Immunotherapy innovations in triple-negative breast cancer: targeting checkpoints, combinations, and biomarkers.

Background: Durable responses of triple negative breast cancer (TNBC) to pembrolizumab (anti-PD-1) or atezolizumab (anti-PD-L1) have been reported in the metastatic setting. Moreover, it is currently being hypothesized that immune checkpoint inhibitors might be more effective in the neoadjuvant setting, due to better preserved anti-tumor immunity in early TN disease. However, biomarkers predictive of response to anti-PD-1 or anti-PD-L1 agents, as well as biomarker-based strategies for testing those drugs in the neoadjuvant setting are still lacking. We evaluated PD-L1 protein expression and the composition of tumor-infiltrating lymphocytes (TILs) in untreated TNBC, in order to get a better insight into the TNBC sub-population(s) which would be suitable for neoadjuvant anti-PD-1 or anti-PD-L1 therapy evaluation. Methods: TNBC patients consecutively treated at the Jean Perrin Comprehensive Cancer Centre (Clermont-Ferrand, France), from 01/01/2010 to 12/31/2014, were included. FFPE tumor tissues were assessed for PD-L1 expression by immunohistochemical (IHC) laboratory-developed test (clone 28-8, Abcam), in tumor cells (tPD-L1) and in TILs. Positivity cut-offs evaluated were ≥1%, ≥5% and ≥10%. The amount CD8+, CD4+, FoxP3+ or PD-1+ TILs was determined by counting those cells, detected by IHC methods, within 5 consecutive HPFs (x400), from tumor invasive front toward tumor center. Clinical disease stage was determined using the TNM system. Results: One hundred and two TNBCs were assessed. There were 28.4%, 23.5% or 16.7% tPD-L1-positive cases (cs), for cut-offs ≥1%, ≥5% or ≥10%, respectively. Similarly, 32.4%, 15.7% or 5.9% of cs were positive for PD-L1 in TILs, using the same cut-offs. With ≥5% as cut-off, positivity for tPD-L1 significantly correlated with the amount of CD8+ (p=0.023), FoxP3+ (p=0.0036) or PD-1+ TILs (p=0.043). The same cut-off, applied to TILs, revealed significant correlations between PD-L1 positivity and the amount of each CD8+, CD4+ or PD-1+ TILs (p=0.025, 0.039 and 0.0042, respectively). Interestingly, when the ≥5% cut-off was used, tumors of T2 size were most frequently tPD-L1+ (11/41 cs, 26.8%), compared with the T1 (3/35 cs, 8.6%) and T3+T4 (3/18 cases, 16.7%) (p=0.04). With regards to TILs, with the ≥5% cut-off, the PD-L1+ cases belonged exclusively to the T1+T2 group (15/76), whereas the T3+T4 group was PD-L1-negative (0/18 cs). Other two cut-offs revealed only occasional correlations. Conclusion: This single-center, real-world TNBC cohort contained a high number of smaller tumors (T1-T2). The IHC-based PD-L1 assessment, with ≥5% as the positivity cut-off, revealed that approximately 1/4 of TNBC could be candidates for neoadjuvant anti-PD-1/anti-PD-L1 approaches. Combined with the amount of CD8+ and PD-1+ TILs, tumor PD-L1 positivity might make an easy-to-use composite biomarker for the 1st-level patient selection for PD-1 or PD-L1 inhibitors in neoadjuvant TNBC treatment. The 2nd level could exploit, for example, the assessment of mutation burden in tumors with low tPD-L1 or amount of CD8+ or PD-1+ TILs. Such tumors might be more frequent among larger TNBC (T3-T4). Citation Format: Finck W, Passildas J, Poirier C, Kwiatkowski F, Abrial C, Durando X, Penault-Llorca F, Radosevic-Robin N. The ≥5% cut-off reveals tumor PD-L1 positivity as potential selection biomarker for patient enrollment into the trials evaluating anti-PD-1 or anti-PD-L1 agents in neoadjuvant treatment of triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-12-09.

As an aggressive subtype of breast cancer, triple-negative breast cancer (TNBC) is associated with poor prognosis and lack of effective therapy, except chemotherapy. In recent years, immunotherapy based on immune checkpoint (IC) inhibition has emerged as a promising therapeutic strategy in TNBC. TNBC has more tumor-infiltrating lymphocytes (TILs) and higher rate of mutation and programmed cell death ligand-1 (PD-L1) expression than other subtypes of breast cancer have. However, previous studies have shown that monotherapy has little efficacy and only some TNBC patients can benefit from immunotherapy. Therefore, it is important to identify biomarkers that can predict the efficacy of IC inhibitors (ICIs) in TNBC. Recently, various biomarkers have been extensively explored, such as PD-L1, TILs and tumor mutational burden (TMB). Clinical trials have shown that PD-L1-positive patients with advanced TNBC benefit from ICIs plus chemotherapy. However, in patients with early TNBC receiving neoadjuvant therapy, PD-L1 cannot predict the efficacy of ICIs. These inconsistent conclusions suggest that PD-L1 is the best to date but an imperfect predictive biomarker for efficacy of ICIs. Other studies have shown that advanced TNBC patients with TMB ≥10 mutations/Mb can achieve clinical benefits from pembrolizumab. TILs also have potential predictive value in TNBC. Here, we select some biomarkers related to ICIs and discuss their potential predictive and prognostic value in TNBC. We hope these biomarkers could help to identify suitable patients and realize precision immunotherapy.

Introduction Triple negative breast cancer (TNBC) is an area of high unmet medical need in terms of new effective treatment strategies. Although breast cancer is traditionally considered a ‘cold’ tumor type, TNBC is the most appropriate subtype for immunotherapeutic strategies; this is due to the high level of tumor infiltrating lymphocytes, PD-L1 expression, and tumor mutational burden compared to other breast cancer subtypes. Areas covered This review examines the available evidence on the use of immunotherapeutic strategies in early and advanced TNBC, discusses the pitfalls and limitations often encountered in clinical research, and summarizes data on novel promising immunomodulatory approaches that have been explored in early-phase trials. Expert opinion PD-1-blockade is approved for stage II/III TNBC and for first-line treatment of PD-L1-positive TNBC patients with metastatic disease and should be considered standard of care. However, question marks and difficulties remain; these include the identification of predictive biomarkers to select patients who benefit from the addition of PD1-blockade and the balance between efficacy and long-term toxicity for an individual patient. Numerous treatment combinations and new immunotherapeutic strategies beyond PD1 blockade are being evaluated, thus reflecting a promising evolution towards a more personalized approach, and extended clinical benefit in TNBC. Abbreviations:Triple-negative breast cancer (TNBC); breast cancers (BCs); estrogen receptor (ER); progesterone receptor (PgR); human epidermal growth factor-2 (HER-2); basal-like 1 (BL1), basal-like 2 (BL2); mesenchymal (MES); mesenchymal stem-like (MSL); immunomodulatory (IM); luminal androgen receptor (LAR); basal-like immunosuppressed (BLIS); basal-like immune-activated (BLIA); tumor-infiltrating lymphocytes (TILs); tumor mutational burden (TMB); immune cells (ICs); immunohistochemistry (IHC); overall response rate (ORR); overall survival (OS); progression-free survival (PFS); intention-to-treat (ITT); hazard ratio (HR); confidence interval (CI); Food and Drug Administration (FDA); European Medicines Agency (EMA); immune checkpoint inhibitors (ICI); Combined Positive Score (CPS); disease control rate (DCR); neoadjuvant chemotherapy (NACT); pathological complete response (pCR); event-free survival (EFS); disease-free survival (DFS); residual cancer burden (RCB); San Antonio Breast Cancer Symposium (SABCS); antibody-drug conjugates (ADCs); PARP inhibitors (PARPi); clinical benefit rate (CBR); Histone deacetylase inhibitors (HDACi); Dendritic cell (DC); talimogene laherparepvec (TVEC); granulocyte-macrophage colony-stimulating factor (GM-CSF); mismatch repair deficiency (dMMR).

Purpose: To date no biomarker has been identified that predicts response to ICI in mTNBC. This study aimed to explore if tumor genomic alterations correlate with efficacy of PD-1/PD-L1 inhibition in patients (pts) with mTNBC. Methods: Demographic, treatment response, and long-term outcome data were collected on patients with mTNBC treated at Dana-Farber Cancer Institute (DFCI) under several clinical trials incorporating PD-1/PD-L1 inhibitors, given as monotherapy or combined with chemotherapy (CT). Pts included in this analysis had available results of targeted exon sequencing performed using Oncopanel, our institutional gene sequencing panel, on archival tumor tissue. TMB was calculated by determining the number of non-synonymous somatic mutations that occur per megabase of exonic sequence data across all genes on the panel. High TMB was defined as 310 mutations/megabase. TMB and gene alterations were correlated with objective response rate (ORR) per RECIST 1.1, progression-free (PFS) and overall survival (OS). Results: A total of 50 pts with mTNBC were included in this analysis. At baseline, the median age was 55.9 years (31.8–75.9), 60% had ECOG 0 and 40% had ECOG 1, 72% had visceral metastasis, and 46% had received 31 prior lines of systemic therapy in the metastatic setting. While 26% of pts received monotherapy [pembrolizumab (n=7, NCT02447003); atezolizumab (n=6; NCT01375842)], 74% received combination with CT [pembrolizumab plus eribulin (n=31; NCT02513472); atezolizumab plus nab-paclitaxel (n=6; NCT01633970)]. PTEN alterations were present in 30% of pts (mutations = 7; one copy number loss = 7; two copy number loss = 1). Median follow-up was 14 months (1–40). Pts with tumors harboring PTEN alterations had lower ORR (7% vs 57%; P<0.001), shorter median PFS (2.3 vs 6.3 months; P=0.027), and shorter median OS (8.1 vs 20.1 months; P=0.012) compared to pts without PTEN alterations. The median TMB was 6.6 mut/Mb (1.2–50.8), and 23% of pts had a high TMB. While high TMB was not associated with higher ORR (P=0.56), it was associated with better median PFS (16.5 vs 2.4 months; P=0.017), and better median OS (not reached vs 13.5 months; P=0.026). Both PTEN status and TMB remained significantly associated with PFS in the multivariable model. Only PTEN status remained significantly associated with OS in the multivariable analysis with the same covariables. Ongoing analysis to better understand if these predictors are specific for predicting benefit to immunotherapy and/or a marker of chemotherapy resistance will be presented at the symposium. Conclusion: PTEN genomic alterations and TMB may impact benefit from PD-1/PD-L1 inhibitors largely administered with chemotherapy in mTNBC. These observations warrant prospective validation and may inform the importance of stratifying pts according to these characteristics in future randomized studies with ICI. Table 1.Multivariable analysis for PFS Hazard ratioConfidence Intervalp-valueCombination therapy0.420.16 – 1.130.009Visceral metastasis1.310.63 – 2.770.46Previous lines of therapy1.020.09 – 0.700.85ECOG 12.11.06 – 1.280.034PTEN altered3.741.65 – 8.440.002Hypermutated tumors0.850.75 – 0.970.011 Citation Format: Barroso-Sousa R, Tyekucheva S, Pernas-Simon S, Exman P, Jain E, Garrido-Castro AC, Hughes M, Bychkovsky B, Di Lascio S, Umeton R, Files J, Lindeman NI, MacConaill LE, Hodi FS, Krop IE, Dillon D, Winer EP, Wagle N, Lin NU, Mittendorf EA, Tolaney SM. PTEN alterations and tumor mutational burden (TMB) as potential predictors of resistance or response to immune checkpoint inhibitors (ICI) in metastatic triple-negative breast cancer (mTNBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-12-02.

Breast cancer (BC) remains a significant global health challenge due to its complex biology, which complicates both diagnosis and treatment. Immunotherapy and cancer vaccines have emerged as promising alternatives, harnessing the body's immune system to precisely target and eliminate cancer cells. However, several key factors influence the selection and effectiveness of these therapies, including BC subtype, tumor mutational burden (TMB), tumor-infiltrating lymphocytes (TILs), PD-L1 expression, HER2 resistance, and the tumor microenvironment (TME). BC subtypes play a critical role in shaping treatment responses. Triple-negative breast cancer (TNBC) exhibits the highest sensitivity to immunotherapy, while HER2-positive and hormone receptor-positive (HR+) subtypes often require combination strategies for optimal outcomes. High TMB enhances immune responses by generating neoantigens, making tumors more susceptible to immune checkpoint inhibitors (ICIs); whereas, low TMB may indicate resistance. Similarly, elevated TIL levels are associated with better immunotherapy efficacy, while PD-L1 expression serves as a key predictor of checkpoint inhibitor success. Meanwhile, HER2 resistance and an immunosuppressive TME contribute to immune evasion, highlighting the need for multi-faceted treatment approaches. Current breast cancer immunotherapies encompass a range of targeted treatments. HER2-directed therapies, such as trastuzumab and pertuzumab, block HER2 dimerization and enhance antibody-dependent cellular cytotoxicity (ADCC), while small-molecule inhibitors, like lapatinib and tucatinib, suppress HER2 signaling to curb tumor growth. Antibody-drug conjugates (ADCs) improve tumor targeting by coupling monoclonal antibodies with cytotoxic agents, minimizing off-target effects. Meanwhile, ICIs, including pembrolizumab, restore T-cell function, and CAR-macrophage (CAR-M) therapy leverages macrophages to reshape the TME and overcome immunotherapy resistance. While immunotherapy, particularly in TNBC, has demonstrated promise by eliciting durable immune responses, its efficacy varies across subtypes. Challenges such as immune-related adverse events, resistance mechanisms, high costs, and delayed responses remain barriers to widespread success. Breast cancer vaccines-including protein-based, whole-cell, mRNA, dendritic cell, and epitope-based vaccines-aim to stimulate tumor-specific immunity. Though clinical success has been limited, ongoing research is refining vaccine formulations, integrating combination therapies, and identifying biomarkers for improved patient stratification. Future advancements in BC treatment will depend on optimizing immunotherapy through biomarker-driven approaches, addressing tumor heterogeneity, and developing innovative combination therapies to overcome resistance. By leveraging these strategies, researchers aim to enhance treatment efficacy and ultimately improve patient outcomes.

Background: Triple negative breast cancer (TNBC) is a heterogeneous disease with several molecular subtypes: basal-like1 (BL-1), basal-like 2 (BL-2), mesenchymal (M), and luminal androgen receptor (LAR). Molecular evolution of TNBC through chemotherapy selection pressure is well recognized but poorly understood. In addition, approximately 20% of TNBCs respond to PD-1 or PD-L1 inhibitors. It has been observed that heavily pre-treated patients may not respond well to immunotherapy. This study was carried out to perform immune profiling of paired primary and recurrent TNBC. Here we report the result of the first 10 paired tissue pilot analysis. Methods: Twenty specimens were identified through an IRB-approved protocol via the City of Hope Biospecimen Repository (2002-2015). Two brain and one bone metastasis specimens were not included due to technical difficulty. Formalin-fixed paraffin embedded (FFPE) sample blocks were cut into 5-mm thick slides and labeled with the following antibodies: CD4, CD8, CD3, FOXP3, CD20, CD33, Pan-CK, and PD-1 using the multiplex IHC opal method. Image acquisition and cell counting were carried out using PerkinElmer Vectra automated quantitative pathology imaging system and inForm software analysis (PerkinElmer, Waltham, MA). mRNA expression profiling was performed using Affymetrix Human Genechip 2.0. Raw data were normalized and processed using Expression Console. Using Vanderbilt TNBC sub-classification tool, we have sub-classified the 20 primary and recurrent TNBC specimens. Tumor mutation burden (TMB) was generated through FoundationOne® platform. Result: A total of 17 samples were analyzed (M, 5; LAR, 3; BL-1, 4; BL-2, 5). M-subtype had a significantly lower tumor-infiltrating CD3+ T cells (p=0.005), CD8+ T cells (p=0.024), CD4+ T cells (p=0.065) and CD4+FOXP3+ Treg cells (p=0.054), irrespective of the site of metastasis. CD20+ B cells were particularly enriched in BL-1 subtype (p=0.0013, 23.5% of 17 samples). Of 17 samples, 8 had TMB. Seven had low TMB (<10 mut/Mb) and one had intermediate TMB (11 mut/Mb, LAR subtype). The tumor with intermediate TMB had the highest quantity of tumor-infiltrating CD3+ T cells, CD8+ T cells, CD8+PD1+ T cells, and CD4+FOXP3+PD1+ Treg cells compared to the 7 tumors with low TMB. Compared with recurrent tumors, primary tumors had a significantly higher percentage of tumor-infiltrating T cells (TIL). To validate multiplexed IHC results, these samples were evaluated by a licensed pathologist at City of Hope using the International TILs Working Group 2014 guidelines, and there was a good correlation between percent of TILs and CD3+ T cells by IHC approach. Conclusion: To our knowledge, this is the first study linking tumor immune cell profiles with the TNBC 4 subtypes. Distinctive immune cell patterns were observed among 4 TNBC subtypes. M subtype had significantly lower TILs, which may indicate poor response to checkpoint inhibitors. Further analysis of a total of 50 paired TNBCs is currently underway. Contact information: Yuan Yuan MD PhD, Email: yuyuan@coh.org Citation Format: He T-F, Yost S, Schmolze D, Wang R, Rosario A, Tu T, Chu P, Lee P, Yuan Y. Immune profiling of paired primary and recurrent triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-02.

Breast cancer is the most commonly diagnosed cancer (estimated 2.3 million new cases in 2020) and the leading cause of cancer death (estimated 685,000 deaths in 2020) in women globally. Breast cancers have been categorized into four major molecular subtypes based on the immunohistochemistry (IHC) expression of classic hormone and growth factor receptors including the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), as well as a proliferation marker Ki-67 protein expression. Triple-negative breast cancer (TNBC), a breast cancer subtype lacking ER, PR, and HER2 expression, is associated with a high metastatic potential and poor prognosis. TNBC accounts for approximately only 15%–20% of new breast cancer diagnoses; it is responsible for most breast cancer–related deaths due to the lack of targeted treatment options for this patient population, and currently, systemic chemotherapy, radiation, and surgical excision remain the major treatment modalities for these patients with TNBC. Although breast cancer patients in general do not have a robust response to the immunotherapy, a subset of TNBC has been demonstrated to have high tumor mutation burden and high tumor-infiltrating lymphocytes, resembling the features observed on melanoma or lung cancers, which can benefit from the treatment of immune checkpoint inhibitors (ICIs). Therefore, the immunogenic nature of this aggressive disease has presented an opportunity for the development of TNBC-targeting immunotherapies. The recent US Food and Drug Administration approval of atezolizumab in combination with the chemotherapeutic agent nab-paclitaxel for the treatment of PD-L1-positive unresectable, locally advanced, or metastatic TNBC has led to a new era of immunotherapy in TNBC treatment. In addition, immunotherapy becomes an active research area, both in the cancer biology field and in the oncology field. In this review, we will extend our coverage on recent discoveries in preclinical research and early results in clinical trials from immune molecule-based therapy including cytokines, monoclonal antibodies, antibody–drug conjugates, bi-specific or tri-specific antibodies, ICIs, and neoantigen cancer vaccines; oncolytic virus-based therapies and adoptive immune cell transfer–based therapies including TIL, chimeric antigen receptor-T (CAR-T), CAR-NK, CAR-M, and T-cell receptor-T. In the end, we will list a series of the challenges and opportunities in immunotherapy prospectively and reveal novel technologies such as high-throughput single-cell sequencing and CRISPR gene editing-based screening to generate new knowledges of immunotherapy.

Background: Hormone-receptor (HR) positive breast cancer is the main subtype of breast cancer. Although overall survival of HR-positive metastatic breast cancer (MBC) patients has improved by various therapies including endocrine therapies, CDK4/6 inhibitors, and cytotoxic chemotherapy, it is still considered incurable. Immune checkpoint inhibitors have rarely been clinically tested in HR-positive breast cancer, despite proving anti-cancer activity in early and metastatic triple-negative breast cancer in various trials. We evaluated efficacy and safety of combined durvalumab and tremelimumab in the HR-positive MBC, which was enriched with high tumor mutational burden (TMB). Methods: HR-positive MBC patients who received prior 1 or more lines of therapy in metastatic setting were prescreened with whole exome sequencing (WES) using metastatic or recurred tumor biopsies. Criterion of high TMB was defined as upper 30%. In the beginning, the criterion of high TMB was 2.1 mutations per Mb, based on the retrospective WES database in Yonsei Cancer Center and this criterion was recalculated every 30 cases. Patients who met upper 30% of TMB were treated with combined durvalumab (1500mg every 4 weeks upto 13 doses) and tremelimumab (75mg every 4 weeks upto 4 doses). Response was evaluated every 2 cycles using RECIST 1.1 and toxicity was evaluated using NCI-CTCAE 4.03. Tumor-infiltrating lymphocyte (TIL) and PD-L1 expression were also analyzed to investigate a correlation with TMB. Results: Biopsies of recurrent or metastatic tumors were taken from a total of 119 patients for WES assay. A median turn-around-time of TMB data was 30.0 days (range, 16~67). Of these 119 patients, a median number of nonsynonymous mutations was 2.0 per Mb (range, 0~21.7) with upper 30% criterion of 3.1. High TMB showed a trend toward old age (P=0.074) and single positivity of estrogen receptor (ER) or progesterone receptor (PR) compared to positivity of both ER and PR (P=0.055). TMB was positively correlated with TILs (r=0.289, P=0.005). Thirty patients with high TMB received study treatment with a median 2 cycles (range, 1~13). A median prior lines of therapies in metastatic setting was 4 (range, 1~9). The objective response and clinical benefit rates were 6.3% (2 PRs of 30) and 20% (2 PRs plus 4 SDs of 30). There was one treatment-related mortality due to pneumonitis. Immune-related adverse events included endocrinopathy (n=3; hypothyroidism in 2, hyperthyroidism in 1), enteritis (n=2), skin rash (n=2), pneumonitis (n=1), and so on. Biomarker analyses are underway. Conclusions: WES-based TMB using metastatic tumor biopsy was a feasible platform to prescreen HR-positive MBC patients. Combined durvalumab and tremelimumab showed a modest activity and good tolerability in heavily treated, HR-positive MBC with high TMB. Citation Format: Yong Wha Moon, Eunyoung Kim, Min Hwan Kim, Gun Min Kim, Seul-Gi Kim, YeeSoo Chae, Jieun Lee, Jae Ho Jeong, Kyung-Hun Lee, Han Jo Kim, Joo Young Jung, Su-Jin Koh, Kyoung Eun Lee, Hee-Jun Kim, Kyong Hwa Park, Seungtaek Lim, Yeon Hee Park, Tae Hoen Kim, Sewha Kim, Yohan Yang, Sangwoo Kim, Joohyuk Sohn. Phase II trial of durvalumab and tremelimumab in the hormone receptor-positive metastatic breast cancer with high tumor mutational burden selected by whole exome sequencing: Korean cancer study group trial (KCSG BR17-04) [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 P1-19-03.

Background: The benefits of immune check point inhibition (ICI) are primarily seen in immunogenic cancer types. Unfortunately, most breast cancers are non-immunogenic, and therefore new approaches that target tumor immunogenicity are key to increasing the efficacy of ICI. Even within a single breast cancer subtype, patients with liver metastases are less responsive to ICI as compared to other metastatic sites. Therefore, the objective of this analysis is to investigate differences in prognostic and predictive ICI related biomarkers such as PD-L1 expression, tumor mutation burden (TMB), and immune-cell populations between primary tumors, liver metastases (LM) and non-liver metastatic (NLM) sites from breast cancer patients. Our goal is to provide an in-depth analysis of current and potential markers of immunogenicity within LM and NLM. Methods: Unpaired samples taken from primary and recurrent breast (BT), liver metastases (LM) and non-liver metastases (NLM) (all identified as breast cancer) were compared using Fisher-exact or Chi2 and corrected for multiple comparison. Tumors were classified as hormone receptor positive (HR+), HER2+ or triple negative breast cancer (TNBC). Breast cancer samples were tested using NextGen DNA sequencing (NextSeq, 592 gene panel) and whole transcriptome RNA sequencing (NovaSeq). PDL1 was scored on immune cells using VENTANA PD-L1 (SP142) assay. TMB was measured by counting somatic non- synonymous missense mutations on the 592 gene panel and ≥ 10 mutations/Megabase (mut/Mb) was considered high. MCP counter was used to evaluate relative cell abundance (in arbitrary units) in TME using transcriptome data. Results: Biopsies from 3166 tumors were queried, 1268 of which were from BT, 495 from LM, and 1403 from NLM, all are unpaired. All histologic subtypes are represented. HR+ breast cancer was the most prevalent among LM. Both metastatic groups (LM and NLM) were significantly more likely to have a high TMB as compared to BT (24.8 & 24.8 vs. 16.6%, p<0.0001). PD-L1 expression in immune cells however, was significantly decreased in LM as compared to BT (12 vs. 34%, p<0.0001) and NLM (12 vs. 28%, p<0.0001), a trend largely driven by HER2+ and TNBC subtypes. Comparison of immune cells within tumor biopsies of LM vs. BT demonstrated significantly fewer cytotoxic CD8+ T cells (1.38 vs. 0.69, p<0.001), B cells (234 vs. 98, p<0.001), and myeloid dendritic cells (DCs) (1.5 vs. 1.02, p<0.001). These observations were most predominant in TNBC and HR+ breast cancers. HR+ LM also had fewer natural killer cells (0.85 vs. 0.03, p<0.005). Cytotoxic T cells and DCs were not significantly altered in HER2+ LM. All immune cell types, except for the monocytic lineage, were significantly (p<0.001) higher within NLM as compared to LM. Investigation of molecular alterations revealed significant differences in LM vs. NLM vs. BT, e.g, LM from all subtypes were enriched for copy number alterations in genes such as CCND1 and FGF19/14, FGFR1. Significant (p<0.001) enrichment of mutations in ESR1 (2.8 vs. 27.1%), HER2 (1.9 vs.6.1%), and GATA3 (7.8 vs> 12.9%), were observed in LM vs. BT. Conclusions: In this patient cohort, immune cells within the TME of LM were less abundant and suggest the liver is a less immunogenic niche. However, LM had increased TMB as compared to breast tumors, suggesting that immunosuppressive cells (i.e. Tregs and MDSCs) or cytokines, may be preventing cytotoxic immune cell infiltration into the TME. Moreover, immune cell populations within LM had decreased PD-L1 expression when compared to breast tumors, suggesting another mechanism that could explain the lack of response to ICI in such patients. Further characterization of the genetic and molecular alterations of breast cancer LM and NLM will help identify additional biomarkers of response and determine their role in defining tumor immune response to ICI. Citation Format: Sofi Castanon, Daniel Flores, Joanne Xiu, Paula R. Pohlmann, Foluso Ademuyiwa, Elia Obeid, Jasgit Sachdev, Michael Simon, Elisa Krill Jackson, Lee Schwartzberg, Antoinette R Tan, Neelima Denduluri, W. Michael Korn, Evanthia T Roussos Torres. The immune microenvironment of liver metastasis as a guide for immunotherapeutic potential in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-02.

Triple-negative breast cancer (TNBC) is a clinically aggressive subtype of breast cancer that represents 15–20% of breast tumors and is more prevalent in young pre-menopausal women. It is the subtype of breast cancers with the highest metastatic potential and recurrence at the first 5 years after diagnosis. In addition, mortality increases when a complete pathological response is not achieved. As TNBC cells lack estrogen, progesterone, and HER2 receptors, patients do not respond well to hormone and anti-HER2 therapies, and conventional chemotherapy remains the standard treatment. Despite efforts to develop targeted therapies, this disease continues to have a high unmet medical need, and there is an urgent demand for customized diagnosis and therapeutics. As immunotherapy is changing the paradigm of anticancer treatment, it arises as an alternative treatment for TNBC patients. TNBC is classified as an immunogenic subtype of breast cancer due to its high levels of tumor mutational burden and presence of immune cell infiltrates. This review addresses the implications of these characteristics for the diagnosis, treatment, and prognosis of the disease. Herein, the role of immune gene signatures and tumor-infiltrating lymphocytes as biomarkers in TNBC is reviewed, identifying their application in patient diagnosis and stratification, as well as predictors of efficacy. The expression of PD-L1 expression is already considered to be predictive of response to checkpoint inhibitor therapy, but the challenges regarding its value as biomarker are described. Moreover, the rationales for different formats of immunotherapy against TNBC currently under clinical research are discussed, and major clinical trials are highlighted. Immune checkpoint inhibitors have demonstrated clinical benefit, particularly in early-stage tumors and when administered in combination with chemotherapy, with several regimens approved by the regulatory authorities. The success of antibody–drug conjugates and research on other emerging approaches, such as vaccines and cell therapies, will also be addressed. These advances give hope on the development of personalized, more effective, and safe treatments, which will improve the survival and quality of life of patients with TNBC.

Breast cancer is the leading cause of cancer-related mortality in women, with triple-negative breast cancer (TNBC) being an aggressive subtype associated with poor prognosis and limited treatment options. TNBC is known for its immunogenic characteristics, including high genetic instability and elevated tumor-infiltrating lymphocytes (TILs). Immune checkpoint inhibitors (ICIs) have shown efficacy in TNBC treatment, but the optimal treatment duration in case of prolonged response remains unclear. This case series here reported presents two patients with metastatic TNBC who demonstrated an excellent response to ICI therapy. The first patient, a 60-year-old, was enrolled in a Phase I clinical trial and received a combination of anti-PD-1, anti-LAG-3, and anti-CSF-1 monoclonal antibodies. The second patient, a 45-year-old with BRCA1-mutated TNBC, participated in a Phase II trial and received a combination of avelumab (anti-PD-L1) and talazoparib (PARP inhibitor). Both patients achieved a complete radiological response (CR), which has been maintained for 5 years. A literature review performed here identified seven additional long-term ICI responders in metastatic TNBC. While ICIs showed significant efficacy in some patients, variability in PD-L1 expression and TILs suggests that other factors may influence response. Two patients in previous studies discontinued ICIs after 2 years without progression, prompting questions about the optimal treatment duration. ICIs optimal treatment duration remains uncertain. Literature on metastatic melanoma suggests that discontinuing ICIs after a complete response rarely leads to recurrence. Prospective studies and emerging biomarkers, such as circulating tumor DNA, may help tailor treatment decisions.

Background: Triple-negative breast cancer (TNBC), a very aggressive breast cancer subtype, exhibits high variability at both molecular and clinical aspects. Loss of function (LOF) germline mutation in BRCA1 has a very close association with this subtype of breast cancer. BRCA1 impairment is associated with homologous recombination deficiency (HRD) and tumor with HRD may benefit from treatment with drugs that induces DNA damage and also with PARP1 inhibitors. We previously detected that a significant fraction of TNBC diagnosed in young Brazilian women exhibits BRCA1 impairment by mechanisms including germline pathogenic mutation and promoter hypermethylation and that this group of tumors presented better overall survival. In the current study our aim is to comprehensively characterize the resistance to chemotherapy (DNA-damage agents) in patients with Hereditary or Sporadic TNBC by investigating somatic mutations in circulating tumor DNA (ctDNA). Methods: A series of 34 TNBC patients were subjected to germline genetic testing using a 26- or 96-gene panel, including homologous recombination-related genes, for classifying the TNBC as Hereditary or Sporadic. Tumor mutation burden (TMB) analysis was assessed in 20 cases using a panel containing 409 cancer-associated genes. Tumor somatic mutations were detected by evaluating DNA from tumor biopsies (Formalin-fixed, paraffin-embedded tissue) and matched leucocyte and were screened in cell-free DNA from 6 serial plasma samples during neoadjuvant treatment and for 6 months after surgery. Results: Pathogenic germline mutations were identified in BRCA1 (17.6% - 6/34), BRCA2 (2.9% -1/34) and TP53 (2.9% - 1/34) genes. Additionally, variants of uncertain significance (VUS) were identified in 21 patients (61.7% - 21/34), being ATM, BUB1B and PMS2 the most affected genes by VUS. Based on the 34 patients that underwent pre-operative chemotherapy and surgery, 47% exhibited pathological complete response (pCR), 50% in hereditary and 48% in sporadic. In terms of somatic variants, tumor mutation burden (TMB) analysis showed that 20% had high and 80% low TMB, with no association with hereditary or sporadic status. Also, we found, on average, three somatic variants per tumor (range 0-7) and used them as tumor marks in the screening of ctDNA in plasma. Somatic mutations in TP53 were identified in 80% (16 of 21) tumor biopsy samples investigated. In DNA from plasma before treatment, confident detection of at least one tumor mutation (ctDNA) was observed in 60% of patients (10 of 16). Persistent ctDNA detection in plasma during treatment was observed in 6 out 10 (60%) patients with residual disease after surgery and only in 1 out of 6 (16%) in patients with pCR. For 3 patients persistent ctDNA was detected after surgery at increasing levels showing clear anticipation of disease progression or metastasis. Serial plasma ctDNA samples showed great association with the clinical response data suggesting that the chemotherapy-resistance mechanisms can be investigated by ctDNA in TNBC. Conclusions: Germline and somatic evaluation of TNBC can provide valuable information for improving the clinical management of patients. For somatic evaluation, development of gene panels optimized for FFPE samples and low DNA input is enabling a comprehensive detection of potentially clinically-actionable somatic variants with better success rate in cancer treatment. Although still challenging, investigation of somatic variants in ctDNA from plasma samples is feasible and presents huge potential in monitoring patients during chemotherapy, especially regarding both the presence of residual disease and monitoring of disease progression. Citation Format: Dirce M Carraro, Rafael C Brianese, Giovana T Torrezan, Marina de Brot, Claudia AA de Paula, Maria NC Formiga, Solange M Sanches, Vladmir CC de Lima, Fabiana BA Makdissi. Circulating tumor DNA (ctDNA) analysis for investigating resistance to chemotherapy with DNA-damage agents in patients with hereditary or sporadic triple-negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-19.

Background: The KEYNOTE-522 (KN-522) regimen [chemotherapy (CT) + immune checkpoint inhibitor (ICI)] is the treatment of choice for patients with triple-negative breast cancer (TNBC) in the neoadjuvant setting. Addition of ICI could be associated with life-threatening immune related adverse events (irAEs). There is an unmet need to identify a subgroup of TNBC patients who would benefit from ICI, while sparing its use in the majority where CT alone may be sufficient. Our study aimed to compare the pathological complete response (pCR) rate between two matched cohorts (CT + ICI vs. CT alone), identify clinicopathologic variables that would predict benefit from ICI and study association of irAE with pCR. Methods: Patients treated with CT + ICI (n=128) were 1:1 matched to patients treated with CT alone (n=128) on age range (10 years), race, clinical-AJCC stage, and histologic type [metaplastic vs. no special type] from four US academic institutions. Additionally, data on Nottingham grade, degree of necrosis, and percentage of tumor infiltrating lymphocytes (TILs) was collected from pre-treatment core needle biopsy (CNB). To identify the clinicopathologic features of patients who could benefit from ICI, multivariable logistic regression models were used. Data on incidence of any grade irAEs was collected and its association with pCR was also analyzed using multivariable logistic regression models. Results: pCR was achieved in 64/128 (50%) patients treated with CT + ICI vs. 46/128 (35.9%) treated with CT alone (p = 0.02). The optimal threshold to predict pCR for TILs was 30% for both CT + ICI and CT alone groups, with AUC of 0.78 and 0.73, respectively (p<0.001 for both). In the CT group, lower TILs in CNB, non-Black race, metaplastic histology and higher degree of necrosis were associated with non-pCR; where the corresponding ROC curve had an AUC of 0.83. Patients who received CT were categorized into quartiles (Q) based on the predicted risk of non-pCR (Q1 representing the lowest risk, Q4 representing the highest risk of non-pCR (34/128 or 26.6%), and Q2 and Q3 representing intermediate levels of risk). In the Q4, the pCR rate with CT alone was only 2.9% (1/46) while it was 20% (6/64) in the CT + IT cohort (p=0.044). The following variables were associated with Q4 vs. Q1-3: non-Black race (96.9% vs. 88%, p=0.04), metaplastic histology (29.7% vs. 1.6%, p<0.01), lymph node (LN) positive disease (54.6% vs. 50%, p<0.01), Nottingham grade 2 (32.8% vs. 6.3%, p<0.01), and lower mean TILs (12.8 +/- 15.0 vs. 36.7 +/- 26.5, p<0.01). The incidence of any grade irAEs was 32% (41/128) in patients treated with CT + ICI. Among patients with an irAE, 63.4% achieved pCR vs. 43.7% without irAE who achieved pCR (p=0.04). There was a higher likelihood of pCR in patients treated with CT + ICI who experienced irAE (OR=3.34, 95% CI 1.17-9.58) while controlling for age, AJCC stage, nottingham grade, race, TILs, and number of ICI doses. Conclusions: In our real-world multicenter study, the pCR rate with neoadjuvant CT + ICI was lower than that reported in KN-522, with a similar irAE rate. We identified a subgroup of patients with TNBC likely to benefit from addition of neoadjuvant ICI to CT: those with metaplastic histology, non-Black race, LN positive disease, nottingham grade 2, or low TILs (<30%). Occurrence of any irAE was associated with increased likelihood of achieving pCR. Our results warrant validation in larger cohorts. Citation Format: Shipra Gandhi, Gary Tozbikian, Oluwole Fadare, Samira Syed, Briana To, Dionisia Quiroga, Song Yao, Kristopher Attwood, Yisheng Fang, Thaer Khoury. Clinicopathological features predictive of neoadjuvant immune checkpoint inhibitor benefit in triple-negative breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P2-11-11.

Context: Triple negative breast cancers (TNBC) constitute about 15% of breast neoplasms. In contrast to estrogen receptor (ER) or human epidermal growth factor receptor (HER2) positive breast cancers, which respond to hormonal therapy (such as tamoxifen) or anti-HER2 therapy (such as trastuzumab), respectively, the main standard therapy in either early or late stage TNBC is chemotherapy. Therefore, it is necessary to find new treatment modalities for TNBC patients. We searched the literature to find published studies on immunotherapy in triple negative breast cancer and the putative biomarkers of response to these treatments. Evidence Acquisition: We searched PubMed, Scopus, and Web of Science Core Collection with these keywords: “Triple negative breast cancer, Immunotherapy, Resistance, Response, Programmed cell death 1 receptor, CTLA-4, Tumor mutation burden, and Immune signature”. Results: TNBC is considered a heterogeneous neoplasm with regard to molecular aberrations. Analysis of genomic expression profile of TNBC has delineated 4 subtypes. TNBC tumors show high genetic instability. Tumor infiltrating lymphocytes (TILs) are detected more in TNBCs, compared to other breast cancer types. It has been shown that the amount of CD8 positive T cells in TNBCs is an independent predictor of overall survival. Up to now, two immunotherapy strategies have been used in clinical trials of TNBC, including immune checkpoint blockers and therapeutic cancer vaccines. Tumor programmed cell death ligand 1 (PDL1) expression is the most widely used immunotherapy biomarker. Tumor mutation burden (TMB) can be a promising biomarker of response to immunotherapy. The more somatic mutations a cancer cell has, the more neoantigens it probably produces. Analysis of TMB can give an estimate of tumor mutation load. Increased somatic mutation load has also been observed in tumors with impaired mismatch repair (MMR). Conclusions: As TNBC is regarded a heterogeneous disease, the discovery of biomarkers of response to immunotherapy will increase the likelihood of response to these therapies. Further in-depth investigations are needed to find novel biomarkers of response to these immunotherapies for the better management of patients with TNBC.

Background: Genomic mechanisms associated with response to ICI in mTNBC are largely unknown. The aim of this work is to assess the genomic and immune profiles of mTNBC samples collected from patients (pts) treated with ICI. Methods: We identified 31 women with mTNBC treated with ICI (pembrolizumab, n=6, NCT02447003; atezolizumab, n=4, NCT01375842; nivolumab + cabozantinib, n = 6, NCT03316586; pembrolizumab + eribulin, n=8, NCT02513472; atezolizumab + nab-paclitaxel, n=7, NCT01633970) who had tumor tissue or blood available for sequencing obtained before and after ICI. Clinical benefit (CB), here defined as any objective response or stable disease (SD) for > 24 weeks, was observed in 20 pts (65%). An extraordinary responder was defined as having CB ≥ 2 yrs; 5 pts were considered extraordinary responders (range 26-60months). Whole exome sequencing (WES) was done on each tumor and on germline DNA from blood (23 pts had successful WES performed on samples collected before ICI; 5 of these had WES on samples taken after disease progression). RNA sequencing (RNAseq) was successfully performed in 18 of the tumors with WES performed on samples before ICI; and 3 of these had RNAseq on samples taken after disease progression. 18 pts had tumors assessed by multiplex immunofluorescence (mIF) panels encompassing CD4, CD8, PD-1, PD-L1, and cytokeratin on samples collected before ICI. WES, deep targeted panel and low coverage whole genome sequencing were performed on serially collected plasma samples from 22 pts to evaluate tumor fraction and specific mutations. The association between biomarkers and clinical benefit to ICI was assessed. Results: 21 of 31 pts (67%) had received ≥1 prior lines of systemic therapy in the metastatic setting before starting ICI. Among the most frequently mutated genes at baseline are: TP53 (57%); PIK3CA (18%); DNAH5, MYH8 (both 13%); KMT2C, AKT1, LAMA2 (all 9%). Pts with CB had a higher tumor mutational burden (TMB) than pts with no CB (p=0.018). Differential expression analysis of RNAseq data revealed an upregulation of several immune-related genes in pts with CB, indicating increased immune infiltration in that group. Gene set enrichment analysis of this expression data using hallmark and canonical pathway gene sets from MSigDB (nominal p-val < 0.05) showed that, compared to samples from pts without CB, extraordinary responders had elevated transcriptional signatures of several cancer-related pathways associated with cell survival, proliferation and metabolism, as well as genes associated with increased immune infiltration and upregulation of inflammatory response programs. The mIF showed that the tumor microenvironment (TME) of pts with CB were enriched in Cytokeratin-negative/PD-L1-positive cells compared to those without CB (p=0.014). Expression of CD4, CD8 and PD-1 was not significantly different between pts with and without CB. Genomic analysis of circulating tumor DNA, and tumor evolutionary analysis for pts with both pre- and post-ICI samples (acquired resistance) will be presented. Conclusions: Clinical benefit to ICI in mTNBC was associated with upregulation of immune-related pathways, enrichment of non-tumoral PD-L1-positive cells in TME, and high TMB. Citation Format: Romualdo Barroso-Sousa, Juliet Forman, Zachary T. Weber, Katherine Collier, Katrina Z. Kao, Edward T. Richardson, III, Tanya Keenan, Ofir Cohen, Michael P. Manos, Ryan C. Brennick, Patrick Ott, F. Steve Hodi, Deborah A. Dillon, Nancy U. Lin, Eliezer E. Van Allen, Scott Rodig, Eric P. Winer, Elizabeth A. Mittendorf, Catherine J. Wu, Daniel Stover, Nikhil Wagle, Sachet Shukla, Sara Tolaney. Comprehensive genomic analysis reveals molecular correlates of response to immune checkpoint inhibitors (ICI) in metastatic triple-negative breast cancer (mTNBC) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-25.