tRNA-Derived RNA Fragments Are Novel Biomarkers for Diagnosis, Prognosis, and Tumor Subtypes in Prostate Cancer
and Shiping Ding 1,*
Round 1
Reviewer 1 Report
In “tRNA-derived RNA fragments are novel biomarkers for diagnosis, prognosis, and tumor subtypes in prostate cancer”, Weigang Liu et. al. performed bioinformatic analyses to investigate the clinical value of 5'-tRFs in PRAD.
I consider that this work contribute to the understanding of PRAD and could help to improve the clinical management of patients.
I have major comments that could improve this manuscript:
1.- In section “2.4. Identification of 5'-tRFs dysregulated in PRAD, the authors explain that they used Wilcoxon rank sum test to compare expression levels of 5'-tRFs between tumor tissues and adjacent normal tissues. However Wilcoxon rank sum test is a nonparametric test to compare a median from one group to a theoretical value. Thus, did the authors performed Wilcoxon matched test? In that case also correct this section.
Also, Wilcoxon matched test is a non parametric paired t-test, so in Results Section “3.1. 5'-tRFs are dysregulated in PRAD”, the sentence “We analyzed small RNA-sequencing data of 499 tumor tissues and 52 adjacent normal tissues of PRAD from TCGA.” Should be corrected since the authors could only used the tumor and adjacent normal paired samples from 52 patients.
2.- Figures 1, 2 and 3 are not sharp, they look blurry, authors should improve the quality.
3.- The paragraph from line 272, describe that “Patients with lower tRF score had significantly longer PFS (log rank test, P=6.89e-9) and DFS (P=1.50e-7) than those with higher tRF score.”, but Figure 3 shows that patients with high tRF score have longer PFS and DFS.
4.- Did the authors evaluate overall survival according to tRF score? They should include this in the manuscript.
5.- Authors claim that in light of results presented, 5'-tRFs could be promising clinical biomarkers for the diagnosis of PRAD. Even when I agree that the results clearly demonstrate that 5'-tRFs have a diagnostic value for PRAD, I wonder how this could be applicable for the clinic, because the detection of the 5'-tRFs needs to perform a biopsy. I think that authors should include a discussion explaining in which cases patients would benefice with 5'-tRFs detection would help? The authors should discuse about the sensitivity and specificity of the regular biopsy and the need of more clinical studies to compare the ROC curves of regular biopsy with the 5'-tRFs.
6.- Also, have 5'-tRFs been detected in circulation or urine? Because in that case, 5'-tRFs could be promising biomarkers for early detection.
Author Response
Below are responses to comments (shown in italics and bold font) from two Reviewers. We greatly appreciate their helpful comments and suggestions, which significantly improved and clarified the presentation of our paper.
Reviewer #1
I consider that this work contribute to the understanding of PRAD and could help to improve the clinical management of patients.
Response: Thank you very much for your comments! We have addressed your comments one by one below.
I have major comments that could improve this manuscript:
1.- In section “2.4. Identification of 5'-tRFs dysregulated in PRAD, the authors explain that they used Wilcoxon rank sum test to compare expression levels of 5'-tRFs between tumor tissues and adjacent normal tissues. However Wilcoxon rank sum test is a nonparametric test to compare a median from one group to a theoretical value. Thus, did the authors performed Wilcoxon matched test? In that case also correct this section.
Also, Wilcoxon matched test is a non parametric paired t-test, so in Results Section “3.1. 5'-tRFs are dysregulated in PRAD”, the sentence “We analyzed small RNA-sequencing data of 499 tumor tissues and 52 adjacent normal tissues of PRAD from TCGA.” Should be corrected since the authors could only used
the tumor and adjacent normal paired samples from 52 patients.
Response: In our study, two-sample Wilcoxon tests were used to detect differentially expressed 5'-tRF between tumor tissues (n=499) and adjacent normal tissues (n=52). To utilize all tumor data, the unpaired test was chosen in the analysis. We implemented this analysis using the function “wilcox.test” (see below) in the R package. We have added this information in the 2.4 Section.
2.- Figures 1, 2 and 3 are not sharp, they look blurry, authors should improve the quality.
Response: As you suggested, we have re-generated Figures 1-3 with high resolution.
3.- The paragraph from line 272, describe that “Patients with lower tRF score had significantly longer PFS (log rank test, P=6.89e-9) and DFS (P=1.50e-7) than those with higher tRF score.”, but Figure 3 shows that patients with high tRF score have longer PFS and DFS.
Response: Thank you for the important reminder and we apologize for the mislabeling in Figure 3. The label in Figure 3 was switched by mistake and we have corrected it in the revised manuscript.
4.- Did the authors evaluate overall survival according to tRF score? They should include this in the manuscript.
Response: We constructed prognostic predictors of 5'-tRFs based on progression-free survival (PFS) of PRAD. We presented the results of PFS and DFS (including prognosis of four 5'-tRFs subtypes) throughout in the paper. The overall survival (OV) rate of PRAD is generally better than other cancers. In PRAD dataset, the large majority of patents (97.9%) are still alive at the end of the observation period. This flattens the OV survival curve, showing no difference between the two tested groups. However, the recurrence rate of PRAD is quite higher, and most patients developed castration-resistant PRAD at advanced stages. Therefore, PFS and DFS seem to be more suitable for evaluating the performance of these clinical biomarkers.
5.- Authors claim that in light of results presented, 5'-tRFs could be promising clinical biomarkers for the diagnosis of PRAD. Even when I agree that the results clearly demonstrate that 5'-tRFs have a diagnostic value for PRAD, I wonder how this could be applicable for the clinic, because the detection of the 5'-tRFs
needs to perform a biopsy. I think that authors should include a discussion explaining in which cases patients would benefice with 5'-tRFs detection would help? The authors should discuse about the sensitivity and specificity of the regular biopsy and the need of more clinical studies to compare the ROC curves of regular biopsy with the 5'-tRFs.
Response: Thanks for your valuable suggestion. We have included a discussion on the applicability and potential challenges of 5 '- tRFs as clinical biomarkers of PRAD in the revised manuscript (page 16). We also completely agree with this reviewer that these new findings require further validation from independent large clinical cohorts before they can be used clinically as diagnostic, prognostic, or subtyping biomarkers for PRAD (see the discussion). It is worth noting that 5 '- tRFs can not only provide potential diagnostic information, but more importantly, they can also provide prognostic and tumor subtyping information for PRAD patients.
6.- Also, have 5'-tRFs been detected in circulation or urine? Because in that case, 5'-tRFs could be promising biomarkers for early detection.
Response: Yes, tRFs are abundant and can be detected in bodily fluids such as serum and urine. We have included this information in the Discussion in the revised manuscript. We stated in the Discussion that “tRFs are abundant and can be detected in bodily fluids such as blood samples, urine, saliva, and exosomes, making them promising non-invasive biomarkers for complex diseases such as cancer (8). Therefore, the clinical value of these 5'-tRFs in the peripheral blood or urine of PRAD patients deserves further evaluation.”
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authors:
The authors analyzed small RNA sequencing data to systematically assess the clinical values of 5'-tRFs in prostate adenocarcinoma. They tried to demonstrate 5'-tRFs as promising clinical biomarkers for the diagnosis, prognosis, and classification of tumor molecular subtypes. They also believe in a strong potential to aid clinicians in developing personalized treatment plans for prostatic cancer patients. The topic is currently interesting and some amendments are required. The paper is well structured and worthy of consideration after a minor revision.
- You mentioned how in recent years, a big number of biomarkers have been tested and searched for the clinical diagnosis and prognosis of prostate cancer. In this regard, worthily of mention is a new finding on a novel urinary biomarker. Urinary levers of 8-OHdG and 8-iso-PGF2α before and after surgical treatments have been shown to predict radicality after RARP and maybe recurrence (DOI: 10.3390/jcm11206102). I strongly believe this should be mentioned and included in your paper.
- More findings and further validation from independent large clinical cohorts are required before clinical and diagnostic application. In this view, please be less incisive in the conclusion.
- Check typos
Author Response
Reviewer #2
The authors analyzed small RNA sequencing data to systematically assess the clinical values of 5'-tRFs in prostate adenocarcinoma. They tried to demonstrate 5'-tRFs as promising clinical biomarkers for the diagnosis, prognosis, and classification of tumor molecular subtypes. They also believe in a strong potential to aid clinicians in developing personalized treatment plans for prostatic cancer patients. The topic is currently interesting and some amendments are required. The paper is well structured and worthy of consideration after a minor revision.
Response: Thank you very much for your comments! We have addressed your comments one by one below.
- You mentioned how in recent years, a big number of biomarkers have been tested and searched for the clinical diagnosis and prognosis of prostate cancer. In this regard, worthily of mention is a new finding on a novel urinary biomarker. Urinary levers of 8-OHdG and 8-iso-PGF2α before and after surgical treatments have been shown to predict radicality after RARP and maybe recurrence (DOI: 10.3390/jcm11206102). I strongly believe this should be mentioned and included in your paper.
- Response: As you suggested, we have described and cited this new finding in the Introduction in the revised manuscript. We stated that “…Of note, urine levels of 8-OHdG and 8-Iso-PGF2α before and after surgery in patients with PRAD can help predict radicality (and possibly local recurrence) after robot-assisted radical prostatectomy…”
- - More findings and further validation from independent large clinical cohorts are required before clinical and diagnostic application. In this view, please be less incisive in the conclusion.
- Response: As you suggested, we have revised our statement in the conclusion. We changed that “… small ncRNAs-tRFs
- as valuable clinical biomarkers .... These findings can not only provide clinicians ...” into “… small ncRNAstRFs
as potential clinical biomarkers .... These findings may not only provide clinicians ...”. - - Check typos
Response: We have gone through our manuscript again and corrected several typos in the revised version.
Reviewer 3 Report
Dear Authors,
Please see my comments below for your kind attention.
1. LINE 75: Mounting evidence suggests that these small ncRNAs play important roles in cancer development and progression [13]---> please describe more with some more papers, and cancers. Then may be focus on prostate cancer. Please refer to the prostate cancer section of this paper (https://www.frontiersin.org/articles/10.3389/fcell.2022.954431/full#B91).
2. Please delete the last paragraph of introduction ("To discover new......."). That is the result which you described there which is not the place to discuss. I will suggest, replace the last paragraph by 2-3 lines with your hypothesis.
3. Can this concept be utilized to explore the potential drug sensitivity to prostate cancer cells or resistance upon progression? In short, are there any studies on utilizing tRNA-derived RNA fragments as a tool to use in deciding treatment options.
E.g. Sun et al. demonstrated that tRF-30-JZOYJE22RR33 and tRF-27-ZDXPHO53KSN were correlated with trastuzumab resistance in breast cancer (Sun C, Yang F, Zhang Y, Chu J, Wang J, Wang Y, et al. tRNA-derived fragments as novel predictive biomarkers for trastuzumab-resistant breast cancer. Cell Physiol Biochem. 2018;49:419–31.)
4. Please highlight if I have missed anywhere, but I am interested to know about the sex hormone-dependent tRNA derived RNA (SHOT-RNA which is highly expressed in hormone-dependent cancers. This should be relevant in breast and prostate cancer
Author Response
Below are responses to comments (shown in italics and bold font) from Reviewer #3. We greatly appreciate their helpful comments and suggestions, which significantly improved and clarified the presentation of our paper.
Reviewer #3
- LINE 75: Mounting evidence suggests that these small ncRNAs play important roles in cancer development and progression [13]---> please describe more with some more papers, and cancers. Then may be focus on prostate cancer. Please refer to the prostate cancer section of this paper
(https://www.frontiersin.org/articles/10.3389/fcell.2022.954431/full#B91).
Response:
As you suggested, we have cited this reference and described more research related to prostate cancer in the Introduction (lines 79-93).
2. Please delete the last paragraph of introduction ("To discover new......."). That is the result which you described there which is not the place to discuss. I will suggest, replace the last paragraph by 2-3 lines with your hypothesis.
Response:
As you suggested, we replaced the last paragraph of the Introduction by the hypothesis of our manuscript (lines 94-98).
3. Can this concept be utilized to explore the potential drug sensitivity to prostate cancer cells or resistance upon progression? In short, are there any studies on utilizing tRNA-derived RNA fragments as a tool to use in deciding treatment options. E.g. Sun et al. demonstrated that tRF-30-JZOYJE22RR33 and tRF-27-ZDXPHO53KSN were correlated with trastuzumab resistance in breast cancer (Sun C, Yang F, Zhang Y, Chu J, Wang J, Wang Y, et al. tRNA-derived fragments as novel predictive biomarkers for trastuzumabresistant breast cancer. Cell Physiol Biochem. 2018;49:419–31.)
Response:
Thanks for your thoughtful consideration. This is another very interesting area that needs further research in the future. We do not have clinical data related to drug resistance, so we cannot determine the relevance of individual tRFs in drug sensitivity of prostate cancer. We have cited and discussed this reference in the
Discussion (lines 475-479).
We have also discussed the implication of tRF subtype in the treatment of PRAD in the Discussion. For example, the tF-2 subtype has a higher TMB and genomic alterations than the other subtypes and therefore may respond better to immunotherapy. In addition, the tF-1 tumor subtype has more frequent ETS family gene fusions (such as ERG and ETV1) than the tF-2 tumor subtype. Therefore, the tRF-1 subtype may be more sensitive to, and therefore preferential for, androgen deprivation therapy than the tRF-2 subtype (Karnes RJ, et al. Cancer Res. 2010, 70:8994-9002). Recent preclinical studies have demonstrated an association between ETS gene fusions and components of the DNA damage response pathway (Feng FY, et al. Clin Cancer Res. 2014, 20:4442-8). Targeting DNA damage response pathways with inhibitors of PARP1, DNAPK, and HDAC1 may also be an alternative therapeutic option for the tRF-1 subtype with frequent EST gene fusions. HRD scores quantify the extent to which double-strand breaks in DNA in tumor cells cannot be repaired. Clinical trials have also shown that high levels of these HRD scores are associated with better responses to PARP inhibitor- or platinum-based therapy in ovarian and breast cancer (Ray-Coquard I, et al. N Engl J Med. 2019, 381:2416-28; Zhao EY, et al. Clin Cancer Res. 2017, 23:7521-30). Therefore, the tRF-2 subtype may respond better to PARP inhibitor- or platinum-based therapy than the other tRF subtypes in PRAD patients.
4. Please highlight if I have missed anywhere, but I am interested to know about the sex hormonedependent tRNA derived RNA (SHOT-RNA which is highly expressed in hormone-dependent cancers. This should be relevant in breast and prostate cancer
Response:
In the 3rd paragraph of the Introduction, we have already described this finding. We stated that “…tRNAhalves from tRNAAsp-GUC, tRNAHis-GUG, and tRNALys-CUU have been noted as promoters of cellular proliferation in breast cancer and PRAD in a sex hormone-dependent manner (Shozo Honda et al. PNAS 2015, 112:E3816-E25)…” We have now added the alias of tRNA-halves, SHOT-RNA, to this description (lines 85-87).
Round 2
Reviewer 1 Report
The manuscript has improved after revision and all comments were answered.
1.- Thank you, now I understand you have performed In our study, two-sample Wilcoxon tests with R (also called Mann-Withney-Wilcoxon test or Wilcoxon rank sum test or Mann-Whitney U test).
2.-
3.- Perfect.
4.- I agree with your explanation.
5.- Perfect.
6.- Perfect.
Author Response
No further comments were raised by Reviewer #1. Thank you!
