics. Inherited mutations of DNA damage repair genes in a racially diverse cohort of men with prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B054.

A rad50 germline mutation induces tumorigenesis and ataxia-telangiectasia phenotype in a transparent medaka model.

The MRE11A-RAD50-NBS1 complex activates the ataxia-telangiectasia mutated (ATM) pathway and plays a central role in genome homeostasis. The association of RAD50 mutations with disease remains unclear; hence, we adopted a medaka rad50 mutant to demonstrate the significance of RAD50 mutation in pathogenesis using the medaka as an experimental animal. A 2-base pair deletion in the rad50 gene was introduced into transparent STIII medaka using the CRISPR/Cas9 system. The mutant was analyzed histologically for tumorigenicity and hindbrain quality, as well as for swimming behavior, to compare with existing ATM-, MRE11A-, and NBS1-mutation-related pathology. Our results revealed that the medaka rad50 mutation concurrently reproduced tumorigenesis (8 out of 10 rad50Δ2/+ medaka), had a decrease in median survival time (65.7 ± 1.1 weeks in control vs. 54.2 ± 2.6 weeks in rad50Δ2/+ medaka, p = 0.001, Welch's t-test), exhibited semi-lethality in rad50Δ2/Δ2 medaka and most of the major ataxia-telangiectasia phenotypes, including ataxia (rheotaxis ability was lower in rad50Δ2/+ medaka than in the control, Mann-Whitney U test, p < 0.05), and telangiectasia (6 out of 10 rad50Δ2/+ medaka). The fish model may aid in further understanding the tumorigenesis and phenotype of ataxia-telangiectasia-related RAD50 germline mutations and in developing novel therapeutic strategies against RAD50 molecular disorders.

Conventional chondrosarcoma of the rib cage and sternum: clinicopathological and molecular analysis of 27 patients treated at a single institution

Conventional chondrosarcoma of the chest wall is rare, accounting for 15% of cases. Our purpose was to document clinicopathological, imaging and outcome results from a novel set of chest wall chondrosarcomas, and to analyze for IDH mutations and novel molecular alterations. Gross and microscopic pathology, imaging and clinical charts were reviewed. Targeted next-generation sequencing was performed to identify somatic mutations and copy number alterations. The cohort consisted of 27 patients: 16 men and 11 women (mean age 51 years; range 23-76). Palpable mass was the most common presentation. Five were discovered incidentally. Among 20 tumors with complete imaging, 15 arose from a rib and 5 from the sternum. Seven rib tumors were central/intramedullary, 5 were periosteal, 2 were secondary peripheral chondrosarcomas, and one was indeterminate. Among sternal tumors, 4 were central/intramedullary and one was periosteal. Half the periosteal tumors arose from the costochondral junctional cartilage (CCJ). Periosteal chondrosarcomas were sometimes mistaken for extraskeletal masses on initial clinical or radiological examinations. Fifty-nine percent of all tumors were grade 1 and 41% were grade 2. None were dedifferentiated chondrosarcomas. Heterozygous IDH1 mutation was detected in one tumor and heterozygous RAD50 mutation in another. Local recurrence(s) happened in 41% and metastasis in 41%. Grade had strong association with local recurrence (25% grade 1 vs. 64% grade 2 [P=.0447]), metastatic recurrence (19% grade 1 vs. 73% grade 2 [P=.0058]), and survival. Although chest wall chondrosarcomas share morphologic and molecular features with other chondrosarcomas, there is a much higher incidence of periosteal chondrosarcomas. IDH mutant tumors are uncommon. Early diagnosis and margin-negative resection is treatment of choice since chondrosarcomas are chemo- and radioresistant.

Risk stratification and molecular heterogeneity of endometrial cancer and expression profile of TIM-3: A retrospective cohort study

ObjectiveThe present study aimed to implement ProMisE classification and risk grouping on a retrospective cohort of 628 patients with endometrial cancer (EC) and determine the molecular heterogeneity across subtypes and subgroups, as well as to investigate the potential beneficiary for TIM-3 checkpoint inhibition in ECs. MethodsProtein expressions of p53, MMR, TIM-3 and CD8 were measured by immunohistochemistry, and massively parallel sequencing was conducted for 128 cancer-related genes. Patients were categorized into four ProMisE subtypes: MMR-deficient (MMRd), POLE-ultramutated (POLEmut), p53-wild type (p53wt), and p53-abnormal (p53abn), and were subjected to risk classification. Results43 (6.9%) patients belonged to POLEmut, 118 (18.8%) to MMRd, 69 (11%) to p53abn, and 398 (63.3%) to p53wt. Compared to the 2016 stratification system, the 2021 ESGO/ESTRO/ESP risk stratification integrated with molecular classification revealed that 11 patients (11/628, 1.8%) were upgraded due to the p53abn signature, whereas 23 patients (23/628, 3.7%) were downgraded due to the POLEmut signature. JAK1 and RAD50 mutations showed higher frequencies in patients with aggressive phenotypes. RAD51B mutation was significantly related to poor RFS of the p53wt subtype but not of the other three molecular subgroups. TIM-3 expression was detected in 30.9% immune cells (ICs) and 29.0% tumor cells (TCs) in ECs, respectively. It was frequently expressed in POLEmut and MMRd ECs as compared to that in the other two molecular subtypes in TCs and ICs. ConclusionsOur study revealed the molecular heterogeneity across subtypes and subgroups. The new risk stratification system changed the risk grouping of some patients due to the integration of molecular features. RAD51B mutation can further stratify the recurrence risk in the p53wt subtype. Patients with MMRd or POLEmut may benefit most from immunotherapy against TIM-3.

The Largest Chinese Cohort Study Indicates Homologous Recombination Pathway Gene Mutations as Another Major Genetic Risk Factor for Colorectal Cancer with Heterogeneous Clinical Phenotypes.

While genetic factors were associated with over 30% of colorectal cancer (CRC) patients, mutations in CRC-susceptibility genes were identified in only 5% to 10% of these patients. Besides, previous studies on hereditary CRC were largely designed to analyze germline mutations in patients with single genetic high-risk factor, which limited understanding of the association between genotype and phenotypes. From January 2015 to December 2018, we retrospectively enrolled 2,181 patients from 8,270 consecutive CRC cases, covering 5 categories of genetic high-risk factors. Leukocyte genomic DNA was analyzed for germline mutations in cancer predisposition genes. The germline mutations under each category were detected and analyzed in association with CRC susceptibility, clinical phenotypes, and prognoses. A total of 462 pathogenic variants were detected in 19.3% of enrolled CRC patients. Mismatch repair gene mutation was identified in 9.1% of patients, most prevalent across all high-risk groups. Homologous recombination (HR) gene mutations were detected in 6.5% of cases, penetrated in early-onset and extra-colonic cancer risk groups. Mutations in HR genes, including BARD1, RAD50, and ATM, were found to increase CRC risk with odds ratios of 2.8-, 3.1-, and 3.1-fold, respectively. CRC patients with distinct germline mutations manifested heterogeneous phenotypes in clinicopathology and long-term prognoses. Thus, germline mutation screenings should be performed for CRC patients with any of those genetic risk factors. This study also reveals that HR gene mutations may be another major driver for increased CRC risk.

Pan-cancer analysis of co-occurring mutations in RAD52 and the BRCA1-BRCA2-PALB2 axis in human cancers.

In human cells homologous recombination (HR) is critical for repair of DNA double strand breaks (DSBs) and rescue of stalled or collapsed replication forks. HR is facilitated by RAD51 which is loaded onto DNA by either BRCA2-BRCA1-PALB2 or RAD52. In human culture cells, double-knockdowns of RAD52 and genes in the BRCA1-BRCA2-PALB2 axis are lethal. Mutations in BRCA2, BRCA1 or PALB2 significantly impairs error free HR as RAD51 loading relies on RAD52 which is not as proficient as BRCA2-BRCA1-PALB2. RAD52 also facilitates Single Strand Annealing (SSA) that produces intra-chromosomal deletions. Some RAD52 mutations that affect the SSA function or decrease RAD52 association with DNA can suppress certain BRCA2 associated phenotypes in breast cancers. In this report we did a pan-cancer analysis using data reported on the Catalogue of Somatic Mutations in Cancers (COSMIC) to identify double mutants between RAD52 and BRCA1, BRCA2 or PALB2 that occur in cancer cells. We find that co-occurring mutations are likely in certain cancer tissues but not others. However, all mutations occur in a heterozygous state. Further, using computational and machine learning tools we identified only a handful of pathogenic or driver mutations predicted to significantly affect the function of the proteins. This supports previous findings that co-inactivation of RAD52 with any members of the BRCA2-BRCA1-PALB2 axis is lethal. Molecular modeling also revealed that pathogenic RAD52 mutations co-occurring with mutations in BRCA2-BRCA1-PALB2 axis are either expected to attenuate its SSA function or its interaction with DNA. This study extends previous breast cancer findings to other cancer types and shows that co-occurring mutations likely destabilize HR by similar mechanisms as in breast cancers.

Prognostic Impact of TP53 Mutations and Tumor Mutational Load in Colorectal Cancer

The DNA damage response (DDR) is critical for maintaining genome stability, and abnormal DDR—resulting from mutations in DNA damage-sensing and repair proteins—is a hallmark of cancer. Here, we aimed to investigate the predictive power of DDR gene mutations and the tumor mutational load (TML) for survival outcomes in a cohort of 22 rectal cancer patients who received pre-operative neoadjuvant therapy. Univariate analysis revealed that TML-high and TP53 mutations were significantly associated with worse overall survival (OS) with TML-high retaining significance in multivariate analyses. Kaplan–Meier survival analyses further showed TML-high was associated with worse disease-free (p = 0.036) and OS (p = 0.024) results in our patient cohort. A total of 53 somatic mutations were identified in 22 samples with eight (36%) containing mutations in DDR genes, including ATM, ATR, CHEK2, MRE11A, RAD50, NBN, ERCC2 and TP53. TP53 was the most frequently mutated gene, and TP53 mutations were significantly associated with worse OS (p = 0.023) in Kaplan–Meier survival analyses. Thus, our data indicate that TML and TP53 mutations have prognostic value for rectal cancer patients and may be important independent biomarkers for patient management. This suggests that prognostic determination for rectal cancer patients receiving pre-operative neoadjuvant therapy should include consideration of the initial TML and tumor genetic status.

Abstract 5766: Pan-cancer analysis revealed characteristics of reversion mutations in homologous recombination repair genes

Abstract Background: Reversion mutations of BRCA1/2 are associated with decreased response to platinum-based chemotherapies and PARP inhibitors. While patient with homologous recombination repair (HRR) genes have proven efficacy to PARP inhibitors, reversion mutations in these genes have not been widely studied. Here, we performed pan-cancer analysis to reveal the characteristics of HRR reversion mutations. Methods: We assessed 1,992 patients with at least one loss of function mutation in BRCA1, BRCA2, ATM, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCL, PALB2, RAD51B, RAD51C, RAD51D, RAD54L, ERCC3, RAD51, WRN, FANCI, MRE11A, ATR, FANCD2, FANCA, FANCC, BLM and NBN. Somatic reversion mutations were indel or missense mutations that restore the open reading frame and potentially protein function fully or partially. Definitive reversion mutations are confirmed secondary mutations in patients with known pathogenic germline HRR gene mutations, or in a subsequent sample of previously detected somatic pathogenic mutants. Other reversion mutations are putative due to lack of matched samples. Results: Overall, we found 31/1,992 (1.6%) tumors with definitive or putative HRR reversion mutations, including 17 with germline and 14 with somatic predisposed pathogenic HRR mutations. Of these, we identified 9 BRCA1 (29%, 2 sBRCA1 and 7 gBRCA1) and 12 BRCA2 (39%, 4 sBRCA2 and 8 gBRCA2). We also found pathogenic germline PALB2 (1, 3%) and RAD41D (1, 3%), and somatic mutations in CDK12 (3, 9%), ATM (2, 6%), WRN (1, 3%), FANCD2 (1, 3%), and ATR (1, 3%). Overall, 43 reversion mutations were detected (37 definitive and 6 putative), with eight patients carrying multiple reversion mutations. Importantly, 40/43 reversion mutations were found within 2bp upstream to the start site of original deleterious variants, and all were unique to currently known database. Missense-induced reversions were found in 74% primary stop-gain mutations, while indel-induced reversions were associated with 59% primary frameshift mutations. These HRR reversion mutations were primarily found in breast cancer (9%, 10/107) and ovarian cancer (6%, 3/54), and also in cases of prostate (1/25, 4%), pancreatic (1/29, 3%), colorectal (2%, 9/418), and non-small cell lung cancer (1%, 6/703). Of note, the incidence of multiple HRR reversion mutations was higher in breast cancer (75%, 6/8). Lastly, reversion mutations were detectable in plasma, pleural and ascites fluid samples, suggesting great clinical utility of ctDNA to monitor reversion mutations. Conclusions: This study analyzed clinical and mutational characteristics of reversion mutations in HRR genes, which could occur as single or multiple variants to restore the function of predisposed pathogenic HRR mutations, resulting in resistance to platinum-based chemotherapies or PARP inhibitors. Monitoring these mutations with tissue or liquid biopsy samples are crucial for treatment guidance. Citation Format: Jing Li, Xiaoying Wu, Yan Shi, Di Wang, Yedan Chen, Yang Shao. Pan-cancer analysis revealed characteristics of reversion mutations in homologous recombination repair genes [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 5766.