Quantitative Immunomorphological Analysis of Heat Shock Proteins in Thyroid Follicular Adenoma and Carcinoma Tissues Reveals Their Potential for Differential Diagnosis and Points to a Role in Carcinogenesis

Round 1

Reviewer 1 Report

This is a study that uses HSP as a marker to distinguish benign and malignant thyroid tumors. This topic is very interesting. However, the number of cases used in the study is somewhat small, and numerical analysis alone may not be reliable. It is not enough to compare the number of positive cells by immunostaining. It is necessary to clarify the difference in immunohistological findings. Immunohistopathological(IHC) findins will support the reliability of research results. Show that IHC findings are morphologically related to the nature of cancer cells. IHC findings should be an association with cell differentiation, proliferation, and invasion.

Introduction,

Line 17: the majority of patients with these lesions are referred to surgery without a precise diagnosis.

Please provide evidence if possible. I don't think it applies at least to all facilities.

Materials and Methods, Samle collection,

Please add clinical information (age, gender, stage), etc. of the case.
When using clinical materials, it is necessary to state that the study is approved by the ethics committee of your institution.

Materials and Methods, Immunohistochemistry,

Please unify the items described in the antibody product information. The description varies depending on the type of antibody.

Line 17: clone H-300

Hsp60 is a polyclonal antibody. Therefore, H-300 is not a clone number.

Line 36: against human Hsp27

Details of the Hsp27 antibody product are unknown. The text says anti-Hsp70. . . Is this product reliable? Is it confirmed to react with human Hsp27?

Line 56: microscope

Information on manufacturing company, location, etc.

Line 58: quantify the percentage of cells

Specify the evaluation criteria for counting immunopositive cells. Whether only epithelial cells are counted, whether connective tissue cells are included, staining intensity identified as positive, whether positive nuclei are counted, whether cytoplasmic positives are counted, etc.

Results,

To add findings of immunohistochemical staining results. Add findings on the relationship between histomorphological features and immunohistochemical findings.

Discussion,

Reflect the results of immunostaining for HSPs in the discussion. The expression pattern of HSPs in tumor tissue will reflect the biological nature of the tumor. Consider the role of HSPs in the tumor tissue based on the findings of immunohistochemistry.

Author Response

Reviewer 1: Authors’ Reply

Comments and Suggestions for Authors

This is a study that uses HSP as a marker to distinguish benign and malignant thyroid tumors. This topic is very interesting. However, the number of cases used in the study is somewhat small, and numerical analysis alone may not be reliable. It is not enough to compare the number of positive cells by immunostaining. It is necessary to clarify the difference in immunohistological findings. Immunohistopathological(IHC) findins will support the reliability of research results. Show that IHC findings are morphologically related to the nature of cancer cells. IHC findings should be an association with cell differentiation, proliferation, and invasion.

Authors’ Reply: We thank the Reviewer for the positive comment on the contents of the manuscript. We have mentioned in the revised version the IHC findings where pertinent.

Introduction,

Line 17: the majority of patients with these lesions are referred to surgery without a precise diagnosis. Please provide evidence if possible. I don't think it applies at least to all facilities.

Authors’ Reply: For the differential diagnosis between follicular adenoma and follicular carcinoma there are many studies in literature about potential immunohistochemical markers, but the diagnosis remains almost exclusively histological as it is based on the infiltration of the capsule and blood vessels.

Materials and Methods, Samle collection,

Please add clinical information (age, gender, stage), etc. of the case.

Authors’ Reply: We added a table in the manuscript.

When using clinical materials, it is necessary to state that the study is approved by the ethics committee of your institution.

Authors’ Reply: The experiments related to this study were conducted as part of the study project that by the Ethics Committee of University Hospital AUOP Paolo Giaccone of Palermo (N° 05/2017 of 05/10/2017). We added this information in the manuscript.

Materials and Methods, Immunohistochemistry,

Please unify the items described in the antibody product information. The description varies depending on the type of antibody.

Authors’ Reply: Thanks for this suggestion; we modified and unified the antibody information in the revised text.

Line 17: clone H-300

Hsp60 is a polyclonal antibody. Therefore, H-300 is not a clone number.

Authors’ Reply: Sorry for this mistake. We corrected it.

Line 36: against human Hsp27

Details of the Hsp27 antibody product are unknown. The text says anti-Hsp70. . . Is this product reliable? Is it confirmed to react with human Hsp27?

Authors’ Reply: Sorry for this mistake. We corrected itt.

Line 56: microscope

Information on manufacturing company, location, etc.

Authors’ Reply: Thank you for this suggestion; we added the pertinent information.

Line 58: quantify the percentage of cells

Specify the evaluation criteria for counting immunopositive cells. Whether only epithelial cells are counted, whether connective tissue cells are included, staining intensity identified as positive, whether positive nuclei are counted, whether cytoplasmic positives are counted, etc.

Authors’ Reply: We thank the Reviewer for this comment. We have added the specifications in the revised manuscript.

Results,

To add findings of immunohistochemical staining results. Add findings on the relationship between histomorphological features and immunohistochemical findings.

Authors’ Reply: Yes, we added pertinent modifications in the revised manuscript..

Discussion,Reflect the results of immunostaining for HSPs in the discussion. The expression pattern of HSPs in tumor tissue will reflect the biological nature of the tumor. Consider the role of HSPs in the tumor tissue based on the findings of immunohistochemistry.

Authors’ Reply: We added pertinent comments in the revised manuscript, but with moderation considering that we have only immunomorphological data.

Author Response File: Author Response.pdf

Reviewer 2 Report

In this study, Pitruzzella et al. investigate the amount of the heat-shock proteins HSP 27, 60, 70 and 90 in tissue of follicular adenoma, carcinoma of the surrounding parenchyma. The experiments are well performed, and the results are clearly presented. However, in fact that the expression of these HSPs is mainly investigated, there are major questions that are not assessed in this study. 

general comments:

the correlation of the findings with other possible tumor markers as thyroid peroxidases, thyroglobulin, and galectin-3 are not investigated the functional outcome of the expression remains completely unclear: did the HSP expression correlate with the patients' outcome or staging? is the HSP expression associated with side-effects as inflammation what therapy was applied to the patients? radiation or chemo could also alter the expression profile! HSPs can act as DAMPs and are recognized by immune cells: The inflammatory cells were not stained and investigated --> possibilities of immunotherapy? HSPs are not investigated in the context of cell death: Is the up-regulation higher in faster proliferating and dying tissue, e.g., carcinoma --> cell death marker staining is needed HE and DAB staining are the only methods that are used, and no validation with another method (as FACS) was performed the selectivity of the staining? As the authors describe the carcinoma grows invasive into the surrounding tissue. How selective is your analysis?

results and their presentation:

the figure legends show +SD and not +-SD; please correct your labeling please follow for the description of your graphs your figure legends were "follicular adenoma" is abbreviated as "FA" and not as the Italian "adenoma follicoloare = AF" a clear description of the visible structures in figure 1 may increase the understanding of the images by the reader... a closeup to distinguish between carcinoma and adenoma cells would also be helpful --> what are the visible differences between both how did the authors make sure that only malignant cells were analyzed? the title says "immunomorphological analysis" but how did you asses the morphology? Is the expression of that HSPs following a morphological distribution? Core or outer tumor cell layers? results section could be extended 

discussion:

you discuss the role of HSP27 in disease while your results show no change in HSP227 expression. This is only mentioned later in the discussion "when abnormal in structure and/or quantity and/or function, and/or location, chaperones can cause disease" - how could you investigate whether your HSPs are functional or unfunctional? you mention especially HSP90 to be associated with increased invasion and proliferation --> does it correlate with, e.g. Ki-67 expression or different invasiveness in your slides? other tumor markers are not discussed immunogenic cell death and HSPs as DAMPs that are recognized by immune cells need to be discussed! 

Author Response

Reviewer 2: Authors’ Reply

Comments and Suggestions for Authors

In this study, Pitruzzella et al. investigate the amount of the heat-shock proteins HSP 27, 60, 70 and 90 in tissue of follicular adenoma, carcinoma of the surrounding parenchyma. The experiments are well performed, and the results are clearly presented. However, in fact that the expression of these HSPs is mainly investigated, there are major questions that are not assessed  in this study. 

Authors’ Reply: Thank you for the positive comments and suggestions.

general comments:

the correlation of the findings with other possible tumor markers as thyroid peroxidases, thyroglobulin, and galectin-3 are not investigated the functional outcome of the expression remains completely unclear: did the HSP expression correlate with the patients' outcome or staging? is the HSP expression associated with side-effects as inflammation what therapy was applied to the patients? radiation or chemo could also alter the expression profile! HSPs can act as DAMPs and are recognized by immune cells: The inflammatory cells were not stained and investigated --> possibilities of immunotherapy? HSPs are not investigated in the context of cell death: Is the up-regulation higher in faster proliferating and dying tissue, e.g., carcinoma --> cell death marker staining is needed HE and DAB staining are the only methods that are used, and no validation with another method (as FACS) was performed the selectivity of the staining? As the authors describe the carcinoma grows invasive into the surrounding tissue. How selective is your analysis?

Authors’ Reply: Thank you for these very insightful comments. Ours is an immunomorphological study with a view on daily practice in a clinical pathology laboratory. The studies suggested by this Reviewer are very pertinent and some of them will be part of our work as we continue this project. This will now be possible and justified since the present work indicates that HSPs do change in quantity and location: the moment has arrived for elucidating how, why, and exactly when (in relation to various other parameters) the changes occur. However, these are topics for future work, which, as said above, now we know it is justified to investigate.

results and their presentation:

the figure legends show +SD and not+-SD; please correct your labeling please follow for the description of your graphs your figure legends were "follicular adenoma" is abbreviated as "FA" and not as the Italian "adenoma follicoloare = AF" a clear description of the visible structures in figure 1 may increase the understanding of the images by the reader... a closeup to distinguish between carcinoma and adenoma cells would also be helpful --> what are the visible differences between both how did the authors make sure that only malignant cells were analyzed? the title says "immunomorphological analysis" but how did you asses the morphology? Is the expression of that HSPs following a morphological distribution? Core or outer tumor cell layers? results section could be extended. 

Authors’ Reply: Thank you for your suggestion. We, regularly, use to show the histograms with +SD to make the reader's observation easier. However we modified the figure legends as requested by this Reviewer.

For "immunomorphology" we mean the use of immunological techniques (i.e.: immunohistochemistry) to study cellular and subcellular presence and localization of molecules that can be detected by monoclonal or polyclonal antibodies. In this paper, we perfomed a research of presence and localization of HSPs in thyroid tissue specimens to identify in which cells (normal or tumoral) they are present and in which compartment (cytosol, nuclei, etc) they are localized. We modified the text to better explain what we assessed in this study.

discussion:

you discuss the role of HSP27 in disease while your results show no change in HSP227 expression. This is only mentioned later in the discussion "when abnormal in structure and/or quantity and/or function, and/or location, chaperones can cause disease" - how could you investigate whether your HSPs are functional or unfunctional? you mention especially HSP90 to be associated with increased invasion and proliferation --> does it correlate with, e.g. Ki-67 expression or different invasiveness in your slides? other tumor markers are not discussed immunogenic cell death and HSPs as DAMPs that are recognized by immune cells need to be discussed!

Authors’ Reply: Thank you for these very insightful comments. As said in one of the responses above, ours is an immunomorphological study with a view on daily practice in a clinical pathology laboratory. The questions raised by this Reviewer are very pertinent and some of them will be part of our work as we continue this project. For example, attempts will be made to elucidate if the HSPs bear post-translational modifications, if their quantitative changes are due to increased gene expression and/or other post-transcriptional mechanisms, and similar experiments. Again, as explained above, these mechanistic studies are now justified since the present work indicates that HSPs do change in quantity and location. However, these are topics beyond the scope of the present work.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The instructions are corrected appropriately.

Reviewer 2 Report

The authors have suitably addressed my comments and I recommend the manuscript for publication.