Spectrum of Biliary Lesions/Neoplasms in Hepatic Parenchyma with Reference to a Precursor of Small Duct-Type Intrahepatic Cholangiocarcinoma: Comprehensive Categorization into Three Groups
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
This is a good assimilation of complex differential criteria aimed at classifying subtypes of small duct intrahepatic cholangiocarcinomas. At a conceptual level, the tripartite differentiation is clear and sensible. The classification structure is introduced early, and reiterated throughout, which makes for the sort of self-reinforcement that helps a reader get through some fairly dense oncology.
Invasive growth markers present the strongest and most consistently defended discriminator, providing rational separation for traditional BLNP vs. dysplastic BLNP vs. low-grade malignant BLNP. The manuscript innovates by convincingly arguing that atypia alone is insufficient for malignancy, with key resolving criteria including structural complexity, increasing Ki-67, and evidence of molecular alterations, as exemplified in dysplastic VMCs and BAF with in situ carcinoma. These molecular alterations are discussed in the context of associative (not necessarily causative) evidence, which is a sensible hedge.
Where clarity begins to erode is at the operational boundary between categories 2 and 3, and within category 2 itself, which functions more as a spectrum than a discrete class. Features such as deep parenchymal location, multiplicity and absence of portal tracts are repeatedly cited as suggestive of neoplastic potential without definitive statistical or rational context, or any clear mechanistic basis. It is worth questioning whether these features imply progression risk instead of reactive heterogeneity.
Also, whereas strong statistical arguments are made for associativity of VMCs and BDAs in background livers of SD-iCCA, the authors seem to alternate between multiple different lines of reasoning, such as field effect, or secondary cancerization, or shared injury milieu. Is it possible to make a reasonable conjecture on whether the lesions, in pathological terms, are precursors, bystanders, or targets of cancerization? If the precise relationship can't be definitively established, this ambiguity should be stated clearly.
Finally, although a range of criterial candidates are identified, and there is qualitative distinction between invasive markers (discriminatory), cytologic atypia (secondary weight) and molecular alterations (associative), the discussion does not establish a clean hierarchy that could be implemented in practice. If the implementational basis for a classification tool still needs to be worked out, this could be stated as a future objective.
Beyond this, I have no technical or scientific reservations. I suspect that other referees with much deeper oncological backgrounds that I could identify factors that the authors might consider instead, but my more limited experience limits my comments to seeking to make a stronger story based on the criteria presented.
Finally, the manuscript is well written, and seems well on the way to publication.
Author Response
A list of revisions by a point-to-point response
The Editor and reviewers, thank you very much for nice and thoughtful inquiries, suggestions and comments. In the following responses, the comments of the Editor and reviewers are written by brown, our responses are written by blue, and the revised parts are written by red and underlined. Sentences of the original manuscript are shown by black. Revised parts are written by red and underlined, and deleted parts are not shown in the revised manuscript.
Reviewer #1
Comments and Suggestions for Authors
This is a good assimilation of complex differential criteria aimed at classifying subtypes of small duct intrahepatic cholangiocarcinomas. At a conceptual level, the tripartite differentiation is clear and sensible. The classification structure is introduced early, and reiterated throughout, which makes for the sort of self-reinforcement that helps a reader get through some fairly dense oncology.
Invasive growth markers present the strongest and most consistently defended discriminator, providing rational separation for traditional BLNP vs. dysplastic BLNP vs. low-grade malignant BLNP. The manuscript innovates by convincingly arguing that atypia alone is insufficient for malignancy, with key resolving criteria including structural complexity, increasing Ki-67, and evidence of molecular alterations, as exemplified in dysplastic VMCs and BAF with in situ carcinoma. These molecular alterations are discussed in the context of associative (not necessarily causative) evidence, which is a sensible hedge.
Our response;
Thank you for kind comments. We think these comments and suggestion do not need our answers.
Where clarity begins to erode is at the operational boundary between categories 2 and 3, and within category 2 itself, which functions more as a spectrum than a discrete class. Features such as deep parenchymal location, multiplicity and absence of portal tracts are repeatedly cited as suggestive of neoplastic potential without definitive statistical or rational context, or any clear mechanistic basis. It is worth questioning whether these features imply progression risk instead of reactive heterogeneity.
Our response;
As the reviewer pointed out, categories 1, 2 and 3 are proposed to function as a spectrum or constellation of hepatic parenchymal lesions reflecting neoplastic or malignant changes or reactive changes but not as a discrete class. Particularly, category 2 is composed of BLNP with “unusual” or “dysplastic” features. that is, some category 2 could be neoplastic, though the others could be simply “not typical” or “unusual” BLNP compared to traditional BLNP and not neoplastic. As the reviewer commented, the features characterizing category 2 do not reflect progression risk but heterogenous components or features. At the moment, there are no objective markers applicable to distinguish between them. Similarly, the boundery between category 2 and 3 could be overlapping, and again, there are no objective markers to distinctively and clearly separate category 2 and 3.
Accordingly, we newly added one para in the revised paper as follows. We also modified Figure 6 to represent the relation of these three categories, according to the comments of this reviewer.
Page 29, 2nd para:
However, these three categories were proposed to function as a spectrum or constellation of hepatic parenchymal lesions reflecting neoplastic or malignant changes or reactive changes but not as a discrete class. Particularly, unusual/dysplastic BLNP was composed of BLNP with “unusual” or “dysplastic” features. That is, while some of unusual/dysplastic BLNP could be neoplastic, the others could be simply “：typical” or “unusual” BLNP compared to traditional BLNP and not neoplastic. So, the features characterizing unusual/dysplastic BLNP do not always reflect progression risk. At the moment, there no objective markers applicable to distinguish between them. Similarly, the boundery between unusual/dysplastic BLNP and low-grade malignant BLNP could be overlapping, and again.
We modified Figure 6.
Page 33, Figure 6. Relation among “traditional biliary lesions/neoplasm in hepatic parenchyma (traditional BLNP)”, “unusual/dysplastic BLNP” and “low-grade malignant BLNP” is schematically depicted. Traditional BLNP lacks cytological atypia and does not seem to relate to small duct-type intrahepatic cholangiocarcinoma (SD-iCCA). Some of unusual/dysplatic BLNP could be overlapped with traditional BLNP. Unusual/dysplatic BLNP may be followed by the development of SD-iCCA, and may be also by the development of low-grade malignant BLNP. Some of unusual/dysplatic BLNP could be overlapped with low-grade malignant BLNP. Low-grade malignant BLNP may be early malignant and may be followed by SD-iCCA. De novo carcinogenesis from hepatic parenchyma to SD-iCCA could be present.
Also, whereas strong statistical arguments are made for associativity of VMCs and BDAs in background livers of SD-iCCA, the authors seem to alternate between multiple different lines of reasoning, such as field effect, or secondary cancerization, or shared injury milieu. Is it possible to make a reasonable conjecture on whether the lesions, in pathological terms, are precursors, bystanders, or targets of cancerization? If the precise relationship can't be definitively established, this ambiguity should be stated clearly.
Our response;
As this reviewer pointed out, there were reportedly strong statistical arguments between associativity of VMCs and BDAs in background livers of SD-iCCA. However, we thought more analyses seem necessary to conlclude this causal relationship. So, we described to alternate between multiple different lines of reasoning, such as field effect, or secondary cancerization, or shared injuries. We could not make a reasonable conjecture on whether the lesions, in pathological terms, are precursors, bystanders, or targets of cancerization and also establish definitely the precise relationship at the moment, so this ambiguity were described clearly in the revised paper.
Accordingly, we newly added two para in the revised paper.
Page 14, 4th para:
While some of dysplastic VMC may reflect precursor progression to iCCA, the other may be secondary cancerization. However, at the moment, objective marker(s) to distinguish these dysplastic VMC into either are not available, so this classification remains a future objective.
Page 23, 3.2.2., 2nd para.
As above mentioned, BDA was frequently identifiable in the background liver of SD-iCCA, and some BDA presented dysplastic changes and BRAFp.V600E mutation, suggesting that some BDA could be a precursor of SD-iCCA. However, as mentioned above, BDA itself could be heterogeneous and the diagnostic criteria of BDA itself was not unified internationally, so the exact progression processes of BDA to SD-iCCA remain to be clarified.
Finally, although a range of criterial candidates are identified, and there is qualitative distinction between invasive markers (discriminatory), cytologic atypia (secondary weight) and molecular alterations (associative), the discussion does not establish a clean hierarchy that could be implemented in practice. If the implementational basis for a classification tool still needs to be worked out, this could be stated as a future objective.
Our response;
As this reviewer pointed out, the implementational basis for a classification tool in practice still needs to be worked out, we described that this classification is comprehensive, ambitious and promising, but still lack a clear hierarchy and remain a future objective, at the moment.
Accordingly, we added the following sentences in the revised paper.
Page 33, 1st para. --- in Fig.6. This classification is comprehensive, ambitious and promising, but still lacks a clear hierarchy and remains a future objective, at the moment. The implementational basis for a classification tool in practice still needs to be worked out.
Beyond this, I have no technical or scientific reservations. I suspect that other referees with much deeper oncological backgrounds that I could identify factors that the authors might consider instead, but my more limited experience limits my comments to seeking to make a stronger story based on the criteria presented.
Our response;
Thank you for your comments. Of course, we have revised our paper similarly according to the comments and suggestions raised by other reviewer, too.
Finally, the manuscript is well written, and seems well on the way to publication.
Our response
Thank you again for kind encouragement.
Reviewer 2 Report
Comments and Suggestions for Authors
Nakanuma et al wrote a comprehensive and ambitious review about the spectrum of benign and borderline biliary lesions in the hepatic parenchyma and their possible relationship to small duct–type intrahepatic cholangiocarcinoma (SD-iCCA). The authors propose a novel and conceptually coherent three-tier classification that integrates morphology, clinicopathologic behavior, and emerging molecular data.
The manuscript reflects deep expertise, extensive familiarity with historical and contemporary literature, and offers original synthesis. Upon revision to modifying following comments, this review would be a valuable reference for hepatobiliary pathologists and researchers.
- The unusual/dysplastic BLNP may represent SD-iCCA precursors, much of the evidence remains associative rather than causative. Authors should more explicitly distinguish hypothesis-generating observations from established precursor–carcinoma sequences. Author should add a paragraph provide some supporting information (morphologic continuity, molecular overlap etc) and missing information (longitudinal studies etc)
- Authors did not distinctly resolve alternate mechanism of dysplastic VMCs. They should provide a schematic representation distinguishing true precursor progression and secondary cancerization.
- Please add a schematic figure of three BLNP categories.
- Authors should be consistent about standard use, such as AI-TCC vs AL-TCC.
- Some citations appear incomplete.
- In table headings, authors should use abbreviations instead of full text, such as in Table 3, ductal plate malformation that should be DPM.
- Some typographical grammatical corrections are required, such as duplicated words.
Comments for author File: 
Comments.pdf
Author Response
A list of revisions by a point-to-point response
The Editor and reviewers, thank you very much for nice and thoughtful inquiries, suggestions and comments. In the following responses, the comments of the Editor and reviewers are written by brown, our responses are written by blue, and the revised parts are written by red and underlined. Sentences of the original manuscript are shown by black. Revised parts are written by red and underlined, and deleted parts are not shown in the revised manuscript.
Reviewer 2
Comments and Suggestions for Authors
Nakanuma et al wrote a comprehensive and ambitious review about the spectrum of benign and borderline biliary lesions in the hepatic parenchyma and their possible relationship to small duct–type intrahepatic cholangiocarcinoma (SD-iCCA). The authors propose a novel and conceptually coherent three-tier classification that integrates morphology, clinicopathologic behavior, and emerging molecular data.
The manuscript reflects deep expertise, extensive familiarity with historical and contemporary literature, and offers original synthesis. Upon revision to modifying following comments, this review would be a valuable reference for hepatobiliary pathologists and researchers.
Our response
Thank you for thoughtful comments. We think these comments and suggestions do not need answers.
1.The unusual/dysplastic BLNP may represent SD-iCCA precursors, much of the evidence remains associative rather than causative. Authors should more explicitly distinguish hypothesis-generating observations from established precursor–carcinoma sequences. Author should add a paragraph provide some supporting information (morphologic continuity, molecular overlap etc) and missing information (longitudinal studies etc)
Our responses
Thank you for critical comments and suggestions. Accordingly, we rewrote 5.4. Dysplastic/unusual BLNC could be a precursor of SD-iCCA and berelated to Low-Grade Malignant BLNP and divided it into two para, and we provided some supporting information and missing information in precursor-carcinoma sequences in 5.4.1. as follows.
Page 32, 2nd para. 5.4.1. Dysplastic/Unusual BLNC Could Be a Precursor of SD-iCCA
While traditional BLNPs are unlikely to be followed by conventional SD-iCCAs, unusual/dysplastic BLNP, particularly VMC and BDA, may represent SD-iCCA precursors and support precursor-carcinoma sequences. For example, unusual/dysplastic BLNPs are associated with conventional SD-iCCAs to a variable frequency [6,25,59,60,91] and unusual/dysplastic VMC/BDA are occasionally observed near or at the peripheral rim of conventional SD-iCCA [22,25,26,39]. Dysplastic VMC showed in situ-like lesions [25,26,31,62], and genetic and molecular alterations suggesting neoplastic changes were reported in dysplastic VMC [37,62]. BDAs were also shown to present p.V600E mutation frequently, and such mutation was actually identifiable in occasional cases of SD-iCCA [61,69,70,74]. It is likely that such unusual/dysplastic BLNPs might have been involved in the development of conventional SD-iCCAs, suggesting the malignant transformation of these BLNP. However, no large-scale epidemiologic data are available to support the causal relationship between these unusual/dysplastic BLNPs (VMC and BDA) and SD-iCCAs, and no clinical follow up studies showing malignant transformation of unusual/dysplastic VMCs and BDAs to SD-iCCAs in the same tumors have been observed. So far, much of the evidence remains associative rather than causative, and precursor–carcinoma sequences of unusual/dysplastic BLNP have not been established. However, if precursor(s) or early malignant lesions of SD-iCCA exist, they could be found in unusual/dysplastic BLNPs. So, it is imperative to narrow down or clarify which lesion(s) of these BLNPs, particularly unusual/dysplastic VMCs or BDAs, are precursor(s).
2.Authors did not distinctly resolve alternate mechanism of dysplastic VMCs. They should provide a schematic representation distinguishing true precursor progression and secondary cancerization.
Our responses
While usual VMC is lined by a single layer of cuboidal and benign appearing and homogeneous biliary epithelia, VMC lined by hyperchromatic and variably atypical nuclear changes and stratified epithelia and some fulfilling carcinoma in situ (dysplastic VMC) (Figure 1d in the revised paper), reflecting precursor progression to CCA. However, not infrequently, atypical epithelia in VMC appeared highly malignant similar or identical to the surrounding invasive carcinoma and showed an abrupt transition to a benign-appearing lining epithelia in the same VMC, and this type of dysplastic VMC deserved to be called cancerization (Figure 1e in the revised paper). While it seems plausible that both extreme dysplastic VMC may reflect true precursor progression and secondary cancerization, respectively, a majority of dysplatstic VMC were between them and objective marker(s) to classify these dysplastic VMC into either are not available at the moment. So, it is difficult to provide a schematic representation distinguishing true precursor progression and secondary cancerization. Instead, we tried to show both extreme dysplastic VMC in figures by adding two new figures in Figure 1 and several sentences were added in the revised paper.
Figure 1. We added two figures (Fig.1c and 1e).
Figure 1 legend. hyperchromatic. HE. (c) One VMC shows variable-sized microcystic lesions and irregularly formed ducts lined by a single layer of cuboidal bening epithelia. Some of the ductules contain bile. HE. (d) The VMC shows a nodular lesion composed of irregularly formed small ducts with hyperchromatic nuclei and nuclear stratification embedded in fibrous stroma. Some ducts contain bile. HE. (e) The VMC (*) adjacent to invasive carcinoma (**) is lined by a single layer of benign biliary epithelia and also highly atypical epithelia similar to surroundinig invasive carcinoma (arrow) with an abrupt transition between them. HE.
Page 15, 4th para
While some of dysplastic VMC may reflect precursor progression to iCCA, the other could be secondary cancerization. At the moment, objective marker(s) to distinguish these dysplastic VMC into either are not available, and this classification remains a future objective.
3.Please add a schematic figure of three BLNP categories.
Our responses
According to your suggestion, we modified Figure 6 to show more clearly and schematically the relation among three BLNP categories. Some sentences were added to explain Figure 6 in the text in the revised paper.
Figure 6. We modified it.
Figure 6. Legend. Relation among “traditional biliary lesions/neoplasm in hepatic parenchyma (traditional BLNP)”, “unusual/dysplastic BLNP” and “low-grade malignant BLNP” is schematically depicted. Traditional BLNP lacks cytological atypia and does not seem to relate to small duct-type intrahepatic cholangiocarcinoma (SD-iCCA). Some of unusual/dysplatic BLNP could be overlapped with traditional BLNP. Unusual/dysplatic BLNP may be followed by the development of SD-iCCA, and may be also by the development of low-grade malignant BLNP. Some of unusual/dysplatic BLNP could be overlapped with low-grade malignant BLNP. Low-grade malignant BLNP may be early malignant and may be followed by SD-iCCA. De novo carcinogenesis from hepatic parenchyma to SD-iCCA could be present.
4.Authors should be consistent about standard use, such as AI-TCC vs AL-TCC.
Our responses
We have checked and used AL-TCC.
5.Some citations appear incomplete.
Our responses
According to your comment, we checked reference citation.
5.In table headings, authors should use abbreviations instead of full text, such as in Table 3, ductal plate malformation that should be DPM.
Our responses
According to your suggestion, we adopted abbreviations instead of full text. Please see the corrected tables in the revised paper.
6.Some typographical grammatical corrections are required, such as duplicated words.
Our responses
We checked again our paper and corrected typographical grammatical errors including duplicated words. Thank you for your comments.
Round 2
Reviewer 2 Report
Comments and Suggestions for Authors
The paper is much improved.
