Primary Melanoma Histologic Subtype: Impact on Survival and Response to Therapy.

BACKGROUND: Two primary histologic subtypes, superficial spreading melanoma (SSM) and nodular melanoma (NM), comprise the majority of all cutaneous melanomas. NM is associated with worse outcomes, which have been attributed to increased thickness at presentation, and it is widely expected that NM and SSM would exhibit similar behavior once metastasized. Herein, we tested the hypothesis that primary histologic subtype is an independent predictor of survival and may impact response to treatment in the metastatic setting. METHODS: We examined the most recent Surveillance, Epidemiology, and End Results (SEER) cohort (n = 118 508) and the New York University (NYU) cohort (n = 1621) with available protocol-driven follow-up. Outcomes specified by primary histology were studied in both the primary and metastatic settings with respect to BRAF-targeted therapy and immunotherapy. We characterized known driver mutations and examined a 140-gene panel in a subset of NM and SSM cases using next-generation sequencing. All statistical tests were two-sided. RESULTS: NM was an independent risk factor for death in both the SEER (hazard ratio [HR] = 1.55, 95% confidence interval [CI] = 1.41 to 1.70, P < .001) and NYU (HR = 1.47, 95% CI = 1.05, 2.07, P = .03) cohorts, controlling for thickness, ulceration, stage, and other variables. In the metastatic setting, NM remained an independent risk factor for death upon treatment with BRAF-targeted therapy (HR = 3.33, 95% CI = 1.06 to 10.47, P = .04) but showed no statistically significant difference with immune checkpoint inhibition. NM was associated with a higher rate of NRAS mutation (P < .001), and high-throughput sequencing revealed NM-specific genomic alterations in NOTCH4, ANK3, and ZNF560, which were independently validated. CONCLUSIONS: Our data reveal distinct clinical and biological differences between NM and SSM that support revisiting the prognostic and predictive impact of primary histology subtype in the management of cutaneous melanoma.

Characterization of MicroRNAs Regulating FOXO Expression.

MicroRNAs are critical post-transcriptional regulators of a majority of genes, of which the FOXO family of transcription factors is no exception. Here, we describe generalizable methods, including 3' UTR reporter assays and western blotting after microRNA manipulation, to test if a candidate miRNA (miR-182) directly targets a candidate (FOXO3) gene product. We also provide guidance on candidate miRNA selection and unbiased miRNA-target identification methods.

A miRNA-Based Signature Detected in Primary Melanoma Tissue Predicts Development of Brain Metastasis.

PURPOSE: Brain metastasis is the major cause of mortality among patients with melanoma. A molecular prognostic test that can reliably stratify patients at initial melanoma diagnosis by risk of developing brain metastasis may inform the clinical management of these patients. EXPERIMENTAL DESIGN: We performed a retrospective, cohort-based study analyzing genome-wide and targeted microRNA expression profiling of primary melanoma tumors of three patient cohorts (n = 92, 119, and 45) with extensive clinical follow-up. We used Cox regression analysis to establish a microRNA-based signature that improves the ability of the current clinicopathologic staging system to predict the development of brain metastasis. RESULTS: Our analyses identified a 4-microRNA (miR-150-5p, miR-15b-5p, miR-16-5p, and miR-374b-3p) prognostic signature that, in combination with stage, distinguished primary melanomas that metastasized to the brain from nonrecurrent and non-brain metastatic primary tumors (training cohort: C-index = 81.4%, validation cohort: C-index = 67.4%, independent cohort: C-index = 76.9%). Corresponding Kaplan-Meier curves of high- versus low-risk patients displayed a clear separation in brain metastasis-free and overall survival (training: P < 0.001; P < 0.001, validation: P = 0.033; P = 0.007, independent: P = 0.021; P = 0.022, respectively). Finally, of the microRNA in the prognostic model, we found that the expression of a key lymphocyte miRNA, miR-150-5p, which is less abundant in primary melanomas metastatic to brain, correlated with presence of CD45(+) tumor-infiltrating lymphocytes. CONCLUSIONS: A prognostic assay based on the described miRNA expression signature combined with the currently used staging criteria may improve accuracy of primary melanoma patient prognoses and aid clinical management of patients, including selection for adjuvant treatment or clinical trials of adjuvant therapies.

Identification of metastasis-suppressive microRNAs in primary melanoma.

BACKGROUND: Surgical management of primary melanoma is curative for most patients with clinically localized disease at diagnosis; however, a substantial number of patients recur and progress to advanced disease. Understanding molecular alterations that influence differential tumor progression of histopathologically similar lesions may lead to improved prognosis and therapies to slow or prevent metastasis. METHODS: We examined microRNA dysregulation by expression profiling of primary melanoma tumors from 92 patients. We screened candidate microRNAs selected by differential expression between recurrent and nonrecurrent tumors or associated with primary tumor thickness (Student's t test, Benjamini-Hochberg False Discovery Rate [FDR] < 0.05), in in vitro invasion assays. We performed in vivo metastasis assays, matrix remodeling experiments, and molecular studies to identify metastasis-regulating microRNAs and their cellular and molecular mechanisms. All statistical tests were two-sided. RESULTS: We identified two microRNAs (hsa-miR-382, hsa-miR-516b) whose expression was lower in aggressive vs nonaggressive primary tumors, which suppressed invasion in vitro and metastasis in vivo (mean metastatic foci: control: 37.9, 95% confidence interval [CI] = 25.6 to 50.2; miR-382: 19.5, 95% CI = 12.2 to 26.9, P = .009; miR-516b: 12.5, 95% CI = 7.7 to 17.4, P < .001, Student's t test). Mechanistically, miR-382 overexpression inhibits extracellular matrix degradation by melanoma cells. Moreover, we identified actin regulators CTTN, RAC1, and ARPC2 as direct targets of miR-382. Depletion of CTTN partially recapitulates miR-382 effects on matrix remodeling, invasion, and metastasis. Inhibition of miR-382 in a weakly tumorigenic melanoma cell line increased tumor progression and metastasis in vivo. CONCLUSIONS: Aberrant expression of specific microRNAs that can functionally impact progression of primary melanoma occurs as an early event of melanomagenesis.

Serum-based miRNAs in the prediction and detection of recurrence in melanoma patients.

BACKGROUND: Identification of primary melanoma patients at the highest risk of recurrence remains a critical challenge, and monitoring for recurrent disease is limited to costly imaging studies. We recently reported our array-based discovery of prognostic serum miRNAs in melanoma. In the current study, we examined the clinical utility of these serum-based miRNAs for prognosis as well as detection of melanoma recurrence. METHODS: Serum levels of 12 miRNAs were tested using qRT-PCR at diagnosis in 283 melanoma patients (training cohort, n = 201; independent validation, n = 82; median follow-up, 68.8 months). A refined miRNA signature was chosen and evaluated. We also tested the potential clinical utility of the miRNAs in early detection and monitoring of recurrence using multiple longitudinal samples (pre- and postrecurrence) in a subset of 82 patients (n = 225). In addition, we integrated our miRNA signature with publicly available Cancer Genome Atlas data to examine the relevance of these miRNAs to melanoma biology. RESULTS: Four miRNAs (miR-150, miR-30d, miR-15b, and miR-425) in combination with stage separated patients by recurrence-free survival (RFS) and overall survival (OS) and improved prediction of recurrence over stage alone in both the training and validation cohorts (training RFS and OS, P < .001; validation RFS, P < .001; OS, P = .005). Serum miR-15b levels significantly increased over time in recurrent patients (P < .001), adjusting for endogenous controls as well as age, sex, and initial stage. In nonrecurrent patients, miR-15b levels were not significantly changed with time (P =.17). CONCLUSIONS: Data demonstrate that serum miRNAs can improve melanoma patient stratification over stage and support further testing of miR-15b to guide patient surveillance.

BRD4 sustains melanoma proliferation and represents a new target for epigenetic therapy.

Metastatic melanoma remains a mostly incurable disease. Although newly approved targeted therapies are efficacious in a subset of patients, resistance and relapse rapidly ensue. Alternative therapeutic strategies to manipulate epigenetic regulators and disrupt the transcriptional program that maintains tumor cell identity are emerging. Bromodomain and extraterminal domain (BET) proteins are epigenome readers known to exert key roles at the interface between chromatin remodeling and transcriptional regulation. Here, we report that BRD4, a BET family member, is significantly upregulated in primary and metastatic melanoma tissues compared with melanocytes and nevi. Treatment with BET inhibitors impaired melanoma cell proliferation in vitro and tumor growth and metastatic behavior in vivo, effects that were mostly recapitulated by individual silencing of BRD4. RNA sequencing of BET inhibitor-treated cells followed by Gene Ontology analysis showed a striking impact on transcriptional programs controlling cell growth, proliferation, cell-cycle regulation, and differentiation. In particular, we found that, rapidly after BET displacement, key cell-cycle genes (SKP2, ERK1, and c-MYC) were downregulated concomitantly with the accumulation of cyclin-dependent kinase (CDK) inhibitors (p21 and p27), followed by cell-cycle arrest. Importantly, BET inhibitor efficacy was not influenced by BRAF or NRAS mutational status, opening the possibility of using these small-molecule compounds to treat patients for whom no effective targeted therapy exists. Collectively, our study reveals a critical role for BRD4 in melanoma tumor maintenance and renders it a legitimate and novel target for epigenetic therapy directed against the core transcriptional program of melanoma.

MicroRNA and cutaneous melanoma: from discovery to prognosis and therapy.

Melanoma incidence and associated mortality continue to increase worldwide. The lack of treatments with durable responses for stage IV melanoma may be due, at least in part, to an incomplete understanding of the molecular mechanisms that regulate tumor initiation and/or progression to metastasis. Recent evidence supports miRNA dysregulation in melanoma impacting several well-known pathways such as the PI3K/AKT or RAS/MAPK pathways, but also underexplored cellular processes like protein glycosylation and immune modulation. There is also increasing evidence that miRNA can improve patient prognostic classification over the classical staging system and provide new therapeutic opportunities. The integration of this recently acquired knowledge with known molecular alterations in protein coding genes characteristic of these tumors (i.e., BRAF and NRAS mutations, CDKN2A inactivation) is critical for a complete understanding of melanoma pathogenesis. Here, we compile the evidence of the functional roles of miRNAs in melanomagenesis and progression, and of their clinical utility as biomarkers, prognostic tools and potential therapeutic targets. Characterization of miRNA alterations in melanoma may provide new angles for therapeutic intervention, help to decipher mechanisms of drug resistance, and improve patient classification for disease surveillance and clinical benefit.

3-Indolyl sultams as selective CRTh2 antagonists.

CRTh2 (DP(2)) is a prostaglandin D(2) receptor implicated in the recruitment of eosinophils and basophils within the asthmatic lung. Here we report the discovery of a novel series of 3-indolyl sultam antagonists with low nM affinity for CRTh2. These compounds proved to be selective over the other primary prostaglandin D(2) receptor (DP1) as well as the thromboxane A(2) receptor (TP).

Isosteric ramatroban analogs: selective and potent CRTH-2 antagonists.

The chemoattractant receptor-homologous molecule expressed on T(H)2 cells (CRTH-2), also found on eosinophils and basophils, is a prostaglandin D2 receptor involved in the recruitment of these cell types during an inflammatory response. In this report, we describe the synthesis and optimization of a ramatroban isostere that is a selective and potent antagonist of CRTH-2 which may be useful in the treatment of certain diseases.