Primary Biliary Cholangitis—The Changing Biomarker Paradigms for Staging Fibrosis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript provides a comprehensive and well-structured review of evolving biomarkers for staging fibrosis in primary biliary cholangitis (PBC). It effectively covers the transition from traditional histopathological staging to contemporary non-invasive approaches, including serum-based, genetic, and imaging biomarkers. The review is clinically relevant and serves as a valuable resource for clinicians and researchers. However, its originality is somewhat limited, and several sections require clarification, expansion, and refinement.

 

1. While the synthesis of existing knowledge is thorough, the review lacks a deep exploration of very recent advances (e.g., studies from 2023-2025) and emerging biomarker classes (e.g., proteomic signatures, microRNAs, extracellular vesicles).
2. The statement that "none of the non-invasive tests fulfill the prerequisites to be the new gold standard as defined by the FDA" is made without specifying what these FDA-defined prerequisites are. This weakens the argument.
3. The authors mentioned biomarker, the supramolecular method should be introduced. Here are some references: Exploration, 2023, 3, 20230070; Coord. Chem. Rev. 2024, 517, 216054.  
4. The conclusion summarizes the present state but does not sufficiently articulate the specific, pressing challenges or prioritized research questions for the future.
5. There are too few figures, which is not suitable for a review article, and the layout is also poor.

 

 

Author Response

This manuscript provides a comprehensive and well-structured review of evolving biomarkers for staging fibrosis in primary biliary cholangitis (PBC). It effectively covers the transition from traditional histopathological staging to contemporary non-invasive approaches, including serum-based, genetic, and imaging biomarkers. The review is clinically relevant and serves as a valuable resource for clinicians and researchers. However, its originality is somewhat limited, and several sections require clarification, expansion, and refinement. Thank you for the comments and feedback. I have penciled in my responses in red font to make it easier to follow.

1. While the synthesis of existing knowledge is thorough, the review lacks a deep exploration of very recent advances (e.g., studies from 2023-2025) and emerging biomarker classes (e.g., proteomic signatures, microRNAs, extracellular vesicles). I completely agree. New sections covering these topics have now been added: Section 8.2 Metabolic reprogramming in primary biliary cholangitis: includes proteomics and lipidomics 3 Non-coding RNAs in primary biliary cirrhosis: includes microRNAs and extracellular vesicles.

To the initial 74 citations, 25 more have been added. Of these, 38 are now from 2023-2026.

2. The statement that "none of the non-invasive tests fulfill the prerequisites to be the new gold standard as defined by the FDA" is made without specifying what these FDA-defined prerequisites are. This weakens the argument. Section 11: The manuscript has been expanded to include the prerequisites.According to the US Food and Drug Administration (FDA), to replace liver histology as a surrogate endpoint in clinical trials, a number of guiding principles have to be fulfilled before a NIT can be ratified for that specific purpose. First prerequisite pertains to the context of utilization, which defines the intended use of the NIT and the decisions that will emanate from the results i.e. the biomarker qualification pathway in which the FDA approves the use of the NIT.   In this particular case, the intended use would be to replace liver biopsy in the assessment of fibrosis for diagnosis, disease monitoring, treatment response and prognostic purposes.  The second important determinant of the NIT is the analytical robustness of the tool or assay to be used, particularly with respect to accuracy and reproducibility.  The third key component is clinical performance i.e. the ability of the NIT to accurately reflect a biological phenomenon, e.g. fibrosis. Lastly, the NIT should demonstrate clinical utility i.e. the benefit to the patient in terms of outperforming what is currently available.   

 

3. The authors mentioned biomarker, thesupramolecular method should be introduced. Here are some references: Exploration, 2023, 3, 20230070; Coord. Chem. Rev. 2024, 517, 216054. Thank you for the citations. This has been introduced under a new section (Section 10: Future directions).
4. The conclusion summarizes the present state but does not sufficiently articulate the specific, pressing challenges or prioritized research questions for the future. This is now covered under a new section (Section 10: Future directions).

 

5. There are too few figures, which is not suitable for a review article, and the layout is also poor. Three diagrams have been added to the illustrations. When I transport the figures to the Manuscript template, they appear to be slightly off-centre. I'm not sure how to fix that. The photomicrographs are from real people, and they are trying to convey specific points of discussion.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

In this review manuscript, the Authors aimed to address the clinically relevant topic of biomarkers to assess and stage fibrosis in primary biliary cholangitis (PBC). The manuscript discusses literature data about cholangiocyte pathophysiology, serum autoantibodies, and treatment response. Importantly, the role of liver biopsy and the histological classification system, as well as non-invasive Liver Disease Assessments, including genetic and imaging biomarkers, are also addressed. Finally, variants of autoimmune liver diseases have been discussed. The manuscript addresses a clinically relevant topic; however, in my opinion, it could be improved by addressing important issues: 1) Serum autoantibodies: although more than 90% of PBC patients are AMA positive, as they stated, a significant number of patients also exhibit antinuclear antibodies (ANA). The authors could add more specifically the important role of ANA in PBC, as they act as surrogate, non-invasive diagnostic markers of PBC when AMA are lacking, they also may provide prognostic information, as anti-gp210 and anti-centromere antibodies are associated with two different disease progression patterns evolving toward cirrhosis and portal hypertension, respectively, as comprehensively discussed (DOI: 10.1586/erm.11.82). Importantly, the ANA positivity due to anti-double-stranded (ds-DNA) antibodies has been reported as a specific serological marker of the overlap syndrome AIH-PBC.

-Overlap and associated disease: the authors should recall the importance of the Pris criteria for the diagnosis of AIH-èBC overlap, and the not-rare association of PBC with celiac disease (DOI: 10.1111/j.1572-0241.2002.06031.x)

 

Author Response

Thank you for the comments and feedback. I have penciled in my responses in red font to make it easier to follow.

In this review manuscript, the Authors aimed to address the clinically relevant topic of biomarkers to assess and stage fibrosis in primary biliary cholangitis (PBC). The manuscript discusses literature data about cholangiocyte pathophysiology, serum autoantibodies, and treatment response. Importantly, the role of liver biopsy and the histological classification system, as well as non-invasive Liver Disease Assessments, including genetic and imaging biomarkers, are also addressed. Finally, variants of autoimmune liver diseases have been discussed. The manuscript addresses a clinically relevant topic; however, in my opinion, it could be improved by addressing important issues: 1) Serum autoantibodies: although more than 90% of PBC patients are AMA positive, as they stated, a significant number of patients also exhibit antinuclear antibodies (ANA). The authors could add more specifically the important role of ANA in PBC, as they act as surrogate, non-invasive diagnostic markers of PBC when AMA are lacking, they also may provide prognostic information, as anti-gp210 and anti-centromere antibodies are associated with two different disease progression patterns evolving toward cirrhosis and portal hypertension, respectively, as comprehensively discussed (DOI: 10.1586/erm.11.82). The section has been re-written to cover these points and also indicate the two different disease progression patterns. Citations have been added. Importantly, the ANA positivity due to anti-double-stranded (ds-DNA) antibodies has been reported as a specific serological marker of the overlap syndrome AIH-PBC. I agree. This has now been added to text and table 4; with citations.

-Overlap and associated disease: the authors should recall the importance of the Paris criteria for the diagnosis of AIH-èBC overlap, and the not-rare association of PBC with celiac disease (DOI: 10.1111/j.1572-0241.2002.06031.x). Both of these points (with citations) have now been added (table 4).

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled “Primary biliary cholangitis – The changing biomarker paradigms for staging fibrosis” is a narrative review that aims to summarize tissue, blood-based, genetic, and imaging biomarkers for fibrosis staging and risk stratification in PBC. While the topic is clinically relevant, several issues should be addressed to improve the overall quality, clarity, and scientific rigor of the work. I recommend major revision.

Major comments

1) The manuscript is formatted as a “Review,” yet it includes IRB approval, a patient consent waiver, and data availability statements referring to raw data, which are not appropriate for a narrative review. Please remove these sections or clarify if original data were analyzed.

2) Please specify the search strategy, selection criteria, and any approach to evidence grading, which is expected even for a narrative review in a biomarker-focused paper. Without this information, the completeness and neutrality of the review cannot be adequately assessed.

3) The author must ensure that all in-text citations match the reference list throughout the manuscript. Please perform a full cross-check of citations against the bibliography. In addition, the reference formatting appears incomplete or non-standard in several places. Please standardize all references to MDPI format and add DOIs.

4) The author should consider condensing the sections on cholangiocyte pathophysiology and general autoantibodies, and reallocating space to fibrosis-relevant biomarkers, prognostic scores, longitudinal changes in biomarkers, and outcome prediction. The current emphasis is not clear for a biomarker review, is difficult to follow, and is not well aligned with the stated objective of fibrosis staging.

5) The author state risk thresholds for VCTE (e.g., <8 kPa vs >15 kPa; alternative <6.5 and >11 kPa). Please define which fibrosis stage and clinical outcome each cut-off predicts, summarize supporting diagnostic accuracy (e.g., sensitivity, specificity, AUROC) where available, and explain why cut-offs differ across studies.

6) Table 2 should be a central element of this review; however, it currently does not match its stated purpose and is not presented in a clinically usable format. The authors are encouraged to redesign Table 2 to include: each biomarker, what it predicts (e.g., F0–F2 vs F3–F4, liver-related events, mortality) included proposed cut-offs, level/quality of evidence, and key limitations or confounders.

Minor comments

1) Abstract Section, Line 9: This sentence is not accurate as written, since AMA-negative PBC occurs in approximately 5–10% of patients. Consider revising to: “Primary biliary cholangitis (PBC) is an autoimmune-mediated disease characterized by chronic, non-suppurative, small-duct lymphocytic cholangitis.”

Line 27: “Machine-learning classifiers can then be used to create decision tree algorithms.” This statement is not supported by the content as currently presented. Please revise.

2) In the Liver biopsy section, Line 138 , Table 1 is not correctly formatted; the “type of biomarker” column is not aligned with the other columns. Please revise the table layout.

3) In the Traditional histologic staging section, Lines 196–197, please add a citation to support this statement; otherwise, it should be removed or rephrased.

4) In the Newer histologic classification section, Lines 213–215: “Quintessentially…” This paragraph is difficult to follow. Please rephrase for clarity and simplify the wording.

Lines 234–236: The statement that necroinflammatory grading may identify patients who would benefit from budesonide + UDCA is not adequately supported. Please provide evidence or revise the claim.

Lines 244–246: The sentence linking NILDA safety profile to “(Table 2)” is not supported by the current Table 2 content. Please revise the cross-reference or restructure Table 2 accordingly.

5) In the Non-invasive liver disease assessments section, Lines 252–255 this paragraph mixes fibrosis NITs (FIB-4, APRI, ELF, TPR) with prognostic-survival models (UK-PBC risk score). Please separate these categories for clarity. In addition, the Bilirubin–Hyaluronic Index (BHI) requires an appropriate citation, it does not appear sufficiently supported.

6) In the Variants of autoimmune liver diseases section, Table 4 spans multiple pages and has uneven formatting (split layout, underlining/formatting artifacts). Consider simplifying and ensuring each row is complete and readable in a single view. Please also verify terminology (e.g., “idiopathic” vs “inflammatory” bowel disease) and ensure punctuation consistency.

7) The abbreviations list contains duplication and formatting issues (e.g., “EUS Endoscopic ultrasound Endoscopic ultrasound”). Please remove duplicates and standardize/alphabetize abbreviations according to journal style.

Author Response

Thank you for the comments and feedback. I have penciled in my responses in red font to make it easier to follow.

 The manuscript entitled “Primary biliary cholangitis – The changing biomarker paradigms for staging fibrosis” is a narrative review that aims to summarize tissue, blood-based, genetic, and imaging biomarkers for fibrosis staging and risk stratification in PBC. While the topic is clinically relevant, several issues should be addressed to improve the overall quality, clarity, and scientific rigor of the work. I recommend major revision.

Major comments

1) The manuscript is formatted as a “Review,” yet it includes IRB approval, a patient consent waiver, and data availability statements referring to raw data, which are not appropriate for a narrative review. Please remove these sections or clarify if original data were analyzed. The manuscript is more of a "Perspective" than a "Review" i.e. it is looking at the pros and cons of various biomarkers, and how this continues to evolve based on technical improvements and new approaches. Using the drop down menu, I have now changed the manuscript type from "Review" to "Perspective". Since the photomicrographs are from case material of real patients (anonymized, patient consent is waivered), I think that it may be appropriate to include IRB approval. However, if there is a strong sense that these sections be removed, that is fine.

2) Please specify the search strategy, selection criteria, and any approach to evidence grading, which is expected even for a narrative review in a biomarker-focused paper. Without this information, the completeness and neutrality of the review cannot be adequately assessed. This follows on from point #1 above. The article is not a "Meta-analysis" which requires a specific search strategy, selection criteria, and timeframe. The paper provides the perspective of the author who has seen the continuing evolution in the deployment of biomarkers for assessing liver fibrosis. It covers historical aspects of staging PBC, starting from the 1960s and the rationale for changes e.g. F0–F2 in traditional PBC staging has more to do with inflammation than 'fibrosis' (in contrast to the staging system for e.g. viral hepatitis where F0-F4 is purely based on fibrosis). This partly explains why the Japanese staging system has gained traction, though it is not perfect.

3) The author must ensure that all in-text citations match the reference list throughout the manuscript. Please perform a full cross-check of citations against the bibliography. In addition, the reference formatting appears incomplete or non-standard in several places. Please standardize all references to MDPI format and add DOIs. The references have now been cross-checked using Google Scholar as the search engine and new ones have been added. This provides a mechanism for cross-referencing into DOIs.

4) The author should consider condensing the sections on cholangiocyte pathophysiology and general autoantibodies, and reallocating space to fibrosis-relevant biomarkers, prognostic scores, longitudinal changes in biomarkers, and outcome prediction. The current emphasis is not clear for a biomarker review, is difficult to follow, and is not well aligned with the stated objective of fibrosis staging. The sections on cholangiocyte pathophysiology and general autoantibodies were expanded based on the initial recommendation that the 'Introduction' was too short. This (Section 2) has now been shortened (please see marked-up copy) with space has now been reallocated to fibrosis-relevant biomarkers; new sections have been added.

5) The author state risk thresholds for VCTE (e.g., <8 kPa vs >15 kPa; alternative <6.5 and >11 kPa). Please define which fibrosis stage and clinical outcome each cut-off predicts, summarize supporting diagnostic accuracy (e.g., sensitivity, specificity, AUROC) where available, and explain why cut-offs differ across studies. These risk thresholds are just examples to illustrate issues that still need to be addressed with LSMs. Different studies provide different cut-offs. The differences in cutoffs can be due to any of several factors: i) the definitions of low-, medium- and high risk can vary depending on the study e.g. F0-F2, F3, and F4 respectively. Other studies: ≤F1, F2-F3, F4 etc. ii) the cut-offs can be influenced by the type of equipment, probe size, calibration, clinical practice guidelines and training, depending on geographic location, iii) histologic staging system that is used for validation e.g. Scheuer, Ludwig, Metavir, Brunt, Kleiner or Japanese system), iv) age and gender [76-80]. There are ongoing efforts to standardize guidelines, but universally accepted guidelines have not yet been established. Section 8.5 has been revised to include these points.

6) Table 2 should be a central element of this review; however, it currently does not match its stated purpose and is not presented in a clinically usable format. The authors are encouraged to redesign Table 2 to include: each biomarker, what it predicts (e.g., F0–F2 vs F3–F4, liver-related events, mortality) included proposed cut-offs, level/quality of evidence, and key limitations or confounders. i) I think that the table format was partially lost in transferring the document to the Template. Hopefully, this can be corrected. ii) The purpose of this table is to be illustrative of the general themes pertaining to biomarkers. For similar reasons as in item #5 above, proposed cut-offs, level/quality of evidence, and limitations or confounders etc., vary depending on the study, location and practice guidelines. Collating all this information could be the basis of a very interesting study e.g. a meta-analysis. However, I think that the scope of the current study is somewhat different.

Minor comments

1) Abstract Section, Line 9: This sentence is not accurate as written, since AMA-negative PBC occurs in approximately 5–10% of patients. Consider revising to: “Primary biliary cholangitis (PBC) is an autoimmune-mediated disease characterized by chronic, non-suppurative, small-duct lymphocytic cholangitis.” Completely agree, thank you.

Line 27: “Machine-learning classifiers can then be used to create decision tree algorithms.” This statement is not supported by the content as currently presented. Please revise. This has been revised (please see 'marked-up' copy.   

2) In the Liver biopsy section, Line 138 , Table 1 is not correctly formatted; the “type of biomarker” column is not aligned with the other columns. Please revise the table layout. This has been revised

3) In the Traditional histologic staging section, Lines 196–197, please add a citation to support this statement; otherwise, it should be removed or rephrased. Citations [42-44] have now been added. Textbooks of Hepatology and Pathology also support this e.g. [Schiff L. Diseases of the Liver. 7^th ed, 1993, p378], MacSween RN, Burt AD, Portmann BC, Ishak KG, Scheuer PJ, Anthony PP. Pathology of the liver. 4^th ed 2002, p465.  

4) In the Newer histologic classification section, Lines 213–215: “Quintessentially…” This paragraph is difficult to follow. Please rephrase for clarity and simplify the wording. The lines have been rephrased for clarity as follows: A more recent histologic classification of PBC has been proposed by Japanese investigators [45-47]. The main difference from the classical systems is that it separates histologic staging from grading. In essence, the fixed lesions (fibrosis, bile duct loss and deposition of copper-binding proteins) (Figure 4) are used for staging while the necroinflammatory lesions (cholangitis activity and hepatitis activity) are used for grading.

Lines 234–236: The statement that necroinflammatory grading may identify patients who would benefit from budesonide + UDCA is not adequately supported. Please provide evidence or revise the claim. Citations (references 39, 47) have now been added. Reference 48 also supports the idea.  

 Lines 244–246: The sentence linking NILDA safety profile to “(Table 2)” is not supported by the current Table 2 content. Please revise the cross-reference or restructure Table 2 accordingly. The sentence has been revised for accuracy (Lines 273-274). Thank you.

5) In the Non-invasive liver disease assessments section, Lines 252–255 this paragraph mixes fibrosis NITs (FIB-4, APRI, ELF, TPR) with prognostic-survival models (UK-PBC risk score). Please separate these categories for clarity. This has been corrected, removing UK-PBC risk score (Lines 276-277). In addition, the Bilirubin–Hyaluronic Index (BHI) requires an appropriate citation, it does not appear sufficiently supported. Reference 3 uses BHI, but most citations use Hyaluronic Acid. Therefore, BHI has been removed.

6) In the Variants of autoimmune liver diseases section, Table 4 spans multiple pages and has uneven formatting (split layout, underlining/formatting artifacts). Consider simplifying and ensuring each row is complete and readable in a single view. We shall try to correct the formatting. Please also verify terminology (e.g., “idiopathic” vs “inflammatory” bowel disease) and ensure punctuation consistency. Idiopathic inflammatory bowel disease (IBD) is being used as the blanket term for ulcerative colitis, Crohn's disease and indeterminate colitis.

7) The abbreviations list contains duplication and formatting issues (e.g., “EUS Endoscopic ultrasound Endoscopic ultrasound”). Please remove duplicates and standardize/alphabetize abbreviations according to journal style. This has been corrected.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The author did not fully address all the questions raised by the reviewers, and it is recommended that the manuscript be revised. For example, the references for Question 3 were not cited.

Reviewer 2 Report

Comments and Suggestions for Authors

In this revised version, the raised issues have been satisfactorily addressed.