Isakoff, Steven J.

536 Background: Folate receptor alpha (FRα) is overexpressed in ~80% of triple-negative breast cancers (TNBC). We previously demonstrated that patients (pts) with breast cancer naturally mount immune responses to FRα. In prior Phase I and II studies, a GM-CSF-adjuvanted FRα peptide vaccine (FRPV) was safe and induced durable FRα-specific T-cell immunity in 83–90% of pts with breast and ovarian cancer. Methods: Eligible pts were women ≥18 years with TNBC (ER/PR ≤10%) with 1+ of the following: stage ≥T1c disease, node-positivity, or residual disease after neoadjuvant chemotherapy. Pts were randomized 2:1 to receive FRPV or placebo (GM-CSF alone) every 28 days for cycles 2-7 and then every six months for 7 booster doses, with cycle 1 consisting of cyclophosphamide 50mg orally twice daily for days 1-7 and 15-21 (in both arms). Randomization was stratified by chemotherapy setting (adjuvant only vs neoadjuvant) and stage (I/II vs III). FRα expression was evaluated by immunohistochemistry. The study was designed to detect (at 30 months after the last pt enrolled) a disease-free survival (DFS) difference of 14% assuming a 3.5-year DFS rate of 70% in the control group and with 78% power. Results: 280 pts were enrolled (median age 52). The majority had stage I or II tumors (n = 219, 78%). There were numerically more node-positive tumors in the vaccine arm, 78 (41%) vs 27 (32%), p = 0.15. Median follow-up was 3.5 years, and 33 (FRPV: 25, placebo: 8) DFS events were observed; 3.5-year DFS rate was 88% with FRPV and 90% with placebo (HR 1.42, 95% CI 0.64–3.15; p = 0.39). Overall survival (OS) also did not differ by arm (HR 2.48, 95% CI 0.55–11.20; p = 0.22). Among node-positive pts (n = 105), DFS did not differ between arms (HR 0.95, 95% CI 0.34–2.66; p = 0.92). However, a numerical DFS event rate improvement (14% for vaccine vs. 26% for placebo) was observed in 61 patients with stage III disease (HR 0.51, 95% CI 0.16–1.68; p = 0.26). DFS did not differ by chemotherapy setting or by receipt of anthracycline, capecitabine, and/or an immune checkpoint inhibitor. FRPV was well tolerated, with predominantly grade 1–2 adverse events (AEs) and no vaccine-related grade ≥4 AEs. FRα expression was available from tumor in 230 pts, with 72% expressing > 0. Overall, DFS was not significantly associated with FRα expression (0 vs > 0; HR 0.55, 95% CI 0.21–1.44; p = 0.21), and no significant differences in DFS by FRα expression were observed between treatment arms. Conclusions: FRPV following oral cyclophosphamide was safe, with no vaccine-related high-grade AEs. While no significant improvement in DFS or OS was observed overall, a numeric trend toward improved DFS was observed in pts with stage III TNBC. This finding is hypothesis-generating and supports further evaluation of FRα-targeted vaccination in higher-risk TNBC. Immune correlative analyses are ongoing to better define vaccine immunogenicity and its relationship to clinical outcomes. Clinical trial information: W81XWH-15-1-0293.

Background Immune exclusion inhibits antitumor immunity and response to immunotherapy, but its mechanisms remain poorly defined. In triple-negative breast cancer (TNBC), an aggressive and generally immune-rich subtype, an immune-cold microenvironment predicts poor prognosis due to a limited response to chemotherapy and immune checkpoint inhibitors. This study aimed to identify mechanisms regulating immune infiltration in TNBC. Methods We performed spatial transcriptomic analysis comparing immune-enriched versus immune-cold treatment-naïve TNBCs. Functional analyses, including loss-of-function and reconstitution experiments, were conducted to investigate the role of trophoblast cell-surface antigen 2 (TROP2), a key target of anticancer antibody drug conjugates (ADCs), in promoting TNBC progression. A humanized TROP2 syngeneic TNBC model was used to assess the effects of TROP2-targeting in combination with anti-programmed cell death protein 1 (PD-1) therapy. Additionally, data from patients treated with immune checkpoint blockade were used to test hypotheses from the preclinical findings. Results We reveal that TROP2 controls barrier-mediated immune exclusion in TNBC through claudin 7 association and tight junction regulation. TROP2 expression is inversely correlated with T-cell infiltration and predicts poor outcomes in TNBC. We demonstrate that TROP2 is sufficient to drive tumor progression in vivo in a CD8 T cell-dependent manner, while its loss deregulates expression and localization of multiple tight junction proteins, enabling T-cell infiltration. We show that TROP2 targeting via hRS7, the antibody component of the ADC sacituzumab govitecan, enhances the anti-PD-1 response and improves T-cell accessibility and effector function. Correspondingly, TROP2 expression is highly associated with lack of response to anti-PD-1 therapy in human breast cancer. Conclusions This study defines a new mechanism of barrier-mediated immune exclusion in cancer controlled by TROP2-dependent tight junctions. This mechanism drives tumor progression but can be targeted via TROP2-directed therapy to activate antitumor immunity and enhance immunotherapy response.

ABSTRACT Background Immune exclusion inhibits anti-tumor immunity and response to immunotherapy, but its mechanisms remain poorly defined. In triple-negative breast cancer (TNBC), an aggressive and generally immune-rich subtype, an immune-cold microenvironment predicts poor prognosis due to a limited response to chemotherapy and immune checkpoint inhibitors. This study aimed to identify mechanisms regulating immune infiltration in TNBC. Methods We performed spatial transcriptomic analysis comparing immune-enriched versus immune-cold treatment-naïve TNBCs. Functional analyses, including loss-of-function and reconstitution experiments, were conducted to investigate the role of Trophoblast Cell-Surface Antigen 2 (TROP2), a key target of anti-cancer Antibody Drug Conjugates (ADCs), in promoting TNBC progression. A humanized TROP2 syngeneic TNBC model was used to assess the effects of TROP2-targeting in combination with anti-PD1 therapy. Additionally, data from patients treated with immune checkpoint blockade were used to test hypotheses from the preclinical findings. Results We reveal that TROP2 controls barrier-mediated immune exclusion in TNBC through Claudin 7 association and tight junction regulation. TROP2 expression is inversely correlated with T cell infiltration and predicts poor outcomes in TNBC. We demonstrate that TROP2 is sufficient to drive tumor progression in vivo in a CD8 T cell-dependent manner, while its loss deregulates expression and localization of multiple tight junction proteins, enabling T cell infiltration. We show that TROP2 targeting via hRS7, the antibody component of the ADC Sacituzumab govitecan (SG), enhances the anti-PD1 response and improves T cell accessibility and effector function. Correspondingly, TROP2 expression is highly associated with lack of response to anti-PD1 therapy in human breast cancer. Conclusions This study defines a new mechanism of barrier-mediated immune exclusion in cancer controlled by TROP2-dependent tight junctions. This mechanism drives tumor progression but can be targeted via TROP2-directed therapy to activate anti-tumor immunity and enhance immunotherapy response. What is already known on this topic Barrier-mediated immune exclusion is emerging as a mechanism of immune evasion in cancer such as triple-negative breast cancer (TNBC), but its molecular underpinnings remain poorly understood. TROP2 is a surface glycoprotein targeted by antibody-drug conjugates such as Sacituzumab govitecan, yet its functional role beyond drug delivery has been unclear. What this study adds This study identifies TROP2 as a key regulator of tight junction-mediated immune exclusion in TNBC, independent of its intracellular signaling function. TROP2 promotes an immune-cold tumor microenvironment by enforcing mechanical barriers that limit T cell infiltration. How this study might affect research, practice or policy These findings establish TROP2 as a functional driver of immune evasion and provide a mechanistic rationale for combining TROP2-targeting therapies with immune checkpoint inhibitors to overcome resistance in immune-excluded tumors.

PURPOSE Long-term outcomes of patients with stage I human epidermal growth factor receptor 2 (HER2)–positive breast cancer receiving adjuvant trastuzumab emtansine (T-DM1) remain undefined, and prognostic predictors represent an unmet need. METHODS In the ATEMPT phase II trial, patients with stage I centrally confirmed HER2-positive breast cancer were randomly assigned 3:1 to adjuvant T-DM1 for 1 year or paclitaxel plus trastuzumab (TH). Coprimary objectives were to compare the incidence of clinically relevant toxicities between arms and to evaluate invasive disease-free survival (iDFS) with T-DM1. Correlative analyses included the HER2DX genomic tool, multiomic evaluations of HER2 heterogeneity, and predictors of thrombocytopenia. RESULTS After a median follow-up of 5.8 years, 11 iDFS events were observed in the T-DM1 arm, consistent with a 5-year iDFS of 97.0% (95% CI, 95.2 to 98.7). At 5 years, the recurrence-free interval (RFI) was 98.3% (95% CI, 97.0 to 99.7), the overall survival was 97.8% (95% CI, 96.3 to 99.3), and the breast cancer-specific survival was 99.4% (95% CI, 98.6 to 100). Comparable iDFS was observed with T-DM1 irrespective of tumor size, hormone receptor status, centrally determined HER2 immunohistochemical score, and receipt of T-DM1 for more or less than 6 months. Although ATEMPT was not powered for this end point, the 5-year iDFS in the TH arm was 91.1%. Among patients with sufficient tissue for HER2DX testing (n = 187), 5-year outcomes significantly differed according to HER2DX risk score, with better RFI (98.1% v 81.8%, hazard ratio [HR], 0.10, P = .01) and iDFS (96.3% v 81.8%, HR, 0.20, P = .047) among patients with HER2DX low-risk versus high-risk tumors, respectively. CONCLUSION Adjuvant T-DM1 for 1 year leads to outstanding long-term outcomes for patients with stage I HER2-positive breast cancer. A high HER2DX risk score predicted a higher risk of recurrence in ATEMPT.

2505 Background: ATRC-101 monoclonal antibody targets tumor-specific ribonucleoprotein complex (RNP) and elicits anti-tumor immunity, innate and adaptive, in preclinical models. Methods: Phase 1b is evaluating ATRC-101 as monotherapy, every 3 (3M) or 2 (2M) weeks (wk), and ATRC-101 in combination with pembrolizumab (3P) in participants (pts) with advanced solid tumors. ATRC-101 target expression is determined by CAP-CLIA IHC assay. For pts described herein, pre-treatment (tx) tumor biopsies were assessed for target expression by IHC retrospectively or prospectively. Enrollment in 3M and 3P is currently open to pts who are target positive (RNP+ve) by IHC with an H-score ≥ 50. Objectives are safety (primary), PK, immunogenicity, recommended dose for expansion, activity by RECIST1.1 and biomarker analyses in pre-tx and on-tx tumors. Results: As of Nov 18th, 2022, 67 pts received ≥1 dose of ATRC-101 (median prior tx 5, range 1-12). Tumor types were colorectal (CRC, 28), ovarian (10), breast (9), melanoma (8), non-small cell lung (NSCLC, 6), head and neck squamous cell (3), urothelial (1), esophageal (1) and small bowel (1). Tx emergent adverse events (TEAEs) Grade ≥3 occurred in 22 (33%) pts and were related to ATRC-101 in 2 (3%). No tx discontinuations or dose reductions related to ATRC-101. RECIST1.1 best overall responses in efficacy evaluable 61 pts included 1 complete response (CR) in RNP+ve melanoma (3P, 10mg/kg), and 1 partial response (PR) in RNP+ve NSCLC (3M, 30mg/kg). Further, disease stabilized (SD) in 24 pts (39%) and 35 pts had disease progression (PD, 57%) with evidence of association between anti-tumor activity and target expression. Of 24 SD, 22 were in monotherapy cohorts and included 9 CRC pts with highly refractory disease. Conclusions: ATRC-101 alone and in combination with pembrolizumab continues to be well tolerated with evidence of anti-tumor activity. Interim data support ongoing enrollment of pts with RNP+ve pre-tx tumors. Updated and new data including durability of disease control will be presented. Clinical trial information: NCT04244552 . [Table: see text]

Abstract Background: The ATEMPT trial primary analysis found that one year of adjuvant trastuzumab emtansine (T-DM1) achieved a 3-year iDFS of 97.8% for patients with stage I HER2+ breast cancer, but was not associated with fewer clinically relevant toxicities (CRTs) compared with paclitaxel and trastuzumab (TH). In this end-of-study analysis, we report 5-year survival outcomes and correlative analyses from the trial. Methods: Patients with stage I centrally confirmed HER2+ breast cancer were randomly assigned 3:1 to adjuvant T-DM1 for one year or TH and received T-DM1 3.6 mg/kg IV every 3 weeks for 17 cycles or paclitaxel 80 mg/m2 IV with weekly trastuzumab IV followed by trastuzumab for 9 months. The co-primary objectives were to compare the incidence of CRTs between the 2 arms and to evaluate iDFS in patients receiving T-DM1. To investigate proteomic correlates of recurrence, spatial proteomic analyses were performed on samples from 13 patients experiencing iDFS events (cases) and 24 matched controls using the NanoString GeoMx Digital Spatial Profiler. The impact of HER2 heterogeneity on outcomes was investigated among 17 cases and 51 matched controls by fluorescence in-situ hybridization (FISH). HER2 genetic heterogeneity was assessed by scrutinizing the whole tumor area and defined as the occurrence of HER2 gene amplification in >5% but < 50% invasive tumor cells. The risk of recurrence was evaluated centrally with the HER2DX genomic assay from 225 primary tumor samples. Germline whole genome sequencing (WGS) was conducted among 55 patients experiencing T-DM1-induced thrombocytopenia and/or bleeding and 55 matched controls to identify genomic correlates for this side effect. Results: A total of 497 patients who initiated protocol therapy were included in this analysis (383 T-DM1 and 114 TH). After a median follow up 5.8 years, among patients receiving T-DM1 there were a total of 11 iDFS events, with 3 distant recurrences. The 5-year iDFS for T-DM1 was 97.0% (95% CI, 95.3-98.8%), the 5-year recurrence-free interval (RFI) was 98.6% (95% CI: 97.4-99.8%) and the 5-year overall survival (OS) for T-DM1 was 97.8 % (95% CI, 96.3-99.3%). Although the study was not powered to evaluate the efficacy of TH, among the 114 patients receiving TH, a total of 9 iDFS events were observed, including 2 distant events; the 5-year iDFS with TH was 91.3% (95% CI: 86.0-96.9%), 5-year RFI was 93.3% (95% CI: 88.6-98.2%) and 5-year OS was 97.9% (95% CI: 95.2-100%). A total of 56 samples were evaluable for heterogeneity analyses, among which 14% (n=8) harbored HER2 genetic heterogeneity. Spatial proteomic analyses found that NF1 (adjusted p=0.72 × 10-6) and CTLA-4 (adjusted p=0.15 × 10-3) were significantly upregulated in primary samples from cases, while cleaved caspase 9, CD25, GITR, ICOS, p53 and PD-L2 were significantly upregulated in controls (all with adjusted p< 0.05). Germline WGS found that the top gene associations with thrombocytopenia and thrombocytopenia or bleeding were ALMS1 (p=0,19 × 10-3) and APBA3 (p=0,23 × 10-3), respectively, although none reaching the threshold for genome wide significance. rs62143195 and rs114169776 were the top single nucleotide polymorphisms associated with thrombocytopenia and thrombocytopenia or bleeding, respectively. Data on the impact of HER2 heterogeneity and of HER2DX score on survival outcomes will be presented. Conclusion: With longer follow-up, adjuvant T-DM1 confirmed outstanding long-term outcomes among patients with stage I HER2+ breast cancer, demonstrating a 5-year RFI of 98.6%. Spatial proteomic analyses identified a potential association between NF1 and CTLA-4 expression with recurrence. Details on the impact of HER2 heterogeneity and HER2DX assay on prognosis will be presented. Citation Format: Paolo Tarantino, Nabihah Tayob, Chau T Dang, Denise Yardley, Steven J. Isakoff, Vicente Valero, Meredith Faggen, Therese Mulvey, Ron Bose, Douglas Weckstein, Antonio C. Wolff, Katherine Reeder-Hayes, Hope Rugo, Bhuvaneswari Ramaswamy, Dan Zuckerman, Lowell Hart, Vijayakrishna K. Gadi, Michael Constantine, Kit Cheng, Audrey Merrill Garrett, Paul K. Marcom, Kathy S. Albain, Patricia DeFusco, Nadine Tung, Blair Ardman, Rita Nanda, Rachel C. Jankowitz, Mothaffar Rimawi, Vandana Abramson, Paula R. Pohlmann, Catherine Van Poznak, Andres Forero-Torres, Minetta C. Liu, Kathryn Ruddy, Yue Zheng, Romualdo Barroso-Sousa, Adrienne Waks, Michelle K. DeMeo, Molly K. DiLullo, Giuseppe Curigliano, Harold Burstein, Ann Partridge, Eric Winer, Giuseppe Viale, Winnie Hui, Elizabeth A. Mittendorf, Bryan P. Schneider, Aleix Prat, Ian Krop, Sara Tolaney. Adjuvant Trastuzumab Emtansine Versus Paclitaxel plus Trastuzumab for Stage I HER2+ Breast Cancer: 5-year results and correlative analyses from ATEMPT (TBCRC033) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD18-01.

The therapeutic options for patients with noninvasive or invasive breast cancer are complex and varied. These NCCN Clinical Practice Guidelines for Breast Cancer include recommendations for clinical management of patients with carcinoma in situ, invasive breast cancer, Paget disease, phyllodes tumor, inflammatory breast cancer, and management of breast cancer during pregnancy. The content featured in this issue focuses on the recommendations for overall management of ductal carcinoma in situ and the workup and locoregional management of early stage invasive breast cancer. For the full version of the NCCN Guidelines for Breast Cancer, visit NCCN.org .

Abstract Background: Survival advantages with neoadjuvant chemotherapy (NACT) have been observed in patients (pts) with locally advanced breast cancer (BC) who achieve pathological complete response (pCR). Predicting risk of early recurrence in pts without pCR remains difficult. Circulating tumor DNA (ctDNA) may be a biomarker of neoadjuvant therapy response and recurrence. This study applied tumor genotyping of peripheral blood in pts with HER2− tumor BRCA-mutated (tBRCAm) BC treated with neoadjuvant niraparib. The relationship between ctDNA and tumor response was also characterized, with a focus on circulating TP53 mutant allelic frequency (MAF). Methods: Blood was collected from 21 pts enrolled in the study (NCT03329937) evaluating neoadjuvant niraparib in pts with localized HER2− tBRCAm BC. Pts received niraparib 200 mg once daily for two 28-day cycles. After 2 cycles, pts underwent surgery or received up to 6 cycles of niraparib and/or NACT prior to surgery. Blood samples were collected at screening, Cycle 1 Day 28 (C1D28), Cycle 2 Day 28 (C2D28), and pre-surgery. Ultrasound (USG) was done after each cycle and MRI after Cycle 2. Next-generation sequencing of cell-free plasma DNA was carried out using a proprietary target capture and analysis (Paweletz, Clin Cancer Res 2016). Results: ctDNA and TP53 mutations (TP53m) were detected at screening in 10/21 pts (47.6%). Across all 21 pts there was a correlation between TP53 MAF and baseline tumor stage (T) (mean [standard deviation], T1 0 [0], T2 0.05 [0.09], and T3 0.21 [0.12]; P=0.005). Linear regression modelling showed a positive correlation between tumor volume and baseline TP53 MAF (R2=0.206, P=0.0386). Baseline TP53 MAF also appeared to be associated with advanced disease stage (Table 1). Depletion of detectable TP53m from baseline was evident by C1D28 and persisted to pre-surgery (Table 2). This corresponded with decreases in tumor volume at C1D28 and further decreases at C2D28. Of 11 pts analyzed, 6 pts with >90% tumor volume decreases by MRI or USG at C2D28 had sustained TP53m depletion. Three pts had pCR; of these, 2 had sustained TP53m depletion (data unavailable for third pCR pt). Two pts with less robust tumor responses had increased pre-surgery TP53 MAF. Conclusions: We report the potential utility of ctDNA as a predictive biomarker for neoadjuvant niraparib response in pts with HER2− tBRCAm BC. Although sample size limited analysis of genes other than TP53, baseline ctDNA strongly correlated with tumor volume, and TP53 MAF was associated with tumor volume and trended towards association with disease stage. Longitudinal analysis of TP53 MAF suggested a correlation with niraparib response. Further study of ctDNA dynamics during NACT and correlation with pCR and long-term clinical outcomes is warranted. Funding: GlaxoSmithKline (GSK) study 213355; NCT03329937. Medical writing support was provided by Fishawack Indicia, part of Fishawack Health, funded by GSK. Table 1.Baseline TP53 MAF association with disease stage and subtypeTP53 MAFDisease Stage (I–III)Disease SubtypeStage IStage IIStage IIIHR+TNBC(n=8)(n=10)(n=3)(n=6)(n=15)Mean (SD)0.01 (0.02)0.07 (0.12)0.10 (0.05)0.02 (0.02)0.06 (0.10)HR, hormone receptor; MAF, mutant allelic frequency; SD, standard deviation; TNBC, triple-negative breast cancer. Table 2.TP53 MAF correlation with tumor volume decrease by ultrasound and MRIOutcomeScreeningCycle 1 Day 28 Cycle 2 Day 28 Pre-Surgeryn11997TP53 MAF, mean, (SD)0.09 (0.11)000.01 (0.02)n11112Percent change in tumor volume from baseline by MRI, mean (SD)-N/A*−72.6 (22.5)N/A*Percent change in tumor volume from baseline by ultrasound, mean (SD)-−60.8 (24.0)−80.6 (21.8)−95.9 (4.3)*MRI was only performed at Cycle 2 Day 28. MAF, mutant allelic frequency; MRI, magnetic resonance imaging; N/A, not applicable; SD, standard deviation. Citation Format: Ming Shan, Laura Spring, Minetta C Liu, Erika Hamilton, Hanna Irie, Cesar A Santa-Maria, Steven J Isakoff, James Reeves, Leif W Ellisen, Lee P Lim, Kavita Garg, Caterina Bertucci, Bin Feng, Hailei Zhang, Kaiming Sun, Julie R Graham, Erin Hofstatter, Hyo Han. Relationship between circulating tumor DNA and response to neoadjuvant niraparib in HER2-negative, BRCA-mutated breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-21.

The NCCN Guidelines for Breast Cancer include up-to-date guidelines for clinical management of patients with carcinoma in situ, invasive breast cancer, Paget disease, phyllodes tumor, inflammatory breast cancer, male breast cancer, and breast cancer during pregnancy. These guidelines are developed by a multidisciplinary panel of representatives from NCCN Member Institutions with breast cancer–focused expertise in the fields of medical oncology, surgical oncology, radiation oncology, pathology, reconstructive surgery, and patient advocacy. These NCCN Guidelines Insights focus on the most recent updates to recommendations for adjuvant systemic therapy in patients with nonmetastatic, early-stage, hormone receptor–positive, HER2-negative breast cancer.