Pembrolizumab in combination with gemcitabine for patients with HER2-negative advanced breast cancer: GEICAM/2015-04 (PANGEA-Breast) study

Pembrolizumab in combination with gemcitabine for patients with HER2-negative advanced breast cancer: GEICAM/2015-04 (PANGEA-Breast) study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Pembrolizumab in combination with gemcitabine for patients with HER2-negative advanced breast cancer: GEICAM/2015-04 (PANGEA-Breast) study Luis de la Cruz-Merino, María Gion, Josefina Cruz, Jose Luis Alonso-Romero, and 23 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1638137/v2 This work is licensed under a CC BY 4.0 License Status: Under Review Version 2 posted 11 You are reading this latest preprint version Show more versions Abstract Background : We evaluated a combination of two immunomodulatory agents considering a synergism that may induce long-term clinical benefit in advanced breast cancer (ABC). Methods : HER2-negative ABC patients received 21-day cycles of pembrolizumab 200 mg (day 1) and gemcitabine (days 1 and 8). A run-in-phase (6+6 design) was planned with two dose levels (DL) of gemcitabine (1,250 mg/m 2 [DL0]; 1,000 mg/m 2 [DL1]) to determine the recommended phase II dose (RP2D). The primary objective was objective response rate (ORR). Tumor infiltrating lymphocytes (TILs) density and PD-L1 expression in tumors and myeloid-derived suppressor cells (MDSCs) levels in peripheral blood were analyzed. Results : Fourteen patients were treated with DL0, resulting in RP2D. Thirty-six patients were evaluated during the first stage of Simon’s design. Recruitment was stopped as statistical assumptions were not met. The median age was 52; 21 (58%) patients had triple-negative disease, 28 (78%) visceral involvement, and 27 (75%) ≥2 metastatic locations. Progression disease was observed in 29 patients. ORR was 15% (95% CI, 5–32). Eight patients were treated ≥6 months before progression. Fourteen patients reported grade ≥3 treatment-related adverse events. No association was found between TILs density and treatment efficacy. Patients with negative PD-L1 expression showed significantly prolonged progression-free survival. MDSCs levels were associated with treatment response. Conclusion : Pembrolizumab 200 mg and gemcitabine 1,250 mg/m 2 were considered as RP2D. The objective of ORR was not met; however, 22% patients were on treatment for ≥6 months. Tumor PD-L1 expression and MDSCs levels suggested a potential predictive role in this population. Trial registration number : ClinicalTrials.gov and EudraCT (NCT03025880 and 2016-001779-54, respectively). Registration dates: 20/01/2017 and 18/11/2016, respectively. Pembrolizumab Chemotherapy HER2-negative advanced breast cancer TILs PD-L1 MDSCs Figures Figure 1 Figure 2 Background Immunotherapy based on immune checkpoint inhibitors (ICIs) has revolutionized treatment of several cancer types. An impact on overall survival (OS) is documented in advanced setting( 1 – 3 ). The potential role of immunotherapy in breast cancer (BC) remains unclear. Efforts have been focused on the triple-negative (TN) BC subtype, as it has high levels of potential immune biomarkers in its microenvironment. Two phase III clinical trials( 4 – 6 ) reported positive results in PD-L1 + metastatic TNBC patients. However, other trials( 7 ) with similar designs have recently reported negative outcomes. Therefore, the real value of immunotherapy for TNBC needs still to be clarified. In this trial, we aimed to test whether the combination of gemcitabine and pembrolizumab could be feasible and provide meaningful responses in pretreated advanced BC (ABC) patients with TNBC or luminal A/B subtype according to St. Gallen recommendations( 8 ), irrespective of their PD-L1 statuses. Gemcitabine was combined with pembrolizumab as immunogenic properties through the elimination of myeloid-derived suppressor cells (MDSCs) and regulatory T lymphocytes (Tregs) were previously suggested in preclinical tumor models( 9 – 12 ). Methods Study design The PANGEA-Breast was an open-label, single-arm, multicenter phase II trial conducted in Spain. Gemcitabine plus pembrolizumab was evaluated in human epidermal growth factor receptor 2 (HER2)-negative ABC patients, with balanced distribution between TN and hormone receptor-positive (HR+) cohorts. In an initial exploratory run-in-phase with a 6 + 6 design, toxicity was evaluated within the first cycle. Fixed doses of pembrolizumab on day 1 and gemcitabine on days 1 and 8 of each 21-day cycle were administered to determine the recommended phase II dose (RP2D) based on the occurrence of any dose-limiting toxicity (DLT). Dose level (DL) 0 comprised pembrolizumab 200 mg and gemcitabine 1,250 mg/m 2 as an intravenous (IV) infusion. De-escalation to DL-1 (gemcitabine 1,000 mg/m 2 ) was planned if DL0 was not tolerable. Once the RP2D was defined, eligible patients were enrolled in phase II. The primary objective was the objective response rate (ORR), defined as the number of patients with ≥ 1 treatment dose and complete response (CR) plus partial response (PR) according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. The secondary objectives were clinical benefit rate (CBR) with stable disease (SD) of ≥ 24 weeks, duration of response (DoR), progression-free survival (PFS), OS, and safety and tolerability according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 4.0. The exploratory objectives were to assess the efficacy based on immune-related response criteria, search for tumor tissue and peripheral blood biomarkers for clinical activity, correlate a set of immune biomarkers with disease evolution and efficacy of the combination, and comparison of this set of biomarkers from cohorts of healthy volunteers and patients from this trial. The study was conducted according to the International Conference on Harmonization Good Clinical Practice Guidelines and Declaration of Helsinki and was approved by the institutional ethical review boards of the participating sites and Spanish health authorities. It was registered at ClinicalTrials.gov and EudraCT (NCT03025880 and 2016-001779-54, respectively). Written informed consent was obtained from all patients before performing any protocol-specific procedures. Patients The key inclusion criteria were women aged ≥ 18 years; HER2-negative ABC by immunohistochemistry (IHC) and/or in situ hybridization based on local testing of the most recent tumor biopsy; ≥10 mm measurable lesion as per the RECIST 1.1; an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0 or 1; prior anthracyclines and taxanes (unless contraindicated), and ≥ 2 endocrine therapy (ET) and ≤ 4 chemotherapy (CT) lines for ABC; patient agreement for a fresh metastatic tumor biopsy at inclusion and progressive disease (PD); adequate organ function; negative pregnancy test for women of child-bearing potential (WCBP) and adequate contraception. Patients with treated brain metastases and stable without steroids were allowed. Healthy controls from the University Hospital Virgen Macarena in Seville, Spain, provided written informed consent to participate in the analysis. Treatment plan Patients received pembrolizumab (200 mg IV) on day 1 and gemcitabine (1,250 mg/m 2 IV) on days 1 and 8 of each 21-day cycle until objective PD, clinical PD (under investigator’s judgment), unacceptable toxicity, death, or consent withdrawal, whichever occurred first. Evaluation procedures Baseline assessments were performed ≤ 28 days from starting treatment. These included tumor assessment using radiological tests accepted by the RECIST 1.1, standard 12-lead electrocardiogram, hematology, biochemistry, coagulation test, thyroid hormones, urinalysis, physical examination, ECOG PS evaluation, and pregnancy test in WCBP. Tumor assessments were performed every 9 weeks until PD. AEs were graded using the NCI-CTCAE v4.0. Other safety endpoints included regular monitoring of vital signs and laboratory tests. Biomarker analyses TILs and PD-L1 assessments Thirty (83%) available pre-treatment metastatic tumor samples were assessed for TILs density, according to the suggested international guidelines( 13 ). Cut-offs explored for TILs evaluation were ≥ 5%, ≥ 10%, and ≥ 20%. PD-L1 expression measured using IHC was assessed in 29 pre-treatment metastatic tumor samples using the mAb anti-PD-L1 clone 22C3 (Merck, Kenilworth, NJ). Immune cell (IC) scores and combined positive score (CPS) were obtained. PD-L1 IC score and CPS ≥ 1 were considered positive. Cut-offs (≥ 5, ≥ 10, and ≥ 20) were also explored for CPS. The logistic and Cox regression models were used to evaluate the association of TILs density and PD-L1 expression with treatment efficacy in terms of ORR, CBR (with SD ≥ 24 weeks) and PFS according to the RECIST 1.1. Immunophenotyping of whole peripheral blood using flow cytometry analysis Blood samples were collected in EDTA-K3 tubes at baseline, before cycles 3 (C3) and 6 (C6), or at the end of treatment (EOT), whichever occurred first, to determine MDSCs counts. Cell populations were determined using flow cytometry of the whole blood using the BD FACSCanto™ system (BD Biosciences, San Jose, CA, USA). Monocytic-MDSCs (M-MDSCs) were defined as CD45 + CD11b + CD33 + HLA-DR − CD14 + CD15 − and granulocytic-MDSCs (G-MDSCs) as CD45 + CD11b + CD33 + HLA-DR − CD14 − CD15+. Monoclonal antibodies Antibodies were obtained from Becton Dickinson Immunocytometry Systems (BD Biosciences, CA, USA) and were used at the manufacturer’s recommended concentrations. Statistical analyses Simon’s minimax two-stage design was employed for the phase II part of the study with the option of early stopping owing to lack of response. The sample size was calculated by testing the null hypothesis (H0) that gemcitabine resulted in an ORR of approximately 20%. With the study combination, the alternative hypothesis was 35% (an absolute increase of 15%); with an alpha error of 0.05 and a power of 80%, 53 evaluable patients were required. The first stage should include 31 evaluable patients, and if at least seven presented a response, recruitment would continue to include 53 evaluable patients. The H0 of 20% was rejected if ≥ 16 responses were observed in 53 patients. The SAS Enterprise Guide (version 7.15) was used for all analyses. Results Patients’ characteristics From June 2017 to May 2018, 36 patients were recruited for the first stage of Simon’s minimax two-stage design (Fig. 1 ); however, only five patients achieved a response, and recruitment was stopped. Their characteristics are presented in Table 1 . Approximately 58% of the tumors were TN. Twenty-three (64%) patients had up to two metastatic locations, and 28 (78%) had visceral involvement. Patients had a median of four prior therapy lines for ABC. Table 1 Patients and disease characteristics Characteristics N = 36 Age, years (median [range]) 51 (31–77) <65, n (%) 33 (91.7) ≥65, n (%) 3 (8.3) Menopausal status, n (%) Postmenopausal 26 (72.2) Premenopausal 10 (27.8) ECOG PS, n (%) 0 23 (63.9) 1 12 (33.3) 2 1 (2.8) Time since 1st BC diagnosis to study inclusion, years (median [range]) 4 (1–37) Time since M1 diagnosis to study inclusion, years (median [range]) 2 (0.2–14) Number of metastatic locations, n (%) 1 9 (25.0) 2 14 (38.9) ≥ 3 13 (36.2) Metastatic locations, n (%) Visceral 28 (77.8) • Liver 25 (69.4) • Lung 7 (19.4) • Adrenal gland, pericardial effusion, or pleural involvement 6 (16.6) Non-visceral only 8 (22.2) • Bone 20 (55.6) • Breast 2 (5.6) • Lymph nodes 13 (36.1) • Skin 4 (11.1) • Soft tissue 5 (13.9) Histological type, n (%) Invasive ductal carcinoma 33 (91.7) Invasive lobular carcinoma 2 (5.6) Invasive squamous carcinoma 1 (2.8) Histological grade, n (%) 1 1 (2.8) 2 14 (38.9) 3 16 (44.4) Unknown 5 (13.9) HR status (local), n (%) Negative 21 (58.3) Positive 15 (41.7) Ki67 expression (local) (%) Median (range) 30 (15–95) < 20%, n (%) 5 (13.9) ≥ 20%, n (%) 21 (58.3) Unknown 10 (27.8) BC subtype by IHC (local), n (%) TN 21 (58.3) HR-positive disease 15 (41.7) Prior therapy, n (%) CT 36 (100.0) • ABC 34 (94.4) ET 26 (72.2) • ABC 19 (52.8) BT 25 (69.4) • ABC 24 (66.7) Number of prior lines for ABC, n (%) None 1 (2.8) 1 9 (25.0) 2 2 (5.6) 3 3 (8.3) 4 5 (13.9) 5 7 (19.4) ≥ 6 9 (25.0) Median (range) 4 (0–11) • TN 2 (1–8) • HR-positive disease 5 (3–11) Abbreviations: n, number of patients; ECOG PS, Eastern Cooperative Oncology Group performance status; BC, breast cancer; M1, metastases or metastatic disease; HR, hormone receptor; IHC, immunohistochemistry; TN, triple negative; CT, chemotherapy; ET, endocrine therapy; BT, biological therapy; ABC, advanced breast cancer. Run-in phase and determination of RP2D Fourteen patients were treated with DL0. Three patients were replaced since they experienced early PD (Supplementary Figure, SF1). Of the first six patients, one experienced DLT of grade 3 tumor pain and grade 2 anemia and thrombocytopenia; six additional patients were recruited at this DL without any reported DLT; therefore, DL0 was declared as the RP2D. Treatment exposure A median of 4 (range, 1–24) cycles for pembrolizumab and 4.5 (range, 1–24) cycles for gemcitabine were administered. The median relative dose intensity at the RP2D was 100% (100–100) and 80% (39–101), respectively. Nine (25%) patients experienced delays in pembrolizumab administration because of AEs in five (13.9%) patients; these AEs included hematological and liver function test alterations, nausea, arthralgia, and infections. Thirty (83.3%) patients experienced dose modifications for gemcitabine, with omissions being the most frequent modification followed by reductions and delays. AEs were the most common reasons for all types of modifications. The main reasons for treatment discontinuation included PD in 29 (80.6%) patients; death in two (5.6%); and AE (grade 3 respiratory failure), patient`s decision, and physician’s decision in one (2.8%) patient each. At the time of analysis, two patients were still on treatment. Efficacy Considering the efficacy population (n = 33), the ORR was 15.2% (n = 5/33; 95% confidence interval [CI] 5.1–31.9), with only PR reported, and the ORR in patients with TN or HR + BC was similar to the ORR in the whole population. Twelve (36.4%) patients achieved SD (one case lasted for > 6 months), and 12 (36.4%) experienced PD (Supplementary SF2). The CBR, including SD of any duration, was 51.5% (n = 17/33; 95% CI 33.5–69.2). The median DoR was 4.3 months (95% CI 2.3–7.4), median PFS was 3.1 months (95% CI 2.0–4.3), and median OS was 7.9 months (95% CI 6.5–10.3). Eight patients were on treatment for ≥ 6 months before PD (11.4 and 16.1 months in two cases). Efficacy based on immune-related response criteria showed similar results. Safety Almost all patients (35 [97.2%]) experienced AE, which was related to the study treatment in 25 (69.4%) patients. AEs led to treatment discontinuation in five (13.9%) patients. Grade 3 AEs were reported in 15 (41.7%) patients and grade 4 AEs in 7 (19.4%), and of these AEs, their relationship with treatment was established by the investigators in 14 (38.9%) patients. Serious AEs were reported in 12 (33.3%) patients and were related to treatment in three cases. No grade 5 AEs related to treatment were reported. Treatment-emergent AEs, irrespective of their causal relationship with the study treatment, are summarized in Table 2 . Table 2 Treatment-emergent adverse events by grade according to the NCI-CTCAE (version 4.03) and with a frequency of at least 5% in any grade Safety Population (n = 36) Adverse Event Term Grade 1, n (%) Grade 2, n (%) Grade 3, n (%) Grade 4, n (%) Patients with any TEAE 27 (75.0) 27 (75.0) 18 (50.0) 7 (19.4) Pyrexia 10 (27.8) 1 (2.8) 0 0 Fatigue 11 (30.6) 10 (27.8) 2 (5.6) 0 Anemia 8 (22.2) 2 (5.6) 3 (8.3) 0 Nausea 6 (16.7) 2 (5.6) 0 0 Decreased appetite 4 (11.1) 1 (2.8) 0 0 Musculoskeletal chest pain 3 (8.3) 0 0 0 Pruritus 3 (8.3) 0 0 0 Upper respiratory tract infection 3 (8.3) 0 0 0 Albuminuria 2 (5.6) 0 0 0 Back pain 2 (5.6) 0 1 (2.8) 0 Hot flush 2 (5.6) 0 0 0 Hypothyroidism 2 (5.6) 0 0 0 Abdominal pain upper 1 (2.8) 2 (5.6) 0 0 Elevated AST level 1 (2.8) 0 2 (5.6) 0 Constipation 1 (2.8) 2 (5.6) 1 (2.8) 0 Diarrhea 1 (2.8) 3 (8.3) 1 (2.8) 0 Dyspnea 1 (2.8) 2 (5.6) 0 0 Painful skin 1 (2.8) 2 (5.6) 0 0 Rash 1 (2.8) 2 (5.6) 0 0 Tachycardia 1 (2.8) 2 (5.6) 0 0 Hypertension 0 4 (11.1) 0 0 Neutrophil count decreased 0 6 (16.7) 10 (27.8) 3 (8.3) Weight decreased 0 2 (5.6) 0 0 Pembrolizumab-related TEAEs included rash, anemia, decreased neutrophil count, diarrhea, and increased AST and ALT levels. Abbreviations: TEAE, treatment-emergent adverse events; NCI-CTCAE, National Cancer Institute Common Terminology Criteria for Adverse Events; n, number of patients; AST, aspartate aminotransferase Association of TILs density and PD-L1 expression with treatment efficacy TILs density values are listed in Table 3 . No significant associations were observed between TILs density and either ORR/CBR or PFS (Table 3 ). Analysis of the median TILs density distribution according to tumor subtype and ORR showed that TNBC patients who achieved ORR tended to present higher levels of lymphocyte infiltration (Supplementary SF3). Table 3 Logistic and Cox regression models to analyze the association between TILs density and PD-L1 expression (IC and CPS scores) with ORR (CR + PR), CBR (CR +PR + SD of >6 months), and PFS ORR p-value Odds ratio (95% CI) CBR p-value Odds ratio (95% CI) PFS p-value Hazard ratio (95% CI) TILs (n = 30) Cut-Off (n, %) ≥5% (24, 80%) <5% (6, 20%) NA 0.556 0.47 (0.04, 5.90) 0.534 1.37 (0.51, 3.64) ≥10% (13, 43%) <10% (17, 57%) 0.773 1.36 (0.17, 11.23) 0.554 0.50 (0.05, 4.98) 0.311 1.51 (0.68, 3.36) ≥20% (6, 20%) <20% (24, 80%) 0.789 1.40 (0.12, 16.46) 0.699 0.56 (0.03, 10.93) 0.073 2.50 (0.92, 6.82) PD-L1 Score (n = 29) Cut-Off (n, %) IC ≥1% (11, 38%) IC <1% (18, 62%) 0.595 1.78 (0.21, 14.86) 0.635 0.57 (0.06, 5.77) 0.321 1.51 (0.67, 3.40) CPS ≥1 (14, 48%) CPS <1 (15, 52%) 0.941 1.08 (0.13, 8.95) 0.668 1.60 (0.19, 13.69) 0.038 2.56 (1.05, 6.23) CPS ≥5 (11, 38%) CPS <5 (18, 62%) 0.571 0.50 (0.05, 5.51) 0.482 2.50 (0.19, 32.19) 0.053 2.34 (0.99, 5.53) CPS ≥10 (8, 28%) CPS <10 (21, 72%) 0.901 0.86 (0.08, 9.69) 0.793 1.43 (0.10, 20.44) 0.173 1.84 (0.77, 4.42) CPS ≥20 (7, 24%) CPS <20 (22, 76%) 0.965 1.06 (0.09, 12.14) 0.793 1.43 (0.10, 20.44) 0.072 2.36 (0.93, 6.01) Abbreviations: TILs, tumor-infiltrating lymphocytes; ORR, objective response rate; CBR, clinical benefit rate; PFS, progression-free survival; IC, immune cell; CPS, combined positive score; CR, complete response; PR; partial response; SD, stable disease; CI, confidence interval Eleven (38%) and 14 (48%) patients showed PD-L1 + IC score and CPS at a cut-off of 1; 10 (28%) of them were positive for both scores. No associations were observed between PD-L1 IC score and ORR, CBR, or PFS. For CPS, we observed that patients with a CPS of ≥ 1 showed significantly worse PFS (hazard ratio [HR] 2.56; 95% CI 1.05–6.23; p = 0.038), and the same tendency was observed with a CPS of ≥ 5 (HR 2.34; 95% CI 0.99–5.53; p = 0.053); this association was not maintained with CPS cut-offs of ≥ 10 and ≥ 20 (HR 1.84; 95% CI 0.77–4.42; p = 0.173, and HR 2.36; 95% CI 0.93–6.01; p = 0.072, respectively) (Table 3 ). No association was observed between CPS, ORR, or CBR. Nine (53%) TN patients were PD-L1 + for IC scores ≥ 1% and nine (53%) for CPS ≥ 1 (seven were positive for both the scores). In the HR + subpopulation, five (38%) patients were positive for CPS, and two (15%) were positive for IC score. PD-L1 + scores were not associated with ORR in either TN or HR+ (data not shown). PD-L1 expression by tumor subtype and ORR is shown in Supplementary SF4. PD-L1 + IC scores seemed to be more frequent in TNBC patients with no ORR, while the majority of HR + patients not achieving ORR showed PD-L1 − IC scores (Supplementary SF4B). Finally, a significant positive correlation between TILs levels and PD-L1 expression was observed (correlation coefficient, 0.567; p = 0.00134) (Supplementary SF5). Association between MDSCs levels and efficacy Patients showed significantly higher concentrations of total MDSCs at baseline than did the healthy control cohort (median values of 44.5 vs. 17.5 cells/µL, respectively, p = 0.0018). This difference was especially marked for M-MDSCs (median values of 33.5 vs. 10.3 cells/µL, p = 0.0010) rather than G-MDSCs (median values of 8.6, vs. 5.0 cells/µl, p = 0.1880) (Fig. 2 ). Analysis of variation in MDSCs levels along the treatment revealed significant differences in M-MDSCs levels associated with CBR between baseline-C3 and baseline-C6/EOT ( p = 0.0313 and 0.0469, respectively) (Table 4 ). A similar trend, although not significant ( p < 0.1), was also observed for differences between baseline and C6/EOT levels for total MDSCs ( p = 0.0781). Table 4 MDSCs levels (cells/µL) according to tumor response MDSCs Total M-MDSCs G-MDSCs CB (SD ≥ 24 weeks) (n = 7) PD (n = 12) CB (SD ≥ 24 weeks) (n = 7) PD (n = 12) CB (SD ≥ 24 weeks) (n = 7) PD (n = 12) Median IR Median IR Median IR Median IR Median IR Median IR Baseline 38.6 (n = 7) 53.0 47.6 (n = 11) 42.8 30.7 (n = 7) 32.8 39.8 (n = 11) 33.2 7.9 (n = 7) 20.9 5.2 (n = 11) 27.9 C3 17.4 (n = 7) 27.0 60.7 (n = 11) 247.4 4.3 (n = 7) 13.9 47.4 (n = 11) 223.1 5.2 (n = 7) 10.3 7.7 (n = 11) 24.3 C6/EOT 12.4 (n = 7) 8.5 23.2 (n = 5) 37.2 6.3 (n = 7) 8.5 8.0 (n = 5) 31.2 4.3 (n = 7) 10.2 12.4 (n = 5) 8.6 Baseline vs. C6/EOT p-value 0.0781 0.8750 0.0469 0.8750 0.6875 0.6250 Baseline vs. C3 p-value 0.2969 0.6953 0.0313 0.7695 0.9375 0.6250 CB vs. PD (C6/EOT) p-value 0.0513 0.7453 0.0348 CB vs. PD (C3) p-value 0.1743 0.0572 0.5261 CB vs. PD (baseline) p-value 0.3651 0.1743 0.9278 Abbreviations: MDSCs, myeloid derived suppressor cells; M-MDSCs, monocytic MDSCs; G-MDSCs, granulocytic MDSCs; CB, clinical benefit; SD, stable disease; PD, progressive disease; IR, interquartile range; C3, cycle 3; C6, cycle 6; EOT, end of treatment Analysis of the difference in MDSCs levels between patients with CBR or PD at different time points showed significant differences only in case of G-MDSCs at C6/EOT ( p = 0.0348). No significant differences ( p < 0.1) were observed at the same time point for total MDSCs levels and at C3 for M-MDSCs levels (Table 4 ). Discussion In the PANGEA-Breast trial, pembrolizumab plus gemcitabine achieved a modest ORR of 15.2%. No long-term responders were observed, although two patients were still alive at study closing data (22/07/2021). Some facts may explain these results. First, BC patients included in our study were heavily pretreated in most cases, with a median of four prior lines for ABC. Second, there was no patient preselection with respect to PD-L1 expression or TILs density, which confirms that an unselected population is probably an adverse scenario for immunotherapy in ABC. Finally, gemcitabine probably does not harbor powerful immunogenic properties for treating ABC, as we expected. Therefore, other drugs that induce immunogenic cell death, such as anthracyclines, may be more appropriate. Some clinically outstanding results as those reported in the TONIC trial (35% ORR with doxorubicin [15 mg/m 2 ] followed by nivolumab in metastatic TNBC patients)( 14 ) or KEYNOTE-522 study( 15 ) in the neoadjuvant setting, support the hypothesis that the combination of immunotherapy and CT matters, and anthracyclines could trigger and boost the immune response better than other chemotherapeutic drugs. Discordant results between the IMpassion-130( 15 ) and IMpassion-131( 7 ) trials can also be partially explained with this theory. The difference among these two trials was the CT used (nab-paclitaxel or conventional paclitaxel, respectively) and premedication with corticosteroids in the IMpassion-131 trial. Additionally, based on tissue immune biomarkers analysis of the PANGEA-Breast trial, we could not identify a subpopulation benefiting from the chemoimmunotherapy combination. Neither TILs density nor PD-L1 expression using CPS revealed a subgroup with a higher probability of response or better survival. The small sample size (n = 36) and highly heterogeneous population (e.g., TN and HR + BC, different previous lines of treatment) represent a major limitation at this point; therefore, our results should be considered cautiously, especially when the IMpassion-130 and KEYNOTE-522 trials have reported favorable results in PD-L1 enriched populations. Regarding the peripheral blood immune biomarker analysis, MDSCs results were intriguing as their levels were clearly elevated in patients than in the healthy cohort ( p = 0.0018), suggesting that an immunosuppressive status is induced by ABC. These results are concordant with previous findings that correlate higher levels of MDSCs with adverse prognostic factors and tumor burden in ABC( 16 ). Interestingly, MDSCs decreased along treatment implementation in the CB group versus PD group with a clear trend at C3 and seem less obvious at C6. MDSCs may represent emerging and valuable biomarkers; however, the limited number of patients and samples in our study jeopardized any major interpretation of our data. Conclusions In summary, this trial reinforces the hypothesis that immunotherapy for ABC could eventually work only in highly selected and enriched populations, ideally for first-line therapy. Pretreated patients, especially those heavily pretreated in our work and in the KEYNOTE-119 trial( 17 ) showed little benefit from ICIs, with a trend of improved efficacy with PD-L1 enrichment. For future clinical trials in this setting, better selection of patients with ABC would be advisable. Additionally, unless new results are available, different original strategies should be tested, as chemoimmunotherapy outcomes in ABC appear globally modest. Approaches aiming to induce the host immune system through effective immunogenic cell death modalities, depletion of immunosuppressive cells such as MDSCs or Tregs( 18 , 19 ), or favoring neoantigen presentation( 20 , 21 ) could widen the spectrum of immunotherapy for ABC. Declarations Ethics approval and consent to participate The study was conducted according to the International Conference on Harmonization Good Clinical Practice Guidelines and Declaration of Helsinki and was approved by the institutional ethical review boards of the participating sites and Spanish health authorities. Written informed consent was obtained from all patients before performing any protocol-specific procedures. Availability of data and material The data that support the findings of this study are available from GEICAM. Data are available from the authors upon reasonable request to the principal investigator Dr. de la Cruz ( [email protected] ) and with permission of the PANGEA-Breast steering committee. Competing Interests L. de la Cruz-Merino received consulting or advisory role fees from Merck Sharp & Dohme (MSD)-Merck, Roche, Bristol-Myers-Squibb (BMS), Pierre-Fabre, Amgen, and Novartis; research funding from MSD-Merck, Roche, and Celgene; speaker’s honoraria from MSD-Merck, Roche, BMS, and Amgen; and grant support from Roche and BMS. M Gion received honoraria from Roche and traveled accommodation from Pfizer. J. Cruz received consulting or advisory role fees from Roche, PharmaMar, Lilly, Novartis, Eisai, Pfizer Amgen, and Celgene; travel accommodation from Roche, Novartis, and PharmaMar. V. Quiroga received speakers’ bureau honoraria from Pfizer, Novartis, and Roche; research funding from Celgene; and travel accommodation from Novartis, Roche, and Pfizer. F. Moreno received an advisory role honoraria from Pfizer, Roche, Novartis MSD, and AstraZeneca; speaker’s bureau honoraria and research funding from Pfizer; and travel accommodation from Pfizer, Roche, and Novartis. R. Andrés received travel accommodation from Roche. M. Santisteban received honoraria from Roche, Novartis, and Pfizer; and consulting or advisory role honoraria from Gilead, MSD, Novartis, Pfizer, and Biomerieux. M. Ramos received speakers’ bureau honoraria from Novartis, Roche, and Pfizer. J. Cortés has stock and other ownership interests in MedSIR and has received honoraria from Novartis, Eisai, Celgene, Pfizer, Roche, SAMSUNG, Lilly, MSD, and Daiichi Sankyo; consulting or advisory role honoraria from Celgene, Cellestia Biotech, AstraZeneca, Biothera, Merus, Roche, Seattle Genetics, Daiichi Sankyo, ERYTECH Pharma, Polyphor, Athenex, Lilly, Servier, MSD , GlaxoSmithKline (GSK), Leuko, Clovis Oncology, Bioasis, Boehringer Ingelheim, and Kyowa Kyrin; research funding from ARIAD, AstraZeneca, Baxalta GMBH/Servier Affaires, Bayer, Eisai Farmaceutica, Guardanth health, MSD, Pfizer, Puma CO, Queen Mary University of London, Roche, and Piqur; and travel accommodation from Roche, Pfizer, Eisai, Novartis, and Daiichi Sankyo. E. López received consulting or advisory role honoraria from AstraZeneca, Pfizer, Roche, and Novartis; and the speaker’s bureau honoraria from Roche, Eisai, Pfizer, and Novartis. A. Cortés received consulting or advisory role honoraria from Lilly, Roche, Clovis, Ferrer, and Pfizer; speakers’ bureau honoraria from MSD, GSK, AstraZeneca, Roche and Pfizer, and research funding and travel accommodation from Pfizer. L. M. Rodríguez received honoraria from Pfizer and Pierre Fabre. I. Ceballos received consulting or advisory role honoraria from Roche and Merk KGaA; speakers’ bureau honoraria from Roche, Pfizer, BMS, and Celgene; and travel accommodation from Roche, Merck, Pfizer, and Novartis. F. Rojo received consulting or advisory role fees from Roche, AstraZeneca, Novartis, BMS, MSD, Lilly, Pfizer, Genomic Health, Guardant Health, Archer, and Pierre-Fabre; speaker bureau/expert testimony fees from Roche, AstraZeneca, Novartis, BMS, MSD, Lilly, Pfizer, and Pierre-Fabre; and travel accommodation from Roche and Novartis His institution received research grant/funding from Roche and Pfizer. The rest of authors declare no conflict of interest. Funding The study was partially supported by a research grant from Merck (MSD in Europe) Investigator Initiated Studies Program, which also supplied pembrolizumab. Authors´ contribution Conceptualization, L.dC., F.R. and R.C.; Methodology, LdC, F.R., SB, SBe, R.C. and M.Ch.; Software and Validation SB and S.Be.; Formal Analysis, M.C.; Investigation and Data Curation, L.dC., F.R., C.J-C., V.S-M., M.G., J.C., V.Q., R.A., F.M., J.L.A-R., M.R., E.H., J.C., E. L-M., F.H., N.P-C., L.M.R., I.C., M.S., A.C., A.S. and A.P. Resources, L.dC., F.R., C.J-C., V.S-M., M.G., J.C., V.Q., R.A., F.M., J.L.A-R., M.R., E.H., J.C., E. L-M., F.H., N.P-C., L.M.R., I.C., M.S., A.C., A.S., A.P., R.C., M.Ch., S.B and S.Be.; Writing – Original Draft Preparation, L.dC., F.R., S.B., M.C., R.C., M.Ch. and S.Be.; Writing – Review & Editing and Visualization, all authors.; Supervision, L.dC. and S.B.; Project Administration, L.dC., S.B. and S.Be.; Funding Acquisition, L.dC., R.C., and S.B. Acknowledgments We acknowledge the investigators (see list below), pathology departments, and other staff of the participant sites, the patients, and the GEICAM staff involved in this trial. We would like to thank Editage (www.editage.com) for English language editing. References Food and Drug Administration (FDA): KEYTRUDA® (pembrolizumab) prescribing information 2019 [Available from: https://www.accessdata.fda.gov/drugsatfda_docs/label/2019/125514Orig1s054lbl.pdf. Food and Drug Administration (FDA): OPDIVO (nivolumab) prescribing information. 2019. Food and Drug Administration (FDA): TECENTRIQ® (atezolizumab) prescribing informatioin. 2019. Schmid P, Adams S, Rugo HS, Schneeweiss A, Barrios CH, Iwata H, et al. Atezolizumab and Nab-Paclitaxel in Advanced Triple-Negative Breast Cancer. N Engl J Med. 2018;379(22):2108-21. Cortes J, Cescon DW, Rugo HS, Nowecki Z, Im S-A, Yusof MM, et al. Pembrolizumab plus chemotherapy versus placebo plus chemotherapy for previously untreated locally recurrent inoperable or metastatic triple-negative breast cancer (KEYNOTE-355): a randomised, placebo-controlled, double-blind, phase 3 clinical trial. The Lancet. 2020;396(10265):1817-28. Rugo HS, Schmid P, Cescon DW, Nowecki Z, Im S-A, Yusof MM, et al. Abstract GS3-01: Additional efficacy endpoints from the phase 3 KEYNOTE-355 study of pembrolizumab plus chemotherapy vs placebo plus chemotherapy as first-line therapy for locally recurrent inoperable or metastatic triple-negative breast cancer. Cancer Res. 2021;81(4 Supplement):GS3-01-GS3-. Miles D AF, Gligorov J, Verma S, Xu B, Cameron D, Barrios C.H, Schneeweiss A, Easton V, Dolado I, O’Shaughnessy J. IMpassion131: Phase III study comparing 1L atezolizumab with paclitaxel vs placebo with paclitaxel in treatment-naive patients with inoperable locally advanced or metastatic triple negative breast cancer (mTNBC). Annals of Oncology. 2017;28(September 2017):105. Goldhirsch A, Winer EP, Coates AS, Gelber RD, Piccart-Gebhart M, Thurlimann B, et al. Personalizing the treatment of women with early breast cancer: highlights of the St Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2013. Ann Oncol. 2013;24(9):2206-23. Srivastava MK, Sinha P, Clements VK, Rodriguez P, Ostrand-Rosenberg S. Myeloid-derived suppressor cells inhibit T-cell activation by depleting cystine and cysteine. Cancer Res. 2010;70(1):68-77. Suzuki E, Kapoor V, Jassar AS, Kaiser LR, Albelda SM. Gemcitabine selectively eliminates splenic Gr-1+/CD11b+ myeloid suppressor cells in tumor-bearing animals and enhances antitumor immune activity. Clin Cancer Res. 2005;11(18):6713-21. Shevchenko I, Karakhanova S, Soltek S, Link J, Bayry J, Werner J, et al. Low-dose gemcitabine depletes regulatory T cells and improves survival in the orthotopic Panc02 model of pancreatic cancer. Int J Cancer. 2013;133(1):98-107. Rettig L, Seidenberg S, Parvanova I, Samaras P, Curioni A, Knuth A, et al. Gemcitabine depletes regulatory T-cells in human and mice and enhances triggering of vaccine-specific cytotoxic T-cells. Int J Cancer. 2011;129(4):832-8. Hendry S, Salgado R, Gevaert T, Russell PA, John T, Thapa B, et al. Assessing Tumor-infiltrating Lymphocytes in Solid Tumors: A Practical Review for Pathologists and Proposal for a Standardized Method From the International Immunooncology Biomarkers Working Group: Part 1: Assessing the Host Immune Response, TILs in Invasive Breast Carcinoma and Ductal Carcinoma In Situ, Metastatic Tumor Deposits and Areas for Further Research. Adv Anat Pathol. 2017;24(5):235-51. Voorwerk L, Slagter M, Horlings HM, Sikorska K, van de Vijver KK, de Maaker M, et al. Immune induction strategies in metastatic triple-negative breast cancer to enhance the sensitivity to PD-1 blockade: the TONIC trial. Nat Med. 2019;25(6):920-8. Schmid P, Cortes J, Pusztai L, McArthur H, Kummel S, Bergh J, et al. Pembrolizumab for Early Triple-Negative Breast Cancer. N Engl J Med. 2020;382(9):810-21. Safarzadeh E, Hashemzadeh S, Duijf PHG, Mansoori B, Khaze V, Mohammadi A, et al. Circulating myeloid-derived suppressor cells: An independent prognostic factor in patients with breast cancer. J Cell Physiol. 2019;234(4):3515-25. Cortés J, Lipatov O, Im SA, Gonçalves A, Lee KS, Schmid P, et al. LBA21 - KEYNOTE-119: Phase III study of pembrolizumab (pembro) versus single-agent chemotherapy (chemo) for metastatic triple negative breast cancer (mTNBC). Ann Oncol. 2019;30:v859-v60. Hodge JW, Ardiani A, Farsaci B, Kwilas AR, Gameiro SR. The tipping point for combination therapy: cancer vaccines with radiation, chemotherapy, or targeted small molecule inhibitors. Semin Oncol. 2012;39(3):323-39. Drake CG. Combination immunotherapy approaches. Annals of Oncology. 2012;23:viii41-viii6. Torimura T, Iwamoto H, Nakamura T, Koga H, Ueno T, Kerbel RS, et al. Metronomic chemotherapy: possible clinical application in advanced hepatocellular carcinoma. Transl Oncol. 2013;6(5):511-9. Garcia-Aranda M, Redondo M. Immunotherapy: A Challenge of Breast Cancer Treatment. Cancers (Basel). 2019;11(12). Additional Declarations Competing interest reported. L. de la Cruz-Merino received consulting or advisory role fees from Merck Sharp & Dohme (MSD)-Merck, Roche, Bristol-Myers-Squibb (BMS), Pierre-Fabre, Amgen, and Novartis; research funding from MSD-Merck, Roche, and Celgene; speaker’s honoraria from MSD-Merck, Roche, BMS, and Amgen; and grant support from Roche and BMS. M Gion received honoraria from Roche and traveled accommodation from Pfizer. J. Cruz received consulting or advisory role fees from Roche, PharmaMar, Lilly, Novartis, Eisai, Pfizer Amgen, and Celgene; travel accommodation from Roche, Novartis, and PharmaMar. V. Quiroga received speakers’ bureau honoraria from Pfizer, Novartis, and Roche; research funding from Celgene; and travel accommodation from Novartis, Roche, and Pfizer. F. Moreno received an advisory role honoraria from Pfizer, Roche, Novartis MSD, and AstraZeneca; speaker’s bureau honoraria and research funding from Pfizer; and travel accommodation from Pfizer, Roche, and Novartis. R. Andrés received travel accommodation from Roche. M. Santisteban received honoraria from Roche, Novartis, and Pfizer; and consulting or advisory role honoraria from Gilead, MSD, Novartis, Pfizer, and Biomerieux. M. Ramos received speakers’ bureau honoraria from Novartis, Roche, and Pfizer. J. Cortés has stock and other ownership interests in MedSIR and has received honoraria from Novartis, Eisai, Celgene, Pfizer, Roche, SAMSUNG, Lilly, MSD, and Daiichi Sankyo; consulting or advisory role honoraria from Celgene, Cellestia Biotech, AstraZeneca, Biothera, Merus, Roche, Seattle Genetics, Daiichi Sankyo, ERYTECH Pharma, Polyphor, Athenex, Lilly, Servier, MSD , GlaxoSmithKline (GSK), Leuko, Clovis Oncology, Bioasis, Boehringer Ingelheim, and Kyowa Kyrin; research funding from ARIAD, AstraZeneca, Baxalta GMBH/Servier Affaires, Bayer, Eisai Farmaceutica, Guardanth health, MSD, Pfizer, Puma CO, Queen Mary University of London, Roche, and Piqur; and travel accommodation from Roche, Pfizer, Eisai, Novartis, and Daiichi Sankyo. E. López received consulting or advisory role honoraria from AstraZeneca, Pfizer, Roche, and Novartis; and the speaker’s bureau honoraria from Roche, Eisai, Pfizer, and Novartis. A. Cortés received consulting or advisory role honoraria from Lilly, Roche, Clovis, Ferrer, and Pfizer; speakers’ bureau honoraria from MSD, GSK, AstraZeneca, Roche and Pfizer, and research funding and travel accommodation from Pfizer. L. M. Rodríguez received honoraria from Pfizer and Pierre Fabre. I. Ceballos received consulting or advisory role honoraria from Roche and Merk KGaA; speakers’ bureau honoraria from Roche, Pfizer, BMS, and Celgene; and travel accommodation from Roche, Merck, Pfizer, and Novartis. F. Rojo received consulting or advisory role fees from Roche, AstraZeneca, Novartis, BMS, MSD, Lilly, Pfizer, Genomic Health, Guardant Health, Archer, and Pierre-Fabre; speaker bureau/expert testimony fees from Roche, AstraZeneca, Novartis, BMS, MSD, Lilly, Pfizer, and Pierre-Fabre; and travel accommodation from Roche and Novartis His institution received research grant/funding from Roche and Pfizer. The rest of authors declare no conflict of interest. Supplementary Files PANGEABreastSM.docx Cite Share Download PDF Status: Under Review Version 2 posted Editorial decision: Major revision 12 Oct, 2022 Reviews received at journal 11 Oct, 2022 Reviewers agreed at journal 30 Sep, 2022 Reviews received at journal 30 Aug, 2022 Reviewers agreed at journal 14 Aug, 2022 Reviewers agreed at journal 08 Aug, 2022 Reviewers invited by journal 08 Jul, 2022 Editor assigned by journal 07 Jul, 2022 Editor invited by journal 07 Jul, 2022 Submission checks completed at journal 07 Jul, 2022 First submitted to journal 22 Jun, 2022 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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1","display":"","copyAsset":false,"role":"figure","size":19492,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStudy flowchart. PD-L1, programmed death-ligand 1; MDSCs, myeloid derived suppressor cell\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-1638137/v2/b9e660f57a6f266861e1d4a0.png"},{"id":24409940,"identity":"cfa2abc0-d8d3-4b8a-b958-571005bb4b5e","added_by":"auto","created_at":"2022-07-27 17:03:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":39137,"visible":true,"origin":"","legend":"\u003cp\u003eBaseline median values of MDSCs (G-, M-, Total; cells/µL) in ABC patients and healthy cohort (HC). MDSCs, myeloid derived suppressor cells; M-MDSCs, monocytic MDSCs; G-MDSCs, granulocytic MDSCs; ABC, advanced breast cancer.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-1638137/v2/4b1645e585079beba9cb0109.png"},{"id":24410451,"identity":"46088af4-8b58-4e77-994c-1d5dda7c599c","added_by":"auto","created_at":"2022-07-27 17:08:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":876098,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1638137/v2/11a0ddb4-fd7c-4552-95c5-25b4b2058b22.pdf"},{"id":24410450,"identity":"ce231e8e-5bb8-4f91-aedf-e57c43669d86","added_by":"auto","created_at":"2022-07-27 17:08:44","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":196867,"visible":true,"origin":"","legend":"","description":"","filename":"PANGEABreastSM.docx","url":"https://assets-eu.researchsquare.com/files/rs-1638137/v2/cd8d0bf082fe4e31875e52ed.docx"}],"financialInterests":"Competing interest reported. L. de la Cruz-Merino received consulting or advisory role fees from Merck Sharp \u0026 Dohme (MSD)-Merck, Roche, Bristol-Myers-Squibb (BMS), Pierre-Fabre, Amgen, and Novartis; research funding from MSD-Merck, Roche, and Celgene; speaker’s honoraria from MSD-Merck, Roche, BMS, and Amgen; and grant support from Roche and BMS. M Gion received honoraria from Roche and traveled accommodation from Pfizer. J. Cruz received consulting or advisory role fees from Roche, PharmaMar, Lilly, Novartis, Eisai, Pfizer Amgen, and Celgene; travel accommodation from Roche, Novartis, and PharmaMar. V. Quiroga received speakers’ bureau honoraria from Pfizer, Novartis, and Roche; research funding from Celgene; and travel accommodation from Novartis, Roche, and Pfizer. F. Moreno received an advisory role honoraria from Pfizer, Roche, Novartis MSD, and AstraZeneca; speaker’s bureau honoraria and research funding from Pfizer; and travel accommodation from Pfizer, Roche, and Novartis. R. Andrés received travel accommodation from Roche. M. Santisteban received honoraria from Roche, Novartis, and Pfizer; and consulting or advisory role honoraria from Gilead, MSD, Novartis, Pfizer, and Biomerieux. M. Ramos received speakers’ bureau honoraria from Novartis, Roche, and Pfizer. J. Cortés has stock and other ownership interests in MedSIR and has received honoraria from Novartis, Eisai, Celgene, Pfizer, Roche, SAMSUNG, Lilly, MSD, and Daiichi Sankyo; consulting or advisory role honoraria from Celgene, Cellestia Biotech, AstraZeneca, Biothera, Merus, Roche, Seattle Genetics, Daiichi Sankyo, ERYTECH Pharma, Polyphor, Athenex, Lilly, Servier, MSD , GlaxoSmithKline (GSK), Leuko, Clovis Oncology, Bioasis, Boehringer Ingelheim, and Kyowa Kyrin; research funding from ARIAD, AstraZeneca, Baxalta GMBH/Servier Affaires, Bayer, Eisai Farmaceutica, Guardanth health, MSD, Pfizer, Puma CO, Queen Mary University of London, Roche, and Piqur; and travel accommodation from Roche, Pfizer, Eisai, Novartis, and Daiichi Sankyo. E. López received consulting or advisory role honoraria from AstraZeneca, Pfizer, Roche, and Novartis; and the speaker’s bureau honoraria from Roche, Eisai, Pfizer, and Novartis. A. Cortés received consulting or advisory role honoraria from Lilly, Roche, Clovis, Ferrer, and Pfizer; speakers’ bureau honoraria from MSD, GSK, AstraZeneca, Roche and Pfizer, and research funding and travel accommodation from Pfizer. L. M. Rodríguez received honoraria from Pfizer and Pierre Fabre. I. Ceballos received consulting or advisory role honoraria from Roche and Merk KGaA; speakers’ bureau honoraria from Roche, Pfizer, BMS, and Celgene; and travel accommodation from Roche, Merck, Pfizer, and Novartis. F. Rojo received consulting or advisory role fees from Roche, AstraZeneca, Novartis, BMS, MSD, Lilly, Pfizer, Genomic Health, Guardant Health, Archer, and Pierre-Fabre; speaker bureau/expert testimony fees from Roche, AstraZeneca, Novartis, BMS, MSD, Lilly, Pfizer, and Pierre-Fabre; and travel accommodation from Roche and Novartis His institution received research grant/funding from Roche and Pfizer. The rest of authors declare no conflict of interest.","formattedTitle":"Pembrolizumab in combination with gemcitabine for patients with HER2-negative advanced breast cancer: GEICAM/2015-04 (PANGEA-Breast) study","fulltext":[{"header":"Background","content":"\u003cp\u003eImmunotherapy based on immune checkpoint inhibitors (ICIs) has revolutionized treatment of several cancer types. An impact on overall survival (OS) is documented in advanced setting(\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The potential role of immunotherapy in breast cancer (BC) remains unclear. Efforts have been focused on the triple-negative (TN) BC subtype, as it has high levels of potential immune biomarkers in its microenvironment. Two phase III clinical trials(\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) reported positive results in PD-L1\u0026thinsp;+\u0026thinsp;metastatic TNBC patients. However, other trials(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) with similar designs have recently reported negative outcomes. Therefore, the real value of immunotherapy for TNBC needs still to be clarified.\u003c/p\u003e \u003cp\u003eIn this trial, we aimed to test whether the combination of gemcitabine and pembrolizumab could be feasible and provide meaningful responses in pretreated advanced BC (ABC) patients with TNBC or luminal A/B subtype according to St. Gallen recommendations(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), irrespective of their PD-L1 statuses. Gemcitabine was combined with pembrolizumab as immunogenic properties through the elimination of myeloid-derived suppressor cells (MDSCs) and regulatory T lymphocytes (Tregs) were previously suggested in preclinical tumor models(\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003eStudy design\u003c/h2\u003e\n \u003cp\u003eThe PANGEA-Breast was an open-label, single-arm, multicenter phase II trial conducted in Spain. Gemcitabine plus pembrolizumab was evaluated in human epidermal growth factor receptor 2 (HER2)-negative ABC patients, with balanced distribution between TN and hormone receptor-positive (HR+) cohorts. In an initial exploratory run-in-phase with a 6\u0026thinsp;+\u0026thinsp;6 design, toxicity was evaluated within the first cycle. Fixed doses of pembrolizumab on day 1 and gemcitabine on days 1 and 8 of each 21-day cycle were administered to determine the recommended phase II dose (RP2D) based on the occurrence of any dose-limiting toxicity (DLT). Dose level (DL) 0 comprised pembrolizumab 200 mg and gemcitabine 1,250 mg/m\u003csup\u003e2\u003c/sup\u003e as an intravenous (IV) infusion. De-escalation to DL-1 (gemcitabine 1,000 mg/m\u003csup\u003e2\u003c/sup\u003e) was planned if DL0 was not tolerable.\u003c/p\u003e\n \u003cp\u003eOnce the RP2D was defined, eligible patients were enrolled in phase II. The primary objective was the objective response rate (ORR), defined as the number of patients with \u0026ge;\u0026thinsp;1 treatment dose and complete response (CR) plus partial response (PR) according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. The secondary objectives were clinical benefit rate (CBR) with stable disease (SD) of \u0026ge;\u0026thinsp;24 weeks, duration of response (DoR), progression-free survival (PFS), OS, and safety and tolerability according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 4.0. The exploratory objectives were to assess the efficacy based on immune-related response criteria, search for tumor tissue and peripheral blood biomarkers for clinical activity, correlate a set of immune biomarkers with disease evolution and efficacy of the combination, and comparison of this set of biomarkers from cohorts of healthy volunteers and patients from this trial.\u003c/p\u003e\n \u003cp\u003eThe study was conducted according to the International Conference on Harmonization Good Clinical Practice Guidelines and Declaration of Helsinki and was approved by the institutional ethical review boards of the participating sites and Spanish health authorities. It was registered at ClinicalTrials.gov and EudraCT (NCT03025880 and 2016-001779-54, respectively). Written informed consent was obtained from all patients before performing any protocol-specific procedures.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003ePatients\u003c/h2\u003e\n \u003cp\u003eThe key inclusion criteria were women aged\u0026thinsp;\u0026ge;\u0026thinsp;18 years; HER2-negative ABC by immunohistochemistry (IHC) and/or \u003cem\u003ein situ\u003c/em\u003e hybridization based on local testing of the most recent tumor biopsy; \u0026ge;10 mm measurable lesion as per the RECIST 1.1; an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0 or 1; prior anthracyclines and taxanes (unless contraindicated), and \u0026ge;\u0026thinsp;2 endocrine therapy (ET) and \u0026le;\u0026thinsp;4 chemotherapy (CT) lines for ABC; patient agreement for a fresh metastatic tumor biopsy at inclusion and progressive disease (PD); adequate organ function; negative pregnancy test for women of child-bearing potential (WCBP) and adequate contraception. Patients with treated brain metastases and stable without steroids were allowed. Healthy controls from the University Hospital Virgen Macarena in Seville, Spain, provided written informed consent to participate in the analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003eTreatment plan\u003c/h2\u003e\n \u003cp\u003ePatients received pembrolizumab (200 mg IV) on day 1 and gemcitabine (1,250 mg/m\u003csup\u003e2\u003c/sup\u003e IV) on days 1 and 8 of each 21-day cycle until objective PD, clinical PD (under investigator\u0026rsquo;s judgment), unacceptable toxicity, death, or consent withdrawal, whichever occurred first.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec6\"\u003e\n \u003ch2\u003eEvaluation procedures\u003c/h2\u003e\n \u003cp\u003eBaseline assessments were performed\u0026thinsp;\u0026le;\u0026thinsp;28 days from starting treatment. These included tumor assessment using radiological tests accepted by the RECIST 1.1, standard 12-lead electrocardiogram, hematology, biochemistry, coagulation test, thyroid hormones, urinalysis, physical examination, ECOG PS evaluation, and pregnancy test in WCBP. Tumor assessments were performed every 9 weeks until PD. AEs were graded using the NCI-CTCAE v4.0. Other safety endpoints included regular monitoring of vital signs and laboratory tests.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003eBiomarker analyses\u003c/h2\u003e\n \u003cp\u003e\u003cstrong\u003eTILs and PD-L1 assessments\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThirty (83%) available pre-treatment metastatic tumor samples were assessed for TILs density, according to the suggested international guidelines(\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e). Cut-offs explored for TILs evaluation were \u0026ge;\u0026thinsp;5%, \u0026ge;\u0026thinsp;10%, and \u0026ge;\u0026thinsp;20%. PD-L1 expression measured using IHC was assessed in 29 pre-treatment metastatic tumor samples using the mAb anti-PD-L1 clone 22C3 (Merck, Kenilworth, NJ). Immune cell (IC) scores and combined positive score (CPS) were obtained. PD-L1 IC score and CPS\u0026thinsp;\u0026ge;\u0026thinsp;1 were considered positive. Cut-offs (\u0026ge;\u0026thinsp;5, \u0026ge;\u0026thinsp;10, and \u0026ge;\u0026thinsp;20) were also explored for CPS.\u003c/p\u003e\n \u003cp\u003eThe logistic and Cox regression models were used to evaluate the association of TILs density and PD-L1 expression with treatment efficacy in terms of ORR, CBR (with SD\u0026thinsp;\u0026ge;\u0026thinsp;24 weeks) and PFS according to the RECIST 1.1.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eImmunophenotyping of whole peripheral blood using flow cytometry analysis\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eBlood samples were collected in EDTA-K3 tubes at baseline, before cycles 3 (C3) and 6 (C6), or at the end of treatment (EOT), whichever occurred first, to determine MDSCs counts. Cell populations were determined using flow cytometry of the whole blood using the BD FACSCanto\u0026trade; system (BD Biosciences, San Jose, CA, USA). Monocytic-MDSCs (M-MDSCs) were defined as CD45\u0026thinsp;+\u0026thinsp;CD11b\u0026thinsp;+\u0026thinsp;CD33\u0026thinsp;+\u0026thinsp;HLA-DR\u0026thinsp;\u0026minus;\u0026thinsp;CD14\u0026thinsp;+\u0026thinsp;CD15\u0026thinsp;\u0026minus;\u0026thinsp;and granulocytic-MDSCs (G-MDSCs) as CD45\u0026thinsp;+\u0026thinsp;CD11b\u0026thinsp;+\u0026thinsp;CD33\u0026thinsp;+\u0026thinsp;HLA-DR\u0026thinsp;\u0026minus;\u0026thinsp;CD14\u0026thinsp;\u0026minus;\u0026thinsp;CD15+.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMonoclonal antibodies\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eAntibodies were obtained from Becton Dickinson Immunocytometry Systems (BD Biosciences, CA, USA) and were used at the manufacturer\u0026rsquo;s recommended concentrations.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec8\"\u003e\n \u003ch2\u003eStatistical analyses\u003c/h2\u003e\n \u003cp\u003eSimon\u0026rsquo;s minimax two-stage design was employed for the phase II part of the study with the option of early stopping owing to lack of response. The sample size was calculated by testing the null hypothesis (H0) that gemcitabine resulted in an ORR of approximately 20%. With the study combination, the alternative hypothesis was 35% (an absolute increase of 15%); with an alpha error of 0.05 and a power of 80%, 53 evaluable patients were required. The first stage should include 31 evaluable patients, and if at least seven presented a response, recruitment would continue to include 53 evaluable patients. The H0 of 20% was rejected if\u0026thinsp;\u0026ge;\u0026thinsp;16 responses were observed in 53 patients. The SAS Enterprise Guide (version 7.15) was used for all analyses.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv class=\"Section2\" id=\"Sec10\"\u003e\n \u003ch2\u003ePatients\u0026rsquo; characteristics\u003c/h2\u003e\n \u003cp\u003eFrom June 2017 to May 2018, 36 patients were recruited for the first stage of Simon\u0026rsquo;s minimax two-stage design (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e); however, only five patients achieved a response, and recruitment was stopped. Their characteristics are presented in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Approximately 58% of the tumors were TN. Twenty-three (64%) patients had up to two metastatic locations, and 28 (78%) had visceral involvement. Patients had a median of four prior therapy lines for ABC.\u003c/p\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePatients and disease characteristics\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCharacteristics\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eN\u0026thinsp;=\u0026thinsp;36\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge, years (median [range])\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e51 (31\u0026ndash;77)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;65, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33 (91.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ge;65, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3 (8.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMenopausal status, n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePostmenopausal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26 (72.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePremenopausal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10 (27.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eECOG PS, n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23 (63.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12 (33.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (2.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTime since 1st \u0026nbsp;BC diagnosis to study inclusion, years (median [range])\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4 (1\u0026ndash;37)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTime\u0026nbsp;since\u0026nbsp;M1 diagnosis\u0026nbsp;to\u0026nbsp;study inclusion, years (median [range])\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (0.2\u0026ndash;14)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of metastatic locations, n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9 (25.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14 (38.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ge;\u0026thinsp;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13 (36.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMetastatic locations, n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVisceral\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28 (77.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026bull; Liver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25 (69.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026bull; Lung\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7 (19.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026bull; Adrenal gland, pericardial effusion, or pleural involvement\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6 (16.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNon-visceral only\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 (22.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026bull; Bone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20 (55.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026bull; Breast\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (5.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026bull; Lymph nodes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13 (36.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026bull; Skin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4 (11.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026bull; Soft tissue\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (13.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eHistological type, n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInvasive ductal carcinoma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33 (91.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInvasive lobular carcinoma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (5.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInvasive squamous carcinoma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (2.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eHistological grade, n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (2.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14 (38.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16 (44.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUnknown\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (13.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eHR status (local), n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21 (58.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15 (41.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eKi67 expression (local) (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMedian (range)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30 (15\u0026ndash;95)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;20%, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (13.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ge;\u0026thinsp;20%, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21 (58.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUnknown\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10 (27.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eBC subtype\u0026nbsp;by\u0026nbsp;IHC (local), n\u0026nbsp;(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21 (58.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHR-positive disease\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15 (41.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrior therapy, n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36 (100.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026bull; ABC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34 (94.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eET\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26 (72.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026bull; ABC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19 (52.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25 (69.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026bull; ABC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24 (66.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of prior lines for ABC, n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (2.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9 (25.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (5.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3 (8.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (13.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7 (19.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ge;\u0026thinsp;6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9 (25.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMedian (range)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4 (0\u0026ndash;11)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026bull; TN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (1\u0026ndash;8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026bull; HR-positive disease\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (3\u0026ndash;11)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\"\u003eAbbreviations: n, number of patients; ECOG PS, Eastern Cooperative Oncology Group performance status; BC, breast cancer; M1, metastases or metastatic disease; HR, hormone receptor; IHC, immunohistochemistry; TN, triple negative; CT, chemotherapy; ET, endocrine therapy; BT, biological therapy; ABC, advanced breast cancer.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cdiv class=\"Section3\" id=\"Sec11\"\u003e\n \u003ch2\u003eRun-in phase and determination of RP2D\u003c/h2\u003e\n \u003cp\u003eFourteen patients were treated with DL0. Three patients were replaced since they experienced early PD (Supplementary Figure, SF1). Of the first six patients, one experienced DLT of grade 3 tumor pain and grade 2 anemia and thrombocytopenia; six additional patients were recruited at this DL without any reported DLT; therefore, DL0 was declared as the RP2D.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec12\"\u003e\n \u003ch2\u003eTreatment exposure\u003c/h2\u003e\n \u003cp\u003eA median of 4 (range, 1\u0026ndash;24) cycles for pembrolizumab and 4.5 (range, 1\u0026ndash;24) cycles for gemcitabine were administered. The median relative dose intensity at the RP2D was 100% (100\u0026ndash;100) and 80% (39\u0026ndash;101), respectively.\u003c/p\u003e\n \u003cp\u003eNine (25%) patients experienced delays in pembrolizumab administration because of AEs in five (13.9%) patients; these AEs included hematological and liver function test alterations, nausea, arthralgia, and infections. Thirty (83.3%) patients experienced dose modifications for gemcitabine, with omissions being the most frequent modification followed by reductions and delays. AEs were the most common reasons for all types of modifications. The main reasons for treatment discontinuation included PD in 29 (80.6%) patients; death in two (5.6%); and AE (grade 3 respiratory failure), patient`s decision, and physician\u0026rsquo;s decision in one (2.8%) patient each. At the time of analysis, two patients were still on treatment.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec13\"\u003e\n \u003ch2\u003eEfficacy\u003c/h2\u003e\n \u003cp\u003eConsidering the efficacy population (n\u0026thinsp;=\u0026thinsp;33), the ORR was 15.2% (n\u0026thinsp;=\u0026thinsp;5/33; 95% confidence interval [CI] 5.1\u0026ndash;31.9), with only PR reported, and the ORR in patients with TN or HR\u0026thinsp;+\u0026thinsp;BC was similar to the ORR in the whole population. Twelve (36.4%) patients achieved SD (one case lasted for \u0026gt;\u0026thinsp;6 months), and 12 (36.4%) experienced PD (Supplementary SF2). The CBR, including SD of any duration, was 51.5% (n\u0026thinsp;=\u0026thinsp;17/33; 95% CI 33.5\u0026ndash;69.2). The median DoR was 4.3 months (95% CI 2.3\u0026ndash;7.4), median PFS was 3.1 months (95% CI 2.0\u0026ndash;4.3), and median OS was 7.9 months (95% CI 6.5\u0026ndash;10.3). Eight patients were on treatment for \u0026ge;\u0026thinsp;6 months before PD (11.4 and 16.1 months in two cases). Efficacy based on immune-related response criteria showed similar results.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec14\"\u003e\n \u003ch2\u003eSafety\u003c/h2\u003e\n \u003cp\u003eAlmost all patients (35 [97.2%]) experienced AE, which was related to the study treatment in 25 (69.4%) patients. AEs led to treatment discontinuation in five (13.9%) patients. Grade 3 AEs were reported in 15 (41.7%) patients and grade 4 AEs in 7 (19.4%), and of these AEs, their relationship with treatment was established by the investigators in 14 (38.9%) patients. Serious AEs were reported in 12 (33.3%) patients and were related to treatment in three cases. No grade 5 AEs related to treatment were reported. Treatment-emergent AEs, irrespective of their causal relationship with the study treatment, are summarized in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u0026nbsp;\u003c/p\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tab2\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eTreatment-emergent adverse events by grade according to the NCI-CTCAE (version 4.03) and with a frequency of at least 5% in any grade\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eSafety Population (n\u0026thinsp;=\u0026thinsp;36)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdverse Event Term\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGrade 1, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGrade 2, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGrade 3, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGrade 4, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePatients with any TEAE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27 (75.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27 (75.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18 (50.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7 (19.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePyrexia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e10 (27.8)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (2.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFatigue\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e11 (30.6)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10 (27.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (5.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAnemia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e8 (22.2)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (5.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3 (8.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNausea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e6 (16.7)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (5.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDecreased appetite\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e4 (11.1)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (2.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMusculoskeletal chest pain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e3 (8.3)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePruritus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e3 (8.3)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUpper respiratory tract infection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e3 (8.3)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlbuminuria\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2 (5.6)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBack pain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2 (5.6)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (2.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHot flush\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2 (5.6)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHypothyroidism\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2 (5.6)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAbdominal pain upper\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (2.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2 (5.6)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eElevated AST level\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (2.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2 (5.6)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eConstipation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (2.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2 (5.6)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (2.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDiarrhea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (2.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e3 (8.3)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (2.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDyspnea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (2.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2 (5.6)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePainful skin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (2.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2 (5.6)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRash\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (2.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2 (5.6)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTachycardia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (2.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2 (5.6)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHypertension\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e4 (11.1)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNeutrophil count decreased\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6 (16.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e10 (27.8)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3 (8.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWeight decreased\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2 (5.6)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003ePembrolizumab-related TEAEs included rash, anemia, decreased neutrophil count, diarrhea, and increased AST and ALT levels.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003eAbbreviations: TEAE, treatment-emergent adverse events; NCI-CTCAE, National Cancer Institute Common Terminology Criteria for Adverse Events; n, number of patients; AST, aspartate aminotransferase\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec15\"\u003e\n \u003ch2\u003eAssociation of TILs density and PD-L1 expression with treatment efficacy\u003c/h2\u003e\n \u003cp\u003eTILs density values are listed in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. No significant associations were observed between TILs density and either ORR/CBR or PFS (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Analysis of the median TILs density distribution according to tumor subtype and ORR showed that TNBC patients who achieved ORR tended to present higher levels of lymphocyte infiltration (Supplementary SF3).\u003c/p\u003e\n \u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e\u003c/p\u003e\n \u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e\u0026nbsp;Logistic and Cox regression models to analyze the association between TILs density and PD-L1 expression (IC and CPS scores) with ORR (CR + PR), CBR (CR +PR + SD of \u0026gt;6 months), and PFS\u003c/strong\u003e\u003c/p\u003e\n \u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"102%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"45.45454545454545%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.181818181818183%\"\u003e\n \u003cp\u003e\u003cstrong\u003eORR\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ep-value\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eOdds ratio (95% CI)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.181818181818183%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCBR\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ep-value\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eOdds ratio (95% CI)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.181818181818183%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePFS\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ep-value\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eHazard ratio (95% CI)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003e\u003cstrong\u003eTILs\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n = 30)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eCut-Off (n, %)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.242424242424242%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026ge;5%\u003c/strong\u003e (24, 80%)\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;5%\u003c/strong\u003e (6, 20%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003e0.556\u003c/p\u003e\n \u003cp\u003e0.47\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(0.04, 5.90)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003e0.534\u003c/p\u003e\n \u003cp\u003e1.37\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(0.51, 3.64)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.76923076923077%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026ge;10%\u003c/strong\u003e (13, 43%)\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;10%\u003c/strong\u003e (17, 57%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.076923076923077%\"\u003e\n \u003cp\u003e0.773\u003c/p\u003e\n \u003cp\u003e1.36\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(0.17, 11.23)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.076923076923077%\"\u003e\n \u003cp\u003e0.554\u003c/p\u003e\n \u003cp\u003e0.50\u003c/p\u003e\n \u003cp\u003e(0.05, 4.98)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.076923076923077%\"\u003e\n \u003cp\u003e0.311\u003c/p\u003e\n \u003cp\u003e1.51\u003c/p\u003e\n \u003cp\u003e(0.68, 3.36)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.76923076923077%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026ge;20%\u003c/strong\u003e (6, 20%)\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;20%\u003c/strong\u003e (24, 80%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.076923076923077%\"\u003e\n \u003cp\u003e0.789\u003c/p\u003e\n \u003cp\u003e1.40\u003c/p\u003e\n \u003cp\u003e(0.12, 16.46)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.076923076923077%\"\u003e\n \u003cp\u003e0.699\u003c/p\u003e\n \u003cp\u003e0.56\u003c/p\u003e\n \u003cp\u003e(0.03, 10.93)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.076923076923077%\"\u003e\n \u003cp\u003e0.073\u003c/p\u003e\n \u003cp\u003e2.50\u003c/p\u003e\n \u003cp\u003e(0.92, 6.82)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePD-L1 Score (n = 29)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eCut-Off (n, %)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.242424242424242%\"\u003e\n \u003cp\u003e\u003cstrong\u003eIC \u0026ge;1%\u003c/strong\u003e (11, 38%)\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eIC \u0026lt;1%\u003c/strong\u003e (18, 62%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003e0.595\u003c/p\u003e\n \u003cp\u003e1.78\u003c/p\u003e\n \u003cp\u003e(0.21, 14.86)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003e0.635\u003c/p\u003e\n \u003cp\u003e0.57\u003c/p\u003e\n \u003cp\u003e(0.06, 5.77)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003e0.321\u003c/p\u003e\n \u003cp\u003e1.51\u003c/p\u003e\n \u003cp\u003e(0.67, 3.40)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.76923076923077%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCPS \u0026ge;1\u003c/strong\u003e (14, 48%)\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eCPS \u0026lt;1\u003c/strong\u003e (15, 52%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.076923076923077%\"\u003e\n \u003cp\u003e0.941\u003c/p\u003e\n \u003cp\u003e1.08\u003c/p\u003e\n \u003cp\u003e(0.13, 8.95)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.076923076923077%\"\u003e\n \u003cp\u003e0.668\u003c/p\u003e\n \u003cp\u003e1.60\u003c/p\u003e\n \u003cp\u003e(0.19, 13.69)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.076923076923077%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.038\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e2.56\u003c/p\u003e\n \u003cp\u003e(1.05, 6.23)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.76923076923077%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCPS \u0026ge;5\u003c/strong\u003e (11, 38%)\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eCPS \u0026lt;5\u003c/strong\u003e (18, 62%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.076923076923077%\"\u003e\n \u003cp\u003e0.571\u003c/p\u003e\n \u003cp\u003e0.50\u003c/p\u003e\n \u003cp\u003e(0.05, 5.51)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.076923076923077%\"\u003e\n \u003cp\u003e0.482\u003c/p\u003e\n \u003cp\u003e2.50\u003c/p\u003e\n \u003cp\u003e(0.19, 32.19)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.076923076923077%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.053\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e2.34\u003c/p\u003e\n \u003cp\u003e(0.99, 5.53)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.76923076923077%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCPS \u0026ge;10\u003c/strong\u003e (8, 28%)\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eCPS \u0026lt;10\u003c/strong\u003e (21, 72%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.076923076923077%\"\u003e\n \u003cp\u003e0.901\u003c/p\u003e\n \u003cp\u003e0.86\u003c/p\u003e\n \u003cp\u003e(0.08, 9.69)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.076923076923077%\"\u003e\n \u003cp\u003e0.793\u003c/p\u003e\n \u003cp\u003e1.43\u003c/p\u003e\n \u003cp\u003e(0.10, 20.44)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.076923076923077%\"\u003e\n \u003cp\u003e0.173\u003c/p\u003e\n \u003cp\u003e1.84\u003c/p\u003e\n \u003cp\u003e(0.77, 4.42)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.76923076923077%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCPS \u0026ge;20\u003c/strong\u003e (7, 24%)\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eCPS \u0026lt;20\u003c/strong\u003e (22, 76%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.076923076923077%\"\u003e\n \u003cp\u003e0.965\u003c/p\u003e\n \u003cp\u003e1.06\u003c/p\u003e\n \u003cp\u003e(0.09, 12.14)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.076923076923077%\"\u003e\n \u003cp\u003e0.793\u003c/p\u003e\n \u003cp\u003e1.43\u003c/p\u003e\n \u003cp\u003e(0.10, 20.44)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.076923076923077%\"\u003e\n \u003cp\u003e0.072\u003c/p\u003e\n \u003cp\u003e2.36\u003c/p\u003e\n \u003cp\u003e(0.93, 6.01)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eAbbreviations: TILs, tumor-infiltrating lymphocytes; ORR, objective response rate; CBR, clinical benefit rate; PFS, progression-free survival; IC, immune cell; CPS, combined positive score; CR, complete response; PR; partial response; SD, stable disease; CI, confidence interval\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eEleven (38%) and 14 (48%) patients showed PD-L1\u0026thinsp;+\u0026thinsp;IC score and CPS at a cut-off of 1; 10 (28%) of them were positive for both scores. No associations were observed between PD-L1 IC score and ORR, CBR, or PFS. For CPS, we observed that patients with a CPS of \u0026ge;\u0026thinsp;1 showed significantly worse PFS (hazard ratio [HR] 2.56; 95% CI 1.05\u0026ndash;6.23; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.038), and the same tendency was observed with a CPS of \u0026ge;\u0026thinsp;5 (HR 2.34; 95% CI 0.99\u0026ndash;5.53; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.053); this association was not maintained with CPS cut-offs of \u0026ge;\u0026thinsp;10 and \u0026ge;\u0026thinsp;20 (HR 1.84; 95% CI 0.77\u0026ndash;4.42; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.173, and HR 2.36; 95% CI 0.93\u0026ndash;6.01; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.072, respectively) (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). No association was observed between CPS, ORR, or CBR. Nine (53%) TN patients were PD-L1\u0026thinsp;+\u0026thinsp;for IC scores\u0026thinsp;\u0026ge;\u0026thinsp;1% and nine (53%) for CPS\u0026thinsp;\u0026ge;\u0026thinsp;1 (seven were positive for both the scores). In the HR\u0026thinsp;+\u0026thinsp;subpopulation, five (38%) patients were positive for CPS, and two (15%) were positive for IC score. PD-L1\u0026thinsp;+\u0026thinsp;scores were not associated with ORR in either TN or HR+ (data not shown). PD-L1 expression by tumor subtype and ORR is shown in Supplementary SF4. PD-L1\u0026thinsp;+\u0026thinsp;IC scores seemed to be more frequent in TNBC patients with no ORR, while the majority of HR\u0026thinsp;+\u0026thinsp;patients not achieving ORR showed PD-L1\u0026thinsp;\u0026minus;\u0026thinsp;IC scores (Supplementary SF4B).\u003c/p\u003e\n \u003cp\u003eFinally, a significant positive correlation between TILs levels and PD-L1 expression was observed (correlation coefficient, 0.567; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00134) (Supplementary SF5).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec16\"\u003e\n \u003ch2\u003eAssociation between MDSCs levels and efficacy\u003c/h2\u003e\n \u003cp\u003ePatients showed significantly higher concentrations of total MDSCs at baseline than did the healthy control cohort (median values of 44.5 vs. 17.5 cells/\u0026micro;L, respectively, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0018). This difference was especially marked for M-MDSCs (median values of 33.5 vs. 10.3 cells/\u0026micro;L, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0010) rather than G-MDSCs (median values of 8.6, vs. 5.0 cells/\u0026micro;l, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.1880) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eAnalysis of variation in MDSCs levels along the treatment revealed significant differences in M-MDSCs levels associated with CBR between baseline-C3 and baseline-C6/EOT (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0313 and 0.0469, respectively) (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). A similar trend, although not significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.1), was also observed for differences between baseline and C6/EOT levels for total MDSCs (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0781).\u003c/p\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tab4\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMDSCs levels (cells/\u0026micro;L) according to tumor response\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"13\"\u003e\n \u003cp\u003eMDSCs\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eM-MDSCs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eG-MDSCs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eCB\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(SD\u0026thinsp;\u0026ge;\u0026thinsp;24 weeks) (n\u0026thinsp;=\u0026thinsp;7)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003ePD (n\u0026thinsp;=\u0026thinsp;12)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eCB\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(SD\u0026thinsp;\u0026ge;\u0026thinsp;24 weeks) (n\u0026thinsp;=\u0026thinsp;7)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003ePD (n\u0026thinsp;=\u0026thinsp;12)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eCB\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(SD\u0026thinsp;\u0026ge;\u0026thinsp;24 weeks) (n\u0026thinsp;=\u0026thinsp;7)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003ePD (n\u0026thinsp;=\u0026thinsp;12)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedian\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eIR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedian\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eIR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedian\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eIR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedian\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eIR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedian\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eIR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedian\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eIR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eBaseline\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.6 (n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47.6\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;11)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.7\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39.8\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;11)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.9\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.2\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;11)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eC3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.4\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60.7\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;11)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e247.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.3\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47.4\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;11)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e223.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.2\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.7\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;11)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eC6/EOT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.4\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.2\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.3\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.0\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.3\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.4\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eBaseline vs. C6/EOT\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003ep-value\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.0781\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.8750\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0469\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.8750\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.6875\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.6250\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eBaseline vs. C3\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003ep-value\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.2969\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.6953\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0313\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.7695\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.9375\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.6250\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCB vs. PD\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(C6/EOT)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003ep-value\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003e0.0513\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003e0.7453\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0348\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCB vs. PD\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(C3)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003ep-value\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003e0.1743\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003e0.0572\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003e0.5261\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCB vs. PD\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(baseline)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003ep-value\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003e0.3651\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003e0.1743\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003e0.9278\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"13\"\u003eAbbreviations: MDSCs, myeloid derived suppressor cells; M-MDSCs, monocytic MDSCs; G-MDSCs, granulocytic MDSCs; CB, clinical benefit; SD, stable disease; PD, progressive disease; IR, interquartile range; C3, cycle 3; C6, cycle 6; EOT, end of treatment\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eAnalysis of the difference in MDSCs levels between patients with CBR or PD at different time points showed significant differences only in case of G-MDSCs at C6/EOT (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0348). No significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.1) were observed at the same time point for total MDSCs levels and at C3 for M-MDSCs levels (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the PANGEA-Breast trial, pembrolizumab plus gemcitabine achieved a modest ORR of 15.2%. No long-term responders were observed, although two patients were still alive at study closing data (22/07/2021). Some facts may explain these results. First, BC patients included in our study were heavily pretreated in most cases, with a median of four prior lines for ABC. Second, there was no patient preselection with respect to PD-L1 expression or TILs density, which confirms that an unselected population is probably an adverse scenario for immunotherapy in ABC. Finally, gemcitabine probably does not harbor powerful immunogenic properties for treating ABC, as we expected. Therefore, other drugs that induce immunogenic cell death, such as anthracyclines, may be more appropriate. Some clinically outstanding results as those reported in the TONIC trial (35% ORR with doxorubicin [15 mg/m\u003csup\u003e2\u003c/sup\u003e] followed by nivolumab in metastatic TNBC patients)(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e) or KEYNOTE-522 study(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) in the neoadjuvant setting, support the hypothesis that the combination of immunotherapy and CT matters, and anthracyclines could trigger and boost the immune response better than other chemotherapeutic drugs. Discordant results between the IMpassion-130(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) and IMpassion-131(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) trials can also be partially explained with this theory. The difference among these two trials was the CT used (nab-paclitaxel or conventional paclitaxel, respectively) and premedication with corticosteroids in the IMpassion-131 trial.\u003c/p\u003e \u003cp\u003eAdditionally, based on tissue immune biomarkers analysis of the PANGEA-Breast trial, we could not identify a subpopulation benefiting from the chemoimmunotherapy combination. Neither TILs density nor PD-L1 expression using CPS revealed a subgroup with a higher probability of response or better survival. The small sample size (n\u0026thinsp;=\u0026thinsp;36) and highly heterogeneous population (e.g., TN and HR\u0026thinsp;+\u0026thinsp;BC, different previous lines of treatment) represent a major limitation at this point; therefore, our results should be considered cautiously, especially when the IMpassion-130 and KEYNOTE-522 trials have reported favorable results in PD-L1 enriched populations.\u003c/p\u003e \u003cp\u003eRegarding the peripheral blood immune biomarker analysis, MDSCs results were intriguing as their levels were clearly elevated in patients than in the healthy cohort (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0018), suggesting that an immunosuppressive status is induced by ABC. These results are concordant with previous findings that correlate higher levels of MDSCs with adverse prognostic factors and tumor burden in ABC(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Interestingly, MDSCs decreased along treatment implementation in the CB group versus PD group with a clear trend at C3 and seem less obvious at C6. MDSCs may represent emerging and valuable biomarkers; however, the limited number of patients and samples in our study jeopardized any major interpretation of our data.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, this trial reinforces the hypothesis that immunotherapy for ABC could eventually work only in highly selected and enriched populations, ideally for first-line therapy. Pretreated patients, especially those heavily pretreated in our work and in the KEYNOTE-119 trial(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) showed little benefit from ICIs, with a trend of improved efficacy with PD-L1 enrichment. For future clinical trials in this setting, better selection of patients with ABC would be advisable. Additionally, unless new results are available, different original strategies should be tested, as chemoimmunotherapy outcomes in ABC appear globally modest. Approaches aiming to induce the host immune system through effective immunogenic cell death modalities, depletion of immunosuppressive cells such as MDSCs or Tregs(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), or favoring neoantigen presentation(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) could widen the spectrum of immunotherapy for ABC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted according to the International Conference on Harmonization Good Clinical Practice Guidelines and Declaration of Helsinki and was approved by the institutional ethical review boards of the participating sites and Spanish health authorities. Written informed consent was obtained from all patients before performing any protocol-specific procedures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from GEICAM. Data are available from the authors upon reasonable request to the principal investigator Dr. de la Cruz ([email protected]) and with permission of the PANGEA-Breast steering committee. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL. de la Cruz-Merino received consulting or advisory role fees from Merck Sharp \u0026amp; Dohme (MSD)-Merck, Roche, Bristol-Myers-Squibb (BMS), Pierre-Fabre, Amgen, and Novartis; research funding from MSD-Merck, Roche, and Celgene; speaker\u0026rsquo;s honoraria from MSD-Merck, Roche, BMS, and Amgen; and grant support from Roche and BMS. M Gion received honoraria from Roche and traveled accommodation from Pfizer. J. Cruz received consulting or advisory role fees from Roche, PharmaMar, Lilly, Novartis, Eisai, Pfizer Amgen, and Celgene; travel accommodation from Roche, Novartis, and PharmaMar. V. Quiroga received speakers\u0026rsquo; bureau honoraria from Pfizer, Novartis, and Roche; research funding from Celgene; and travel accommodation from Novartis, Roche, and Pfizer. F. Moreno received an advisory role honoraria from Pfizer, Roche, Novartis MSD, and AstraZeneca; speaker\u0026rsquo;s bureau honoraria and research funding from Pfizer; and travel accommodation from Pfizer, Roche, and Novartis. R. Andr\u0026eacute;s received travel accommodation from Roche. M. Santisteban received honoraria from Roche, Novartis, and Pfizer; and consulting or advisory role honoraria from Gilead, MSD, Novartis, Pfizer, and Biomerieux. M. Ramos received speakers\u0026rsquo; bureau honoraria from Novartis, Roche, and Pfizer. J. Cort\u0026eacute;s has stock and other ownership interests in MedSIR and has received honoraria from Novartis, Eisai, Celgene, Pfizer, Roche, SAMSUNG, Lilly, MSD, and Daiichi Sankyo; consulting or advisory role honoraria from Celgene, Cellestia Biotech, AstraZeneca, Biothera, Merus, Roche, Seattle Genetics, Daiichi Sankyo, ERYTECH Pharma, Polyphor, Athenex, Lilly, Servier, MSD , GlaxoSmithKline (GSK), Leuko, Clovis Oncology, Bioasis, Boehringer Ingelheim, and Kyowa Kyrin; research funding from ARIAD, AstraZeneca, Baxalta GMBH/Servier Affaires, Bayer, Eisai Farmaceutica, Guardanth health, MSD, Pfizer, Puma CO, Queen Mary University of London, Roche, and Piqur; and travel accommodation from Roche, Pfizer, Eisai, Novartis, and Daiichi Sankyo. E. L\u0026oacute;pez received consulting or advisory role honoraria from AstraZeneca, Pfizer, Roche, and Novartis; and the speaker\u0026rsquo;s bureau honoraria from Roche, Eisai, Pfizer, and Novartis. A. Cort\u0026eacute;s received consulting or advisory role honoraria from Lilly, Roche, Clovis, Ferrer, and Pfizer; speakers\u0026rsquo; bureau honoraria from MSD, GSK, AstraZeneca, Roche and Pfizer, and research funding and travel accommodation from Pfizer. L. M. Rodr\u0026iacute;guez received honoraria from Pfizer and Pierre Fabre. I. Ceballos received consulting or advisory role honoraria from Roche and Merk KGaA; speakers\u0026rsquo; bureau honoraria from Roche, Pfizer, BMS, and Celgene; and travel accommodation from Roche, Merck, Pfizer, and Novartis. F. Rojo received consulting or advisory role fees from Roche, AstraZeneca, Novartis, BMS, MSD, Lilly, Pfizer, Genomic Health, Guardant Health, Archer, and Pierre-Fabre; speaker bureau/expert testimony fees from Roche, AstraZeneca, Novartis, BMS, MSD, Lilly, Pfizer, and Pierre-Fabre; and travel accommodation from Roche and Novartis His institution received research grant/funding from Roche and Pfizer. The rest of authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was partially supported by a research grant from Merck (MSD in Europe) Investigator Initiated Studies Program, which also supplied pembrolizumab.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026acute; contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, L.dC., F.R. and R.C.; Methodology, LdC, F.R., SB, SBe, R.C. and M.Ch.; Software and Validation SB and S.Be.; Formal Analysis, M.C.; Investigation and Data Curation, L.dC., F.R., C.J-C., V.S-M., M.G., J.C., V.Q., R.A., F.M., J.L.A-R., M.R., E.H., J.C., E. L-M., F.H., N.P-C., L.M.R., I.C., M.S., A.C., A.S. and A.P. Resources, L.dC., F.R., C.J-C., V.S-M., M.G., J.C., V.Q., R.A., F.M., J.L.A-R., M.R., E.H., J.C., E. L-M., F.H., N.P-C., L.M.R., I.C., M.S., A.C., A.S., A.P., R.C., M.Ch., S.B and S.Be.; Writing \u0026ndash; Original Draft Preparation, L.dC., F.R., S.B., M.C., R.C., M.Ch. and S.Be.; Writing \u0026ndash; Review \u0026amp; Editing and Visualization, all authors.; Supervision, L.dC. and S.B.; Project Administration, L.dC., S.B. and S.Be.; Funding Acquisition, L.dC., R.C., and S.B.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge the investigators (see list below), pathology departments, and other staff of the participant sites, the patients, and the GEICAM staff involved in this trial. We would like to thank Editage (www.editage.com) for English language editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFood and Drug Administration (FDA): KEYTRUDA\u0026reg; (pembrolizumab) prescribing information 2019 [Available from: https://www.accessdata.fda.gov/drugsatfda_docs/label/2019/125514Orig1s054lbl.pdf.\u003c/li\u003e\n\u003cli\u003eFood and Drug Administration (FDA): OPDIVO (nivolumab) prescribing information. 2019.\u003c/li\u003e\n\u003cli\u003eFood and Drug Administration (FDA): TECENTRIQ\u0026reg; (atezolizumab) prescribing informatioin. 2019.\u003c/li\u003e\n\u003cli\u003eSchmid P, Adams S, Rugo HS, Schneeweiss A, Barrios CH, Iwata H, et al. Atezolizumab and Nab-Paclitaxel in Advanced Triple-Negative Breast Cancer. N Engl J Med. 2018;379(22):2108-21.\u003c/li\u003e\n\u003cli\u003eCortes J, Cescon DW, Rugo HS, Nowecki Z, Im S-A, Yusof MM, et al. Pembrolizumab plus chemotherapy versus placebo plus chemotherapy for previously untreated locally recurrent inoperable or metastatic triple-negative breast cancer (KEYNOTE-355): a randomised, placebo-controlled, double-blind, phase 3 clinical trial. The Lancet. 2020;396(10265):1817-28.\u003c/li\u003e\n\u003cli\u003eRugo HS, Schmid P, Cescon DW, Nowecki Z, Im S-A, Yusof MM, et al. Abstract GS3-01: Additional efficacy endpoints from the phase 3 KEYNOTE-355 study of pembrolizumab plus chemotherapy vs placebo plus chemotherapy as first-line therapy for locally recurrent inoperable or metastatic triple-negative breast cancer. Cancer Res. 2021;81(4 Supplement):GS3-01-GS3-.\u003c/li\u003e\n\u003cli\u003eMiles D AF, Gligorov J, Verma S, Xu B, Cameron D, Barrios C.H, Schneeweiss A, Easton V, Dolado I, O\u0026rsquo;Shaughnessy J. IMpassion131: Phase III study comparing 1L atezolizumab with paclitaxel vs placebo with paclitaxel in treatment-naive patients with inoperable locally advanced or metastatic triple negative breast cancer (mTNBC). Annals of Oncology. 2017;28(September 2017):105.\u003c/li\u003e\n\u003cli\u003eGoldhirsch A, Winer EP, Coates AS, Gelber RD, Piccart-Gebhart M, Thurlimann B, et al. Personalizing the treatment of women with early breast cancer: highlights of the St Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2013. Ann Oncol. 2013;24(9):2206-23.\u003c/li\u003e\n\u003cli\u003eSrivastava MK, Sinha P, Clements VK, Rodriguez P, Ostrand-Rosenberg S. Myeloid-derived suppressor cells inhibit T-cell activation by depleting cystine and cysteine. Cancer Res. 2010;70(1):68-77.\u003c/li\u003e\n\u003cli\u003eSuzuki E, Kapoor V, Jassar AS, Kaiser LR, Albelda SM. Gemcitabine selectively eliminates splenic Gr-1+/CD11b+ myeloid suppressor cells in tumor-bearing animals and enhances antitumor immune activity. Clin Cancer Res. 2005;11(18):6713-21.\u003c/li\u003e\n\u003cli\u003eShevchenko I, Karakhanova S, Soltek S, Link J, Bayry J, Werner J, et al. Low-dose gemcitabine depletes regulatory T cells and improves survival in the orthotopic Panc02 model of pancreatic cancer. Int J Cancer. 2013;133(1):98-107.\u003c/li\u003e\n\u003cli\u003eRettig L, Seidenberg S, Parvanova I, Samaras P, Curioni A, Knuth A, et al. Gemcitabine depletes regulatory T-cells in human and mice and enhances triggering of vaccine-specific cytotoxic T-cells. Int J Cancer. 2011;129(4):832-8.\u003c/li\u003e\n\u003cli\u003eHendry S, Salgado R, Gevaert T, Russell PA, John T, Thapa B, et al. Assessing Tumor-infiltrating Lymphocytes in Solid Tumors: A Practical Review for Pathologists and Proposal for a Standardized Method From the International Immunooncology Biomarkers Working Group: Part 1: Assessing the Host Immune Response, TILs in Invasive Breast Carcinoma and Ductal Carcinoma In Situ, Metastatic Tumor Deposits and Areas for Further Research. Adv Anat Pathol. 2017;24(5):235-51.\u003c/li\u003e\n\u003cli\u003eVoorwerk L, Slagter M, Horlings HM, Sikorska K, van de Vijver KK, de Maaker M, et al. Immune induction strategies in metastatic triple-negative breast cancer to enhance the sensitivity to PD-1 blockade: the TONIC trial. Nat Med. 2019;25(6):920-8.\u003c/li\u003e\n\u003cli\u003eSchmid P, Cortes J, Pusztai L, McArthur H, Kummel S, Bergh J, et al. Pembrolizumab for Early Triple-Negative Breast Cancer. N Engl J Med. 2020;382(9):810-21.\u003c/li\u003e\n\u003cli\u003eSafarzadeh E, Hashemzadeh S, Duijf PHG, Mansoori B, Khaze V, Mohammadi A, et al. Circulating myeloid-derived suppressor cells: An independent prognostic factor in patients with breast cancer. J Cell Physiol. 2019;234(4):3515-25.\u003c/li\u003e\n\u003cli\u003eCort\u0026eacute;s J, Lipatov O, Im SA, Gon\u0026ccedil;alves A, Lee KS, Schmid P, et al. LBA21 - KEYNOTE-119: Phase III study of pembrolizumab (pembro) versus single-agent chemotherapy (chemo) for metastatic triple negative breast cancer (mTNBC). Ann Oncol. 2019;30:v859-v60.\u003c/li\u003e\n\u003cli\u003eHodge JW, Ardiani A, Farsaci B, Kwilas AR, Gameiro SR. The tipping point for combination therapy: cancer vaccines with radiation, chemotherapy, or targeted small molecule inhibitors. Semin Oncol. 2012;39(3):323-39.\u003c/li\u003e\n\u003cli\u003eDrake CG. Combination immunotherapy approaches. Annals of Oncology. 2012;23:viii41-viii6.\u003c/li\u003e\n\u003cli\u003eTorimura T, Iwamoto H, Nakamura T, Koga H, Ueno T, Kerbel RS, et al. Metronomic chemotherapy: possible clinical application in advanced hepatocellular carcinoma. Transl Oncol. 2013;6(5):511-9.\u003c/li\u003e\n\u003cli\u003eGarcia-Aranda M, Redondo M. Immunotherapy: A Challenge of Breast Cancer Treatment. Cancers (Basel). 2019;11(12).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Pembrolizumab, Chemotherapy, HER2-negative, advanced breast cancer, TILs, PD-L1, MDSCs","lastPublishedDoi":"10.21203/rs.3.rs-1638137/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1638137/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: We evaluated a combination of two immunomodulatory agents considering a synergism that may induce long-term clinical benefit in advanced breast cancer (ABC). \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: HER2-negative ABC patients received 21-day cycles of pembrolizumab 200 mg (day 1) and gemcitabine (days 1 and 8). A run-in-phase (6+6 design) was planned with two dose levels (DL) of gemcitabine (1,250 mg/m\u003csup\u003e2\u003c/sup\u003e [DL0]; 1,000 mg/m\u003csup\u003e2\u003c/sup\u003e [DL1]) to determine the recommended phase II dose (RP2D). The primary objective was objective response rate (ORR). Tumor infiltrating lymphocytes (TILs) density and PD-L1 expression in tumors and myeloid-derived suppressor cells (MDSCs) levels in peripheral blood were analyzed. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Fourteen patients were treated with DL0, resulting in RP2D. Thirty-six patients were evaluated during the first stage of Simon’s design. Recruitment was stopped as statistical assumptions were not met. The median age was 52; 21 (58%) patients had triple-negative disease, 28 (78%) visceral involvement, and 27 (75%) ≥2 metastatic locations. Progression disease was observed in 29 patients. ORR was 15% (95% CI, 5–32). Eight patients were treated ≥6 months before progression. Fourteen patients reported grade ≥3 treatment-related adverse events. No association was found between TILs density and treatment efficacy. Patients with negative PD-L1 expression showed significantly prolonged progression-free survival. MDSCs levels were associated with treatment response.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: Pembrolizumab 200 mg and gemcitabine 1,250 mg/m\u003csup\u003e2\u003c/sup\u003e were considered as RP2D. The objective of ORR was not met; however, 22% patients were on treatment for ≥6 months. Tumor PD-L1 expression and MDSCs levels suggested a potential predictive role in this population.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eTrial registration number\u003c/strong\u003e: ClinicalTrials.gov and EudraCT (NCT03025880 and 2016-001779-54, respectively). Registration dates: 20/01/2017 and 18/11/2016, respectively.\u003c/p\u003e","manuscriptTitle":"Pembrolizumab in combination with gemcitabine for patients with HER2-negative advanced breast cancer: GEICAM/2015-04 (PANGEA-Breast) study","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2022-07-27 17:03:41","doi":"10.21203/rs.3.rs-1638137/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-10-12T05:29:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-10-11T15:49:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"26cf0792-1b87-4c4f-a1a4-9df1d66a5d53","date":"2022-09-30T16:57:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-08-31T02:45:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"ef9c097a-e8a3-4222-88ee-085de3696add","date":"2022-08-14T15:31:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"7521de12-1e86-4a5b-8987-9171f71b05d5","date":"2022-08-08T16:55:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-07-08T23:12:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-07-07T17:09:22+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2022-07-07T17:04:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-07-07T12:33:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2022-06-22T15:20:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"afba163d-8b3d-42d2-a932-51137298e60e","owner":[],"postedDate":"July 27th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-11-24T07:14:25+00:00","versionOfRecord":[],"versionCreatedAt":"2022-07-27 17:03:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-1638137","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1638137","identity":"rs-1638137","version":["v2"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}