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

Some melanocytic lesions do not present enough clinical and dermoscopic features to allow ruling out a possible melanoma diagnosis. These "doubtful melanocytic lesions" pose a very common and challenging scenario in clinical practice and were selected at this study for reflectance confocal microscopy evaluation and subsequent surgical excision for histopathological diagnosis. The study included 110 lesions and three confocal features were statistically able to distinguish benign melanocytic lesions from melanomas: "peripheral hotspot at dermo-epidermal junction", "nucleated roundish cells at the dermo-epidermal junction" and "sheet of cells". The finding of a peripheral hotspot (atypical cells in 1mm2) at the DEJ is highlighted because has not been previously reported in the literature as a confocal feature related to melanomas.

Introduction

The diagnosis of melanocytic skin lesions based only on clinical and dermoscopic evaluation can be challenging, even for experienced dermatologists. Some melanocytic lesions do not present enough clinical and dermoscopic features to establish a definitive diagnosis with reliability, imposing the need for biopsy and histopathological evaluation.

The use of additional non-invasive imaging techniques, like in vivo reflectance confocal microscopy (RCM) permits a cytoarchitectural evaluation of the epidermis, the dermo-epidermal junction (DEJ) and the upper dermis. Cellular atypia and pleomorphism can also be visualized in vivo to aid the diagnosis [1]. As a result, RCM represents a sensitive and specific tool for the early detection of melanomas and other skin tumors [1].

However, only few articles in the literature have described the RCM features present in melanocytic lesions. This study aimed to describe in detail the RCM features of melanocytic lesions with a doubtful diagnosis after clinical and dermoscopic evaluation, and search for new RCM features capable to differentiate them.

Materials and methods

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. A total of 96 patients from the Cutaneous Oncology Department of AC Camargo Cancer Center, São Paulo, Brazil, with 110 doubtful clinical and dermoscopic melanocytic lesions were selected. 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.

The dermoscopic diagnostic method used was the Pattern Analysis, applying the following criteria: eccentric pigmentation, abrupt network loss, poorly defined network, enlarged/atypical pigment network, multiple brown or dark globules with irregular shape and distribution, peppering, multiple colors, negative network, blue-white veil, radial streaks, pseudopods and structureless areas [2]. 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”.

These “doubtful melanocytic lesions” were submitted to RCM examination by an experienced dermatologist using the VivaScope® 1500 confocal microscope (Lucid-Tech, Rochester, New York, USA). The examination was carried out in a step-by-step manner, with complete image documentation for subsequent analyses blinded from anatomopathological results. The RCM evaluation was based on features previously described (Table 1) [3-5].

Table 1

Description of RCM features.

RCM features	Description
Honeycomb pattern at suprabasal epidermis: Typical/Atypical	Typical: normal or “preserved” honeycomb pattern; keratinocytes are well-demarcated, “visible”. Atypical: “partial loss” of honeycomb pattern, keratinocytes demarcation are “poorly or not visible”
Atypical cells at epidermis: Presence/Absence	Atypical cells are large, bright and pleomorphic cells
Atypical cells at epidermis: Nucleated roundish/Dendritic	If present, atypical cells were classified: a) nucleated roundish cells—dark nucleus and bright cytoplasm, frequently twice the size of keratinocytes; b) dendritic cells—elongated branching structure extending from the cell body, usually present in melanocytes and Langerhans cells
Hotspot at epidermis: Presence/Absence	Hotspot was defined as the 1 x 1 mm area of the lesion that presents more atypia
Atypical cells at hotspot (epidermis): ≤ 10 or > 10	If present, hotspots were classified according to the number of atypical cells (≤ 10 or > 10 cells)
Location of hotspot at epidermis: Central/Peripheral	If present, hotspots were classified according to their predominantly location at the lesion: central or peripheral
Cobblestone pattern at basal cells: Typical/Atypical	Typical: uniform basal cells distribution; no variation in brightness or cellular outline between individual cells. Atypical: basal cells are not uniformly distributed
Papillae at DEJ: Edged/Non-edged	Edged: demarcated by a rim of bright basal cells (confluent cells). Non-edged: absence of a demarcated rim of bright cells, but separated by a series of large reflecting cells
General atypia at DEJ: Presence/Absence	Present if the normal architecture of the DEJ is partially or completely lost. Described as present if some RCM findings were noted: atypical meshwork pattern, atypical cells (dendritic or roundish cells), sheet of cells and “mitochondria-like structures”
Meshwork pattern at DEJ: Presence/Absence	Characterized by small dark holes surrounded by thickened interpapillary spaces
Meshwork pattern at DEJ: Typical/Atypical	If present, meshwork pattern was classified: a) typical—clearly thickened interpapillary spaces; b) atypical—irregular and enlarged interpapillary spaces by the presence of atypical cells
Atypical cells at DEJ: Presence/Absence	Atypical cells are large, bright and pleomorphic cells
Atypical cells at DEJ: Nucleated roundish/Dendritic	If present, atypical cells were classified: a) nucleated roundish cells—isolated round to oval refractive cells with a dark nucleus, located in the papillary dermis; b) dendritic cells—elongated dendritic cells around dermal papillae
Hotspot at DEJ: Presence/Absence	Hotspot was defined as the 1 x 1 mm area of the lesion that presents more atypia
Atypical cells at hotspot (DEJ): ≤ 10 or > 10	If present, hotspots were classified according to the number of atypical cells (≤ 10 or > 10 cells)
Location of hotspot at DEJ: Central/Peripheral	If present, hotspots were classified according to their predominantly location at the lesion: central or peripheral
Junctional nests: Presence/Absence	Oval compact cellular aggregates, bulging within the dermal papillae connected with the epidermal basal cell layer
Dense and sparse nests: Presence/Absence	Roundish nonreflecting structures with a well-demarcated border, containing isolated round to oval cells with dark nucleus and reflecting cytoplasm; sometimes presenting in a multilobate configuration
Dense (homogeneous) nests: Presence/Absence	Compact aggregates with sharp margin and similar cells in morphology and refractivity
Atypical nests: Presence/Absence	Dense and sparse nests composed by pleomorphic atypical cells
Peripheral nests: Presence/Absence	Enlarging nevus characterized by bulging junctional nests at the lesion periphery
Sheet of cells: Presence/Absence	Atypical pleomorphic melanocytes distributed in sheet-like structures
“Mitochondria-like structures”: Presence/Absence	Elongated dendritic cells crowded around dermal papillae, some of them forming bridges that resembles the mitochondrial aspect
Short interconnections: Presence/Absence	Junctional thickenings and nests surrounding the papillae
Inflammatory cells: Presence/Absence	Bright particles within the papillae
Melanophages: Presence/Absence	Plump irregularly shaped bright cells with ill-defined borders and usually no visible nucleus in single units or in clusters
Coarse collagen fibers: Presence/Absence	Collagen fibers are packed together forming a coarse web-like architecture

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 Figs 1 and 2.

Fig 1

Superficial spreading melanoma in situ.

(A): Dermoscopy shows atypical network and peppering. (B): RCM mosaic image (8.0 x 8.0 mm) at the spinous layer; visual inspection positioned the hotspot at a central location (1.0 x 1.0 mm square area is marked with yellow dashed outline and better shown in C). (C): Atypical honeycomb pattern and widespread roundish cells (yellow arrows) and dendritic cells (red arrows) in sheet of cells distribution. (D): RCM mosaic image (8.0 X 8.0 mm) at DEJ; visual inspection positioned the hotspot at a peripheral location (1.0 x 1.0 mm square area is marked with yellow dashed outline and better shown in E). (E): Non-edged papillae, roundish cells at DEJ (yellow arrows) and dendritic cells (red arrows) in sheet of cells distribution. (F): Histopathology confirms a superficial spreading melanoma in situ (H&E, original magnification x200), with disarrangement of the rete ridge and increased number of atypical melanocytes affecting the adnexae.

Fig 2

Superficial spreading melanoma.

(A): Dermoscopy shows atypical network and peppering. (B): RCM mosaic image (8.0 x 8.0 mm) at epidermis (suprabasal layer); visual inspection positioned the hotspot at a central location (1.0 x 1.0 mm square area is marked with yellow dashed outline and better shown in C). (C): Atypical cobblestone pattern. (D): RCM mosaic image (8.0 X 8.0 mm) at DEJ; visual inspection positioned the hotspot at a peripheral location (1.0 x 1.0 mm square area is marked with yellow dashed outline and better shown in E). (E): Non-edged papillae, roundish cells at DEJ (yellow arrow) and dendritic cells (red arrows). (F): Histopathology confirms a superficial spreading melanoma (H&E, original magnification x200), with Breslow = 0.55 and presence of pagetoid cells.

Subsequently, all lesions were excised for histopathological diagnosis, which was considered the gold standard for final diagnosis, and categorized into benign melanocytic lesions (common melanocytic nevi and atypical melanocytic nevi) or melanomas. Statistical analysis by simple and multiple logistic regressions were conducted using SPSS software (version 21) and p value ≤ 0.05 was considered significant.

Results

All 110 melanocytic lesions were subjected to histopathological examination, and their final diagnoses were: 31 common nevi (28%), 53 atypical (dysplastic) nevi (48%) and 26 melanomas (24%). Intense atypia was present in 3 (6%) dysplastic nevi. Among the 26 melanomas, 18 (69%) were in situ, 7 (27%) were thin (Breslow < 1 mm) and 1 (4%) had Breslow of 1.42 mm. Seven (27%) melanomas appeared from a pre-existing nevus.

The features found in the RCM examination were analyzed according to the final histopathological diagnoses (Table 2).

Table 2

Frequency of RCM features in common nevi, atypical nevi and melanomas.

RCM features	Common nevus (31)	Atypical nevus (53)	Melanoma (26)
Typical Honeycomb	31 (100%)	48 (91%)	23 (43%)
Atypical Honeycomb	0 (0%)	5 (9%)	3 (12%)
Atypical cells (nucleated roundish or dendritic cells) at epidermis	07 (23%)	32 (60%)	23 (88%)
Nucleated roundish cells at epidermis	0 (0%)	2 (4%)	4 (15%)
Dendritic cells at epidermis	6 (19%)	31 (58%)	20 (77%)
Hotspot ≤ 10 atypical cells at epidermis	6 (19%)	33 (62%)	9 (35%)
Hotspot > 10 atypical cells at epidermis	0 (0%)	15 (28%)	14 (54%)
Absent of hotspot at epidermis	25 (81%)	5 (09%)	3 (12%)
Central hotspot at epidermis	5 (16%)	44 (83%)	16 (62%)
Peripheral at epidermis	1 (3%)	05 (6%)	07 (27%)
Typical cobblestone	31 (100%)	42 (79%)	12 (46%)
Atypical cobblestone	0 (0%)	11 (21%)	14 (54%)
Edged papillae	29 (94%)	24 (45%)	6 (23%)
Non-edged papillae	2 (6%)	29 (55%)	20 (77%)
DEJ general atypia	2 (6%)	39 (74%)	26 (100%)
Absent of meshwork pattern	11 (35%)	23 (43%)	13 (50%)
Typical meshwork pattern	20 (65%)	17 (32%)	4 (15%)
Atypical meshwork pattern	0 (0%)	13 (25%)	9 (35%)
Atypical cells (nucleated roundish or dendritic cells) at DEJ	2 (6%)	29 (55%)	20 (77%)
Nucleated roundish cells at DEJ	0 (0%)	6 (11%)	2 (8%)
Dendritic cells at DEJ	2 (6%)	27 (51%)	17 (65%)
Hotspot ≤ 10 atypical cells at DEJ	2 (6%)	17 (32%)	3 (12%)
Hotspot > 10 atypical cells at DEJ	0 (0%)	12 (23%)	15 (58%)
Absent of hotspot at DEJ	29 (94%)	24 (45%)	8 (31%)
Central hotspot at DEJ	2 (6%)	20 (38%)	12 (46%)
Peripheral at DEJ	0 (0%)	8 (15%)	6 (23%)
Junctional nests	23 (74%)	47 (89%)	23 (88%)
Dense and sparse nests	11 (39%)	15 (35%)	9 (44%)
Dense (homogeneous) nests	27 (30%)	51 (40%)	25 (60%)
Atypical nests	0 (0%)	3 (10%)	6 (33%)
Peripheral nests	10 (36%)	17 (45%)	5 (28%)
Sheet of cells	0 (0%)	2 (6%)	7 (6%)
Mitochondria-like structures”	0 (0%)	6 (11%)	9 (35%)
Short interconnections	4 (13%)	16 (30%)	11 (42%)
Inflammatory cells	14 (45%)	37 (70%)	22 (85%)
Melanophages	13 (42%)	26 (49%)	16 (62%)
Melanophages isolated	13 (42%)	16 (30%)	16 (62%)
Melanophages clusters	4 (13%)	15 (28%)	09 (35%)
Coarse collagen fibers	31 (100%)	52 (98%)	25 (96%)

Statistical analyses of RCM results were performed by simple and multiple logistic regressions. Several RCM findings at the DEJ occurred simultaneously and were labeled as “DEJ general atypia” to better differentiate the groups: atypical meshwork pattern, presence of atypical cells (dendritic or roundish cells), sheet of cells and “mitochondria-like structures”.

After multiple logistic regression, the presence of three of these features remained statistically significant to differentiate benign melanocytic lesions (common and atypical melanocytic nevi) from melanomas: atypical roundish nucleated cells at DEJ (p = 0.048, 95% CI = 1.01–47.96), peripheral hotspot at DEJ (p = 0.032, 95% CI = 1.18–42.88) and sheet of cells (p = 0.04, 95% CI = 1.09–42.35) (Table 3). Only the presence of peripheral hotspot at DEJ was significantly related to melanoma diagnosis, instead predominantly central location of atypical cells at the DEJ hotspot was not.

Table 3

Simple and multiple logistic regressions comparing RCM criteria in common and atypical nevi versus melanomas.

Simple logistic regression
RCM features	Category	OR	95% CI	P value
Honeycomb	Typical or atypical pattern	2.872	0.71–11.61	0.139
Cobblestone	Typical pattern	1.0	2.86–21.00	<0.0001
Cobblestone	Atypical pattern	7.742	2.86–21.00	<0.0001
Atypical cells at epidermis	Nucleated roundish cells	7.455	1.28–43.39	0.025
Atypical cells at epidermis	Dendritic cells	4.234	1.54–11.61	0.005
Atypical cells at epidermis	Nucleated roundish cells or dendritic cells	8.846	2.47–31.73	0.001
Hotspot at epidermis	Absent	NA	NA	NA
Hotspot at epidermis	≤ 10 cells	5.423	1.35–21.81	0.017
Hotspot at epidermis	> 10 cells	19.939	4.87–81.61	<0.0001
Hotspot location at epidermis	Central	7.667	2.06–2.05	0.002
Hotspot location at epidermis	Peripheral	17.889	3.62–88.41	<0.0001
Papillae at DEJ	Non-edged papillae	5.699	2.07–15.71	0.001
DEJ general atypia	Presence	NA	NA	NA
Meshwork	Absent	NA	NA	NA
Meshwork	Typical pattern	0.283	0.08–0.95	0.041
Meshwork	Atypical pattern	1.811	0.63–5.24	0.27
Atypical cells at DEJ	Nucleated roundish cells	12.3	2.31–65.55	0.003
Atypical cells at DEJ	Dendritic cells	3.582	1.42–9.03	0.007
Atypical cells at DEJ	Nucleated roundish cells or dendritic cells	5.699	2.07–15.71	0.001
Hotspot at DEJ	Absent	NA	NA	NA
Hotspot at DEJ	≤10 cells	1.046	0.25–4.36	0.951
Hotspot at DEJ	> 10 cells	8.281	2.86–23.96	<0.0001
Hotspot location at DEJ	Central	3.682	1.32–10.24	0.012
Hotspot location at DEJ	Peripheral	5.062	1.39–18.45	0.014
Junctional nest	Presence	1.533	0.40–5.82	0.53
Dense and homogeneous nests	Presence	0.446	0.17–1.17	0.102
Dense and sparse nests	Presence	1.181	0.47–2.10	0.726
Atypical nests	Presence	8.1	1.86–35.22	0.005
Location of nests	DEJ /dermis	0.603	0.24–1.52	0.284
Location of nests	Peripheral	0.503	0.17–1.48	0.211
Sheet of cells	Presence	15.105	2.90–78.56	0.001
"Mitochondria-like structures"	Presence	6.882	2.16–21.92	0.001
Short interconnections	Presence	2.347	0.93–5.92	0.071
Inflammatory cells	Presence	3.559	1.13–11.26	0.031
Melanophages	Presence	1.846	0.75–4.54	0.181
Melanophages	Isolated	1.846	0.75–4.54	0.181
Melanophages	Clusters	1.811	0.70–4.71	0.223
Coarse collagen fibers	Presence	3.322	0.20–55.02	0.402
Multiple logistic regression *
RCM features	Category	OR	95% CI	P value
Atypical cells at DEJ	Nucleated roundish cells	6.973	1.01–47.96	0.048
Hotspot location at DEJ	Peripheral	7.106	1.18–42.88	0.032
Sheet of cells	Presence	6.792	1.09–42.35	0.04

NA = not applicable

* shows only RCM features with p ≤ 0.05.

Based on the final histological diagnosis, each different combination of these statistically significant RCM features was related to the probability of the lesion being a melanoma. The absence of these criteria confers a probability of 5.5%, while lesions that presented only one positive criterion presented a 31.5% to 37.5% probability, the lesions with two positive criteria presented 79.5% to 83.9% probability and the lesions that had three positive features presented 97.5% probability of being melanoma (Table 4).

Table 4

Probability of melanoma diagnosis, related to the presence of RCM features.

RCM features	Presence/Absence
Sheet of cells	✗	✔	✗	✗	✔	✔	✗	✔
Nucleated roundish cells at DEJ	✗	✗	✔	✗	✔	✗	✔	✔
Peripheral hotspot at DEJ	✗	✗	✗	✔	✗	✔	✔	✔
Melanoma diagnosis probability	5.5%	31.5%	33.5%	37.5%	79.5%	82.4%	83.9%	97.5%

✗ = absent, ✔ = present.

Discussion

The main advantage of RCM is increasing the sensitivity and specificity of melanoma diagnosis and differentiating them from common and atypical nevi [4, 7, 8]. “Featureless melanomas”, a term used in literature to describe melanomas without clinical and dermoscopy signs of malignancy, and melanomas originating from pre-existing nevi, which may have only focal atypia [9], might also benefit from RCM evaluation [10].

With these advantages, RCM can be used with clinical examination and dermoscopy to decrease the number of unnecessary excisions. The ratio of surgically excised benign lesions to reach a single melanoma diagnosis has been related to examination method, with 45:1 by clinical examination [7-10], 17:1 by dermoscopy [10], 4–7:1 by dermoscopy associated to digital monitoring [7-10], and 2:1 by RCM [11].

Some algorithms including RCM evaluation have been proposed in the literature to reach melanoma diagnosis and were related to different sensitivity (86.1% - 93%) and specificity (57–95.3%) [8, 12, 13]. However, the majority of these studies included several subtypes of melanomas, as invasive and amelanotic melanomas.

In our study, we decided to include only melanocytic lesions with few or faint dermoscopy features related to melanoma diagnosis and lacked enough criteria for benign lesion, termed here as “doubtful melanocytic lesions”, because they pose a very common and challenging scenario in clinical practice. Melanocytic lesions that had enough clinical and dermoscopy features to be classified with high probability of being benign or malignant were excluded because their management is already defined (follow-up or excision, respectively). As a result, our approach was highly effective to detect melanomas at their initial stage, with the majority of our melanomas identified as in situ (18 out of 26). After simple and multiple logistic regressions, three confocal features were statistically able to distinguish benign melanocytic lesions from melanomas.

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.

The finding of a peripheral hotspot (atypical cells in 1mm2) at the DEJ was revealed as a specific confocal feature for melanoma diagnosis, however with low sensitivity. On the other hand, the presence of a central hotspot was not statistically more frequent in melanomas. Central hotspots were seen in 12 melanomas (46%) and in 22 (26%) benign melanocytic lesions, while peripheral hotspots were noted in six melanomas (23%) and not in any benign melanocytic lesions. The description of this RCM feature has not been previously reported in the literature.

The detection of atypical nucleated roundish cells at the DEJ have already been reported by other studies and related to 15 times greater risk for a lesion being malignant. The presence of cells distributed in sheet-like structures disrupting the papillary architecture of the basal layer was also reported as highly specific, but with low sensitivity for melanoma diagnosis [7]. These findings were confirmed in our study: atypical nucleated roundish cells at the DEJ was present in 6 (23.08%) melanomas and in 2 (2.38%) benign lesions (p = 0.048, 95% CI = 1.01–47.96); and “sheet of cells” was present in 7 (26.92%) melanomas and in 2 (2.38%) benign lesions (p = 0.04, 95% CI = 1.09–42.35).

Conclusion

Our study is focused in RCM evaluation of doubtful melanocytic lesions and identified three confocal features statistically able to distinguish benign melanocytic lesions from melanomas. Among them, the finding of a peripheral hotspot (atypical cells in 1mm2) at the DEJ is highlighted because has not been previously reported in the literature. 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.

Dermoscopy features of all lesions included at the study.

(XLSX)

Click here for additional data file.

Confocal microscopy features of all lesions included at the study.

(XLSX)

Click here for additional data file.

21 Jul 2021

PONE-D-21-14949

A NEW REFLECTANCE CONFOCAL MICROSCOPY ALGORITHM TO DIFFERENTIATE CHALLENGING MELANOCYTIC SKIN LESIONS

PLOS ONE

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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.

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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.

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Reviewers' comments:

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Reviewer #1: No

Reviewer #2: Partly

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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.

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Reviewer #1: No

Reviewer #2: No

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1 Sep 2021

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

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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.

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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.”

Submitted filename: Answers for revision.docx

Click here for additional data file.

28 Jan 2022

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.

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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.

**********

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Reviewer #1: No

4 Feb 2022

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.

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