Balko, J. M.

Abstract Background: In MBC, CTC enumeration is highly prognostic for both progression free survival (PFS) and overall survival (OS). Multiparametric technologies enable for expanded phenotypic profiling of CTCs, including ER and HER2 assessment. Limited information is available on the concordance of CTC ER and HER2 levels compared to synchronously obtained tissue. We assessed the prognostic performance of CTC enumeration and phenotypic profiling of ER and HER2 on CTCs. Methods: The prospective, multi-institutional Aurora study enrolled pts with MBC of any subtype either at diagnosis of metastatic disease or at time of progression on any line of systemic therapy. Pts underwent a metastatic site biopsy, consented to archival primary tissue collection and donated blood samples serially every 3-4 months until progression. CTCs, from blood collected at study entry, were identified as nucleated, EpCAM+/ cytokeratin+/ CD45- cells using an automated imaging system (RareCyte, Seattle). ER and HER2 expression levels were measured by mean fluorescence intensity (MFI), with thresholds above 80 (ER) and 160 (HER2) considered positive. Comparisons were made to total RNAseq from paired metastatic tissue biopsies. PFS and OS were calculated from the time of study entry to last follow up or death. Results: Baseline CTC enumeration was performed in 154 pts. The most common clinical subtype was HR+/HER2- (59.7%), followed by HER2-positive (16.8%) and TNBC (15.5%). The median lines of prior therapy were 1 (range 0-10). At least 1 CTC was identified in 68/154 pts (44%), 30 (19%) had ≥ 5 CTCs, and 15 (9.7%) had CTC clusters. The median CTC value was 0 (0-877). The presence of CTCs (≥1 or ≥5) and CTC clusters was adversely associated with PFS and OS (Table 1 Table 1. CTC survival analyses Number Median PFS PFS HR (95% CI) Median OS OS HR (95% CI) Presence of CTC 0 CTC 86 7.37 (0.03 - 43.1) reference 20.72 (0.03 - 50.6) reference ≥ 1 CTC 68 4.7 (0.43 - 19.5) 1.65 (1.16-2.36) 12.04. (0.43-44.1) 2.13 (1.44-3.16) Presence of ≥ 5 CTC <5 CTC 124 7.7 (0.03 - 43.1) reference 19.31 (0.03 - 50.6) reference ≥ 5 CTC 30 2.88 (0.43 - 18.7) 1.77 (1.15 - 2.71) 5.74 (0.43 - 29.3) 3.59 (2.26-5.70) Presence of clusters 0 clusters 139 7.37 (0.03 - 43.1) reference 18.06 (0.03 - 50.6) reference ≥ 1 cluster 15 1.91 (0.43 - 18.7) 2.06 (1.16 - 3.68) 3.42 (0.43 - 18.7) 5.44 (2.94-10.09) ). Across all subtypes, the median CTC ER-MFI across was 36.2 (range 4.2-10,016.6) and the median HER2-MFI was 139.6 (range 4.2-2480.5). We observed high intra-patient heterogeneity of ER-MFI (CV: 0.65 [0.34-1.25]) and HER2-MFI (CV: 0.79 [0.42-1.54]). Clinical metastatic subtype from metastasis at study entry was available in 54/68 pts with CTCs. Pts with clinical ER+ MBC had a higher median ER-MFI compared to clinical TNBC (111.9 vs 68.6, p = 0.02). However, 11 pts (26.8%) with clinical ER+ MBC had all CTCs with an ER-MFI <80. Lower ER expression was observed in CTCs collected after prior exposure to a SERD (median ER-MFI: post-SERD [n=8] 27 vs post-aromatase inhibitor [n=4] 252, p<2.22e-16). No correlations were observed between the median CTC ER-MFI or HER2-MFI and paired tissue ESR1 or ERBB2 RNA expression (n=66). Conclusions: CTCs are strongly predictive of PFS and OS. CTC ER- and HER2-MFI were highly heterogeneous without clear correlation with bulk tissue RNA expression. The clinical impact of discordant clinical receptor status and CTC receptor status warrants further exploration. Citation Format: K. V. Giridhar, W. C. Nenad, H. Ye, G. L. Wheeler, T. Hinoue, A. R. Michmerhuizen, B. M. Felsheim, U. R. Chandran, B. J. Kelly, E. R. Mardis, P. W. Laird, J. M. Balko, A. C. Garrido-Castro, S. Coppens, J. Bowen, R. Nanda, N. U. Lin, C. K. Anders, C. Isaacs, J. J. Tao, J. D. Anampa, N. Jahan, L. Huppert, A. DeMichele, B. H. Park, S. Vinayak, A. L. Delson, M. M. Magbanua, M. Balic, H. A. Parsons, A. V. Lee, L. A. Carey, L. Norton, A. C. Wolff, N. E. Davidson, I. Krop, T. A. King, C. M. Perou, K. A. Hoadley, Aurora US Metastases Network. Characterizing circulating tumor cell (CTC) enumeration and phenotypic profiling in blood with synchronously obtained tissue in metastatic breast cancer (MBC) in TBCRC /AURORA US [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 PS2-07-17.

Abstract Background: PALLAS (PALbociclib CoLlaborative Adjuvant Study, NCT02513394) is a randomized phase III trial comparing two years of the CDK4/6 inhibitor palbociclib combined with the physician’s choice of adjuvant endocrine therapy, versus endocrine therapy alone for patients with Stage II-III hormone receptor-positive, HER2-negative (HR+/HER2-) breast cancer. Molecular residual disease (MRD) detection via circulating tumor DNA (ctDNA) testing was a predefined biomarker analysis to identify patients at highest risk of recurrence. Methods: PALLAS participants with available tumor block, at least a baseline (pre-study treatment) plasma sample, and either whole blood or buffy coat were randomly selected from the overall study population. Plasma samples on cycle 1 day 1 (C1D1), cycle 6 day 1 (C6D1), and at the end of treatment (EOT) were collected, processed, and stored. Nucleic acids were extracted from primary tumor tissue or, when primary tissue was insufficient, from post-neoadjuvant tumor tissue, provided the sample had sufficient material and tumor cellularity (>25 mm2 tissue area with ≥20% tumor nuclei). Data from next generation sequencing of tumor tissue and matched normal DNA were used to create a personalized ctDNA SignateraTM assay (Natera, Inc.) for MRD assessment in the peripheral blood. ctDNA was reported as mean tumor molecules/mL (MTM/mL) of plasma. The primary endpoint was distant recurrence-free interval (DRFI) based on ctDNA status. Cox Proportional Hazards models were used to evaluate prognosis and predictive interactions. To minimize bias, the biorepository and laboratory teams were blinded to participant identity. Results: In total, 1280 participants were randomly selected for inclusion in this preplanned ctDNA sub-study. WES and WGS are being conducted on tissue, and Signatera results will be generated. Results for association of MRD status (positive/negative) and MTM/mL with DRFI in the context of 7 year median follow up of this study cohort will be reported. Conclusions: The planned analysis is ongoing and the presented work will provide data from the first randomized phase 3 adjuvant study in HR+/HER2- breast cancer to report MRD status by treatment arm. Citation Format: H. A. Parsons, K. Ballman, E. Heitzer, M. Watson, M. Balic, D. Hlauschek, D. Renner, E. Kalashnikova, G. Steger, J. M. Balko, Y. Novik, M. Martin, A. A. Rodriguez, Z. Dayao, A. Chan, E. Nili Gal-Yam, M. C. Liu, C. Isaacs, M. Los, M. Gil Gil, B. Felder, C. Denkert, P. Fasching, F. Liu, T. O'Donnell, E. L. Mayer, M. Gnant, W. F. Symmans, A. DeMichele. Tumor-informed circulating tumor DNA analysis to assess molecular residual disease for prognosis and prediction of benefit from palbociclib in the PALLAS trial [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 RF3-04.

Abstract Background PALLAS trial compared palbociclib plus endocrine therapy (ET) vs. ET alone in stage II-III HR+/HER2- breast cancer. SETER/PR index quantifies transcriptional activity related to estrogen and progesterone receptors and serves as a measure of endocrine sensitivity. In CALGB 9741, low SETER/PR (< 0.75) - reflecting endocrine resistance - predicted survival benefit from adjuvant anthracycline/taxane-based treatment. In PALLAS low SETER/PR was associated with worse prognosis and early recurrence. Early recurrences driven by endocrine-resistant tumors may obscure the benefit of palbociclib by disproportionately contributing events from a more resistant population. We hypothesized that palbociclib efficacy might vary by endocrine sensitivity and prior chemotherapy regimen, with greatest benefit in patients with intermediate SETER/PR index. Methods Invasive disease-free survival (iDFS) was summarized with Kaplan-Meier plots and the treatment arms were compared with a log-rank test. Cox models estimated the hazard ratio (HR) with 95% confidence interval (95%CI) (significance level used was a two-sided 0.05). SETER/PR index was successfully obtained for 3,090 PALLAS patients (TransPALLAS ITT cohort, Metzger et al. ASCO 2024). SETER/PR index cut points (0.75 and 1.50) were proposed from previous studies comparing different adjuvant chemotherapy regimens. Results Table 1 summarizes the efficacy of adjuvant palbociclib by prior (neo)adjuvant chemotherapy regimen. Palbociclib was associated with longer iDFS in the group who received prior anthracycline-paclitaxel chemotherapy (AC-P), though the chemo regimen x palbociclib interaction term was not significant (Table 1). In the 1,132 patients from the TransPALLAS cohort who received AC-P chemotherapy, the 518 (46%) patients with intermediate SETER/PR (0.75≤ SETER/PR <1.50) had longer iDFS with palbociclib (HR 0.58; 95% CI: 0.40-0.84; p=0.004; 7-yr iDFS 80.2% vs. 68.4%), but this subset did not predict palbociclib benefit relative to all others, i.e., 206 (18%) patients with low SETER/PR (<0.75) and 408 (36%) high SETER/PR (≥1.50); P interaction=0.11. Exploratory analysis of SETER/PR index as a continuous variable in patients who received prior AC-P chemotherapy revealed a non-linear relationship with the hazard ratio favoring palbociclib observed in the subset with SETER/PR values between 1.1 and 1.8, compared to all others (P interaction=0.01). Conclusions In PALLAS, we observed a numerical benefit favoring palbociclib among pts treated with adjuvant AC-P; in this subset a moderate level of endocrine transcriptional activity (SETER/PR index between 1.1 and 1.8) predicted palbociclib benefit. These results are hypothesis-generating. Citation Format: O. Metzger, P. O'Brien, K. Ballman, M. Gnant, M. Watson, E. Chen, K. Tran, D. Hlauschek, M. Martin, J. Balko, Z. Nowecki, O. Hahn, C. Denkert, C. Curtis, F. Liu, A. Dueck, C. Fesl, E. Mayer, A. De Michele, W. Symmans. Exploratory analysis of palbociclib benefit in the PALLAS trial by SETERPR index and prior chemotherapy regimens (ABCSG-42/AFT-05/BIG-14-13) [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 PS3-08-13.

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 Background: Patients with metastatic breast cancer (MBC) often have short survival, and clinical disease management is challenging due to tumor heterogeneity, treatment resistance, and unique contributions of organ-specific metastatic microenvironments. Methods: The retrospective and prospective phases of the AURORA US Metastasis Project have profiled samples from 220 MBC patients using RNA-seq, DNA-seq, and DNA methylation arrays. RNA-seq has been performed on 392 specimens from 143 primary tumors and 249 metastases. In total, there are 162 primary-metastasis pairs from 134 patients (13 patients have 2+ metastases profiled). The 249 metastatic samples include 116 liver, 29 lymph node, 17 lung, 14 bone, 10 brain, and 8 skin metastases. Each specimen was assayed to determine its clinical and molecular subtypes. Here we investigate the molecular causes of clinical and PAM50 subtype switching in primary tumor-metastasis pairs using multi-platform data. Results: Of the patients with paired data in the AURORA dataset, 38/134 (28%) had clinical subtype (HR+/HER2+, HR-/HER2+, HR+/HER2-, TNBC) switches between the primary tumor and paired metastasis. The most common clinical subtype switch was loss of hormone receptor expression (16/38 patients) in the metastatic tumor. Many of these observations have been confirmed by concomitant RNA and/or DNA changes. In addition, 59/134 (44%) patients had PAM50 molecular subtype switches. 26/59 (44%) patients had a Luminal A primary tumor which switched either into Luminal B (16/26) or HER2-Enriched (7/26) subtypes, suggesting recurrent progression mechanisms during metastasis. For example, patient ARRP had a Luminal A primary tumor and a HER2-Enriched bone metastasis. Using DNA clonality data, we identified a clonal mutation in the DNA binding domain of FOXA1 (H247L) as well as mutations in CDH1 (frameshift) and NLRC3 (Q650H) that originated in the primary tumor. A distinct, dominant clone arose in the metastasis, harboring oncogenic activating mutations in PIK3CA (H1047R) and ERBB2 (L869R). There was also an increase in RNA expression of ERBB2 and FGFR4 but no change in ERBB2 copy number. This is in alignment with previous publications demonstrating a shift to the HER2-Enriched subtype, often without gain of HER2 amplification. Interestingly, there were 21 patients that had both clinical and PAM50 subtype switches. Patient ANFC had a HR+/HER2- primary tumor (Luminal A) and a TNBC liver metastasis (HER2-Enriched). Notably, the metastasis lost clinical expression of ER and PR (by IHC), and this was also observed through a decrease in ESR1 and PGR mRNA expression. Using DNA clonality data, we identified 5 new clones in the metastasis that were not present in the primary tumor. Finally, we observed that basal-like primary tumors with a PAM50 subtype switch in the paired metastasis had a significantly lower correlation to the basal-like centroid compared to primary tumors with a basal-like paired metastasis (p=0.0053). This suggests that either these tumors had significant intra-tumoral heterogeneity in the primary tumor and the metastasis originated from a non-basal-like portion of the tumor, or the switches from a basal-like primary tumor are a byproduct of the PAM50 bioinformatics algorithm (i.e. the tumor is very close to 2 subtype centroids). Ongoing analyses will precisely determine which subtype switches can be attributed to genetic or epigenetic causes, organ site biases, and/or bioinformatic methodologies. Conclusions: Using multi-platform molecular analyses, we identified cases of clinical and molecular subtype switching in primary-metastasis pairs. These findings begin to elucidate the underlying molecular drivers of tumor progression and subtype switching in patients with MBC, which may have implications for clinical treatment. Citation Format: A. R. Michmerhuizen, B. M. Felsheim, G. L. Wheeler, B. J. Kelly, M. Singha, T. Hinoue, W. C. Nenad, A. C. Garrido-Castro, J. M. Balko, K. Giridhar, U. Chandran, A. V. Lee, L. A. Carey, T. A. King, K. A. Hoadley, P. W. Laird, E. R. Mardis, C. M. Perou, AURORA US Metastasis Network. Molecular causes of subtype switching in primary and metastatic breast tumor pairs from the AURORA US Project [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 PD2-06.