TP53 Abnormalities and MMR Preservation in 5 Cases of Proliferating Trichilemmal Tumours

Round 1

Reviewer 1 Report

This manuscript presents immunostaining of 5 proliferating trichilemmal tumours (PTTs) for p53 and four mismatch repair (MMR) enzymes, MLH1, PMS2, MSH6 and MSH2. None of the tumours exhibited abnormal expression of the MMR enzymes, while p53 expression varied in regions of the tumours, as has been demonstrated previously. Due to the low number of cases included in this study (5), these results cannot be extrapolated to represent all PTTs. Although the manuscript is very clearly written and includes a good introduction to and discussion of this tumour type, I feel that in its present form, the article does not contain sufficient new information to warrant publication at this time. The authors could consider the following questions and comments.

1. English language is generally very good, however there are a number of minor grammatical and syntax errors that require amendment. In addition, there are numerous words that have been erroneously written as hyphenated syllables (e.g. seba-ceous, isth-mus, etc). All of these require correction.
2. The rationale for assessment of MMR enzyme immunohistochemistry is not evident when it is first presented in the Abstract, and although Muir-Torre syndrome is mentioned in the second half of the Introduction, the justification for this approach is weak and largely based on current knowledge that MMR defects are evident in greater or lesser proportions in a variety of cancer types. Stronger justification for the investigators’ approach, which may involve rearrangement of information presented in the Abstract and/or Introduction would help to emphasise the important features of the study.
3. I do not feel that the approach of the investigators addresses the topic sufficiently. MMR enzyme defects do not simply involve loss of expression of MLH1/PMS2 or MSH6/MSH2. Abnormal immunohistochemical staining for these MMR enzymes requires supporting evidence from molecular studies, which the authors have not reported. In addition, microsatellite instability may be present without loss of expression of any of these four enzymes, and there are other enzymes and proteins in the DNA mismatch repair pathways that the authors have not investigated. Nor have these pathways been discussed. Finally, because only five tumours have been included in this study, it is not appropriate to propose that results are representative of this tumour type. It remains a possibility that a proportion of PTTs are associated with MMR enzyme defects. (Many of these comments would not apply if abnormal MMR enzyme expression was identified. However, as immunostaining was normal, no conclusions can be made in the absence of supporting evidence).
4. I feel that the quality of immunohistochemistry images is not suitable for publication. Focus is poor on many of the images, colours are washed out and image magnification (or image size) could be improved for some images. Size bars should also be added to immunohistochemistry figures.
5. There are several instances where comments or conclusions are overstated in relation to either the authors’ or previously published results (e.g. lines 208-210 where the authors are discussing the relationship between UVB and MMR proteins as “proved”). It is suggested that language is modified to match the number and comprehensiveness of previous studies. (Note that there are UV-specific molecular signatures that support hypotheses of UV-related tumours, however I am unable to find examples for PTTs – this may be a study for the future to further investigate this tumour type).

Author Response

Thank you very much for your effort in the review. Your comments and proposals on the manuscript are highly valued and we agree with them. We have addressed all, altogether with other reviewers´s, and we now feel the new manuscript has been considerably improved.

1. Hyphenated syllables were corrected; it was a typing error produced in the copy / paste proccess between original manuscript document and the specific template for the journal. The text has been gramatically revised and some gramatical flaws were detected. As long as the text has automac hyphefenation, I hope the new version is correct in this respect.
2. Actually there was a sentence missing in the abstract. We included it to make the abstract brief and enough informative. It is true that the justification for studying MMR and p53 is different in the abstract and the introduction; modifications have been included on both sections to better reflect it and strengthen the justification for the study.
3. We understand the reserves regarding the representativeness of the study. The mismatch repair pathway, which most clinical relevance is colorectal cancer, is currently under investigation. Although is true that many different proteins may take part on the mismatch repair pathway, the ones clinically employed for diagnostic purposes are MSH2, MSH6, MLH1 and PMS2, as long as many mutations lead to diminished (or absent) expression on these ones. This pathway can also be explored with MSI molecular technique that reveals predisposition to mutations affecting key elements MMR. MSI has an additional clinical relevance on itself when altered (MSI-H vs MSI-L), but both immunohistochemistry for MMR and MSI have very high sensitivity and specifity. An advantage of immunohistochemistry technique is that morphogy is not lost in the procedure, as is on a PCR. Our lab have considered at the moment to keep using immunohistochemistry instead of changing to a MSI platform (that was an affordable option, as we have many other molecular studies performed though different platforms). This information was stated in introduction section.
4. At last, the appreciations made in all the study applies to the small described series, please consider PTT is not frequent and literature concerning this tumor is mainly based on case reports and small series. Of course, this work is not sufficient to completely rule out the significant subset of patients related with FCCTX syndrome that do not demonstrate conventional MMR alteration.These comments have been included in the text at the end of the discussion section.
5. Figures were transferred from Power Point directly to the Word template. There was a huge loss of quality regarding the photographs; most of each single images had 27 Mb, the Power Point with combined figures had 50 Mb and the journal´s template including all figures was only 1,3 Mb. Please, trust me if I say original images are very good. The colour washing is probrably referred to the PMS2 column, and is caused by a slightly thinner tissue section on the slide. As long as the four MMR slides are serial sectioned, there was performed a subtle color correction in an attempt to improve this issue. We tried several ways to improve quality of images on the MS Word template in order to solve these issues. Figure 3 was enlarged and bars are added as requested. MS Word document size has notably increased (more than 50 Mb now) and to our eyes images are now very good regarding their quality.
6. The aforementioned affirmation was nuanced according to reviewer´s suggestion. The whole manuscript has been restructured, trying to avoid overstated affirmations.

Reviewer 2 Report

The authors Martín-Sanz et al. present an interesting case series about the underlying factors of proliferative ability of the condition of proliferating trichilemmal tumors (PTT). Although the number of studied cases is small, most available literature on this entity is based on case reports and comparably small case series. Therefore, this article may be of interest for a dermatohistopathological audience. The studied topic regarding the role of mismatch repair proteins (MMR) is pathophysiologically relevant given its major signifcance in tumor-syndroms involving tumors of sebaceaous differentiation (Muir-Torre-Syndrome).

The article includes a well written introduction and thoroughly describes materials, methods and results. The figures and images are of high quality and are coherent by themselves. The article includes important literature and includes citations on all major aspects discussed.

Major remarks:

- The discussion should be restructured and rephrased for a better apprehensibility. For example, the authors might first discuss the role of UVB, p53 and MMR proteins in general and in regard to PTT. Then they might discuss their own results in light of these considerations. The discussion should conclude with the proposed models of distinct mutation profiles in different sebacious carcinomas and a short summary.

Minor remarks:

- The article needs minor editing for English grammar and a spell check

please pay attention to consistently use past tense throughout the article (e.g. page 4 line 128: „all the studied tumors have“

further examples for minor flaws:

e.g. page 1 line 32: „accounts a 2%“

e.g. page 1 line 39 „hace differential diagnosis“

e.g. page 1 line 42 „demonstrated and intermediate“

e.g. page 2 line 67-76: the paragraph should be rephrased as it is difficult to understand the authors‘ intention

e.g. page 5 line 150 „in the panoramic view can be identified the tumor“

e.g. page 6 line 173: „we don‘t think“

e.g. page 6 line 178 „away from the head“

e.g. page 6 line 182-186: please rephrase or clarify „both have added effect reducing chemotherapy effect“

- There is contradiction regarding figure 2f: page 4 line 123 „and no granular layer“ and page 4 line 125 „a prominent granular layer“, please correct

- The title of the article should reflect the results of the study instead of raising an open question

- Figure 1 legend: please include a remark on which case is depicted in each photograph

Author Response

Thank you very much for the effort in reviewing our paper. We consider your kind comments and remarks on the manuscript really valuable and are highly appreciated. We have addressed all of them and we think the manuscript has been considerably improved thanks to reviewers´ suggestions.

1. References to morphological aspects in the discussion were removed, and discussion is now more focused on UV, p53 and MMR regarding skin tumours and PTT in particular, as proposed. Discussion of our results was jointed together for MMR and p53. They are displayed after discussing the general role of these elements, too, following the proposed scheme for discussion section. We feel discussion is more solid this way.
2. Some flaws are present in the text. We are sincerelly grateful for the help in their detection. Of course they were addressed and sometimes the language was modified.
3. Mentioned paragraph on page 2 was removed. The topic was written again. Other confusing sentences were modified.
4. The first paragraph of the results was considerably reduced, also according to other reviewer´s comments, referring to figure legend. We now specify Figure 2f illustrates the focal parakeratotic change in the right side, and conventional trichilemmal keratinisation on the left.
5. Title was changed to better correspond with the text.
6. Photographs in figures were named according to table1.

Reviewer 3 Report

The aim of this paper, entitled “Can DNA Mismatch Repair Protein Explain the Proliferative Ability of Proliferating Trichilemmal Tumours?” is to clarify the potential role of MMR and p53 in the pathogenesis of Proliferating Trichilemmal Tumours.

In view of the not yet complete knowledge of this neoplasm, the topic may be of interest. However, the work has some critical issues.

The main criticisms are as follows:

The title is redundant and should be changed.

The introduction section is excessively long and disorganized. it should be focused on the topic of trichilemmal tumor, and not cover different areas. In particular, the parts relating to Muir -Torre syndrome do not seem relevant.

Also, some parts of the discussion may be omitted, in particular those concerning the sites most frequently affected. On the other hand, the potential role of p53 should be discussed more extensively

Image 1b is not necessary. Regarding the case presented in image 1c, please provide better quality images.

The microscopic description presented in the results section is too long and must be shortened, referring to image 2 and its legend.

It is not clear if the IHC results presented and illustrated in Figure 3 refer to a single sample or were observed in all, and ifthe extent and distribution are superimposable. Please specify better. A table showing the different expression in the various samples would be useful.

There are some English and typing errors that need to be corrected

Author Response

Thank you very much for your effort reviewing the paper. Your kind indications are highly appreciated and we have addressed all of them. We feel the new manuscript has been considerably improved.

1. The title was changed to resume the whole manuscript.
2. Introduction was modified, including a more consistent justification for the study (also proposed by other reviewer). Information regarding Muir Torre syndrome and some non relevant poins were reduced and simplified.
3. Some sections of the discussion were also eliminated to focus on molecular events. Discussion regarding p53 was enhanced.
4. Image 1b was included as part of 1d. We included a closer photograph in figure 1c. We consider that previous image 1b has sense connecting the wide resection piece with the clinical image.
5. Image quality substantially worsened from the original images to the journal´s MS Word template. We have tried to maintain as much as possible the original quality in this new version: to our eyes images have been substatially improved.
6. Microscopic descriptions were reduced on the manuscript, referring as suggested to figure legends.
7. Results regarding figure 3 are superimposable to other cysts. A table was included, although it is presented with average values that undervalue the nuances of the morphological assessment.

Round 2

Reviewer 1 Report

In this amended manuscript, the authors have addressed some of the reviewers’ comments, however I feel that the content and the quality of the text is not suitable for publication. Although the authors have made some effort to improve English language, there are numerous errors in syntax that make the document difficult to read and interpret. There are also a number of inaccuracies in the text that require more careful attention. I remain of the opinion that simply performing immunohistochemical stains that do not definitively support or reject a hypothesis is insufficient for publication (i.e. the results presented cannot confirm or rule out the involvement of microsatellite instability or p53 mutations in the aetiology or growth of proliferating trichilemmal tumours (PTTs)). Some of these issues are described in greater detail below.

1. I do not feel that the amended title of the manuscript is suitable for an academic publication. The language is quite awkward and it overstates the content of the study (general conclusions about a pathological entity cannot be made on the basis of 5 cases).
2. There are numerous simple English language errors that make the document (and the Introduction in particular) difficult to read. For example, small words (‘the’, ‘a’, etc) are either missing or inserted in inappropriate places, incorrect use of ‘on’ instead of ‘in’ in many places, and other instances of unusual word usage. Grammatical errors in phrasing (e.g. ‘There have been described…’, ‘There is depicted…’, etc) also make the text difficult to read and understand. Comprehensive professional English language editing would enable the document to undergo proper review and be understood by readers.
3. The final statement in the Abstract contains English language errors and is difficult to understand. It does not seem to be supported by the literature or reflect the authors’ own conclusions. This should be amended.
4. The use of references to support statements made in the first paragraph on page 2 (lines 49-59) does not seem appropriate, Very little mutation analysis has been performed on PTTs, and results from trichilemmal cysts, other low grade skin tumours or animal/ cell line models cannot be directly applied to PTTs. General statements also cannot be legitimately made on the basis of very few specimens.
5. Immunohistochemical analysis of p53 is not sufficient to hypothesise mutation status as p53 mutations may be associated with lack of, unchanged or elevated (p53) protein expression. p53 is normally expressed in some skin cells and its expression may be induced in other cell and tissue processes such as tissue remodelling, which may be relevant in the context of tumour growth. In addition, there are ‘signature’ p53 mutations in skin cells that are highly associated with UV damage and these have not been investigated by the authors. Aneuploidy associated with loss of 17p, the chromosomal arm containing the TP53 gene similarly may not be specifically associated with elevated or reduced p53 expression. (Deletion of all TP53 copies will of course be associated with a lack of p53 expression).
6. The accuracy of some statements is questionable. For example, in lines 65-68, the authors state that MMR immunohistochemistry and MSI testing have high sensitivity and specificity for detection of Lynch syndrome. This statement is not true. These tests cannot differentiate Lynch syndrome from the more common sporadic forms of MSI – specificity is poor.
7. In line 81-82, the authors state that both MMR and p53 are associated with UV radiation damage, in particular UVB. What is their reference for this (specifically MMR and UVB)? There have been several studies in plants and using msh2 null mice, however the type of support required for such a general statement would include significant evidence of MMR enzyme abnormalities and/or evidence of MSI in UVB associated tumours.
8. I do not feel that there is justification for grouping PTTs with other skin cancers that are known to be associated with sun exposure or UV damage. Apart from the lack of molecular data indicating UV-associated DNA mutations in PTTs, clinical or epidemiological data to support this assertion, for example a higher incidence in fair-skinned/fair-haired or highly sun-exposed individuals, are lacking. In the absence of this type of evidence, discussion around this topic should be speculative rather than it being presented as an accepted fact.
9. In Table 1, the term ‘Female’ is generally used for gender, not ‘Woman’.
10. I notice that the Institutional Review Board response is ‘Not applicable’. Due to the nature of the personal and clinical information provided, I do not know why the authors assert that this is not applicable. Although it is stated that patients approved use of images, the study as a whole, and the presentation of patients’ personal information would normally trigger an institutional review. Could this please be clarified.
11. A general note: The type and scope of experiments reported in manuscripts published in the 1990s are frequently not sufficient for current publication standards due to the progress and wider availability of techniques and resources that were novel or still under development 25 years ago.

Author Response

We would like to specially recognize the effort of Reviewer 1 in the revision(s) of this manuscript. We consider the manuscript was dramatically improved thanks to the quality of the reviews and suggestions made. We addressed all the issues described. Thank you very much.

1. Title was changed to be less pretentious, to include the limitation of the study and, at the same time, to include the main conclusions. We also tried to be brief (11 words).
2. There are some errors due to neglect. We revised the small words and sentence construction. Several sentences were modified to simplify the read. We hope it is grammatically fine now.
3. Final statement in the abstract was reviewed and modified to best suit the reality of the study. We performed a FISH study of p53 indicating deletion in many cells. The whole manuscript was modified.
4. The referred paragraph introduces the molecular mechanisms related to both TC and PTT. We consider them lesions with close relationship, and for this reason TCs are introduced and discussed. We guess the mentioned use of inappropiate references refers to the PTT ones and, probably, this comment agrees with comment 11 criticizing the use of 1990s references. We take reference 18 only for the PTT, not for other low grade cutaneous tumors; I can´t see animal or cell line models in this paragraph. Very little mutation analysis has been performed on PTTs, and that´s the reason to include this “old” biobliography in introduction. It will also be discussed in the discussion section, with more recent bibliography. Mentioned general statements have been modified to introduce some degree of uncertainty.
5. We performed a FISH technique directed to p53. This way, deletion of p53 was demonstrated in a majority of cells in 4 of 5 cases. This is a quite interesting finding and we have to specially thank Reviewer 1 for the emphasis on performing additional techniques. Of course this means the addition of a new figure and various modifications in text and table. We will focus on the topic on next year, with more cases and a wider molecular analysis.
6. The referred statement is, indeed, only applicable to sensitivity. It was modified.
7. A new paragraph included to clarify the statement, with various references.
8. Our intention was to compare some UV-related tumours with PTT, but not to group these entities. They are mentioned only for reference. Anyway the text was modified in many points, as long as some affirmations are not accurate.
9. This was a typho. We have replaced woman for female. Thank you.
10. The institutional review was included. Anyway there are no patients contacted here, no patients identifiable in the figures and no risk for any patient.
11. The provided bibliography counts with 4 references prior to 1990, 11 references of the 90s decade, 15 references of the 2000 decade, 29 references of the 2010s and 15 references of the current 2020 decade. Of course articles published in the 1990s have limitations, but we think that they still have value in many aspects.  In any case older references are present for reasons including their novelty, high number of included cases, absence of more recent papers addressing a topic, etc. These references support the manuscript, are cited by others and have scientific soundness at least in the cited point. If you consider there is (or there are) references to be removed, please, could you be more precise?

Reviewer 3 Report

Author Response

Dear Reviewer 3, thank you very much for the revision performed on the manuscript. We revised again the English language. The manuscript was dramatically changed due to Reviewer 1 indications, as we performed FISH to evaluate the presence of p53 deletions, with positive results. Hope this new version is now suitable for publication. Best regards.