Large-Scale Profiling of Extracellular Vesicles Identified miR-625-5p as a Novel Biomarker of Immunotherapy Response in Advanced Non-Small-Cell Lung Cancer Patients
, Michele Iuliani 1,*, Sonia Simonetti 1, Paolo Manca 3, Andrea Napolitano 1, Simone Tiberi 4, Marco Russano 1, Fabrizio Citarella 1, Simone Foderaro 1, Elisabetta Vulpis 5, Alessandra Zingoni 5, Laura Masuelli 6, Roberto Bei 7, Giulia Ribelli 1, Marzia Del Re 8, Romano Danesi 8, Bruno Vincenzi 1, Giuseppe Perrone 2, Giuseppe Tonini 1,†and add
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Round 1
Reviewer 1 Report
The research paper written by Pantano et al. is very interesting. The statistical analysis is fine with a good cohort of patient. The clinicians could identify, through your work, the patients who will have little or no benefit from ICI therapy despite the high PDL-1 expression.
Major comments.
None
Minor comments.
I suggest to the authors to have a better image quality in figure.
Please, correct the word microvesicles line 414; the word variable lines 458 and 459.
In the conclusion, please, precise the type of treatment to be combined (or not) with ICIs in case of PDL1>50% and miR-625-5p high. (An exemple of a concrete clinical application would be appreciated).
Author Response
The research paper written by Pantano et al. is very interesting. The statistical analysis is fine with a good cohort of patient. The clinicians could identify, through your work, the patients who will have little or no benefit from ICI therapy despite the high PDL-1 expression.
Major comments.
None
Minor comments.
I suggest to the authors to have a better image quality in figure.
Reply: we provided to improve images quality
Please, correct the word microvesicles line 414; the word variable lines 458 and 459.
Reply: we provided to correct the mistakes
In the conclusion, please, precise the type of treatment to be combined (or not) with ICIs in case of PDL1>50% and miR-625-5p high. (An exemple of a concrete clinical application would be appreciated).
Reply: we specified in conclusion section that patients were treated with pembrolizumab or nivolumab as monotherapy
Reviewer 2 Report
In their manuscript „Large-scale profiling of extracellular vesicles identified miR-625-5p as a novel biomarker of immunotherapy response in advanced non-small cell lung cancer patients“ Pantano et al report analysis of micro RNA from extracellular vesicles (EV) obtained in a cohort of 88 out of 218 NSCLC patients who received anti-PD1 treatment. The authors applied strict inclusion criteria and analyzed the presence of a panel of 799 miRNAs. Tey subsequently performed extensive and rigorous statistical analyses revealing that exclusively miR625-5p suffices as independent prognostic biomarker.
Despite obvious deficiencies in the manuscript (the finding is limited, experimental or prospective validation is missing), this reviewer recommends that the work be accepted for publication provided that minor changes have been made.
Specific points:
Figures must be improved in quality and regarding labeling:
- Individual charts in figures deserve a title
- axis labels should designate the depicted variable
- in figure legends skip “panel” and just indicate “a)” or “b)”
line 23: grammar: repeatedly occurring throughout the text)
line 35-36: what is “biological” or “clinical” significance? How can a biomarker capture interaction?
Line 45-46: add e.g. “abundance of” miRNA625-5p was found to be correlated
Line 80: EV-miRNA profiles – delete “s” in “EV-miRNAs”
Line 108: “(deaths)ranged” insert space
Line 131-134: It seems a verb is missing and a full stop is missing before “Progression free survival”
Line 138: “did not passed” – delet “ed”
Line 145-148: redundant description – please simplify
Line 163-165: rephrase, sentence is incomplete
Line 172: Typo
Line 181: delete “)”
Line 190-191: rephrase, a variable is unlikely to be “significant” rather then e.g. “significantly different in group a as compared to group b).
Line 232: “Panel A).Scatter” – space missing
Figure 2: some obsolete pixels above “has-miR-625-5p Class”
Figure 3: please add chart title, correct axis label
Line 262: delete “described”
Figure 4: please improve labelling
Line 290: odd sentence
Line 302: delete “o” from Violino
Line 315-316: This appears to be the major finding and deserves more confidence
Figure 6: Quality of text is low. Would it be possible to include text rather than converting text into pixels?
Line 324: rephrase “software”, e.g. programmes, tools, packages, ….
Line 331: shown rather than showed
Line 332: Unclear “Targets mRNAs wereranked” – please rephrase
Line 343-344: odd sentence “more investigated” - please rephrase
Line 344: “to not exclude” equals “include”
Line 355-363: The authors should directly compare specific features of studies.
Line 386: odd: “more expressed”
Line 395 change “suggest” to “suggests”
Line 410: unclear: “…limited by this detection and immortal time biases“ - please rephrase
Supplementary table 6: *) abbreviations not explained;
**) Lines 5&6: double “Current Smoker”
***) “Node Met” – please use explicit phrases
Author Response
In their manuscript „Large-scale profiling of extracellular vesicles identified miR-625-5p as a novel biomarker of immunotherapy response in advanced non-small cell lung cancer patients“ Pantano et al report analysis of micro RNA from extracellular vesicles (EV) obtained in a cohort of 88 out of 218 NSCLC patients who received anti-PD1 treatment. The authors applied strict inclusion criteria
and analyzed the presence of a panel of 799 miRNAs. Tey subsequently performed extensive and rigorous statistical analyses revealing that exclusively miR625-5p suffices as independent prognostic biomarker.
Despite obvious deficiencies in the manuscript (the finding is limited, experimental or prospective validation is missing), this reviewer recommends that the work be accepted for publication provided that minor changes have been made.
Specific points:
Figures must be improved in quality and regarding labeling:
Individual charts in figures deserve a title
Reply: we provided to include a title for each figure.
axis labels should designate the depicted variable
Reply: we checked it in all figures
in figure legends skip “panel” and just indicate “a)” or “b)”
Reply: we deleted “panel” in the text
line 23: grammar: repeatedly occurring throughout the text)
Reply: we checked grammar across the text
line 35-36: what is “biological” or “clinical” significance? How can a biomarker capture
interaction?
Reply: we modified this sentence to clarify the meaning
Line 45-46: add e.g. “abundance of” miRNA625-5p was found to be correlated
Reply: we added “abundance of” as suggested
Line 80: EV-miRNA profiles – delete “s” in “EV-miRNAs”
Reply: we provided to delete it
Line 108: “(deaths)ranged” insert space
Reply: we provided to correct.
Line 131-134: It seems a verb is missing and a full stop is missing before “Progression free survival”
Reply: we provided to add a full stop.
Line 138: “did not passed” – delet “ed”
Reply: we provided to make it
Line 145-148: redundant description – please simplify
Reply: we simplified the sentence
Line 163-165: rephrase, sentence is incomplete
Reply: we provided to complete the sentence.
Line 172: Typo - Reply: we provided to correct it
Line 181: delete “)”
Reply: we provided to delete it
Line 190-191: rephrase, a variable is unlikely to be “significant” rather then e.g. “significantly different in group a as compared to group b).
Reply: we modified as suggested
Line 232: “Panel A).Scatter” – space missing
Reply: we added the space
Figure 2: some obsolete pixels above “has-miR-625-5p Class”
Reply: we modified the figure
Figure 3: please add chart title, correct axis label
Reply: we modified the figure
Line 262: delete “described”
Reply: we provided to delete it
Figure 4: please improve labelling
Reply: we improved it
Line 290: odd sentence
Reply: we modified the sentence
Line 302: delete “o” from Violino
Reply: we provided to delete it
Line 315-316: This appears to be the major finding and deserves more confidence
Reply: we modified the sentence
Figure 6: Quality of text is low. Would it be possible to include text rather than converting text into pixels?
Reply: we improved it
Line 324: rephrase “software”, e.g. programmes, tools, packages, …
Reply: we provided to rephrase “software”
Line 331: shown rather than showed
Reply: we provided to correct it
Line 332: Unclear “Targets mRNAs wereranked” – please rephrase
Reply: we modified the sentence
Line 343-344: odd sentence “more investigated” - please rephrase
Reply: we modified the sentence
Line 344: “to not exclude” equals “include”
Reply: we modified the sentence
Line 355-363: The authors should directly compare specific features of studies.
Reply: we modified this paragraph directly comparing specific features
Line 386: odd: “more expressed”
Reply: we modified the sentence
Line 395 change “suggest” to “suggests”
Reply: we provided to correct it
Line 410: unclear: “…limited by this detection and immortal time biases“ - please rephrase
Reply: we modified the sentence
Supplementary table 6: *) abbreviations not explained;
**) Lines 5&6: double “Current Smoker”
***) “Node Met” – please use explicit phrases
Reply: we modified the supplementary table
Reviewer 3 Report
The article presented for review is a good example of valuable research study, very modern and of importance for science and clinical practice. It is evidence that there is a need for search for biomarkers and predictive factors for solid tumors immunotherapy. Identification of EVs and miRNA seems to help in this however, with low chance to routine use. Thus, for better present the results and to facilitate its understanding I suggest some corrections:
- Introduction- please explain in some sentences how : “ circulating EV-associated miRNAs (EV-miRNAs) could provide relevant information regarding not only cancer cell biology, but also tumor microenvironment including tumor-immune system interactions”, please cite some of the works of Whiteside T group.
- Paragraph- Sample size- Here I need a help of statistician to state if such a plan brings a risk of not objective qualification and or if allow to perform an objective study?
- 2- please correct this paragraph by division to patients and methods, separately. The inclusion criteria should be presented. Line 121- “inclusion criteria are patients…”- no, please correct. The table with patients characteristic should be presented in the main document, not supplement. Please show what was the stage of lung cancer.
- My main doubt concerns the influence of previous treatment on the results. I suggest to clearly explain that you are sure that only ICIs are responsible for the results.
Author Response
The article presented for review is a good example of valuable research study, very modern and of importance for science and clinical practice. It is evidence that there is a need for search for biomarkers and predictive factors for solid tumors immunotherapy. Identification of EVs and miRNA seems to help in this however, with low chance to routine use. Thus, for better present the
results and to facilitate its understanding I suggest some corrections:
Introduction- please explain in some sentences how : “ circulating EV-associated miRNAs (EVmiRNAs) could provide relevant information regarding not only cancer cell biology, but also tumor microenvironment including tumor-immune system interactions”, please cite some of the works of Whiteside T group.
Reply: we provided to discuss briefly this point including some papers of Whiteside T research group.
Paragraph- Sample size- Here I need a help of statistician to state if such a plan brings a risk of not
objective qualification and or if allow to perform an objective study?
Reply: sample size was calculated to be adequately powered in order to perform an objective study (Response Rate and Overall Survival)
2- please correct this paragraph by division to patients and methods, separately. The inclusion criteria should be presented. Line 121- “inclusion criteria are patients…”- no, please correct. The table with patients characteristic should be presented in the main document, not supplement. Please show what
was the stage of lung cancer.
Reply: we provided to add a specific paragraph on inclusion and exclusion criteria. We moved the table with patients characteristics in main document
My main doubt concerns the influence of previous treatment on the results. I suggest to clearly explain that you are sure that only ICIs are responsible for the results.
Reply: according to multivariate analysis (figure 4), EV-miR-625-5p expression retains its performance in identifying patients who mostly benefit from ICI in term of objective response, overall survival and progression free survival independently from the treatment type and line.
