Challenges for New Adopters in Pre-Surgical Margin Assessment by Handheld Reflectance Confocal Microscope of Basal Cell Carcinoma; A Prospective Single-center Study Nina Anika Richarz1,2,3, Aram Boada 1,2,3, Ane Jaka1,2,3, Julio Bassas1,2,3, Carlos Ferrándiz1,2,3, José Manuel Carrascosa1,2,3, Oriol Yélamos 4,5 1 Dermatology Department, University Hospital Germans Trial i Pujol, Universitat Autònoma de Barcelona, Badalona, Spain. 2 Institut d’investigació Germans Trias Badalona, Barcelona, Spain. 3 Universitat Autònoma de Barcelona, Barcelona, Spain. 4 Dermatology Department, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Spain. 5 Dermatology Department, Centro Médico Teknon - Quirónsalud, Barcelona, Spain. Key words: reflectance confocal microscopy, basal cell carcinoma, micrographic Mohs surgery, margin control Citation: Richarz NA, Boada A, Jaka A, et al. UV Irradiation of Nevi: Impact on Performance of Electrical Impedance Spectroscopy and a Convolution Neural Network. Dermatol Pract Concept. 2022;12(4):e2022162. DOI: https://doi.org/10.5826/dpc.1204a162 Accepted: February 9, 2022; Published: October 2022 Copyright: ©2022 Richarz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution- NonCommercial License (BY-NC-4.0), https://creativecommons.org/licenses/by-nc/4.0/, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original authors and source are credited. Funding: None. Competing interests: None. Authorship: All authors have contributed significantly to this publication. Corresponding author: Nina A. Richarz, MD, Department of Dermatology University Hospital Germans Trias i Pujol. Address: Carretera de Canyet s/n, 08916 Badalona, Barcelona, Spain. Phone: +34 934651200, E-mail: richarznina@gmail.com ABSTRACT Introduction: In vivo reflectance confocal microscopy (RCM) is a useful tool for assessing pre-­surgical skin tumor margins when performed by a skilled, experienced user. The technique, however, poses significant challenges to novice users, particularly when a handheld RCM (HRCM) device is used. Objectives: To evaluate the performance of an HRCM device operated by a novice user to delineate basal cell carcinoma (BCC) margins before Mohs micrographic surgery (MMS). Methods: Prospective study of 17 consecutive patients with a BCC in a high-risk facial area (the H zone) in whom tumor margins were assessed by HRCM and dermoscopy before MMS. ­Predicted surgical ­defect areas (cm2) were calculated using standardized photographic digital documentation and com- pared to final defect areas after staged excision. Results: No significant differences were observed between median HRCM-predicted and observed surgical defect areas (2.95 cm2 [range: 0.83–17.52] versus 2.52 cm2 [range 0.71–14.42]; P = 0.586). Dermoscopy, by contrast, produced significantly underestimated values (median area of 1.34 cm2 Review | Dermatol Pract Concept. 2022;12(4):e2022162 1 [0.41-4.64] versus 2.52 cm2 [range 0.71-14.42]; P < 0.001). Confounders leading to poor agreement between predicted and observed areas were previous treatment (N = 5), a purely infiltrative subtype (N = 1), and abundant sebaceous hyperplasia (N = 1). Conclusions: Even in the hands of a novice user, HRCM is more accurate than dermoscopy for delin- eating lateral BCCs margins in high-risk areas and performs well at predicting final surgical defects. Introduction Methods Basal cell carcinoma (BCC) is the most common skin can- We prospectively included consecutive patients with cer and its incidence is rising worldwide due to chronic UV non-pigmented, ill-defined, biopsy-proven BCCs located in exposure and aging [1]. While the vast majority of BCCs at high-risk areas of the face treated with MMS at our der- sites such as the trunk and extremities can be removed by matology department between August 2020 and September simple excision or local destructive therapies, tumors located 2021. The study was approved by the hospital ethics com- in high-risk areas such as the H zone (central area of the mittee, and informed written consent was obtained from all face, around the eyes, nose, lips, and ears) are at greater risk participants prior to enrolment. The study was conducted of destructive local spread and recurrence. Effective surgical according to the principles of the Declaration of Helsinki. treatment is essential for guaranteeing tumor-free margins The target lesions were imaged using the Vivascope 3000 and maximal functional and cosmetic outcomes in BCCs HRCM device (MAVIG/Caliber ID), which has a horizon- that are clinically ill defined and those in high-risk areas, tal resolution of ~1 μm, optical sectioning of ~3 μm, and a with an aggressive histologic subtype (micronodular, mor- field of view of 0.75 × 0.75 mm. The images were captured pheaform, basosquamous, infiltrative), or a history of in- in vivo before surgery by an investigator (NR) with 1-year complete excision. experience who had attended an RCM course and received Mohs micrographic surgery (MMS) with rapid intraop- 2 months practical training at a reference unit. erative histologic confirmation of full tumor margins offers The clinical margins were first determined by dermos- the highest success rates in the excision of facial BCCs [2]. copy and marked on the skin using a silver paint marker MMS, however, requires advanced surgical and histopatho- (Edding 780 creative 0.8 mm, Edding International. These logic skills and support from histotechnicians with expe- markings facilitated RCM navigation and margin calculation rience in this procedure. There may also be financial and since ­silver ink can be visualized by RCM [6]. The margins resource-related obstacles. Reflectance confocal microscopy were then determined using HRCM and the original silver (RCM) might facilitate BCC margin mapping prior to MMS markings readjusted to the distance of one field of view (0.75 as it can be used to delineate lateral tumor margins that can- x 0.75) between the last inside tumor island to the internal not be determined clinically or by dermoscopy [3,4]. Studies side of the silverpen delineation. Using a method previously of the use of RCM in this setting, however, have involved described by a member of our team (OY) [12], we produced users with more than 5-years experience in image navigation standardized photographic documentation containing digi- and interpretation [3,5-9]. In addition, most of the lesions tal images of the lesions before and after dermoscopy, before investigated were located on flat, even surfaces on the face and after RCM, and during and after MMS. These images or trunk, where natural skin folds and bony prominences, were then calibrated in ImageJ (NIH, available from http:// such as those found in the H zone, do not interfere with nav- imagej.nih.gov/ij/) using anthropometric measurements and igation. With the recent approval of new Current Procedural a surgical ruler placed in the image field. The same software Terminology (CPT) codes for RCM imaging and evaluation was used to calculate surgical defect areas predicted by der- in the United States and the advent of lower-cost RCM de- moscopy and HRCM. A 3-mm margin was added to the pre- vices, the number of users is expected to increase [10,11]. dicted values as the MMS protocol at our hospital requires histologic clearance of at least 3 mm. Images of the final Objectives surgical defect were obtained before surgical reconstruc- tion and the area was re-measured to compare it with the The aim of this study was to investigate the performance of a dermoscopy- and HRCM-predicted areas. handheld RCM (HRCM) device operated by a user with 1-year The surgeons who performed MMS (AJ, JB, GC) were experience in this technique for lateral margin a­ ssessment in blinded to the dermoscopy and HRCM calculations, and, BCCs in high-risk locations in a real-life c­ linical setting. as per protocol, extended the dermoscopic margin by 3 mm 2 Review | Dermatol Pract Concept. 2022;12(4):e2022162 during the first excision stage. The excised specimen was fro- Conclusions zen and sectioned for microscopic examination of lateral and deep margins and, where necessary, the process was r­ epeated The main limitation of this study is its small sample size until achievement of full histologic clearance. (17 consecutive patients), which was partly due to the COVID-19 pandemic, as fewer operations were performed Statistical Analysis and fewer patients agreed to participate in the study due to Descriptive statistics were used to describe the characteristics fear of infection by severe acute respiratory syndrome coro- of the cohort, lesion size, and predicted and observed surgi- navirus 2. Another notable limitation is the decreased resolu- cal defect areas. Normal distribution was checked using the tion offered by RCM at depths of greater than 200-250 μm. Kolmogorov-Smirnoff test. Since most of the variables were Deep margin assessment with this technique is thus subopti- non-normally distributed, non-parametric tests were used. The mal, particularly for purely infiltrative tumors, deep tumors, Wilcoxon test was used to compare the final surgical defect and tumors located under scar tissue [13]. Finally, since this area and the areas predicted by dermoscopy and HRCM. All was a real-life bedside study involving live imaging, we were analyses were performed in SPSS version 22 (IBM corporation). unable to assess the difficulty of each case and compare the performance of the novice confocalist with that of an expert. Another limitation is that there is yet no follow-up of the Results cases available. Seventeen consecutive patients (9 men and 8 women) agreed The use of in vivo RCM imaging for the bedside diagnosis to participate in the study and underwent complete BCC ex- and histologic subtyping of BCC and other skin cancers has cision by MMS (Table 1). The median age at the time of gained popularity in the past decade [13-15]. Its usefulness HRCM examination was 70 years (range: 46–86 years). in the assessment of lateral margins has been demonstrated Thirteen tumors were located on the nose, 2 on the temple, in nodular and superficial BCCs located on flat surfaces such and 1 each on the ear and inner canthus of the eye ­(Figure 1). as the cheek, forehead, and trunk [3,5,6]. Candidates for Four patients had previously undergone conventional sur- MMS, however, usually have lesions in high-risk areas of the gery at the same site and had had positive margins or ex- face, where performance of wide-probe RCM is complicated perienced recurrence. Another 4 patients had been treated by skin elasticity and the presence of concave and convex with cryotherapy, curettage, or topical imiquimod and 2 had surfaces. HRCM, by contrast, offers advantages in uneven undergone radiotherapy. Salvage MMS was performed in locations, as it allows for free-form navigation. It also pres- one patient in whom oral vismodegib had been discontin- ents challenges, however, especially for new users working ued after 4 months due to adverse effects. Fourteen patients in real-life settings, as unlike wide-probe RCM, it does not (82.3%) had an infiltrative BCC component on histology, 2 have mosaicking capabilities. Image acquisition is therefore had a nodular/superficial subtype, and 1 had a superficial, heavily user dependent. undetermined subtype (Table 1). This prospective study analyzed the use of HRCM in the A median of 2 MMS stages (range: 1–4) were needed to assessment of BCC margins prior to MMS in clinical prac- achieve tumor-free margins after margin delineation with der- tice at a single institution. Our results suggest that, even in moscopy (Table 1). Dermoscopy underestimated the f­inal sur- the hands of novice operators and in challenging locations gical defect area by a median of 1.18 cm2 (1.34 cm2 [range: such as the nose, HRCM outperforms dermoscopy. In more 2 0.41–4.64] versus 2.52 cm [range: 0.71–14.42]; P < 0.001). complex cases, however, such as tumors previously treated There were no statistical differences between the HRCM-pre- with radiotherapy, surgery, or imiquimod, it may produce 2 dicted area and the final area (2.95 cm [range: 0.83–17.52] less accurate results due to the presence of scar tissue im- versus 2.52 cm2 [range: 0.71–14.42]; P = 0.586). HRCM, peding visualization of tumor structures. Other difficulties however, overestimated defect size in three cases and underesti- include recognition of infiltrative BCC components or BCC mated it in four (Figure 2). Of the 3 patients with overestimated mimickers, such as sebaceous hyperplasia, hair follicles, and defect areas, 1 had been previously treated by radiotherapy eccrine glands (Table 2). Novice users need to be familiar (patient #12), another had a purely infiltrative component with normal skin structures and aware of the limitations (patient #14), and another had prominent sebaceous hyper- of HCRM. We believe that image-reading challenges can plasia mistaken for tumor islands by the confocalist (patient be overcome by ensuring that training programs, in addi- #3). Of the 4 patients with underestimated defect areas, 1 had tion to focusing on pathologic characteristics of tumors, been treated with conventional surgery (patient #11), 1 with include content on normal skin structures, mimickers, and curettage (patient #16), 1 with radiotherapy (patient #9), and RCM limitations. This knowledge should shorten the learn- 1 with imiquimod (patient #5). These treatments had resulted ing curve. Rapid feedback from experienced confocalists via in scarring at different levels of the epidermis and/or dermis. image-sharing platforms in cases of doubt could also be very Review | Dermatol Pract Concept. 2022;12(4):e2022162 3 4 Table 1. Summary of demographic and BCC characteristics and comparison of surgical defect areas predicted by HRCM and dermoscopy and observed after MMS. Dermoscopy- Final predicted HRCM-predicted surgical No. of surgical defect surgical defect area defect area MMS Case Sex Age (y) BCC location BCC subtype Treatment before RCM area (cm2 ) (cm2) (cm2) stages 1 M 83 Nasal tip Nodular /infiltrative Surgery in 2012, 4.727 4.687 4.364 1 vismodegib for 4 months in 2017 2 M 42 Nasal ala Nodular/adenoid/ Surgery with positive 1.082 0.803 0.818 2 micronodular margins in 2019 3 M 86 Nose Superficial multifocal/ 2.114 4.148 2 1 nodular/infiltrative 4 M 76 Nose Superficial/nodular 2.191 3.326 3.468 3 5 M 68 Tip/dorsum of nose Superficial multifocal/ Imiquimod 3.614 2.718 3.968 2 nodular 6 F 68 Nasal ala Infiltrative/micronodular 2.725 2.947 2.874 1 7 M 84 Ear Nodular/infiltrative Cryotherapy 2.149 2.249 2.125 2 8 F 71 Nose Infiltrative 1.72 1.87 1.794 1 9 M 70 Nasal ala Infiltrative/micronodular Radiotherapy 2.612 3.261 7.647 4 10 F 46 Temple Infiltrative/superficial Surgery in 2015 6.351 8.426 8.423 2 cryotherapy, imiquimod 11 M 82 Nose Infiltrative/superficial/ Curettage + 6.559 5.379 7.124 1 nodular electro-coagulation 12 F 52 Nasal tip Superficial/undefined Radiotherapy 0.977 6.83 0.71 1 13 F 68 Nasal ala Micronodular 2.509 2.505 2.357 3 14 F 73 Temple Micronodular/infiltrative 5.13 17.519 14.416 4 15 F 78 Nasal tip Infiltrative/micronodular 1.044 1.06 1.105 3 16 M 60 Inner canthus of eye Infiltrative Surgery in 2020 1.753 1.832 2.52 1 17 F 53 Nasal ala Infiltrative 1.106 1.115 1.03 1 BCC = basal cell carcinoma; F = female; HRCM = handheld reflectance confocal microscopy; M = male; MMS = micrographic Mohs surgery. Review | Dermatol Pract Concept. 2022;12(4):e2022162 valuable. Scouting biopsies are useful in complex cases as they can help differentiate tumors from benign structures or scar tissue, especially when working at depths of greater than 200-250 µm. A summary of the above challenges and proposed solutions is given in Table 2. Although HRCM has cellular resolution, it presents some technical challenges. Navigation in the horizontal plane, for example, can be problematic due to loss of reference points and the impossibility of building an overall mosaic of the lesion. Visualization thus is restricted to a small field of vi- sion (0.75 × 0.75 mm or 1 × 1 mm depending on the gener- ation of microscope). Patient breathing and movement can also result in abrupt motion changes that can distort im- ages. Distortion can also occur when navigating skin folds or bony prominences where it is impossible to establish a flat contact (Table 2). Newer multimodal systems such as combined RCM and optical coherence tomography (OCT) and line-field confocal OCT allow for deeper tissue imaging and improved accuracy in the delineation of lateral and deep tumor margins [10,17]. The problem of reference point loss could be overcome by using in vivo wide-field imaging to Figure 1. Locations of basal cell carcinomas (red circles) guide the horizontally moving device over the skin surface included in the study. High-risk areas are shown in orange. Figure 2. Representative cases of basal cell carcinoma (BCC) showing good agreement between surgical defect areas predicted by handheld reflectance confocal microscopy (HRCM) and the defect areas after Mohs micrographic surgery (MMS) (A-D), overestimated defect areas (E-H), and underestimated defect areas (I-L). (A) Similarity between the HRCM-predicted surgical defect area (blue line) and the final area (B) in a BCC with evident tumor islands seen by RCM (C) and a nodular subtype identified in the intraoperative frozen section (D). (E) HRCM-predicted surgical defect area in a BCC previously treated with radiotherapy that was significantly larger than the final defect area after MMS (F). HRCM images of radiation-induced dermal fibrosis (G) were mistaken for collagen surrounding deep tumor islands, but these were ruled out by histology (H). The HRCM–predicted surgical defect area in (I) was significantly smaller than the final area (J) in a superficial, infiltrative BCC visible by HRCM (K) and histology (L). Review | Dermatol Pract Concept. 2022;12(4):e2022162 5 Table 2. Basal cell carcinoma margin delineation by HRCM: limitations, challenges for new users, and proposed solutions. Image-reading challenges/limitations Proposed solutions Infiltrative subtypes (no clear tumor islands or clefting but dark Training in image reading silhouettes that appear as imprints embedded in bright collagen) Scouting biopsies Combined use of OCT Platforms for consulting with RCM experts Limited penetration depth for delineating deep margins Combined use of OCT to explore vertical planes Prominent sebaceous hyperplasia, hair follicles, and eccrine glands Training in image reading that can be mistaken for tumor islands in facial BCC Scouting biopsies Platforms for consulting with RCM experts Tissue distortion due to previous biopsies and treatments Training in image reading Scouting biopsies Technical challenges associated with horizontal plane navigation Proposed solutions Loss of reference points due to small field of vision (0.75 × 0.75 mm Integration of videomosaicking algorithm into or 1 × 1 mm) and lack of automated mosaicking capabilities native HRCM software Use of in vivo wide-field imaging to guide HRCM navigation Use of a robotic arm Image distortion due to patient breathing and movement Use of a a robotic arm Artificial intelligence image modeling to restore image aberrations Loss of direct contact with HRCM device over natural skin folds Smaller-diameter HRCM lens to facilitate direct and bony prominences contact with the skin Use of a robotic arm FOV = field of view; HRCM = handheld reflectance confocal microscopy; OCT = optical coherence tomography; RCM = reflectance confocal microscopy. 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