**4. Discussion**

The current data indicate that a higher TST level at the stage of CRPC predicted a favorable response to Enza. When patients treated with Enza and Abi were combined, serum TST levels remained as independent prognostic factors for PFS. Moreover, the characteristics of the prognostic factors showed distinct differences between Enza and Abi. The response to Enza was more closely related to the response to first-line therapy, while the response to Abi was rather more independent from the response to first-line therapy. On the basis of the current data, TST level and the PFS of first-line therapy may be considered when choosing the treatment strategy for CRPC patients.

The reason why a higher TST level was associated with a favorable response to Enza and Abi may be related to the AR dependency of prostate cancer. When comparing clinical factors between higher and lower TST groups, higher TST was related to a relatively higher initial PSA (low TST 58.57 ng/mL vs high TST 81.65 ng/mL), although it was not significant. Since the expression of PSA is mediated by the transcriptional activity of nuclear AR, a higher initial PSA value may represent a higher basal AR activity inside the tumor microenvironment [18]. As both Enza and Abi work through AR, the higher AR dependency of the tumor may predict a higher response to AR-targeted drugs.

Another reason may be that a TST level of 13 ng/dL itself represents the remaining potential to target the AR-related pathway. The clinical significance of lowering the TST level below 50 ng/dL has been described in several reports [4,19]. Our group and others have previously reported that patients who achieved nadir TST < 20 ng/dL survived longer than those who did not [4,20–22]. Klotz et al. reported that patients with first-year nadir testosterone consistently >20 ng/dL had significantly higher risks of dying of prostate cancer [21]. These data indicated the clinical significance of lowering TST to <20 ng/dL in the tumor microenvironment [4]. However, these data were obtained when Enza and Abi were not on the market or were had a very limited use.

On the contrary, our data indicate that higher serum TST > 13 ng/dL represented the longer PFS for Enza and Abi groups, which is a novelty. The current cut-off value of serum TST of 13 ng/dL may represent the remaining AR dependency of the tumor that can only be blocked by treating with novel AR-targeted therapy. If tumor relapse occurs with a TST level >13 ng/dL, then this tumor may contain more AR-dependent cells, compared to tumors with a relapse occurring with TST < 13 ng/dL. Thus, even with a TST of 20 ng/dL, intensive blockade of the AR pathway through novel AR-targeted drugs would be effective, especially among patients with a higher TST level.

The clinical advantage of high TST was also reported by Ryan et al. [17]. Although the TST cut-off value was even lower (>8.6 ng/dL) compared to that in the present study, these authors found a higher TST to be related to a better response to Abi. On the other hand, in our study, a clinical advantage of high TST was not found for Abi-treated patients but was found for Enza-treated patients.

Interestingly enough, the higher TST group (TST ≥ 13 ng/dL) showed a relatively higher ALP, initial PSA, and PSA at the start of treatment with a similar rate of the high-volume tumor. The rate of visceral metastases was relatively higher in the TST ≥ 13 ng/dL group. Since the rate of visceral metastases was low (10–26%), it will be necessary to objectively assess the clinical significance of visceral metastases in a large number of patients. However, the present data may indicate that the response to novel AR-targeted drugs may not be related to a high or low tumor burden but may rather depend on the AR dependency of the tumor.

The current data indicate that the first-line PFS (12 months) predicted a favorable response to Enza (*p* = 0.0046), while the first-line PFS showed no association with the response to Abi (*p* = 0.6051). These data are in line with the findings of a previous report. Loriot et al. reported that the first-line ADT period predicted the response to novel AR-targeted drugs, mainly Enza [23]. Bellmunt et al. reported that the response to Abi was not related to the first-line ADT period [24]. The reason for this difference is not clear. However, one of the reasons may be that Enza shares a common mechanism with bicalutamide as an AR antagonist. Enza blocks AR with over a 30-fold higher affinity compared to bicalutamide [25]. The majority of the patients received CAB with bicalutamide and LH-RH agonist/antagonist as first-line ADT. Therefore, the patients who responded well to bicalutamide may also represent the patients who respond well to a potent AR antagonist, namely Enza. On the other hand, Abi works through Cyp17 inhibition, so the mechanism is distinct from that of bicalutamide and Enza. This mechanistic difference between Abi and Enza may represent the difference in response to the first-line therapy between Enza and Abi.

The present study provides several important findings. (1) Higher TST (≥13 ng/dL) at the initiation of drug administration was associated with a favorable response to novel AR-targeted drugs, especially Enza; (2) The response to Enza was affected by the PFS of the first-line ADT, while the response to Abi was not affected by the PFS of the first-line ADT.

There are several limitations associated with this study. First, the sample size was rather small, which limits the reliability of the analysis, especially when assessing Abi and Enza independently. Second, because of the limited follow-up periods and limited outcomes regarding OS, the assessment of prognostic factors was mainly based on PFS. Third, because of the limited number of patients, the analysis was not divided in pre- or post-chemo settings. Although TST levels remain as predictors even among the factors that included the previous usage of chemotherapy, the response rate may also be affected by the previous usage of chemotherapy. Fourth, because of the prior introduction of Enza in the Japanese healthcare system, the majority of patients in the Enza group (92%) received the drug as a first-line novel AR-targeted drug, while half of the patients (54%) received Abi as a first-line AR-targeted drug. The different results observed for Enza and Abi, including association with TST levels, may possibly be affected by the background of the patients who received the drugs. We are currently performing a prospective study to re-assess the role of TST in the initial usage of Enza and Abi. The effects of the differences in the clinical features of the patients that induced the treatment choice in the current manuscript will be answered in the near future.
