Useful Predictor for Exacerbation of Esophagogastric Varices after Hepatitis C Virus Eradication by Direct-Acting Antivirals

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript examined useful predictor for exacerbation of esophagogastric varices after hepatitis C virus eradication by direct-acting antivirals. However, there are drawbacks in this study. 1) there are no data presented to demonstrate the HCV eradication. 2) There are no control groups in this study, which makes it hard to evaluated the usefulness of the markers the authors found. 3) Fig. 1 should show data points which are missing. Fig 1 has low quality. 4) Fig. 2 have varied patients in different groups. Some groups have small size of populations. 5) Fig. 3b has low resolution. 

Author Response

Response to Reviewer 1

This manuscript examined useful predictor for exacerbation of esophagogastric varices after hepatitis C virus eradication by direct-acting antivirals. However, there are drawbacks in this study. 1) there are no data presented to demonstrate the HCV eradication. 2) There are no control groups in this study, which makes it hard to evaluated the usefulness of the markers the authors found. 3) Fig. 1 should show data points which are missing. Fig 1 has low quality. 4) Fig. 2 have varied patients in different groups. Some groups have small size of populations. 5) Fig. 3b has low resolution.

Major problems

1) there are no data presented to demonstrate the HCV eradication.

Response: Thank you for your comments. As noted, we did not present data demonstrating HCV eradication, but in recent years, these DAA regimens have resulted in SVR rates of greater than 95% in Japan among patients with compensated disease ^1-9). Furthermore, DAA therapy for decompensated cirrhosis patients has recently been approved in Japan with an SVR rate reported to be 91.3% ¹⁰⁾. Therefore, we expect to achieve good SVR rates in the future, and more patients may be included. We would like to emphasize that careful follow-up is necessary even after SVR is achieved in patients with cirrhosis, including decompensated cirrhosis, because complications related to portal hypertension may develop.

We have added the DAA treatment regimens to Table 1.

1.Kumada H, Suzuki Y, Ikeda K, Toyota J, Karino Y, Chayama K, et al. Daclatasvir plus asunaprevir for chronic HCV genotype 1b infection. Hepatology. 2014; 59:2083-91.

2.Mizokami M, Yokosuka O, Takehara T, Sakamoto N, Korenaga M, Mochizuki H, et al. Ledipasvir and sofosbuvir fixed-dose combination with and without ribavirin for 12 weeks in treatment-naive and previously treated Japanese patients with genotype 1 hepatitis C: an open-label, randomised, phase 3 trial. Lancet Infect Dis. 2015; 15:645-53.

3.Omata M, Nishiguchi S, Ueno Y, Mochizuki H, Izumi N, Ikeda F,et al. Sofosbuvir plus ribavirin in Japanese patients with chronic genotype 2 HCV infection: an open-label, phase 3 trial. Journal of Viral Hepatitis, 2014; 21: 762-768.

4.Kumada H, Chayama K, Rodrigues L Jr, Suzuki F, Ikeda K, Toyoda H, et al. Randomized phase 3 trial of ombitasvir/paritaprevir/ritonavir for hepatitis C virus genotype 1b-infected Japanese patients with or without cirrhosis. Hepatology. 2015; 62:1037-46.

5.Chayama K, Notsumata K, Kurosaki M, Sato K, Rodrigues L Jr, Setze C, et al. Randomized trial of interferon- and ribavirin-free ombitasvir/paritaprevir/ritonavir in treatment-experienced hepatitis C virus-infected patients. Hepatology. 2015; 61:1523–32.

6.Kumada H, Suzuki Y, Karino Y, Chayama K, Kawada N, Okanoue T, et al. The combination of elbasvir and grazoprevir for the treatmentof chronic HCV infection in Japanese patients: a randomizedphase II/III study. J Gastroenterol. 2017; 52:520-533.

7.Toyota J, Karino Y, Suzuki F, Ikeda F, Ido A, Tanaka K, et al. Aclatasvir/asunaprevir/beclabuvir fixed-dose combinationin Japanese patients with HCV genotype 1 infection. J Gastroenterol. 2017; 52:385-395.

8.Chayama K, Suzuki F,Karino Y, Kawakami Y, Sato K Atarashi T, et al. Efficacy and safety of glecaprevir/pibrentasvir in Japanese patients with chronic genotype 1 hepatitis C virus infection with and without cirrhosis. J Gastroenterol. 2018; 53:557-565.

9.Toyoda H, Chayama K, Suzuki F, Sato K, Atarashi T, Watanabe T, et al. Efficacy and Safety of Glecaprevir/Pibrentasvir in Japanese Patients With Chronic Genotype 2 Hepatitis C Virus Infection. Hepatology. 2018; 67:505-513.

10. Tahata Y, Hikita H, Mochida S, Sato K, Atarashi T, Watanabe T,et al. Post treatment liver function, but not baseline liver function stratifies patient survival after direct-acting antiviral treatment in decompensated cirrhosis with hepatitis C virus.

J Gastroenterol. 2023; 58:1211-1221.

2) There are no control groups in this study, which makes it hard to evaluated the usefulness of the markers the authors found.

Response: As the reviewer noted, there are no control groups in this study, which makes it hard to evaluate the usefulness of the markers. As you pointed out, we should set up a control group and conduct a prospective study in the future. However, in this study, we focused on patients with compensated cirrhosis. At present, there are no clear criteria for determining whether patients with cirrhosis, who are prone to complications due to portal hypertension, will develop portosystemic encephalopathy or exacerbations of esophagogastric varices.

Therefore, we conducted a retrospective study for now, but based on the results, we believe that a controlled study may be possible in the future. In addition, the Baveno VI criteria propose criteria that do not necessarily require endoscopic examination (LSM, platelet count), but in our study, exacerbations of esophagogastric varices were observed even outside of the proposed criteria. Therefore, we believe that it is necessary to conduct prospective studies with a larger number of cases in the future.

3) Fig. 1 should show data points which are missing. Fig 1 has low quality.

Response: We provided a higher-quality version of Fig. 1.

4) Fig. 2 have varied patients in different groups. Some groups have small size of populations.

Response: As the reviewer noted, some groups have small population sizes. In the group without EGV aggravation, the number of cases involving tests for liver stiffness three years after the end was small. One possible reason for this is that liver stiffness has been declining year by year, and it was judged that there was little risk of aggravation of EGV, so evaluation was based solely on blood tests. Ideally, liver stiffness should be measured along with blood tests. On the other hand, in the group with EGV aggravation, even when SVR was achieved, there was little improvement in liver stiffness, and examination of LSM was continued in many cases after DAA therapy.  Regarding the variability in case numbers, we believe this is a very important point and plan to take it into account in future retrospective studies.

5) Fig. 3b has low resolution.

Response: Following the reviewer's suggestion, we created a higher-resolution version of Figure 3b and added the rate of EGV aggravation by LSM to the Results section (page 14, lines 302-304). We created a revised Figure 3b and described the changes in ammonia levels in patients with or without experience of encephalopathy after SVR in the RESULTS section as the reviewer suggested (page 7, lines 224-238).

The cumulative aggravated EGV rates at 1, 3, and 5 years were 14%, 63%, and 74% for the group with a pretreatment LSM ≥30 kPa, 10%, 31%, and 31% for the group with an LSM of 20–30 kPa, and 4%, 16%, 16% for the group with an LSM of 15–20 kPa, and 0%, 0%, and 7% for the group with an LSM of 10–15 kPa, respectively (Figure 3b).

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript of Yuko Nagaoki and collaborators investigates a relevant issue regarding the increased risk of esophagogastric varices (EGV) in hepatitis C patients (HCV) with cirrhosis who achieved sustained virological response (SVR) through direct-acting antiviral (DAA) therapy. The study used a cohort of 167 patients and conducted a multivariant analysis to identify several independent risk factors that could be used to predict the high risk of EGV aggravation after SVR. While the study's findings are not entirely novel, the authors highlight the significance of incorporating serum bile acids into the multivariate model for better patient stratification with a higher risk of EGV aggravation. Although the research question addressed in the article is clinically relevant, there are some aspects that are unclear to the reviewer, requiring further revision and improvement to meet the publication standards of Livers.

·       In the Introduction section, several references are lacking throughout the main text. For instance, the information stated in the sentences of lines 61 and 65 is not supported by any references to other studies.

·       The sentences of lines 69 and 70 should be integrated into the previous paragraphs in order to enhance the readability of the manuscript.

·       It is not clear to the reviewer if the study included patients with decompensated cirrhosis. Although this information is indicated in the first paragraph of the Materials and Methods section, no further information about this is provided in the rest of the manuscript.

·       In the Materials and Methods section, the content of the subheading ‘clinical assessments’ should be changed. Currently, there is an overlap in the content of this subheading and the subheading ‘measurement of liver stiffness’, ‘endoscopic examination for assessing EGV’, and ‘ CT examination for portal hypertension’.

·       It would be beneficial for the manuscript if more information related to liver function tests is included in the Materials and Methods section. The authors should at least indicate which parameters were evaluated as this information is currently only provided in the Results section.

·       There are some inconsistencies in the manner the authors described the instruments used for measuring certain variables in the Materials and Methods section. For instance, the model and brand of the liver stiffness instrument are indicated, but no information about the endoscope used or the dynamic computed machine is provided.

·       It would be beneficial for the manuscript if the authors indicated how the data were expressed in the subheading ‘Statistical analysis’. For instance, continuous variables were expressed as median and range.

·       There are inconsistencies between the data presented in the main text of the manuscript and the tables. For example, in line 133, it is stated that the LSM range was 3.6-44.2, while in the table, it is 5.6-44.2. This information should be revised.

·       The title of Table 1 is unclear and difficult to understand without context. It would be beneficial for the manuscript if this title is revised and changed.

·       The reviewer is wondering about the rationale behind the cut-off of the LSM used for the stratification of the patients in Figure 3. Is there any scientific reason? If so, literature data that supports this selection should be indicated as a reference in the main text of the manuscript. Linked to this, why the cut-offs for stratifying the patients in Figure 3A is different from those in Figure 3B?

·       A literature reference that supports the information provided in the line 214-215 is missing.

·       It is not clear to the reviewer when the tests were performed at the end of the treatment (EOT) or after achieving SVR. This should be clarified in the manuscript.

Comments on the Quality of English Language

       The manuscript contains several typographical mistakes and inconsistencies, including incorrect abbreviations. It is recommended that the authors carefully review and revise the manuscript to address these issues

Author Response

Response to Reviewer 2

The manuscript of Yuko Nagaoki and collaborators investigates a relevant issue regarding the increased risk of esophagogastric varices (EGV) in hepatitis C patients (HCV) with cirrhosis who achieved sustained virological response (SVR) through direct-acting antiviral (DAA) therapy. The study used a cohort of 167 patients and conducted a multivariant analysis to identify several independent risk factors that could be used to predict the high risk of EGV aggravation after SVR. While the study's findings are not entirely novel, the authors highlight the significance of incorporating serum bile acids into the multivariate model for better patient stratification with a higher risk of EGV aggravation. Although the research question addressed in the article is clinically relevant, there are some aspects that are unclear to the reviewer, requiring further revision and improvement to meet the publication standards of Livers.

Major points

1) In the Introduction section, several references are lacking throughout the main text. For instance, the information stated in the sentences of lines 61 and 65 is not supported by any references to other studies.

Response: Thank you for pointing out this oversight. Following the reviewer's suggestion, we corrected the sentences around line 61 in the Introduction section (page 2, lines 58-63).

Portal hypertension is a major consequence of cirrhosis and is responsible for its most severe complications, including ascites, bleeding from esophagogastric varices (EGV), and portosystemic encephalopathy. SVR achievement by direct-acting antiviral (DAA) therapy was reported to decrease portal venous pressure [3-6], which is expected to reduce the risk of portal hypertension in liver cirrhosis patients with HCV infection.

Following the reviewer's suggestion, we also corrected the sentences following line 65 in the Introduction section (page 2, lines 66-73). In addition, the study referred to in reference 8 was an interferon-based study and has been removed from this list.

We have previously reported that patients with HCV-related cirrhosis who had already developed collateral vessels may experience aggravation of EGV or develop portosystemic encephalopathy after they have achieved SVR [8]. Tsuji et al. also reported that even if SVR is achieved with DAA therapy, patients with HCV-related compensated cirrhosis who had already developed portosystemic shunt showed little improvement in liver function [9]. Therefore, careful follow-up is necessary for liver cirrhosis patients with portal hypertension even after achieving SVR.

2) The sentences of lines 69 and 70 should be integrated into the previous paragraphs in order to enhance the readability of the manuscript.

Response: Thank you for pointing this out. Following the reviewer's suggestion, the paragraphs have been merged (page 2, lines 73-74).

3) It is not clear to the reviewer if the study included patients with decompensated cirrhosis. Although this information is indicated in the first paragraph of the Materials and Methods section, no further information about this is provided in the rest of the manuscript.

Response: Thank you for noting this oversight. We added the following information to the Clinical and laboratory assessments section of the Materials and Methods.

2.1.2 Clinical and laboratory assessments

This study included patients with compensated cirrhosis. Cirrhosis was assessed based on liver imaging tests or prior liver biopsy showing F4. Patients with Child-Pugh class A without a history of decompensated events were considered to have compensated cirrhosis, and patients with Child-Pugh class B or C or patients with a history of decompensated events were considered to have decompensated cirrhosis. This study did not include patients with decompensated cirrhosis. Laboratory assessment was performed before treatment and 24 weeks and 1, 2, and 3 years after EOT. In addition to general biochemical tests, autotaxin and bile acids were also included, and the albumin-bilirubin (ALBI) score and fibrosis-4 (FIB-4) index, which serve as surrogate markers of hepatic spare ability and liver fibrosis, respectively, were calculated as previously reported [12,13].

4) In the Materials and Methods section, the content of the subheading ‘clinical assessments’ should be changed. Currently, there is an overlap in the content of this subheading and the subheading ‘measurement of liver stiffness’, ‘endoscopic examination for assessing EGV’, and ‘CT examination for portal hypertension’.

Response: Thank you for pointing out this overlap. We corrected the Materials and Methods section, accordingly.

2.1.3 Measurement of liver stiffness

We measured the severity of liver stiffness measurement (LSM) before DAA therapy, and 24 weeks (SVR achievement), 1, 2, and 3 years after DAA therapy using a vibration controlled transient elastography (VCTE). Patients were placed in a supine position with the right hand at the most abducted position for scanning right lobe of the liver [14]. When at least 10 valid measurements were obtained with valid measurements at ≥60% and in-terquartile range of <30%, such measurements were considered valid and the median value of these measurements was used for analysis.

2.1.4 Endoscopic examination for assessing EGV

We evaluated the endoscopic findings of EGV based on the classification of the Japanese Society for Portal Hypertension and Esophageal Varices [15]. The form (F) of EGV was classified as follows: F0 was treated and completely treated, with no varices; F1 was straight and relatively thin; F2 was beaded and moderately thick; and F3 was thick, nodular, or mass-like. There are three types of Red Color (RC) sign: red wale marking, cherry red spot, and hematocystic spot. RC1 is observed only in one-line varices, RC2 is observed between RC1 and RC3, and RC3 is observed in all circumferential varices. Endoscopy was performed within 6 months before starting antiviral therapy and was evaluated at least once each following year. Compared with baseline findings on follow-up endoscopy, worsening of F and RC signs was defined as aggravation of EGV. Endoscopy results were confirmed by two expert endoscopists.

2.1.5 CT Examination for portal hypertension

All patients underwent CT examination at 24 weeks from EOT achievement. CT was performed in the high-quality scanning mode. We focused on the left gastric vein (LGV) and splenorenal shunt as portosystemic collateral vessels, and these vessels were evaluated by dynamic CT, measuring the vessel diameter and recording the widest part of the vessel in all cases in this study.

5) It would be beneficial for the manuscript if more information related to liver function tests is included in the Materials and Methods section. The authors should at least indicate which parameters were evaluated as this information is currently only provided in the Results section.

Response: We added the following information to the Clinical and laboratory assessments section in the Materials and Methods.

2.1.2 Clinical and laboratory assessments

This study included patients with compensated cirrhosis. Cirrhosis was assessed based on liver imaging tests or prior liver biopsy showing F4. Patients with Child-Pugh class A without a history of decompensated events were considered to have compensated cirrhosis, and patients with Child-Pugh class B or C or patients with a history of decom-pensated events were considered to have decompensated cirrhosis. This study did not in-clude patients with decompensated cirrhosis. Laboratory assessments was examination before treatment and 24 weeks and 1, 2, and 3 years from EOT, in addition to general biochemical tests, autotaxin and bile acids were also included, and the albumin-bilirubin (ALBI) score and fibrosis-4 (FIB-4) index, serving as surrogate markers of hepatic spare ability and liver fibrosis, respectively, were calculated as previously reported [12,13].

6) There are some inconsistencies in the manner the authors described the instruments used for measuring certain variables in the Materials and Methods section. For instance, the model and brand of the liver stiffness instrument are indicated, but no information about the endoscope used or the dynamic computed machine is provided.

Response: Thank you for alerting us to these omissions. We corrected the Measurement of liver stiffness section in the Materials and Methods.

2.1.3 Measurement of liver stiffness

We measured the severity of liver stiffness measurement (LSM) before DAA therapy, and 24 weeks (SVR achievement), 1, 2, and 3 years after DAA therapy using vibration controlled transient elastography (VCTE). Patients were placed in a supine position with the right hand at the most abducted position for scanning the right lobe of the liver [14]. When at least 10 valid measurements were obtained with valid measurements at ≥60% and interquartile range of <30%, such measurements were considered valid and the median value of these measurements was used for analysis.

7) It would be beneficial for the manuscript if the authors indicated how the data were expressed in the subheading ‘Statistical analyses. For instance, continuous variables were expressed as median and range.

Response: We added the following text to the Statistical analysis section in the Materials and Methods.

2.1.6 Statistical analysis

Continuous variables were expressed as median and range. Continuous variables were analyzed using the Mann-Whitney U-test. Aggravated EGV was calculated using the Kaplan–Meier method, and differences between groups were assessed using a log-rank test. Multivariate analysis was performed using a Cox proportional hazard model with a stepwise selection of variables or two logistic regression analyses. Receiver operating characteristic curves were used to determine the cutoff values for predicting the aggravated EGV-related events in the patients. All statistical analyses were performed using IBM SPSS version 23.0. P < 0.05 was considered significant.

8) There are inconsistencies between the data presented in the main text of the manuscript and the tables. For example, in line 133, it is stated that the LSM range was 3.6-44.2, while in the table, it is 5.6-44.2. This information should be revised.

Response: Thank you for pointing out these discrepancies. We corrected this in the Baseline characteristics of the patients section (page 3, lines 155).

The median FIB-4 index was 5.98 (range 3.27–26.09), the ALBI score was –2.56 (range –3.43 to –1.28), and the LSM was 18.9 (range 5.6–44.2) kPa.

9) The title of Table 1 is unclear and difficult to understand without context. It would be beneficial for the manuscript if this title is revised and changed.

Response: Thank you for pointing this out. Following the reviewer's suggestion, we added this sentence to the description for Table 1.

Table 1. Clinical characteristics of 167 HCV-related compensated cirrhosis patients who achieved SVR by DAA therapy.

10) The reviewer is wondering about the rationale behind the cut-off of the LSM used for the stratification of the patients in Figure 3. Is there any scientific reason? If so, literature data that supports this selection should be indicated as a reference in the main text of the manuscript. Linked to this, why the cut-offs for stratifying the patients in Figure 3A is different from those in Figure 3B?

Response: Thank you for pointing this out. Following the reviewer's suggestion, we added the following sentences in the Discussion section (page 10, lines 301-314).

In this study, the LSM cutoff in Fig. 3 was used as a criterion for liver stiffness, and the analysis was performed using the LSM presented in Baveno VII guideline as a reference [20]. Previously, according to the Baveno VI criteria, if liver stiffness by VCTE is LSM ≤ 15.0 kPa and platelet count ≥15 × 10⁴/mL, clinically significant portal hypertension is excluded. However, in compensated cirrhosis patients whose liver stiffness with VCTE is LSM ≥20 kPa and platelet count ≤15 × 10⁴/mL, the presence of clinically significant portal hypertension cannot be ruled out, proposed to perform upper gastrointestinal endoscopy [10]. Indeed, as shown in Figure 3a, when liver stiffness has already been higher before DAA therapy, drastic reduction of liver stiffness is not expected after SVR because a long time is needed to improve liver stiffness, suggesting a high risk of EGV worsening. Furthermore, aggravation of EGV has been observed even when LSM was 10–15 kPa, so liver stiffness and platelets alone may not be sufficient, therefore, it is necessary to combine several risk factors to identify the conditions that aggravate of EGV.

And, we added the result in Changes in liver stiffness after achieving SVR and aggravated EGV after eradicating HCV by pretreatment LSM (page 7, lines 224-228).

The cumulative aggravated EGV rates at 1, 3, and 5 years were 14%, 63%, and 74% for the group with a pretreatment LSM ≥30 kPa, 10%, 31%, and 31% for the group with an LSM of 20–30 kPa, and 4%, 16%, 16% for the group with an LSM of 15–20 kPa, and 0%, 0%, and 7% for the group with an LSM of 10–15 kPa, respectively (Figure 3b).

11) A literature reference that supports the information provided in the line 214-215 is missing.

Response: Following the reviewer's suggestion, we have removed it from this paragraph as it is already mentioned in the Discussion section.

12) It is not clear to the reviewer when the tests were performed at the end of the treatment (EOT) or after achieving SVR. This should be clarified in the manuscript.

Response: We apologize for the confusion. The examination was before treatment and 24 weeks and 1, 2, and 3 years after EOT, which we have clarified in the Materials and Methods section.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript describes some useful predictors (Liver function, liver fibrosis, LSM, and bile acids) for exacerbation of EGV after HCV eradication with DAA drugs. The authors included 167 patients with HCV-related compensated cirrhosis and followed them for nearly 10 years. Despite achieving SVR, EGV worsened in 42 patients (25%). The cumulative EGV worsening rates at 1, 3, 5, and 10 years were 7%, 23%, 25%, and 27%, respectively. After analysis, the authors concluded that the following independent risk factors for worsening EGV after SVR were identified: platelet count <11.0 × 104/mL, LSM ≥18.0 kPa, total bile acid≥33.0 mmol/L, and LGV diameter ≥5.0 mm. These findings provide further support for the Baveno VII guidelines. I really admire the author's long-term and continuous research. This paper is well writing and represents an interesting work.

As such, I recommend this manuscript minor revision as below:

  1.  "Introduction" and "2.1.2 Measurement of liver stiffness" part, the author can expand it appropriately;
  2. Line 60 and 96, references need to be added;
  3. Table 1, “past history of HCC, yes/no 89/78”. Can the author give more explanation?
  4. Regarding Serum bile acid, did the author take into account the corresponding patient weight?

Author Response

Response to Reviewer 3

This manuscript describes some useful predictors (Liver function, liver fibrosis, LSM, and bile acids) for exacerbation of EGV after HCV eradication with DAA drugs. The authors included 167 patients with HCV-related compensated cirrhosis and followed them for nearly 10 years. Despite achieving SVR, EGV worsened in 42 patients (25%). The cumulative EGV worsening rates at 1, 3, 5, and 10 years were 7%, 23%, 25%, and 27%, respectively. After analysis, the authors concluded that the following independent risk factors for worsening EGV after SVR were identified: platelet count <11.0 × 104/mL, LSM ≥18.0 kPa, total bile acid≥33.0 mmol/L, and LGV diameter ≥5.0 mm. These findings provide further support for the Baveno VII guidelines. I really admire the author's long-term and continuous research. This paper is well writing and represents an interesting work.

As such, I recommend this manuscript minor revision as below:

Minor problems

1) "Introduction" and "2.1.2 Measurement of liver stiffness" part, the author can expand it appropriately;

Response: Thank you for pointing this out. We added the following text to the Introduction and Measurement of liver stiffness sections.

Introduction

Sustained virologic response (SVR) after treatment of hepatitis C virus (HCV) infection is associated with improved patient survival rates [1], with a reduction in the risk for liver decompensation and hepatocellular carcinoma (HCC). Achieving SVR is generally associated with several long-term changes in the liver: normalized hepatic enzyme levels; regression of hepatic liver necrosis, inflammation, and fibrosis; and improved hepatic function [2]. Portal hypertension is a major consequence of cirrhosis and is responsible for its most severe complications, including ascites, bleeding from esophagogastric varices (EGV), and portosystemic encephalopathy. SVR achievement by direct-acting antiviral (DAA) therapy was reported to decrease their portal venous pressures [3-6], expecting to avoid progression of portal hypertension in liver cirrhosis patients with HCV infection.

However, when starting DAA therapy, if portal hypertension or EGV have already developed or if collateral vessels are dilated, symptoms associated with portal hypertension may be difficult to improve even if SVR is achieved. We have previously reported that patients with HCV-related cirrhosis who had already developed collateral vessels may experience aggravation of EGV or develop portosystemic encephalopathy after they achieved SVR [8]. Tsuji et al. also reported that even if SVR was achieved with DAA therapy, patients with HCV-related compensated cirrhosis who had already developed portosystemic shunt showed little improvement in liver function [9]. Therefore, careful follow-up is necessary for liver cirrhosis patients with portal hyperten-sion even after achieving SVR. On the other hand, risk factors for complications associated with worsening portal hypertension after SVR remain unclear.

In portal hypertension, it is important to determine the status of portal venous pressure, and hepatic venous pressure gradient (HVPG) is used as an estimate [10]. However, HVPG is an invasive examination. Therefore, we decided to use non-invasive testing to elucidate the risk factors for worsening portal hypertension after SVR with HCV-related cirrhosis. In addition to liver stiffness, platelet count, and diameter of portosystemic collateral vessels, we also analyzed autotaxin and bile acid levels as liver fibrosis markers. Especially, although autotaxin has recently been reported as a new liver fibrosis marker that increases from the early stage of fibrosis and has a high diagnostic ability [11], there are few reports on the relationship between bile acid and complications in portal hypertension.

In this study, we retrospectively analyzed the risk of portal hypertension in patients with HCV-related cirrhosis after eradicating HCV.

2.1.3 Measurement of liver stiffness

We measured the severity of liver stiffness measurement (LSM) before DAA therapy, and 24 weeks (SVR achievement), 1, 2, and 3 years after DAA therapy using a vibration-controlled transient elastography (VCTE). Patients were placed in a supine position with the right hand at the most abducted position for scanning right lobe of the liver [14]. When at least 10 valid measurements were obtained with valid measurements at ≥60% and interquartile range of <30%, such measurements were considered valid and the median value of these measurements was used for analysis.

2) Line 60 and 96, references need to be added;

Response: Thank you for pointing out these oversights. We revised the text as follows in the Introduction section (page 2, lines 58-63).

Portal hypertension is a major consequence of cirrhosis and is responsible for its most severe complications, including ascites, bleeding from esophagogastric varices (EGV), and portosystemic encephalopathy. SVR achievement by direct-acting antiviral (DAA) therapy was reported to decrease their portal venous pressures [3-6], expecting to avoid progression of portal hypertension in liver cirrhosis patients with HCV infection.

We also added references in the Measurement of liver stiffness section.

2.1.3 Measurement of liver stiffness

We measured the severity of liver stiffness measurement (LSM) before DAA therapy, and 24 weeks (SVR achievement), 1, 2, and 3 years after DAA therapy using a vibration-controlled transient elastography (VCTE). Patients were placed in a supine position with the right hand at the most abducted position for scanning right lobe of the liver [14]. When at least 10 valid measurements were obtained with valid measurements at ≥60% and interquartile range of <30%, such measurements were considered valid and the median value of these measurements was used for analysis.

14.Sandrin, L.; Fourquet, B.; Hasquenoph, JM.; Yon, S.; Fournier, C.; Mal, F.; Christidis, C.; Ziol, M.; Poulet, B.; Kazemi, F.; Beaugrand, M.; Palau, R. Transient elastography: a new noninvasive method for assessment of hepatic fibrosis. Ultrasound Med Biol. 2003, 29, 1705–13.

3) Table 1, “past history of HCC, yes/no 89/78”. Can the author give more explanation?

Response: We added a note explaining the phrase “past history of HCC treatment” in Tables 1 and 2, as follows: “DAA therapy was received after curative treatment for HCC.”

 4) Regarding Serum bile acid, did the author take into account the corresponding patient weight?

Response: Thank you for this observation. Indeed, it has been reported that total bile acids change depending on weight. However, we were unable to take weight into account in this study, but we will be sure to do so in future studies.