Dermatology: Practical and Conceptual Original Article | Dermatol Pract Concept. 2022;12(4):e2022207 1 Reflectance Confocal Microscopy Follow-up of Multifocal Superficial Basal Cell Carcinomas Treated With Imiquimod 5% Cream Yunmin Zou1, Xiaohong Zhu1, Rushan Xia1 1 Department of Dermatology, The Affiliated Wuxi No.2 People’s Hospital of Nanjing Medical University Wuxi, Jiangsu Province, China Key words: superficial basal cell carcinoma, imiquimod 5% cream, reflectance confocal microscopy, multifocal Citation: Zou Y, Zhu X, Xia R. Reflectance confocal microscopy follow-up of multifocal superficial basal cell carcinomas treated with imiquimod 5% cream. Dermatol Pract Concept. 2022;12(4):e2022207. DOI: https://doi.org/10.5826/dpc.1204a207 Accepted: March 28, 2022; Published: October 2022 Copyright: ©2022 Zou 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: Scientific Research Project of Wuxi Municipal Health Commission. (Q202141) Competing interests: None. Authorship: All authors have contributed significantly to this publication. Corresponding author: Rushan Xia, Department of Dermatology, The Affiliated Wuxi No.2 People’s Hospital of Nanjing Medical University, 68 Zhongshan Road, Wuxi, 214002, Jiangsu Province, China E-mail: wxeyxrs@163.com Introduction: Patients with multifocal superficial basal cell carcinomas (sBCC) require a non-in- vasive treatment and follow-up with a non-invasive technique. Imiquimod 5% cream is a new non-invasive therapy for BCC. Reflectance confocal microscopy (RCM) is a non-invasive, real-time imaging technique. Objectives: To evaluate and describe the feasibility and efficacy of imiquimod 5% cream for the treat- ment of multifocal sBCC using RCM. Methods: The efficacy of imiquimod 5% cream for the treatment of multifocal sBCC was evaluat- ed, as well as the potential of RCM for assessing therapeutic effects. We reported four patients with 34 sBCC lesions were treated with imiquimod 5% cream. RCM was performed in the baseline and at 12 weeks, 24 weeks and 52 weeks after starting treatment. Results: Of 34 lesions treated with imiquimod 5%, 32 responded to the treatment and showed com- plete clinical clearing. Two subclinical BCC lesions were identified by RCM. The complete tumor clear- ance rate was 88.2%, and the efficiency rate was 97.1%. No lesion recurred at 24-month follow-up. RCM identified previously described confocal features of BCC and was more sensitive than clinical examination. Local skin reactions were relieved after expectant treatment. Conclusions: Imiquimod 5% cream may be useful for the treatment of multifocal sBCC, and its side effects are easy to manage. RCM can be used for non-invasive monitoring of treatment response and improved the tumor clearance rate. ABSTRACT 2 Original Article | Dermatol Pract Concept. 2022;12(4):e2022207 Introduction Basal cell carcinoma (BCC) is the most common skin cancer worldwide. Although surgical treatment of BCC is the gold standard, patients may be reluctant to accept  invasive treat- ment by reason of multifocal BCC-lesions. In recent years, non-invasive therapies have been widely used for the treatment of superficial (s)BCC [1,2]. Imiquimod is a new local treatment that can remove tumor tissues without injuring the surround- ing normal tissues. However, histological evidence of tumor clearance is difficult to obtain. In vivo reflectance confocal mi- croscopy (RCM) is a non-invasive, real-time imaging technique that provides near histologic level resolution cross-sectional images of superficial layers of the skin. RCM can be used to diagnose and monitor treatment effectiveness in BCC [3-5]. Objectives The aim of this study was to evaluate and describe the feasi- bility and efficacy of imiquimod 5% cream for the treatment of multifocal sBCC using RCM. Methods Four patients (3 females and 1 male) with diagnoses of sBCC, with 2 or more lesions, and aged from 27 to 76 years (mean 56 years) were included in the study. The patients presented with 34 sBCC appearing as mild reddish-brown patches and pigmented patches (Figure 1A). The patients were overall healthy and had no other symp- toms associated with Gorlin Syndrome. All lesions were di- agnosed clinically, and BCC features were confirmed under RCM. All the lesions were confirmed as superficial (s)BCC histologically (Figure 1B). Each lesion was treated with 5% imiquimod cream (Aldara®; 3M Pharmaceuticals). Imiquimod 5% cream was applied once daily for 5 days/ week for 12 weeks. Treatment visit was performed once ev- ery  two weeks. Treatment  ended when there was no clini- cal or RCM evidence. If there is no response or insufficient response, treatment was continued and reevaluated every 2 weeks, until complete clinical response was obtained. The imiquimod cream was applied before going to bed and re- moved with soap and water approximately 8 hours later. The treatment area was not covered except in cases of bleeding or excessive discharge from the wound. Each treatment field encompassed the lesion and 1.0 cm margins surrounding the visible BCC lesion. Each lesion was followed up by RCM (Vivascope 1500, Lucid Technologies) at 12 weeks, 24 weeks and 52 weeks af- ter the start of imiquimod treatment. Baseline and follow-up images were collected. The RCM images were evaluated by two experienced physicians. If there was discordance between the two evaluators, a consistent interpretation was obtained from a third reviewed. Therapeutic effects were determined by tumor size combined with RCM imaging features. Results A total of 34 BCC lesions were included in the analysis. The tumor size ranged from 0.5 to 3 cm in the maximum diam- eter (mean, 1.4 cm). Lesions were located in the neck area (N = 3), scalp (N = 2), chest (N = 8), abdomen (N = 5), back (N = 5), lower extremities (N = 6), and upper extremities (N = 5). The durations of the drug application period var- ied from 8 weeks to 28 weeks, with an average of 18.6 ± 6.9 weeks. Figure 1. (A) Multifocal BCCs in the chest area before treatment. (B) Histopathology showed the basaloid cords and basaloid island connected to the epidermis (H&E, magnification 40×). Original Article | Dermatol Pract Concept. 2022;12(4):e2022207 3 Reflectance Confocal Microscopy Analysis RCM imaging details are provided in Table 1. RCM crite- ria previously described were used for the diagnosis of BCC [6-8]. The RCM criteria for the diagnosis of BCC include principal  criteria (≧ 2 principal  criteria present): tumor is- lands, elongated and polarized nuclei, peripheral palisading; and  secondary  criterion (≧ 3 secondary  criterion present): keratinocyte atypia, cords connected to the epidermis, bright particles, peri-tumoral clefting, increased vascularization, and dendritic cells inside tumor islands (Figure 2).  At the 12-week, 24-week and 52-week follow-up evaluations, there was no RCM evidence in 22 lesions, 26 lesions, and 30 lesions, respectively (Figure 3). At the 52-week follow-up, residual BCC was detected by RCM in 4 of 34 sites (11.8%), and there was no clinical manifestation in two lesions. The area of three lesions was obviously reduced, but no effect was observed in one lesion. The complete tumor clear- ance  rate  was 88.2%, and the efficiency rate was 97.1%. In the residual lesion, treatment with imiquimod 5% cream was discontinued, and surgical treatment was performed. No lesion recurred at 24-month follow-up. Local Skin Reactions The patient is still under clinical and RCM monitoring. All lesions had good cosmetic outcomes except hypopigmen- tation in eight lesions (23.5%). Local skin reactions (LSR) Table 1. Reflectance confocal microscopy characteristics of basal cell carcinomas before (baseline) treatment with imiquimod and at 12, 24 and 52 weeks after treatment. Reflectance confocal microscopy Baseline, % (No.) 12-week follow-up, % (No.) 24-week follow-up, % (No.) 52-week follow-up, % (No.) Tumor islands 82.4 (28) 23.5 (8) 17.6 (6) 2.9 (1) Elongated and polarized nuclei 79.4 (27) 23.5 (8) 14.7 (5) 2.9 (1) Peripheral palisading 52.9 (18) 20.6 (7) 8.8 (3) 0 (0) Keratinocyte atypia 100 (34) 35.3 (12) 23.5 (8) 11.8 (4) Peri-tumoral clefting 17.6 (6) 5.9 (2) 0 (0) 0 (0) Dendritic cells inside tumor islands 97.1 (33) 35.3 (12) 20.6 (7) 8.8 (3) Cords connected to the epidermis 94.1 (32) 32.4 (11) 17.7 (6) 5.9 (2) Increased vascularization 82.4 (28) 26.5 (9) 5.9 (2) 0 (0) Bright particles 70.6 (24) 35.3 (12) 23.5 (8) 5.9 (2) Figure 2. Reflectance confocal microscopy (RCM) image of basal cell carcinoma (BCC). (A) Keratinocyte atypia (blue arrowheads). (B) Increased vascularization (blue arrowheads). (C) Peri-tumoral clefting (blue arrowheads). (D)Tumor islands (blue arrowheads) and bright particles (red arrowheads). (E) Elongated and polarized nuclei (red arrowheads). (F) Dendritic cells inside tumor islands (blue arrowheads). (G) Cords connected to the epidermis (blue arrowheads). (H) Peripheral palisading (blue arrowheads). 4 Original Article | Dermatol Pract Concept. 2022;12(4):e2022207 bright particles in the epidermis (Figure 3F) after 12 weeks of imiquimod application indicate that imiquimod 5% has initiated an immune response and is a reliable biomarker for predicting treatment response. Surgery is the gold standard for treatment of BCC. How- ever, in some cases such as elderly patients who are too frail to withstand excisional surgery, or patients with multifocal BCC-lesions, non-invasive therapies are needed. Studies eval- uating BCCs of the trunk or extremities suggest that imiqui- mod is as effective as surgical treatment regarding clinical cure rates and it achieves good to excellent cosmetic out- comes [10-11]. In this study, we also confirmed that imiqui- mod 5% is effective for the treatment of multiple sBCCs. The complete tumor clearance rate was 88.2%, higher than those of documents reported [1,2,12], which may be credited to RCM surveillance. The therapeutic regimens were differ- ent in the previous studies [12-15]. Consistent with previous studies [16-17], the imiquimod dosing regimen of 5 times per week for 12 weeks is an effective treatment for sBCC. In this study, the most common side effects were ery- thema and hypopigmentation, and recovery was gradual. The erythema may be a sign of an immune response in the epidermis. LSRs mainly occurred during the first 3 months of therapy. The present study had some limitations. This was an open study with no control group and it included only four patients. Larger clinical studies with longer follow-up peri- ods are necessary. In conclusion, we confirmed the efficacy including erythema, edema, dryness, crusting, and erosion were present in nine lesions (26.5%). They were relieved af- ter expectant treatment with moisturizer. Treatment was in- terrupted for 1 week or 2 weeks for recovery in serious cases. Conclusions RCM provides a useful, noninvasive tool  for the diagno- sis of BCC. The presence of two or more RCM criteria is 100% sensitive for the diagnosis of BCC, and with 4 or more RCM criteria present the specificity was 95.7% [9]. In this study, 5 criteria or more present could be diagnosed as BCC, by which diagnostic coincidence rate was  100% comparing with pathological outcome. Our study confirmed that imiquimod 5% cream is highly effective for the treat- ment of multifocal sBCC. Notably, RCM features were not present after treatment with imiquimod 5% cream in 30 of 34 lesions. Before treatment, the RCM criteria for the di- agnosis of BCC were detected as described in Table 1. At 12 weeks after treatment, these RCM parameters were re- duced by approximately 64.7 % of the initial values. At the 52-week follow-up, clinical evaluation showed that 94.1% (32 of 34 lesions) achieved complete clearance, whereas RCM showed that 88.2% (30 of 34 lesions) of lesions had lost BCC features. Residual BCC was identified upon RCM examination in two lesions that did not present clinical man- ifestation. RCM improved the tumor clearance rate during follow-up. The presence of increased dendritic structures and Figure 3. Clinical and confocal images before treatment and during follow-up on the second lesion. (A) reddish-brown and pigmented patches before treatment. (B) At the 12-week follow-up, the lesion presents with erythema and crusting induced by treatment. (C) At the 24-week follow-up, the lesion presents as a small residual tumor on the border. (D) At the 52-week follow-up, complete clearance, with no sign of recurrence. (E)Before treatment, RCM shows tumor islands with peripheral palisading. (F) The12-week RCM follow-up shows increased high-refractive particles inside the tumor. (G) The 24-week RCM follow-up shows irregular tumor islands and increased dendritic cells inside tumor islands. (H) At the 52-week RCM follow-up, RCM showed regularly spaced cells composing a regular honeycomb pattern. Original Article | Dermatol Pract Concept. 2022;12(4):e2022207 5 of imiquimod 5% for the treatment of multiple sBCC and showed that RCM is a valuable tool for non-invasive moni- toring of treatment response. References 1. Williams HC, Bath-Hextall F, Ozolins M, et al. Surgery Ver- sus Imiquimod for Nodular and Superficial Basal Cell Carci- noma (SINS) Study Group. Surgery Versus 5% Imiquimod for Nodular and Superficial Basal Cell Carcinoma: 5-Year Results of the SINS  Randomized Controlled Trial. J Invest Dermatol. 2017;137(3):614-619. DOI: 10.1016/j.jid.2016.10.019. PMID: 27932240. 2. 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