Citations

Abstract Background: While there are abundant data on the surgical management of BC after neoadjuvant chemotherapy (NAC), there is less guidance regarding surgical management of BC after NET, particularly with respect to lymph node (LN) surgery. While pathologic complete response (pCR) is common with NAC, it is a rare event with NET. In this analysis we assess the surgeons’ approach in the context of NET. Methods: The ALTERNATE trial is a phase III study that randomized postmenopausal patients (pts) with clinical stage II/III ER+ HER2- BC to receive neoadjuvant anastrozole (A), fulvestrant (F), or both for 6 mos. before surgery. The primary objective was to determine if neoadjuvant F or A+F increases the rate of pCR or ypT1-2N0/N1mic/Ki67 <2.7% residual disease over A alone. Breast surgery was per standard of care. Sentinel lymph node biopsy (SLNB) with or without axillary lymph node dissection (ALND) was required to determine preoperative endocrine prognostic index PEPI score and residual cancer burden (RCB). Surgeons were required to indicate if the patient would be eligible for breast- conserving surgery (BCS) prior to NET. Patient accrual occurred from 2014-2018. The surgical outcomes are reported for the entire group. Results: Among the 3 treatment arms, 933 patients completed 6 cycles of NET. The clinical T stage at diagnosis was 73.6% T2, 22.6% T3and 3.8% T4. Initial tumor size was assessed by mammogram and ultrasound. Primary tumor histology was ductal in 64.8% and lobular in 25.4%.568 pts (60.9%) were clinically node negative (cN0), 36.2% cN1, 2.6% cN2 with 0.3% cN3. LNs were palpable in 25.2%. 383 had LN needle biopsy pre NET, of which 80.9% were positive. The surgeons’ initial impression of the patient’s eligibility for BCS was 74% while 0.6% were considered inoperable.75.5% of pts had post-NET imaging performed. Of those with tumor size recorded, 52.9% were T1 or not visible. Overall, 69.9% of the 933 patients had BCS. Of those 330 pts without post-NET imaging or who had post-NET imaging without a size noted, 64.8% underwent BCS. For pts undergoing BCS, 80.2% required only one surgery for margin clearance. Of the 690 pts deemed eligible for BCS pre NET, 79% had BCS. Of the 240 pts deemed ineligible for BCS pre-NET, 43.8% had BCS. Of the pre-NET cT2 tumors, 79% had BCS while only 45.5% of T3 tumors and 37.1% of T4 tumors had BCS. On surgical pathology, 474 (50.8%) pts were LN+ compared to pre-NET cN+ 39.1% and needle biopsy-proven 33.2%. For pts with pN0, 93.3% had SLNB alone. 6.7 % of pN0 had ALND alone or SLNB+ALND. For those with pN1-3 , 36.3% had SLN only; 37.3% SLN+ ALND; 26.4% ALND alone. Only 0.8% of the patients had no ALND or SLNB performed. Pts undergoing BCS were more likely to have SLNB alone for a +SLN compared to mastectomy pts (62.9% vs. 37.6%; chi-square p<0.0001). For pts with pre-NET +LN on needle biopsy, only 26.8% underwent SLNB alone, while 37.4% underwent ALND alone. Of the 204 LN-positive pts having SLNB alone, 55.9% had only 1 + LN and 27% had only 2+ LNs. For pN+ pts who underwent SLNB +ALND, 42.1% had 1-2 + LNs. Conclusion: With NET, 69.9% of pts achieved BCS, including 43.8% deemed ineligible pre-NET. Given the low rate of pCR to NET, there should not be the expectation of pCR in +LNs. Based on the procedures performed in pts with +SLNs, it appears that surgeons were applying ACOSOG Z0011 criteria for pts having BCS, but were less likely to omit ALND for +LN in pts having mastectomy. The finding of only 1-2 +LNs in many of the LN+ pts suggests that omission of ALND may be a reasonable approach post-NET. Longer follow-up is required to assess local-regional recurrence. Support: U10CA180821, U10CA180882; https://acknowledgments.alliancefound.org. Clinical Trials.gov Identifier: NCT01953588 Citation Format: A. M. Leitch, T. Dockter, V. suman, A. Weiss, G. W. Unzeitig, J. Guenther, S. Sanati, K. Vij, J. Hoog, A. Caudle, A. Tiersten, M. Mita, W. Razaq, T. J. Hieken, Y. Wang, M. Rimawi, M. J. Ellis, E. Winer, K. K. Hunt, A. H. Partridge, L. A. Carey. Surgical outcomes in the ALTERNATE trial (Alliance A011106) -a randomized phase 3 neoadjuvant endocrine therapy (NET) trial in postmenopausal women with clinical stage II/III estrogen receptor positive (ER+) and HER2 negative (HER2-) breast cancer (BC) [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 RF1-02.

Abstract Breast cancer is the most common female malignancy in the U.S. and the leading cause of cancer-related deaths in women worldwide. Nearly 80% of breast tumors express estrogen receptor alpha (ERα) which drives tumor progression. While CDK4/6 inhibitors in combination with endocrine therapy improve overall survival in the metastatic setting and disease-free survival in the adjuvant setting, many patients eventually relapse/progress with resistant forms of disease. In the post-CDK4/6 inhibitor setting, second line endocrine therapy responses are limited, highlighting the need for alternative treatment approaches. Through a genome-wide CRISPR knockout screen, we identified Lemur Tail Kinase 2 (LMTK2) as the top hit exhibiting synthetic lethality in the presence of the tamoxifen metabolite, endoxifen, in both endocrine-sensitive and -resistant cell line models. We further studied multiple other endocrine resistant models and identified that LMTK2 mRNA and protein expression were both increased in ERα cell lines resistant to estrogen deprivation, tamoxifen, fulvestrant, abemaciclib and palbociclib, as well as in endocrine resistant patient derived models. We then studied LMTK2 in patient samples and identified copy number gain/amplification in 67% of ERα+ human metastatic breast tumors, while only 1% of primary tumors harbored these genomic alterations. From a prognostic standpoint, elevated tumoral LMTK2 expression was associated with worse recurrence-free survival and overall survival in early-stage breast cancer. Through proteomic studies, we identified the MET-PI3K-AKT pathway as overactivated in LMTK2 overexpressing cells. Unlike activating PIK3CA mutations that drive AKT signaling, LMTK2 was found to employ an alternative mechanism to activate AKT signaling by inhibiting PP1α, an AKT-inhibitory phosphatase. Further, LMTK2 overexpression was sufficient to drive endocrine and CDK4/6 inhibitor resistance in vitro and resulted in dramatic increases in AKT and ERα phosphorylation and activity. While knockout of LMTK2 in treatment naïve ERα+ breast cancer cells neither altered AKT/ERα phosphorylation/activity nor endocrine/CDK4/6 inhibitor responsiveness, attempts to knockout LMTK2 in endocrine/CDK4/6 inhibitor-resistant models failed, further confirming its essentiality in this setting. In summary we have uncovered LMTK2 as a novel biomarker of endocrine/CDK4/6 inhibitor resistance that is mechanistically linked to the AKT pathway. LMTK2 may be a novel drug target for the effective treatment of resistant and advanced forms of ERα+ breast cancer. Citation Format: A. M. Detry, S. Marigliano, J. Heinen, A. Bass, C. Jones, R. S. Muthyala, M. P. Goetz, J. R. Hawse. Lmtk2: a novel driver of endocrine resistance in ER+ breast cancer [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 PS2-11-01.

Abstract Background: We have identified two potentially predictive signatures of palbociclib resistance: the RBsig, composed of E2F1/E2F2 dependent genes, which is associated with genetic loss of RB1, and the ratio between the gene expression levels of CCNE1 to RB1 (CCNE1/RB1). Both signatures have been previously analyzed in vitro and in neoadjuvant and metastatic studies with palbociclib. The present analysis aims to explore the role of RBsig and CCNE1/RB1 in the phase III adjuvant PALLAS trial, which randomized patients to palbociclib plus endocrine therapy, versus endocrine therapy alone. Materials and methods: Gene expression data from PALLAS were generated using the HTG EdgeSeq Oncology BM Panel. Of the 87 genes composing RBsig, 46 were available within the EdgeSeq dataset and were used for the analyses; CCNE1 and RB1 were both available. RBsig was calculated as the mean of the Z-score scaled gene expression (log) of the 46 genes; CCNE1/RB1 was computed as the log ratio between the mRNA expression of CCNE1 and RB1. The prognostic/predictive effect of the continuous signatures in terms of invasive disease-free survival (IDFS), distant recurrence-free survival (DRFS), and overall survival (OS) was evaluated using Cox proportional hazard models. Results: HTG data were available from 2,587 of 5,748 patients from the entire PALLAS intention-to-treat (ITT) cohort. As RBsig and CCNE1/RB1 levels are affected by pre-surgical treatments, we limited the analyses to 1612 pre-treatment samples (530 core biopsies and 1082 surgical specimens) (analysis population). Compared with the entire PALLAS ITT cohort, the analysis population was significantly enriched for patients with low clinical risk (41.3% vs 59.4%) who did not receive prior chemotherapy (17.5% vs 55.9%). In keeping with the PALLAS ITT cohort, no significant benefit from the addition of palbociclib was observed in the analysis population. Higher baseline RBsig or CCNE/RB1 values were significantly and independently associated with worse IDFS (hazard ratio [HR] 1.63, 95% confidence interval [CI] 1.3-2.0; HR 1.22, 95%CI 1.1-1.3, respectively), DRFS (HR 1.94, 95%CI 1.5-2.5; HR 1.26, 95%CI 1.1-1.4, respectively) and OS (HR 1.79, 95% CI 1.3-2.4; HR 1.26, 95%CI 1.1-1.4, respectively) in the analysis population. No predictive effect of palbociclib benefit was observed for either signature in the analysis population, nor according to clinical risk or chemotherapy receipt. Interestingly, higher RBsig was strongly prognostic in patients who did not receive adjuvant chemotherapy (HR 1.83, 95%CI 1.4-2.3) but not in those who received it (HR 1.01, 95%CI 0.6-1.6) (interaction p=0.02) suggesting a potential predictive role of RBsig for chemotherapy benefit. Conclusions: In the PALLAS trial, higher baseline RBsig or CCNE1/RB1 values are associated with worse prognosis, but are not predictive of benefit from palbociclib in the adjuvant setting. RBsig might be associated with differential benefit from chemotherapy. Further study of these biomarkers may strengthen their role in risk stratification.https://acknowledgments.alliancefound.org Citation Format: L. Malorni, D. Hlauschek, E. Mayer, M. Benelli, L. Biganzoli, I. Migliaccio, A. Kermanidis, G. Pfeiler, E. P. Mamounas, A. M. Brufsky, M. Bellet-Ezquerra, K. Clifton, G. Rubovszky, T. Foukakis, M. Goetz, S. Lee, M. Ruiz-Borrego, W. Symmans, K. V. Ballman, F. Liu, J. Machacek-Link, C. Denkert, A. DeMichele, M. Gnant. Prognostic and predictive role of RBsig and CCNE1/RB1 gene-expression signatures for patients with early breast cancer treated with endocrine therapy with or without palbociclib in the PALLAS trial (ABCSG-42, AFT-05, BIG 14-03) [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 PS2-07-13.

Abstract Background: Microtubule inhibitors remain a standard chemotherapy in the management of HER2-negative metastatic breast cancer (MBC). CALGB (Alliance) 40502 was a phase 3 randomized study involving 799 patients with MBC receiving first-line chemotherapy, to determine the optimal chemotherapeutic agent among paclitaxel, nab-paclitaxel, or ixabepilone (with or without bevacizumab). Secondary analysis suggested inferior PFS with nab-paclitaxel specifically among patients with hormone receptor positive (HR+)/HER2-negative breast cancer. Correlative analysis demonstrated significant association of stromal tumor infiltrating lymphocytes (sTILs) with improved progression-free (PFS) and overall survival (OS) in CALGB40502. We hypothesized that immune activation would be associated with differential benefit among distinct microtubule agents. To address this, we evaluated the sTILs and RNA-based immune features association with clinical outcomes, including evaluation of nab-paclitaxel versus paclitaxel. Methods: To limit heterogeneity, analyses focused on pre-treatment primary tumor breast samples with central review/pathologist-enumerated sTILs in accordance with International TILs Working Group methods and RNAseq (n = 280). PAM50 molecular subtypes were assigned based on transcriptomic features. RNAseq data were used to generate immune deconvolution estimates from 5 distinct algorithms (CIBERSORT, xCell, ABIS, ConsensusTME, ImmuCellAI). 1,018 curated breast cancer gene expression signatures GES) were derived from previously published studies. Associations between sTILs and GES were evaluated with sTILs being a categorical variable with the thresholds <5% (low) and (≥5%) high. Results: In the evaluable population, basal-like PAM50 subtype was enriched for high sTILs category (p=1e-4), as anticipated. T-cell and B-cell immune signatures and immune deconvolution estimates were among most highly associated with sTILs. In addition to the expected prognostic association of high T-cell GES, high B-cell-related IgG signature score was also significantly associated with improved overall survival (OS) among triple-negative breast cancer (TNBC) in CALGB 40502 (HR = 0.50,95% CI:0.30-0.86, log-rank p=0.01) but not in HR+/HER2-. In an exploratory analysis of young patients (age at study entry <50 years), high sTILs were associated with improved OS among TNBC patients but worse OS among those with HR+/HER2- MBC (HR = 2.19, 95% CI:1.10-4.36, log-rank p=0.022). High (above median) RNA-based Xcell total immune score was significantly associated with worse OS in both CALGB 40502 and a validation dataset of primary breast cancers, METABRIC (OS HR = 2.12, 95% CI:1.01-4.45, log-rank p=0.042). To further investigate the clinical trial finding of inferior PFS for nab-paclitaxel compared with paclitaxel in patients with HR+/HER2- MBC, there was a significant enrichment of T-cell signatures associated with poor outcome in patients receiving nab-paclitaxel (Fisher exact p<0.001). Conclusion: In this translational analysis of sTILs and RNAseq from a large phase III clinical trial, sTILs and immune signatures were not uniformly associated with better outcome. Rather, high sTILs or immune signatures were associated with worse OS in young patients with HR+/HER2- breast cancer and higher T-cell signatures were associated with inferior PFS with nab-paclitaxel among HR+/HER2- MBC patients. These data reinforce the importance of context/subtype-specific interrogation of the tumor-immune microenvironment Support: U10CA180821, U10CA180882; https://acknowledgments.alliancefound.org. Bristol Myers Squibb (BMS); Clinicaltrials.gov Identifier: NCT00785291 Citation Format: E. K. Blige, K. V. Ballman, A. Michmerhuizen, M. Vater, L. A. Carey, A. H. Partridge, W. F. Symmans, M. A. Watson, C. M. Perou, D. G. Stover, H. S. Rugo. Rna-based immune features associated with benefit from distinct microtubule inhibitor therapy for metastatic her2-negative breast cancer: a post hoc analysis of the calgb 40502 (alliance) phase iii randomized clinical 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 PS1-13-17.

Abstract Background: In the treatment of ER+/HER2- metastatic breast cancer (MBC), randomized phase II/III trials testing selective estrogen receptor degraders (SERDs), proteolysis-targeting chimeras (PROTACs), complete estrogen receptor antagonists (CERANs) and selective ER modulators (SERMs) (mostly compared to fulvestrant) have demonstrated modest (1-2 months) improvements in progression-free survival (PFS), mainly limited to patients with ESR1-mutated MBC. In these studies, the median PFS (mPFS) ranges from 3 to 6 months, with shorter mPFS in patients with prior progression on CDK 4/6 inhibitor (CDK 4/6i). Here we report the final results of a randomized phase II study that compared Z-endoxifen (Endx) 80 mg/day with tamoxifen (Tam) 20 mg/day in postmenopausal women with estrogen receptor (ER)- positive/ HER2-negative MBC, and report outcomes according to ESR1, PIK3CA, PTEN and AKT alterations (Alliance A011203; NCT02311933). Methods: Postmenopausal women with unlimited prior endocrine regimens (including CDK 4/6i) and up to two chemotherapy regimens for MBC, with tumor biopsy confirmation of ER+ (>10% nuclear staining) and negative for HER2 amplification/overexpression were eligible. Prior adjuvant Tam was allowed if relapse was > 1 year following Tam completion. Patients randomized to Tam could cross over to Endx at progression. The primary end point was PFS analyzed by the interval censored data analytic approach. ctDNA was analyzed for ESR1 and PIK3CA/PTEN/AKT alterations using Guardant360 CDx 74-gene next-generation sequencing-based assay (Guardant Health). Results: Among 108 women who preregistered, 76 (Endx: 40; Tam: 36) were randomized and eligible. Patients were heavily pretreated; all patients had prior exposure to an aromatase inhibitor (AI), 50% had ≥ 2 AI based regimens in the metastatic setting and 50% in each arm had received fulvestrant. Approximately half on each arm received at least one chemotherapy regimen in the metastatic setting. Prior CDK 4/6i therapy was imbalanced (48% on Endx and 28% on Tam). Endx did not significantly prolong PFS compared to Tam (median PFS [mPFS] 4.3 vs 1.7 months; HR 0.72, p=0.168). Because arms were imbalanced according to prior CDK 4/6i exposure, we performed an unplanned analysis demonstrating that Endx improved PFS relative to Tam in CDK 4/6i-naïve patients (mPFS 8.3 vs 2.1 months; HR 0.39, p=0.0097) but not in patients treated with prior CDK 4/6i (mPFS 3.0 vs 1.5 months). In the subset of patients who consented for ctDNA testing (n=28), the mPFS for those with either an activating alteration in the PIK3CA/PTEN/AKT pathway or with an ESR1 mutation was similar as the overall trial. For the 24 patients who progressed on Tam and crossed-over to Endx, the median PFS was 3.5 months (range 1.5-15.0 months) with a 6-month clinical benefit rate of 33.3% (8/24; 90% CI: 17.8-52.1%). Conclusions: Endx did not meet the prespecified threshold for concluding that it significantly improved PFS relative to Tam in a cohort of heavily pretreated MBC. However, the observations of a > 6-month improvement in mPFS in CDK 4/6i-naïve patients, anti-tumor activity following crossover to Tam, and mPFS estimates similar to other novel endocrine agents in development all support the ongoing development of Endx. Support: U10CA180821, U10CA180882, U24CA196171, U10CA180820 (ECOG-ACRIN) and 5P50CA116201. Clinicaltrials.gov Id: NCT02311933. https://acknowledgments.alliancefound.org. Citation Format: M. P. Goetz, V. Suman, J. Reid, T. Dockter, C. Strand, W. F. Symmans, J. Hawse, A. Storniolo, J. Doroshow, J. M. Collins, H. Streicher, M. M. Ames, A. Tarnower, O. M. Hahn, J. N. Ingle, L. Carey, A. Partridge. Final results from the Randomized Phase II study of Z-Endoxifen and Tamoxifen in Endocrine Refractory, Estrogen Receptor Positive, HER2 negative Metastatic Breast Cancer (Alliance A011203) [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 PS1-08-07.

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.

Abstract Background: Despite its widespread adoption, there is a lack of data as to whether breast MRI for local staging and surgical planning of newly diagnosed breast cancer patients improves oncologic outcomes. The Alliance A011104 phase III trial was designed to test the hypothesis that MRI detection of mammographically occult disease helps to tailor surgical intervention thereby reducing local recurrence and improving local regional control. Here we report on the primary outcome of 5-year local regional recurrence. Methods: Patients with newly diagnosed clinical stage I-II, ER/PR- and HER2- negative (triple- negative) or ER/PR-negative and HER2-positive breast cancer, deemed eligible for breast- conserving surgery (BCS) based on clinical exam, mammography +/- ultrasound were randomized to undergo staging breast MRI or no further local staging. Patients with multifocal tumors that could be encompassed in a single operative resection were eligible. Patients who were carriers of gBRCA 1/2, or who had bilateral breast cancer, or a prior history of breast cancer were excluded. Initial eligibility was limited to patients not scheduled to receive neoadjuvant chemotherapy; however, a subsequent amendment allowed for enrollment after completion of neoadjuvant chemotherapy. Categorical variables were compared between arms with a chi-square or Fisher’s exact test. LRR, distant recurrence and overall survival (OS) were estimated with Kaplan-Meier point estimates and 95% confidence intervals (CIs). Hazard ratios (HRs) and 95% CIs were obtained from Cox proportional hazards models. Results: Of 319 patients enrolled from 2/2014-12/2019,161 were randomized to undergo breast MRI and 158 to no breast MRI evaluation. The mean age at enrollment was 58.9 years (range 29-85). Most patients (72.1%) had T1 tumors, 93.4% were cN0 and 19.7% were HER2 positive. Systemic chemotherapy was utilized in 85% with 17.6% receiving treatment neoadjuvantly; receipt of systemic therapy was unknown for 15 patients (4.7%). Two hundred and ninety-eight patients (93.4%) underwent surgical intervention; 91.9% underwent BCS as initial surgical procedure with no significant differences noted between groups (92.7% in the no MRI group and 91.9% in the MRI group [p=0.62]). Of patients who underwent breast surgery, 293 underwent axillary evaluation with the majority (270 patients, 92%) undergoing sentinel lymph node biopsy as the only axillary intervention. Among patients who received neoadjuvant chemotherapy, the overall pathologic complete response rate was 39.3%, 29.0%in the MRI arm vs. 52.0% no MRI arm (p=0.10). Receipt of adjuvant radiation was comparable between arms, 85.4% and 85.0% receiving radiation in the no MRI and MRI arms, respectively. Among 298 patients evaluable for the primary endpoint, with median follow-up of 61.1 months (0.2, 68.1), there were no differences in rates of LRR; 5-year local regional control was 93.2% (89.0-97.6%) in the MRI group and 95.7% (92.3-99.1%) in the no MRI group (HR: 1.1, 95% CI: 0.3, 3.9). For the entire cohort, 5-year distant recurrence-free rate and OS were 94.3% (91.7, 97.1) and 92.2% (89.1, 95.4), respectively, with no differences between groups. Conclusions: With multimodality therapy, rates of 5-year LRR in patients with early stage triple- negative and HER2-positive breast cancer are very low. Breast MRI for local staging and surgical planning in this setting does not result in improved local regional control. Support: U10CA180821, U10CA180882; UG1CA189823; U10CA180868 (NRG Oncology); ClinicalTrials.gov Identifier: NCT01805076; https://acknowledgments.alliancefound.org Citation Format: I. Bedrosian, K. Ballman, L. M. McCall, C. E. Comstock, G. W. Unzeitig, T. Yen, A. Weiss, B. G. Haffty, J. M. Bensenhaver, S. M. DeSnyder, J. J. Liu, A. H. Partridge, L. A. Carey, K. K. Hunt.Effect of Preoperative Breast MRI Staging on Local Regional Recurrence (LRR) in Early Stage Breast cancer: Alliance A011104/ACRIN 6694 [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 GS2-07.

BACKGROUND: With pulmonary hypertension (PH), a pulmonary artery wedge pressure (PAWP)>15 mm Hg is used to diagnose left heart dysfunction, but some patients with adjudicated group 1 PH demonstrate PAWP>15 mm Hg. The primary objective of the study was to evaluate group 1 PH with high PAWP>15 mm Hg. METHODS: Patients with adjudicated group 1 PH from PVDOMICS between 2016 and 2019 were separated into high PAWP(>15 mm Hg) or normal PAWP and compared with adjudicated combined pre- and postcapillary (Cpc) PH related to heart failure with preserved ejection fraction (HFpEF). Participants underwent dynamic right heart catheterization and metabolomics. Findings were validated in 3 independent cohorts with adjudicated group 1 PH (validation cohorts 1 and 2 with exercise right heart catheterization and validation cohort 3 with resting right heart catheterization). RESULTS: Of 325 patients with group 1 PH (73% women, mean age 53.0±14.3 years), 15% (n=48) had high PAWP. Group 1 PH+high PAWP demonstrated greater obesity, left ventricular hypertrophy ( P <0.0002), left ventricular strain impairment ( P <0.0001), and decreased left ventricular compliance ( P <0.0001) compared with group 1 PH+normal PAWP, with changes comparable to Cpc-PH HFpEF (n=75). Compared with Cpc-PH HFpEF, left atrial function was better in group 1 PH+high PAWP with lower PAWP V wave, higher left atrial compliance, and left atrial ejection fraction ( P <0.0001 for all). Metabolomics demonstrated little difference between group 1 PH with high versus normal PAWP but large differences between group 1 PH+high PAWP versus Cpc-PH HFpEF (100 metabolites altered at false discovery rate P <0.05). Elevated PAWP was also observed in 18% of group 1 PH in the validation cohort 1 (n=402), with exercise PAWP response intermediately abnormal in group 1 PH+high PAWP relative to Cpc-PH HFpEF and group 1 PH+normal PAWP (interaction P <0.0001). Elevated PAWP was similarly observed in 22% and 19% of group 1 PH in validation cohorts 2 (n=55) and 3 (n=787), respectively. CONCLUSIONS: Approximately 1 in 5 adjudicated patients with group 1 PH has elevation in resting PAWP despite severe pulmonary vascular dysfunction and metabolomics consistent with traditionally defined group 1 PH. Despite resting PAWP elevation, these patients with group 1 PH were metabolomically and biologically distinct from Cpc PH HFpEF, with better left atrial function and diastolic reserve during exercise. These data emphasize the limitations of using resting PAWP alone to separate group 1 PH from HFpEF and call for development of more integrated clinical diagnostic criteria.

Abstract Intravital imaging studies have provided insights into the spatial and temporal variations of platelet activation and thrombin generation that occur during hemostasis; however, these studies are generally limited to small vessels due to the practical limitations of imaging in thicker tissues. Recent advances in cleared tissue fluorescence imaging as well as volume electron microscopy (vEM) coupled with machine learning–based image segmentation provide an opportunity for analysis of the 3-dimensional structure of complex tissues. We utilized these technologies to examine hemostatic plugs from murine jugular veins and carotid arteries to investigate the spatial distribution of platelet activation and biochemical responses in these disparate physiologic contexts. Both venous and arterial hemostatic plugs had a heterogeneous structure with regions of sparsely and densely packed platelets. Despite similar injury sizes, arterial hemostatic plugs were at least an order of magnitude larger than venous plugs. The difference in plug size was primarily due to a 19-fold increase in the population of densely packed platelets in the extravascular compartment. Venous plugs displayed significant platelet aggregation extending into the vessel lumen and developed distinctive fibrin and red blood cell–filled cavities. Complementary fluorescence microscopy revealed that platelet activation was spatially heterogeneous in both contexts, with α-granule secretion and phosphatidylserine exposure confined to specific microenvironments, highlighting tightly regulated thrombin activity. Overall, our findings reveal both conserved and distinct mechanisms of hemostatic thrombus formation in different physiologic contexts. They also demonstrate the power of vEM coupled with machine learning–based image segmentation for the quantitative analysis of large imaging data sets from complex tissues.