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Every cell in the human body has a unique set of somatic mutations, but it remains difficult to comprehensively genotype an individual cell1. Here we describe ways to overcome this obstacle in the context of normal human skin, thus offering a glimpse into the genomic landscapes of individual melanocytes from human skin. As expected, sun-shielded melanocytes had fewer mutations than sun-exposed melanocytes. However, melanocytes from chronically sun-exposed skin (for example, the face) had a lower mutation burden than melanocytes from intermittently sun-exposed skin (for example, the back). Melanocytes located adjacent to a skin cancer had higher mutation burdens than melanocytes from donors without skin cancer, implying that the mutation burden of normal skin can be used to measure cumulative sun damage and risk of skin cancer. Moreover, melanocytes from healthy skin commonly contained pathogenic mutations, although these mutations tended to be weakly oncogenic, probably explaining why they did not give rise to discernible lesions. Phylogenetic analyses identified groups of related melanocytes, suggesting that melanocytes spread throughout skin as fields of clonally related cells that are invisible to the naked eye. Overall, our results uncover the genomic landscapes of individual melanocytes, providing key insights into the causes and origins of melanoma.
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Genoma Humano/genética , Genómica , Salud , Melanocitos/citología , Melanoma/genética , Análisis de la Célula Individual , Piel/citología , Análisis Mutacional de ADN , Femenino , Genotipo , Humanos , Masculino , Melanocitos/metabolismo , Melanocitos/patología , Melanoma/patología , Mutación , Piel/patología , Flujo de TrabajoRESUMEN
As healthcare systems are improving and thereby the life expectancy of human populations is increasing, cancer is representing itself as the second leading cause of death. Although cancer biologists have put enormous effort on cancer research so far, we still have a long way to go before being able to treat cancers efficiently. One interesting approach in cancer biology is to learn from natural resistance and/or predisposition to cancer. Cancer-resistant species and tissues are thought-provoking models whose study shed light on the inherent cancer resistance mechanisms that arose during the course of evolution. On the other hand, there are some syndromes and factors that increase the risk of cancer development, and revealing their underlying mechanisms will increase our knowledge about the process of cancer formation. Here, we review natural resistance and predisposition to cancer and the known mechanisms at play. Further insights from these natural phenomena will help design future cancer research and could ultimately lead to the development of novel cancer therapeutic strategies.
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Neoplasias , Humanos , Neoplasias/tratamiento farmacológico , Susceptibilidad a Enfermedades , Genotipo , Inmunidad InnataRESUMEN
Gene expression profiling technologies have revolutionized cell biology, enabling researchers to identify gene signatures linked to various biological attributes of melanomas, such as pigmentation status, differentiation state, proliferative versus invasive capacity, and disease progression. Although the discovery of gene signatures has significantly enhanced our understanding of melanocytic phenotypes, reconciling the numerous signatures reported across independent studies and different profiling platforms remains a challenge. Current methods for classifying melanocytic gene signatures depend on exact gene overlap and comparison with unstandardized baseline transcriptomes. In this study, we aimed to categorize published gene signatures into clusters based on their similar patterns of expression across clinical cutaneous melanoma specimens. We analyzed nearly 800 melanoma samples from six gene expression repositories and developed a classification framework for gene signatures that is resilient against biases in gene identification across profiling platforms and inconsistencies in baseline standards. Using 39 frequently cited published gene signatures, our analysis revealed seven principal classes of gene signatures that correlate with previously identified phenotypes: Differentiated, Mitotic/MYC, AXL, Amelanotic, Neuro, Hypometabolic, and Invasive. Each class is consistent with the phenotypes that the constituent gene signatures represent, and our classification method does not rely on overlapping genes between signatures. To facilitate broader application, we created WIMMS (what is my melanocytic signature, available at https://wimms.tanlab.org/), a user-friendly web application. WIMMS allows users to categorize any gene signature, determining its relationship to predominantly cited signatures and its representation within the seven principal classes.
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Significance: Measuring changes in cellular structure and organelles is crucial for understanding disease progression and cellular responses to treatments. A label-free imaging method can aid in advancing biomedical research and therapeutic strategies. Aim: This study introduces a computational darkfield imaging approach named quadrant darkfield (QDF) to separate smaller cellular features from large structures, enabling label-free imaging of cell organelles and structures in living cells. Approach: Using a programmable LED array as illumination source, we vary the direction of illumination to encode additional information about the feature size within cells. This is possible due to the varying level of directional scattering produced by features based on their sizes relative to the wavelength of light used. Results: QDF successfully resolved small cellular features without interference from larger structures. QDF signal is more consistent during cell shape changes than traditional darkfield. QDF signals correlate with flow cytometry side scatter measurements, effectively differentiating cells by organelle content. Conclusions: QDF imaging enhances the study of subcellular structures in living cells, offering improved quantification of organelle content compared to darkfield without labels. This method can be simultaneously performed with other techniques such as quantitative phase imaging to generate a multidimensional picture of living cells in real-time.
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Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematological malignancies but has been clinically less effective in solid tumors. Engineering macrophages with CARs has emerged as a promising approach to overcome some of the challenges faced by CAR-T cells due to the macrophage's ability to easily infiltrate tumors, phagocytose their targets, and reprogram the immune response. We engineered CAR-macrophages (CAR-Ms) to target chondroitin sulfate proteoglycan 4 (CSPG4), an antigen expressed in melanoma, and several other solid tumors. CSPG4-targeting CAR-Ms exhibited specific phagocytosis of CSPG4-expressing melanoma cells. Combining CSPG4-targeting CAR-Ms with CD47 blocking antibodies synergistically enhanced CAR-M-mediated phagocytosis and effectively inhibited melanoma spheroid growth in 3D. Furthermore, CSPG4-targeting CAR-Ms inhibited melanoma tumor growth in mouse models. These results suggest that CSPG4-targeting CAR-M immunotherapy is a promising solid tumor immunotherapy approach for treating melanoma. STATEMENT OF SIGNIFICANCE: We engineered macrophages with CARs as an alternative approach for solid tumor treatment. CAR-macrophages (CAR-Ms) targeting CSPG4, an antigen expressed in melanoma and other solid tumors, phagocytosed melanoma cells and inhibited melanoma growth in vivo . Thus, CSPG4-targeting CAR-Ms may be a promising strategy to treat patients with CSPG4-expressing tumors.
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Basal cell carcinoma (BCC) is classified histologically into subtypes that determine treatment decisions. MicroRNAs (miRs) are short noncoding RNAs that may serve as diagnostic biomarkers. We investigated if particular miRs could distinguish BCC subtypes. We sequenced miRs from 55 archival BCC and 9 control skin specimens and then validated these miRs by qRT-PCR assay on a second BCC cohort (18 superficial, 16 nodular, 15 infiltrative) and control skin (n = 12). Expression values for individual miRs were normalized to miR-16-5p, which was the least variant among the control skin and BCC samples. We found that (i) miR-383-5p and miR-145-5p are downregulated in all BCC subtypes compared with control skin, (ii) miR-181c-5p is downregulated in superficial compared with invasive (nodular/infiltrative) BCC, and (iii) miR-22-5p and miR-708-5p are upregulated in infiltrative compared with superficial/nodular BCC and miR-30c-5p is downregulated in infiltrative compared with nodular BCC. Receiver operating characteristic analysis demonstrated excellent capacity of these miRs to discriminate between BCC and control skin (area under the curve, 0.94-0.98), whereas the capacity to discriminate between superficial and invasive subtypes was less robust (area under the curve, 0.7-0.8). Future prospective studies may determine the utility of these miRs as diagnostic biomarkers to guide biopsy and treatment of BCC.
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Aberrant activation of the PI3K-AKT pathway is common in many cancers, including melanoma, and AKT1, 2 and 3 (AKT1-3) are bona fide oncoprotein kinases with well-validated downstream effectors. However, efforts to pharmacologically inhibit AKT have proven to be largely ineffective. In this study, we observed paradoxical effects following either pharmacologic or genetic inhibition of AKT1-3 in melanoma cells. Although pharmacological inhibition was without effect, genetic silencing of all three AKT paralogs significantly induced melanoma cell death through effects on mTOR. This phenotype was rescued by exogenous AKT1 expression in a kinase-dependent manner. Pharmacological inhibition of PI3K and mTOR with a novel dual inhibitor effectively suppressed melanoma cell proliferation in vitro and inhibited tumor growth in vivo. Furthermore, this single-agent-targeted therapy was well-tolerated in vivo and was effective against MAPK inhibitor-resistant patient-derived melanoma xenografts. These results suggest that inhibition of PI3K and mTOR with this novel dual inhibitor may represent a promising therapeutic strategy in this disease in both the first-line and MAPK inhibitor-resistant setting.
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Melanoma , Proteínas Proto-Oncogénicas c-akt , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Línea Celular Tumoral , Serina-Treonina Quinasas TOR/metabolismo , Proliferación Celular , Muerte CelularRESUMEN
Acral melanoma (AM) is an aggressive melanoma variant that arises from palmar, plantar, and nail unit melanocytes. Compared to non-acral cutaneous melanoma (CM), AM is biologically distinct, has an equal incidence across genetic ancestries, typically presents in advanced stage disease, is less responsive to therapy, and has an overall worse prognosis. Independent analysis of published genomic and transcriptomic sequencing identified that receptor tyrosine kinase (RTK) ligands and adapter proteins are frequently amplified, translocated, and/or overexpressed in AM. To target these unique genetic changes, a zebrafish acral melanoma model was exposed to a panel of narrow and broad spectrum multi-RTK inhibitors, revealing that dual FGFR/VEGFR inhibitors decrease acral-analogous melanocyte proliferation and migration. The potent pan-FGFR/VEGFR inhibitor, Lenvatinib, uniformly induces tumor regression in AM patient-derived xenograft (PDX) tumors but only slows tumor growth in CM models. Unlike other multi-RTK inhibitors, Lenvatinib is not directly cytotoxic to dissociated AM PDX tumor cells and instead disrupts tumor architecture and vascular networks. Considering the great difficulty in establishing AM cell culture lines, these findings suggest that AM may be more sensitive to microenvironment perturbations than CM. In conclusion, dual FGFR/VEGFR inhibition may be a viable therapeutic strategy that targets the unique biology of AM.
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MicroRNAs are non-coding RNAs fundamental to metazoan development and disease. Although the aberrant regulation of microRNAs during mammalian tumorigenesis is well established, investigations into the contributions of individual microRNAs are wrought with conflicting observations. The underlying cause of these inconsistencies is often attributed to context-specific functions of microRNAs. We propose that consideration of both context-specific factors, as well as underappreciated fundamental concepts of microRNA biology, will permit a more harmonious interpretation of ostensibly diverging data. We discuss the theory that the biological function of microRNAs is to confer robustness to specific cell states. Through this lens, we then consider the role of miR-211-5p in melanoma progression. Using literature review and meta-analyses, we demonstrate how a deep understating of domain-specific contexts is critical for moving toward a concordant understanding of miR-211-5p and other microRNAs in cancer biology.
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The combination of multiple imaging modalities in a single microscopy system can enable new insights into biological processes. In this work, we describe the construction and rigorous characterization of a custom microscope with multimodal imaging in a single, cost-effective system. Our design utilizes advances in LED technology, robotics, and open-source software, along with existing optical components and precision optomechanical parts to offer a modular and versatile design. This microscope is operated using software written in Arduino and Python and has the ability to run multi-day automated imaging experiments when placed inside of a cell culture incubator. Additionally, we provide and demonstrate methods to validate images taken in brightfield and darkfield, along with validation and optimization for differential phase contrast (DPC) quantitative phase imaging.
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Quantitative phase imaging (QPI) is a label-free, wide-field microscopy approach with significant opportunities for biomedical applications. QPI uses the natural phase shift of light as it passes through a transparent object, such as a mammalian cell, to quantify biomass distribution and spatial and temporal changes in biomass. Reported in cell studies more than 60 years ago, ongoing advances in QPI hardware and software are leading to numerous applications in biology, with a dramatic expansion in utility over the past two decades. Today, investigations of cell size, morphology, behavior, cellular viscoelasticity, drug efficacy, biomass accumulation and turnover, and transport mechanics are supporting studies of development, physiology, neural activity, cancer, and additional physiological processes and diseases. Here, we review the field of QPI in biology starting with underlying principles, followed by a discussion of technical approaches currently available or being developed, and end with an examination of the breadth of applications in use or under development. We comment on strengths and shortcomings for the deployment of QPI in key biomedical contexts and conclude with emerging challenges and opportunities based on combining QPI with other methodologies that expand the scope and utility of QPI even further.
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Microscopía , Neoplasias , Animales , Humanos , Microscopía/métodos , Programas Informáticos , Tamaño de la Célula , MamíferosRESUMEN
Despite significant progress in the development of treatment options, melanoma remains a leading cause of death due to skin cancer. Advances in our understanding of the genetic, transcriptomic, and morphologic spectrum of benign and malignant melanocytic neoplasia have enabled the field to propose biomarkers with potential diagnostic, prognostic, and predictive value. While these proposed biomarkers have the potential to improve clinical decision making at multiple critical intervention points, most remain unvalidated. Clinical validation of even the most commonly assessed biomarkers will require substantial resources, including limited clinical specimens. It is therefore important to consider the properties that constitute a relevant and clinically-useful biomarker-based test prior to engaging in large validation studies. In this review article we adapt an established framework for determining minimally-useful biomarker test characteristics, and apply this framework to a discussion of currently used and proposed biomarkers designed to aid melanoma detection, staging, prognosis, and choice of treatment.
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Mucosal melanoma (MM) is a rare melanoma subtype that originates from melanocytes within sun-protected mucous membranes. Compared with cutaneous melanoma (CM), MM has worse prognosis and lacks effective treatment options. Moreover, the endogenous or exogenous risk factors that influence mucosal melanocyte transformation, as well as the identity of MM precursor lesions, are ambiguous. Consequently, there remains a lack of molecular markers that can be used for early diagnosis, and therefore better management, of MM. In this review, we first summarize the main functions of mucosal melanocytes. Then, using oral mucosal melanoma (OMM) as a model, we discuss the distinct pathologic stages from benign mucosal melanocytes to metastatic MM, mapping the possible evolutionary trajectories that correspond to MM initiation and progression. We highlight key areas of ambiguity during the genetic evolution of MM from its benign lesions, and the resolution of which could aid in the discovery of new biomarkers for MM detection and diagnosis. We outline the key pathways that are altered in MM, including the MAPK pathway, the PI3K/AKT pathway, cell cycle regulation, telomere maintenance, and the RNA maturation process, and discuss targeted therapy strategies for MM currently in use or under investigation.
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In humans, epidermal melanocytes are responsible for skin pigmentation, defence against ultraviolet radiation and the deadliest common skin cancer, melanoma. Although there is substantial overlap in melanocyte development pathways between different model organisms, species-dependent differences are frequent and the conservation of these processes in human skin remains unresolved. Here, we used a single-cell enrichment and RNA-sequencing pipeline to study human epidermal melanocytes directly from the skin, capturing transcriptomes across different anatomical sites, developmental age, sexes and multiple skin tones. We uncovered subpopulations of melanocytes that exhibit anatomical site-specific enrichment that occurs during gestation and persists through adulthood. The transcriptional signature of the volar-enriched subpopulation is retained in acral melanomas. Furthermore, we identified human melanocyte differentiation transcriptional programs that are distinct from gene signatures generated from model systems. Finally, we used these programs to define patterns of dedifferentiation that are predictive of melanoma prognosis and response to immune checkpoint inhibitor therapy.
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Epidermis/metabolismo , Melanocitos/metabolismo , Melanoma/metabolismo , Neoplasias Cutáneas/metabolismo , Diferenciación Celular/fisiología , Humanos , Piel/metabolismo , Neoplasias Cutáneas/genética , Rayos Ultravioleta , Melanoma Cutáneo MalignoRESUMEN
Benign melanocytic nevi frequently emerge when an acquired BRAFV600E mutation triggers unchecked proliferation and subsequent arrest in melanocytes. Recent observations have challenged the role of oncogene-induced senescence in melanocytic nevus formation, necessitating investigations into alternative mechanisms for the establishment and maintenance of proliferation arrest in nevi. We compared the transcriptomes of melanocytes from healthy human skin, nevi, and melanomas arising from nevi and identified a set of microRNAs as highly expressed nevus-enriched transcripts. Two of these microRNAs-MIR211-5p and MIR328-3p-induced mitotic failure, genome duplication, and proliferation arrest in human melanocytes through convergent targeting of AURKB. We demonstrate that BRAFV600E induces a similar proliferation arrest in primary human melanocytes that is both reversible and conditional. Specifically, BRAFV600E expression stimulates either arrest or proliferation depending on the differentiation state of the melanocyte. We report genome duplication in human melanocytic nevi, reciprocal expression of AURKB and microRNAs in nevi and melanomas, and rescue of arrested human nevus cells with AURKB expression. Taken together, our data describe an alternative molecular mechanism for melanocytic nevus formation that is congruent with both experimental and clinical observations.
Lots of people have small dark patches on their skin known as moles. Most moles form when individual cells known as melanocytes in the skin acquire a specific genetic mutation in a gene called BRAF. This mutation causes the cells to divide rapidly to form the mole. After a while, most moles stop growing and remain harmless for the rest of a person's life. Melanoma is a type of skin cancer that develops from damaged melanocytes. The same mutation in BRAF that is found in moles is also present in half of all cases of melanoma. Unlike in moles, the melanoma-causing mutation makes the melanocytes divide rapidly to form a tumor that keeps on growing indefinitely. It remains unclear why the same genetic mutation in the BRAF gene has such different consequences in moles and melanomas. To address this question, McNeal et al. used genetic approaches to study melanocytes from moles and melanomas. The experiments identified some molecules known as microRNAs that are present at higher levels in moles than in melanomas. Increasing the levels of two of these microRNAs in melanocytes from human skin stopped the cells from growing and dividing by inhibiting a gene called AURKB. This suggested that these microRNAs are responsible for halting the growth of moles. Introducing the mutated form of BRAF into melanocytes also stopped cells from growing and dividing by inhibiting AURKB. However, changing the environment surrounding the cells reversed this effect and allowed the melanocytes to resume dividing. In this way the mutated form of BRAF acts like a switch that allows melanocytes in skin cancers to start growing again under certain conditions. Further experiments found that a drug called barasertib is able to inhibit the growth of melanoma cells with the mutant form of BRAF. Future work will investigate whether it is possible to use this drug and other tools to stop skin cancer tumors from growing, and possibly even prevent skin tumors from forming in the first place.
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Aurora Quinasa B/genética , Melanocitos/fisiología , MicroARNs/metabolismo , Mitosis/genética , Proteínas Proto-Oncogénicas B-raf/genética , Aurora Quinasa B/metabolismo , Humanos , Proteínas Proto-Oncogénicas B-raf/metabolismo , Transducción de SeñalRESUMEN
To date, over 1000 melanocytic neoplasms, spanning all stages of tumorigenesis, have been sequenced, offering detailed views into their -omic landscapes. This has coincided with advances in genetic engineering technologies that allow molecular biologists to edit the human genome with extreme precision and new mouse models to simulate disease progression. In this review, we describe how these technologies are being harnessed to provide insights into the evolution of melanoma at an unprecedented resolution, revealing that prior models of melanoma evolution, in which pathways are turned 'on' or 'off' in a binary fashion during the run-up to melanoma, are oversimplified.
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Carcinogénesis/genética , Sistema de Señalización de MAP Quinasas/genética , Melanocitos/patología , Melanoma/genética , Neoplasias Cutáneas/genética , Animales , Carcinogénesis/patología , Puntos de Control del Ciclo Celular/genética , Proliferación Celular/genética , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Dosificación de Gen , Regulación Neoplásica de la Expresión Génica , Ingeniería Genética , Humanos , Melanoma/patología , Ratones , Ratones Transgénicos , Mutación , Proteína de Retinoblastoma/metabolismo , Neoplasias Cutáneas/patologíaRESUMEN
The loss of primary cilia on melanocytes is a useful biomarker for the distinction of melanoma from conventional melanocytic nevi. It is unknown whether ciliation status is beneficial for diagnosing spitzoid tumors-a subclass of melanomas that present inherently ambiguous histology and are challenging to classify. We evaluated the Ciliation Index (CI) in 68 cases of spitzoid tumors ranging from Spitz nevi and atypical Spitz tumors to spitzoid melanoma. We found a significant decrease in CI within the spitzoid melanoma group when compared with either the Spitz nevi or atypical Spitz tumors groups. In addition, we used a machine-learning-based algorithm to determine the value of CI when considered in combination with other histopathologic and molecular features commonly used for diagnosis. We found that a low CI was consistently ranked as a top predictive feature in the diagnosis of malignancy. Predictive models trained on only the top four predictive features (CI, asymmetry, hyperchromatism, and cytologic atypia) outperformed standard histologic assessment in an independent validation cohort of 56 additional cases. The results provide an alternative approach to evaluate diagnostically challenging melanocytic lesions, and further support the use of CI as an ancillary diagnostic test.
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Biomarcadores de Tumor/metabolismo , Melanocitos/patología , Melanoma/diagnóstico , Nevo de Células Epitelioides y Fusiformes/diagnóstico , Neoplasias Cutáneas/diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Algoritmos , Niño , Preescolar , Cilios/metabolismo , Hibridación Genómica Comparativa , Diagnóstico Diferencial , Reacciones Falso Positivas , Femenino , Humanos , Aprendizaje Automático , Masculino , Melanoma/genética , Melanoma/patología , Persona de Mediana Edad , Nevo de Células Epitelioides y Fusiformes/genética , Nevo de Células Epitelioides y Fusiformes/patología , Valor Predictivo de las Pruebas , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Adulto Joven , Melanoma Cutáneo MalignoRESUMEN
The use of microRNAs as biomarkers has been proposed for many diseases, including the diagnosis of melanoma. Although hundreds of microRNAs have been identified as differentially expressed in melanomas as compared to benign melanocytic lesions, a limited consensus has been achieved across studies, constraining the effective use of these potentially useful markers. In this study, we applied a machine learning-based pipeline to a dataset consisting of genetic features, clinical features, and next-generation microRNA sequencing from micro-dissected formalin-fixed paraffin embedded melanomas and their adjacent benign precursor nevi. We identified patient age and tumor cellularity as variables that frequently confound the measured expression of potentially diagnostic microRNAs. By employing the ratios of microRNAs that were either enriched or depleted in melanoma compared to the nevi as a normalization strategy, we developed a model that classified all the available published cohorts with an area under the receiver operating characteristic curve of 0.98. External validation on an independent cohort classified lesions with 81% sensitivity and 88% specificity and was uninfluenced by the tumor content of the sample or patient age.
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Biomarcadores de Tumor/genética , Melanocitos/fisiología , Melanoma/diagnóstico , MicroARNs/genética , Nevo/diagnóstico , Neoplasias Cutáneas/diagnóstico , Conjuntos de Datos como Asunto , Diagnóstico Diferencial , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Aprendizaje Automático , Pronóstico , Curva ROC , Sensibilidad y Especificidad , Análisis de Secuencia de ARNRESUMEN
Importance: Use of prognostic gene expression profile (GEP) testing in cutaneous melanoma (CM) is rising despite a lack of endorsement as standard of care. Objective: To develop guidelines within the national Melanoma Prevention Working Group (MPWG) on integration of GEP testing into the management of patients with CM, including (1) review of published data using GEP tests, (2) definition of acceptable performance criteria, (3) current recommendations for use of GEP testing in clinical practice, and (4) considerations for future studies. Evidence Review: The MPWG members and other international melanoma specialists participated in 2 online surveys and then convened a summit meeting. Published data and meeting abstracts from 2015 to 2019 were reviewed. Findings: The MPWG members are optimistic about the future use of prognostic GEP testing to improve risk stratification and enhance clinical decision-making but acknowledge that current utility is limited by test performance in patients with stage I disease. Published studies of GEP testing have not evaluated results in the context of all relevant clinicopathologic factors or as predictors of regional nodal metastasis to replace sentinel lymph node biopsy (SLNB). The performance of GEP tests has generally been reported for small groups of patients representing particular tumor stages or in aggregate form, such that stage-specific performance cannot be ascertained, and without survival outcomes compared with data from the American Joint Committee on Cancer 8th edition melanoma staging system international database. There are significant challenges to performing clinical trials incorporating GEP testing with SLNB and adjuvant therapy. The MPWG members favor conducting retrospective studies that evaluate multiple GEP testing platforms on fully annotated archived samples before embarking on costly prospective studies and recommend avoiding routine use of GEP testing to direct patient management until prospective studies support their clinical utility. Conclusions and Relevance: More evidence is needed to support using GEP testing to inform recommendations regarding SLNB, intensity of follow-up or imaging surveillance, and postoperative adjuvant therapy. The MPWG recommends further research to assess the validity and clinical applicability of existing and emerging GEP tests. Decisions on performing GEP testing and patient management based on these results should only be made in the context of discussion of testing limitations with the patient or within a multidisciplinary group.