Genome-wide Loss Of Heterozygosity Research Articles

Pan-Cancer Analysis of Copy-Number Features Identifies Recurrent Signatures and a Homologous Recombination Deficiency Biomarker to Predict Poly (ADP-Ribose) Polymerase Inhibitor Response.

Copy-number (CN) features reveal the molecular state of cancers and may have predictive and prognostic value in the treatment of cancer. We sought to apply published CN analysis methods to a large pan-cancer data set and characterize ubiquitous CN signatures across tumor types, including potential utility for treatment selection. We analyzed the landscape of CN features in 260,333 pan-cancer samples. We examined the association of 10 signatures with genomic alterations and clinical characteristics and trained a machine learning classifier using CN and insertion and deletion features to detect homologous recombination deficiency signature (HRDsig) positivity. Clinical outcomes were assessed using a real-world clinicogenomic database (CGDB) of comprehensive genomic profiling linked to deidentified, electronic health record-derived clinical data. CN signatures were prevalent across cancer types and associated with diverse processes including focal tandem duplications, seismic amplifications, genome-wide loss of heterozygosity (gLOH), and HRD. Our novel HRDsig outperformed gLOH in predicting BRCAness and effectively distinguished biallelic BRCA and homologous recombination-repair wild-type (HRRwt) samples pan-tumor, demonstrating high sensitivity to detect biallelic BRCA in ovarian (93%) and other HRD-associated cancers (80%-87%). Pan-tumor prevalence of HRDsig was 6.4%. HRRwt cases represented a significant fraction of the HRDsig-positive cohort, likely reflecting a population with nongenomic mechanisms of HRD. In ovarian and prostate CGDBs, HRDsig identified more patients than gLOH and had predictive value for poly (ADP-ribose) polymerase inhibitor (PARPi) benefit. Tumor CN profiles are informative, revealing diverse processes active in cancer. We describe the landscape of 10 CN signatures in a large pan-cancer cohort, including two associated with HRD. We trained a machine learning-based HRDsig that robustly identified BRCAness and associated with biallelic BRCA pan-tumor, and was predictive of PARPi benefit in real-world ovarian and prostate data sets.

Determining PARP Inhibition as a Treatment Strategy in Melanoma Based on Homologous Recombination Deficiency–Related Loss of Heterozygosity

There is a lack of effective treatments for immunotherapy-refectory melanoma. Although PARP inhibitors (PARPi) are an effective treatment strategy in cancers with homologous recombination deficiency (HRD), determining HRD status is challenging in melanoma. Here, we chart the longitudinal relationship between PARPi response and HRD scores derived from genome-wide loss of heterozygosity (LOH) in 4 patients with metastatic melanoma. When next examining 933 melanoma cases, using an updated threshold, we observed HRD-related LOH (HRD-LOH) in nearly one-third of all cases compared with <10% using traditional gene panels. Taken together, HRD-LOH in refractory melanoma is both a common occurrence and a potential biomarker for response to PARPi.

Penile squamous cell carcinoma (PSCC) with elevated tumor mutational burden (TMB): A genomic landscape study.

5044 Background: TMB has emerged as a novel biomarker of immune checkpoint inhibitor (ICPI) response in a wide variety of malignancies, but not yet in PSCC. Methods: 397 clinically advanced PSCC underwent hybrid capture based comprehensive genomic profiling (CGP) to evaluate all classes of genomic alterations (GA). TMB was determined on up to 1.1 Mb of sequenced DNA and categorized into three groups: <10 mutations/Megabase [muts/Mb] (low), 10-19 muts/Mb (high), and >20 muts/Mb (very high). MSI was determined on 114 loci. Genome-wide loss of heterozygosity (gLOH) was determined using validated pipelines and PD-L1 expression by IHC (TPS and Dako 22C3). Statistical comparisons utilized the Bonferonni correction method. Results: There were 339 (85.4%) TMB low, 40 (10.1%) TMB 10-19 and 18 (4.5%) TMB very high PSCC cases. The mean age of PSCC with very high TMB at 70.1 yrs was older than TMB low at 63.4 yrs (p=.08). The GA per tumor frequencies ranged from 5.4 to 5.5 in the 3 groups. There were no significant differences in genomic ancestry among the 3 groups. The TMB 10-19 and TMB very high tended to feature an APOBEC genomic mutational signature more than the TMB low PSCC cases (74 and 76% vs 44%). MSI high status was absent in the TMB low PSCC, but was present in 7.5% in the TMB 10-19 and 11.8% in the TMB very high cases. gLOH levels above 16% were similar in all 3 groups and ranged from 6.2 to 9.4%. GA associated with differences in TMB status in the PSCC cases included higher PIK3CA GA in TMB 10-19 (40.0%) vs TMB low (18.3%; p=.035) and TMB very high (66.7%) vs low (p=.0002). CDKN2A GA were higher in TMB low (45.7%) than the combined TMB 10-19 + very high (25.9%; p=.049). GA in KMT2D were higher in the combined TMB 10-19 + very high (29.3%) than TMB low (7.7%; p=0002). FGFR3 GA were similar in all 3 groups. In total, 6.5% of all GA were predicted to be of germline nature. The highest proportions of germline GA were seen for HRR genes ATM and CHEK2 (both 40%) and BRCA2 (37.5%) and PMS2 (40%) linked to Lynch syndrome. PD-L1 expression was not significantly different among the 3 groups with TMB low (78.3%), TMB 10-19 (64.2%) and TMB very high (54.5%). HPV identification was more frequent as TMB increased: 28.3% for TMB low, 50.0% for TMB high and 58.8% for TMB very high groups. Conclusions: The evaluation of PSCC by CGP based on TMB levels reveals significant differences in biomarkers for the near 15% of cases that have TMB ≥10 muts/Mb. TMB and MSI status can also serve as biomarkers of response to SOC anti-PD1 based on the current FDA indications. These data may be useful to provide rationale for inclusion of PSCC in basket or umbrella trials testing novel monotherapies or combination therapies. [Table: see text]

Role of BRCA1 promotor methylation (BRCA1meth) in homologous recombination deficiency (HRD) in high-grade epithelial ovarian cancer.

e17592 Background: In TCGA data BRCA1meth was found to be inferior to BRCAmut regarding prognosis in high-grade ovarian cancer. This was explained by a probably lower sensitivity to platinum-based chemotherapy. Methods: In 151 high-grade epithelial ovarian cancers (HGOC) tested for BRCA status, BRCA DNA methylation was determined using MethyLight PCR. Additionally, genome-wide loss of heterozygosity (LOH) and Aneuploidy Normalized Telemetric imbalance (ANTI) by means of SNP-Array analysis (GSA-24 v 3.0/Ilumina) as surrogates for “genomic scaring” in BRCA-independent HRD. For both parameters a “clinical-threshold” was calculated on the basis of platinum-response determined at the first recurrence according to the platinum-free interval. Both variables were integrated in a common HRD-Score with a cut-off value of ≥ 10.3 for HRD positivity. Results: In the present cohort PFS of BRCA1meth HGOC (n = 15; 10%) was not revealed to be inferior to BRCAmut HGOC. Worst PFS was observed in the subgroup of BRCAwt-unmethylated HGOC (p = 0.013) as compared to HGOC with genetic and epigenetic BRCA alterations. In line, neither LOH nor ANTI and consecutively also HRD-Score did prove to be statistically different in BRCAmut - and BRCA1meth-HGOC. However, BRCAwt-unmethylated HGOC exhibited significantly lower HRD-, LOH- and ANTI-Scores when compared with BRCAmut (p < 0.001) and BRCA1meth-HGOC (p < 0.005). All but one of the HGOC classified as platinum-refractory or -resistant at first relapse (92%) were BRCAwt-unmethylated. None of the BRCA1meth cancers were rated platinum-refractory or -resistant at progression and only one of the BRCAmut cancers was associated with platinum-resistant first relapse. 99% of the recurrences from HGOC with genetically or epigenetically altered BRCA genes were platinum sensitive, from which 78% were classified as “high-sensitive”. In multivariate analysis, HRD-Score was found to be of independent prognostic significance regarding PFS (p = 0.022); this was mainly due to the prognostic power of ANTI-Score (multivariate: p = 0.014) but not to that of genome wide LOH (univariate: p = 0.771). Conclusions: HGOC without genetic or epigenetic BRCA alterations exhibit the most unfavorable PFS. This is related to impaired platinum sensitivity. From a clinical point of view BRCA1 promotor methylation seems to confer an equivalent detrimental effect on the homologous recombination DNA repair mechanism than do pathogenic mutations of the BRCA genes. This is corroborated by a nearly identical extent of genome-wide scaring in both cancer subgroups.

Abstract P5-02-45: An HRD scoring system based on long-focal copy number alterations predictive of PARP inhibitor response

Abstract Homologous recombination deficiency (HRD) is characterized by a defective double-stranded DNA repair mechanism due to alterations in the homologous recombination (HR) pathway. Deleterious mutations in HR pathway genes can help identify potential responders to platinum-based chemotherapies and PARP inhibitors (PARPi), which promote apoptosis in HRD cells. Until recently, the use of PARPi in breast cancer (BC) was limited to advanced or metastatic disease with pathogenic or likely pathogenic germline BRCA (gBRCA) mutations. However, the use of PARPi has expanded to treat early-stage HER2-negative BC with a high risk of recurrence and gBRCA mutations, HER2-negative BC with somatic BRCA mutations, and triple-negative BC. To date, no universal method for HRD scoring is accepted; therefore, biomarkers are needed to stratify patients into PARPi responders and nonresponders more effectively. Because PARPi are used to treat HRD-driven cancers, we aimed to identify genomic consequences of HRD. For HRD-specific features, we calculated the proportion of long-focal total copy number alterations (LF-tCNA), which estimates amplification events for long-focal segments. Aneuploidy is especially prevalent in medullary, metaplastic, and invasive micropapillary BCs. These copy number variations are not necessarily consequences of HRD but can influence HRD scores. Genome-wide loss of heterozygosity (gwLOH) can be a result of HRD and used as a biomarker for HRD. We developed several scoring methods based on LF-tCNA, gwLOH, and aneuploidy scores. These scores were tested as predictors of HRD, defined as known loss-of-function germline mutations in BRCA1, BRCA2, PALB2, or BARD1 (TCGA-BRCA cohort, n = 1,032). To calculate the optimal HRD score, we used multivariate logistic regression analysis with HRD as an outcome and LF-tCNA, LOH, and ploidy as predictors. Based on the results of logistic regression, LF-tCNA and ploidy were selected to calculate HRD status. The positive and negative predictive values (PPV, NPV) were used to set the upper and lower thresholds, respectively. Samples with HRD scores above 7 (PPV) have the greatest potential for PARPi and platinum-based therapy response, while scores below -1 (NPV) were considered HRD wild type (WT). To validate the developed HRD score, we tested the score by combining BC cohorts (MSK_NCI and MET500 cohorts, n = 164) and used gBRCA1/2 pathogenic mutations as an outcome. Our HRD scoring system distinguished HRD WT from HRD-positive samples (AUC = 0.81) more effectively than a previously reported score based on LOH, telomeric-allelic imbalance (TAI), and large-scale state transitions (LST) (AUC = 0.72). We tested our HRD scoring system across tumor microenvironment molecular subtypes. The median HRD score in the basal-like subtype was increased compared to other subtypes, supporting the prevalence of gBRCA1/2 pathogenic mutations in this subtype. Moreover, the number of HRD-positive patients and the percent positive agreement, defined as the proportion of positive test results from our HRD also positive for HR mutations, were calculated for each BC subtype. Only 22% of basal-like HRD-positive samples also carried germline HR pathway mutations, which indicates 78% of HRD-positive patients who might benefit from PARPi or platinum treatment would have been missed by germline HR gene panels. This BC HRD scoring method is a promising tool for identifying HRD patients who may respond to PAPRi and platinum-based therapies, but additional studies are required for clinical validation. Citation Format: Maria Gurileva, Nikita Kotlov, Anna Love, Alexandra Melnikova, Krystle Nomie, Nathan Fowler, Alexander Bagaev. An HRD scoring system based on long-focal copy number alterations predictive of PARP inhibitor response [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 P5-02-45.

Penile squamous cell carcinoma (PSCC) with elevated tumor mutational burden (TMB): A genomic landscape study.

4 Background: TMB has emerged as a major biomarker of efficacy in immune checkpoint inhibitor (ICPI) therapies in the neoadjuvant, adjuvant and metastatic disease setting in a wide variety of malignancies, but not in PSCC. Methods: 397 clinically advanced (local major recurrence and/or metastatic disease) PSCC underwent hybrid capture-based comprehensive genomic profiling (CGP) to evaluate all classes of genomic alterations (GA). Tumor mutational burden (TMB) was determined on up to 1.1 Mb of sequenced DNA and microsatellite instability (MSI) was determined on up to 114 loci. Trinucleotide mutation signatures were evaluated (Alexandrov, et al. 2013). Genome-wide loss of heterozygosity (gLOH) was determined using validated pipelines and excluding whole-arm and whole-chromosome events. TMB was categorized into three cohorts: <10 mutations/Megabase [muts/Mb] (low), 10-19 muts/Mb (high), and >20 muts/Mb (very high). Tumor cell PD-L1 expression was determined by IHC (Dako 22C3) and defined as tumor proportion score (TPS) >1. The presence of HPV16/18 was determined by next generation sequencing (NGS). Statistical comparisons were corrected for multiple comparisons using the Bonferonni method. Results: There were 339 (85.4%) TMB low, 40 (10.1%) TMB 10-19 and 18 (4.5%) TMB very high PSCC cases in this study. The mean age of PSCC with very high TMB at 70.1 yrs was older than for TMB low at 63.4 yrs (p=.08). There were no significant differences in genomic ancestry among the 3 groups. The TMB 10-19 and TMB very high tended to feature an APOBEC genomic mutational signature more than the TMB low PSCC cases (74 and 76% vs 44%). MSI high status was absent in the TMB low PSCC, but was present in 7.5% of the TMB 10-19 and 11.8% of the TMB very high cases. gLOH levels above 16% were similar in all 3 groups and ranged from 6.2 to 9.4%. GA associated with differences in TMB status in the PSCC cases included higher PIK3CA GA in TMB 10-19 (40.0%) vs TMB low (18.3%; p=.035) and TMB very high (66.7%) vs TMB low (p=.0002). CDKN2A GA were higher in TMB low (45.7%) than in the combined TMB 10-19 + very high (25.9%; p=.049). GA in KMT2D were higher in the combined TMB 10-19 + very high (29.3%) than the TMB low PSCC (7.7%; p=0002). FGFR3 GA were similar in all 3 groups. PD-L1 expression was not significantly different among the 3 groups with TMB low (78.3%), TMB 10-19 (64.2%) and TMB very high (54.5%). HPV identification was more frequent as TMB increased: 28.3% for the TMB low, 50.0% for the TMB high and 58.8% for the TMB very high groups. Conclusions: The evaluation of PSCC by CGP based on TMB levels revels significant differences in biomarkers for the near 15% of cases that have TMB >10 muts/Mb. Further study of TMB as a biomarker in ICPI-based clinical trials for advanced PSCC appear warranted.

Clinical and molecular characteristics of ARIEL3 patients who derived exceptional benefit from rucaparib maintenance treatment for high-grade ovarian carcinoma

ObjectiveARIEL3 (NCT01968213) is a placebo-controlled randomized trial of the poly(ADP-ribose) polymerase inhibitor rucaparib as maintenance treatment in patients with recurrent high-grade ovarian carcinoma who responded to their latest line of platinum therapy. Rucaparib improved progression-free survival across all predefined subgroups. Here, we present an exploratory analysis of clinical and molecular characteristics associated with exceptional benefit from rucaparib. MethodsPatients were randomized 2:1 to receive rucaparib 600 mg twice daily or placebo. Molecular features (genomic alterations, BRCA1 promoter methylation) and baseline clinical characteristics were evaluated for association with exceptional benefit (progression-free survival ≥2 years) versus progression on first scan (short-term subgroup) and other efficacy outcomes. ResultsRucaparib treatment was significantly associated with exceptional benefit compared with placebo: 79/375 (21.1%) vs 4/189 (2.1%), respectively (p < 0.0001). Exceptional benefit was more frequent among patients with favorable baseline clinical characteristics and with carcinomas harboring molecular evidence of homologous recombination deficiency (HRD). A comparison between patients who derived exceptional benefit from rucaparib and those in the short-term subgroup revealed both clinical markers (no measurable disease at baseline, complete response to latest platinum, longer penultimate platinum-free interval) and molecular markers (BRCA1, BRCA2, RAD51C, and RAD51D alterations and genome-wide loss of heterozygosity) significantly associated with exceptional benefit. ConclusionsExceptional benefit in ARIEL3 was more common in, but not exclusive to, patients with favorable clinical characteristics or molecular features associated with HRD. Our results suggest that rucaparib can deliver exceptional benefit to a diverse set of patients with recurrent high-grade ovarian carcinoma.

Mitonuclear genotype remodels the metabolic and microenvironmental landscape of Hürthle cell carcinoma.

Hürthle cell carcinomas (HCCs) display two exceptional genotypes: near-homoplasmic mutation of mitochondrial DNA (mtDNA) and genome-wide loss of heterozygosity (gLOH). To understand the phenotypic consequences of these genetic alterations, we analyzed genomic, metabolomic, and immunophenotypic data of HCC and other thyroid cancers. Both mtDNA mutations and profound depletion of citrate pools are common in HCC and other thyroid malignancies, suggesting that thyroid cancers are broadly equipped to survive tricarboxylic acid cycle impairment, whereas metabolites in the reduced form of NADH-dependent lysine degradation pathway were elevated exclusively in HCC. The presence of gLOH was not associated with metabolic phenotypes but rather with reduced immune infiltration, indicating that gLOH confers a selective advantage partially through immunosuppression. Unsupervised multimodal clustering revealed four clusters of HCC with distinct clinical, metabolomic, and microenvironmental phenotypes but overlapping genotypes. These findings chart the metabolic and microenvironmental landscape of HCC and shed light on the interaction between genotype, metabolism, and the microenvironment in cancer.

PARP Inhibitor Insensitivity to BRCA1/2 Monoallelic Mutations in Microsatellite Instability-High Cancers.

PURPOSETo examine the overlap of homologous recombination deficiency (HRD) and microsatellite instability high (MSI-H) status, and to dissect driver versus bystander status of BRCA1/2 mutations (BRCAm) in this context.METHODSA pan-cancer comprehensive genomic profiling cohort (n = 213,199) was examined for overlap between BRCAm and MSI-H status. BRCA1/2 variant zygosity was examined and correlated with MSI-H status, tumor mutational burden, and genome-wide loss of heterozygosity (gLOH). Clinical histories of two patients with prostate cancer with co-occurring BRCAm and MSI-H are described.RESULTSHRD and MSI-H phenotypes were generally mutually exclusive events (P < .001). BRCAm that co-occurred together with high tumor mutational burden or MSI-H were predominantly monoallelic bystander alterations. In breast, ovarian, and pancreatic cancers, very few BRCAm occurred in the context of MSI-H; however, in prostate cancer, 12.8% of BRCA1 and 3.4% of BRCA2 alterations co-occurred with MSI-H. In these BRCA-associated cancers, co-occurring BRCAm were generally monoallelic and were not associated with elevated gLOH. Two patients with prostate cancer with co-occurring BRCAm and MSI-H showed resistance to poly (ADP-ribose) polymerase inhibition but sensitivity to subsequent anti–programmed cell death protein 1 therapy.CONCLUSIONMSI-H status and HRD are generally mutually exclusive phenomena across cancer types, but may rarely co-occur, especially in prostate cancer. Although MSI-H samples had a higher BRCAm prevalence relative to microsatellite-stable tumors, these BRCA1/2 mutations were generally monoallelic and were not associated with elevated gLOH. Our findings suggest that most BRCAm coexisting with microsatellite instability are likely bystander events that may not result in sensitivity to poly (ADP-ribose) polymerase inhibitors.

Performance and clinical utility of homologous recombination deficiency (HRD) determined by genome-wide loss of heterozygosity (LOH).

e15025 Background: Homologous recombination deficiency (HRD) status plays an important role in identifying patients with ovarian cancer likely to benefit from Poly (ADP-ribose) polymerase inhibitor (PARPi) and platinum-based chemotherapies. Methods: ACTHRD is an NGS-based assay was designed to determine the homologous recombination deficiency (HRD) status based on the extent of genome-wide loss of heterozygosity (gLOH) and the detection of BRCA1/2 genes alteration. More than that, ACTHRD also assesses the mutational status of 22 homologous recombination repair (HRR) genes. We evaluated the performance and clinical utility of the ACTHRD and compared it with the FDA-approved companion test myChoice CDx. Results: Of the 36 specimens assayed, 24 were determined to be HRD, and 10 were determined to be homologous recombination repair proficient (HRP). The results showed high concordance with myChoice CDx with 100% positive percent agreement (PPA) and 90.91% negative percent agreement (NPA) in the HRD status estimation. Except one case showed HRD in the ACTHRD but showed homologous recombination proficiency (HRP) in the comparator panel. This patient responded to platinum-based chemotherapy and remained disease-free for up to 56 months. One BRCA1/2 wild-type patient was identified as HRD by myChoice CDx but not eligible for ACTHRD test due to low tumor cellularity experienced refractory 13 months after the initiation of the treatment. We further found that ovarian cancer patients identified as HRD by ACTHRD had significantly prolonged progression-free survival (PFS) after being treated with platinum-containing chemotherapies, regardless of BRCA1/2 mutational status. Conclusions: The ACTHRD assay provides high accuracy for identifying deleterious BRCA1/2 alterations and determining the HRD status of ovarian cancers. The analytical robustness of the test has been validated and indicates its suitability for clinical use. Notably, the prognosis data demonstrates that ACTHRD can predict eligible patients for platinum-based chemotherapy and may also be sensitive to PARPi treatment.

Abstract PD4-09: Comprehensive assessment of the genomic landscape of breast cancer brain metastases reveals targetable alterations and genomic signatures relevant to immune-checkpoint and PARP inhibitors

Abstract Background: Central nervous system (CNS) disease secondary to breast cancer (BC) is a growing clinical problem and a cause of significant morbidity and mortality. A better understanding of the genomic landscape is key to understanding its cause and to developing novel treatments. In this study, we examined the genomic landscape of a large cohort of breast cancer brain metastases (BMs) and compared them to a cohort of local breast cancers (BCs) and non-CNS metastases (N-CNS). Material and Methods: We analyzed 822 BMs and compared them to 11,988 local, breast-biopsied BCs and 15,516 N-CNS (unpaired samples) with comprehensive genomic profiling (CGP) for all classes of alterations in at least 324 genes (Foundation Medicine, Cambridge, MA, USA). Homologous recombination deficiency detected by genome-wide loss of heterozygosity (HRD-gLOH; cutoff 16%), tumour mutation burden (TMB; cutoff 10 mutations/Mb), microsatellite instability (MSI) and PD-L1 prevalence and expression by VENTANA SP142 immune cell immunohistochemistry (IHC; cutoff 1%) was also investigated within and across the cohorts. Results: As compared to local BCs, 31 genes were significantly enriched for genomic alterations in BMs with BM prevalence &amp;gt;3% and false-discovery rate (FDR) &amp;lt;0.1. The most enriched genes were: TP53 (71.8%), MYC (25.9%), ERBB2 (24.6%), PTEN (16.7%), CDKN2A and RB1 (10.3% each), NF1 (8.6%), GNAS (7.9%), BRCA1, CDKN2B and ZNF217 (7.8% each), ARID1A (7.3%), CCNE1 (6.7%), ESR1 (6.4%), MAP2K4 (5.8%), AURKA (5.5%) and PIK3R1 (5.1%). Of these 31 genes, 26 were also enriched between BM vs N-CNS. The BM-enriched. genes that lost significance in comparison to N-CNS were: ZNF217, ARID1A, MAP2K4, AURKA and ARFRP1. PIK3CA prevalence was BMs: 30.2%, BCs: 31.8% and N-CNS: 38.9% (no significant difference between cohorts). HRD-gLOH was higher in BMs: 52.0% vs BCs: 33.7% and N-CNS: 31.5%. The prevalence of immune checkpoint inhibition (ICPI) biomarkers in BM were as follows: TMB-High 15.5%, MSI-High 2.0%, PD-L1 (CD274) 3.5% and its paralogue PDCD1LG2 (PD-L2, CD273) 3.4%. All were significantly higher in the BM vs primary BC and N-CNS group (p&amp;lt;0.05, FDR&amp;lt;0.1 for all comparisons). PD-L1 copy number (CN) variation was also higher in BMs: 3.4% vs BCs: 1.6% and N-CNS: 1.1%. PD-L1 positive expression was lower in BMs: 38.7% vs BCs: 54.3% but higher than N-CNS: 26.2%. Higher PD-L1 positive expression was observed in ER-/HER2- BMs: 48.3% vs BCs: 50.0% vs N-CNS: 21.4%. Table 1 summarizes the genomic signatures and PD-L1 positivity in the BC, BM and N-CNS cohorts and within different receptor subtypes. Conclusion: A high prevalence of clinically-relevant genomic alterations in BM is identified in comparison to the local BCs and/or N-CNS disease. A high proportion of BMs have high HRD-gLOH, TMB, MSI and PD-L1 alterations rendering them potentially amenable to treatment with ICPIs and PARP inhibitors. These data reinforce the importance of acquiring BM material and/or cerebrospinal fluid for CGP to enable rationale treatment decisions as well for enrolment into genomically driven BM studies of targeted agents. Table 1.Prevalence (%) of the genomic signatures HRD-gLOH, TMB, MSI and PD-L1+ expression.Site% HRD-gLOH% TMB-H% MSI-H% PDL1+ALLlocal BC33.70%. (2573/7636)5.04%. (527/10464)0.37%. (39/10464)54.25%. (1092/2013)BM52.01%. (362/696)15.51%. (116/748)2.01%. (15/748)38.73%. (55/142)N-CNS31.55%. (3237/10260)9.82%. (1395/14210)0.23%. (32/14210)26.21%. (615/2346)ER+/HER2-local BC31.48%. (119/378)3.41%. (19/557)0.36%. (2/557)40.00%. (14/35)BM43.17%. (79/183)11.40%. (22/193)1.04%. (2/193)23.08%. (9/39)N-CNS29.65%. (126/425)8.92%. (51/572)0.17%. (1/572)12.99%. (10/77)ER+/HER2+local BC19.57%. (9/46)2.78%. (2/72)0.00%. (0/72)42.86%. (3/7)BM26.67%. (16/60)9.68%. (6/62)0.00%. (0/62)27.27%. (3/11)N-CNS47.37%. (18/38)19.57%. (9/46)0.00%. (0/46)14.29%. (1/7)ER-/HER2+local BC32.26%. (10/31)8.70%. (4/46)0.00%. (0/46)100.00%. (5/5)BM33.33%. (23/69)21.33%. (16/75)2.67%. (2/75)57.14%. (4/7)N-CNS34.38%. (11/32)4.26%. (2/47)0.00%. (0/47)42.86%. (3/7)ER-/HER2-local BC52.25%. (186/356)2.70%. (14/518)0.19%. (1/518)50.00%. (30/60)BM70.45%. (174/247)17.41%. (47/270)2.59%. (7/270)48.33%. (29/60)N-CNS43.52%. (84/193)10.04%. (27/269)0.74%. (2/269)21.43%. (6/28) Citation Format: Athina Giannoudis, Ethan Sokol, Shakti H Ramkissoon, Talvinder Bhogal, Kimberly McGregor, Allison Clark, Evangelia D Razis, Rupert Bartsch, Richard SP Huang, Carlo Palmieri. Comprehensive assessment of the genomic landscape of breast cancer brain metastases reveals targetable alterations and genomic signatures relevant to immune-checkpoint and PARP inhibitors [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD4-09.

Homologous recombination repair pathway alterations and their relationship to homologous recombination deficiency in advanced pancreatic cancer patients.

609 Background: Homologous recombination repair (HRR) is critical for limiting DNA damage arising from double strand breaks. Disrupting HRR has emerged as a therapeutic strategy in pancreatic cancer given the development of PARP inhibitors that can specifically target tumors with homologous recombination deficiency (HRD). While HRD is strongly associated with loss-of-function mutations in BRCA1/2, a broad range of pathogenic alterations have been identified in other HRR genes. Identification of pathogenic alterations that predict response to PARP inhibition remains a critical knowledge gap in the field. Methods: We retrospectively analyzed de-identified records from 895 patients with advanced pancreatic cancer that underwent next generation sequencing (NGS) with the Tempus|xT assay (DNA-seq of 648 genes at 500x coverage, matched normal, full transcriptome RNA-seq). HRD-positivity and genome-wide loss of heterozygosity (LOH) were compared across groups, which were defined based on alterations to BRCA1/2 or other HRR pathway genes. Results: We identified 293/895 (33%) pancreatic cancer patients to be HRD-positive. Among HRD positive patients, 23/895 (2.6%) tumors harbored two alterations in BRCA1/2 (BRCA++), 179/895 (20%) harbored a single alteration in BRCA1/2 (BRCA+), 25/895 (2.8%) harbored two alterations in at least one other non-BRCA HRR gene (other HRR++), 367/895 (41%) harbored a single alteration in one other HRR gene (other HRR+), and 143/895 (16%) had pathogenic alterations in neither BRCA1/2 nor other HRR genes. In terms of HRD status, 23/23 (100%) of BRCA++ cases, 179/337 (53%) of BRCA+ cases, 4/25 (16%) of other HRR++ cases, and 87/367 (24%) of other HRR+ cases were HRD-positive. We observed no cases that were HRD-positive without an alteration in either BRCA1/2 or other HRR gene. Within the other HRR+ group, we found that single alterations in CHEK1/2, FANCA/L, MRE11, PALB2, and RAD51B were all significantly associated with HRD-positivity whereas CDK12, for instance, was not (Table). Conclusions: Comprehensive genomic profiling demonstrates that approximately 1 in 4 patients without BRCA alterations may potentially benefit from PARP inhibition due to alterations in other HRR genes. Future studies may examine the role of PARP inhibition in this population of HRR+ pancreatic cancer patients.[Table: see text]

Pan-cancer Analysis of Homologous Recombination Repair-associated Gene Alterations and Genome-wide Loss-of-Heterozygosity Score.

To study associations across tumor types between genome-wide loss of heterozygosity (gLOH) and alterations in homologous recombination repair (HRR)-associated genes beyond BRCA1 and BRCA2. Genomic profiling using a targeted next-generation sequencing assay examining 324-465 genes (FoundationOne, FoundationOne Heme, and FoundationOne CDx; Foundation Medicine, Inc.) was performed in a cohort of 160,790 samples across different tumor types. Zygosity predictions and gLOH status were calculated and linked with alterations in 18 HRR-associated genes (BRCA1, BRCA2, PALB2, BARD1, ATR, ATRX, ATM, BAP1, RAD51B, RAD51C, RAD51D, BRIP1, NBN, CHEK1, CHEK2, FANCA, FANCC, MRE11) and other genomic features, using Fisher's exact test and Mann-Whitney U tests. We identified a strong correlation between elevated gLOH and biallelic alterations in a core set of HRR-associated genes beyond BRCA1 and BRCA2, such as BARD1, PALB2, FANCC, RAD51C, and RAD51D (particularly in breast, ovarian, pancreatic, and prostate cancer). Monoallelic/heterozygous alterations in HRR-associated genes were not associated with elevated gLOH. gLOH was also independently associated with TP53 loss. Co-occurrence of TP53 loss and alterations in HRR-associated genes, and combined loss of TP53-PTEN or TP53-RB1, was associated with a higher gLOH than each of the events separately. Biallelic alterations in core HRR-associated genes are frequent, strongly associated with elevated gLOH, and enriched in breast, ovarian, pancreatic, and prostate cancer. This analysis could inform the design of the next generation of clinical trials examining DNA repair-targeting agents, including PARP inhibitors.

Integrated, Integral, and Exploratory Biomarkers in the Development of Poly(ADP-Ribose) Polymerase Inhibitors.

In this article, we highlight biomarkers for poly(ADP-ribose) polymerase inhibitor (PARPi) sensitivity and resistance and discuss their implications for the clinic. We review the predictive role of a range of DNA repair genes, genomic scars, mutational signatures, and functional assays available or in development. The biomarkers used for patient selection in the specific Food and Drug Administration-approved indications for breast, ovarian, prostate, and pancreatic cancer vary across tumor type and likely depend on disease-specific DNA repair deficiencies but also the specifics of the individual clinical trials that were conducted. Mutations in genes involved in homologous recombination and/or replication fork protection are synthetic lethal with PARPi. Cancers with homologous recombination deficiency exhibit high genomic instability, characterized by genome-wide loss of heterozygosity, among other genomic aberrations. Next-generation sequencing can identify multiple patterns of genomic changes including copy number variations, single-nucleotide variations, insertions/deletions, and structural variations rearrangements characteristic of homologous recombination deficiency. Clinical trial evidence supports the use of BRCA mutation testing for patient selection, and for ovarian cancer, there are 3 commercial assays available that additionally incorporate genomic instability for identifying subgroups of patients that derive different magnitudes of benefit from PARPi therapy. Finally, we summarize new strategies for extending the benefit of PARPi therapy toward broader populations of patients through the use of novel biomarkers. Ultimately, design of a composite biomarker test combining multiple mutational signatures or development of a dynamic assay for functional assessments of homologous recombination may help improve the test accuracy for future patient stratification.

Characterization of patients with long-term responses to rucaparib treatment in recurrent ovarian cancer

ObjectiveTo describe molecular and clinical characteristics of patients with high-grade recurrent ovarian carcinoma (HGOC) who had long-term responses to the poly(ADP-ribose) polymerase (PARP) inhibitor rucaparib. MethodsThis post hoc analysis pooled patients from Study 10 (NCT01482715; Parts 2A and 2B; n = 54) and ARIEL2 (NCT01891344; Parts 1 and 2; n = 491). Patients with investigator-assessed complete or partial response per RECIST were classified based on duration of response (DOR): long (≥1 year), intermediate (6 months to <1 year), or short (<6 months). Next-generation sequencing was used to detect deleterious mutations and loss of heterozygosity (LOH) in tumors. ResultsOverall, 25.3% (138/545) of enrolled patients were responders. Of these, 27.5% (38/138) had long-term responses; 28.3% (39/138) were intermediate- and 34.8% (48/138) were short-term responders. Most of the long-term responders harbored a BRCA1 or BRCA2 (BRCA) mutation (71.1%, 27/38), and BRCA structural variants were most frequent among long-term responders (14.8%; 4/27). Responders with HGOC harboring a BRCA structural variant (n = 5) had significantly longer DOR than patients with other mutation types (n = 81; median not reached vs 0.62 years; HR, 0.21; 95% CI, 0.10–0.43; unadjusted p = 0.014). Among responders with BRCA wild-type HGOC, most long- and intermediate-term responders had high genome-wide LOH: 81.8% (9/11) and 76.9% (10/13), respectively, including 7 with deleterious RAD51C, RAD51D, or CDK12 mutations. ConclusionAmong patients who responded to rucaparib, a substantial proportion achieved responses lasting ≥1 year. These analyses demonstrate the relationship between DOR to PARP inhibitor treatment and molecular characteristics in HGOC, such as presence of reversion-resistant BRCA structural variants.

Homologous recombination deficient endometrial serous carcinoma: implications for PARPi therapy

<h3>Objectives:</h3> Endometrial serous carcinoma (EMSC) is an aggressive variant of uterine cancer with limited therapeutic options. We analyzed the genomics of the largest EMSC cohort to date (n=2,159) to determine the extent of homologous recombination deficiency (HRD) and potential for targeted therapies. <h3>Methods:</h3> EMSC was assayed via hybrid capture-based comprehensive genomic profiling. HRD status was determined using genome wide loss of heterozygosity (gLOH) scores with validated methods for PARP inhibitor effectiveness in ovarian carcinoma. gLOH-high was defined as a score of ≥16%, similar to previously described for ovarian carcinomas. Tumor mutational burden (TMB) was determined on 0.8Mbp of sequenced DNA, and TMB-high was defined as ≥10mut/Mb using an FDA-approved companion diagnostic cutoff for pembrolizumab in patients with unresectable or metastatic solid tumor based on the KEYNOTE-158 trial. Microsatellite instability (MSI) was determined on 95 loci. Clinicopathological and genomic data was centrally re-reviewed. <h3>Results:</h3> EMSC is characterized by <i>TP53</i> mutations (92%), microsatellite stability (99%), low tumor mutational burden (96%), and less frequent recurrent alterations in <i>PIK3CA</i> (35%), <i>PPP2R1A</i> (25%), <i>CCNE1</i> (19%), <i>ERBB2</i> (17%), <i>FBXW7</i> (17%) and <i>MYC</i> (12%). Evidence for HRD via high gLOH was identified in 21% of EMSC. There was no difference in age, MSI and TMB-high status between gLOH-high vs gLOH-low EMSC. However, alterations in <i>BRCA1/2</i> and HRD genes as a group were significantly enriched in gLOH-high EMSC (14% vs 2% and 23% vs 9%, both p<1E-8). gLOH-high EMSC exhibited significantly higher frequency of solid, pseudo-endometrioid or transitional-like (i.e. SET) variant morphology (52% vs 24%, p<0.05). Genomic ancestry calling revealed similar ancestry prevalence in HRD vs. non-HRD subgroups; however, in EMSC patients of African descent, there was an overall higher prevalence of <i>CCNE1</i> amplification (26%) and lower prevalence of <i>PIK3CA</i> (24%) and <i>PPP2R1A</i> (18%) alterations compared with patients of non-African descent (p<0.05). Finally, EMSC exhibited significantly higher frequency of gLOH-high (21% vs 8%, p=19E-24) as well as different genomic signatures (MSI-H 1% vs 28%; TMB-high 4% vs 33%, and distinct alteration frequencies of 134 genes, all p<0.05) compared with endometrioid-type endometrial carcinomas (n=2,346). <h3>Conclusions:</h3> EMSC has overall distinct genomic signatures compared to endometrioid endometrial carcinomas with a significant proportion of gLOH-high EMSC, compatible with HRD. Our study provides the framework for future studies to test the susceptibility of EMSC to PARP inhibitor therapy in a molecularly defined EMSC subgroup with evidence for HRD.

Abstract 2062: Comprehensive molecular profiling of DNA damage response (DDR) deficiencies in advanced solid tumors using a real-world genomic database

Abstract Purpose: DNA damage response (DDR) deficiency is a hallmark of cancer and can be exploited with therapeutic interventions such as the use of PARP inhibitors and PD-1/PD-L1 immune checkpoint inhibitors. To understand the relationship between clinically actionable DDR genomic alterations (GA) and DDR deficiency biomarkers, we investigated the distribution of the GA in the most common DDR genes and explored the association with DDR deficiency biomarkers by utilizing the Foundation Medicine comprehensive genomic database across different cancer types. These data may provide essential information to refine and define the clinical translational strategies for innovative cancer therapies in clinical development. Methods: Molecular profiles from a total of 269,371 clinically advanced and diverse malignancies were analyzed using FoundationInsightsTM. Genomic alterations including short variants, copy number alterations and rearrangements in 35 DDR genes, more complex biomarkers (tumor mutation burden [TMB], genome-wide loss of heterozygosity [gLOH], microsatellite instability [MSI] status) and PD-L1 expression were studied. Only functional alterations (known or likely oncogenic) were analyzed. Results: DDR gene alterations are frequent and non-uniformly distributed by type and frequency across cancer types including breast, ovarian, colorectal, esophagus, biliary track, gastric, head and neck, pancreas, melanoma, lung, bladder, and prostate. TP53 is the most frequently altered DDR gene across all cancers as expected (59%), followed by ARID1A (9%), ATM (4%), SMARCA4 (3%), MDM2 (3%), BRCA2 (3%), BRCA1 (2%), CHK2 (2%), MUTYH (2%), whereas many other DDR genes are rarely altered (≤1%). Excluding TP53 from the analysis, bladder cancer has the highest cumulative rate of cases with mutations in DDR genes (46.59%), while other cancers have rates ranging between 20-40%. Further analyses including the relative distribution of TMB, MSI, gLOH and PD-L1 expression by tumor type and their association with DDR GA are ongoing. Conclusion: Identifying associations of DDR alterations and immuno-oncology associated predictive biomarkers (TMB, PD-L1) as well as assessment of signatures of genomic instability (MSI, gLOH) are critical for enabling biomarker-driven precision oncology. We systematically analyzed somatic alterations using the Foundation Medicine comprehensive genomic database and provide a comprehensive molecular profiling of DDR deficiencies in advanced solid tumors. It is anticipated that this data will collectively contribute to identify molecularly defined tumor subsets, where single and/or combination therapies such as DDR inhibitors, immunotherapies, targeted therapies etc. may result in increased clinical benefit for patients with cancer. Citation Format: Danyi Wang, Brian Elenbaas, Karthikeyan Murugesan, Lee A. Albacker, Julien Doudement, Juergen Scheuenpflug, Giuseppe Locatelli, Zheng Feng. Comprehensive molecular profiling of DNA damage response (DDR) deficiencies in advanced solid tumors using a real-world genomic database [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2062.

Assessment of predictive biomarker prevalence in molecularly defined adult-type ovarian granulosa cell tumors.

5567 Background: The FDA has separately approved pembrolizumab for all advanced solid tumors with either microsatellite instability or high tumor mutational burden (≥10 mutations per megabase), and homologous recombination deficiency is an established biomarker for response to poly ADP ribose polymerase inhibitors in epithelial ovarian cancer. The detection of these predictive biomarkers in adult-type ovarian granulosa cell tumors could identify novel treatment strategies in this rare disease. The primary objective of this study was to determine the prevalence of established predictive biomarkers among molecularly defined adult-type ovarian granulosa cell tumors. Methods: With institutional review board approval, we performed a cross-sectional study examining de-identified FoundationOne companion diagnostic molecular profiles for 423 women with molecularly defined (FOXL2 c.C402G positive) adult type ovarian granulosa cell tumors. The dataset was comprised of coding variants for up to 406 genes as well as genomic signatures including microsatellite instability, tumor mutational burden, and genome wide loss of heterozygosity. PD-L1 expression by immunohistochemistry was also available for a subset of tumors. Descriptive statistics were used for comparison between groups and all statistical tests were two-sided. Results: Women in this cohort had a mean age of 57 years (range 24-87) at the time of sample submission for molecular profiling. The median tumor mutational burden was 1.3 mutations per megabase [mut/Mb] (range 0-8.8 mut/Mb). TP53-mutated aGCT had a higher tumor mutation burden than TP53 non-mutated tumors (median 2.4 mut/Mb, 95% CI 1.7-3.0 mut/Mb vs median 1.3 mut/Mb, 95% CI 1.5-1.9 mut/Mb; P=.02). All 384 tumors with available microsatellite instability testing were microsatellite stable. Sixty-seven tumors had PD-L1 expression measured and of these 94% (63/67) were negative with the remainder “low positive.” No tumors were positive for genome wide loss of heterozygosity. Apart from FOXL2 c.C402G, the most frequent short variants were TERT promoter mutations (-124C&gt;T: 190/423, 45.0%; -146C&gt;T: 39/423, 9.2%). Other frequently observed variants included truncating mutations in KMT2D/MLL2 (71/423, 16.8%), pathogenic TP53 mutations (35/423, 8.3%), CDKN2A/B deletions (43/423, 10.2%), and activating PIK3CA mutations (23/423, 5.4%). Conclusions: No women with molecularly defined adult-type ovarian granulosa cell tumors in this large cross-sectional study would be eligible for FDA-approved pembrolizumab based on either microsatellite instability or high tumor mutational burden. No tumors exhibited evidence of homologous recombination deficiency and molecularly targetable mutations were rare. The development of novel precision treatment options remains a critical unmet need for this rare disease.

Accumulation of genome-wide somatic loss of heterozygosity (LOH) as a prominent feature of advanced malignant soft tissue tumors and association with the BRCAness status, suppression of immune responses, and lower survival rates.

11533 Background: Malignant soft tissue tumor is a rare cancer with few therapeutic options. Recent genomic analysis revealed widespread CNA and the cumulative burden of cancer-related pathogenic germline mutations/variants, their clinical and therapeutic significance were unknown. Methods: We recruited 155 patients with advanced malignant soft tissue tumors (135 female and 20 male, mean age 51 at analysis, 100 LMS, 19 LPS, 4 ESS, 3 UPS, 3 AS, 3 MPT, 3 GIST and others) with confirmed metastasis/recurrence and information on familial cancer burden. Whole exome sequencing was performed in both blood and tumor samples as described in 2018ASCO. The copy number of BRCA2 gene was determined by the MLPA method. Tumor immune microenvironment was assessed by immunohistochemistry. The MSI status was analysed by PCR. Results: We analyzed the LOH status in 595 COSMIC genes and found that genome-wide accumulation of somatic LOH of polygenic germline mutations/variants. Patients with more than 33% LOH genes (n=102) in the total of somatic and LOH mutations showed significantly lower OS rates compared with those (n=53) with less LOH genes (5-year survival rates; 49 vs 75%, p=0.010), which constitute 78% of LMS (n=78/100) and 26% of LPS (n=5/19). Total of 41 patients (26%, n=41/155) including 33 LMS (33%, n=33/100) showed LOH in the BRCA2 locus with hemizygous VUS. Those patients with BRCA2 LOH (n=41) showed significantly lower OS rates compared with those without BRCA2 LOH (n=114) (5-year survival rates; 43 vs. 64%, p=0.019). Neither TMB nor the MSI status was associated with LOH. In contrast, accumulation of somatic LOH (mean LOH values of 71.7 vs. 15.7%) was clearly and negatively associated with CD8+T-cell immune infiltrates (T-cells; 44±23 vs 555±180/mm2, n=7, p=0.016), CD20+B-cell accumulation in tertiary lymphoid structures (TLS) (TLS; 0.57±0.43 vs. 20.1±6.1/tumor, n=7, p=0.008) and low levels of neutrophil-to-lymphocyte ratio (NLR) (NLR; 3.63±0.45 vs. 1.71±0.17, n=7, p=0.002), hallmarks of the immunological response to tumors. Conclusions: This study suggests that in advanced malignant soft tissue tumors, accumulation of genome-wide LOH of germline mutations/variants is associated with the BRCAness status and suppression of the immune responses to tumors, and thus influences therapeutic response and survival of the patients.

Clinical and molecular characteristics of ARIEL3 patients who derived exceptional benefit from rucaparib maintenance treatment for high-grade ovarian cancer (HGOC).

5537 Background: ARIEL3 is a placebo-controlled randomized trial of the PARP inhibitor (PARPi) rucaparib as maintenance treatment in HGOC patients (pts) who responded to the latest line of platinum therapy (NCT01968213). Rucaparib improved progression-free survival (PFS) across all predefined subgroups. Here, we present an exploratory analysis of clinical and molecular characteristics associated with exceptional benefit from rucaparib. Methods: Pts were randomized 2:1 to receive rucaparib 600 mg BID or placebo. At the data cutoff of Dec 31, 2019, 33/375 (9%) and 1/189 (0.5%) pts were still ongoing and receiving rucaparib or placebo, respectively. Molecular features (genomic alterations, BRCA1 promoter methylation) and baseline clinical characteristics were compared between pts who derived exceptional benefit (PFS ≥2 yrs), and those with disease progression on first scan (≈12 wks; the short-term [ST] subgroup) within each treatment arm. Results: Of 564 pts, 83 (15%) showed exceptional benefit: 79/375 (21%) in the rucaparib arm and 4/189 (2%) in the placebo arm. Within the rucaparib arm, exceptional benefit pts had more favorable clinical prognostic factors at baseline compared with the ST subgroup (Table). While BRCA mutations were enriched in the rucaparib exceptional benefit subgroup, 34/79 (43%) of these pts were BRCA wild type. Among other biomarkers, RAD51C/D mutations were associated with exceptional benefit; low genome-wide loss of heterozygosity was enriched within the ST subgroup; and high BRCA1 methylation was present at similar fractions. Trends were similar in the placebo arm (Table). Conclusions: Exceptional benefit in ARIEL3 was more common in, but not exclusive to, pts with favorable clinical characteristics and known mechanisms of PARPi sensitivity. Our results suggest that rucaparib can deliver exceptional benefit to a diverse set of HGOC pts. Clinical trial information: NCT01968213. [Table: see text]