TPS618 Background: GP2 is a biologic nine amino acid peptide of the HER2/ neu protein delivered in combination with Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) that stimulates an immune response targeting HER2/neu expressing cancers, the combination known as GLSI-100. Of the 146 patients that have been treated with GLSI-100 over 4 clinical trials, GLSI-100 was well-tolerated and no serious adverse events observed were considered related to the immunotherapy. Methods: This Phase III trial is a prospective, randomized, double-blinded, multi-center study. After 1 year of trastuzumab-based therapy, 6 intradermal injections of GLSI-100 or placebo will be administered over the first 6 months and 5 subsequent boosters will be administered over the next 2.5 years. The participant duration of the trial will be 3 years treatment plus 1 additional year follow-up. Study Size – Interim Analysis: Approximately 498 patients will be enrolled. To detect a hazard ratio of 0.3 in invasive breast cancer free survival (IBCFS), 28 events will be required. An interim analysis for superiority and futility will be conducted when at least 14 events have occurred. This sample size provides 80% power if the annual rate of events in placebo patients is 2.4% or greater. Up to 250 non-HLA-A*02 subjects will be enrolled in an open-label arm. Eligibility Criteria: The patient population is defined by these key eligibility criteria: 1) HER2/neu positive and HLA-A*02, 2) Residual disease or High risk pCR (Stage III at presentation) post neo-adjuvant therapy, 3) Exclude Stage IV, and 4) Completed at least 90% of planned trastuzumab-based therapy. Trial Objectives: The trial objectives are to: 1) Determine if GP2 therapy increases IBCFS, 2) Assess the safety profile of GP2, and 3) Monitor immunologic responses to treatment and assess relationship to efficacy and safety. Study Status: The study is actively recruiting and enrolling patients in the US and Europe at up to 150 sites. Contact Information: Greenwich LifeSciences, Inc., Stafford, TX; Email: Flamingo-01@greenwichlifesciences.com ; Website: greenwichlifesciences.com Funding: This trial is supported by Greenwich LifeSciences. Clinical trial information: NCT05232916 .

Abstract Background: This Phase III trial is a prospective, randomized, double-blinded, multi-center study (NCT05232916) in HLA-A*02 patients at approximately 140 sites in the US and Europe. A third non-randomized arm of approximately 250 non-HLA-A*02 patients is now fully enrolled and preliminary immune response data is presented below. GP2 is a biologic nine amino acid peptide of the HER2/neu protein delivered in combination with Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) that stimulates an immune response targeting HER2/neu expressing cancers, the combination known as GLSI-100. Methods: After standard of care neoadjuvant and adjuvant therapy, 6 intradermal injections of GLSI-100 will be administered over the first 6 months and 5 subsequent boosters will be administered over the next 2.5 years. The participant duration of the trial will be 3 years treatment plus 1 additional year follow-up. Immune responses to GP2 were measured over time using delayed-type-hypersensitivity (DTH) skin tests and injection site reactions (ISRs). The patient population is defined by these key eligibility criteria: 1) HER2/neu positive and HLA, 2) Residual disease or High risk pCR (Stage III at presentation) post neo-adjuvant therapy, 3) Exclude Stage IV, and 4) Completed at least 90% of planned trastuzumab-based therapy. Results: All patients (n=247) were vaccinated with GLSI-100 and continue in treatment and follow-up. A DTH reaction (redness) was used to assess in vivo immune responses in patients. The DTH orthogonal mean was measured 48-72 hours after injection. In this preliminary data analysis, there was a significant increase in percentage of subjects experiencing a DTH reaction in month 4 or month 6 compared to baseline. The frequency of DTH reactions increased by approximately 4x from 5.2% of the patients experiencing a DTH reaction at baseline, prior to any GLSI-100 administration, to 20.4% of the patients experiencing a DTH reaction in month 4 or month 6 (McNemar, p < 0.001). The study is ongoing and data collection and cleaning continue so final results may vary. Conclusions: The increase in the incidence of DTH reactions over time found in this preliminary analysis of GLSI-100 treated non-HLA-A*02 patients shows that GLSI-100 treatment should not be limited to the HLA-A*02 genotype. Subjects treated with GLSI-100 were increasingly able to mount an immune response to GP2 as evidenced in this preliminary data. Future investigations may explore the use of immune responses to assess: correlation of DTH to ISRs, immunogenicity of GLSI-100 by specific HLA type, timing of boosters to sustain immunity, clinical site performance, and the discontinuation of treatment for non-responders. Funding: This trial is supported by Greenwich LifeSciences. Citation Format: Snehal S. Patel, Jaye Thompson, F. Joseph Daugherty, Francois-Clement Bidard, William J. Gradishar, Marcus Schmidt, Miguel Martin, Joyce A. O’Shaughnessy, Hope S. Rugo, Cesar A. Santa-Maria, Laura M. Spring, Mothaffar F. Rimawi. Preliminary delayed-type-hypersensitivity immune response results from open-label arm of on-going Phase III study to evaluate the efficacy and safety of GLSI-100 (GP2 + GM-CSF) in breast cancer patients with residual disease or high-risk PCR after both neo-adjuvant and postoperative adjuvant anti-HER2 therapy, Flamingo-01 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr CT138.

BACKGROUND:Tumor-infiltrating lymphocytes (TILs), assessed by visual examination, are prognostic and predictive in early-stage triple-negative breast cancer. Computational assessment may provide a complementary approach. We evaluated the prognostic value of TILs by visual examination and computational assessment. METHODS:Cisplatin vs Paclitaxel for Triple Negative Breast Cancer (TBCRC030; ClinicalTrials.gov identifier NCT01982448) was a randomized phase 2 trial enrolling patients with BRCA1/2-proficient stage I to III triple-negative breast cancer to receive preoperative cisplatin or paclitaxel. The primary endpoint was pathological response at surgery. The TILs were visually scored on digitized pretreatment biopsies per International TILS Working Group recommendations. Computational assessment used the 4D Path QPOR platform to generate TILs, an immune heterogeneity index, and a combined immune/cell cycle biomarker (CmbI). Predictive performance for residual cancer burden 0/1 was assessed using receiver operating characteristic curves and odds ratios (ORs) with 95% CIs; all statistical tests were 2-sided. RESULTS:Of 139 response-evaluable patients, 121 had matched visual examination and computational assessment data (59 on cisplatin, 62 on paclitaxel). Median visual examination TILs were higher in responders (40.0% vs. 10.0%; P = .002) and predicted response (OR = 1.86, 95% CI = 1.24 to 2.87; area under the curve = 0.69, 95% CI = 0.57 to 0.80). Computational assessment CmbI differed by response group and predicted residual cancer burden 0/1 (OR = 3.20, 95% CI = 1.05 to 11.07; area under the curve = 0.62, 95% CI = 0.51 to 0.73). Computational assessment TILs and immune heterogeneity index were not predictive. Visual examination TILs and computational assessment CmbI predicted response to paclitaxel (OR = 2.91, 95% CI = 1.56 to 6.14; OR = 9.17, 95% CI = 2.01 to 66.39, respectively) but not to cisplatin. CONCLUSION:Visual examination TILs and computational assessment CmbI were each associated with response to neoadjuvant chemotherapy in triple-negative breast cancer in the overall cohort and the paclitaxel arm. Computational assessment CmbI did not outperform visual assessment. Further validation is needed before clinical implementation of computational approaches.

Abstract Introduction: CDK4/6 inhibitors (CDK4/6i) are the standard of care for hormone receptor-positive/HER2-negative breast cancer (HR+/HER2- BC). Yet resistance is common and often characterized by loss or dysfunction of the Retinoblastoma (Rb) protein or inadequate suppression of its phosphorylation. Circulating tumor cells (CTCs) offer minimally invasive means to monitor tumor biology. Here, we describe the analytical performance of a multiplex immunofluorescence (mIF) assay integrated with an image-analysis pipeline to quantify Rb and phospho-Rb (pRb) expression on CTCs and evaluation of cell recovery efficiency by CTCeptor, a microfluidic CTC isolation platform. Methods: The mIF assay utilized antibodies against Rb (AF595), pRb (AF488), cytokeratin (CK, AF647), and CD45 (AF555), with DAPI for nuclear staining. Antibody specificity and minimal non-specific binding were verified using single-marker staining and isotype controls. Assay performance (sensitivity, specificity, signal-to-noise ratio (SNR), and dynamic range) was evaluated using positive (MCF7, T47D) and negative (T47D-PalboR, MDA-MB-468, or leukocytes) control cell lines. Healthy donor leukocytes were spiked with control cell lines to simulate patient-derived matrices. Various blocking buffers, wash buffers, and cell attachment methods were compared. Cell recovery was evaluated after processing blood spiked with MCF7 cells using CTCeptor followed by staining with anti-CK-AF647, anti-CD45-AF555, and DAPI in HyPICC chamber. Imaging was performed on Leica STED SP8 or Nikon N-STORM microscopes. Quantitative analysis of mean fluorescence intensity (MFI) was conducted using FIJI, and statistical analysis was performed using GraphPad Prism v10. Results: The optimized mIF with integrated image analysis pipeline demonstrated high analytical sensitivity (Rb: 84%, pRb: 83%) and specificity (Rb: 96%, pRb: 100%). SNRs were 5 for Rb and 50 for pRb, with dynamic range exceeding two log units for both markers. Single-marker staining and isotype controls confirmed antibody specificity and minimal non-specific binding. Based on comparative analyses, 1% BSA + 0.5% goat serum was selected as the blocking buffer, and Triton-based buffer as the wash buffer. RareCyte® cell attachment method yielded superior cell retention (79%). Recovery rate after processing the spiked blood with MCF7 cells using CTCeptor followed by staining in HyPICC chamber ranged from 40-60%. Conclusion: We have developed and optimized a quantitative mIF assay and image analysis workflow to detect Rb and pRb expression on CTCs. Future studies will assess the predictive biomarker utility of the assay by measuring Rb and pRb dynamics on CTCs in metastatic HR+/HER2- BC patients before and after CDK4/6i therapy. Acknowledgement: This research was funded by CPRIT (RP210148) and NCATS CTSA pilot grant (1UM1TR004539-01A1). Citation Format: Mantasha Tabassum, Shivaani Suresh Kanna, Wangjia Cao, Suneel Kumar, Mothaffar F. Rimawi, George Miles, Meghana Trivedi. Method development and workflow optimization of a CTC-based biomarker assay to predict response to CDK4/6 inhibitors in HR+/HER2- breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5250.

Abstract Background: GP2 is a biologic nine amino acid peptide of the HER2/neu protein delivered in combination with Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) that stimulates an immune response targeting HER2/neu expressing cancers, the combination known as GLSI-100. Methods: This Phase III trial is a prospective, randomized, double-blinded, multi-center study (NCT05232916). After 1 year of trastuzumab-based therapy, 6 intradermal injections of GLSI-100 or placebo will be administered over the first 6 months and 5 subsequent boosters will be administered over the next 2.5 years. The participant duration of the trial will be 3 years treatment plus 1 additional year follow-up. Study Size - Interim Analysis: Approximately 498 patients will be enrolled. To detect a hazard ratio of 0.3 in invasive breast cancer free survival (IBCFS), 28 events will be required. An interim analysis for superiority and futility will be conducted when at least 14 events have occurred. This sample size provides 80% power if the annual rate of events in placebo patients is 2.4% or greater. Up to 250 non-HLA-A*02 subjects will be enrolled in an open-label arm. Eligibility Criteria: The patient population is defined by these key eligibility criteria: 1) HER2/neu positive and HLA-A*02, 2) Residual disease or High risk pCR (Stage III at presentation) post neo-adjuvant therapy, 3) Exclude Stage IV, and 4) Completed at least 90% of planned trastuzumab-based therapy. Trial Objectives: The trial objectives are to: 1) Determine if GP2 therapy increases IBCFS, 2) Assess the safety profile of GP2, and 3) Monitor immunologic responses to treatment and assess relationship to efficacy and safety. Study Status: The study is actively recruiting and enrolling patients in the US and Europe at up to 150 sites. Contact Information: Greenwich LifeSciences, Inc. Stafford, TX Email: Flamingo-01@greenwichlifesciences.com Website: greenwichlifesciences.com Funding: This trial is supported by Greenwich LifeSciences. Citation Format: Jaye Thompson, Snehal Patel, Mira Patel, Anu Tammareddi, F. Joseph Daugherty, Mothaffar F. Rimawi. Phase III study to evaluate the efficacy and safety of GLSI-100 (GP2 + GM-CSF) in breast cancer patients with residual disease or high-risk PCR after both neo-adjuvant and postoperative adjuvant anti-HER2 therapy, Flamingo-01 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr CT227.

PURPOSE:In TBCRC 022 (NCT01494662), neratinib and ado-trastuzumab emtansine (T-DM1) demonstrated intracranial activity among patients with HER2-positive breast cancer brain metastases. However, gastrointestinal (GI) toxicities-particularly diarrhea-were common, potentially impacting quality of life. Clinician-reported adverse event (CTCAE) grading may underestimate the patient experience. We report GI toxicities using patient-reported outcomes (PROs) in TBCRC's neratinib-T-DM1 cohort. METHODS:Patients received neratinib (160 mg daily) and T-DM1 (3.6 mg/kg IV every 21 days). A pre-planned analysis assessed patient-reported GI toxicities during Cycles 1-4 using the Patient-reported Outcomes Measurement Information System (PROMIS) GI Diarrhea scale, Systemic Therapy-Induced Diarrhea Assessment Tool (STIDAT), and PRO-CTCAE. Descriptive statistics and linear mixed effects models evaluated symptom trajectories over time. We evaluated agreement for PRO and clinician-reported data. RESULTS:Forty-four patients enrolled; all completed ≥1 PRO. GI symptom burden increased over Cycles 1-3. PROMIS scores worsened significantly by Cycle 2, with a peak mean increase of 6.62 (95% confidence interval (CI): 2.67-10.59; p = 0.002) at Cycle 3. STIDAT scores also worsened by Cycle 3 (mean change 0.50; 95%CI: 0.11-0.90; p = 0.015). Based on maximum PRO-CTCAE scores, moderate-to-severe symptoms were reported by 20.5% (diarrhea), 24.4% (appetite loss), and 41.0% (constipation). Agreement between PRO-CTCAE and clinician-reported CTCAE was low (kappa <0.2) with clinicians reporting less toxicity. PROMIS and STIDAT scores were significantly correlated. CONCLUSION:This GI-focused PRO analysis highlights the value of PROs in capturing patient-experienced toxicities that impact quality of life yet are underestimated by clinicians. Incorporating PROs into clinical trials can inform supportive care and prophylactic strategies.

Abstract Background: Endocrine therapy (ET) in combination with CDK4/6 inhibitors (i) is the standard-of-care for estrogen receptor (ER)+ metastatic breast cancer (BC). Though effective, resistance is inevitable. Aurora A kinase (AURKA) drives mitotic entry and spindle assembly for proper cell division. Clinically, high AURKA levels have been linked to cancer aggressiveness, poor prognosis, and CDK4/6i resistance. A recent phase II trial (NCT02860000) reported clinically meaningful benefit of the AURKAi, alisertib (ALS), in advanced, endocrine-resistant ER+ BC. Identifying biomarkers and key signaling pathways involved in AURKA inhibition is essential to optimize ALS use and guide effective combination strategies to enhance sensitivity and overcome resistance in ER+ BC. Methods: ER+ BC cell models naïve or resistant (R) to various ET (EndoR) and/or palbociclib (PalboR, a CDK4/6i) were used. ALS-resistant (ALSR) derivatives were developed via long-term exposure to gradually increasing ALS concentrations (>150 nM). Molecular changes associated with growth and survival signaling, senescence, and apoptosis were assessed by Western blot upon short-term ALS treatment (72 hr) and at resistance. Signaling profiles guided the choice of specific drug combinations, which were then evaluated by cell growth assays. Results: Morphological changes, characterized by flattened, enlarged giant cells, slowed growth, senescence, and elevated p21, γH2AX, and c-PARP levels were observed in the ALSR derivatives of EndoR and/or PalboR models. In general, induction of various components of the HER/PI3K/MAPK pathways was seen across all ALSR models, with a specific increase in phosphorylated AKT levels in some models. Indeed, AKTi plus ALS was the most effective combination to overcome resistance in the ET-naïve/PalboR and the estrogen-deprivation (ED)R/PalboR/ALSR models. In the tamoxifenR/PalboR model and its ALSR derivative, in line with the signaling changes, EGFRi, pan-HERi (Neratinib, Nrb), or MEKi together with tamoxifen+Palbo+ALS showed strong efficacy. In the dual fulvestrantR/PalboR model, ALS+Nrb was highly effective, and adding Palbo further enhanced this effect, while in its ALSR derivative, fulvestrant+Palbo+ALS+Nrb regimen was needed to achieve substantial growth inhibition. Conclusions: Our findings highlight the potential of combining ALS with AKT, MAPK, and HER pathway inhibitors, to enhance efficacy or overcome ALS resistance in ET- and CDK4/6i-resistant ER+ BC, inferred by ALS-induced signaling changes. The presence of flattened and enlarged giant cells may reflect ongoing endoreplication, potentially indicating a dormant or persister cell state that may eventually support relapse and disease progression. Additional studies are underway to determine how these cell states can be targeted to induce cell death and overcome resistance. Citation Format: Chia-Chia Liu, Alekya Raghavan, Lanfang Qin, Sarmistha Nanda, Fu-Tien Liao, Georg F. Bischof, Mothaffar F. Rimawi, C. Kent Osborne, Jamunarani Veeraraghavan, Rachel Schiff. Targeting Aurora A kinase together with alternative survival signaling overcomes resistance to endocrine and CDK4/6 therapies in ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7126.

Abstract Background: The use of CDK4/6 inhibitors (CDK4/6i) in combination with endocrine therapy (ET) has revolutionized treatment practices for advanced and high-risk ER+/HER2- breast cancer (BC) via effective inhibition of cell cycle activation. However, the vast majority of patients eventually progress on these therapies, underscoring the need for new treatment strategies to select patients, monitor response, and target novel therapeutic vulnerabilities. We previously reported that high expression of MCM2 and its origin licensing complex (LC) is enriched in primary tumors refractory to CDK4/6i (SABCS22-P2-03-10). LC factors are transcriptionally regulated by E2F/MYC-driven programs, which are activated by diverse drivers of resistance. We propose that MCM2/LC expression may be a biomarker reflecting multiple ET and CDK4/6i resistance mechanisms, due to its role in cell cycle activation. Methods: A retrospective review was conducted on a real-world, treatment-naive ER+/HER2- BC cohort of 1,960 largely metastatic patients with biopsies from breast or other sites (∼50% each) that underwent NGS profiling (DNA: 592-gene panel/whole-exome; RNA: whole-transcriptome) at Caris Life Sciences. Tumors were stratified by MCM2 or LC gene set mRNA into Q4 (high) versus Q1 (low; n=490 each), balanced for ET/CDK4/6i exposure, MSI-H, and TMB-H. Histology, PAM50, mutations (mut), gene amplifications(amp) and deletions(del), and differential gene expression were analyzed. Chi-square, Fisher’s Exact, or Mann-Whitney U tests were performed with Benjamini-Hochberg FDR for molecular variables (q<0.05). Results: MCM2-high vs -low tumors were LumB-dominant (LumB 85.5% vs 39.8%; LumA 5.3% vs 56.3%, p<1e-63) with less lobular histology (6.1% vs 17.1%, p=0.0001). Transcriptomically, PI3K/AKT/mTOR, E2F/MYC targets, G2M checkpoint, and DNA repair hallmark pathways were enriched in MCM2-high (all FDR q<0.05). Genomically, MCM2-high harbored a higher frequency of TP53mut (32.6% vs 16.6%, q=4e-6), RB1mut (5.8% vs 1.2%, q=0.052), CCND1amp (26% vs 6%, q=4.5e-11), FGF3/4/19amp (∼25% vs ∼6%, q≤2.2e-10), MYCamp (5.4% vs 0.8%, q=0.0136), and CDKN2A/Bdel (CDKN2A 29.3% vs 15.5%, q=6.9e-4; CDKN2B 17.5% vs 7.3%, q=1.38e-3). Notably, MCM2 and MKI67 gene expression were highly correlated (Spearman’s ρ=0.77, p<0.05). Very similar results were found for LC-high vs -low tumors. Conclusions: In ER+/HER2- BC, high MCM2/LC is associated with LumB subtype, E2F/MYC and cell cycle activation, selection of TP53 and RB1 mutations, and CCND1/MYC/FGF amplifications. Our findings suggest that MCM2 or LC mRNA may act as a surrogate marker for a network of oncogenic pathways previously identified to drive endocrine and CDK4/6i resistance. Future studies will evaluate MCM2/LC beyond proliferation, as a predictive biomarker for CDK4/6i-based regimens, as we have previously shown in the primary ET-treated setting (SABCS24-P1-03-20). Citation Format: Alekya Raghavan, Rosemary N. Plagens, Cynthia X. Ma, Stephanie Graff, Maryam Lustberg, Andrew Elliott, George W. Sledge, C. Kent Osborne, Mothaffar F. Rimawi, Ahmed Elkhanany, Rachel Schiff. MCM2 and the origin licensing complex: A putative node of ER+/HER2- breast cancer therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6647.

Stakeholders have made major advances in generation of real-world evidence (RWE); however, core challenges remain, limiting its value and utility in guiding treatment decision-making. In particular, persisting lack of industry consensus on standardized, clinically meaningful, patient-centered endpoints was considered a critical area of interest for the multidisciplinary Think tank on RWE In the US from Multiple Perspectives in Healthcare (TRIUMPH). Collaborating in a group forum, TRIUMPH assembled and reviewed a first draft of potential endpoints that may be more consistently incorporated into RWE research. Among this list, time to next treatment, adherence/persistence, psychosocial well-being, and frailty were prioritized for subsequent discussion on ways to enable their broader adoption. Across the four endpoints, key opportunities for improving adoption included additional education for researchers, standardization of algorithms, and exploration of new approaches for derivation in real-world data. Over the course of discussion, TRIUMPH also identified frailty and area/neighborhood socioeconomic status as key baseline characteristics that may guide stratified analyses of the prioritized endpoints, to increase patient-centric value of the resulting RWE. In the future, other endpoints and characteristics (beyond those in focus for TRIUMPH) will also likely emerge, and should similarly be incorporated into routine care and research, to continue growing the utility of RWE in better informing treatment decision-making.

Treatments for leptomeningeal metastasis (LM) are limited and prognosis is poor. In this phase 2, nonrandomized, single-arm, multicenter study, we evaluated a tucatinib-trastuzumab-capecitabine regimen in patients with newly diagnosed LM and human epidermal growth factor receptor 2-positive (HER2+) breast cancer. The primary endpoint was overall survival; secondary endpoints included central nervous system progression-free survival, LM objective response, neurological symptom improvement, pharmacokinetics and safety. The trial met its prespecified interim efficacy threshold and exceeded the historical control of 4.4 months. Among 17 enrolled women, all had magnetic resonance imaging-confirmed LM, 15 (88%) were symptomatic and 8 (47%) had abnormal cerebrospinal fluid cytology. For a median follow-up of 18 months (range 9.0-26.7 months), 6 of 17 (41%) remained alive. Tucatinib reached therapeutic levels in the cerebrospinal fluid. The median overall survival was 10 months (95% confidence interval 4.1 months, not reached). The median time to central nervous system progression was 6.9 months (95% confidence interval 2.8, 13.8 months). Of 13 response-evaluable patients, 5 (38%) achieved composite LM objective response. Of 12 evaluable patients, 7 (58%) had improved neurological deficits. This prospective study suggests clinical benefit with a systemic regimen for HER2+ LM including objective responses, improved symptoms and extended survival. These data support systemic therapy as an approach in HER2+ breast cancer LM. ClinicalTrials.gov registration: NCT03501979 .

Supplementary Figure S1. Study design and tissue availability for biomarker analysis.

Supplementary Figure S4. Decision tree algorithm to construct the multiparameter classifier of response in TBCRC023.

ADCs have revolutionized the therapeutic landscape in oncology. Three ADCs are US FDA-approved in MBC: sacituzumab govitecan (SG), trastuzumab deruxtecan (T-DXd) and trastuzumab emtansine (T-DM1), with many others in development. Despite these advances, ADC resistance mechanisms remain unknown. To discern biomarkers of therapeutic resistance, we evaluated genomic and transcriptomic differences in MBC before and after ADC treatment (tx). We analyzed next-generation sequencing (NGS) data from patients (pts) in the Tempus Database that received SG, T-DXd or T-DM1 pre or post biopsy (bx) (nSG=157, nT-Dxd=120, nT-DM1=84; 32.4% TNBC, 27.1% HR+/HER2-, 20.2% HER2+, 20.2% NOS). Pre-tx groups included pts with bx collected < 1 year before or < 15 days after the ADC start date. Post-tx groups included pts who had bx collected < 3 months (mo) after ADC end date. Acquired resistance (AcqRes) was analyzed by comparison of pre- and post-tx bx for pts treated with ADC for > 3 mo. Primary resistance (PrRes) was defined as pre- and post-tx bx for pts treated with ADC for < 3 mo. We evaluated resistance based on ADC mechanisms of action (MoA), including genes related to antigen expression, processing mechanism, payload effect and efflux pumps. Somatic differences were compared using Pearson’s Chi-squared test or Fisher’s exact test, as appropriate. In this exploratory analysis, all p-values are reported as uncorrected. The median log2(TPM) gene expression was compared between groups using the Wilcoxon rank sum exact test. In the AcqRes cohort, there were no significant differences in mutation frequency or expression of the target antigen for pre/post-SG or T-DXd bx, though in the pre- vs post-T-DM1 bx there was a decrease in ERBB2 alterations (69% vs 40%, p=0.047) and expression (10.9 vs. 8.4, p=0.024). A trend toward higher expression across efflux pump genes was seen in pts with AcqRes for SG (ABCB1: 2.7 vs 3.4, p=0.08; ABCC2: 2.4 vs 3.4, p=0.2) and T-DXd (ABCC1: 6.3 vs. 6.7, p=0.016; ABCB1: 2.77 vs. 3.44, p=0.4), with mixed changes in T-DM1 (ABCB1: 3.3 vs. 2.7, p=0.08; ABCC1: 6.3 vs. 6.8, p=0.05; ABCC2: 3.19 vs. 3.18, p=0.2). A trend of higher efflux pump gene expression was associated with PrRes to SG and T-DXd. Overall, there were no significant differences in expression of genes associated with ADC processing or DNA damage repair, though rare mutations in TOP1 and ATM were associated with PrRes to T-DXd. Genomic and transcriptomic analysis identified potential mechanisms of AcqRes and PrRes to SG and T-DXd, including higher drug efflux pump expression. This might be related to the ADC MoA, particularly the payload release and bystander effect. Additional research is needed to validate these novel findings and the molecular underpinnings mediating resistance to ADCs. Samer Alkassis, Marla Lipsyc-Sharf, Catherine Hegarty-Traverso, Jacob Mercer, Binyam Yilma, Stamatina Fragkogianni, Yuan Yuan, Funda Meric-Bernstam, Hope Rugo, Joyce O’Shaughnessy, Mothaffar Rimawi, Rachel Schiff, David Elashoff, John Glaspy, Martina McDermott, Amy Cummings, Calvin Chao, Aditya Bardia. Genomic and transcriptomic mediators of resistance to antibody-drug conjugates (ADCs) in metastatic breast cancer (MBC): A comprehensive multi-center study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1711.

Supplementary Figure S3. Distribution of intrinsic subtypes in (A) TBCRC023 and (B) PAMELA cohort.

Investigator list and Supplementary Table 1

BACKGROUND:Early metabolic change on PET/CT was predictive of response to neoadjuvant trastuzumab/pertuzumab (HP) in TBCRC026. We hypothesized that a composite biomarker incorporating PET/CT and HER2 tissue-based biomarkers could improve biomarker performance. METHODS:83 patients with estrogen receptor-negative/HER2-positive breast cancer received neoadjuvant HP alone [pathologic complete response (pCR) 22 %]. PET/CT was performed at baseline and 15 days post initiation of therapy (C1D15). Promising imaging biomarkers included ≥40 % SULmax decline between baseline and C1D15, and C1D15 SULmax ≤3. Baseline tissue-based biomarkers included HER2-enriched intrinsic subtype (72 %, 46/64; NanoString), tumor HER2 protein abundance (median log2 13.5, range log2 7.1-15.9; NanoString DSP), and HER2 3+ (83 %, 64/77; immunohistochemistry). Logistic regressions were fitted to predict pCR with HER2/PET-CT biomarkers. The C statistic assessed overall prediction power. The optimal composite score cut-off was determined by maximizing Youden's index. RESULTS:Factors most predictive for pCR in single predictor models included C1D15 SULmax (OR 0.43; p = 0.007, c = 0.77), % reduction in SULmax (OR 1.03, p = 0.006, c = 0.72) and tumor HER2 protein abundance (OR 1.75; p = 0.01, c = 0.76). The composite of C1D15 SULmax and % reduction in SULmax and their interaction term, had improved probability (c = 0.89 from c = 0.78), with high sensitivity (100 %) and negative predictive value (100 %). The addition of tumor HER2 protein did not further improve prediction power (c = 0.90). CONCLUSION:The HER2/PET-CT biomarker had high prediction power for pCR, however was not superior to the prediction power of PET/CT alone. Non-invasive PET/CT biomarkers may facilitate a response-guided approach to neoadjuvant therapy, allowing intensification and de-intensification of treatment, pending further evaluation.

HER2-positive (+) breast cancer is an aggressive disease with poor prognosis, a narrative that changed drastically with the advent and approval of trastuzumab, the first humanized monoclonal antibody targeting HER2. In addition to another monoclonal antibody, more classes of HER2-targeted agents, including tyrosine kinase inhibitors, and antibody-drug conjugates were developed in the years that followed. While these potent therapies have substantially improved the outcome of patients with HER2+ breast cancer, resistance has prevailed as a clinical challenge ever since the arrival of targeted agents. Efforts to develop new treatment regimens to treat/overcome resistance is futile without a primary understanding of the mechanistic underpinnings of resistance. Resistance could be attributed to mechanisms that are either specific to the tumor epithelial cells or those that emerge through changes in the tumor microenvironment. Reactivation of the HER receptor layer due to incomplete blockade of the HER receptor layer or due to alterations in the HER receptors is one of the major mechanisms. In other instances, resistance may occur due to deregulations in key downstream signaling such as the PI3K/AKT or RAS/MEK/ERK pathways or due to the emergence of compensatory pathways such as ER, other RTKs, or metabolic pathways. Potent new targeted agents and approaches to target key actionable drivers of resistance have already been identified, many of which are in early clinical development or under preclinical evaluation. Ongoing and future translational research will continue to uncover additional therapeutic vulnerabilities, as well as new targeted agents and approaches to treat and/or overcome anti-HER2 treatment resistance.