Circulating miR-618 Has Prognostic Significance in Patients with Metastatic Colon Cancer

Round 1

Reviewer 1 Report

In this study, Radanova et al. found the overexpression of miR-618 in the serum of metastatic colorectal cancer (CRC) patients is associated with improved overall survival. They also found that this miRNA expression is higher in the CRC patients' sera than the normal subjects. They further did the genotypic analysis on circulating miR-618. They observed that the patients harboring AC rs2682818 genotype have a decreased risk for CRC compared to patients with CC and AA genotypes.

The strengths of this manuscript being that it employed a decent number of patients, i.e., 104. Though miR-618 has been widely studied in different cancer types, including colorectal cancer but here, the authors, for the first time, showed the 'serum level' association of miR-618 in patients with metastatic CRC towards overall survival. Coming to the genotype part, the authors showed that in the caucasian population, the AC genotype had decreased risk of CRC than individuals with CC/AA genotypes. This observation is similar to what Chen et al. found in the Chinese population, which the authors discussed. Finally, as the authors mentioned, miRNA-618 could be useful as a prognostic biomarker in CRC based on their data.

Something that I couldn't get my head around is the main basis of the paper, i.e., in the discussion line 264, the authors mentioned, based on this study, the 'tumor suppressor' role of mir-618, which is evident from the improved overall survival of CRC patients where it is overexpressed in their sera. Why, then, if it has a tumor suppressor role, is overexpressed in the sera of cancer patients compared with the healthy ones? Rationally thinking, it should be underexpressed in the CRC owing to its tumor-suppressing properties. Are there similar studies out there where cancer patients have a higher expression of a particular miRNA, and the overexpression is linked with better survival? In line 279, authors mentioned from their other study of 10 CRC patients, "We found lower expression of miR-618 in tumor colon tissues in comparison to colon tissues from healthy people". This actually makes sense and can be linked with why lower miR-618 is associated with better survival but what the authors showed for its expression in sera is the opposite. So, I want the authors to give me the example of other similar studies and explain the biological and rational predisposition and oddity behind this. The authors tend to explain it in the discussion. Still, they do not explain it well, and what readers will also find unconventional is that the patients have higher miRNA in sera compared to healthy counterparts. However, this higher expression is linked with better survival. 

Author Response

Comment:

Something that I couldn't get my head around is the main basis of the paper, i.e., in the discussion line 264, the authors mentioned, based on this study, the 'tumor suppressor' role of mir-618, which is evident from the improved overall survival of CRC patients where it is overexpressed in their sera. Why, then, if it has a tumor suppressor role, is overexpressed in the sera of cancer patients compared with the healthy ones? Rationally thinking, it should be underexpressed in the CRC owing to its tumor-suppressing properties. Are there similar studies out there where cancer patients have a higher expression of a particular miRNA, and the overexpression is linked with better survival? In line 279, authors mentioned from their other study of 10 CRC patients, "We found lower expression of miR-618 in tumor colon tissues in comparison to colon tissues from healthy people". This actually makes sense and can be linked with why lower miR-618 is associated with better survival but what the authors showed for its expression in sera is the opposite. So, I want the authors to give me the example of other similar studies and explain the biological and rational predisposition and oddity behind this. The authors tend to explain it in the discussion. Still, they do not explain it well, and what readers will also find unconventional is that the patients have higher miRNA in sera compared to healthy counterparts. However, this higher expression is linked with better survival.

Author’s Reply:

We would like to thank you for your thoughtful comments, questions and constructive suggestions, which helped to improve the quality of this manuscript. All incorporated changes are presented using the ‘tracked changes’ function in the revised manuscript.

We agree with you that there is some kind of contradiction in our results, but we repeated the experiment and confirmed that CRC patients with a high and intermediate expression levels of miR-618 live longer and these levels are significantly higher than healthy controls (median of expression levels in healthy control was 0.1460 and median - of CRC patients - 2.392). Our explanation is hypothetical, ad-hoc and has to be proven in subsequent experiments. At this stage of our study, we suggest that our results are a manifestation of the dual nature of miR-618. Ivanovic et al., 2018 [reference 12 in our study] for the first time show this for miR-618 and define it as context-dependent miRNA.

Several studies report that miRNA levels in plasma and tumor tissue showed significant differences. A strong correlation between expression levels of miRNA in colon tumor tissue and levels of fecal miRNAs were found, but not between levels of miRNAs in colon tumor and plasma miRNAs (Fukada et al., 2020, Cui and Cui, 2020). This suggests that plasma regulated miRNAs maybe not actively secreted from colorectal tumor cells. These studies also cannot find answers to questions such as: where the body fluid miRNAs are from and what their functions are, or why sometimes there is no correlation between expression levels of miRNAs in different human body fluids and in tumor tissue.

We think that miR-148a could be considered as an example of miRNA, which has a behavior similar to miR-618. For miR-148a Tsai et al., 2013 found that its overexpression inhibited colon cancer cell proliferation and migration. Moreover, lower levels of miRNA-148a expression in advanced CRC tissues were associated with significantly poorer overall survival rates (Takahashi et al., 2012, Tsai et al., 2013). At the same time, miR-148 is strongly upregulated in the serum of CRC patients in a study of Gmerek et al., 2019. Unfortunately, the authors do not provide data, whether this overexpression is linked with better survival in patients. We cannot compare our data with the results of other authors because the expression of miR-618 has not been studied in the circulation of any solid tumor.

Both Takahashi et al., 2012, Tsai et al., 2013 report that miR-148a expression is higher in early stage II CRC tissues than in advanced stages and are commensurate with levels of miR-618 in healthy controls.

We also plan to see the levels of expression of miR-618 in II and III stages of the disease. So far we have prospectively collected only 28 patients in I and II stages and this is one of our next tasks. At this point we analyzed the TCGA dataset (http://ualcan.path.uab.edu/index.html) for the intratumoral expression of miR-618 in patients with colon cancer (Fig. 1).

Fig. 1. Expression of miR-618 in the colon (COAD) cancer tissue in patients in different stages. These data are available on http://ualcan.path.uab.edu/index.html (TCGA database).

These data do not show a difference in miR-618 expression in different stages of disease in colon cancer. But there is a trend towards high expression levels in stages I and II colon cancer patient which might become significant in a larger cohort. These results will be very important because will give us the answer whether miR-618 could be a good diagnostic marker. If its expression is similar to healthy controls it is not suitable for an indication of early stages of colon cancer.

If we accept that miR-618 is tumor suppressor in CRC and its expression levels are high in tissue and low in plasma in healthy and in stage II CRC patient, but patients in advanced CRC have high levels in plasma and low in tumor tissue, we may suggest that miR-618 is probably inhibited in tumor tissue in advanced CRC and its expression in blood is increased compensatory from another source outside the tumor in an attempt to suppress the tumor progression. Our statement that overexpression of miRNA-618 in the blood is probably a result of the internal stimuli in acute inflammatory or advanced disease is a hypothesis in the discussion. Unfortunately, we have no experimental evidence in this regard, so we are frugal in explaining this seeming contradiction in the discussion.

We made a major revision of the "Discussion" section.

We are hoping that we answered your questions and comments.

References:

Fukada M, Matsuhashi N, Takahashi T, Sugito N, Heishima K, Akao Y, Yoshida K. Tumor Tissue MIR92a and Plasma MIRs21 and 29a as Predictive Biomarkers Associated with Clinicopathological Features and Surgical Resection in a Prospective Study on Colorectal Cancer Patients. J Clin Med. 2020 Aug 4;9(8):2509. doi: 10.3390/jcm9082509. PMID: 32759718; PMCID: PMC7465950.

Cui C, Cui Q. The relationship of human tissue microRNAs with those from body fluids. Sci Rep. 2020 Mar 27;10(1):5644. doi: 10.1038/s41598-020-62534-6. PMID: 32221351; PMCID: PMC7101318.

Gmerek L, Martyniak K, Horbacka K, Krokowicz P, Scierski W, Golusinski P, Golusinski W, Schneider A, Masternak MM. MicroRNA regulation in colorectal cancer tissue and serum. PLoS One. 2019 Aug 30;14(8):e0222013. doi: 10.1371/journal.pone.0222013. PMID: 31469874; PMCID: PMC6716664.

Tsai HL, Yang IP, Huang CW, Ma CJ, Kuo CH, Lu CY, Juo SH, Wang JY. Clinical significance of microRNA-148a in patients with early relapse of stage II stage and III colorectal cancer after curative resection. Transl Res. 2013 Oct;162(4):258-68. doi: 10.1016/j.trsl.2013.07.009. Epub 2013 Aug 7. PMID: 23933284.

Takahashi M, Cuatrecasas M, Balaguer F, Hur K, Toiyama Y, Castells A, Boland CR, Goel A. The clinical significance of MiR-148a as a predictive biomarker in patients with advanced colorectal cancer. PLoS One. 2012;7(10):e46684. doi: 10.1371/journal.pone.0046684. Epub 2012 Oct 3. PMID: 23056401; PMCID: PMC3463512.

Author Response File: Author Response.pdf

Reviewer 2 Report

This is an interesting study where miR-618 in serum was evaluated as possible biomarker of prognosis of metastatic colon cancer. The authors claim that circulating miR-618 may be a promissing biomarker showing that patients with metastatic colon cancer have higher miR-618 expression when compared with healthy individuals and longer mean overall survival. Moreover, they also demonstrate that patients harboring AC rs2682818 genotype have a decreased risk for colon cancer in comparison with patients with CC and AA genotypes. In the end, they conclude that this was the first report on the expression of miR-618 in colon cancer patients serum.

This work has a valid clinical question about finding a novel and non-invasive biomarker to predict the prognosis of colon cancer. In this work, the authors highlight a possible miRNA with prognostic relevance in clinical setting by using a good sample size of patients serum samples in their work. Although I recognize the relevance of this work and understood the aim of theses studies, there are still major issues and questions regarding the manuscript, experimental design and the conclusions that i would like to address.

Comments

Comment 1) There are English grammatical problems along the text and figures, so please read it carefully and edit the manuscript. Be careful and uniformize words and expressions such as “qPCR or qRT-PCR or RT-qPCR”. Replace “sera” by “serum” in all manuscript. Be careful with italic and upperline words “p value”, “in vivo”, “in vitro” and “33^th percentile or 2^-∆∆Ct method”.  Spell out some abbreviations and then use it along the text when needed.

Comment 2) Sometimes you use colon cancer and other times CRC. Be aware that colorectal cancer consists of colon and rectal cancer together, two types of cancer with different treatment options and therefore different miRNA expression and outcomes. For what I understood all your patients had metastatic colon cancer but not rectal cancer so you should change “CRC” to “colon cancer or colon cancer (CC)” in the manuscript.

Comment 3) The abstract lacks the background information and it should be more concise. Besides, Line 23-26 give the same information. Please revise.

Comment 4) In the Material and Methods section you should provide information on how the patients blood was collected and stored. In here, you also mentioned CRC patients but did you had or not patients with only colon and/or rectal cancer?

Comment 5) Line 69: Replace “Participants” for “Patients”

Comment 6) Line 74: Spell out the abbreviations “VEGF” and “EGFR”

Comment 7) Line 74-83: This information would be easier to read in a table. You should also add information regarding the patients with each rs2682818 variants. Besides, in the results regarding rs2682818 genotype you mention that your study population is fully caucasian. If this is relevant for your work you should also add this information in the material and methods section.

Comment 8) Line 86: Please change the sentence to “This was a retrospective study with two approvals …”

Comment 9) Line 90: Please change the sentence to “The authors declare that all research was conducted in accordance with the regulations, …”

Comment 10) Line 98: Spell out “qRT-PCR”

Comment 11) Line 99 – “Small RNA fraction <200 nt” ..can it be replaced by “miRNAs”?

Comment 12) Line 110: Replace “ three repeats” by “in triplicate”

Comment 13) Which system was used for the qPCR?

Comment 14) Line 139: Spell out “ROC”

Comment 15) Line 172: Figure 2 is writen 2x

Comment 16) Figure 3A: Please provide the number of patients (n) in each column. If you put the statiscical test used here, you have to put it also in figure 1. Correct the figure - CA .

Comment 17) Line 231: Explain the sentence “The test has moderate sensitivity and specificity”.

Comment 18) Line 228-235 should be put after L261 and revised with the information written from L262-292. Please revise the discussion section.

Comment 19) Lines 228 and 316: where it reads “in patients with CRC” it should read “in patients with metastatic colon cancer”.

Questions

Question1) You used a cohort of healthy volunteers samples as the control group - “90 healthy volunteers, age and sex matched to the patients, were included as a control group for the miR-618 expression and SNP genotyping analyses”. Do you think that these samples are the best control to compare the expression of miR-618 as a biomarker? In general miRNAs, specially circulating miRNAs, are not specific of a tissue/disease. Do you have additional information on other clinical characteristics even pathological ones that the healthy volunteers might have that could be relate or not to miR-618 expression.

Question 2) Regarding the previous question you say that “Probably, miR-618 is not only cancer-related miRNA, but its overexpression in the blood is a result of the internal stimuli in acute inflammatory or advanced disease.”(L290-292) In this sense, do you know more clinical information about your cancer and healthy patients?

Question 3) You have shown that miR-618 is highly expressed in cancer patients when compared with healthy individuals. However, you also show that patients with higher miR-618 expression are also the ones with longer overall survival. Do you think this result makes sense in accordance with the previous one?  Did you check for miRNA levels of these patients after treatment? What about miRNA levels in tissue samples?

Question 4) In the results and discussion you also mentioned that there are no studies among Caucasians populations for the association between presence of rs2682818 and CRC susceptibility and that this is the first study for Caucasians. Did all your 104 samples were from Caucasians patients? Did you select only Caucasians samples in the beginning of your design study? Are your healthy controls also caucasians? Otherwise you can not conclude that by making comparisons among heterogeneous population. Nevertheless, if you want to highlight on the prognostic significant of rs2682818 specifically in caucasian population you have to put this information and explain it in the objetives of the work and in the experimental design.

Question 5) How did you detect the concentration of miRNA and purity? And how much (quantity) of miRNA extracted was used for cDNA synthesis? You should add this information in M&M.

Question 6) In figure 1A the data is standard deviation (SD) or standard error of the mean (SEM)? You should add this information on the legend. Did you saw if the expression values had a normal distribution? All patients and healthy control samples had mir-618 amplification? If not provide the number of patients in each column and explain. Legend says “HV” and not “HC”.

Question 7) In the discussion section (Line 230 -231) you wrote that “…circulating miR-618 is a biomarker, allowing to distinguish a person with or without metastatic colon cancer.” This is not demonstrated in this work. As you describe in the title, circulating miR-618 seems to have a prognostic significance in patients with Metastatic Colon Cancer but it can not be considered yet a biomarker only by the results presented in the manuscript. In addition, you do not show that this miRNA allows to differentiate among a person with or without metastatic colon cancer since you did not had patients with non-metastatic colon cancer in your study.

Question 8) You said in the discussion that “In our research, we found that high and intermediate expression of circulating miR- 262 in metastatic CRC patients was associated with a better outcome than in patients with low levels of miR-618…..it could explain that miR-618 acts as tumor-suppressor miRNA in patients ..” (L262-266). Although you have a better outomce in patients with higher miR-618 expression you can not conclude (at least in your study conditions) that miR-618 is a tumour supressor when you have a significant result showing that your cancer patients have a higher mir-618 expression comparing to the healthy volunteers (figure 1). If this miRNA is overexpressed in the disease vs control it can not be acting as a tumour supressor unless this was not the best control to compare your expression. Maybe there is also a relation between miRNA expression and treatment and besides the quantity of circulating miRNAs may vary a lot in the blood.

Question 9) Discussion Line 276-287: This seems to be very relevant to explain the results of your work regarding miR-618 expression vs outcome even if its only an preliminary study and so you should add this data in your results section and then discuss it in the discussion section. In this sense, you also have to add this to the M&M regarding the patients samples used etc and clarify if these patients were CRC or only colon patients.

Question 10) L296-298: “To explore whether low levels of miR-618 are a 296 result of the presence of rs2682818 polymorphism we performed SNP genotyping of CRC patients and healthy controls.” Did you also explore in healthy controls? This is not in the results 3.4 section.

Author Response

Reviewer 2

We would like to thank you for your thoughtful comments, questions and constructive suggestions, which helped to improve the quality of this manuscript.

All incorporated changes are presented using the ‘tracked changes’ function in the revised manuscript. To facilitate your work we cited the line numbers and exact changes that were made in the revised manuscript.

Comment 1: There are English grammatical problems along the text and figures, so please read it carefully and edit the manuscript. Be careful and uniformize words and expressions such as “qPCR or qRT-PCR or RT-qPCR”. Replace “sera” by “serum” in all manuscript. Be careful with italic and upperline words “p value”, “in vivo”, “in vitro” and “33th percentile or 2-∆∆Ct method”.  Spell out some abbreviations and then use it along the text when needed.

Author’s Reply:

Thank you for your comments. Your recommendations were considered and accepted. The changes were made in the text of the manuscript.

Comment 2: Sometimes you use colon cancer and other times CRC. Be aware that colorectal cancer consists of colon and rectal cancer together, two types of cancer with different treatment options and therefore different miRNA expression and outcomes. For what I understood all your patients had metastatic colon cancer but not rectal cancer so you should change “CRC” to “colon cancer or colon cancer (CC)” in the manuscript.

Author’s Reply:

Thank you for your comment. You are absolutely right. We apologize for this rough mistake. Our group of patients consist of colon cancer patients. That is why we changed everywhere “CRC patients” to “colon cancer patients” or “CC patients” “and metastatic CRC” to “metastatic CC” or “mCC”.

Comment 3: The abstract lacks the background information and it should be more concise. Besides, Line 23-26 give the same information. Please revise.

Author’s Reply:

We edited the text of the abstract significantly, but the journal has a restriction of 200 words in the "Abstract" section.

We do not quite agree that between Line 23 and Line 26 information is the same (in revised version Lines 24-28). By log-rank test, we receive information about the difference in mean overall survival (OS) between groups of patients – with a high and intermediate expression of miR-618 and with low expression. While through Cox regression analysis we provide an estimate of the hazard ratio and its confidence interval.

The text between Lines 23-26 was corrected this way:

Patients with high and intermediate expression of miR-618 had a significantly longer mean overall survival (OS) of 21 months than patients with low expression – 16 months. In addition, multivariate Cox regression analysis confirmed the association between high/intermediate levels of miRNA-618 and longer OS, HR=0.51, 95% CI: 0.30-0.86, p=0.012.

Comment 4: In the Material and Methods section you should provide information on how the patients blood was collected and stored. In here, you also mentioned CRC patients but did you had or not patients with only colon and/or rectal cancer?

Author’s Reply:

All serum samples from mCC patients and healthy controls used in this study were obtained from peripheral blood and stored at -80°C until use. This information was added in the manuscript text, “Materials and Methods” section (in revised version Lines 109-110).

All patients included in the study were with colon cancer as we replied to your comment 2.

Comment 5: Line 69: Replace “Participants” for “Patients”

Author’s Reply:

Thank you for your comment. “Participants” was replaced with “Patients and healthy controls” (in revised version Line 72), because the section describes both groups of participants in our study.

Comment 6: Line 74: Spell out the abbreviations “VEGF” and “EGFR”

Author’s Reply:

The meaning of the abbreviations “VEGF” and “EGFR” were added in the “Materials and methods” section (in revised version Lines 78-79).

Comment 7: Line 74-83: This information would be easier to read in a table. You should also add information regarding the patients with each rs2682818 variants. Besides, in the results regarding rs2682818 genotype you mention that your study population is fully caucasian. If this is relevant for your work you should also add this information in the material and methods section.

Author’s Reply:

Please, allow us to not agree with your proposal to make a table with the information presented in the description of the cohort because it would be a repeat of the information from Table 1.

Information about distribution of genotypes and allele frequencies of rs2682818 variant in mCC patients and healthy controls was provided in Table 3 in details.

Our cohort consists of only Caucasians and this information was added in the “Materials and methods” section (in revised version Line 73 and Line 91).

Comment 8: Line 86: Please change the sentence to “This was a retrospective study with two approvals …”

Author’s Reply:

The change was made (in revised version Line 95).

Comment 9: Line 90: Please change the sentence to “The authors declare that all research was conducted in accordance with the regulations, …”

Author’s Reply:

The change was made (in revised version Lines 101-106).

Comment 10: Line 98: Spell out “qRT-PCR”

Author’s Reply:

We apologize for our mistake. The term “qRT-PCR” was remained from other our text. We substituted it with “qPCR” which is quantitative PCR.

Comment 11: Line 99 – “Small RNA fraction <200 nt” ..can it be replaced by “miRNAs”?

Author’s Reply:

We have already inserted clarifications in the manuscript text in “Materials and methods” section, 2.2. RNA extraction, cDNA synthesis and qPCR (in revised version Line 113). In addition, we gave more details in the answer of your Question 5).

Comment 12: Line 110: Replace “ three repeats” by “in triplicate”

Author’s Reply:

The change was made (in revised version Line 126).

Comment 13: Which system was used for the qPCR?

Author’s Reply:

7500 Real-Time PCR System was used for all qPCR and SNP analyses. This detail was added in “Materials and methods” 2.2. RNA extraction, cDNA synthesis and qPCR (in revised version Line 127).

Comment 14: Line 139: Spell out “ROC”

Author’s Reply:

The description of the ROC analysis was missed in the "Materials and Methods section, 2.4. Statistical analysis.

We corrected our omission and we added the following text (in revised version Lines 144-149):

Specificity and sensitivity of serum miRNA-618 expression levels to distinguish mCC patients from healthy controls were evaluated with receiver operating curve (ROC) analysis. The diagnostic accuracy of circulating miR-618 was determined by evaluation of the largest possible area under the curve (AUC) in ROC analysis. Tests with AUC values greater than 0.9 had high accuracy, between 0.7–0.9 had moderate accuracy and between 0.5–0.7 - low accuracy [23].

Reference:

23. Swets JA. Measuring the accuracy of diagnostic systems. Science. 1988 Jun 3;240(4857):1285-93. doi: 10.1126/science.3287615. PMID: 3287615.

Comment 15: Line 172: Figure 2 is writen 2x

Author’s Reply:

We apologize for our technical mistake.

Comment 16: Figure 3A: Please provide the number of patients (n) in each column. If you put the statiscical test used here, you have to put it also in figure 1. Correct the figure - CA .

Author’s Reply:

The changes were made.

Comment 17: Line 231: Explain the sentence “The test has moderate sensitivity and specificity”.

Author’s Reply:

We explained it in "Materials and Methods section, 2.4. Statistical analysis (in revised version Lines 148-149).

Comment 18: Line 228-235 should be put after L261 and revised with the information written from L262-292. Please revise the discussion section.

Author’s Reply:

We made the recommended changes in response to your comments and questions in the discussion.

Comment 19: Lines 228 and 316: where it reads “in patients with CRC” it should read “in patients with metastatic colon cancer”.

Author’s Reply:

The phrases “patients with CRC” or “CRC patients” were substituted with “patients with mCC” or “mCC patients” respectively.

Question1: You used a cohort of healthy volunteers samples as the control group - “90 healthy volunteers, age and sex matched to the patients, were included as a control group for the miR-618 expression and SNP genotyping analyses”. Do you think that these samples are the best control to compare the expression of miR-618 as a biomarker? In general miRNAs, specially circulating miRNAs, are not specific of a tissue/disease. Do you have additional information on other clinical characteristics even pathological ones that the healthy volunteers might have that could be relate or not to miR-618 expression.

Author’s Reply:

Our main aim was to evaluate the expression of miR-618 in the serum of mCC patients to find a less invasive biomarker for evaluation of disease. The investigated samples are baseline serum samples as we focused on the events prior to therapy and we cannot compare them to anything other than samples from healthy controls. That is why we have used healthy individuals, who were matched to our patients in ethnicity, sex and age, as a control group. We think that the selected 90 healthy volunteers are sufficient and relatively good control to compare the expression of miR-618 also because they had no first-line relatives with a history of colon cancer and no clinical evidence of any other disease. We believe that miR-618 expression in the group gave a clear baseline expression to compare to the miR-618 expression in patients with metastatic colon cancer. Moreover, we are not the only one that have used samples from healthy individuals as a control group. Many studies compare the miRNA expression in fluid biopsy from gastrointestinal cancers patients with the expression in healthy individuals (So et al., 2020; Cojocneanu et al., 2020; Zanutto et al., 2014).

We agree that many questions remain unanswered regarding the specificity of circulating miRNAs and their ability to reflect tumor tissue changes. However, we found many studies that support the potential of miRNAs as specific biomarkers in different cancer types (Lan et al., 2015, Takahashi et al., 2015, Nowicka et al., 2019, Ingenito et al., 2019, Hasanzadeh et al., 2019).

References:

So JBY, Kapoor R, Zhu F, Koh C, Zhou L, Zou R, Tang YC, Goo PCK, Rha SY, Chung HC, Yoong J, Yap CT, Rao J, Chia CK, Tsao S, Shabbir A, Lee J, Lam KP, Hartman M, Yong WP, Too HP, Yeoh KG. Development and validation of a serum microRNA biomarker panel for detecting gastric cancer in a high-risk population. Gut. 2020 Oct 7:gutjnl-2020-322065. doi: 10.1136/gutjnl-2020-322065. Epub ahead of print. PMID: 33028667.

Cojocneanu R, Braicu C, Raduly L, Jurj A, Zanoaga O, Magdo L, Irimie A, Muresan MS, Ionescu C, Grigorescu M, Berindan-Neagoe I. Plasma and Tissue Specific miRNA Expression Pattern and Functional Analysis Associated to Colorectal Cancer Patients. Cancers (Basel). 2020 Mar 31;12(4):843. doi: 10.3390/cancers12040843. PMID: 32244548; PMCID: PMC7226631.

Zanutto S, Pizzamiglio S, Ghilotti M, Bertan C, Ravagnani F, Perrone F, Leo E, Pilotti S, Verderio P, Gariboldi M, Pierotti MA. Circulating miR-378 in plasma: a reliable, haemolysis-independent biomarker for colorectal cancer. Br J Cancer. 2014 Feb 18;110(4):1001-7. doi: 10.1038/bjc.2013.819. Epub 2014 Jan 14. PMID: 24423916; PMCID: PMC3929896.

Lan H, Lu H, Wang X, Jin H. MicroRNAs as potential biomarkers in cancer: opportunities and challenges. Biomed Res Int. 2015;2015:125094. doi: 10.1155/2015/125094. Epub 2015 Mar 22. PMID: 25874201; PMCID: PMC4385606.

Takahashi RU, Miyazaki H, Ochiya T. The Roles of MicroRNAs in Breast Cancer. Cancers (Basel). 2015 Apr 9;7(2):598-616. doi: 10.3390/cancers7020598. PMID: 25860815; PMCID: PMC4491673.

Nowicka Z, Stawiski K, Tomasik B, Fendler W. Extracellular miRNAs as Biomarkers of Head and Neck Cancer Progression and Metastasis. Int J Mol Sci. 2019 Sep 27;20(19):4799. doi: 10.3390/ijms20194799. PMID: 31569614; PMCID: PMC6801477.

Ingenito F, Roscigno G, Affinito A, Nuzzo S, Scognamiglio I, Quintavalle C, Condorelli G. The Role of Exo-miRNAs in Cancer: A Focus on Therapeutic and Diagnostic Applications. Int J Mol Sci. 2019 Sep 21;20(19):4687. doi: 10.3390/ijms20194687. PMID: 31546654; PMCID: PMC6801421.

Hasanzadeh M, Movahedi M, Rejali M, Maleki F, Moetamani-Ahmadi M, Seifi S, Hosseini Z, Khazaei M, Amerizadeh F, Ferns GA, Rezayi M, Avan A. The potential prognostic and therapeutic application of tissue and circulating microRNAs in cervical cancer. J Cell Physiol. 2019 Feb;234(2):1289-1294. doi: 10.1002/jcp.27160. Epub 2018 Sep 7. PMID: 30191988.

Question 2: Regarding the previous question you say that “Probably, miR-618 is not only cancer-related miRNA, but its overexpression in the blood is a result of the internal stimuli in acute inflammatory or advanced disease.”(L290-292) In this sense, do you know more clinical information about your cancer and healthy patients?

Author’s Reply:

Our patients were with metastatic colon cancer and did not have significant comorbidity.

All healthy volunteers had no first-line relatives with a history of colon cancer and no clinical evidence of any other disease. This information about healthy volunteers was added to the text of the manuscript in the "Materials and Methods" section (in revised version Lines 92-94).

Question 3: You have shown that miR-618 is highly expressed in cancer patients when compared with healthy individuals. However, you also show that patients with higher miR-618 expression are also the ones with longer overall survival. Do you think this result makes sense in accordance with the previous one?  Did you check for miRNA levels of these patients after treatment? What about miRNA levels in tissue samples?

Author’s Reply:

If miR-618 has tumor-suppression function it's high expression is good for fighting with disease and is expected to suppress tumor progression. The levels of miRNAs in the circulation and in the tumor tissue are not comparable (Fukada et al., 2020, Cui and Cui, 2020). We believe that miR-618 is probably inhibited in tumor tissue in advanced CRC (by cicr-RNA, for example) and its expression in blood is increased compensatory from another source outside the tumor in an attempt to suppress the tumor progression.

In the revised "Discussion" section we developed our hypothesis and presented an example of another tumour-suppressor miRNA (miR-148a) that have similar to miR-618 behaviour.

We evaluated the expression level of miR-618 in a small number of available colon tissue samples from 10 healthy volunteers and tumor tissue samples from 10 mCC patients as a part of a preliminary study. These results were presented in the first submitted variant of the manuscript.

References:

Fukada M, Matsuhashi N, Takahashi T, Sugito N, Heishima K, Akao Y, Yoshida K. Tumor Tissue MIR92a and Plasma MIRs21 and 29a as Predictive Biomarkers Associated with Clinicopathological Features and Surgical Resection in a Prospective Study on Colorectal Cancer Patients. J Clin Med. 2020 Aug 4;9(8):2509. doi: 10.3390/jcm9082509. PMID: 32759718; PMCID: PMC7465950.

Cui C, Cui Q. The relationship of human tissue microRNAs with those from body fluids. Sci Rep. 2020 Mar 27;10(1):5644. doi: 10.1038/s41598-020-62534-6. PMID: 32221351; PMCID: PMC7101318.

Question 4: In the results and discussion you also mentioned that there are no studies among Caucasians populations for the association between presence of rs2682818 and CRC susceptibility and that this is the first study for Caucasians. Did all your 104 samples were from Caucasians patients? Did you select only Caucasians samples in the beginning of your design study? Are your healthy controls also caucasians? Otherwise you can not conclude that by making comparisons among heterogeneous population. Nevertheless, if you want to highlight on the prognostic significant of rs2682818 specifically in caucasian population you have to put this information and explain it in the objetives of the work and in the experimental design.

Author’s Reply:

The Bulgarians are Caucasians and all of our participants - patients and healthy volunteers were Caucasians. This information was added to the text of the manuscript in the "Materials and Methods" section (in revised version Line 73 and Line 91).

There a couple of genetics analyses on Bulgarians which shows that our population is not heterogeneous. According to Hellenthal et al., 2014 the Bulgarian autosomal genetic legacy is Mediterranean, about the half of which resembles the Caucasian and Middle Eastern (Hellenthal et al., 2014). But Ivanova et al., 2002 proved that Bulgarians are more closely related to Macedonians, Greeks, and Romanians (all Caucasians) than to other European populations and Middle Eastern people living near the Mediterranean. Moreover, Karachanak et al., 2013 found that Bulgarians were close to Macedonian Greeks, but relatively far from Turks.

References:

Hellenthal G, Busby GBJ, Band G, Wilson JF, Capelli C, Falush D, Myers S. A genetic atlas of human admixture history. Science. 2014 Feb 14;343(6172):747-751. doi: 10.1126/science.1243518. PMID: 24531965; PMCID: PMC4209567.

Ivanova M, Rozemuller E, Tyufekchiev N, Michailova A, Tilanus M, Naumova E. HLA polymorphism in Bulgarians defined by high-resolution typing methods in comparison with other populations. Tissue Antigens. 2002 Dec;60(6):496-504. doi: 10.1034/j.1399-0039.2002.600605.x. PMID: 12542743.

Karachanak S, Grugni V, Fornarino S, Nesheva D, Al-Zahery N, Battaglia V, Carossa V, Yordanov Y, Torroni A, Galabov AS, Toncheva D, Semino O. Y-chromosome diversity in modern Bulgarians: new clues about their ancestry. PLoS One. 2013;8(3):e56779. doi: 10.1371/journal.pone.0056779. Epub 2013 Mar 6. PMID: 23483890; PMCID: PMC3590186.

Question 5: How did you detect the concentration of miRNA and purity? And how much (quantity) of miRNA extracted was used for cDNA synthesis? You should add this information in M&M.

Author’s Reply:

We have used Synergy 2 BioTek System to measure spectrophotometrically the concentration of extracted RNAs, which are less than 200 nt in length in the samples. The determination of the purity of RNAs was based on A260/A280 ratios. The same samples were used for cDNA synthesis with initial quantity of 90 ng of extracted RNAs in accordance with the manufacturer’s instructions of RevertAid First Strand cDNA Synthesis kit (ThermoFisher Scientific). These details are already added in "Materials and Methods" section as you advised us (in revised version Line 115).

To increase the total yield concentration of miRNAs we kept strictly the recommendations regarding the volume of elution buffer during the elution procedure.

Question 6: In figure 1A the data is standard deviation (SD) or standard error of the mean (SEM)? You should add this information on the legend. Did you saw if the expression values had a normal distribution? All patients and healthy control samples had mir-618 amplification? If not provide the number of patients in each column and explain. Legend says “HV” and not “HC”.

Author’s Reply:

On Figure 1. (a) are shown the mean value with the range of miR-618 expression in two groups – healthy controls and patients with colon cancer. On Figure 3. (a) are shown the mean value with the range of miR-618 expression in patients with different genotype of rs2682818 SNP.

The expression of miR-618 was not normaly distributed, that is why we used non parametric test for subsequent analysis.

All included in the study patients and healthy controls had a value of miR-618 expression.

The discrepancy between legend where healthy controls are “HC” and indication in Figure 1 (a) for healthy controls -“HV” was corrected.

Question 7: In the discussion section (Line 230 -231) you wrote that “…circulating miR-618 is a biomarker, allowing to distinguish a person with or without metastatic colon cancer.” This is not demonstrated in this work. As you describe in the title, circulating miR-618 seems to have a prognostic significance in patients with Metastatic Colon Cancer but it can not be considered yet a biomarker only by the results presented in the manuscript. In addition, you do not show that this miRNA allows to differentiate among a person with or without metastatic colon cancer since you did not had patients with non-metastatic colon cancer in your study.

Author’s Reply:

Thank you for your comment. You are right that we can not to claim that this miR-618 allows to differentiate a person with or without metastatic colon cancer because we did not compare these two group. The part of sentence in Lines 230 - 231 was corrected (in revised version Lines 301-302). We added in the description of the limitations of our work that we have not investigated miR-618 expression in stage II and III CC patients.

Question 8: You said in the discussion that “In our research, we found that high and intermediate expression of circulating miR- 262 in metastatic CRC patients was associated with a better outcome than in patients with low levels of miR-618…..it could explain that miR-618 acts as tumor-suppressor miRNA in patients ..” (L262-266). Although you have a better outomce in patients with higher miR-618 expression you can not conclude (at least in your study conditions) that miR-618 is a tumour supressor when you have a significant result showing that your cancer patients have a higher mir-618 expression comparing to the healthy volunteers (figure 1). If this miRNA is overexpressed in the disease vs control it can not be acting as a tumour supressor unless this was not the best control to compare your expression. Maybe there is also a relation between miRNA expression and treatment and besides the quantity of circulating miRNAs may vary a lot in the blood.

Author’s Reply:

Thank you very much for your discussion.

You are right that in our study conditions we cannot claim that miR-618 is a tumor suppressor we only suggest it in an analogy of other studies for this miRNA. High levels of miRNAs in circulation do not exclude their tumor-suppressive function. We present an example in the revised version of the manuscript (“Discussion” section) about another miRNA with high expression in plasma and also with tumor-suppressive function. We believe that variation in levels of miRNAs like miR-618 in circulation is connected more to disease activity than to therapy. We plan to check both possibilities and these are part of our next tasks. We are already prospectively collecting samples from patients I and II stages of CRC and have planed to investigated levels of miR-618 in some of our patients for whom there are available samples after therapy on 1st evaluation.

Question 9: Discussion Line 276-287: This seems to be very relevant to explain the results of your work regarding miR-618 expression vs outcome even if its only an preliminary study and so you should add this data in your results section and then discuss it in the discussion section. In this sense, you also have to add this to the M&M regarding the patients samples used etc and clarify if these patients were CRC or only colon patients.

Author’s Reply:

Please, allow us to not agree with your proposal to add the data for tissue expression of miR-618 in “Result” section.

This is part of our new study for which we are prospectively collecting samples since August 2020 after an accepted change in the approval of the Commission for Scientific Research Ethics” of Medical University of Varna, Bulgaria (protocol №83/16.05.2019). In the "Discussion" section here we present preliminary results in a try to defend our hypothesis of the behaviour of miR-618.

We found a trend towards but not a significant difference between the miR-618 expressions in colon tissues from healthy people compared to the tumor tissue from CRC patients (Fig. 1).

Fig. 1. Levels of miR-618 in colon tissues samples from healthy controls (HC, n=10) and tumor tissues samples from CRC patients (n=10). The expression levels were measured using qPCR. Relative gene expression was calculated using 2^-∆∆Ct method.

Our results were compared with data in two databases - miR-TV (http://mirtv.ibms.sinica.edu.tw/) and OncoMir Cancer Database (https://www.oncomir.umn.edu/omcd/basic_search.php) where is revealed that the expression of miR-618 in the colon adenocarcinoma tissue was significantly decreased in comparison to the surrounding healthy tissue.

Further analyses in a larger group of patients are needed. At this time we can not be able to realize that kind of analyses.

Question 10: L296-298: “To explore whether low levels of miR-618 are a 296 result of the presence of rs2682818 polymorphism we performed SNP genotyping of CRC patients and healthy controls.” Did you also explore in healthy controls? This is not in the results 3.4 section.

Author’s Reply:

If we right understand your question - yes we explore the presence of the rs2682818 polymorphism in healthy controls and this is shown as result in "Results" section, 3.5. Rs2682818 AC genotype was associated with a decreased risk of mCC and Table 3. In Table 3 you can see genotypes distribution and allele frequencies of rs2682818 in healthy controls.

We are hoping that we answered your questions and comments.

Author Response File: Author Response.pdf

Reviewer 3 Report

In this manuscript by Radanova et al., the authors have investigated the prognostic significance of circulating miRNA-618 in colorectal cancer (CRC). They found that the miRNA-618 was overexpressed in the sera of CRC patients and correlated with longer over-all survival. Patients harboring AC rs2682818 genotype was found to have a decreased risk for CRC. This is an interesting study. But, the following points should be addressed before reconsidering the manuscript for publication.

1. The novelty and limitations of the study should be clearly emphasized in the introduction section.
2. In table 1, the statistical significance was not reached between miRNA-618 expression and clinicopathological features including metastasis. But, the title of the manuscript states that the miRNA-618 has prognostic significance in metastatic colon cancer. The authors should explain. 
3. The authors should analyze the correlation between miRNA-618 and disease-free survival of patients.
4. The effect of miRNA-618 on invasion and migration of colorectal cancer cells should be investigated in vitro.

Author Response

Reviewer 3

We would like to thank you for your thoughtful comments, questions and constructive suggestions, which helped to improve the quality of this manuscript.

All changes are presented using the ‘tracked changes’ function in the revised manuscript. We have incorporated text about the limitations of our study and to facilitate your work we cited the line numbers and exact changes that were made in the revised manuscript.

Comment 1:

1. The novelty and limitations of the study should be clearly emphasized in the introduction section.

Author’s Reply:

Thank you for your comment. We have incorporated in the text information about the limitations of our study but in the "Discussion" section. We apologies that we could not found the appropriate place for this in the "Introduction" section.

We added the following text in "Discussion" section(in revised version Lines 390-394):

Several limitations were established in our study. First, our study is retrospective and cross-sectional. It is also descriptive with an absence of in vitro analyses of the effect of miR-618 on tumor progression. We investigated expression levels of miR-618 in tissue in a small number of normal and tumor colon tissue samples. Another limitation is that we have not investigated miR-618 expression in stage II and III CRC patients.

Comment 2:

1. In table 1, the statistical significance was not reached between miRNA-618 expression and clinicopathological features including metastasis. But, the title of the manuscript states that the miRNA-618 has prognostic significance in metastatic colon cancer. The authors should explain.

Author’s Reply:

We agree with you that the association between the expression of miR-618 and clinicopathological features could provide information about the prognostic significance of miR-618. But, such associations can be expected when comparing patients in a more heterogeneous group - patients with different TNM stage, T pathological category, tumor size, patients with or without lymph node metastasis and with or without distal metastasis. Our study was included only patients with unresectable metastatic colorectal cancer. Therefore the prognostic significance of miR-618 in the present study was evaluated by survival analysis such as – Kaplan-Meier plots, log-rank tests, and Cox proportional hazards regression model, which are presented in Fig. 2. and Table 2.

Comment 3:

1. The authors should analyze the correlation between miRNA-618 and disease-free survival of patients.

Author’s Reply:

Usually disease-free survival (DFS) is defined as the interval from the date of treatment to the date of local recurrence, distal recurrence or death, and it is typically used in the adjuvant treatment setting (Korn and Freidlin, 2020).

We estimated progression-free survival (PFS) for our metastatic patients and found that patients with a low level of expression of miR-618 in serum showed a trend towards shorter mean PFS of 6.15 months (95% CI: 4.41-7.88) as compared to those with high/intermediate expression of miR-618 – 7.84 months (95%, CI: 6.80-8.86) (log-rank test, p=0.061, data not shown). This result was presented in the "Results" section - 3.3. Low miR-618 levels in serum were associated with short survival

Reference:

Korn E.L., Freidlin B. 18 - Clinical Trial Designs in Oncology, Editor(s): John E. Niederhuber, James O. Armitage, Michael B. Kastan, James H. Doroshow, Joel E. Tepper, Abeloff's Clinical Oncology (Sixth Edition), Elsevier, 2020, Pages 296-307.e2, ISBN 9780323476744, https://doi.org/10.1016/B978-0-323-47674-4.00018-9.

Comment 4:

1. The effect of miRNA-618 on invasion and migration of colorectal cancer cells should be investigated in vitro.

Author’s Reply:

Thank you very much for your comment. We agree with you that investigation of the effect of miRNA-618 on invasion and migration of colorectal cancer cells in vitro would make the story behind miR-618 function in CRC more complete.

The aim of our current manuscript is to evaluate the expression in plasma of non-investigated miRNA in colorectal cancer. Our study reports for the first time miR-618 in colon cancer patients and make the first attempt to evaluate its prognostic significance in a non-small number of patient (n=104). Elucidation of the role of miR-618 in tumorigenesis of CRC is already planned as an aim of our next study. We schedule to study in details the mechanism of activity of miR-618 in advanced CRC stages. Our hypothesis is that the low expression of miR-618 in tumor tissue is caused by oncogenic cicrCCDC6 sponging. Targets for miR-618 in colon cancer are also unknown.

We are hoping that we answered your questions and comments.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors managed to address the contradiction in the manuscript and can now be considered!

Author Response

Thank you!

Reviewer 2 Report

This version of the manuscript is much better, specially the discussion section. I also understood your explanations regarding my previous comments and questions and the fact that you are currently working in new studies to further investigate the role of miR-618. For now, there are still some minor comments that i will adress.

Comment 1) Please revise the “p” in the manuscript and figures. It is not in italic.

Comment 2) Change the keywords to metastatic colon cancer instead of CRC

Comment 3) Line 73: Change “104 patients all Caucasians” to “104 caucasians patients”

Comment 4) Delete the “100%” in figure 1B

Comment 5) In both figures 1A and 3A legend you added “…are shown the mean value with the range of miR-618 expression in two groups”. But what you should also add is if the error bars are a representation of standard deviation (SD) or standard error of the mean (SEM) values?

Comment 6) Line 387-394: I think you can edit this part of the discussion to something like: “In this study, we recognized the significance of miR-618 however there are still several limitations in our study. First, our study is retrospective and cross-sectional. It is also descriptive with an absence of in vitro analyses of the effect of miR-618 on tumor progression. Therefore, more research is needed to confirm our findings and assumptions and to explore the potential mechanisms of action of miR-618 in mCC. Moreover, the expression levels of miR-618 should be investigated in a larger cohort of tissue samples and also in stage II and III CC patients.”

Author Response

Reviewer 2

We would like to thank you for your attentive comments and useful suggestion, which helped us improve the “Discussion” section of the manuscript. All changes are presented with “tracked changes”.

Comment 1: Please revise the “p” in the manuscript and figures. It is not in italic.

Author’s Reply:

The changes were made in the text and the figures of the manuscript.

Comment 2: Change the keywords to metastatic colon cancer instead of CRC

Author’s Reply:

The change was made in the keywords of the manuscript on Line 34.

Comment 3: Line 73: Change “104 patients all Caucasians” to “104 caucasians patients”

Author’s Reply:

The change was made on Line 73 in “Materials and Methods” section, 2.1. Patients and healthy controls.

Comment 4: Delete the “100%” in figure 1B

Author’s Reply:

Please, allow us to not agree with your proposal to delete the “100%” in figure 1B because as far as we know the X-axis represents False positive rate which is calculated as follows:

False positive fraction = FP/(FP+TN))

where  FP is False Positive; TN is True Negative,

respectively represented on ROC curve analysis graph as 100%-specificity or 1-specificity.

We deleted “%”  in “100%” from X-axis description – “100%-Specificity%” on Figure 1 (b).

Comment 5: In both figures 1A and 3A legend you added “…are shown the mean value with the range of miR-618 expression in two groups”. But what you should also add is if the error bars are a representation of standard deviation (SD) or standard error of the mean (SEM) values?

Author’s Reply:

In Figure 1 (a) description the sentence “In the Figure 1. (a) are shown the mean value with the range of miR-618 expression in two groups” was deleted and “data are presented as mean ±SD” was added on Line 166.

In the Figure 3 (a) description the sentence “In the Figure 3. (a) are shown the mean value with the range of miR-618 expression in patients with different genotype” was deleted and “data are presented as mean ±SD” was added on Line 229.

On both graphs, we showed the ±SD of the mean value of expression instead of the mean value with the range of expression.

On the old graph of Figure 3 (a) one data point was left outside the Y-axis limits. We corrected this in the graph of Figure 3 (a) in this revised version.

Comment 6: Line 387-394: I think you can edit this part of the discussion to something like: “In this study, we recognized the significance of miR-618 however there are still several limitations in our study. First, our study is retrospective and cross-sectional. It is also descriptive with an absence of in vitro analyses of the effect of miR-618 on tumor progression. Therefore, more research is needed to confirm our findings and assumptions and to explore the potential mechanisms of action of miR-618 in mCC. Moreover, the expression levels of miR-618 should be investigated in a larger cohort of tissue samples and also in stage II and III CC patients.”

Author’s Reply:

The correction that you suggest describes advantages and limitations of our study in a more precise manner. Therefore we replaced the text on Line 387-394 with yours. Thank you very much!

Author Response File: Author Response.docx

Reviewer 3 Report

The authors have addressed all the comments and the manuscript can be accepted for publication.

Author Response

Thank you!