Cellular, Molecular and Proteomic Characteristics of Early Hepatocellular Carcinoma

Round 1

Reviewer 1 Report

Dear all,

Thank you for the opportunity to review the manuscript entitled "Cellular, molecular and proteomic characteristics of early hepatocellular carcinoma".

The authors aimed to evaluate diagnostic markers for the detection of early HCC based on proteome analysis, miRNAs and circulating tumor cells (CTCs) in the blood of patients with cirrhosis, early HCC, and advanced HCC. They found that patients with advanced HCC had higher proportion of detectable CTCs. Also, patients with early and advanced HCC exhibited a significant increase of miR-200b when compared to cirrhotic patients. Proteome analysis indicated that early HCC patients present a significant upregulation of APOA2, APOC3 proteins when compared to cirrhotic patients.

The manuscript is well-written, but I have some concerns.

Comments:

-          I encourage the authors to provide a brief explanation about the calculation of sample size for their study.

-          The authors should explain the role of the five healthy participants who were included in the study.

-          The small sample size of the study limits the study and should be discussed in the manuscript.

-      Demographic and clinical parameters of the collective (age, gender of the patients, tumor size and stage (TNM classification), presence of microvascular invasion and Grade of the tumors) should be reported in the manuscript.

-          Did the authors analyze cutoffs by measuring the biomarkers quantitatively? Moreover, some markers, such as CTCs, Vimentin and miR-222, have been reported to be associated with advanced and metastatic HCC (Clin Cancer Res. 2010 Feb 1;16(3):867-75; Cell Physiol Biochem. 2015;37(2):629-40; Oncogene. 2004 Jan 8;23(1):298-302). These should be discussed in the manuscript.

Best regards!

Author Response

Please see attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

In the present manuscript, Armakolas and colleagues investigated novel potential biomarkers for hepatocellular carcinoma (HCC). Different methodological approaches were adopted, including proteomic analysis, detection of selected miRNAs and circulating tumor cells (CTC). Interestingly, the authors observed specific CTC patterns in patients with HCC, and particularly in patients with advanced stage HCC as compared to early tumor; furthermore, they identified a two proteins (namely APOA2 and APOC3) that combined with the assessment of miR-200b expression provided an accuracy of 100% for the identification of patients with early HCC as compared to patients with cirrhosis without tumor. Overall, the manuscript is well written and worthy of consideration. However, there are some issues that need to be addressed before final acceptance.

1)  Introduction. Patients with cirrhosis and HCV, even if cured for HCV infection, maintain a significant risk of HCC development (REF: 35159094 and 34738664). I suggest highlighting this aspect in the introduction section (lines 50-54).

2) The study included 117 patients (89 HCC and 28 cirrhotics) but not all of them were tested for proteomic analysis, miRNAs and CTC; sometimes, it is difficult to comprehend how many patients underwent 1, 2 or all 3 analyses. For this reason, I recommend to add a flow chart of the study highlighting how many patients underwent the specific investigations.

3) Please provide a table summarizing the demographic, clinical, and biochemical characteristics of the patients included in the study. Can the authors provide BCLC classification for patients with HCC?

4) qRT-PCR for miRNAs quantitation was carried out using a SYBR Green protocol. Can you provide some details regarding assay specificity?

5) Can the authors provide a REF for lines 162-163?

6) Table 2 is difficult to understand. I suggest to add more explanation on how “to read” the table (a legend or so).

7)  Looking at Figure 4, it seems that ROC curve analysis has been performed using dichotomous data. I suggest to perform the analysis using biomarkers continuous variables. Then, cut-off values could be calculated based on the results of this analysis (on Yuden index or other appropriate criteria).

8) 2 out of 28 patients with cirrhosis had detectable CTCs. Is the clinical follow up (FU) available for these patients? It would be very important and interesting to know whether cirrhotic patients with detectable CTCs developed HCC during FU.

9)  Authors are encouraged to add a paragraph concerning the limitations of the study (for instance, lack of validation cohort).

10) References format should be amended according to MDPI journals’ style.

Author Response

Please see attacment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Dear,

The revised version of the article entitled "Cellular, molecular and proteomic characteristics of early hepatocellular carcinoma" was evaluated.

After the revision, the quality of the manuscript has been increased significantly. In the current format, the study could be interesting and useful for the readers.

 

Regards,

 

Reviewer 2 Report

The authors improved the manuscript as requested. Please