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Background: The CLEOPATRA trial established trastuzumab, pertuzumab and a taxane (THP) as a standard of care for first line metastatic, HER2-positve breast cancer with median progression-free survival (PFS) of 18.7 months and median OS of 57 months. NRG BR004 was a phase III, placebo-controlled trial designed to determine whether the addition of the PD-L1 inhibitor, atezolizumab, to THP would improve progression-free survival (PFS), relative to THP/placebo in patients with newly documented HER2-positive measurable metastatic breast cancer. Methods: BR004 was designed to detect an improvement in the primary endpoint of PFS in patients with measurable disease from 16.5 to 22.5 months with addition of atezolizumab (HR 0.733). A sample size of 600 would provide 80% power with a type I error rate of 0.05 to detect such an improvement when 326 PFS events had been reported. Monthly accrual was projected at 30 patients per month with completion of accrual in 24 months. In addition to routine monitoring of safety data by the IDMC every 6 months, a formal analysis of the toxicity data was to be performed 16 weeks after the 100th patient had been randomized with review by the IDMC. Results: First patient was randomized on May 1, 2019, and after 37 months 190 patients had been randomized. Several amendments were not successful in addressing the low accrual rate. The IDMC began regular monitoring of safety and accrual data in July 2020 and reviewed the formal safety analysis in February 2022. As of the February 2022 IDMC meeting, four Grade 5 adverse events (AEs) had been reported (2 occurring in 2020 and 2 in 2021), one of which occurred in a patient with evolving liver failure due to rapid disease progression at the start of therapy. The recommendation was to continue without modification, but notice was given the Grade 5 AEs had occurred on the same treatment arm without unblinding. When additional Grade 5 AEs occurred on 3/4/2022 and 4/27/2022 both on the same study arm with none reported on the other arm, accrual was held until the IDMC could review updated safety data, narratives of the Grade 5 AEs and the overall context of the trial. There was no evidence of clinically important imbalances between Grade 3 and Grade 4 AEs between the arms., Based on an uncertain but material safety signal, the ongoing accrual challenges, and determination that the clinical question being addressed was no longer sufficiently compelling, the IDMC recommended that the trial should be permanently closed to further enrollment. Summary safety data from 187 treated patients are provided in the Table. A decision was made to discontinue atezolizumab/placebo in patients receiving the investigational component of the trial therapy and unblind investigators and patients. The study will continue to collect information on PFS events, deaths and late immune AEs through April of 2024 when PFS and OS will be analyzed. Conclusions: The imbalance in Grade 5 AEs which occurred on BR004 coupled with continued poor accrual and the changing landscape in HER2+ MBC resulted in early closure of enrollment and unblinding of patients. Follow-up continues to assess PFS, OS and monitor for delayed immune AEs. Support: U10CA180868, -189867, -180822; U24CA196067; and Genentech. Citation Format: Charles E. Geyer, Jr, Gong Tang, Priya Rastogi, Vicente Valero, Stephen K. Chia, Erin F. Cobain, Elias Obeid, David B. Page, Andrew S. Poklepovic, William J. Irvin, Jr., Adam M. Brufsky, Irene L. Wapnir, Jennifer M. Suga, Eleftherios (Terry) Mamounas, Norman Wolmark. Safety Analyses of NRG BR004: A Randomized, Double-blind, Phase III Trial of Taxane/Trastuzumab/Pertuzumab with Atezolizumab or Placebo in First-line HER2-Positive Metastatic Breast Cancer (MBC) [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 OT2-16-05.

Background: Racial disparities among patients with breast cancer in the United States are well documented, with Black patients having a 40% higher mortality than White patients. Socioeconomic status, tumor biology differences, and treatment access are known contributing factors. Additionally, despite having a higher incidence of TNBC, Black patients are substantially less likely to receive gBRCA1/2mut testing than White patients. TNBC is an aggressive subtype, accounting for 15% of all breast cancers diagnosed in the United States. TNBC disproportionately affects BRCA mutation carriers and Black women. With the advent of poly ADP-ribose polymerases inhibitors (PARPi), gBRCA1/2mut are considered actionable biomarkers. Limited data are available on the impact of race on clinical outcomes among patients with gBRCA1/2-mutated advanced TNBC in the United States. Methods: US oncologists retrospectively reviewed patient charts (July 2019-June 2020) of a quasirandom selection of patients aged ≥18 years with gBRCA1/2-mutated advanced TNBC who received ≥1 cytotoxic chemotherapy (CT) regimen for advanced TNBC between Jan 2013-April 2018. Patients were categorized into 2 mutually exclusive cohorts of White and Black (based on self-identification). Descriptive analysis was performed for treatment patterns for the first 2 lines of therapy (LOT). Clinical outcomes (progression-free survival [PFS] by LOT and survival rates) were estimated with the Kaplan-Meier method. A log-rank test was used to assess differences in PFS and survival rates between White and Black patients. Results: Among 182 patients with gBRCA1/2-mutated advanced TNBC, 99.5% were women and 76.4% were White. The median age was 57.2 years (range, 48.7-64.7 years), and 30.2% had no known family history of BRCA-related cancer. Treatments for White patients with advanced TNBC included first-line (n=139) nonplatinum-based CT (61.9%) and platinum-based CT (38.1%) and second-line (n=90) PARPi (40.0%), nonplatinum-based CT (43.3%), platinum-based CT (11.1%), and other (5.6%). Treatments for Black patients with advanced TNBC included first-line (n=43) nonplatinum-based CT (60.5%) and platinum-based CT (39.5%) and second-line (n=19) PARPi (47.4%), nonplatinum-based CT (31.6%), platinum-based CT (10.5%), and other (10.5%). Across treatment types, no significant difference in 2-year survival rates was observed between White and Black patients (73.8% vs 73.2%, P=0.89). Median PFS rates by LOT were not statistically different across White and Black patients (Table 1). Conclusion: In this real-world study, no significant differences in clinical outcomes were observed between White and Black patients with gBRCA1/2-mutated advanced TNBC. This observation may be because this sample reflects a select patient population with a known genetic test result of a gBRCA1/2mut who were treated by oncologists that understood the value of genetic testing, and provided appropriate treatment options. The findings suggest that when all patients are provided with equitable care (inclusive of genetic testing), racial disparities in breast cancer may be minimized. Further trials are needed to validate these findings. Funding: Pfizer Inc Table 1.PFS (Months) by LOT and Race Among Patients With gBRCA1/2-Mutated Advanced TNBCWhite PatientsBlack PatientsP ValueFirst-line, n 13642PFS, median (95% CI)10.7 (9.3−12.6)15.6 (9.7−NE)0.078Second-line, n 5310PFS, median (95% CI)7.2 (5.9−11.4)9.2 (2.8−NE)0.406NE=not estimatable. Citation Format: Kristen Whitaker, Rohan Parikh, Elizabeth Esterberg, Bhakti Arondekar, Abigail Hitchens, Lillian Shahied Arruda, Alexander Niyazov, Elias Obeid. Impact of race on clinical outcomes among patients with advanced triple negative breast cancer (TNBC) and Germline BRCA1/2 mutation(s) (gBRCA1/2mut): Results from a US real-world study [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 P2-09-08.

Background: Inflammatory breast cancer (IBC) is characterized by a young age at diagnosis, high metastatic potential, and poor overall survival. To better understand the etiology of this aggressive disease, we performed a retrospective study to evaluate genomic alterations and tumor microenvironment in IBC patients. Methods: Targeted next generation sequencing (NGS) was performed using blood cell-free DNA (cfDNA) (n=32), paired DNA from tumor tissues (n=29) and DNA from whole blood or normal tissue (n=20). We also characterized the tumor infiltrating lymphocytes (TILs) in tumor tissue biopsies by studying several markers (CD4, CD8, FoxP3, CD20, PD-1 and PD-L1) through immunohistochemistry (IHC) staining. Results: Our results shown a high incidence of germline variants in patients with IBC that could be associated with an increased risk of developing the disease. Germline variants were found more frequently in patients with TN (38.9%) than non-TN IBC (28.6%). IHC staining revealed that in 7 of 24 (29%) patients, tumor biopsies were positive for PD-L1 and PD-1 expression on TILs. Patients with PD-L1&PD-1 positive biopsies had a lower OS (31 months) than patients with PD-L1&PD-1 negative (38 months) tumors, though the difference was not statistically significant. PD-L1&PD-1 positive tumors had significantly more CD8+ (cytotoxic T-cells), CD20+ (B-cells) and FoxP3+ (Tregs) cells than PD-L1&PD-1 negative ones. Furthermore, most IBC patients showed to have deficiencies in the homologous recombination repair (HRR) pathway (BRCA1, BRCA2, PALB2, RAD51C, ATM, BARD1) making them sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors. All tumor biopsies showed positive staining for p-FAK1(Tyr 397). Conclusion: A subset of IBC patients (29%) expressed both PD-L1 and PD-1 on tumor immune cells infiltrates, providing rationale for testing PD-1/PD-L1 blocking therapies. IBC patients could have benefits from PD-1/PD-L1 blocking therapies alone or in combination with PARP or FAK (Tyr397) kinase inhibitors, both having the capacity of altering the tumor microenvironment. Citation Format: Maria F. Arisi, Yulan Gong, Rajeswari Nagarathinam, Lorenzo Gerratana, Jianming Pei, Kathy Q. Cai, Jennifer Winn, Zachary Hasse, Elias Obeid, Julio Noriega, Christopher Sebastiano, Eric Ross, R. Katherine Alpaugh, Massimo Cristofanilli, Sandra V Fernandez. Genetic variants and tumor microenvironment in inflammatory breast cancer: Clues for targeted therapies [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 P1-24-02.

Background: gBRCA1/2mut represents~5% of ABC. Recently poly ADP-ribose polymerases inhibitors (PARPi) have demonstrated improved clinical outcomes, advantageous side effect profile, and favorable patient reported outcomes vs. chemotherapy(CT) in gBRCA1/2mut HER2- ABC patients. Optimal treatment sequencing has not been established. We assessed real-world treatment patterns and clinical outcomes by line of treatment (LOT) among adult patients with gBRCA1/2mut HER2- ABC. Methods: US Oncologists retrospectively reviewed charts (July 2019-June 2020) of quasi-random selected patients ≥18 y, with gBRCA1/2mut HER2- ABC who received ≥1 cytotoxic chemotherapy (CT)regimen(s) for ABC between Jan 2013-April 2018. Descriptive analyses were performed for treatment patterns for the first 3 LOTs. Clinical outcomes (PFS by LOT and survival rates) were estimated using the Kaplan-Meier method. PARPi clinical outcomes data was immature given its recent launch. Additional analyses evaluating outcomes in patients receiving PARPi are planned. Results: 305 HER2- gBRCA ABC patients were included: 99.7% were women, 76.4% were white, 11.5% were of Ashkenazi Jewish descent. Median age was 57.3 yrs, 36.4% had hormone receptor (HR)+/HER2-ABC, and 63.6% had advanced triple-negative BC (TNBC). Treatments for HR+/HER2-included: 1st line - CT (78.4%),endocrine based therapy (EBT) (19.8%), PARPi (0.9%), other (0.9%); 2ndline - EBT (51.2%), CT (31.0%), PARPi (13.1%),other (4.8%); 3rd line - CT (50.0%), EBT (27.8%), PARPi (19.4%). Across treatment types, 77.0% of patients were alive at 2 years after 1stCT. Treatment for TNBC included: 1st line - non-platinum based CT(60.8%), platinum based CT (39.2%), non-platinum based CT (60.8%); 2ndline - PARPi (42.0%), non-platinum based CT (40.2%), platinum-based CT (10.7%),other (7.1%); 3rd line - PARPi (43.5%), non-platinum based CT (39.1%), platinum-based CT (6.5%). Across treatment types, 73.9% of patients were alive at 2 years after 1stCT. Median PFS for HR+/HER2- and TNBC are reported in Table 1. Conclusion: CT was frequently used among both HR+ and HR- gBRCA1/2mut HER2-ABC. Recent clinical trials have demonstrated use of chemotherapy-sparing disease specific targeted treatments (e.g., PARPi) improved clinical, and patient reported outcomes, and had a manageable side effect profile. Such disease specific targeted therapies should possibly be considered in earlier LOTs in this patient population and is an area that could benefit from well-designed studies, particularly addressing this important research question. Therefore, future studies assessing treatment sequencing and the associated clinical outcomes including targeted treatments (i.e. PARPi) in patients with gBRCA1/2mut are warranted. Funding: Pfizer Table 1. PFS (months) by LOT and HR status among gBRCA1/2mut HER2- ABC patients.HR+/HER2-TNBC1st line (n)110190PFS, median no. (95% CI)12.1 (9.1-14.3)11.6 (9.9-13.6)2nd line (n)6864PFS, median no. (95% CI)14.2 (10.7-29.5)9.0 (6.2-11.4)3rd line (n)2926PFS, median no. (95% CI)6.5 (4.4-9.3)5.9 (3.5-not estimable) Citation Format: Elias Obeid, Rohan C Parikh, Elizabeth Esterberg, Bhakti Arondekar, Abigail Hitchens, Lillian Shahied Arruda, Alexander Niyazov. Treatment patterns and clinical outcomes among patients (pts) with HER2- advanced breast cancer (ABC) and germline BRCA1/2 mutation(s) (gBRCA1/2mut): results from a US real-world study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS11-42.

Background: gBRCA1/2mut ABC represents ~5% of all breast cancer (BC), although a fair estimate of gBRCA1/2mut HER2+ BC remains to be determined. HER2-targeted therapy is the current standard of care for these patients with HER2+ BC. While PARP inhibitors (PARPi) are approved for the treatment of gBRCA1/2mut HER2- ABC, recently, NCCN updated its guidelines (v6.2020) to additionally consider the use of PARPi in patients with gBRCA1/2mut HER2+ metastatic BC. However, clinical trials have not evaluated the efficacy of PARPi among patients with gBRCA1/2mut HER2+ metastatic BC. In order to establish a baseline reference point, we assessed real-world treatment patterns and clinical outcomes among patients with gBRCA1/2mut HER2+ ABC. Methods: US Oncologists retrospectively reviewed charts (July 2019-June 2020) of randomly selected patients ≥18 y, with gBRCA1/2mut ABC who received ≥1 cytotoxic chemotherapy (CT) regimen(s) for ABC between Jan 2013-April 2018. A descriptive analysis was performed for the HER2+ patients including 1st line ABC treatment patterns. Clinical outcomes (1st line ABC PFS rates) were estimated using the Kaplan-Meier method. PARPi clinical outcomes data were immature given its recent launch. Additional analyses evaluating outcomes in patients receiving PARPi are planned. Results: Of the 387 patients with gBRCA1/2mut ABC included in the study, 82 (21%) female patients had HER2+ disease. Of the gBRCA1/2mut HER2+ patients, median age was 56y, 72% were white. Clinical characteristics: 62% HR+/HER2+, 37% HR-/HER2+, and 1% had unknown HR/HER2+ ABC. Treatments in the 1st line setting for HR+/HER2+ ABC patients (n=51) included CT (55%) and CT + HER2-targeted therapy (43%). First-line treatments used for HR-/HER2+ ABC patients (n=30) included CT + HER2-targeted therapy (77%) and CT (20%). 12-month PFS rate for 1st line HR+/HER2+ patients was 77% and for HR-/HER2+ patients was 64%. Later line treatments will be presented. Conclusion: In this analysis of HER2+ patients with gBRCA1/2mut ABC, unexpectedly low rates of HER2-targeted therapy were observed in patients with HR+/HER2+ disease. Appropriately high rates of HER2-targeted therapy with CT was observed among gBRCA1/2mut HR-/HER2+ patients. Treatment patterns among patients with non-gBRCA1/2mut HER2+ ABC in a real-world setting need to be evaluated. Clinical outcome findings from this study demonstrate the need to better understand best treatment strategies for this patient population. As PARPi are not yet approved for patients with gBRCA1/2mut HER2+ ABC, studies assessing efficacy of PARPi +/- HER2-targeted therapy are warranted. Funding: Pfizer Citation Format: Elias Obeid, Rohan C Parikh, Elizabeth Esterberg, Bhakti Arondekar, Abigail Hitchens, Lillian Shahied Arruda, Alexander Niyazov. Treatment Patterns and Clinical Outcomes Among Patients with germline BRCA1/2 mutated (gBRCA1/2mut) HER2+ Advanced Breast Cancer (ABC): Results from a US Real-world Study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS10-53.

Background: Recently, somatic genetic information is being increasingly usedin clinical decisions for treatment of metastatic diseases, including breastcancer. Somatic mutations in the BRCA1/2 genes are not frequently seen.The poly ADP-ribose polymerases inhibitors (PARPi) have been approved astreatments for patients with germline (gBRCA1/2mut) HER2- ABC. Real-world practice shows that these agents arealso being utilized for patients who have sBRCA1/2mut. While we await data from large clinicaltrials such as MATVH and TAPUR, currently, limited information is available onthe effectiveness of these agents in patients with sBRCA1/2mut andestablishing a reference point is necessary to assess the unknown potentialclinical benefit. We assessed real-world treatment patterns and clinicaloutcomes among patients with HER2- sBRCA1/2mut without associatedgermline mutation and who had received treatment for ABC. Methods: US oncologists retrospectivelyreviewed charts (July 2019-June 2020) of quasi-randomly selected patients ≥18y, who received ≥1 cytotoxic chemotherapy (CT) regimen(s) for ABC between Jan2013-April 2018. Descriptive analyses were performed for patients with sBRCA1/2mut(without associated germline mutation), which included evaluation of treatment patternsand safety events for the first 3 lines of therapy. Clinical outcomes(progression free survival [PFS] and overall survival rates) were estimatedusing the Kaplan-Meier method for 1st line of therapy. Given therelatively recent launch of PARPi, clinical outcomes for these agents wereimmature. Results: 32 patients were included who only had sBRCA1/2mut: 96.9%were female, 68.8% were white, and 28.1% were black. Median age was 53.0 yrs. Advanced triplenegative breast cancer was observed in 40.6% of patients and 59.4% had hormonereceptor (HR)+/HER2- ABC. Common treatments for HR+/HER2- sBRCA1/2mutdisease included: 1st line (n=19), cyclophosphamide + doxorubicin (10.5%);2nd line (n=17), docetaxel (11.8%), PARPi monotherapy (11.8%), CDK4/6i + letrozole (11.8%); 3rd line- (n=7), eribulin (42.9%). Treatments for triplenegative sBRCA1/2mut breast cancer included: 1st line (n=13), cyclophosphamide + doxorubicin (30.8%);2nd line (n=9), PARPi (22.2%); 3rd line (n=7),atezolizumab based (71.4%). Median PFS across both subtypes was 10.8 months,95% CI 8.5 - 15.0. Overall, 78.9% of patients were alive at 2 years after 1stCT. Safety data will be presented. Conclusion: In this analysis of sBRCA1/2mut HER2- ABC, an individualapproach to select treatment regimens such as CT was frequently used, withoutadequate knowledge on best sequential therapy to include tumor genetic directedintervention. Poor clinical outcomes highlight the need for more efficacioustreatment options. Further studies assessingclinical outcomes among patients with sBRCA1/2mut ABC receivingPARPi are warranted. Citation Format: Elias Obeid, Rohan C Parikh, Elizabeth Esterberg, Bhakti Arondekar, Abigail Hitchens, Lillian Shahied Arruda, Alexander Niyazov. Treatment patterns and clinical outcomes among patients (pts) with HER2- advanced breast cancer (ABC) and somatic BRCA1/2 mutation(s) (sBRCA1/2mut): Results from a US real-world study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS10-36.

Background: PD-1 blockade has shown promising clinical activity in mTNBC. We designed an investigator-initiated multi-center clinical trial consisting of a Safety run-in, into a randomized phase II trial of combination pembrolizumab (P) with carboplatin (C) and gemcitabine (G) in patients with mTNBC. A detailed characterization of peripheral immune cell changes may help in understanding responses in mTNBC to immune checkpoint inhibition. Correlative studies for the purpose of understanding the effect of combining chemotherapy (CT) simultaneously with checkpoint inhibition on the peripheral immune response are planned as part of this clinical trial. Methods: Patients with a diagnosis of mTNBC are recruited to this trial with a Safety Run-in (N = 6 subjects), followed by a randomized design of C + G with/without P (2:1 randomization, N = 75). Treatment consists of P 200 mg on day 1 of each 21-day cycle, and C (AUC2) + G (800mg/m2) on days 1 and 8 for cohort A, and C (AUC2) + G (800mg/m2) for cohort B. Patients are consented for a peripheral blood (PB) collection pre-treatment on day 1 of cycle 1, and post-treatment on day 1 of cycle 3, in order to phenotype immune changes by flow-cytometry. Results: A total of 60 patients have been consented, 42 patients were enrolled (6 on safety run-in, 36 on the randomized part II). Of those on the randomized part II, blood samples from 17 patients have been analyzed from cycle 1 day 1 and cycle 3 day 3. A decreased expression of PD-1 on effector and memory subsets of both CD4+ and CD8+ T cells from C1D1 to C3D1 was observed in blood from both cohorts, especially cohort A. We found an increased % NK, CD4+ T and CD8+ T cell subsets expressing the activation marker, CD69, in cohort A, but not cohort B, reflecting activation by pembrolizumab.​ There was a parallel increased expression of the proliferative marker, Ki67, in CD56bright (immature) NK cells and effector memory CD8+ T cells in blood from C1D1 to C3D1 in cohort A. We also found an increased % CD56bright (immature) NK and effector memory CD4+ T cells expressing the exhaustion marker, CD39, which was consistent with increased expression of the activation marker, CD69. Conclusions: Although comprising a limited number of patients in this early analysis, our correlative studies found evidence for effective immune stimulation upon combining CT with the PD-1 blockade. The randomized nature of this trial, with our planned pre and post treatment blood collection will be helpful in understanding response to immune check point inhibition combined with chemotherapy in mTNBC. Correlation with absolute lymphocyte counts and outcomes data is planned and will be presented. Citation Format: Elias Obeid, Judy Fang, Alex W McFarlane, IV, Irina Shchaveleva, Shauna Souchuck, Kerry S Campbell, Lori J Goldstein. Title:peripheral blood immuneimpactsin arandomizedphase iiclinical trial ofprogrammed death 1 (pd-1) blockadecombined with platinum-based chemotherapy in patients with metastatic triple negative breast cancer (mtnbc) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS12-19.

Background: Invasive lobular carcinoma (ILC) is the second most common type of invasive breast cancer and accounts for 10-15% of all cases. Though ILC has distinct clinical, prognostic and molecular features, studies are limited and include few patients. ILCs show a decreased response to neoadjuvant chemotherapy and an increased resistance to endocrine therapy. Thus, there is a great need to identify alternative therapies, such as immunotherapy, that could improve overall survival. Success of immunotherapy largely depends on tumor immunogenicity which varies with histologic type. Determination of predictive and prognostic biomarkers for ILC will help determine who can benefit the most. Our study investigates canonical markers of immunogenicity - PD-L1 expression and Tumor Mutational Burden (TMB) - in patients with ILC compared to invasive ductal carcinoma (IDC). We further correlate these markers in different subtypes i.e. hormone receptor positive (HR+), HER2 positive (HER2+), and in triple negative breast cancers (TNBC). We also analyze differences in immune cell profiles constituting the tumor microenvironment (TME) and differences in genomic alterations between ILC and IDC. Methods: A retrospective data analysis was performed to identify breast cancer tumors with ILC or IDC histology profiled at Caris Life Sciences (Phoenix, AZ) that were tested for PD-L1 by SP142 assay and had whole transcriptome sequencing data available. ILC and IDC cases were further subtyped as HR+ and HER2+ and TNBC. PD-L1 expression in immune cell was assessed using Ventana PD-L1 (SP142) histochemical assay and PD-L1 expression in tumor cell was assessed by laboratory developed test using SP142 clone with staining higher than 2+ considered positive. TMB was measured by counting somatic non-synonymous missense mutations on the 592 gene panel (Nextseq) next generation sequencing (NGS) assay, and ≥ 10 mutations/megabase (mut/Mb) was considered high. Using the whole transcriptome RNA sequencing (NovaSeq) data we analyzed the difference in immune cell profiles constituting the TME using a computational RNA deconvolution approach. NGS was used to identify significant differences in genomic alterations between ILC and IDC. Results: We identified 1,359 breast cancer patients (ILC: 194 vs IDC: 1,165). Among ILC, 79% were HR +, 11% TNBC, 10% ILC: 1% vs. IDC: 5% p=0.017). PD-L1 expression in tumor cells was also lower in ILC (ILC 1% vs IDC 5%; p =0.0136). All subtypes of ILC had lower PD-L1 expression in immune and tumor cells when compared to IDC. TMB was similar and comparable in IDC and ILC (median TMB 7 mut/Mb). Assessment of TME showed a significantly increased abundance of cytotoxic lymphocytes and monocytic cells in IDC, and stromal endothelial cells in ILC. Androgen receptor (AR) expression, and mutations in CDH1 (ILC 76% vs IDC 2%) and PIK3CA (ILC 44% vs IDC 30%) were more common in ILC. CDH1 mutation was significantly higher in both TN ILC (ILC 60% vs IDC 1%; p Citation Format: Upama Giri, Joanne Xiu, Tatiana Karadimitriou, Sofi Castanon, Foluso Ademuyiwa, Paula Pohlmann, Elias Obeid, Jasgit Sachdev, Elisa Krill-Jackson, Michael Simon, Antoinette Tan, Neelima Denduluri, Lee Schwartzberg, Michael Korn, Evanthia T Roussos Torres. Molecular evaluation of immunogenicity and genomic alterations in invasive lobular breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-27.

Background: Up to 10% of breast cancers are hereditary. Pathogenic variants (PV) in the BRCA1/2 genes are responsible for two thirds of hereditary breast cancer (BC). Additional non-BRCA genes (ATM, CHEK2, PALB2, etc.) have been identified as predisposing for BC. While genetic testing(GT) for hereditary BC traditionally involved testing mostly for mutations in the BRCA1/2 genes, next generation sequencing has enabled multiple BC genes to be simultaneously tested at a cost often lower than testing for BRCA1/2 alone. Consequently, MGP testing has been rapidly replacing BRCA1/2 only testing. Studies have demonstrated that MGP testing doubles the detection of PV. Because prior studies examining MGP findings have been mostly conducted in predominantly white populations, it is unknown if the use of MGP testing in African-American (AA) patients also results in increased detection of non-BRCA mutations and whether there are significant differences in the prevalence of PV between AA and white women with invasive BC. In this study, we sought to compare the rate and spectrum of PV between AA and White women with invasive BC undergoing MGP testing. Methods: In this retrospective study, we assessed the prevalence of PV and spectrum of mutations among a cohort of 680 racially diverse women diagnosed with invasive BC who underwent GT with a MGP between 2014 and 2019 at a single cancer center. This analysis included participants who had a documented diagnosis of invasive BC and completed MGP testing. Fisher’s exact tests were used to analyze differences between racial groups. Subjects previously signed informed consent to participate in research. Results: 680 women with invasive BC were included in the study. The study population was 79.4% non-Hispanic white, 10.6% AA, and 10% other. PV were identified in 13.4 % of the overall population. The prevalence of PV was 15.5% (84/540) in AA compared to 8.3% (6/72) in white women, but this difference was not statistically significant (p=0.14). In white women, the prevalence of PV was 5.9% (32/540) in BRCA1/BRCA2 and 9.6% (52/540) in non-BRCA genes. MGP testing allowed for the detection of 52 non-BRCA PV, including BC genes (CHEK2-17, ATM-7, PALB2-4, CDH1-2, BARD1-1, TP53-1, RAD50-1) and non-BC genes (MUTYH-8, NTHL1-2, BRIP1-2, PMS2-2, RAD51C-1, RAD51D-1, MSH6-1, FH-1, MITF-1). In AA patients, the prevalence of PV was 6.9% in BRCA1/2 and 1.4% in non-BRCA genes. MGP testing in AA women with invasive BC detected only one non-BRCA mutation in the gene MUTYH. VUS rates were higher in AA, 29.2% compared to whites, 19.3% with a trend towards a statistically significant difference (p=0.07). Conclusions: While MGP testing more than doubled the detection rate of PV in white patients with BC by detecting various non-BRCA PV, for AA patients the use of MGP testing did not result in many additional non-BRCA PV being identified in this analysis. However, MGP testing did result in a high VUS rate, 19.3%, without the added value of detecting additional PV that could be helpful to the patient and their family members by expanding opportunities for cancer prevention and early detection. We acknowledge that in this study sample of 10.6% AA women, this is an under-representation. Large population-specific studies should be done to confirm those findings. Given the known psychological harms to the patient and their family members in response to the uncertainty of VUS findings as well as the possible mismanagement of VUS results, well-designed studies should investigate the selection of who to consider for MGP testing rather than BRCA1/2 testing for hereditary BC to ensure that the benefits of MGP testing over BRCA1/2 only testing outweigh the risks associated with panel testing. Citation Format: Kristen Whitaker, Nicole Ventriglia, Karthik Devarajan, Elias Obeid. Differences in the benefit of multi-gene panel (MGP) genetic testing between African American and white women with invasive breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS8-16.

Purpose: To define the prevalence of HER2 positive breast cancer (HER2+ BC) in women with gBRCA1/2-BC and to determine whether there are significant differences in the prevalence of HER2 positivity in BRCA1 mutation carriers compared to BRCA2 mutation carriers. Background: It is estimated that 5-10% of patients with breast cancer have a BRCA1 or BRCA2 mutation. Two poly ADP ribose polymerase inhibitors (PARPi) have been approved for metastatic HER2-negative gBRCA1/2-BC, as trials excluded those with HER2+ BC and gBRCA1/2 mutations. Most often, breast tumors in BRCA1 carriers lack the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 receptor or gene amplification (HER2). BRCA2 tumors have similar rates of ER positivity as sporadic controls, but are less likely to be HER2+. The prevalence of HER2 positivity among BRCA1 and BRCA2 mutation carriers has been variably reported in the literature. The prevalence of HER2 positivity has generally been reported as lower among BRCA1 carriers than BRCA2 carriers. The prevalence of HER2 positivity in BRCA1 carriers and BRCA2 carriers has ranged from 0-10% and 0-13% respectively. The true prevalence of HER2+ breast cancer in gBRCA1/2-BC remains poorly defined. Methods: In this study, we assessed the prevalence of HER2 positivity among a cohort of 137 BRCA1 and BRCA2 mutation carriers who consented for participation in a single institution risk assessment program registry between 2009 and 2019. To be eligible for this study, participants had to have a documented diagnosis of invasive breast cancer and be a confirmed BRCA1 or BRCA2 mutation carrier. Participants with all stages of invasive breast cancer were included. Fisher’s exact tests and univariate logistic regression were used to analyze differences between BRCA1 and BRCA2 mutation carriers. Results: 69 BRCA1 mutation carriers and 68 BRCA2 carriers were included in the study. Only 38 BRCA1 mutation carriers and 41 BRCA2 mutation carriers had complete HER2 data and were included in this analysis for prevalence. 12.6 % (10/79) of the study participants had HER2+ invasive breast cancer; 3 of these participants were BRCA1 carriers and 7 were BRCA2 carriers. The prevalence of HER2 positivity in BRCA1 carriers was 7.9%. The prevalence of HER2 positivity among BRCA2 carriers was 17.0% (7/41). In this small sample, the prevalence of HER2 positivity in BRCA 2 mutation carriers was numerically higher than that in BRCA1 mutation carriers, although this difference was not statistically significant (p= 0.3145). Conclusion: In this analysis, the rates of HER2 positivity among BRCA1 mutation carriers are consistent with the range of rates previously reported (0-10%). However, the rates of HER2 positivity in BRCA2 mutation carriers (17.0%) are higher than those previously reported (0-13%). This study included participants who received breast cancer treatment at our institution where their HER2 status was verified with a pathology report, but also participants who received their breast cancer treatment at outside institutions. In the latter, HER2 status most often was obtained by verbal self-reporting. It is possible that this led to over-reporting of positive HER2 status in either BRCA1 or BRCA2 mutation carriers. Thus, estimates of HER2 positivity might be slightly overestimated. Despite some variation in HER2 positivity rates among BRCA1 and BRCA2 mutation carriers, this study demonstrates that HER2 positive breast cancers may not be as rare in gBRCA1/2-BC as previously reported in initial studies (0-3% in either BRCA1 or BRCA2 carriers). Given that BRCA1/2 mutation carriers could potentially derive therapeutic benefit from drugs, such as PARPi, it is important that future studies include gBRCA1/2-BC with HER2 positive breast cancer. Citation Format: Kristen Whitaker, Elias Obeid, Lori Goldstein, Mary Daly. Prevalence of HER2 positivity in germline BRCA 1/2-associated breast cancers (gBRCA1/2-BC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-08-21.

Background: Recent data indicate a promising response to immune checkpoint inhibition in patients with metastatic TNBC. Ample research showed that PD-L1, a PD-1 ligand, is expressed in multiple tumor types, including TNBC, and may be a predictor of response to PD-1/PD-L1 blockade. Quantification of the stromal composition, particularly PD-1 and PD-L1 expression, continues to be controversial in its relationship to immune checkpoint inhibition in several cancer types, and it remains unclear whether PD-L1 expression is necessary to predict response. Here, we aimed to determine the distribution of PD-1 and PD-L1 in a large set of centrally ascertained specimens of TNBC. Methods: The study cohort consisted of 993 tumor samples (both primary and metastatic TNBC) analyzed for either PD-1 or PD-L1 expression in one laboratory (Caris Life Sciences; Phoenix, AZ). Estrogen receptor and progesterone receptor status was assessed by immunohistochemistry (IHC). HER2/Neu expression or amplification was assessed by either IHC or in-situ hybridization. PD-1 and PD-L1 expression were confirmed using IHC with validated antibodies. For PD-L1, clone SP142 (Roche Diagnostics) was utilized and a sample was considered positive if there was > 5% membranous staining of tumor cells. For PD-1, clone EH21.1 (BD Biosciences) was used. Tumor infiltrating lymphocytes (TILs) expressing PD-1 were counted and a sample was considered positive if there was at least one PD-1 positive TIL per 40x microscopic field. Results: The median age in this cohort was 56 years (range: 22 – 88). A total of 363 TNBC specimens were tested for PD-1 via IHC. One hundred fifty eight (158; 43.5%) were negative for PD-1 expression. Two hundred five (205; 56.5%) were positive for PD-1. Of those that were PD-1 positive, 116 (56.6%), were in samples from a primary site (breast) and 89 (43.4%) in samples from a metastatic site. A total of 630 TNBC specimens were tested for PD-L1 via IHC. Five hundred seventy four (574; 91.1%) were negative for PD-L1. Fifty-six (56; 8.9%) were positive for PD-L1. Of those that were PD-L1 positive, were equally distributed between primary site and metastatic sites (28/324, and 28/306, respectively). Conclusion: In this retrospective analysis, we describe, to the best of our knowledge, the distribution of PD-1 and PD-L1 expression in one of the largest datasets reported in TNBC. Unlike prior reports showing a high PDL-1 expression in excess of 50% in TNBC, this analysis show a low distribution of PD-L1 positivity. Our cohort represents a biased sample as those were unselected patients with recurrent breast cancer. Additionally, other factors can be implicated, including a change in the antibody used. These findings call for future standardization of the PD-L1 assay, particularly if further exploration showed PD-L1 to be a predictive or prognostic biomarker in mTNBC, particularly in relationship to therapy with immune checkpoint blockade. Citation Format: Baker TM, Gatalica Z, Goldstein LJ, Obeid E. Programmed death 1 (PD-1) and PD-1 ligand (PD-L1) distribution in triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-05-09.

Background: Quadruple-negative breast cancer (QNBC) is a subgroup of triple-negative breast cancer (TNBC) that lacks androgen receptor (AR) expression. While TNBC patients with AR expression have shown a promising response to AR-targeted therapies, QNBC patients' treatment options remain limited, with no targeted therapy We investigated the biomarker profiles of large cohorts of AR+TNBC and QNBC to identify their molecular differences. Method: TNBC tumors (defined as negative by IHC for ER, PR, Her2 and ISH for Her2) referred to Caris Life Sciences (Phoenix, AZ) between 2009 and 2015 were evaluated by board-certified pathologists with a combination of immunohistochemistry (AR, cKIT, cMET, EGFR, ER, ERCC1, Her2, MGMT, PD-1, PD-L1, PGP, PR, PTEN, RRM1, SPARC, TLE3, TOPO2A, TOPO1, TS and TUBB3), fluorescent/chromogenic in-situ hybridization (cMET, EGFR, Her2, TOP2A), and sequencing (Next-generation and Sanger). Tumors evaluated included a mix of primary tumors and metastases. QNBC tumors were defined as TNBC tumors that showed negative AR expression ( Results: Among 2,071 TNBC tumors identified, 1,952 tumors had AR IHC performed, out of which 1,612 (83%) were QNBC and 340 (17%) were AR+ TNBC tumors. Tumor expression of PD-L1 (Ab: SP142, Spring Bioscience/130021, R&D Systems, cutoff used: 2+, 5%) was significantly higher in QNBC compared to AR+TNBC tumors (18% vs. 8%, p=0.01), while PD-1 (Ab: NAT105, Ventana) expression on tumor-infiltrating lymphocytes was comparable between the two cohorts (60% vs. 62%). QNBC tumors were significantly more likely to express proteins of cKIT (26% vs. 15%, p=0.01), EGFR (69% vs. 56%, p=0.03), TS (49% vs. 33%, p Conclusion: Biomarker comparisons between two molecular subgroups of the TNBC tumors confirm the molecular heterogeneity of this aggressive type of breast cancer. Our biomarker results suggests that for AR+TNBC tumors, future clinical trial design can consider fluoropyrimidines, taxanes, and agents targeting PI3K/AKT/mTOR pathway as well as pan-HER inhibitors, and those agents may be combined with anti-androgen therapies. On the other hand, clinical trials for immune checkpoint inhibitors, TOP2A inhibitors, as well as agents that target cKIT and EGFR should be considered for QNBC tumors. Our findings highlight the molecular differences that should be considered in the design of future clinical trial strategies, warranting further investigation for improving targeted therapy and outcomes in TNBC. Citation Format: Xiu J, Obeid E, Gatalica Z, Reddy S, Goldstein LJ, Link J, Waisman J. Biomarker comparison between androgen receptor – Positive-triple-negative breast cancer (AR+ TNBC) and quadruple-negative breast cancer (QNBC). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-26.

Background: Triple-negative breast cancer (TNBC) is one type of breast cancer that remains challenging because of its aggressive nature and the lack of effective targeted therapy for it. Molecular profiling has revealed different subtypes, indicating a potential for promising targeted therapy such as androgen blockade and PARP inhibition in some TNBCs. The purpose of this study is to identify differences in BRCA1/2 mutated and non-mutated TNBC to shed light on potential therapeutic options in both subtypes, utilizing a multiplatform approach. Methods: A cohort of 386 triple-negative breast cancer specimens were tested via a multiplatform profiling service (Caris Life Sciences, Phoenix, AZ) consisting of gene sequencing (next generation sequencing [NGS]), protein expression (immunohistochemistry [IHC]) and gene amplification (fluorescence or chromogenic in situ hybridization [FISH or CISH]). Primary and metastatic specimens were evaluated. Tumor specimens with any BRCA1 and/or BRCA2 mutation (i.e. pathogenic or variant of unknown significance) were categorized as "BRCA1/2-mutated", while all others were considered "BRCA1/2 non-mutated". Results: In our cohort, 16.3% (63/386) of specimens were BRCA1/2-mutated while 83.7% (323/386) had no BRCA1/2 alteration detected. Amongst the highest rates of protein expression in BRCA1/2-mutated and non-mutated specimens were biomarkers like TOPO1 (63.5% and 63.4%), EGFR (65.2% and 67.4%), and the immune checkpoint biomarker, PD-1 (65.1% and 61.9%), with non-statistically significant differences. Differences noted between BRCA1/2-mutated and non-mutated specimens were detected by IHC in AR (11.1% versus 22.0%, p=0.0585) and PTEN (47.6% versus 59.6%, p=0.0941), with both trending but not achieving statistical significance. The highest overall mutation rate in both BRCA1/2-mutated and non-mutated were TP53 (80.6% and 73.1%, p=0.2659). Differences were also noted between BRCA1/2-mutated and non-mutated specimens by NGS in APC (6.3% versus 1.9%, p = 0.0644) and PIK3CA (11.1% versus 25.8%, p = 0.0137), with PIK3CA being statistically significant. Conclusion: Multiplatform tumor profiling identified differences in molecular profiles between BRCA1/2 mutated and BRCA1/2 non-mutated TNBC. Our findings raise the possibility for future investigation of potential combination therapeutic targeted therapy. Increased AR overexpression in BRCA1/2 non-mutated specimens is consistent with reports from other institutions. Further studies utilizing tumor profiling to elucidate the biological differences in in TNBC subtypes are warranted, to optimally include patients on clinical trials with specific targeted therapy and possibly improve treatment options. Citation Format: Arguello D, Abbott B, Reddy S, Gatalica Z, Obeid E, Goldstein L. Molecular profiling comparison of BRCA1/2-mutated and BRCA1/2 non-mutated triple-negative breast cancer (TNBC). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-30.

In order to better understand the etiology of inflammatory breast cancer (IBC), we performed a retrospective study to evaluate genomic alterations in 32 IBC patients using circulating DNA from blood. In 20 patients, mutation analyses were also conducted on tumor tissue or tumor cells from pleural effusions; in 15 of these patients, blood and tissue or pleural effusions were collected at the same time. Next generation sequencing (NGS) and a panel of 93 breast cancer related genes (Qiagen) were used for these studies. We found that of the 32 total patients, 11 (34.4%) carried at least one putative pathogenic germline variant, and another 8 patients (25%) carried at least one likely pathogenic germline variant. Ten patients (31.3%) carried only germline variants of uncertain significance and in 3 patients (9.4%) germline variants were not detected. Putative germline pathogenic variants were observed in the BRCA2 (2/32), TP53 (2/32), BRCA1 (1/32), PALB2 (1/32), CHEK2 (1/32), ATM (1/32), PMS1 (1/32), MUTYH (1/32), and MSH2 (1/32) genes. In addition, putative germline likely pathogenic variants were observed in the BARD1 (10/32), RB1 (2/32), APC (1/32), FANCC (1/32), AR (1/32), RAD51C (1/32), RAD51D (1/32) and CASP8 (1/32) genes. Most of the patients in our study had a family history of cancer and seventeen of the patients (53.1%) had family history of breast cancer in particular. Of those patients, 7 had a family history of breast cancer at a young age ( Citation Format: Jennifer S. Winn, Zachary Hasse, Jianming Pei, Michael Slifker, Sebastian M. Arisi-Fernandez, Jacqueline N. Talarchek, Elias Obeid, Karthik Devarajan, Hong Wu, Yulan Gong, Massimo Cristofanilli, R.Katherine Alpaugh, Sandra Viviana Fernandez. Germline and somatic variants in patients with inflammatory breast cancer (IBC) detected using cell-free DNA [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-03-11.

Background: Breast cancer (BC) is a heterogeneous disease. It is estimated that 5 to 10% of all BC to have a germline genetic predisposition. A 50-gene assay (PAM50) identifies 5 intrinsic molecular subtypes (IMS): Luminal A, Luminal B, HER2-enriched, Basal-like, and Normal-like. Basal-like breast cancers are enriched for BRCA1/2 germline mutations. Deleterious mutations in BRCA1/2 or other DNA-damage repair (DDR) genes may increase tumor mutational burden (TMB), a biomarker for response to checkpoint inhibition therapy. We sought to determine the spectrum of germline mutations in molecular BC subtypes (IMS), and their relation to somatic TMB. Methods: We performed a retrospective analysis of data from NantHealth database. RNAseq was used to classify breast tumors into IMS. Germline variants within putative driver genes (COSMIC v.76) were detected in analysis of 181 whole-genomes and 89 whole-exomes sequenced using Illumina chemistry. Classification of germline variants into potentially pathogenic variants (pPv) was determined using ClinVar database annotation. Patients were categorized as TMB-high by thresholding on >200 non-synonymous exonic somatic mutations as was previously reported. Results: A total of 270 BC patients with comprehensive omics profiling (germline DNAseq, somatic DNAseq, and somatic RNAseq) were available for this analysis. The mean age (±SD) was 56.4 (± 12.5) years (range 20.8-86.5). Forty-six patients (17.0%) were classified TMB-high. The IMS distribution was 40.7% Luminal A, 31.5% Luminal B, 5.9% HER2-enriched, 21.5% Basal-like, and 0.37% Normal-like. Over 200 unique germline variants were detected of which 98 were pPv according to ClinVar annotation. These pPv spanned 21 genes, 7 of which are directly related to DDR. One hundred and four patients had ≥1 pPv (78 had only 1 pPv, and 26 had >1 pPv). The most common pPv were APC (5.9%), BRCA2 (5.2%), TSC2 (4.4%), BRCA1 (3.7%), SDHB (3.3%), SDHD (3.3%), TSC1 (3.0%), PMS2 (3.0%), MUTYH (2.6%), MSH2 (1.5%) and MSH6 (1.5%). BRCA1 and especially BRCA2 pPv were mostly seen in the basal-like patients. Luminal B had distinctly more germline pPv in PMS2, BRCA1 & BRCA2 than Luminal A. TMB-high patients were not significantly enriched for germline pPv (OR 0.73, p=0.41), even when limited to pPv in DDR genes (OR 0.69, p=0.52). TMB-high patients were present in all 4 major IMS types; Her2-enriched 37.5%, Luminal B 23.5%, Basal-like 17.2%, and Luminal A 9.1%. Conclusion: We identified differential distribution of germline pPv in BC IMS. Of the pPv found, APC was the most commonly detected pPv across subtypes, while BRCA1/2 pPv were clustered in Basal-like subtype, and PMS2 in Luminal B subtype. 17% of all patients had a pPv within at least one DDR gene, that potentially may benefit from targeted therapy. Despite IMS types having distinct germline pPv profiles especially in DDR genes, there was no association with subsequent somatic TMB. This suggests that either 1. somatic events are the primary drivers of TMB, or 2. that germline variants with either unknown or benign significance need to be revisited. Future analysis in a larger demographically well-annotated dataset (commercial data, ExAC, other) or via functional studies should be considered. Citation Format: Obeid E, Reddy SB, Goldstein LJ, Daly MB, Benz SC, Hall MJ, Szeto C. Germline potentially pathogenic variants in breast cancer intrinsic molecular subtypes are not associated with somatic TMB [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-03-03.

Up to 40% of breast cancers have been shown to express the glucocorticoid receptor (GR), and activation of the GR is associated with poor prognosis in ER-negative breast cancer. We hypothesize that GR activation in breast cancer cells initiates anti-apoptotic signaling contributing to chemotherapy resistance. Based on our compelling preclinical data demonstrating that GR antagonism with mifepristone (mif) increases paclitaxel (pac) induced breast cancer cell death in vitro and in vivo, we conducted the first clinical trial of the combination of anti-GR therapy and chemotherapy in patients (pts) with metastatic breast cancer (MBC). Because the combination of mif and nab-paclitaxel (nab) had not previously been administered to pts, and because nab can result in cumulative neurotoxicity, a randomized, placebo-controlled, phase I design was used. Mif is known to inhibit CYP2C8, an enzyme involved in the metabolism of pac, thus plasma pac levels were monitored to evaluate for significant changes in clearance. A 3+3 dose escalation scheme was planned, with an initial nab dose of 100mg/m2 weekly and a mif dose of 300 mg/d for 2 days (day prior to and of nab infusion). For each dose level (DL), patients were randomized 3:2 to mif:placebo (p) for cycle 1 (C1). After C1, pts randomized to p were crossed over to receive mif at their assigned DL for the duration of study treatment. DL1 was deemed intolerable due to neutropenia, so the nab dose was decreased to 80 mg/m2 for DL2, and dose escalation of mif beyond 300 mg was halted. Serum cortisol and ACTH levels—biomarkers of effective GR blockade—were measured before and after mif treatment. Archival tumor was collected to determine tumor GR expression, and peripheral blood lymphocytes (PBLs) were collected to evaluate GR-target gene expression after mif. 9 pts were enrolled. Median age was 56 yrs (range 47-74). Median number of prior therapies for MBC was 2 (range 0-3). 8 pts had triple-negative breast cancer (TNBC), and 1 had ER+ disease. 8 of 9 pts had recurred after taxane-based therapy. 1 pt had a CR, 4 pts a PR, and 4 pts POD. 4 of 5 pts who responded to nab plus mif had previously relapsed after taxane-based therapy. 4 pts were treated at DL1 (2 mif, 2 p). Both pts randomized to mif experienced a dose-limiting toxicity (DLT) of neutropenia during C1. 5 pts were treated at DL2 (3 mif, 2 p), and 2 of the 3 pts randomized to mif for C1 experienced a DLT (neutropenia). Plasma pac levels were consistent with delayed clearance of nab when co-administered with mif for most pts. All pts were found to have a 2-fold or greater rise in their serum cortisol levels, demonstrating effective adrenal GR inhibition. Mif delays clearance of nab in most pts, and results in DLT. Given the inter-patient variability in delay of nab clearance by mif, co-administration produces unpredictable toxicity. However, given the responses seen in taxane-pretreated TNBC pts, GR antagonism is a promising approach for treating aggressive TNBCs. As neither carboplatin nor gemcitabine are metabolized, mif is unlikely to affect clearance of these agents. As they are widely used for advanced TNBC, both agents represent attractive chemotherapy partners for future clinical investigation. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-16-21.

Background: ESR1 mutations and fusions arise in hormone receptor positive (ER+ and/or PR+) breast cancer (HR+ BC) patients after aromatase inhibitor (AI) therapy (low estrogen states), to become constitutively active in a ligand-independent manner. Patients with ESR1 mutations exhibit worse prognosis and outcome with no particular benefit of chemotherapy vs tamoxifen treatment (Augusto, et al. ASCO 2016). A retrospective analysis of the ESR1 mutation frequency and co-occurring alterations that could guide subsequent therapy approaches was investigated. Methods: Molecular profiles of 416 breast cancer patients [HR+ (n=237), HER2+ (n=29) and TNBC (n=139)] were assessed. Protein expression (IHC) and gene amplification (ISH) were performed. Genomic testing included 592-gene hybrid-capture NGS [NextSeq Illumina platforms] and ArcherDx fusion assay based on anchored multiplex PCR (AMP) FusionPlex Solid Tumor. ESR1 variant (ESR1var) (mutation/fusion) profiles and HR+ BC patients lacking genomic ESR1 alterations (ESR1 WT) were compared; Pearson's chi-squared test was used to test for significant differences. Results: An ESR1 mutation (point mutations, insertion-deletions [n=49] and fusions [n=4]) was detected in 13% (53 /416) of the specimens, and this constitutes 21% (50/237) of all HR+ breast cancers. Two TNBC patients exhibited ESR1 variants (H398Y in exon 7 and A491S in exon 9, both are classified as variants of unknown significance). ESR1 mutations were not detected in HER2+ BC. Seventy-seven percent of patients with ESR1 mutations were detected in metastatic specimens (p=0.03), with liver (19/53 or 36%) and bone (8/53 or 15%) specimens as the most frequent sources for ESR1 variant (ESR1var) positivity. The most common alleles detected were: D538G (24%), Y537S (18%), E380Q (14%) and L536H (4%); 15 other additional alleles were detected (each 2%). ESR1 fusions were detected in 4 ESR1 WT patients: ESR1-ATP2B2, ESR1-MKL1/ESR1-TNRC6B, ESR1-ARNT2 and ESR1-C6ORRF211. We next compared ESR1var (mutation/fusion) profiles to HR+ breast patients lacking genomic ESR1 alterations (ESR1 WT). ER expression was present in 100% and 96% of ESR1var and ESR1 WT BC, respectively, however expression of PR was negative in 22% and 38% of ESR1var and WT BC, respectively (p=0.05). Significantly higher rates of other gene amplification events observed in ESR1var vs. ESR1 WT BC included: c11orf30 [EMSY, BRCA2 interacting transcriptional repressor] (20% vs. 7%), CCND1 (51% vs.28%), CCND2 (6% vs. 0%), FGF3 (37% vs. 16%), FGF4 (40% vs. 12%) and FGF19 (40% vs. 15%), whereas cMYC was more frequently amplified in ESR1 WT BC (17% vs. 2%); all p-values Conclusions: ESR1 mutations and fusions are detected in 21% of HR+ BC, the majority of which are in metastatic sites. Amplifications of genes involved in downstream regulatory pathways were present and may contribute to the poor prognosis of ESR1var HR+ BC. Correlation with antecedent therapy is currently underway. Citation Format: Tan A, Feldman R, Pohlmann P, Lynce F, Swain S, Nunes MR, Gatalica Z, Ghazalpour A, Swensen J, Vidal G, Obeid E, Vacirca J, Isaacs C, Schwartzberg L. ESR1 mutations, ESR1 fusions and co-occurring alterations assessed in breast cancer tumors [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-01.

Purpose: To develop a novel approach for early detection of breast cancer and examine molecular features of screen detected cancers in prospectively ascertained high-risk women undergoing semi-annual dynamic contrast-enhanced breast magnetic resonance imaging (DCE-MRI) for women at high genetic risk. Background: Women with a personal or family history of breast cancer and genetic mutation carriers of BRCA1 and BRCA2 have a higher than normal risk of breast cancer. An intensified screening surveillance regimen is an early detection strategy in high-risk women. The American Cancer Society recommends annual DCE-MRI in addition to annual mammogram based off several pivotal screening studies that demonstrated improved sensitivity and cancer detection rates and decreased interval cancer rates with the addition of annual DCE-MRI. Questions remain regarding the optimal screening modality and interval regimen in these high-risk women. Methods: Between 2004 and 2016, we assembled a prospective cohort of high-risk women undergoing semi-annual DCE-MRI and annual mammography. To be eligible, women had a lifetime breast cancer risk >20% and/or tested positive for a pathogenic mutation using a cancer gene panel including BRCA1, BRCA2, CDH1, PALB2, CHEK2 and other cancer susceptibility genes in the DNA repair pathway. Somatic mutation events in screen-detected tumors were investigated using UW-OncoPlex cancer gene panel using DNA extracted from FFPE shavings. Results: 295 women were recruited to the study; 44% of the study participants had pathogenic mutations in BRCA1 or BRCA2 genes. At a median follow-up of 3.3 years (range 0-12 years), 3 DCIS and 13 early stage invasive breast cancers were detected, of which 14 occurred in subjects with identifiable pathogenic mutations (11 BRCA1, 2 BRCA2, 1 CDH1). The incidence rate is 1.3% in all subjects, but 3.5 % per year in BRCA1 carriers. DCE-MRI identified all 13 invasive cancers at a mean size of 0.61 cm (range 0.1-1.0 cm); none had lymph node metastasis. No interval cancers occurred. In addition, 7 of the breast cancers were detected on DCE-MRI imaging obtained at the 6 months screening interval; they would be interval cancers if only annual screening were implemented. There was very little DNA for somatic mutation testing in the majority of cases. However, as expected, there was heterogeneity in the spectrum of mutations but the most commonly somatically mutated gene in the early cancers was TP53. Conclusions: DCE-MRI every 6 months performed well for early detection of invasive breast cancer in high-risk women, accomplishing the ultimate goal of breast cancer screening—detecting node-negative, invasive tumors less than 1 cm. Semi-annual DCE-MRI performed especially well in BRCA1 mutation carriers at risk for the most aggressive subtype of breast cancer. Further interventional studies evaluating this novel screening approach are warranted to personalize breast cancer risk assessment and prevention. Citation Format: Whitaker K, Guindalini R, Abe H, Sheeth D, Huo D, Hong S, Churpek J, Verp M, Obeid E, Zheng Y, Amico A, Yoshimatsu T, Olopade O. Breast cancer surveillance in high-risk women with dynamic contrast-enhanced magnetic resonance imaging every 6 months: Results from a single institution study [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-02-10.

Background: Recent data indicate a promising response to immune check point blockade (ICB) in patients with breast cancer. Pembrolizumab, a humanized monoclonal antibody against programmed death 1 (PD-1) receptor and one of several ICB agents in development, was given an FDA approval for all MSI (microsatellite instability)-high solid tumors. MSI incidence in breast cancer is not fully elucidated. Other biomarkers being explored in possible relationship to ICB activity include PD-1 ligand (PD-L1) status and tumor mutational load (TML). In this study, we aimed to explore the incidence of these biomarkers in invasive breast cancers. Methods: A retrospective data analysis of patients profiled by commercial next-generation sequencing (NGS) at Caris Life Sciences was performed. MSI results were either high, stable, or equivocal. MSI was calculated by comparing repeat-insertions or deletions across over 7,000 microsatellite sequences in the patient sample to the hg19 reference genome. Samples with repeat variances in more than 45 microsatellites were classified as MSI-High PD-L1 expression was evaluated using immunohistochemical analysis (IHC), with clone SP-142 (Roche Diagnostics). A sample was considered positive if there was >5% membranous staining of tumor cells. Tumor mutational load was calculated as a total number of non-synonymous somatic mutations identified per megabase of the genome coding area with high being greater than or equal to 17. Results: A total of 9,627 breast cancer cases were queried from the Caris Life Sciences database. The mean age (±SD) was 56.8 ± 12.4 years (range 20-90). The tumor distribution was 60.7% hormone receptor (HR) positive (ER and/or PR) and HER2 negative, 9.5% HER2 positive (with HR negative or positive), and 29.8% triple negative (negative for ER, PR and HER2). Of all cases, there were 5,203 tested for PD-L1 status, 354 (6.8%, 95% CI 6.2-7.5%) were positive. Of 1,440 tumors tested for MSI status, 15 (1.04%, 95% CI 0.58-1.71%) were either high (8) or equivocal (7), the rest were MSI-low. Tumor mutational load (TML) was available on 1,766 tumors, of which 55 (3.1%, 95% CI 2.4-4.0%) were high. Seven out of the 8 MSI-high tumors were also TML-high. Four out of the 8 MSI-high breast cancers were triple negative. Conclusion: In this large dataset of molecularly profiled breast cancer, MSI was observed in about 1% of the breast tumors tested. Overall, modest positivity of TML, PD-L1, and MSI of all invasive breast cancers was observed. The percentage of patients that had at least one of these biomarkers that may confer responsiveness to ICB is planned and will be further stratified by subtype. MSI-high breast cancers mostly overlapped with those that were TML-high. Future research is needed to show the clinical utility of these biomarkers in response to ICB. Updated data will be presented. Citation Format: Obeid E, Ellerbrock A, Handorf E, Goldstein L, Gatalica Z, Arguello D, Swain SM, Isaacs C, Vacirca J, Tan A, Schwartzberg L. Distribution of microsatellite instability, tumor mutational load, and PD-L1 status in molecularly profiled invasive breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD6-03.

Background: Inflammation and angiogenesis in the tumor microenvironment play a role in human breast cancer. We previously showed that tumors with high M2 macrophages and high microvessel density (MVD) have a shorter overall survival. The antitumor effect of Toll-like receptor 7 (TLR7) has been previously described whereby its activation can alter the tumor microenvironment to adopt an antitumorigenic (Th-1) immune response and inhibit angiogenesis, with a potential immune mediated benefit in breast cancer. To our knowledge, there are no studies that evaluated TLR7 and its relationship to macrophages and angiogenesis in human breast cancer. Methods: Breast tumors were obtained from the University of Chicago Breast Cancer SPORE tissue bank under IRB approved protocols. Tissue microarrays were constructed and molecular subtype was assigned based on immunohistochemical (IHC) staining into the following groups: luminal A (ER+, PR+, HER2-), luminal B (ER+, HER2+ or ER+, PR-), HER2-like (ER-, HER2+) and basal-like (ER-, HER2-, EGFR+ and/or CK5/6+). Macrophage density was determined using double staining with CD68/CD163. Microvessel density (MVD) was measured by IHC staining using anti-CD34. Staining quantification was performed by a trained pathologist and scoring performed independently by two pathologists. Based on median distribution, we assigned M2 macrophage content and MVD into high vs. low, and the TLR7 expression into faint vs. strong. To evaluate the association between M2 macrophages and TLR7, Spearman's rho correlation coefficients were calculated. Survival analysis was done using Tarone ware statistics. Results: After removal of duplicate cores, we had 100 tumors available for scoring. There was an equal distribution of M2 macrophage content, MVD, and TLR7 expression (p = 0.074, p = 0.908, p = 0.145 respectively). The tumor subtypes were 53.2% luminal A, 34.0% basal-like, 7.4%, luminal B, and 5.3% Her2-like. We found a significant correlation between high number of M2 macrophages and high MVD (spearman's rho correlation coefficient = 0.343, p = 0.004). There was an association between M2 macrophages and TLR7 expression (Pearson χ2 = 4.425, p = 0.025). Compared to tumors with faint TLR7 expression, those with strong TLR7 expression exhibited a longer overall survival (Tarone ware statistic 5.053, p = 0.025) and this continued to be significant when adjusted for M2 macrophage content (Tarone ware statistic = 4.049, p = 0.044) and for MVD (Tarone ware statistic = 4.042, p = 0.044). There was no statistically significant association between TLR7 expression and molecular subtypes of breast cancer. Conclusion: Tumor associated macrophages (M2 macrophages) and angiogenesis (high MVD) are known to contribute to worse outcomes in human breast cancer. Our results suggest that activation of TLR7 can have a protective effect in human breast cancer even in tumors harboring features of more aggressive disease. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-01-04.