CD64 Staining in Dermatofibroma: A Sensitive Marker Raising the Question of the Cell Differentiation Lineage of This Neoplasm

Round 1

Reviewer 1 Report

Review of manuscript titled “CD64 staining in dermatofibroma: a sensitive marker raising the question of line of differentiati

I thank the academic editor for giving me the opportunity to review this manuscript of which I recommend a major revision due to some defects to be corrected.on of this neoplasm”.

Please, in MDPI style, the points have been placed after the brackets. Correct.

Line 43-44: please, reformulate this sentence because it is not written in a right way. Thanks.

Line 48-49: “We have stained 47 cases of dermatofibroma CD64, CD34, CD14, CD163 and CD68 48 antibodies”: this is lack of  a word like “with”. Please, add.

Line 54: “onto” is not correct. Please, correct.

Line 59: please, formulate a sentence for the Table 1 and not only “Table 1” without any link with the text.

Line 91-94: “…; CD34 (usually negative in DF, although could be expressed in 6 to 20% of DF) [9] [2], extracellular matrix glycoprotein, strome- 92 lysin3 (usually positive in DF) and the high mobility group A (HMGA2 y 2) (positive in 93DF), apolipoprotein D (negative), nestin (negative) and CD117 (negative)”.

This sentence is confusing and written in a bad way. Please reformulate being careful to the points and the references (for example 2 before than 9).

The authors confuse factor XII and factor XIIIa. Please correct. Furthermore, CD163 cannot be said to be positive in AFX if 0% positivity is then reported. Review this very confusing part.

Finally, It would be interesting that the authors discuss about data of literature related to other variants of DF, such as granular cell DF. I suggest to add this paper:

Cazzato G, Colagrande A, Cimmino A, Marrone M, Stellacci A, Arezzo F, Lettini T, Resta L, Ingravallo G. Granular Cell Dermatofibroma: When Morphology Still Matters. Dermatopathology (Basel). 2021 Aug 13;8(3):371-375. doi: 10.3390/dermatopathology8030041. PMID: 34449582; PMCID: PMC8395898.

Author Response

RESPONSE TO REVIEWERS

REVIEWER 1

I thank the academic editor for giving me the opportunity to review this manuscript of which I recommend a major revision due to some defects to be corrected.

Please, in MDPI style, the points have been placed after the brackets. Correct.

Response: We have made this correction throughout the text.

Line 43-44: please, reformulate this sentence because it is not written in a right way. Thanks.

Response: We have corrected this sentence trying to clarify the aims of our study.

Line 48-49: “We have stained 47 cases of dermatofibroma CD64, CD34, CD14, CD163 and CD68 48 antibodies”: this is lack of a word like “with”. Please, add.

Response: We have added “with” in the proper place.

Line 54: “onto” is not correct. Please, correct.

Response: We have corrected it.

Line 59: please, formulate a sentence for the Table 1 and not only “Table 1” without any link with the text.

Response: We have included a phrase referring to the information summarized in Table 1.

Line 91-94: “…; CD34 (usually negative in DF, although could be expressed in 6 to 20% of DF) [9] [2], extracellular matrix glycoprotein, stroma- 92 lysin3 (usually positive in DF) and the high mobility group A (HMGA2 y 2) (positive in 93DF), apolipoprotein D (negative), nestin (negative) and CD117 (negative)”.

This sentence is confusing and written in a bad way. Please reformulate being careful to the points and the references (for example 2 before than 9).

Response: We have rewritten the phrase. We hope that now the meaning is clearer. We have also corrected the points and the citation of references.

The authors confuse factor XII and factor XIIIa. Please correct. Furthermore, CD163 cannot be said to be positive in AFX if 0% positivity is then reported. Review this very confusing part.

Response: Regarding FXIIa in this sentence, it was a misspelling since we were actually meaning FXIIIa. We have corrected this mistake. Regarding CD163 expression we agree with the reviewer that there is an error about expression in AFX, which is actually negative. We have corrected this error and rephrased the entire paragraph, which we believe is now clearer.

Finally, It would be interesting that the authors discuss about data of literature related to other variants of DF, such as granular cell DF. I suggest to add this paper:

Cazzato G, Colagrande A, Cimmino A, Marrone M, Stellacci A, Arezzo F, Lettini T, Resta L, Ingravallo G. Granular Cell Dermatofibroma: When Morphology Still Matters. Dermatopathology (Basel). 2021 Aug 13;8(3):371-375. doi: 10.3390/dermatopathology8030041. PMID: 34449582; PMCID: PMC8395898.

Response: We have included granular cell DF in the discussion and added the proposed article as a reference.

Reviewer 2 Report

Thank you I enjoyed reading this paper but have a couple of suggestions.

Concise, well written paper with good English and photos which deserves publication. JMP readers I feel would find it of interest and the findings are convincing. However, I feel it could be improved.

My main reservation which has been omitted, is that I remain unclear why the authors state by introduction "immunohistochemical markers such as FXIII, which stains dermal dendrocytes, is the classic one. [1]" and in later in the discussion "Dermal dendritic cells forming dermatofibroma that are factor XIIIa+ and/or CD163 have been postulated to arise in the bone marrow as monocytes and to settle after circulating in the blood [3] ", however, they have chosen not included this routine important marker in their panel for comparison. In my view, as the authors have demonstrated that CD64 staining in dermatofibroma is a sensitive marker surely readers would want also to know if CD64 mirrors or it is superior to Factor XIIIa. Lastly, the authors could also expand on the specificity, significance, nature and cell identification of CD64  in relation with M2 macrophage differentiation in skin.

Author Response

REVIEWER 2

Thank you I enjoyed reading this paper but have a couple of suggestions.

Concise, well written paper with good English and photos which deserves publication. JMP readers I feel would find it of interest and the findings are convincing. However, I feel it could be improved.

My main reservation which has been omitted, is that I remain unclear why the authors state by introduction "immunohistochemical markers such as FXIIIa, which stains dermal dendrocytes, is the classic one. [1]" and in later in the discussion "Dermal dendritic cells forming dermatofibroma that are factor XIIIa+ and/or CD163 have been postulated to arise in the bone marrow as monocytes and to settle after circulating in the blood [3] ", however, they have chosen not included this routine important marker in their panel for comparison. In my view, as the authors have demonstrated that CD64 staining in dermatofibroma is a sensitive marker surely readers would want also to know if CD64 mirrors or it is superior to Factor XIIIa. Lastly, the authors could also expand on the specificity, significance, nature and cell identification of CD64 in relation with M2 macrophage differentiation in skin.

Response. Thanks a lot for your kind comments. We agree with you, we have not compared in this subset of cases both staining, but in our experience CD64 is as good as Factor XIIIa. We consider it better, as the immunostaining in our lab is cleaner. We decided not to include FXIIIa as we wanted to determine cell lineage differentiation of DF, and FXIIIa is not a specific marker of monocytes or myeloid cells despite being useful in diagnosis to distinguish between DF and DFSP, mostly when combined with CD34.  Therefore, Factor XIIIa staining was not performed in all the cases included in this study, thereby precluding a detailed comparison with CD64. Nevertheless, we have added to the manuscript our impression on this comparison based on the few cases stained with factor XIIIa.

Regarding significance of CD64, CD163 and macrophage differentiation, we have included additional comments in the discussion in order to clarify this point.

We hope that the included sentences will make it clearer to the readers.

Reviewer 3 Report

This is an immunohistochemical study that evaluates several histiocytic markers in a series of dermatofibromas (DFs).

The finding of expression of CD64 in DFs appears to be novel and therefore merits publication.

Overall, I believe the paper would benefit from an improvement of readability and weeding out of typos.

Specific comments:

1. In the Materials and Methods section, the methodology of immunohistochemistry is described in detail up to the step of incubation with the antibody, but the remainder of the procedure is not addressed at all. What was the readout, e.g., autostainer, commercial kit, etc.

2. In the legend to Figure 1, “HE x10” and “HE x20” appear to give the magnifications of the microscope objectives? What should be given are the overall magnifications (probably 20x first row, 100x second row and and 200x third row?).

3. In the discussion (lines 118 to 129), the authors try to identify a cell of origin for DFs based on the immunohistochemical phenotype reported in Table 2. They focus on an immature monocyte-lineage cell type because references 12 and 13 show that this type expresses CD64. To this reviewer, what seems more important is that CD64 is used as a marker for M1 polarization and CD163 as a marker for M2 polarization of macrophages.  Because Table 1 shows that much more DFs are positive for CD64 than for CD163, could that support a macrophage M1 phenotype for DFs?  In some sentences the authors mention M1 and M2, but that concept should be stated more clearly and be supported with pertinent references.

Author Response

REVIEWER 3

This is an immunohistochemical study that evaluates several histiocytic markers in a series of dermatofibromas (DFs).

The finding of expression of CD64 in DFs appears to be novel and therefore merits publication.

Overall, I believe the paper would benefit from an improvement of readability and weeding out of typos.

Response: Thanks a lot for your kind words. We have reviewed the English, correcting typos and making the text clearer.

Specific comments:

1. In the Materials and Methods section, the methodology of immunohistochemistry is described in detail up to the step of incubation with the antibody, but the remainder of the procedure is not addressed at all. What was the readout, e.g., autostainer, commercial kit, etc.

Response: We have added the information on how the immunostainings were processed after incubation with antibodies.

2. In the legend to Figure 1, “HE x10” and “HE x20” appear to give the magnifications of the microscope objectives? What should be given are the overall magnifications (probably 20x first row, 100x second row and 200x third row?).

Response: We agree with the reviewer on this. We have changed the legend to figure 1 to include de overall magnification of the images as indicated.

3. In the discussion (lines 118 to 129), the authors try to identify a cell of origin for DFs based on the immunohistochemical phenotype reported in Table 2. They focus on an immature monocyte-lineage cell type because references 12 and 13 show that this type expresses CD64. To this reviewer, what seems more important is that CD64 is used as a marker for M1 polarization and CD163 as a marker for M2 polarization of macrophages.  Because Table 1 shows that much more DFs are positive for CD64 than for CD163, could that support a macrophage M1 phenotype for DFs?  In some sentences the authors mention M1 and M2, but that concept should be stated more clearly and be supported with pertinent references.

Response: We have modified the discussion to make it clearer that our immunostaining results point towards an M1 proinflammatory macrophage phenotype as predominant in DF. We have clarified the concept of M1 and M2 phenotypes adding pertinent references. We completely agree with the reviewer that it is worth highlighting that CD64 supports M1 polarization while CD163 supports M2 polarization and DF seems to be predominantly M1 polarized based on our immunohistochemical study. Thanks a lot for such valuable comments.

Round 2

Reviewer 1 Report

The authors addressed all my suggestion. The manuscript is worthy to be published.

Author Response

Thanks a lot for your kind words.

Reviewer 3 Report

The paper has been significantly improved relative to the original version.

One "minor" thing: In the text the authors state twice that DFs were positive for CD64 in all but two cases (lines 87 and 142), but Table 2 reports that 100% of DFs were positive. I hope that can be resolved.

Author Response

All dermatofibromas were positive with CD64. All of them diffusely and intense but two, positive with CD64 but in a focal distribution (cases 33 and 3). We have added this information to clarify it in the text.