Hotspot analysis by confocal microscopy can help to differentiate challenging melanocytic skin lesions

Raquel de Paula Ramos Castro; Juliana Casagrande Tavoloni Braga; Mariana Petaccia de Macedo; Clóvis Antonio Lopes Pinto; José Humberto Tavares Guerreiro Fregnani; Gisele Gargantini Rezze

doi:10.1371/journal.pone.0263819

Peer Review History

Original SubmissionMay 7, 2021
21 Jul 2021 Decision Letter - Nikolas K. Haass, Editor PONE-D-21-14949 A NEW REFLECTANCE CONFOCAL MICROSCOPY ALGORITHM TO DIFFERENTIATE CHALLENGING MELANOCYTIC SKIN LESIONS PLOS ONE Dear Dr. Castro, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. This manuscript is showing an interesting and relevant observation which may represent a novelty in the field: the identification of "hotspots" in RCM evaluation of melanocyte features for melanoma identification. In contrast, the development of another diagnostic algorithm does not appear to add much value to the field. In agreement with the expert reviewers, especially reviewer 1, this manuscript will benefit significantly from changing its focus to the definition and description of the new parameter ("hotspot") and its diagnostic value, positioned in the context of literature. Please submit your revised manuscript by Sep 04 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. 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Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Nikolas K. Haass, MD/PhD Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for including your ethics statement: "The study was approved by the institution’s ethical committee (01524/11). Patients included agreed to participate and signed the informed consent document. A total of 96 patients from the Cutaneous Oncology Department of AC Camargo Cancer Center, São Paulo, Brazil. The form of the consent was written.". Please amend your current ethics statement to include the full name of the ethics committee/institutional review board(s) that approved your specific study. Once you have amended this statement in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”). For additional information about PLOS ONE ethical requirements for human subjects research, please refer to http://journals.plos.org/plosone/s/submission-guidelines#loc-human-subjects-research. 3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. 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Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ Additional Editor Comments (if provided): This manuscript is showing an interesting and relevant observation which may represent a novelty in the field: the identification of "hotspots" in RCM evaluation of melanocyte features for melanoma identification. In contrast, the development of another diagnostic algorithm does not appear to add much value to the field. In agreement with the expert reviewers, especially reviewer 1, this manuscript will benefit significantly from changing its focus to the definition and description of the new parameter ("hotspot") and its diagnostic value, positioned in the context of literature. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: No Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Dear Authors the paper proposes a new algorithm for melanoma diagnosis. There are some limitations: Number of cases is little compared to recent literature. In a list of so many parameters selection of some and their combination is affected by a relevant statistical instability. The "hotspot" parameter is newly introduced and not clearly described Most of the parameters are likely to be each-other correlated (for example, if hotspot is an area where atypical cells are more densely packed, it should be assumed that there is correspondence with presence of atypical cells). Lack of distinction of subgroups of lesions (palpable/nodular vs. fact; facial vs. body ..) where different patterns are typically used for differential diagnosis I feel that the value of this paper is more in the identification of a new descriptor than in the introduction of another algorithm. Thus my recommendation is to reconsider extensively the value of the paper, concentrating on the definition and description of the new parameter ("hotspot"), making easy and clear its identification, considering situation where there are numerous cells everywhere (would you call this hotspot or not? any situation with diffused atypical cells is also presenting an hotspot?), considering relative values (a melanoma LM type is usually presenting dendritic clells infiltrating the follicles: could be this an hotspot?) Then, compare the value of "hotspot" in diagnosis of melanoma, considering this feature per se, comparing with previously proposed algorithms, and as an addition to other already used algorithms. Only if it could make sense, create your own algorithm. Reviewer #2: Authors claim that their algorithm is better than the previous ones for melanoma. However, there is no proof to that. I would suggest the authors to perform diagnostic accuracy by their expert readers on the same used in the study using Pellacani’ s and other algorithm. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". 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Revision 1
1 Sep 2021 Author Response Dear editor and reviewers, We thank for the careful revision of our article and all comments. We accepted all suggestions and made changes accordingly. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf AUTHORS: The manuscript was revised and modifications were made to meet the style requirements. 2. Thank you for including your ethics statement: "The study was approved by the institution’s ethical committee (01524/11). Patients included agreed to participate and signed the informed consent document. A total of 96 patients from the Cutaneous Oncology Department of AC Camargo Cancer Center, São Paulo, Brazil. The form of the consent was written.". Please amend your current ethics statement to include the full name of the ethics committee/institutional review board(s) that approved your specific study. Once you have amended this statement in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”). For additional information about PLOS ONE ethical requirements for human subjects research, please refer to http://journals.plos.org/plosone/s/submission-guidelines#loc-human-subjects-research. AUTHORS: The full name of the ethics committee/institutional review board(s) that approved your specific study was included at the Materials and Methods section of the manuscript: Line 104: “This retrospective study was approved by the Fundação Antônio Prudente ethical committee (01524/11) and all patients included agreed to participate and signed the informed consent document.” 3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. AUTHORS: The minimal data set underlying the results described in our manuscript were attached as supplemental files. Table 5 has the dermoscopy features of all lesions included at the study and Table 6 has the RCM features of all lesions included at the study. 4. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ AUTHORS: An ORCID iD for the corresponding author was created, and this information was included at the Title Page. Additional Editor Comments (if provided): This manuscript is showing an interesting and relevant observation which may represent a novelty in the field: the identification of "hotspots" in RCM evaluation of melanocyte features for melanoma identification. In contrast, the development of another diagnostic algorithm does not appear to add much value to the field. In agreement with the expert reviewers, especially reviewer 1, this manuscript will benefit significantly from changing its focus to the definition and description of the new parameter ("hotspot") and its diagnostic value, positioned in the context of literature. AUTHORS: We agree with the comment, and the manuscript was modified to highlight the identification and importance of "peripheral hotspots at DEJ" in RCM evaluation for melanoma identification. Also, the development of another diagnostic algorithm was excluded from the manuscript. We believe that these modifications made, the manuscript is more concise, address more directly and is focused in your main contribution for the literature. Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: No Reviewer #2: Partly AUTHORS: Modifications were made in the manuscript to facilitate replication studies. At the methods section, the “hotspot feature” is better explained and figures were included to depict our finding. Also, all results form the 110 melanocytic lesions were included as supplemental files. Lines 117-118: “Melanocytic lesions with few or faint dermoscopy features related to melanoma diagnosis and lacked enough criteria for benign lesion were termed as “doubtful melanocytic lesions”. Lines 126-136: “In superficial spreading melanoma, junctional aggregates of melanocytes are commonly found, with high shape and size variability, composed of highly atypical melanocytes and with a tendency to confluence (6). Due to the non-uniform distribution of these atypical melanocytes aggregates throughout the lesion and in order to quantify the degree and location of higher atypia inside a given lesion, hotspot analyses were included. A hotspot was defined as the 01 x 01 mm area of the lesion where the atypical cells are more aggregated, and was searched at the epidermis and DEJ levels. The selection of the hotspot was defined subjectively by the dermatologist after careful visual inspection of RCM mosaic images from the epidermis and DEJ levels, delimiting the 01 x 01 mm area with higher atypia and classifying its location as central or peripheral. The degree of atypia in a hotspot was based on the number of atypical cells inside its 01 x 01 mm area (absent, ≤ 10 or > 10 atypical cells). Illustrative cases of hotspot location and quantification are shown in figures 1 and 2”. 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes • AUTHORS: Our statistical analyses were performed by an statistician, and all results were also included as supplemental files (Tables 5 and 6). 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes AUTHORS: In order to make all data underlying our findings fully available, supplemental files were included: Table 5: Dermoscopy features of all lesions included at the study, Table 6: Confocal microscopy features of all lesions included at the study. 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No Reviewer #2: Yes AUTHORS: The manuscript text was revised and typographical or grammatical errors were corrected. 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Dear Authors The paper proposes a new algorithm for melanoma diagnosis. There are some limitations: Number of cases is little compared to recent literature. In a list of so many parameters selection of some and their combination is affected by a relevant statistical instability. The "hotspot" parameter is newly introduced and not clearly described. Most of the parameters are likely to be each-other correlated (for example, if hotspot is an area where atypical cells are more densely packed, it should be assumed that there is correspondence with presence of atypical cells). Lack of distinction of subgroups of lesions (palpable/nodular vs. fact; facial vs. body ..) where different patterns are typically used for differential diagnosis- I feel that the value of this paper is more in the identification of a new descriptor than in the introduction of another algorithm. Thus my recommendation is to reconsider extensively the value of the paper, concentrating on the definition and description of the new parameter ("hotspot"), making easy and clear its identification, considering situation where there are numerous cells everywhere (would you call this hotspot or not? any situation with diffused atypical cells is also presenting an hotspot?), considering relative values (a melanoma LM type is usually presenting dendritic clells infiltrating the follicles: could be this an hotspot?) Then, compare the value of "hotspot" in diagnosis of melanoma, considering this feature per se, comparing with previously proposed algorithms, and as an addition to other already used algorithms. Only if it could make sense, create your own algorithm. AUTHORS: We thank you for the careful revision and comments. -We agree the number of lesions included is limited and from a single institution. We decided to remove from the manuscript the development of another diagnostic algorithm and focused on the description of the RCM features found at the lesions. This limitation is highlighted at the conclusions section: Lines 250-252: “Our study included a limited number of cases from a single institution and further research is still necessary to validate to importance and impact of this RCM feature to improve melanoma diagnosis accuracy. “ -At the methods section, the “hotspot feature” is better explained and figures were included to depict this finding. Lines 126-136: “In superficial spreading melanoma, junctional aggregates of melanocytes are commonly found, with high shape and size variability, composed of highly atypical melanocytes and with a tendency to confluence (6). Due to the non-uniform distribution of these atypical melanocytes aggregates throughout the lesion and in order to quantify the degree and location of higher atypia inside a given lesion, hotspot analyses were included. A hotspot was defined as the 01 x 01 mm area of the lesion where the atypical cells are more aggregated, and was searched at the epidermis and DEJ levels. The selection of the hotspot was defined subjectively by the dermatologist after careful visual inspection of RCM mosaic images from the epidermis and DEJ levels, delimiting the 01 x 01 mm area with higher atypia and classifying its location as central or peripheral. The degree of atypia in a hotspot was based on the number of atypical cells inside its 01 x 01 mm area (absent, ≤ 10 or > 10 atypical cells). Illustrative cases of hotspot location and quantification are shown in figures 1 and 2.” -All lesions were analyzed for several RCM features, and some parameters are each-other correlated. However, only three RCM features were statistically able to distinguish benign melanocytic lesions from melanomas and the manuscript focused on their description and importance. If readers are also interested in more detail of all RCM features analyzed, we included all data as supplemental files. -Different subtypes of melanomas were excluded because they have other dermoscopy and confocal parameters. The inclusion and exclusion criteria were better explained in the methods section: Lines 107-112: “Only lesions suspicious of superficial spreading melanomas were included because other melanomas subtypes have other dermoscopy and confocal parameters (i.e. lentigo maligna, amelanotic melanoma, nodular melanomas and acral melanomas). Also, lesions located at sites where the Vivascope 1500 RCM device could not be adapted and lesions located at special sites such as the face, scalp and digits were excluded.” -We agree with the recommendations and made modifications accordingly. This made the manuscript more concise and addressed more directly its main contribution for the literature (description of the “peripheral hotspot at the DEJ” feature). This feature is also better explained, to clarify where the hotspot should be located (“where the atypical cells are more aggregated”): Lines 227-234: “The distribution of melanocytes forming aggregates is a frequently pattern found in melanocytic lesions. In benign melanocytic lesions, these aggregates are generally uniform spread throughout the lesion and constituted by non-atypical melanocytes (6). In superficial spreading melanoma, the melanocyte aggregates are haphazardly distributed, with often highly variable shape and size, constituted by atypical melanocytes, tendency to confluence and located usually at the dermal epidermal junction and / or within the mid-portion and upper levels of the epidermis (14). Our methods included the hotspot location to study the non-uniform distribution of these atypical melanocytes aggregates at the doubtful melanocytic lesions and also its degree of atypia.” -At discussion, our findings are compared to other studies and algorithms of the literature. We decided to exclude the algorithm due to the limited number of cases. Reviewer #2: Authors claim that their algorithm is better than the previous ones for melanoma. However, there is no proof to that. I would suggest the authors to perform diagnostic accuracy by their expert readers on the same used in the study using Pellacani’ s and other algorithm. AUTHORS: We thank the reviewer for the comment and decided to exclude the algorithm due to the limited number of cases. Also, at discussion, our findings are compared to other studies and algorithms of the literature. This made the manuscript more concise and addressed more directly its main contribution for the literature (description of the “peripheral hotspot at the DEJ” feature). This feature was also better detailed: Lines 126-136: “In superficial spreading melanoma, junctional aggregates of melanocytes are commonly found, with high shape and size variability, composed of highly atypical melanocytes and with a tendency to confluence (6). Due to the non-uniform distribution of these atypical melanocytes aggregates throughout the lesion and in order to quantify the degree and location of higher atypia inside a given lesion, hotspot analyses were included. A hotspot was defined as the 01 x 01 mm area of the lesion where the atypical cells are more aggregated, and was searched at the epidermis and DEJ levels. The selection of the hotspot was defined subjectively by the dermatologist after careful visual inspection of RCM mosaic images from the epidermis and DEJ levels, delimiting the 01 x 01 mm area with higher atypia and classifying its location as central or peripheral. The degree of atypia in a hotspot was based on the number of atypical cells inside its 01 x 01 mm area (absent, ≤ 10 or > 10 atypical cells). Illustrative cases of hotspot location and quantification are shown in figures 1 and 2.” Attachments Attachment Submitted filename: Answers for revision.docx https://doi.org/10.1371/journal.pone.0263819.r002
28 Jan 2022 Decision Letter - Nikolas K. Haass, Editor Hotspot analysis by confocal microscopy can help to differentiate challenging melanocytic skin lesions PONE-D-21-14949R1 Dear Dr. Castro, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Nikolas K. Haass, MD/PhD Academic Editor PLOS ONE Additional Editor Comments (optional): The authors have addressed all comments. Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: All requests have been adequately addressed. No further comments or anything to add. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No https://doi.org/10.1371/journal.pone.0263819.r003
Formally Accepted
4 Feb 2022 Acceptance Letter - Nikolas K. Haass, Editor PONE-D-21-14949R1 Hotspot analysis by confocal microscopy can help to differentiate challenging melanocytic skin lesions Dear Dr. Castro: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Prof Nikolas K. Haass Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0263819.r004

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