Highlights d FOXA1 mutations are enriched in metastatic tumors d FOXA1 mutations are associated with worse outcome to aromatase inhibitors d Wing2 mutations promote an enhanced estrogen response upon estrogen d SY242CS induces alternative chromatin states by binding to a non-canonical motif

Background: Germline genetic alterations are established mediators of breast carcinogenesis, often giving rise to specific forms of genomic instability. BRCA1/2 pathogenic variants (PVs) are emblematic of this phenomenon through their induction of homologous recombination deficiency. While specific patterns of genomic instability may sensitize cancers to therapies such as PARP inhibitors (PARPi) or platinum chemotherapy, their implications for lineage-directed therapies such as endocrine therapy (ET) or CDK4/6 inhibitors (CDK4/6i) are unknown. Herein, we systematically investigated the patterns of association of germline alterations with specific somatic alterations and explored the resulting effect on clinical outcomes. Methods: Patients who underwent germline and matched tumor tissue sequencing utilizing MSK-IMPACT from April 2014 to May 2021 and had available germline analysis results were included. The final analysis presented at SABCS will include 6000 tumors from 5,150 patients, anonymized according to established institutional IRB guidelines to allow for germline analysis on the full cohort. We analyzed genomic data to inform the full spectrum of somatic and germline mutations, ploidy, and allele-specific copy number to determine loss of heterozygosity (LOH). We performed gene- and pathway-level enrichment analyses between somatic variants and germline PVs. Univariable and multivariable Cox proportional hazards models were constructed to assess the association of therapy-specific progression-free survival (PFS) with select germline PVs and germline-somatic interactions. Results: The preliminary analysis includes 2,798 tumors from 2,242 patients with germline and somatic sequencing results. The most frequent germline PVs were: BRCA2 (n = 81), BRCA1 (n = 67), CHEK2 (n = 57), ATM (n = 32), PALB2 (n = 19). The cohort robustly confirmed previously established relationships such as mutual exclusivity of gATM and TP53 variants (OR 0.10, 95% CI 0.032 - 0.33, q = 0.005). Alterations of TP53 were seen in 83% (56/67) of gBRCA1 patients; however, this did not achieve significance when adjusted for receptor subtype (OR 3.90, 95% CI 1.34-11.38, q = 0.15). The size of the cohort allowed discovery of several novel relationships. For instance, gBRCA2 loss was associated with alterations in TGF-B pathway components (OR 3.58, 95% CI 1.70 - 7.56, q = 0.002), potentially relevant to metastatic disease progression. PIK3CA mutations were significantly less prevalent in both gBRCA2 (OR 0.52, 95% CI 0.31 - 0.87, q = 0.063) and gBRCA1 PVs (OR 0.21, 95% CI 0.085 - 0.51, q = 0.014). Our analysis uncovered a strong association between gBRCA2 and somatic RB1 pathogenic alterations (OR 3.58, 95% CI 1.70 - 7.56, q = 0.011), with most variants (80%) encountered in metastatic gBRCA2 tumors. Given the essential role of RB1 in CDK4/6i response, we investigated the effect of BRCA2 status on clinical efficacy of CDK4/6i-ET. Strikingly, gBRCA2 PVs were significantly associated with inferior PFS (HR 2.17, 95% CI 1.46-3.22, p < 0.001) on first line treatment with CDK4/6i-ET. We posited the enrichment of somatic RB1 loss as a potential mechanism of resistance to CDK4/6i. Given the proximity of RB1 to BRCA2 on chromosome 13, we hypothesized that co-LOH of BRCA2 and RB1 predisposes the cancer cells to bi-allelic loss under therapeutic pressure of CDK4/6i. Indeed, 18/26 gBRCA2 (69.2%) tumors evaluable for allele-specific copy number had evidence of RB1 LOH. Discussion: Analysis of germline-somatic interactions yielded novel associations relevant to breast cancer progression and treatment resistance. Among these, we demonstrated BRCA2 carriers to have inferior outcomes to first line CDK4/6i-ET with potential implications for optimal first line therapy and sequencing of CDK4/6i vs PARPi in this patient population. Citation Format: Anton Safonov, Chai Bandlamudi, Paulino Tallón de Lara, Emanuela Ferraro, Fatemeh Derakhshan, Marie Will, Mark Donoghue, Pier Selenica, Joshua Drago, Ezra Rosen, Carlos dos Anjos, Elaine Walsh, Elizabeth A Comen, Mehnaj Ahmed, Barbara Acevedo, Ahmet Zehir, Michael F Berger, David Solit, Larry Norton, Ronglai Shen, Zsofia Stadler, Simon Powell, Jorge S Reis-Filho, Sarat Chandarlapaty, Mark Robson, Pedram Razavi. Comprehensive genomic profiling of patients with breast cancer identifies germline-somatic interactions mediating therapy resistance [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 GS4-08.

Background: HER2 positive (HER2+) breast cancers harboring downstream MAPK or PI3K pathway alterations manifest persistent downstream signaling on anti-HER2 inhibitors with metastatic patients having worse outcomes on first line trastuzumab and pertuzumab (HP) therapy. However, HER2 antibody-drug conjugates (ADCs) are not as dependent upon potent signal transduction inhibition to exert their antitumor effects. To further investigate, we sought to determine whether MAPK and/or PI3K alterations affect the biologic or clinical outcomes of patients and models receiving HER2 ADCs. Methods: We performed prospective genomic sequencing using MSK-IMPACT on patients with advanced HER2+ breast cancer who received trastuzumab emtansine (T-DM1) in the metastatic setting between March 2013 and July 2021. We collected detailed information on clinical outcomes and correlates through our institutional IRB-approved retrieval process. HER2/ER/PR status at the time of metastatic recurrence were defined as per ASCO/CAP guidelines. Cox proportional hazard models were used to determine the association between MAPK and PI3K pathways alterations and progression-free survival (PFS) on T-DM1. Common mutations associated with outcomes were modeled in HER2+ breast cancer cell lines using short hairpin RNAs and CRISPR/Cas9, and the sensitivity to HER2 ADC was evaluated via cell proliferation and xenograft assays. Results: We identified 185 HER2+ breast cancer patients treated with T-DM1 at any line (median: 5) whose primary (N=65) or metastatic (N=120) tumor samples were sequenced. Median age was 55 (range: 20-87). The majority of the patients received T-DM1 in 2nd or 3rd line (52%) and received prior trastuzumab or HER2 TKI in metastatic setting (96%). 74/185 (40%) had de novo metastatic breast cancer and 119/185 (64%) had ER/PR+/HER2+ disease. Pathogenic activating alterations involving the MAPK pathway were observed in 14% of patients with the most frequent alterations being ERBB2 activating mutations (42%) and NF1 loss (34%). PI3K pathway alterations were identified in 42% of the patients, the majority being activating mutations of PIK3CA (87%). MAPK alterations were significantly enriched in the metastatic tumors compared to the treatment-naïve primaries (20% vs 3%, p=0.001), while PI3K alterations were not (44% vs 40%, p=0.6). To reduce the possible confounding resistance mechanisms induced by prior treatment, we restricted the survival analyses to patients who received T-DM1 up to 3rd line of therapy (N=100). On multivariable analysis adjusted for ER/PR status (positive vs negative), stage at the presentation of metastatic disease (de novo metastatic vs recurrence), treatment line and type of sequenced sample (primary vs metastatic), patients with MAPK (N=14) and PI3K (N= 38) alterations had similar PFS compared to wild type (HR 1.20, 95%CI 0.62-2.30, p=0.6 and HR 1.23, 95%CI 0.77-1.95, p=0.4, respectively). Similar results were found in the combined analysis including alterations in either pathway (N=48, HR 1.28, 95%CI 0.81-2.04, p=0.3). To verify the antiproliferative effect of HER2 ADCs on breast cancer cells with MAPK pathway activation, we depleted NF1 in a panel of HER2+ breast cancer cell lines. Consistently, MAPK-altered cell lines were sensitive to FDA-approved HER2 ADCs including trastuzumab deruxtecan (T-DXd). Conclusions: In contrast to H/P therapy, T-DM1 therapy was equally effective in tumors with downstream PI3K or MAPK alterations and wild type tumors. Expanded analysis on a larger cohort, including a subgroup of patients treated with novel HER2 ADCs such as T-DXd will be presented. The characterization of PI3K and MAPK pathways status in metastatic HER2+ breast cancer may inform prioritization of treatment options. Citation Format: Emanuela Ferraro, Anton Safonov, Yuan Chen, Charlie White, Antonio Marra, Mehnaj Ahmed, Barbara Acevedo, Chau T Dang, Shanu Modi, David B. Solit, Larry Norton, Mark E. Robson, Jorge Reis-Filho, Sarat Chandarlapaty, Pedram Razavi. Efficacy of HER2 ADCs against HER2 inhibitor resistance alterations in the PI3K and MAPK pathways in HER2-positive breast cancer [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 P4-02-01.

Background: Oncogenic mutations involving PIK3CA gene (PI3Kα) are observed in over 40% of patients with hormone receptor-positive (HR+) advanced breast cancer (ABC). Clinical efficacy of PI3K inhibition was demonstrated in a recent trial, but data on the natural history of disease by PIK3CA status are limited in ABC. We assessed patient and disease characteristics, treatment patterns, and clinical outcomes among patients with ABC treated at the Memorial Sloan Kettering Cancer Center in the United States, prior to the clinical availability of alpelisib (a PI3Kα inhibitor). Methods: A retrospective medical record review of adult patients diagnosed with HR+ and human epidermal growth factor receptor 2-negative (HER2-) ABC between January 2014 and December 2018 was conducted. Data collection is ongoing and updated results will be presented. Cohorts were determined by eligible patients’ PIK3CA gene status (i.e., mutant or wild-type [WT]), and were frequency matched to have an equal distribution of the year of ABC diagnosis between cohorts. Study measures were descriptively summarized, and the Kaplan-Meier method was used to estimate progression-free survival (PFS) and left truncation-adjusted overall survival (OS), in months. To assess the association of PIK3CA mutation status on OS, a multivariate Cox proportional hazard model adjusted for age, race, stage at diagnosis, menopausal status, visceral disease, bone only disease, and Charlson Comorbidity Index score was computed. Additional adjustment on treatment modalities is planned. Results: Data for 249 PIK3CA-mutant and 143 PIK3CA-WT patients was analyzed. For the mutant cohort, the mean (SD) age was 59.0 (11.2) years, 80.7% were White, 30.9% were diagnosed with de novo ABC, and 54.6% were postmenopausal at initial BC diagnosis. Visceral- and bone only-disease were observed among 41.4% and 21.7% of patients, respectively. Among patients with a single PIK3CA mutation (n=220, 88.4%), the most common mutations were H1047R (37.3%), E545K (20.5%), and E542K (12.7%). Multiple PIK3CA mutations were observed in 29 (11.6%) patients. For the WT cohort, the mean (SD) age was 56.3 (12.1) years, 77.6% were White, 39.9% were diagnosed with de novo ABC, and 45.5% were postmenopausal at initial BC diagnosis. Visceral- and bone-only disease were observed among 51.8% and 16.8% of patients, respectively. Most common 1st-line treatments for ABC were cyclin-dependent kinase 4 and 6 inhibitor (CDK4/6i) + aromatase inhibitor (AI) (mutant: 38.6%; WT: 40.6%), AI only (mutant: 21.3%; WT:22.4%), CDK4/6i + fulvestrant (mutant: 15.7%; WT: 7.7%), and chemotherapy-based regimens (mutant: 9.6%; WT: 11.2%). Chemotherapy-based regimens were the most common 2nd- (mutant: 25.6%; WT: 21.3%) and 3rd-line (mutant: 41.4%; WT: 33.9%) therapies for both cohorts. Results for PFS and OS are presented in Table 1, and the survival risk was similar for both cohorts (adjusted HR: 1.18, 95% CI: 0.88-1.60). Conclusion: In this preliminary analysis of HR+/HER2- ABC, CDK4/6i-based regimens were the most common 1st-line therapy, and PFS to 1st-3rd line therapies and OS were not statistically significantly different. The recent availability of PI3K inhibitor therapy may improve clinical outcomes among patients with PIK3CA-mutant tumors. Table 1.Progression-free Survival and Overall Survival Among Patients with HR+/HER2- ABC.PIK3CA MutantPIK3CA Wild-typeProgression-free Survival (months)1st line – all patients (n)249143Median (95% CI)12.4 (10.4-15.2)13.5 (10.3-16.0)1st line – CDK4/6i + AI (n)9658Median (95% CI)21.0 (17.0-27.9)19.1 (9.4-28.6)2nd line – all patients (n)211127Median (95% CI)7.3 (6.3-10.4)6.8 (5.4-8.8)2nd line – chemotherapy-based regimens (n)5427Median (95% CI)5.1 (3.7-6.3)5.2 (3.3-6.3)3rd line – all patients (n)186115Median (95% CI)4.5 (3.7-5.4)5.7 (4.7-8.9)3rd line – chemotherapy-based regimens (n)7739Median (95% CI)4.3 (3.0-5.1)4.7 (3.0-7.7)Overall Survival (months)Median (95% CI)47.4 (42.8-52.4)55.5 (44.9-61.9) Citation Format: Pedram Razavi, Rohan C Parikh, Barbara Acevedo, Abigail Hitchens, Mehnaj Ahmed, Anton Safonov, Emanuela Ferraro, Komal Jhaveri, Michelle Sidel, Stacey Simmons, Mark Robson, Sarat Chandarlapaty, Sanjeev Balu. Real-world natural history data among patients with PIK3CA-mutant and PIK3CA-wild-type advanced breast cancer [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 P1-18-18.