Title: An elevated percentage of CD4#CD25#CD127low regulatory T cells in peripheral blood
indicates a poorer prognosis in hepatocellular carcinoma after curative hepatectomy
Version: 2 Date: 04 Apr 2025
Reviewer's report:
This retrospective observational study investigates the prognostic relevance of circulating
CD4+CD25+CD127^low^ regulatory T cells (Tregs) in 77 hepatocellular carcinoma (HCC)
patients undergoing curative hepatectomy. Using flow cytometry, the authors quantified
peripheral Tregs levels and stratified patients based on an X-tile-determined cut-off value
(5.85%). High Tregs levels were significantly associated with early recurrence, poor recurrence-
free survival (RFS), and overall survival (OS). Multivariate Cox regression confirmed Tregs
as an independent predictor of recurrence and mortality. Kaplan-Meier and subgroup analyses
further supported the prognostic role of Tregs, even in traditionally low-risk subsets (e.g.,
AFP < 400 ng/ml, absence of MVI, well-differentiated tumors). The study is methodologically
well-structured and statistically robust, with well-presented figures and comprehensive tables.
However, external generalizability is restricted due to its single-center, HBV-predominant
cohort, and some areas require clarification, including the biological interpretation of Tregs’
prognostic role and post-recurrence treatment stratification.
Major Comments
1. Scientific Rationale and Novelty
While the prognostic significance of Tregs in tumor microenvironments has been previously
explored, the authors add value by focusing specifically on preoperative circulating
CD4+CD25+CD127^low^ Tregs in HCC. The novelty lies in their use of a pre-treatment, blood-
based biomarker. However, the manuscript would benefit from deeper contextualization of how
peripheral versus intra-tumoral Tregs differentially impact HCC progression.
2. Patient Cohort and Generalizability
The inclusion/exclusion criteria are clearly defined, and surgical management appears
standardized. Yet, the cohort predominantly includes HBV-related HCC, limiting applicability
to etiologically diverse populations (e.g., HCV, NAFLD). Furthermore, while the study period
is sufficient, no external validation or multi-center recruitment is provided to support broader
clinical application.
3. Statistical Rigor and Methodology
The use of X-tile software to determine an optimal cut-off is methodologically appropriate.
Multivariate Cox regression is correctly applied and adjusted for relevant covariates. Subgroup
analyses are comprehensive, reinforcing the consistency of findings. However, the proportional
hazards assumption is not discussed, and confidence intervals for certain variables (e.g., MVI)
are wide, indicating instability in estimates.
4. Biological Interpretation and Mechanistic Insight
The study demonstrates strong statistical associations but lacks mechanistic discussion.
The dual role of Tregs in immune modulation (tumor-promoting vs. tumor-suppressive)
is acknowledged, but a deeper integration of how peripheral Tregs mechanistically reflect
intratumoral immunosuppression is lacking.
5. Post-Recurrence Treatment Analysis
Table 5 attempts to stratify outcomes by post-recurrence treatment modalities; however, the
low number of recurrent cases per subgroup (e.g., re-hepatectomy, TACE) and absence of
stratification by Tregs level within each treatment limit interpretability. The clinical implications
of Tregs in guiding treatment decisions remain underexplored.
Minor Comments
• The manuscript requires language refinement. Several grammatical inconsistencies and
awkward phrasings affect clarity.
• Several abbreviations (e.g., ALB, TB) are used inconsistently or defined late. Uniform
definition at first mention would aid readability.
• Figure legends could be more descriptive, especially in summarizing key findings from each
panel.
• The role of liver function markers (e.g., ALB, TB) in the multivariate model warrants brief
elaboration.
• Ethical approval and consent are addressed, but the declaration of funding support could be
more transparently detailed.
• Data on antiviral treatment in HBV-positive patients is not presented, despite its known
prognostic significance.
Limitations
• Single-center, retrospective design introduces selection and referral biases; prospective
validation is necessary.
• Small sample size, especially in high Tregs and subgroup analyses, may reduce statistical
power and increase risk of type II error.
• HBV predominance restricts applicability to HCV- or NAFLD-associated HCC populations.
• Lack of external validation cohort limits generalizability and clinical adoption.
• Limited mechanistic exploration of Tregs biology in peripheral blood versus tumor
microenvironment.
• Post-recurrence therapy outcomes are underpowered and not stratified by Tregs level, limiting
clinical utility in treatment stratification.
Title: An elevated percentage of CD4#CD25#CD127low regulatory T cells in peripheral blood
indicates a poorer prognosis in hepatocellular carcinoma after curative hepatectomy
Version: 2 Date: 04 Apr 2025
Reviewer's report:
This retrospective observational study investigates the prognostic relevance of circulating
CD4+CD25+CD127^low^ regulatory T cells (Tregs) in 77 hepatocellular carcinoma (HCC)
patients undergoing curative hepatectomy. Using flow cytometry, the authors quantified
peripheral Tregs levels and stratified patients based on an X-tile-determined cut-off value
(5.85%). High Tregs levels were significantly associated with early recurrence, poor recurrence-
free survival (RFS), and overall survival (OS). Multivariate Cox regression confirmed Tregs
as an independent predictor of recurrence and mortality. Kaplan-Meier and subgroup analyses
further supported the prognostic role of Tregs, even in traditionally low-risk subsets (e.g.,
AFP < 400 ng/ml, absence of MVI, well-differentiated tumors). The study is methodologically
well-structured and statistically robust, with well-presented figures and comprehensive tables.
However, external generalizability is restricted due to its single-center, HBV-predominant
cohort, and some areas require clarification, including the biological interpretation of Tregs’
prognostic role and post-recurrence treatment stratification.
Major Comments
1. Scientific Rationale and Novelty
While the prognostic significance of Tregs in tumor microenvironments has been previously
explored, the authors add value by focusing specifically on preoperative circulating
CD4+CD25+CD127^low^ Tregs in HCC. The novelty lies in their use of a pre-treatment, blood-
based biomarker. However, the manuscript would benefit from deeper contextualization of how
peripheral versus intra-tumoral Tregs differentially impact HCC progression.
2. Patient Cohort and Generalizability
The inclusion/exclusion criteria are clearly defined, and surgical management appears
standardized. Yet, the cohort predominantly includes HBV-related HCC, limiting applicability
to etiologically diverse populations (e.g., HCV, NAFLD). Furthermore, while the study period
is sufficient, no external validation or multi-center recruitment is provided to support broader
clinical application.
3. Statistical Rigor and Methodology
The use of X-tile software to determine an optimal cut-off is methodologically appropriate.
Multivariate Cox regression is correctly applied and adjusted for relevant covariates. Subgroup
analyses are comprehensive, reinforcing the consistency of findings. However, the proportional
hazards assumption is not discussed, and confidence intervals for certain variables (e.g., MVI)
are wide, indicating instability in estimates.
4. Biological Interpretation and Mechanistic Insight
The study demonstrates strong statistical associations but lacks mechanistic discussion.
The dual role of Tregs in immune modulation (tumor-promoting vs. tumor-suppressive)
is acknowledged, but a deeper integration of how peripheral Tregs mechanistically reflect
intratumoral immunosuppression is lacking.
5. Post-Recurrence Treatment Analysis
Table 5 attempts to stratify outcomes by post-recurrence treatment modalities; however, the
low number of recurrent cases per subgroup (e.g., re-hepatectomy, TACE) and absence of
stratification by Tregs level within each treatment limit interpretability. The clinical implications
of Tregs in guiding treatment decisions remain underexplored.
Minor Comments
• The manuscript requires language refinement. Several grammatical inconsistencies and
awkward phrasings affect clarity.
• Several abbreviations (e.g., ALB, TB) are used inconsistently or defined late. Uniform
definition at first mention would aid readability.
• Figure legends could be more descriptive, especially in summarizing key findings from each
panel.
• The role of liver function markers (e.g., ALB, TB) in the multivariate model warrants brief
elaboration.
• Ethical approval and consent are addressed, but the declaration of funding support could be
more transparently detailed.
• Data on antiviral treatment in HBV-positive patients is not presented, despite its known
prognostic significance.
Limitations
• Single-center, retrospective design introduces selection and referral biases; prospective
validation is necessary.
• Small sample size, especially in high Tregs and subgroup analyses, may reduce statistical
power and increase risk of type II error.
• HBV predominance restricts applicability to HCV- or NAFLD-associated HCC populations.
• Lack of external validation cohort limits generalizability and clinical adoption.
• Limited mechanistic exploration of Tregs biology in peripheral blood versus tumor
microenvironment.
• Post-recurrence therapy outcomes are underpowered and not stratified by Tregs level, limiting
clinical utility in treatment stratification.
Please review this paper.
user
Title: An elevated percentage of CD4#CD25#CD127low regulatory T cells in peripheral blood
indicates a poorer prognosis in hepatocellular carcinoma after curative hepatectomy
Version: 2 Date: 04 Apr 2025
Reviewer's report:
This retrospective observational study investigates the prognostic relevance of circulating
CD4+CD25+CD127^low^ regulatory T cells (Tregs) in 77 hepatocellular carcinoma (HCC)
patients undergoing curative hepatectomy. Using flow cytometry, the authors quantified
peripheral Tregs levels and stratified patients based on an X-tile-determined cut-off value
(5.85%). High Tregs levels were significantly associated with early recurrence, poor recurrence-
free survival (RFS), and overall survival (OS). Multivariate Cox regression confirmed Tregs
as an independent predictor of recurrence and mortality. Kaplan-Meier and subgroup analyses
further supported the prognostic role of Tregs, even in traditionally low-risk subsets (e.g.,
AFP < 400 ng/ml, absence of MVI, well-differentiated tumors). The study is methodologically
well-structured and statistically robust, with well-presented figures and comprehensive tables.
However, external generalizability is restricted due to its single-center, HBV-predominant
cohort, and some areas require clarification, including the biological interpretation of Tregs’
prognostic role and post-recurrence treatment stratification.
Major Comments
1. Scientific Rationale and Novelty
While the prognostic significance of Tregs in tumor microenvironments has been previously
explored, the authors add value by focusing specifically on preoperative circulating
CD4+CD25+CD127^low^ Tregs in HCC. The novelty lies in their use of a pre-treatment, blood-
based biomarker. However, the manuscript would benefit from deeper contextualization of how
peripheral versus intra-tumoral Tregs differentially impact HCC progression.
2. Patient Cohort and Generalizability
The inclusion/exclusion criteria are clearly defined, and surgical management appears
standardized. Yet, the cohort predominantly includes HBV-related HCC, limiting applicability
to etiologically diverse populations (e.g., HCV, NAFLD). Furthermore, while the study period
is sufficient, no external validation or multi-center recruitment is provided to support broader
clinical application.
3. Statistical Rigor and Methodology
The use of X-tile software to determine an optimal cut-off is methodologically appropriate.
Multivariate Cox regression is correctly applied and adjusted for relevant covariates. Subgroup
analyses are comprehensive, reinforcing the consistency of findings. However, the proportional
hazards assumption is not discussed, and confidence intervals for certain variables (e.g., MVI)
are wide, indicating instability in estimates.
4. Biological Interpretation and Mechanistic Insight
The study demonstrates strong statistical associations but lacks mechanistic discussion.
The dual role of Tregs in immune modulation (tumor-promoting vs. tumor-suppressive)
is acknowledged, but a deeper integration of how peripheral Tregs mechanistically reflect
intratumoral immunosuppression is lacking.
5. Post-Recurrence Treatment Analysis
Table 5 attempts to stratify outcomes by post-recurrence treatment modalities; however, the
low number of recurrent cases per subgroup (e.g., re-hepatectomy, TACE) and absence of
stratification by Tregs level within each treatment limit interpretability. The clinical implications
of Tregs in guiding treatment decisions remain underexplored.
Minor Comments
• The manuscript requires language refinement. Several grammatical inconsistencies and
awkward phrasings affect clarity.
• Several abbreviations (e.g., ALB, TB) are used inconsistently or defined late. Uniform
definition at first mention would aid readability.
• Figure legends could be more descriptive, especially in summarizing key findings from each
panel.
• The role of liver function markers (e.g., ALB, TB) in the multivariate model warrants brief
elaboration.
• Ethical approval and consent are addressed, but the declaration of funding support could be
more transparently detailed.
• Data on antiviral treatment in HBV-positive patients is not presented, despite its known
prognostic significance.
Limitations
• Single-center, retrospective design introduces selection and referral biases; prospective
validation is necessary.
• Small sample size, especially in high Tregs and subgroup analyses, may reduce statistical
power and increase risk of type II error.
• HBV predominance restricts applicability to HCV- or NAFLD-associated HCC populations.
• Lack of external validation cohort limits generalizability and clinical adoption.
• Limited mechanistic exploration of Tregs biology in peripheral blood versus tumor
microenvironment.
• Post-recurrence therapy outcomes are underpowered and not stratified by Tregs level, limiting
clinical utility in treatment stratification.
assistant