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Histopathology for tumor margin assessment is time-consuming and expensive. High-resolution full-field optical coherence tomography (FF-OCT) images fresh tissues rapidly at cellular resolution and potentially facilitates evaluation. Here, we define FF-OCT features of normal and neoplastic skin lesions in fresh ex vivo tissues and assess its diagnostic accuracy for malignancies. For this, normal and neoplastic tissues were obtained from Mohs surgery, imaged using FF-OCT, and their features were described. Two expert OCT readers conducted a blinded analysis to evaluate their diagnostic accuracies, using histopathology as the ground truth. A convolutional neural network was built to distinguish and outline normal structures and tumors. Of the 113 tissues imaged, 95 (84%) had a tumor (75 BCCs and 17 SCCs). The average reader diagnostic accuracy was 88.1%, with, a sensitivity of 93.7%, and a specificity of 58.3%. The AI model achieved a diagnostic accuracy of 87.6%±5.9%, sensitivity of 93.2%±2.1%, and specificity of 81.2%±9.2%. A mean intersection-over-union of 60.3%±10.1% was achieved when delineating the nodular BCC from normal structures. Limitation of the study was the small sample size for all tumors, especially SCCs. However, based on our preliminary results, we envision FF-OCT to rapidly image fresh tissues, facilitating surgical margin assessment. AI algorithms can aid in automated tumor detection, enabling widespread adoption of this technique.

Conventional tissue sampling can lead to misdiagnoses and repeated biopsies. Additionally, tissue processed for histopathology suffers from poor nucleic acid quality and/or quantity for downstream molecular profiling. Targeted micro-sampling of tissue can ensure accurate diagnosis and molecular profiling in the presence of spatial heterogeneity, especially in tumors, and facilitate acquisition of fresh tissue for molecular analysis. In this study, we explored the feasibility of performing 1–2 mm precision biopsies guided by high-resolution reflectance confocal microscopy (RCM) and optical coherence tomography (OCT), and reflective metallic grids for accurate spatial targeting. Accurate sampling was confirmed with either histopathology or molecular profiling through next generation sequencing (NGS) in 9 skin cancers in 7 patients. Imaging-guided 1–2 mm biopsies enabled spatial targeting for in vivo diagnosis, feature correlation and depth assessment, which were confirmed with histopathology. In vivo 1-mm targeted biopsies achieved adequate quantity and high quality of DNA for next-generation sequencing. Subsequent mutational profiling was confirmed on 1 melanoma in situ and 2 invasive melanomas, using a 505-gene mutational panel called Memorial Sloan Kettering-Integrated mutational profiling of actionable cancer targets (MSK-IMPACT). Differential mutational landscapes, in terms of number and types of mutations, were found between invasive and in situ melanomas in a single patient. Our findings demonstrate feasibility of accurate sampling of regions of interest for downstream histopathological diagnoses and molecular pathology in both in vivo and ex vivo settings with broad diagnostic, therapeutic and research potential in cutaneous diseases accessible by RCM-OCT imaging.

Ex vivo confocal microscopy (EVCM) generates digitally colored purple-pink images similar to HE without time-consuming tissue processing. It can be used during Mohs surgery for rapid detection of basal cell carcinoma (BCC); however, reading EVCM images requires specialized training. An automated approach using a deep learning algorithm for BCC detection in EVCM images can aid in diagnosis. A total of 40 BCCs and 28 negative (not-BCC) samples were collected at Memorial Sloan Kettering Cancer Center to create three training datasets: (i) EVCM image dataset (663 images), (ii) HE image dataset (516 images), and (iii) a combination of the two datasets. A total of seven BCCs and four negative samples were collected to create an EVCM test dataset (107 images). The model trained with the EVCM dataset achieved 92% diagnostic accuracy, similar to the HE model (93%). The area under the receiver operator characteristic curve was 0.94, 0.95, and 0.94 for EVCM-, HE-, and combination-trained models, respectively. We developed an algorithm for automatic BCC detection in EVCM images (comparable accuracy to dermatologists). This approach could be used to assist with BCC detection during Mohs surgery. Furthermore, we found that a model trained with only HE images (which are more available than EVCM images) can accurately detect BCC in EVCM images.

Immunotherapies have shown unprecedented clinical benefits in several malignancies1–3. However, clinical responses remain variable and unpredictable, indicating the need to develop predictive platforms that can improve patient stratification4. Phenotyping of tumors into hot, altered, or cold5 based on assessment of only T-cell infiltration in static tumor biopsies provides suboptimal prediction of immunotherapy response6,7. In vivo dynamic mechanisms within the tumor microenvironment such as tumor angiogenesis and leukocyte trafficking5,8,9 also play a central role in modulating anti-tumor immunity and therefore immunotherapy response. Here, we report novel tumor immune microenvironment (TiME) phenotyping in vivo in patients with non-invasive spatially-resolved cellular-level imaging based on endogenous contrast. Investigating skin cancers as a model, with reflectance confocal microscopy (RCM) imaging10, we determined four major phenotypes with variable prevalence of vasculature (Vasc) and inflammation (Inf) features: VaschiInfhi, VaschiInflo, VascloInfhi and Vascmed/hiInflo. The VaschiInfhi phenotype correlates with high immune activation, exhaustion, and vascular signatures while VaschiInflo with endothelial anergy and immune exclusion. Automated quantification of TiME features demonstrates moderate-high accuracy and correlation with corresponding gene expression. Prospectively analyzed response to topical immunotherapy show highest response in VascloInfhi, and reveals the added value of vascular features in predicting treatment response. Our novel in vivo cellular-level imaging and phenotyping approach can potentially advance our fundamental understanding of TiME, develop robust predictors for immunotherapy outcomes and identify novel targetable pathways in future.

Reflectance confocal microscopy (RCM) with endogenous backscattered contrast can noninvasively image basal cell carcinomas (BCCs) in skin. However, BCCs present with high nuclear density, and the relatively weak backscattering from nuclei imposes a fundamental limit on contrast, detectability, and diagnostic accuracy. We investigated PARPi-FL, an exogenous nuclear poly(adenosine diphosphate ribose) polymerase (PARP1)–targeted fluorescent contrast agent, and fluorescence confocal microscopy toward improving BCC diagnosis. Methods: We tested PARP1 expression in 95 BCC tissues using immunohistochemistry, followed by PARPi-FL staining in 32 fresh surgical BCC specimens. The diagnostic accuracy of PARPi-FL contrast was evaluated in 83 surgical specimens. The optimal parameters for permeability of PARPi-FL through intact skin was tested ex vivo on 5 human skin specimens and in vivo in 3 adult Yorkshire pigs. Results: We found significantly higher PARP1 expression and PARPi-FL binding in BCCs than in normal skin structures. Blinded reading of RCM–and–fluorescence confocal microscopy images by 2 experts demonstrated a higher diagnostic accuracy for BCCs with combined fluorescence and reflectance contrast than for RCM alone. Optimal parameters (time and concentration) for PARPi-FL transepidermal permeation through intact skin were successfully determined. Conclusion: Combined fluorescence and reflectance contrast may improve noninvasive BCC diagnosis with confocal microscopy.

Towards improving non-invasive BCC diagnosis, we demonstrate successful PARPi-FL (PARP1-targeted nuclear contrast) labeling in tumors, permeability through stratum corneum, and high diagnostic accuracy with combined PARPi-FL and reflectance contrast.

In vivo phenotyping of tumor-immune microenvironment (TiME) for predicting response to immunotherapy in patients using non-invasive reflectance confocal microscopy (RCM) demonstrates high molecular and treatment response correlation.

Dermoscopy and reflectance confocal microscopy (RCM) are two noninvasive, optical imaging tools used to facilitate clinical diagnosis. A biopsy technique that produces exact correlation with optical imaging features is not previously reported. To evaluate the applications of a novel feature-focused ‘precision biopsy’ technique that correlates clinical–dermoscopy–RCM findings with histopathology. This was a prospective case-series performed during August 2017 and June 2019 at a tertiary care cancer. We included consecutive patients requiring a precise dermoscopy–RCM–histopathologic correlation. We performed prebiopsy dermoscopy and both wide probe and handheld RCM of suspicious lesions. Features of interest were isolated with the aid of paper rings and a 2 mm punch biopsy was performed in the dermoscopy- or RCM-highlighted area. Tissue was processed either en face or with vertical sections. One-to-one correlation with histopathology was obtained. Twenty-three patients with 24 lesions were included in the study. The mean age was 64.6 years (range 22–91 years); there were 16 (69.6%) males, 14 (58.3%) lesions biopsied were on head and neck region. We achieved tissue-conservation diagnosis in 100% (24/24), 13 (54.2%) were clinically equivocal lesions, six (25%) were selected for ‘feature correlation’ of structures on dermoscopy or RCM, and five (20.8%) for ‘correlation of new/unknown’ RCM features seen on follow-up. The precision biopsy technique described herein is a novel method that facilitates direct histopathological correlation of dermoscopy and RCM features. With the aids of optical imaging devices, accurate diagnosis may be achieved by minimally invasive tissue extraction.

BACKGROUND: Ex vivo confocal microscopy (CM) works under two modes, fluorescence and reflectance, allowing the visualization of different structures. Fluorescence CM (FCM) requires a contrast agent and has been used for the analysis of basal cell carcinomas (BCC) during Mohs surgery. Conversely, reflectance CM (RCM) is mostly used for in vivo diagnosis of equivocal skin tumours. Recently, a new, faster ex vivo confocal microscope has been developed which simultaneously uses both lasers (fusion mode). OBJECTIVES: To describe the BCC features identified on reflectance, fluorescence and fusion modes using this novel device. To determine the best mode to identify characteristic BCC features. To develop a new staining protocol to improve the visualization of BCC under the different modes. METHODS: From September 2016 to June 2017, we prospectively included consecutive BCCs which were excised using Mohs surgery in our department. The lesions were evaluated using ex vivo CM after routine Mohs surgery. The specimens were first stained with acridine orange and then stained using both acetic acid and acridine orange. RESULTS: We included 78 BCCs (35 infiltrative, 25 nodular, 12 micronodular, 6 superficial). Most features were better visualized with the fusion mode using the double staining. We also identified new CM ex vivo features, dendritic and plump cells, which have not been previously reported. CONCLUSIONS: Our results suggest that nuclei characteristics are better visualized in FCM but cytoplasm and surrounding stroma are better visualized in RCM. Thus, the simultaneous evaluation of reflectance and fluorescence seems to be beneficial due to its complementary effect.

In vivo Reflectance confocal microscopy (RCM) of tumor-immune microenvironment (TiME) and microvasculature enables visualization and quantification of dynamic correlations between microvascular density, leukocyte-trafficking frequency, and spatio-temporal distribution of immune cell types for prognostic estimations.

Investigating the dynamic crosstalk between the tumor-immune microenvironment (TiME) and microvasculature in vivo in patients can lead to important insights into the underlying biology, help identify tumor phenotypes and reveal attractive druggable targets. Dynamic non-invasive label-free imaging of TiME and microvasculature in real-time directly in patients using reflectance confocal microscopy (RCM) was investigated on 60 skin cancer patients (basal cell carcinoma, BCC; squamous cell carcinoma, SCC), followed by automated and machine-learning based quantification of TiME and microvasculature features such as vascular density, leukocyte trafficking and immune cell density. Manual (two readers) and histopathological evaluation (dermatopathologist) of these features was also performed. Molecular correlation of imaging features and phenotypes was performed using anti-CD3/anti-CD20 IHC staining for tertiary lymphoid structures (TLS) and total lymphocyte density (n=33), flow cytometry for immune cells (n=3), and differential RNA expression (n=14). Correlation of RCM features and phenotypes at baseline (before treatment) with treatment response was also evaluated on 9 cancer lesions undergoing topical immunotherapy imiquimod. High agreement for feature presence on RCM and Histology, and manual and automated RCM features was observed. Unsupervised clustering on total TiME and microvasculature features on RCM using principal component analysis (PCA) indicates four distinct tumor phenotypes (PCA 1). The phenotype with high inflammation, high trafficking and higher density of vessels or the denoted ‘hot' phenotype correlated with higher activated CD8+ Granzyme B+ cells (67% of total CD8+cells). The clustering pattern on RCM was compared to TLS and lymphocyte density (PCA 2) and gene expression following CIBERSORT analysis (PCA 3). The clustering in RCM correlated better with gene expression (PCA 1 and 3, 100% agreement) than TLS and lymphocyte density (PCA 1 and 2, 86% agreement). The ‘hot' phenotype in RCM correlated with higher immune gene signatures and higher TLS/lymphocyte density. Increased plasma, CD8, activated CD4 memory and activated NK cells, M1 macrophages and monocytes, along with up-regulation of JAK-STAT, chemokine and cell adhesion signaling cascade were found in the ‘hot' RCM phenotype. Statistical modeling for correlating phenotypes with treatment outcomes was performed using principal component-linear discriminant analysis (PC-LDA). Two responders with tumor regression were predicted as ‘hot' phenotype while the non-responding patients (remaining 7) were classified as cold phenotype; suggesting that RCM 'hot' phenotype correlates with better treatment response. Thus, we demonstrate the potential utility of noninvasive RCM imaging in identifying ‘hot' and ‘cold' tumor phenotypes directly in patients. Citation Format: Aditi Sahu, Melissa Gill, Miguel Cordova, Anthony Santella, Kivanc Kose, Teguru Tembo, Anabel Alfonso, Pratik Chandrani, Christi Fox, Salvador Gonzalez, Nicholas Kurtansky, Melissa Pulitzer, William Phillips, Madison Li, Kimeil King, Stephen Dusza, Shuaitong Liu, Ning Yang, Haaris Jilani, Paras Mehta, Ashfaq Marghoob, Allan Halpern, Anthony Rossi, Liang Deng, Chih-Shan Jason Chen, Milind Rajadhyaksha. Dynamic imaging of tumor-immune microenvironment (TiME) and microvasculature identifies ‘hot' and ‘cold' tumor phenotypes in vivo in patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2814.

A diminished-staining artifact is observed in some Mohs frozen sections that are stained in toluidine blue (T-blue). Such an artifact, not yet described in the literature, may interfere with a Mohs surgeon's accurate reading. The authors hypothesize that topical hemostatic agents, aluminum chloride, and Monsel's solution are the causative factors.To evaluate the aforementioned topical hemostatic agents as a potential cause of the nonstaining artifact, to propose the mechanism associated with this phenomenon, and to develop a method to prevent or rectify the problem.Leftover Mohs frozen sections and specimens were treated with aluminum chloride or Monsel's solution and processed with routine Mohs histology.Nonstaining artifact is reproduced in aluminum chloride or Monsel's solution-treated ex vivo skin specimens. The authors found that ethylenediaminetetraacetic acid (EDTA), a chelating agent, can reverse the staining blockage. Such a finding suggests that aluminum or ferric cations bind to tissue and subsequently inhibit T-blue from interacting with the tissue. Direct binding of ferric cations to the tissue section is demonstrated with Prussian blue iron staining.By rinsing Mohs frozen sections in an EDTA solution before T-blue staining, the authors could prevent hemostatic agent-induced nonstaining. Applying an EDTA wash and restaining the slides can correct the same artifact.

The limited sampling of biopsy and histopathology can lead to incomplete and/or inaccurate assessment of basal cell carcinomas (BCCs), subtypes and depth, which can affect diagnosis and treatment outcome. Reflectance confocal microscopy (RCM) combined with optical coherence tomography (OCT) can help achieve comprehensive 3-dimensional sampling in vivo, which may improve the diagnostic accuracy and margin assessment of BCCs. In a clinical study, we tested a combined RCM-OCT probe on 85 patients, with either clinically-suspicious (n=60, in intact skin) or biopsy-proven BCCs (n=25, in scarred skin). We correlated BCC features in RCM and OCT images with histopathology, calculated diagnostic accuracy and correlated depth predicted by OCT with histopathologically measured depth. The main features were small tumors extending from the basal cell layer at the dermal-epidermal junction; small and large tumor nests; in dermis; dark silhouettes; dilated blood vessels; horn cyst and bright peritumoral stroma. Deeper features such as necrosis and intratumoral mucin pools were correlated on OCT and histology. Higher sensitivity and negative predictive value (100%) and comparable specificity (48% vs 56% on RCM) and positive predictive value (82.19 vs 84.59 % on RCM) were observed for the combined RCM-OCT device for diagnosis of all lesions (n=85). Relatively higher specificity (94.1%) and positive predictive value (75%) were observed in the clinically suspicious lesions (n=60, in intact skin). High correlation was observed (R=0.86) between the OCT predicted depth and histopathologically measured depth. Therefore, RCM-OCT imaging may be prospectively used to comprehensively diagnose suspicious BCC lesions, determine subtype and triage for treatment.

Background Merkel cell carcinoma (MCC) is a neuroendocrine carcinoma, associated with Merkel cell polyomavirus. MCC admixed with squamous cell carcinoma (SCC) is unassociated with polyomavirus, and is genetically distinct. Objective We sought to distinguish clinically and dermoscopically between MCC and SCC/MCC. Methods We compared patient data for SCC/MCC (n = 26) and MCC (n = 20), and reviewed clinical and dermoscopic images (n = 9) of SCC/MCC. Results Patients with SCC/MCC were older (median 76.5 vs 69 years) and more often male (77% vs 60%), and had more nonmelanoma skin cancer (85% vs 25%), malignant extracutaneous tumors (25% vs 5%), lymphoproliferative disorders (23% vs 10%), and immunodeficient/proinflammatory states (77% vs 35%). In all, 58% of SCC/MCC versus 10% of MCC were clinically diagnosed nonmelanoma skin cancer. Patients with SCC/MCC had more metastases (77% vs 40%), more treatment failures (53% vs 45%), shorter survival (41 vs 54 months), and more death from disease (50% vs 40%). SCC/MCC demonstrated marked scale (7/9), and telangiectasia (1/9). Dermoscopically, small dotted and short linear irregular peripheral vessels and central milky-red areas with large-diameter arborizing vessels were seen. Limitations The rarity of SCC/MCC limits available data. Conclusions SCC/MCC is aggressive, arising within elderly patients' chronically ultraviolet-exposed skin, often in the setting of immunosuppression or inflammation. Dermoscopically, polymorphous vessels in lesions suspicious for nonmelanoma skin cancer are suggestive.

Background Facing rising incidence of basal cell carcinoma (BCC) and increasing pressure to contain health care spending, physicians need to contemplate cost-effective paradigms for managing BCC. Objective We sought to perform a cost analysis comparing the traditional BCC management scheme with a simplified detect-and-treat scheme that eliminates the biopsy before initiating definitive treatment. Methods A decision analytic model was developed to compare the costs of traditional BCC management with the detect-and-treat scheme, under which qualifying lesions diagnosed clinically were either treated with shave removal or referred to Mohs micrographic surgery for on-site histologic check. Values for model parameters were based on literature and our institutional data analysis. Costs were based on 2014 Medicare fee schedule. Results The average cost per lesion with detect-and-treat scheme was $449 for non-Mohs micrographic surgery–indicated lesions (vs $566 with traditional management, $117 in savings) and $819 for Mohs micrographic surgery–indicated lesions (vs $864 with traditional management, $45 in savings). The combined weighted average savings per case was $95 (15% of total average cost). Conclusions were similar under various plausible scenarios. Limitations Model parameter values may vary based on individual practices. Conclusions A simplified management strategy eliminating routine pretreatment biopsy can reduce BCC treatment cost without compromising quality of care.

Laser ablation offers a procedure for precise, fast, and minimally invasive removal of superficial and early nodular basal cell carcinomas (BCCs). However, the lack of histopathological confirmation has been a limitation toward widespread use in the clinic. A reflectance confocal microscopy (RCM) imaging-guided approach offers cellular-level histopathology-like feedback directly on the patient, which may then guide and help improve the efficacy of the ablation procedure. Following an ex vivo benchtop study (reported in our earlier papers), we performed an initial study on 44 BCCs on 21 patients in vivo, using a pulsed erbium:ytterbium aluminum garnet laser and a contrast agent (aluminum chloride). In 10 lesions on six patients, the RCM imaging-guided detection of either presence of residual tumor or complete clearance was immediately confirmed with histopathology. Additionally, 34 BCCs on 15 patients were treated with RCM imaging-guided laser ablation, with immediate confirmation for clearance of tumor (no histopathology), followed by longer-term monitoring, currently in progress, with follow-up imaging (again, no histopathology) at 3, 6, and 18 months. Thus far, the imaging resolution appears to be sufficient and consistent for monitoring efficacy of ablation in the wound, both immediately postablation and subsequently during recovery. The efficacy results appear to be promising, with observed clearance in 19 cases of 22 cases with follow-ups ranging from 6 to 21 months. An additional 12 cases with 1 to 3 months of follow-ups has shown clearance of tumor but a longer follow-up time is required to establish conclusive results. Further instrumentation development will be necessary to cover larger areas with a more automatically controlled instrument for more uniform, faster, and deeper imaging of margins.

Laser ablation offers a procedure for precise, fast and minimally invasive removal of superficial and early nodular basal cell carcinomas (BCCs). However, the lack of histopathological confirmation has been a limitation toward widespread use in the clinic. A reflectance confocal microscopy (RCM) imaging-guided laser ablation approach offers cellular-level histopathology-like feedback directly on the patient, which may guide and help improve the efficacy of this procedure. We performed an initial study on 44 BCCs on 21 patients in vivo (based in an ex vivo bench-top study reported in our earlier papers), using a pulsed erbium: ytterbium aluminum garnet laser and a contrast agent (aluminum chloride). Initial 10 lesions, the RCM imaging-guided detection of either presence of residual tumor or complete clearance was immediately confirmed with histopathology. Additionally, 34 BCCs on 15 patients were treated with RCM imaging-guided laser ablation, and the clearance of tumor is currently being monitored with follow-up imaging (i. e., no histopathology) at 3, 6 and 18 months. Thus far, the imaging resolution appears to be sufficient and consistent for monitoring efficacy in the wound, both immediately post-ablation and subsequently during recovery. The efficacy appears to be promising. However, further investigation and optimization to image over the entire wound (without missing any areas) need to be investigated.

We present a multimodality mapping approach to guide staged excision for challenging lentigo maligna (LM) and lentigo maligna melanoma (LMM) lesions. LM/LMM often present with ill-defined clinical borders and subclinical tumor extension on the head and neck, where preservation of functionality and cosmesis can be critical. In our approach, pre-surgical evaluation of clinical margins is defined with the use of Wood’s Lamp, dermoscopy, confocal microscopy, and scouting punch biopsies. Once pre-operative margin mapping has been completed, a staged excision with complete pathologic assessment prior to final excision and subsequent closure is performed. Our approach ensures complete resection of these difficult-to-manage LM/LMM cases while preserving healthy tissue in order to minimize the impact on cosmesis and functionality.

Laser ablation offers precise and fast removal of superficial and early nodular types of basal cell carcinomas (BCCs). Nevertheless, the lack of histological confirmation has been a limitation. Reflectance confocal microscopy (RCM) imaging combined with a contrast agent can offer cellular-level histology-like feedback to detect the presence (or absence) of residual BCC directly on the patient. We conducted an ex vivo bench-top study to provide a set of effective ablation parameters (fluence, number of passes) to remove superficial BCCs while also controlling thermal coagulation post-ablation to allow uptake of contrast agent. The results for an Er:YAG laser (2.9 um and pulse duration 250us) show that with 6 passes of 25 J/cm 2 , thermal coagulation can be effectively controlled, to allow both the uptake of acetic acid (contrast agent) and detection of residual (or absence) BCCs. Confirmation was provided with histological examination. An initial in vivo study on 35 patients shows that the uptake of contrast agent aluminum chloride) and imaging quality is similar to that observed in the ex vivo study. The detection of the presence of residual tumor or complete clearance was confirmed in 10 wounds with (additional) histology and in 25 lesions with follow-up imaging. Our results indicate that resolution is sufficient but further development and use of appropriate contrast agent are necessary to improve sensitivity and specificity. Advances in RCM technology for imaging of lateral and deep margins directly on the patient may provide less invasive, faster and less expensive image-guided approaches for treatment of BCCs.

Mohs micrographic surgery is often considered the treatment of choice for a variety of skin malignancies. In recent years, the application of immunostaining techniques has facilitated the successful removal of a number of common and less common cutaneous malignancies, including basal cell carcinoma, squamous cell carcinoma, malignant melanoma, dermatofibrosarcoma protuberans, microcystic adnexal carcinoma, sebaceous carcinoma, atypical fibroxanthoma, extramammary Paget's disease, and even sarcomas. Immunostains highlight the tumor cells and allow the Mohs surgeons to pinpoint and eliminate the residual tumor at the surgical margin. It is especially helpful when a tumor presents with subtle or nonspecific histologic features or when a tumor is masked in a pocket of dense inflammation. However, the cost, the labor, and the time consumption are of concern to many of our peers, as are the diversity of antigens, which may overwhelm some. This article serves as a review of the literature on current uses of immunostaining in Mohs micrographic surgery and as a summary of their realistic applications in the dermatologic surgeon's practice. We conclude that immunohistochemical technique has played an important role in Mohs surgery advancement. With greater use and more cost-effective staining methods, we believe that the use of immunostains in a Mohs practice will become routine.

Importance Laser ablation is a rapid and minimally invasive approach for the treatment of superficial skin cancers, but efficacy and reliability vary owing to lack of histologic margin control. High-resolution reflectance confocal microscopy (RCM) may offer a means for examining margins directly on the patient. Observations We report successful elimination of superficial and early nodular basal cell carcinoma (BCC) in 2 cases using RCM imaging to guide Er:YAG laser ablation. Three-dimensional (3D) mapping is feasible with RCM to delineate the lateral border and thickness of the tumor. Thus, the surgeon may deliver laser fluence and passes with localized control—ie, by varying the ablation parameters in sublesional areas with specificity that is governed by the 3D topography of the BCC. We further demonstrate intraoperative detection of residual BCC after initial laser ablation and complete removal of remaining tumor by additional passes. Both RCM imaging and histologic sections confirm the final clearance of BCC. Conclusions and Relevance Confocal microscopy may enhance the efficacy and reliability of laser tumor ablation. This report represents a new translational application for RCM imaging, which, when combined with an ablative laser, may one day provide an efficient and cost-effective treatment for BCC.

The plasminogen activator (PA) proteolytic cascade comprises two enzymes known as urokinase PA (uPA) and tissue PA (tPA), both of which activate plasminogen to plasmin. In normal human epidermis uPA is the predominant PA. In lesional epidermis from patients with a variety of cutaneous diseases, including psoriasis, pemphigus foliaceous, pemphigus vulgaris, bullous pemphigoid, and benign chronic pemphigus, however, tPA is selectively elevated and becomes the predominant PA activity. The enhanced tPA is likely to be a reaction to the disease challenge rather than an initiating event in these clinically and etiologically diverse lesions. In the present study, cultured human keratinocytes, propagated under serum-free conditions, have been shown to respond to the addition of bovine or human serum with an increase in tPA activity and antigeri. Furthermore, tPA is found predominantly in the suprabasal keratinocytes both in lesional epidermis and in stratified cultures that have been incubated for approximately 8 d in the presence of serum. These results suggest a possible mechanism by which epidermal tPA may be increased in diverse cutaneous lesions: The plasma infiltrate, into lesional epidermis may stimulate the suprabasal keratinocytes in vivo to express tPA.

A 61-year-old white woman with a history of melanoma presented with an enlarging and asymptomatic lesion on her left arm for several months. Physical examination of the left deltoid revealed a 1.9 0.7-cm asymmetric, erythematous, and faintly brown plaque that was partially firm (Figure 1). Under dermoscopy, a patchy network and dotted blood vessels were observed. An excisional biopsy was performed for diagnosis (Figure 2 and Figure 3). What is your diagnosis?

Mohs micrographic surgery is often considered the treatment of choice for a variety of skin malignancies. In recent years, the application of immunostaining techniques has facilitated the successful removal of a number of common and less common cutaneous malignancies, including basal cell carcinoma, squamous cell carcinoma, malignant melanoma, dermatofibrosarcoma protuberans, microcystic adnexal carcinoma, sebaceous carcinoma, atypical fibroxanthoma, extramammary Paget's disease, and even sarcomas. Immunostains highlight the tumor cells and allow the Mohs surgeons to pinpoint and eliminate the residual tumor at the surgical margin. It is especially helpful when a tumor presents with subtle or nonspecific histologic features or when a tumor is masked in a pocket of dense inflammation. However, the cost, the labor, and the time consumption are of concern to many of our peers, as are the diversity of antigens, which may overwhelm some. This article serves as a review of the literature on current uses of immunostaining in Mohs micrographic surgery and as a summary of their realistic applications in the dermatologic surgeon's practice. We conclude that immunohistochemical technique has played an important role in Mohs surgery advancement. With greater use and more cost-effective staining methods, we believe that the use of immunostains in a Mohs practice will become routine.

Knowledge of the accurate margins of a lentigo maligna melanoma (LMM) is crucial in the presurgical evaluation of the patient. Towards this end clinicians have utilized the Wood's lamp and dermoscopy to help delineate the borders of the LMM. However, many LMMs arise on photodamaged skin, making it difficult to determine the border of the LMM and separate it from the background lentiginous skin. We present a case of a patient with a recurrent LMM on the scalp that developed in a background of photodamage with diffuse melanocytic atypia and lentigines, making it virtually impossible to determine the precise margins of the LMM by clinical, Wood's lamp or dermoscopic examination. To avoid subjecting the patient to multiple staged excisions we attempted to determine the margins of the LMM by utilizing in vivo confocal laser scanning reflectance microscopy. Using this, it was apparent that there were increased numbers of atypical/dendritic intraepidermal melanocytes in all layers of the epidermis within the LMM. In contrast, skin not involved with the LMM, as viewed under confocal laser examination, had normal honeycomb architecture and no abnormal melanocytes. The confocally determined border was further confirmed by obtaining multiple punch biopsies that were evaluated by haematoxylin and eosin histology and immunohistochemistry. Based on this information, the presurgical margins were marked and the tumour excised accordingly. The excised tissue was examined with multiple-step sections and the margins were determined to be clear. There has been no evidence of tumour recurrence after 1 year. In conclusion, this case illustrates that confocal reflectance microscopy, in conjunction with other in vivo optical instruments, can be utilized to enhance the accuracy for the presurgical margin mapping of LMM.

For the removal of superficial and nodular basal cell carcinomas (BCCs), laser ablation provides certain advantages relative to other treatment modalities. However, efficacy and reliability tend to be variable because tissue is vaporized such that none is available for subsequent histopathological examination for residual BCC (and to confirm complete removal of tumor). Intra-operative reflectance confocal microscopy (RCM) may provide a means to detect residual tumor directly on the patient and guide ablation. However, optimization of ablation parameters will be necessary to control collateral thermal damage and preserve sufficient viability in the underlying layer of tissue, so as to subsequently allow labeling of nuclear morphology with a contrast agent and imaging of residual BCC. We report the results of a preliminary study of two key parameters (fluence, number of passes) vis-à-vis the feasibility of labeling and RCM imaging in human skin ex vivo, following ablation with an erbium:yttrium aluminum garnet laser.