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Proxalutamide in patients with AR-positive metastatic breast cancer: Results from an open-label multicentre phase Ib study and biomarker analysis

  • Author Footnotes
    1 Contributed equally as first authors.
    Hanfang Jiang
    Footnotes
    1 Contributed equally as first authors.
    Affiliations
    Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Breast Oncology, Peking University Cancer Hospital & Institute, Beijing, China
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  • Author Footnotes
    1 Contributed equally as first authors.
    Quchang Ouyang
    Footnotes
    1 Contributed equally as first authors.
    Affiliations
    Department of Breast Oncology, Hunan Cancer Hospital, Changsha, Hunan, China
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  • Author Footnotes
    1 Contributed equally as first authors.
    Yongmei Yin
    Footnotes
    1 Contributed equally as first authors.
    Affiliations
    Department of Breast Oncology, Jiangsu Province Hospital, Nanjing, Jiangsu, China
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  • Author Footnotes
    1 Contributed equally as first authors.
    Zhongshen Tong
    Footnotes
    1 Contributed equally as first authors.
    Affiliations
    Department of Breast Oncology, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China
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  • Kunwei Shen
    Affiliations
    Department of Breast Oncology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
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  • Zhongyu Yuan
    Affiliations
    Department of Breast Oncology, Sun-Yat-sen University Cancer Center, Guangzhou, Guangdong, China
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  • Cuizhi Geng
    Affiliations
    Department of Breast Centre, The Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei, China
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  • Yaxin Liu
    Affiliations
    Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Breast Oncology, Peking University Cancer Hospital & Institute, Beijing, China
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  • Guohong Song
    Affiliations
    Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Breast Oncology, Peking University Cancer Hospital & Institute, Beijing, China
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  • Ran Ran
    Affiliations
    Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Breast Oncology, Peking University Cancer Hospital & Institute, Beijing, China
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  • Wei Li
    Affiliations
    Department of Breast Oncology, Jiangsu Province Hospital, Nanjing, Jiangsu, China
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  • Qing Qu
    Affiliations
    Department of Breast Oncology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
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  • Meiyu Wang
    Affiliations
    Suzhou Kintor Pharmaceuticals, Inc., Suzhou, Jiangsu, China
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  • Luping Meng
    Affiliations
    Suzhou Kintor Pharmaceuticals, Inc., Suzhou, Jiangsu, China
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  • Youzhi Tong
    Correspondence
    Corresponding author: Suzhou Kintor Pharmaceuticals, Inc., No. 20 Songbei Road, Suzhou Industrial Park, Jiangsu 215123, China.
    Affiliations
    Suzhou Kintor Pharmaceuticals, Inc., Suzhou, Jiangsu, China
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  • Huiping Li
    Correspondence
    Corresponding author: Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Breast Oncology, Peking University Cancer Hospital & Institute, No. 52nd Fucheng Rd, Haidian District, Beijing 100142, China.
    Affiliations
    Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Breast Oncology, Peking University Cancer Hospital & Institute, Beijing, China
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  • Author Footnotes
    1 Contributed equally as first authors.
Open AccessPublished:September 28, 2022DOI:https://doi.org/10.1016/j.ejca.2022.08.025

      Highlights

      • Proxalutamide showed promising activity in heavily pretreated AR+ mBC patients.
      • Proxalutamide showed an acceptable safety profile in heavily pretreated AR+ mBC.
      • Recommended phase II dose of proxalutamide was defined as 200 mg once daily.
      • AR expression, cell-free DNA yield, CNV might be associated with response.
      • Patients with PIK3CA pathogenic mutation showed longer progression-free survival.

      Abstract

      Aim

      Proxalutamide is a novel second-generation non-steroidal androgen receptor (AR) antagonist. This study aimed to evaluate the preliminary efficacy and safety of proxalutamide in patients with AR-positive metastatic breast cancer (AR+ mBC).

      Methods

      In this open-label, dose-expansion, multicentre phase Ib trial, patients with AR+ mBC (immunohistochemistry [IHC] ≥1%) received proxalutamide orally once daily. Two proxalutamide dose cohorts (cohort A: 200 mg; cohort B: 300 mg) were sequentially investigated. Primary endpoints were disease control rate (DCR) at 8 and 16 weeks and recommended phase II dose (RP2D).

      Results

      Forty-five patients with three median lines (range, 1–13) prior systemic therapy were enrolled (cohort A, n = 30; cohort B, n = 15). Among 39 evaluable patients, DCR at 8 and 16 weeks was 25.6% (95% confidence interval [CI], 11.9–39.4%), with 26.9% in cohort A and 23.1% in cohort B. No patient achieved partial response or complete response. Proxalutamide 200 mg/day was determined as RP2D. The 6-month progression-free survival (PFS) rate was 19.6% (95% CI, 10.2–37.5%). In the triple-negative subgroup, DCR at 8 weeks was 38.5%, with median PFS of 9.1 months (95% CI, 7.8–NA) in those who achieved response at 8 weeks (n = 5). Most common grade 3/4 adverse events were aspartate aminotransferase increase (8.9%) and γ-glutamyltransferase increase (8.9%). By biomarker analysis, patients with moderate AR expression of IHC (26%–75%), PIK3CA pathogenic mutations, or <60 ng/ml cell-free DNA yield showed longer PFS.

      Conclusion

      Proxalutamide showed promising anti-tumour activity with good tolerability in patients with heavily pretreated AR+ mBC, supporting further investigation.

      Trial registration

      This clinical study was prospectively registered at chinadrugtrials.org.cn (Identifier: CTR20170757) and clinical trials.gov (Identifier: NCT04103853).

      Keywords

      1. Introduction

      Metastatic breast cancer (mBC) remains a largely incurable disease in most patients [
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      ]. No regimen is considered as the ‘gold standard’ for metastatic cases, and most patients develop resistance to standard-of-care treatments [
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      Breast cancer.
      ]. Thus, any novel therapy likely to provide a survival advantage in patients is valuable.
      In recent years, the androgen receptor (AR) has been found to be commonly expressed in the majority of breast cancer (BC) cases (60–80%), including in up to 90% of hormone receptor-positive (HR+) subtype, 60–80% of human epidermal growth factor receptor 2-positive (HER2+) subtype, and 30–55% of triple-negative breast cancer (TNBC) cases [
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      ]. AR pathway cross-talks with the oestrogen receptor (ER) [
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      Targeting androgen receptor in estrogen receptor-negative breast cancer.
      ], and other key signalling pathways, such as the phosphoinositide 3-kinase (PI3K)/serine–threonine kinase (Akt)/mammalian target of rapamycin (mTOR) pathway [
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      Targeting the PI3K/AKT pathway for the treatment of prostate cancer.
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      PIK3CA mutations in androgen receptor-positive triple negative breast cancer confer sensitivity to the combination of PI3K and androgen receptor inhibitors.
      ]. Targeting AR therapy, including AR antagonists and agonists, has renewed the interest in BC treatment [
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      ]. AR antagonists show promising anti-tumour activity and tolerable safety, thus being chemo-free alternatives to treat chemo-unresponsive patients with BC [
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      ]. However, the clinical benefits provided by these agents are limited with a clinical benefit rate (CBR) of about 20%, including 19% for bicalutamide [
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      ]. No AR antagonists have been approved for BC treatment. Therefore, novel AR antagonists are urgently needed to improve AR-targeted therapies for mBC.
      Proxalutamide is an oral, newly developed, second-generation non-steroidal AR antagonist [
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      ]. Proxalutamide has a mechanism similar to enzalutamide, but a higher binding affinity (3.4-fold) to AR and stronger potency (2- to 5-fold) to inhibit AR-mediated gene transcription, induce AR downregulation than enzalutamide, and also inhibit endogenous metabolism in prostate cancer cells [
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      ]. Proxalutamide has shown preliminary anti-tumour activity and acceptable safety in castration-resistant prostate cancer [
      • Zhou T.
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      ]. For patients with castration-resistant prostate cancer, there is an ongoing phase I/II trial in the United States of America (NCT03899467) and an ongoing phase III trial in China (CTR20180849). We previously reported that proxalutamide can effectively inhibit AR+ BC growth in mouse xenograft tumour models, and observed that the CBR at 16 weeks was 23.1% (3/13) in patients with mBC in a phase Ia dose-escalation study using a traditional ‘3 + 3’ design [
      • Li H.
      • Song G.
      • Zhou Q.
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      • et al.
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      ]. No dose-limiting toxicities were observed at the five dose levels from 100 to 500 mg, and the maximum tolerated dose was not reached. However, patients in the 400 and 500 mg cohorts complained of fatigue. Among the six evaluable patients with AR-positive (AR+) mBC, three patients showed a response: one in the 500 mg cohort (received 8 weeks of treatment but withdrew because of fatigue) and two in the 200 mg cohort (received over 23 months of treatment). Based on these results, two dose levels of proxalutamide, 200 and 300 mg/day, were selected for dose expansion. Therefore, we designed a phase Ib dose-expansion study to evaluate the preliminary efficacy and safety of proxalutamide monotherapy in patients with pretreated AR+ mBC and determine the recommended phase II dose (RP2D). Exploratory translational analyses were also performed to identify the potential predictive biomarkers of clinical response to proxalutamide.

      2. Materials and methods

      2.1 Study design and patients

      This was an open-label, 7-centre, single-arm, dose-expansion phase Ib study designed to evaluate the preliminary efficacy and safety of proxalutamide monotherapy in patients with pretreated AR+ mBC and to determine the RP2D of proxalutamide. Patients who completed two or more cycles of proxalutamide treatment were eligible for exploratory translational analyses. Eligible patients were women aged ≥18 years with histologically or cytologically confirmed AR+ mBC (immunohistochemistry [IHC] ≥1%) that were refractory or intolerant to standard anti-tumour therapy prior to enrolment. The patients had not used enzalutamide or other AR inhibitors during previous treatment. Patients with central nervous system metastases were excluded from this study. Detailed inclusion and exclusion criteria are described in the Supplemental Methods. The AR-positive status of patients with mBC was assessed using tumour tissue via IHC staining (Supplemental Methods). AR was sub-classified by the intensity of IHC staining into 3 categories: 1–25% (low), 26–75% (moderate), and >75% (high).
      This study was approved by the independent ethics committee of each study centre and was conducted in accordance with the International Ethical Guidelines for Biomedical Research Involving Human Subjects, Good Clinical Practice guidelines, and the Declaration of Helsinki. Written informed consent was obtained from all patients prior to enrolment. This clinical study was prospectively registered at chinadrugtrials.org.cn (Identifier: CTR20170757) and later at ClinicalTrials.gov (Identifier: NCT04103853).

      2.2 Procedures

      Two dose levels of proxalutamide (200 or 300 mg once daily) were used according to the results of the phase I dose-escalation study [
      • Li H.
      • Song G.
      • Zhou Q.
      • Ran R.
      • Jiang H.
      • Zhang R.
      • et al.
      Activity of preclinical and phase I clinical trial of a novel androgen receptor antagonist GT0918 in metastatic breast cancer.
      ]. Thirty eligible patients were enrolled in cohort A and received 200 mg proxalutamide orally once daily for one cycle of 28 d. After completing enrolment in cohort A, fifteen patients were enrolled in cohort B and received 300 mg proxalutamide orally once daily during the 28-day cycle. Patients were not allowed to receive any other anti-tumour treatment, including Chinese medicine, during this trial. Proxalutamide was discontinued in cases of disease progression, unacceptable toxicity, or any other reason. Dose interruptions or modifications were permitted because of treatment-related toxicities. Detailed dose interruption, modification, and discontinuation criteria are presented in Supplemental Methods.

      2.3 Study objectives

      The primary objective was to evaluate the disease control rate (DCR) at 8 and 16 weeks and determine the RP2D of proxalutamide. DCR was defined as the proportion of patients with a complete response (CR), partial response (PR), or stable disease (SD), as per the response evaluation criteria in solid tumours (RECIST) 1.1. The secondary endpoints included overall response rate (ORR), progression-free survival (PFS), safety, and tolerability. Subgroup analyses of the efficacy among different molecular subtypes (HER2+, hormone receptor-positive [HR+]/HER2, and TNBC) were also performed. Detailed definitions of the molecular subtypes are described in Supplemental Methods. The exploratory endpoint was to identify potential predictive biomarkers associated with PFS.

      2.4 Assessments

      Tumour assessments were performed via radiologic evaluation at baseline and every 2 cycles until disease progression or end of treatment and assessed by investigators according to RECIST 1.1. Safety was assessed according to the Common Terminology Criteria for Adverse Events (CTCAE version 4.03) at every visit.
      Peripheral blood samples for exploratory biomarker assessments were collected at baseline, at the end of every 2 cycles, and at the study exit visit. Cell-free DNA (cfDNA) was extracted from the plasma to detect gene mutations and copy number variations (CNVs). The cfDNA yield was quantified at the scheduled visit time during treatment to monitor dynamic changes. The process is described in the Supplemental Methods.

      2.5 Statistical analysis

      As the phase Ib dose-expansion study was an exploratory study, no formal hypothesis testing or sample size calculation was predefined for decision making; 45 patients were planned to be recruited. Demographic and clinical characteristics of the patients, safety, and tumour response were summarised using descriptive statistics. Efficacy analyses were performed for patients who received at least one dose of proxalutamide and had at least one assessment of tumour response. Safety was assessed in all patients who received at least one dose of proxalutamide. The 95% confidence intervals (CIs) of the best tumour response were calculated using the Clopper–Pearson method. PFS was estimated using the Kaplan–Meier method and compared among subgroups using the log-rank test. Statistical analysis was performed using the SAS (Statistical analysis system) software (version 9.4, SAS Institute Inc., Cary, NC, USA).

      3. Results

      3.1 Patient characteristics

      Seventy-three patients were screened for tumour AR expression and 60 (82.2%) were found to be AR+. Of the patients with AR+ tumours, 15 (25%) were excluded from enrolment because of withdrawal of consent (n = 2) or screening failure (n = 13). Finally, 45 eligible patients were enrolled and treated (Fig. 1). Thirty eligible patients were enrolled in cohort A (200 mg orally once daily) from April 19, 2018, to March 7, 2019. Fifteen patients were recruited into cohort B (300 mg orally once daily) between March 11, 2019, and April 16, 2019.
      Fig. 1
      Fig. 1CONSORT diagram. AR, androgen receptors; IHC, immunohistochemistry; N, number of patients.
      The median age of the patients was 54 years (range, 34–79 years) (Table 1). The majority (62.2%) of patients had an AR staining intensity >25%, with AR high 35.6% (n = 16), moderate 26.7% (n = 12), and low 37.8% (n = 17). Fourteen (31.1%) patients had TNBC, eight (17.8%) had HER2+ cancer, and 23 (51.1%) had HR+/HER2 cancer. The most common sites of metastasis were the lymph nodes (64.4%), bones (51.1%), liver (46.7%), and soft tissues (46.7%). Most patients (68.9%) presented with visceral metastasis, but 21 patients (46.7%) had ≥3 metastatic sites. All patients were heavily pretreated, with a median of three (range, 1–13) prior lines of systemic therapy in the metastatic setting.
      Table 1Patient demographics and clinical characteristics at baseline.
      CharacteristicCohort A: 200 mg

      n = 30
      Cohort B: 300 mg

      n = 15
      All patients

      n = 45
      Median age, years (range)56 (38–79)51 (34–71)54 (34–79)
      ECOG PS status, n (%)
       016 (53.3)8 (53.2)24 (53.3)
       114 (46.7)7 (46.7)21 (46.7)
      Receptor status, n (%)
       HER2+3 (10.0)5 (33.3)8 (17.8)
       PgR+9 (30.0)10 (66.7)19 (42.2)
       ER+16 (53.3)12 (80.0)28 (62.2)
      Subtypes, n (%)
      Molecular subtypes were stratified into 3 types according to the ASCO/CAP guidelines.
       HR+/HER215 (50.0)8 (53.3)23 (51.1)
       HER2+3 (10.0)5 (33.3)8 (17.8)
       TNBC12 (40.0)2 (13.3)14 (31.1)
      AR expression, n (%)
      AR expression was stratified by the intensity of IHC staining into 3 categories: 1–25% (low), 26-75% (moderate), and >75% (high).
       Low (1–25%)12 (40.0)5 (33.3)17 (37.8)
       Moderate (26–75%)7 (23.3)5 (33.3)12 (26.7)
       High (>75%)11 (36.7)5 (33.3)16 (35.6)
      Metastatic site, n (%)
       Bone16 (53.3)7 (46.7)23 (51.1)
       Lymph nodes21 (70.0)8 (53.3)29 (64.4)
       Soft tissue15 (50.0)6 (60.0)21 (46.7)
       Skin2 (6.7)1 (6.7)3 (6.7)
       Liver12 (40.0)9 (60.0)21 (46.7)
       Lung10 (33.3)6 (40.0)16 (35.6)
      Site of disease, n (%)
       Visceral19 (63.3)12 (80.0)31 (68.9)
       Nonvisceral11 (36.7)3 (20.0)14 (31.1)
      Number of metastatic sites, n (%)
       14 (13.3)3 (20.0)7 (15.6)
       212 (40.0)5 (33.3)17 (37.8)
       ≥314 (46.7)7 (46.7)21 (46.7)
      Histological type, n (%)
       Invasive ductal30 (100)15 (100)45 (100)
      Prior therapy for primary tumour
       Surgical resection29 (96.7)15 (100)44 (97.8)
       Radiotherapy11 (36.7)6 (40.0)17 (37.8)
      Number of prior lines of therapy in the metastatic setting median (range)3 (1, 13)3 (1, 11)3 (1, 13)
      Previous therapy in the metastatic setting, n (%)30 (100)15 (100)45 (100)
       Hormonal therapy14 (46.7)10 (66.7)24 (53.3)
       Chemotherapy25 (83.3)15 (100)40 (88.9)
       Targeted therapy9 (30.0)7 (46.7)16 (35.6)
      Prior lines of chemotherapy in the metastatic setting, median (range)2 (0, 8)2 (1, 4)2 (0, 8)
      Lines of chemotherapy in the metastatic setting
       05 (16.7)05 (11.1)
       16 (20.0)7 (46.7)13 (28.9)
       28 (26.7)3 (20.0)11 (24.4)
       ≥311 (36.7)5 (33.3)16 (35.6)
      Data are expressed as n (%) or median (range).
      Abbreviations: ECOG PS, Eastern Cooperative Oncology Group performance status; TNBC, triple-negative breast cancer; HR, hormone receptor; HER2, human epidermal growth factor receptor 2; PgR, progesterone receptor; ER, oestrogen receptor; FISH, fluorescence in situ hybridisation; AR, androgen receptor.
      a Molecular subtypes were stratified into 3 types according to the ASCO/CAP guidelines.
      b AR expression was stratified by the intensity of IHC staining into 3 categories: 1–25% (low), 26-75% (moderate), and >75% (high).
      At the time of data cut-off on January 21, 2020, eight (17.8%) patients were still receiving treatment [7(23.3%) in cohort A; 1 (6.7%) in cohort B], while 36 (80.0%) patients [23 (65.7%) in cohort A and 13 (86.7%) in cohort B] had discontinued treatment. The most common reason for discontinuation was disease progression.

      3.2 Efficacy and RP2D

      Among the 39 evaluable patients, none achieved a PR or CR. The ORR was 0%. Ten patients (25.6%) had SD as their best response in the overall population, with seven (26.9%) in cohort A and three (23.1%) in cohort B. The DCRs at 8 and 16 weeks were both 25.6% (95% CI, 11.9–39.4%) in the overall population, with 26.9% (95% CI, 9.9–44.0%) in cohort A and 23.1% (95% CI, 0.2–46.0%) in cohort B (Table 2). Thus, the proxalutamide 200 mg once daily was confirmed as the RP2D. Fig. 2A and B provide additional details regarding the depth and duration of the response.
      Table 2Anti-tumour activity by cohorts in evaluable patients.
      Cohort A: 200 mg (n = 26)Cohort B: 300 mg (n = 13)All evaluable patients (n = 39)
      8 weeks16 weeks8 weeks16 weeks8 weeks16 weeks
      Best objective response
       Complete response000000
       Partial response000000
       Stable disease7 (26.9)7 (26.9)3 (23.1)3 (23.1)10 (25.6)10 (25.6)
       Progressive disease19 (73.1)19 (73.1)10 (76.9)10 (76.9)29 (74.4)29 (74.4)
      Disease control
      Disease control defined as complete response, partial response, or stable disease.
      7 (26.9; 9.9–44.0)7 (26.9; 9.9–44.0)3 (23.1; 0.2–46.0)3 (23.1; 0.2–46.0)10 (25.6; 11.94–39.35)10 (25.6; 11.94–39.35)
      Data are expressed as n (%; 95% CI) or median (95% CI).
      a Disease control defined as complete response, partial response, or stable disease.
      Fig. 2
      Fig. 2Tumour response and progression-free survival data. (A) Waterfall plot of maximum percent change in the target lesion size from baseline among evaluable patients as per the response evaluation criteria in solid tumours (RECIST)-1.1. (B) Time to response and duration of treatment. Each bar represents one patient. (C) Progression-free survival stratified by treatment cohorts. (D) Progression-free survival and (E) best tumour response stratified by molecular subtypes. Molecular subtypes were stratified into three categories: triple-negative breast cancer (TNBC), human epidermal growth factor receptor 2 (HER2)+, and hormone receptor (HR)+/HER2. (F) Progression-free survival stratified by androgen receptor (AR) expression. ∗AR expression was stratified by the intensity of immunohistochemical (IHC) staining into three categories: 1–25% (low), 26–75% (moderate), and >75% (high). AR, androgen receptor; HER2, human epidermal growth factor receptor 2; TNBC, triple-negative breast cancer; HR, hormone receptor; SD, stable disease; PD, disease progression.
      The 3-month and 6-month PFS rates of all evaluable patients were 25.2% (95% CI, 14.6–43.5%) and 19.6% (95% CI, 10.2–37.5%), respectively. There was no significant difference in the median PFS between cohorts A and B (p = 0.963, Fig. 2C).
      The efficacy by molecular subtype and AR expression was explored in 39 evaluable patients. DCRs at 8 weeks in TNBC, HER2+, and HR+/HER2 subgroups were 38.5% (5/13) (95% CI, 12.0–64.9%), 20.0% (1/5) (95% CI, 0.0–55.1%), and 19.0% (4/21) (95% CI, 2.3%–35.8%), respectively (p = 0.431, Fig. 2E). Although there was no correlation with the subtypes detected, TNBC patients tended to have better DCR than HER2+ patients (38.5% versus 20.0%, p = 0.615) and HR+/HER2 patients (38.5% versus 19.0%, p = 0.254). The median PFS did not show significantly statistical differences among the three subgroups (p = 0.212, Fig. 2D). Of note, the median PFS was 9.1 months (95% CI, 7.8-not reached) in the triple-negative subgroup that achieved a response at 8 weeks (n = 5), and 7.2 months (95% CI, 3.6-not reached) in the HR+/HER2 subgroup who achieved a response at 8 weeks (n = 4). Interestingly, we found that patients with moderate AR expression (26–75%) had a longer median PFS than those with low (1–25%) or high (>75%) expression (p = 0.043, Fig. 2F).

      3.3 Safety and tolerability

      All 45 patients were evaluable for safety. Overall, 43 patients (95.6%) experienced at least one AE of any grade (29 [96.7%] in cohort A; 14 [93.3%] in cohort B, Table 3). The most common toxicities of any grade were fatigue (46.7%), aspartate aminotransferase (AST) increase (33.3%), anorexia (22.2%), urinary tract infection (20.0%), and alanine aminotransferase (ALT) increase (20.0%). The most common grade 3 or 4 AEs were AST increase (8.9%) and γ-glutamyltransferase increase (8.9%). Among all patients, only seven (15.6%) had serious AE (six [20.0%] in cohort A; one [6.7%] in cohort B). Grade 3 or more serious AEs only occurred in two patients in cohort A (one heart failure and pneumonitis; one platelet count decreased) and one in cohort B (abdominal pain). AEs that occurred in 38 (84.4%) patients were determined as related to proxalutamide (25 [83.3%] in cohort A; 13 [86.7%] in cohort B). No treatment-related deaths or dose reductions occurred in either cohort.
      Table 3Treatment-related adverse events (≥10% or any toxicities with grade 3 or higher).
      Cohort A: 200 mg (n = 30)Cohort B: 300 mg (n = 15)All patients (n = 45)
      All gradesGrade 3Grade 4All gradesGrade 3Grade 4All gradesGrade 3Grade 4
      Any events
      Any events were defined as all treatment-emergent adverse events regardless of relationship to study drug.
      29 (96.7)7 (23.3)7 (23.3)14 (93.3)3 (20.0)043 (95.6)10 (22.2)7 (15.6)
      Any serious events
      Serious events were defined as events that result in death, hospital admission or prolongation of a hospital admission, persistent or significant disability/incapability, congenital anomaly/birth defect, or life-threatening, or any other medically important events.
      6 (20.0)02 (6.7)1 (6.7)1 (6.7)07 (15.6)1 (2.2)2 (4.4)
      Any drug-related events
      Drug-related events were defined as any events identified by the investigator as possibly, probably, or definitely related to proxalutamide.
      25 (83.3)5 (16.7)2 (6.7)13 (86.7)2 (13.3)038 (84.4)7 (15.6)2 (4.4)
       Haematologic toxicity
      Neutrophil count decreased7 (23.3)02 (6.7)0007 (15.6)02 (4.4)
      White blood cell decreased6 (20.0)2 (6.7)01 (6.7)007 (15.6)2 (4.4)0
      Anaemia8 (26.7)1 (3.3)01 (6.7)009 (20.0)1 (2.2)0
      Platelet count decreased3 (10.0)1 (3.3)1 (3.3)0003 (6.7)1 (2.2)1 (2.2)
       Non-haematologic toxicity
      Fatigue14 (46.7)1 (3.3)07 (46.7)0021 (46.7)1 (2.2)0
      AST increased9 (30.0)4 (13.3)06 (40.0)0015 (33.3)4 (8.9)0
      Anorexia9 (30.0)2 (6.7)01 (6.7)0010 (22.2)2 (4.4)0
      Urinary tract infection7 (23.3)002 (13.3)009 (20.0)00
      ALT increased5 (16.7)2 (6.7)04 (26.7)009 (20.0)2 (4.4)0
      Cholesterol high6 (20.0)002 (13.3)008 (17.8)00
      Constipation6 (20.0)000006 (13.3)00
      γ-GT increased4 (13.3)1 (3.3)2 (6.7)3 (20.0)1 (6.7)07 (15.6)2 (4.4)2 (4.4)
      Weight loss4 (13.3)003 (20.0)007 (15.6)00
      LDH increased4 (13.3)003 (20.0)007 (15.6)00
      Hypertriglyceridemia3 (10.0)003 (20.0)006 (13.3)00
      Alkaline phosphatase increased3 (10.0)1 (3.3)02 (13.3)005 (11.1)1 (2.2)0
      Bilirubin increased4 (13.3)01 (3.3)1 (6.7)005 (11.1)01 (2.2)
      Cholesterol high4 (13.3)1 (3.3)01 (6.7)005 (11.1)1 (2.2)0
      Nausea4 (13.3)002 (13.3)006 (13.3)00
      Insomnia2 (6.7)003 (20.0)005 (11.1)00
      Vomiting5 (16.7)000005 (11.1)00
      Diarrhoea3 (10.0)001 (6.7)004 (8.9)00
      Fever1 (3.3)003 (20.0)004 (8.9)00
      Dizziness1 (3.3)002 (13.3)003 (6.7)00
      Palpitation1 (3.3)002 (13.3)003 (6.7)00
      Proteinuria2 (6.7)002 (13.3)004 (8.9)00
      Spasticity0002 (13.3)002 (4.4)00
      Cough3 (10.0)001 (6.7)004 (8.9)00
      Pleural effusion3 (10.0)000003 (6.7)00
      Pain3 (10.0)1 (3.3)00003 (6.7)00
      Heart failure001 (3.3)000001 (2.2)
      Pneumonitis001 (3.3)000001 (2.2)
      Abdominal pain00001 (6.7)001 (2.2)0
      Data are expressed as n (%).
      AST, aspartate aminotransferase. γ-GT, γ-glutamyltransferase; LDH, lactic dehydrogenase; ALT, alanine aminotransferase.
      a Any events were defined as all treatment-emergent adverse events regardless of relationship to study drug.
      b Serious events were defined as events that result in death, hospital admission or prolongation of a hospital admission, persistent or significant disability/incapability, congenital anomaly/birth defect, or life-threatening, or any other medically important events.
      c Drug-related events were defined as any events identified by the investigator as possibly, probably, or definitely related to proxalutamide.
      Nine patients (20.0%) [5 (16.7%) in cohort A; 4 (26.7%) in cohort B] occurred dose interruptions. Of these, patients interrupted treatment because of AEs in cohort A and forgetting doses in cohort B. Three patients (10.0%) in cohort A discontinued treatment due to AEs.

      3.4 Exploratory analyses

      Of the 39 evaluable patients, 31 (20 in cohort A and 11 in cohort B) had available blood samples for circulating tumour DNA sequencing and were included in the translational sub-study. AR splicing variants (AR-V3 and AR-V7) were detected in five (16.1%) patients but showed no association with clinical outcomes (Supplementary Table 1). We identified 19 patients (61.3%) with TP53 mutation. However, there was no significant difference in PFS between the TP53 mutant population and the TP53 wild-type population (p = 0.65; Fig. 3A). PIK3CA mutations were detected in 17 patients (54.9%) (100% [9/9] TNBC, 33.3% [6/18] HR+/HER2, 50% [2/4] HER2+) and 14 patients (45.2%) (77.7% [7/9] TNBC, 33.3% [6/18] HR+/HER2, and 25% [1/4] HER2+) with pathogenic mutations in PIK3CA. The p.E542K mutation in the PIK3CA gene was identified in two long-responders (PFS, 6.6 and 9.2 months, respectively). PIK3CA p.H1047R was identified in three patients with a PFS of nearly two months. Overall, patients with PIK3CA pathogenic mutations achieved a survival advantage compared to others with PIK3CA wild-type (p = 0.048; Fig. 3B). Additionally, we found that CNVs in the AKT, CCND1, FEFR1, MYC, and NF2 genes were frequently detected in the short-responder (PFS<1.84 months) patients both at baseline (9.7%, 6.5%, 12.9%, 6.5%, and 3.2%, respectively) and the end of treatment (12.9%, 19.4%, 16.1%, 12.9%, and 12.9%, respectively), indicating that CNVs might be associated with a poor response to proxalutamide therapy (Fig. 3C).
      Fig. 3
      Fig. 3Exploratory analysis for potential genomic biomarkers for response to therapy. (A–B) Kaplan–Meier survival plots of progression-free survival stratified by (A) TP53 mutation status or (B) phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) pathogenic mutation status in 31 evaluable patients with available blood samples; (C) Copy number variations at baseline and end of treatment (EOT) in 31 evaluable patients with available blood samples; (D) Dynamic change in cell-free DNA (cfDNA) yield in five long-responder patients; (E) cfDNA yield at baseline and EOT in patients with TNBC. cfDNA, Cell-free DNA; EOT, end of treatment; PFS, progression-free survival; TNBC, triple-negative breast cancer.
      In five long-responder patients (PFS>7 months), cfDNA yield was maintained at low levels (<60 ng/ml) [
      • Keup C.
      • Storbeck M.
      • Hauch S.
      • Hahn P.
      • Sprenger-Haussels M.
      • Tewes M.
      • et al.
      Cell-free DNA variant sequencing using CTC-depleted blood for comprehensive liquid biopsy testing in metastatic breast cancer.
      ] or tended to decline from baseline to the end of treatment (Fig. 3D). Especially in the majority (7/10, 70%) of TNBC patients, the relatively longer PFS was generally accompanied by low cfDNA yield (<60 ng/ml) during treatment (Fig. 3E).

      4. Discussion

      This phase Ib expansion study is part of the first prospective trial of proxalutamide in patients with AR+ mBC. This study demonstrated the promising anti-tumour activity of proxalutamide with an acceptable safety profile in heavily pretreated patients with AR+ mBC. Notably, there was no difference in efficacy between patients administered 200 mg and 300 mg orally once daily. Thus, the RP2D of proxalutamide was determined as 200 mg orally once daily. This finding was similar to the recommended dose of proxalutamide for the phase III trial in patients with metastatic castration-resistant prostate cancer, which is 200 mg once daily [
      • Zhou T.
      • Ye D.
      • Sun Z.
      • Meng Q.
      • He D.
      • Shi G.
      • et al.
      A phase II, multicenter, randomized, open-label study to evaluate the safety and tolerability of proxalutamide (GT0918) in subjects with metastatic castrate-resistant prostate cancer (mCRPC).
      ].
      This study demonstrated that proxalutamide had a comparable DCR (25.6%) at both 8 and 16 weeks in the overall population to the results of our previous phase I dose-escalation study (DCR of 23.1% at 16 weeks) [
      • Li H.
      • Song G.
      • Zhou Q.
      • Ran R.
      • Jiang H.
      • Zhang R.
      • et al.
      Activity of preclinical and phase I clinical trial of a novel androgen receptor antagonist GT0918 in metastatic breast cancer.
      ]. Meanwhile, the response to proxalutamide in our study was slightly higher than the findings from other AR antagonists under investigation in patients with AR+ mBC (approximately CBR of 20%) previously reported [
      • Gucalp A.
      • Tolaney S.
      • Isakoff S.J.
      • Ingle J.N.
      • Liu M.C.
      • Carey L.A.
      • et al.
      Phase II trial of bicalutamide in patients with androgen receptor-positive, estrogen receptor-negative metastatic Breast Cancer.
      ,
      • Traina T.A.
      • Miller K.
      • Yardley D.A.
      • Eakle J.
      • Schwartzberg L.S.
      • O'Shaughnessy J.
      • et al.
      Enzalutamide for the treatment of androgen receptor-expressing triple-negative breast cancer.
      ,
      • Bonnefoi H.
      • Grellety T.
      • Tredan O.
      • Saghatchian M.
      • Dalenc F.
      • Mailliez A.
      • et al.
      A phase II trial of abiraterone acetate plus prednisone in patients with triple-negative androgen receptor positive locally advanced or metastatic breast cancer (UCBG 12-1).
      ]. This preliminary anti-tumour activity was encouraging, considering the sample size. However, no patients achieved ORR in this phase Ib study, while CR or PR were observed in patients treated with enzalutamide in the MDV3100-11 trial [
      • Traina T.A.
      • Miller K.
      • Yardley D.A.
      • Eakle J.
      • Schwartzberg L.S.
      • O'Shaughnessy J.
      • et al.
      Enzalutamide for the treatment of androgen receptor-expressing triple-negative breast cancer.
      ]. This discrepancy might be explained by the different sample sizes and different subjects in these two trials. The MDV3100-11 trial was a single-arm, phase II trial in which enzalutamide-treated 118 AR+ metastatic TNBC patients received one median line of prior therapy (range, 0–7) [
      • Traina T.A.
      • Miller K.
      • Yardley D.A.
      • Eakle J.
      • Schwartzberg L.S.
      • O'Shaughnessy J.
      • et al.
      Enzalutamide for the treatment of androgen receptor-expressing triple-negative breast cancer.
      ], while our study was a phase Ib trial that enrolled 45 patients with AR+ mBC with three median lines of prior systemic therapy (range, 1–13), only including 14 TNBC (31.1%). Importantly, the subgroup analysis in our study showed that TNBC patients tended to have a better DCR of 38.5%. Furthermore, we found that the median PFS was 9.1 months for five TNBC patients who achieved a response at 8 weeks. This finding highlighted that proxalutamide might provide a long-term survival advantage for TNBC patients who have achieved preliminary efficacy. Moreover, as a cross-sectional analysis reported, BC in Asian women is more aggressive than that in Caucasians [
      • Tea M.K.
      • Fan L.
      • Delancey J.W.
      • Staudigl C.
      • Steurer S.
      • Lang C.
      • et al.
      Is breast cancer in young Asian women more aggressive than in Caucasians? A cross-sectional analysis.
      ].
      In this study, we observed that CBR at 8 weeks in HR+/HER2 subgroup was 19.0% (4/21), with 2 patients sustaining disease stability over 9 months, indicating that proxalutamide as an AR antagonist may have modest anti-tumour activity against heavily pretreated HR+/HER2 mBC. Based on these promising but modest data, we are conducting a phase Ic trial of proxalutamide in combination with fulvestrant in patients with AR+/ER+/HER2 mBC who had progressed on an aromatase inhibitor (CTR20191063). This phase Ic trial completed recruitment. Rough analyses showed a notably good clinical outcome (data will be disclosed). Interestingly, enobosarm as an AR agonist showed promising anti-tumour activity in patients with heavily pretreated AR+/ER+ mBC in a phase II study [
      • Palmieri C.
      • Linden H.M.
      • Birrell S.
      • Lim E.
      • Schwartzberg L.S.
      • Rugo H.S.
      • et al.
      Efficacy of enobosarm, a selective androgen receptor (AR) targeting agent, correlates with the degree of AR positivity in advanced AR+/estrogen receptor (ER)+ breast cancer in an international phase 2 clinical study.
      ]. These clinical findings are consistent with the preclinical data observed in in vivo ER+/AR+ breast cancer models, in which AR antagonists and agonists both inhibited tumour growth [
      • Venema C.M.
      • Bense R.D.
      • Steenbruggen T.G.
      • Nienhuis H.H.
      • Qiu S.Q.
      • van Kruchten M.
      • et al.
      Consideration of breast cancer subtype in targeting the androgen receptor.
      ]. Therefore, further investigation is warranted.
      Thus far, the predictive value of AR expression for therapeutic response remains controversial. We observed that 62.2% of patients had an AR staining intensity >25%, and patients with moderate AR IHC staining had a relatively longer median PFS, suggesting that AR expression by IHC might be a potential predictive biomarker for use of proxalutamide. However, AR expression intensity by IHC was not associated with the benefit of enzalutamide in patients with AR+ TNBC [
      • Lehmann B.D.
      • Abramson V.G.
      • Sanders M.E.
      • Mayer E.L.
      • Haddad T.C.
      • Nanda R.
      • et al.
      TBCRC 032 IB/II multicenter study: molecular insights to AR antagonist and PI3K inhibitor efficacy in patients with AR(+) metastatic triple-negative breast cancer.
      ] or AR+/HR+/HER2 mBC [
      • Krop I.
      • Abramson V.
      • Colleoni M.
      • Traina T.
      • Holmes F.
      • Garcia-Estevez L.
      • et al.
      A randomized placebo controlled phase II trial evaluating exemestane with or without enzalutamide in patients with hormone receptor-positive breast cancer.
      ]. AR expression by IHC ≥40% was found to be associated with the benefit of enobosarm in patients with AR+/ER+ mBC [
      • Palmieri C.
      • Linden H.M.
      • Birrell S.
      • Lim E.
      • Schwartzberg L.S.
      • Rugo H.S.
      • et al.
      Efficacy of enobosarm, a selective androgen receptor (AR) targeting agent, correlates with the degree of AR positivity in advanced AR+/estrogen receptor (ER)+ breast cancer in an international phase 2 clinical study.
      ], which is very similar to our findings. Considering the small sample size and possible selection bias, further studies with larger cohorts are warranted to identify which patients are likely to benefit most from proxalutamide.
      In the present study, the majority of AEs were grade 1 or 2, with no grade 5 AEs. The most common AEs of any grade were fatigue (46.7%), with only one (2.2%) case of grade 3, and AST increase (33.3%). The most common grade 3 or 4 AEs were AST increase (8.9%) and γ-glutamyltransferase increase (8.9%). These frequent events were usually mild, tolerable, reversible, and resolved soon with dose interruptions and supportive care. The safety profile of proxalutamide in patients with mBC is also similar to that observed in prostate cancer [
      • Zhou T.
      • Xu W.
      • Zhang W.
      • Sun Y.
      • Yan H.
      • Gao X.
      • et al.
      Preclinical profile and phase I clinical trial of a novel androgen receptor antagonist GT0918 in castration-resistant prostate cancer.
      ,
      • Zhou T.
      • Ye D.
      • Sun Z.
      • Meng Q.
      • He D.
      • Shi G.
      • et al.
      A phase II, multicenter, randomized, open-label study to evaluate the safety and tolerability of proxalutamide (GT0918) in subjects with metastatic castrate-resistant prostate cancer (mCRPC).
      ,
      • Vogelzang N.J.
      • Levin R.
      • Rezazadeh A.
      • Park C.H.
      • Bolemon B.H.
      • Gabrail N.Y.
      • et al.
      Preliminary analysis of a U.S. phase II study of the safety and tolerability of proxalutamide (GT0918) in subjects with mCRPC who had progressed on either abiraterone (Abi) or enzalutamide (Enza).
      ] or other AR antagonists (e.g., enzalutamide) [
      • Traina T.A.
      • Miller K.
      • Yardley D.A.
      • Eakle J.
      • Schwartzberg L.S.
      • O'Shaughnessy J.
      • et al.
      Enzalutamide for the treatment of androgen receptor-expressing triple-negative breast cancer.
      ]. Generally, from a safety perspective, proxalutamide had an acceptable safety profile in this population, without novel safety signals.
      We conducted another exploratory analysis to identify potential predictive biomarkers of treatment response. Recently, AR-V7 was identified in patients with BC and may lead to AR inhibitor therapy resistance [
      • Lehmann B.D.
      • Abramson V.G.
      • Sanders M.E.
      • Mayer E.L.
      • Haddad T.C.
      • Nanda R.
      • et al.
      TBCRC 032 IB/II multicenter study: molecular insights to AR antagonist and PI3K inhibitor efficacy in patients with AR(+) metastatic triple-negative breast cancer.
      ,
      • Ferguson D.C.
      • Mata D.A.
      • Tay T.K.
      • Traina T.A.
      • Gucalp A.
      • Chandarlapaty S.
      • et al.
      Androgen receptor splice variant-7 in breast cancer: clinical and pathologic correlations.
      ], similar to that in prostate cancer [
      • Armstrong A.J.
      • Halabi S.
      • Luo J.
      • Nanus D.M.
      • Giannakakou P.
      • Szmulewitz R.Z.
      • et al.
      Prospective multicenter validation of androgen receptor splice variant 7 and hormone therapy resistance in high-risk castration-resistant prostate cancer: the PROPHECY study.
      ]. In our study, although AR splicing variants (AR-V3 and AR-V7) were detected in five (16.1%) patients, they showed no association with clinical outcomes. This clinical finding might support preclinical evidence that proxalutamide is active in AR splicing variants, both resistance mutations and reverting mutations [
      • Zhou T.
      • Xu W.
      • Zhang W.
      • Sun Y.
      • Yan H.
      • Gao X.
      • et al.
      Preclinical profile and phase I clinical trial of a novel androgen receptor antagonist GT0918 in castration-resistant prostate cancer.
      ].
      Activating mutations in PIK3CA are found in approximately 30%–40% of patients with BC, making them one of the most common genetic aberrations in BC [
      • Kalinsky K.
      • Jacks L.M.
      • Heguy A.
      • Patil S.
      • Drobnjak M.
      • Bhanot U.K.
      • et al.
      PIK3CA mutation associates with improved outcome in breast cancer.
      ,
      Comprehensive molecular portraits of human breast tumours.
      ,
      • Cizkova M.
      • Susini A.
      • Vacher S.
      • Cizeron-Clairac G.
      • Andrieu C.
      • Driouch K.
      • et al.
      PIK3CA mutation impact on survival in breast cancer patients and in ERα, PR and ERBB2-based subgroups.
      ,
      • Di Leo A.
      • Johnston S.
      • Lee K.S.
      • Ciruelos E.
      • Lønning P.E.
      • Janni W.
      • et al.
      Buparlisib plus fulvestrant in postmenopausal women with hormone-receptor-positive, HER2-negative, advanced breast cancer progressing on or after mTOR inhibition (BELLE-3): a randomised, double-blind, placebo-controlled, phase 3 trial.
      ], and are more frequent in the HR+/HER2 subtype (42%), followed by the HER2+ subtype (31%) and TNBC subtype (16%) [
      • Martínez-Sáez O.
      • Chic N.
      • Pascual T.
      • Adamo B.
      • Vidal M.
      • González-Farré B.
      • et al.
      Frequency and spectrum of PIK3CA somatic mutations in breast cancer.
      ]. We observed that 45.2% (14/31) of patients (77.7% [7/9] TNBC; 33.3% [6/18] HR+/HER2; 25% [1/4] HER2+) had PIK3CA pathogenic mutations, which was inconsistent, probably due to the small sample size. In our study, patients with PIK3CA pathogenic mutations presented better PFS than patients with PIK3CA wild-type. These findings may be explained by the potentially good prognostic implication of the PIK3CA mutation, which translates into improved clinical outcomes [
      • Kalinsky K.
      • Jacks L.M.
      • Heguy A.
      • Patil S.
      • Drobnjak M.
      • Bhanot U.K.
      • et al.
      PIK3CA mutation associates with improved outcome in breast cancer.
      ]. We also observed that cfDNA yield was maintained at low levels or decreased steadily post-treatment in five long-responder patients receiving proxalutamide. A recent study showed that the total cfDNA level is a predictor of response and PFS in patients with mBC [
      • Fernandez-Garcia D.
      • Hills A.
      • Page K.
      • Hastings R.K.
      • Toghill B.
      • Goddard K.S.
      • et al.
      Plasma cell-free DNA (cfDNA) as a predictive and prognostic marker in patients with metastatic breast cancer.
      ], which was comparable to our findings. For note, cfDNA monitors tumour burden limited by tumour heterogeneity in mBC [
      • García-Saenz J.A.
      • Ayllón P.
      • Laig M.
      • Acosta-Eyzaguirre D.
      • García-Esquinas M.
      • Montes M.
      • et al.
      Tumor burden monitoring using cell-free tumor DNA could be limited by tumor heterogeneity in advanced breast cancer and should be evaluated together with radiographic imaging.
      ]. Thus, we assumed that cfDNA might be a surrogate biomarker for monitoring changes in tumour burden and response to proxalutamide in mBC. In contrast, CNVs were frequently detected in short-responder patients in our study, indicating that CNVs might also have the ability to predict patient response to proxalutamide. Overall, although the translational sub-study is preliminary and exploratory, the important findings further encourage the assessment of genomic profiling in patients with AR+ mBC to identify those who may benefit from proxalutamide in future studies.
      This study has several limitations. First, our early phase clinical trial study was limited by the small sample size. The efficacy and biomarker analysis should be interpreted with caution and considered exploratory. Second, the population comprises patients with various molecular subtypes of BC, who usually present different prognoses. Third, this study recruited only Chinese female patients. Hence, these findings need to be validated in future studies.

      5. Conclusions

      Proxalutamide demonstrated promising anti-tumour activity with an acceptable safety profile in patients with heavily pretreated AR+ mBC, particularly in the TNBC subgroup. And, this study determined the RP2D to be proxalutamide 200 mg once daily. Furthermore, we identified that AR expression, CNVs, and cfDNA yield may be associated with the response to proxalutamide, highlighting the importance of conducting genomic profiling in patients with AR+ mBC to identify those likely to benefit from proxalutamide treatment. Based on these results, a confirmatory trial will be planned in the future.

      Author contributions

      H. Jiang: Investigation, formal analysis, methodology, writing-original draft, writing-review and editing. Q. Ouyang: Investigation, writing-review and editing. Y. Yin: Investigation, writing-review and editing. Z. Tong: Investigation, writing-review and editing. K. Shen: Resources. Z. Yuan: Resources. C. Geng: Resources, investigation. Y. Liu: Investigation, data curation, and validation. G. Song: Investigation, data curation, and validation. R. Ran: Investigation, data curation, and validation. W. Li: Resources, investigation. Q. Zhai: Resources, investigation. M. Wang: Formal analysis. L. Meng: Supervision, project administration, and formal analysis. Y. Tong: Conceptualisation, supervision, project administration, and funding acquisition. H. Li: Conceptualisation, methodology, resources, investigation, supervision, writing-original draft, writing-review and editing.

      Funding

      This work was supported by Suzhou Kintor Pharmaceuticals, Inc. by providing proxalutamide and funds for this study. But, the source of funding had no role in the study design, data collection, analysis, interpretation, or preparation of the manuscript.

      Conflict of interest statement

      The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
      M. Wang, L. Meng, and Y. Tong are employed by Suzhou Kintor Pharmaceuticals, Inc. No disclosures were reported by the other authors.

      Appendix A. Supplementary data

      The following are the Supplementary data to this article:

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