Binghe Xu,Qingyuan Zhang,Xichun Hu,Qing Li,Tao Sun,Wei Li,Quchang Ouyang,Jingfen Wang,Zhongsheng Tong,Min Yan,Huiping Li,Xiaohua Zeng,Changping Shan,Xian Wang,Xi Yan,Jian Zhang,Yue Zhang,Jiani Wang,Liang Zhang,Ying Lin,Jifeng Feng,Qianjun Chen,Jian Huang,Lu Zhang,Lisong Yang,Ying Tian,Hongyan Shang
Entinostat, a class I selective histone deacetylase inhibitor, plus exemestane for Chinese patients with hormone receptor-positive advanced breast cancer: A multicenter, randomized, double-blind, placebo-controlled, phase 3 trial
Advanced breast cancer,Hormone receptor-positive,Histone deacetylase inhibitors,Phase 3 clinical trial
Entinostat plus exemestane in hormone receptor-positive (HR+) advanced breast cancer (ABC) previously showed encouraging outcomes. This multicenter phase 3 trial evaluated the efficacy and safety of entinostat plus exemestane in Chinese patients with HR + ABC that relapsed/progressed after ≥1 endocrine therapy. Patients were randomized (2:1) to oral exemestane 25 mg/day plus entinostat (n = 235) or placebo (n = 119) 5 mg/week in 28-day cycles. The primary endpoint was the independent radiographic committee (IRC)-assessed progression-free survival (PFS). The median age was 52 (range, 28–75) years and 222 (62.7%) patients were postmenopausal. CDK4/6 inhibitors and fulvestrant were previously used in 23 (6.5%) and 92 (26.0%) patients, respectively. The baseline characteristics were comparable between the entinostat and placebo groups. The median PFS was 6.32 (95% CI, 5.30–9.11) and 3.72 (95% CI, 1.91–5.49) months in the entinostat and placebo groups (HR, 0.76; 95% CI, 0.58–0.98; P = 0.046), respectively. Grade ≥3 adverse events (AEs) occurred in 154 (65.5%) patients in the entinostat group versus 23 (19.3%) in the placebo group, and the most common grade ≥3 treatment-related AEs were neutropenia [103 (43.8%)], thrombocytopenia [20 (8.5%)], and leucopenia [15 (6.4%)]. Entinostat plus exemestane significantly improved PFS compared with exemestane, with generally manageable toxicities in HR + ABC (ClinicalTrials.gov #NCT03538171).
Entinostat, a class I selective histone deacetylase inhibitor, plus exemestane for Chinese patients with hormone receptor-positive advanced breast cancer: A multicenter, randomized, double-blind, placebo-controlled, phase 3 trial

Entinostat plus exemestane in hormone receptor-positive (HR+) advanced breast cancer (ABC) previously showed encouraging outcomes. This multicenter phase 3 trial evaluated the efficacy and safety of entinostat plus exemestane in Chinese patients with HR + ABC that relapsed/progressed after ≥1 endocrine therapy. Patients were randomized (2:1) to oral exemestane 25 mg/day plus entinostat (n = 235) or placebo (n = 119) 5 mg/week in 28-day cycles. The primary endpoint was the independent radiographic committee (IRC)-assessed progression-free survival (PFS). The median age was 52 (range, 28–75) years and 222 (62.7%) patients were postmenopausal. CDK4/6 inhibitors and fulvestrant were previously used in 23 (6.5%) and 92 (26.0%) patients, respectively. The baseline characteristics were comparable between the entinostat and placebo groups. The median PFS was 6.32 (95% CI, 5.30–9.11) and 3.72 (95% CI, 1.91–5.49) months in the entinostat and placebo groups (HR, 0.76; 95% CI, 0.58–0.98; P = 0.046), respectively. Grade ≥3 adverse events (AEs) occurred in 154 (65.5%) patients in the entinostat group versus 23 (19.3%) in the placebo group, and the most common grade ≥3 treatment-related AEs were neutropenia [103 (43.8%)], thrombocytopenia [20 (8.5%)], and leucopenia [15 (6.4%)]. Entinostat plus exemestane significantly improved PFS compared with exemestane, with generally manageable toxicities in HR + ABC (ClinicalTrials.gov #NCT03538171).

Breast cancer (BC) is the most common malignancy among women, accounting for nearly one-third of all new diagnoses. Hormone receptor-positive (HR+) BC is frequently seen, representing about two-thirds of the cases,. Endocrine therapy is often used as a systemic treatment in HR+ BC4, 5, 6. Treatment with selective estrogen receptor (ER) down-regulators and CDK4/6 inhibitors improved patient prognosis; however, HR+ BC has developed resistance to endocrine drugs, with 15%–20% of tumors being intrinsically resistant and 30%–40% acquiring resistance over time,. Altered gene expression by epigenetic modifications causes the development of primary and secondary resistance to endocrine therapy,. Histone deacetylase (HDAC) enzyme is involved in epigenetic modifications. HDAC inhibitors can reverse the epigenetic modifications that cause resistance to endocrine therapies, and induce antitumor immune responses. The ACE trial showed superior progression-free survival (PFS) with endocrine therapy combined with HDAC inhibitors compared to endocrine therapy alone in Chinese patients with HR+ BC that progressed after endocrine therapy. Nevertheless, <10% of patients had prior fulvestrant treatment, and no patient previously received CDK4/6 inhibitors in the ACE study. Therefore, additional evidence of the efficacy and safety of HDAC inhibitors is required in Chinese patients with HR+ BC who previously received CDK4/6 inhibitors and/or fulvestrant and other endocrine therapies after tumor progression. Entinostat is a synthetic benzamide derivative HDAC inhibitor with oral bioavailability, which selectively inhibits Class I and Class IV HDAC enzymes. It possesses a unique characteristic of a long half-life ranging from 33 to 150 h. Entinostat could upregulate ER and aromatase expression and restore cellular sensitivity to nonsteroidal aromatase inhibitors (NSAIs),. A phase 1 study showed tolerability and antitumor activity of exemestane in combination with entinostat in HR+ advanced BC (ABC). The phase 2 (ENCORE301) trial enrolled patients with HR+ ABC that progressed after endocrine therapy without prior CDK4/6 inhibitor treatment, and the exemestane plus entinostat-treated group showed a significant improvement in PFS and overall survival (OS) compared with the exemestane alone group. The phase 3 E2112 trial analyzed patients with HR+ ABC that progressed after endocrine therapy, some of whom were previously treated with CDK4/6 inhibitors or fulvestrant, and the PFS and OS with exemestane plus entinostat treatment did not significantly differ from exemestane therapy alone. However, the ENCORE301 and E2112 trials enrolled patients mainly from North America, and evidence for the efficacy and safety of entinostat in Chinese patients with HR+ ABC is lacking. Therefore, this study aimed to evaluate the efficacy and safety of exemestane plus entinostat in Chinese patients with HR+ ABC that progressed after endocrine therapy.

This randomized, double-blind, placebo-controlled, phase 3 trial was conducted at 35 clinical sites in China. The study was approved by the Ethics Committee of each participating center and conducted in accordance with the principles of Good Clinical Practice, the Declaration of Helsinki, and regulations from China. This study was registered with ClinicalTrials.gov (#NCT03538171). Written informed consent was obtained from every patient before screening and enrollment. Eligible patients were women who were 18–75 years old, with HR+ and human epidermal growth factor receptor-2 negative (HER2–) BC, whose disease had relapsed or progressed after at least one endocrine therapy in an advanced or adjuvant setting, with at least one measurable lesion or bone lesions only, ECOG performance status of 0–1, and life expectancy of ≥12 weeks. HR+ was defined as ER-positive and progesterone receptor-negative or positive. A receptor was considered positive when ≥1% of cells were stained by immunohistochemistry. HER2-positivity was defined as a 3+ score by IHC and/or a positive ISH result. Adjuvant treatment with exemestane was allowed for those with a disease-free interval of >12 months after discontinuation of exemestane. Patients were allowed to undergo ≤1 line of prior chemotherapy (not including neoadjuvant and adjuvant chemotherapies). Key exclusion criteria were history or presence of metastases to the central nervous system, leptomeningeal disease, uncontrollable or severe cardiovascular disorders, clinically significant gastrointestinal abnormalities, and active infection within 14 days of the onset of the study treatment. The full inclusion and exclusion criteria are provided in the appendix.

The participants were randomly assigned at a ratio of 2:1 to receive oral exemestane plus entinostat or exemestane plus placebo, respectively. The subjects were randomized using an interactive web-response system, and the minimization method was utilized to balance the following covariates: previous use of CDK4/6 inhibitors (yes vs. no), fulvestrant (yes vs. no), chemotherapy (yes vs. no), and the presence of visceral metastases (yes vs. no). Patients, investigators, study center staff, the local and central pathologists who reviewed imaging, and the study sponsor were masked to the treatment assignment.

Each treatment cycle had a duration of 28 days. All participants received exemestane tablets 25 mg once daily and placebo or entinostat tablets 5 mg once weekly. Premenopausal/perimenopausal females were required to use luteinizing hormone-releasing hormone agonist goserelin starting from at least 4 weeks before randomization and during the study. The treatment continued until the subject had confirmed progressive disease (PD), intolerable adverse events (AEs), or other conditions judged by the investigator that required drug discontinuation. If grade 3 or 4 toxicities occurred, entinostat/placebo was discontinued for 2 weeks. The administration of entinostat/placebo was restarted at a dose of 3 mg/week if the hematological, gastrointestinal, and metabolic/nutritional toxicities recovered to grade 2 or below, and other non-hematological toxicities recovered to grade 1 or below; otherwise, the study treatment was permanently discontinued. Entinostat/placebo dosage adjustment guidelines are presented in Supporting Information Table S1. Dose adjustment for exemestane (including dose interruption or permanent discontinuation) was conducted in accordance with the approved label.

Tumor assessment was performed every 8 weeks according to the RECIST version 1.1. The primary endpoint was PFS assessed by the independent radiographic committee (IRC), defined as the time from randomization to either the first documented disease progression (PD) or death from any cause. Secondary endpoints were investigator-assessed PFS, OS (defined as the time from randomization to death from any cause), IRC- and investigator-assessed objective response rate [ORR, defined as the proportion of patients with a complete response (CR) or a partial response (PR)], IRC- and investigator-assessed clinical benefit rate (defined as the proportion of patients with an objective response or stable disease (SD) that lasted 24 weeks or longer), and safety. Safety evaluation included laboratory tests (blood routine test, blood biochemical assays, and urine routine test), electrocardiography, physical examination, vital signs, and ECOG performance status. These examinations were performed at the time of screening, the end of each treatment cycle, and the end of treatment. AEs were recorded until 30 days after the last dose or before initiating a new antitumor therapy (whichever occurred first) and graded according to the National Cancer Institute's Common Terminology Criteria for Adverse Events (CTCAE) version 4.03.

A total of 260 PFS events (disease progression or death) in the two groups were required to achieve 90% power to detect a 2.2-month difference in PFS, assuming 4.1 months for the placebo group and 6.3 months for the entinostat group [corresponding to a hazard ratio (HR) of 0.65], with a two-sided 5% level of significance from a log-rank test. Assuming 18 months of recruitment period, 6 months of follow-up time, randomization at a ratio of 2:1 between the entinostat and placebo groups, and 5% loss to follow-up per year, a total of 347 patients (231 subjects in the entinostat group and 116 subjects in the placebo group) were required. Efficacy analyses were performed based on the full analysis set (FAS), comprising all randomized patients. The safety set (SS) included all patients who received at least one dose of the study drug. The Kaplan-Meier method was utilized to construct survival plots and estimate median PFS. The differences in the treatment effects were tested using the permutation test. The HR and its 95% confidence interval (CI) were estimated using univariate and multivariate Cox proportional hazard regression models. All statistical analyses were performed using SAS version 9.4 (SAS Institute, Cary, NC, USA). Two-sided P < 0.05 was considered statistically significant.

From April 16, 2019, to May 13, 2020, 354 patients were enrolled and randomized to the entinostat (n = 235) and placebo (n = 119) groups. The entinostat and placebo groups had 216 (91.9%) and 112 (94.1%) patients with distant metastasis, and 162 (68.9%) and 82 (68.9%) patients with visceral diseases, respectively. The median age was 53 and 51 in the entinostat and placebo groups, respectively, and the median number of previous endocrine therapies for advanced disease was 1 (range, 0–3) in both groups. There were 53 (22.6%) and 32 (26.9%) patients who received entinostat and placebo plus exemestane as the first-line therapy for advanced disease, respectively. Fifteen (6.4%) and eight (6.7%) patients previously received CDK4/6 inhibitors in the entinostat and placebo groups, respectively. Fulvestrant was previously administered to 62 (26.4%) participants in the entinostat group and 30 (25.2%) participants in the placebo group. All 354 participants received at least one dose of the study drug and were included in the FAS and SS (Fig. 1). The characteristics of the participants are shown in Table 1.

The data cut-off date was May 4, 2021, and the median follow-up was 15.7 (range, 0.4−24.0) months. Disease progression or death events occurred in 149 participants in the entinostat group and 88 in the placebo group. Thirty-seven participants in the entinostat group and 14 in the placebo group were still receiving the study drugs. The median IRC-assessed PFS was 6.32 (95% CI, 5.30−9.11) months in the entinostat group and 3.72 (95% CI, 1.91−5.49) months in the placebo group (HR 0.73; 95% CI, 0.56−0.95 from univariate Cox regression analysis, HR 0.76; 95% CI, 0.58−0.98 from multivariate Cox regression analysis; P = 0.046) (Fig. 2A). The median investigator-assessed PFS was 5.76 (95% CI, 5.43−9.11) months in the entinostat group and 4.05 (95% CI, 3.59−5.53) months in the placebo group (Fig. 2B). The results from the subgroup analyses of PFS based on ECOG, visceral metastasis, treatment line for advanced disease, primary endocrine resistance, previous chemotherapy, previous treatment with CDK4/6 inhibitors, and previous fulvestrant treatment were consistent with those from the overall population (Fig. 3). The OS data were immature at the time of analysis. Seventy-one (30.2%) deaths occurred in the entinostat group and 41 (34.5%) occurred in the placebo group (Fig. 2C). Based on the assessment by IRC, the ORRs were 15.7% in the entinostat group and 10.1% in the placebo group (P = 0.192), and the clinical benefit rates were 37.4% and 32.8% (P = 0.413), respectively. Based on the investigator assessment, the ORRs were 17.4% in the entinostat group and 10.9% in the placebo group (P = 0.119), and the clinical benefit rates were 45.1% and 31.9%, respectively (P = 0.022) (Table 2).

The median duration of exemestane treatment was 145 (range, 8−736) days in the entinostat group and 112 (range, 20−676) days in the placebo group. The median treatment duration of entinostat and placebo was 20 (range, 1−106) and 16 (range, 1−97) weeks, respectively. Thirty-three (14.0%) participants in the entinostat group and six (5.0%) in the placebo group discontinued entinostat/placebo due to AEs [AEs (≥1%) leading to entinostat discontinuation included: neutropenia (n = 22, 9.4%), elevated γ-glutamyl transferase (n = 4, 1.7%), and anemia (n = 3, 1.3%)]. Entinostat/placebo dose interruption or reduction was reported in 132 (56.2%) and 8 (6.7%) patients in the entinostat group and placebo groups, respectively. The incidence rate of any grade AE was 99.1% (233/235) in the entinostat group and 88.2% (105/119) in the placebo group. Grade ≥3 AEs occurred in 154 (65.5%) patients in the entinostat group and 23 (19.3%) patients in the placebo group. The most common grade ≥3 treatment-related hematological AEs in the entinostat group were neutropenia [103 (43.8%)], thrombocytopenia [20 (8.5%)], and leukopenia [15 (6.4%)], while the most common grade ≥3 non-hematological treatment-related AE (TRAE) was increased aspartate aminotransferase [6 (2.6%); Table 3]. Grade 5 AEs occurred in five participants in the entinostat group and one in the placebo group. Among the five grade 5 AEs in the entinostat group, two cases were considered possibly related to entinostat treatment by the investigator confounded by cancer progression and concomitant diseases (one autoimmune encephalitis and one febrile neutropenia), and one case of death with exact cause not confirmed (possibly related to disease progression); the other two deaths were not related to entinostat treatment as assessed by the investigator.

This study evaluated the efficacy and safety of entinostat plus exemestane treatment in Chinese patients with HR + ABC that progressed after endocrine therapy. The median IRC-assessed PFS was 6.32 months (95% CI, 5.30−9.11) in the entinostat group and 3.72 months (95% CI, 1.91−5.49) in the placebo group (HR 0.76; 95% CI, 0.58−0.98). Exemestane plus entinostat significantly improved PFS in patients with HR+/HER2− ABC previously treated with endocrine therapy (P = 0.046). The treatment was generally safe, with manageable adverse events, and no new safety signals were observed. The results were consistent with the outcome of the ENCORE301 trial. Epigenetic alterations contribute to endocrine resistance in ER + HER2− ABCs. The repression of ER-α is often related to epigenetic aberration. When treated with entinostat, ER-negative breast cancer cells displayed an increased response to endocrine treatment with aromatase inhibitors in a dose-dependent manner, i.e., re-sensitizing tumor cells to endocrine therapy. The phase 2 ENCORE301 trial showed that exemestane plus entinostat improved PFS in HR+ ABC patients who progressed after NSAI treatment, with a median PFS of 4.3 months compared to 2.3 months in the placebo group; the median OS was also 28.1 months with entinostat compared to 19.8 months with the placebo. The phase 3 ACE trial showed that an HDAC inhibitor (tucidinostat) plus exemestane resulted in a superior PFS than a placebo plus exemestane (7.4 vs. 3.8 months). On the other hand, the phase 3 E2112 trial with exemestane plus entinostat and exemestane plus placebo showed a median PFS of 3.3 and 3.1 months and a median OS of 23.4 and 21.7 months, respectively. Several factors may have been responsible to the inconsistent results between our study and the E2112 trial. First, the baseline characteristics of the patients were different. 1) Different ethnicities. Differences in genetic and non-genetic features of BC were seen between Westerners and East Asians27, 28, 29, and these differences could have influenced the response to entinostat. A meta-analysis found a correlation between HDAC1 overexpression and better OS in Asian patients with breast cancer, but this correlation was not found in European populations. Differences in HDAC expression due to various ethnicities were reported in esophageal carcinoma. Therefore, the role of HDAC in resistance to endocrine therapy might be different among different ethnic groups. Further translational studies will be performed to determine any difference in genetic characteristic between this study and E2112. 2) Age. The median age of the patients in this study was younger than that in the E2112 trial (53 vs. 63 years). In the ACE trial, there appears to be a differential treatment effect in the younger subgroup (<65 years of age) (HR 0.71) relative to the older subgroup (≥65 years) (HR 1.10), although no statistical significance was reached. 3) Prior use of CDK4/6i. The proportion of patients with prior treatment with CDK4/6 inhibitors was relatively lower in this study (6.4%) than that in the E2112 trial (37.2%). The resistance mechanisms to CDK4/6 inhibitors were mainly categorized as cell cycle-specific and cell cycle-nonspecific. A retrospective analysis revealed that patients with prior treatment with CDK4/6 inhibitors exhibited significantly higher tumor mutation burden than those without previous CDK4/6 inhibitors treatment. We speculate that prior treatment with CDK4/6 inhibitors might have been related to a poorer prognosis. Second, the median number of treatment cycles was only three (range, 1−43) in the E2112 trial, and 70% of the participants who received entinostat had dose modiﬁcation (discontinuation, delay, missed, or reduced), indicating relatively low drug exposure. Third, tumor status was assessed every 12 weeks in E2112, in contrast to 8 weeks in this study. Long tumor assessment intervals might fail to capture the differences between the treatment groups. Fourth, E2112 was an investigator-initiated trial with a large number of participating centers (>100 sites) and a very long study period (approximately 6 years). These factors might have posed significant challenges to the trial conduct. Fulvestrant and CDK4/6 inhibitors are currently marketed in China. Some of the patients in this study were previously treated with fulvestrant and/or CDK4/6 inhibitors (both reagents are frequently applied as first-line therapy in current treatment strategy for HR+ HER2− ABC patients). As far as we know, this is the first study to determine the efficacy and safety of HDAC inhibitors in these patients. Subgroup analyses of PFS in Encore 301, ACE, and this trial were all consistent with the results in the overall population, with all P-values for the subgroup interaction of >0.05. The sample sizes of these subgroups are generally small, thus it is difficult to clearly identify the populations that will most likely to benefit from entinostat combination therapy. Further studies are warranted to explore tumor biomarkers and analyze the efficacy and safety of combination therapy in patients with different biomarkers. The present study's safety profile was similar to those observed in the ENCORE301 and E2112 trials,. In the present trial, drug withdrawal due to AEs was observed in 14% of the participants in the entinostat group, close to 11% and 16% in the ENCORE301 and E2112 trials, respectively,. In both previous trials, neutropenia, hypophosphatemia, anemia, leukopenia, fatigue, and diarrhea were the most common grade 3–4 AEs. The most common AEs related to tucidinostat were neutropenia, thrombocytopenia, and leukopenia as well. No new safety signals were observed in the present trial. This trial had limitations as well. It was conducted only in Chinese patients, preventing direct comparisons among races. Second, the proportion of patients with prior CDK4/6 inhibitor therapy was small, and the exact impact of prior therapy on the efficacy of entinostat was unknown. Finally, OS data at the time of data cut-off were not mature enough to support full assessment, and further follow-up for OS is required.

Entinostat plus exemestane significantly improved PFS compared with exemestane alone in patients with HR+/HER2– ABC that progressed after endocrine therapy. The safety profile was generally manageable with the hematological disorders being the most common toxicities. Entinostat plus exemestane could be a new treatment option for Chinese patients with ABC.