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Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype and has the highest rate of recurrence. The predominant standard of care for advanced TNBC is systemic chemotherapy with or without immunotherapy, however responses are typically short-lived. Thus, there is an urgent need to develop more effective treatments. PI3K pathway components represent plausible therapeutic targets, as approximately 40% of TNBCs have PIK3CA/AKT1/PTEN alterations. However, unlike hormone receptor-positive tumors, it is still unclear if or how PI3K pathway inhibitors will be effective in triple-negative disease. Here we identify a promising AKT inhibitor-based therapeutic combination for TNBC. Specifically, we show that AKT inhibitors potently synergize with agents that suppress the histone methyltransferase, EZH2, and promote robust tumor regression in multiple TNBC models in vivo. AKT and EZH2 inhibitors exert these effects by first cooperatively driving basal-like TNBC cells into a more differentiated, luminal-like state, which cannot be effectively induced by either agent alone. More importantly, once differentiated, these agents kill TNBCs by hijacking signals that normally drive mammary gland involution. Together these findings identify a promising therapeutic strategy for this highly aggressive tumor type and illustrate how deregulated epigenetic enzymes can insulate tumors from oncogenic vulnerabilities. These studies also reveal how developmental tissue-specific cell death pathways may be co-opted for therapeutic benefit. Citation Format: Amy Schade, Naiara Perurena, Marina Watanabe, Carrie L. Rodriguez, Patrick Loi, Natalie Pilla, Rachel A. Davis, Kaia Mattioli, Dongxi Xiang, Jason J. Zoeller, Zhe Li, Ana C. Garrido-Castro, Sara Tolaney, Karen Cichowski. AKT and EZH2 inhibitors kill TNBCs by hijacking mechanisms of involution [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 P4-08-11.

Background: The introduction of immune checkpoint inhibitors (ICI) to standard neoadjuvant chemotherapy regimens has been shown to significantly improve outcomes in patients with triple negative breast cancer and is being investigated for high-risk hormone receptor-positive (HR+)/human epidermal growth factor-2 negative (HER2-) breast cancer. Preclinical evidence suggests radiation therapy (RT) can stimulate intra-tumoral T cell infiltration and enhance the expression and immune detection of tumor-specific neoantigens. This phase II pilot randomized study (NCT04443348) aims to evaluate the safety and efficacy of two different doses of preoperative primary tumor RT boost when combined with neoadjuvant pembrolizumab, then followed by standard neoadjuvant chemotherapy. Dual co-primary endpoints include determining the pathologic complete response (pCR) rate in the non-irradiated and pathologically confirmed metastatic axillary lymph node(s) in each treatment arm and quantifying tumor-infiltrating T lymphocytes in on-treatment (C1D14) tumor biopsies. We hypothesize that high-dose RT will increase the proportion of tumors with high T cell infiltration (i.e., top quartile) from 25% to 55%. Secondary endpoints include measuring residual cancer burden, evaluating tolerability of the regimen, and assessing quality of life. Exploratory endpoints include evaluation of treatment-associated changes in the tumor immune microenvironment, circulating immune cell analyses, and circulating tumor DNA kinetics. Methods: The study plans to enroll 128 participants with either triple negative (n=80) or high-risk HR=/HER2- (n=48) breast cancer who will be randomized to receive no, low (9 Gy), or high (24 Gy) dose of preoperative RT boost, after which 24 participants of either breast cancer subtype will be enrolled to an exploratory high dose proton therapy boost cohort. The eligibility criteria include patients who have biopsy-proven, axillary lymph node-positive breast cancer that is either triple negative (defined as ER< 10%, PR< 10%, and HER2-negative) or high-risk HR+/HER2- (grade III or having a high-risk genomic assay score). Study treatment is given in 6-week cycles, with 400 mg Pembrolizumab given on day 1 of each cycle. For those participants randomized to receive a preoperative RT boost, treatment is delivered in 3 fractions (3 × 3 Gy or 3 × 8 Gy) over consecutive business days, where one of the fractions is given on the same day as C1D1 Pembrolizumab. Standard neoadjuvant chemotherapy begins on C1D15 with paclitaxel (plus carboplatin for triple negative) administered weekly for 12 weeks, and then starting on C3D15, dose-dense doxorubicin/cyclophosphamide is administered every 2 weeks for 8 weeks. Following neoadjuvant treatment, participants will receive standard breast surgery (including removal of the pathologically confirmed metastatic lymph node) followed by adjuvant pembrolizumab, radiation therapy, and standard-of-care systemic therapy as clinically indicated. Tissue samples from the primary tumor and biopsy-proven lymph node are taken at baseline, C1D14, and at the time of surgery. There are eleven blood collection timepoints throughout the neoadjuvant and adjuvant settings. Participants will be followed for 2 years after surgery to assess safety and durability of responses. Results: This study has accrued 12 participants to date, including 10 with triple negative breast cancer and 2 with high-risk HR+/HER2- breast cancer. Formal results for this study are forthcoming, as the trial is actively accruing at 6 institutions, with plans to open at 3 more within the year. For persons with a specific interest in this trial, please contact Joseph Connolly, Multi-Center Coordinator, at jconnolly28@mgh.harvard.edu. Citation Format: Joseph J. Connolly, Laura M. Spring, Alphonse G. Taghian, Michele Gadd, Laura Warren, Ana C. Garrido-Castro, Tari King, Elizabeth A. Mittendorf, Jose P. Leone, Dana L. Casey, Lisa Carey, Tiffany A. Traina, Yara Abdou, Atif Khan, George Plitas, Jean Wright, Cesar Augusto Santa-Maria, Lisa Jacobs, Rachel Blitzblau, E Shelley Hwang, Carey Anders, Ian Krop, Antonio C. Wolff, Alastair M. Thompson, Elyssa Denault, Gaorav Gupta, Alice Ho. TBCRC-053: P-RAD: A Randomized Study of Preoperative Chemotherapy, Pembrolizumab and No, Low or High Dose RADiation in Node-Positive, HER2-Negative 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 OT3-15-01.

Background: About half of all breast cancers exhibit low HER2 expression. Despite lack of ERBB2 amplification, HER2-low tumors respond to trastuzumab deruxtecan (T-DXd), leading to the NCCN recommendation of T-DXd both for patients with HER2+ and HER2-low metastatic breast cancer (MBC). It remains however unclear if HER2-low represents a distinct molecular entity, as compared to HER2-0 MBC. Here, we compare the genomic landscape of HER2-low versus HER2-0 breast cancers in a large, single institution cohort. Methods: We identified consecutive patients with MBC seen at Dana-Farber Cancer Institute between 07/2013 and 12/2020. Patients were included if they had HER2-negative MBC per ASCO/CAP Guidelines and had undergone next generation sequencing (NGS) testing with a targeted, tumor-only platform (OncoPanel). Based on the HER2 status of the specimen tested by NGS, patients were divided into 2 groups: (i) HER2-low if immunohistochemistry (IHC) 1+ or 2+ non-amplified, or (ii) HER2-0 if IHC 0. Mutations of interest detected on NGS were classified as oncogenic using the OncoKB tool and additional annotation. Genomic profiles of HER2-low and HER2-0 tumors were compared using Chi-Square and Kruskal-Wallis tests. To determine genomic event enrichment between the two HER2 groups, logistic regression models were used, accounting for background rate and estrogen receptor (ER) expression. ERBB2 copy counts were calculated for tumors with recorded histology-estimated purities and copy-number segmentation using a simple model of allelic gain/loss. Results: Among 1847 patients with HER2-negative MBC, 1043 underwent NGS testing on a HER2-low (n=489, 47%) or HER2-0 sample (n=554, 53%). Most samples were metastatic (71%, n=743) while 29% (n=300) were from primary tumors. 73% had ductal histology, 13% were lobular and 14% had mixed or other histology. ER expression was enriched among HER2-low vs. HER2-0 tumors (76% vs. 60%; p< 0.001). Focusing on the most commonly occurring genetic mutations, no major differences were observed in HER2-low vs. HER2-0 tumors, after correcting for ER status (Table 1). Among all mutational events, any mutation in MPL, CYLD, and MAP3K and oncogenic mutations in TP53 and NF1 were more common in HER2-0, while any mutation in MTOR, RAD21, DNMT3A, and PDGFRA were enriched in HER2-low patients, when controlling for ER status and background mutational rate (p< 0.05). However, no mutation reached significance after accounting for multiple hypothesis testing. Similarly, no deep deletion or high amplification CNV events reached significance for either group. Analysis of tumor mutational burden in HER2-low vs. HER-0 tumors revealed no significant differences (median: 7.26 muts/Mb vs. 7.60 muts/Mb, p=1.00), including when accounting for ER status. Finally, among tumors with sufficient tumor purity for ERBB2 copy count analysis (n=374 and 419 for HER2-low and HER2-0, respectively), HER2-low tumors had a significantly higher number of ERBB2 alleles as compared to HER2-0 (< 2 copies, 15.0% vs. 30.9%, 2 copies 67.4% vs. 60.5%, and >2 copies, 17.6% vs. 8.6%; p< 0.001 by Kruskal-Wallis). Conclusions: To our knowledge, this is the largest comprehensive genomic analysis of HER2-low MBC to date. In our cohort of patients with HER2-negative MBC, the genomic landscape of HER2-low and HER2-0 tumors did not differ significantly, apart from a higher number of ERBB2 alleles. These data further support the notion that HER2-low, as currently defined, is not a distinct molecular subtype of breast cancer. Citation Format: Paolo Tarantino, Hersh V. Gupta, Melissa E. Hughes, Janet L. Files, Sarah Strauss, Gregory Kirkner, Anne-Marie Feeney, Yvonne Y. Li, Ana C. Garrido-Castro, Romualdo Barroso-Sousa, Brittany Bychkovsky, Laura MacConaill, Neal Lindeman, Bruce Johnson, Matthew Meyerson, Sheheryar Kabraji, Rinath Jeselsohn, Xintao Qiu, Rong Li, Henry W. Long, Eric Winer, Deborah A. Dillon, Giuseppe Curigliano, Andrew Cherniack, Sara Tolaney, Nancy U. Lin. HER2-05 Comprehensive genomic characterization of HER2-low 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 HER2-05.

Background. Patients (pts) who develop MBC at older ages are underrepresented in clinical trials, are less likely to be included in comprehensive biomarker characterization studies, and experience worse breast cancer-specific survival than their younger counterparts. Elucidating genomic underpinnings of MBC and possible therapeutic targets for older breast cancer patients are critical priorities. Methods. We identified pts age >70 years at MBC diagnosis and a younger cohort (ages 50-69; age < 50), who were treated for MBC at a single center and who had their metastatic (or if not available, the primary) tumor, assessed by a targeted, tumor-only next generation sequencing (NGS) platform (OncoPanel) between 2013-2020. The NGS panel included mutations, copy number variation, tumor mutational burden (TMB), and hypermutation (HM) status, with mutations classified as oncogenic using the OncoKB tool and additional annotation. Copy number events were selected as being “oncogenic” if a high amplification was called for an oncogene or a deep deletion for a tumor suppressor. We compared findings for older (age >70) vs. younger (age < 50 and ages 50-69) MBC pts using Chi-Square and Kruskal-Wallis tests. To determine genomic event enrichment, logistic regression (LR) models were used, controlling for age (continuous), background rate, and tumor subtype (those with unknown subtype [n=27] were excluded from models). False discovery rate (FDR) was used to correct for multiple hypothesis testing. Results. The final analytic cohort included 2,380 pts. The median age at MBC diagnosis was 54.1 years overall (range 18.5- 91.9) and 73.6 years for those age >70. A total of 137 metastatic and 76 primary tumors were sequenced in pts age >70; in those age < 70, 1383 metastatic and 784 primary tumors were sequenced (for age < 50 [n=857] and 50-69 [n=1310]). Older pts were more likely to present with HR+/HER2- tumors (70.9% v. 62.4% v. 52.4%), and less likely to present with HER2+ (9.4% v. 14.4% v. 22.8%) or triple-negative breast cancer (TNBC) (18.8% v. 21.9% vs. 24.0%) at MBC diagnosis (listed >70, 50-69, < 50; P=1e-7). Older pts had higher average TMB vs. younger pts (9.57 in pts > 70, 8.56 in ages 50-69, 7.34 in ages < 50; P=3.5e-5). This was due to older pts having a higher incidence of hypermutation status as defined as TMB >10: 26.3% in age >70, 23.2% in ages 50-69, 16.8% in age < 50. Using q=0.1 as the threshold of significance, the presence of CDH1, PIK3CA, MAP3K1, TET2, and AKT oncogenic mutations were also enriched in older pts, while the presence of oncogenic GATA3, BRCA2, and TP53 mutations, as well as any mutation in BRCA1 were enriched in younger pts (too few oncogenic BRCA1 mutations were present for accurate modeling). The frequency of oncogenic PIK3CA mutations in HR+/HER2- tumors was highest in the oldest pts (44.4% in pts age >70 v. 31.6% in age 50-69 v. 26.7% in age < 50). Of pts who had oncogenic BRCA1/2 mutations identified on tumor-only NGS testing and underwent clinical germline testing (n=7 v. 60 v. 67, oldest to youngest), older pts had the lowest incidence of germline BRCA pathogenic variants (14.3% vs. 47.2.% vs. 67.2%; p=0.01); most BRCA mutations identified on NGS testing in older patients were considered likely somatic. When assessing enrichment in copy number events, ERBB2, RAD21, and BRIP1 amplifications were all significantly less frequent in older pts (q< 0.1), even when accounting for tumor subtype. Conclusions. In a large cohort of pts with MBC, the mutational and copy number landscape for older pts differs from that in younger pts, even after controlling for tumor subtype. Key actionable findings include a higher proportion of high TMB and PIK3CA-mutated tumors, emphasizing the importance of genomic profile testing in this pt population and further exploration of efficacy and tolerability of relevant therapies in those age >70 years. Citation Format: Hersh V. Gupta, Rachel Freedman, Melissa E. Hughes, Yvonne Y. Li, Gregory Kirkner, Janet L. Files, Sarah Strauss, Ana C. Garrido-Castro, Lauren Buckley, Romualdo Barroso-Sousa, Brittany Bychkovsky, Sara Tolaney, Laura MacConaill, Neal Lindeman, Bruce Johnson, Matthew Meyerson, Eric Winer, Deborah A. Dillon, Andrew Cherniack, Nancy U. Lin. Tumor Genomic Landscape in Older Women with 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 P5-14-06.

Background: With the development of novel antibody drug conjugates (ADC), it is increasingly important to understand the changes that occur in cell-surface targets over time from early-stage to metastatic breast cancer. Discordance in HER2 expression per immunohistochemistry (IHC) has been reported between primary and metastatic tumors in patients (pts) with HER2-negative breast cancer defined per ASCO/CAP guidelines, with both gain and loss of expression described. Representation of HR-negative (TNBC) tumors has been limited in these studies, and HER2 status at multiple time points in TNBC has not been described. Here we report changes in HER2 IHC in patients diagnosed with TNBC, using a collection of matched tumor samples over time. Methods: Pts were identified from two sources: 1) an institutional database including all consecutive pts who underwent surgery for stage I-III breast cancer at Dana-Farber/Brigham Cancer Center between 2015-2018, and 2) a prospective research biopsy protocol for pts with known or suspected metastatic breast cancer. Pts were included in the present analysis if they received neoadjuvant chemotherapy (NAC) for stage I-III TNBC (eTNBC), or if they were diagnosed with any stage TNBC and ultimately developed metastatic TNBC (mTNBC). Clinical pathology records were reviewed for HER2 IHC results of samples collected at: initial diagnosis (DX); residual disease (RD) post-NAC, if applicable; and at recurrence (M). HER2 IHC was classified as HER2-0 if HER2 IHC 0, and HER2-low if 1+ or 2+ (and ISH non-amplified). For matched comparisons, if IHC was performed in more than one DX sample (e.g., breast, node), only the breast was considered; if more than one M sample, only the first M biopsy with available IHC was considered. Results: Among 110 pts in this cohort, 101 were initially diagnosed with eTNBC (79 received NAC; 22 underwent surgery as first intervention) and 9 with de novo mTNBC. Median age was 48.7 years (range 19.8-71.6). Among all pts, a total of 292 samples (136 DX, 53 RD, 103 M) had available HER2 IHC scores. When restricting to one sample per time point, HER2-low prevalence was 49/102 (48.0%) in DX breast tumors, 21/53 (39.6%) in RD, and 13/58 (22.4%) in first M samples (with all remaining samples HER2-0, except one HER2 3+ sample). In eTNBC pts, HER2 IHC scores were available for 50 paired DX and RD, and 48 paired DX and M samples. Among 50 pts with paired DX and RD, HER2 IHC was discordant in 56% (28/50) (Table 1). Of the 21 HER2-0 DX tumors, 23.8% (5/21) became HER2-low at RD. Of the 29 HER2-low DX tumors, 51.7% (15/29) became HER2-0 at RD. Among 48 eTNBC pts who recurred and had paired DX and M samples, HER2 IHC was discordant in 50% (24/48) (Table 1). Change from HER2-0 to low was 12.5% (3/24), and from HER2-low to HER2-0 was 66.7% (16/24). Among 9 de novo mTNBC pts, 5 had HER2 IHC available in paired DX breast and M prior to starting therapy; 3 were concordant (IHC 0, n=2; IHC 1+, n=1), one had IHC 0 in DX breast and IHC 2+ in M (liver), one had IHC 1+ in DX breast and IHC 0 in M (node). Conclusions: HER2 IHC classification was discordant in about half of the TNBC cases we examined, with more frequent rates of conversion from HER2-low to HER2-0 in both paired DX/RD post-NAC, and paired DX/M samples. Additional analyses will be presented exploring HER2 IHC changes among multiple metastases per patient. Genomic and molecular analysis, including whole exome sequencing, RNA sequencing, and methylation profiling, are underway in these samples to further elucidate HER2 evolutionary dynamics. Citation Format: Ana C. Garrido-Castro, Lan D. Ngo, Edward T. Richardson, Allison Frangieh, Ayesha Mohammed-Abreu, Melissa E. Hughes, Jorge Gomez Tejada Zanudo, John Navarro, Paolo Tarantino, Elizabeth A. Mittendorf, Sara Tolaney, Tari King, Eric Winer, Nancy U. Lin, Nikhil Wagle. HER2-10 Dynamics of HER2-low expression in triple-negative 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 HER2-10.