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Peer-Review Record

Higher Serum Testosterone Levels Associated with Favorable Prognosis in Enzalutamide- and Abiraterone-Treated Castration-Resistant Prostate Cancer

Reviewer 1: Anonymous
Reviewer 2: Anonymous
J. Clin. Med. 2019, 8(4), 489; https://doi.org/10.3390/jcm8040489
Received: 1 March 2019 / Revised: 5 April 2019 / Accepted: 8 April 2019 / Published: 11 April 2019

Round  1

Reviewer 1 Report

Substitute "running Head" with "running title"
Introduction section should be improved focusing the attention also on androgen and estrogen receptors that exert a pivotal role in PC initiation and progression. To obtain useful information, authors could read the following manuscripts . doi: 10.3389/fonc.2018.00002, doi: 10.18632/oncotarget.6220.
Tables 1 and 2 are lacking. Did the authors refer to supplementary Tables?
There are no information about the Gleason's Score.
I think that the authors analyzed the Androgen receptors or other proteins that are currently  screened, but there are no information.
Authors should emphasize the clinical features of the patients that induced the treatment choice.
 "Definition of high-volume tumor" seems a very strange definition.  Did You performed any measure of tumor size?
In the list of abbreviations different acronyms are lacking.

Author Response

Reviewer 1

 Point 1

Substitute "running Head" with "running title"

Response 1

Thank you very much. We have changed running as suggested.

 Point 2

Introduction section should be improved focusing the attention also on androgen and estrogen receptors that exert a pivotal role in PC initiation and progression.

To obtain useful information, authors could read the following manuscripts . doi: 10.3389/fonc.2018.00002, doi: 10.18632/oncotarget.6220.

Response 2

We have reconstructed and cited the indicated Ref in the introduction part.

 Point 3

Tables 1 and 2 are lacking. Did the authors refer to supplementary Tables?

Response 3

We are so sorry for the mistake in the upload of tables. We will add the table 1-4.

 Point 4 

There are no information about the Gleason's Score.

Response 4

We have included Gleason's Score in proportional hazard analysis in Table 1-4.

As shown in table, Gleason's Score did not remain as an independent prognostic factor.

 Point 5

I think that the authors analyzed the Androgen receptors or other proteins that are currently  screened, but there are no information.

 Response 5

The point is really important. We are currently studying the association AR mutation and amplification in cell free DNA and prognosis in patients received novel AR targeted drugs. AR amplification seems to associated with serum testosterone. However, the contents will be included in the next paper. The current manuscripts we would like to focus on the serum testosterone.

 Point 6

Authors should emphasize the clinical features of the patients that induced the treatment choice.

Response 6

Thank you very much. We have listed the patients back ground in Table 1. As reviewer suggested, majority of patients received Enza as a fist line. So we have described in the limitation section as follows “Fourth, due to the prior introduction of Enza in the Japanese healthcare system, the majority of Enza group (92%) received the drugs as a 1st line novel AR-targeted drugs, while of a half patients (54%) received Abi as a 1st line AR target drugs. The different result observed between Enza and Abi, including association with serum TST level, may possibly be affected by the background of patients received the drugs. We are currently performing a prospective study to re-assess the role of serum TST in the initial usage of Enza and Abi. The effect of the difference in the clinical features of the patients that induced the treatment choice in the current manuscripts will be answered in the near future.”

 Point 7

 "Definition of high-volume tumor" seems a very strange definition.  Did You performed any measure of tumor size?

Response 7

The size of the tumor was not measured. The number of metastasis and location was counted. The definition of high volume was defined base on the land mark paper of CHAARTED trial [defined as the presence of visceral metastases or ≥4 bone lesions with ≥1 beyond the vertebral bodies and pelvis]. (N Engl J Med 2015; 373:737-746 DOI: 10.1056/NEJMoa1503747)

 Point 8

In the list of abbreviations different acronyms are lacking.

Response 8

We have added list of abbreviations as indicated in abbreviations section on page 2.

Reviewer 2 Report

This study investigated the association between total TST and PFS in CRPC patients. It is interesting that TST could be the prognostic factor for relapsed prostate cancer patients. In addition, the difference between ENZ and Abi can be of help for determining the drugs for CRPC patients. However, several points should be improved.

1) I could not find table 1-4  in the manuscript.

2) Introduction is poorly described. The authors should write the background of prostate cancer research, problems in clinical setting and past reports about the diagnostic value of TST measurements.

3) The authors mentioned the diagnostic value of TST before the treatment. To understand the difference of effects between ENZ and Abi, the response of TST or PSA to these treatments should also shown.

3) Ln126: It is quite confusing that TST > 12 is used as a cut-off value. In Ln122, TST > 13 was used.

4) High TST (>13) does not have a prognostic value for overall survival of patients (Fig. 1d). How do the authors explain this result?

5) Discussion: Ln199-208. I could not understand what the authors intended to mean in this paragraph. They should illustrate clearly what is shown by each report they cited.

6) Ln215-218: The reference of Ryan et al is not appropriate. The result of Ryan et al is not consistent with that of this study because Ryan et al showed that high TST is associated with the patients treated with Abi not ENZ. In addition, the discussion in Ln.226- 239 is also contradicted with this report.

7) Overall, total TST has been previously reported to be associated with prognosis of CRPC patients in several studies. The authors should clearly focus on the novel aspect of this study.

Author Response

Reviewer 2

Thank you very much for the valuable comments. We will answer respective points.

1) I could not find table 1-4  in the manuscript.

1) We are so sorry for the mistake in the upload of tables. We will add the table 1-4.

Table   1. Patient’s backgrounds


ValueRange / %
Enza as an initial   therapy82
Abi as an initial   therapy25Total 107
Enza as a second line   therapy7
Abi as a second line   therapy21Total 28
Median Age73.054-88
Median BMI (kg/m2)23.416.09-34.06
Median TST at biopsy   (ng/dL)457.5228-847
Median PSA at Biopsy   (ng/ml)79.53.43-15332
Median PSA at   Enza/Abi/total (ng/ml)30.1/41.1/34.10.59-5942.62/3.52-13296/0.59-13296
Median TST at   Enza/Abi/total (ng/dL)13/12/132-92/3-31/2-92
Median Previous   treatment course number31 to 5
Median EOD Score  0/1/2/3/427/23/18/28/15
Median Follow up   Period (month)68.311.81-241.60
Median Enza/Abi PFS   period (month)3.9/2.10-16.50/0-13.37
Median 1st PFS (month)15.90.50-171.40
Gleason Score sum (N)

≤644.17%
71515.63%
82526.04%
≥95254.17%
Bone Mets8983.18%
Lymph Mets3431.78%
Lung Mets98.41%
Liver Mets65.61%
No Mets1211.21%
Patients with   death2321.50%
Pre/Post Docetaxel43/6440.19/59.81%
Steroid Use4340.19%
Estramustine Use5046.73%
Enzalutamide Dose160mg/80mg84/5
Abiraterone Dose1000mg/750mg45/1
BMI; body mass index, PFS; progression free survival, Mets;   metastasis, PSA:prostate-specific antigen , TST;  Testosterone
Table 2.    Predictive factors of PFS in Enzalutamide and Abiraterone






Univariate   Analysis
Multivariate   Analysis

Cut offHRCOIP
HRCOIP
Age720.750.48 -1.160.1932




GS90.930.59-1.480.7654




1st   line PFS (m)15.40.600.37-0.950.0309
0.850.215-3.250.8126
Previous   Docetaxel+/-2.441.575 -3.81<.0001< td="">
1.500.43-5.360.5222
Liver   Mets+/-1.690.70-3.460.2186




Visceral   Mets+/-1.510.83-2.580.1682




Lymph   Mets01.671.07 -2.630.0249
2.320.71-7.900.1607
High   Volume+/-1.731.09-2.830.0198
2.470.45-13.740.2927
EOD   Score22.791.52-5.470.0004
2.790.65-14.570.1721
ALP   (ng/dL)2541.200.78-1.860.4057




ICTP   (ng/ml)6.61.400.71-2.780.3307




Hb   (g/dL)11.90.650.42-1.010.0538




LDH   (mg/dL)2120.980.64 -1.520.9446




Alb   (g/dL)3.90.850.54 -1.350.5001




CRP   (mg/dL)0.151.871.11 -3.300.0173
0.670.17-3.100.5871
NLR   (ng/dL)2.61.130.66-1.940.6593




PSA   (ng/ml)34.11.731.12 -2.710.0143
1.240.44-3.660.6899
TST   (ng/dL)130.260.13-0.51<.0001< td="">
0.310.10-0.930.0365*
Steroid   use+/-2.241.44 -3.540.0003
1.100.40-2.950.8484
Estramustine   use+/-1.691.09-2.650.0179
1.410.46-4.390.5442
BMI;  body mass index, Pre-treatment course; previous treatment   course,   Mets; metastasis, ALP;   Alkaline phosphatase, ICTP; I collagen  telopeptide, LDH; Lactate   Dehydrogenase ,  Alb; albumin, CRP;    C-reactive protein , NLR; Neutrophil / Lymphocyte ratio,    PSA:prostate-specific antigen , TST;    Testosterone, * Statistical  significancer p<0.05< td="">
Table 3. Comparison of clinical factors   between TST<13ng/dL and TST≥13ng/dL


TST< 13TST≥13



Median(Average)Median(Average)P-value

Age71.50(68.74)70.00(70.88)0.2298
1st PFS (month)29.40(36.33)14.63(32.755)0.3576
††
Pre-Docetaxel42.11%(16/38)23.53%(8/34 )0.1336
†††
GS≥941.94%(13/31)50%(16/30)0.6159
†††
Lymph   Mets54.29%(19/35)38.24%(13/34 )0.2301
†††
Bone   Mets92.11% (35/38 )88.24% (30/34 )0.7002
†††
Liver   Mets5.56% (2/36 )14.71% (5/34 )0.2533
†††
Visceral   Mets8.33% (3/36 )23.53% (8/34)0.1062
†††
High   Volume65.71% (23/35)50.00% (17/34 )0.2270
†††
EOD   Score2(2.24)2(1.97)0.3965
BSI0.48(2.16)0.59(2.33)0.9342
††
ALP   (ng/dL)209(647.87)263.50(603.50)0.0158*††
PSA  (ng/ml)33.34(710.73)39.66(110.72)0.8391
††
CRP  (mg/dL)0.42(1.09)0.50(1.53)0.7866
††
PSA   at biopsy   (ng/ml)58.57(473.01)72.95(563.79)0.3903
††
TST   at biopsy  (ng/dL)4.42(4.22)3.41(3.32)0.1544
TST   nadir at 1st line  (ng/dL)9(38.69)12(13.23)0.8974
††
PFS;  progression free survival, GS; gleason score, Mets;   metastasis, EOD;  extent of disease,    BSI; bone scan index, ALP; Alkaline phosphatase,  ICTP; I collagen   telopeptide, LDH; Lactate Dehydrogenase , CRP;  C-reactive protein ,   PSA:prostate-specific antigen , TST;     Testosterone,  * Statistical   significancer p<0.05< td="">
† welch, †† wilcoxon,†††   fisher




Table 4. Predictive factors of OS in   Enzalutamide and Abiraterone





Univariate   Analysis
Multivariate   Analysis

Cut offHRCOIP
HRCOIP
Age720.940.54-1.660.8371




GS90.810.45-1.460.4801




1st   line PFS  (m)150.700.36-1.230.2134




Previous   Docetaxel+/-2.381.35-4.310.0025
3.041.41-7.1750.0038*
Liver   Mets+/-4.351.87-8.920.0014
0.350.07-1.9050.2079
EOD   Score22.871.305-7.560.0069
1.640.56-5.580.3778
Visceral   Mets+/-2.901.45-5.400.0038
7.881.62-29.520.0139*
Lymph   Mets+/-2.131.21-3.800.0091
1.600.77-3.290.2041
High   Volume+/-2.651.38-5.630.0028
1.600.64-4.500.3266
ALP   (ng/dL)2543.041.71-5.660.0001
2.511.25-5.2650.0090*
ICTP   (ng/ml)6.62.261.87-6.510.0941




Hb   (g/dL)11.90.490.27-0.850.0109
0.870.39-1.860.7144
LDH   (mg/dL)2121.921.10-3.430.0210
2.951.43-6.400.0033*
Alb   (g/dL)3.90.610.33-1.100.0990




CRP   (mg/dL)0.151.930.97-4.280.0607




NLR   (ng/dL)2.61.930.93-4.180.0790




PSA   (ng/ml)34.13.111.72-5.970.0001
1.760.88-3.690.1101
TST   (ng/dL)130.990.44-2.550.8750




Steroid   use+/-1.110.64-1.950.7011




Estramustine   use+/-0.950.55-1.660.8567




BMI;  body mass index, Pre-treatment course; previous treatment   course,   Mets; metastasis, ALP;   Alkaline phosphatase, ICTP; I collagen  telopeptide, LDH; Lactate   Dehydrogenase ,  Alb; albumin, CRP;    C-reactive protein , NLR; Neutrophil / Lymphocyte ratio,    PSA:prostate-specific antigen , TST;    Testosterone, * Statistical  significancer p<0.05< td="">










2)  Introduction is poorly described. The authors should write the  background of prostate cancer research, problems in clinical setting and  past reports about the diagnostic value of TST measurements.

2) Thank you very much. We have reconstructed the introduction as shown in red colors.

3)  The authors mentioned the diagnostic value of TST before the treatment.  To understand the difference of effects between ENZ and Abi, the  response of TST or PSA to these treatments should also shown.

3)  Thank you very much for the comments. This is an important point. We  have added the supplementary table 5 showing the PSA response rate in  the sequential usage of Enza or Abi.

Supplementary Table 5. PSA response in the   sequential use of novel AR targeted drugs.

All patients1st AbiEnza→Abi p-Value1st EnzAbi→Enzap-Value
30%PSA response






% (median)42.70%42.86%26.32%0.158942.62%28.57%0.3331
50%PSA response






% (median)37.10%35.71%21.05%0.186237.70%28.57%0.5208
PFS






week (median)12.415.79.930.07641211.10.2726
95% CI15.5-20.912.2-24.38.47-16.0
15.1-21.37.5-19.3
PFS; prgression free survival, Enza; Enzalutamide, Abi;   Abiraterone, 1st Abi/Enza; Abi/Enz as 1st line therapy, Enz→Abi; Abi use after Enz, Abi→Enza; Enza use after Abi.

In  terms of serum testosterone, due to the regulation of national  healthcare system, we could not follow the value of TST after the  treatment with Enza or Abi. However, in the limited patients, Enza usage  caused increased TST value (10-20%), while in Abi, TST reduced to the  undetectable value.

4) Ln126: It is quite confusing that TST > 12 is used as a cut-off value. In Ln122, TST > 13 was used.

4)  Thank you very much. I know this may be confusing. The median TST at  the initiation of Enza was 13 ng/dL, and the median TST at the  initiation of Abi was 12 ng/dL. The median of Enza and Abi overall was  13ng/dL. So, we have used TST 13ng/dL and 12ng/dL. In order to be  objective, to avoid any subjective intention, we have used median value  throughout the study in all the clinical factors.

5)  High TST (>13) does not have a prognostic value for overall survival  of patients (Fig. 1d). How do the authors explain this result?

5) This is a very important point. There are several reasons that we speculate. 1st,  as shown in table 3, TST>13ng/dL group showed the significantly  higher ALP and relatively higher liver mets, GS>9. Although there  seems to be an advantage in the PFS, high ALP, liver mets and GS>9  components may resulted in the limited response to the following  treatments such as docetaxel. 2nd, median PFS of Abi or Enza was 3-4 month, that limited the advantage of PFS on OS.

6)  Discussion: Ln199-208. I could not understand what the authors intended  to mean in this paragraph. They should illustrate clearly what is shown  by each report they cited.

6)  Thank you very much. We have reconstructed the discussion as shown in  red color on page 7. What we try to describe was that, even at lower TST  level<20ng high="" serum="" tst="">13ng/dL may represent the  remaining AR dependency that can only be targeted by novel AR targeted  drugs.

7)  Ln215-218: The reference of Ryan et al is not appropriate. The result  of Ryan et al is not consistent with that of this study because Ryan et  al showed that high TST is associated with the patients treated with Abi  not ENZ.

7) Thank you very much. We have reconstructed the discussion as follows on page 7.

The present data correlated with the findings of The clinical advantage of high serum TST was also reported by Ryan et al.10  Although the TST cut-off value was even lower (>8.6 ng/dL) compared  to the present study, they found a higher serum TST to be related to a  better response to Abi. On the other hand, in our study, the clinical advantage of high serum TST was not found in Abi but found in Enza.

8) In addition, the discussion in Ln.226- 239 is also contradicted with this report.

8) Thank you very much. We found long 1st  line PFS was related to the favorable PFS in Enza but not in Abi. We  think the result was similar to the finding of the Loriot et al., in Eur J Cancer 2015, 51 (14), 1946-52 that indicated TTCRPC>12 month was related to the good PFS in Enza.

9)  Overall, total TST has been previously reported to be associated with  prognosis of CRPC patients in several studies. The authors should  clearly focus on the novel aspect of this study.

9) Thank you very much. We totally agree. We have changed the conclusion part as follows.

Conclusion

A higher serum TST level (≥13 ng/dL) was associated with a favorable prognosis in Enza and/or Abi-treated patients. Serum TST level 13ng/dL may represent the sign of favorable response to novel AR targeted drugs in CRPC patients.

Round  2

Reviewer 1 Report

Authors improved the quality of the manuscript according to reviewers suggestions.

Author Response

Thank you very much for help us improve the manuscript. 

Reviewer 2 Report

The authors responded to my concerns.

In Introduction, the authors added a description about estrogen receptor (ER) function in CRPC. It is interesting that female hormone is also important in CRPC treatment. However, I can not understood how this description is associated with this study. I wonder if the authors expect TST is affected by estrogen signals. I could not find other description regarding the selection of ER-blocker in clinical data the authors presented. How did the atuhros select ER-blokcker, AR-blocker (Abi and Enz) or combination of both?

Author Response

Thank you so much for the bring up the very important points

We will try to answere the question from the reviewer. 

Question 

It is interesting that female hormone is also important in CRPC treatment. However, I can not understood how this description is associated with this study. I wonder if the authors expect TST is affected by estrogen signals. I could not find other description regarding the selection of ER-blocker in clinical data the authors presented. How did the atuhros select ER-blokcker, AR-blocker (Abi and Enz) or combination of both? 

Answer

 We at first treat with androgen signal bocker, however, after became refractory to the AR targeted drugs, we swich to the ER stimulator such as Estramustine. It actually work in some goup of patients. That is why 46.7% of patients previously received Estramustine in table 1. 

As listed in table 2, Estramustine use was related to resistant to the Enza and Abi response as indicated as HR 1.69 P=0.0179. 

I do not know exact mechanism, however, there seems to be cross talk between ER and AR signaling. 


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