Dermatology: Practical and Conceptual Original Article | Dermatol Pract Concept. 2024;14(2):e2024090 1 Combining Reflectance Confocal Microscopy, Optical Coherence Tomography and Ex-Vivo Fluorescence Confocal Microscopy for Margin Assessment in Basal Cell Carcinoma Excision Simone Michelini1, Victor Desmond Mandel2, Marco Ardigò2, Silvana Ciardo3, Carlo Cota2, Anna Maria Cesinaro4, Elena Rossi3, Barbara Ferrari3, Shaniko Kaleci3, Marco Di Fraia1, Camilla Chello1, Carmen Cantisani1, Federica Trovato1, Caterina Longo3,5, Giovanni Pellacani1 1 Dermatologic Unit, Department of Clinical Internal, Anesthesiological and Cardiovascular Sciences, La Sapienza University of Rome, Rome, Italy 2 Porphyria and Rare Diseases Unit, San Gallicano Dermatological Institute - IRCCS, Rome, Italy 3 Dermatology Unit, Surgical, Medical and Dental Department of Morphological Sciences related to Transplant, Oncology and Regenerative Medicine, University of Modena and Reggio Emilia, Modena, Italy 4 Department of Anatomic Pathology, Azienda Ospedaliero-Universitaria Policlinico, Modena, Italy 5 Centro Oncologico ad Alta Tecnologia Diagnostica, Azienda Unità Sanitaria Locale - IRCCS, Reggio Emilia, Italy Key words: BCC, margin assessment, RCM, OCT, FCM ex-vivo Citation: Michelini S, Mandel VD, Ardigò M, et al. Combining Reflectance Confocal Microscopy, Optical Coherence Tomography and Ex-Vivo Fluorescence Confocal Microscopy for Margin Assessment in Basal Cell Carcinoma Excision. Dermatol Pract Concept. 2024;14(2):e2024090. DOI: https://doi.org/10.5826/dpc.1402a90 Accepted: October 22, 2023; Published: April 2024 Copyright: ©2024 Michelini 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. Simone Michelini and Victor Desmond Mandel equally contributed to this manuscript and should be considered co-first authors. Corresponding Author: Dr. Simone Michelini, Dermatologic Unit, Department of Clinical Internal, Anesthesiological and Cardiovascular Sciences, La Sapienza University of Rome, Viale del Policlinico n° 155, zip code 00161, Rome, Italy. e-mail: simone.michelini@uniroma1.it Introduction: Recent developments of noninvasive, high-resolution imaging techniques, such as re- flectance confocal microscopy (RCM) and optical coherence tomography (OCT), have enhanced skin cancer detection and precise tumor excision particularly in highly aggressive and poorly defined basal cell carcinomas (BCCs). Objectives: The aim of this pilot study is to assess the feasibility and reproducibility of a systematic clinical workflow combining noninvasive (RCM-OCT) and invasive fluorescence confocal microscopy (FCM) imaging modalities in pre- and intra-surgical evaluations of the lateral and deep margins of ABSTRACT 2 Original Article | Dermatol Pract Concept. 2024;14(2):e2024090 BCC. Methods: Superficial incisions were made 2 mm beyond the clinical-dermoscopic BCC margins. Lateral margins were then explored with OCT and RCM. In positive margins, a further cut was made 2 mm distal from the previous. A final RCM/OCT-based double-negative margin was drawn around the entire perimeter of the lesion before referring to surgery. The freshly excised specimen was then examined with FCM (ex-vivo) for the evaluation of the deep margin. Histopathologic examination eventually confirmed margin involvement. Results: The study included 22 lesions from 13 patients. At the end of the study, 146 margins—106 negative (73%) and 40 positive (27%) at RCM/OCT—were collected. The RCM/OCT margin evalu- ation showed an overall sensitivity of 100% and a specificity of 96.3%. The overall positive margins diagnostic accuracy was 98.2%. Reproducibility was evaluated on recorded images and the raters showed a substantial inter-observer agreement on both RCM (κ = 0.752) and OCT images (κ = 0.724). Conclusions: The combined RCM/OCT/FCM ex-vivo approach noninvasively facilitates the presurgi- cal and intrasurgical lateral and deep margin assessment of poorly defined BCCs. Introduction Basal cell carcinoma (BCC) is a widely diffused neo- plasm in western countries with an increasing incidence as a consequence of inappropriate sun exposure and increased longevity of the population. Although many different min- imal to noninvasive procedures have been proposed and applied in selected cases [1], surgical excision remains the recommended treatment option achieving average 5-year disease-free rates of over 98% for BCCs [2]. According to the National Comprehensive Cancer Net- work (NCCN), the recommended lateral margin for BCCs is 4 mm which should be extended in case of high-risk BCCs, such as sclerosing, infiltrative or micronodular BCCs, be- cause of the higher rate of recurrence. Mohs micrographic surgery (MMS) in its original form or in its variants (i.e. spaghetti technique, Tubingen torte, slow-Mohs) represents the best treatment option in terms of margin clearance and recurrence rate in these clinical situations [3]. However, due to MMS highly specialized and expensive requirements, this procedure is not available everywhere. In the last decades, the development of non-invasive, high resolution imaging techniques, such as reflectance con- focal microscopy (RCM) and optical coherence tomography (OCT), allowed the possibility to explore the tissue in vivo at nearly histologic resolution, significantly improving skin cancer diagnostic accuracy. As a consequence, due to shal- low imaging penetration, the use of these techniques has also been proposed for lateral margin assessment in lentigo maligna and BCC [4,5-10]. Additionally, ex-vivo fluores- cence confocal microscopy (FCM) is an emerging imaging technique that allows real-time microscopic examination of freshly excised cutaneous tissue. Thanks to its procedural simplicity and digital histopathologic acquisition rapidity, this tool is mainly applied to intra-operative analysis of the surgical margins of BCC in a MMS-like setting, as it is able to observe the entire skin specimen and both superficial and deeper margins with a very high accuracy [11]. The combined use of highly performing invasive and non-invasive imaging methods may enhance the capabilities for skin cancer detection and precise tumor excision partic- ularly useful in highly aggressive and poorly defined BCCs in order to guarantee radical treatment whilst saving proce- dural time and costs. Objectives The aim of this pilot study is to assess the feasibility and reproducibility of an organized and systematic clinical workflow combining non-invasive (RCM-OCT) and inva- sive (FCM) imaging modalities in the pre- and intra-surgical evaluation of lateral and deep margins. Methods Patients presenting lesions with a confirmed clinical, der- moscopic and RCM diagnosis of BCC were recruited from the outpatient dermatology clinics of San Gallicano Derma- tological Institute of Rome and University of Modena and Reggio Emilia. Inclusion criteria were to present (i) at least one lesion with clinical, dermoscopic or RCM diagnosis of primary BCC; (ii) poorly defined lateral borders and/or clinical features sug- gesting sclerosing or infiltrating forms; (iii) lesion fully acces- sible for examination with RCM and OCT; (iv) patients >18 years old (v) patient willingness to participate. Exclusion criteria were: (i) crusted or ulcerated lesions, (ii) local relapses or previously treated lesions; (iii) lesions lo- cated on anatomical sites not allowing a proper evaluation Original Article | Dermatol Pract Concept. 2024;14(2):e2024090 3 with RCM/OCT (eg nose wings, eyelid margins, auricles etc.); (iv) incapability to understand and sign the informed consent. Written informed consent was collected from all the participants. Imaging Procedure Prior to mapping procedure, all patients underwent a clinical, dermoscopic (Dermlite HR, DL4W magnification 10x) and hand-held RCM (Vivascope 3000® Vivascope GmbH) evalua- tion to confirm BCC diagnosis. The lesion mapping procedure consisted of 4 steps, adapted on the “SMART” approach pre- viously proposed for skin tumor mapping, as follows [12,13]: Step 1. Clinical dermoscopic margin marking. After lesion inspection, visible BCC margins were delimited in hexagonal or rhomboidal shaped margins around the tumor (depending on the size and shape) to facil- itate the subsequent surgical procedure, and marked with an ink pen 2 mm beyond the clinically and der- moscopically determined borders. Step 2. Margin superficial cut. After 1 hour of occlusive ap- plication of topical anesthetic, (lidocaine 25 mg/g + prilocaine 25 mg/g), a superficial cut was made with a scalpel (blade#15), overlying the dermo-graphic pen ink. In case of bleeding, it was readily arrested with sterile gauze soaked in tranexamic acid. Step 3. Lateral Margin exploration with non-invasive techniques. Margins were assessed by combining the infor- mation from both OCT and RCM. OCT imaging was carried out by Vivosight D-OCT (Michelson Diagnostics) as previously described [14,15]. RCM margins were explored with a hand-held RCM Vi- vascope 3000 in live mode [12,13]. The imaging procedure started from the center of the lesion out- wards in a radial direction up to the visualization of the superficial cut for each margin with both techniques. A margin was considered “positive” if presenting OCT or RCM BCC specific features less than 1mm inward or outward from the cut. OCT BCC positive features corresponded to the “Berlin Score” system [16], and RCM ones corresponded to the features enlisted by Longo et al. (dark sil- houettes, bright tumor islands/cords, cleft-like dark spaces, dendritic cells, increased vascularization) [6]. In case of a positive margin, a further cut was made 2 mm distal from the previous or at an es- timated 2 mm distance from the outermost visible BCC structure, repeating the procedure in case of Figure 1. The procedure in clinical (A) and dermoscopic (B) detail. With an ink pen, visible BCC margins were defined around the tumor in a hexagonal or rhomboidal form 2 mm beyond the clinically and dermoscopically confirmed limits. A shallow incision was made over the dermographic pen ink. (C) An OCT scan shows basaloid islands (asterisks) extending beyond the first incision (red arrow). The margin has then been advanced by 2 mm (white arrow). 4 Original Article | Dermatol Pract Concept. 2024;14(2):e2024090 Figure 2. RCM exploration confirmed the presence of basaloid islands (asterisk) near the margin (white arrow). a further positive margin. A final RCM/OCT-based double negative margin was drawn around the entire perimeter of the lesion before referring to surgery. All OCT and RCM margin imaging were ac- quired as a multilayer tiff file and an AVI video file, respectively, for reproducibility study (Figures 1 and 2). Step 4. Surgical procedure and deep margin check. Patients proceeded to surgery following the RCM/OCT annotated margins. After specimen excision, the freshly excised specimen was prepared for the FCM (ex vivo) imaging procedure for intra-operatory margin evaluation: - FCM of deep margin: FCM imaging was per- formed with VivaScope 2500 4th Gen® (MA- VIG GmbH) following the previously described procedure. [17] Along the side of the polygonal shaped specimen thin transversal sections were cut from the epidermal surface to the bottom of the excised specimen. The remaining central portion of the specimen and the lateral sections were prepared for FCM imaging. The bottom of the central portion was first imaged for the eval- uation of the deep margin. Subsequently, each lateral section was imaged positioning the spec- imen facing the internal side, on the device glass plate. A board-certified dermatologist (M.A.), experienced in reading FCM imaging, evaluated BCC margin involvement. In case of BCC positive FCM positive margin, the surgical cut was selec- tively enlarged in the positive sector. After negative FCM margin confirmation, surgical breach closure is performed. Histopathologic examination was sent to a board-certified pathologist (C.C, A.M.C.) to confirm the diagnosis and margin involvement (Fig3). Follow-up study. After 1 year from excision, patients un- derwent clinical and dermoscopic examination of the scar and its peripheral area in order to identify possible BCC recurrence. Reproducibility Study To validate reproducibility of RCM/OCT reading pro- cedure, all the RCM imaging videos and the OCT images from all the margins evaluated were randomized and retro- spectively evaluated by two external readers, blinded to any dermoscopic, clinical and histopathologic information. The external readers were asked to evaluate RCM and OCT margin as positive or negative, separately. Statistical Analysis As descriptive statistics, absolute numbers and percent- ages of true positive, true negative, false positive and false negative margins have been reported along with sensitivity and specificity values. The diagnostic positive margins per- formance is evaluated on the receiver operating characteris- tic (ROC) curve and the area under the curve. The Cohen kappa (κ) statistic has been used to measure the agreement between the histologic positive margin and the two “in vivo” instruments. Moreover, κ was also calcu- lated in the evaluation of the agreement between the final operator positive margins and histological positive margins. We evaluated inter-observer agreement for positive margins in both RCM and OCT evaluations in relation to the golden standard. The interpretation of agreement adopted here is less than chance agreement (κ < 0), slight agreement (κ = 0.01-0.20), fair agreement (κ = 0.21-0.40), moderate agree- ment (κ = 0.41-0.60), substantial agreement (κ = 0.61-0.80), and almost perfect agreement (κ = 0.81-0.99). The inter- pretation of reproducibility adopted is marginal (κ = 0.00- 0.40), good (κ = 0.40-0.75) and excellent (κ >0.75). For all analyses, a P < 0.001 was considered statistically significant. STATA program version 14 (StataCorp) was used to perform statistical analysis. Results The study included a total of 146 margins from 22 lesions from 13 patients, 4 females (30.8%) 9 males (69.2%), median age 71.4 years (range: 47-90 years), enrolled be- tween June 2021 and November 2021 at the San Gallicano Original Article | Dermatol Pract Concept. 2024;14(2):e2024090 5 Figure 3. (A) Introperative axial FCM image of an excisional biopsy in fluorescence mode. Reflectance mode (B) and combined mode (C) shoving multiple basaloid islands of a preauricular BCC. En face view highlighting the superfi- cial cut (white arrows) in Fluorescence mode (D) reflectance mode (E) and combined mode (F). Deep margin showed no BCC feature in FCM. (G) Histology image displaying the margin cut (black arrow) close to the BCC. 6 Original Article | Dermatol Pract Concept. 2024;14(2):e2024090 The RCM/OCT margin evaluation showed an overall sen- sitivity of 100% and a specificity of 96.3% and an overall positive margins diagnostic accuracy was 98.2%. Concerning diagnostic accuracy, the percentages of agree- ment with histopathology was higher for the first rater, reaching 95.7% accuracy for RCM (κ = 0.89) and 95.1% for OCT (κ = 0.87), than the second one, reaching 91.5% and 89.4% (κ = 0.76 and κ = 0.71), respectively (Table 1). Reproducibility was evaluated on recorded images, and the raters showed a substantial inter-observer agreement on both RCM (κ = 0.751) and OCT images (κ = 0.724) (Table 2). Ex vivo FCM deep margin check. All deep margins re- sulted negative in histopathology as well as in ex vivo FCM imaging. Follow-up study. After one-year follow-up no recurrences have been observed in clinical and dermoscopic evaluation. Conclusions The aim of our study was the evaluation of the impact of the in vivo tumor lateral margin assessment in a presurgical phase using RCM/OCT method combined and the ex-vivo Dermatological Institute of Rome (N = 7) and Dermatology Department of Modena (N = 6). Out of the 22 treated lesions, 12 (54.6%) were localized on the trunk, 3 (13.6%) on the limbs and 7 (31.8%) on the face. The size of the lesion’s major diameter ranged between 5-15 mm (mean of 8 mm). Histological predominant subtypes resulted in 7 (31.8%) nodular BCC, 8 (36.3%) superficial BCC followed by 3 micronodular BCC (13.6%) and 4 infil- trative BCC (18.2%). Lesions were framed into 4 margins (rhombus) in 13 cases (59.1%) or 6 margins (hexagon) in 9 cases (40.9%) depend- ing on the size and the shape of the lesions, thus resulting in a total of 106 first stage margins. 39 margins (36%) were positive, leading to a second stage margin that resulted nega- tive in all cases but one. At the end of the study 146 margin, 106 negative (73%) and 40 positive (27%) at RCM/OCT, were collected. 4 margins were excluded from the study be- cause of poor quality imaging and thus not suitable for the reproducibility study. Concerning histopathology, 33 out of 142 margins were pos- itive. Eight of 22 lesions (36.4%) had all negative margins, 5 (22.7%) had 1 positive margin, 3 (13,6%) had 2 positive margins and 4 (18,2%) had 3 positive margins. Table 1. Lateral Margin Exploration With Non-Invasive Techniques, Correlation With Histopathology and Reproducibility Histology a Sensitivity SpecificityNegative Positive % of correct diagnosis K-value Level of agreement AUC RCM Negative 105 0 97.2 0.924 Almost perfect 0.982 100 96.3 Positive 4 33 OCT Negative 105 0 97.2 0.924 Almost perfect 0.982 100 96.3 Positive 4 33 Rater 1 RCM Negative 103 0 95.7 0.888 Almost perfect 0.972 100 94.5 Positive 6 33 OCT Negative 103 1 95.1 0.868 Almost perfect 0.957 96.9 94.5 Positive 6 32 Rater 2 RCM Negative 104 7 91.5 0.758 Substantial 0.871 78.8 95.4 Positive 5 26 OCT Negative 100 6 89.4 0.713 Substantial 0.867 81.8 91.7 Positive 9 27 a4 margins histologically result not evaluable. AUC = area under the curve; OCT = optical coherence tomography; RCM reflectance confocal microscopy. First and second rater evaluation for RCM and OCT of margins compared to histological diagnoses, the percentage of correct diagnoses, κ value, the level of agreement, the sensitivity, the specificity, and ROC area for both raters. Original Article | Dermatol Pract Concept. 2024;14(2):e2024090 7 Table 2. Agreement Between Operators RCM rater 1 Level of agreementNegative Positive K-value RCM rater 2 Negative 101 11 0.751 Substantial Positive 3 31 OCT rater 1 OCT rater 2 Negative 98 9 0.724 Substantial Positive 7 32 RCM rater 1 OCT rater 1 Negative 102 3 0.916 Almost perfect Positive 2 39 RCM rater 2 OCT rater 2 Negative 100 7 0.653 Substantial Positive 12 27 OCT = optical coherence tomography; RCM reflectance confocal microscopy. tumor deep margin check in the intra-operative phase by means of ex vivo FCM, on BCC excision. Our experience disclosed that the combined approach, in vivo OCT/RCM + ex vivo FCM represents a promising new approach to BCC margins identification. Achieving clear narrow margins and attaining the recommended wide safety margins may be complex in some cases, relying only on clin- ical and dermoscopic criteria. For this reason, we selected a series of BCC showing unclear clinical and dermoscopic margins. BCC subtypes with aggressive histologic characteristics, poorly defined clinical margins and sites in certain areas, including the H region of the face have been linked to an increased risk of recurrence. For these reasons, Mohs surgery was developed for locally aggressive tumors [18-20]. In cases of poorly defined BCCs, Mohs surgery showed great effectiveness. Primary BCC recurrence rates following rou- tine excision versus MMS are 10% and 1%, respectively. In a randomized trial, the 10-year cumulative probabilities for recurrence in primary BCCs were 12.2% versus 4.4% with standard excision and MMS, respectively [21]. However, the application of Mohs is limited in several healthcare systems due to technological issues, costs and availability of a dedicated pathologist. As a result, several methods have been developed, especially in Europe, to use noninvasive methods to detect lateral tu- mor margins. In a recent meta-analysis, dermoscopy revealed no statistically significant differences in the proportion of complete margin clearance on the first MMS stage between BCCs treated with dermoscopy-guided MMS and those who underwent curettage or visual inspection. However, lateral margin involvement was significantly lower in BCCs that had dermoscopy-guided MMS [22]. The precise assessment of the dermoscopic margins of infil- trative BCC may be very difficult given that these tumors are often more amelanotic and less heavily pigmented than less aggressive subtypes [23]. In one study, RCM demonstrated good global accuracy for primary BCC lateral margin detection with a sensitivity and specificity of 95%. However, the study has been done only on superficial BCC-type [24]. To note, in our study diffi- cult BCC in terms of clinically definable margins has been included. OCT displayed a sensitivity of 88.9%-92.6% and a speci- ficity of 96.8%-98.4% on examining BCC-involved margin in 40 BCCs [25]. The major limit of this study is that the histological BCC subtypes were not reported. However, each of these approaches has its limitations. RCM has excellent lateral resolution (~0.1-0.8 μm) but low tis- sue penetration power (100–200 μm) while OCT has lower lateral resolution (~5-7.5 μm) but higher penetration power (~1mm). The combined use of RCT and OCT seems to have multiple advantages: OCT displays very quickly (~10 sec/acquisition) the entire volume of the lesion with a stack of orthogonally oriented images, each of FOV 2 mm. OCT imaging detects dark hypoechoic areas, which indicate the potential presence of BCC. RCM confirms OCT data by visualizing BCC fea- tures with cellular resolution. However, none of the non-invasive techniques currently in use enable the vision of deep margin involvement, which is crucial for the possible recurrence of BCC since it might re- sult in infiltration and tumor development in deep tissues. FCM has been selected for the detection of positive deep margins after surgical excision as RCM and OCT lack to 8 Original Article | Dermatol Pract Concept. 2024;14(2):e2024090 methods of sterilization used for surgical devices. Integration of RCM/OCT imaging in Mohs surgery could be considered in a presurgical stage potentially able to save time by reduc- ing the required number of Mohs stages. Line-field confocal optical coherence tomography (LC- OCT) is a novel technique that combines the technological advantages of reflectance confocal microscopy with OCT in a single instrument. Compared to the procedures used independently, it has a lower resolution, but it allows for a quicker switch between diagnostic techniques, facilitating the diagnosis. However, LC-OCT is unable to provide information on the involve- ment of the deep margin in non-superficial BCC [31,32]. The information provided by the RCM/OCT/FCM combined procedure has all the potential for routinely applications in the presurgical and intrasurgical assessment of adequate lat- eral and deep margin in BCC. This procedure is likely to be most beneficial for difficult BCC of particular areas like the face, where wide margins may be difficult to attain. Moreover, potentially the FCM can be reserved in very deep BCC in which the deep silhouette is not clearly visible when assessed with in-vivo techniques (RCM, OCT, LC-OCT). This approach could potentially lead to a positive impact on the patient’s surgical experience, satisfaction, and improve the physician’s decision-making process. This can decrease patient anxiety, reduce cost by reducing the number of re- currences and improve pre-operative surgical planning by discussing appropriate reconstruction options and potential non-invasive treatment options. In summary, the combined approach RCM/OCT/FCM ex vivo noninvasively facilitates both diagnosis and depth assessment, and consequently BCCs may be treated through a “one-stop shop” approach with no need for a biopsy. References 1. Peris K, Fargnoli MC, Garbe C, et al. Diagnosis and treatment of basal cell carcinoma: European consensus-based interdisciplin- ary guidelines. Eur J Cancer. 2019;118:10-34. DOI: 10.1016/j. ejca.2019.06.003. PMID: 31288208. 2. Rhodes LE, de Rie MA, Leifsdottir R, et al. Five-year follow-up of a randomized, prospective trial of topical methyl amino- levulinate photodynamic therapy vs surgery for nodular basal cell carcinoma. Arch Dermatol. 2007;143(9):1131-1136. DOI: 10.1001/archderm.143.9.1131. PMID: 17875873. 3. 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