†These authors contributed equally.
Background: This paper sought to investigate the association
between androgen receptor splice variant 7 (AR-V7) status in circulating tumors
cells (CTCs) and resistance to abiraterone (Abi) and docetaxel (Doc) in men with
metastatic castration-resistant prostate cancer (mCRPC).
Methods: This was a prospective clinical study, newly confirmed mCRPC
patients who were randomized going to receive Abi or Doc with age
Androgen deprivation therapy (ADT) is the most commonly used treatment for advanced prostate cancer (PCa); however, most patients still progress to castration-resistant prostate cancer (CRPC) . Several agents have emerged for the treatment of CRPC, including docetaxel (Doc), abiraterone (Abi), Enzalutamide, Cabazitaxel, Radium-233 and Sipuleucel-T, these drugs not only significantly inhibit tumor progression but actually improve OS [2, 3, 4]. Among these agents, Doc and Abi are most commonly used in China due to their recent availability . However, a proportion of patients are still not sensitive to Doc, Abi or Enzalutamide, and all eventually acquire secondary resistance; however, the mechanism of this remains unclear . One plausible mechanism for agent resistance is the existence of androgen receptor variants (AR-Vs); androgen receptor splice variant 7 (AR-V7) is the most common subtype of AR-Vs, its activation didn’t require the combination of androgen with androgen receptor leading to the therapy resistance , and it is much more common in CRPC than in localized PCa . Our clinical study sought to investigate the association between AR-V7 status with resistance to Abi and Doc as well as time-to event outcomes in men with metastatic castration-resistant prostate cancer (mCRPC), which is one of the first studies regarding that.
We prospectively enrolled patients with metastatic castration-resistance
prostate cancer (mCRPC) who were beginning first-line treatment in our center
between January 2016 and December 2017. Inclusion criteria: (1) age
Peripheral blood samples for analysis of CTCs were obtained from eligible
patients before receiving Abi or Doc. CTCs collection protocol: 5 mL venous blood
was collected by a vacuum blood collection vessel containing EDTA; the vessels
were immediately sent to laboratory or stored in a 4
AR-V7 mRNA detection in CTCs: Materials required: (1) QIAGEN-74034 RNeasy Plus Micro Kit: RNA extraction Kit (QIAGEN Biology, Cat.n.74034, Dusseldorf, Germany); (2) Vazyme HisScript ® II Q RT SuperMix for qPCR (+ gDNA wipers): Reverse transcription kit (Vazyme Botech Co, Cat. No. R233-01, Nanjing, Jiangsu province, China); (3) applied biosystems TaqMan ® Universal Master Mix II: PCR amplification reagents (Thermofisher, Cat. No. 4440040, Waltham, MA, USA). In this study, RNA was extracted from the collected samples for qPCR amplification after reverse transcriptionAR-V7 status was classified as positive or negative according to the literature [10, 11]. Sequencing results: AR-V7 probe CCGGGTTGGCAATTGCAA; GAPDH probe TCCCGTTCTCAGCCTTGA.
In patients where CTCs were detected, a computer- based random distribution was used to randomized patients to receive either Abi or Doc, ADT therapy was continued in all the patientsAR-V7 mRNA in CTCs was detected before patients received Abi or Doc. Total prostate specific antigen (tPSA), free PSA, f/t PSA ratio (the ratio of free PSA to total PSA) and serum testosterone was detected every 1–2 months. Emission-computed tomography (ECT) bone scan, magnetic resonance imaging (MRI) and computerized tomography (CT), including chest-, abdomen- and pelvis- or prostate-specific membrane antigen (PSMA) PET-CT were performed every 2 to 3 months to determine the number and diameter of bone and soft tissue lesions (10 cases with PSMA PET, 129 with ECT bone scan, MRI and CT).
Follow-up data was assessed after enrollment: (1) PSA RR—proportion of
patients with PSA response, and patients with PSA response were defined as PSA
Statistical analyses were performed in two parts: (1) clinical data were assessed and compared between AR-V7-positive and negative cases in patients receiving Abi and Doc, respectively; (2) clinical data were assessed and compared between Abi and Doc in AR-V7-positive and AR-V7-negative patients, respectively.
Measurement data conforming to normal distribution analyzed by Shapiro–Wilk
test are represented as mean
From January 2016 to December 2017, 155 patients were screened and 139 (89.7%
yield) with detectable CTCs were enrolled, of whom, 67 received Abi and 72
received Doc. Among patients enrolled, 111 (79.86%) with only bone metastases,
14 (10.07%) with only visceral metastases and 14 (10.07%) with bone and
visceral metastases (Table 1). Median age was 67.15 years (range, 56–83) and
median follow-up time was 36.37 months (range, 28–52). There was no difference
in median age or follow up time between the Abi and Doc groups. A total of
35.25% (49 of 139) patients were AR-V7-positive, and 35.8% (24 of 67) of the
Abi-treated patients and 34.7% (25 of 72) of the Doc-treated patients were
AR-V7-positive. The Gleason Score of the AR-V7-positive patients was
significantly higher than for the AR-V7-negative patients (8.63
|Variables||Bone only||Visceral only||Bone and visceral||p value|
|AR-V7 (+)||37||Abi 18||5||Abi 3||7||Abi 3||0.469|
|n (%)||(75.5)||Doc 19||(10.2)||Doc 2||(14.3)||Doc 4|
|AR-V7 (–)||74||Abi 36||9||Abi 5||7||Abi 2|
|n (%)||(82.8)||Doc 38||(10)||Doc 4||(7.8)||Doc 5|
|There is no statistically difference of distribution of metastatic sites between AR-V7 positive and AR-V7 negative patients.|
|Abi, n = 67||Doc, n = 72|
|AR-V7 (+)||AR-V7 (–)||p||AR-V7 (+)||AR-V7 (–)||p|
|Population, n (%), n = 139||24 (35.80)||43 (64.2)||25 (34.70)||47 (65.3)|
|Baseline PSA Level, ng/mL||4.68
|PSA RR, n (%)||5 (20.80)||28 (65.10)||0.001||12 (48)||23 (48.90)||0.940|
|PSA RT, days||139.75
|PSA PFS, days||139
|Clinical PFS, days||241.8
|Radiographic PFS, days||214.83
|Abi, abiraterone; Doc, docetaxel; HSDT, hormone-sensitive duration time; PSA RR, PSA responding rate; PSA RT, PSA responding time (time since patients began receiving Abi or Doc to PSA response); PFS, progression-free survival time; CSS, cancer-specific survival.|
|AR-V7 positive, n = 49||AR-V7 negative, n = 90|
|Population, n = 139||24||25||43||47|
|PSA RR, n (%)||5 (20.80)||12 (48)||0.046||28 (65.10)||23 (48.90)||0.120|
|PSA RT, days||139.75
|PSA PFS, days||139
|Clinical PFS, days||321.50
|Radiographic PFS, days||214.83
|Abi, abiraterone; Doc, docetaxel; HSDT, hormone-sensitive duration time; PSA RR, PSA responding rate; PSA RT, PSA responding time (time since patients began receiving Abi or Doc to PSA response); PFS, progression-free survival; CSS, cancer-specific survival; PSA, prostate specific antigen.|
In Abi-treated patients, the PSA RR of AR-V7-positive patients was significantly
lower than that among AR-V7-negative patients (p = 0.001), while the PSA
RT was significantly longer (p
Kaplan–Meier analysis of
prostate specific antigen (PSA) progression free survival (PFS), clinical PFS,
radiographic PFS and cancer specific survival (CSS) according to androgen
receptor splice variant 7 (AR-V7) status in abiraterone (Abi) treated patients.
(A) PSA PFS of AR-V7-negative patients was longer than AR-V7-positive patients
by log-rank test, p
|Variables||Multivariable Cox regression analysis|
|HR||95% CI||p value|
|PSA PFS||AR-V7 (–)|||||
|Clinical PFS||AR-V7 (–)|||||
|Ref, reference; each multivariable model included 5 variables (AR-V7 status, baseline PSA level, Gleason Score, age and vesical metastasis). AR-V7 positive was the independent factor predicting the outcome of PSA PFS, clinical PFS and rPFS in patients treated with Abi.|
Among 72 Doc-treated patients, PSA RR, PSA RT, PSA PFS, clinical PFS, radiographic PFS and CSS were assessed and compared between AR-V7-positive and -negative patients; no statistically significant differences were found (Table 2).
Among 49 AR-V7-positive patients, PSA RR was significantly lower in Abi-treated patients than in Doc-treated patients, while PSA RT was significantly longer (Table 3). PSA PFS and radiographic PFS of Abi were significantly shorter than Doc; clinical PFS and CSS of Abi were shorter but the difference was not statistically significant (Table 3). A Kaplan–Meier analysis model was used to evaluate the association between agents and time-to-event outcomes in AR-V7-positive patients, and Abi was associated with shorter PSA PFS and radiographic PFS but not clinical PFS and CSS (Fig. 2). In a multivariable COX model adjusted for the baseline PSA level, Gleason Score, age and vesical metastasis, Abi remained an independent risk factor associated with shorter PSA PFS and radiographic PFS (Table 5, Ref. ).
Kaplan–Meier analysis and comparison of PSA PFS, clinical PFS, radiographic PFS and CSS between Doc and Abi. (A) PSA PFS of docetaxel (Doc)-treated patients was longer than Abi by log-rank test, p = 0.002. (B) No significant difference in clinical PFS between Doc and Abi by log-rank test, p = 0.107. (C) Radiographic PFS of Doc-treated patients was longer than Abi by log-rank test, p = 0.004. (D) No significant difference in CSS between Doc- and Abi-treated patients by log rank test, p = 0.485.
|Variables||Multivariable Cox regression analysis|
|HR||95% CI||p value|
|Ref, reference; each multivariable model included 5 variables (AR-V7 status, baseline PSA level, Gleason Score, age and vesical metastasis). Abi was the independent factor associated with the outcome of PSA PFS and clinical PFS in AR-V7 positive patients.|
Among 90 AR-V7-negative patients, PSA RR, PSA RT, PSA PFS, clinical PFS, radiographic PFS and CSS were compared between Abi- and Doc-treated patients, and differences were not statistically significant (Table 3).
This study evaluated the association of AR-V7 status and Abi resistant in mCPRC patients. According to multivariable COX model we concluded that AR-V7 was the independent factor associated with Abi resistant and might be used as a biomarker guiding treatment strategy, the result was consistent with results of previous studies ; we also found that in AR-V7 positive patients, the HSDT time and PSA RR was much lower while PSA RT was much longer than that in AR-V7 negative patients, AR-V7 might be associated with the rapid tumor progression in mHSPC phase, and influence the therapy efficiency of Abi in mCRPC phase due the longer PSA RT and lower PSA RR, to our knowledge, there is little or no information in the literature regarding that. Since the mechanism of CRPC is complicated and not well understood, recently some studies suggested that AR-Vs might be associated with CRPC . As a subtype of AR-Vs, studies found that AR-V7 is more common in CRPC than in mHSPC and local PCa, it might be associated with tumor progression [14, 15]. Another study shows the same conclusion that the presence of AR-V7 is responsible for tumor progression . Abi and Doc are the most commonly used agents for CRPC in China. However, the response rate for Doc is about 50%, yet half of men do not benefit from it. The mechanism of agent resistance has been detected by many studies [15, 16, 17, 18, 19] but the mechanism is still unknown. In recent years, several studies began to detect the association of AR-V7 status and therapeutic outcomes of Doc. Research in 2015 analyzing AR-V7 mRNA in CTCs in 37 CRPC patients who received Doc has shown that AR-V7 positive is not associated with PSA RR and PSA PFS . Thadani Mulero, et al.  claimed thatAR-V7 is associated with Doc resistance while AR-V567es is not. Another study suggested that AR-V7 could affect the efficiency of Doc only when above a certain level .
The association of AR-V7 with Doc resistant is still controversial. In our study, clinical data support the claim that AR-V7 status is not associated with the therapeutic efficiency of Doc. As for Abi, our study supports a growing body of literature suggesting that PCa patients with AR-V7 positive were resistant to Abi [7, 23, 24]. Hu R, et al.  claimed that AR-V7 is commonly expressed in CRPC and the response rate of Abi is low. Another clinical study claims that there is a strong association between the presence of AR-V7 in CTCs and resistance to Abi , and a subsequent study shows that, in AR-V7-positive patients, Doc is more beneficial than Abi or Enzalutamide; however, the scale of this study is small, with only 37 patients included . A prospective study enrolled 118 CRPC patients treated with Abi or Enzalutamide, showing that AR-V7 positivity is associated with shorter PFS and OS . Our study supports the claim that AR-V7 positivity is associated with Abi resistance in mCRPC patients as previous study reported, and clinical data suggest that AR-V7 positivity is strongly associated with a higher Gleason Score, lower PSA RR, and shorter HSDT and PFS (PSA, clinical and radiographic) which all suggested a poor prognostic of AR-V7 positive patients. In fact, the frequency of AR-V7-positive patients was higher in our cohort than in previous work , raising the possibility of an association between AR-V7 positivity and ethnicity. From the results of our research, the role of AR-V7 in guiding treatment strategy was reliable. Thus, tremendous time and money could be saved as a recent study presumed that USD 1.5 billion could be saved if AR-V7 detection were to be used for agent selection in CRPC .
This study still has some limitations. First, the short follow-up time; second, not all CRPC patients have deteactable CTCs, which may limit the role of AR-V7 in CTCs as predictive marker for agent resistance; third, inconsistent imaging methods may cause bias; fourth, not all the factors (i.e., comorbidities) related to the prognostic of PCa were included, this may cause bias. Further studies are required.
In conclusion, our study confirmed that AR-V7-positive was the independent factor associated with Abi resistance in mCRPC but is not associated with the effectiveness of Doc, the predictive value of AR-V7 in guiding treatment strategy is reliable; we also found that in AR-V7 positive patients, the HSDT time was much shorter than that in AR-V7 negative patients, AR-V7 might be associated with the rapid tumor progression in mHSPC phase.
PD and SW designed the study. SW, YC and XT made the same contributions to this study as the first co-author. SW, YC, XT, JM, ZY, YY and XY performed the study and analyzed the data. PD and SW wrote the manuscript draft and revised the manuscript. All authors have read and agreed to the published version of the manuscript.
This study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of Peking University Cancer Hospital and Institution in April 2018 (protocol code 2018KT27). All the patients signed informed consent before enrollment.
This research was funded by Capital Clinical Characteristics and Application Research Project. Project No: Z18110700170000.
The authors declare no conflict of interest.
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy of patients.