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Long follow-up time is needed for overall survival (OS) data to mature for early-stage melanoma. This retrospective study aimed to describe the relationships between OS and two intermediate endpoints - real-world recurrence-free survival (rwRFS) and real-world distant metastasis-free survival (rwDMFS) - for patients with stage IIB or IIC melanoma that was completely resected from 1 January 2008 to 31 December 2017, with follow-up to 31 December 2020. We used three different approaches to describe the relationships: estimates of correlation using Kendall τ rank correlation; comparisons of all-cause survival with/without recurrence or distant metastasis using adjusted Cox proportional hazard models; and landmark analyses of all-cause survival stratified by recurrence status at 1-5 years. During a 39-month median follow-up from surgical resection, 223/567 patients (39%) experienced recurrence, among whom 171/567 patients (30%) developed distant metastasis. Median OS from surgical resection was 117.6 months [95% confidence interval (CI), 104.7-not reached], median rwRFS was 49.8 months (95% CI, 39.6-61.0), and median rwDMFS was 70.9 months (95% CI, 58.4-89.1). We observed strong correlations between rwRFS and OS, and between rwDMFS and OS (Kendall τ of 0.73 and 0.82, respectively). Risk of death was significantly greater after recurrence (all-cause survival adjusted hazard ratio [HR], 7.48; 95% CI, 4.55-12.29) or distant metastasis (adjusted HR, 11.00; 95% CI, 6.92-17.49). Risk of death remained significantly elevated with recurrence or distant metastasis by landmark years 1, 3, and 5 after surgical resection. These findings support the use of recurrence/rwRFS and distant metastasis/rwDMFS as surrogate endpoints for OS after complete resection of stage IIB or IIC melanoma., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported. Pembrolizumab adjuvant therapy was shown to significantly improve recurrence-free survival (RFS) and distant metastasis-free survival (DMFS) in patients with resected stage IIB or IIC melanoma in earlier analyses of the randomized, double-blind, phase III KEYNOTE-716 study (ClinicalTrials.gov identifier: NCT03553836). We report results of the protocol-specified final analysis of DMFS for KEYNOTE-716. Overall, 976 patients were randomly allocated to pembrolizumab (n = 487) or placebo (n = 489). As of January 4, 2023, median follow-up was 39.4 months (range, 26.0-51.4 months). The median DMFS was not reached in either treatment group, and the estimated 36-month DMFS was 84.4% for pembrolizumab and 74.7% for placebo (hazard ratio [HR], 0.59 [95% CI, 0.44 to 0.79]). The median RFS was not reached in either treatment group, and the estimated 36-month RFS was 76.2% for pembrolizumab and 63.4% for placebo (HR, 0.62 [95% CI, 0.49 to 0.79]). DMFS and RFS results were consistent across most prespecified subgroups, including stage IIB and stage IIC melanoma. The safety profile of pembrolizumab was manageable and consistent with previous reports. These results continue to support the use of pembrolizumab adjuvant therapy in patients with resected stage IIB or IIC melanoma.

Introduction: Pembrolizumab was approved in the US as adjuvant treatment of patients with stage IIB or IIC melanoma post-complete resection, based on prolonged recurrence-free survival vs. placebo in the Phase 3 KEYNOTE-716 trial. This study aimed to evaluate the cost-effectiveness of pembrolizumab vs. observation as adjuvant treatment of stage IIB or IIC melanoma from a US health sector perspective., Methods: A Markov cohort model was constructed to simulate patient transitions among recurrence-free, locoregional recurrence, distant metastasis, and death. Transition probabilities from recurrence-free and locoregional recurrence were estimated via multistate parametric modeling based on patient-level data from an interim analysis (data cutoff date: 04-Jan-2022). Transition probabilities from distant metastasis were based on KEYNOTE-006 data and network meta-analysis. Costs were estimated in 2022 US dollars. Utilities were based on applying US value set to EQ-5D-5L data collected in trial and literature., Results: Compared to observation, pembrolizumab increased total costs by $80,423 and provided gains of 1.17 quality-adjusted life years (QALYs) and 1.24 life years (LYs) over lifetime, resulting in incremental cost-effectiveness ratios of $68,736/QALY and $65,059/LY. The higher upfront costs of adjuvant treatment were largely offset by reductions in costs of subsequent treatment, downstream disease management, and terminal care, reflecting the lower risk of recurrence with pembrolizumab. Results were robust in one-way sensitivity and scenario analyses. At a $150,000/QALY threshold, pembrolizumab was cost-effective vs. observation in 73.9% of probabilistic simulations that considered parameter uncertainty., Conclusion: As an adjuvant treatment of stage IIB or IIC melanoma, pembrolizumab was estimated to reduce recurrence, extend patients' life and QALYs, and be cost-effective versus observation at a US willingness-to-pay threshold., (© 2023. The Author(s).)

Understanding pigmentation regulations taking into account the original skin color type is important to address pigmentary disorders. Biological models including adult melanocytes from different phenotypes allow to perform fine-tuned explorative studies and support discovery of treatments adapted to populations' skin color. However, technical challenges arise when trying to not only isolate but also amplify melanocytes from highly pigmented adult skin. To bypass the initial isolation and growth of cutaneous melanocytes, we harvested and expanded fibroblasts from light and dark skin donors and reprogrammed them into iPSC, which were then differentiated into melanocytes. The resulting melanocyte populations displayed high purity, genomic stability, and strong proliferative capacity, the latter being a critical parameter for dark skin cells. The iPSC-derived melanocyte strains expressed lineage-specific markers and could be successfully integrated into reconstructed skin equivalent models, revealing pigmentation status according to the native phenotype. In both monolayer cultures and 3D skin models, the induced melanocytes demonstrated responsiveness to promelanogenic stimuli. The data demonstrate that the iPSC-derived melanocytes with high proliferative capacity maintain their pigmentation genotype and phenotypic properties up to a proper integration into 3D skin equivalents, even for highly pigmented cells., (© 2022 The Authors. Pigment Cell & Melanoma Research published by John Wiley & Sons Ltd.)

Background: Patients with stage IIB or IIC melanoma who undergo surgery alone are at a substantial risk for disease recurrence. Adjuvant pembrolizumab significantly improved recurrence-free survival versus placebo in stage IIB or IIC melanoma in the first interim analysis of the KEYNOTE-716 trial. Here, we report results from the secondary endpoint of distant metastasis-free survival (prespecified third interim analysis), and recurrence-free survival with longer follow-up., Methods: KEYNOTE-716 is a multicentre, double-blind, placebo-controlled, crossover or rechallenge, randomised, phase 3 trial done at 160 academic medical centres and hospitals across 16 countries. Eligible patients were aged 12 years and older with newly-diagnosed, completely resected, and histologically confirmed stage IIB (T3b or T4a) or IIC (T4b) cutaneous melanoma; negative sentinel lymph node biopsy; and an Eastern Cooperative Oncology Group performance status of 0-1. Patients were randomly assigned (1:1) to receive either 200 mg of pembrolizumab (2 mg/kg up to a maximum of 200 mg in paediatric patients) or placebo, both intravenously, every 3 weeks for 17 cycles (part 1) or until disease recurrence or unacceptable toxicity. Eligible patients with disease recurrence could receive further treatment with pembrolizumab in the part 2 crossover or rechallenge phase. Randomisation was done using an interactive response technology system and stratified by T category and paediatric status. The primary endpoint was investigator-assessed recurrence-free survival (assessed here with longer follow-up), and we report the prespecified third interim analysis of distant metastasis-free survival (secondary endpoint). Efficacy analyses were done in the intention-to-treat population (all patients who were randomly assigned, according to assigned group) and safety was assessed in all patients who were randomly assigned and received at least one dose of trial treatment, according to the treatment received. KEYNOTE-716 is registered at ClinicalTrials.gov, NCT03553836, and has completed recruitment., Findings: Between Sept 23, 2018, and Nov 4, 2020, 976 patients were randomly assigned to receive pembrolizumab (n=487) or placebo (n=489). At a median follow-up of 27·4 months (IQR 23·1-31·7), median distant metastasis-free survival was not reached (95% CI not reached [NR]-NR) in either group. Pembrolizumab significantly improved distant metastasis-free survival (hazard ratio [HR] 0·64, 95% CI 0·47-0·88, p=0·0029) versus placebo. Median recurrence-free survival was 37·2 months (95% CI NR-NR) in the pembrolizumab group and not reached in the placebo group (95% CI NR-NR). The risk of recurrence remained lower with pembrolizumab versus placebo (HR 0·64, 95% CI 0·50-0·84). The most common grade 3 or worse adverse events were hypertension (16 [3%] of 483 patients in the pembrolizumab group vs 17 [4%] of 486 patients in the placebo group), diarrhoea (eight [2%] vs one [<1%]), rash (seven [1%] vs two [<1%]), autoimmune hepatitis (seven [1%] vs two [<1%]), and increased lipase (six [1%] vs eight [2%]). Treatment-related serious adverse events occurred in 49 (10%) patients in the pembrolizumab group and 11 (2%) patients in the placebo group. No treatment-related deaths were reported., Interpretation: Adjuvant pembrolizumab is an efficacious treatment option for resected stage IIB and IIC melanoma, with significant improvement in distant-metastasis free survival versus placebo and continued reduction in the risk of recurrence with an adverse event profile consistent with previous studies of pembrolizumab. The overall benefit-risk of pembrolizumab continues to be positive in the adjuvant setting., Funding: Merck Sharp & Dohme, a subsidiary of Merck & Co., Competing Interests: Declaration of interests GVL reports research funding to their institution from Merck Sharp & Dohme (MSD), a subsidiary of Merck & Co; fees for consulting or advisory roles for Agenus, Amgen, Array Biopharma, Boehringer Ingelheim International, Bristol Myers Squibb, Evaxion Biotech, Hexal (Sandoz Company), Highlight Therapeutics, MSD (Australia), Novartis, Oncosec Medical Australia, Pierre Fabre, Provectus, Qbiotics, and Regeneron Pharmaceuticals; and honoraria for speaker's bureau from Bristol-Myers Squibb (BMS) and Pierre Fabre. JJL reports research funding to the institution for clinical studies from MSD, AbbVie, Agios, Array, Astellas, AstraZeneca, BMS, Corvus, Day One, EMD Serono, Fstar, Genmab, Ikena, Immatics, Incyte, Kadmon, KAHR, Macrogenics, Moderna, Nektar, Next Cure, Numab, Palleon, Pfizer, Replimmune, Rubius, Servier, Scholar Rock, Synlogic, Takeda, Trishula, Tizona, and Xencor; membership on data safety monitoring board for AbbVie and Immutep; membership on scientific advisory boards for 7 Hills, Bright Peak, Fstar, Inzen, RefleXion, and Xilioo; stocks for Actym, Alphamab Oncology, Arch Oncology, Kanaph, Mavu, NeoTx, Onc.AI, Pyxis, and Tempest; compensation for consultancy with AbbVie, Alnylam, Bayer, BMS, CheckMate, Codiak, Crown, Cstone, Day One, Eisai, EMD Serono, Flame, Genentech, Gilead, HotSpot, Kadmon, KSQ, Janssen, Ikena, Immunocore, Incyte, Macrogenics, Merck, Mersana, Nektar, Novartis, Pfizer, Regeneron, Ribon, Rublus, Silicon, Synlogic, Synthekine, TRex, Werewold, and Xencor; and patents (provisional) for cancer immunotherapy (PCT/US18/36052; microbiome biomarkers for Anti-PD-1/PD-L1 responsiveness: diagnostic, prognostic and therapeutic uses thereof). MAK, J-JG, FdG, and CHY report research funding to their institution for clinical studies from MSD. LdlCM reports research funding to their institution for clinical studies from Celgene, MSD, and Roche; reimbursement for travel expenses from Roche; and consultant or advisory roles for BMS, MSD, Novartis, and Roche. MDV reports research funding to their institution for clinical studies from MSD and honoraria as a consultant or advisor for Novartis, BMS, MSD, and Pierre Fabre. PR reports research funding to their institution from MSD and Pfizer; honoraria for lectures and advisory board participation for MSD, BMS, Novartis, Pierre Fabre, Sanofi, Merck, Philogen, and Blueprint Medicines. FS reports research funding to their institution for clinical studies from MSD; and fees for consulting and honoraria for speakers bureaus from MSD, Novartis, Pierre Fabre, Philogen, Sun Pharma, Merck, and BMS. JM reports research funding to their institution for clinical studies from MSD; honoraria from BMS, MSD, Roche, Novartis, and Pierre Fabre; and consultant or advisory roles for BMS and MSD. VC-S reports research funding to their institution for clinical studies from MSD; reimbursement for travel and accommodation for medical congress from Novartis and Pierre Fabre; and honoraria for advisory board participation for Novartis, Pierre Fabre, BMS, and Merck-Serono. JMK reports research funding to their institution from MSD, Amgen, BMS, Checkmate Pharmaceuticals, Immunocore, Iovance Biotherapies, Novartis Pharmaceuticals, Castle Biosciences, and Merck; fees from Amgen, Ankyra Therapeutics, Axios Research Instil Bio, BMS, Checkmate Pharmaceuticals, DermTech, Elsevier, Harbour BioMed, Immunocore, Iovance Biotherapies, Istari Oncology, Millenium Pharmaceuticals or Takeda Pharmaceuticals, Natera, Novartis Pharmaceutical, OncoCyte Corporation, OncoSec Medical, Pfizer, Scopus BioPharma, and Merck. CR reports research funding to their institution for clinical studies from MSD and fees as a consultant or advisor for Amgen, AstraZeneca, BMS, MSD, Novartis, Pfizer, Pierre Fabre, Roche, and Sanofi. DS reports research finding to their institution for clinical studies from BMS, MSD, Novartis, Amgen, and Array; patient fees to their institution from BMS, MSD, Novartis, Merck–EMD, Philogen, Pfizer, Array, InflarX, OncoSec, Replimune, Neracare, Nektar, Sun Pharma, Sandoz, and UltimoVacs; reimbursement for travel from BMS, Merck, Novartis, Merck-EMD, Pfizer, Pierre Fabre, InflarX, NeraCare, and Nektar; and non-financial support from BMS, MSD, Novartis, Merck-EMD, Pierre-Fabre, InFlarX, Neracare, Nektar, Sun Pharma, and Sandoz. MSC reports research funding to their institution for clinical studies for MSD and honoraria for consulting from BMS, Eisia, IDEAYA Biosciences, Merck Serono, MSD, Novartis, Oncosec, Pierre Fabre, Qbiotics, Roche, and Sanofi. PM reports research funding to their institution for clinical studies from MSD, Amgen, Johnson & Johnson, Merck Serono, Novartis, Pfizer, Sanofi, and Sun Pharma; honoraria from Amgen, BMS, Merck, MSD, Novartis, Pierre Fabre, Genentech (Roche Group), and Sanofi; consulting or advisory roles for Amgen, BMS, Merck, MSD, Novartis, Pierre Fabre, Roche, and Sanofi; fees for speakers bureau from Amgen, BMS, MSD, Novartis, Roche, and Sanofi; and reimbursement for travel, accommodations, and expenses from Amgen, BMS, MSD, Novartis, Pierre Fabre, Roche, Sanofi, and Sun Pharma. RD reports research funding to their institution for clinical studies from MSD and consulting or advisory roles with MSD, Novartis, Roche, BMS, Amgen, Takeda, Pierre Fabre, Sun Pharma, Sanofi, Catalym, Second Genome, Regeneron, Alligator, T3 Pharma, MaxiVAX, Pfizer, and touchIME. JEG reports research funding to their institution for clinical studies from Merck; royalties for content contribution to UpToDate; consulting or advisory roles for Merck, Regeneron, Syndax, and Bristol-Myers Squibb; fees for speakers bureau for Banner MD Anderson Phoenix; reimbursement for travel to meetings from American Joint Committee on Cancer (AJCC), American College of Surgeons, Melanoma Research Alliance, and Bridges Melanoma meeting; and leadership roles for AJCC and Melanoma Research Alliance. XLW, MF-K, and CK are employees of and own stock in MSD. AMME reports research funding to their institution for clinical studies from MSD; fees for advisory board membership for Merck, Agenus, Biocad, BioInvent, BioNTech, BMS, CatalYm, Ellipses, Galecto, GSK, IO Biotech, ISA Pharmaceuticals, Merck, MSD, Nektar, Novartis, Pfizer, Sellas, SkylineDX, TigaTx, and TxDiscovery; fees for independent data monitoring committee for Biocad, GSK, Pfizer, and Novartis;fees for speakers bureau from Biocad, BMS, and Merck; and equity in IO Biotech and SkylineDX. PAA reports research funding to their institution for clinical studies by MSD, BMS, Genentech (Roche Group), Sanofi, and Pfizer or Array BioPharma; fees as a consultant or advisor from BMS, Genentech (Roche Group), MSD, Novartis, Merck Serono, Pierre Fabre, Sun Pharma, Sanofi, Idera, Sandoz, 4SC, Nektar, Italfarmaco, Pfizer or Array BioPharma, Lunaphore, Medicenna, Bio-Al Health, and ValoTx; and participation on data safety monitoring boards or advisory boards for BMS, Genentech (Roche Group), MSD, Novartis, AstraZeneca, Immunocore, Boehringer-Ingelheim, Eisai, Regeneron, Daiichi Sankyo, Oncosec, and ITeos., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Adjuvant pembrolizumab significantly improved recurrence-free survival (RFS) versus placebo in resected stage IIB and IIC melanoma in the phase 3 KEYNOTE-716 study. Health-related quality of life (HRQoL) results are reported., Methods: Patients were randomly assigned 1:1 to pembrolizumab 200 mg (2 mg/kg, patients ≥12 to <18 years) Q3W or placebo for ≤17 cycles or until disease recurrence, unacceptable toxicity, or withdrawal. Change from baseline in EORTC QLQ-C30 global health status (GHS)/quality of life (QoL) was a prespecified exploratory end point. Change in EORTC QLQ-C30 functioning, symptom, and single-item scales, and EQ-5D-5L visual analog scale (VAS) were also summarized. Primary analyses were performed at week 48 to ensure adequate completion/compliance. The HRQoL population comprised patients who received ≥1 dose of treatment and completed ≥1 assessment., Results: The HRQoL population included 969 patients (pembrolizumab, n = 483; placebo, n = 486). Compliance at week 48 was ≥80% for both instruments. EORTC QLQ-C30 GHS/QoL, physical functioning, role functioning, and EQ-5D-5L VAS scores were stable from baseline to week 48 in both arms, with no clinically meaningful decline observed. Scores did not differ significantly between pembrolizumab and placebo. EORTC QLQ-C30 GHS/QoL, physical functioning, role functioning, and EQ-5D-5L VAS scores remained stable through week 96 in both arms., Conclusions: HRQoL was stable with adjuvant pembrolizumab, with no clinically meaningful decline observed. Change from baseline in HRQoL was similar between arms. These results, in conjunction with the improved RFS and manageable safety previously reported, support the use of adjuvant pembrolizumab for high-risk stage II melanoma., Competing Interests: Conflict of interest statement The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: MK has no conflicts of interest to declare. JJL reports advisory/consultancy roles with AbbVie, Immutep, Evaxion, 7 Hills, Bright Peak, Exo, Fstar, Inzen, RefleXion, Xilio, Actym, Alphamab Oncology, Arch Oncology, Kanaph, Mavu, NeoTx, Onc.AI, OncoNano, Pyxis, STipe, Tempest, Bayer, Bristol Myers Squibb, Castle, Checkmate, Codiak, Crown, Day One, Duke St, EMD Serono, Endeavor, Flame, Genentech, Gilead, Glenmark, HotSpot, Kadmon, Janssen, Ikena, Immunocore, Incyte, IO Biotech, Macrogenics, Merck, Nektar, Novartis, Partner, Pfizer, Regeneron, Roivant, Servier, STINGthera, Synlogic, and Synthekine; stock ownership in Actym, AlphamabOncology, Arch Oncology, Kanaph, Mavu, NeoTx, Onc.AI, OncoNano, Pyxis, STipe, and Tempest; research grants/funding to their institution from AbbVie, Astellas, AstraZeneca, Bristol Myers Squibb, Corvus, Day One, EMD Serono, Fstar, Genmab, Ikena, Immatics, Incyte, Kadmon, KAHR, Macrogenics, Merck, Moderna, Nektar, Next Cure, Numab, Palleon, Pfizer, Replimmune, Rubius, Servier, Scholar Rock, Synlogic, Takeda, Trishula, Tizona, and Xencor; travel, accommodation, and/or expenses from Castle; and the following provisional patents: Serial #15/612,657 (Cancer Immunotherapy), PCT/US18/36052 (Microbiome Biomarkers for Anti-PD-1/PD-L1 Responsiveness: Diagnostic, Prognostic and Therapeutic Uses Thereof). GVL is consultant advisor for Agenus, Amgen, Array Biopharma, Boehringer Ingelheim, Bristol Myers Squibb, Evaxion, Hexal AG (Sandoz Company), Highlight Therapeutics S.L., Innovent Biologics USA, Merck Sharpe & Dohme, Novartis, OncoSec, PHMR Ltd, Pierre Fabre, Provectus, Qbiotics, Regeneron. PAA reports grants or contracts from Bristol Myers Squibb, Roche-Genentech, Pfizer/Array, and Sanofi; consulting fees from Bristol Myers Squibb, Roche-Genentech, MSD, Novartis, Merck Serono, Pierre-Fabre, Sun Pharma, Sanofi, Idera, Sandoz, 4SC, Italfarmaco, Nektar, Pfizer/Array, Lunaphore, and Medicenna.Bio-Al Health; and participation on a data safety monitoring board or advisory board for Bristol Myers Squibb, Roche-Genentech, MSD, Novartis, AstraZeneca, Immunocore, Boehringer Ingelheim, Eisai, Regeneron, Daiichi Sankyo, Oncosec, Nouscom, Seagen, and iTeos. PR reports advisory/consultancy roles with MSD, BMS, Merck, Sanofi, Pierre Fabre, Blueprint Medicines, and Philogen; speaker bureau for BMS, MSD, Novartis, Pierre Fabre, Merck, and Sanofi; research grant/funding to their institution from Pfizer; and officer/board of director roles with ASCO, ESMO, and the Polish Oncological Society. DS reports advisory/consultancy roles with 4SC, Amgen, Array Biopharma, AstraZeneca, BMS, Daiichi Sankyo, Haystack, Immunocore, InFlarX, Innovent, Labcorp, Merck Serono, MSD, Nektar, Neracare, Novartis, OncoSec, Pfizer, Philogen, Pierre Fabre, Replimune, Roche, Sandoz, Sanofi/Regeneron, and Sun Pharma; speaker bureau for BMS, Merck/MSD, Merck Serono, Novartis, Roche, Sanofi, and Sun Pharma; research grant/funding to their institution from Amgen, Array/Pfizer, BMS, MSD, Novartis, and Roche; travel, accommodation, and/or expenses from BMS, Merck Serono, MSD, Novartis, and Sanofi; officer/board of director role with WTZ; and the following nonremunerated activities: EORTC-MG, member of board of directors; coordinating PI for 4SC, BMS, MSD, Nektar, Novartis, and Pierre Fabre; local PI for Philogen, Roche, and Sanofi. CR worked in a consulting/advisory role for BMS, Roche, Amgen, Novartis, Pierre Fabre, MSD, Sanofi, Biothera, CureVac, and Merck. MAP worked in a consulting/advisory role for BMS, Merck, Eisai, Novartis, Incyte, NewLink Genetics, Aduro, and Pfizer; received research/grant support from BMS, Merck, Novartis, Array BioPharma, RGenix, Infinity, and AstraZeneca. JJG reports advisory/consultancy roles with BMS, MSD, Novartis, Roche, Philogen, Pierre Fabre, Sanofi, Amgen, Merck, and Pfizer; and travel, accommodations, and/or expenses from BMS and Pierre Fabre. LDLCM reports advisory/consultancy roles with Roche, BMS, and MSD-Merck; research grant/funding to their institution from Roche, Celgene, and MSD; and travel, accommodation, and/or expenses from Gilead. MDV reports honoraria and advisory/consultancy roles with Bristol Myers Squibb, Novartis, MSD, and Pierre Fabre. FS reports honoraria from BMS, Novartis, MSD, Pierre Fabre, Sanofi, Sun Pharma, Merck; and advisory/consultancy roles with Novartis, MSD, Pierre Fabre, Sun Pharma, and Philogen. JM reports honoraria from MSD, Bristol Myers Squibb, Pierre Fabre, Roche, Novartis; advisory/consultancy roles with MSD and Bristol Myers Squibb; and travel, accommodations, and/or expenses from MSD, Bristol Myers Squibb, Pierre Fabre, Roche, and Novartis. VCS reports travel, accommodation, and/or expenses from Pierre-Fabre and Novartis; and nonremunerated activities for BMS, Pierre-Fabre, and Merck-Serono. MSC reports honoraria from Bristol Myers Squibb, MSD, and Novartis; and advisory/consultancy roles with Amgen, Bristol Myers Squibb, Eisai, Ideaya, MSD, Nektar, Novartis, Oncosec, Pierre Fabre, Qbiotics, Regeneron, and Roche. PM reports honoraria and advisory/consultancy roles with Pierre Fabre, GSK, MSD, Merck Germany, Roche, BMS, Novartis, Sanofi, and Immunocore; research grant/funding from BMS, Novartis, and MSD; and travel, accommodation, and/or expenses from Pierre Fabre, MSD, Merck Germany, and Roche. FDG reports advisory/consultancy roles with BMS and Novartis; and research grant/funding to their institution from Novartis. MR reports advisory/consultancy roles with MSD; and travel, accommodation, and/or expenses from MSD. ZE reports advisory/consultancy roles with Pfizer, Novartis, Genentech, Regeneron, OncoSec, Natera, and Eisai; and research grant/funding to their institution from Pfizer and Novartis. KC is an employee of Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ USA. RJ, MFK, and CK are employees of Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ USA, and are stockholders of Merck & Co., Inc., Rahway, NJ USA. AE reports honoraria from Agenus, Biocad, BioInvent, BioNTech, Brenus, CatalYm, Clover, Ellipses, Galecto, GSK, IO Biotech, IQVIA, Merck/MSD, Nektar, Pfizer, Pierre Fabre, Sairopa, Sellas, SkylineDx, TigaTx, and TTxDiscovery; advisory/consultancy roles with Agenus, Biocad, BioInvent, BioNTech, Brenus, CatalYm, Clover, Ellipses, Galecto, GSK, IO Biotech, IQVIA, Merck/MSD, Nektar, Pfizer, Pierre Fabre, Sairopa, Sellas, SkylineDx, TigaTx, and TTxDiscovery; speaker bureau for Biocad, BMS, and Merck/MSD; a leadership role with the European Academy of Cancer Sciences; stock ownership in IO Biotech and SkylineDx; and nonremunerated activities for the European Academy of Cancer Sciences (EACS) and the Fondation Cancer (FOCA). JMK reports advisory/consultancy roles with Applied Clinical Intelligence LLC, Amgen Inc., Ankyra Therapeutics, Axio Research/Instil Bio, Becker Pharmaceutical Consulting, Bristol Myers Squibb, Checkmate Pharmaceuticals, DermTech, Fenix Group International, Harbour BioMed, Immunocore LLC, Intellisphere LLC/Cancer Network, Iovance Biotherapeutics, IQVIA, Istari Oncology, Merck, Millennium Pharmaceuticals/Takeda Pharmaceutical, Natera Inc, Novartis Pharmaceuticals, OncoCyte Corporation, OncoSec Medical Inc., Pfizer, Replimune, Scopus BioPharma, and SR One Capital Management; and research grant/funding to their institution from Amgen Inc., Bristol Myers Squibb, Castle Biosciences Inc, Checkmate Pharmaceuticals, Harbour BioMed, Immvira Pharma Co., Immunocore LLC, Iovance Biotherapeutics, Merck, Novartis Pharmaceuticals, Schering-Plough, Takeda, and Verastem Inc., (Copyright © 2022 The Author(s), Merck Sharp & Dohme LLC., a subsidiary of Merck & Co., Inc.,. Published by Elsevier Ltd.. All rights reserved.)

Aim: To describe clinical outcomes after complete surgical resection of stage IIB and IIC melanoma. Methods: Adult patients (n = 567) with stage IIB or IIC cutaneous melanoma initially diagnosed and completely resected from 2008-2017 were identified using data from a US community-based oncology network. Results: Median patient follow-up was 38.8 months from melanoma resection to death, last visit or data cut-off (31 December 2020). For stage IIB (n = 375; 66%), Kaplan-Meier median real-world recurrence-free survival (rwRFS) was 58.6 months (95% CI, 48.6-69.5). For stage IIC (n = 192; 34%), median rwRFS was 29.9 months (24.9-45.5). Overall, 44% of patients had melanoma recurrence or died; 30% developed distant metastases. Conclusion: Melanoma recurrence was common, highlighting the need for effective adjuvant therapy for stage IIB and IIC melanoma.

The upregulation of the adaptor protein NUMB triggers melanocytic differentiation from multipotent skin stem cells, which share many properties with aggressive melanoma cells. Although NUMB acts as a tumor suppressor in various human cancer types, little is known about its role in melanoma. In this study, we investigated the role of NUMB in melanoma progression and its regulatory mechanism. Analysis of The Cancer Genome Atlas melanoma datasets revealed that high NUMB expression in melanoma tissues correlates with improved patient survival. Moreover, NUMB expression is downregulated in metastatic melanoma cells. NUMB knockdown significantly increased the invasion potential of melanoma cells in a three-dimensional collagen matrix in vitro and in the lungs of a mouse model in vivo; it also significantly upregulated the expression of the NOTCH target gene CCNE. Previous studies suggested that Wnt signaling increases NUMB expression. By mimicking Wnt stimulation through glycogen synthase kinase-3 inhibition, we increased NUMB expression in melanoma cells. Furthermore, a glycogen synthase kinase-3 inhibitor reduced the invasion of melanoma cells in a NUMB-dependent manner. Together, our results suggest that NUMB suppresses invasion and metastasis in melanoma, potentially through its regulation of the NOTCH‒CCNE axis and that the inhibitors that upregulate NUMB can exert therapeutic effects in melanoma., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Metastatic melanoma is challenging to clinically address. Although standard-of-care targeted therapy has high response rates in patients with BRAF-mutant melanoma, therapy relapse occurs in most cases. Intrinsically resistant melanoma cells drive therapy resistance and display molecular and biologic properties akin to neural crest-like stem cells (NCLSC) including high invasiveness, plasticity, and self-renewal capacity. The shared transcriptional programs and vulnerabilities between NCLSCs and cancer cells remains poorly understood. Here, we identify a developmental LPAR1-axis critical for NCLSC viability and melanoma cell survival. LPAR1 activity increased during progression and following acquisition of therapeutic resistance. Notably, genetic inhibition of LPAR1 potentiated BRAFi ± MEKi efficacy and ablated melanoma migration and invasion. Our data define LPAR1 as a new therapeutic target in melanoma and highlights the promise of dissecting stem cell-like pathways hijacked by tumor cells. SIGNIFICANCE: This study identifies an LPAR1-axis critical for melanoma invasion and intrinsic/acquired therapy resistance., (©2021 The Authors; Published by the American Association for Cancer Research.)

Anti-PD-1 therapy is used as a front-line treatment for many cancers, but mechanistic insight into this therapy resistance is still lacking. Here we generate a humanized (Hu)-mouse melanoma model by injecting fetal liver-derived CD34 + cells and implanting autologous thymus in immune-deficient NOD-scid IL2Rγ null (NSG) mice. Reconstituted Hu-mice are challenged with HLA-matched melanomas and treated with anti-PD-1, which results in restricted tumor growth but not complete regression. Tumor RNA-seq, multiplexed imaging and immunohistology staining show high expression of chemokines, as well as recruitment of FOXP3 + Treg and mast cells, in selective tumor regions. Reduced HLA-class I expression and CD8 + /Granz B + T cells homeostasis are observed in tumor regions where FOXP3 + Treg and mast cells co-localize, with such features associated with resistance to anti-PD-1 treatment. Combining anti-PD-1 with sunitinib or imatinib results in the depletion of mast cells and complete regression of tumors. Our results thus implicate mast cell depletion for improving the efficacy of anti-PD-1 therapy.

Melanoma cells share many biological properties with neural crest stem cells. Here we show that the homeodomain transcription factor MSX1, which is significantly correlated with melanoma disease progression, reprograms melanocytes and melanoma cells toward a neural crest precursor-like state. MSX1-reprogrammed normal human melanocytes express the neural crest marker p75 and become multipotent. MSX1 induces a phenotypic switch in melanoma, which is characterized by an oncogenic transition from an E-cadherin-high nonmigratory state toward a ZEB1-high invasive state. ZEB1 up-regulation is responsible for the MSX1-induced migratory phenotype in melanoma cells. Depletion of MSX1 significantly inhibits melanoma metastasis in vivo. These results show that neural crest-like reprogramming achieved by a single factor is a critical process for melanoma progression., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

In melanoma, therapies with inhibitors to oncogenic BRAF V600E are highly effective but responses are often short-lived due to the emergence of drug-resistant tumor subpopulations. We describe here a mechanism of acquired drug resistance through the tumor microenvironment, which is mediated by human tumor-associated B cells. Human melanoma cells constitutively produce the growth factor FGF-2, which activates tumor-infiltrating B cells to produce the growth factor IGF-1. B-cell-derived IGF-1 is critical for resistance of melanomas to BRAF and MEK inhibitors due to emergence of heterogeneous subpopulations and activation of FGFR-3. Consistently, resistance of melanomas to BRAF and/or MEK inhibitors is associated with increased CD20 and IGF-1 transcript levels in tumors and IGF-1 expression in tumor-associated B cells. Furthermore, first clinical data from a pilot trial in therapy-resistant metastatic melanoma patients show anti-tumor activity through B-cell depletion by anti-CD20 antibody. Our findings establish a mechanism of acquired therapy resistance through tumor-associated B cells with important clinical implications.Resistance to BRAFV600E inhibitors often occurs in melanoma patients. Here, the authors describe a potential mechanism of acquired drug resistance mediated by tumor-associated B cells-derived IGF-1.

The discovery of activating BRAF mutations in approximately 50% of melanomas has led to the development of MAPK pathway inhibitors, which have transformed melanoma therapy. However, not all BRAF-V600E melanomas respond to MAPK inhibition. Therefore, it is important to understand why tumors with the same oncogenic driver have variable responses to MAPK inhibitors. Here, we show that concurrent loss of PTEN and activation of the Notch pathway is associated with poor response to the ERK inhibitor SCH772984, and that co-inhibition of Notch and ERK decreased viability in BRAF-V600E melanomas. Additionally, patients with low PTEN and Notch activation had significantly shorter progression free survival when treated with BRAF inhibitors. Our studies provide a rationale to further develop combination strategies with Notch antagonists to maximize the efficacy of MAPK inhibition in melanoma. Our findings should prompt the evaluation of combinations co-targeting MAPK/ERK and Notch as a strategy to improve current therapies and warrant further evaluation of co-occurrence of aberrant PTEN and Notch activation as predictive markers of response to therapy.

In the vertebrate embryo, melanocytes arise from the neural crest, migrate to and colonize the basal layer within the skin and skin appendages. Post-migratory melanocytes are securely attached to the basement membrane, and their morphology, growth, adhesion, and migration are under control of neighboring keratinocytes. Melanoma is a malignant tumor originated from melanocytes or their progenitor cells. During melanocyte transformation and melanoma progression, melanocytes lose their interactions with keratinocytes, resulting in uncontrolled proliferation and invasion of the malignant cells. Melanoma cells at the advanced stages often lack melanocytic features and resemble multipotent progenitors, which are a potential melanocyte reservoir in human skin. In this mini-review, we will summarize findings on cell-cell interactions that are responsible for normal melanocyte homeostasis, stem cell self-renewal, and differentiation. Our ultimate goal is to define molecules and pathways, which are essential for normal cell-cell interactions but deregulated in melanoma formation and progression.

Technological advances often dictate progress in cancer research. Melanoma research has been considerably influenced by implementation of novel techniques and has contributed to our understanding of the mechanism of tumor progression. The three-dimensional (3D) human skin reconstruct is an ideal model to dissect each step of melanoma development and progression. Reconstructed human skin consists of fibroblast-contracted collagen gels as a dermal compartment and a stratified epidermal compartment. The epidermis comprises keratinocytes and normal melanocytes or melanoma cells from different stages. Normal melanocytes in skin reconstructs remain singly distributed at the basement membrane within the basal layer of keratinocytes. The radial growth phase (RGP) melanoma cells grow as cell clusters in the epidermis. The vertical growth phase (VGP) melanoma cells invade the dermis of reconstructs. Metastatic melanoma cells aggressively invade deep into the dermis. Grafting melanoma skin reconstructs onto mice induces local tumor formation and metastatic foci in distant organs such as lungs. The growth patterns and the range of metastases reflect proliferation and metastatic capacity of the original tumors. Skin reconstruct as xenografts enable us to observe to which organs melanoma cells spread. In this chapter, we describe the usefulness of the model in studying not only melanocyte physiology but also pathophysiological conditions such as melanocyte transformation and melanoma progression. A better understanding of these processes will benefit the entire melanoma field.

US28, a constitutively active G-protein-coupled receptor encoded by the human cytomegalovirus, leads to mechanistically unknown programmed cell death. Here we show that expression of wild-type US28 in human melanoma cells leads to apoptotic cell death via caspase 3 activation along with reduced cell proliferation. Reduced tumor growth upon US28 expression was observed in a xenograft mouse model. The signaling mute US28R129A showed a reduced antiproliferative effect. On evaluating different G-proteins coupled to US28 for signal transduction, Gα13 was identified as the main G-protein executing the apoptotic effect. Silencing of Gα13 but not Gαq resulted in a substantial increase in cell survival. Overexpression of Gα13 but not Gαq and their GTPase deficient forms Gα13Q226L and GαqQ209L, respectively, confirmed the requirement of Gα13 for US28 mediated cell death. Increasing expression of Gα13 alone induced cell death underscoring its relay function for US28 mediated decreased cell viability. Further reduced expression of Gα13 in melanoma cell lines isolated from advanced lesions and melanoma tissue was observed. These findings identified Gα13 as crucial for US28-induced cell death, substantiating that the effect of US28 on cell fate depends on preferred G-protein binding., (© 2015 UICC.)

Multipotent stem cells with neural crest-like properties have been identified in the dermis of human skin. These neural crest stem cell (NCSC)-like cells display self-renewal capacity and differentiate into neural crest derivatives, including epidermal pigment-producing melanocytes. NCSC-like cells share many properties with aggressive melanoma cells, such as high migratory capabilities and expression of the neural crest markers. However, little is known about which intrinsic or extrinsic signals determine the proliferation or differentiation of these neural crest-like stem cells. Here we show that, in NCSC-like cells, Notch signaling is highly activated, similar to melanoma cells. Inhibition of Notch signaling reduced the proliferation of NCSC-like cells, induced cell death, and downregulated noncanonical Wnt5a, suggesting that the Notch pathway contributes to the maintenance and motility of these stem cells. In three-dimensional skin reconstructs, canonical Wnt signaling promoted the differentiation of NCSC-like cells into melanocytes. This differentiation was triggered by the endogenous Notch inhibitor Numb, which is upregulated in the stem cells by Wnt7a derived from UV-irradiated keratinocytes. Together, these data reveal a cross talk between the two conserved developmental pathways in postnatal human skin, and highlight the role of the skin microenvironment in specifying the fate of stem cells.

Background: Melanocytes originate from the neural crest and migrate ventrally from the dorsal neural tube during embryogenesis. How human melanocytes locate at their suitable positions during embryogenesis, however, is unclear. Although a growing body of evidence indicates that melanocytes, melanoblasts, and melanocyte stem cells are closely related to hair follicles, little is known about volar skin., Objective: The aim of this study was to observe skin development during human fetal period and clarify the site-specific migration process of human fetal sole melanocytes., Methods: We obtained 4-mm punch biopsies from the scalp, back, abdomen, and right sole of 36 aborted fetuses (gestational age 12-21 weeks). We compared the migration process between hairly areas and volar areas by immunohistochemical staining., Results: Immunohistochemical examination revealed that gp100 (HMB-45) sensitively detects human melanocytes in embryogenesis. Melanocytes were present at the epidermal base, where hair placodes/buds form at 12-15 weeks gestation. Fetal melanocytes in hair follicles are supplied from the epidermis. In volar skin, melanocytes originally localize only in the acrosyringium, where they migrate deeper into with gland development at 16-18 weeks gestation. Palmoplantar melanocyte migration and maturation processes differ considerably from those of the other hairy skin sites., Conclusion: Eccrine sweat glands seem to have a central role in the palmoplantar melanocyte migration process, similar to the role of hair follicles in hairy sites., (Copyright © 2015 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Cutaneous malignant melanomas originate primarily within epidermal melanocytic cells. Melanoma cells share many characteristics with melanocyte precursors, suggesting that melanoma cells utilize the developmental programs of their normal counterpart for their own progression. The pigmentation system provides an advantageous model to assess survival pathway interactions in the melanocytic lineage, as genetic alterations controlling melanocyte development can be easily detectable by coat color phenotype that do not affect the viability of an animal. By integrating combinatorial gene knockout approaches, cell-based assays and immunohistochemical observations, recent studies have illustrated several genes and pathways that play important roles both in melanocyte specification and maintenance and in melanoma formation and progression. We are reviewing those genes and pathways to understand the connection between normal and cancerous development and to reveal therapeutic potential of targeting developmental pathways for melanoma therapy.

Despite success with BRAFV600E inhibitors, therapeutic responses in patients with metastatic melanoma are short-lived because of the acquisition of drug resistance. We identified a mechanism of intrinsic multidrug resistance based on the survival of a tumor cell subpopulation. Treatment with various drugs, including cisplatin and vemurafenib, uniformly leads to enrichment of slow-cycling, long-term tumor-maintaining melanoma cells expressing the H3K4-demethylase JARID1B/KDM5B/PLU-1. Proteome-profiling revealed an upregulation in enzymes of mitochondrial oxidative-ATP-synthesis (oxidative phosphorylation) in this subpopulation. Inhibition of mitochondrial respiration blocked the emergence of the JARID1B(high) subpopulation and sensitized melanoma cells to therapy, independent of their genotype. Our findings support a two-tiered approach combining anticancer agents that eliminate rapidly proliferating melanoma cells with inhibitors of the drug-resistant slow-cycling subpopulation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Skin, as the body's largest organ, has been extensively used to study adult stem cells. Most previous skin-related studies have focused on stem cells isolated from hair follicles and from keratinocytes. Here we present a protocol to isolate multipotent neural crest stem-like dermis-derived stem cells (termed dermal stem cells or DSCs) from human neonatal foreskins. DSCs grow like neural spheres in human embryonic stem cell medium and gain the ability to self-renew and differentiate into several cell lineages including melanocytes, neuronal cells, Schwann cells, smooth muscle cells, adipocytes, and chondrocytes. These cells express neural crest stem cell markers (NGFRp75 and nestin) as well as an embryonic stem cell marker (OCT4).

It has been shown that a side population (SP), which is characterized by high chemical efflux capacity, is present in human melanoma cell lines. However, it was not clear whether patients' samples contain the same subpopulation. In this issue, Luo et al. (2012) report that they have isolated SP cells directly from patients' melanomas. SP cells are resistant to paclitaxel because of the upregulation of ABCB1 and ABCB5. Notably, these cells are also resistant to temozolomide, which is not a substrate for ATP-Binding Cassette (ABC) transporters, in an interleukin (IL)-8-dependent manner. This study provides novel clues for understanding how a small, but critical, subpopulation within melanomas is resistant to therapies.

Human melanocytes have been extensively studied, but a melanocyte stem cell reservoir in glabrous skin has not yet been found. Human dermis contains cells that are nonpigmented but can differentiate to several different cell types. We have recently shown that multipotent dermal stem cells isolated from human neonatal foreskins are able to differentiate to multiple cell lineages, including pigmented melanocytes. The dermal stem cells grow as three-dimensional spheres in human embryonic stem cell medium and express some neural crest stem cell and embryonic stem cell markers. Melanocytes derived from dermal stem cells express melanocytic markers and act the same way as mature epidermal melanocytes. Dermal spheres, embedded in the reconstructed dermis consisting of collagen with fibroblasts, can migrate to the basement membrane, where they become pigmented in the same way as epidermal melanocytes suggesting that dermal stem cells can give rise to epidermal melanocytes.

Mouse and human somatic cells can either be reprogrammed to a pluripotent state or converted to another lineage with a combination of transcription factors suggesting that lineage commitment is a reversible process. Here we show that only one factor, the active intracellular form of Notch1, is sufficient to convert mature pigmented epidermal-derived melanocytes into functional multipotent neural crest (NC) stem-like cells. These induced NC stem cells (iNCSCs) proliferate as spheres under stem cell media conditions, re-express NC-related genes, and differentiate into multiple NC-derived mesenchymal and neuronal lineages. Moreover, iNCSCs are highly migratory and functional in vivo. These results demonstrate that mature melanocytes can be reprogrammed toward their primitive NC cell precursors through the activation of a single stem cell-related pathway. Reprogramming of melanocytes to iNCSCs may provide an alternate source of NCSCs for neuroregenerative applications., (Copyright © 2011 AlphaMed Press.)

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

The utility of different models to identify cancer stem cells continues to be a subject of intense debate. Here, we summarize recent efforts to characterize intra-tumoral heterogeneity of melanoma and delineate key questions for future studies. Within a developing or already established tumor microenvironment, we propose that continuous tumor maintenance is assured by specific sub-populations whose phenotype is not static but instead is dynamically regulated. These small and temporarily distinct sub-populations likely have critical roles in tumor progression. They are important therapeutic targets, but only in the context of combination therapies, that also eliminate the bulk of the tumor.

Human multipotent dermal stem cells (DSCs) have been isolated and propagated from the dermal region of neonatal foreskin. DSCs can self-renew, express the neural crest stem cell markers NGFRp75 and nestin, and are capable of differentiating into a wide variety of cell types including mesenchymal and neuronal lineages and melanocytes, indicative of their neural crest origin. When placed in the context of reconstructed skin, DSCs migrate to the basement membrane zone and differentiate into melanocytes. These findings, combined with the identification of NGFRp75-positive cells in the dermis of human foreskin, which are devoid of hair, suggest that DSCs may be a self-renewing source of extrafollicular epidermal melanocytes. In this review, we discuss the properties of DSCs, the pathways required for melanocyte differentiation, and the value of 3D reconstructed skin to assess the behavior and contribution of DSCs in the naturalized environment of human skin. Potentially, DSCs provide a link to malignant melanoma by being a target of UVA-induced transformation., (2011 John Wiley & Sons A/S.)

BRAF is an attractive target for melanoma drug development. However, resistance to BRAF inhibitors is a significant clinical challenge. We describe a model of resistance to BRAF inhibitors developed by chronic treatment of BRAF(V)⁶⁰⁰(E) melanoma cells with the BRAF inhibitor SB-590885; these cells are cross-resistant to other BRAF-selective inhibitors. Resistance involves flexible switching among the three RAF isoforms, underscoring the ability of melanoma cells to adapt to pharmacological challenges. IGF-1R/PI3K signaling was enhanced in resistant melanomas, and combined treatment with IGF-1R/PI3K and MEK inhibitors induced death of BRAF inhibitor-resistant cells. Increased IGF-1R and pAKT levels in a post-relapse human tumor sample are consistent with a role for IGF-1R/PI3K-dependent survival in the development of resistance to BRAF inhibitors., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Melanomas are highly heterogeneous tumors, but the biological significance of their different subpopulations is not clear. Using the H3K4 demethylase JARID1B (KDM5B/PLU-1/RBP2-H1) as a biomarker, we have characterized a small subpopulation of slow-cycling melanoma cells that cycle with doubling times of >4 weeks within the rapidly proliferating main population. Isolated JARID1B-positive melanoma cells give rise to a highly proliferative progeny. Knockdown of JARID1B leads to an initial acceleration of tumor growth followed by exhaustion which suggests that the JARID1B-positive subpopulation is essential for continuous tumor growth. Expression of JARID1B is dynamically regulated and does not follow a hierarchical cancer stem cell model because JARID1B-negative cells can become positive and even single melanoma cells irrespective of selection are tumorigenic. These results suggest a new understanding of melanoma heterogeneity with tumor maintenance as a dynamic process mediated by a temporarily distinct subpopulation., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Melanocytes sustain a lifelong proliferative potential, but a stem cell reservoir in glabrous skin has not yet been found. Here, we show that multipotent dermal stem cells isolated from human foreskins lacking hair follicles are able to home to the epidermis to differentiate into melanocytes. These dermal stem cells, grown as three-dimensional spheres, displayed a capacity for self-renewal and expressed NGFRp75, nestin and OCT4, but not melanocyte markers. In addition, cells derived from single-cell clones were able to differentiate into multiple lineages including melanocytes. In a three-dimensional skin equivalent model, sphere-forming cells differentiated into HMB45-positive melanocytes, which migrated from the dermis to the epidermis and aligned singly among the basal layer keratinocytes in a similar fashion to pigmented melanocytes isolated from the epidermis. The dermal stem cells were negative for E-cadherin and N-cadherin, whereas they acquired E-cadherin expression and lost NGFRp75 expression upon contact with epidermal keratinocytes. These results demonstrate that stem cells in the dermis of human skin with neural-crest-like characteristics can become mature epidermal melanocytes. This finding could significantly change our understanding of the etiological factors in melanocyte transformation and pigmentation disorders; specifically, that early epigenetic or genetic alterations leading to transformation may take place in the dermis rather than in the epidermis.

Cells within the normal human colonic epithelium undergo a dynamic cycle of growth, differentiation, and death. The organotypic culture system of human fetal colonic epithelial cells seeded on top of collagen gels with embedded colonic fibroblasts allowed prolonged culture of the colonic epithelial cells (Kalabis J, Patterson MJ, Enders GM, Marian B, Iozzo RV, Rogler G, Gimotty PA, Herlyn M. FASEB J 17: 1115-1117, 2003). Herein, we have evaluated the role of endothelin-3 (ET3) and both cognate endothelin receptors (ETRA, ETRB) for human colonic epithelial cell growth and survival. ET3 was produced continuously by the fibroblasts as a result of adenovirus-mediated gene transfer. The presence and function of the endothelin receptors (ETRs) in epithelial cells was evaluated by [(3)H]thymidine incorporation using primary epithelial cells in monoculture and by immunohistochemistry on human fetal and adult paraffin-embedded tissues. In organotypic culture, ET3 increased the number of goblet cells but not of enteroendocrine cells. The increase in goblet cells was caused by prolonged cell survival and differentiation. The inhibition of both ETRA and ETRB significantly decreased the number of goblet cells and proliferation in epithelial cells, whereas the number of enteroendocrine cells remained unchanged. ET3 induced activation of IkappaB and MAPK in the epithelial cells, suggesting that these signaling pathways mediate its proproliferation and prosurvival activities. Our results demonstrate that ET3 is involved in regulating human colonic epithelial cell proliferation and survival, particularly for goblet cells, and may be an important component of colonic homeostasis.

Matricellular proteins are modulators of cell-matrix interactions and cellular functions. The group includes thrombospondin, osteopontin, osteonectin/SPARC, tenascin, disintegrins, galectins and CCN proteins. The production of matricellular proteins such as osteopontin, SPARC or tenascin is highly upregulated in melanoma and other tumors but little is known about their functions in tumor growth, survival, and metastasis. The distribution pattern of CCN3 differs from most other matricellular proteins, such that it is produced abundantly by normal melanocytes, but is not significantly expressed in melanoma cells. CCN3 is known to inhibit melanocyte proliferation and stimulate adhesion to collagen type IV, the main component of the basement membrane. CCN3 has a unique role in securing adhesion of melanocytes to the basement membrane distinct from other melanoma-produced matricellular proteins which act as de-adhesive molecules and antagonists of focal adhesion. Qualitative and quantitative changes in matricellular protein expression contribute to melanoma progression similar to the E-cadherin to N-cadherin class switch, allowing melanoma cells to escape from keratinocyte control.

The matricellular protein SPARC (secreted protein acidic and rich in cysteine) has diverse functions in melanoma cells. Because this secreted protein is produced not only in tumor cells but also in stromal cells, the question has been asked whether paracrine effects of stroma-derived SPARC contribute to melanoma progression. In this issue, Prada et al. (2007) begin to answer this question by demonstrating that SPARC produced by melanoma, but not stromal cells, is essential to regulate melanoma cell growth.

Melanocytes reside within the basal layer of the human epidermis, where they attach to the basement membrane and replicate at a rate proportionate to that of keratinocytes, maintaining a lifelong stable ratio. In this study, we report that coculturing melanocytes with keratinocytes up-regulated CCN3, a matricellular protein that we subsequently found to be critical for the spatial localization of melanocytes to the basement membrane. CCN3 knockdown cells were dissociated either upward to the suprabasal layers of the epidermis or downward into the dermis. The overexpression of CCN3 increased adhesion to collagen type IV, the major component of the basement membrane. As the receptor responsible for CCN3-mediated melanocyte localization, we identified discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase that acts as a collagen IV adhesion receptor. DDR1 knockdown decreased melanocyte adhesion to collagen IV and shifted melanocyte localization in a manner similar to CCN3 knockdown. These results demonstrate an intricate and necessary communication between keratinocytes and melanocytes in maintaining normal epidermal homeostasis.