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Background Non-physician practitioners (NPPS), including nurse practitioners (NPs) and physician assistants (PAs) are expanding their scope of practice outside of primary care and performing more procedures in dermatology. Objective To understand the scope and geographic pattern of practice by NPs and PAs in dermatology in the US. Methods Cross-sectional retrospective cohort analysis of dermatology practices in the 2014 Medicare Physician/Supplier Procedure Summary Master File, which reflects Part B carrier and durable medical equipment fee-for-service claims in the US. Results Over 4 million procedures were billed independently by NPs and PAs, which accounts for 11.51% of all. Injection, simple repair, and biopsy were the most commonly billed by non-physician practitioners, but complex procedures were also increasingly billed independently by NPs and PAs. Proportions of their claims are higher in the East Coast, Midwest, and Mountain states. Limitations Data is at the state level, limited to Medicare beneficiaries, and doesn’t include billing incident-to physicians. Conclusions This study demonstrated the increasing scope of practice of NPs and PAs in dermatology, despite limited training and lack of uniform regulations. To ensure quality and safety of care, it is prudent to set benchmarks for proper supervision and utilization of procedures in dermatology.

Background Lentigo maligna/lentigo maligna melanoma (LM/LMM) can present with subclinical extension that may be difficult to define preoperatively and lead to incomplete excision and potential recurrence. Preliminarily studies have used reflectance confocal microscopy (RCM) to assess LM/LMM margins. Objective To evaluate the correlation of LM/LMM subclinical extension defined by RCM compared to the gold standard histopathology. Methods Prospective study of LM/LMM patients referred for dermatologic surgery. RCM was performed at the clinically-defined initial surgical margin followed by margin-controlled staged excision with paraffin-embedded tissue and histopathology was correlated with RCM results. Results Seventy-two patients were included. Mean age was 66.8 years (SD 11.1; 38 – 89 years); 69.4% were males. 70/72 (97.2%) lesions were located on the head neck with mean largest clinical diameter of 1.3cm (0.3 – 5 cm). Diagnostic accuracy for detection of residual melanoma in the tumor debulk (after biopsy) had a sensitivity of 96.7% and a specificity of 66.7% when compared to the histopathology. RCM margin assessment revealed an overall agreement with final histopathology of 85.9% (kappa 0.71; p Limitations No RCM imaging beyond initial planned margins was performed. Conclusion RCM showed moderate to excellent overall agreement between RCM imaging of LM/LMM and histopathology of staged excision margins.

Mohs micrographic surgery (MMS) is the treatment of choice for high-risk basal cell carcinoma (BCC). However, there are no evidence-based recommendations regarding which biopsy type is more appropriate to obtain tumour samples prior to MMS. Shave or punch biopsies are performed depending on the clinical characteristics of the tumour, surgeon experience and local protocols. However, biopsy type might result in difficult histopathological interpretation and influence the practical implementation of MMS. We performed a retrospective study on 208 consecutive BCCs treated with MMS. Of the 208 BCC biopsies, 42 (20.2%) were obtained by the shave method and 166 (79.8%) via punch. Those obtained with the shave technique had a mean of 1.64 stages vs. 1.69 stages with the punch technique (P = 0.130). These findings suggest biopsy type does not affect Mohs surgery performance. The biopsy type of choice is the one deemed adequate for each specific case to obtain a diagnosis and tumour subtyping.

Keratinocyte carcinomas are amenable to many treatments, including radiotherapy (RT). Electronic skin surface brachytherapy (ESSB) enables the precise delivery of radiation without radioisotopes.In this prospective multicenter clinical trial, we characterized early outcomes of ESSB prospectively through both patient- and clinician-reported measures. To corroborate the cosmesis observations, we also assessed patient-reported quality of life (QoL) and adverse events.Patients ≥60 years old with stage TCosmesis and QoL were collected at 97% (99/102) of possible follow-up patient-time points. By 12 weeks post-treatment, 93.9% (31/33) of patient-reported and 96.9% (31/32) of clinician-reported cosmesis outcomes were "good." Compared to baseline, total Skindex-16 score significantly deteriorated at 2 weeks post-treatment (10.5 vs 24.5, p0.001), but significantly improved at 6 weeks (10.5 vs 4.7, p=.014) and 12 weeks (10.5 vs 2.1, p=.001) post-treatment. Total SCI score significantly improved from baseline to 6 weeks (78.4 vs 89.0, p=.001) post-treatment. The most frequent adverse events were radiation dermatitis, skin pain, and pruritus. All adverse events resolved to Grade ≤1 by 12 weeks post-treatment.This prospective, multicenter study demonstrated that ESSB is associated with a high rate of "good" early patient-reported cosmesis and increasing QoL and satisfaction with time. Validated assessments demonstrated a significant improvement in quality of life and resolution of moderate early adverse events by 6-12 weeks after treatment and corroborate the observation of favorable cosmesis.

Lymphedema is a frequent debilitating condition among cancer patients. Daily supportive treatment may be necessary without long-term improvement. We describe two cases with chronic refractory lymphedema treated with fractional 10,600 nm CO2 laser. A 61-year-old female with locally advanced cervical cancer presented with postsurgical edematous swelling of the vulva and mons pubis and recurring cellulitis due to chronic lymphangiectasia. After six treatments of fractional CO2 laser, she noticed an 80% reduction of lymphorrea, swelling, and frequency of cellulitis. A 32-year old melanoma patient presented with refractory right lower leg lymphedema post right inguinal lymph node dissection and radiation. After fractional CO2 laser, she noted increased softness of her inguinal scar and a decrease of the lower leg edema. Fractional CO2 laser may be useful in addressing chronic refractory lymphedema. Further research should confirm our findings to consider fractional laser as a standard method in the treatment of chronic lymphedema.

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.

Prone positioning, a postural adjuvant therapy for improving ventilation, has been widely used to treat COVID-19 pneumonia complicated by acute respiratory distress syndrome that can reduce mortality when used for at least 12 hours a day.1 The extensive time needed for prone positioning to be effective may result in prolonged pressure points on the face leading to facial ulcers. Before the COVID-19 pandemic, facial pressure ulcers due to prone positioning in the setting of acute respiratory distress syndrome had already been described.1 These facial ulcers frequently occur at the level of the bony structures, including the forehead, cheekbone, and chin.1 It has been hypothesized that in the context of severe COVID-19 pneumonia, hypoxemia, microvascular injury, and thrombosis may increase the risk of pressure ulcers.1 Once healed, these wounds can have a lasting impact on the patient as they may lead to scarring, dyspigmentation, and atrophy. Patients find hypopigmented scars to be more bothersome than hyperpigmented scars.2 These scars are particularly stigmatizing distressing when in the cervicofacial region and in patients of color who represent more than 60% of the world's population.2, 3, 4 Hypopigmented scars are difficult for dermatologists to treat because there are limited effective and practical treatment options available that yield long-lasting results. Treatment options implicated in the management of hypopigmented scars include cosmetic camouflage, microneedling, dermabrasion, chemical peels, skin excision, and laser therapy. Herein, we report our success in achieving repigmentation in the setting of atrophic, depigmented, and hypopigmented scars using CO2 laser drug delivery of bimatoprost and microneedling.

BACKGROUND: There are multiple modalities for patient education ranging from written to audiovisual formats. However, little is known regarding which modality is optimal.OBJECTIVE: To assess patient preference for educational materials about scar care following surgery for facial skin cancer using the FACE-Q Skin Cancer patient reported outcome measure.MATERIALS AND METHODS: On the day of Mohs surgery, patients were given a written handout or viewed a 3-minute animation video regarding best practices in scar improvement. Afterward, patients received the FACE-Q Skin Cancer-Satisfaction with Information: Appearance scale. Three months later, patients were called and given the same scale and additional questions regarding scar care.RESULTS: A total of 75 patients were enrolled. There was no difference between the 2 groups' preoperative information scores (p = .85) and the three-month postoperative scores (p = .37). The change in preoperative and postoperative score showed no significant difference between the 2 groups (p = .21); but there was a trend of higher satisfaction in the video group on the day of Mohs surgery. After the 3-month timepoint, there was a higher satisfaction trend observed with the written handout group.CONCLUSION: Patient preferences in information delivery and accessibility will contribute to greater information retention and satisfaction.

During the approximately 5 days of Drosophila neurogenesis (late embryogenesis to the beginning of pupation), a limited number of neural stem cells produce approximately 200,000 neurons comprising hundreds of cell types. To build a functional nervous system, neuronal types need to be produced in the proper places, appropriate numbers, and correct times. We discuss how neural stem cells (neuroblasts) obtain so-called area codes for their positions in the nervous system (spatial patterning) and how they keep time to sequentially produce neurons with unique fates (temporal patterning). We focus on specific examples that demonstrate how a relatively simple patterning system (Notch) can be used reiteratively to generate different neuronal types. We also speculate on how different modes of temporal patterning that operate over short versus long time periods might be linked. We end by discussing how specification programs are integrated and lead to the terminal features of different neuronal types.

Background There are no well-defined clinical factors to predict the risk of occult invasion in melanoma of the lentigo maligna type (LM) before complete histopathologic analysis. Objective To evaluate whether clinical size was a predictor of invasion in LM and subclinical extension. Methods Consecutive cases of LM were recorded in a prospectively maintained database from 2006 to 2019. Patient and tumor data were recorded during initial evaluation. The LM clinical area was calculated in square millimeters (length × width). All patients were treated with staged excision. Results We included 600 patients. The mean age was 65.9 years (standard deviation, 12.3; range, 27-95 years); 62.8% (n = 377) were men. The mean LM clinical area was 128.32 mm2 for in situ lesions versus 200.14 mm for invasive lesions (P = .1). Based on quantile regression, the median margin required for complete removal increased with LM clinical area. Limitations The study was performed in a tertiary cancer center with possible referral bias and more complex cases. Conclusions LM can present with variable clinical size, which may correlate with subclinical extension; however, the presence of invasion is not well estimated by LM clinical area.

The second article in this continuing medical education series reviews the evidence regarding the intraoperative and postoperative risks for patients and health care workers. We share the most up-to-date recommendations for risk management and postoperative complication management to ensure optimal surgical efficacy and patient safety.

Metastatic primary cutaneous extramammary Paget disease (EMPD) is a rare clinical entity with a 5-year survival

While telemedicine has been utilized with more frequency over the past two decades, there remained significant barriers to its broad implementation. The COVID-19 global pandemic served as a stimulus for rapid expansion and implementation of telemedicine services across medical institutions worldwide in order to maximize patient care delivery, minimize exposure risk among healthcare providers and patients alike, and avoid overcrowding of patient care facilities. In this experience report, we highlight the teledermatology initiatives executed by the Dermatology Service at Memorial Sloan Kettering Cancer Center in New York City, with particular emphasis on image ingestion and potential for future automation and improvement.

BACKGROUND Extramammary Paget disease (EMPD) poses treatment challenges. Invasive and noninvasive treatment modalities exist with variable success reported. Reflectance confocal microscopy (RCM) is emerging as an adjuvant diagnostic tool. OBJECTIVE To evaluate the treatment of EMPD patients and the role of RCM. METHODS Prospective study. Demographic and tumor characteristics were recorded. Handheld-RCM was performed and correlated with histology. Treatment, clearance, pathology, and follow-up were all recorded. RESULTS Thirty-six EMPD lesions in 33 patients were included. Mean age was 71.7 years, and 23 were men. Mean number of surgical stages needed to clear margins was 1.9 (SD, 0.9; 1.0-3.0 stages), and mean margin needed to clear was 1.8 cm. Reflectance confocal microscopy correlated well with scouting punch biopsies (kappa, 0.93; p < .001). Disruption of the dermoepidermal junction was associated with invasive EMPD versus in situ (83.3% vs 25.9%) on histology (p = .01). LIMITATIONS Relatively small sample size. CONCLUSION Extramammary Paget disease is challenging, and lesion demarcation is of the utmost importance. Using a staged surgical excision approach, the mean margins needed were 1.8 cm, less than previously reported. Nonsurgical modalities, including radiation therapy, imiquimod, or photodynamic therapy can be considered if surgery is not pursued. Reflectance confocal microscopy is a valuable noninvasive imaging modality for the management of EMPD.

The brain consists of thousands of neuronal types that are generated by stem cells producing different neuronal types as they age. In Drosophila, this temporal patterning is driven by the successive expression of temporal transcription factors (tTFs)(1-3) We used single-cell mRNA sequencing to identify the complete series of tTFs that specify most Drosophila optic lobe neurons. We verify that tTFs regulate the progression of the series by activating the next tTF(s) and repressing the previous one(s), and also identify more complex regulations. Moreover, we establish the temporal window of origin and birth order of each neuronal type in the medulla and provide evidence that these tTFs are sufficient to explain the generation of all the neuronal diversity in this brain region. Finally, we describe the first steps of neuronal differentiation. We find that terminal differentiation genes, such as neurotransmitter-related genes, are present as transcripts, but not as proteins, in immature larval neurons; we show that these steps are conserved in humans. This comprehensive analysis of a temporal series of tTFs in the optic lobe offers mechanistic insights into how tTF series are regulated, and how they can lead to the generation of a complete set of neurons.

BACKGROUND: Squamous cell carcinoma in situ (SCCIS) of the nail unit is a complex malignancy; with little understanding of rate of upstaging or occult invasion in these patients. OBJECTIVES: We sought to evaluate the rate of upstaging in nail unit SCCIS after Mohs micrographic surgery (MMS). METHODS: Retrospective review of 346 patients who referred for and underwent MMS for biopsy proven SCCIS on the hands and feet between January 1, 2000 and December 30, 2019. Only cases in the nail unit were included. Clinical, surgical details, histopathological features, HPV status and rate of upstaging were recorded. RESULTS: Thirty-one cases met the inclusion criteria and were analyzed. Twenty-four patients were male (77.4%). Mean age was 55 years (SD 17.26, range 27–84). Mean clinical size was 9.9 mm; 19 cases tested for HPV, 15/19 were positive (78.9%) and 8/19 (42.1%) were associated with high-risk HPV. Three patients (9.7%) were upstaged to invasive on either MMS margins or tumor-debulking. Limitations included a relatively small sample size and retrospective in nature. CONCLUSION: Rate of upstaging of SCCIS in the nail unit is not frequent, and when upstaging occurred it was focal, superficial, and with no PNI or bone invasion.

INTRODUCTION: Recently, a combined reflectance confocal microscopy (RCM)-optical coherence tomography (OCT) has been tested for the diagnosis of basal cell carcinoma (BCC). Evaluating the role of RCM-OCT in management of complex BCCs has not been studied. The objective of the study was to investigate the utility of a new combined RCM-OCT device in the evaluation and management of complex BCCs in a descriptive study. METHODS: Prospective study of consecutive cases (July 2018- June 2019) of biopsy proven ‘complex’ BCC defined as BCC in the head and neck area with multiple high risk criteria such as large size in the mask area, multiple recurrences, and infiltrative subtype. All cases were evaluated with a combined RCM-OCT device that provided simultaneous image viewing on a screen. Lesions were evaluated bedside with RCM-OCT according to previously described criteria. RESULTS: Ten patients with complex head and neck BCCs had mean age of 73.1±13.0 years. Six (60%) patients were males. Mean BCC clinical size was 1.9±1.2 cm (range 0.6–4.0 cm). RCM detected residual BCC in 8 out of 10 cases (80%) and OCT detected residual BCC in all 10 cases (100%). Six BCCs (60%) had a depth estimate of >1000 μm under OCT. In 5 cases, (50%) RCM-OCT imaging results led to a change/modification in BCC management. CONCLUSION: The use of a combined RCM-OCT device may help in the evaluation of complex head and neck BCCs by guiding treatment selection and defining the extent of surgery.

BACKGROUND Men represent a growing segment of the facial aesthetic market. OBJECTIVE To evaluate investigator-assessed efficacy, patient-reported outcomes, and safety after onabotulinumtoxinA treatment of forehead lines (FHL) in men. METHODS Subjects with moderate to severe FHL received onabotulinumtoxinA (frontalis: 20 U; glabellar complex: 20 U, with/without 24 U in crow's feet regions) or placebo in 6-month, double-blind periods of 2 pivotal trials. Results for men were pooled. RESULTS Men comprised 12% (140/1,178) of subjects. Day 30 male responder rates for achieving at least 1-grade Facial Wrinkle Scale (FWS) improvement at maximum eyebrow elevation and at rest were 98.2% and 93.3%, respectively; a significant difference in responder rates was maintained versus placebo (p < .05) through Day 150. Despite men having proportionately more severe FHL at baseline, 81.8% and 79.8% achieved Day 30 FWS ratings of none or mild at maximum eyebrow elevation and at rest, respectively (p < .05); significance versus placebo was maintained through Day 120. Men reported high satisfaction rates and improved psychological impacts. No new safety signals were detected. CONCLUSION Standard dosing and administration of onabotulinumtoxinA significantly improved static and dynamic FHL appearance, despite men having proportionately more severe FHL at baseline. Men reported high satisfaction and appearance-related psychological impact improvements.

Extramammary Paget disease (EMPD) is a frequently recurring malignant neoplasm with metastatic potential that presents in older adults on the genital, perianal, and axillary skin. Extramammary Paget disease can precede or occur along with internal malignant neoplasms.To develop recommendations for the care of adults with EMPD.A systematic review of the literature on EMPD from January 1990 to September 18, 2019, was conducted using MEDLINE, Embase, Web of Science Core Collection, and Cochrane Libraries. Analysis included 483 studies. A multidisciplinary expert panel evaluation of the findings led to the development of clinical care recommendations for EMPD.The key findings were as follows: (1) Multiple skin biopsies, including those of any nodular areas, are critical for diagnosis. (2) Malignant neoplasm screening appropriate for age and anatomical site should be performed at baseline to distinguish between primary and secondary EMPD. (3) Routine use of sentinel lymph node biopsy or lymph node dissection is not recommended. (4) For intraepidermal EMPD, surgical and nonsurgical treatments may be used depending on patient and tumor characteristics, although cure rates may be superior with surgical approaches. For invasive EMPD, surgical resection with curative intent is preferred. (5) Patients with unresectable intraepidermal EMPD or patients who are medically unable to undergo surgery may receive nonsurgical treatments, including radiotherapy, imiquimod, photodynamic therapy, carbon dioxide laser therapy, or other modalities. (6) Distant metastatic disease may be treated with chemotherapy or individualized targeted approaches. (7) Close follow-up to monitor for recurrence is recommended for at least the first 5 years.Clinical practice guidelines for EMPD provide guidance regarding recommended diagnostic approaches, differentiation between invasive and noninvasive disease, and use of surgical vs nonsurgical treatments. Prospective registries may further improve our understanding of the natural history of the disease in primary vs secondary EMPD, clarify features of high-risk tumors, and identify superior management approaches.

Background and objectives Many cutaneous drug-delivery techniques rely on passive diffusion to deliver topical compounds to the skin. When attempting to deliver drugs to thicker lesions, such as skin tumors, modalities that do not rely on diffusion may serve as a better drug-delivery method. In this histological study, we aim to investigate the cutaneous delivery patterns of an electronic pneumatic needle-free injection device. Study design/materials and methods Needle-free-injection was investigated in 24 ex vivo porcine skin samples and one basal cell carcinoma (BCC) tissue sample. A needle-free injection device with a nozzle size of 200 μm delivered 80 μl compound ink (0.1 cc black ink: 5.0 cc saline) at low (30%/3.1 bar; n = 6 porcine skin; n = 1 BCC tissue), medium (50%/3.9 bar; n = 6 porcine skin), high (65%/4.6 bar; n = 6 porcine skin), and stacked (30 + 50%/3.1 + 3.9 bar; n = 6 porcine skin) pressures. Depth, width, and depth of maximum width of ink deposition were evaluated on histological slides. Results Depositions with small ink-lined vacuoles were seen intra-dermally in all samples, including the BCC tissue. Deposition depth was similar at low and medium pressures (924 vs. 994 μm; P = 0.873) but increased significantly with high pressure (1,564 μm; P = 0.010). When injections were stacked (3.1 + 3.9 bar), the depth remained similar to that of a single injection (931 μm; P = 1.000). The width of the deposition stayed comparable for low, medium, and high pressures when a single needle-free injection was performed (30% = 2,394 μm; 50% = 2,226 μm; and 65% = 2,757 μm; P = 0.09), but increased significantly with stacking (2,979 μm; P = 0.037). The depth of maximal width was superficially located in the papillary dermis at low and medium pressures (321 and 305 μm; P = 0.748) but shifted to the deeper reticular dermis with high pressure (950 μm; P = 0.004) and with stacking (734 μm; P = 0.004). Conclusions In conclusion, with an electronically controlled, pneumatic needle-free injector, depth and width of a cutaneous deposition can be influenced by pressure and stacking, respectively. The pneumatic needle-free injection can potentially serve as a viable drug-delivery technique for cutaneous pathologies where dermal deposition is essential. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

SummaryIntratumoral (IT) delivery of immune-activating viruses can serve as an important strategy to turn “cold” tumors into “hot” tumors, resulting in overcoming resistance to immune checkpoint blockade (ICB). Modified vaccinia virus Ankara (MVA) is a highly attenuated, non-replicative vaccinia virus that has a long history of human use. Here we report that IT recombinant MVA (rMVA), lacking E5R encoding an inhibitor of the DNA sensor cyclic GMP-AMP synthase (cGAS), expressing a dendritic cell growth factor, Fms-like tyrosine kinase 3 ligand (Flt3L), and a T cell co-stimulator, OX40L, generates strong antitumor immunity, which is dependent on CD8+ T cells, the cGAS/STING-mediated cytosolic DNA-sensing pathway, and STAT1/STAT2-mediated type I IFN signaling. Remarkably, IT rMVA depletes OX40hi regulatory T cells via OX40L/OX40 interaction and IFNAR signaling. Taken together, our study provides a proof-of-concept for improving MVA-based cancer immunotherapy, through modulation of both innate and adaptive immunity.One Sentence SummaryIntratumoral delivery of recombinant MVA for cancer immunotherapy

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.

BACKGROUND The rate of occult invasive disease within biopsy-proven squamous cell carcinoma in situ (SCCIS) is not well defined. OBJECTIVE To examine the rate of occult invasion in SCCIS. METHODS An institutional-wide pathology database was retrospectively searched for "squamous cell carcinoma in situ" over a 6-year period, and the treatment modality and final pathology results were analyzed for evidence of invasion. In addition, consecutive tumor blocks from Mohs micrographic surgery (MMS) cases of SCCIS were prospectively analyzed for invasion. RESULTS The rate of occult invasion for biopsy-proven SCCIS treated with excision was 4.0% (N = 49) and for the MMS margins was 3.5% (N = 310). For the prospective MMS tumor block analysis, the rate of occult invasion was 10.1% (N = 69). No clinical factors (age, sex, location, or size) correlated with increased risk of invasion. All invasive SCCs detected were superficial and well differentiated. CONCLUSION The rate of occult invasion for biopsy-proven SCCIS ranged from 3.5% to 10.1%. The detected rate of invasion varies based on the method of excision and pathology processing of specimens. The rate of invasion should be considered when managing SSCIS.

Background Basal cell carcinoma (BCC) treatment modalities can be stratified based on tumor subtype and recurrence risk. The main limitation of non-surgical treatment modalities is the lack of histopathological confirmation. Reflectance confocal microscopy (RCM) is a non-invasive imaging device that provides quasi-histological images. Objective To evaluate the feasibility and efficacy of RCM-guided carbon dioxide (CO2) laser ablation of low-risk BCCs. Methods Prospective study with biopsy-proven low-risk BCCs imaged with RCM. RCM was performed on these sites and ablated; if residual tumor was found, a new series of laser passes were performed. The patients were then followed for recurrence clinically and with RCM. Results Twenty-two tumor sites in nine patients were imaged and treated. Median age was 59±12.9 years (range 30 – 74). Male-to-female ratio was 5:4. Mean tumor size was 7.7 mm (range 5 – 10 mm). Residual tumor was identified in 5/22 cases (22.7%) under RCM on immediate first pass post-ablation sites, prompting additional laser passes. Median follow-up was 28.5 month (22 – 32 months) with no recurrences found. Conclusions Addition of RCM to laser ablation workflow can detect subclinical persistent tumor after initial ablation and may serve as an aid to increase the efficacy of laser ablation.