**3. Results**

The study population included 107 patients, of whom 89 were treated with Enza, and 46 were treated with Abi. Twenty-eight patients received sequential therapy with the novel AR-targeted drugs. The median follow-up time was 68.3 months from first-line ADT. The patients' characteristics are shown in Table 1. The patients' median age was 73.0 years. The median PSA levels were 30.1 ng/mL for Enza and 41.1 ng/dL for Abi. The rates of lymph node, lung, liver, and bone metastases were 31.78%, 8.41%, 5.61%, and 83.18%, respectively. The rates of previous steroid use and estramustine use in Enza and Abi were 40.19% and 46.73%, respectively. The median TST at the initiation of Enza was 13 ng/dL, and that of Abi was 12 ng/dL. Further information is shown in Table 1.


**Table 1.** Patient's backgrounds.


**Table 1.** *Cont.*

Enza: enzalutamide; Abi: abiraterone; BMI: body mass index; PFS: progression-free survival; Mets: metastasis; PSA: prostate-specific antigen; TST: target serum testosterone; EOD: extent of disease.

Table 2 shows the results of the univariate and multivariate analyses of the prognostic factors of PFS in Enza and Abi overall. By univariate analysis, first-line PFS ≥ 15.4 months (Hazard Ratio (HR) 0.60, *p* = 0.0309), previous docetaxel (HR 2.44, *p* = <.0001), high volume (HR 1.73, *p* = 0.0198), C-reactive protein (CRP) ≥ 0.15 mg/dL (HR 1.87, *p* = 0.0173), extent of disease (EOD) score (HR 2.79, *p* = 0.0004), PSA ≥ 34.1 ng/mL (HR 1.73, *p* = 0.0143), TST ≥ 13 ng/dL (HR 0.26, *p* = <.0001), steroid use (HR 2.24, *p* = 0.0003), and estramustine use (HR 1.69, *p* = 0.0179) were found to be significant prognostic factors. By multivariate analysis, only TST ≥ 13 ng/dL (HR 0.31, *p* = 0.0365) remained as an independent prognostic factor.

**Table 2.** Predictive factors of PFS in enzalutamide- and abiraterone- treated patients.


Pre-treatment course: previous treatment course; GS: Gleason Score; ALP: alkaline phosphatase; ICTP: I collagen telopeptide; Hb: hemoglobin; LDH: Lactate dehydrogenase; Alb: albumin; CRP: C-reactive protein; NLR: Neutrophil/Lymphocyte ratio; PSA: prostate-specific antigen; \* Statistical significance *p* < 0.05.

The prognostic value of TST was also confirmed in each Enza and Abi group.

As shown in Supplementary Table S1, TST ≥ 13 ng/dL (median) was a significant factor in univariate (HR 0.28, *p* = 0.0044) and multivariate analysis (HR0.08, *p* = 0.0032) in Enza-treated patients. However, as shown in Supplementary Table S2, TST ≥ 12 ng/dL (median) was not significant in univariate analysis (HR0.40, *p* = 0.0891) in Abi-treated patients.

Furthermore, the effect of te TST on the response to Enza and Abi was studied by the Kaplan–Meier model. As shown in Figure 1, higher TST levels (≥13 ng/dL) were related to significantly better PFS in Enza and Abi groups overall (*p* < 0.0001) and in Enza-treated patients (*p* = 0.0032) (Figure 1a,b), while higher TST levels (≥12 ng/dL) showed no prognostic difference in Abi-treated patients (*p* = 0.0881) (Figure 1c).

**Figure 1.** Progression-free survival according to TST levels. (**a**) Progression-free survival according to TST level <13 ng/dL or ≥13 ng/dL as the cut-off values in enzalutamide- and abiraterone-treated patients. (**b**) Progression-free survival according to TST level <13 ng/dL or ≥13 ng/dL as the cut-off values in enzalutamide-treated patients. (**c**) Progression-free survival according to TST level <12 ng/dL or ≥12 ng/dL as the cut-off values in abiraterone-treated patients. (**d**) Overall survival according to TST level <13 ng/dL or ≥13 ng/dL as the cut-off values in enzalutamide- and abiraterone-treated patients.

When the PFS periods were stratified by the TST level (8 to 20 ng/dL), higher TST levels were related to longer PFS in Enza and Abi groups, with a correlation coefficient of 0.32 (*p* = 0.0067) (Figure 2a,b).

To study the patients' characteristics, two groups (TST <13 ng/dL and TST ≥ 13 ng/dL) were compared with respect to various clinical factors. A level of TST ≥ 13 ng/dL was related to higher alkaline phosphatase (ALP) levels (*p* = 0.0158) (Table 3).

**Figure 2.** Progression-free survival based on the serum TST cut off level. (**a**) Progression-free survival according to TST level <8 ng/dL, ≥8 ng/dL, ≥13 ng/dL, or ≥20 ng/dL as the cut-off values in enzalutamide- and abiraterone-treated patients. (**b**) Correlation of serum TST level and progression-free survival in enzalutamide- and abiraterone-treated patients. The red circle indicates 95% of data used for the correlation analysis, to avoid errors.



BSI: bone scan index; \* Statistical significancer *p* < 0.05; † welch, †† wilcoxon, ††† fisher.

Table 4 shows the results of the univariate and multivariate analyses of the prognostic factors of overall survival (OS) in Enza and Abi groups overall. By multivariate analysis, previous docetaxel (HR 3.04, *p* = 0.0038), visceral mets (HR 7.88, *p* = 0.0139), ALP (HR 2.51, *p* = 0.0090) and lactate dehydrogenase (LDH) (HR 2.95, *p* = 0.0033) remained as independent prognostic factors. On the other hand, TST was not a significant factor neither in univariate analysis (HR 0.99, *p* = 0.8750) (Table 4), nor in Kaplan–Meier analysis (*p* = 0.8748) (Figure 1d). When patients who received initial AR-targeted therapy were selected, TST (HR 0.39, *p* = 0.0086) and CRP (HR 2.03, *p* = 0.0487) remained significant predictive factor for PFS in multivariate analysis (Supplementary Table S3), while, for OS, no factor remained significant in multivariate analysis (Supplementary Table S4).


**Table 4.** Predictive factors of overall survival (OS) in enzalutamide- and abiraterone-treated patients.

 Statistical significance *p* < 0.05.

\*

Next, the effects of the first-line therapy on the responses to Enza and Abi were evaluated. As shown in Figure 3a, first-line PFS (median ≥ 15.4 months) was related to a favorable response (*p* = 0.0273) for Enza and Abi overall (Figure 3a). However, the first-line PFS did not affect PFS in Abi-treated patients (median ≥ 12.6 months) (*p* = 0.6105), while they significantly affected PFS in Enza-treated patients (median ≥ 17.9 months) (*p* = 0.0429) (Figure 3b,c). Since the median first-line PFS in the Abi group was around 12 months, the patients were divided on the basis of the first-line PFS of 12.0 months for Enza. It was evident that a long first-line PFS ( ≥12.0 months) was related to significantly longer PFS in the Enza group (*p* = 0.0046) (Figure 3d).

The results of the sequential use of Enza and Abi were also examined. Although it was not significant, the subsequent usage of novel AR-targeted drugs reduced the PSA response rate in patients treated with both drug (Supplementary Table S5). Regarding PFS, a reduction in the response period was more evident in patients receiving Abi after Enza (first Abi 15.7 weeks vs. Enza before Abi 9.93 weeks) than in patients administered Enza after Abi (1st Enza 12 weeks vs Abi before Enza 11.1 weeks).

**Figure 3.** *Cont.*

**Figure 3.** Progression-free survival according to the progression-free survival of first-line therapy. (**a**) Progression-free survival according to a median progression-free survival of first-line therapy <15.4 months or ≥15.4 months as the cut-off values in enzalutamide- and abiraterone-treated patients. (m, months). (**b**) Progression-free survival according to the progression-free survival of first-line therapy <12.0 months or ≥12.0 months as the cut-off values in enzalutamide-treated patients. (**c**) Progression-free survival according to a median progression-free survival of first-line therapy <17.9 months or ≥17.9 months as the cut-off values in enzalutamide-treated patients. (**d**) Progression-free survival according to a median progression-free survival of first-line therapy <12.6 months or ≥12.6 months as the cut-off values in abiraterone-treated patients.
