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Melanomas from gynecologic sites (MOGS) are rare and have poor survival. MicroRNAs (miRs) regulate gene expression and are dysregulated in cancer. We hypothesized that MOGS would display unique miR and mRNA expression profiles. The miR and mRNA expression profile in RNA from formalin fixed, paraffin embedded vaginal melanomas (relative to vaginal mucosa) and vulvar melanomas (relative to cutaneous melanoma) were measured with the Nanostring Human miRNA assay and Tumor Signaling mRNA assay. Differential patterns of expression were identified for 21 miRs in vaginal and 47 miRs in vulvar melanoma (fold change >2, p<0.01). In vaginal melanoma, miR-145-5p (tumor suppressor targeting TLR4, NRAS) was downregulated and miR-106a-5p, miR-17-5p, miR-20b-5p (members of miR-17-92 cluster) were upregulated. In vulvar melanoma, known tumor suppressors miR-200b-3p and miR-200a-3p were downregulated, and miR-20a-5p and miR-19b-3p, from the miR-17-92 cluster, were upregulated. Pathway analysis showed an enrichment of "proteoglycans in cancer". Among differentially expressed mRNAs, topoisomerase IIα (TOP2A) was upregulated in both MOGS. Gene targets of dysregulated miRs were identified using publicly available databases and Pearson correlations. In vaginal melanoma, suppressor of cytokine signaling 3 (SOCS3) was downregulated, was a validated target of miR-19b-3p and miR-20a-5p and trended toward a significant inverse Pearson correlation with miR-19b-3p (p = 0.093). In vulvar melanoma, cyclin dependent kinase inhibitor 1A (CDKN1A) was downregulated, was the validated target of 22 upregulated miRs, and had a significant inverse Pearson correlation with miR-503-5p, miR-130a-3p, and miR-20a-5p (0.005 < p < 0.026). These findings support microRNAs as mediators of gene expression in MOGS. [ABSTRACT FROM AUTHOR]

Sentinel lymph node biopsy (SLNB) is a standard practice for staging cutaneous melanoma. High false-negative rates have an increased interest in adjunctive techniques for localizing SLNs. Mobile gamma cameras (MGCs) represent potential tools to enhance SLNB performance. An institutional review board approval was obtained for this study (ClinicalTrials.gov ID NCT01531608). After obtaining informed consent, 20 eligible melanoma patients underwent 99mTc sulfur colloid injection and standard lymphoscintigraphy with a fixed gamma camera (FGC). A survey using a 20 cm square MGC, performed immediately preoperatively by the study surgeon, was used to establish an operative plan while blinded to the FGC results. Subsequently, SLNB was performed using a gamma probe and a novel 6 cm diameter handheld MGC. A total of 24 SLN basins were detected by FGC. Prior to unblinding, all 24 basins were identified with the preoperative MGC and the operative plan established by preoperative MGC imaging was confirmed accurate by review of the FGC images. All individual sentinel lymph nodes were identified during intraoperative MGC imaging, and in 5/24 (21%) cases, surgeon-reported additional clinically useful information was obtained from the MGC. Preoperative MGC images provide information consistent with FGC images for planning SLNB and in some cases provide additional information that aided in surgical decision-making. [ABSTRACT FROM AUTHOR]

Inguinal complete lymph node dissection (CLND) for metastatic melanoma exposes the femoral vein and artery. To protect femoral vessels while preserving the sartorius muscle, we developed a novel sartorius and adductor fascial flap (SAFF) technique for coverage. The SAFF technique includes dissection of fascia off sartorius and/or adductor muscles, rotation over femoral vasculature, and suturing into place. Patients who underwent inguinal CLND with SAFF for melanoma at our institution were identified retrospectively from a prospectively-collected database. Patient characteristics and post-operative outcomes were obtained. Multivariate logistic regression assessed associations of palpable and non-palpable disease with wound complications. From 2008 to 2019, 51 patients underwent CLND with SAFF. Median age was 62 years, and 59% were female. Thirty-one (61%) patients were presented with palpable disease and 20 (39%) had non-palpable disease. Fifty-five percent (95% confidence interval CI: 40%-69%) experienced at least one wound complication: wound infection was most common (45%; 95% CI: 31%-60%), while bleeding was the least (2%; 95% CI: 0.05%-11%). Complications were similar, with and without palpable disease. The SAFF procedure covers femoral vessels, minimizes bleeding, preserves the sartorius muscle, and uses standard surgical techniques easily adoptable by surgeons who perform inguinal CLND. • Sartorius and adductor fascial flap can be useful after inguinal dissection. • Fascial flap provides adequate coverage of femoral vessels. • Outcomes after fascial flap are consistent with other CLND operations. • Bleeding complications were minimal after fascial flap technique. [ABSTRACT FROM AUTHOR]

β-catenin (βcat) is an important downstream effector in the Wnt signaling pathway and plays important roles in the development and progression of many cancers including melanoma. βcat expression is regulated by GSK-3β-mediated phosphorylation at positions 33, 37 and 41. In normal cells, phosphorylation at these sites triggers proteasomal degradation, which prevents accumulation of free cytoplasmic βcat. In cancer cells, stabilized β-catenin translocates into the nucleus, where it associates with TCF/Lef proteins to activate transcription of genes that promote tumorigenesis and metastasis, including PD-L1. It has been suggested that nuclear phospho-βcat (pβcat) staining may be diagnostically useful in differentiating primary from metastatic melanoma. Also, a pβcat peptide (residues 30–39, with only S33 phosphorylated) is naturally presented by melanoma cells as a T-cell target. We evaluated expression of pS33-βcat in primary and metastatic melanomas by immunohistochemistry and found its expression varied widely but was most commonly cytoplasmic. Nuclear staining was identified in only 18% of metastatic melanomas. Staining with antibodies to pS33-βcat and pS33/37/T41-βcat was most intense in mitotic melanoma cells; however, pS33-βcat intensity was not significantly associated with AJCC stage, tumor location, BRAF mutation status, or immune infiltrates. Yet, PD-L1 and PD-L2 expression by tumor cells were significantly higher in tumors with high pS33-βcat expression. The low rate of nuclear pS33-βcat expression suggests that pS33-βcat may have limited utility for identifying metastatic melanomas. However, high expression in dividing cells and strong associations with PD-L1 and PD-L2 expression may inform future personalized therapies for tumors with high pS33-βcat expression. [ABSTRACT FROM AUTHOR]

Background: Immune cells in the tumor microenvironment have prognostic value. In preclinical models, recruitment and infiltration of these cells depends on immune cell homing (ICH) genes such as chemokines, cell adhesion molecules, and integrins. We hypothesized ICH ligands CXCL9-11 and CCL2-5 would be associated with intratumoral T-cells, while CXCL13 would be more associated with B-cell infiltrates.Methods: Samples of human melanoma were submitted for gene expression analysis and immune cells identified by immunohistochemistry. Associations between the two were evaluated with unsupervised hierarchical clustering using correlation matrices from Spearman rank tests. Univariate analysis performed Mann-Whitney tests.Results: For 119 melanoma specimens, analysis of 78 ICH genes revealed association among genes with nonspecific increase of multiple immune cell subsets: CD45+, CD8+ and CD4+ T-cells, CD20+ B-cells, CD138+ plasma cells, and CD56+ NK-cells. ICH genes most associated with these infiltrates included ITGB2, ITGAL, CCL19, CXCL13, plus receptor/ligand pairs CXCL9 and CXCL10 with CXCR3; CCL4 and CCL5 with CCR5. This top ICH gene expression signature was also associated with genes representing immune-activation and effector function. In contrast, CD163+ M2-macrophages was weakly associated with a different ICH gene signature.Conclusion: These data do not support our hypothesis that each immune cell subset is uniquely associated with specific ICH genes. Instead, a larger set of ICH genes identifies melanomas with concordant infiltration of B-cell and T-cell lineages, while CD163+ M2-macrophage infiltration suggesting alternate mechanisms for their recruitment. Future studies should explore the extent ICH gene signature contributes to tertiary lymphoid structures or cross-talk between homing pathways. [ABSTRACT FROM AUTHOR]

The strength and durability of systemic anti-tumor immune responses induced by cancer vaccines depends on adjuvants to support an immunogenic vaccine site microenvironment (VSME). Adjuvants include water-in-oil emulsions with incomplete Freund's adjuvant (IFA) and combinations of toll-like receptor (TLR) agonists, including a preparation containing TLR4 and TLR9 agonists with QS-21 (AS15). IFA-containing vaccines can promote immune cell accumulation at the VSME, whereas effects of AS15 are largely unexplored. Therefore, we assessed innate and adaptive immune cell accumulation and gene expression at the VSME after vaccination with AS15 and compared to effects with IFA. We hypothesized that AS15 would promote less accumulation of innate and adaptive immune cells at the VSME than IFA vaccines. In two clinical trials, patients with resected high-risk melanoma received either a multipeptide vaccine with IFA or a recombinant MAGE-A3 protein vaccine with AS15. Vaccine site biopsies were obtained after one or multiple vaccines. T cells accumulated early after vaccines with AS15, but this was not durable or of the same magnitude as vaccination in IFA. Vaccines with AS15 increased durable expression of DC- and T cell-related genes, as well as PD-L1 and IDO1, suggesting complex activation and regulation of innate and adaptive immune function with AS15. These changes were generally greater with vaccines containing IFA, but IFA induced reduction in myeloid suppressor cells markers. Evidence of tertiary lymphoid structure (TLS) formation was observed with both adjuvants. Our findings highlight adjuvant-dependent changes in immune features at the VSME that may impact systemic immune responses. [ABSTRACT FROM AUTHOR]

There is a need for better classification and understanding of tumor-infiltrating lymphocytes (TILs). Here, we applied advanced functional genomics to interrogate 9,000 human tumors and multiple single-cell sequencing sets using benchmarked T cell states, comprehensive T cell differentiation trajectories, human and mouse vaccine responses, and other human TILs. Compared with other T cell states, enrichment of T memory/resident memory programs was observed across solid tumors. Trajectory analysis of single-cell melanoma CD8+ TILs also identified a high fraction of memory/resident memory-scoring TILs in anti-PD-1 responders, which expanded post therapy. In contrast, TILs scoring highly for early T cell activation, but not exhaustion, associated with non-response. Late/persistent, but not early activation signatures, prognosticate melanoma survival, and co-express with dendritic cell and IFN-γ response programs. These data identify an activation-like state associated to poor response and suggest successful memory conversion, above resuscitation of exhaustion, is an under-appreciated aspect of successful anti-tumoral immunity. [Display omitted] • Improved global TIL classification methods are required to deconvolve cell states • αPD-1 non-responder TILs and dysfunctional TILs score for T activation, not exhaustion • αPD-1 response and patient survival associates with late T cell memory/T RM scoring • Persistent programs co-express with DC maturation and IFN-γ response programs Jaiswal et al. highlight the need for improved tumor-infiltrating lymphocyte (TIL) classification by showing that current transcriptome assignments may misclassify early activated/effector TILs as exhausted. The study surveys 9,000 solid tumors, multiple single-cell RNA sequencing sets, mouse and human models, and scoring methods to reclassify TILs and associate melanoma survival to T cell memory/resident memory. [ABSTRACT FROM AUTHOR]