Dermatology: Practical and Conceptual Research Letter | Dermatol Pract Concept. 2025;15(1):5031 1 Line-field Confocal Optical Coherence Tomography Image Markers of Basal Cell Carcinoma in Excised Mohs Micrographic Surgery Tissue: A Case Series Kevin Jacobsen1, Emily Wenande1, Martin Glud1, Merete Haedersdal1,2, Vinzent Kevin Ortner1 1 Department of Dermatology, Copenhagen University Hospital – Bispebjerg and Frederiksberg, Copenhagen, Denmark 2 Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark Key words: Line-field confocal optical coherence tomography, Image markers, Basal cell carcinoma, Mohs micrographic surgery, Ex vivo imaging Citation: Jacobsen K, Wenande E, Glud M, Haedersdal M, Ortner VK. Line-field Confocal Optical Coherence Tomography Image Markers of Basal Cell Carcinoma in Excised Mohs Micrographic Surgery Tissue: A Case Series. Dermatol Pract Concept. 2025;15(1):5031. DOI: https://doi.org/10.5826/dpc.1501a5031 Accepted: December 4, 2024; Published: January 2025 Copyright: ©2024 Jacobsen 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: Kevin Jacobsen, MD, PhD fellow, Department of Dermatology, Copenhagen University Hospital – Bispebjerg and Frederiksberg, Nielsine Nielsens Vej 17, DK-2400, Bispebjerg, Denmark. Email: kevin.jacobsen@regionh.dk Introduction Line-field confocal optical coherence tomography (LC-OCT) is an evolving tool for in vivo skin cancer diagnostics [1]. As reviewed by Jacobsen et al. [2], studies have shown that LC-OCT improves the detection of clinically challenging basal cell carcinoma (BCC) lesions compared to dermoscopy alone [3]. LC-OCT is also expanding into other domains of skin cancer management; for instance, we recently published a case report using LC-OCT to visualize infiltrative BCC for precise margin adjustment during surgical planning [4]. While perioperative use of LC-OCT for in vivo BCC imaging is evolving, its applications in ex vivo Mohs micrographic surgery (MMS) remains unexplored. As a starting point for its potential integration in the context of MMS, we present a case series describing the use of LC-OCT to detect the pres- ence of three key BCC image markers—lobules, clefting, and collagen alterations—in excised MMS tissue specimens from six patients with BCC. Case Presentation Cases were selected from six different MMS patients with con- firmed BCC. Of these, six were nodular BCCs, one of which had an infiltrative BCC component. Tissue samples were ex- cised from the nasal tip, nasal alae, or lower eyelid during the first stage of MMS and had sizes ranging from 8 mm to 15 mm in diameter (Table 1). Prior to LC-OCT examination, speci- mens were stored at -80°C and thawed at room temperature. For LC-OCT examination, we used the handheld probe (deepLive, DAMAE Medical, Paris, France) to acquire im- ages from the subcutaneous surface of each tissue specimen with the skin surface facing downwards. On the subcutane- ous tissue surface, targeted regions corresponding to areas of BCC identified in H&E histopathological sections were im- aged (white boxes in Figure 1A-F). An experienced LC-OCT user (K.J.), who was blinded to the histopathological results, assessed the images and deemed them to be of sufficient quality for evaluation. 2 Research Letter | Dermatol Pract Concept. 2025;15(1):5031 Table 1. Tumor Characteristics and Number of Mohs Micrographic Surgery (MMS) Stages of Included Tissue Specimens. Patient # BCC Subtype Anatomical Site Size, mm # of MMS Stages 1 Nodular Scalp 15x15mm 1 stage 2 Nodular Nose, left ala 15x6 mm 2 stages 3 Nodular Right lower eyelid 10x6 mm 4 stages 4 Nodular Nose, left ala 11x13 mm 2 stages 5 Nodular Nasal tip 8x8 mm 2 stages 6 Nodular and infiltrative Nose, right ala 13x10 mm 2 stages Abbreviation: BCC: basal cell carcinoma. Figure 1. Key basal cell carcinoma (BCC) image markers in line-field confocal optical coherence tomography (LC-OCT) from six ex vivo Mohs micrographic surgery (MMS) tissue specimens of confirmed BCC. (A-F) Clinical, (G-L) cor- responding H&E histopathology, and (M-R) en face LC-OCT images from the same targeted area (white boxes in clinical images) are presented on the same horizontal plane in the figure. Lobules (arrows) are shown in (G-L) and (M-R). Clefting (star) and collagen alterations (triangle) are prominent in (M). Scale bar in H&E histopathology and LC-OCT images: 100 microns. Research Letter | Dermatol Pract Concept. 2025;15(1):5031 3 Overall, our ex vivo findings align with previously iden- tified key in vivo LC-OCT markers for BCC (Figure 2) [2]. In all six MMS tissue specimens, LC-OCT examination— confirmed by H&E histopathology (Figure 1G-L)— consistently revealed the presence of lobules, clefting, and collagen alterations (Figure 1M-R). Assessment of images (N=13) showed that lobules were the most prominent marker, appearing in 100% of vertical images and in 92.3% of en face images. Clefting and collagen alterations were evenly distributed in vertical images and en face images (84.6% and 92.3%, respectively). For reference, Figure 2 il- lustrates a side-by-side comparison of the LC-OCT ex vivo BCC images collected in the present study with previously obtained LC-OCT in vivo BCC images from other patients. Conclusion LC-OCT was able to visualize key BCC image markers in six MMS tissue specimens, suggesting there is potential for LC-OCT in an ex vivo context. The described method of subcutaneous imaging with LC-OCT may be valuable in a perioperative context to visualize deeper BCC regions that are currently beyond LC-OCT’s dermal scan depth. Further studies are warranted to validate the findings from this case series in a larger cohort. References 1. Dubois A, Levecq O, Azimani H, et al. Line-field confocal optical coherence tomography for high-resolution noninvasive imaging of skin tumors. J Biomed Opt. 2018;23(10). DOI: 10.1117/1 .JBO.23.10.106007. 2. Jacobsen K, Ortner VK, Wenande E, Sahu A, Paasch U, Haedersdal M. Line-field confocal optical coherence tomogra- phy in dermato-oncology: A literature review towards harmo- nized histopathology-integrated terminology. Exp Dermatol. 2024;33(4). DOI: 10.1111/exd.15057. 3. Gust C, Schuh S, Welzel J, et al. Line-Field Confocal Optical Coherence Tomography Increases the Diagnostic Accuracy and Confidence for Basal Cell Carcinoma in Equivocal Lesions: A Prospective Study. Cancers. 2022 Jan;14(4). DOI: 10.3390 /cancers14041082. 4. Jacobsen K, Wenande E, Ortner VK, Schmidt G, Haedersdal M. Surgical planning with line-field confocal optical coherence tomography for recurrent infiltrative basal cell carcinoma: visu- alizing subclinical tumor for margin adjustment. J Dtsch Derma- tol Ges. 2024;22(3). DOI: 10.1111/ddg.15330. Figure 2. Line-field confocal optical coherence tomography (LC-OCT) images of basal cell carcinoma (BCC), comparing (A) in vivo skin sur- face and (B) an ex vivo subcutaneous tissue images (B) from Mohs micrographic surgery tissue. Both images (A) and (B) reveal key LC-OCT BCC markers: lobules (blue circle), clefting (green arrow), and collagen alterations (white arrow) in the vertical (top) and en face views (bottom). Intralobular cystic structure (orange star) is also observed in (B). Scale bar in LC-OCT images: 100 microns.