The Impact of Next-generation Sequencing on Interobserver Agreement and Diagnostic Accuracy of Desmoplastic Melanocytic Neoplasms.

Next-generation sequencing (NGS) is increasingly being utilized as an ancillary tool for diagnostically challenging melanocytic neoplasms. It is incumbent upon the pathology community to perform studies assessing the benefits and limitations of these tools in specific diagnostic scenarios. One of the most challenging diagnostic scenarios faced by skin pathologists involves accurate diagnosis of desmoplastic melanocytic neoplasms (DMNs). In this study, 20 expert melanoma pathologists rendered a diagnosis on 47 DMNs based on hematoxylin and eosin sections with demographic information. After submitting their diagnosis, the experts were given the same cases, but this time with comprehensive genomic sequencing results, and asked to render a diagnosis again. Identification of desmoplastic melanoma (DM) improved by 7%, and this difference was statistically significant ( P <0.05). In addition, among the 15 melanoma cases, in the pregenomic assessment, only 12 were favored to be DM by the experts, while after genomics, this improved to 14 of the cases being favored to be DM. In fact, some cases resulting in metastatic disease had a substantial increase in the number of experts recognizing them as DM after genomics. The impact of the genomic findings was less dramatic among benign and intermediate-grade desmoplastic tumors (BIDTs). Interobserver agreement also improved, with the Fleiss multirater Kappa being 0.36 before genomics to 0.4 after genomics. NGS has the potential to improve diagnostic accuracy in the assessment of desmoplastic melanocytic tumors. The degree of improvement will be most substantial among pathologists with some background and experience in bioinformatics and melanoma genetics.

Tumorigenesis driven by the BRAFV600E oncoprotein requires secondary mutations that overcome its feedback inhibition of migration and invasion.

BRAFV600E mutation occurs in 46% of melanomas and drives high levels of ERK activity and ERK-dependent proliferation. However, BRAFV600E is insufficient to drive melanoma in GEMM models, and 82% of human benign nevi harbor BRAFV600E mutations. We show here that BRAFV600E inhibits mesenchymal migration by causing feedback inhibition of RAC1 activity. ERK pathway inhibition induces RAC1 activation and restores migration and invasion. In cells with BRAFV600E, mutant RAC1, overexpression of PREX1, PREX2, or PTEN inactivation restore RAC1 activity and cell motility. Together, these lesions occur in 48% of BRAFV600E melanomas. Thus, although BRAFV600E activation of ERK deregulates cell proliferation, it prevents full malignant transformation by causing feedback inhibition of cell migration. Secondary mutations are, therefore, required for tumorigenesis. One mechanism underlying tumor evolution may be the selection of lesions that rescue the deleterious effects of oncogenic drivers.

BRAF Mutated and Morphologically Spitzoid Tumors, a Subgroup of Melanocytic Neoplasms Difficult to Distinguish From True Spitz Neoplasms.

Drivers of Spitz neoplasms include activating point mutations in HRAS and Spitz-associated genomic fusions. It has become evident that some BRAF -mutated melanocytic neoplasms can morphologically mimic Spitz tumors (STs). These have been termed BRAF mutated and morphologically spitzoid (BAMS). In this study, 17 experts from the International Melanoma Pathology Study Group assessed 54 cases which included 40 BAMS and 14 true STs. The participants reviewed the cases blinded to the genomic data and selected among several diagnostic options, including BAMS, ST, melanoma, and other. A total of 38% of all diagnostic selections in the BAMS cases were for BAMS, whereas 32% were for ST. In 22 of the BAMS cases, the favored diagnosis was BAMS, whereas in 17 of the BAMS cases, the favored diagnosis was ST. Among the 20 cases in the total group of 54 with the highest number of votes for ST, half were BAMS. Of BAMS, 75% had a number of votes for ST that was within the SD of votes for ST seen among true ST cases. There was poor interobserver agreement for the precise diagnosis of the BAMS (kappa = 0.16) but good agreement that these cases were not melanoma (kappa = 0.7). BAMS nevi/tumors can closely mimic Spitz neoplasms. Expert melanoma pathologists in this study favored a diagnosis of ST in nearly half of the BAMS cases. There are BAMS cases that even experts cannot morphologically distinguish from true Spitz neoplasms.

A TCF4-dependent gene regulatory network confers resistance to immunotherapy in melanoma.

To better understand intrinsic resistance to immune checkpoint blockade (ICB), we established a comprehensive view of the cellular architecture of the treatment-naive melanoma ecosystem and studied its evolution under ICB. Using single-cell, spatial multi-omics, we showed that the tumor microenvironment promotes the emergence of a complex melanoma transcriptomic landscape. Melanoma cells harboring a mesenchymal-like (MES) state, a population known to confer resistance to targeted therapy, were significantly enriched in early on-treatment biopsies from non-responders to ICB. TCF4 serves as the hub of this landscape by being a master regulator of the MES signature and a suppressor of the melanocytic and antigen presentation transcriptional programs. Targeting TCF4 genetically or pharmacologically, using a bromodomain inhibitor, increased immunogenicity and sensitivity of MES cells to ICB and targeted therapy. We thereby uncovered a TCF4-dependent regulatory network that orchestrates multiple transcriptional programs and contributes to resistance to both targeted therapy and ICB in melanoma.

Ultra-sensitive molecular residual disease detection through whole genome sequencing with single-read error correction.

While whole genome sequencing (WGS) of cell-free DNA (cfDNA) holds enormous promise for molecular residual disease (MRD) detection, its performance is limited by WGS error rate. Here we introduce AccuScan, an efficient cfDNA WGS technology that enables genome-wide error correction at single read level, achieving an error rate of 4.2×10 -7 , which is about two orders of magnitude lower than a read-centric de-noising method. When applied to MRD detection, AccuScan demonstrated analytical sensitivity down to 10 -6 circulating tumor allele fraction at 99% sample level specificity. In colorectal cancer, AccuScan showed 90% landmark sensitivity for predicting relapse. It also showed robust MRD performance with esophageal cancer using samples collected as early as 1 week after surgery, and predictive value for immunotherapy monitoring with melanoma patients. Overall, AccuScan provides a highly accurate WGS solution for MRD, empowering circulating tumor DNA detection at parts per million range without high sample input nor personalized reagents. One Sentence Summary: AccuScan showed remarkable ultra-low limit of detection with a short turnaround time, low sample requirement and a simple workflow for MRD detection.

A bedside to bench study of anti-PD-1, anti-CD40, and anti-CSF1R indicates that more is not necessarily better.

BACKGROUND: Stimulating inflammatory tumor associated macrophages can overcome resistance to PD-(L)1 blockade. We previously conducted a phase I trial of cabiralizumab (anti-CSF1R), sotigalimab (CD40-agonist) and nivolumab. Our current purpose was to study the activity and cellular effects of this three-drug regimen in anti-PD-1-resistant melanoma. METHODS: We employed a Simon's two-stage design and analyzed circulating immune cells from patients treated with this regimen for treatment-related changes. We assessed various dose levels of anti-CSF1R in murine melanoma models and studied the cellular and molecular effects. RESULTS: Thirteen patients were enrolled in the first stage. We observed one (7.7%) confirmed and one (7.7%) unconfirmed partial response, 5 patients had stable disease (38.5%) and 6 disease progression (42.6%). We elected not to proceed to the second stage. CyTOF analysis revealed a reduction in non-classical monocytes. Patients with prolonged stable disease or partial response who remained on study for longer had increased markers of antigen presentation after treatment compared to patients whose disease progressed rapidly. In a murine model, higher anti-CSF1R doses resulted in increased tumor growth and worse survival. Using single-cell RNA-sequencing, we identified a suppressive monocyte/macrophage population in murine tumors exposed to higher doses. CONCLUSIONS: Higher anti-CSF1R doses are inferior to lower doses in a preclinical model, inducing a suppressive macrophage population, and potentially explaining the disappointing results observed in patients. While it is impossible to directly infer human doses from murine studies, careful intra-species evaluation can provide important insight. Cabiralizumab dose optimization is necessary for this patient population with limited treatment options. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03502330.

Manipulating mitochondrial electron flow enhances tumor immunogenicity.

Although tumor growth requires the mitochondrial electron transport chain (ETC), the relative contribution of complex I (CI) and complex II (CII), the gatekeepers for initiating electron flow, remains unclear. In this work, we report that the loss of CII, but not that of CI, reduces melanoma tumor growth by increasing antigen presentation and T cell-mediated killing. This is driven by succinate-mediated transcriptional and epigenetic activation of major histocompatibility complex-antigen processing and presentation (MHC-APP) genes independent of interferon signaling. Furthermore, knockout of methylation-controlled J protein (MCJ), to promote electron entry preferentially through CI, provides proof of concept of ETC rewiring to achieve antitumor responses without side effects associated with an overall reduction in mitochondrial respiration in noncancer cells. Our results may hold therapeutic potential for tumors that have reduced MHC-APP expression, a common mechanism of cancer immunoevasion.

IL-7R licenses a population of epigenetically poised memory CD8+ T cells with superior antitumor efficacy that are critical for melanoma memory.

Recurrence of advanced melanoma after therapy is a major risk factor for reduced survival, and treatment options are limited. Antitumor immune memory plays a critical role in preventing melanoma recurrence and memory T cells could be a potent cell-based therapy, but the identity, and functional properties of the required immune cells are incompletely understood. Here, we show that an IL-7Rhi tumor-specific CD8+ population is critical for antitumor memory and can be epigenetically augmented to drive powerful antitumor immune responses. Using a model of functional antimelanoma memory, we found that high IL-7R expression selectively marks a CD8+ population in lymphoid organs that plays critical roles in maintaining tumor remission after immunotherapy or surgical resection. This population has intrinsic cytotoxic activity, lacks markers of exhaustion and has superior antitumor efficacy. IL-7Rhi cells have a functionally poised epigenetic landscape regulated by DNA methylation, which can be augmented by hypomethylating agents to confer improved survival and complete melanoma clearance in naive mice. Importantly, greater than 95% of tumor-specific T cells in draining lymph nodes after therapy express high levels of IL-7R. This overlap between IL-7Rhi and antigen-specific T cells allows for enrichment of a potent functional CD8+ population without determining antigen-specificity, which we demonstrate in a melanoma model without a known antigen. We identify that IL-7R expression in human melanoma is an independent prognostic factor of improved survival. These findings advance our basic understanding of antitumor memory and suggest a cell-based therapy using high IL-7R expression to enrich for a lymph node population with superior antitumor activity that can be augmented by hypomethylating agents.

Combinatorial Immunotherapy with Agonistic CD40 Activates Dendritic Cells to Express IL12 and Overcomes PD-1 Resistance.

Checkpoint inhibitors have revolutionized cancer treatment, but resistance remains a significant clinical challenge. Myeloid cells within the tumor microenvironment can modulate checkpoint resistance by either supporting or suppressing adaptive immune responses. Using an anti-PD-1-resistant mouse melanoma model, we show that targeting the myeloid compartment via CD40 activation and CSF1R blockade in combination with anti-PD-1 results in complete tumor regression in a majority of mice. This triple therapy combination was primarily CD40 agonist-driven in the first 24 hours after therapy and showed a similar systemic cytokine profile in human patients as was seen in mice. Functional single-cell cytokine secretion profiling of dendritic cells (DC) using a novel microwell assay identified a CCL22+CCL5+ IL12-secreting DC subset as important early-stage effectors of triple therapy. CD4+ and CD8+ T cells are both critical effectors of treatment, and systems analysis of single-cell RNA sequencing data supported a role for DC-secreted IL12 in priming T-cell activation and recruitment. Finally, we showed that treatment with a novel IL12 mRNA therapeutic alone was sufficient to overcome PD-1 resistance and cause tumor regression. Overall, we conclude that combining myeloid-based innate immune activation and enhancement of adaptive immunity is a viable strategy to overcome anti-PD-1 resistance.

Setdb1 -loss induces type-I interferons and immune clearance of melanoma.

Despite recent advances in the treatment of melanoma, many patients with metastatic disease still succumb to their disease. To identify tumor-intrinsic modulators of immunity to melanoma, we performed a whole-genome CRISPR screen in melanoma and identified multiple components of the HUSH complex, including Setdb1 , as hits. We found that loss of Setdb1 leads to increased immunogenicity and complete tumor clearance in a CD8+ T-cell dependent manner. Mechanistically, loss of Setdb1 causes de-repression of endogenous retroviruses (ERVs) in melanoma cells and triggers tumor-cell intrinsic type-I interferon signaling, upregulation of MHC-I expression, and increased CD8+ T-cell infiltration. Furthermore, spontaneous immune clearance observed in Setdb1 -/- tumors results in subsequent protection from other ERV-expressing tumor lines, supporting the functional anti-tumor role of ERV-specific CD8+ T-cells found in the Setdb1 -/- microenvironment. Blocking the type-I interferon receptor in mice grafted with Setdb1 -/- tumors decreases immunogenicity by decreasing MHC-I expression, leading to decreased T-cell infiltration and increased melanoma growth comparable to Setdb1 wt tumors. Together, these results indicate a critical role for Setdb1 and type-I interferons in generating an inflamed tumor microenvironment, and potentiating tumor-cell intrinsic immunogenicity in melanoma. This study further emphasizes regulators of ERV expression and type-I interferon expression as potential therapeutic targets for augmenting anti-cancer immune responses.

Determinants of overall survival in patients with metastatic uveal melanoma.

BACKGROUND: Despite improvements in the treatment of primary uveal melanoma (UM), patients with metastatic disease continue to exhibit poor survival. METHODS: A retrospective review of metastatic UM patients at Yale (initial cohort) and Memorial Sloan Kettering (validation cohort) was conducted. Cox proportional hazards regression was used to determine baseline factors that are associated with overall survival, including sex, Eastern Cooperative Oncology Group (ECOG) Performance Status Scale, laboratory measurements, metastasis location, and use of anti-CTLA-4 and anti-PD-1 therapies. Differences in overall survival were analyzed using Kaplan-Meier analysis. RESULTS: A total of 89 patients with metastatic UM were identified; 71 and 18, in the initial and validation cohorts, respectively. In the initial cohort, median follow-up was 19.8 months (range, 2-127 months) and median overall survival was 21.8 months (95% CI, 16.6-31.3). Female sex, anti-CTLA-4, and anti-PD-1 therapy were associated with better survival outcomes with adjusted death hazard ratios (HRs) of 0.40 (95% CI, 0.20-0.78), 0.44 (0.20-0.97), and 0.42 (0.22-0.84), respectively, whereas development of hepatic metastases and ECOG score ≥1 (per 1 U/L) were associated with worse survival outcomes with HRs of 2.86 (1.28-7.13) and 2.84 (1.29-6.09), respectively. In both the initial and validation cohorts, use of immune checkpoint inhibitors was associated with improved overall survival after adjusting for sex and ECOG score, with death HRs of 0.22 (0.08-0.56) and 0.04 (0.002-0.26), respectively. CONCLUSIONS: Development of extrahepatic-only metastases, ECOG of 0, immune checkpoint therapy, and female sex were each associated with more than 2-fold reductions in risk of death. PLAIN LANGUAGE SUMMARY: Metastatic uveal melanoma patients face limited treatment options and poor survival rates. Results from this retrospective analysis indicate that immune checkpoint inhibitors, such as anti-CTLA-4 and anti-PD-1 therapies, were associated with improved survival outcomes. Factors such as extrahepatic-only metastases, better baseline performance status, and female sex contributed to a more than 2-fold reduction in death risk. These findings highlight the potential of immunotherapy in treating metastatic uveal melanoma.

Cooperative sensing of mitochondrial DNA by ZBP1 and cGAS promotes cardiotoxicity.

Mitochondrial DNA (mtDNA) is a potent agonist of the innate immune system; however, the exact immunostimulatory features of mtDNA and the kinetics of detection by cytosolic nucleic acid sensors remain poorly defined. Here, we show that mitochondrial genome instability promotes Z-form DNA accumulation. Z-DNA binding protein 1 (ZBP1) stabilizes Z-form mtDNA and nucleates a cytosolic complex containing cGAS, RIPK1, and RIPK3 to sustain STAT1 phosphorylation and type I interferon (IFN-I) signaling. Elevated Z-form mtDNA, ZBP1 expression, and IFN-I signaling are observed in cardiomyocytes after exposure to Doxorubicin, a first-line chemotherapeutic agent that induces frequent cardiotoxicity in cancer patients. Strikingly, mice lacking ZBP1 or IFN-I signaling are protected from Doxorubicin-induced cardiotoxicity. Our findings reveal ZBP1 as a cooperative partner for cGAS that sustains IFN-I responses to mitochondrial genome instability and highlight ZBP1 as a potential target in heart failure and other disorders where mtDNA stress contributes to interferon-related pathology.

InterMEL: An international biorepository and clinical database to uncover predictors of survival in early-stage melanoma.

INTRODUCTION: We are conducting a multicenter study to identify classifiers predictive of disease-specific survival in patients with primary melanomas. Here we delineate the unique aspects, challenges, and best practices for optimizing a study of generally small-sized pigmented tumor samples including primary melanomas of at least 1.05mm from AJTCC TNM stage IIA-IIID patients. We also evaluated tissue-derived predictors of extracted nucleic acids' quality and success in downstream testing. This ongoing study will target 1,000 melanomas within the international InterMEL consortium. METHODS: Following a pre-established protocol, participating centers ship formalin-fixed paraffin embedded (FFPE) tissue sections to Memorial Sloan Kettering Cancer Center for the centralized handling, dermatopathology review and histology-guided coextraction of RNA and DNA. Samples are distributed for evaluation of somatic mutations using next gen sequencing (NGS) with the MSK-IMPACTTM assay, methylation-profiling (Infinium MethylationEPIC arrays), and miRNA expression (Nanostring nCounter Human v3 miRNA Expression Assay). RESULTS: Sufficient material was obtained for screening of miRNA expression in 683/685 (99%) eligible melanomas, methylation in 467 (68%), and somatic mutations in 560 (82%). In 446/685 (65%) cases, aliquots of RNA/DNA were sufficient for testing with all three platforms. Among samples evaluated by the time of this analysis, the mean NGS coverage was 249x, 59 (18.6%) samples had coverage below 100x, and 41/414 (10%) failed methylation QC due to low intensity probes or insufficient Meta-Mixed Interquartile (BMIQ)- and single sample (ss)- Noob normalizations. Six of 683 RNAs (1%) failed Nanostring QC due to the low proportion of probes above the minimum threshold. Age of the FFPE tissue blocks (p<0.001) and time elapsed from sectioning to co-extraction (p = 0.002) were associated with methylation screening failures. Melanin reduced the ability to amplify fragments of 200bp or greater (absent/lightly pigmented vs heavily pigmented, p<0.003). Conversely, heavily pigmented tumors rendered greater amounts of RNA (p<0.001), and of RNA above 200 nucleotides (p<0.001). CONCLUSION: Our experience with many archival tissues demonstrates that with careful management of tissue processing and quality control it is possible to conduct multi-omic studies in a complex multi-institutional setting for investigations involving minute quantities of FFPE tumors, as in studies of early-stage melanoma. The study describes, for the first time, the optimal strategy for obtaining archival and limited tumor tissue, the characteristics of the nucleic acids co-extracted from a unique cell lysate, and success rate in downstream applications. In addition, our findings provide an estimate of the anticipated attrition that will guide other large multicenter research and consortia.

Mouse models for immuno-oncology.

Realizing the clinical promise of cancer immunotherapy is hindered by gaps in our knowledge of in vivo mechanisms underlying treatment response as well as treatment limiting toxicity. Preclinical in vivo model systems and technologies are required to address these knowledge gaps and to surmount the challenges faced in the clinical application of immunotherapy. Mice are commonly used for basic and translational research to support development and testing of immune interventions, including for cancer. Here, we discuss the advantages and the limitations of current models as well as future developments.

Discovery of decreased ferroptosis in male colorectal cancer patients with KRAS mutations.

Aberrant tumor metabolism is a hallmark of cancer in which metabolic rewiring can support tumor growth under nutrient deficient conditions. KRAS mutations occur in 35-45% of all colorectal cancer (CRC) cases and are difficult to treat. The relationship between mutant KRAS and aberrant metabolism in CRCs has not been fully explored and could be a target for intervention. We previously acquired non-targeted metabolomics data from 161 tumor tissues and 39 normal colon tissues from stage I-III chemotherapy naïve CRC patients. In this study, we revealed that only in male patients, tumors with KRAS mutations had several altered pathways that suppress ferroptosis, including glutathione biosynthesis, transsulfuration activity, and methionine metabolism. To validate this phenotype, MC38 CRC cells (KRASG13R) were treated with a ferroptosis inducer; RAS-selected lethal (RSL3). RSL3 altered metabolic pathways in the opposite direction to that seen in KRAS mutant tumors from male patients confirming a suppressed ferroptosis metabolic phenotype in these patients. We further validated gene expression data from an additional CRC patient cohort (Gene Expression Omnibus (GEO)), and similarly observed differences in ferroptosis-related genes by sex and KRAS status. Further examination of the relationship between these genes and overall survival (OS) in the GEO cohort showed that KRAS mutant tumors are associated with poorer 5-year OS compared to KRAS wild type tumors, and only in male patients. Additionally, high compared to low expression of GPX4, FTH1, FTL, which suppress ferroptosis, were associated with poorer 5-year OS only in KRAS mutant tumors from male CRC patients. Additionally, low compared to high expression of ACSL4 was associated with poorer OS for this group. Our results show that KRAS mutant tumors from male CRC patients have suppressed ferroptosis, and gene expression changes that suppress ferroptosis associate with adverse outcomes for these patients, revealing a novel potential avenue for therapeutic approaches.

Type 2 Dendritic Cells Orchestrate a Local Immune Circuit to Confer Antimetastatic Immunity.

The progression of transformed primary tumors to metastatic colonization is a lethal determinant of disease outcome. Although circulating adaptive and innate lymphocyte effector responses are required for effective antimetastatic immunity, whether tissue-resident immune circuits confer initial immunity at sites of metastatic dissemination remains ill defined. Here we examine the nature of local immune cell responses during early metastatic seeding in the lung using intracardiac injection to mimic monodispersed metastatic spread. Using syngeneic murine melanoma and colon cancer models, we demonstrate that lung-resident conventional type 2 dendritic cells (DC2) orchestrate a local immune circuit to confer host antimetastatic immunity. Tissue-specific ablation of lung DC2, and not peripheral DC populations, led to increased metastatic burden in the presence of an intact T cell and NK cell compartment. We demonstrate that DC nucleic acid sensing and transcription factors IRF3 and IRF7 signaling are required for early metastatic control and that DC2 serve as a robust source of proinflammatory cytokines in the lung. Critically, DC2 direct the local production of IFN-γ by lung-resident NK cells, which limits the initial metastatic burden. Collectively, our results highlight, to our knowledge, a novel DC2-NK cell axis that colocalizes around pioneering metastatic cells to orchestrate an early innate immune response program to limit initial metastatic burden in the lung.

Discovery of decreased ferroptosis in male colorectal cancer patients with KRAS mutations.

Aberrant tumor metabolism is a hallmark of cancer in which metabolic rewiring can support tumor growth under nutrient deficient conditions. KRAS mutations occur in 35-45% of all colorectal cancer (CRC) cases and are difficult to treat. The relationship between mutant KRAS and aberrant metabolism in CRCs has not been fully explored and could be a target for intervention. We previously acquired non-targeted metabolomics data from 161 tumor tissues and 39 normal colon tissues from stage I-III chemotherapy naïve CRC patients. In this study, we revealed that tumors from male patients with KRAS mutations only, had several altered pathways that suppress ferroptosis, including glutathione biosynthesis, transsulfuration activity, and methionine metabolism. To validate this phenotype, MC38 CRC cells (KRAS G13R ) were treated with a ferroptosis inducer; RAS-selected lethal (RSL3). RSL3 altered metabolic pathways in the opposite direction to that seen in KRAS mutant tumors from male patients confirming a suppressed ferroptosis metabolic phenotype in these patients. We further validated gene expression data from an additional CRC patient cohort (Gene Expression Omnibus (GEO), and similarly observed differences in ferroptosis-related genes by sex and KRAS status. Further examination of the relationship between these genes and overall survival (OS) in the GEO cohort showed that KRAS mutant tumors are associated with poorer 5-year OS compared to KRAS wild type tumors, and only in male patients. Additionally, high compared to low expression of GPX4, FTH1, FTL , which suppressed ferroptosis, were associated with poorer 5-year OS only in KRAS mutant tumors from male CRC patients. Low compared to high expression of ACSL4 was associated with poorer OS for this group. Our results show that KRAS mutant tumors from male CRC patients have suppressed ferroptosis, and gene expression changes that suppress ferroptosis associate with adverse outcomes for these patients, revealing a novel potential avenue for therapeutic approaches.

PTEN phosphatase inhibits metastasis by negatively regulating the Entpd5/IGF1R pathway through ATF6.

PTEN encodes a tumor suppressor with lipid and protein phosphatase activities whose dysfunction has been implicated in melanomagenesis; less is known about how its phosphatases regulate melanoma metastasis. We demonstrate that PTEN expression negatively correlates with metastatic progression in human melanoma samples and a PTEN-deficient mouse melanoma model. Wildtype PTEN expression inhibited melanoma cell invasiveness and metastasis in a dose-dependent manner, behaviors that specifically required PTEN protein phosphatase activity. PTEN phosphatase activity regulated metastasis through Entpd5. Entpd5 knockdown reduced metastasis and IGF1R levels while promoting ER stress. In contrast, Entpd5 overexpression promoted metastasis and enhanced IGF1R levels while reducing ER stress. Moreover, Entpd5 expression was regulated by the ER stress sensor ATF6. Altogether, our data indicate that PTEN phosphatase activity inhibits metastasis by negatively regulating the Entpd5/IGF1R pathway through ATF6, thereby identifying novel candidate therapeutic targets for the treatment of PTEN mutant melanoma.

Validation of the Prognostic Usefulness of the Gene Expression Profiling Test in Patients with Uveal Melanoma.

PURPOSE: To validate the prognostic usefulness of gene expression profile (GEP) testing in patients with uveal melanoma. To determine whether combining tumor size with the GEP classification provides additional prognostic value. DESIGN: Retrospective analysis. PARTICIPANTS: Patients with a diagnosis of choroidal melanoma examined at Yale New Haven Hospital; University of California, San Diego; and Memorial Sloan Kettering Cancer Center. METHODS: Patients' demographic and clinical data and tumor characteristics were collected. Univariate and multivariate Cox hazard regression analysis were used to assess the association between tumor characteristics and GEP classification with metastasis as an outcome. MAIN OUTCOME MEASURES: Metastasis-free survival (MFS). RESULTS: Of the 337 individuals included in the study, 87 demonstrated metastases. The mean follow-up time was 37.2 (standard deviation [SD], 40.2) months for patients with metastases and 55.0 (SD, 49.3) months for those without metastases. Tumors of larger thickness and GEP class 2 (vs. class 1) were associated significantly with increased risk of metastasis. Tumor thickness showed better prognostic usefulness than GEP classification (Wald statistic, 40.7 and 24.2, respectively). Class 2 tumors with a thickness of 7.0 mm or more were associated with increased risk of metastasis than tumors with a thickness of < 7.0 mm (hazard ratio [HR], 3.23; 95% confidence interval [CI], 1.61-6.51), whereas class 1 tumors with a thickness of 9.0 mm or more were associated with increased risk of metastasis than tumors with a thickness of < 9.0 mm (HR, 2.07; 95% CI, 0.86-4.99). No difference in MFS was found between patients with class 1A tumors compared with those with class 1B tumors (P = 0.8). Patients with class 2 tumors showed an observed 5-year MFS of 47.5% (95% CI, 36.0%-62.8%). CONCLUSIONS: Tumor size was the most significant predictor of metastasis and provided additional prognostic value independent of GEP classification. In addition, rates of metastasis for class 2 tumors were lower than estimates reported by Castle Bioscience, and no difference in rates of metastasis were found between class 1A and 1B tumors. This indicates that tumor size should be accounted for when relying on GEP for prognostication and that patients with GEP class 1A or 1B tumors may benefit from the same metastatic surveillance protocols. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.