Mardis, ER

Abstract Introduction: With emerging antibody drug conjugates (ADC) as treatment of metastatic breast cancer (mBC), it is key to understand the mechanisms that drive resistance to inform optimal ADC selection and sequencing. The AURORA (AUR) US Network was established to characterize paired primary and metastatic (met) samples via multiplatform profiling, with a prospective phase of met tissue collection that included pre and post-ADC samples. Methods: Fresh frozen or FFPE met and archival primary tumors were analyzed across 3 platforms: DNA low pass whole genome and exome sequencing (WES), DNA methylation arrays (Methyl) and RNA sequencing (RNA seq). The molecular cohort included pts with data from ≥1 platform. Pairwise gene expression comparisons were performed in R using t-tests, and pathway analysis with GSEA. Results: A total of 168 pts comprised the molecular cohort: WES, 168; Methyl, 163; RNA seq, 168. Median age at mBC diagnosis was 56 (28-88). 21 pts (12.5%) had known germline BRCA1/2 mutations; 37 (22.0%) presented with de novo mBC. Subtype at mBC (if not available, at primary) diagnosis was: 108 (64.3%) HR+/HER2-, 22 (13.1%) HER2+, 33 (19.6%) TNBC and 5 (3.0%) unknown. Among 139 pts with known subtype in the first AUR met collected, 95 (68.3%) were HR+/HER2-, 17 (12.2%) HER2+ and 27 (19.4%) TNBC. 72 pts received ≥1 ADC with 1 (0-4) median line of chemotherapy for mBC prior to ADC1. As ADC1, 42 pts received HER2 ADC (35 trastuzumab deruxtecan, T-DXd; 7 trastuzumab emtansine), 29 TROP2 ADC (28 sacituzumab govitecan, SG; 1 datopotamab deruxtecan) and 1 NECTIN4 ADC. 22 pts received ≥2 ADC (16 immediate sequence; 6 intervening therapy); 11 T-DXd and 11 SG as ADC2. Median duration of ADC1 was 141 days (6-522); 81 (7-425) for ADC2. Progressive disease was the most common reason for ADC discontinuation, denoting resistance in post-ADC samples. 99 samples were collected pre-ADC1 and 24 post-ADC1, of which 5 were pre-ADC2. ERBB2 expression did not differ between samples after HER2 (n=9) vs non-HER2 ADC (n=8) (p=0.55), nor did TACSTD2 after TROP2 (n=8) vs non-TROP2 ADC (n=9) (p=0.16). In pts with HER2+ subtype at mBC diagnosis or AUR collection who received HER2 ADC, ERBB2 expression significantly decreased in post (n=4) vs pre-treatment (n=7) samples (p=0.043). ERBB2 downregulation was observed in 5 pts with paired pre vs post-HER2 ADC samples. In pts who received TROP2 ADC, TACSTD2 expression did not differ in post (n=10) vs pre-treatment (n=47) samples (p=0.63). GSEA of post-ADC samples revealed significant pathway differences by payload, including actin cytoskeleton in post-microtubule inhibitor (MTi) vs non-MTi ADC samples (p=0.001) and ATM-dependent DNA damage response after topoisomerase I inhibitor (TOP1i) vs non-TOP1i ADCs (p=0.043). Lysosomal vesicle biogenesis (p=0.014) and calcium release (p=0.05) pathways significantly differed between ADC naïve vs treated metastases. ERBB2 hypermethylation was not detected in the cohort. TACST2 promoter methylation was observed in 10 samples (β value >0.3) without association with TROP2 ADC exposure. ERBB2 or TACSTD2 mutations were not identified in the ADC-treated cohort. A novel loss-of-function (LOF) mutation in MED12 was detected in one pt immediately post-T-DXd. MED12 LOF has been reported to promote PARP inhibitor and platinum resistance in BRCA-deficient tumors and may represent a novel resistance mechanism to TOP1i. Conclusions: Multiplatform molecular characterization of mBC treated with ADC revealed potential mechanisms of resistance related to downregulation of target expression, payload (e.g., microtubule formation, DNA damage repair) and lysosomal processing. Additional analyses integrating these multimodal data are ongoing. Strategies to overcome resistance, e.g. modifying ADC target, are being evaluated in clinical trials. Citation Format: A. C. Garrido-Castro, W. C. Nenad, J. M. Balko, T. Hinoue, G. L. Wheeler, B. J. Kelly, M. Singha, B. M. Felsheim, A. R. Michmerhuizen, U. Chandran, A. V. Lee, L. A. Carey, T. A. King, E. Mardis, P. W. Laird, K. V. Giridhar, K. A. Hoadley, C. M. Perou. Molecular characterization of resistance to antibody drug conjugates in metastatic breast cancer: a prospective analysis from the AURORA US Network [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PD3-09.

Abstract Support: Alliance U10CA180821; Alliance Statistical Center grant U10CA180882; ACOSOG grant U10CA76001 HER2 gene amplification and its corresponding overexpression are present in approximately 12% of invasive breast cancers. While HER2-targeted agents (e.g. trastuzumab, pertuzumab, and lapatinib) are effective treatments, resistance remains a major cause of death from HER2-positive breast cancer. Mechanisms of resistance are poorly understood. Without a molecular understanding of these mechanisms, therapeutic advances will be delayed. We have generated molecular profiles of primary HER2-positive breast cancers treated on a neoadjuvant clinical trial, and compared features associated with response to treatment. The American College of Surgeons Oncology Group (ACOSOG) Z1041 trial in HER2-positive breast cancer was designed to compare the pathologic complete response (pCR) rate of a regimen of paclitaxel and trastuzumab, followed by trastuzumab administered with fluorouracil, epirubicin, and cyclophosphamide (FEC-75) to a regimen of FEC-75 alone followed by paclitaxel and trastuzumab. The trial identified no difference in pCR rates between the regimens (Buzdar et al., The Lancet Oncology 2013). In supplement to the tissues obtained from 37 of the patients enrolled in the Z1041 trial, an additional 11 cases were obtained from a single institution study (201101961) of patients treated with neoadjuvant trastuzumab that had pre-treatment core biopsies suitable for genomic studies. We have extracted genomic DNA from both pretreatment tumor biopsies and blood samples of these 48 patients and performed whole genome (WGS) and exome sequencing. Coincident with these efforts, we have extracted high quality RNA from 42 of the 48 biopsies, and have processed RNA-seq profiles of the tumors. Among patients in this cohort, 24 (50%) achieved a pCR. Because no difference was observed between arms of the Z1041 trial, patients with or without a pCR were directly compared without adjusting for treatment regimen. On average, each tumor and normal sample pair were sequenced to a depth of 49.4x and 32.5x by WGS respectively. In total, 15,027 candidate somatic variants were identified in known genes, including 11,606 missense, 860 nonsense, and 418 frameshift insertions or deletions. Preliminary results identified mutations in HER2 that were associated with the failure to achieve pCR in several cases. Furthermore, tumors assigned to the HER2-enriched subtype by RNA-seq analysis were more likely to achieve a pCR (19 compared to 8) than tumors with genomic features indicative of either the luminal or basal-like subtypes (3 compared to 12); a significant difference in the proportion of cases that achieve pCR (Fisher's exact test p-value = 0.0032). The identification of these features suggests that it may be possible to predict, at the time of diagnosis, those patients who will not respond to the current standard of care for HER2-positive breast cancer. Citation Format: Lesurf R, Griffith O, Griffith M, Watson MA, Hoog J, Ellis MJ, Ota D, Suman VJ, Meric-Bernstam F, Leitch AM, Boughey JC, Unzeitig G, Buzdar AU, Hunt KK, Mardis ER. The genomics of response to neoadjuvant trastuzumab and chemotherapy in HER2-positive breast cancer – Results from the ACOSOG Z1041 (Alliance) trial. [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 PD6-02.

Abstract Background: Intrinsic breast cancer subtypes determined by the PAM50 assay are reported to be prognostic independent of standard clinicopathological variables. The CALGB (Alliance) 9741 adjuvant trial randomized treatment in a 2×2 factorial design to (i) 2-wk (dose dense; DD) vs 3-wk therapy and (ii) sequential vs concurrent doxorubicin, cyclophosphamide, paclitaxel (A>T>C vs AC>T). DD therapy improved disease-free survival and overall survival (OS) (Citron et al. JCO 2003.). A significant interaction between intrinsic breast cancer subtypes and the use of DD therapy was hypothesized. Methods: C9741 was conducted in collaboration with ECOG, NCCTG, and SWOG. Biospecimens were collected from 1652 of 2005 subjects. Multiplexed gene expression profiling (NanoString Technologies, Inc) generated PAM50 subtype calls (luminal A, luminal B, HER2-enriched, basal-like), risk of relapse (ROR) score, and proliferation score for 1321 cases (80%). Excluded samples were due to insufficient tumor (n = 181) or RNA (n = 150). The primary endpoint was relapse-free survival (RFS). The primary analysis to determine if the clinical benefit of DD therapy is dependent on intrinsic subtype was conducted as a test of interaction in a Cox proportional hazards model (3 degrees of freedom, df; alpha=0.05). Similar analyses were performed for ROR score and proliferation score as continuous measures of risk (1 df). PAM50 subtype, ROR score, and proliferation score were evaluated in separate multivariate models adjusting for number of positive nodes, menopausal status, dose density, and sequence of therapy. Results: Improved outcomes for DD therapy in the evaluable subset mirrored results from the complete dataset (HR = 1.20; 95%CI 0.99–1.44) with a median follow-up of 12.3 yrs. Subtype analysis identified 32% luminal A (n = 417), 26% luminal B (n = 341), 20% HER2-enriched (n = 267), and 22% basal-like (n = 296). Intrinsic subtypes were prognostic of RFS (p < 0.0001) irrespective of treatment assignment with HRs relative to the luminal A subgroup of 1.50 (luminal B), 1.70 (HER2-enriched) and 1.66 (basal-like). No subtype specific treatment effect on RFS was identified (p = 0.44). ROR scores (range 0–100, median 60) were also prognostic of RFS (HR = 1.12 for a 10-unit change; 95% CI 1.07–1.18; p < 0.0001), but no association with DD therapy benefit was seen (p = 0.58). Similar results were obtained for the proliferation score, for OS as a secondary endpoint, and from multivariate models with clinical covariates. Conclusion: The prognostic value of PAM50 subtype, ROR score, and proliferation score remains highly significant in patients treated with contemporary adjuvant anthracycline and taxane based chemotherapy. Intrinsic subtype did not predict for improved survival with the 2-wk DD vs 3-wk treatment schedule; we hypothesize that this is because the prognostic differences by subtype outweighed the modest but significant clinical benefit of DD chemotherapy administration for the overall population. Planned clinical validation studies will assess the ability of PAM50 and other gene signatures to stratify patients and individualize systemic therapy regimens based on the expected risk of distant recurrence. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-10-01.