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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.

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Supplementary Methods

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Purpose:In contrast to recurrence after initial diagnosis of stage I–III breast cancer [recurrent metastatic breast cancer (rMBC)], de novo metastatic breast cancer (dnMBC) represents a unique setting to elucidate metastatic drivers in the absence of treatment selection. We present the genomic landscape of dnMBC and association with overall survival (OS).Experimental Design:Targeted DNA sequencing (OncoPanel) was prospectively performed on either primary or metastatic tumors from 926 patients (212 dnMBC and 714 rMBC). Single-nucleotide variants, copy-number variations, and tumor mutational burden (TMB) in treatment-naïve dnMBC primary tumors were compared with primary tumors in patients who ultimately developed rMBC, and correlated with OS across all dnMBC.Results:When comparing primary tumors by subtype, MYB amplification was enriched in triple-negative dnMBC versus rMBC (21.1% vs. 0%, P = 0.0005, q = 0.111). Mutations in KMTD2, SETD2, and PIK3CA were more prevalent, and TP53 and BRCA1 less prevalent, in primary HR+/HER2− tumors of dnMBC versus rMBC, though not significant after multiple comparison adjustment. Alterations associated with shorter OS in dnMBC included TP53 (wild-type: 79.7 months; altered: 44.2 months; P = 0.008, q = 0.107), MYC (79.7 vs. 23.3 months; P = 0.0003, q = 0.011), and cell-cycle (122.7 vs. 54.9 months; P = 0.034, q = 0.245) pathway genes. High TMB correlated with better OS in triple-negative dnMBC (P = 0.041).Conclusions:Genomic differences between treatment-naïve dnMBC and primary tumors of patients who developed rMBC may provide insight into mechanisms underlying metastatic potential and differential therapeutic sensitivity in dnMBC. Alterations associated with poor OS in dnMBC highlight the need for novel approaches to overcome potential intrinsic resistance to current treatments.

Background: Despite advances in treatment options, outcomes remain poor for many pts with breast cancer brain metastases (BCBMs). Identifying genomic predictors of brain metastasis from primary tumors could lead to better stratification of pts at risk and drive the development of preventative strategies. The objective of this study was to describe the landscape of genomic alterations in primary tumors from pts with MBC who subsequently did or did not develop BCBMs. Methods: We performed a case control study to identify somatic alterations in primary tumors associated with a higher incidence of brain metastases. We reviewed outcomes for 2562 unique MBC patients from a single institution who underwent targeted next-generation DNA sequencing of > 280 cancer-related genes (OncoPanel) from their tumor between July 1, 2013 and December 31, 2020. Pts were included in this analysis if they had at least 2 years of follow-up from date of metastatic diagnosis and OncoPanel testing on a primary breast tumor. We compared single nucleotide variants (oncogenic or likely oncogenic), copy number variation (amplification and deep deletions) and tumor mutation burden in the primary tumors of pts in this cohort. Copy number variation was corrected for Panel version and tumor purity. Wilcoxon rank sum test and Fisher exact test was used to compare genomic differences between groups. False discovery rate was used to correct for multiple hypothesis testing and q < 0.1 was considered significant Results: A total of 369 pts were included in the final analytic cohort. Of these, 115 were diagnosed with brain mets (cases, BM group) and 224 were not (controls, nBM group). The BM group was enriched for patients with HER2-positive breast cancer (33 vs 12.5%), consistent with previous work. In the whole cohort, the most common and clinically significant somatic alterations (oncogenic single nucleotide variants or copy number high amplification or two copy deletion) are shown in Table 1. When adjusting for subtype there were no significantly enriched SNVs in BM vs nBM group. When adjusting for subtype, FGFR1 amplification was significantly enriched in hormone receptor positive HER2 negative (HR+ HER2-) patients with BM (log2 odds ratio 1.22, q < 0.1). Tumor mutation burden was not significantly different in primary tumors between the BM and nBM groups (median TMB 7.3 vs 6.1, Wilcoxon p = 0.08). Pathway analysis combining all subtypes revealed that RTK_RAS pathway (log2 odds ratio 1.64, q value < 0.1) and TP53 pathway (log2 odds ratio 1.15, q value < 0.1) gene sets were significantly enriched in the BM group. When controlling for subtype, pathway analysis revealed that RTK_RAS pathway gene set was significantly enriched in HR+ HER2- BM group (log2 odds ratio 1.36 q < 0.1). Conclusions: In this case control series of patients with metastatic breast cancer with or without brain metastases, we found that primary tumors that are enriched for somatic alterations in the RTK_RAS and TP53 pathway may be associated with higher risk of developing brain metastases. Further validation in larger cohorts is warranted. Table 1. Frequency of somatic alterations in primary tumor by brain metastasis outcome. Citation Format: Sheheryar Kabraji, Yvonne Y. Li, Melissa E. Hughes, Hersh V. Gupta, Lauren Buckley, Janet L. Files, Ayesha Mohammed-Abreu, Anne-Marie Feeney, Greg Kirkner, Ashka Patel, Ana C. Garrido-Castro, Romualdo Barroso-Sousa, Brittany Bychkovsky, Matthew Meyerson, Sara Tolaney, Deborah A. Dillon, Bruce Johnson, Eric Winer, Andrew Cherniack, Nancy U. Lin. Somatic alterations in primary tumors of patients (pts) with metastatic breast cancer (MBC) may predict likelihood of brain metastasis [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 PD7-07.

Purpose: In contrast to recurrence after initial diagnosis of stage I–III breast cancer [recurrent metastatic breast cancer (rMBC)], de novo metastatic breast cancer (dnMBC) represents a unique setting to elucidate metastatic drivers in the absence of treatment selection. We present the genomic landscape of dnMBC and association with overall survival (OS). Experimental Design: Targeted DNA sequencing (OncoPanel) was prospectively performed on either primary or metastatic tumors from 926 patients (212 dnMBC and 714 rMBC). Single-nucleotide variants, copy-number variations, and tumor mutational burden (TMB) in treatment-naïve dnMBC primary tumors were compared with primary tumors in patients who ultimately developed rMBC, and correlated with OS across all dnMBC. Results: When comparing primary tumors by subtype, MYB amplification was enriched in triple-negative dnMBC versus rMBC (21.1% vs. 0%, P = 0.0005, q = 0.111). Mutations in KMTD2, SETD2, and PIK3CA were more prevalent, and TP53 and BRCA1 less prevalent, in primary HR+/HER2− tumors of dnMBC versus rMBC, though not significant after multiple comparison adjustment. Alterations associated with shorter OS in dnMBC included TP53 (wild-type: 79.7 months; altered: 44.2 months; P = 0.008, q = 0.107), MYC (79.7 vs. 23.3 months; P = 0.0003, q = 0.011), and cell-cycle (122.7 vs. 54.9 months; P = 0.034, q = 0.245) pathway genes. High TMB correlated with better OS in triple-negative dnMBC (P = 0.041). Conclusions: Genomic differences between treatment-naïve dnMBC and primary tumors of patients who developed rMBC may provide insight into mechanisms underlying metastatic potential and differential therapeutic sensitivity in dnMBC. Alterations associated with poor OS in dnMBC highlight the need for novel approaches to overcome potential intrinsic resistance to current treatments.