Size matters: integrating tumour volume and immune activation signatures predicts immunotherapy response.

Immune checkpoint inhibitors (ICIs) have transformed cancer treatment, providing significant benefit to patients across various tumour types, including melanoma. However, around 40% of melanoma patients do not benefit from ICI treatment, and accurately predicting ICI response remains challenging. We now describe a novel and simple approach that integrates immune-associated transcriptome signatures and tumour volume burden to better predict ICI response in melanoma patients. RNA sequencing was performed on pre-treatment (PRE) tumour specimens derived from 32 patients with advanced melanoma treated with combination PD1 and CTLA4 inhibitors. Of these 32 patients, 11 also had early during treatment (EDT, 5-15 days after treatment start) tumour samples. Tumour volume was assessed at PRE for all 32 patients, and at first computed tomography (CT) imaging for the 11 patients with EDT samples. Analysis of the Hallmark IFNγ gene set revealed no association with ICI response at PRE (AUC ROC curve = 0.6404, p = 0.24, 63% sensitivity, 71% specificity). When IFNg activity was evaluated with tumour volume (ratio of gene set expression to tumour volume) using logistic regression to predict ICI response, we observed high discriminative power in separating ICI responders from non-responders (AUC = 0.7760, p = 0.02, 88% sensitivity, 67% specificity); this approach was reproduced with other immune-associated transcriptomic gene sets. These findings were further replicated in an independent cohort of 23 melanoma patients treated with PD1 inhibitor. Hence, integrating tumour volume with immune-associated transcriptomic signatures improves the prediction of ICI response, and suggest that higher levels of immune activation relative to tumour burden are required for durable ICI response.

Employment, work hours, and wages in adults with myelin oligodendrocyte glycoprotein antibody disease: An international cohort study.

OBJECTIVES: The objectives were to understand the employment impacts of myelin oligodendrocyte glycoprotein-associated antibody disease (MOGAD) on adults in an international cohort by determining lost employment, work hours, and wages. BACKGROUND: Clinically, MOGAD can be associated with significant disability; however, its socioeconomic consequences for adults are barely reported. METHODS: Participants of potential working age (18-70 years old) with neurologist-diagnosed MOGAD were recruited from clinical sites in 13 countries, April 2022 to August 2023. Each participant completed a one-time survey. Regression models assessed associations with post-MOGAD (1) unemployment and (2) work hours. RESULTS: A total of 117 participants (66.7% female), mean age 39.7 years, median disease duration 3 years (25th, 75th percentile: 1, 7) were analyzed. Employment post-MOGAD reduced from 74 (63.2%) to 57 (48.7%) participants. Participants employed pre-diagnosis reduced their work hours, on average, from 31.6 hours/week to 19.5 hours/week post-diagnosis. Residence in a high-income country was statistically significantly associated with post-diagnosis employment and higher weekly work hours. Depressed mood was associated with unemployment. MOGAD-related pain and history of myelitis were independently associated with lost work hours. CONCLUSION: MOGAD can have significant impacts on adult employment, particularly in non-high-income countries. Depressed mood and pain are potentially modifiable factors related to socioeconomic status in MOGAD.

Single-cell RNA sequencing reveals melanoma cell state-dependent heterogeneity of response to MAPK inhibitors.

BACKGROUND: Melanoma is a heterogeneous cancer influenced by the plasticity of melanoma cells and their dynamic adaptations to microenvironmental cues. Melanoma cells transition between well-defined transcriptional cell states that impact treatment response and resistance. METHODS: In this study, we applied single-cell RNA sequencing to interrogate the molecular features of immunotherapy-naive and immunotherapy-resistant melanoma tumours in response to ex vivo BRAF/MEK inhibitor treatment. FINDINGS: We confirm the presence of four distinct melanoma cell states - melanocytic, transitory, neural-crest like and undifferentiated, and identify enrichment of neural crest-like and undifferentiated melanoma cells in immunotherapy-resistant tumours. Furthermore, we introduce an integrated computational approach to identify subsets of responding and nonresponding melanoma cells within the transcriptional cell states. INTERPRETATION: Nonresponding melanoma cells are identified in all transcriptional cell states and are predisposed to BRAF/MEK inhibitor resistance due to pro-inflammatory IL6 and TNFɑ signalling. Our study provides a framework to study treatment response within distinct melanoma cell states and indicate that tumour-intrinsic pro-inflammatory signalling contributes to BRAF/MEK inhibitor resistance. FUNDING: This work was supported by Macquarie University, Melanoma Institute Australia, and the National Health and Medical Research Council of Australia (NHMRC; grant 2012860, 2028055).

Multi-actor system dynamics in access to disease-modifying treatments for multiple sclerosis in Southeast Asia: A regional survey and suggestions for improvement.

BACKGROUND: Despite the global availability of multiple sclerosis (MS) treatments, accessing and financing them in Southeast Asia (SEA) remains a challenge. This descriptive survey-based study aimed to describe the current state of MS treatment access and local access dynamics within this region. METHODS: The survey questionnaire, comprising of 15 closed-ended and five open-ended questions, was developed by three neurologists with expertise in MS and routine MS patient management, or had training in neuroimmunology. Questionnaire development was guided by the recent Atlas of MS and in alignment with the Access to Treatment framework, focusing on MS diagnosis and treatment issues in SEA. Fifteen neurologists experienced in managing MS across the region were identified as key informants for this study. RESULTS: All fifteen neurologists participated in the survey via email and videoconferencing between January 2020 and February 2023, which included the following countries: Brunei, Cambodia, Indonesia, Malaysia, Myanmar, Lao PDR, Philippines, Singapore, Thailand, Timor-Leste, and Vietnam. All had at least five years of experience in managing MS patients and six had previously completed a neuroimmunology fellowship programme. SEA countries showed disparities in healthcare financing, availability of neurologists, MS treatments, and investigative tools. Access to MS disease-modifying treatments (DMTs) is hindered by high cost, lack of MS specialists, and weak advocacy efforts. On-label DMTs are not listed as essential medicines regionally except for interferon beta1a and teriflunomide in Malaysia. On-label monoclonals are available only in Malaysia, Singapore, and Thailand. Generic on-label DMTs are unavailable due to lack of distributorship and expertise in using them. Off-label DMTs (azathioprine, methotrexate, and rituximab) predominate in most SEA countries. Other challenges include limited access to investigations, education, and knowledge about DMTs among general neurologists, and absence of registries and MS societies. Patient champions, communities, and MS organisations have limited influence on local governments and pharmaceutical companies. Despite its increasing prevalence, there is a lack of concerted priority setting due to MS being perceived as a rare, non-communicable disease. CONCLUSION: This study highlights the distinct dynamics, challenges, and research gaps within this region, and provides suggestions to improve MS diagnosis, education, and medicine access.

Efficacy and Safety of Ripretinib in Advanced Gastrointestinal Stromal Tumors within an Expanded Access Program: A Cohort Study.

Ripretinib, a novel tyrosine kinase inhibitor used in advanced gastrointestinal stromal tumors (GIST) resistant to standard therapies, was assessed in the United Kingdom (UK) within an Expanded Access Program (EAP). A retrospective review of patients treated between January 2020 and October 2021 within the ripretinib EAP in our Institution was conducted. Clinician-documented and mRECIST 1.1 assessments were collected. The primary endpoints were progression-free survival (PFS) and time to treatment discontinuation (TTD). Treatment beyond progression (TBP), overall survival (OS), objective response rates and safety data were also analyzed. Survival curves were constructed using the Kaplan-Meier method, and univariate and multivariate Cox regression analyses were performed. All analyses were performed with R software. Overall, forty-five patients were included. After a median follow-up of 24.2 (95% CI 19.7-29.7) months, the median PFS of the group receiving 150 mg ripretinib once daily (OD) was 7.9 (95% CI 5.6-19.3) months. In the cohort of 22 patients with dose escalation upon tumor progression to 150 mg ripretinib twice daily (BD), the median PFS from BD was 5.4 (95% CI 2.8-9.3) months. Overall, median PFS and OS values for patients on ripretinib were 9.7 (95% CI 8.3-18.1) and 14.0 (95% CI 9.9-NA) months, respectively. TTD was similar to PFS. TBP was observed in about one third of all patients. Objective responses to ripretinib OD and BD treatments were observed in 16.7% and 10.0% of the patients, respectively. No new safety signals were identified. In conclusion, patients with advanced GIST receiving ripretinib in the UK within the EAP reported prolonged benefits, in line with the recent phase III clinical trials.

PKC-independent PI3K signalling diminishes PKC inhibitor sensitivity in uveal melanoma.

Protein kinase C (PKC) is activated downstream of gain-of-function GNAQ or GNA11 (GNAQ/GNA11) mutations in over 90% of uveal melanoma (UM). Phase I clinical trials of PKC inhibitors have shown modest response rates with no survival benefit in metastatic UM. Although PKC inhibitors actively suppress mitogen-activated protein kinase (MAPK) signalling in UM, the effect on other UM signalling cascades is not well understood. We examined the transcriptome of UM biopsies collected pre- and post-PKC inhibitor therapy and confirmed that MAPK, but not PI3K/AKT signalling, was inhibited early during treatment with the second-generation PKC inhibitor IDE196. Similarly, in GNAQ/GNA11-mutant UM cell models, PKC inhibitor monotherapy effectively suppressed MAPK activity, but PI3K/AKT signalling remained active, and thus, concurrent inhibition of PKC and PI3K/AKT signalling was required to synergistically induce cell death in a panel of GNAQ/GNA11-mutant UM cell lines. We also show that re-activation of MAPK signalling has a dominant role in regulating PKC inhibitor responses in UM and that PI3K/AKT signalling diminishes UM cell sensitivity to PKC inhibitor monotherapy. Thus, combination therapies targeting PKC and PKC-independent signalling nodes, including PI3K/AKT activity, are required to improve responses in patients with metastatic UM.

Single-cell RNA sequencing in melanoma: what have we learned so far?

Over the past decade, there have been remarkable improvements in the treatment and survival rates of melanoma patients. Treatment resistance remains a persistent challenge, however, and is partly attributable to intratumoural heterogeneity. Melanoma cells can transition through a series of phenotypic and transcriptional cell states that vary in invasiveness and treatment responsiveness. The diverse stromal and immune contexture of the tumour microenvironment also contributes to intratumoural heterogeneity and disparities in treatment response in melanoma patients. Recent advances in single-cell sequencing technologies have enabled a more detailed understanding of melanoma heterogeneity and the underlying transcriptional programs that regulate melanoma cell diversity and behaviour. In this review, we examine the concept of intratumoural heterogeneity and the challenges it poses to achieving long-lasting treatment responses. We focus on the significance of next generation single-cell sequencing in advancing our understanding of melanoma diversity and the unique insights gained from single-cell studies.

Post-COVID-19 Condition Characterization: Insights From a Cross-Sectional Study in a Malaysian Rehabilitation Center.

Although post-COVID-19 condition (PCC) is a major public health concern, studies on PCC in Southeast Asia are lacking. This study aimed to describe PCC symptoms and its functional impact among COVID-19 survivors undergoing outpatient rehabilitation in Malaysia. We evaluated 3037 patients with confirmed COVID-19, referred between November 2020 and September 2022, 3 to 6 months after infection. PCC was diagnosed in 71.1%. Fatigue and dyspnea were the most common symptoms. The PCC patients had reduced respiratory, ambulatory, and musculoskeletal function, and higher fatigue and pain scores, and were less likely to return to work (odds ratio [OR] = 0.55) compared with non-PCC patients. Recognition of PCC symptoms and its functional impact can guide early, tailored, rehabilitation interventions.

Natalizumab Treatment for Relapsing Multiple Sclerosis Stabilises Normal-Appearing White Matter Microstructure: A One-Year Prospective Ultra-High-Field Quantitative Imaging Study.

(1) Background: Natalizumab dramatically reduces relapses and MRI inflammatory activity (new lesions and enhancing lesions) in multiple sclerosis (MS). Chemical exchange saturation transfer (CEST) MRI can explore brain tissue in vivo with high resolution and sensitivity. We investigated if natalizumab can prevent microstructural tissue damage progression measured with MRI at ultra-high field (7 Tesla) over the first year of treatment. (2) Methods: In this one-year prospective longitudinal study, patients with active relapsing-remitting MS were assessed clinically and scanned at ultra-high-field MRI at the time of their first natalizumab infusion, at 6 and 12 months, with quantitative imaging aimed to detect microstructural changes in the normal-appearing white matter (NAWM), including sequences sensitive to magnetisation transfer (MT) effects from amide proton transfer (MTRAPT) and the nuclear Overhauser effect (MTRNOE). (3) Results: 12 patients were recruited, and 10 patients completed the study. The difference in the T1 relaxation times at month 6 and month 12 of natalizumab treatment was not significant, suggesting the lack of accumulation of tissue damage, while improvements were seen in MTR (MTRAPT and MTRNOE measures) at month 12, suggesting a tissue repair effect. This paralleled the expected lack of clinical and radiological worsening of conventional MRI measures of disease activity (new lesions or gadolinium-enhancing lesions). (4) Conclusion: Natalizumab prevents microstructural brain damage and has effects suggesting an improved white matter microstructure measured at ultra-high field during the first year of treatment.

The molecular and functional landscape of resistance to immune checkpoint blockade in melanoma.

Resistance to immune checkpoint inhibitor therapies in melanoma is common and remains an intractable clinical challenge. In this study, we comprehensively profile immune checkpoint inhibitor resistance mechanisms in short-term tumor cell lines and matched tumor samples from melanoma patients progressing on immune checkpoint inhibitors. Combining genome, transcriptome, and high dimensional flow cytometric profiling with functional analysis, we identify three distinct programs of immunotherapy resistance. Here we show that resistance programs include (1) the loss of wild-type antigen expression, resulting from tumor-intrinsic IFNγ signaling and melanoma de-differentiation, (2) the disruption of antigen presentation via multiple independent mechanisms affecting MHC expression, and (3) immune cell exclusion associated with PTEN loss. The dominant role of compromised antigen production and presentation in melanoma resistance to immune checkpoint inhibition highlights the importance of treatment salvage strategies aimed at the restoration of MHC expression, stimulation of innate immunity, and re-expression of wild-type differentiation antigens.

IFN-γ Signaling Sensitizes Melanoma Cells to BH3 Mimetics.

Immunotherapy targeting PD-1 and/or CTLA4 leads to durable responses in a proportion of patients with melanoma. However, many patients will not respond to these immune checkpoint inhibitors, and up to 60% of responding patients will develop treatment resistance. We describe a vulnerability in melanoma driven by immune cell activity that provides a pathway towards additional treatment options. This study evaluated short-term melanoma cell lines (referred to as PD1 PROG cells) derived from melanoma metastases that progressed on PD-1 inhibitor-based therapy. We show that the cytokine IFN-γ primes melanoma cells for apoptosis by promoting changes in the accumulation and interactions of apoptotic regulators MCL-1, NOXA, and BAK. The addition of pro-apoptotic BH3 mimetic drugs sensitized PD1 PROG melanoma cells to apoptosis in response to IFN-γ or autologous immune cell activation. These findings provide translatable strategies for combination therapies in melanoma.

Longitudinal clinical study of patients with iron rim lesions in multiple sclerosis.

BACKGROUND: Iron rims (IRs) surrounding white matter lesions (WMLs) are suggested to predict a more severe disease course. Only small longitudinal cohorts of patients with and without iron rim lesions (IRLs) have been reported so far. OBJECTIVE: To assess whether the presence and number of IRLs in patients with clinically isolated syndrome (CIS) and multiple sclerosis (MS) are associated with long-term disability or progressive disease. METHODS: Ninety-one CIS/MS patients were recruited between 2008 and 2013 and scanned with 7 T magnetic resonance imaging (MRI). Expanded Disability Status Scale (EDSS) was used to calculate Age-related Multiple Sclerosis Severity Score (ARMSS) at the time of scan and at the latest clinical follow-up after 9 years. WMLs were assessed for the presence of IRL using Susceptibility weighted imaging (SWI)-filtered phase images. RESULTS: In all, 132 IRLs were detected in 42 patients (46%); 9% of WMLs had IRs; 54% of the cohort had no rims, 30% had 1-3 rims and 16% had ⩾4. Patients with IRL had a higher EDSS and ARMSS. Presence of IRL was also a predictor of long-term disability, especially in patients with ⩾4 IRLs. IRLs have a greater impact on disability compared to the WML number and volume. CONCLUSION: The presence and number of perilesional IR on MRI hold prognostic value for long-term clinical disability in MS.

Association of temporomandibular disorders severity with otologic and concomitant pain symptoms in Asian youths.

OBJECTIVE: To investigate the presence/severity of TMDs among Asian youths and examine the associations between TMD severity, otologic, and concomitant pain symptoms. METHODS: Youths (17-24 years old) were recruited from a local polytechnic. The presence/severity of TMDs was determined with the Fonseca Anamnestic Index (FAI), while otologic/concomitant pain symptoms were appraised with the Maciel's Symptoms Checklist (MSC). Demographic, FAI, and MSC data were evaluated using Kruskal Wallis, chi-square, and relevant post-hoc tests (α = 0.05). RESULTS: Among the participants (n = 200) enrolled, 40.5% had no TMD, whereas mild, moderate, and severe TMD were present in 43.5%, 12.5%, and 3.5%, respectively. Participants with moderate/severe TMDs had significantly more otalgia, tinnitus, vertigo, dizziness, ear pruritus, hearing loss, ear fullness, headache, eye, neck, and back pain than those with no TMDs (p < 0.001). CONCLUSION: Otologic and concomitant pain symptoms were associated with TMDs and appear to increase with progressive TMD severity.

Young-onset stroke complicating ulcerative colitis.

A 16-year-old girl developed a proximal occlusion of the right middle cerebral artery during a flare-up of acute ulcerative colitis. Although mechanical thrombectomy led to successful middle cerebral artery recanalisation, she required an immediate second thrombectomy due to reocclusion of the same arterial segment. She developed a second ischaemic event 7 days later despite intravenous heparin infusion, later switched to low-molecular-weight heparin, and a third event after 3 days despite the addition of aspirin. We discuss stroke risks in people with inflammatory bowel disease and the uncertainties around anticoagulation and antiplatelet regimens in such cases.

Melanoma Cell State-Specific Responses to TNFα.

Immune checkpoint inhibitors that target the programmed cell death protein 1 (PD1) pathway have revolutionized the treatment of patients with advanced metastatic melanoma. PD1 inhibitors reinvigorate exhausted tumor-reactive T cells, thus restoring anti-tumor immunity. Tumor necrosis factor alpha (TNFα) is abundantly expressed as a consequence of T cell activation and can have pleiotropic effects on melanoma response and resistance to PD1 inhibitors. In this study, we examined the influence of TNFα on markers of melanoma dedifferentiation, antigen presentation and immune inhibition in a panel of 40 melanoma cell lines. We report that TNFα signaling is retained in all melanomas but the downstream impact of TNFα was dependent on the differentiation status of melanoma cells. We show that TNFα is a poor inducer of antigen presentation molecules HLA-ABC and HLA-DR but readily induces the PD-L2 immune checkpoint in melanoma cells. Our results suggest that TNFα promotes dynamic changes in melanoma cells that may favor immunotherapy resistance.

Tumor MHC Expression Guides First-Line Immunotherapy Selection in Melanoma.

Immunotherapy targeting T-cell inhibitory receptors, namely programmed cell death-1 (PD-1) and/or cytotoxic T-lymphocyte associated protein-4 (CTLA-4), leads to durable responses in a proportion of patients with advanced metastatic melanoma. Combination immunotherapy results in higher rates of response compared to anti-PD-1 monotherapy, at the expense of higher toxicity. Currently, there are no robust molecular biomarkers for the selection of first-line immunotherapy. We used flow cytometry to profile pretreatment tumor biopsies from 36 melanoma patients treated with anti-PD-1 or combination (anti-PD-1 plus anti-CTLA-4) immunotherapy. A novel quantitative score was developed to determine the tumor cell expression of antigen-presenting MHC class I (MHC-I) molecules, and to correlate expression data with treatment response. Melanoma MHC-I expression was intact in all tumors derived from patients who demonstrated durable response to anti-PD-1 monotherapy. In contrast, melanoma MHC-I expression was low in 67% of tumors derived from patients with durable response to combination immunotherapy. Compared to MHC-I high tumors, MHC-I low tumors displayed reduced T-cell infiltration and a myeloid cell-enriched microenvironment. Our data emphasize the importance of robust MHC-I expression for anti-PD-1 monotherapy response and provide a rationale for the selection of combination immunotherapy as the first-line treatment in MHC-I low melanoma.

Genetic Alterations in the INK4a/ARF Locus: Effects on Melanoma Development and Progression.

Genetic alterations in the INK4a/ARF (or CDKN2A) locus have been reported in many cancer types, including melanoma; head and neck squamous cell carcinomas; lung, breast, and pancreatic cancers. In melanoma, loss of function CDKN2A alterations have been identified in approximately 50% of primary melanomas, in over 75% of metastatic melanomas, and in the germline of 40% of families with a predisposition to cutaneous melanoma. The CDKN2A locus encodes two critical tumor suppressor proteins, the cyclin-dependent kinase inhibitor p16INK4a and the p53 regulator p14ARF. The majority of CDKN2A alterations in melanoma selectively target p16INK4a or affect the coding sequence of both p16INK4a and p14ARF. There is also a subset of less common somatic and germline INK4a/ARF alterations that affect p14ARF, while not altering the syntenic p16INK4a coding regions. In this review, we describe the frequency and types of somatic alterations affecting the CDKN2A locus in melanoma and germline CDKN2A alterations in familial melanoma, and their functional consequences in melanoma development. We discuss the clinical implications of CDKN2A inactivating alterations and their influence on treatment response and resistance.

Platelet function/reactivity testing and prediction of risk of recurrent vascular events and outcomes after TIA or ischaemic stroke: systematic review and meta-analysis.

BACKGROUND: The prevalence of ex vivo 'high on-treatment platelet reactivity (HTPR)' and its relationship with recurrent vascular events/outcomes in patients with ischaemic cerebrovascular disease (CVD) is unclear. METHODS: A systematic review and meta-analysis was performed in accordance with the PRISMA statement. MEDLINE, EMBASE and Cochrane Library were searched for completed manuscripts until May 2019 on TIA/ischaemic stroke patients, ≥ 18 years, treated with commonly-prescribed antiplatelet therapy, who had platelet function/reactivity testing and prospective follow-up data on recurrent stroke/TIA, myocardial infarction, vascular death or other cerebrovascular outcomes. Data were pooled using random-effects meta-analysis. Primary outcome was the composite risk of recurrent stroke/TIA, myocardial infarction or vascular death. Secondary outcomes were recurrent stroke/TIA, severe stroke (NIHSS > 16) or disability/impairment (modified Rankin scale ≥ 3) during follow-up. RESULTS: Antiplatelet-HTPR prevalence was 3-65% with aspirin, 8-56% with clopidogrel and 1.8-35% with aspirin-clopidogrel therapy. Twenty studies (4989 patients) were included in our meta-analysis. There was a higher risk of the composite primary outcome (OR 2.93, 95% CI 1.90-4.51) and recurrent ischaemic stroke/TIA (OR 2.43, 95% CI 1.51-3.91) in patients with vs. those without 'antiplatelet-HTPR' on any antiplatelet regimen. These risks were also more than twofold higher in patients with vs. those without 'aspirin-HTPR' and 'dual antiplatelet-HTPR', respectively. Clopidogrel-HTPR status did not significantly predict outcomes, but the number of eligible studies was small. The risk of severe stroke was higher in those with vs. without antiplatelet-HTPR (OR 2.65, 95% CI 1.00-7.01). DISCUSSION: Antiplatelet-HTPR may predict risks of recurrent vascular events/outcomes in CVD patients. Given the heterogeneity between studies, further prospective, multi-centre studies are warranted.

Transcriptional downregulation of MHC class I and melanoma de- differentiation in resistance to PD-1 inhibition.

Transcriptomic signatures designed to predict melanoma patient responses to PD-1 blockade have been reported but rarely validated. We now show that intra-patient heterogeneity of tumor responses to PD-1 inhibition limit the predictive performance of these signatures. We reasoned that resistance mechanisms will reflect the tumor microenvironment, and thus we examined PD-1 inhibitor resistance relative to T-cell activity in 94 melanoma tumors collected at baseline and at time of PD-1 inhibitor progression. Tumors were analyzed using RNA sequencing and flow cytometry, and validated functionally. These analyses confirm that major histocompatibility complex (MHC) class I downregulation is a hallmark of resistance to PD-1 inhibitors and is associated with the MITFlow/AXLhigh de-differentiated phenotype and cancer-associated fibroblast signatures. We demonstrate that TGFß drives the treatment resistant phenotype (MITFlow/AXLhigh) and contributes to MHC class I downregulation in melanoma. Combinations of anti-PD-1 with drugs that target the TGFß signaling pathway and/or which reverse melanoma de-differentiation may be effective future therapeutic strategies.