Biallelic Alterations Research Articles

Clonal competition assays identify fitness signatures in cancer progression and resistance in multiple myeloma.

Multiple myeloma (MM) is a genetically heterogeneous disease and the management of relapses is one of the biggest clinical challenges. TP53 alterations are established high-risk markers and are included in the current disease staging criteria. KRAS is the most frequently mutated gene affecting around 20% of MM patients. Applying Clonal Competition Assays (CCA)byco-culturing color-labeled genetically modified cell models, we recently showed that mono- and biallelic alterations in TP53 transmit a fitness advantage to the cells. Here, we report a similar dynamic for two mutations in KRAS (G12A and A146T), providing a biological rationale for the high frequency of KRAS and TP53 alterations at MM relapse. Resistance mutations, on the other hand, did not endow MM cells with a general fitness advantage but rather presented a disadvantage compared to the wild-type. CUL4B KO and IKZF1 A152T transmit resistance against immunomodulatory agents, PSMB5 A20T to proteasome inhibition. However, MM cells harboring such lesions only outcompete the culture in the presence of the respective drug. To better prevent the selection of clones with the potential of inducing relapse, these results argue in favor of treatment-free breaks or a switch of the drug class given as maintenance therapy. In summary, the fitness benefit of TP53 and KRAS mutations was not treatment-related, unlike patient-derived drug resistance alterations that may only induce an advantage under treatment. CCAs are suitable models for the study of clonal evolution and competitive (dis)advantages conveyed by a specific genetic lesion of interest, and their dependence on external factors such as the treatment.

Abstract LB373: Pilot whole-exome sequencing data of formalin-fixed paraffin embedded endometrial tumors in the Epidemiology of Endometrial Cancer Consortium (E2C2)

Abstract Background: Endometrial cancer (EC) is the most common gynecologic cancer in the U.S.: incidence and mortality are rising. Black women face 2-fold higher mortality compared with other racial groups. To understand the role of tumor biology underlying these health inequities, we are sequencing the tumor exomes of 1,000 Black and 2,000 non-Black women in the Epidemiology of Endometrial Cancer Consortium. To inform this effort, we have completed whole-exome tumor sequencing in 35 EC patients. We present early sample performance and mutational insights of this pilot. Methods: We extracted DNA from formalin-fixed paraffin-embedded (FFPE) tumor tissues (cores, scroll) and matched normal specimens (blood, tissue) from 35 women diagnosed with EC in the Louisiana Tumor Registry, Nurses’ Health Study, and Polish Endometrial Cancer Study. Whole-exome sequencing was conducted by the Center for Inherited Disease Research at Johns Hopkins University, which called somatic alterations using matched normal samples as the germline profile (NovaSeq6000). We excluded two contaminated core samples and one paired scroll with extremely low coverage, leaving 33 women in our analytic sample. We examined the mutational landscape of EC overall and separately in 7 Black and 26 non-Black patients. For 23 women, we obtained both an FFPE scroll of the full block and FFPE tumor cores enabling a direct comparative analysis of DNA extraction yield and sequencing performance by sample source. We ran a published allele-specific copy number and clonal heterogeneity analysis tool (FACETS) in these 23 women to calculate tumor purity by tissue type. Results: Results from our pilot study revealed the most frequently mutated genes in our pilot population were PTEN (61%), ARID1A (48%), and PIK3CA (39%). We observed similar mutation frequencies for PIK3CA in both non-Black and Black women (38% vs 43%). TP53 was more frequently mutated in Black women (57% vs 16%), while ARID1A mutation was more common in non-Black women (58% vs 14%). 10 (30%) patient samples had chromosome 10 copy-neutral loss of heterozygosity. Of these, 8 (80%) had PTEN bi-allelic alterations. Of participants with both core and scroll tissue available, both sample types were successfully sequenced, although cores showed modestly better performance: 74% of core samples (vs 26% for scrolls) had a higher percentage of bases read at a target depth of 100x, and the median tumor purity for cores estimated using FACETS was higher than scrolls (53% vs 42%). Tumor purity could not be calculated for seven tissue samples due to either tumor diploidy or purity being too low: of these samples the majority were scrolls (86%). We observed greater concordance between core and scroll variant allele frequency when tumor purity was ≥30%: the benefits of cores vs scrolls matter at lower tumor purity. ConclusionsIn line with TCGA data, these pilot findings suggest potential differences in tumor mutational landscape by race and we will be interrogating these in our larger study population. FFPE core tissue generally performed better than FFPE scroll tissue in whole-exome sequencing, but our data suggest scrolls remain a viable option for sequencing when cores are unavailable. This has the potential to offer valuable insight for conducting population-based next-generation studies, which mostly rely on FFPE-derived tissues. Citation Format: Noah C. Peeri, Megan A. Clarke, Immaculata De Vivo, Mengmeng Du, Lucio Miele, Jeffin Naduparambil, Kathryn L. Penney, Venkat Seshan, Ronglai Shen, Veronica W. Setiawan, Nicolas Wentzensen, Xiao-Cheng Wu, Arnold Zea, Wenyu Zhang. Pilot whole-exome sequencing data of formalin-fixed paraffin embedded endometrial tumors in the Epidemiology of Endometrial Cancer Consortium (E2C2) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB373.

Pathogenic germline variants in non-BRCA1/2 homologous recombination genes in ovarian cancer: Analysis of tumor phenotype and survival

ObjectiveTo define molecular features of ovarian cancer (OC) with germline pathogenic variants (PVs) in non-BRCA homologous recombination (HR) genes and analyze survival compared to BRCA1/2 and wildtype (WT) OC. MethodsWe included patients with OC undergoing tumor-normal sequencing (MSK-IMPACT) from 07/01/2015–12/31/2020, including germline assessment of BRCA1/2 and other HR genes ATM, BARD1, BRIP1, FANCA, FANCC, NBN, PALB2, RAD50, RAD51B, RAD51C, and RAD51D. Biallelic inactivation was assessed within tumors. Progression-free (PFS) and overall survival (OS) were calculated from pathologic diagnosis using the Kaplan-Meier method with left truncation. Whole-exome sequencing (WES) was performed in a subset. ResultsOf 882 patients with OC, 56 (6.3%) had germline PVs in non-BRCA HR genes; 95 (11%) had BRCA1-associated OC (58 germline, 37 somatic); and 59 (6.7%) had BRCA2-associated OC (40 germline, 19 somatic). High rates of biallelic alterations were observed among germline PVs in BRIP1 (11/13), PALB2 (3/4), RAD51B (3/4), RAD51C (3/4), and RAD51D (8/10). In cases with WES (27/35), there was higher tumor mutational burden (TMB; median 2.5 [1.1–6.0] vs. 1.2 mut/Mb [0.6–2.6]) and enrichment of HR-deficient (HRD) mutational signatures in tumors associated with germline PALB2 and RAD51B/C/D compared with BRIP1 PVs (p < 0.01). Other features of HRD, including telomeric-allelic imbalance (TAI) and large-scale state transitions (LSTs), were similar. Although there was heterogeneity in PFS/OS by gene group, only BRCA1/2-associated OC had improved survival compared to WT OC (p < 0.01). ConclusionsOCs associated with germline PVs in non-BRCA HR genes represent a heterogenous group, with PALB2 and RAD51B/C/D associated with an HRD phenotype.

Genomic profiling of primary and metastatic thyroid cancers.

The genetic repertoire of primary thyroid cancers (TCs) is well documented, but there is a considerable lack of molecular profiling in metastatic TCs. Here, we retrieved and analyzed the molecular and clinical features of 475 primary and metastatic TCs subjected to targeted DNA sequencing, from the cBioPortal database. The cohort included primary and metastatic samples from 276 papillary thyroid carcinomas (PTCs), 5 follicular thyroid carcinomas, 22 Hürthle cell carcinomas (HCCs), 127 poorly differentiated thyroid carcinomas (PDTCs), 30 anaplastic thyroid carcinomas (ATCs) and 15 medullary thyroid carcinomas. The ATCs had the highest tumor mutational burden and the HCCs the highest fraction of the genome altered. Compared to primary PTCs, the metastases had a significantly higher frequency of genetic alterations affecting TERT (51% vs 77%, P < 0.001), CDKN2A (2% vs 10%, P < 0.01), RET (2% vs 7%, P < 0.05), CDKN2B (1% vs 6%, P < 0.05) and BCOR (0% vs 4%, P < 0.05). The distant metastases had a significantly lower frequency of BRAF (64% vs 85%, P < 0.01) and a significantly higher frequency of NRAS (13% vs 3%, P < 0.05) hotspot mutations than the lymph node metastases. Metastases from HCCs and PDTCs were found to be enriched for NF1 (29%) and TP53 (18%) biallelic alterations, respectively. The frequency of subclonal mutations in ATCs was significantly higher than in PTCs (43% vs 25%, P < 0.01) and PDTCs (43% vs 22%, P < 0.01). Metastatic TCs are enriched in clinically informative genetic alterations such as RET translocations, BRAF hotspot mutations and NF1 biallelic losses that may be explored therapeutically.

Functional Characterization of GPRC5D alteration and Its Impact on Talquetamab Resistance in Relapsed/ Refractory Multiple Myeloma

Background T-cell engaging (TCE) antibodies show unprecedented efficacy in heavily pretreated penta-refractory multiple myeloma (MM). Responses however are often not durable as resistance mechanisms can arise despite the initial absence of resistance-conferring mutations. Biallelic loss of GPRC5D on chr.12p is a rare, yet well-established mechanism of acquired resistance to the first-in-class anti-GPRC5D directed TCE talquetamab (Lee et al., 2022). At the same time, monoallelic GPRC5D frameshift and missense mutations are much more common and occur in up to 15% of MM patients (Truger et al., 2021). In this study, we investigated the functional relevance of monoallelic GPRC5D alterations and their impact on talquetamab resistance. Methods GPRC5D alterations were modeled in vitro by using CRISPR-Cas9 editing in the OPM-2 MM cell line. The bioluminescence-based cytotoxic assay was performed using PBMCs as effector cells to measure the resistance to talquetamab. Quantitative PCR (qPCR), immunohistochemistry, bulk whole genome sequencing (WGS), and whole genome bisulfite sequencing (WGBS) were performed for molecular, genomic, and epigenetic characterization in a patient with acquired talquetamab resistance. Results Using CRISPR-Cas9, we generated cell line models with mono- vs. biallelic deletions in the GPRC5D gene. Clones with biallelic alterations (OPM-2 Del/Del) displayed a 96.4%, reduced expression of GPRC5D ( p &amp;lt; .0001), leading to in vitro resistance to talquetamab. GPRC5D expression levels in monoallelic models (OPM-2 Wt/Del) decreased by 57.4% ( p = .0004) compared to the wild type. Sensitivity to talquetamab in these clones was equally impaired, however, less pronounced than in the biallelic knock-outs. To assess the impact of monoallelic GPRC5D deletions in the clinic, we next investigated the case of a heavily pretreated 58-year-old patient with lambda light-chain MM treated at our institution with talquetamab in his 7th line of therapy. qPCR was used to measure GPRC5D expression in CD138+ MM cells from the bone marrow of this patient before and at the time of relapse to talquetamab after 15 months of treatment (best response: CR). At the time of relapse, we observed a 94.0% ( p &amp;lt; .0001) decrease in GPRC5D expression upon qPCR assessment as compared to the pre-treatment time point which was confirmed by immunohistochemistry. WGS revealed a novel monoallelic loss of chr.12p encompassing GPRC5D in talquetamab-resistant CD138+ MM cells. Since monoallelic alterations seem unlikely as the sole drivers of resistance, we hypothesized that epigenetic modifications may have contributed to talquetamab resistance in this patient. To this end, WGBS was performed and uncovered a significant shift towards hypermethylation of two regulatory regions of GPRC5D, most likely resulting in the silencing of the second GPRC5D allele. Time-course experiments are currently ongoing to explore the methylation landscape of our CRISPR-Cas9-generated clones with monoallelic GPRC5D deletions. Data for these validation experiments will be presented at the meeting. Conclusions Our study provides the first evidence of the functional impact of monoallelic GPRC5D alterations resulting in relative resistance to talquetamab in vitro. We further report on epigenetic regulation as a potential second-hit mechanism conferring clinical resistance to anti-GPRC5D-directed TCE therapy. This data suggests that patients with monoallelic GPRC5D deletions may be at particular risk for developing resistance to talquetamab.

Microsatellite Instability and Mismatch Repair Deficiency Define a Distinct Subset of Lung Cancers Characterized by Smoking Exposure, High Tumor Mutational Burden, and Recurrent Somatic MLH1 Inactivation

IntroductionMicrosatellite instability (MSI) and mismatch repair (MMR) deficiency represent a distinct oncogenic process and predict response to immune checkpoint inhibitors (ICIs). The clinicopathologic features of MSI-high (MSI-H) and MMR deficiency (MMR-D) in lung cancers remain poorly characterized. MethodsMSI status from 5171 patients with NSCLC and 315 patients with SCLC was analyzed from targeted next-generation sequencing data using two validated bioinformatic pipelines. ResultsMSI-H and MMR-D were identified in 21 patients with NSCLC (0.41%) and six patients with SCLC (1.9%). Notably, all patients with NSCLC had a positive smoking history, including 11 adenocarcinomas. Compared with microsatellite stable cases, MSI-H was associated with exceptionally high tumor mutational burden (37.4 versus 8.5 muts/Mb, p < 0.0001), MMR mutational signatures (43% versus 0%, p < 0.0001), and somatic biallelic alterations in MLH1 (52% versus 0%, p < 0.0001). Loss of MLH1 and PMS2 expression by immunohistochemistry was found in MLH1 altered and wild-type cases. Similarly, the majority of patients with MSI-H SCLC had evidence of MLH1 inactivation, including two with MLH1 promoter hypermethylation. A single patient with NSCLC with a somatic MSH2 mutation had Lynch syndrome as confirmed by the presence of a germline MSH2 mutation. Among patients with advanced MSI-H lung cancers treated with ICIs, durable clinical benefit was observed in three of eight patients with NSCLC and two of two patients with SCLC. In NSCLC, STK11, KEAP1, and JAK1 were mutated in nonresponders but wild type in responders. ConclusionsWe present a comprehensive clinicogenomic landscape of MSI-H lung cancers and reveal that MSI-H defines a rare subset of lung cancers associated with smoking, high tumor mutational burden, and MLH1 inactivation. Although durable clinical benefit to ICI was observed in some patients, the broad range of responses suggests that clinical activity may be modulated by co-mutational landscapes.

A Novel HRD Signature Is Predictive of FOLFIRINOX Benefit in Metastatic Pancreatic Cancer.

Pancreatic cancer (PC) represents an aggressive disease with median overall survival (OS) of less than 1 year in the front-line setting. FOLFIRINOX and gemcitabine and paclitaxel (GP) are standard of care options for these patients; however, optimal selection of therapy is challenging. Comprehensive genomic profiling was performed on 8358 PC patients. Outcomes were available for 1149 metastatic PC patients treated with 1L FOLFIRINOX or GP. A scar-based measure of HRD was called using a machine learning-based algorithm incorporating copy number and indel features. A scar-based HRD signature (HRDsig) was identified in 9% of patients. HRDsig significantly co-occurred with biallelic alterations in BRCA1/2, PALB2, BARD1, and RAD51C/D, but encompassed a larger population than that defined by BRCA1/BRCA2/PALB2 (9% vs. 6%). HRDsig was predictive of 1L FOLFIRNOX chemotherapy benefit with doubled OS relative to gemcitabine and paclitaxel (GP) (rwOS aHR 0.37 [0.22-0.62]), including 25% of the population with long-term (2 year+) survival in a real-world cohort of patients. Less benefit from FOLFIRINOX was observed in the HRDsig(-) population. Predictive value was seen in both the BRCA1/2/PALB2 mutant and wildtype populations, suggesting additional value to mutational profiling. A scar-based HRD biomarker was identified in a significant fraction of PC patients and is predictive of FOLFIRINOX benefit. Incorporating a biomarker like HRDsig could identify the right patients for platinum chemotherapy and potentially reduce FOLFIRINOX use by over 40%, minimizing toxicities with similar survival outcomes. Confirmatory studies should be performed.

Exploring the prognostic and predictive impact of genomic loss of heterozygosity (LOH) in patients with metastatic colorectal cancer (mCRC).

3576 Background: Genomic LOH consists in the loss of chromosomal regions and is associated with the homologous recombination repair (HRR) system deficiency (dHRR). In ovarian cancer, LOH-high predicts benefit from platinum-based chemotherapy and PARP inhibitors. In mCRC, the role of LOH has been poorly investigated. Methods: An NGS-based assay (FoundationOne CDx; Foundation Medicine, Inc. Cambrige, MA) was used to determine the percentage of genomic LOH and the presence of pathogenetic mutations in HRR-related genes in archival chemo-naïve tumor tissues of mCRC patients included in a real-world registry and in the AtezoTRIBE (NCT03721653) and AVETRIC (NCT04513951) studies. Both these trials assessed the combination of an anti-PDL1 (atezolizumab or avelumab) with the triplet FOLFOXIRI plus bevacizumab or cetuximab. The prespecified cut-off of ≥14.08 for LOH-high described for mCRC (Sokol et al., JCO Precis Oncol 2020) was adopted. Tumors with at least one biallelic alteration in any of the 27 genes involved in the HRR pathway (Riaz et al., Nat Commun 2017) included in the FoundationOne CDx panel (BARD1, BRCA1, BRCA2, BRIP1, MRE11A, NBN, PALB2, RAD51, RAD51B, RAD51C, RAD51D, RAD52, RAD54L, RBBP8, XRCC2, ABL1, ATM, ATR, BAP1, CDK12, DNMT3A, ERCC4, FANCA, FANCC, FANCG, FANCL, PARP1) were defined dHRR. Results: Overall, 196 samples were analysed. None of 7 MSI-H tumors were classified as LOH-high. Fourteen (7%) and 6 (3%) of 189 MSS tumors were classified as LOH-high and dHRR, respectively. In particular, in LOH-high subgroup, 3 (21%) tumors were defined dHRR, while 3 (50%) tumors were classified LOH-high among dHRR tumors. LOH-high tumors were more frequently BRAF mutated (p = 0.019) and dHRR (p = 0.006) compared to LOH-low. Among patients receiving triplet chemotherapy +/- biologic agent alone (N = 55) or with an anti-PDL1 (N = 58), an interaction effect was shown between the effect of the addition of the checkpoint inhibitor and LOH with higher benefit in the LOH-high subgroup (N = 10) in terms of both PFS (pinteraction= 0.002) and OS (pinteraction&lt; 0.001). In the cohort of patients not receiving the anti-PDL1, longer PFS was observed in patients with LOH-low (N = 125) respect to LOH-high (N = 6) tumors (12.1 vs 5.1 months, HR: 0.11, 95%CI: 0.04-0.26, p &lt; 0.001). No differences in PFS or OS were reported between patients treated with first-line oxaliplatin- (N = 40) vs irinotecan-based doublets (N = 25) or with the triplet FOLFOXIRI (N = 55) vs doublets (N = 65) according to LOH status. No prognostic or predictive impact of HRR deficiency was shown. Conclusions: In MSS mCRC, LOH-high was associated with biallelic alterations in the HRR system, worse prognosis and higher benefit from the addition of anti-PDL1 agents to chemotherapy. Considering the low number of LOH-high tumors in our study, these results deserve confirmation in larger cohorts.

Germline variants in non-BRCA1/2 homologous recombination-related genes and ovarian cancer: Analysis of tumor phenotype and survival.

5565 Background: BRCA1/2-associated ovarian cancer (OC) exhibits homologous recombination (HR)-deficiency (HRD) and a better prognosis than BRCA1/2-wildtype (WT) OC. However, it is unclear if patients with germline pathogenic variants (gPV) in other HR-related genes have a similar tumor phenotype. We sought to define OC molecular features from patients with gPV in other HR genes and analyze survival compared to patients with BRCA1/2 and WT OC. Methods: We identified patients with OC treated at our institution who underwent tumor-normal sequencing using MSK-IMPACT targeting 341-505 cancer-related genes with germline analysis of ≥76 genes from 7/2015-12/2020. Biallelic inactivation was inferred via assessments of loss of heterozygosity (LOH) at gPV in non- BRCA1/2 HR-related genes ( ATM, BARD1, BRIP1, FANCA, FANCC, NBN, PALB2, RAD50, RAD51B, RAD51C, and RAD51D). Clinical characteristics were compared to patients with BRCA1/2-associated and WT OC using non-parametric tests. Progression-free (PFS) and overall-survival (OS) were calculated from date of pathologic diagnosis using the Kaplan-Meier method. Left truncation at date of MSK-IMPACT consent was applied. Whole-exome sequencing (WES) was performed in a subset of OCs. Results: Of 882 patients with OC, 56 (6.3%) had gPV in non- BRCA1/2 HR-related genes compared to 95 (10.8%) patients with BRCA1-associated OC (58 germline, 37 somatic) and 59 (6.7%) patients with BRCA2-associated OC (40 germline, 19 somatic). Patients with a deleterious genetic alteration were diagnosed with OC at younger age and more likely to have high-grade serous histology compared to the WT group (p &lt; 0.01). High rates of biallelic alterations were observed amongst gPV in BRIP1 (11/13), PALB2 (3/4), RAD51B (3/4), RAD51C (3/4), and RAD51D (8/10), and WES was performed in a subset (n = 27) of tumors from these patients with adequate tumor purity (&gt;30%). We observed a higher tumor mutational burden (TMB), median 2.5 (1.1-6.0) vs. 1.2 (0.6-2.6) mut/Mb, and enrichment of HRD mutational signatures in tumors associated with PALB2 and RAD51B/C/D compared with BRIP1 (p &lt; 0.01), although markers of telomeric-allelic imbalance (TAI), large-scale state transitions (LST) and fraction of genome altered (FGA) were similar. PFS and OS varied by gene group with best survival in BRCA1/2-associated OC, even after adjustment for clinical covariates in multivariable models (p &lt; 0.01). Although we observed heterogeneity in PFS and OS for those with gPV in other HR-related genes by biallelic status and HRD phenotype, none had significantly better survival than those with WT OC. Conclusions: OCs associated with gPV in non- BRCA1/2 HR-related genes represent a heterogenous group. OCs in those with gPV in PALB2 and RAD51B/C/D preferentially harbored biallelic alterations and displayed an HRD phenotype.

Molecular Profiling Provides Clinical Insights Into Targeted and Immunotherapies as Well as Colorectal Cancer Prognosis

Tumor genetic testing is indispensable in the management of primary and metastatic colorectal cancer (mCRC), yet the indications for genomics-guided precision medicine and immunotherapy need to be better understood and defined. We prospectively sequenced tumors from 869 Chinese colorectal cancer patients by a large panel and evaluated the clinical significance of single gene somatic mutations and co-occurring events in mCRC, as well as their functional effects and tumorigenic mechanisms. We systematically assessed the heterogeneity of the tumor immune microenvironment in different genomic contexts through the combined analysis of Immunoscore, multiplex immunostaining, whole-exome sequencing, transcriptome, and single-cell sequencing. Single-gene somatic mutations in BRAF or RBM10 were associated with shorter progression-free survival (PFS) in mCRC patients. Functional studies suggested RBM10 acts as a tumor suppressor in CRC development. Co-mutations of KRAS/AMER1 or KRAS/APC were enriched in the metastatic cohort, which had poor PFS and did not benefit from bevacizumab due to accelerated drug metabolism. Forty patients (4.6%) carried pathogenic or likely pathogenic germline alterations in the DNA damage repair pathway, and 37.5% of these tumors had secondary-hit events with loss of heterozygosity or biallelic alterations. A high tumor indel burden with MSI-high suggested immunogenicity with numerous activated tumor-infiltrating lymphocytes, whereas POLE-mutation with TMB-ultrahigh indicated a relatively quiescent immunophenotype. The heterogeneous genomic-immunological interactions were reflected in the divergent neoantigen presentation and depletion, immune checkpoint expression, PD-1/PD-L1 interaction, and T-cell responsiveness to pembrolizumab. Our integrated analysis provides insights into CRC prognostic stratification, drug response, and personalized genomics-guided targeted and immunotherapies.

Abstract 2149: A whole genome sequencing classifier of homologous recombination deficiency

Abstract Homologous recombination deficiency (HRd) is a DNA repair defect prevalent in but not exclusive to breast and ovarian cancer most commonly associated with BRCA1 or BRCA2 alterations. HRd results in accumulation of small and large scale genetic alterations across the genome, including allele specific copy number alterations (aCNAs), small nucleotide variants (SNVs), deletions, and structural variants (SVs). Detection of HRd in tumors predicts response to genotoxic drugs such as PARP inhibitors and platinum. Genome wide aCNAs such as large state transitions (LST), loss of heterozygosity (LOH), and telomeric allelic imbalances (TAI) in conjunction with BRCA1/2 mutation detection have been implemented in routine diagnostic testing to identify HRd in tumors. However, these features represent a subset of the genetic signatures predictive of HRd, and we hypothesize that a significant portion of tumors with HRd are missed using these existing assays. Whole genome sequencing (WGS) enables the detection of the full spectrum of genetic lesions that arise in an HRd tumor in a single assay. To demonstrate the added value of WGS to identify HRd, we trained and validated a pan-cancer classifier of HRd. A tumor/normal matched cohort of 321 cancer patients sequenced by WGS was assembled and analyzed as part of a retrospective study, representing 62 tumor types. An unbiased analysis of HRd associated SV signatures revealed the top quartile of samples harboring tandem duplications (Dups) and deletions (Dels) in the size range of 1-10kbp were enriched with BRCA1, BRCA2, and RAD51C/D alterations. Through curating Dels, Dups, HRd SNV/InDel signatures, and alteration of HRd associated genes, 37 unique patients were found to have high confidence HRd, out of which 13% had no alterations in BRCA1, BRCA2, or other HRd genes. We then trained a random forest classifier to identify HRd tumors. The most important predictive features were WGS-specific, namely small deletions with microhomology, SV Dels, and SV Dups. The HRd classifier was validated using an independent cohort of 556 samples from the Pan-Cancer Analysis of Whole Genomes (PCAWG) study. Of 46 samples with biallelic BRCA1/2 alterations, the classifier achieved high areas under receiver-operator characteristic (AUROC, 0.99) and precision recall curves (AUPRC, 0.96). The aCNA score, the number of segments harboring LST, LOH, and TAI, had similar AUROC (0.96) but lower AUPRC (0.87). There were 11 BRCA1/2 non-altered cases predicted to be HRd with the classifier which were not identified by CNA scores, in which 10 had at least 1 alteration in an HRd gene, including RAD51C, CHEK2 biallelic alterations and SVs in PALB2, Fanconi pathway genes, and ATM/ATR. We conclude that a classifier incorporating the additional mutational features which can only be detected using WGS can achieve superior precision in identifying HRd tumors and, in the future, uncover additional patients for therapeutic options. Citation Format: Kevin Hadi, Gunes Gundem, Max F. Levine, Aditya Deshpande, Minal Patel, Stan Skzrypczak, Majd Al Assaad, Juan Miguel Mosquera, Olivier Elemento, Andrew L. Kung, Juan S. Medina-Martínez, Elli Papaemmanuil. A whole genome sequencing classifier of homologous recombination deficiency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2149.

Abstract P2-23-15: Histologic, immunohistochemical and genomic comparison between classic Invasive lobular carcinomas and lobular-like invasive ductal carcinomas

Abstract Background: Invasive lobular carcinomas (ILCs) are the most frequent special histologic subtype of breast cancer, accounting for up to 15% of all breast cancer cases. ILCs are characterized by a distinctive discohesive growth pattern, with cells arranged in single cell infiltrative file and dispersed throughout the stroma, which stems from the loss of E-cadherin expression due to bi-allelic inactivation of the CDH1 gene. A subset of breast cancers display a similar single cell infiltrative growth pattern but, in contrast to classic ILC, display diffuse strong membranous E-cadherin reactivity and membranous p120 expression. We refer to such cases as “lobular-like invasive ductal carcinoma” (LLIDC), but it is unclear if this terminology is appropriate and if such cases show biallelic inactivation of CDH1, similarly to ILCs. Here, we sought to define whether LLIDCs would harbor bi-allelic alterations of CDH1 and to perform an exploratory, hypothesis generating analysis of the repertoire of somatic genetic alterations of LLIDCs and classic ILCs. Materials and methods: Representative H&amp;Es, as well as sections subjected to E-cadherin and p120 immunohistochemistry from seven classic ILCs and seven bona fide “lobular-like invasive ductal carcinomas” were retrieved and independently reviewed by two pathologists with experience and expertise in breast pathology. DNA samples were extracted from representative sections from tumor and normal breast tissue from each patient and subjected to an FDA-approved targeted sequencing assay comprising the coding regions and selected regulatory elements of 515 genes. Somatic single nucleotide variants (SNVs) were detected with MuTect, indels with Strelka, Varscan2, Scalpel and Lancet. All mutations were manually inspected using the Integrative Genomics Viewer (IGV). The cancer cell fraction (CCF) of each mutation was inferred, as well as clonal probability, using ABSOLUTE. Copy number alterations and loss of heterozygosity were determined using FACETS. Mutational signatures were inferred using SigMA based on all synonymous and nonsynonymous somatic mutations. Results: Based on the histopathologic evaluation, of the 14 cases analyzed, seven were classified as ILC, and the other seven were classified as LLIDC. Sequencing analysis revealed that the classic ILCs harbored 16q LOH and CDH1 mutations (7/7), of which five were frameshift indel and two were splice site mutations consistently coupled with loss-of-heterozygosity (LOH) of the wild-type allele. Conversely, five of the seven LLIDCs did not harbor CDH1 mutations or genomic rearrangements. CDH1 mutations were identified in 2 LLIDCs: one harbored a subclonal CDH1 in-frame indel mutation coupled with LOH. This case displayed membranous E-cadherin and p120 expression with areas of aberrant expression. The other CDH1-mutated LLIDC harbored a complex in-frame indel with subclonal LOH. This case displayed membranous E-cadherin and p120 expression. The comparative analysis of the repertoire of somatic genetic alterations and mutational signatures present in LLIDCs and classic ILCs did not reveal any significant differences. Conclusion: Despite the histologic similarities, LLIDCs differ from classic lobular carcinomas based on the lack of CDH1 bi-allelic inactivation and the patterns of expression of E-cadherin and p120 catenin. Further whole-genome sequencing analyses are warranted to define the molecular basis of the discohesive cancer cells of LLIDC display. Citation Format: Edaise M. da Silva, Thais Basili, Jing Yu, Juan Blanco-Heredia, Pier Selenica, Qiqi Ye, Arnaud da Cruz Paula, Higinio Dopeso, Antonio Marra, Steffi Oesterreich, Jorge Reis-Filho, Rohit Bhargava. Histologic, immunohistochemical and genomic comparison between classic Invasive lobular carcinomas and lobular-like invasive ductal carcinomas [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-23-15.

Comprehensive analysis of germline drivers in endometrial cancer.

We sought to determine the prevalence of germline pathogenic variants (gPVs) in unselected patients with endometrial cancer (EC), define biallelic gPVs within tumors, and describe their associations with clinicopathologic features. Germline assessment of at least 76 cancer predisposition genes was performed in patients with EC undergoing clinical tumor-normal Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) sequencing from January 1, 2015, to June 30, 2021. In patients with gPVs, biallelic alterations in ECs were identified through analysis of loss of heterozygosity and somatic PVs. Clinicopathologic variables were compared using nonparametric tests. Of 1625 patients with EC, 216 (13%) had gPVs, and 15 patients had 2 gPVs. There were 231 gPVs in 35 genes (75 [32%] high penetrance; 39 [17%] moderate penetrance; and 117 [51%] low, recessive, or uncertain penetrance). Compared with those without gPVs, patients with gPVs were younger (P = .002), more often White (P = .009), and less obese (P = .025) and had differences in distribution of tumor histology (P = .017) and molecular subtype (P < .001). Among 231 gPVs, 74 (32%) exhibited biallelic inactivation within tumors. For high-penetrance gPVs, 63% (47 of 75) of ECs had biallelic alterations, primarily affecting mismatch repair (MMR) and homologous recombination related genes, including BRCA1,BRCA2, RAD51D, and PALB2. Biallelic inactivation varied across molecular subtypes with highest rates in microsatellite instability-high (MSI-H) or copy-number (CN)-high subtypes (3 of 12 [25%] POLE, 30 of 77 [39%] MSI-H, 27 of 60 [45%] CN-high, 9 of 57 [16%] CN-low; P < .001). Of unselected patients with EC, 13% had gPVs, with 63% of gPVs in high-penetrance genes (MMR and homologous recombination) exhibiting biallelic inactivation, potentially driving cancer development. This supports germline assessment in EC given implications for treatment and cancer prevention.

Short- and long-interval intracortical inhibition in EPM1 is related to genotype.

Progressive myoclonic epilepsy type 1 (EPM1) is caused by biallelic alterations in the CSTB gene, most commonly dodecamer repeat expansions. Although transcranial magnetic stimulation (TMS)-induced long-interval intracortical inhibition (LICI) was previously reported to be normal in EPM1, short-interval intracortical inhibition (SICI) was reduced. We explored the association between these measures and the clinical and genetic features in a separate group of patients with EPM1. TMS combined with electromyography was performed under neuronavigation. LICI was induced with an inter-stimulus interval (ISI) of 100 ms, and SICI with ISIs of 2 and 3ms, and their means (mSICIs) were expressed as the ratio of conditioned to unconditioned stimuli. LICI and mSICI were compared between patients and controls. Nonparametric correlation was used to study the association between inhibition and parameters of clinical severity, including the Unified Myoclonus Rating Scale (UMRS); among patients with EPM1 due to biallelic expansion repeats, also the association with the number of repeats was assessed. The study protocol was completed in 19 patients (15 with biallelic expansion repeats and 4 compound heterozygotes), and 7 healthy, age- and sex-matched control participants. Compared to controls, patients demonstrated significantly less SICI (median mSICI ratio 1.18 vs 0.38; p < .001). Neither LICI nor SICI was associated with parameters of clinical severity. In participants with biallelic repeat expansions, the number of repeats in the more affected allele (greater repeat number [GRN]) correlated with LICI (rho=0.872; p < .001) and SICI (rho=0.689; p=.006). Our results strengthen the finding of deranged γ-aminobutyric acid (GABA)ergic inhibition in EPM1. LICI and SICI may have use as markers of GABAergic impairment in future trials of disease-modifying treatment in this condition. Whether a higher number of expansion repeats leads to greater GABAergic impairment warrants further study.

Integrating Mosaic Chromosomal Alterations and Single-Nucleotide Variants to Predict Evolution of Clonal Hematopoiesis

Background: Clonal hematopoiesis (CH) is an age-related expansion of mutated hematopoietic stem cells. Multiple studies have previously described distinct types of CH, CHIP (mutations in genes commonly associated with myeloid malignancies) as well as mosaic chromosomal alterations (mCA), that have been associated with increased risk of hematologic malignancies and cardiovascular morbidity. We hypothesized that the presence of both mCA and CHIP mutations may have an additive impact vis-à-vis risk of hematologic malignancy. Methods: Data on 91,335 patients from the BioVU repository, a Vanderbilt University Medical Center biobank with linked de-identified EHRs, spanning 2006 to 2021 were included in the analysis. Peripheral blood-derived DNA of 91,335 subjects from BioVU were genotyped on the Illumina Multi-Ethnic Global Array (MEGAEX) platform, which contains more than 2 million SNPs. CHIP mutation calls (DNMT3A R882/JAK2 V617F) were made by deconvoluting the MEGAEX array intensity data. Predetermined array intensity scores greater than a defined threshold for DNMT3A R882 and JAK2 V617F mutations were highly predictive of the corresponding mutation, so these mutations were included in the analysis. Individuals with a diagnosis of hematologic malignancy (HM) prior to blood draw were excluded, while those that went on to develop HM post-blood draw were included in the analyses. The relationship between independent variables and disease progression and death was assessed using Cox proportional hazards model. All statistical analyses were performed using R. Results: In this large cohort (n=91,335), CH is strongly age-related with an increased frequency in the elderly, as has been previously shown (eg., Bick et al, Nature 2021; Jaiswal et al NEJM 2014). In total, we identified mCA in 765 (0.8%) patients. DNMT3A R882 and JAK2 V617F SNV/indels were identified in 1167 (1.3%) and 347 (0.4%) patients, respectively. Most frequent mCA detected in this cohort include 9p CN-LOH (n=105, 6.9%), del (5q) (n=99, 6.5%), del (20q) (n=98, 6.4%), del (7q) (n=94, 6.2%), and del (13q) (n=67, 4.4%). Combined mCA and DNMT3A R882/JAK2 V617F mutations were reported in 147 (0.2%). Co-occurring JAK2 and 9p CN-LOH (n= 100, OR=5658, q<0.001) are consistent with the well-known mechanisms of biallelic alterations involved in the pathogenesis of HM. There were significant correlations between abnormal blood counts and CH lesions, for example, high platelet count had a higher frequency of JAK2 V617F and 9p CN-LOH (OR=2.57, q=3.41e-141). Individuals with mCA are associated with lower WBC and platelet count, and any blood count abnormality (Figure 1A). Finally, we looked at how presence of both mCA and JAK2/DNMT3A impact the risk of HM. In total, 1615 cases in the entire BioVU cohort were reported to develop HM: 947 myeloid, AML (n=210), CML (n=73), MDS (n=315), MPN (n=349) and 668 lymphoid, ALL (n=72), B-NHL (n=252), T-NHL (n=22), MM (n=322). The presence of mCA was associated with a higher incidence of HM (hazard ratio (HR) = 6.85, 95% confidence interval (CI) = 5.9-7.95, P < 0.001), and DNMT3A/JAK2 was associated with higher incidence of HM ( HR=5.7, 95% CI= 4.75-6.85, P<0.001), and presence of both (mCA and DNMT3A/JAK2) were significantly associated with higher incidence of HM (HR=26.67, 95% CI= 19.48-36.5, P<0.001) than observed in no CH patients (Figure 1B). Conclusion: In this large cohort of patients, the presence of CH (both mCA and DNMT3A/JAK2) in apparently healthy individuals positively correlated with blood count abnormalities and increased incidence of hematologic malignancy. The results from this large cohort of patients emphasize the significance of detecting both mCA and SNV/indels in clonal hematopoiesis. These results should be validated in large datasets to determine if detecting both mCAs and CH can augment clinical care and outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Retrotransposon insertion as a novel mutational cause of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder resulting from biallelic alterations of the SMN1 gene: deletion, gene conversion or, in rare cases, intragenic variants. The disease severity is mainly influenced by the copy number of SMN2, a nearly identical gene, which produces only low amounts of full-length (FL) mRNA. Here we describe the first example of retrotransposon insertion as a pathogenic SMN1 mutational event. The 50-year-old patient is clinically affected by SMA type III with a diagnostic odyssey spanning nearly 30years. Despite a mild disease course, he carries a single SMN2 copy. Using Exome Sequencing and Sanger sequencing, we characterized a SINE-VNTR-Alu (SVA) type Fretrotransposon inserted in SMN1 intron 7. Using RT-PCR and RNASeq experiments on lymphoblastoid cell lines, we documented the dramatic decrease of FL transcript production in the patient compared to subjects with the same SMN1 and SMN2 copy number, thus validating the pathogenicity of this SVA insertion. We described the mutant FL-SMN1-SVA transcript characterized by exon extension and showed that it is subject to degradation by nonsense-mediated mRNA decay. The stability of the SMN-SVA protein may explain the mild course of the disease. This observation exemplifies the role of retrotransposons in human genetic disorders.

356P Biallelic MUTYH germline inactivation in patients with colon polyposis and subjects with sporadic colorectal cancer

MUTYH-associated polyposis (MAP) is a rare recessive condition typically characterized by the presence of 10-100 colorectal polyps and the elevated risk of colorectal cancer (CRC). CRCs arising in biallelic carriers of MUTYH mutation often contain KRAS G12C substitution and frequently arise without concomitant polyposis. This study included 74 consecutive patients with colon polyposis and 3486 consecutive patients with CRC referred to the N.N. Petrov Institute of Oncology (St. Petersburg, Russia) for molecular testing. Normal DNA obtained from subjects with polyposis was analyzed by NGS using custom panel for hereditary cancer genes. CRC patients were screened for recurrent MUTYH pathogenic alleles p.Y179C, p.R245H and p.G396D. The status of the remaining allele in MUTYH-heterozygous CRC cases was analyzed by Sanger sequencing. Biallelic MUTYH mutations were identified in 8/74 (11%) patients with polyposis. MAP was the second most common hereditary polyposis syndrome after familial adenomatous polyposis (47/74, 64%), with Peutz-Jeghers syndrome being in the third place (3/74, 5%). MUTYH biallelic alterations were detected in 9/3486 (0.3%) consecutive CRC patients. Somatic KRAS G12C substitution was observed in 223/3486 (6%) CRCs. Germ-line MUTYH inactivation was identified in 7/223 (3%) KRAS G12C-positive CRCs vs. 2/3253 (0.06%) patients without this mutation (p < 0.00001). Germ-line MUTYH biallelic mutations are significant contributors to the incidence of polyposis, while their impact in CRC morbidity is moderate. However, MUTYH gene analysis is highly feasible in CRC patients whose tumor harbors KRAS G12C substitution.

TRIT1 deficiency: Two novel patients with four novel variants

TRIT1 encodes a tRNA isopentenyl transferase that allows a strong interaction between the mini helix and the codon. Recent reports support the TRIT1 bi-allelic alterations as the cause of an autosomal recessive disorder, named combined oxydative phophorylation deficiency 35, with microcephaly, developmental disability, and epilepsy. The phenotype is due to decreased mitochondrial function, with deficit of i6A37 in cytosolic and mitochondrial tRNA. Only 10 patients have been reported. We report on two new patients with four novel variants, and confirm the published clinical TRIT1 deficient phenotype stressing the possibility of both very severe, with generalized pharmaco-resistant seizures, and mild phenotypes.

Abstract 1249: Development of a pan-cancer algorithm to predict homologous recombination deficiency and sensitivity to PARPi therapy

Abstract Background: PARP inhibitors (PARPi) are approved for multiple indications with ongoing trials to explore broader utility. However, identifying the right patients for these therapies across multiple disease types remains a challenge. In ovarian cancer, genomic-scar based measures for homologous recombination deficiency (HRD) are approved diagnostics (genome-wide LOH [gLOH] and genomic instability score [GIS]); however, broader utility has not been established. Methods: A pan-cancer genomic profiling dataset (n = 202,472; Foundation Medicine, Cambridge, MA) was split 70:30 for training and validation of an HRD signature using an XGB machine learning model (mlHRD). A broad set of copy number (Macintyre 2018) and indel features (Alexandrov 2020) were used to identify signatures of HRD. gLOH (Coleman 2017) and GIS (Timms 2014) were calculated using copy number profiles. Biallelic alterations were predicted using a computational zygosity algorithm (Sun, 2018). The nationwide, de-identified Flatiron Health-Foundation Medicine ovarian and prostate clinico-genomic databases (FH-FMI CGDB) were utilized for outcomes analysis. The de-identified data originated from approximately 280 US cancer clinics (~800 sites of care). Time to therapy discontinuation (TTD) was estimated with Kaplan-Meier analysis. Hazard ratios were calculated using unadjusted Cox proportional Hazard models. Results: We developed an algorithm to predict HRD status using indel and copy number features (see methods). Across the pan-cancer dataset, the rate of mlHRD was 6.4% with the highest frequency in fallopian tube (30%), ovarian (30%), peritoneal (23%), breast (16%), and prostate cancers (15%). Sensitivity to detect biallelic BRCA1/2 alterations was high across tumors [ovary (93%), prostate (87%), breast (85%), pancreas (80%)]. Beyond BRCA1/2, mlHRD positivity was associated with biallelic alterations in RAD51D (OR = 24, p&amp;lt;1E-10), PALB2 (OR = 23, p&amp;lt;1E-10), BARD1 (OR = 23, p&amp;lt;1E-10), and RAD51C (OR = 19, p&amp;lt;1E-10). In the FH-FMI CGDB ovarian cancer cohort, 150 patients were treated with PARPi (mlHRD positive = 73; negative = 77); mlHRD positivity was associated with improved TTD (median 8.9 mo v 3.9 mo; HR = 0.49 [0.34-0.71], p &amp;lt; 0.001), with similar predictive power to gLOH &amp;gt;16% (HR = 0.55 [0.38-0.79], p = 0.001) and GIS &amp;gt;42 (HR = 0.59 [0.41-0.86], p = 0.006). For 62 patients with prostate cancer treated with PARPi (mlHRD positive = 27; negative = 35), mlHRD was associated with prolonged TTD on PARPi (median 6.8 mo v 3.4 mo; HR = 0.56 [0.30-1.03], p = 0.064), trending more predictive than gLOH &amp;gt;8.29% (Sokol 2020) and GIS &amp;gt;42 (HR = 0.64 [0.29-1.40] and 0.80 [0.37-1.73], respectively; p&amp;gt;0.05). Conclusion: These findings suggest that HRD is associated with genomic scarring beyond ovarian cancer. Additional retrospective and prospective analyses in clinical datasets are needed to explore the utility of this signature. Citation Format: Emmanuel Antonarakis, Jay Moore, Dexter Jin, Tim Chen, Justin Newberg, Zoe Fleischmann, Karthikeyan Murugesan, Garrett Frampton, David Fabrizio, Russell Madison, Ethan Sokol. Development of a pan-cancer algorithm to predict homologous recombination deficiency and sensitivity to PARPi therapy [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 1249.

Abstract 1260: Nonclinical evaluation of rucaparib in tumors with mutations in non-BRCA1/2 homologous recombination repair (HRR) genes

Abstract Background: Rucaparib is a PARP inhibitor approved to treat ovarian cancer (OC) and metastatic castration-resistant prostate cancer (mCRPC). Deficiencies in HRR resulting from genetic or epigenetic alterations in BRCA1/2 sensitize tumors to rucaparib through synthetic lethality. Inactivation of other HRR genes may also confer sensitivity to PARP inhibition. Here, we evaluated rucaparib efficacy in preclinical models with genomic or epigenetic alterations in an HRR gene panel. Methods: BRCA1/2 and 14 additional genes were selected for their role in DNA repair and mutation frequency in OC and mCRPC. Small interfering RNA (siRNA) knockdowns were performed in OC (OVCAR-3, OAW28, SK-OV-3) and mCRPC (DU145, PC-3) cell lines to model their inactivation and assess impact on rucaparib sensitivity (IC50) in cell viability assays. Patient-derived xenografts (PDX) harboring deleterious alterations in BRCA1/2, BARD1, BRIP1, NBN, PALB2, RAD51B, RAD51C, or RAD51D, or BRCA1/RAD51C promoter hypermethylation were treated with up to 150 mg/kg rucaparib BID for 25-42 days and tumor growth was monitored. Mutational status was confirmed by next-generation sequencing. Results: In addition to BRCA1/2 knockdown, depletion of BARD1, PALB2, or RAD51 resulted in potent rucaparib activity in OC and mCRPC cells, where the rucaparib IC50 was reduced by &amp;gt;70% vs control in at least 4 of the 5 cell lines tested. Rucaparib also displayed greater potency in cells with siRNA-mediated decreased expression of FANCA, NBN, RAD51C, or RAD54L, where at least 1 cell line had &amp;gt;50% decrease in IC50. Rucaparib treatment in 19/33 HRR-deficient PDX models resulted in significant tumor growth inhibition (TGI). These included BRCA1/2, PALB2, NBN, RAD51B, RAD51C, or RAD51D-mutated and BRCA1/RAD51C-hypermethylated models. Efficacy was strongly associated with biallelic inactivation of BRCA1/2, PALB2, RAD51C, or RAD51D with 87% vs 29% mean TGI for monoallelic BRCA1/2 alterations (P&amp;lt;0.0001). In addition, a single PDX model each for the exploratory genes NBN and RAD51B had 101% and 80% TGI, respectively. No significant difference was observed between PDX models with BRCA1/2 vs non-BRCA1/2 (PALB2, NBN, RAD51B, RAD51C, or RAD51D) biallelic alterations (98% vs 80% mean TGI, respectively; P=0.21). In support of these nonclinical findings, we highlight a patient with RAD51B-mutant mCRPC treated with rucaparib in the TRITON2 trial (NCT02952534) who had confirmed radiographic and PSA responses. Conclusions: Cell lines and PDX models with biallelic alterations in HRR genes other than BRCA1/2, including RAD51B, RAD51C, RAD51D, PALB2, and NBN, are comparably sensitive to rucaparib. Rucaparib is being evaluated in the LODESTAR trial (NCT04171700) in patients with tumors associated with deleterious alterations in BRCA1/2, PALB2, RAD51C, and RAD51D (cohort A) and BARD1, BRIP1, FANCA, NBN, RAD51, and RAD51B (exploratory cohort B). Citation Format: Liliane Robillard, Kevin K. Lin, Andrea Loehr, Tanya Kwan, Rachel Dusek, Andrew D. Simmons, Thomas C. Harding, Brieuc Sautois, Minh Nguyen. Nonclinical evaluation of rucaparib in tumors with mutations in non-BRCA1/2 homologous recombination repair (HRR) 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 1260.