PONE-D-21-28792Features of patients who developed hepatocellular carcinoma after direct-acting antiviral treatment for hepatitis C VirusPLOS ONE

Dear Dr. Mawatari,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please comply with all points raised by the reviewers. In particular, please address the issue of  underdiagnosis of cirrhosis as it will very likely impact accurate identification of factors associated with HCC as well as development of models for HCC risk assessment. As suggested, colinearity between HA and chirrosis/advance fibrosis should be explored and included into the dataset. Furthermore, the issues raised in respect to confounding factors in the cohort should be addressed.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In the current study, Mawatari et aimed to shed light on the features of HCV-infected patients developing HCC upon DAA treatment. After studying a cohort of 1494 DAA-SVR patients, they found that 60 of them (=4%) developed HCC within 47.6 months and in these patients. The main conclusion is that AFP>5.3 ng/mL at the end of the DAA treatment (EOT) can be useful for predicting HCC development after an SVR. Eventually, the combination of the following markers is associated for predicting HCC: EOT-AFP>5.3 ng/mL, males aged >73 years, hyaluronic acid >75 ng/mL, EOT-albumin <3.9 g/dL.

The study is essentially descriptive and statistically sound. However, I have some concerns about the usefulness of these markers in predicting HCC in DAA treated patients:

1. These markers have not been evaluated in a cohort without DAA treatments (par ex. NASH, NAFLD)

2. I have doubts about the age and sex considered as risk factors in this study since higher HCC rates are encountered in older males irrespectively of HCC etiology.

3. The authors state that EOT-AFP is an important indicator for HCC development, but at the same time they state in table 5 that in 37.3% of the patients who developed HCC, the AFP (and DCP) values were within the normal limits. This is confusing. Also, DCP has not been explained and commented.

4. Other markers that could be of interest upon HCV clearance such as miRNA-122 have not been studied and discussed. Immune markers such as TGF-beta, VEGF, IL-6, etc. have not been addressed neither.

Reviewer #2: In this manuscript, Mawatari and colleagues analyzed the features of patients with chronic C Hepatitis successfully treated with DAAs who developed HCC over time.

He conducted a well-designed prospective study, with a large study population and considerable follow-up.

Although the parameters (including AFP levels) associated with HCC development after DAAs therapy have been already analyzed and described by several authors, changes in AFP levels over time after DAAs were not considered before and represent therefore a novel parameter of interest.

The study has a rationale and has been carried out with an adequate statistical approach.

The article address a an important topic, considering that HCV infection is now easily cured with IFN-free therapies, but HCC risk still remains after achieving SVR. The authors analyzed the risk factors for HCC and created a 5- factors score that could be use to estimate the cumulative HCC risk over time, categorizing the patients in low-moderate-high risk. The laboratory parameters used in this score system are routinely tested in clinics, therefore the score, if further validated, could be suggested as tool in clinical practice.

The fact that liver fibrosis was not diagnosed by biopsy or transient elastography represents a limitation, but the authors acknowledged it. A validation of this score will be important in order to understand its potential role in clinical practice in earlier identify subjects at higher risk.

Major comments:

• In the introduction, the authors explain the concept of RASs in relationship to non-response to DAAs. I don’t find this particular topic related to the main subject of the manuscript since only the patients who achieved SVR were enrolled. This paragraph should be removed.

• The authors do not mention HIV-coinfected patients, who have overall a higher risk for cancer. Were they excluded from the study population? If so, the authors should mention that in the Methods. Otherwise, they should consider this variable.

• Is the study still ongoing? It is well specified when the observation started but it’s not clear to me if the observation period is over.

• Patients with EOT-AFT<5.3 ng/ml did not show any changes in AFP levels at HCC onset. Did the author observe any peculiar characteristics (size, number of nodules, vascularization?) in HCC occurred in this subgroup of patients?

Minor comments:

Material and Methods:

-“observation” should be replaced by “observational”

- Instead of “The study population is shown in Figure 1”, I would rather write “Study population enrollment is shown…” I would suggest also to modify Figure 1 title consistently with that, specifying that it represents a flow chart of the study population enrollment.

- Titles of Table 3 and 4 are the same. Authors should be differentiate.

Reviewer #3: This is a Japanese multicentre cohort study of n=1494 post-SVR patients following DAA treatment. The study followed the cohort (median 47-months) and described the factors associated with the development of HCC (N=60). They identify: age, males, hyaluronic acid, AFP and albumin as factors associated with HCC. They then developed a risk stratification score based on these factors.

The manuscript is well structured and clear. A strength of the study is the exploration of AFP dynamics. This explores the impact of change in AFP and then relationship to DCP. This supports the data that AFP is not a great screening tool.

Comments:

•The major issue with this study is the categorisation of cirrhosis. It is clear from multiple studies that cirrhosis is a major aetiological factor in HCC development and the vast majority of HCCs occur in the context of cirrhosis. In this study only 55% of HCC was associated with cirrhosis which suggests that cirrhosis was underdiagnosed. It is likely the result of suboptimal assessment of liver fibrosis. This would impact any analysis of factors associated with HCC, especially if they are factors that are also related to fibrosis/cirrhosis. Key to this is hyaluronic acid which is a strong marker of fibrosis. The authors were not clear about their definition of cirrhosis and this needs to be stated clearly in the methods. Specifically, cut-offs for FIB4, clinical and biochemical criteria etc. It is not acceptable to state “other factors”.

•Hyaluronic acid (HA) is related to liver fibrosis. It is a part of many non-invasive algorithms of liver fibrosis. Given this, it is likely closely related to cirrhosis. Potentially it is collinear with cirrhosis. In the 2-year follow-up and 3-year follow-up models HA and cirrhosis are alternatively significant. Collinearity between HA and cirrhosis and advanced fibrosis must be explored.

•Gender and age differences in HA may also have an impact. Could this be analysed in the cohort?

•HA should also form a part of the discussion.

•Usually, to analyse de novo HCC, any HCC exclusion that developed within 3-6 months post treatment should be excluded. Could this be done to see if this changes the analysis of factors.

•A weakness is co-factors which could not be addressed including: alcohol, metabolic syndrome, and aspirin use. This would lead to confounding of results.

•This is a Japanese population with different screening protocols compared with other international guidelines. Here, even non-cirrhotic patients were surveyed and cirrhotics were evaluated at a 3-4 month interval when most international guidelines are 6-months US. This should be made clear in the discussion as it will impact generalisability.

Minor:

•P15 – The comparison is cirrhosis to no cirrhosis (not chronic hepatitis as the HCV has been cleared).

•Table 3: Aetiology should be cirrhosis (present or not)

•Table 3: In the Advanced fibrosis multivariable analysis, why are insignificant outcomes presented?

•Figure 2 should change the y-axis to small scale.

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Reviewer #1: No

Reviewer #2: Yes: Viola Guardigni

Reviewer #3: Yes: mark danta

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Features of patients who developed hepatocellular carcinoma after direct-acting antiviral treatment for hepatitis C Virus

PONE-D-21-28792R1

Dear Dr. Mawatari,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Birke Bartosch

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have improved the manuscript according to the majority of the referees' comments and their article is now better structured and scientifically sound. The authors now clearly show the limitation of their study and the results/conclusions are therefore better interpreted.

Reviewer #2: The authors have addressed each of my previuos comments exhaustively. I have no further comment to add concerning the contents. I think that the manuscript could be accepted for publication on PLOS ONE.

Reviewer #3: The authors have addressed the issues that were raised as best they could. There are still issues around confounding and classification of cirrhosis which could not be addressed in the study design.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Viola Guardigni

Reviewer #3: Yes: A/Prof Mark Danta