Abstract OT1-08-01: A phase Ib trial of sequential combinations of BN-brachyury, entinostat, ado-trastuzumab emtansine (T-DM1) and bintrafusp alfa (M7824) in advanced stage breast cancer (BrEAsT)

Background Breast cancer (BC) is considered to be the second highest cause of cancer-related death in women, accounting for 15% of all female cancer-related deaths globally, despite major advancements in early detection and treatment methods. Traditionally, BC has not been considered as an immunogenic tumour, especially when compared to other solid tumours like lung or melanoma. However, a rapidly expanding body of evidence indicates significant immunogenic variability among various BC subtypes, with hormone receptor-negative BC being typically more immunogenic than hormone receptor-positive cancers. Programmed death ligand-1 (PD-L1) is a transmembrane protein, found in a variety of normal tissue cells and also in a wide range of epithelial malignancies, including breast carcinoma. Binding of PD-L1, expressed on tumor cells, to its receptor PD-1, expressed on activated T cells and B cells, disrupts effector immune functions. Antibodies targeting PD-L1 have been approved for treating a wide range of malignancies including breast cancer especially unresectable and metastatic triple negative breast cancer (TNBC). However, PD-L1 expression and its prognostic role in BC is still the target of several researches in order to maximize its therapeutic role in different clinicopathological settings. Objectives to evaluate immunohistochemical expression of PD-L1 in tumour cells and tumour infiltrating lymphocytes (TILs) in different types of breast carcinoma and to correlate between PD-L 1 expression and clinicopathological variables. Material and Methods Ninety cases of breast carcinoma mastectomy specimens were collected and stained immunohistochemically for PD-L1. PD-L1 expression was evaluated in TCs and TILs in the two setting of cases; (Group A) in which the patients didn’t receive preoperative neoadjuvant chemotherapy (NAC) and (Group B) in which the patients received preoperative NAC. The expression of PD-L1 was correlated with clinicopathological parameters. Survival analysis was conducted to correlate disease-free survival (DFS) with PD-L1 expression. Results In cases of Group A (Those didn’t receive preoperative NAC), (31.1%) of cases showed PD-L1 expression by tumor cells [PD-L1 (TCs)] and (47.5%) showed PD-L1 expression by tumor infiltrating lymphocytes [PD-L1 (TILs)]. PD-L1 (TCs) and PD-L1 (TILs) expression were significantly associated with histologic type of invasive duct carcinoma, NST, grade 2, presence of carcinoma in situ, negative hormone receptors, high proliferation index (Ki-67), molecular subtype of TNBC and high level of TILs. In cases received preoperative NAC (Group B), (13.8%) of cases showed PD-L1 expression by tumor cells and (41.4%) showed PD- L1 expression by tumor infiltrating lymphocytes. PD-L1 (TCs) expression was significantly associated with high level of TILs. While PD-L1 (TILs) expression was significantly associated with presence of carcinoma in situ, negative hormone receptors, high proliferation index (Ki-67) and molecular subtype of TNBC. The correlation between PD-L1 expression in TCs and TILs and DFS was statistically insignificant. Conclusion PD-L1 immunohistochemical expression in breast carcinoma is much more prevalent in TILs than TCs and both PD-L1 (TCs) and PD- L1 (TILs) are significantly associated with high proliferation index (K i-67), non-luminal subtypes (TNBC and HER2 Positive) and high TILs level, suggesting that they may be important candidates for anti-PD- 1/PD-L1 therapy.

Background: Programmed cell death-ligand 1 (PD-L1) is of great interest in breast cancer (BC). PD-L1 IHC 22C3 pharmDx (SK006) is an approved companion diagnostic assay for patients with triple-negative breast cancer (TNBC). Multiple sub-types of BC are identified by the binary expression status (positive/negative) of human epidermal growth factor 2 (HER2) and the hormone receptors (HRs) estrogen receptor (ER) and progesterone receptor (PR). The ability of the assay to reliably identify PD-L1 expression in TNBC (ER-/PR-/HER2-) tissues was previously demonstrated. However, the sensitivity of the assay on HR+/HER2- specimens (including ER+/PR+, ER+/PR-, and ER-/PR+; non-TNBC) is not as well-known. The data presented here demonstrates the ability of the assay to reliably identify the dynamic range of PD-L1 expression on non-TNBC specimens when assessed using the Combined Positive Score (CPS). Methods: Primary and metastatic BC specimens, sourced from multiple anatomical sites, were prepared and stained according to the assay’s Instructions for Use (IFU). The PD-L1 IHC 22C3 pharmDx immunohistochemical assay uses Monoclonal Mouse Anti-PD-L1, Clone 22C3, for detection of PD-L1 protein in formalin-fixed, paraffin-embedded tissues. Stained sections were scored using CPS, which is the number of PD-L1 staining cells (tumor cells, lymphocytes, macrophages) divided by the total number of viable tumor cells, multiplied by 100. CPS totals are displayed as integers with a maximum CPS of 100. Additional cut sections from the same specimens were also prepared and stained using Agilent ER, PR and HER2 reagents, where positivity or status was based upon the IFU for each reagent. Each specimen was then grouped into non-TNBC or TNBC sub-types. Prevalence was assessed to demonstrate that the assay will detect the target molecule (PD-L1 protein) in BC specimens across the dynamic range of PD-L1 expression (CPS 0–100). For the purposes of this abstract, scores were reported according to the CPS ≥ 1 and CPS ≥ 10 cutoffs. Results: Prevalence data of Agilent’s internal BC tissue bank was evaluated. At the time of the assessment, two categories of BC specimens and their totals were grouped: 149 non-TNBC and 184 TNBC. When examining the CPS ranges for the non-TNBC specimens, 55/149 (36.9%) were found to have a CPS ≥ 1 and 17/149 (11.4%) had a CPS ≥ 10. When examining the CPS for the TNBC specimens, 115/184 (62.5%) were found to have a CPS ≥ 1 and 57/184 (31.0%) had a CPS ≥ 10. For all specimens of each cohort (non-TNBC and TNBC), the assay identified PD-L1 expression across the dynamic range. This analysis indicates that the dynamic range of PD-L1 expression is present across all BC tissues regardless of HR status, though a higher prevalence is noted in TNBC specimens at the CPS ≥ 1 cutoff. Conclusions: Overall, the assay detected PD-L1 protein across the dynamic expression range (CPS 0–100) for both non-TNBC and TNBC specimens. In this small specimen cohort, retrospective analysis demonstrated that HR status may correlate to a difference in PD-L1 prevalence in HER2- breast cancer specimens. This analysis further demonstrated that PD-L1 IHC 22C3 pharmDx can identify PD-L1 expression in the dynamic range within non-TNBC specimens, specifically. Further studies should be conducted within different combinations of HR status and PD-L1 prevalence to expand on this data, to address gaps in knowledge, and to learn if HR status may inform PD-L1 immune checkpoint inhibitor therapy benefit for BC patients. Citation Format: Joseph Barreto, Jaret Quiroz, Emily Olander, Donna Kell, Siena Tabuena-Frolli, Camilla Recke, Kelly Martyniuk. Hormone receptor status and PD-L1 expression prevalence in breast cancer when assessed with PD-L1 IHC 22C3 pharmDx and Combined Positive Score [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 P3-03-18.

Recent and emerging data have generated great excitement about manipulating the patient’s own immune system to attack and eliminate malignant tumors. Although the majority of success has not been in breast cancer, recent studies have increased enthusiasm about this therapeutic modality. The immune system plays a complex role in cancer biology involving both in promotion and eradication of disease. A series of immune checkpoints are hardwired into the immune system in order to modulate the duration and amplitude of the immune response, reducing collateral normal tissue damage. These immune checkpoint pathways serve as a mechanism for tumor immune invasion, particularly against T-cells generated against tumor antigens. Recently several monoclonal antibodies specifically targeted at blocking immune checkpoint receptor-ligand interactions have demonstrated unprecedented anti-tumor responses in a wide variety of immunogenic tumors that has led to U.S. FDA approval of a variety of agents, including those blocking the programmed cell death protein 1 (PD1) receptor and its ligand programmed cell death-ligand 1 (PD-L1). Unlike many solid tumors, breast cancer has not typically been responsive to modulation of the immune system. The success of checkpoint inhibitors to date has generally relied on the immunogenicity and high mutational burden which is thought to be important in generating an antitumor immune response to neoantigens that can then be reactivated by immune checkpoint inhibitors. In breast cancer, proliferative subsets such as TNBC and HER2+ disease have a higher mutational burden, and could be more amenable to immunotherapy. Higher levels of tumor infiltrating lymphocytes (TILs) are found in TNBC and HER2 positive breast cancer and have been associated with improved outcome in both early and late stage disease settings. Expression of PD-L1 has been shown to be associated with lack of ER expression, higher TIL counts and the triple negative phenotype. Chemotherapy may increase mutational burden or expression of neoantigens through cell death, potentially increasing tumor immunogenicity and making combination studies very appealing. Four phase I monotherapy expansion trials in patients with metastatic breast cancer have demonstrated low but durable single agent responses to PD-1 and PD-L1 inhibitors, ranging from 4.8 to 19%. Two trials included patients with TNBC, one trial included patients with any breast cancer subtype, and one trial included only patients with HR+ disease. Higher response rates are seen in TNBC, compared to hormone receptor positive disease. Variability in requirements for tumor PD-L1 expression, and variations in testing complicate cross trial comparisons. Two large single agent trials enrolling over 100 patients have recently been presented evaluating either atezolizumab or pembrolizumab as single agent therapy for TNBC. Response rates ranged from 5 to 10%, but in both trials the response rate was much higher in patients treated in the first line setting at 23-26%, potentially due to multiple mechanisms of resistance in the second or greater line setting and bringing up the question of clinical trial design. Although rare durable responses have been seen, it is clear that combination therapy is a critical next step in the study of immunotherapy for breast cancer. Both pre-clinical and clinical data suggest that chemotherapy can enhance the host immune response, acting as an ‘immune agonist.’ A 5th phase Ib trial reported a 38% response rate in metastatic TNBC treated with atezolizumab and nab-paclitaxel in combination. Several phase III trials combining immune checkpoint inhibitors with chemotherapy as first line therapy for metastatic TNBC are ongoing, as well as a number of adjuvant and post-neoadjuvant trials. One small trial suggested that response to checkpoint inhibition might be improved with induction therapy with either a short course of radiation or chemotherapy. Additional studies are evaluating combinations with immune agonists. Given data suggesting improved response in less heavily pretreated disease, there has been great interest in combining immune check-point inhibition with chemotherapy as neoadjuvant therapy for high risk breast cancer. Recent data from the multi-arm I-SPY2 phase II neoadjuvant trial demonstrated a marked and significant improvement in estimated pathologic complete remission when pembrolizumab was added to weekly paclitaxel before anthracyclines in both triple negative and hormone receptor positive disease. Longer exposure to pembrolizumab is currently being evaluated. Several randomized trials in the neoadjuvant setting are planned or ongoing as well. Treatment with checkpoint inhibitors is generally well tolerated, with low rates of immune toxicity including hypothyroidism, pneumonitis, hepatitis, colitis, and hypophysitis, occurring even months after the end of therapy. In the neoadjuvant I-SPY2 trial, higher rates of adrenal insufficiency were noted, although these responded quickly to treatment. Further evaluation is needed to understand this difference. Ongoing studies are evaluating combinations with chemotherapy, as well as a number of other targeted agents such as immune agonists, MEK inhibitors, HDAC inhibitors, HER2 targeted therapy and cyclin dependent kinase 4/6 inhibitors. 1.Mittendorf EA, Philips AV, Meric-Bernstam F, et al. PD-L1 Expression in Triple-Negative Breast Cancer. Cancer Immunol Res. 2014;2;361. 2.Chen G, Emens LA. Chemoimmunotherapy: reengineering tumor immunity. Cancer Immunol Immunother. 2013;62(2):203-216. 3.Nanda R, Chow LQ, Dees EC, et al. Pembrolizumab in Patients With Advanced Triple-Negative Breast Cancer: Phase Ib KEYNOTE-012 Study. J Clin Oncol. 2016;34(21):2460-2467. 4.Dirix LY, Takacs I, Nikolinakos P, et al. Avelumab (MSB0010718C), an anti-PD-L1 antibody, in patients with locally advanced or metastatic breast cancer: A phase Ib JAVELIN solid tumor trial. SABCS. 2015. 5.Rugo HS, Delord JP, Im SA, et al. Preliminary efficacy and safety of pembrolizumab (MK-3475) in patients with PD-L1–positive, estrogen receptor-positive (ER+)/HER2-negative advanced breast cancer enrolled in KEYNOTE-028. Cancer Res 2016;76 (4 suppl):abstr S5-07. 6.Emens LA, Braiteh FS, Cassier P, et al. Inhibition of PD-L1 by MPDL3280A leads to clinical activity in patients with metastatic triple-negative breast cancer. SABCS. 2014:PD1-6. 7.Schmid P, Cruz C, Braiteh FS, et al. Atezolizumab in metastatic TNBC (mTNBC): Long-term clinical outcomes and biomarker analyses. Proc AACR 2017:Abst. 2. 8.Adams S, Schmid P, Rugo HS, et al. Phase 2 study of pembrolizumab (pembro) monotherapy for previously treated metastatic triple-negative breast cancer (mTNBC): KEYNOTE-086 cohort A. JCO. 2017;35, (no. 15_suppl):1008. 9.Adams S, Diamond JR, Hamilton EP, et al. Phase Ib trial of atezolizumab in combination with nab-paclitaxel in patients with metastatic triple-negative breast cancer (mTNBC). JCO. 2016;34, (no. 15_suppl):1009. 10.Nanda R, Liu MC, Yau C, et al. Pembrolizumab plus standard neoadjuvant therapy for high-risk breast cancer (BC): Results from I-SPY 2. J Clin Oncol 2017;35: abstr 506. Citation Format: Hope S. Rugo. Setting the stage for immunotherapy in breast cancer: where are we and where are we going in the clinic? [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr IA24.

Background: Approximately 10%-20% of breast cancers (BCs) are classified as triple-negative breast cancer (TNBC). TNBC lacks targeted treatment options and has a poor prognosis. Bintrafusp alfa (M7824) is an innovative first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor (a TGF-β “trap”) fused to a human IgG1 mAb blocking PD-L1. We report interim results in patients with advanced TNBC treated with bintrafusp alfa in an expansion cohort of an ongoing, open-label, phase 1 trial (NCT02517398). Methods: Eligible patients had confirmed TNBC (ER: IHC <1%; PR: IHC <1%; HER2: FISH non-amplified, IHC 0/1+, or IHC 2+ and FISH non-amplified) that progressed during or after first-line chemotherapy with an ECOG performance status of ≤1 and measurable disease by RECIST 1.1. Prior treatment with immune checkpoint inhibitors was not permitted. Patients received bintrafusp alfa 1200 mg every 2 weeks until confirmed progression, unacceptable toxicity, or trial withdrawal. The primary endpoint was confirmed best overall response (BOR) as assessed by independent review committee (IRC) according to RECIST 1.1. Key secondary endpoints included investigator-assessed BOR and safety. Exploratory endpoints included an extensive integrated biomarker evaluation of patient samples. These data are presented in a separate, submitted abstract. Results: As of August 24, 2018, 33 patients with heavily pretreated TNBC (54.5% of patients had ≥4 prior regimens) were enrolled in this study of bintrafusp alfa. Patients received a median of 3 doses of bintrafusp alfa (range, 1-24). Confirmed responses as assessed by IRC and investigator occurred in 3 patients (1 complete response and 2 partial responses as assessed by IRC; ORR, 9.1% [95% CI, 1.9%-24.3%]); disease control was achieved in a total of 5 patients (15.2% [95% CI, 5.1%-31.9%]) by both IRC and investigator read. The median progression-free survival per IRC was 1.3 months (95% CI, 1.2-1.4 months), and the median overall survival was 7.8 months (95% CI, 2.1-12.8 months). Importantly, a biomarker related to TGF-β biology was identified via RNAseq analysis of tumor samples with 32.0-fold higher expression in patients who experienced disease control (response or stable disease) compared with those who had progressive disease (biomarker results for this expansion cohort are presented in a separate abstract). PD-L1 expression was not associated with response to treatment. In the entire cohort, 6 patients (18.2%) experienced ≥1 grade 3 treatment-related adverse event (TRAE), including anemia (n=3), asthenia, decreased appetite, generalized rash, hypophysitis, and increased transaminases (all n=1). No grade 4 TRAEs were reported. Three patients discontinued treatment due to TRAEs, including 1 death that was assessed by the investigator as related to treatment. Dyspnea, hemolysis, and thrombocytopenia were reported as grade 5 TRAEs in this patient, who had extensive disease at trial entry and was noted to have multiple pulmonary emboli, progressive disease, and expanding pleural effusion after 3 doses. No autoantibodies mediating hemolysis or thrombocytopenia were identified on workup. Conclusions: In summary, bintrafusp alfa was well tolerated, with a safety profile consistent with expectations in this heavily pretreated, advanced TNBC cohort. We identified high expression of a potential predictive biomarker of response in patients with advanced TNBC treated with bintrafusp alfa. Detailed biomarker data are presented in the separate abstract and warrant further investigation of bintrafusp alfa in TNBC. Citation Format: Alexander Spira, Ahmad Awada, Nicolas Isambert, David Lorente Estellés, John Nemunaitis, Nicolas Penel, Laureen S Ojalvo, Christoph Helwig, Christian Borel. Bintrafusp alfa (M7824), a bifunctional fusion protein targeting transforming growth factor-β and programmed death ligand 1, in advanced triple-negative breast cancer: Preliminary results from a phase 1 cohort [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 P3-09-06.

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer. Identification of biomarkers that are predictive of response to investigational therapies will help identify patients that are more likely to clinically benefit from treatment. Bintrafusp alfa (M7824) is an innovative first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor (a TGF-β “trap”) fused to a human IgG1 mAb blocking PD-L1. In this expansion cohort of a global, phase 1, open-label trial (NCT02517398), an extensive tumor biomarker analysis was conducted to identify potential markers correlating with efficacy in patients with advanced TNBC treated with bintrafusp alfa. Methods: Patients received infusions of bintrafusp alfa 1200 mg every 2 weeks until confirmed progression, unacceptable toxicity, or trial withdrawal. Tumor archival material was collected from each patient for biomarker analysis. All biomarker analyses were exploratory. PD-L1 expression and immune phenotype were determined by immunohistochemistry. Tumor samples were also processed for RNA sequencing (RNAseq) and resulting data were analyzed to identify potential associations between gene expression and efficacy of bintrafusp alfa treatment. Results: As of August 24, 2018, 33 patients with heavily pretreated TNBC were treated with bintrafusp alfa (54.5% of patients received ≥4 prior regimens). The objective response rate was 9.1% (n=3) and the disease control rate was 15.2% in a total of 5 patients. PD-L1 expression levels in tumor cells and in the tumor microenvironment were generally low in this cohort with the majority of patients having 0% PD-L1+ tumor cells and/or ≤10% PD-L1+ immune cells in their tumor microenvironment. Moreover, several patients had an immune desert phenotype. PD-L1 expression was not associated with response to treatment. Importantly, exploratory analysis of RNAseq data (n=26 passing quality control) identified differential expression of the high mobility group AT-hook 2 (HMGA2) gene with 32.0-fold higher expression (as computed by DESeq2; q=2.23e−13) in patients who experienced disease control (response or stable disease) compared to those who had progressive disease. HMGA2 is a transcriptional regulator whose expression is upregulated by TGF-β signaling. Furthermore, HMGA2 is known to be an important factor in mediating TGF-β-induced epithelial-mesenchymal transition. In The Cancer Genome Atlas dataset, approximately 12% of subjects annotated as TNBC have high expression of HMGA2. Clinical efficacy and safety data for this expansion cohort are presented in a separate abstract. Conclusions: We identified high expression of HMGA2 as a potential predictive biomarker of response in TNBC patients treated with bintrafusp alfa. HMGA2 assay development is on-going to confirm this finding in a future clinical trial. Citation Format: George Locke, Yue Zhang, Laureen S Ojalvo, Christoph Helwig, Alex Rolfe, Olaf Christensen, Isabelle Dussault. Identification of a tumor biomarker in advanced triple-negative breast cancer that predicts response to bintrafusp alfa (M7824), a bifunctional fusion protein targeting transforming growth factor-β and programmed death ligand 1 [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 P3-09-13.

Breast tumors commonly harbor low mutational burden, low PD-L1 expression, defective antigen processing/presentation, and an immunosuppressive tumor microenvironment (TME). In a malignancy mostly refractory to checkpoint blockade, there is an unmet clinical need for novel combination approaches that increase tumor immune infiltration and tumor control. Preclinical data have guided the development of this clinical trial combining 1) BN-Brachyury (a poxvirus vaccine platform encoding the tumor associated antigen brachyury), 2) bintrafusp alfa (a bifunctional protein composed of the extracellular domain of the TGF-βRII receptor (TGFβ “trap”) fused to a human IgG1 anti-PD-L1), 3), entinostat (a class I histone deacetylase inhibitor), and 4) T-DM1 (ado-trastuzumab emtansine, a standard of care antibody-drug conjugate targeting HER2). We hypothesize that this tetratherapy will induce a robust immune response against HER2+ breast cancer with improved response rates through 1) expanding tumor antigen-specific effector T cells, natural killer cells, and immunostimulatory dendritic cells, 2) improving antigen presentation, and 3) decreasing inhibitory cytokines, regulatory T cells, and myeloid-derived suppressor cells. In an orthotopic HER2+ murine breast cancer model, tetratherapy induced high levels of antigen-specific T cell responses, tumor CD8+ T cell/Treg ratio, and augmented the presence of IFNγ- or TNFα-producing CD8+ T cells and IFNγ/TNFα bifunctional CD8+ T cells with increased cytokine production. Similar effects were observed in tumor CD4+ effector T cells. Based on this data, a phase 1b clinical trial evaluating the stepwise addition of BN-Brachyury, bintrafusp alfa, T-DM1 and entinostat in advanced breast cancer was designed. Arm 1 (TNBC) receives BN-Brachyury + bintrafusp alfa. Arm 2 (HER2+) receives T-DM1 + BN-Brachyury + bintrafusp alfa. After safety is established in Arm 2, Arm 3 (HER2+) will receive T-DM1 + BN-Brachyury + bintrafusp alfa + entinostat. Reimaging will occur every 2 cycles (1 cycle = 21 days). Arms 2 and 3 undergo research biopsies at baseline and after 2 cycles to evaluate changes within the TME. Peripheral immune responses will be evaluated. Co-primary objectives are response rate and safety. All arms employ a safety assessment in the initial six patients and a 2-stage Simon design for clinical efficacy (Arm 1 if ≥ three responses of eight then expand to 13 patients; Arms 2 and 3 if ≥ four responses of 14 then expand to 19 patients per arm). Secondary objectives include progression-free survival and changes in tumor infiltrating lymphocytes. Exploratory analyses include changes in peripheral immune cells and cytokines. To our knowledge, the combination of a vaccine, an anti-PD-L1 antibody, entinostat, and T-DM1 has not been previously evaluated in the preclinical or clinical setting. This trial (NCT04296942) is open at the National Cancer Institute (Bethesda, MD).

e15262 Background: The clinical benefit of immune checkpoint inhibitors (ICIs) targeting the programmed death-1/programmed death ligand 1 (PD-L1) pathway has previously been demonstrated across a range of tumor types, including in PD-L1+ patients with metastatic triple-negative breast cancer (TNBC). Various PD-L1 immunohistochemistry (IHC) assays and scoring algorithms are being investigated to select patients with breast cancer (BC) most likely to respond to ICIs. Scoring algorithms include PD-L1 expression on tumor cells, immune cells (ICs), or both. We compared the analytical concordance of 3 PD-L1 IHC assays and evaluated PD-L1+ prevalence, using combined positive score (CPS) and % IC scoring algorithms in commercially procured TNBC and hormone receptor–positive, HER2-negative (HR+/HER2−) BC samples. Methods: PD-L1 expression was assessed by HistoGeneX (Naperville, IL) in 163 commercially procured, surgically resected, formalin-fixed, paraffin-embedded BC samples (mostly stage I–III) using the Ventana PD-L1 (SP142) and Dako PD-L1 IHC 28-8 and 22C3 pharmDx assays in conjunction with the CPS (28-8 and 22C3 assays) and % IC algorithms (SP142 and 28-8 assays). PD-L1+ prevalence with each assay and concordance between assays were calculated using CPS ≥ 1 and IC ≥ 1% cutoffs, with a single pathologist assigned to each scoring algorithm. Results: 93 HR+/HER2− BC and 70 TNBC samples were evaluable for PD-L1 expression across all assays and algorithms. Overall concordance was higher between the 28-8 and 22C3 assays (CPS cutoff of 1) than between the 28-8 and SP142 assays (IC cutoff of 1%). PD-L1+ prevalence was similar with the 28-8 and 22C3 assays (CPS ≥ 1) and higher with the 28-8 assay (IC ≥ 1%) than with the SP142 assay (IC ≥ 1%). PD-L1+ samples identified by the SP142 assay (IC ≥ 1%) were mostly included within PD-L1+ sample sets defined by the 28-8 assay (IC ≥ 1% and CPS ≥ 1). PD-L1+ prevalence was higher in TNBC vs HR+/HER2− BC for all assays (Table). No clear trend in PD-L1+ prevalence was observed across tumor grade and stage. Conclusions: High analytical concordance was observed between the 28-8 and 22C3 assays (CPS cutoff of 1) in both HR+/HER2− BC and TNBC samples. PD-L1+ prevalence varied according to IHC assay, scoring algorithm, and cutoff used. Further studies are needed to select the most appropriate PD-L1 assay and scoring algorithm for BC clinical trials. [Table: see text]

BackgroundInhibitors of programmed cell death 1 (PD-1)/programmed cell death ligand 1(PD-L1) checkpoint have been approved for metastatic triple negative breast cancer (mTNBC) in patients positive for PD-L1 expression. Negative results from the recent phase III trials (IMPassion131 and IMPassion132) have raises questions on the efficacy of PD-1/PD-L1 checkpoint inhibitors and the predictive value of PD-L1 expression. Here we attempt to systematically analyze the biomarker value of PD-L1 expression for predicting the response of PD-1/PD-L1 checkpoint inhibitors in mTNBC.Materials and methodsPubMed database was searched until Dec 2021 for studies evaluating PD-1/PD-L1 checkpoint inhibitors plus/minus chemotherapy in mTNBC. Outcome of interest included objective response rate (ORR), progression-free survival (PFS), and overall survival (OS). Review Manager (RevMan) version 5.4. was used for data-analysis.ResultsIn total, 20 clinical trials comprising 3962 mTNBC patients (ICT: 2665 (67%); CT: 1297 (33%) were included in this study. Overall ORR was 22% (95%CI, 14-30%) and significant improvement was observed for PD-L1+ patients (ORR 1.78 [95%CI, 1.45-2.19], p<0.00001) as compared to PD-L1- cohort. Pooled outcome also indicated a significant 1-year PFS and 2-year OS advantage for patients with PD-L1 expression (1-year PFS: ORR 1.39 [95%CI, 1.04-1.85], p=0.02; I2 = 0%; 2-year OS: (ORR 2.47 [95%CI, 1.30-4.69], p=0.006; I2 = 63%). Subgroup analysis indicated that PD-L1 expression can successfully predict tumor response and 2-year OS benefit in mTNBC patients regardless of the type of investigating agent, line of treatment administration, and to some extent the type of treatment. Biomarker ability of PD-L1 expression to predict 1-year PFS was slightly better with pembrolizumab (p=0.09) than atezolizumab (p=0.18), and significantly better when treatment was administered in the first-line setting (OR 1.38 [95%CI, 1.02-1.87], p=0.04) and chemotherapy was added (OR 1.38 [95%CI, 1.02-1.86], p=0.03). Immune-related toxicity of any grade and grade≥3 was 39% (95%CI, 26%-52%) and 10% (95%CI, 8%-13%), respectively.ConclusionsPD-L1 expression can predict objective response rate and 2-year OS in mTNBC patients receiving PD-1/PD-L1 checkpoint inhibitors. One-year PFS is also predicted in selected patients. PD-L1 expression can be a useful biomarker of efficacy of PD-1/PD-L1 checkpoint inhibitors in mTNBC.

PD-L1 (B7-H1) expression and the immune tumor microenvironment in primary and metastatic breast carcinomas

Purpose: Recent studies have shown that local immune environment revealed with programmed death 1(PD-1)/programmed death ligand 1(PD-L1) and tumor infiltrating lymphocytes(TIL) affects the tumor-growth and prognosis. In this study, we evaluated the tumor local immune environments using immunohistological staining for analysis of PD-1/PD-L1 expression and TIL of tumor local in operable early-stage breast cancer. Method: A total of 100 surgical specimens of stageI-III invasive breast carcinoma paraffinembedded between 1995 and 2005 were analyzed. Immunohistological staining for PD-1, PD-L1, PTEN, CD3, CD8, and CD163 were performed by the conventional PAP method. In addition, intratumoral and intrastromal TILs and macrophages were simultaneously stained by anti-CD3, CD8, CD163 antibodies and measured by 'Win ROOF' computer software (version 5.7, Mitani Corporation, Japan). Results: Intratumoral PD-1 expressed significantly higher in triple negative breast cancer (TNBC) compared to other subtype BC (p=0.0094), intratumoral and intrastromal CD3+ lymphocytes and CD163+ macrophages were also significantly higher in TNBC, respectively (CD3: p=0.0002; 0.0139 and CD163: p=0.0043; 0.0270). PTEN loss was also more frequently observed in TNBC (p=0.0475). In addition, after a median 5-year follow-up, patients of luminal A subtype with lower PD-L1 and PTEN expression showed better disease free survival (DFS) with a significant difference (p=0.0148, p=0.0475). Conclusion: Local expression of PD-1/PD-L1 antigens on tumor cells, CD3+ lymphocytes, CD163+ macrophages infiltration singnificantly increased in early-stage TNBC. PTEN expression on tumor local might be associated with DFS in patients with early-stage BC. Citation Format: Okabe M, Toh U, Iwakuma N, Mishima M, Kawahara A, Kage M, Itoh K, Akagi Y. Local immunologic environment related with tumor infiltrating lymphocytes (TIL) and PD-1/PD-L1 expression in early stage breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-08-16.

Background The programmed death ligand 1 (PD-L1) has recently emerged as a target immunotherapy in Triple-negative breast cancer (TNBC). However, the tumor-intrinsic role of PD-L1 and its pathway still needs to be fully clarified. Recently, a close association between CD73, PD-L1, cancer cell steaminess and epithelial-to-mesenchymal transition phenotypes is emerging. In addition, we have previously demonstrated that miR-320a and miR-145 target PD-L1, whereas miR-30 family is known to target CD73. In this study, we aimed at investigating the role of these miRNAs as prognostic and predictive dynamic biomarkers for immunotherapy in TNBC. Additionally, we investigated whether PD-L1 exerts cellular autonomic functions in TNBC beyond its role in the immune checkpoint. Results CD73 and PD-L1 expression was assessed in a panel of breast cancer lines and the non-malignant breast cells MCF-10. We showed a significantly higher CD73 and PD-L1 expression in MDA-MB-231 (TNBC, Basal B) than in MCF-10. Conversely, in MCF-7 cells (HR +, Luminal A), CD73 and PD-L1 expression is lower than in MCF-10. Furthermore, we showed that low PDL1 expression is correlated with high levels of miR-320a and miR-145, and CD73 expression is inversely related to miR-30 expression. These putative biomarkers were also validated in two patients with different PD-L1 status. From the analysis of the data obtained by real-time qRT-PCR, we found high plasma level of CD73 and low plasma level of miR-320a, miR-145 and miR-30 in the patient with PD-L1 positive BC. On contrary, in the patient with PD-L1-negative BC, we found low plasma level of CD73 and high plasma level of miR-320a, miR-145 and miR-30. Furthermore, to explore whether PD-L1 could also have an intrinsic role in tumor development and invasiveness, we used stably MDA–MB-231 PD-L1-silenced cells. We found that cell growth, colony formation, migration rate and the ability to form spheres are consistently reduced upon shRNA-mediated PDL1 silencing. In particular, we observed a reduction in the number, diameter and volume of the 3D spheres compared to the control cells. To further demonstrate the role of PD-L1, we treated the PDL1-high expression cells MDA-MB-231, PD-L1 silenced MDA-MB-231 clones, and PD-L1 low expression cells MCF-7 with Durvalumab, an anti-PD-L1. Interestingly, we observed that in PD-L1 silenced clones and MCF-7 cells, the sphere-forming ability was increased in presence of Durvalumab treatment. On the contrary, in MDA-MB-231, Durvalumab inhibited the sphere-forming ability. Conclusion Our data confirm that PD-L1 and CD73 are targets of miR-320a/miR-145 and miR-30 respectively. These could be potential predictive dynamic biomarkers for chemotherapy, ICIs and anti-CD73 therapeutic approach. Future validations of these biomarkers in an extensive series of TNBC patients are needed to support their use in clinical practice. Here, we further characterized the cellular autonomic role of PD-L1 in breast cancer and showed a differential role of basal PD-L1 expression in PD-L1 checkpoint inhibitors treatment efficacy. This suggests a potential role in monitoring PD-L1 expression indirect biomarkers (i.e. miR-320a, miR-145 and CD73) during ICIs treatment.

Background: Indoleamine 2,3-dioxygenase (IDO) is an immune checkpoint that has been shown to play a key immunomodulatory role in various solid tumors, including breast cancer (BC). Although increased IDO expression has been previously observed in some BC subtypes, mainly triple-negative BC (TNBC), the clinical relevance of this protein across the entire range of BC and its exact correlations with other immune checkpoints remain to be elucidated. We herein aimed to further investigate the differential expression patterns of IDO and programmed death-ligand 1 (PD-L1) in variable BC subtypes and in distinct compartments of breast cancer tissue, and to explore their potential associations with standard patient- and tumor-related clinicopathological parameters as well as prognosis. Methods: This was a retrospective multi-center cohort study of 150 female patients with BC. The clinicopathological parameters analyzed were retrieved from the medical records of patients while sections from archival formalin-fixed, paraffin-embedded (FFPE) tissue blocks were also obtained for the performance of immunohistochemistry. The expression of IDO and PD-L1 was evaluated separately on tumor cells (IDO/CA, PD-L1/CA), lymphocytes (IDO/L, PD-L1/L) and stromal cells (IDO/S, PD-L1/S) and the results were correlated with the remaining clinical and pathological features of patients, as well as with local recurrence, metastasis and survival. Results: The mean age of patients was 59.5 years (SD = 13.4 years). Positive expression of IDO/CA, IDO/L and IDO/S was found in 6%, 93.3% and 90.7% of tissue samples, respectively, while 4%, 11.2% and 6.7% of tumors were positive for PD-L1/CA, PD-L1/L and PD-L1/S, respectively. A significantly higher rate of positive IDO/CA expression was observed in triple-negative BC (TNBC) patients (p = 0.037). Positive expression of IDO-CA was also significantly associated with positivity for PD-L1/L and PD-L1/S (p = 0.001 and p = 0.015, respectively). Multivariable logistic regression analysis showed independent correlations between IDO/CA and IDO/L and the presence of invasive ductal carcinoma (IDC) (OR = 1.10; p = 0.026) and N1 status (OR = 10.93; p = 0.039), respectively, IDO/S and both N1 (OR = 14.64; p = 0.018) and positive HER2 status (OR = 6.11; p = 0.019), PD-L1/L and high Ki67 (OR = 7.96; p = 0.001) as well as negative ER (OR = 0.08; 0.003) and PR status (OR = 0.09; p = 0.002), PD-L1/S and both NST (no special type) histology (OR = 4.68; p = 0.032) and negative ER status (OR = 0.21; p = 0.044). No statistically significant associations were observed between the expression patterns of the examined biomarkers and recurrence, metastasis or survival. Conclusions: In our study, IDO expression on tumor cells was predominantly observed in TNBC and was found to correlate with PD-L1 expression in the lymphocytic and stromal compartments. Furthermore, expression of PD-L1 among lymphocytes was found to independently correlate with unfavorable clinicopathological parameters, including high proliferation rate and negative hormone receptor status.

BackgroundImmune checkpoint inhibitors play a vital role in triple-negative breast cancer (TNBC) immunotherapy. A recent study showed that chemokine-like factor (CKLF)-like MARVEL transmembrane domain containing 6 (CMTM6) has a crucial role in programmed death-ligand 1 (PD-L1) stability. The aim of this study was to investigate the relationship between CMTM6 and PD-L1 in TNBC and the association with clinical characteristics.MethodsA total of 143 patients, including 75 with human epidermal growth factor receptor 2 (HER2)-driven breast cancer and 68 with TNBC, were included in this study. In 83 paired primary breast cancers (PBCs) and metastatic breast cancers (MBC) comprising 45 HER2-driven breast cancers and 38 TNBC, CMTM6 and PD-L1 were detected based on immunohistochemistry (IHC) with FFPE tissues. Another 60 PBCs comprising 30 HER2-driven breast cancers and 30 TNBC in order to detect CMTM6 and PD-L1 mRNA expressions based on real-time polymerase chain reaction (RT-PCR) using frozen tissues. Furthermore, 153 patients comprising 30 TNBC and 123 HER2-driven breast cancer based on The Cancer Genome Atlas (TCGA) database were used to confirm the difference mRNA expression.ResultsThe expression of CMTM6 in patients with TNBC was significantly higher than in those with HER2-driven PBC (IHC, P=0.036, mRNA, P=0.036, TCGA dataset, P=0.039). CMTM6 was correlated with PD-L1 based on IHC in triple-negative MBC (P=0.004); the same result was found based on mRNA data in triple- negative PBC (P=0.021). Moreover, a high expression of CMTM6 in TNBC was associated with poor progression-free survival (PFS) (P=0.030, 95% CI: 1.08–4.57, HR =2.22). After multiple Cox regression analysis, CMTM6 in TNBC emerged as an independent risk factor for PFS (P=0.027, 95% CI: 1.11–5.20, HR =2.40). The expression of PD-L1 was negatively correlated with lymph node metastasis (P=0.026) and was not associated with PFS.ConclusionsThe expression of CMTM6 was higher in TNBC than in HER2-driven breast cancer. In TNBC, CMTM6 was correlated with PD-L1 expression, and potentially could be used as an independent risk factor for predicting PFS.

Background: Breast cancer (BC) evolution is influenced by tumor microenvironment. Presence of CD8+ cytotoxic T lymphocytes (TILs) has been proposed as surrogate marker of adaptive immune response, and programmed death ligand-1 (PD-L1) is a negative regulator of the tumor immune microenvironment. However, whether PD-L1 expression adversely affects breast cancer outcome is unknown (Oncotarget 2014; 6:5449). Tumor-associated macrophages (TAMs) in the tumor microenvironment may contribute to BC progression and metastagenicity. We assessed the potential correlations between PD-L1 expression, the presence of TAMs and TILs, and BC outcomes. Methods: 59 primary BCs (16 HR+, 16 HER2+, and 27 TNBC) with known clinical and pathological features and patient (pt) follow-up for a median of 3.9 years were evaluated by immunohistochemistry for expression of CD8, CD68, and PD-L1 within tumor and stroma. The average number of CD8+ cells within 10 high power fields was determined separately for invasive tumor cell nests and for stroma within each sample, and the median number of CD8+ cells within tumor vs stroma was calculated (Breast Cancer Res Treat 2011 128:703–711). Non-lymphocyte mononuclear cells in tumor and stroma were used in counting CD68+ TAMs. BCs were positive for CD8+ TILs (Cell Marque clone #C8144B) or CD68+ TAMs (Cell Marque clone #KP1) if the number of cells positive in the sample was greater than the median. PD-L1 (Dako 28-8 pharmDx) was positive if at least 1% of tumor cells expressed PD-L1. The log-rank was used to compare the survival and progression free survival between groups and Spearman's rank-order correlation tests were conducted to determine associations between CD8, CD68, and PD-L1. Results: 57% of TN, 26% of HER2+ and 13% of HR+ BCs expressed PD-L1 in tumor. TNBC pts received anthracycline/ taxane chemotherapy (62%) or taxane therapy alone (22%). HR+ and HER2+ pts received standard endocrine therapy and trastuzumab-based therapy. Stromal CD8+ TILs were associated with improved OS in the overall population (log rank p=0.026). In TNBC, PD-L1 expression was positively correlated with the presence of TILs (p=0.0002) and TAMs (p=0.0005) as well as with improved 3 year PFS and OS (log rank p=0.04 and p=0.03, respectively). Furthermore, stromal CD8+ TILs and stromal CD68+ TAMs correlated positively with each other in the TNBC group (p=0.0094). Conclusions: PD-L1 expression correlated with TILs and TAMs in TNBC and was associated with a favorable outcome. PD-L1+ TNBCs with high levels of TILs and TAMs may be primed for exceptional response to immunogenic chemotherapy alone. Whether some pts with PD-L1+ TNBCs with high TILs/TAMs will benefit additionally from an anti-PD-L1/PD-1 agent is being investigated currently. Citation Format: Mardones MA, Grosser D, Levin MK, Daoud Y, Palucka K, O'Shaughnessy J, Osborne C. PD-L1 expression in triple negative breast cancer (TNBC) is associated with improved outcomes [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-04-20.

Programmed death-ligand 1 (PD-L1) is an immune checkpoint protein expressed on tumor and immune cells that promotes immune evasion by inhibiting T-cell activation. In advanced triple-negative breast cancer (TNBC), a subtype with aggressive behavior and limited targeted treatment options, PD-L1 expression has emerged as a predictive biomarker for response to immune checkpoint inhibitors. However, most studies have focused on predominantly European-ancestry populations, leaving gaps in understanding PD-L1’s expression and clinical relevance among women of African ancestry, who are disproportionately affected by TNBC. Given TNBC’s molecular heterogeneity across ancestral groups, examining PD-L1 expression in diverse populations is critical for equitable immunotherapy and understanding ancestry-specific tumor biology.The International Center for the Study of Breast Cancer Subtypes (ICSBCS) biobank provides a unique ancestrally diverse tissue repository, enabling biomarker studies across historically underrepresented populations. Leveraging this resource, we are conducting a large-scale immunohistochemical (IHC) analysis of PD-L1 on formalin-fixed paraffin-embedded (FFPE) TNBC tissue sections. The study aims to evaluate PD-L1 in 1,000 TNBC samples, equally distributed among Ghanaian, Ethiopian, African American (AA), and European American (EA) patients. Here, we report initial findings.To date, 301 cases have been processed (AA n = 47, Ethiopian n = 12, Ghanaian n = 205, EA n = 37). PD-L1 expression, measured as percent positive tumor cells by IHC, showed means (± SD, 95% CI) of 7.25% (± 6.28, 3.34-11.16) in Ethiopian, 6.55% (± 6.28, 4.36-8.73) in EA, 5.32% (± 6.28, 3.39-7.25) in AA, and 1.13% (± 6.28, 0.20-2.06) in Ghanaian cases. Tukey-Kramer HSD comparisons revealed significant differences between Ethiopian and Ghanaian (p=0.0156), EA and Ghanaian (p&amp;lt;0.0001), and AA and Ghanaian (p=0.0008). Corresponding medians (IQR) were 9% (9.13%) for Ethiopian, 1% (4.5%) for EA, 2% (9.5%) for AA, and 0% (0.5%) for Ghanaian cases, reflecting heterogeneity and skewed PD-L1 expression. Initial analyses suggest differences in PD-L1 expression across self-reported race and ancestry groups. Comprehensive clinical data and additional tumor microenvironment measures, including tumor-infiltrating lymphocyte (TIL) scores, are being collected for integrative assessment of PD-L1 and its clinical significance. Continued analysis will advance understanding of population-specific tumor immunobiology and inform equitable immunotherapy approaches in TNBC.These preliminary findings underscore the value of the ICSBCS biobank in enabling ancestry-informed biomarker research in TNBC. By systematically evaluating PD-L1 across Ghanaian, Ethiopian, AA, and EA populations, this study aims to uncover differences in immune checkpoint activation to inform tailored therapies. These data are preliminary, with limitations including small sample sizes in some ancestry groups, particularly Ethiopian and EA cohorts, which may affect precision. As analysis progresses toward the full 1,000-case cohort, continued integration of clinical, pathological, and molecular data will be essential for advancing population-specific tumor immunobiology and improving equity in breast cancer outcomes. Citation Format: B. Stonaker, E. Adjei, S. Demaria, R. Martini, M. B. Davis, L. Newman. Programmed death-ligand 1expression in triple negative breast cancer: insights from an African ancestry-enriched multinational cohort [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS2-12-05.