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A Prospective Study of the Relationship Between Clinical Outcomes After Enzalutamide and Serum Androgen Levels Measured via Liquid Chromatography-tandem Mass Spectrometry in Patients with Castration-resistant Prostate Cancer
Corresponding author. Department of Urology, Gunma University Graduate School of Medicine, 3-9-22 Showa-machi, Maebashi, Gunma 371-8511, Japan. Tel. +81 27 2208306; Fax: +81 27 2208307.
Enzalutamide (ENZ) is used to treat patients with castration-resistant prostate cancer (CRPC). However, the kinetics of serum androgens before and after ENZ treatment are unknown.
Objective
To elucidate the kinetics of serum androgens and explore the possibility of identifying a useful marker for predicting the effects of ENZ.
Design, setting, and participants
We conducted a prospective study from 2014 to 2018 at Gunma University Hospital and related facilities. Data were analyzed for 104 patients with CRPC treated with ENZ.
Outcome measurements and statistical analysis
We measured serum androgen levels using liquid chromatography-tandem mass spectrometry. Relationships with outcomes were assessed using multivariable Cox regression and log-rank analyses.
Results and limitations
The median age of the patients was 73 yr. Median serum testosterone, dihydrotestosterone (DHT), androstenedione, and dehydroepiandrosterone sulfate levels were 49.0, 5.8, 222.2, and 326.3 pg/ml, respectively. We performed multivariate analysis using Cox regression to predict prostate-specific antigen progression–free survival (PSA-PFS) and overall survival (OS). Hemoglobin level (≥12.5 vs <12.5 g/dl), docetaxel treatment history (no vs yes), and DHT level (≥5.9 vs <5.9 pg/ml) were significant predictors of PSA-PFS (p < 0.05). Eastern Cooperative Oncology Group performance status (0 vs. 1–2), hemoglobin level (≥12.5 vs <12.5 g/dl), presence of visceral metastasis (no vs yes), amount of bone metastasis (extent of disease 0–2 vs 3–4), and docetaxel treatment history (no vs yes) were significant predictors of OS (p < 0.05). Binomial logistic analysis of the predictors of any grade of anorexia, malaise, and fatigue showed that the presence of visceral metastasis and a low DHT level (<5.9 pg/ml) were significant.
Conclusions
Our results suggest that serum androgen levels before ENZ treatment may be useful for predicting efficacy, prognosis, and the incidence of adverse events.
Patient summary
We measured blood levels of testosterone and other male hormones before treatment with enzalutamide among men with prostate cancer resistant to castration. We found that the levels of these hormones may be useful for predicting the efficacy of enzalutamide treatment, prognosis, and the occurrence of adverse side effects.
]. The treatment of metastatic castration-resistant prostate cancer (CRPC) has changed dramatically with the advent of novel androgen receptor (AR)-targeting agents (ARTAs) and a new class of taxane agents [
]. The pivotal PREVAIL and AFFIRM clinical trials showed that use of these drugs was associated with significant improvements in overall survival (OS), radiographic progression-free survival (rPFS), prostate-specific antigen progression–free survival (PSA-PFS), quality of life (as measured with the Functional Assessment of Cancer Therapy-Prostate questionnaire), and skeletal-related events [
Effect of enzalutamide on health- related quality of life, pain, and skeletal-related events in asymptomatic and minimally symptomatic, chemotherapy-naive patients with metastatic castration resistant prostate cancer (PREVAIL): results from a randomised, phase 3 trial.
Effect of enzalutamide on time to first skeletal-related event, pain, and quality of life in men with castration-resistant prostate cancer: results from the randomised, phase 3 AFFIRM trial.
ENZ has proven efficacy against CRPC and is widely used in this setting. No definitive biomarker predicts the effects of ENZ. It is well known that serum testosterone (T) levels before and during androgen deprivation therapy (ADT) are prognostic for the success of primary ADT [
Nadir testosterone within first year of androgen-deprivation therapy (ADT) predicts for time to castration-resistant progression: a secondary analysis of the PR-7 trial of intermittent versus continuous ADT.
]. Several retrospective studies in CRPC found that patients with higher serum T levels before ARTA treatment experienced better therapeutic effects than patients with lower T levels did [
Serum testosterone level as possible predictive marker in androgen receptor axis-targeting agents and taxane chemotherapies for castration-resistant prostate cancer.
]. We thus decided to elucidate the kinetics of androgen levels after ENZ administration. In addition, we prospectively examined the effects of ENZ on initial serum androgen concentrations among patients with CRPC.
2. Patients and methods
2.1 Patients and study design
We prospectively evaluated 104 patients with CRPC treated in the urology department of Gunma University Hospital and affiliated hospitals from 2014 to 2018. The primary endpoint was the PSA response rate (50% and 90%), and the secondary endpoints were OS, PSA-PFS, the objective tumor response, and the relationship between serum androgen levels and clinical outcomes. Extent of disease (EOD) was used as a semiquantitative grading system according to the extent of bone metastasis on bone scans as follows: 0, normal; 1, fewer than six bony metastases, each of which is <50% of the size of a vertebral body; 2, between six and 20 bony metastases; 3, more than 20 bony metastases but less than a “super scan”; and 4, “superscan” or bony metastases involving more than 75% of the ribs, vertebrae, and pelvic bones [
]. We collected blood samples before treatment, at 3 and 6 mo after the commencement of treatment, and at the end of treatment. The correlation coefficients for automated immunoassays are relatively poor when T concentrations are low (<4.0 nmol/l) [
]. Therefore, we measured serum androgen levels via liquid chromatography-tandem mass spectrometry (LC-MS/MS), which was carried out by Asuka Pharmaceutical (Tokyo, Japan). Serum T, dihydrotestosterone (DHT), androstenedione (A-dione), and dehydroepiandrosterone sulfate (DHEA-S) levels were measured. The lower limit of detection for T, DHT, A-dione, and DHEA-S was 0.5, 0.5, 1.0, and 1.0 pg/ml, respectively.
The eligibility criteria for the study were age >20 yr, histologically confirmed prostate cancer, surgical or medical castration, a diagnosis of CRPC, Eastern Cooperative Oncology Group performance status (ECOG PS) score of 0–2, and consent to participate. The exclusion criteria were a history of seizures (such as epilepsy); a history of brain metastasis, organic brain disease, or brain injury; severe liver dysfunction; allergy to ENZ; and any contraindication to ENZ treatment as judged by a doctor.
2.2 Disease progression and treatment efficacy
OS and PSA-PFS were assessed using Prostate Cancer Working Group 2 criteria [
Design and end points of clinical trials for patients with progressive prostate cancer and castrate levels of testosterone: recommendations of the Prostate Cancer Clinical Trials Working Group.
]. PSA failure was defined according to the same group as a rise in PSA >2 ng/ml above the nadir that was ≥25% of the nadir, as confirmed by a second test performed at least 3 wk later, accompanied by a castration level of T (<50 ng/dl). The objective tumor response was evaluated using Response Evaluation Criteria for Solid Tumors (RECIST) version 1.1.
2.3 Statistical analysis
Student t test, the Welch t test, the Fisher test, and the Wilcoxon signed-rank test were used as appropriate to assess associations between androgen levels and other clinical variables. We calculated Pearson correlation coefficients among hormone levels. Univariate and multivariate Cox proportional-hazard models and the Kaplan–Meier method were used for statistical analyses. Patient inclusion and data collection were prospective, but the multivariate analysis was conducted on a post hoc basis. Multivariate analysis of factors predicting OS and PFS was conducted on an exploratory basis. Factors with p < 0.05 on univariate analysis were included in a Cox proportional-hazards multivariate analysis. SPSS version 25.0 (IBM Corp., Armonk, NY, USA) was used for all statistical analyses. A p value <0.05 was considered indicative of significance.
2.4 Institutional ethics approval
We explained the nature of the study to all participants. All participants agreed in writing to participate. The study was approved by the institutional review board (IRB) of Gunma University Hospital (approval no. 1177). The IRB also approved the inclusion of patients from other facilities.
3. Results
Patient characteristics are summarized in Table 1. Although 104 patients were enrolled, there were two patients for whom measurement could not be completed owing to the poor condition of the sample before treatment and three patients who could not continue follow-up owing to relocation. Therefore, the final cohort for analysis was 99 cases, for which we explored the relationships between serum androgen levels and prognosis. Table 2 shows the blood test data and serum androgen levels before ENZ treatment. The median patient age was 73 yr and the median PSA level was 27.6 ng/ml. Seventeen patients (16.3%) had a history of curative treatment and 44 (42.3%) a history of DOC treatment. The proportion of patients with a 50% and 90% PSA decline was 62.5% and 25.0%, respectively (Fig. 1).
Table 1Patient characteristics
Variable
N
%
TNM classification at diagnosis
T stage
T1
2
1.9
T2
11
10.6
T3
46
44.2
T4
40
38.5
Tx
5
4.8
N stage
N0
44
42.3
N1
53
51.0
Nx
7
6.7
M stage
M0
31
29.8
M1a
7
6.7
M1b
52
50.0
M1c
10
9.6
Mx
4
3.8
Eastern Cooperative Oncology Group performance status
0
73
70.2
1
22
21.2
2
6
5.8
Unknown
3
2.9
Gleason score
6
2
1.9
7
13
12.5
8
17
16.3
9
48
46.2
10
19
18.3
Unknown
5
4.8
Metastasis at study baseline
Metastasis negative
3
2.9
Metastasis positive
101
97.1
Metastasis site
Regional lymph nodes
35
33.7
Distant lymph nodes
31
29.8
Bone
80
79.6
Lung
10
9.6
Liver
1
1.0
Other
2
1.9
Extent of disease at study baseline
1
17
16.3
2
13
12.5
3
14
13.5
4
5
4.8
Unknown
31
29.8
Previous radical treatment
Yes
17
16.3
Surgery
12
11.5
Radiation
5
4.8
No
87
83.7
Previous docetaxel treatment
No
60
57.7
Yes
44
42.3
Time to castration-resistant prostate cancer from initial ADT
On analysis of 91 cases for whom objective tumor responses could be evaluated, the response rate (complete response [CR] + partial response [PR]) was 35.2% and the clinical benefit (CR + PR + stable disease) was 81.3%, as assessed using the RECIST criteria (Table 3). Pre-DOC PSA-PFS and OS were significantly higher than the post-DOC results (PSA-PFS: 12.3 vs 2.8 mo; p < 0.001; OS: not yet reached vs 17.2 mo; p < 0.001; Fig. 2). Mean serum T, DHT, A-dione, and DHEA-S levels were 49.0, 5.8, 222.2, and 326.3 pg/ml, respectively. The median time to treatment discontinuation was 40.0 wk (range 3–203 wk, standard deviation 58.0). Of the 99 cases, ENZ was discontinued in 12 by 12 wk because of progression and in a further 41 by 24 wk. For 58 cases, ENZ treatment continued for ≥24 wk. Mean serum levels of T, DHT, and A-dione were significantly higher at 12 and 24 wk after treatment compared to pretreatment levels (all p < 0.05; Fig. 3). There was a positive correlation between pretreatment T, DHT, A-dione, and DHEA-S levels. Pearson’s correlation coefficients revealed significant positive correlation of pretreatment levels for each pair of hormones (Supplementary Fig. 1).
Table 3Objective tumor response rate according to Response Evaluation Criteria in Solid Tumors version 1.1 among the 91 patients
Fig. 2Kaplan-Meier curve of PSA-PFS and OS for patients with and without docetaxel treatment. A log rank test was performed to determine the significance of differences.
Fig. 3Kinetics of serum androgen levels during enzalutamide treatment. Mean serum levels of testosterone, dihydrotestosterone (DHT), and androstenedione (A-dione) were significantly higher at 12 and 24 wk after treatment compared to before treatment (Pre; * p < 0.05).
The study was conducted using previously reported prognostic factors such as baseline PSA, Gleason score, presence or absence of visceral metastasis, and extent of metastasis. Patients were divided into two groups according to the median value for each blood test and hormone, and we assessed whether these were significant factors. Of the 99 patients, 51 died during the observation period. PSA-PFS and OS were significantly longer for patients with androgen levels greater than the median (log-rank test, p < 0.05). We performed multivariate Cox regression analysis for prediction of OS and PSA-PFS. We found that hemoglobin (≥11.4 vs <11.4 g/dl), a history of DOC treatment (no vs yes), and DHT (≥5.8 vs <5.8 pg/ml) were significant predictors of PSA-PFS (all p < 0.05; Table 4). In addition, ECOG PS (0 vs 1–2), hemoglobin (≥11.4 vs <11.4 g/dl), visceral metastasis status (no vs yes), extent of bone metastasis (0–2 vs 3–4), and history of DOC treatment (no vs yes) were significant predictors of OS (all p < 0.05; Table 5).
Table 4Cox regression analysis of PSA progression–free survival
The grade 1–2 adverse events observed were anorexia in 22 patients (21.1%), malaise in 22 (21.1%), fatigue in 13 (12.5%), nausea in seven (6.7%), dysgeusia in five (4.8%), and hot flushes in three (2.9%). The grade 3 adverse events were hypertension, anorexia, fatigue, elevated liver enzyme levels, and retinal artery occlusion. No adverse events of grade ≥4 were observed. The most common adverse events (>20% of cases; 33/99) were anorexia, malaise, and fatigue; we sought factors predictive of these events using binomial logistic analysis. Visceral metastasis and a DHT level below the median (<5.8 pg/ml) significantly predicted any grade of these three events (Table 6).
Table 6Binomial logistic analysis of the predictors of any grade of anorexia, malaise, and fatigue
We explored serum androgen kinetics in patients with metastatic CRPC receiving ENZ. The therapeutic effects of ENZ were significantly better in the group with higher androgen levels. On univariate analysis, OS was also better, but significance was lacking for the relationship with androgen levels on multivariate analysis. The incidence of common side effects (anorexia, malaise, and fatigue) was significantly lower for patients with high androgen levels. To the best of our knowledge, this is the first prospective examination of the relationship between ENZ therapy and blood androgen levels in Japanese patients with metastatic CRPC.
Several earlier retrospective studies reported that higher T levels before treatment with AR-targeting agents such as ENZ and abiraterone were beneficial [
Serum testosterone level as possible predictive marker in androgen receptor axis-targeting agents and taxane chemotherapies for castration-resistant prostate cancer.
] reported that changes in (circulatory) AR gene copy number and T levels <0.09 nmol/l were associated with poor prognosis and poor response to ENZ and abiraterone. Our prospective study confirmed these findings. We found that high blood DHT levels predicted PSA-PFS, but not OS, on multivariate analysis. Shiota et al [
Serum testosterone level as possible predictive marker in androgen receptor axis-targeting agents and taxane chemotherapies for castration-resistant prostate cancer.
] found that PFS for patients with serum T >0.05 ng/ml was significantly inferior to that for patients with serum T <0.05 ng/ml. DOC chemotherapy for these patients was associated with poorer OS in the former group [
Serum testosterone level as possible predictive marker in androgen receptor axis-targeting agents and taxane chemotherapies for castration-resistant prostate cancer.
] reported that higher serum T levels were predictive of poor prognosis for patients with CRPC treated with DOC. As cross-resistance to abiraterone treatment may develop after ENZ treatment [
Optimal sequencing of enzalutamide and abiraterone acetate plus prednisone in metastatic castration-resistant prostate cancer: a multicentre, randomised, open-label, phase 2, crossover trial.
], DOC is often selected if the drug has not previously been prescribed, and cabazitaxel otherwise. The effects of taxane-based drugs are poorer for patients with higher T levels, and the drugs did not significantly enhance OS in the present study.
] reported on the relationships between changes in blood and bone-marrow androgen levels after treatment with ENZ or abiraterone, as well as the therapeutic effects. T concentrations declined to less than picogram-per-milliliter levels, and remained at that level, during treatment with abiraterone [
]. In contrast to the inhibition of androgen biosynthesis by abiraterone, T levels in blood and bone marrow increased, accompanied by a nuclear-to-cytoplasmic AR shift, following 8 wk of ENZ therapy [
]. T levels increased after 8 wk of treatment in most patients for whom paired samples of both blood (40/51, 78%) and bone marrow aspirate (34/44, 77%) were available [
]. Similarly, in the present study, T, DHT, DHEA-S, and A-dione levels tended to increase after ENZ administration, and T, DHT, and A-dione levels were significantly higher by 12 and 24 wk after treatment initiation. Efstathiou et al [
] suggested that such increases reflect physiological feedback. It is unclear whether such androgen signaling contributes to treatment resistance and, if so, to what extent. A future study should explore whether androgen kinetics affect prognosis and ENZ efficacy.
Fatigue, anorexia, and malaise are relatively common with ENZ treatment [
]. The incidence of these adverse events was higher in the group of patients with higher DHT levels before ENZ treatment. Fatigue is common during prostate cancer treatment and may persist even when therapy is complete. The underlying mechanism remains poorly understood. Feng et al [
] showed that combination ADT and radiation therapy worsened fatigue and was associated with anemia and mitochondrial dysfunction. By contrast, Bandara et al [
] used multivariate models with fatigue as the outcome and found that neither hemoglobin nor T levels had a significant within-patient effect on fatigue developing during ADT/radiation therapy. Further study on this issue is necessary.
Our work has several limitations. One of the main limitations of the study is the low number of patients included, so the multivariate analysis has limited meaning. The multivariable analysis in our study was considered as exploratory and hypothesis-generating. In terms of blood androgen measurements, it was not possible to collect samples from patients exhibiting early progression (within 12 or 24 wk). In addition, the inclusion of patients with and without prior DOC therapy might have affected the outcomes of ENZ treatment. ENZ was approved in Japan only as our study commenced. The number and degree of adverse events were lower than in previous reports [
], and may have affected of the relationship between androgen levels and fatigue, anorexia, and malaise. LC-MS/MS measurement of androgen levels is very costly, precluding routine clinical application of this approach.
5. Conclusions
In conclusion, we studied androgen kinetics during ENZ administration. Pretreatment androgen levels usefully predicted treatment efficacy, prognosis, and the incidence of adverse events for patients with mCRPC treated with ENZ.
Author contributions: Yoshiyuki Miyazawa had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design: Miyazawa, Suzuki.
Acquisition of data: Miyazawa, Nakamura, Takezawa, Shimizu, Matsuo, Ogura, Takei.
Analysis and interpretation of data: Miyazawa, Arai, Sekine.
Drafting of the manuscript: Miyazawa.
Critical revision of the manuscript for important intellectual content: Miyazawa.
Statistical analysis: Miyazawa.
Obtaining funding: Suzuki.
Administrative, technical, or material support: Suzuki.
Supervision: Suzuki.
Other: None.
Financial disclosures: Yoshiyuki Miyazawa certifies that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript (eg, employment/affiliation, grants or funding, consultancies, honoraria, stock ownership or options, expert testimony, royalties, or patents filed, received, or pending), are the following: Kazuhiro Suzuki has acted as a consultant for Takeda Pharmaceutical, Astellas Pharma, Daiichi-Sankyo, AstraZeneca, Sanofi, Janssen, and Bayer, and has received grants from Takeda Pharmaceutical, Astellas Pharma, Daiichi-Sankyo, and Ono Pharmaceutical. The remaining authors have nothing to disclose.
Funding/Support and role of the sponsor: Our research funded by Astellas Pharma (Tokyo, Japan). The sponsor played a role in approval of the manuscript.
Ethics statement: We explained the study to all participants and all agreed in writing to participate. This study was approved by the institutional review board at Gunma University Hospital (approval no. 1177). There are no administrative permissions or licenses to access the data to formally note. The study was conducted according to the International Conference on Harmonization/Good Clinical Practice (ICH/GCP) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Appendix A. Supplementary data
The following is Supplementary data to this article:
Correlation between each blood androgens levels in the scatter plot. Significantly positive correlations were found between T, DHT, A-dione, and DHEA-S. “r” represents the Pearson product-moment correlation coefficient. Abbreviations: DHT; dihydrotestosterone, A-dione; androstenedione, DHEA-S; dehydroepiandrosterone-sulfate.
Effect of enzalutamide on health- related quality of life, pain, and skeletal-related events in asymptomatic and minimally symptomatic, chemotherapy-naive patients with metastatic castration resistant prostate cancer (PREVAIL): results from a randomised, phase 3 trial.
Effect of enzalutamide on time to first skeletal-related event, pain, and quality of life in men with castration-resistant prostate cancer: results from the randomised, phase 3 AFFIRM trial.
Nadir testosterone within first year of androgen-deprivation therapy (ADT) predicts for time to castration-resistant progression: a secondary analysis of the PR-7 trial of intermittent versus continuous ADT.
Serum testosterone level as possible predictive marker in androgen receptor axis-targeting agents and taxane chemotherapies for castration-resistant prostate cancer.
Design and end points of clinical trials for patients with progressive prostate cancer and castrate levels of testosterone: recommendations of the Prostate Cancer Clinical Trials Working Group.
Optimal sequencing of enzalutamide and abiraterone acetate plus prednisone in metastatic castration-resistant prostate cancer: a multicentre, randomised, open-label, phase 2, crossover trial.
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