MicroRNA: A Signature for the Clinical Progression of Chronic Lymphocytic Leukemia

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The Authors of the manuscript undertook a study of the expression of selected miRNAs in the bone marrow and lymph nodes cells of a group of patients with chronic lymphocytic leukemia (CLL). They found that the expression of miR-26b, 34a and 150 was significantly increased  in bone marrow cells in the patients with CLL as compared to noncancerous bone marrow samples, and  the expression of other ones (20a, 26b, 34a and 451a) was decreased in CLL lymph nodes as compared to samples of patients with lymphadenopathy. Moreover they found, that miRNA-34a and 150 in bone marrow cells was the highest in the CLL patients requiring treatment with anemia, lower in treatment requiring patients without anemia, even lower in patients without treatment requirement and the lowest in non-cancerous bone marrow samples. Those results, although interesting, are only crude observations. One of the aims of the study was to determine the role of miRNAs in biological pathways associated with leukemogenesis in CLL. However, the Authors did not make any attempt to test a possbible impact of altered expression of miRNAs studied on intracellular pathways which those miRNAs may be involved in, or on the expression of genes targeted by the miRNAs studied. The analysis of the bibliographic data on this issue is not enough for considering this goal as achieved. The analysis of a relationship between the expression of the miRNAs analyzed and the clinical and biological features of the patients included into the study had to be carried out. The only finding concerning a possible impact of deregulated expression of the miRNAs on the clinical picture of CLL was that the miRNA-34a and 150 were upregulated in the bone marrow stronglier in progressive, i.e. requiring the treatment, than in stable cases. The Authors analyzed separately progressive cases with and without anemia, and found that the relative expression of both miRNA in the patients with anemia was higher than in the absence of anemia.  However, the anemia in CLL may have very different causes, not only medullary suppression of hematopoiesis,  like autoimmune phenomena, hypersplenism, chronic blood loss relative to thrombocytopenia etc. The Authors did not provide any information regarding the mechanism(s) of anemia in their patients and its relationhsip to CLL. The analysis of the relationship between the expression of miRNAs and the occurence of anemia regardless its cause may not justify valid conclusions. The sentence „We noted a significant dynamic upregulation of miRNA-150 and miRNA-34a during clinical progression of CLL” is not supported by the results reported in the manuscript. To make such statement the Authors would have performed sequential miRNA expression measures during the observation period of the patients, and not only the single one during the disease progression.

There is a serious methodological concern regarding the control groups: bone marrow from patients with non cancerous blood diseases and lymph nodes from patients with lymphadenopathy. There is no information what neoplastic diseases it was about, and what was the cause of those lymphadenopathies – inflammatory? infectious? undetermined?  There is a the lack of information about the bone marrow composition of the patients’ and control groups. In normal bone marrow the lymphatic cells usually make up about 10-20% of all nucleated cells, but in the bone marrow of CLL patients the percentage of lymphoid cells is much higher, and sometimes is close even to 100%. Therefore, it is highly probably that the Authors compared the expression of miRNA obtained from cells belonging to completely different lineage: lymphoid cells in CLL bone marrows, and a mixture of myeloid, lymphoid and erythroid cells in control marrows. The same doubt arises regarding the comparison of progressive and stable CLL patients. It cannot be excluded that the percentage of CLL lymphocytes was higher in the bone marrow of patients with progressive diseases than in patients with the stable one, and this difference accounted for, at least partially, the differences of the expression of miRNA-34a and 150.

At last, one wonders why the Authors decided to carry out the study on the bone marrow and lymph nodes cells, and not on peripheral blood CLL lymphocytes, which would have been the easiest to obtain and the purest as neoplastic cells population.

In conclusion, I regret to express the opinion that the manuscript in its current form is not suitable for publication in Lymphatics

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The article by Veryaskina and collaborators is well written, argued and developed, and could be advisable for publication with minor revision:

1. The possible limitations of the study carried out and future studies to be carried out should be briefly expanded.

2. Finally, there is an interesting reference on microRNA signatures in pathology that could be cited:

Legaz et al. MicroRNAs as Potential Graft Rejection or Tolerance Biomarkers and Their Dilemma in Clinical Routines Behaving like Devilish, Angelic, or Frightening Elements. Biomedicines. 2024 Jan 5;12(1):116. doi: 10.3390/biomedicines12010116

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors identify the miRNA expression profile in CLL and determine the role of miRNAs in biological pathways associated with leukemogenesis in CLL. The author presents a novel approach by dynamically profiling the expression levels of specific miRNA-34a and miRNA-150 in CLL patients and correlating these levels with clinical progression and therapy needs. This dynamic aspect offers a deeper understanding of miRNA roles over time, which is less commonly addressed in similar studies. Albeit, I consider these findings to provide new insight into leukemia-related fields, I still have some suggestions.

1, Most figures are highly professional; however, the authors should guide the readers to the meaning of the images appropriately; otherwise, it will likely cause misunderstandings. Therefore, I suggest the author consider revising these figures and legends again.

2, For Figure 1, the author employed the Mann-Whitney U test to evaluate the differences between two independent groups. The author may need to use other statistical analyses, such as ANOVA to calculate the P-value for three or more groups of data, and please update the “Statistical Analysis” of the Method during further revision (PMID: 37274638, 34329194, 34400890).

3, LINE 137: An analysis of expression levels of miRNA-150, -20a, -26b, -34a, -451a, and -96  demonstrated a unique miRNA expression profile in CLL in BM and LN samples. However, it would be much better if the authors could provide some Workflow or Scheme for this analysis, I suggest that they can take a look at the recent paper in MDPI (PMID:  35327353, 34834441,35563422).

4, In Table 2, the author identified target genes participating in hematopoiesis and biological processes involving lymphocytes. However, I suggest the authors can validate their data via Proteinatlas or cBioportal database. (PMID: 17008526, 22588877, 34202143).

5, There are few typo issues for the authors to pay attention to; please also unify the writing of scientific terms. “Italic, capital”? Please double-check the superscripts and subscripts for the whole manuscript. 

6, Most references are out of date, the author needs to discuss the recent paper as well as the analysis methods in this manuscript.
7, For Figure 2, the font is too small for the current figures; meanwhile, the manuscript also needs English proofreading.

Comments on the Quality of English Language

 Minor editing of English language required

Author Response

Please see the attachment

Author Response File: Author Response.pdf