Purpose: The heterogeneity of tumor cells presents a major challenge to cancer diagnosis and therapy. Cutaneous T-cell lymphomas (CTCL) are a group of T lymphocyte malignancies that primarily affect skin. Lack of highly specific markers for malignant lymphocytes prevents early diagnosis, while only limited treatment options are available for patients with advanced stage CTCL. Droplet-based single-cell transcriptome analysis of CTCL skin biopsies opens avenues for dissecting patient-specific T lymphocyte heterogeneity, providing a basis for identifying specific markers for diagnosis and cure of CTCL. Experimental Design: Single-cell RNA-sequencing was performed by Droplet-based sequencing (10X Genomics), focusing on 14,056 CD3 þ lymphocytes (448 cells from normal and 13,608 cells from CTCL skin samples) from skin biopsies of 5 patients with advanced-stage CTCL and 4 healthy donors. Protein expression of identified genes was validated in advanced stage CTCL skin tumors by immunohistochemistry and confocal immunofluorescence microscopy. Results: Our analysis revealed a large inter-and intratumor gene expression heterogeneity in the T lymphocyte subset, as well as a common gene expression signature in highly proliferating lymphocytes that was validated in multiple advanced-stage skin tumors. In addition, we established the immunologic state of reactive lymphocytes and found heterogeneity in effector and exhaustion programs across patient samples. Conclusions: Single-cell analysis of CTCL skin tumor samples reveals patient-specific landscapes of malignant and reactive lymphocytes within the local microenvironment of each tumor, giving an unprecedented view of lymphocyte heterogeneity and identifying tumor-specific molecular signatures, with important implications for diagnosis and personalized disease treatment.

Talimogene laherparepvec (T-VEC) is the first OV approved for the treatment of melanoma and presents new challenges as it enters the clinical setting. Several other OVs are at various stages of clinical and pre-clinical development for the treatment of melanoma. Reports from phase Ib-III clinical trials combining T-VEC with checkpoint blockade are encouraging and demonstrate potential added benefit of combination immunotherapy. OVs have recently emerged as a standard treatment option for patients with advanced melanoma. Several OVs and therapeutic combinations are in development. Immunooncolytic virotherapy combined with immune checkpoint inhibitors is promising for the treatment of advanced melanoma.

◥Patients with resected stage II-III melanoma have approximately a 35% chance of death from their disease. A deeper understanding of the tumor immune microenvironment (TIME) is required to stratify patients and identify factors leading to therapy resistance. We previously identified that the melanoma immune profile (MIP), an IFN-based gene signature, and the ratio of CD8 þ cytotoxic T lymphocytes (CTL) to CD68 þ macrophages both predict disease-specific survival (DSS). Here, we compared primary with metastatic tumors and found that the nuclei of tumor cells were significantly larger in metastases. The CTL/macrophage ratio was significantly different between primary tumors without distant metastatic recurrence (DMR) and metastases. Patients without DMR had higher degrees of clustering between tumor cells and CTLs, and between tumor cells and HLA-DR þ macrophages, but not HLA-DR À macrophages. The HLA-DR À subset coexpressed CD163 þ CSF1R þ at higher levels than CD68 þ HLA-DR þ macrophages, consistent with an M2 phenotype. Finally, combined transcriptomic and multiplex data revealed that densities of CD8 and M1 macrophages correlated with their respective cell phenotype signatures. Combination of the MIP signature with the CTL/macrophage ratio stratified patients into three risk groups that were predictive of DSS, highlighting the potential use of combination biomarkers for adjuvant therapy.Significance: These findings provide a deeper understanding of the tumor immune microenvironment by combining multiple modalities to stratify patients into risk groups, a critical step to improving the management of patients with melanoma.

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BACKGROUND A growing body of literature suggests that Mohs micrographic surgeons can safely and successfully perform complex eyelid reconstruction. Given that up to 10 percent of all skin cancers occur on the periorbital skin, it is imperative that Mohs surgeons understand form and function to properly assess the defect and select the appropriate reconstruction method for a variety of eyelid defects. OBJECTIVE Our objective is to provide a thorough understanding of eyelid anatomy with an emphasis on form and function, provide a framework for defect analysis, and an algorithmic approach to defect analysis and appropriate selection of repair. METHODS AND MATERIALS A review of the literature on eyelid reconstruction was performed with specific reference to defect analysis and appropriately choosing repairs that are applicable to Mohs micrographic surgeons. CONCLUSION Mohs micrographic surgeons can safely and successfully perform complex eyelid repairs. An understanding of eyelid anatomy is the first step toward the best surgical outcome, and there are various methods for reconstructing eyelid defects. Defect size, location and analysis of the anterior lamella, posterior lamella, and the canthal regions helps to create an organized operative plan.