Faculty Bibliography

Primary cutaneous squamous cell carcinoma (cSCC) is the second most common human cancer with a rising incidence of about 1.8 million in the United States annually. Primary cSCC is usually curable by surgery; however, in some cases, cSCC eventuates in nodal metastasis and death from disease specific death. cSCC results in up to 15,000 deaths each year in the United States. Until recently, non-surgical options for treatment of locally advanced or metastatic cSCC were largely ineffective. With the advent of checkpoint inhibitor immunotherapy, including cemiplimab and pembrolizumab, response rates climbed to 50%, representing a vast improvement over chemotherapeutic agents used previously. Herein, we discuss the phenotype and function of SCC associated Langerhans cells, dendritic cells, macrophages, myeloid derived suppressor cells and T cells as well as SCC-associated lymphatics and blood vessels. Possible role(s) of SCC-associated cytokines in progression and invasion are reviewed. We also discuss the SCC immune microenvironment in the context of currently available and pipeline therapeutics.

Deliberate practice-based medical education has demonstrated superiority in trainee acquisition and maintenance of skills in several surgical subspecialties. In an effort to highlight the impact of a deliberate practice-based surgical curriculum on the technical proficiency of dermatology residents, a prospective cohort study including first- and second-year dermatology residents was performed. A total of 87.5% (7 of 8) first-year dermatology residents completed three hands-on simulations at 6-week intervals. Additionally, six of eight (75.0%) second-year dermatology residents at the same institution were evaluated at a single point-in-time session without accessing the surgical curriculum prior. A 5-point global rating scale (GRS) was used to assess resident performance on six core surgical techniques. Nonparametric ANOVA statistical methods using the Kruskal-Wallis test was performed. The residents' overall GRS increased from a median of 1-2.75 after completion of the curriculum (p < 0.01). There was a significant improvement in the median scores of each tested surgical technique. The first-year residents had a greater overall GRS after completion of the curriculum compared to the second-year residents (median of 2.13 versus 1.88, p < 0.001). Limitations include the small sample size and lack of a synchronized control group. Our study highlights the use of deliberate practice-based strategies as an effective modality in teaching surgical skills to dermatology residents.

Background/Purpose: The skin is recognized as a window into the immunopathogenic mechanisms driving the vast phenotypic spectrum of psoriatic disease.
Method(s): To better decipher the cellular landscape of both healthy and psoriatic skin, we employed spatial transcriptomics (ST), a ground-breaking technology that precisely maps gene expression from histologically-intact tissue sections (Fig. 1A).
Result(s): Findings gleaned from computationally integrating our 23 matched lesional and non-lesional psoriatic and 7 healthy control samples with publicly-available single-cell ribonucleic acid (RNA) sequencing datasets established the ability of ST to recapitulate the tissue architecture of both healthy and inflamed skin (Fig. 1B) and highlighted topographic shifts in the immune cell milieu, from a predominantly perifollicular distribution in steady-state skin to the papillary and upper reticular dermis in psoriatic lesional skin. We also incidentally discovered that ST's ability to ascertain gene expression patterns from intact tissue rendered it particularly conducive to studying the transcriptome of lipid-laden cells such as dermal adipose tissue and sebaceous glands (Fig. 1C), whose expression profiles are typically lost in the process of tissue handling and dissociation for bulk and single-cell RNA seq. Unbiased clustering of pooled healthy and psoriatic samples identified two epidermal clusters and one dermal cluster that were differentially expanded in psoriatic lesional skin (p values <=0.05) (Fig. 1D); pathway analysis of these clusters revealed enrichment of known psoriatic inflammatory pathways (Fig. 1E). Unsupervised classification of skin-limited psoriasis and psoriatic arthritis samples revealed stratification by cutaneous disease severity or Psoriasis Area and Severity Index (PASI) score and not by presence or absence of concomitant systemic/synovial disease (Fig. 1F). Remarkably, this PASI-dependent segregation was also evident in distal, non-lesional samples and was driven by the dermal macrophage and fibroblast cluster and the lymphatic endothelium (Fig. 2A). Inquiry into the mechanistic drivers of this observed stratification yielded enrichment of pathways associated with key T cell and innate immune cell activation, B cells, and metabolic dysfunction (Fig. 2B). Finally, tissue scale computational cartography of gene expression revealed differences in regional enrichment of specific cell types across phenotypic groups, most notably upward extension of fibroblasts to the upper dermis in both lesional and non-lesional samples from mild psoriasis and restriction to the lower dermis in the moderate-to-severe psoriasis samples (Fig. 2C), suggesting that disease severity stratification may be driven by emergent cellular ecosystems in the upper dermis. Fig. 1. (A) Schematic of spatial transcriptomics study workflow. Four mm skin punch biopsies were obtained from healthy volunteers (n=3) and lesional and non-lesional skin from patients with psoriatic disease (n=11). Ten micron-thick sections were then placed on capture areas on the ST microarray slide, each containing molecularly barcoded, spatially encoded spots with a diameter of 50 microns and a center-to-center distance of 100 microns. (B) Side-by-side comparison of a hematoxylin-eosin (H&E) stained section of representative healthy, lesional, and non-lesional skin samples and the corresponding ST plots showed concordance of unbiased gene expression-based clustering with histologic tissue architecture. (C) Pathway analysis of the adipose cluster in healthy skin (cluster 2) confirmed upregulation of lipid-associated processes. Inset: Spots corresponding to the adipose cluster highlighted in yellow. (D) Wilcoxon rank sum test (results displayed as box plots) yielded statistically significant expansion of three clusters in lesional skin compared to both non-lesional and healthy skin-inflamed suprabasal epidermis (cluster 4), epidermis 2 (cluster 7), and inflamed dermis (cluster 10). HC=healthy control, L=lesional psoriatic skin, NL=non-lesional psoriatic skin. (E) Pathways enriched in clusters 4, 7, and 10. (F) Principal component analysis (PCA) plots demonstrating segregation of samples by severity of cutaneous disease in both lesional and non-lesional samples along the first principal component (right) that was not seen in the samples categorized according to presence or absence of arthritis (left). PsA=psoriatic arthritis, PsO=skin-limited psoriasis. Fig. 2. (A) PCA of lesional and non-lesional samples colored by disease severity in spatial clusters 1 (left) and 12 (right) revealed more discrete clustering. (B) Pathways significantly enriched in clusters 1 (left) and 12 (right) showed enrichment of pathways associated with key T cell and innate immune cell activation, B cells, and metabolic dysfunction (highlighted in red). (C) SpaceFold one dimension projection of cell distribution from an independently-generated single-cell RNA seq data set on aggregated ST lesional and non-lesional samples from mild (PASI-low) and moderate-severe (PASI-high) samples. Y-axis represents tissue position, starting with the lower dermis marked as position 0 to suprabasal epidermis marked as position 1. Dashed line represents epidermal-dermal junction, discerned by cell types in the basal epidermal layer (melanocytes and Langerhans cells). Fibroblast signatures (red arrows) were largely relegated to the lower dermis in the PASI-high group, but extended to the upper dermis in the PASI-low group. This striking difference in fibroblast localization was also noted in non-lesional PASI-high vs. PASI-low groups. In addition to fibroblasts, lymphatic, endothelial, myeloid, and T cells signatures (black arrows) were also observed in the upper dermis of lesional PASI-low samples, but were much lower in the dermis of PASI-low non-lesional and all samples in the PASI-high group. Interfollicular epidermis (IFE), hair follicle and infundibulum (HF/IFN), n= number of individual biopsies.
Conclusion(s): Thus, we have been able to successfully leverage ST integrated with independently-generated single-cell RNA seq data to spatially define the emergent cellular ecosystems of healthy and matched psoriatic lesional and non-lesional skin and in so doing, demonstrated the value of ST in unearthing the genetic groundwork at both the site of inflammation and in distal, clinically-uninvolved skin

Background: The immune tumor microenvironment (TME) is key to controlling disease progression in skin cancer. We aim to compare the TME in these cancers to better inform prognosis and therapeutic decision making.
Method(s): CD8+ tumor infiltrating lymphocytes (TILs) obtained from fresh immunocompetent SCC (n=5) and BCC (n=12) tumors and solid-organ transplant recipient SCC ("TSCC", n=6) tumors were subject to single-cell RNA profiling. Data were analyzed using iCellR.
Result(s): CD8+ TILs were clustered based on gene expression: cytotoxic (GZMA, GZMB, IFN-gamma, PRF1); naive (CCR7, LEF1, TCF7, IL7R, OX40); exhausted (BTLA, CTLA4, PDCD1, TIM3, LAG3, STAT3, 4-1BB); and regulatory (FOXP3, TIM3, OX40); additional subtypes were observed for naive, exhausted, and regulatory T cells. Similar percentages of cytotoxic TILs were observed in SCC and BCC, significantly greater than in TSCC. Conversely, TSCC had the highest percentage of exhausted TILs. TSCC exhausted cells also tended to be more terminally exhausted, while those in SCC and BCC tended to be more progenitor exhausted. While the overall percentage of the regulatory CD8 compartment was similar across all three groups, SCC had a significantly greater proportion of a more suppressive subtype.
Conclusion(s): Many significant differences in the CD8 TME were observed between SCC, TSCC, and BCC. TSCC was associated with a proportionally larger and extensively terminally exhausted CD8 compartment. BCC was also associated with a significantly greater exhausted compartment than SCC, possibly correlating to the relatively slow-growing clinical course of BCC. Additionally, SCC was associated with a significantly greater proportion of more suppressive regulatory CD8s, possibly correlating to the greater clinical aggressiveness of SCC.
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Background: TCR repertoire of TILs is increasingly used as a fingerprint for identifying unique immune responses to cancer and as a biomarker for prognosticating clinical course and response to immunotherapy.
Method(s): CD8+ tumor infiltrating lymphocytes (TILs) obtained from fresh basal cell carcinoma ("BCC") tumor specimens from nodular subtype ("nBCC", n=6) versus infiltrative subtype ("iBCC", n=6) were subject to single-cell VDJ profiling. Data were analyzed using iCellR and ImmunArch.
Result(s): Pooled CD8+ TILs from nBCC and iBCC generated a significantly more diverse TCR repertoire than CD8+ T cells from healthy skin, but a significantly less diverse repertoire than CD8+ from healthy PBMCs. TCR gene bias analysis revealed that nBCC and iBCC TILs expressed a differential preference for certain V genes on both the alpha and beta CDR3 chain. Clonotype analysis revealed the top 10 most expanded clonotypes among iBCC TILs represented 25% of the TCR repertoire versus 20% of the nBCC TIL repertoire; also, iBCC TIL singletons represented 37% of the TCR repertoire while nBCC TIL singletons represented 50% of the TCR repertoire, indicating a greater degree of clonality among iBCC TILs. Additionally, cytotoxic TILs recovered from nBCC possessed a more diverse TCR repertoire than those from iBCC. Moreover, cytotoxic TILs from nBCC possessed a distinct TCR repertoire with little overlap with exhausted TILs; cytotoxic TILs from iBCC possess a TCR repertoire that highly overlapped with exhausted TILs.
Conclusion(s): TILs from nBCC generally possessed a less clonal TCR repertoire than iBCC TILs, possibly contributing to observed relative clinical aggressiveness of these tumors. Additionally, TCR repertoire in exhausted TILs in iBCC overlapped more with cytotoxic TILs, informing clinical decision-making with regards to check point inhibitor immunotherapy.
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Dermatomyositis (DM) is an autoimmune connective tissue disease that most commonly affects skin and muscles. Clinical manifestations can be protean, with some patients experiencing skin-limited disease while others develop serious systemic complications including interstitial lung disease (ILD). While both T cell-mediated and humoral immune dysregulation have been purported to play a role in manifesting said end-organ damage, our understanding of immunophenotypic prognostic factors associated with the development of ILD in DM patients remains limited. Thus, we sought to identify potential biomarkers that might be differentially expressed in lesional skin obtained from DM patients with versus without ILD. Utilizing NanoString technology, we assessed differential mRNA expression of 800 immune-related genes in formalin-fixed, paraffin-embedded lesional skin samples obtained from five DM patients with ILD compared to five DM patients without ILD. Among 42 highly regulated genes, 10 were significantly (p<0.05) upregulated in DM patients with ILD: CD44, NFKBIZ, CD24, EGR1, DEFB103B, CCL18, CCL22, CXCL2, S100A8 and S100A9. Notably, serum levels of S100A8/A9 heterodimer (an endogenous ligand of Toll-like receptor 4) are known to correlate with clinical severity in patients with DM-associated ILD, thereby supporting NanoString's utility in identifying salient genes associated with end-organ damage in DM. We also identified several novel genes downregulated in patients with DM-associated ILD that play a role in autophagy and adaptive immune activation: CLEC7, LEF1, KLRC2, BTLA, MUC1, and TCF7. Taken together, our results begin to characterize an immune-related gene expression signature in lesional DM skin that may be associated with comorbid ILD. While validation experiments are ongoing, the identification of biomarkers of systemic disease in lesional DM skin not only has the potential for risk stratifying DM patients at the time of tissue diagnosis but may provide novel targets for early intervention against DM-associated ILD.
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Background: Immune response is key in defense against basal cell carcinoma (BCC). We aim to better define the immune tumor microenvironment in this cancer.
Method(s): CD8+ tumor infiltrating lymphocytes (TILs) obtained from fresh BCC tumor specimens from nodular subtype ("nBCC", n=6) versus infiltrative subtype ("iBCC", n=6) were subject to single-cell RNA profiling. Data were analyzed using iCellR.
Result(s): CD8+ TILs were clustered based on gene expression as follows: cytotoxic (GZMA, GZMB, IFN-gamma, PRF1); naive (CCR7, LEF1, TCF7, IL7R, OX40); exhausted (BTLA, CTLA4, PDCD1, TIM3, LAG3, STAT3, 4-1BB); and regulatory (FOXP3, TIM3, OX40); additional subtypes were observed for naive, exhausted, and regulatory T cells. A significantly greater percentage of cytotoxic TILs and lesser percentage of naive TILs were observed in iBCC compared to nBCC. Further analysis revealed a unique, pro-tumor-controlling naive subtype in nBCC and a unique, highly suppressive regulatory subtype in iBCC; moreover, regulatory TILs in nBCC exclusively clustered in a low suppressive activity subtype. Additionally, percentage of all exhausted TILs was not significantly different across tumor subtype; however, there was a significantly greater proportion of pro-tumor-controlling progenitor exhausted and terminally exhausted TILs in nBCC.
Conclusion(s): Two significantly greater proportions of pro-tumor controlling exhausted subtypes and one significantly lower proportion of highly suppressive regulatory subtype were identified in nBCC compared to iBCC, possibly contributing to observed relative aggressiveness of these tumors. Additionally, more exhausted TILs from nBCC tended to cluster in a progenitor exhausted phenotype that may be more responsive to immune checkpoint blockade therapy.
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