Mutations In DNA Repair Genes Research Articles

Abstract B029: Repurposing Digital PCR to Diagnose and Create a Novel Genomic Signature for Homologous Recombination Deficiency in Pancreatic Adenocarcinoma

Abstract Introduction: Germline mutations in DNA repair genes are often associated with increased risk of cancer. Mutations that affect the homologous recombination (HR) DNA repair pathway ( e.g., BRCA 1 and BRCA2) are known drivers of pancreatic adenocarcinoma. Early detection relies on identification of pathogenic mutations in patients with a family history of cancer. Next generation sequencing panels often use a select set of pathogenic variants for diagnosis, meaning that individuals with variants of uncertain significance may be missed, limiting testing of other family members for the potentially disease-causing variant. Here we describe a digital PCR assay developed to detect BRCA1 and BRCA2 variant that may eventually serve as a rapid, cost-effective alternative to sequencing that costs less than $5.00 per sample with results in 24 hours. This study highlights the need for better diagnostic tools, which will become critically important as we begin to study pathogenic mutations in more diverse populations. Methods: We have collected blood from 4 BRCA1 mutants and 5 BRCA2 mutants who have already undergone germline sequencing from commercially available assays. Clinicopathologic data has been collected as well. We designed two fluorescently labelled TaqMan probes, one that recognizes the wild-type BRCA alleles and one specific for mutant BRCA alleles. Briefly, 1 ng of genomic DNA is mixed with amplicon primers and fluorescent Taq-Man probes, then partitioned into 20,000 separate 517 picoliter wells. Quantification and detection are achieved based upon the fluorescent signal measured from each well. Results: We have successfully confirmed pathologic BRCA1 and BRCA2 mutations previously identified with next generation sequencing in 100% of samples analyzed. All three BRCA2 mutations generate a premature stop codon leading to a truncated protein and are known to be pathogenic. Two related patients have the same BRCA1 frameshift mutation that results in a premature stop codon, and one patient has a duplicated cytosine that leads to a frameshift mutation that is one of three characterized Ashkenazi Jewish founder mutations that increases cancer risk in this population . Conclusions: Here we report an innovative assay using digital PCR (dPCR) that provides a cost-effective, time-efficient method to identify MMR mutants and characterize VUS in patients and their families. Citation Format: Jennifer Brooke Valerin, Sophie Hasson, Valeria Rangel, Nicholas Pannunzio. Repurposing Digital PCR to Diagnose and Create a Novel Genomic Signature for Homologous Recombination Deficiency in Pancreatic Adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B029.

Abstract PR-04: Phase 2 Trial Testing The PARP Inhibitor Niraparib In Patients With Advanced Pancreatic Cancer with Pathogenic Variants In BRCA 1, BRCA2, PALB2, ATM and CHEK2

Abstract Background: Poly-(adenosine diphosphate ribose) polymerase (PARP) inhibitors have demonstrated efficacy in patients with platinum-sensitive pancreatic cancer who harbor germline BRCA 1/2 pathogenic variants (PVs). Whether PARP inhibitors are effective in a broader population of pancreatic cancer patients is still under investigation. Methods: This was a multicenter, single arm, open-label phase II trial (NCT03601923) investigating the use of the PARP inhibitor niraparib in patients with advanced pancreatic cancer. Eligible patients were required to have germline or somatic mutations in BRCA1, BRCA2, PALB2, ATM and/or CHEK2. Prior treatment with PARP inhibitors and prior progression on a platinum chemotherapy were not allowed, though patients could be included if they had progression on non-platinum based therapy. Patients were treated with weight-adjusted niraparib (200 mg or 300 mg) once daily on a 28-day cycle. The primary endpoint was 6-month progression free survival (PFS) rate (binary). Secondary endpoints were PFS, overall survival (OS), objective response rate (ORR) by RECIST v1.1, and safety. Serial plasma samples were collected for circulating tumor DNA analyses. Results: Thirty-two patients (10 females [31%]; median age 67) were evaluable for the primary endpoint. Patients had a germline or somatic alteration in one or more of the following genes: ATM (n=14), BRCA2 (n=10), PALB2 (n=3), CHEK2 (n=4), or BRCA1 (n=2). There were 10 patients who received niraparib as maintenance therapy and 12 patients whose tumors previously progressed on a non-platinum-based chemotherapy. The 6-month PFS rate was 25% (90% CI: 13-41), rejecting the null hypothesis of a 17% 6-month PFS rate. The median PFS for the entire population was 2.0 months (90% CI: 1.7-3.7) and median OS was 10.2 months (90% CI: 8.2-13.2). The objective response rate was 14% (90% CI: 5-30%); 2 patients had a complete response, 2 patients had a partial response. Patients who never previously progressed on chemotherapy had a significantly longer median PFS (p=0.02) and OS (p=0.003) than patients who previously progressed on chemotherapy. Three patients with biallelic inactivation of ATM, who never progressed on prior lines of chemotherapy, were on niraparib for over 1 year (range 1 – 4 years). Reversion mutations restoring the reading frame in BRCA2 were identified in cell-free DNA in 3 patients with BRCA2 pathogenic frameshift variants. Additional putative resistance mutations in other DNA repair genes were identified and will be reported. Conclusions: Niraparib demonstrated a promising 6-month PFS rate and provided clinically meaningful benefit in patients with pancreatic cancer. The observation of prolonged benefit of three patients with biallelic ATM inactivation warrants further investigation. Acknowledgment: Funding for this study was provided by GSK. GSK was provided the opportunity to review a preliminary version of this abstract for factual accuracy. The authors are solely responsible for final content and interpretation. Citation Format: Brandon M. Huffman, Jett Crowdis, Matthew B. Yurgelun, Nora Horick, Yvonne Li, Huy Nguyen, Kalindi Parmar, Bose S. Kochupurakkal, Andrea Enzinger, Marios Giannakis, Nadine McCleary, Kimmie Ng, Anuj K. Patel, Kimberly J. Perez, Douglas A. Rubinson, Benjamin L. Schlechter, Harshabad Singh, Helen Lam, Kaitlyn Ramsey, Elizabeth Andrews, Leigh Culnane, Andrea Bullock, Ahmed Rattani, Jeffrey Clark, Mary Linton Peters, Brian M. Wolpin, Alan D. D'Andrea, Geoffrey I. Shapiro, Andrew J. Aguirre, James M. Cleary. Phase 2 Trial Testing The PARP Inhibitor Niraparib In Patients With Advanced Pancreatic Cancer with Pathogenic Variants In BRCA 1, BRCA2, PALB2, ATM and CHEK2 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-04.

Molecular differentiation between complete and incomplete responders to neoadjuvant therapy in rectal cancer.

Neoadjuvant chemoradiotherapy (nCRT) is the standard treatment for locally advanced rectal cancer, but only 20-40% of patients completely respond to this treatment. To define the molecular features that are associated with response to nCRT, we generated and collected genomic and transcriptomic data from 712 cancers prior to treatment from our own data and from publicly available data. We found that patients with a complete response have decreased risk of both local recurrence and future metastasis. We identified multiple differences in DNA mutations and transcripts between complete and incomplete responders. Complete responder tumors have a higher tumor mutation burden and more significant co-occurring mutations than the incomplete responder tumors. In addition, mutations in DNA repair genes (across multiple mechanisms of repair) were enriched in complete responders and they also had lower expression of these genes indicating that defective DNA repair is associated with complete response to nCRT. Using logistic regression, we identified three significant predictors of complete response: tumor size, mutations within specific network genes, and the existence of three or more specific co-occurrent mutations. In incompletely responder tumors, abnormal cell-cell interaction and increased cancer associated fibroblasts were associated with recurrence. Additionally, gene expression analysis identified a subset of immune hot tumors with worse outcomes and upregulated of immune checkpoint proteins. Overall, our study provides a comprehensive understanding of the molecular features associated with response to nCRT and the molecular differences in non-responder tumors that later reoccur. This knowledge may provide critical insight for the development of precision therapy for rectal cancer.

Endometrioid ovarian carcinoma landscape: pathological and molecular characterization.

Endometrioid ovarian cancers (EOvC) are usually managed as serous tumors. In this study, we conducted a comprehensive molecular investigation to uncover the distinct biological characteristics of EOvC. This retrospective multicenter study involved patients from three European centers. We collected clinical data and formalin-fixed paraffin-embedded (FFPE) samples for analysis at the DNA level using panel-based next-generation sequencing and array-comparative genomic hybridization. Additionally, we examined mRNA expression using NanoString nCounter® and protein expression through tissue microarray. We compared EOvC with other ovarian subtypes and uterine endometrioid tumors. Furthermore, we assessed the impact of molecular alterations on patient outcomes, including progression-free survival (PFS) and overall survival (OS). Preliminary analysis of clinical data from 668 patients, including 86 (12.9%) EOvC, revealed more favorable prognosis for EOvC compared with serous ovarian carcinoma (5-year OS of 60% versus 45%; P = 0.001) driven by diagnosis at an earlier stage. Immunohistochemistry and copy number alteration (CNA) profiles of 43 cases with clinical data and FFPE samples available indicated that EOvC protein expression and CNA profiles were more similar to endometrioid endometrial tumors than to serous ovarian carcinomas. EOvC exhibited specific alterations, such as lower rates of PTEN loss, mutations in DNA repair genes, and P53 abnormalities. Survival analysis showed that patients with tumors harboring loss of PTEN expression had worse outcomes (median PFS 19.6 months vs. not reached; P = 0.034). Gene expression profile analysis confirmed that EOvC differed from serous tumors. However, comparison to other rare subtypes of ovarian cancer suggested that the EOvC transcriptomic profile was close to that of ovarian clear cell carcinoma. Downregulation of genes involved in the PI3K pathway and DNA methylation was observed in EOvC. In conclusion, EOvC represents a distinct biological entity and should be regarded as such in the development of specific clinical approaches.

Effect of ctDNA whole-exome sequencing in pancreatic adenocarcinoma on prognostic value, actionable alterations, and organ tropisms in metastatic disease.

4149 Background: The dismal prognosis associated with pancreatic ductal adenocarcinoma (PDAC) needs a deeper understanding of progression mechanisms and personalized treatment. While the prognostic value of ctDNA has already been established, this study purposes to apply plasma whole-exome sequencing (WES) to identify molecular alterations beyond KRAS. This approach aims to predict survival, uncover progression mechanisms, and identify therapeutic targets. Methods: Eighty metastatic PDAC patients were prospectively recruited and divided into two consecutive cohorts: discovery (n=37) and validation (n=43). Tumor and plasma obtained at diagnosis were analyzed using WES. The variant allele frequency (VAF) of KRASmutations was quantified by ddPCR in plasma at baseline and at response from all patients (n=80). Results: Plasma WES identified at least one pathogenic variant across all individuals, which can be categorized into four pathways: oncogenic mechanisms, DNA repair, microsatellite instability and TGFb pathway alterations. 7 unique druggable mutations were detected in tissue and 11 in plasma. Other targetable mutations ( CDK12, NF1, BRCA2, and TSC2), were prevalent in plasma but absent in tissue. Patients with survival less than 11 months showed enrichment in cellular organization pathways and exclusive targetable CDK12 mutations. Longer survival cases exhibited exclusive mutations in DNA regulation and repair genes. Patients with liver metastasis displayed unique mutations not just in KRAS but also in the adaptive immune response pathway genes. Baseline plasma KRAS mutations detected by ddPCR was associated with worse progression-free survival. (HR=2.315, CI 95%= 1.027-5.217, p = 0.038; and HR= 3.022, CI 95% = 1.299 - 7.030, p= 0.007 in the discovery and validation cohort, respectively). A significant risk of progression (p = 0.006) was observed if KRAS VAF at response assessment did not decrease, at least, 84.75% in both cohorts. Conclusions: ctDNA WES unveils molecular signatures of rapid progression, potential actionable alterations, and liver metastasis-specific mutations in the adaptive immune response pathway. KRAS detection at baseline and its evolution during treatment emerge as prognostic biomarkers. [Table: see text]

A first-in-human (FIH), phase 1/2, dose-escalation, dose-optimization, and dose-expansion study of PARP1-selective inhibitor IMP1734 in participants with advanced solid tumors.

TPS3191 Background: PARP inhibitors (PARPi) selectively kill tumor cells harboring genetic mutations in critical DNA repair genes (e.g., BRCA1/2). While several approved nonselective PARPi have provided robust anti-tumor activity, they are associated with significant hematologic toxicities that limit dose intensity and clinical benefit. Drugs that selectively inhibit PARP1 but spare PARP2 may improve the risk-benefit profile for this class of therapies by retaining anti-tumor activity while avoiding PARP2-related toxicities. IMP1734 (EIK1003) is a potent PARP1-selective inhibitor that inhibits tumor growth in nonclinical models and may widen the therapeutic index in tumors where non-selective PARPi have demonstrated efficacy. Methods: Study EIK1003-001 (IMP1734-101) is a global, multi-center, Phase 1/2 study evaluating the safety and potential antitumor activity of IMP1734 in participants with advanced solid tumors. The study has 3 parts: Part 1 (FIH; Dose Escalation), Part 2 (Dose Optimization), and Part 3 (Dose Expansion at the recommended dose for expansion [RDE]). Part 1 of this study is currently enrolling and consists of dose escalation of IMP1734 in study participants with advanced/recurrent/metastatic ovarian, breast, or prostate cancer with suspected deleterious/deleterious mutations in a pre-specified panel of homologous recombination repair (HRR) genes (n = 70). Eligible participants must be ≥ 18 years of age, have histologically or cytologically confirmed tumors as indicated in each study part, and at least 1 RECIST v1.1-measurable lesion and/or pre-specified serum tumor-specific marker. All participants must have a deleterious or suspected deleterious mutation in select HRR genes. Primary endpoints include safety and tolerability (evaluation of dose-limiting toxicities and determining the maximum tolerated dose, maximum achievable dose, and RDE). Secondary endpoints include evaluation of the pharmacokinetic parameters of IMP1734 and measures of efficacy, including overall response rate, duration of response, progression-free survival, and overall survival. Exploratory endpoints include analysis of pharmacodynamic biomarkers, serial ctDNA measurements, and patient quality-of-life measures. This study opened on 11 Dec 2023. Clinical trial information: NCT06253130 .

Abstract IA003: Molecular subtypes and clinical benefit from neoadjuvant chemotherapy: A ten-year update

Abstract Ten years ago TCGA and two private groups reported that muscle-invasive bladder cancers (MIBCs) could be grouped into basal and luminal molecular subtypes that shared features with the corresponding subtypes of breast cancer. These and earlier studies by the group at Lund University demonstrated that molecular subtype membership was prognostic: patients whose tumors were assigned to basal/squamous subtypes had shorter survival than did patients whose tumors were assigned to the luminal subtypes, and these relationships were confirmed in subsequent studies. In addition, one of the studies included tumors from patients treated with neoadjuvant cisplatin-based combination chemotherapy (NAC), where patients whose tumors were infiltrated with smooth muscle cells and fibroblasts (the MD Anderson p53-like subtype) had the lowest response rates. Parallel comparisons of patients who either did or did not receive NAC concluded that patients with basal tumors derived the most clinical benefit in terms of disease-specific or overall survival. Thus, in addition to being prognostic, there was a suggestion that molecular subtypes might also be used to predict resistance to NAC. This work led to the design of two large collaborative projects that were designed to validate these observations and the conclusion that inactivating mutations in several DNA damage and repair (DDR) genes were also associated with response. One leveraged the S1314 Phase-2 “CoXEN” trial that was designed to prospectively validate another gene expression-based biomarker, and the other exploited pretreatment samples from the CALGB/Alliance 90601 Phase-3 trial of gemcitabine/cisplatin with or without bevacizumab in patients with advanced or metastatic disease. Comparisons of response rates as a function of molecular subtype membership in S1314 failed to validate the original link between the MD Anderson p53-like subtype and resistance, and patients with basal/squamous tumors in 90601 did not have better outcomes than did patients with luminal tumors. Thus, while the prognostic value of molecular subtype membership was confirmed, the predictive value was not. Developing biomarkers to predict response to chemotherapy remains a challenge not only for bladder cancer but also for other solid and hematopoietic malignancies. It seems likely that success will require the integration of measurements using multiple platforms, including liquid biopsies and others. The exciting results of the EV-302 clinical trial of enfortumab vedotin plus pembrolizumab have shifted the focus of biomarker work to these and other novel agents. Citation Format: David J McConkey. Molecular subtypes and clinical benefit from neoadjuvant chemotherapy: A ten-year update [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr IA003.

G-Quadruplex Forming DNA Sequence Context Is Enriched around Points of Somatic Mutations in a Subset of Multiple Myeloma Patients.

Multiple myeloma (MM) is the second most common hematological malignancy, which remains incurable despite recent advances in treatment strategies. Like other forms of cancer, MM is characterized by genomic instability, caused by defects in DNA repair. Along with mutations in DNA repair genes and genotoxic drugs used to treat MM, non-canonical secondary DNA structures (four-stranded G-quadruplex structures) can affect accumulation of somatic mutations and chromosomal abnormalities in the tumor cells of MM patients. Here, we tested the hypothesis that G-quadruplex structures may influence the distribution of somatic mutations in the tumor cells of MM patients. We sequenced exomes of normal and tumor cells of 11 MM patients and analyzed the data for the presence of G4 context around points of somatic mutations. To identify molecular mechanisms that could affect mutational profile of tumors, we also analyzed mutational signatures in tumor cells as well as germline mutations for the presence of specific SNPs in DNA repair genes or in genes regulating G-quadruplex unwinding. In several patients, we found that sites of somatic mutations are frequently located in regions with G4 context. This pattern correlated with specific germline variants found in these patients. We discuss the possible implications of these variants for mutation accumulation and specificity in MM and propose that the extent of G4 context enrichment around somatic mutation sites may be a novel metric characterizing mutational processes in tumors.

Prevalence of DNA-Repair Gene mutations in Mexican men with prostate cancer

Introduction and objectiveMexico reported 26,742 new cases of prostate cancer in 2020. Different risk factors have been identified in the pathogenesis of prostate cancer. Among them, genetic factors and alterations or mutations in specific genes have been described in different ethnic groups worldwide. The aim of our study is to report the prevalence of germline DNA-repair gene mutations in Mexican patients with prostate cancer. Material and methodWe performed germline genetic testing in 50 patients with localized prostate cancer and 50 patients with metastatic prostate cancer. Demographic, clinical, and histopathological data were collected. ResultsThirty-seven germline mutations were identified in 32 patients. The most commonly affected genes were ATM in 6%, followed by FANCA (5%), and ATR (4%). BRCA2 mutations were identified in 3%. The frequency of mutations was higher in the metastatic group. Discussion and conclusionThe results of our study show different mutations from those reported in different populations or regions. The use of PARP inhibitors is indicated in patients with germline mutations, specifically BRCA2, showing improvement in overall survival and progression free survival. To our knowledge, this is the first study reporting the prevalence of mutations in DNA-repair genes in Mexican patients with prostate cancer.

Synergistic Activity of DNA Damage Response Inhibitors in Combination with Radium-223 in Prostate Cancer.

Radium-223 (223Ra) and Lutetium-177-labelled-PSMA-617 (177Lu-PSMA) are currently the only radiopharmaceutical treatments to prolong survival for patients with metastatic-castration-resistant prostate cancer (mCRPC); however, mCRPC remains an aggressive disease. Recent clinical evidence suggests patients with mutations in DNA repair genes associated with homologous recombination have a greater clinical benefit from 223Ra. In this study, we aimed to determine the utility of combining DNA damage response (DDR) inhibitors to increase the therapeutic efficacy of X-rays, or 223Ra. Radiobiological responses were characterised by in vitro assessment of clonogenic survival, repair of double strand breaks, cell cycle distribution, and apoptosis via PARP-1 cleavage. Here, we show that DDR inhibitors increase the therapeutic efficacy of both radiation qualities examined, which is associated with greater levels of residual DNA damage. Co-treatment of ATM or PARP inhibition with 223Ra increased cell cycle arrest in the G2/M phase. In comparison, combined ATR inhibition and radiation qualities caused G2/M checkpoint abrogation. Additionally, greater levels of apoptosis were observed after the combination of DDR inhibitors with 223Ra. This study identified the ATR inhibitor as the most synergistic inhibitor for both radiation qualities, supporting further pre-clinical evaluation of DDR inhibitors in combination with 223Ra for the treatment of prostate cancer.

Prevalencia de mutaciones en genes de reparación en hombres mexicanos con cáncer de próstata

Introduction and objectiveMexico reported 26,742 new cases of prostate cancer in 2020. Different risk factors have been identified in the pathogenesis of prostate cancer. Among them, genetic factors and alterations or mutations in specific genes have been described in different ethnic groups worldwide. The aim of our study is to report the prevalence of germline DNA-repair gene mutations in Mexican patients with prostate cancer. Material and methodWe performed germline genetic testing in 50 patients with localized prostate cancer and 50 patients with metastatic prostate cancer. Demographic, clinical, and histopathological data were collected. ResultsThirty-seven germline mutations were identified in 32 patients. The most commonly affected genes were ATM in 6%, followed by FANCA (5%), and ATR (4%). BRCA2 mutations were identified in 3%. The frequency of mutations was higher in the metastatic group. Discussion and ConclusionThe results of our study show different mutations from those reported in different populations or regions. The use of PARP inhibitors is indicated in patients with germline mutations, specifically BRCA2, showing improvement in overall survival and progression free survival. To our knowledge, this is the first study reporting the prevalence of mutations in DNA-repair genes in Mexican patients with prostate cancer.

Effect of inactivating heterozygous mutations in DNA repair genes on experimental lung carcinogenesis in mice

Background. Inactivating mutations in Chek2 and Gprc5a genes are known to be associated with cancer development. Experimental carcinogenesis studies in genetically modified mice generate new data on their influence on pathology development.Aim. In the present study in a model of lung carcinogenesis, survival parameters as well as tumor multiplicity and size in mice with Chek2 and Gprc5a heterozygous inactivating mutations were evaluated.Material and methods. F2 hybrid mice from crosses between CBAB6F1 males heterozygous for the studied mutations and wild-type BALB / c females were used: Chek2dAA-carriers (76 males and 64 females) and Gprc5ainsA-carriers (60 males and 42 females). Starting at four months of age, mice received urethane (ethyl carbamate) intraperitoneally at a dose of 600 mg / kg weekly for 6 weeks. After genotyping by allele-specific PCR, animals were allocated to groups. Carcinogenesis parameters were evaluated 40 weeks after the beginning of the experiment.Results. The proportion of mice with mutations surviving to the age of three months roughly followed the Mendelian distribution (35 / 41 males and 33 / 31 females) for the offspring of males heterozygous for Chek2dAA and was significantly lower in the case of Gprc5ainsA (20 / 40 males and 17 / 25 females, p = 0.043). The death of Gprc5ainsA carriers during the experiment was also higher than in the control group (p = 0.0506 in females). Synchronous lung and thymus neoplasms were found in 2 out of 4 Gprc5ainsA females that died before the end of the experiment, which were not found in other groups. At the end of the experiment, no significant differences in tumor multiplicity, mean linear size, and volume were found between the groups of mice with and without mutations.Conclusion. It was found that heterozygous inactivating mutation Chek2dAA does not affect early age development and does not modify the parameters of induced lung carcinogenesis in mice. Heterozygous carriage of Gprc5ainsA mutation in mice increases the risk of early death and sensitivity to the toxic and carcinogenic effects of urethane.

Abstract 6414: Molecular differentiation between complete and incomplete responders to neoadjuvant therapy in rectal cancer

Abstract Neoadjuvant chemoradiotherapy (nCRT) is the standard treatment for locally advanced rectal cancer, but only 20-40% of patients completely respond to this treatment. This study aims to identify the molecular features associated with a response to nCRT. We generated and collected genomic and transcriptomic data from 712 cancers prior to treatment from our own data and from publicly available data. A comprehensive integrated analysis was performed. We found that patients with a complete response have decreased risk of both local recurrence and future metastasis. We identified multiple molecular differences between complete and incomplete responders. Complete responders have a higher tumor mutation burden and more significant co-occurring mutations than the incomplete responders. In addition, mutations in DNA repair genes were enriched in complete responders and they also had lower expression of these genes indicating that defective DNA repair is associated with complete response to nCRT. Using logistic regression, we identified three significant predictors of complete response: tumor size, mutations within specific network genes, and the existence of three or more specific co-occurrent mutations. In incompletely responder tumors, abnormal cell-cell interaction and increased cancer associated fibroblasts were associated with recurrence. Additionally, gene expression analysis identified a subset of immune hot tumors with worse outcomes and upregulation of immune checkpoint proteins. Our study directly informs the clinical management of rectal cancer by elucidating the molecular differences between tumors with complete and incomplete responses to neoadjuvant chemoradiotherapy (nCRT), as well as the features of tumors prone to recurrence. We have pinpointed critical molecular markers and clinical indicators—such as tumor size, specific gene mutations, and particular mutation patterns—that are significant predictors of treatment response. These findings offer valuable biomarkers for personalizing treatment strategies. Additionally, the observed mutation enrichment in DNA repair genes among complete responders suggests new avenues for therapeutic innovation. By highlighting the distinct molecular and immune profiles of non-responder tumors at higher recurrence risk, our research supports the advancement of precision oncology approaches, enhancing prognosis and treatment customization in rectal cancer care, ultimately striving to bridge the gap between scientific discovery and clinical application. Citation Format: Fengyuan Huang, M. Chandler McLeod, Regina Kay Irwin, Mary Smithson, Zongliang Yue, Min Gao, Karin M. Hardiman, Zechen Chong. Molecular differentiation between complete and incomplete responders to neoadjuvant therapy in rectal cancer [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 6414.

Sensing chemical-induced DNA damage using CRISPR/Cas9-mediated gene-deletion yeast-reporter strains

Microorganism-based genotoxicity assessments are vital for evaluating potential chemical-induced DNA damage. In this study, we developed both chromosomally integrated and single-copy plasmid–based reporter assays in budding yeast using a RNR3 promoter–driven luciferase gene. These assays were designed to compare the response to genotoxic chemicals with a pre-established multicopy plasmid–based assay. Despite exhibiting the lowest luciferase activity, the chromosomally integrated reporter assay showed the highest fold induction (i.e., the ratio of luciferase activity in the presence and absence of the chemical) compared with the established plasmid-based assay. Using CRISPR/Cas9 technology, we generated mutants with single- or double-gene deletions, affecting major DNA repair pathways or cell permeability. This enabled us to evaluate reporter gene responses to genotoxicants in a single-copy plasmid–based assay. Elevated background activities were observed in several mutants, such as mag1Δ cells, even without exposure to chemicals. However, substantial luciferase induction was detected in single-deletion mutants following exposure to specific chemicals, including mag1Δ, mms2Δ, and rad59Δ cells treated with methyl methanesulfonate; rad59Δ cells exposed to camptothecin; and mms2Δ and rad10Δ cells treated with mitomycin C (MMC) and cisplatin (CDDP). Notably, mms2Δ/rad10Δ cells treated with MMC or CDDP exhibited significantly enhanced luciferase induction compared with the parent single-deletion mutants, suggesting that postreplication and for nucleotide excision repair processes predominantly contribute to repairing DNA crosslinks. Overall, our findings demonstrate the utility of yeast-based reporter assays employing strains with multiple-deletion mutations in DNA repair genes. These assays serve as valuable tools for investigating DNA repair mechanisms and assessing chemical-induced DNA damage.Key points• Responses to genotoxic chemicals were investigated in three types of reporter yeast.• Yeast strains with single- and double-deletions of DNA repair genes were tested.• Two DNA repair pathways predominantly contributed to DNA crosslink repair in yeast.

The effect of NBS1 heterozygous inactivating mutation in a model of 7,12-dimethylbenz[a]anthracene-induced carcinogenesis in mice

BACKGROUND: The creation of new experimental models carrying various heterozygous inactivating mutations in DNA repair genes and their carcinogenesis studies could expand our understanding of the spectrum of possible neoplasms that patients with such mutations are at risk of developing.
 AIM: The study of the effect of NBS1 inactivating heterozygous mutation (с.1971insT, p.Arg658Stop) in a model of 7,12-dimethylbenz[a]anthracene (DMBA) induced carcinogenesis in mice.
 MATERIALS AND METHODS: Carcinogenesis was induced in 2-month-old female mice with and without NBS1 heterozygous mutation by intragastric administration of 7,12-DMBA (dose of 50 mg/kg once a week, for 8 weeks) on a high-fat diet. Lifetime observation was conducted, and parameters of lifespan and carcinogenesis were evaluated.
 RESULTS: NBS1 inactivating heterozygous mutation had no significant effect on the incidence of tumor pathology induced by 7,12-DMBA, but somewhat accelerated the rate of tumor development and, consequently, the death of animals. The presence of the mutation increased the sensitivity to carcinogen toxic effect, which was expressed in a statistically significant increase in death rate (by 35%) in the early stages of the study. The mean lifespan in female NBS1 mutant mice was reduced by 14% compared to mutation-free animals.
 CONCLUSION: Carrying NBS1 inactivating heterozygous mutation (с.1971insT, p.Arg658Stop) worsens the prognosis of survival in the model of chemically induced 7,12-DMBA carcinogenesis in mice.

Abstract LB_A03: Proximity ligation sequencing reveals novel and recurrent structural genomic variants in FFPE pancreatic ductal adenocarcinoma samples

Abstract Activating KRAS mutations, homologous recombination deficiency (HRD), deficient mismatch repair (dMMR), and other prognostic or predictive biomarkers are seen in fewer than half of pancreatic ductal adenocarcinoma (PDAC) cases. Structural variants (SV), such as gene fusions, translocations, deletions, duplications, etc, are known to play a role in oncogenesis, metastasis, and/or treatment resistance. In solid tumors, SV discovery and validation has been limited, often due to a fresh or live tissue requirement in cytogenomic analyses. Recent innovations in processing formalin-fixed paraffin-embedded (FFPE) samples for “next-generation” chromatin capture and long-range sequencing platforms (i.e. Hi-C) have overcome this barrier. Hi-C is a genome-wide technology which surveys rearrangements, generating genomic contiguity data from highly fragmented chromatin. Unlike targeted (e.g. hybrid capture) assays, Hi-C can survey the landscape of genomic aberrations in an unbiased manner. Archived FFPE has previously been an obstacle to Hi-C analysis of solid tumors. We present here, to our knowledge, the first Hi-C analysis of FFPE PDAC samples using a previously described, proprietary Hi-C platform. Thirty-one samples from 20 unique patients diagnosed and treated at the University of Colorado Comprehensive Cancer Center between 2014-2022 were processed and analyzed. Twenty-two samples were surgical resections paired with later biopsies of metastatic recurrences. Nine were unpaired surgical resections or metastatic biopsies. Seven of these cases had somatic KRAS mutations, five had germline mutations in DNA repair genes, and none had evidence of dMMR or MSI-high. Fifty percent of samples had sufficient tumor content for processing and successful library generation. Notably, two paired samples from 2014 were successfully processed and sequenced demonstrating the applicability of this technology to archival samples. In successfully analyzed samples, the vast majority of SVs observed in metastases were also seen in paired primary tumors. Several duplications and deletions recurred across two or more patients. Furthermore, at least three SVs from this cohort were observed in other tumor types from a separate cohort analysis using this technology. Cases with relatively more SVs demonstrated a longer time to metastatic recurrence than cases with fewer SVs. Cases with germline-mutated cancer-predisposing genes demonstrated a similar SV signature and time to metastatic recurrence as some cases with or without unknown germline and somatic tumor mutation status. Overall, our findings show that this novel platform can successfully profile archived FFPE PDAC samples. Some paired samples show a shared SV signature suggesting the presence of metastatic clones at the time of initial diagnosis. Following these results, we plan to process and analyze a larger cohort of PDAC cases enriched for wild-type KRAS and HRD carriers. We plan to compare our findings to Hi-C results from other tumors and further focus on any SVs recurrent across tumor types. Citation Format: Abhishek Pandey, Stephen Eacker, Adrie Van Bokhoven, Kathleen Torko, Christopher H Lieu. Proximity ligation sequencing reveals novel and recurrent structural genomic variants in FFPE pancreatic ductal adenocarcinoma samples [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A03.

Liquid Biopsy as a Powerful Therapeutic Tool in Veteran Patients with Prostate Carcinoma

Abstract Introduction/Objective The National Comprehensive Cancer Network (NCCN) guidelines currently recommend somatic tumor molecular testing for alterations in homologous recombination (HR) DNA repair genes in patients with metastatic prostate cancer, which can be performed on either the sampled tumor tissue itself or by liquid biopsy. Particularly when the sampled tumor tissue may be limited, liquid biopsy with next generation sequencing is a recently available attractive alternative that is minimally invasive (requiring just phlebotomy). Based on the information gathered from circulating tumor cells (CTC), a tumor molecular profile can be obtained that can impact treatment, particularly if a HR DNA repair mutation is found as that may predict response to a poly (ADP-ribose) polymerase (PARP) enzyme inhibitor such as Olaparib. However, the literature of metastatic prostate cancers targeted by liquid biopsies and the mutations found is sparse, particularly for the veteran population. Methods/Case Report A retrospective review was performed of all patients with a submitted liquid biopsy specimen at a Veteran Affairs Medical Center (VAMC) October 23rd, 2019, to March 3rd, 2023 to examine those patients with metastatic prostate cancer. Information on mutations detected was collected. Results (if a Case Study enter NA) There were a total of 64 patient specimens with a history of metastatic prostate cancer, from which 56 had an available liquid biopsy report with the first liquid biopsy collection occurring on October 23rd, 2019. HR DNA repair mutations found included CHEK2 (6/56), ATM (4/56), and BRCA1 (1/56). Outside of HR DNA repair genes, relatively common detected mutations were found in TP53 (25/56), PTEN (6/56), BRAF (4/56), and an AR amplification (4/56). 7 of the 56 reports had no reportable genomic alterations detected. Conclusion Liquid biopsies continues to be a minimally invasive, highly useful powerful therapeutic tool in veteran patients with metastatic prostate carcinoma for molecular profiling. Not uncommonly in the Veteran population, these molecular findings include mutations in HR DNA repair genes that as noted by the NCCN may predict response to PARP inhibitor chemotherapy.

DNA damage response and repair gene mutations are associated with tumor mutational burden and outcomes to platinum-based chemotherapy/immunotherapy in advanced NSCLC patients

BackgroundDNA damage response and repair (DDR) genes are crucial for maintaining the integrity of the genome. This study aims to explore the correlation of DDR gene mutations with TMB, clinical characteristics, and outcomes to platinum-based chemotherapy and platinum-based chemotherapy/immunotherapy in non-small cell lung cancer (NSCLC) without EGFR and ALK alterations.MethodsTumor tissue from 49 patients with stage III or IV NSCLC who were without EGFR and ALK alterations were analyzed using targeted next-generation sequencing (NGS). Among them, 13 patients received first-line platinum-based chemotherapy, 32 patients received first-line platinum-based chemotherapy/immunotherapy.ResultsIn these NSCLC patients without EGFR and ALK alterations, the frequently mutated genes included TP53, KMT2D and KRAS, the most frequently mutated DDR gene was FANCG, DDR gene mutations were detected in 20 patients. The mutation frequency of homologous recombination (HR) pathway was significantly higher in lung squamous cell carcinoma (LUSC) than that in lung adenocarcinoma (LUAD) (30.8% vs. 5.7%). Among DDR positive patients, a lower percentage exhibited metastasis. Patients with DDR gene mutations, cell-cycle checkpoint pathway mutations, and BER pathway mutations had significantly higher TMB compared to those without corresponding mutations. In the patients receiving platinum-based chemotherapy/immunotherapy, the disease control rate was significantly lower in the DDR-positive group compared with that in the DDR-negative group (55.6% vs. 100.0%). Among LUAD patients receiving platinum-based chemotherapy/immunotherapy, we observed a worse overall survival (OS) in DDR-positive group, as well as poorer progression-free survival(PFS)and OS in BER-positive and FANCG mutated group.ConclusionsDDR gene mutations are associated with tumor metastasis, TMB, and outcomes to platinum-based chemotherapy/immunotherapy in advanced NSCLC patients.

The relationship of prostate cancer with mutations in tumour suppressor genes

Genetic testing plays an increasing role in the diagnosis of various diseases every year. Special attention is paid to genes with an increased risk of cancer in case of mutation. Hereditary prostate cancer is usually more aggressive and is most often associated with mutations in DNA repair genes. These mutations carriers have an increased risk of metastasis and a shorter life expectancy. The study of mutations in the BRCA1/BRCA2 genes is most often used in clinical practice, while there are many other genes responsible for DNA repair processes that have not been sufficiently studied. In this paper, we report a review of literature sources studying the cellular mechanisms of functioning of DNA repair genes, the effect of such mutations on the disease state and oncological outcomes.

Correction to: ORCHID: A phase II study of Olaparib in Metastatic Renal Cell Carcinoma Patients HarborIng a BAP1 or Other DNA Repair Gene Mutations.

Correction to: ORCHID: A phase II study of Olaparib in Metastatic Renal Cell Carcinoma Patients HarborIng a BAP1 or Other DNA Repair Gene Mutations.