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Aims: To describe the health-related quality of life (HRQoL) of melanoma brain metastasis (MBM) patients throughout the first 18 weeks of ipilimumab–nivolumab or nivolumab treatment. Materials & methods: HRQoL data (European Organisation for Research and Treatment of Cancer's Core Quality of Life Questionnaire, additional Brain Neoplasm Module, and EuroQol 5-Dimension 5-Level Questionnaire) were collected as a secondary outcome of the Anti-PD1 Brain Collaboration phase II trial. Mixed linear modeling assessed changes over time, whereas the Kaplan–Meier method was used to determine median time to first deterioration. Results: Asymptomatic MBM patients treated with ipilimumab–nivolumab (n = 33) or nivolumab (n = 24) maintained baseline HRQoL. MBM patients with symptoms or leptomeningeal/progressive disease treated with nivolumab (n = 14) reported a statistically significant trend toward improvement. Conclusion: MBM patients treated with either ipilimumab–nivolumab or nivolumab did not report a significant deterioration in HRQoL within 18 weeks of treatment initiation. Clinical trial registration: NCT02374242 ( ClinicalTrials.gov )

Targeted therapy (BRAF inhibitor plus MEK inhibitor) is now among the possible treatment options for patients with BRAF mutation-positive stage III or stage IV melanoma. This makes prompt BRAF mutation testing an important step in the management of patients diagnosed with stage III or IV melanoma; one that can help better ensure that the optimal choice of systemic treatment is initiated with minimal delay. This article offers guidance about when and how BRAF mutation testing should be conducted when patients are diagnosed with melanoma in Australia. Notably, it recommends that pathologists reflexively order BRAF mutation testing whenever a patient is found to have American Joint Committee on Cancer (AJCC)/Union for International Cancer Control (UICC) stage III or IV melanoma (i.e., any metastatic spread beyond the primary tumour) and that patient's BRAF mutation status is hitherto unknown, even if BRAF mutation testing has not been specifically requested by the treating clinician (in Australia, Medicare-subsidised BRAF

BRAF V600 mutations (BRAF mut ) are associated with more pigmentation in primary melanomas, but data on melanin content of metastases are limited. This study compares signal characteristics of BRAF mut and BRAF-wildtype (BRAF wt ) intracranial melanoma metastases (IMM). MRI brain examinations at first diagnosis of IMM were identified, all performed at 3-Tesla including 1 mm volumetric pre- and postcontrast T1-weighted imaging and susceptibility-weighted imaging (SWI). Individual metastases were assessed by a neuroradiologist, stratified by size (≥10 mm, 'larger', vs. 2-9 mm, 'small'; up to 10 per group); presence of intrinsic T1-hyperintensity (T1H) and, if present, whether confidently attributable to melanin as opposed to haemorrhage; evidence of haemorrhage; presence of central necrosis. A total of 267 IMM in 73 patients were assessed (87 larger IMM, 180 small). The proportion of larger IMM was similar in both groups (31% BRAF mut and 36% BRAF wt ). In small IMM, MRI evidence of melanin was more common in BRAF mut patients (42% vs. 26%; P = 0.038). Haemorrhage was more common in larger IMM (51%, vs. 20% of small IMM; P0.0001), but did not differ based on BRAF status. Central necrosis was more common in larger IMM (44% vs. 7%; P0.0001) and in BRAF mut IMM (23% vs. 11%; P = 0.011). In the BRAF mut cohort, central necrosis was more common in patients without previous anti-BRAF therapy (33% vs. 7%; P = 0.0001). T1H attributable to melanin is only slightly more common in BRAF mut IMM than BRAF wt . Higher rates of central necrosis in BRAF mut patients without previous anti-BRAF therapy suggest that anti-BRAF therapy may affect the patterns of IMM growth.

Purpose: The randomized phase III coBRIM study (NCT01689519) demonstrated improved progression-free survival (PFS) and overall survival (OS) with addition of cobimetinib to vemurafenib compared with vemurafenib in patients with previously untreated BRAFV600 mutation–positive advanced melanoma. We report long-term follow-up of coBRIM, with at least 5 years since the last patient was randomized. Patients and Methods: Eligible patients were randomized 1:1 to receive either oral cobimetinib (60 mg once daily on days 1–21 in each 28-day cycle) or placebo in combination with oral vemurafenib (960 mg twice daily). Results: 495 patients were randomized to cobimetinib plus vemurafenib (n = 247) or placebo plus vemurafenib (n = 248). Median follow-up was 21.2 months for cobimetinib plus vemurafenib and 16.6 months for placebo plus vemurafenib. Median OS was 22.5 months (95% CI, 20.3–28.8) with cobimetinib plus vemurafenib and 17.4 months (95% CI, 15.0–19.8) with placebo plus vemurafenib; 5-year OS rates were 31% and 26%, respectively. Median PFS was 12.6 months (95% CI, 9.5–14.8) with cobimetinib plus vemurafenib and 7.2 months (95% CI, 5.6–7.5) with placebo plus vemurafenib; 5-year PFS rates were 14% and 10%, respectively. OS and PFS were longest in patients with normal baseline lactate dehydrogenase levels and low tumor burden, and in those achieving complete response. The safety profile remained consistent with previously published reports. Conclusions: Extended follow-up of coBRIM confirms the long-term clinical benefit and safety profile of cobimetinib plus vemurafenib compared with vemurafenib monotherapy in patients with BRAFV600 mutation–positive advanced melanoma.

BackgroundAcral melanoma is a rare melanoma subtype with poor prognosis. Importantly, these patients were not identified as a specific subgroup in the landmark melanoma trials involving ipilimumab and the anti-programmed cell death protein-1 (PD-1) agents nivolumab and pembrolizumab. There is therefore an absence of prospective clinical trial evidence regarding the efficacy of checkpoint inhibitors (CPIs) in this population. Acral melanoma has lower tumor mutation burden (TMB) than other cutaneous sites, and primary site is associated with differences in TMB. However the impact of this on the effectiveness of immune CPIs is unknown. We examined the efficacy of CPIs in acral melanoma, including by primary site.MethodsPatients with unresectable stage III/IV acral melanoma treated with CPI (anti-PD-1 and/or ipilimumab) were studied. Multivariable logistic and Cox regression analyses were conducted. Primary outcome was objective response rate (ORR); secondary outcomes were progression-free survival (PFS) and overall survival (OS).ResultsIn total, 325 patients were included: 234 (72%) plantar, 69 (21%) subungual and 22 (7%) palmar primary sites. First CPI included: 184 (57%) anti-PD-1, 59 (18%) anti-PD-1/ipilimumab combination and 82 (25%) ipilimumab. ORR was significantly higher with initial anti-PD-1/ipilimumab compared with anti-PD-1 (43% vs 26%, HR 2.14, p=0.0004) and significantly lower with ipilimumab (15% vs 26%, HR 0.49, p=0.0016). Landmark PFS at 1 year was highest for anti-PD-1/ipilimumab at 34% (95% CI 24% to 49%), compared with 26% (95% CI 20% to 33%) with anti-PD-1 and 10% (95% CI 5% to 19%) with ipilimumab. Despite a trend for increased PFS, anti-PD-1/ipilimumab combination did not significantly improve PFS (HR 0.85, p=0.35) or OS over anti-PD-1 (HR 1.30, p=0.16), potentially due to subsequent therapies and high rates of acquired resistance. No outcome differences were found between primary sites.ConclusionWhile the ORR to anti-PD-1/ipilimumab was significantly higher than anti-PD-1 and PFS numerically higher, in this retrospective cohort this benefit did not translate to improved OS. Future trials should specifically include patients with acral melanoma, to help determine the optimal management of this important melanoma subtype.

Resistance to targeted therapies is a major challenge in cancer care and occurs via genetic and non-genetic mechanisms. In this issue of Cancer Cell, Marin-Bejar et al. demonstrate that melanomas recurrently select genetic or non-genetic resistance trajectories and that targeting neural crest stem cell-like cells prevents non-genetic, but not genetic, resistance.

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Purpose: To report the 5-year overall survival (OS) landmark and the long-term safety profile of vemurafenib plus cobimetinib (BRAF plus MEK inhibition, respectively) in the BRIM7 study. Patients and Methods: This phase Ib, dose-finding, and expansion study evaluated combination treatment with vemurafenib and cobimetinib in two cohorts of patients with advanced BRAFV600-mutated melanoma: patients who were BRAF inhibitor (BRAFi)-naïve (n = 63) or patients who had progressed on prior treatment with BRAFi monotherapy [vemurafenib monotherapy–progressive disease (PD); n = 66]. Patients in the dose-escalation phase received vemurafenib at 720 or 960 mg twice daily in combination with cobimetinib at 60, 80, or 100 mg/d for 14 days on/14 days off, 21 days on/7 days off, or continuously. Two regimens were selected for expansion: vemurafenib (720 and 960 mg twice daily) and cobimetinib (60 mg/d 21/7). Results: Median OS was 31.8 months [95% confidence interval (CI), 24.5–not estimable] in the BRAFi-naïve cohort. The landmark OS rate plateaued at 39.2% at years 4 and 5 of follow-up. In the vemurafenib monotherapy–PD cohort, the median OS was 8.5 months (95% CI, 6.7–11.1), and the landmark OS rate plateaued at 14.0% from 3 years of follow-up. No increase was observed in the frequency and severity of adverse events with long-term follow-up. No new toxicities were detected, and there was no increase in the frequency of symptomatic MEK inhibitor class-effect adverse events. Conclusions: A subset of patients with advanced BRAFV600-mutated melanoma treated with a combination regimen of vemurafenib and cobimetinib achieve favorable long-term outcomes.

We provide an updated review of clinical trials evaluating the combination of BRAF/MEK inhibitors with anti-PD-(L)1 therapy (triplet therapy) for patients with advanced BRAF-mutant melanoma, accompanied by a summary of the biological evidence supporting this combination.Resistance to BRAF/MEK inhibition and comparatively low response rates to immune checkpoint inhibitors remain clinical challenges in the treatment of melanoma. Preclinical data demonstrates that targeted therapy is immune-modulatory and synergises with immune checkpoint inhibition. Several randomised controlled trials have evaluated the combination of targeted therapy with immune checkpoint inhibition. Triplet therapy has shown improvements in progression-free survival and durability of response compared to BRAF/MEK inhibition alone; however, questions remain regarding the best clinical scenario for implementation of this regimen in the era of front-line immunotherapy.

Simple Summary Targeted anti-cancer therapies have revolutionised melanoma patient care; however, cures remain uncommon due to acquired drug resistance that results in disease relapse. Recent insights from the clinic and experimental settings have identified a key role for metabolic plasticity, defined as the flexibility to utilise different nutrients and process them in different ways, in both disease progression and response to targeted therapies. Here, we discuss how this plasticity creates a moving target with important implications for identifying new combination therapies. Abstract Resistance to therapy continues to be a barrier to curative treatments in melanoma. Recent insights from the clinic and experimental settings have highlighted a range of non-genetic adaptive mechanisms that contribute to therapy resistance and disease relapse, including transcriptional, post-transcriptional and metabolic reprogramming. A growing body of evidence highlights the inherent plasticity of melanoma metabolism, evidenced by reversible metabolome alterations and flexibility in fuel usage that occur during metastasis and response to anti-cancer therapies. Here, we discuss how the inherent metabolic plasticity of melanoma cells facilitates both disease progression and acquisition of anti-cancer therapy resistance. In particular, we discuss in detail the different metabolic changes that occur during the three major phases of the targeted therapy response—the early response, drug tolerance and acquired resistance. We also discuss how non-genetic programs, including transcription and translation, control this process. The prevalence and diverse array of these non-genetic resistance mechanisms poses a new challenge to the field that requires innovative strategies to monitor and counteract these adaptive processes in the quest to prevent therapy resistance.

BackgroundMelanoma brain metastases (MBMs) are a challenging clinical problem with high morbidity and mortality. Although first-line dabrafenib–trametinib and ipilimumab–nivolumab have similar intracranial response rates (50%–55%), central nervous system (CNS) resistance to BRAF-MEK inhibitors (BRAF-MEKi) usually occurs around 6 months, and durable responses are only seen with combination immunotherapy. We sought to investigate the utility of ipilimumab–nivolumab after MBM progression on BRAF-MEKi and identify mechanisms of resistance.MethodsPatients who received first-line ipilimumab–nivolumab for MBMs or second/third line ipilimumab–nivolumab for intracranial metastases with BRAFV600 mutations with prior progression on BRAF-MEKi and MRI brain staging from March 1, 2015 to June 30, 2018 were included. Modified intracranial RECIST was used to assess response. Formalin-fixed paraffin-embedded samples of BRAFV600 mutant MBMs that were naïve to systemic treatment (n=18) or excised after progression on BRAF-MEKi (n=14) underwent whole transcriptome sequencing. Comparative analyses of MBMs naïve to systemic treatment versus BRAF-MEKi progression were performed.ResultsTwenty-five and 30 patients who received first and second/third line ipilimumab–nivolumab, were included respectively. Median sum of MBM diameters was 13 and 20.5 mm for the first and second/third line ipilimumab–nivolumab groups, respectively. Intracranial response rate was 75.0% (12/16), and median progression-free survival (PFS) was 41.6 months for first-line ipilimumab–nivolumab. Efficacy of second/third line ipilimumab-nivolumab after BRAF-MEKi progression was poor with an intracranial response rate of 4.8% (1/21) and median PFS of 1.3 months. Given the poor activity of ipilimumab–nivolumab after BRAF-MEKi MBM progression, we performed whole transcriptome sequencing to identify mechanisms of drug resistance. We identified a set of 178 differentially expressed genes (DEGs) between naïve and MBMs with progression on BRAF-MEKi treatment (p value ConclusionsSecond-line ipilimumab–nivolumab for MBMs after BRAF-MEKi progression has poor activity. MBMs that are resistant to BRAF-MEKi that also conferred resistance to second-line ipilimumab–nivolumab showed enrichment of the IPRES gene signature.

Significance Targeting CDK4/6 shows great promise in the treatment of many solid cancers, including melanoma. This study has uncovered a mechanism of action of CDK4/6 inhibitors in regulating the MDM4 oncogene and the tumor suppressor, p53. In melanoma, palbociclib activates p53 via modulating PRMT5-dependent alternate MDM4 pre-mRNA splicing, which results in a robust decrease in MDM4 protein expression. Loss of palbociclib regulation of the PRMT5–MDM4 axis leads to drug resistance. Dual inhibition of CDK4/6 and PRMT5 potently suppresses melanoma tumor growth and is well tolerated in vivo. Our findings not only have immediate implications for the advancement of CDK4/6 inhibition for treating melanoma, but also more generally offer insights into CDK4/6 mechanism of action., Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are an established treatment in estrogen receptor-positive breast cancer and are currently in clinical development in melanoma, a tumor that exhibits high rates of CDK4 activation. We analyzed melanoma cells with acquired resistance to the CDK4/6 inhibitor palbociclib and demonstrate that the activity of PRMT5, a protein arginine methyltransferase and indirect target of CDK4, is essential for CDK4/6 inhibitor sensitivity. By indirectly suppressing PRMT5 activity, palbociclib alters the pre-mRNA splicing of MDM4, a negative regulator of p53, leading to decreased MDM4 protein expression and subsequent p53 activation. In turn, p53 induces p21, leading to inhibition of CDK2, the main kinase substituting for CDK4/6 and a key driver of resistance to palbociclib. Loss of the ability of palbociclib to regulate the PRMT5–MDM4 axis leads to resistance. Importantly, combining palbociclib with the PRMT5 inhibitor GSK3326595 enhances the efficacy of palbociclib in treating naive and resistant models and also delays the emergence of resistance. Our studies have uncovered a mechanism of action of CDK4/6 inhibitors in regulating the MDM4 oncogene and the tumor suppressor, p53. Furthermore, we have established that palbociclib inhibition of the PRMT5–MDM4 axis is essential for robust melanoma cell sensitivity and provide preclinical evidence that coinhibition of CDK4/6 and PRMT5 is an effective and well-tolerated therapeutic strategy. Overall, our data provide a strong rationale for further investigation of novel combinations of CDK4/6 and PRMT5 inhibitors, not only in melanoma but other tumor types, including breast, pancreatic, and esophageal carcinoma.

The benign melanocytic nevus is the most common tumor in humans and rarely transforms into cutaneous melanoma. Elucidation of the nevus genome is required to better understand the molecular steps of progression to melanoma. We performed whole genome sequencing on a series of 14 benign melanocytic nevi consisting of both congenital and acquired types. All nevi had driver mutations in the MAPK signaling pathway, either BRAF V600E or NRAS Q61R/L. No additional definite driver mutations were identified. Somatic mutations in nevi with higher mutation loads showed a predominance of mutational signatures 7a and 7b, consistent with UVR exposure, whereas nevi with lower mutation loads (including all three congenital nevi) had a predominance of the ubiquitous signatures 1 and 5. Two nevi had mutations in promoter regions predicted to bind E26 transformation-specific family transcription factors, as well as subclonal mutations in the TERT promoter. This paper presents whole genome data from melanocytic nevi. We confirm that UVR is involved in the etiology of a subset of nevi. This study also establishes that TERT promoter mutations are present in morphologically benign skin nevi in subclonal populations, which has implications regarding the interpretation of this emerging biomarker in sensitive assays.

Background We conducted a retrospective analysis to evaluate the impact of concomitant acid-reducing agents (ARAs) and vemurafenib dose on the efficacy of vemurafenib in patients with BRAFV600 mutation–positive unresectable or metastatic melanoma treated with vemurafenib or cobimetinib plus vemurafenib. Methods Data were pooled for patients treated with vemurafenib or cobimetinib plus vemurafenib in the BRIM-2, BRIM-3, BRIM-7, and coBRIM studies. The primary end-points were progression-free survival and overall survival across patient subgroups defined by vemurafenib dose (full vs reduced) and concomitant ARA use (yes vs no). Objective response rate (ORR) was also analysed. Steady-state vemurafenib concentrations were evaluated according to vemurafenib dosing and concomitant ARA use across treatment cohorts in a subset of patients from BRIM-7 and coBRIM with available concentration data. Results Efficacy analyses included 920 patients: 641 in the vemurafenib cohort and 279 in the cobimetinib plus vemurafenib cohort. Overall, no significant differences in survival outcomes were observed across subgroups according to vemurafenib dose and ARA use, with or without adjustment for known prognostic covariates, in both treatment cohorts. ORR was also similar across subgroups in both treatment cohorts. Steady-state vemurafenib concentrations were analysed in 389 patients (193 in the vemurafenib cohort and 196 in the cobimetinib plus vemurafenib cohort) and were generally similar across vemurafenib dose subgroups, regardless of ARA use in both treatment cohorts. Conclusions Results of this retrospective pooled analysis suggest that ARAs can be used concomitantly with vemurafenib, alone or in combination with cobimetinib, without compromising the efficacy of vemurafenib.

Melanoma is usually driven by mutations in BRAF or NRAS, which trigger hyperactivation of MAPK signaling. However, MAPK-targeted therapies are not sustainably effective in most patients. Accordingly, characterizing mechanisms that co-operatively drive melanoma progression is key to improving patient outcomes. One possible mechanism is the Hippo signaling pathway, which regulates cancer progression via its central oncoproteins YAP and TAZ, although is thought to be only rarely affected by direct mutation. As YAP hyperactivation occurs in uveal melanoma, we investigated this oncogene in cutaneous melanoma. YAP protein expression was elevated in most benign nevi and primary cutaneous melanomas but present at only very low levels in normal melanocytes. In patient-derived xenografts and melanoma cell lines, we observed variable reliance of cell viability on Hippo pathway signaling that was independent of TAZ activity and also of classical melanoma driver mutations such as BRAF and NRAS. Finally, in genotyping studies of melanoma, we observed the first ever hyperactivating YAP mutations in a human cancer, manifest as seven distinct missense point mutations that caused serine to alanine transpositions. Strikingly, these mutate four serine residues known to be targeted by the Hippo pathway and we show that they lead to hyperactivation of YAP. Implications: Our studies highlight the YAP oncoprotein as a potential therapeutic target in select subgroups of melanoma patients, although successful treatment with anti-YAP therapies will depend on identification of biomarkers additional to YAP protein expression.

Advances in the treatment of metastatic melanoma have improved responses and survival. However, many patients continue to experience resistance or toxicity to treatment, highlighting a crucial need to identify biomarkers and understand mechanisms of response and toxicity. Neoadjuvant therapy for regional metastases might improve operability and clinical outcomes over upfront surgery and adjuvant therapy, and has become an established role for drug development and biomarker discovery in other cancers (including locally advanced breast cancer, head and neck squamous cell carcinomas, gastroesophageal cancer, and anal cancer). Patients with clinically detectable stage III melanoma are ideal candidates for neoadjuvant therapy, because they represent a high-risk patient population with poor outcomes when treated with upfront surgery alone. Neoadjuvant therapy is now an active area of research for melanoma with numerous completed and ongoing trials (since 2014) with disparate designs, endpoints, and analyses under investigation. We have, therefore, established the International Neoadjuvant Melanoma Consortium with experts in medical oncology, surgical oncology, pathology, radiation oncology, radiology, and translational research to develop recommendations for investigating neoadjuvant therapy in melanoma to align future trial designs and correlative analyses. Alignment and consistency of neoadjuvant trials will facilitate optimal data organisation for future regulatory review and strengthen translational research across the melanoma disease continuum.

Purpose: Previous investigations identified transcriptional signatures associated with innate resistance to anti–programmed cell death protein 1 therapy in melanoma. This analysis aimed to increase understanding of the role of baseline genetic features in the variability of response to BRAF and MEK inhibitor therapy for BRAFV600-mutated metastatic melanoma. Patients and Methods: This exploratory analysis compared genomic features, using whole-exome and RNA sequencing, of baseline tumors from patients who had complete response versus rapid progression (disease progression at first postbaseline assessment) on treatment with cobimetinib combined with vemurafenib or vemurafenib alone. Associations of gene expression with progression-free survival or overall survival were assessed by Cox proportional hazards modeling. Results: Whole-exome sequencing showed that MITF and TP53 alterations were more frequent in tumors from patients with rapid progression, while NF1 alterations were more frequent in tumors from patients with complete response. However, the low frequency of alterations in any one gene precluded their characterization as drivers of response/resistance. Analysis of RNA profiles showed that expression of immune response–related genes was enriched in tumors from patients with complete response, while expression of keratinization-related genes was enriched in tumors from patients who experienced rapid progression. Conclusions: These findings suggest that enriched immune infiltration might be a shared feature favoring response to both targeted and immune therapies, while features of innate resistance to targeted and immune therapies were distinct.

BackgroundVaginal melanoma (VM) is a rare cancer and has a poor response to immune checkpoint blockade (ICB). CD8+Tissue Resident Memory (TRM) T cells proliferate in response to ICB and correlate with longer survival in metastatic cutaneous melanoma. However, their capacity to respond to VM and their neoantigens is not known.MethodsUsing longitudinal samples, we explored the evolution of VM mutations by whole-exome sequencing and RNAseq, we also defined the immune context using multiplex immunohistochemistry and nanostring pan cancer immune profile. Then using fresh single cell suspensions of the metastatic samples, we explored VM T cells via mass cytometry and single cell RNAseq and T cell receptor sequencing (TCRseq). Finally, we investigated TRM, pre-TRM and exhausted T cell function against melanoma neo-antigens and melanoma differentiation antigens in vitro.ResultsPrimary VM was non-inflamed and devoid of CD8+ TRM cells. In contrast, both metastases showed proliferating CD8+ TRM were clustered at the tumor margin, with increased numbers in the second ICB-refractory metastasis. The first metastasis showed dense infiltration of CD8+ T cells, the second showed immune exclusion with loss of melanoma cell Major histocompatibility complex (MHC)-I expression associated with downregulation of antigen presentation pathway gene expression. CD8+ TRM from both metastases responded to autologous melanoma cells more robustly than all other CD8+ T cell subsets. In addition, CD8+ TRM shared TCR clones across metastases, suggesting a response to common antigens, which was supported by recognition of the same neoantigen by expanded tumor infiltrating lymphocytes.ConclusionsIn this study, we identified TRM clusters in VM metastases from a patient, but not primary disease. We showed TRM location at the tumor margin, and their superior functional response to autologous tumor cells, predicted neoantigens and melanoma differentiation antigens. These CD8+ TRM exhibited the highest tumor-responsive potential and shared their TCR with tumor-infiltrating effector memory T cells. This suggests VM metastases from this patient retain strong antitumor T cell functional responses; however, this response is suppressed in vivo. The loss of VG MHC-I expression is a common immune escape mechanism which was not addressed by anti-PD-1 monotherapy; rather an additional targeted approach to upregulate MHC-I expression is required.

Purpose The treatment of advanced BRAFV600-mutated melanomas with BRAF inhibitors (BRAFi) has improved survival, but the efficacy of BRAFi varies among individuals and the development of acquired resistance to BRAFi through reactivation of mitogen-activated protein kinase (MAPK) signaling is common. We performed an exploratory, retrospective analysis to investigate the effects of BRAFV600 allelic balance, coexisting oncogene mutations, cell proliferation signaling levels, and loss of PTEN expression on progression-free survival (PFS) in patients in the phase III coBRIM study, which compared the combination of the MEK inhibitor cobimetinib with the BRAFi vemurafenib versus vemurafenib as monotherapy. Methods Baseline tumor samples from the intention-to-treat population were analyzed by targeted deep sequencing at a median coverage of 3,600× and by immunohistochemistry for cell proliferation markers, BRAFV600E, and PTEN. The association of these biomarkers with PFS was assessed by Cox proportional hazards modeling. Gene expression in relation to loss of PTEN was profiled by RNA sequencing in 205 patient samples and 42 BRAFV600-mutated melanoma cell lines. Results Neither BRAFV600 allelic balance nor coexisting mutations in the RAS/RAF/RTK pathway affected PFS in either treatment group. Increased baseline MAPK signaling and cell proliferation did not affect PFS in patients treated with cobimetinib combined with vemurafenib. PTEN loss was associated with reduced PFS in patients treated with vemurafenib alone but not in patients treated with cobimetinib combined with vemurafenib. Conclusion Deeper inhibition of the MAPK pathway through targeting of both MEK and BRAF may override the effects of tumor heterogeneity and improve PFS in all patients with advanced BRAFV600 melanoma.

Although melanoma is initiated by acquisition of point mutations and limited focal copy number alterations in melanocytes-of-origin, the nature of genetic changes that characterise lethal metastatic disease is poorly understood. Here, we analyze the evolution of human melanoma progressing from early to late disease in 13 patients by sampling their tumours at multiple sites and times. Whole exome and genome sequencing data from 88 tumour samples reveals only limited gain of point mutations generally, with net mutational loss in some metastases. In contrast, melanoma evolution is dominated by whole genome doubling and large-scale aneuploidy, in which widespread loss of heterozygosity sculpts the burden of point mutations, neoantigens and structural variants even in treatment-naïve and primary cutaneous melanomas in some patients. These results imply that dysregulation of genomic integrity is a key driver of selective clonal advantage during melanoma progression., The genetic changes that occur in late stage metastatic melanoma are not well delineated. Here, the authors use rapid autopsy samples from metastatic melanoma patients and show that the late stage in the disease is characterised by whole genome doubling and aneuploidy.

BACKGROUND: Melanoma brain metastases (MBM) have a poor prognosis. Systemic treatments that have improved outcomes in advanced melanoma have been shown to have an intracranial (IC) effect. We studied the efficacy and outcomes of combined immune checkpoint inhibitor ipilimumab/nivolumab (Combi-ICI) or targeted therapy (Combi-TT) as first-line treatment in MBM. METHODS: MBM patients treated with Combi-ICI or Combi-TT within 3 months after MBM diagnosis. Endpoints were progression-free survival (PFS) and overall survival (OS). RESULTS: 53 patients received Combi-ICI, 32% had symptomatic MBM and 33.9% elevated LDH. 71.7% required local treatment. The disease control rate was 60.3%. IC response rate (RR) was 43.8% at 3-months with durable responses at 6- (46.5%) and 12-months (53.1%). Extracranial (EC) RR was 44.7% at 3-months and 50% at 12-months. Median PFS was 9.6 months (95% CI 3.6-NR) and median overall survival (mOS) 44.8 months (95% CI; 26.2-NR). 63 patients received Combi-TT, 55.6% of patients had symptomatic MBM, 57.2% of patients had elevated LDH and 68.3% of patients required local treatment. The disease control rate was 60.4%. ICRR was 50% at 3-months, but dropped at 6-months (20.9%). ECRR was 69.2% at 3-months and 17.6% at 12-months. Median PFS was 5.8 months (95% CI 4.2-7.6) and mOS 14.2 months (95% CI 8.99-26.8). In BRAFV600 patients, 26.7% of patients received Combi-ICI and 73.3% Combi-TT with OS (p = 0.0053) and mPFS (p = 0.03) in favour to Combi-ICI. CONCLUSION: Combi-ICI showed prolonged mOS with sustainable IC and EC responses. Despite the initially increased efficacy, Combi-TT responses at 12 months were low. Combi-ICI appeared superior to Combi-TT for OS and PFS in BRAFV600 patients. Other clinical factors are determinants for first-line treatment choice.

Simple Summary Cyclin-dependent kinases 4 and 6 (CDK4/6) are key enzymes controlling the cell cycle. CDK4/6 inhibitors are being tested in multiple clinical trials for a range of cancers including melanoma, and a deeper understanding of how they interact with other therapies is vital for their clinical development. Beyond the cell cycle, CDK4/6 regulates cell metabolism, which is a critical factor determining response to standard-of-care mitogen-activated protein kinase (MAPK) pathway therapies in melanoma. Here, we show that CDK4/6 inhibitors increase glutamine and fatty acid-oxidation-dependent mitochondrial metabolism in melanoma cells, but they do not alter the metabolic response to MAPK inhibitors. These observations shed light on how CDK4/6 inhibitors impinge on the regulation of metabolism and how they interact with other therapies in the setting of melanoma. Abstract Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are being tested in numerous clinical trials and are currently employed successfully in the clinic for the treatment of breast cancers. Understanding their mechanism of action and interaction with other therapies is vital in their clinical development. CDK4/6 regulate the cell cycle via phosphorylation and inhibition of the tumour suppressor RB, and in addition can phosphorylate many cellular proteins and modulate numerous cellular functions including cell metabolism. Metabolic reprogramming is observed in melanoma following standard-of-care BRAF/MEK inhibition and is involved in both therapeutic response and resistance. In preclinical models, CDK4/6 inhibitors overcome BRAF/MEK inhibitor resistance, leading to sustained tumour regression; however, the metabolic response to this combination has not been explored. Here, we investigate how CDK4/6 inhibition reprograms metabolism and if this alters metabolic reprogramming observed upon BRAF/MEK inhibition. Although CDK4/6 inhibition has no substantial effect on the metabolic phenotype following BRAF/MEK targeted therapy in melanoma, CDK4/6 inhibition alone significantly enhances mitochondrial metabolism. The increase in mitochondrial metabolism in melanoma cells following CDK4/6 inhibition is fuelled in part by both glutamine metabolism and fatty acid oxidation pathways and is partially dependent on p53. Collectively, our findings identify new p53-dependent metabolic vulnerabilities that may be targeted to improve response to CDK4/6 inhibitors.

BackgroundMucosal melanoma is a rare subtype of melanoma originating from mucosal tissues (1), metastases are very aggressive and respond poorly to therapy, including immune checkpoint inhibitors (ICI) such as anti-CTLA4 and anti-PD1 antibodies (2–5). CD8+ T cells constitute the most abundant immune infiltrate in metastatic melanoma, of which the Tissue Resident Memory subset (TRM) is of particular interest (6). CD8+ TRM cells express the highest levels of immune checkpoint receptors, proliferate in response to ICI and correlate with longer disease-free and overall survival (6–8). The immune landscape in mucosal melanoma remains poorly characterized. We aimed to: 1) phenotype CD8+ T cells and TRM infiltrating metastatic mucosal melanoma, 2) characterize the clonality of TRM in relation to other CD8+ T cell subsets and 3) define the capacity of CD8+ T cells and TRM to respond to melanoma cells and to in vivo and in vitro anti-PD1 treatment.MethodsWe investigated the CD8+ T and TRM cells infiltrating two temporally- and spatially-distant subcutaneous metastases, these originated from a primary vaginal mucosal melanoma. One metastasis was excised prior to anti-PD1 treatment and one was anti-PD1 refractory, having progressed on treatment. We used mass cytometry and single-cell RNA and TCR sequencing to characterise the phenotype and clonality of the T cells, multiplex immunohistochemistry to define their spatial relationship with tumour cells and other T cells, and functional assays to determine TRM response to tumour cells (figure 1).ResultsCD8+ TRM frequency increased with time and anti-PD1 treatment, forming clusters at the tumour margin. T cells in the anti-PD1 refractory lesion were more activated than T cells in the first tumour and were bound by anti-PD1 antibody in vivo. T cells could not be stimulated by anti-PD1 directly ex vivo. Both metastatic lesions shared common T cell clusters including TRM. Furthermore, TRM in each tumour shared T cell clones, suggesting the presence of common antigens between metastatic sites. Indeed, the two metastases had a similar mutational profile. In vitro expanded tumour infiltrating lymphocytes from both lesions recognized tumour cells from both lesions and the same neoantigen generated from a single point mutation in the gene CDKN1C. Finally, tumour cells stimulated TRM cells more robustly than other T cells subsets.Abstract 548 Figure 1Graphical depiction of the methods used to characterise T cells in mucosal metastatic melanomaConclusionsIn this patient with vaginal mucosal melanoma, subsequent melanoma metastases of clonal origin attracted CD8+ T cells of similar specificity, among which TRM cells responded more vigorously to tumour cells than other T cells subsets.AcknowledgementsThe authors would like to acknowledge imCORE La Hoffmann- Roche Ltd. for funding.Ethics ApprovalPatients diagnosed with stage 3 or 4 metastatic melanoma and undergoing clinically indicated surgery were enrolled in prospective studies approved by the Peter MacCallum Cancer Centre human ethics research committee (13/141). All experimental protocols have been approved and clinical data has been collected prospectively.ReferencesCarvajal RD, Hamid O, Ariyan C. Mucosal Melanoma. [cited 2020 Apr 1]; Available from: https://www.uptodate.com/contents/mucosal-melanomaDel Vecchio M, Di Guardo L, Ascierto PA, Grimaldi AM, Sileni VC, Pigozzo J, et al. Efficacy and safety of ipilimumab 3 mg/kg in patients with pretreated, metastatic, mucosal melanoma. Eur J Cancer Oxf Engl 1990; 2014 Jan;50(1):121–7.Postow MA, Luke JJ, Bluth MJ, Ramaiya N, Panageas KS, Lawrence DP, et al. Ipilimumab for patients with advanced mucosal melanoma. The Oncologist 2013 Jun;18(6):726–32.D’Angelo SP, Larkin J, Sosman JA, Lebbe C, Brady B, Neyns B, et al. Efficacy and safety of nivolumab alone or in combination with ipilimumab in patients with mucosal melanoma: a pooled analysis. J Clin Oncol Off J Am Soc Clin Oncol. 2017 Jan 10;35(2):226–35.Hamid O, Robert C, Ribas A, Hodi FS, Walpole E, Daud A, et al. Antitumour activity of pembrolizumab in advanced mucosal melanoma: a post-hoc analysis of KEYNOTE-001, 002, 006. Br J Cancer 2018;119(6):670–4.Boddupalli CS, Bar N, Kadaveru K, Krauthammer M, Pornputtapong N, Mai Z, et al. Interlesional diversity of T cell receptors in melanoma with immune checkpoints enriched in tissue-resident memory T cells. JCI Insight [Internet]. 2016 Dec 22 [cited 2019 Apr 24];1(21). Available from: https://insight.jci.org/articles/view/88955Edwards J, Wilmott JS, Madore J, Gide TN, Quek C, Tasker A, et al. CD103+ Tumor-resident CD8+ T cells are associated with improved survival in immunotherapy-naive melanoma patients and expand significantly during anti-PD-1 treatment. Clin Cancer Res Off J Am Assoc Cancer Res 2018 Jul 1;24(13):3036–45.Savas P, Virassamy B, Ye C, Salim A, Mintoff CP, Caramia F, et al. Single-cell profiling of breast cancer T cells reveals a tissue-resident memory subset associated with improved prognosis. Nat Med 2018 Jul;24(7):986–93.

Identification of mechanisms underlying sensitivity and response to targeted therapies, such as the BRAF inhibitor vemurafenib, is critical in order to improve efficacy of these therapies in the clinic and delay onset of resistance. Glycolysis has emerged as a key feature of the BRAF inhibitor response in melanoma cells, and importantly, the metabolic response to vemurafenib in melanoma patients can predict patient outcome. Here, we present a multiparameter genome-wide siRNA screening dataset of genes that when depleted improve the viability and glycolytic response to vemurafenib in BRAFV600 mutated melanoma cells. These datasets are suitable for analysis of genes involved in cell viability and glycolysis in steady state conditions and following treatment with vemurafenib, as well as computational approaches to identify gene regulatory networks that mediate response to BRAF inhibition in melanoma., Measurement(s) cell viability • glycolytic process Technology Type(s) immunofluorescence microscopy assay • absorbance Factor Type(s) drug treatment • gene silencing Sample Characteristic - Organism Homo sapiens Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.12885593

Background We sought to identify patient subgroups with distinct postprogression overall survival (ppOS) outcomes and investigate the impact of original treatment assignment and initial postprogression treatment (ppRx) on ppOS. Methods Recursive partitioning analysis (RPA) was performed to model relationships between prespecified covariates and ppOS in patients with BRAFV600-mutated metastatic melanoma who had experienced progressive disease (PD) following treatment with cobimetinib plus vemurafenib, vemurafenib monotherapy, or dacarbazine in the BRIM-2, BRIM-3, BRIM-7, and coBRIM studies. Prognostic subgroups identified by RPA were then applied to pooled treatment cohorts. The primary endpoint was ppOS, defined as time from first PD to death from any cause. Results RPA identified baseline lactate dehydrogenase (LDH), baseline disease stage, Eastern Cooperative Oncology Group performance status at PD, and ppRx as significant prognostic factors for ppOS. Median ppOS was longest in patients with normal baseline LDH, stage M1c disease at baseline, and ppRx with immunotherapy or targeted therapy (12.2 months; 95% CI 10.3–16.1) and shortest in those with elevated baseline LDH > 2 × upper limit of normal (2.3 months; 95% CI 1.8–2.7). Original treatment assignment did not impact ppOS. Across treatment cohorts, patients treated with immunotherapy or targeted therapy after PD had better ppOS than those given other treatments. Conclusion A combination of factors at baseline (LDH, disease stage) and PD (performance status, ppRx) impact ppOS outcomes. ppRx with immunotherapy or targeted therapy is an independent prognostic factor for improved overall survival following progression regardless of original treatment. Trial registration The trials included in this analysis are registered with ClinicalTrials.gov: NCT00949702 (BRIM-2), NCT01006980 (BRIM-3), NCT01271803 (BRIM-7), and NCT01689519 (coBRIM).

Introduction The brain is a common site of metastatic disease for patients with advanced melanoma. Brain metastasis portends a poor prognosis, often causing deterioration in neurological function and quality of life, and leading to neurological death. Treatment approaches including surgery, radiotherapy and systemic therapy can lead to better control of this problem. Therefore, appropriate guidelines for the management of melanoma brain metastases need to be established, with regular updating when new treatment options become available. Methods A multidisciplinary working party established by Cancer Council Australia has produced up-to-date, evidence-based clinical practice guidelines for the management of melanoma. After selecting key clinical questions, a comprehensive literature search for relevant studies was conducted, followed by systematic review of those studies. Data were summarised and the evidence was assessed, leading to the development of recommendations. Main recommendations Symptomatic lesions are best treated with surgery, when possible; this provides safe and effective local control. For patients with single or a small number of asymptomatic brain metastases, stereotactic radiotherapy is recommended, but in asymptomatic patients who have not previously received systemic treatment, drug therapy can be considered as a first-line treatment option. Whole brain radiotherapy may provide palliative benefits in patients with multiple brain metastases. Whenever possible, melanoma patients with brain metastases should be managed by a multidisciplinary team of melanoma specialists that considers the optimal combination and sequencing of surgery, radiotherapy and systemic therapy.

BackgroundDecreased cancer incidence and reported changes to clinical management indicate that the COVID-19 pandemic will result in diagnostic and treatment delays for cancer patients. We aimed to develop a flexible model to estimate the impact of delayed diagnosis and treatment initiation on survival outcomes and healthcare costs based on a shift in the disease stage at treatment initiation.MethodsThe stage-shift model estimates population-level health economic outcomes by weighting disease stage-specific outcomes by the distribution of stages at treatment initiation, assuming delays lead to stage-progression. It allows for extrapolation of population-level survival data using parametric distributions to calculate the expected survival in life years. The model was demonstrated based on an analysis of the impact of 3 and 6-month delays for stage I breast cancer, colorectal cancer and lung cancer patients, and for T1 melanoma, based on Australian data. In the absence of patient-level data about time to stage progression, two approaches were explored to estimate the proportion of patients that would experience a stage shift following the delay: 1) based on the relation between time to treatment initiation and overall survival (breast, colorectal and lung cancer), and 2) based on the tumour growth rate (melanoma). The model is available on http://stage-shift.personex.nl/.ResultsA shift from stage I to stage II due to a 6-month delay is least likely for colorectal cancer patients, with an estimated proportion of 3% of the stage I patients diagnosed in 2020 progressing to stage II, resulting in 11 excess deaths after 5 years and a total of 96 life years lost over a 10-year time horizon. For breast and lung cancer, progression from stage I to stage II due to a 6-month delay were slightly higher at 5% (breast cancer) and 8% (lung cancer), resulting in 25 and 43 excess deaths after 5 years, and 239 and 373 life years lost over a 10-year time horizon, respectively. For melanoma, with 32% of T1 patients progressing to T2 disease following a 6-month delay, the model estimated 270 excess death after 5 years and 2584 life years lost over a 10-year time horizon.ConclusionsUsing a conservative 3-month delay in diagnosis and treatment initiation due to the COVID-19 pandemic, this study predicts nearly 90 excess deaths and $12 million excess healthcare costs in Australia over 5 years for the in 2020 diagnosed patients for 4 cancers. If the delays increase to 6 months, excess mortality and cost approach nearly 350 deaths and $46 million in Australia. More accurate data on stage of disease during and after the COVID-19 pandemic are critical to obtain more reliable estimates.

Background Immune checkpoint blockade such as ipilimumab and anti-PD1 monoclonal antibodies have significantly improved survival in advanced melanoma. Biomarkers are urgently needed as a majority of patients do not respond, despite treatment-related toxicities. We analysed pre-treatment 18F-fluorodeoxyglucose positron emission tomography/computerised tomography (FDG PET/CT) parameters to assess its correlation with patient outcome. Methods This retrospective study evaluated pre-treatment FDG PET/CT scans in a discovery cohort of patients with advanced melanoma treated with ipilimumab or anti-PD1. Pre-treatment scans were assessed for maximum tumoral standardised uptake value (SUVmax), metabolic tumour volume (MTV) and spleen to liver ratio (SLR). Progression-free survival (PFS) and overall survival (OS) were characterised and modelled using univariable and multivariable analyses. Correlation of SLR and OS was validated in an independent cohort. Blood parameters and stored sera of patients from the discovery cohort was analysed to investigate biological correlates with SLR. Results Of the 90 evaluable patients in the discovery cohort: 50 received ipilimumab monotherapy, 20 received anti-PD1 monotherapy, and 20 patients received ipilimumab followed by anti-PD1 upon disease progression. High SLR > 1.1 was associated with poor PFS (median 1 vs 3 months; HR 3.14, p = 0.008) for patients treated with ipilimumab. High SLR was associated with poor OS after ipilimumab (median 1 vs 21 months; HR 5.83, p = 0.0001); as well as poor OS after first line immunotherapy of either ipilimumab or anti-PD1 (median 1 vs 14 months; HR 3.92, p = 0.003). The association of high SLR and poor OS after ipilimumab was validated in an independent cohort of 110 patients (median 2.3 months versus 11.9 months, HR 3.74). SLR was associated with poor OS in a multi-variable model independent of stage, LDH, absolute lymphocyte count and MTV. Conclusions Pre-treatment Spleen to liver ratio (SLR) > 1.1 was associated with poor outcome after ipilimumab in advanced melanoma. This parameter warrants prospective evaluation.

Combined inhibition of BRAF, MEK, and CDK4/6 is currently under evaluation in clinical trials for patients with melanoma harboring a BRAFV600 mutation. While this triple therapy has potent tumor-intrinsic effects, the impact of this combination on antitumor immunity remains unexplored. Here, using a syngeneic BrafV600ECdkn2a−/−Pten−/− melanoma model, we demonstrated that triple therapy promoted durable tumor control through tumor-intrinsic mechanisms and promoted immunogenic cell death and T-cell infiltration. Despite this, tumors treated with triple therapy were unresponsive to immune checkpoint blockade (ICB). Flow cytometric and single-cell RNA sequencing analyses of tumor-infiltrating immune populations revealed that triple therapy markedly depleted proinflammatory macrophages and cross-priming CD103+ dendritic cells, the absence of which correlated with poor overall survival and clinical responses to ICB in patients with melanoma. Indeed, immune populations isolated from tumors of mice treated with triple therapy failed to stimulate T-cell responses ex vivo. While combined BRAF, MEK, and CDK4/6 inhibition demonstrates favorable tumor-intrinsic activity, these data suggest that collateral effects on tumor-infiltrating myeloid populations may impact antitumor immunity. These findings have important implications for the design of combination strategies and clinical trials that incorporate BRAF, MEK, and CDK4/6 inhibition with immunotherapy for the treatment of patients with melanoma.

IMspire150 aimed to evaluate first-line combination treatment with BRAF plus MEK inhibitors and immune checkpoint therapy in BRAFIMspire150 was a randomised, double-blind, placebo-controlled phase 3 study done at 112 institutes in 20 countries. Patients with unresectable stage IIIc-IV, BRAFBetween Jan 13, 2017, and April 26, 2018, 777 patients were screened and 514 were enrolled and randomly assigned to the atezolizumab group (n=256) or control group (n=258). At a median follow-up of 18·9 months (IQR 10·4-23·8), progression-free survival as assessed by the study investigator was significantly prolonged with atezolizumab versus control (15·1 vs 10·6 months; hazard ratio [HR] 0·78; 95% CI 0·63-0·97; p=0·025). Common treatment-related adverse events (30%) in the atezolizumab and control groups were blood creatinine phosphokinase increased (51·3% vs 44·8%), diarrhoea (42·2% vs 46·6%), rash (40·9%, both groups), arthralgia (39·1% vs 28·1%), pyrexia (38·7% vs 26·0%), alanine aminotransferase increased (33·9% vs 22·8%), and lipase increased (32·2% vs 27·4%); 13% of patients in the atezolizumab group and 16% in the control group stopped all treatment because of adverse events.The addition of atezolizumab to targeted therapy with vemurafenib and cobimetinib was safe and tolerable and significantly increased progression-free survival in patients with BRAFF Hoffmann-La Roche and Genentech.

BackgroundTo compare the clinical efficacy of New York Esophageal squamous cell carcinoma-1 (NY-ESO-1) vaccine with ISCOMATRIX adjuvant versus ISCOMATRIX alone in a randomized, double-blind phase II study in participants with fully resected melanoma at high risk of recurrence.MethodsParticipants with resected stage IIc, IIIb, IIIc and IV melanoma expressing NY-ESO-1 were randomized to treatment with three doses of NY-ESO-1/ISCOMATRIX or ISCOMATRIX adjuvant administered intramuscularly at 4-week intervals, followed by a further dose at 6 months. Primary endpoint was the proportion free of relapse at 18 months in the intention-to-treat (ITT) population and two per-protocol populations. Secondary endpoints included relapse-free survival (RFS) and overall survival (OS), safety and NY-ESO-1 immunity.ResultsThe ITT population comprised 110 participants, with 56 randomized to NY-ESO-1/ISCOMATRIX and 54 to ISCOMATRIX alone. No significant toxicities were observed. There were no differences between the study arms in relapses at 18 months or for median time to relapse; 139 vs 176 days (p=0.296), or relapse rate, 27 (48.2%) vs 26 (48.1%) (HR 0.913; 95% CI 0.402 to 2.231), respectively. RFS and OS were similar between the study arms. Vaccine recipients developed strong positive antibody responses to NY-ESO-1 (p≤0.0001) and NY-ESO-1-specific CD4+and CD8+responses. Biopsies following relapse did not demonstrate differences in NY-ESO-1 expression between the study populations although an exploratory study demonstrated reduced (NY-ESO-1)+/Human Leukocyte Antigen (HLA) class I+double-positive cells in biopsies from vaccine recipients performed on relapse in 19 participants.ConclusionsThe vaccine was well tolerated, however, despite inducing antigen-specific immunity, it did not affect survival endpoints. Immune escape through the downregulation of NY-ESO-1 and/or HLA class I molecules on tumor may have contributed to relapse.

Background Recent therapeutic strategies, particularly MAP kinase pathway inhibitors (BRAF, MEK) and immune checkpoint blockers (CTLA-4, PD-1), have been put on the test for their differential impact on long-term survival of metastatic melanoma patients. Various agents, dose regimens and combinations have been tested against each other vigorously within these two therapy groups. However, results from prospective head-to-head comparative trials comparing both strategies against each other are still lacking. Methods We performed an exploratory analysis of survival data from selected clinical trials representative for these two treatment strategies in advanced metastatic melanoma. 84 Kaplan–Meier survival curves from 26 trials were digitised and grouped by therapy strategy and treatment line. For each of these groups, mean survival curves were generated for progression-free (PFS) and overall survival (OS) by weighted averaging. Results Survival curves grouped together by therapy strategy revealed a high concordance, with a larger extent in the first-line setting compared to higher treatment lines. In first-line therapy, the averaged 3-year OS proportions were 41.3% for BRAF plus MEK inhibition, 49.9% for PD-1 inhibition, and 58.4% for CTLA-4 plus PD-1 inhibition. Comparison of the mean PFS and OS curves of kinase inhibition and checkpoint blockade revealed a superiority of combined BRAF plus MEK inhibition within the first 12 months, later changing to a superiority of PD-1 blockers alone or in combination with CTLA-4 blockade. In second-line or higher, BRAF plus MEK inhibition was superior to anti-PD-1 monotherapy throughout the first three years; averaged 3-year OS proportions were 42.4% for BRAF plus MEK inhibition, and 40.1% for PD-1 inhibition. Conclusions and relevance: These results need confirmation by head-to-head comparative randomised clinical trials.

Background A proportion of patients develop recurrence following a tumour-negative sentinel lymph node biopsy (SLNB). This study aimed to explore whether melanoma patients with BRAF or NRAS mutant tumours have an increased risk of developing disease recurrence following a negative SLNB compared to patients with wild-type tumours. Methods Prospective cohort study of melanoma patients at three tertiary referral centres in Melbourne, who underwent SLNB. Clinical, pathological and molecular characteristics and recurrence data were prospectively recorded. Multivariate Cox proportional hazards regression models estimated the adjusted hazard ratio (aHR) and corresponding 95% confidence interval (CI) for the association between mutation status and development of recurrence following a negative-SLNB. Results Overall, 344/477 (72.1%) patients had a negative SLNB. Of these, 54 (15.7%) developed subsequent recurrence. The risk of disease recurrence following a negative SLNB was increased for patients with either a BRAF or NRAS mutant tumour compared to wild-type tumours (aHR 1.92, 95% CI: 1.02–3.60, p = 0.04). Conclusion Melanoma patients with BRAF or NRAS mutant tumours had an increased risk compared to patients with BRAF/NRAS wild-type tumours of developing disease recurrence following a tumour-negative SLNB. The findings also confirm the importance of continued surveillance to monitor for disease recurrence among SLNB-negative patients.

This study aimed to determine the frequency and concordance of BRAF and NRAS mutation in tumours arising in patients with multiple primary melanoma (MPM). Patients with MPM managed at one of three tertiary referral centres in Melbourne, Australia, from 2010 to 2015 were included. Incident and subsequent melanomas underwent mutation testing. Cohen's kappa (κ) coefficient assessed agreement between incident and subsequent primary melanomas for both BRAF and NRAS mutation status (mutant versus wild-type). Mutation testing of at least two primary tumours from 64 patients was conducted. There was poor agreement for both BRAF and NRAS mutation status between incident and subsequent melanomas (κ = 0.10, 95% CI -0.10 to 0.42; κ = 0.06, 95% CI -0.10 to 0.57, respectively). In view of the low concordance in BRAF mutation status between incident and subsequent melanomas, mutational analysis of metastatic tissue, rather than of a primary melanoma, in patients with MPM should be used to guide targeted therapy.

Simple Summary Adoptive cell transfer (ACT) is a potentially robust treatment option for patients with advanced melanoma that is resistant to immune checkpoint inhibitors. The addition of cyclin-dependent kinase 4/6 inhibitors to combination BRAF-MEK inhibitors can also greatly improve the duration of response against melanoma. The aim of our study was to investigate adoptive cell transfer with combination BRAF-MEK and CDK4/6 inhibitors. We show triplet targeted therapy is highly efficacious against BRAFV600 melanoma in YOVAL1.1 and the BRAFi resistant SM1WT1 model. Combination ACT with BRAF-MEK-CDK4/6i led to prolonged and deep anti-tumor responses in YOVAL1.1. This work provides additional evidence for BRAF-MEK-CDK4/6i in clinical trials and in combination with ACT. Abstract Despite the success of immune checkpoint inhibitors that target cytotoxic lymphocyte antigen-4 (CTLA-4) and programmed-cell-death-1 (PD-1) in the treatment of metastatic melanoma, there is still great need to develop robust options for patients who are refractory to first line immunotherapy. As such there has been a resurgence in interest of adoptive cell transfer (ACT) particularly derived from tumor infiltrating lymphocytes. Moreover, the addition of cyclin dependent kinase 4/6 inhibitors (CDK4/6i) have been shown to greatly extend duration of response in combination with BRAF-MEK inhibitors (BRAF-MEKi) in pre-clinical models of melanoma. We therefore investigated whether combinations of BRAF-MEK-CDK4/6i and ACT were efficacious in murine models of melanoma. Triplet targeted therapy of BRAF-MEK-CDK4/6i with OT-1 ACT led to sustained and robust anti-tumor responses in BRAFi sensitive YOVAL1.1. We also show that BRAF-MEKi but not CDK4/6i enhanced MHC Class I expression in melanoma cell lines in vitro. Paradoxically CDK4/6i in low concentrations of IFN-γ reduced expression of MHC Class I and PD-L1 in YOVAL1.1. Overall, this work provides additional pre-clinical evidence to pursue combination of BRAF-MEK-CDK4/6i and to combine this combination with ACT in the clinic.

9508 Background: Preliminary data from the ABC (76 pts) and CheckMate 204 (94 pts) trials showed that nivo and nivo+ipi have activity in active melanoma brain metastases, with durable responses in a subset of pts. Here, we report updated 5-yr data from all pts enrolled on the ABC trial (NCT02374242). Methods: This open-label ph2 trial enrolled 3 cohorts of pts with active melanoma brain mets naïve to anti-PD1/PDL1/PDL2/CTLA4 from Nov 2014-Apr 2017. Pts with asymptomatic brain mets with no prior local brain therapy were randomised to cohort A (nivo 1mg/kg + ipi 3mg/kg, Q3Wx4, then nivo 3mg/kg Q2W) or cohort B (nivo 3mg/kg Q2W). Cohort C (nivo 3mg/kg Q2W) had brain mets i) that failed local therapy, ii) with neuro symptoms and/or iii) with leptomeningeal disease. Prior BRAF inhibitor (BRAFi) was allowed. The primary endpoint was best intracranial response (ICR) ≥wk12. Key secondary endpoints were IC PFS, overall PFS, OS, & safety. Results: A total of 76 pts (med f/u 54 mo) were enrolled; median age 59y, 78% male. For cohorts A, B and C: elevated LDH 51%, 58% and 19%; V600BRAF 54%, 56% and 81%; prior BRAFi 23%, 24%, 75%. Efficacy and toxicity are shown in the table. There were no treatment-related deaths. 1/17 deaths in cohort A & 4/16 in cohort B were due to IC progression only. Conclusions: Nivo monotherapy and ipi+nivo are active in melanoma brain mets, with durable responses in the majority of patients who received ipi+nivo upfront. A study of upfront ipi+nivo+/-SRS is underway (NCT03340129). Clinical trial information: NCT02374242. [Table: see text]

Answer questions and earn CME/CNE To update the melanoma staging system of the American Joint Committee on Cancer (AJCC) a large database was assembled comprising >46,000 patients from 10 centers worldwide with stages I, II, and III melanoma diagnosed since 1998. Based on analyses of this new database, the existing seventh edition AJCC stage IV database, and contemporary clinical trial data, the AJCC Melanoma Expert Panel introduced several important changes to the Tumor, Nodes, Metastasis (TNM) classification and stage grouping criteria. Key changes in the eighth edition AJCC Cancer Staging Manual include: 1) tumor thickness measurements to be recorded to the nearest 0.1 mm, not 0.01 mm; 2) definitions of T1a and T1b are revised (T1a

Purpose: Encorafenib, a selective BRAF inhibitor (BRAFi), has a pharmacologic profile that is distinct from that of other clinically active BRAFis. We evaluated encorafenib in a phase I study in patients with BRAFi treatment-naïve and pretreated BRAF-mutant melanoma.Experimental Design: The pharmacologic activity of encorafenib was first characterized preclinically. Encorafenib monotherapy was then tested across a range of once-daily (50–700 mg) or twice-daily (75–150 mg) regimens in a phase I, open-label, dose-escalation and -expansion study in adult patients with histologically confirmed advanced/metastatic BRAF-mutant melanoma. Study objectives were to determine the maximum tolerated dose (MTD) and/or recommended phase II dose (RP2D), characterize the safety and tolerability and pharmacokinetic profile, and assess the preliminary antitumor activity of encorafenib.Results: Preclinical data demonstrated that encorafenib inhibited BRAF V600E kinase activity with a prolonged off-rate and suppressed proliferation and tumor growth of BRAF V600E–mutant melanoma models. In the dose-escalation phase, 54 patients (29 BRAFi-pretreated and 25 BRAFi-naïve) were enrolled. Seven patients in the dose-determining set experienced dose-limiting toxicities. Encorafenib at a dose of 300 mg once daily was declared the RP2D. In the expansion phase, the most common all-cause adverse events were nausea (66%), myalgia (63%), and palmar–plantar erythrodysesthesia (54%). In BRAFi-naïve patients, the overall response rate (ORR) and median progression-free survival (mPFS) were 60% and 12.4 months [95% confidence interval (CI), 7.4–not reached (NR)]. In BRAFi-pretreated patients, the ORR and mPFS were 22% and 1.9 months (95% CI, 0.9–3.7).Conclusions: Once-daily dosing of single-agent encorafenib had a distinct tolerability profile and showed varying antitumor activity across BRAFi-pretreated and BRAFi-naïve patients with advanced/metastatic melanoma. Clin Cancer Res; 23(18); 5339–48. ©2017 AACR.

The treatment of metastatic melanoma is still undergoing a process of major change. The two most important novel therapeutic strategies, selective kinase inhibitors and immune checkpoint blockers, both significantly prolong survival times of patients with advanced metastatic disease. Different agents, dose regimens and combinations have been tested against each other vigorously within these two groups. However, results from prospective head-to-head comparative studies of both strategies are still lacking. We performed an exploratory analysis of survival data from selected clinical trials representative for the new treatment strategies in advanced metastatic melanoma. Eighty-three Kaplan-Meier survival curves from 25 trials were digitised and grouped by therapeutic strategy and treatment line. For each of these groups, mean survival curves were generated for progression-free (PFS) and overall survival (OS) by weighted averaging. Survival curves grouped together by therapeutic strategy revealed a high concordance, particularly in the first-line setting. For kinase inhibitors, the most favourable PFS and OS in all therapy lines were observed for combined BRAF plus MEK inhibition. For immune checkpoint inhibitors, combined PD-1 plus CTLA-4 inhibition demonstrated the best survival outcome in all categories except for OS in first-line therapy. For the latter, combined PD-1 plus CTLA-4 inhibition showed similar outcomes as single-agent PD-1 inhibition. Comparison of kinase inhibitors and checkpoint blockers revealed a superiority of combined BRAF plus MEK inhibition within the first 6 months, later changing to a superiority of PD-1 blockers alone or in combination with CTLA-4 blockers. These results need confirmation by prospective clinical trials.

Purpose: The association of tumor gene expression profiles with progression-free survival (PFS) outcomes in patients with BRAFV600-mutated melanoma treated with vemurafenib or cobimetinib combined with vemurafenib was evaluated. Experimental Design: Gene expression of archival tumor samples from patients in four trials (BRIM-2, BRIM-3, BRIM-7, and coBRIM) was evaluated. Genes significantly associated with PFS (P < 0.05) were identified by univariate Cox proportional hazards modeling, then subjected to unsupervised hierarchical clustering, principal component analysis, and recursive partitioning to develop optimized gene signatures. Results: Forty-six genes were identified as significantly associated with PFS in both BRIM-2 (n = 63) and the vemurafenib arm of BRIM-3 (n = 160). Two distinct signatures were identified: cell cycle and immune. Among vemurafenib-treated patients, the cell-cycle signature was associated with shortened PFS compared with the immune signature in the BRIM-2/BRIM-3 training set [hazard ratio (HR) 1.8; 95% confidence interval (CI), 1.3–2.6, P = 0.0001] and in the coBRIM validation set (n = 101; HR, 1.6; 95% CI, 1.0–2.5; P = 0.08). The adverse impact of the cell-cycle signature on PFS was not observed in patients treated with cobimetinib combined with vemurafenib (n = 99; HR, 1.1; 95% CI, 0.7–1.8; P = 0.66). Conclusions: In vemurafenib-treated patients, the cell-cycle gene signature was associated with shorter PFS. However, in cobimetinib combined with vemurafenib-treated patients, both cell cycle and immune signature subgroups had comparable PFS. Cobimetinib combined with vemurafenib may abrogate the adverse impact of the cell-cycle signature. Clin Cancer Res; 23(17); 5238–45. ©2017 AACR.

Cutaneous melanoma can metastasise haematogenously and/or lymphogenously to form satellite/in-transit, lymph node or distant metastasis. This study aimed to determine if BRAF and NRAS mutant and wild-type tumours differ in their site of first tumour metastasis and anatomical metastatic pathway. Prospective cohort of patients with a histologically confirmed primary cutaneous melanoma at three tertiary referral centres in Melbourne, Australia from 2010 to 2015. Multinomial regression determined clinical, histological and mutational factors associated with the site of first metastasis and metastatic pathway. Of 1048 patients, 306 (29%) developed metastasis over a median 4.7 year follow-up period. 73 (24%), 192 (63%) and 41 (13%) developed distant, regional lymph node and satellite/in-transit metastasis as the first site of metastasis, respectively. BRAF mutation was associated with lymph node metastasis (adjusted RRR 2.46 95% CI 1.07–5.69, P=0.04) and sentinel lymph node positivity (adjusted odds ratio [aOR] OR 1.55, 95% CI 1.14–2.10, P=0.005). BRAF mutation and NRAS mutation were associated with increased odds of developing liver metastasis (aOR 3.09, 95% CI 1.49–6.42, P=0.003; aOR 3.17, 95% CI 1.32–7.58, P=0.01) and central nervous system (CNS) metastasis (aOR 4.65, 95% CI 2.23–9.69, P

Anti-programmed death (PD)-1 and PD-ligand (L)-1 checkpoint inhibitors have revolutionized the therapy of several cancers. Immunotherapy of cancer can offer long-term durable benefit to patients, is active regardless of tumour histology, has a unique immune-related safety profile, and can be used in combination with other cancer treatments. In addition, recent research has shown that immune-based therapy can be used as adjuvant therapy, that outcomes may be influenced by dose, and that clinical activity is observed in patients with brain metastases. Despite our increased understanding of these agents, there are still several important questions that need to be answered. These include strategies to overcome primary and acquired resistance, the influence of mutational status on treatment outcomes, the optimal duration of treatment, and the need to identify novel combination regimens that offer increased anti-tumour potency and/or reduced toxicity. Here we review recent developments in these areas, with particular focus on new data reported at the 2017 ASCO Annual Meeting.

Background Serous chorioretinopathy has been associated with MEK inhibitors, including cobimetinib. We describe the clinical features of serous retinopathy observed with cobimetinib in patients with BRAFV600-mutated melanoma treated in the Phase III coBRIM study. Methods In the coBRIM study, 493 patients were treated in two randomly assigned treatment groups: cobimetinib and vemurafenib (n = 247) or vemurafenib (n = 246). All patients underwent prospective ophthalmic examinations at screening, at regular intervals during the study, and whenever ocular symptoms developed. Patients with serous retinopathy were identified in the study database using a group of relevant and synonymous adverse event terms. Results Eighty-six serous retinopathy events were reported in 70 patients (79 events in 63 cobimetinib and vemurafenib-treated patients vs seven events in seven vemurafenib-treated patients). Most patients with serous retinopathy identified by ophthalmic examination had no symptoms or had mild symptoms, among them reduced visual acuity, blurred vision, dyschromatopsia, and photophobia. Serous retinopathy usually occurred early during cobimetinib and vemurafenib treatment; median time to onset was 1.0 month. Most events were managed by observation and continuation of cobimetinib without dose modification and resolved or were resolving by the data cutoff date (19 Sept 2014). Conclusions Cobimetinib treatment was associated with serous retinopathy in patients with BRAFV600-mutated melanoma. Retinopathy was generally asymptomatic or mild. Periodic ophthalmologic evaluations at regular intervals and at the manifestation of any visual disturbance are recommended to facilitate early detection and resolution of serous retinopathy while patients are taking cobimetinib. Trial Registration Clinicaltrials.gov (NCT01689519). First received: September 18, 2012

The objective of this study was to describe health care resource utilization and health care costs associated with the management of advanced melanoma patients receiving ipilimumab., Background. There are limited real‐world data on health care resource utilization (HCRU) among advanced melanoma patients. The objective of this study was to describe HCRU and health care costs associated with the management of advanced melanoma patients receiving ipilimumab. Methods. This retrospective multinational, observational study included advanced melanoma patients from Australia, Germany, Italy, and Spain who had received at least 1 dose of ipilimumab. Data extracted from medical charts included inpatient admissions, outpatient visits, surgical procedures, laboratory investigations, radiation therapy, imaging studies, and concomitant medications. Cost estimates were based on unit costs from country‐specific standard reimbursement sources. Subgroup analyses were performed for BRAF mutation status and ipilimumab refractory patients, who had disease progression within 24 weeks of their last dose of ipilimumab. Results. Mean age of 362 enrolled patients was 60.6 years (standard deviation [SD] 14.4). During a median follow‐up period of 30.2 weeks, 57% of patients were admitted to hospital and 16% underwent surgery. Health care resource utilization rates varied substantially across countries and were highest in Germany. Concomitant medications to treat adverse events were commonly used. Subgroup analyses showed higher utilization rates among ipilimumab refractory and BRAF mutant patients. Mean weekly total costs associated with HCRU were lower in the pre‐progression period (€107; 95% confidence interval (CI): 79–145) than in the post‐progression period (€216; 95% CI: 180–259). Conclusion. Health care resource utilization pattern and associated costs among patients treated with ipilimumab varied greatly among countries and between pre‐ and post‐progression periods. There is a high economic burden associated with ipilimumab refractory melanoma. Implications for Practice. Metastatic melanoma patients treated with the anti‐CTLA‐4 inhibitor ipilimumab have a high utilization of various types of health care services, such as inpatient hospital stays or doctor visits. There are differences across countries regarding patterns of health care utilization and economic burden of the disease. Health care services are used more frequently after patients experience progression of their disease. The study highlights that better therapies leading to durable response in patients with metastatic melanoma have the potential to decrease health care costs and patient burden in terms of hospitalizations and other health care services.