Early-stage Triple-negative Breast Cancer Research Articles

HORMAD1 overexpression predicts response to anthracycline-cyclophosphamide and survival in triple-negative breast cancers.

Triple negative breast cancers (TNBCs) represent 15-20% of all breast cancers and are associated with higher recurrence and distant metastasis rate. Standard of care for early stage TNBC is anthracyclines combined with cyclophosphamide (AC) followed by taxanes, in the neo-adjuvant or adjuvant setting. This work aimed to identify predictive biomarkers of AC response in patient-derived xenograft (PDX) models of TNBC and to validate them in the clinical setting. By gene and protein expression analysis of 39 PDX with different responses to AC, we found that high expression of HORMAD1 was associated with better response to AC. Both gene and protein expression were associated with promoter hypomethylation. In a cohort of 526 breast cancer patients, HORMAD1 was overexpressed in 71% of TNBC. In a second cohort of 186 TNBC patients treated with AC, HORMAD1 expression was associated with longer metastasis-free survival (MFS). In summary, HORMAD1 overexpression was predictive of an improved response to AC in PDX and is an independent prognostic factor in TNBC patients treated with AC.

Immunotherapy Targeting PD-1/PD-L1 in Early-Stage Triple-Negative Breast Cancer.

The advent of immunotherapy, especially immune checkpoint inhibitors (ICIs), has revolutionized antitumor therapy. Programmed cell death receptor 1 (PD-1) and programmed cell death ligand 1 (PD-L1) are among the most promising targets for encouraging the immune system to eliminate cancer cells. PD-1/PD-L1 have made clinical remission for numerous solid tumors, including metastatic triple-negative breast cancer (TNBC). In recent years, integrating PD-1/PD-L1 inhibitors into existing treatments in early-stage TNBC has attracted wide attention. Herein, we summarize the clinical benefit of PD-1/PD-L1 inhibitors plus neoadjuvant chemotherapy, adjuvant chemotherapy, and targeted therapy in early-stage TNBC. Possible immunotherapy biomarkers, immune-related adverse events (irAEs), and the key challenges faced in TNBC anti-PD-1/PD-L1 therapy are also concluded. Numerous studies on immunotherapy are ongoing, and PD-1/PD-L1 inhibitors have demonstrated great clinical prospects in early-stage TNBC. To maximize the efficacy of anti-PD-1/PD-L1 therapy, further research into the challenges which still exist is necessary.

P002 Adjuvant chemotherapy for resected early-stage triple negative breast cancer

P002 Adjuvant chemotherapy for resected early-stage triple negative breast cancer

Abstract HER2-01: HER2-01 Clinical and Molecular Characteristics of HER2-low/zero Early Stage Triple-Negative Breast Cancer

Abstract Background: In the metastatic setting, low HER2 expression is associated with clinical benefit from trastuzumab deruxtecan, a HER2-targeting antibody drug conjugates. However, little is known about the biological significance of low HER2 expression in patients with early stage triple-negative breast cancer (TNBC) receiving neoadjuvant therapy (NAT). Methods: Out of 595 patients with stage I-III TNBC enrolled on the prospective ARTEMIS trial (NCT02276443) from 2015-2021, we identified 367 patients with available HER2 immunohistochemistry (IHC) results on pre-NAT tumor tissue (HER2-zero: n=218; HER2-low [IHC 1+, 2+]: n=149). All patients were treated with anthracycline-based NAT. In cases where sufficient pre-NAT tumor tissue were available, additional IHC and/or RNAseq were performed. Differential gene expression (DGE) and pathway analysis were performed using DEseq2. Gene set enrichment analysis (GSEA) was performed using the Hallmark gene sets. Deconvolution analyses were performed using CIBERSORT. We controlled for multiple hypothesis using a false discovery rate (FDR) threshold with the Benjamini-Hochberg method, accepting as significant genes with at least a 2-fold change and < 5% FDR. Results: Table 1 summarizes baseline clinicopathological features of the 367 patients. Compared to HER2-zero tumors, HER2-low tumors were less likely of metaplastic histology (p=0.001), associated with lower Ki67 (p=0.017) and were more likely to be androgen receptor (AR)-positive (p=0.01). There were no significant differences in tumor-infiltrating lymphocytes (TILs) infiltration and PD-L1 expression between HER2-zero and HER2-low tumors. Among the 226 patients with sufficient pre-NAT tissue for RNAseq, DGE analyses demonstrated upregulation of genes involved in fatty acid metabolism (ACSM1) and steroid hormone metabolism (DHRS2, UGT2B28) in HER2-low tumors compared with HER2-zero tumors. Deconvolution analyses revealed no significant differences between predicted proportions of immune cell subpopulations between HER2-low and HER2-zero tumors. Although rates of pCR were not significantly different between patients with HER2-zero (46%) and HER2-low tumors (40%) (p=0.34), non-pCR in patients with HER2-low tumors was associated with increased expression of EREG, which encodes an EGFR ligand, while non-pCR in patients with HER2-zero tumors was associated with downregulation in genes involved in immune response pathways. GSEA further identified the Hallmark allograft rejection (FDR q=0.001), interferon gamma response (FDR q=0.002), and interferon alpha response pathways (FDR q=0.007) as the 3 most significantly downregulated pathways in HER2-zero tumors from patients experiencing a non-pCR relative to HER2-zero tumors from patients experiencing a pCR. Conclusion: In early stage TNBC, low HER2 expression is associated with increased AR expression and upregulation of genes associated with fatty acid and steroid hormone metabolism. Gene expression analyses suggest that drivers of resistance to NAT differ between HER2-low and HER2-zero tumors. Biological differences between HER2-zero and HER2-low tumors exist and may influence future personalized treatment for patients with early stage TNBC. Citation Format: Clinton Yam, Ziyi Li, Anil Korkut, Wencai Ma, Elisabeth Kong, Holly A. Hill, Hussein Abbas, Sausan Abouharb, Beatriz Adrada, Banu K. Arun, Carlos H. Barcenas, Ajit Bisen, Daniel Booser, Aman Buzdar, Rosalind Candelaria, Junjie Chen, Alyson Clayborn, Senthil Damodaran, Qingqing Ding, Haven Garber, Gabriel N. Hortobagyi, Kelly K. Hunt, Nuhad K. Ibrahim, Adaeze Iheme, Meghan S. Karuturi, Kimberly Koenig, Rachel M. Layman, Jangsoon Lee, Jennifer K. Litton, Melissa Mitchell, Giancarlo Moscol, Jason Mouabbi, Rashmi K. Murthy, Oluchi Oke, Paula Pohlmann, David Ramirez, Elizabeth Ravenberg, Sadia Saleem, Mediget Teshome, Vicente Valero, Jason White, Madison Williams, Wendy Woodward, Chasity Yajima, Naoto T. Ueno, Ken Chen, Gaiane Rauch, Lei Huo, Debu Tripathy. HER2-01 Clinical and Molecular Characteristics of HER2-low/zero Early Stage Triple-Negative Breast Cancer [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 HER2-01.

Abstract P3-06-03: Trilaciclib induces immune changes within the tumor microenvironment in early-stage triple-negative breast cancer

Abstract Background: In early-stage triple-negative breast cancer (TNBC), there is accumulating evidence of a correlation between tumor-infiltrating lymphocytes in tumor tissue and favorable clinical outcomes, with a high CD8+/regulatory T-cell (Treg) ratio after neoadjuvant chemotherapy being predictive of overall survival and associated with pathologic complete response (Ladoire S, et al. Br J Cancer. 2011; Park YH, et al. Nat Commun. 2020). Trilaciclib is a transient inhibitor of cyclin-dependent kinase 4/6 that is administered intravenously prior to chemotherapy. In preclinical studies, trilaciclib has been shown to have immune-enhancing effects by differentially arresting CD8+ T-cell and Treg subsets, which is followed by the faster recovery of CD8+ T cells than Tregs in the tumor microenvironment. Methods: This phase 2, single-arm, open-label study aims to evaluate neoadjuvant, single-dose trilaciclib followed by trilaciclib plus dose-dense anthracycline/cyclophosphamide and taxane in patients with early-stage TNBC (NCT05112536). Patients with previously untreated, non-metastatic, confirmed TNBC and a primary tumor ≥ 1.5 cm of any nodal status receive a single dose of trilaciclib 240 mg/m2 during the lead-in phase, followed by 4 cycles of doxorubicin 60 mg/m2 plus cyclophosphamide 600 mg/m2, and 12 weekly cycles of paclitaxel 80 mg/m2. Trilaciclib 240 mg/m2 is administered prior to the first chemotherapy dose of each cycle. Pembrolizumab 400 mg every 6 weeks starting on day 1, cycle 1, and/or carboplatin AUC 1.5 every week starting on day 1, cycle 5, is allowed per investigator discretion. Tumor biopsies and peripheral blood samples are collected prior to any treatment, 7 days ± 1 day post administration of trilaciclib, and during surgery, with an additional blood sample collection on day 1, cycle 2. The primary objective is to evaluate the immune-based mechanism of action of trilaciclib after a single dose of trilaciclib, as measured by changes in the CD8+/Treg ratio in tumor tissue. Pathologic complete response, safety and tolerability, and additional exploratory immune biomarker endpoints will also be assessed. Results: As of June 3, 2022, 9 patients with early-stage TNBC had been enrolled and 8 patients had received the trilaciclib lead-in dose and initiated doxorubicin/cyclophosphamide. Patients had a median age of 53.0 years, and all had stage II tumors at diagnosis, with 7 having ductal carcinoma. The median number of chemotherapy cycles received was 3 (range 1–6), and all 8 patients received pembrolizumab. Seven patients continue study treatment; 1 patient discontinued due to disease progression. Five patients had an adverse event (AE) related to any study treatment, including 4 patients with ≥ 1 trilaciclib-related AE. There were no grade ≥ 3 treatment-related AEs or serious AEs. On-treatment, post-trilaciclib monotherapy biopsies were available for 4 patients. Following neoadjuvant trilaciclib treatment, the median density of stromal CD8+ T cells increased from 103.1/mm2 at baseline to 229.8/mm2 at day 7. The median CD8+/Treg ratio increased in 2 patients from 1.85 at baseline to 1.90 at day 7. Conclusions: Preliminary analysis of on-treatment tumor biopsies from 4 patients suggests that a single dose of trilaciclib may modulate the immune cell composition in the tumor microenvironment to support antitumor immune responses. The increase in CD8+ T cells following 7-day neoadjuvant treatment with trilaciclib supports previous data suggesting a role in T-cell infiltration. The complete dataset from all patients (estimated enrollment: N ≈ 24) and additional biomarker analyses will be presented. Citation Format: Michael Danso, Joyce O’Shaughnessy, Lisa S. Wang, Kailash Mosalpuria, Sara Hurvitz, Shom Goel, Sarah Ahn, Subing Cao, John S. Yi, Taofik Oyekunle, Amanda Jacobson, Andrew Beelen, Jeremy Force. Trilaciclib induces immune changes within the tumor microenvironment in early-stage triple-negative breast cancer [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 P3-06-03.

Abstract P2-20-01: Patient-derived triple-negative breast cancer organoids as a platform for glucocorticoid receptor- mediated mechanisms of immunotherapy response and resistance

Abstract Background: In early-stage triple-negative breast cancer (TNBC), high glucocorticoid receptor (GR) expression correlates with worse relapse-free survival across TNBC subtypes, induces oncogenic gene expression and tumor cell survival, and potentiates anti-inflammatory or an anti-immunogenic phenotype. Immune checkpoint therapy has increased pathologic complete response rates in the neoadjuvant setting, however response to immunotherapy remains modest overall and the explanatory mechanisms for this phenomenon are incompletely understood. We sought a three-dimensional model of the TNBC immune microenvironment using patient-derived organoids (PDOs) that capture tumor heterogeneity, partially recapitulates tumor microenvironment, and can be established for longer-term in vitro/in-vivo translational study after exposure to immunotherapy. We hypothesize that during chemoimmunotherapy treatment of early-stage triple-negative breast cancer, high glucocorticoid receptor activity mediates an immunosuppressive phenotype and glucocorticoid receptor modulation can restore anti-tumor immunity. Methods and Results: We are successfully growing 5 PDOs out of 8 surgically resectedion samples tumors from patients with early-stage triple-negative breast cancer treated with chemotherapy vs. chemoimmunotherapy as per KEYNOTE-522. Interrogation of complementary TNBC cell line data revealed that GR activation downregulates the expression of immune checkpoint genes (PD-L1, B7-H3, B7-H4), while GR knockdown or treatment with a selective GR modulator restores expression of these immune checkpoint genes. TNBC-intrinsic GR activation increases the proportion of regulatory T cells vs. CD8+ T cells. In a coculture system of TNBC spheroids and T cells, GR activation in TNBC results in decreased activated CD8+ CD137+ T cells and use of the selective GR modulator increases the proportion of activated CD8+ CD137+ T cells. Conclusions and Future Directions: In early-stage triple-negative breast cancer, GR activation downregulates expression of immune checkpoint genes and reduction of GR activity restores expression. In a coculture system that recapitulates T cell repertoire found in triple-negative breast cancer, tumor-intrinsic GR activation results in increased regulatory T cell population and reduction of GR activity increases activated CD8+ CD137+ T cells. Ongoing analyses of TNBC organoid-immune coculture systems modeling high GR states will be used to (i) define cooperativity amongst immune checkpoint proteins in costimulation vs. coinhibition of anti-tumor responses and (ii) define novel pairs of receptor-ligands amongst TNBC:CD8+ vs. TNBC:Treg vs. CD8+:Treg as discovery of potential new therapeutic targets. Citation Format: Christine Shiang, Candace Frerich, Ishrat Durdana, Cheryl Lewis, Lynda B. Bennett, Carlos Arteaga, Suzanne D. Conzen. Patient-derived triple-negative breast cancer organoids as a platform for glucocorticoid receptor- mediated mechanisms of immunotherapy response and resistance [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 P2-20-01.

Abstract PD5-03: PD5-03 Comparison of a personalized sequencing assay and digital PCR for circulating tumor DNA based Molecular Residual Disease detection in early-stage triple negative breast cancer in the cTRAK-TN trial

Abstract Background: Detection of circulating tumour DNA (ctDNA) in patients (pts) who have completed treatment for early-stage breast cancer is associated with a high risk of future relapse. Identifying those at high risk of subsequent relapse may allow tailoring of further therapy to delay or prevent recurrence. Previous analysis of this cohort showed that tools capable of detecting ctDNA at lower concentrations are needed to increase sensitivity and lengthen the lead time between ctDNA detection and relapse. We compared ctDNA detection via a personalised sequencing assay to dPCR in patients from the cTRAK TN clinical trial. Methods: The cTRAK-TN trial recruited 161 pts into prospective ctDNA surveillance with dPCR, with ctDNA positive pts randomised to 1) CT staging plus pembrolizumab therapy for patients without relapse or 2) observation. Pts had serial post-treatment surveillance plasma samples collected every 3 months for up to 2 years. Whole exome sequencing (WES) was performed on tumor DNA from FFPE samples to design personalised Residual Disease and Recurrence (RaDaR®) multiplex PCR based NGS assays. Retrospectively, plasma DNA extracted from a minimum of 2mls banked plasma, was sequenced with personalised RaDaR assays, and ctDNA detection identified with a proprietary algorithm. dPCR assays tracked 1-2 mutations, as previously described. Primary endpoint was rate of positive ctDNA detection by 12 months from start of surveillance in both assays. Secondary endpoints were agreement in ctDNA detection between RaDaR and dPCR assays and lead-time between ctDNA detection and disease recurrence. Results: Overall, 147 pts and 241 tissue samples were subject to WES, and RaDaR assays were developed for 142 pts with sufficient plasma for testing. RaDaR assays tracked a median of 47 variants (range 33-56) per patient, and a total of 907 timepoints were analysed (median 6 timepoints per pt, range 1-11). With RaDaR, 39.4% (56/142) patients tested ctDNA positive during follow-up, with a median ctDNA detected level of 0.081% estimated variant allele fraction (eVAF). With dPCR, 35.2% (50/142) pts tested ctDNA positive. The ctDNA detection rate by 12 months from the start of ctDNA surveillance was 36.2% (95% CI; 27.6% – 43.7%) with RaDaR and 29.9% (95%CI; 21.6% – 37.3%) with dPCR. The overall test agreement between RaDaR and dPCR assays was 92.7% (95%CI; 90.7% – 94.4%). From a patient perspective, 58.7% pts were ctDNA negative for both assays, 32.9% ctDNA were positive for both assays and 8.6% presented discrepancies. ctDNA was detected by RaDaR but not by dPCR in 9 pts and it was detected by dPCR but not by RaDaR in 3 pts. Among ctDNA positive pts, 55.2% were first detected positive by RaDaR, 5.2% by dPCR, and 39.6% were detected at the same time-point (test of proportions, p< 0.001). The median lead time from ctDNA detection to relapse was 7.1 months (95% CI 5.9 – 15.9%) with RaDaR and 5.7 months (95% CI 3.2% – 7.4%) with dPCR. Conclusion: The RaDaR personalised multi-mutation sequencing assay detected MRD with a longer median lead time prior to relapse, and with higher sensitivity, than dPCR mutation tracking assays. These findings have implications for the choice of ctDNA assay in clinical trials designed to treat patients at the point of MRD detection. Citation Format: Maria Coakley, Prithika Sritharan, Guillermo Villacampa, Claire Swift, Kathryn Dunne, Lucy Kilburn, Katie Goddard, Patricia Rojas, Andy Joad, Warren Emmett, Charlene Knape, Karen Howarth, Peter S. Hall, Catherine Harper-Wynne, Tamas Hickish, Iain Macpherson, Alicia F. Okines, Andrew M. Wardley, Duncan Wheatley, Simon Waters, Rosalind Cutts, Isaac Garcia-Murillas, Judith Bliss, Nicholas Turner. PD5-03 Comparison of a personalized sequencing assay and digital PCR for circulating tumor DNA based Molecular Residual Disease detection in early-stage triple negative breast cancer in the cTRAK-TN trial [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 PD5-03.

Abstract P6-01-09: Efficacy of platinum-based chemotherapy and germline mutational status in early-stage triple-negative breast cancer: a unicenter retrospective analysis with long-term follow-up

Abstract BACKGROUND: In early-stage triple negative breast cancer (TNBC), the addition of carboplatin (CBDCA) to neoadjuvant chemotherapy (CT) increases pathologic complete response (pCR) and relapsed-free survival. However, it is unclear whether CBDCA improves overall survival (OS). In addition, the prognostic and/or predictive role of pathogenic germline variants (PGV) in BRCA1/2 genes and other cancer risk in this setting is not fully understood. Here, we assessed the efficacy of (neo)adjuvant CBDCA and the prognostic and predictive role of a panel of 14 genes in patients (pts) with eTNBC. METHODS: This is a retrospective study on 117 pts diagnosed with early-stage TNBC between 2000-2021 at Hospital Clinic of Barcelona. Eighty-one pts (69%) were candidates for PGV testing. Overall, 14 genes (PGV) (BRCA1, BRCA2, PALB2, BRIP1, CHEK2, TP53, ATM, RAD51C, RAD51D, BARD1, MLH1, MSH2, MSH6 and PMS2) were assessed using the TruSight hereditary cancer panel (Illumina MySeq platform) according to local guidelines. Univariable and multivariable logistic regression and Cox regression analyses were performed to identify clinical and molecular predictors of pCR and relapse-free survival (RFS), respectively. Chi-squared or Fisher’s exact tests were used to assess characteristics’ distribution as appropriate. RESULTS: Of 117 pts, 83 (71%) received CT in the neoadjuvant setting and 28 (24%) in the adjuvant setting. CBDCA was added to standard CT in 68 pts (82%) in the neoadjuvant cohort and 7 pts (6%) in the adjuvant cohort. Among pts with germline testing, 32/81 (39%) harbored PGV. BRCA1 was the most frequently mutated gene (18/32, 56%), followed by BRCA2 (5/32, 16%), PALB2 (4/32, 13%), BRIP1 (2/32, 6%), CHEK2, TP53 and PMS2 (3/32, 6%). Percentages of pts receiving CBDCA were similar between patients with and without PGV (14/16,87% and wild type (53/67,79%)(p=0.120). In the neoadjuvant cohort (n=83), CBDCA was the only variable significantly associated with pCR at both univariate (pCR rates of 58.5% with CBCDA and 14.3% without CBCDA; odds ratio [OR]=8.2 [95% CIs 2.0-55.7], p=0.008) and remained statistically significant after adjusting for PGV, tumor size and nodal status (OR=6.9 [95% CIs 1.4-53.0], p=0.028). pCR rates according to PGV are reported in Table 1. In terms of RFS, addition of CBDCA to neoadjuvant therapy (hazard ratio [HR]=0.2 [0.1-0.45], p< 0.001), PGV (HR=0.2 [0.05-0.9], p=0.048), pCR (HR=0.2 [0.1-0.6], p=0.004) and nodal status (HR=5.1 [1.7-15.2], p< 0.003) were significantly associated with RFS in univariate analyses. In a multivariable model, CBDCA remained an independent predictor of improved RFS along with pCR. When the neoadjuvant and adjuvant cohort were pooled together (n=117), platinum-based CT remained significantly associated with better RFS (HR=0.2 [0.14-0.86], p=0.021) regardless of time of administration (i.e., neoadjuvant or adjuvant). With a median follow-up of 5 years, CBCDA use was not found associated with OS (HR=0.7 [0.3-1.8], p= 0.560). CONCLUSIONS: The addition of CBDCA to standard CT was significantly associated with pCR and RFS but not OS, consistent with the phase III data. The benefit in terms of RFS was independent of the presence and the type of pathogenic germline alterations. Table 1. pCR rates according to PGV Citation Format: Adela Rodríguez Hernández, Benedetta Conte, Laia Fernández, Fara Brasó-Maristany, Belén Pastor, Miriam Potrony, Lorena Moreno, Elia Grau, Joan Antón Puig-Butillé, Aurora Sánchez, Blanca González-Farré, Esther Sanfeliu, Olga Martínez-Sáez, Claudette Falato, Maria Vidal, Nuria Chic, Tomás Pascual, Francesco Schettini, Montserrat Muñoz, Francesc Balaguer, Aleix Prat, Barbara Adamo. Efficacy of platinum-based chemotherapy and germline mutational status in early-stage triple-negative breast cancer: a unicenter retrospective analysis with long-term follow-up [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 P6-01-09.

Abstract PD5-02: PD5-02 An Organoid Model System to Study Resistance Mechanisms, Predictive Biomarkers, and New Strategies to Overcome Therapeutic Resistance in Early-Stage Triple-Negative Breast Cancer

Abstract Background: While new treatments and improved subtyping schemas are anticipated to improve treatment response in triple-negative breast cancer (TNBC) patients, therapeutic resistance remains a significant challenge. Moreover, there is an urgent need for additional research model systems to study resistance and residual disease in breast cancer, including aggressive subtypes of breast cancer. Organoid culture is a promising technology used for growing breast cancer cells with high efficiency; however, the extent to which treatment resistance can be modeled using this system is unknown. This research used patient-derived organoid cultures in the context of computational analyses of large molecular and clinical datasets to study resistance mechanisms, biomarkers, and alternative treatment strategies to overcome drug resistance in early-stage TNBC. Methods: Organoid cultures were derived from breast tumor samples (taken from lumpectomy, mastectomy, or core biopsy samples), digested to the organoid level using collagenase, and grown in three dimensional cultures using a basement membrane extract and a fully-defined organoid medium (Dekkers et al. Nat Protoc 2021). An evaluation of all available I-SPY2 biomarker data (Wolf et al. Cancer Cell 2022) was performed focusing on genes, proteins, and pathways associated with resistance. These were then used to study whether resistance biomarkers identified in patient tumors are also present in organoids propagated from breast cancer post-treatment residual disease. To this end, bulk RNA sequencing data of organoids were normalized and merged with the TCGA dataset (Hoadley et al. Cell 2018) to enable analysis in a larger context, and immunofluorescence staining of organoids was performed. A high-throughput 386 anti-cancer drug compound screen and subsequent synergy testing with the most promising compounds were performed to identify and predict alternative treatment strategies. Additional assays to explore kinome activity in this organoid model are in progress. Results: A TNBC organoid biobank was established (n=31), which was enriched for inflammatory breast cancer (IBC; n=15), an aggressive form of breast cancer. Most organoids were derived from residual disease after neoadjuvant therapy. Bulk RNA sequencing analysis performed on 10 TNBC organoids revealed 3 subsets that were characterized predominantly by either normal-like/luminal androgen receptor or basal-like features or expressed distinct gene expression profiles, with IBC cases present in all 3 subsets. Intriguingly, the IBC organoids were characterized by higher expression of a number of immune-related signatures, despite an absence of clear immune cells in culture. A residual disease IBC/TNBC organoid resistant to chemotherapy was used to perform the 386-drug compound screen. The organoid model showed resistance to veliparib-cisplatin, consistent with the expression of gene/protein biomarkers predictive of drug resistance found in I-SPY2 (low PARPi7 levels and high pFOXO1 and pMEK1/2 expression). In addition, the screen identified multiple classes of inhibitors as promising synergistic/additive candidates for overcoming resistance to cisplatin. Conclusion: In this proof-of-principle study, we demonstrated the utility of matching I-SPY2 resistance biomarkers and signatures to residual disease tumor organoid cultures. We show that tumor organoid cultures modeling drug resistance states are a useful complement to existing research models of breast cancer and can be used for compound testing. We have developed a pipeline to propagate residual tumors from patients enrolled in I-SPY2 into organoid cultures to create a broader platform for preclinical drug testing informed by tumor biology with the ultimate goal of enabling faster, more successful translational studies and increased treatment options for resistant disease. Citation Format: Tam Binh V. Bui, Denise M. Wolf, Kaitlin Moore, Isaac S. Harris, Pravin Phadatare, Christina Yau, Lamorna A. Brown Swigart, Laura J. Esserman, Jean-Philippe Coppe, Julia Wulfkuhle, Emanuel F. Petricoin, Michael Campbell, Laura M. Selfors, Deborah A. Dillon, Beth Overmoyer, Filipa Lynce, Laura Van ’t Veer, Jennifer Rosenbluth. PD5-02 An Organoid Model System to Study Resistance Mechanisms, Predictive Biomarkers, and New Strategies to Overcome Therapeutic Resistance in Early-Stage Triple-Negative Breast Cancer [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 PD5-02.

Abstract PD11-07: PD11-07 Association of TNBC-DX scores with outcomes in triple-negative breast cancer (TNBC) treated with neoadjuvant pembrolizumab and chemotherapy: a correlative analysis from NeoPACT and NeoSTOP trials

Abstract Introduction: The TNBC-DX risk score includes the 14-gene immunoglobulin (IGG) immune signature, tumor size, and nodal status and has shown prognostic value for survival in early-stage TNBC (B. Conte et al., ESMO Breast 2021). However, currently unknown are the value of the TNBC-DX risk score and IGG immune signature in 1) predicting pathologic complete response (pCR) following neoadjuvant therapy, and 2) predicting outcomes the context of neoadjuvant anti-PD1 treatment. Here, we assessed the IGG signature and the TNBC-DX risk score in patients with TNBC treated with neoadjuvant chemoimmunotherapy (NeoPACT; NCT03639948) and neoadjuvant chemotherapy without immunotherapy (NeoSTOP; NCT02413320). Methods: NeoPACT trial enrolled 120 patients with stage I-III TNBC who received carboplatin (AUC 6) + docetaxel (75 mg/m2) + pembrolizumab (200 mg) every 21 days x 6 cycles. NeoSTOP randomized 100 patients with stage I-III TNBC to two chemotherapy regimens; Arm B of NeoSTOP was included in this correlative study as the chemotherapy regimen was identical to NeoPACT. RNA isolated from pretreatment tumor tissue was subjected to next-generation sequencing. The 14-gene IGG immune signature and TNBC-DX risk score were calculated in silico as previously described. Evaluation of stromal tumor-infiltrating lymphocytes (sTILs) was performed as previously described. Markers were tested for prediction of pCR. Logistic regression analysis was used to examine the effect of multiple variables. Event-free survival (EFS) curves were assessed by the Kaplan-Meier method and groups compared by the log-rank test, followed by Cox regression analysis. Results: In this analysis, 112 patients were treated with chemoimmunotherapy on NeoPACT (node-positive = 38%, pCR rate = 58%). In the NeoPACT trial, the 14-gene IGG signature (as a continuous variable) was significantly associated with improved pCR (odds ratio [OR]=1.105, 95% CI 1.019-1.197, P=0.015 for every 0.2 increment). The pCR rates in IGG-high (≥ median) and IGG-low (< median) groups were 71% and 44%, respectively (OR=3.152, 95% CI 1.420-6.996, P=0.005). In terms of EFS, the 14-gene IGG signature was not prognostic (hazard ratio [HR]=0.507, 95% CI 0.148-1.735, p=0.269). In contrast, TNBC-DX risk score was strongly associated with EFS (HR=5.684, 95% CI 1.226-26.356, P=0.012), even when adjusted for sTILs and pCR status (HR=8.415, 95% CI 1.054-67.169, P=0.044). Estimated 3-year EFS rates in TNBC-DX high and low risk groups (above and below median) were 77% and 89%, respectively (P=0.012). In 43 NeoSTOP patients treated with neoadjuvant chemotherapy only (node-positive = 33%, pCR rate = 53%), no association of IGG signature with pCR or TNBC-DX score with EFS was observed. Finally, we observed a moderate correlation between IGG signature and sTILs in both trial datasets combined (r=0.642, P< 0.001). Conclusions: High expression of the 14-gene IGG immune signature in baseline pretreatment tumor samples in early-stage TNBC is significantly associated with pCR following pembrolizumab-based neoadjuvant chemotherapy. The combination of this signature with tumor burden as assessed by TNBC-DX is prognostic for long-term outcomes. Availability of biomarkers that can predict both pathological response and survival with chemoimmunotherapy can optimize this therapy, and evaluation of this biomarker in larger studies is warranted. Citation Format: Priyanka Sharma, Shane R. Stecklein, Rachel Yoder, Joshua M. Staley, Roberto Salgado, Laia Paré, Benedetta Conte, Fara Brasó-Maristany, Anne O’Dea, Lauren Nye, Manana Elia, Deepti Satelli, Gregory Crane, Richard McKittrick, Qamar Khan, Andrew K. Godwin, Aleix Prat. PD11-07 Association of TNBC-DX scores with outcomes in triple-negative breast cancer (TNBC) treated with neoadjuvant pembrolizumab and chemotherapy: a correlative analysis from NeoPACT and NeoSTOP trials [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 PD11-07.

Abstract P1-04-12: Systemic immune response to a Phase I/II trial of Durvalumab concomitant with neoadjuvant chemotherapy in early stage TNBC

Abstract Background Peripheral blood cells and secreted products are important regulators of the systemic immune response. Circulating cytokines have been shown to predict severity of immune-related toxicity in melanoma patients that received combination anti-PD-1-based immunotherapy. In this study, we evaluated 38 serum cytokines and the peripheral blood t cell receptor (TCR) immune repertoire of 66 patients with TNBC for associations with pCR, treatment phase (PRE; POST), and immune related treatment emergent adverse events (TEAEs). Methods Serum and peripheral blood buffy coats were collected at pre-treatment (week 0) and at post-treatment (~ week 24). MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel was run in duplicate and read on the Luminex platform. Genomic DNA was isolated using QIAGEN Kits per manufacturer’s instructions. TCR immune repertoire profiling was performed using the Immunoseq at Adaptive Biotechnologies. Statistical analysis was performed in R. P-values < 0.05 for serum cytokines and P-values < 0.05 for TCR were considered significant. Results Pielou’s diversity index showed no difference between patient groups for TCR (P>0.319). Baseline samples had increased sCD40L, EGF, and IL-10 in patients with RD compared to pCR (P< 0.05). Baseline samples had decreased FGF2 and IFN gamma in patients with immune related TEAEs compared to those with no immune related TEAEs (P< 0.05). Comparison of Pre- vs Post-treatment revealed that EGF, MIP1 alpha, IL-1 alpha, IL-8, and MDC were increased in patients with pCR compared to those with RD. Comparison of Pre- vs. Post- also revealed increased levels of cytokines (EGF, IL-7, IFN gamma, GM-CSF) in samples in patients with immune related TEAEs (P< 0.05) compared to those patients without immune-related TEAEs. It also revealed that there was an increase in a subset of cytokines (IL-7, IL-4, MCP3, and IL-1 alpha) in patients that presented with serious immune related TEAEs (P< 0.05) compared to those patients without serious immune-related TEAEs. Conclusions There are subsets of circulating inflammatory cytokines that may be associated with treatment emergent adverse events in patients with TNBC treated with Durvalumab concomitant with standard of care chemotherapy. Citation Format: Kim Blenman, Michal Marczyk, Julia Foldi, Vignesh Gunasekharan, Andrea L.M. Silber, Lajos Pusztai. Systemic immune response to a Phase I/II trial of Durvalumab concomitant with neoadjuvant chemotherapy in early stage TNBC [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 P1-04-12.

Abstract P3-06-06: Real-world analysis of adverse events of patients with triple negative breast cancer receiving therapy per KEYNOTE-522

Abstract Background: KEYNOTE-522 was a randomized, double-blind, placebo-controlled phase 3 trial which resulted in the FDA approval of pembrolizumab with neoadjuvant chemotherapy for patients (pts) with newly diagnosed, high-risk, early-stage triple negative breast cancer (TNBC). Given the improvement in pathological complete response (pCR) and event-free survival rates, this regimen has emerged as standard-of-care (SOC) therapy. Adverse events in pts on this treatment regimen in clinical practice is unknown and understanding the real-world toxicity of this regimen is critical. Methods: In this IRB approved retrospective, single-center study we examined pts with early-stage TNBC who received planned treatment per KEYNOTE-522 per SOC from 2021-present. This regimen includes a year of pembrolizumab combined with 4 cycles of neoadjuvant carboplatin/paclitaxel followed by 4 cycles of doxorubicin/cyclophosphamide. Number and length of treatment delays, treatment related toxicities of all grades, and pCR rate were collected from the electronic medical record. Results: Of the 87 identified pts, 2 were excluded due to locally recurrent or metastatic disease and 6 did not receive immunotherapy due to concerns for toxicity or patient preference. Of the 79 pts who initiated treatment with chemotherapy and immunotherapy, median age of the cohort was 52 (27-77). 9 pts had a BRCA1 mutation and 1 pt had a BRCA2 mutation. 41 (51.9%) had T1-2 disease and 38 (48.1%) had T3-4 disease. 37 (46.8%) pts had N0 disease and 42 (53.2%) had N1-3 disease. 15 pts had baseline comorbidities, including heart disease, kidney disease, type II DM, and/or peripheral neuropathy. 68 pts (86.1%) had baseline ECOG 0, 9 (11.4%) had ECOG 1, and 2 (2.5%) had ECOG 2. At the time of analysis, 70 pts (88.6%) were receiving active treatment, of which 47 (67.1%) had completed ≥50% of the planned neoadjuvant therapy. Of pts completing ≥50% of planned neoadjuvant therapy and pts off therapy (N=56), 31 (55.4%) had 1 or more hospitalizations and 23 (41.1%) had 1 or more emergency room visits. 30 pts had treatment delays (53.6%) and 21 pts (37.5%) had dose reductions. Rates of adverse events are presented in Table 1. Of the 79 analyzed pts, 35 have undergone surgery. pCR rate was 45.7% (N=16). 8 (22.9%) pts had RCB-I, 4 (11.4%) pts had RCB-II, 3 (8.6%) pts had RCB-III, and 4 (11.4%) pts had residual disease without RCB calculation. Updated analysis will be included at time of presentation. Conclusions: In this single-center retrospective study of pts receiving chemoimmunotherapy for TNBC, we found higher rates of grade 3 toxicity, including nausea, fatigue, neutropenia, diarrhea, peripheral neuropathy, and hypothyroidism, and lower pCR rate than was reported in the KEYNOTE-522 trial. This may reflect a more heterogeneous population of pts treated in routine clinical practice who are typically less fit than pts on clinical trials. Additionally, pts in this study had higher T stages (48.1% with T3-4 disease vs 26.0% in trial) and node positive disease (53.7% with N1-3 disease vs 48.8% in trial). Limitations include immaturity of data and small sample size; however, these data warrant validation through longer-term follow-up and multicenter validation. Adverse Events in pts receiving Keynote-522 regimen as SOC and on clinical trial Citation Format: Mara Hofherr, Jennifer Hedgecorth, Foluso O. Ademuyiwa, Lindsay L. Peterson, Nusayba A. Bagegni, Rama Suresh, Ashley Frith, Ron Bose, Katherine Weilbaecher, Cynthia Ma, Andrew A. Davis, Katherine K. Clifton. Real-world analysis of adverse events of patients with triple negative breast cancer receiving therapy per KEYNOTE-522 [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 P3-06-06.

Abstract P4-06-11: Pathologic Complete Response in Triple Negative Breast Cancer of Black vs White Patients in the Post-Keynote 522 Era

Abstract Pathologic Complete Response in Triple Negative Breast Cancer of Black vs White Patients in the Post-Keynote 522 Era Melanie Sheen MD, Victoria Chung DO, Ruby Maini MD, Michael Duggan BS, Julia Levy BS Background Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer that lacks estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2) gene amplification. Women with TNBC have worse survival outcomes and increased rates of relapse and distant metastasis as compared to women with non-TNBC. Treatment of TNBC has recently been focused on neoadjuvant chemotherapy (NACT) with a goal of achieving a pathologic complete response (pCR) which is associated with longer event-free survival and overall survival. The KEYNOTE-522 trial was presented at the San Antonio Breast Cancer Symposium in December 2019 and found that patients who received pembrolizumab, an anti–programmed death 1 (PD-1) monoclonal antibody, plus NACT were more likely to achieve pCR than women who received placebo plus NACT. On July 26, 2021, the FDA approved the use of pembrolizumab in combination with NACT for high risk, early stage TNBC. KEYNOTE-522 did not collect race as a baseline demographic characteristic, and since TNBC disproportionately affects younger women and Black women, confirming the efficacy of achieving pCR in these groups is essential. Further investigation of the factors that may contribute to achieving pCR in women treated with pembrolizumab for TNBC is warranted. We set out to perform a retrospective analysis examining the rates of pCR in Black versus White patients with TNBC since the initial revelation of the KEYNOTE-522 data. Methods This retrospective chart review of a regional health care network included patients who had been diagnosed with Stage II/III TNBC, documented race as Black/African-American or White, and received treatment with pembrolizumab in the NACT setting. Exclusion criteria included ER-positivity, PR-positivity, HER2-positivity, or unknown receptor status, no pembrolizumab in the neoadjuvant setting, absence of documented race, and age < 18 years-old. Data was collected using Epic SlicerDicer program. Results 118 patients met inclusion criteria. 59 (50.0%) were Black/African American, 59 (50.0%) were white. There were 57 (48.3%) patients who had pCR status identified through pathology reports while the remaining 61 (51.7%) are still undergoing NACT awaiting surgery. 34 Black women and 17 white women have not had surgery yet. Of women who underwent surgery, 12 Black women achieved pCR compared to 15 White women. 13 Black women and 16 White women did not have pCR Discussion Analysis of the data demonstrates an equal number of Black and White women receiving NACT with pembrolizumab. Of those women, there was relatively equal number of pCR between Black and White women. This data shows no appreciable difference in outcomes of Black and White women in terms of response to therapy. Conclusion Given the known association of pCR with increased survival, pembrolizumab should be considered in the treatment regimen for both Black and White women with stage II/III TNBC. This retrospective study is limited by a small patient population. Continued data collection is underway and will be updated. Citation Format: Melanie Sheen, Victoria Chung, Ruby Maini, Michael Duggan, Julia Levy. Pathologic Complete Response in Triple Negative Breast Cancer of Black vs White Patients in the Post-Keynote 522 Era [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 P4-06-11.

Abstract P2-11-25: A 10-gene signature to predict the prognosis of early-stage triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is highly heterogeneous cancer and the most challenging subtype of breast cancer. And its prognosis is poor compared to the other subtypes. Unlike luminal type cancers, there is no valid biomarker to predict the prognosis of patients with early TNBC. To establish an elaborate therapeutic strategy for TNBC, biomarkers that accurately predict the prognosis and response to treatment are needed. Method: 184 patients with early stage TNBC (training cohort, n = 76; validation cohort, n = 108) were enrolled. Median Follow-up period was 51.5 months (range: 4.6-230.8) for training cohort and 58.3 months (range: 6.6-99.8) for validation cohort. Of the patients in training cohort, 13 patients had recurrence or metastasis. Of the patients in validation cohort, 23 patients had recurrence or metastasis. Using a HiSeq sequencer, RNA sequencing was conducted to analyze the gene expression profiles of tumor samples from TNBC patients. Gene signature was analyzed by combination of DEGs which found in gene expression profiles. Cross validation and meta-analysis were conducted as pre-validation. Meta-analysis was conducted using CBS probePINGS. To compare gene signature and other methods, PAM 50 call and TCR diversity analysis were investigated. Statistical analyses were conducted using R language (v.3.4.3). Result: To predict prognosis of recurrence or metastasis for TNBC patients, we identified the 10-gene signature (DGKH, GADD45B, KLF7, LYST, NR6A1, PYCARD, ROBO1, SLC22A20P, SLC24A3 and SLC45A4) that stratified patients with TNBC by risk score (sensitivity = 92.31%; specificity = 92.06%; accuracy = 92.11%) and validated in a cohort of separate institutions. Meta-analysis supported the biological relevance of the 10-gene signature to well-known driving pathways in TNBC. When compared with other potential biomarkers like PAM 50 call and T-cell receptor β diversity, the 10-gene signature was the only independent factor that can predict prognosis for invasive disease-free survival in multivariate analysis. Conclusion: Our novel findings can contribute to solving the diagnostic challenges in TNBC and the 10-gene signature may serve as a novel biomarker for risk-based patient care. Citation Format: Chang Min Kim, Kyong Hwa Park, Yun Suk Yu, Ju Won Kim, Jin Young Park, Jeong Eon Lee, Sung Hoon Sim, Bo Kyoung Seo, Jin Kyeoung Kim, Eun Sook Lee, Yeon Hee Park, Sun-Young Kong. A 10-gene signature to predict the prognosis of early-stage triple-negative breast cancer [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 P2-11-25.

Abstract P3-06-07: Phase Ib/II study to evaluate safety and tolerability of cabiralizumab in combination with nivolumab and neoadjuvant chemotherapy in patients with localized triple-negative breast cancer

Abstract Background: Neoadjuvant immune checkpoint inhibition (ICI) in combination with chemotherapy is approved for patients with high-risk, early-stage triple-negative breast cancer (TNBC) based on improved outcomes in the KEYNOTE-522 trial. However, some patients have primary resistant disease and do not achieve a pathological complete response (pCR), while others experience significant toxicity. Tumor-associated macrophages (TAMs) are a potential resistance mechanism for ICIs and are dependent on colony-stimulating factor 1 receptor (CSF1R). Therefore, we examined the addition of cabiralizumab, a CSF1R inhibitor, to neoadjuvant paclitaxel, carboplatin, and nivolumab to assess the safety, tolerability, and changes in the tumor microenvironment (TME) in patients with early-stage TNBC. Methods: This is a phase Ib/II, single-institution, randomized controlled clinical trial (NCT04331067) in patients with newly diagnosed Stage II-III TNBC. The primary endpoints include: (1) to determine the safety of a 12-week neoadjuvant regimen of paclitaxel (80 mg/m2 IV q week) + carboplatin (AUC5 IV q3 weeks) + nivolumab (240 mg IV q2 weeks) with or without cabiralizumab (4 mg/kg IV q2 weeks) and (2) to evaluate the effect of cabiralizumab on TAMs and changes in tumor infiltrating lymphocytes (TILs) in the TME between baseline and an on-treatment biopsy after 4 weeks of therapy. Adjuvant treatment is per investigator’s choice. Secondary objectives include evaluation of pCR rate and recurrence-free survival. Paired tissue and bone marrow biopsies are collected for evaluation of the TME and disseminated tumor cells, respectively. The study was designed to enroll 50 patients, including a 12-patient safety lead-in cohort. Here, we report the planned interim analysis of the safety lead-in cohort. Results: Between December 2020 and May 2022, we enrolled 12 patients to the safety lead-in, including 6 patients in each arm. 5 of 12 patients (41.7%) enrolled are underrepresented minorities, including 4 Black patients and 1 Hispanic patient. 2 of 6 patients in the nivolumab arm experienced grade 3 severe toxicity, including 1 patient who developed sepsis and 1 who developed peripheral neuropathy. 3 of 6 patients in the nivolumab + cabiralizumab arm developed grade 3 severe toxicity including 2 patients who experienced myositis and 1 patient who developed periorbital edema. Of the first 10 patients enrolled, 5 had a pCR (2 pCR in cabiralizumab arm, 3 pCR in non-cabiralizumab arm) and 3 had non-pCR (1 RCB-1 and 1 RCB-3 in cabiralizumab arm, 1 RCB-1 in non-cabiralizumab arm). 2 patients came off study prior to surgery (1 due to toxicity and 1 due to missing study visits). Data from the final 2 patients still on treatment will be available at the time of presentation. Discussion: Full safety, pathologic, and clinical response data in the safety lead-in cohort for patients with early-stage TNBC receiving neoadjuvant chemotherapy + nivolumab with or without cabiralizumab, will be presented. Citation Format: Andrew A. Davis, Leonel Hernandez-Aya, Jingqin Luo, Mateusz Opyrchal, Foluso O. Ademuyiwa, Nusayba A. Bagegni, Katherine K. Clifton, Jill Anderson, Trish Hammerschmidt, Leslie Nehring, David DeNardo, Mark Watson, Rebecca Aft, Cynthia Ma, Katherine Weilbaecher. Phase Ib/II study to evaluate safety and tolerability of cabiralizumab in combination with nivolumab and neoadjuvant chemotherapy in patients with localized triple-negative breast cancer [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 P3-06-07.

Abstract P2-11-34: Mitotic spindle hotspot counting using deep learning networks is highly associated with clinical outcomes in patients with early-stage triple-negative breast cancer who did not receive systemic therapy

Abstract Background: Triple-negative breast cancers (TNBC) exhibit high rates of recurrence and mortality. However, recent studies suggest that a subset of patients (pts) with early-stage TNBC enriched in tumor-infiltrating lymphocytes (TILs) have excellent clinical outcomes even in the absence of systemic therapy. Additional histological biomarkers that could identify pts for future systemic therapy escalation/de-escalation strategies are of great interest. TNBC are frequently highly proliferative with abundant mitoses. However, classic markers of proliferation (manual mitosis counting and Ki-67) appear to offer no prognostic value. Here, we evaluated the prognostic effects of automated mitotic spindle hotspot (AMSH) counting on RFS in independent cohorts of systemically untreated early-stage TNBC. Methods: AMSH counting was conducted with a state-of-the-art deep learning algorithm trained on the detection of mitoses within 2 mm2 areas with the highest mitotic density (i.e. hotspots) in digital H&E images. Details of the development, training and validation of the algorithm were published previously [1] in a cohort of unselected TNBC. We obtained AMSH counts in a centrally confirmed TNBC cohort from Mayo Clinic [2] and focused our analysis on pts who received locoregional therapy but no systemic therapy. Using a fractional polynomial analysis with a multivariable proportional hazards regression model, we confirmed the assumption of linearity in the log hazard for the continuous variable AMSH and evaluated whether AMSH counts were prognostic of RFS. We corroborated our findings in an independent cohort of systemically untreated TNBC pts from the Radboud University Medical Center in the Netherlands (Radboud Cohort). Results are reported at a median follow-up of 8.1 and 6.7 years for the Mayo and Netherlands cohorts, respectively. Results: Among 182 pts with who did not receive systemic therapy in the Mayo Cohort, 140 (77%) with available AMSH counts were included. The mean age was 61 (range: 31-94), 71% were postmenopausal, 67% had tumors ≤ 2cm, and 83% were node-negative. As expected, most tumors were Nottingham grade 3 (84%) and had a high Ki-67 proliferation index (54% with Ki-67 >30%). Most tumors (73%) had stromal TILs ≤ 30%. The median AMSH count was 18 (IQR: 8, 42). AMSH counts were linearly associated with grade and tumor size, with the proportion of pts with grade 3 tumors and size > 2 cm increasing as the AMSH counts increased (p=0.007 and p=0.059, respectively). In a multivariate model controlling for nodal status, tumor size, and stromal TILs, AMSH counts were independently associated with RFS (p< 0.0001). For every 10-point increase in the AMSH count, we observed a 17% increase in the risk of experiencing an RFS event (HR 1.17, 95% CI 1.08-1.26). We corroborated our findings in the Radboud Cohort (n=126). The mean age was 68 (range: 40-96), and 81% were node-negative. While the median AMSH count was 36 (IQR: 16-63), higher than in the Mayo Cohort (p=0.004), the prognostic impact was similar, with a significant association between AMSH count and RFS (p=0.028) in a multivariate model corrected for nodal status, tumor size, and stromal TILs. For every 10-point increase in the AMSH count in the Netherlands cohort, we observed a 9% increase in the risk of experiencing an RFS event (HR 1.09, 95% CI 1.01-1.17). RFS rates according to AMSH counts for both cohorts are shown in the Table. Conclusions: AMSH counting is a new proliferation biomarker that provides prognostic value independent of nodal status, tumor size, and stromal TILs in systemically untreated early-stage TNBC. Plans are underway to evaluate AMSH counts in additional cohorts of systemically untreated TNBC, and in other disease settings such as prior to neoadjuvant systemic therapy. If validated, this biomarker should be prospectively evaluated as a potential selection biomarker in clinical trials of systemic therapy de-escalation.

Abstract P5-10-01: Phase 2 study of anlotinib combined with taxanes and lobaplatin in the neoadjuvant treatment of triple-negative breast cancer: efficacy, safety and biomarker analysis from the SWH-B006 (neoALTALL) trial

Abstract Background: Anlotinib, a novel multi-target tyrosine kinase inhibitor that effectively inhibits VEGFR, PDGFR, FGFR, c-KIT, c-MET, and RET, monotherapy has been proven effective in HER-2 negative metastatic breast cancer, but its efficacy in early-stage triple-negative breast cancer (TNBC) is unknown. This phase 2 study aims to evaluate the efficacy and safety of adding anlotinib to neoadjuvant chemotherapy in patients (pts) with primary TNBC. Methods: Pts with clinical stage II/III TNBC were to be treated with 5 cycles of anlotinib (12mg, d1-14, q3w) plus 6 cycles of taxanes (docetaxel 75 mg/m2 or nab-paclitaxel 125 mg/m2, d1 and d8, q3w) and lobaplatin (30 mg/m2, d1, q3w), followed by surgery. The primary endpoint was pathological complete response (pCR) in the breast and axilla (tpCR; ypT0/is ypN0) and the secondary endpoints include pCR in the breast (bpCR; ypT0/is), event-free survival (EFS), invasive disease-free survival (iDFS), overall survival (OS), and safety. Exploratory study included biomarker analysis and efficacy comparation based on FUSCC classification (IHC-based). Results: From Jan 2021 to Feb 2022, a total of 24 pts were enrolled. The median age was 50 years (range, 26-64), 54% were postmenopausal, 75% were nodal involved, 29% had stage III, and 79% were Ki-67 high (≥30%). At the data cut off time of 30th Jun 2022, all 24 pts received at least one dose of study treatment and underwent surgery. Overall, 21 pts received five courses of anlotinib. Two pts discontinued anlotinb for safety reason, and one pt discontinued anlotinb due to missed dose in cycle 4. After surgery, 14 out of 24 pts achieved a tpCR (58.3%; 95% CI, 36.6%–77.9%), and the bpCR rate was also 58.3% (14/24). Of the 18 pts with the node-positive disease at diagnosis, 15/18 (83.3%) became ypN0. Based on the FUSCC IHC-based subtypes, the tpCR rates were 66.7% (6/9) for BLIS subtype, 80% (4/5) for IM subtype and 0% (0/4) for LAR subtype, respectively. Next-generation sequencing revealed that the most commonly mutated genes in these pts were TP53 (19/21, 90.5%), MYC (7/21, 33.3%), BRCA1 (5/21, 23.8%), PIK3CA (4/21, 19.0%), BCL2L11 (4/21, 19.0%), and RB1 (3/21, 14.3%). Subgroup analysis showed that the tpCR were 71.4% (5/7) and 42.9% (6/14) in MYC-amplified and wild-type pts, respectively, and 80% (4/5) and 43.8% (7/16) in BRCA1-mutated and wild-type pts, respectively. All of 24 pts in the safety population showed at least one treatment emergent adverse events (TEAEs). Grade 3 or 4 TEAEs occurred in 14 pts (58.3%), and the most common events were leucopenia (29.2%; n=7), neutropenia (29.2%; n=7), thrombocytopenia (20.8%; n=5), anemia (16.7%; n=4), hypertension (12.5%; n=3), and oral mucositis (8.3%; n=2), respectively. No treatment-related deaths occurred. Conclusions: The addition of anlotinib to neoadjuvant chemotherapy showed manageable toxicity and promising antitumor activity for pts with early-stage TNBC. The study is still ongoing, and the enrollment has been completed. Clinical trial information: ChiCTR2100043027. Funding: Chia Tai Tianqing Pharmaceutical Group Co., Ltd. L. Corresponding author: Dr. Xiaowei Qi, qxw9908@foxmail.com. Department of Breast and Thyroid Surgery, Southwest Hospital, Army Medical University, Chongqing. Citation Format: Yan Liang, Jing Liu, Tao Luo, Jia Ge, Hao Tian, Guozhi Zhang, Linjun Fan, Lin Ren, Li Chen, Peng Tang, Kai Zhu, Xiuwu Bian, Jun Jiang, Yi Zhang, Xiaowei Qi, Peng Tang, Kai Zhu, Xiuwu Bian, Jun Jiang, Yi Zhang, Xiaowei Qi. Phase 2 study of anlotinib combined with taxanes and lobaplatin in the neoadjuvant treatment of triple-negative breast cancer: efficacy, safety and biomarker analysis from the SWH-B006 (neoALTALL) trial [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 P5-10-01.

Abstract PD9-05: Stromal tumor-infiltrating lymphocytes identify early-stage triple-negative breast cancer patients with favorable outcomes at 10-year follow-up in the absence of systemic therapy: a pooled analysis of 1835 patients

Abstract Background: The prognostic value of stromal tumor-infiltrating lymphocytes (TILs) as a biomarker for triple-negative breast cancer (TNBC) has been extensively demonstrated in patients (pts) receiving (neo)adjuvant systemic therapy. In addition, several small studies suggest that a subset of pts with early-stage TNBC and high TILs have excellent long-term outcomes, even in the absence of systemic therapy [1-3]. However, data on the absolute risk of TNBC recurrence according to TIL levels in the absence of systemic therapy are limited and critical to inform the design of future systemic therapy de-escalation clinical trials. Methods: We conducted an individual patient data pooled analysis of 12 international cohorts of pts with TNBC treated with locoregional therapy but no systemic therapy. TNBC was defined as tumors with estrogen and progesterone receptor of < 1% and HER2 negative (IHC 0, 1+ or IHC 2+ and FISH negative) per local evaluation. TILs were locally assessed in hematoxylin & eosin-stained slides according to the International Immuno-Oncology Biomarker Working Group guidelines (www.tilsinbreastcancer.org). We used the Kaplan-Meier method to assess survival outcomes according to prespecified TIL thresholds: 30% and 50%. Confidence intervals (CI) for survival probabilities were calculated using a percentile bootstrap method. The primary endpoint was invasive disease-free survival (iDFS, STEEP 2.0 definition). Key secondary outcomes included recurrence-free survival (RFS), distant disease-free survival (DDFS) and overall survival (OS). Results: 1,835 pts diagnosed with TNBC between 1982 and 2017 who did not receive systemic therapy were included. The median age at diagnosis was 56 (IQR 38-71). Menopausal status was known in 1,184 women, of whom 78% were post-menopausal. The median tumor size was 2.0 cm (IQR 1.2-2.6). Most pts (87%) had no axillary lymph node involvement (N0). Most tumors were invasive ductal carcinoma (74%) and grade 3 (70%). The median level of TILs was 15% (IQR 5-40). The median duration of follow-up was 30.4 years (95% CI 29.9, 31.1). A total of 950 (52%) iDFS, 828 (45%) RFS, 767 (42%) DDFS events, and 604 (33%) deaths were observed. In multivariable analyses, higher TILs were independently associated with improved iDFS, RFS, DDFS, and OS beyond clinicopathological factors (likelihood ratio p< 10e-6). Each 10% increment in stromal TILs was associated with an 8% (95% CI: 6-11), 10% (95% CI: 7-13), and 13% (95% CI: 10-15) reduction in the risk of experiencing an iDFS, RFS or DDFS event, and with a 12% (95% CI: 9-15) reduction in the risk of death. iDFS, RFS, DDFS and OS rates according to different TIL thresholds and nodal status are shown in the Table. Of note, the RFS estimates (which exclude second non-breast primaries and contralateral breast cancers) were consistently higher than the iDFS counterparts (which include both), consistent with a high rate of contralateral breast cancers and second primary tumors in this cohort. Notably, patients with node-negative—and especially stage I—TNBC with high TILs had excellent survival rates at 10-year follow-up. Conclusion: TILs are highly prognostic in pts with systemically untreated early-stage TNBC. Pts with pN0 (and especially stage I) TNBC with high TILs exhibited very favorable long-term outcomes even in the absence of systemic therapy. These data define the natural history of TIL-rich TNBC pts and are crucial to identifying the optimal patient population for future chemotherapy and immunotherapy de-escalation clinical trials.

Abstract P5-06-02: Circulating tumor cells in metastatic breast cancer highlight potential role of copy number evolution in late-stage cancer mutational profile

Abstract Background: Structural variation is a hallmark of breast cancer. Previous single-cell genomic analysis of 10 untreated early-stage primary TNBC tumors identified only one or two subclones per tumor, found that copy number variation (CNV) occurs early in the evolution of the tumor, and asserted that CNV changes do not contribute to genomic variation at later time points of a tumor’s growth. This PCNE model (punctuated copy number evolution) is analogous to the big-bang theory of colorectal cancer. However, cancer exists in a dynamic environment, and systemic chemotherapy provides evolutionary pressure, making stasis quite unlikely. An investigation of late-stage cancers could shed light on CNV evolution. Methods: We performed a longitudinal, prospective observational study of over 130 patients with metastatic breast cancer, with regular follow-up and detailed treatment histories. CTCs were enumerated by CellSearch, and samples with >20 CTCs in 7.5ml of whole blood were further processed for single CTC isolation via DEPArray, single-cell library construction, and whole-genome sequencing. CNV counts for each cell were obtained using previously published binning methods (Ginkgo). Three mathematical models were used to evaluate the evolution of CNVs: a punctuated model, a gradual model, and a gradual-on-punctuated hybrid model. Results: In total, 150 patient-years of data were collected. Among samples with sufficient CTC counts for study inclusion, a total of nine patients and 376 cells were isolated for sequencing. A mean sequencing depth of 0.8× was achieved from each single cell. The degree of CNV heterogeneity varies from patient to patient. CNV changes occurred over time in a gradual manner on a background of punctuated changes. The adjusted R2 value for the punctuated model was 0.46, the adj R2 value for the gradual model was 0.76, and the hybrid model achieved an adj R2 of 0.99. Patient samples with large CNV heterogeneity across single cells tend to be from patients with longer treatment histories and from the last blood draw (i.e., the blood collection closest to patient’s point of death). We also identified multiple whole-genome duplication (WGD) events from the same patient, in contrast to previous findings that WGD events are usually early clonal events in most instances. Finally, X loss events are among the most frequent chromosomal aberrations identified in CTCs (in up to 80% of CTCs in some patients), which supports previous literature on the loss of the Barr body in hastening metastatic spread. Discussion and Conclusion: In contrast to the previous study, our findings support a gradual-on-punctuated hybrid model, and this model can explain how copy number changes can be a source from which tumors gain resistance to systemic therapies. The model explains how CNV heterogeneities are seen in patients with longer treatment histories, suggesting that CNV continues to be a source of genetic variation at the metastatic stage of the disease course. Citation Format: Weelic Chong, Rui Luo, Zhenchao Zhang, Maysa Abu-Khalaf, Daniel Silver, Frederick Fellin, Rebecca Jaslow, AnaMaria Lopez, Terrence Cescon, Ronald Myers, Mariel Becker, Qiang Wei, Bingshan Li, Chun Wang, Hushan Yang. Circulating tumor cells in metastatic breast cancer highlight potential role of copy number evolution in late-stage cancer mutational profile [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 P5-06-02.

Abstract OT2-10-02: Phase I trial of an alpha-lactalbumin vaccine in patients with operable triple-negative breast cancer (TNBC)

Abstract Background: Triple-negative breast cancer (TNBC) is the subtype of breast cancer with the worst prognosis and is the subtype most often associated with germline mutations of BRCA1 as well as other genes. Alpha-lactalbumin (aLA) is a milk protein expressed in lactating breasts but not at other times or in other normal tissues. Expression of aLA is found in approximately 70% of TNBC (Cancers PMID: 27322324) so is an attractive immunologic target for TNBC based on the “retired protein hypothesis” (Semin Immunol PMID: 31926646). Pre-clinical studies have shown that vaccination with aLA inhibits the growth of established breast tumors and provides potent protection from development of autochthonous tumors in transgenic murine models of breast cancer and against 4T1 transplantable breast cancer in BALB/c mice (Nat Med PMID: 20512124). Specific Aims: We are conducting a Phase I trial of vaccination with an aLA vaccine in patients with early stage TNBC to demonstrate the safety of this approach and to document the ability to produce a relevant immunologic response to aLA. Trial Design: Patients are being entered into a Phase I trial of alpha-lactalbumin with GMP-grade zymosan adjuvant in Montanide ISA 51 VG vehicle. Subjects receive a total of 3 vaccinations administered once every 2 weeks. Toxic events of Common Terminology Criteria for Adverse Events (CTCAE) grade 2 or greater are considered dose-limiting. Dose levels being tested in the current protocol are alpha-lactalbumin/zymosan 0.01/0.01, 0.1/0.01, 0.5/0.01, 0.5/0.03, and 0.5 mg/0.06 mg. Patients are being monitored for toxicity until 84 days after the first vaccination or resolution of toxicity, whichever is later. Blood is being drawn prior to therapy and 14, 28, and 56 days after the first vaccination to assess cellular response using enzyme-linked immunosorbent spot (ELISpot) assays of interferon-gamma and interleukin-17 production in response to aLA. Humoral response to aLA vaccination is being assessed by enzyme-linked immunosorbent assay (ELISA). Eligibility Criteria: Patients with ER-negative, PR-negative, HER2-negative breast cancer of pathologic stage II-III or who had residual disease after standard pre-operative systemic therapy. Participants must be within 3 years of initiation of treatment and have no evidence of recurrence. Dose Selection: Doses are being escalated using a 3+3 trial design to determine the maximum tolerated dose, the lowest immunologic dose and the optimal immunologic dose Present Accrual and Target Accrual: Dependent on toxicity, 18-30 patients will be treated. Future Plans and Contact Information: After identification of the maximum tolerated dose and expansion of the dose levels associated with effective tumor immunity we plan to enroll 2 additional cohorts of patients: 1) patients who have completed pre-operative chemo-immunotherapy and are receiving pembrolizumab as standard of care therapy, and 2) patients without cancer planning to undergo prophylactic bilateral mastectomy. Interested parties may contact Dr. Budd at buddg@ccf.org. Funding Source: Department of Defense (W81XWH-17-1-0592 and W81XWH-17-1-0593) Citation Format: George Budd, Justin M. Johnson, Emily Rhoades, Halle Moore, Megan L. Kruse, Erin Roesch, Jame Abraham, Brenna Elliott, Elena Haury, Vincent K. Tuohy. Phase I trial of an alpha-lactalbumin vaccine in patients with operable triple-negative breast cancer (TNBC) [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 OT2-10-02.