miR-494 in Extracellular Vesicles as a Potent Biomarker of Chronic Myeloid Leukemia Treatment with Tyrosine Kinase Inhibitors
Round 1
Reviewer 1 Report
In the manuscript "MiR-494 in extracellular vesicles as a potent biomarker of chronic myeloid leukemia treatment with tyrosine kinase inhibitors", the authors described the involvement of miR-494 in EVs in chronic myeloid leukemia (CML).
Despite the authors mentioning that the main goal of the manuscript is the applicability of miRNAs in EVS for CML clinical diagnosis, the structure of the work and the conclusions do not reflect that goal. The manuscript needs a substantial revision of concepts and a clarification of multiple essential methodological aspects.
In the introduction section, some aspects related with TKI treatment and monitoring analysis need to be extensively revised. For example, the authors refer “Recently, they have begun to be selected as the first line for CML-CP” (line 38), but Nilotinib and Dasatinib are approved as first-line treatments for over 10 years. In line 48, “High-sensitive test using peripheral blood is required for CML monitoring”, currently the monitoring is already performed in peripheral blood with high sensitivity allowing the classification of MR5.0. Additional some terms used are not appropriate for hematological cancer as CML, as monitor leukemic cells mass or very small lesions. In introduction should also mention the works in CML and EVs for a better context of the data in this pathology. Relating to miRs, the authors refer “miRNAs in tissue or cell-lineage specific purified EVs is recommended for more accurate and ealry diagnosis” (line 68 and 69), my question is how is that possible to identify the CML specific EVs in peripheral blood?
The aims of the manuscript need to be revised based on the methods applied and samples use.
In the material and methods section, I have some concerns and extensive revision/clarification is needed to ensure the quality of the manuscript and the proper interpretation of results:
- The authors need to better explain the differences between the two CML cell lines used regarding cytogenetic alterations. Since KU812 present some alterations described as High-risk additional chromosomal abnormalities.
- The doses of TKI selected need to be justified and based on each work. Authors must clarify the following sentence “inhibit the cell proliferation adequately and did not induce excess cell death.” (line 86 and 87)
- In patients sample section is mention “Five patients were diagnosed CML disease in chronic and nontreated at Takagi hospital, and five patients were treated by IM continu-ously. Healthy volunteers were confirmed by medical interview and physical examination. (line 93-95). But in table 2 and the results section, the patients are classified as CML-AP/BC or CML-CP, and the healthy as Normal. The description in methods does not match with CML-AP/BC or CML-CP classification. For the authors, the group five patients were treated by IM continu-ously corresponds to CML-AP/BC? And the non treated to CML-CP? If so, this is completely wrong and the group categories need to be revised. Another aspect, the author must clarify the classification system used for patients classification (WHO? ELN?) and introduce treatment information.
- Table 2 is not mentioned in the text and the WBC units need to be revised.
- In the subsection 2.4 and 2.5 the authors must state the amount of RNA used for each technique, in order to allow a proper reproducibility of methods used. More scientific rigor must be uses and vague sentences as “Extracted miRNAs were separated into two tubes, that is one tube for micro-array assay and another tube for qRT-PCR.” (lines 124 and 125) should be avoid
- Statistical analysis must be complete: the normality test used, the analysis done to analyze WB data…
Results section:
- In the 3.1 results section, the authors refer that “EVs in supernatant of CML cell lines (K562 and KU812) were collected by polyethylene glycol (PEG) sedimentation” (lines 148 and 149) this procedure was not described in methods section. The EVs collection was performed only one time (n=?)? The purity for K562 is represented by the histogram in figure 1A, but KU812 data is missing.
- In Figure 2, the volcano plots should be on the same scale to be comparable between the two cell lines. In the present form, the plots seem to indicate that the expression is similar in both cell lines, but in fact since the scales are different between the graph that is not true. Additional these plots refer to IM treated samples or DS treated samples?
- In section 3.3 the data are from three independent samples? The results represented in figure 3 are expressed in mean or median? the authors must include the variation data (standard deviation or SEM) for each measure.
- In section 3.4 and in figure 4 the authors need to refer to the dispersion of data ( median and 95% CI or range).
In the same line as asked in the introduction, the concepts of CML classification, treatment, and guidelines need to be revised in the discussion:
- The scoring systems currently applied in CML are more than those SOKAL and EURO (Hasford) mentioned and exist guidelines more recently than 2013.
- Authors must add the references that support the BCR-ABLINs35bp data.
- In discussion, the authors point-out some of the advantages of use EVs for early cancer detection. However some question need to be further discussed as what is the markers that allow to identify and purify EVS from specific cell-origin? Was this done in CML or in other cancers? The advantage that cancer cells produce more EVs than normal cells is a fact at diagnosis, but during the treatment still easy to have sufficient material to study? Since the ratio between tumor to normal cells is expected to be reduced with good treatment results as TKIs.
- “Furthermore, the levels of cellular miR-494 by microarray were 1.06 (IM) and 1.26 (DS) in KU812, 1.03 (IM) and 1.04 (DS) in K562, so the amounts of miRNA in cells did not significantly change with TKIs treatment.” (lines 248 to 250) This resulst should be presented at results section and not in discussion.
- The authors need to revise the relation between PTEN, AKT and Myc, and revise the following sentences in accordance “These reports suggest the function of miR-494 differs from cell types.”
The conclusions “our data suggest the utilization of miR-494 in circulating EVs as a biomarker of CML staging and the evaluation of the effects of TKI treatments” need to be reformulated. The data presented regarding treatment are only obtained by two cell lines with a small exposure to the drug, very different from the patients reality. Moreover, this is very preliminary data with only 5 samples per group that are not well defined. Furthermore, sentences like “It may lead to development of a less-invasive and high-sensitivity biomarker for CML patients.” Need to be reformulated, since the authors approach is no less invasive than the current use for biomarker monitoring and diagnosis because in both cases is used peripheral blood samples.
The authors must perform a careful reading of the manuscript, including figure legends, to improve the writing quality and correct multiple typing errors.
Author Response
Dear Reviewer 1,
We give many thanks for your worthy comments.
We have done revisions according to your comments and suggestions. Please see the attachment.
We sincerely hope that revised version is now ready for your consideration.
Sincerely Yours,
Tatsuki Shibuta, Ph.D.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Shibuta et al., have presented a case for considering miR-494 in extracellular vehicles (EVs) as a biomarker for CML treatment with TKIs. EVs have been an emerging field in the past decade and are involved in ferrying a wide variety of biomolecules, their role in cancer is much appreciated in the last few years. Authors have done in vitro experiments in CML cell lines and showed that EVs are indeed released and contain miR-494 and miR-373. When interrogated with TKI inhibitors, indeed the amount of these miRNAs decreased significantly. Authors have conducted microarray and quantitative RNA analysis to prove their hypothesis. Finally, they have isolated EVs from CML patients and found that the amount of these miRNAs indeed go down upon treatment with TKIs (CML-AP/BC).
Major Comments: The experimental design is very well and the results support their hypothesis. However, the presentation needs to be improved. In Fig. 4B, show NS where there is no significance. Particularly, in the right side figure show if there is any significance between Normal and CML-AP/BC.
Minor: In multiple places typos are observed eg: L-104, it is mentioned After 72 incubation, hours are missing. In the same line its media instead of mediums. Please check all the typos.
- L-86 ,there should be a space between 1 and h
- L131, space between number and molarity.
Author Response
Dear Reviewer 2,
We give many thanks for your worthy comments.
We have done revisions according to your comments and suggestions. Please see the attachment.
We sincerely hope that revised version is now ready for your consideration.
Sincerely Yours,
Tatsuki Shibuta, Ph.D.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
I would like to thank the author for the answers to the comments. Despite the great improvement in the manuscript, some points need to be clarified:
- In the abstract, the author mentions 5 patients in CML-CP, 5 in CML-AP and 5 in CML-BC, but according to methods and results in total are only 10 patients (5 CML-CP plus 5 CML-AP/BC). Please clarify.
- The authors add the following sentence "Patients with CML-AP/BC were not treated with TKIs.”. What was the treatment? could that influence the miR levels? The authors need to clarify the treatment since the main results were obtained in this group.
Author Response
Dear Reviewer 1,
We give many thanks for your worthy comments again.
We have done revisions according to your comments. Please see the attachment.
We hope that revised version is now ready for your consideration.
Sincerely Yours,
Tatsuki Shibuta, Ph.D.
Author Response File: Author Response.pdf
