Author Correction: PAK signalling drives acquired drug resistance to MAPK inhibitors in BRAF-mutant melanomas.

In this Letter, two relevant references were omitted; see the accompanying Amendment for details. The original Letter has not been corrected.

Corrigendum: Rare cell variability and drug-induced reprogramming as a mode of cancer drug resistance.

This corrects the article DOI: 10.1038/nature22794.

Rare cell variability and drug-induced reprogramming as a mode of cancer drug resistance.

Therapies that target signalling molecules that are mutated in cancers can often have substantial short-term effects, but the emergence of resistant cancer cells is a major barrier to full cures. Resistance can result from secondary mutations, but in other cases there is no clear genetic cause, raising the possibility of non-genetic rare cell variability. Here we show that human melanoma cells can display profound transcriptional variability at the single-cell level that predicts which cells will ultimately resist drug treatment. This variability involves infrequent, semi-coordinated transcription of a number of resistance markers at high levels in a very small percentage of cells. The addition of drug then induces epigenetic reprogramming in these cells, converting the transient transcriptional state to a stably resistant state. This reprogramming begins with a loss of SOX10-mediated differentiation followed by activation of new signalling pathways, partially mediated by the activity of the transcription factors JUN and/or AP-1 and TEAD. Our work reveals the multistage nature of the acquisition of drug resistance and provides a framework for understanding resistance dynamics in single cells. We find that other cell types also exhibit sporadic expression of many of these same marker genes, suggesting the existence of a general program in which expression is displayed in rare subpopulations of cells.

PAK signalling drives acquired drug resistance to MAPK inhibitors in BRAF-mutant melanomas.

Targeted BRAF inhibition (BRAFi) and combined BRAF and MEK inhibition (BRAFi and MEKi) therapies have markedly improved the clinical outcomes of patients with metastatic melanoma. Unfortunately, the efficacy of these treatments is often countered by the acquisition of drug resistance. Here we investigated the molecular mechanisms that underlie acquired resistance to BRAFi and to the combined therapy. Consistent with previous studies, we show that resistance to BRAFi is mediated by ERK pathway reactivation. Resistance to the combined therapy, however, is mediated by mechanisms independent of reactivation of ERK in many resistant cell lines and clinical samples. p21-activated kinases (PAKs) become activated in cells with acquired drug resistance and have a pivotal role in mediating resistance. Our screening, using a reverse-phase protein array, revealed distinct mechanisms by which PAKs mediate resistance to BRAFi and the combined therapy. In BRAFi-resistant cells, PAKs phosphorylate CRAF and MEK to reactivate ERK. In cells that are resistant to the combined therapy, PAKs regulate JNK and ß-catenin phosphorylation and mTOR pathway activation, and inhibit apoptosis, thereby bypassing ERK. Together, our results provide insights into the molecular mechanisms underlying acquired drug resistance to current targeted therapies, and may help to direct novel drug development efforts to overcome acquired drug resistance.

Pembrolizumab versus placebo as adjuvant therapy in resected stage IIB or IIC melanoma (KEYNOTE-716): distant metastasis-free survival results of a multicentre, double-blind, randomised, phase 3 trial.

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.

Real-world clinical outcomes of patients with stage IIB or IIC cutaneous melanoma treated at US community oncology clinics.

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.

New treatments are now available that decrease tumor recurrence when administered after surgery to remove melanoma skin tumors that are graded as stage IIB or IIC (i.e., with no cancer spread to the local lymph nodes). We studied 567 'real-world' patients at clinics in the USA who had stage IIB or IIC melanoma tumors removed in 2008­2017, before these new postsurgical treatments were widely available, to evaluate their survival and tumor recurrence. We found that almost half of these patients (44%) had melanoma recurrence or had died, and a third (30%) had tumor spread beyond the original site, by the end of 2020. These findings highlight the need for more effective treatments after surgical removal of stage IIB and IIC melanoma.

Adjuvant pembrolizumab versus placebo in resected stage III melanoma (EORTC 1325-MG/KEYNOTE-054): distant metastasis-free survival results from a double-blind, randomised, controlled, phase 3 trial.

BACKGROUND: The European Organisation for Research and Treatment of Cancer (EORTC) 1325/KEYNOTE-054 trial assessed pembrolizumab versus placebo in patients with resected high-risk stage III melanoma. At 15-month median follow-up, pembrolizumab improved recurrence-free survival (hazard ratio [HR] 0·57 [98·4% CI 0·43-0·74], p<0·0001) compared with placebo, leading to its approval in the USA and Europe. This report provides the final results for the secondary efficacy endpoint, distant metastasis-free survival and an update of the recurrence-free survival results. METHODS: This double-blind, randomised, controlled, phase 3 trial was done at 123 academic centres and community hospitals across 23 countries. Patients aged 18 years or older with complete resection of cutaneous melanoma metastatic to lymph node, classified as American Joint Committee on Cancer staging system, seventh edition (AJCC-7) stage IIIA (at least one lymph node metastasis >1 mm), IIIB, or IIIC (without in-transit metastasis), and with an Eastern Cooperative Oncology Group performance status of 0 or 1 were eligible. Patients were randomly assigned (1:1) via a central interactive voice response system to receive intravenous pembrolizumab 200 mg or placebo every 3 weeks for up to 18 doses or until disease recurrence or unacceptable toxicity. Randomisation was stratified according to disease stage and region, using a minimisation technique, and clinical investigators, patients, and those collecting or analysing the data were masked to treatment assignment. The two coprimary endpoints were recurrence-free survival in the intention-to-treat (ITT) population and in patients with PD-L1-positive tumours. The secondary endpoint reported here was distant metastasis-free survival in the ITT and PD-L1-positive populations. This study is registered with ClinicalTrials.gov, NCT02362594, and EudraCT, 2014-004944-37. FINDINGS: Between Aug 26, 2015, and Nov 14, 2016, 1019 patients were assigned to receive either pembrolizumab (n=514) or placebo (n=505). At an overall median follow-up of 42·3 months (IQR 40·5-45·9), 3·5-year distant metastasis-free survival was higher in the pembrolizumab group than in the placebo group in the ITT population (65·3% [95% CI 60·9-69·5] in the pembrolizumab group vs 49·4% [44·8-53·8] in the placebo group; HR 0·60 [95% CI 0·49-0·73]; p<0·0001). In the 853 patients with PD-L1-positive tumours, 3·5-year distant metastasis-free survival was 66·7% (95% CI 61·8-71·2) in the pembrolizumab group and 51·6% (46·6-56·4) in the placebo group (HR 0·61 [95% CI 0·49-0·76]; p<0·0001). Recurrence-free survival remained longer in the pembrolizumab group 59·8% (95% CI 55·3-64·1) than the placebo group 41·4% (37·0-45·8) at this 3·5-year follow-up in the ITT population (HR 0·59 [95% CI 0·49-0·70]) and in those with PD-L1-positive tumours 61·4% (56·3-66·1) in the pembrolizumab group and 44·1% (39·2-48·8) in the placebo group (HR 0·59 [95% CI 0·49-0·73]). INTERPRETATION: Pembrolizumab adjuvant therapy provided a significant and clinically meaningful improvement in distant metastasis-free survival at a 3·5-year median follow-up, which was consistent with the improvement in recurrence-free survival. Therefore, the results of this trial support the indication to use adjuvant pembrolizumab therapy in patients with resected high risk stage III cutaneous melanoma. FUNDING: Merck Sharp & Dohme.

Adjuvant pembrolizumab versus placebo in resected stage III melanoma (EORTC 1325-MG/KEYNOTE-054): health-related quality-of-life results from a double-blind, randomised, controlled, phase 3 trial.

BACKGROUND: The European Organisation for Research and Treatment of Cancer (EORTC) 1325-MG/KEYNOTE-054 trial in patients with resected, high-risk stage III melanoma demonstrated improved recurrence-free survival with adjuvant pembrolizumab compared with placebo (hazard ratio 0·57 [98·4% CI 0·43-0·74]; p<0·0001). This study reports the results from the health-related quality-of-life (HRQOL) exploratory endpoint. METHODS: This double-blind, randomised, controlled, phase 3 trial was done at 123 academic centres and community hospitals across 23 countries. Patients aged 18 years or older with previously untreated histologically confirmed stage IIIA, IIIB, or IIIC resected cutaneous melanoma, and an Eastern Cooperative Oncology Group performance status score of 1 or 0 were eligible. Patients were randomly assigned (1:1) using a central interactive voice-response system on the basis of a minimisation technique stratified for stage and geographic region to receive intravenously 200 mg pembrolizumab or placebo. Treatment was administered every 3 weeks for 1 year, or until disease recurrence, unacceptable toxicity, or death. The primary endpoint of the trial was recurrence-free survival (reported elsewhere). HRQOL was a prespecified exploratory endpoint, with global health/quality of life (GHQ) over 2 years measured by the EORTC QLQ-C30 as the primary analysis. Analyses were done in the intention-to-treat population. This study is registered with ClinicalTrials.gov, NCT02362594, and EudraCT, 2014-004944-37, and long-term follow-up is ongoing. FINDINGS: Between Aug 26, 2015, and Nov 14, 2016, 1019 patients were assigned to pembrolizumab (n=514) or placebo (n=505). Median follow-up was 15·1 months (IQR 12·8-16·9) at the time of this analysis. HRQOL compliance was greater than 90% at baseline, greater than 70% during the first year, and greater than 60% thereafter for both groups. Because of low absolute compliance numbers at later follow-up, the analysis was truncated to week 84. Baseline GHQ scores were similar between groups (77·55 [SD 18·20] in the pembrolizumab group and 76·54 [17·81] in the placebo group) and remained stable over time. The difference in average GHQ score between the two groups over the 2 years was -2·2 points (95% CI -4·3 to -0·2). The difference in average score during treatment was -1·1 points (95% CI -3·2 to 0·9) and the difference in average score after treatment was -2·2 points (-4·8 to 0·4). These differences are within the 5-point clinical relevance threshold for the QLQ-C30 and are therefore clinically non-significant. INTERPRETATION: Pembrolizumab does not result in a clinically significant decrease in HRQOL compared with placebo when given as adjuvant therapy for patients with resected, high-risk stage III melanoma. These results support the use of adjuvant pembrolizumab in this setting. FUNDING: Merck Sharp & Dohme.

Outcomes of retreatment with anti-PD-1 monotherapy after response to first course in patients with cutaneous melanoma.

Aim: To determine outcomes of retreatment with anti-PD-1 monotherapy for melanoma. Materials & methods: This retrospective study included adults with unresectable cutaneous melanoma who achieved stable disease (SD) or better after anti-PD-1 monotherapy and were retreated with anti-PD-1 monotherapy after ≥90-day gap. We determined overall survival and real-world tumor response. Results: For 21 eligible patients, from retreatment initiation, median follow-up was 14.4 months (range, 2.6-34.5); median overall survival was 30.0 months (95% CI: 14.4-not reached); 1-year survival was 100% (95% CI: 100-100%); 2-year survival was 83% (48-96%). Of 16 patients with recorded best real-world tumor response, ten (63%) responded (complete/partial response); three achieved SD; three had progressive disease. Conclusion: Patients with advanced melanoma achieving SD/better after first-course anti-PD-1 monotherapy may benefit from retreatment.

Pembrolizumab versus ipilimumab in advanced melanoma (KEYNOTE-006): post-hoc 5-year results from an open-label, multicentre, randomised, controlled, phase 3 study.

BACKGROUND: Pembrolizumab improved progression-free survival and overall survival versus ipilimumab in patients with advanced melanoma and is now a standard of care in the first-line setting. However, the optimal duration of anti-PD-1 administration is unknown. We present results from 5 years of follow-up of patients in KEYNOTE-006. METHODS: KEYNOTE-006 was an open-label, multicentre, randomised, controlled, phase 3 study done at 87 academic institutions, hospitals, and cancer centres in 16 countries. Patients aged at least 18 years with Eastern Cooperative Oncology Group performance status of 0 or 1, ipilimumab-naive histologically confirmed advanced melanoma with known BRAFV600 status and up to one previous systemic therapy were randomly assigned (1:1:1) to intravenous pembrolizumab 10 mg/kg every 2 weeks or every 3 weeks or four doses of intravenous ipilimumab 3 mg/kg every 3 weeks. Treatments were assigned using a centralised, computer-generated allocation schedule with blocked randomisation within strata. Exploratory combination of data from the two pembrolizumab dosing regimen groups was not protocol-specified. Pembrolizumab treatment continued for up to 24 months. Eligible patients who discontinued pembrolizumab with stable disease or better after receiving at least 24 months of pembrolizumab or discontinued with complete response after at least 6 months of pembrolizumab and then progressed could receive an additional 17 cycles of pembrolizumab. Co-primary endpoints were overall survival and progression-free survival. Efficacy was analysed in all randomly assigned patients, and safety was analysed in all randomly assigned patients who received at least one dose of study treatment. Exploratory assessment of efficacy and safety at 5 years' follow-up was not specified in the protocol. Data cutoff for this analysis was Dec 3, 2018. Recruitment is closed; the study is ongoing. This study is registered with ClinicalTrials.gov, number NCT01866319. FINDINGS: Between Sept 18, 2013, and March 3, 2014, 834 patients were enrolled and randomly assigned to receive pembrolizumab (every 2 weeks, n=279; every 3 weeks, n=277), or ipilimumab (n=278). After a median follow-up of 57·7 months (IQR 56·7-59·2) in surviving patients, median overall survival was 32·7 months (95% CI 24·5-41·6) in the combined pembrolizumab groups and 15·9 months (13·3-22·0) in the ipilimumab group (hazard ratio [HR] 0·73, 95% CI 0·61-0·88, p=0·00049). Median progression-free survival was 8·4 months (95% CI 6·6-11·3) in the combined pembrolizumab groups versus 3·4 months (2·9-4·2) in the ipilimumab group (HR 0·57, 95% CI 0·48-0·67, p<0·0001). Grade 3-4 treatment-related adverse events occurred in 96 (17%) of 555 patients in the combined pembrolizumab groups and in 50 (20%) of 256 patients in the ipilimumab group; the most common of these events were colitis (11 [2%] vs 16 [6%]), diarrhoea (ten [2%] vs seven [3%]), and fatigue (four [<1%] vs three [1%]). Any-grade serious treatment-related adverse events occurred in 75 (14%) patients in the combined pembrolizumab groups and in 45 (18%) patients in the ipilimumab group. One patient assigned to pembrolizumab died from treatment-related sepsis. INTERPRETATION: Pembrolizumab continued to show superiority over ipilimumab after almost 5 years of follow-up. These results provide further support for use of pembrolizumab in patients with advanced melanoma. FUNDING: Merck Sharp & Dohme.

Relationships between survival and real-world recurrence-free survival or distant metastasis-free survival among patients with completely resected stage IIB or IIC melanoma.

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.

Pembrolizumab Versus Placebo as Adjuvant Therapy in Resected Stage IIB or IIC Melanoma: Final Analysis of Distant Metastasis-Free Survival in the Phase III KEYNOTE-716 Study.

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.

Pembrolizumab versus placebo as adjuvant therapy in resected stage IIB or IIC melanoma: Outcomes in histopathologic subgroups from the randomized, double-blind, phase 3 KEYNOTE-716 trial.

BACKGROUND: Adjuvant pembrolizumab significantly improved recurrence-free survival (RFS) and distant metastasis-free survival (DMFS) versus placebo in the phase 3 KEYNOTE-716 study of resected stage IIB or IIC melanoma. At the prespecified third interim analysis (data cut-off, January 4, 2022), the HR for RFS in the overall population was 0.64 (95% CI, 0.50 to 0.84) and the HR for DMFS was 0.64 (95% CI, 0.47 to 0.88). We present a post hoc analysis of efficacy by subtypes defined by histopathologic characteristics. METHODS: Patients aged ≥12 years with newly diagnosed, resected stage IIB or IIC melanoma were randomly assigned (1:1) to pembrolizumab 200 mg every 3 weeks (2 mg/kg up to 200 mg for pediatric patients) or placebo. The primary end point was RFS per investigator review; DMFS per investigator review was secondary. Subgroups of interest were melanoma subtype (nodular vs non-nodular), tumor thickness (≤4 mm vs >4 mm), presence of ulceration (yes vs no), mitotic rate (<5 per mm2 (median) vs ≥5 per mm2), and presence of tumor-infiltrating lymphocytes (TILs; absent vs present). RESULTS: Between September 23, 2018, and November 4, 2020, 976 patients were assigned to pembrolizumab (n=487) or placebo (n=489). Median follow-up was 27.4 months (range, 14.0-39.4). The HR (95% CI) for RFS was 0.54 (0.37 to 0.79) for nodular and 0.77 (0.53 to 1.11) for non-nodular melanoma; 0.57 (0.37 to 0.89) for thickness ≤4 mm and 0.69 (0.50 to 0.96) for >4 mm; 0.66 (0.50 to 0.89) for ulceration and 0.57 (0.32 to 1.03) for no ulceration; 0.57 (0.35 to 0.92) for mitotic rate <5 per mm2 and 0.57 (0.40 to 0.80) for ≥5 per mm2; and 0.89 (0.52 to 1.54) for TILs absent and 0.51 (0.34 to 0.76) for TILs present. DMFS results were similar. In a Cox multivariate analysis, treatment arm, tumor thickness, and mitotic rate were significant independent factors for RFS, and treatment arm and mitotic rate were significant independent factors for DMFS. CONCLUSIONS: In this post hoc analysis, the benefit of pembrolizumab was largely consistent with the overall study population regardless of histopathologic characteristics. These results support the use of adjuvant pembrolizumab in patients with resected stage IIB or IIC melanoma. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov, NCT03553836.

Safety of pembrolizumab as adjuvant therapy in a pooled analysis of phase 3 clinical trials of melanoma, non-small cell lung cancer, and renal cell carcinoma.

BACKGROUND: The safety profile of adjuvant pembrolizumab was evaluated in a pooled analysis of 4 phase 3 clinical trials. METHODS: Patients had completely resected stage IIIA, IIIB, or IIIC melanoma per American Joint Committee on Cancer, 7th edition, criteria (AJCC-7; KEYNOTE-054); stage IIB or IIC melanoma per AJCC-8 (KEYNOTE-716); stage IB, II, or IIIA non-small cell lung cancer per AJCC-7 (PEARLS/KEYNOTE-091); or postnephrectomy/metastasectomy clear cell renal cell carcinoma at increased risk of recurrence (KEYNOTE-564). Patients received adjuvant pembrolizumab 200 mg (2 mg/kg up to 200 mg for pediatric patients) or placebo every 3 weeks for approximately 1 year. Adverse events (AEs) were summarized for patients who received ≥ 1 dose of treatment. RESULTS: Data were pooled from 4125 patients treated with pembrolizumab (n = 2060) or placebo (n = 2065). Median (range) duration of treatment was 11.1 months (0.0-18.9) with pembrolizumab and 11.2 months (0.0-18.1) with placebo. Treatment-related AEs occurred in 78.6 % (1620/2060) of patients in the pembrolizumab group (grade 3-5, 16.3 % [336/2060]) and 58.7 % (1212/2065) in the placebo group (grade 3-5, 3.5 % [72/2065]). Immune-mediated AEs (e.g. adrenal insufficiency, hypophysitis, and thyroiditis) occurred in 36.2 % (746/2060) of patients in the pembrolizumab group (grade 3-5, 8.6 % [177/2060]) and 8.4 % (174/2065) in the placebo group (grade 3-5, 1.1 % [23/2065]). Of patients with ≥ 1 immune-mediated AE or infusion reaction, systemic corticosteroids were required for 35.2 % (268/761) and 20.2 % (39/193) of patients in the pembrolizumab and placebo groups, respectively. CONCLUSIONS: Adjuvant pembrolizumab demonstrated a manageable safety profile that was comparable to prior reports in advanced disease.

Cost-Effectiveness Analysis of Pembrolizumab as an Adjuvant Treatment of Resected Stage IIB or IIC Melanoma in the United States.

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.

Seven-Year Follow-Up of the Phase III KEYNOTE-006 Study: Pembrolizumab Versus Ipilimumab in Advanced Melanoma.

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.Immune checkpoint inhibitors have led to unprecedented prolongation of overall survival (OS) for patients with advanced melanoma. Five-year follow-up of KEYNOTE-006 showed pembrolizumab prolonged survival versus ipilimumab. Efficacy results with 7-year follow-up are presented. At data cutoff (April 19, 2021), median follow-up was 85.3 months (range, 0.03-90.8 months). Median OS was 32.7 months for pembrolizumab versus 15.9 months for ipilimumab (hazard ratio [HR], 0.70; 95% CI, 0.58 to 0.83); 7-year OS was 37.8% and 25.3%, respectively. OS HRs favored pembrolizumab regardless of BRAF status or prior BRAF/MEK-inhibitor treatment and prognostic characteristics (elevated lactate dehydrogenase, large tumor size, and brain metastasis). Median modified progression-free survival (mPFS) was 9.4 months for pembrolizumab versus 3.8 months for ipilimumab; 7-year mPFS was 23.8% and 13.3%, respectively. In patients who completed ≥94 weeks of pembrolizumab, the 5-year OS was 92.9% and the 5-year mPFS was 70.1%. The objective response rate with second-course pembrolizumab (n = 16) was 56% (95% CI, 30 to 80) and the 2-year mPFS was 62.5%. These findings confirm that pembrolizumab provides long-term survival benefit in advanced melanoma.

A distinct pattern of growth and RAC1 signaling in melanoma brain metastasis cells.

BACKGROUND: Melanoma, the deadliest of skin cancers, has a high propensity to form brain metastases that are associated with a markedly worsened prognosis. In spite of recent therapeutic advances, melanoma brain lesions remain a clinical challenge, biomarkers predicting brain dissemination are not clear and differences with other metastatic sites are poorly understood. METHODS: We examined a genetically diverse panel of human-derived melanoma brain metastasis (MBM) and extracranial cell lines using targeted sequencing, a Reverse Phase Protein Array, protein expression analyses, and functional studies in vitro and in vivo. RESULTS: Brain-specific genetic alterations were not detected; however, MBM cells in vitro displayed lower proliferation rates and MBM-specific protein expression patterns associated with proliferation, DNA damage, adhesion, and migration. MBM lines displayed higher levels of RAC1 expression, involving a distinct RAC1-PAK1-JNK1 signaling network. RAC1 knockdown or treatment with small molecule inhibitors contributed to a less aggressive MBM phenotype in vitro, while RAC1 knockdown in vivo led to reduced tumor volumes and delayed tumor appearance. Proliferation, adhesion, and migration were higher in MBM vs nonMBM lines in the presence of insulin or brain-derived factors and were affected by RAC1 levels. CONCLUSIONS: Our findings indicate that despite their genetic variability, MBM engage specific molecular processes such as RAC1 signaling to adapt to the brain microenvironment and this can be used for the molecular characterization and treatment of brain metastases.

Phase II LEAP-004 Study of Lenvatinib Plus Pembrolizumab for Melanoma With Confirmed Progression on a Programmed Cell Death Protein-1 or Programmed Death Ligand 1 Inhibitor Given as Monotherapy or in Combination.

PURPOSE: Effective treatments are needed for melanoma that progresses on inhibitors of programmed cell death protein-1 (PD-1) or its ligand (PD-L1). We conducted the phase II LEAP-004 study to evaluate the combination of the multikinase inhibitor lenvatinib and the PD-1 inhibitor pembrolizumab in this population (ClinicalTrials.gov identifier: NCT03776136). METHODS: Eligible patients with unresectable stage III-IV melanoma with confirmed progressive disease (PD) within 12 weeks of the last dose of a PD-1/L1 inhibitor given alone or with other therapies, including cytotoxic T-cell lymphocyte-associated antigen 4 (CTLA-4) inhibitors, received lenvatinib 20 mg orally once daily plus ≤ 35 doses of pembrolizumab 200 mg intravenously once every 3 weeks until PD or unacceptable toxicity. The primary end point was objective response rate (ORR) per RECIST, version 1.1, by independent central review. RESULTS: A total of 103 patients were enrolled and treated. The median study follow-up was 15.3 months. ORR in the total population was 21.4% (95% CI, 13.9 to 30.5), with three (2.9%) complete responses and 19 (18.4%) partial responses. The median duration of response was 8.3 months (range, 3.2-15.9+). ORR was 33.3% in the 30 patients with PD on prior anti-PD-1 plus anti-CTLA-4 therapy. The median progression-free survival and overall survival in the total population were 4.2 months (95% CI, 3.8 to 7.1) and 14.0 months (95% CI, 10.8 to not reached), respectively. Grade 3-5 treatment-related adverse events occurred in 47 (45.6%) patients, most commonly hypertension (21.4%); one patient died from a treatment-related event (decreased platelet count). CONCLUSION: Lenvatinib plus pembrolizumab provides clinically meaningful, durable responses in patients with advanced melanoma with confirmed PD on prior PD-1/L1 inhibitor-based therapy, including those with PD on anti-PD-1 plus anti-CTLA-4 therapy. The safety profile was as expected. These data support lenvatinib plus pembrolizumab as a potential regimen for this population of high unmet need.

Baseline and post-treatment biomarkers of resistance to anti-PD-1 therapy in acral and mucosal melanoma: an observational study.

BACKGROUND: Immunotherapies targeting programmed cell death-1 (PD-1) and its ligands have improved clinical outcomes for advanced melanoma. However, many tumors exhibit primary resistance or acquire secondary resistance after an initial positive response. The mechanisms of resistance are not well understood, and no validated predictive biomarkers are available. This exploratory study aimed to characterize baseline differences and molecular changes arising during treatment in acral and mucosal melanomas that exhibited primary or secondary resistance to anti-PD-1 monotherapy. METHODS: This was an observational retrospective study of 124 patients who had been treated for metastatic acral or mucosal melanoma with anti-PD-1 monotherapy. Tumor samples were collected at baseline (all patients) and post-treatment (resistant tumors only) and were assayed by immunohistochemistry, whole-exome sequencing, and RNA sequencing. RESULTS: At baseline, more non-progressor than resistant tumors exhibited expression of PD-L1, immune cell infiltration, and high tumor mutational burden (TMB); baseline PD-L1 expression was also more common in secondary-resistant than in primary-resistant tumors as well as in late versus early secondary-resistant tumors. Non-progressor tumors also had higher median baseline expression of an 18-gene T cell-inflamed gene expression profile (TcellinfGEP). Among resistant tumors, the proportion of PD-L1-positive melanomas and the expression of the TcellinfGEP mRNA signature increased during treatment, while the expression of mRNA signatures related to WNT and INFA1 signaling decreased. There was evidence for greater changes from baseline in secondary-resistant versus primary-resistant tumors for some markers, including expression of RAS-related and WNT-related mRNA signatures and density of CD11c+ and FOXP3+ T cells. Greater changes in CD11c+ cell density were observed in early compared with late secondary-resistant tumors. CONCLUSIONS: Our findings suggest that TcellinfGEP and PD-L1 expression, TMB, immune cell infiltration, and RAS and WNT signaling warrant further investigation as potential mechanisms and/or biomarkers of anti-PD-1 therapy resistance in acral and mucosal melanomas. Confirmation of these findings in larger populations is needed.

Persister state-directed transitioning and vulnerability in melanoma.

Melanoma is a highly plastic tumor characterized by dynamic interconversion of different cell identities depending on the biological context. Melanoma cells with high expression of the H3K4 demethylase KDM5B (JARID1B) rest in a slow-cycling, yet reversible persister state. Over time, KDM5Bhigh cells can promote rapid tumor repopulation with equilibrated KDM5B expression heterogeneity. The cellular identity of KDM5Bhigh persister cells has not been studied so far, missing an important cell state-directed treatment opportunity in melanoma. Here, we have established a doxycycline-titratable system for genetic induction of permanent intratumor expression of KDM5B and screened for chemical agents that phenocopy this effect. Transcriptional profiling and cell functional assays confirmed that the dihydropyridine 2-phenoxyethyl 4-(2-fluorophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexa-hydro-quinoline-3-carboxylate (termed Cpd1) supports high KDM5B expression and directs melanoma cells towards differentiation along the melanocytic lineage and to cell cycle-arrest. The high KDM5B state additionally prevents cell proliferation through negative regulation of cytokinetic abscission. Moreover, treatment with Cpd1 promoted the expression of the melanocyte-specific tyrosinase gene specifically sensitizing melanoma cells for the tyrosinase-processed antifolate prodrug 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG). In summary, our study provides proof-of-concept for a dual hit strategy in melanoma, in which persister state-directed transitioning limits tumor plasticity and primes melanoma cells towards lineage-specific elimination.