Search

Immune checkpoint inhibitors (ICIs) have transformed cancer treatment, providing significant benefit to patients across various tumour types, including melanoma. However, around 40% of melanoma patients do not benefit from ICI treatment, and accurately predicting ICI response remains challenging. We now describe a novel and simple approach that integrates immune-associated transcriptome signatures and tumour volume burden to better predict ICI response in melanoma patients. RNA sequencing was performed on pre-treatment (PRE) tumour specimens derived from 32 patients with advanced melanoma treated with combination PD1 and CTLA4 inhibitors. Of these 32 patients, 11 also had early during treatment (EDT, 5-15 days after treatment start) tumour samples. Tumour volume was assessed at PRE for all 32 patients, and at first computed tomography (CT) imaging for the 11 patients with EDT samples. Analysis of the Hallmark IFNγ gene set revealed no association with ICI response at PRE (AUC ROC curve = 0.6404, p = 0.24, 63% sensitivity, 71% specificity). When IFNg activity was evaluated with tumour volume (ratio of gene set expression to tumour volume) using logistic regression to predict ICI response, we observed high discriminative power in separating ICI responders from non-responders (AUC = 0.7760, p = 0.02, 88% sensitivity, 67% specificity); this approach was reproduced with other immune-associated transcriptomic gene sets. These findings were further replicated in an independent cohort of 23 melanoma patients treated with PD1 inhibitor. Hence, integrating tumour volume with immune-associated transcriptomic signatures improves the prediction of ICI response, and suggest that higher levels of immune activation relative to tumour burden are required for durable ICI response., (© 2024. The Author(s).)

Background: Melanoma is a heterogeneous cancer influenced by the plasticity of melanoma cells and their dynamic adaptations to microenvironmental cues. Melanoma cells transition between well-defined transcriptional cell states that impact treatment response and resistance., Methods: In this study, we applied single-cell RNA sequencing to interrogate the molecular features of immunotherapy-naive and immunotherapy-resistant melanoma tumours in response to ex vivo BRAF/MEK inhibitor treatment., Findings: We confirm the presence of four distinct melanoma cell states - melanocytic, transitory, neural-crest like and undifferentiated, and identify enrichment of neural crest-like and undifferentiated melanoma cells in immunotherapy-resistant tumours. Furthermore, we introduce an integrated computational approach to identify subsets of responding and nonresponding melanoma cells within the transcriptional cell states., Interpretation: Nonresponding melanoma cells are identified in all transcriptional cell states and are predisposed to BRAF/MEK inhibitor resistance due to pro-inflammatory IL6 and TNFɑ signalling. Our study provides a framework to study treatment response within distinct melanoma cell states and indicate that tumour-intrinsic pro-inflammatory signalling contributes to BRAF/MEK inhibitor resistance., Funding: This work was supported by Macquarie University, Melanoma Institute Australia, and the National Health and Medical Research Council of Australia (NHMRC; grant 2012860, 2028055)., Competing Interests: Declaration of interests JHL has received honorarium from Merck, Sharp & Dohme and AstraZeneca, received conference support from Pfizer and Merck, Sharp & Dohme, and consulting fees from Boxer Capitol. JHL is a consultant for Merck, Sharp & Dohme, Sanofi, Cancer Council and EviO. RAS has received fees for professional services from MetaOptima Technology Inc., F. Hoffmann-La Roche Ltd, Evaxion, Provectus Biopharmaceuticals Australia, Qbiotics, Novartis, Merck Sharp & Dohme, NeraCare, AMGEN Inc., Bristol-Myers Squibb, Myriad Genetics, GlaxoSmithKline, IO Biotech ApS and SkylineDX B.V. GVL is consultant advisor for Agenus, Amgen, Array Biopharma, AstraZeneca, Bayer Health Care, BioNTech, Boehringer Ingelheim, Bristol Myers Squibb, Evaxion, Hexal AG (Sandoz Company), Highlight Therapeutics S.L., IO Biotech, Immunocore, Innovent Biologics USA, Merck Sharpe & Dohme, Novartis, PHMR Ltd, Pierre Fabre, Qbiotics, Regeneron, Scancell Limited and SkylineDX B.V. GVL has received conference support from Bristol Myers Squibb and Pierre Fabre. All other authors declare no conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Neoadjuvant therapy improves recurrence-free survival (RFS) in resectable stage III cutaneous melanoma. However, accurately predicting individual recurrence risk remains a significant challenge. We investigated circulating tumour DNA (ctDNA) as a biomarker for recurrence in measurable stage IIIB/C melanoma patients undergoing neoadjuvant immunotherapy., Methods: Plasma samples were collected pre-neoadjuvant treatment, pre-surgery and/or six weeks post-surgery from 40 patients enrolled in the OpACIN-neo and PRADO clinical trials. Patients received two cycles of ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) before surgery. Cell free DNA (cfDNA) underwent unbiased pre-amplification followed by tumour-informed mutation detection using droplet digital polymerase chain reaction (ddPCR) with the Bio-Rad QX600 PCR system., Results: Pre-treatment ctDNA was detectable in 19/40 (48%) patients. Among these, 17/19 (89%) zero-converted within six weeks of surgery and none recurred. Positive ctDNA post-surgery (N = 4), irrespective of pre-treatment ctDNA status, was 100% predictive of recurrence (sensitivity 44%, specificity 100%). Furthermore, ctDNA cleared prior to surgery in 7/9 (78%) patients who did not recur, warranting further investigation into ctDNA-guided surgical management., Conclusion: Post-surgery ctDNA positivity and zero-conversion are highly predictive of recurrence, offering a window for personalised modification of adjuvant therapy., (© 2024. The Author(s).)

The identification and therapeutic targeting of actionable gene mutations across many cancer types has resulted in improved response rates in a minority of patients. The identification of actionable mutations is usually not sufficient to ensure complete nor durable responses, and in rare cancers, where no therapeutic standard of care exists, precision medicine indications are often based on pan-cancer data. The inclusion of functional data, however, can provide evidence of oncogene dependence and guide treatment selection based on tumour genetic data. We applied an ex vivo cancer explant modelling approach, that can be embedded in routine clinical care and allows for pathological review within 10 days of tissue collection. We now report that ex vivo tissue modelling provided accurate longitudinal response data in a patient with BRAF V600E -mutant papillary thyroid tumour with squamous differentiation. The ex vivo model guided treatment selection for this patient and confirmed treatment resistance when the patient's disease progressed after 8 months of treatment., (© 2023 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Protein kinase C (PKC) is activated downstream of gain-of-function GNAQ or GNA11 (GNAQ/GNA11) mutations in over 90% of uveal melanoma (UM). Phase I clinical trials of PKC inhibitors have shown modest response rates with no survival benefit in metastatic UM. Although PKC inhibitors actively suppress mitogen-activated protein kinase (MAPK) signalling in UM, the effect on other UM signalling cascades is not well understood. We examined the transcriptome of UM biopsies collected pre- and post-PKC inhibitor therapy and confirmed that MAPK, but not PI3K/AKT signalling, was inhibited early during treatment with the second-generation PKC inhibitor IDE196. Similarly, in GNAQ/GNA11-mutant UM cell models, PKC inhibitor monotherapy effectively suppressed MAPK activity, but PI3K/AKT signalling remained active, and thus, concurrent inhibition of PKC and PI3K/AKT signalling was required to synergistically induce cell death in a panel of GNAQ/GNA11-mutant UM cell lines. We also show that re-activation of MAPK signalling has a dominant role in regulating PKC inhibitor responses in UM and that PI3K/AKT signalling diminishes UM cell sensitivity to PKC inhibitor monotherapy. Thus, combination therapies targeting PKC and PKC-independent signalling nodes, including PI3K/AKT activity, are required to improve responses in patients with metastatic UM., (© 2024. The Author(s).)

Over the past decade, there have been remarkable improvements in the treatment and survival rates of melanoma patients. Treatment resistance remains a persistent challenge, however, and is partly attributable to intratumoural heterogeneity. Melanoma cells can transition through a series of phenotypic and transcriptional cell states that vary in invasiveness and treatment responsiveness. The diverse stromal and immune contexture of the tumour microenvironment also contributes to intratumoural heterogeneity and disparities in treatment response in melanoma patients. Recent advances in single-cell sequencing technologies have enabled a more detailed understanding of melanoma heterogeneity and the underlying transcriptional programs that regulate melanoma cell diversity and behaviour. In this review, we examine the concept of intratumoural heterogeneity and the challenges it poses to achieving long-lasting treatment responses. We focus on the significance of next generation single-cell sequencing in advancing our understanding of melanoma diversity and the unique insights gained from single-cell studies., Competing Interests: Declaration of interests All authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Treatment of melanomas with targeted and immunotherapies has proven effective, but resistance to both treatments is a common outcome leaving a high proportion of patients without effective alternative treatment options. Replication stress is a common feature of melanomas, and this is effectively targeted using a combination of checkpoint kinase 1 (CHK1) inhibitor and low-dose hydroxyurea (LDHU). This combination also promotes inflammatory and anti-tumour immune responses in vivo. Melanoma cell lines resistant to BRAF inhibitor (BRAFi) or immune checkpoint inhibitors (ICI) retain their sensitivity to CHK1i + LDHU, with sensitivity similar to that of parental tumours. In vivo, BRAFi-resistant and BRAFi-sensitive parental tumours produce an identical immune response with treatment., (© 2023 The Authors. Pigment Cell & Melanoma Research published by John Wiley & Sons Ltd.)

Purpose: This study characterizes intratumoral macrophage populations within baseline melanoma biopsies from patients with advanced melanoma who received either anti-PD-1 monotherapy or a combination with anti-CTLA-4. Particularly, FcγRIIIa (CD16)-expressing macrophage densities were investigated for associations with response and progression-free survival., Experimental Design: Patients with advanced melanoma who received either anti-PD-1 monotherapy or combination anti-PD-1 and anti-CTLA-4 were retrospectively identified. Macrophage populations were analyzed within baseline melanoma biopsies via multiplex IHC in relation to treatment outcomes., Results: Patients who responded to combination immune checkpoint inhibitor contained higher CD16+ macrophage densities than those who did not respond (196 vs. 7 cells/mm2; P = 0.0041). There was no diffidence in CD16+ macrophage densities in the PD-1 monotherapy-treated patients based on response (118 vs. 89 cells/mm2; P = 0.29). A significantly longer 3-year progression-free survival was observed in combination-treated patients with high intratumoral densities of CD16+ macrophages compared with those with low densities (87% vs. 42%, P = 0.0056, n = 40). No association was observed in anti-PD-1 monotherapy-treated patients (50% vs. 47%, P = 0.4636, n = 50). Melanoma biopsies with high densities of CD16+ macrophages contained upregulated gene expression of critical T-cell recruiting chemokines (CXCL9, CXCL10, and CXCL11)., Conclusions: Our data demonstrate that tumor microenvironments enriched with CD16+ macrophages are favorable for response to combination anti-PD-1 and anti-CTLA-4 therapy but not anti-PD-1 monotherapy. These data provides a potential biomarker of response for combination immunotherapies in patients with metastatic melanoma. See related commentary by Smithy and Luke, p. 2345., (©2023 American Association for Cancer Research.)

Immunotherapy targeting PD-1 and/or CTLA4 leads to durable responses in a proportion of patients with melanoma. However, many patients will not respond to these immune checkpoint inhibitors, and up to 60% of responding patients will develop treatment resistance. We describe a vulnerability in melanoma driven by immune cell activity that provides a pathway towards additional treatment options. This study evaluated short-term melanoma cell lines (referred to as PD1 PROG cells) derived from melanoma metastases that progressed on PD-1 inhibitor-based therapy. We show that the cytokine IFN-γ primes melanoma cells for apoptosis by promoting changes in the accumulation and interactions of apoptotic regulators MCL-1, NOXA, and BAK. The addition of pro-apoptotic BH3 mimetic drugs sensitized PD1 PROG melanoma cells to apoptosis in response to IFN-γ or autologous immune cell activation. These findings provide translatable strategies for combination therapies in melanoma., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Resistance to immune checkpoint inhibitor therapies in melanoma is common and remains an intractable clinical challenge. In this study, we comprehensively profile immune checkpoint inhibitor resistance mechanisms in short-term tumor cell lines and matched tumor samples from melanoma patients progressing on immune checkpoint inhibitors. Combining genome, transcriptome, and high dimensional flow cytometric profiling with functional analysis, we identify three distinct programs of immunotherapy resistance. Here we show that resistance programs include (1) the loss of wild-type antigen expression, resulting from tumor-intrinsic IFNγ signaling and melanoma de-differentiation, (2) the disruption of antigen presentation via multiple independent mechanisms affecting MHC expression, and (3) immune cell exclusion associated with PTEN loss. The dominant role of compromised antigen production and presentation in melanoma resistance to immune checkpoint inhibition highlights the importance of treatment salvage strategies aimed at the restoration of MHC expression, stimulation of innate immunity, and re-expression of wild-type differentiation antigens., (© 2023. The Author(s).)

Immunotherapy has transformed the management of patients with advanced melanoma, with five-year overall survival rates reaching 52% for combination immunotherapies blocking the cytotoxic T-lymphocyte-associated antigen-4 (CTLA4) and programmed cell death-1 (PD1) immune axes. Yet, our understanding of local and systemic determinants of immunotherapy response and resistance is restrained by the paucity of preclinical models, particularly those for anti-PD1 monotherapy. We have therefore generated a novel murine model of melanoma by integrating key immunotherapy response biomarkers into the model development workflow. The resulting YUMM3.3 UVR c34 ( BrafV600E; Cdkn2a -/-) model demonstrated high mutation burden and response to interferon (IFN)γ, including induced expression of antigen-presenting molecule MHC-I and the principal PD1 ligand PD-L1, consistent with phenotypes of human melanoma biopsies from patients subsequently responding to anti-PD1 monotherapy. Syngeneic immunosufficient mice bearing YUMM3.3 UVR c34 tumors demonstrated durable responses to anti-PD1, anti-CTLA4, or combined treatment. Immunotherapy responses were associated with early on-treatment changes in the tumor microenvironment and circulating T-cell subsets, and systemic immunological memory underlying protection from tumor recurrence. Local and systemic immunological landscapes associated with immunotherapy response in the YUMM3.3 UVR c34 melanoma model recapitulate immunotherapy responses observed in melanoma patients and identify discrete immunological mechanisms underlying the durability of responses to anti-PD1 and anti-CTLA4 treatments.

Uveal melanoma (UM) is a rare cancer arising from melanocytes in the uveal tract of the eye. Despite effective primary treatment, there is no approved therapy for metastatic UM and prognosis and survival remain poor. Over 90% of UM are driven by mutations affecting the Gα subunits encoded by the GNAQ and GNA11 genes. These mutations activate downstream and targetable signaling pathways, including the protein kinase C (PKC) cascade. PKC inhibitors have been used in clinical trials for metastatic UM but have shown limited efficacy. In this study, we examined the signaling and functional effects of two PKC inhibitors (AEB071 and IDE196) in a panel of UM cell models. In response to PKC inhibition, all UM cell lines showed potent suppression of PKC activity, but this was not sufficient to predict PKC inhibitor sensitivity and only two UM cell lines showed substantial PKC inhibitor-induced cell death. The differences in UM cell responses to PKC inhibition were not attributable to the degree or timing of PKC suppression or inhibition of the downstream mitogen-activated protein kinase (MAPK) or phosphatidylinositol-3-kinase (PI3K) pathways. Instead, UM cell show complex, PKC-independent signaling pathways that contribute to their survival and resistance to targeted therapies., (© 2022. The Author(s).)

Blue nevi are benign, melanocytic neoplasms that show a range of clinical and morphologic patterns and include common/dendritic, cellular, and atypical cellular subtypes. Like other nevi, they most commonly occur in skin but can occasionally involve lymph nodes where they may be misinterpreted as representing metastatic melanoma. Moreover, whether benign blue nevi can metastasize to lymph nodes and their natural history and prognostic significance has been the subject of great controversy. To date, few cases of nodal blue nevi have been reported in the literature, and those reports have had limited clinical follow-up and supporting molecular data. This study sought to determine the clinical, pathologic, and molecular features of blue nevi involving lymph nodes, clarify their clinical significance, provide evidence for understanding their pathogenesis, and highlight potential pitfalls in the interpretation of lymph nodes with an ultimate aim of improving patient care. Thirteen cases of blue nevi involving lymph nodes were identified in the archives of Royal Prince Alfred Hospital, Sydney, Australia (1984-2018). A detailed assessment of the clinical and pathologic features of each case was performed, including an evaluation of all available immunohistochemical stains. Extended clinical follow-up was available for 9 patients. Droplet digital polymerase chain reaction for GNAQ Q209L, Q209P and GNA11 Q209L mutations was performed on 7 cases of blue nevi within lymph nodes together with matching cutaneous (presumed primary) blue nevi in 2 cases. All cases showed typical histologic features of blue nevi. BAP1 was retained in all cases (n=7). There were no recurrence or metastasis of blue nevus in any case on long-term clinical follow-up (n=9, median follow-up, 12 y). The majority of cases (n=5 of 7 evaluated) had GNAQ and GNA11 driver mutations. The 2 patients with a matched primary cutaneous blue nevus and regionally associated nodal blue nevus had the same GNAQ Q209L mutation in both sites in each patient. We conclude that blue nevi can involve lymph nodes and are associated with benign clinical behavior, and probably represent so-called "benign" metastasis. Awareness of these lesions is important when evaluating lymph nodes to avoid misdiagnosis as metastatic melanoma., Competing Interests: Conflicts of Interest and Source of Funding: A.J.C. was supported as a Jani Haenke Melanoma Pathology fellow through the BB & A Miller Foundation. R.A.S., J.F.T., and G.V.L. are recipients of a National Health and Medical Research Council of Australia (NHMRC) Program Grant (APP1093017) G.V.L. and R.A.S. are supported by NHMRC Practitioner Fellowships. J.S.W. is supported by a NHMRC Investigator grant. G.V.L. is also supported by the University of Sydney Medical Foundation. R.A.S. has received fees for professional services from F. Hoffmann-La Roche Ltd, Evaxion, Provectus Biopharmaceuticals Australia, Qbiotics, Novartis, MSD Sharp & Dohme, NeraCare, AMGEN Inc., Bristol-Myers Squibb, Myriad Genetics, GlaxoSmithKline. G.V.L. is consultant advisor for Aduro Biotech Inc., Amgen Inc., Array Biopharma Inc., Boehringer Ingelheim International GmbH, Bristol-Myers Squibb, Highlight Therapeutics S.L., Merck Sharpe & Dohme, Novartis Pharma AG, QBiotics Group Limited, Regeneron Pharmaceuticals Inc., SkylineDX B.V. J.F.T. has received honoraria for advisory board participation from BMS Australia, MSD Australia, GSK, and Provectus Inc.; and travel and conference support from GSK, Provectus Inc., and Novartis. For the remaining authors none were declared., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Colorectal cancer (CRC) is the third most diagnosed and the second lethal cancer worldwide. Approximately 30-50% of CRC are driven by mutations in the KRAS oncogene, which is a strong negative predictor for response to anti-epidermal growth factor receptor (anti-EGFR) therapy. Examining the phenotype of KRAS mutant and wild-type (WT) CRC cells in response to anti-EGFR treatment may provide significant insights into drug response and resistance. Herein, surface-enhanced Raman spectroscopy (SERS) assay was applied to phenotype four cell surface proteins (EpCAM, EGFR, HER2, HER3) in KRAS mutant (SW480) and WT (SW48) cells over a 24-day time course of anti-EGFR treatment with cetuximab. Cell phenotypes were obtained using Raman reporter-coated and antibody-conjugated gold nanoparticles (SERS nanotags), where a characteristic Raman spectrum was generated upon single laser excitation, reflecting the presence of the targeted surface marker proteins. Compared to the KRAS mutant cells, KRAS WT cells were more sensitive to anti-EGFR treatment and displayed a significant decrease in HER2 and HER3 expression. The SERS results were validated with flow cytometry, confirming the SERS assay is promising as an alternative method for multiplexed characterization of cell surface biomarkers using a single laser excitation system., Competing Interests: There is no conflict of interest to declare., (© 2022 The Authors. Exploration published by Henan University and John Wiley & Sons Australia, Ltd.)

The development of resistance to treatments of melanoma is commonly associated with an upregulation of the MAPK pathway and the development of an undifferentiated state. Previous studies have suggested that melanoma with these resistance characteristics may be susceptible to innate death mechanisms such as pyroptosis triggered by the activation of inflammasomes. In this study, we have taken cell lines from patients before and after the development of resistance to BRAF V600 inhibitors and exposed the resistant melanoma to temozolomide (a commonly used chemotherapy) with and without chloroquine to inhibit autophagy. It was found that melanoma with an inflammatory undifferentiated state appeared susceptible to this combination when tested in vitro and in vivo against xenografts in nonobese diabetic scid gamma mice. Translation of the latter results into patients would promise durable responses in patients treated by the combination. The inflammasome and death mechanism involved appeared to vary between melanoma and involved either AIM2 or NLRP3 inflammasomes and gasdermin D or E. These preliminary studies have raised questions as to the selectivity for different inflammasomes in different melanoma and their selective targeting by chemotherapy. They also question whether the inflammatory state of melanoma may be used as biomarkers to select patients for inflammasome-targeted therapy., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Detection of melanoma mutations using circulating tumor DNA (ctDNA) is a potential alternative to using genomic DNA from invasive tissue biopsies. To date, mutations in the GC-rich TERT promoter region, which is commonly mutated in melanoma, have been technically difficult to detect in ctDNA using next-generation sequencing (NGS) panels. In this study, we developed a custom melanoma NGS panel for detection of ctDNA, which encompasses the top 15 gene mutations in melanoma including the TERT promoter. We analyzed 21 stage III and IV melanoma patient samples who were treatment-naïve or on therapy. The overall detection rate of the custom panel, based on BRAF / NRAS / TERT promoter mutations, was 14/21 (67%) patient samples which included a TERT C250T mutation in one BRAF and NRAS mutation negative sample. A BRAF or NRAS mutation was detected in the ctDNA of 13/21 (62%) patients while TERT promoter mutations were detected in 10/21 (48%) patients. Co-occurrence of TERT promoter mutations with BRAF or NRAS mutations was found in 9/10 (90%) patients. The custom ctDNA panel showed a concordance of 16/21 (76%) with tissue based-detection and included 12 BRAF / NRAS mutation positive and 4 BRAF/NRAS mutation negative patients. The ctDNA mutation detection rate for stage IV was 12/16 (75%) and for stage III was 1/5 (20%). Based on BRAF , NRAS and TERT promoter mutations, the custom melanoma panel displayed a limit of detection of ~0.2% mutant allele frequency and showed significant correlation with droplet digital PCR. For one patient, a novel MAP2K1 H119Y mutation was detected in an NRAS/BRAF/TERT promoter mutation negative background. To increase the detection rate to >90% for stage IV melanoma patients, we plan to expand our custom panel to 50 genes. This study represents one of the first to successfully detect TERT promoter mutations in ctDNA from cutaneous melanoma patients using a targeted NGS panel., Competing Interests: GL receives consultant service fees from Amgen, BMS, Array, Pierre-Fabre, Novartis, Merck Sharp & Dohme (MSD), Qbiotics, Roche and Sandoz. AM is an advisory board member for BMS, MSD, Novartis, Roche, Pierre Fabre and Qbiotics. MC is an advisory board member for MSD, BMS, Novartis, Pierre-Fabre, Roche and Amgen. RAS has received fees for professional services from Evaxion, Provectus Biopharmaceuticals Australia, Qbiotics Group Limited, Novartis Pharma AG, MSD Sharp & Dohme (Australia), NeraCare, AMGEN Inc., Bristol-Myers Squibb, Novartis Pharmaceuticals Australia Pty Limited, Myriad Genetics GmbH, GlaxoSmithKline Australia. RPMS has received honoraria for advisory board participation from MSD, Novartis and Qbiotics and speaking honoraria from BMS. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Diefenbach, Lee, Stewart, Menzies, Carlino, Saw, Stretch, Long, Scolyer and Rizos.)

We concurrently examine the whole genome, transcriptome, methylome, and immune cell infiltrates in baseline tumors from 77 patients with advanced cutaneous melanoma treated with anti-PD-1 with or without anti-CTLA-4. We show that high tumor mutation burden (TMB), neoantigen load, expression of IFNγ-related genes, programmed death ligand expression, low PSMB8 methylation (therefore high expression), and T cells in the tumor microenvironment are associated with response to immunotherapy. No specific mutation correlates with therapy response. A multivariable model combining the TMB and IFNγ-related gene expression robustly predicts response (89% sensitivity, 53% specificity, area under the curve [AUC], 0.84); tumors with high TMB and a high IFNγ signature show the best response to immunotherapy. This model validates in an independent cohort (80% sensitivity, 59% specificity, AUC, 0.79). Except for a JAK3 loss-of-function mutation, for patients who did not respond as predicted there is no obvious biological mechanism that clearly explained their outlier status, consistent with intratumor and intertumor heterogeneity in response to immunotherapy., Competing Interests: Declaration of interests I.P.S. has received travel support by BMS and MSD, and speaker fee by Roche, BMS, MSD, and Novartis. J.F.T. has received honoraria for advisory board participation from Merck Sharpe Dohme Australia and Bristol Myers Squibb Australia and received honoraria and travel expenses from GSK and Provectus Inc. R.A.S. has received fees for professional services from Evaxion, Provectus Biopharmaceuticals Australia, Qbiotics Group Limited, Novartis Pharma AG, MSD Sharp & Dohme (Australia), NeraCare, AMGEN Inc., Bristol-Myers Squibb, Novartis Pharmaceuticals Australia Pty Limited, Myriad Genetics GmbH, and GlaxoSmithKline Australia. G.V.L. is consultant advisor for Aduro Biotech Inc, Agenus Inc, Amgen Inc, Array Biopharma inc, Boehringer Ingelheim International GmbH, Bristol-Myers Squibb, Evaxion Biotech A/S, Hexel AG, Highlight Therapeutics S.L., Merck Sharpe & Dohme, Novartis Pharma AG, OncoSec, Pierre Fabre, QBiotics Group Limited, Regeneron Pharmaceuticals Inc, SkylineDX B.V., Specialised Therapeutics Australia Pty Ltd. R.P.M.S has participated in advisory boards for MSD, Novartis, and Qbiotics and received speaking honoraria from BMS. J.V.P. and N.W. are founders and shareholders of genomiQa Pty Ltd, and members of its Board. M.S.C. is a consultant advisor to MSD, BMS, Novartis, Roche, Pierre Fabre, Sanofi, Merck Serono, Nektar, Eisia, and Ideaya and received honoraria from MSD, BMS, and Novartis. A.M.M. has participated in advisory boards for BMS, MSD, Novartis, Roche, and Pierre-Fabre. C.U.B. has served on advisory boards for BMS, MSD, Roche, Novartis, GSK, AZ, Pfizer, Lilly, GenMab, Pierre Fabre, and Third Rock Ventures; received research funding from BMS, MSD, 4SC, Novartis, and NanoString; has stock ownership in Uniti Cars; and is a cofounder of Immagene BV. The remaining authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Therapy resistance arises from heterogeneous drug-tolerant persister cells or minimal residual disease (MRD) through genetic and nongenetic mechanisms. A key question is whether specific molecular features of the MRD ecosystem determine which of these two distinct trajectories will eventually prevail. We show that, in melanoma exposed to mitogen-activated protein kinase therapeutics, emergence of a transient neural crest stem cell (NCSC) population in MRD concurs with the development of nongenetic resistance. This increase relies on a glial cell line-derived neurotrophic factor-dependent signaling cascade, which activates the AKT survival pathway in a focal adhesion kinase (FAK)-dependent manner. Ablation of the NCSC population through FAK inhibition delays relapse in patient-derived tumor xenografts. Strikingly, all tumors that ultimately escape this treatment exhibit resistance-conferring genetic alterations and increased sensitivity to extracellular signal-regulated kinase inhibition. These findings identify an approach that abrogates the nongenetic resistance trajectory in melanoma and demonstrate that the cellular composition of MRD deterministically imposes distinct drug resistance evolutionary paths., Competing Interests: Declaration of interest The authors declare no competing interests. J-C.M. and F.R. are authors on a patent application related to this work., (Copyright © 2021 Elsevier Inc. All rights reserved.)

It is now well accepted that the immune system can control cancer growth. However, tumors escape immune-mediated control through multiple mechanisms and the downregulation or loss of major histocompatibility class (MHC)-I molecules is a common immune escape mechanism in many cancers. MHC-I molecules present antigenic peptides to cytotoxic T cells, and MHC-I loss can render tumor cells invisible to the immune system. In this review, we examine the dysregulation of MHC-I expression in cancer, explore the nature of MHC-I-bound antigenic peptides recognized by immune cells, and discuss therapeutic strategies that can be used to overcome MHC-I deficiency in solid tumors, with a focus on the role of natural killer (NK) cells and CD4 T cells.

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

The prognosis for patients with UM is poor, and recent clinical trials have failed to prolong overall survival (OS) of these patients. Over 95% of UM harbor activating driver mutations, and this allows for the investigation of ctDNA. In this study, we investigated the value of ctDNA for adaptive clinical trial design in metastatic UM. Longitudinal plasma samples were analyzed for ctDNA in 17 metastatic UM patients treated with PKCi-based therapy in a phase 1 clinical trial setting. Plasma ctDNA was assessed using digital droplet PCR (ddPCR) and a custom melanoma gene panel for targeted next generation sequencing (NGS). Baseline ctDNA strongly correlated with baseline lactate dehydrogenase (LDH) ( p < 0.001) and baseline disease burden ( p = 0.002). Early during treatment (EDT) ctDNA accurately predicted patients with clinical benefit to PKCi using receiver operator characteristic (ROC) curves (AUC 0.84, [95% confidence interval 0.65-1.0, p = 0.026]). Longitudinal ctDNA assessment was informative for establishing clinical benefit and detecting disease progression with 7/8 (88%) of patients showing a rise in ctDNA and targeted NGS of ctDNA revealed putative resistance mechanisms prior to radiological progression. The inclusion of longitudinal ctDNA monitoring in metastatic UM can advance adaptive clinical trial design.

Resistance to BRAF/MEK inhibitor treatment occurs in most patients with advanced BRAF V600 -mutant melanoma. Intermittent drug dosing had been proposed as a strategy to circumvent resistance. In a clinical trial published in Nature Medicine, continuous dosing of BRAF/MEK inhibitors produced superior progression-free survival compared to intermittent dosing in BRAF V600 -mutant melanoma patients., Competing Interests: Declaration of Interests J.H.L. has received honoraria from AstraZeneca and BMS and travel support from BioRad, Novartis, and BMS. M.S.C. has served on advisory boards for Bristol-Myers Squibb, MSD, Amgen, Novartis, Pierre Fabre, Roche, Sanofi, Merck, Ideaya, Regeneron, Nektar, Eisai, Q biotics, and Oncosec and honoraria from Bristol-Myers Squibb, MSD, and Novartis., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

Purpose: We evaluated the predictive value of pretreatment ctDNA to inform therapeutic outcomes in patients with metastatic melanoma relative to type and line of treatment., Experimental Design: Plasma circulating tumor DNA (ctDNA) was quantified in 125 samples collected from 110 patients prior to commencing treatment with immune checkpoint inhibitors (ICIs), as first- ( n = 32) or second-line ( n = 27) regimens, or prior to commencing first-line BRAF/MEK inhibitor therapy ( n = 66). An external validation cohort included 128 patients commencing ICI therapies in the first- ( N = 77) or second-line ( N = 51) settings., Results: In the discovery cohort, low ctDNA (≤20 copies/mL) prior to commencing therapy predicted longer progression-free survival (PFS) in patients treated with first-line ICIs [HR, 0.20; 95% confidence interval (CI) 0.07-0.53; P < 0.0001], but not in the second-line setting. An independent cohort validated that ctDNA is predictive of PFS in the first-line setting (HR, 0.42; 95% CI, 0.22-0.83; P = 0.006), but not in the second-line ICI setting. Moreover, ctDNA prior to commencing ICI treatment was not predictive of PFS for patients pretreated with BRAF/MEK inhibitors in either the discovery or validation cohorts. Reduced PFS and overall survival were observed in patients with high ctDNA receiving anti-PD-1 monotherapy, relative to those treated with combination anti-CTLA-4/anti-PD-1 inhibitors., Conclusions: Pretreatment ctDNA is a reliable indicator of patient outcome in the first-line ICI treatment setting, but not in the second-line ICI setting, especially in patients pretreated with BRAF/MEK inhibitors. Preliminary evidence indicated that treatment-naïve patients with high ctDNA may preferentially benefit from combined ICIs., (©2020 American Association for Cancer Research.)

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

The analysis of plasma circulating tumour nucleic acids provides a non-invasive approach to assess disease burden and the genetic evolution of tumours in response to therapy. BRAF splicing variants are known to confer melanoma resistance to BRAF inhibitors. We developed a test to screen cell-free RNA (cfRNA) for the presence of BRAF splicing variants. Custom droplet digital PCR assays were designed for the detection of BRAF splicing variants p61, p55, p48 and p41 and then validated using RNA from cell lines carrying these variants. Evaluation of plasma from patients with reported objective response to BRAF/MEK inhibition followed by disease progression was revealed by increased circulating tumour DNA (ctDNA) in 24 of 38 cases at the time of relapse. Circulating BRAF splicing variants were detected in cfRNA from 3 of these 38 patients; two patients carried the BRAF p61 variant and one the p55 variant. In all three cases the presence of the splicing variant was apparent only at the time of progressive disease. BRAF p61 was also detectable in plasma of one of four patients with confirmed BRAF splicing variants in their progressing tumours. Isolation and analysis of RNA from extracellular vesicles (EV) from resistant cell lines and patient plasma demonstrated that BRAF splicing variants are associated with EVs. These findings indicate that in addition to plasma ctDNA, RNA carried by EVs can provide important tumour specific information., Competing Interests: CONFLICTS OF INTEREST The following authors have received travel support from: MAK [Merck Sharp and Dohme (MSD), Bristol-Myers Squibb (BMS) and Merck Serono], TMM [BMS, Novartis, AstraZeneca (AZ)] and ESG [MSD]. The following authors sit on advisory boards for: TMM [BMS, MSD, Novartis, AZ]; MSC [Sanofi, Nektar, Merck Serono BMS, MSD, Novartis, Amgen, Pierre Fabre, Ideaya]; AMM [BMS, MSD, Novartis, Roche, Pierre-Fabre], GVL [Aduro, Amgen, Array, BMS, MSD, Novartis, Pierre-Fabre, Oncosec, Roche] and MM [BMS, AZ, Roche, MSD]., (Copyright: © 2020 Clark et al.)

The treatment of melanoma has been markedly improved by the introduction of targeted therapies and checkpoint blockade immunotherapy. Unfortunately, resistance to these therapies remains a limitation. Novel anticancer therapeutics targeting the MCL1 anti-apoptotic protein have shown impressive responses in haematological cancers but are yet to be evaluated in melanoma. To assess the sensitivity of melanoma to new MCL1 inhibitors, we measured the response of 51 melanoma cell lines to the novel MCL1 inhibitor, S63845. Additionally, we assessed combination of this drug with inhibitors of the bromodomain and extra-terminal (BET) protein family of epigenetic readers, which we postulated would assist MCL1 inhibition by downregulating anti-apoptotic targets regulated by NF-kB such as BCLXL, BCL2A1 and XIAP, and by upregulating pro-apoptotic proteins including BIM and NOXA. Only 14% of melanoma cell lines showed sensitivity to S63845, however, combination of S63845 and I-BET151 induced highly synergistic apoptotic cell death in all melanoma lines tested and in an in vivo xenograft model. Cell death was dependent on caspases and BAX/BAK. Although the combination of drugs increased the BH3-only protein, BIM, and downregulated anti-apoptotic proteins such as BCL2A1, the importance of these proteins in inducing cell death varied between cell lines. ABT-199 or ABT-263 inhibitors against BCL2 or BCL2 and BCLXL, respectively, induced further cell death when combined with S63845 and I-BET151. The combination of MCL1 and BET inhibition appears to be a promising therapeutic approach for metastatic melanoma, and presents opportunities to add further BCL2 family inhibitors to overcome treatment resistance., (© 2020 UICC.)

Genetic alterations in the INK4a/ARF (or CDKN2A ) locus have been reported in many cancer types, including melanoma; head and neck squamous cell carcinomas; lung, breast, and pancreatic cancers. In melanoma, loss of function CDKN2A alterations have been identified in approximately 50% of primary melanomas, in over 75% of metastatic melanomas, and in the germline of 40% of families with a predisposition to cutaneous melanoma. The CDKN2A locus encodes two critical tumor suppressor proteins, the cyclin-dependent kinase inhibitor p16 INK4a and the p53 regulator p14 ARF . The majority of CDKN2A alterations in melanoma selectively target p16 INK4a or affect the coding sequence of both p16 INK4a and p14 ARF . There is also a subset of less common somatic and germline INK4a/ARF alterations that affect p14 ARF , while not altering the syntenic p16 INK4a coding regions. In this review, we describe the frequency and types of somatic alterations affecting the CDKN2A locus in melanoma and germline CDKN2A alterations in familial melanoma, and their functional consequences in melanoma development. We discuss the clinical implications of CDKN2A inactivating alterations and their influence on treatment response and resistance.

Immunotherapies blocking immune inhibitory receptors programmed cell death-1 (PD-1) and cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) on T-cells have dramatically improved patient outcomes in a range of advanced cancers. However, the lack of response, and the development of resistance remain major obstacles to long-term improvements in patient outcomes. There is significant interest in the clinical use of biomarkers to improve patient selection, and the expression of PD-1 ligand 1 (PD-L1) is often reported as a potential biomarker of response. However, accumulating evidence suggests that the predictive value of PD-L1 expression in tumor biopsies is relatively low due, in part, to its complex biology. In this review, we discuss the biological consequences of PD-L1 expression by various cell types within the tumor microenvironment, and the complex mechanisms that regulate PD-L1 expression at the genomic, transcriptomic and proteomic levels.

Molecular diagnostic testing of KRAS and BRAF mutations has become critical in the management of colorectal cancer (CRC) patients. Some progress has been made in liquid biopsy detection of mutations in circulating tumor DNA (ctDNA), which is a fraction of circulating cell-free DNA (cfDNA), but slow analysis for DNA sequencing methods has limited rapid diagnostics. Other methods such as quantitative PCR and more recently, droplet digital PCR (ddPCR), have limitations in multiplexed capacity and the need for expensive specialized equipment. Hence, a robust, rapid and facile strategy is needed for detecting multiple ctDNA mutations to improve the management of CRC patients. To address this significant problem, herein, we propose a new application of multiplex PCR/SERS (surface-enhanced Raman scattering) assay for the detection of ctDNA in CRC, in a fast and non-invasive manner to diagnose and stratify patients for effective treatment. Methods: To discriminate ctDNA mutations from wild-type cfDNA, allele-specific primers were designed for the amplification of three clinically important DNA point mutations in CRC including KRAS G12V, KRAS G13D and BRAF V600E. Surface-enhanced Raman scattering (SERS) nanotags were labelled with a short and specific sequence of oligonucleotide, which can hybridize with the corresponding PCR amplicons. The PCR/SERS assay was implemented by firstly amplifying the multiple mutations, followed by binding with multicolor SERS nanotags specific to each mutation, and subsequent enrichment with magnetic beads. The mutation status was evaluated using a portable Raman spectrometer where the fingerprint spectral peaks of the corresponding SERS nanotags indicate the presence of the mutant targets. The method was then applied to detect ctDNA from CRC patients under a blinded test, the results were further validated by ddPCR. Results: The PCR/SERS strategy showed high specificity and sensitivity for genotyping CRC cell lines and plasma ctDNA, where as few as 0.1% mutant alleles could be detected from a background of abundant wild-type cfDNA. The blinded test using 9 samples from advanced CRC patients by PCR/SERS assay was validated with ddPCR and showed good consistency with pathology testing results. Conclusions: With ddPCR-like sensitivity yet at the convenience of standard PCR, the proposed assay shows great potential in sensitive detection of multiple ctDNA mutations for clinical decision-making., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Detection of melanoma-associated mutations using circulating tumor DNA (ctDNA) from plasma is a potential alternative to using genomic DNA from invasive tissue biopsies. In this study, we developed a custom melanoma next-generation sequencing (NGS) panel which includes 123 amplicons in 30 genes covering driver and targetable mutations and alterations associated with treatment resistance. Analysis of a cohort of 74 stage III and IV treatment-naïve melanoma patients revealed that sensitivity of ctDNA detection was influenced by the amount of circulating-free DNA (cfDNA) input and stage of melanoma. At the recommended cfDNA input quantity of 20 ng (available in 28/74 patients), at least one cancer-associated mutation was detected in the ctDNA of 84% of stage IV patients and 47% of stage III patients with a limit of detection for mutant allele frequency (MAF) of 0.2%. This custom melanoma panel showed significant correlation with droplet digital PCR (ddPCR) and provided a more comprehensive melanoma mutation profile. Our custom panel could be further optimized by replacing amplicons spanning the TERT promoter, which did not perform well due to the high GC content. To increase the detection rate to 90% of stage IV melanoma and decrease the sensitivity to 0.1% MAF, we recommend increasing the volume of plasma to 8 mL to achieve minimal recommended cfDNA input and the refinement of poorly performing amplicons. Our panel can also be expanded to include new targetable and treatment resistance mutations to improve the tracking of treatment response and resistance in melanoma patients treated with systemic drug therapies.

Purpose: Brain involvement occurs in the majority of patients with metastatic melanoma. The potential of circulating tumor DNA (ctDNA) for surveillance and monitoring systemic therapy response in patients with melanoma brain metastases merits investigation., Experimental Design: This study examined circulating BRAF, NRAS , and c-KIT mutations in patients with melanoma with active brain metastases receiving PD-1 inhibitor-based therapy. Intracranial and extracranial disease volumes were measured using the sum of product of diameters, and response assessment performed using RECIST. Longitudinal plasma samples were analyzed for ctDNA over the first 12 weeks of treatment (threshold 2.5 copies/mL plasma)., Results: Of a total of 72 patients, 13 patients had intracranial metastases only and 59 patients had concurrent intracranial and extracranial metastases. ctDNA detectability was 0% and 64%, respectively, and detectability was associated with extracranial disease volume ( P < 0.01). Undetectable ctDNA on-therapy was associated with extracranial response ( P < 0.01) but not intracranial response. The median overall survival in patients with undetectable ( n = 34) versus detectable ( n = 38) ctDNA at baseline was 39.2 versus 10.6 months [HR, 0.51; 95% confidence interval (CI), 0.28-0.94; P = 0.03] and on-therapy was 39.2 versus 9.2 months (HR, 0.32; 95% CI, 0.16-0.63; P < 0.01)., Conclusions: ctDNA remains a strong prognostic biomarker in patients with melanoma with brain metastases, especially in patients with concurrent extracranial disease. However, ctDNA was not able to detect or monitor intracranial disease activity, and we recommend against using ctDNA as a sole test during surveillance and therapeutic monitoring in patients with melanoma., (©2020 American Association for Cancer Research.)

Uveal melanoma (UM) is the most common intraocular tumour in adults and despite surgical or radiation treatment of primary tumours, ~50% of patients progress to metastatic disease. Therapeutic options for metastatic UM are limited, with clinical trials having little impact. Here we perform whole-genome sequencing (WGS) of 103 UM from all sites of the uveal tract (choroid, ciliary body, iris). While most UM have low tumour mutation burden (TMB), two subsets with high TMB are seen; one driven by germline MBD4 mutation, and another by ultraviolet radiation (UVR) exposure, which is restricted to iris UM. All but one tumour have a known UM driver gene mutation (GNAQ, GNA11, BAP1, PLCB4, CYSLTR2, SF3B1, EIF1AX). We identify three other significantly mutated genes (TP53, RPL5 and CENPE).

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

Nanotechnology-based RNA interference (RNAi) has shown great promise in overcoming the limitations of traditional clinical treatments for glioblastoma (GBM). However, because of the complexity of brain physiology, simple blood-brain barrier (BBB) penetration or tumor-targeting strategies cannot entirely meet the demanding requirements of different therapeutic delivery stages. Herein, we developed a charge conversional biomimetic nanoplatform with a three-layer core-shell structure to programmatically overcome persistent obstacles in siRNA delivery to GBM. The resulting nanocomplex presents good biocompatibility, prolonged blood circulation, high BBB transcytosis, effective tumor accumulation, and specific uptake by tumor cells in the brain. Moreover, red blood cell membrane (RBCm) disruption and effective siRNA release can be further triggered elegantly by charge conversion from negative to positive in the endo/lysosome (pH 5.0-6.5) of tumor cells, leading to highly potent target-gene silencing with a strong anti-GBM effect. Our study provides an intelligent biomimetic nanoplatform tailored for systemically siRNA delivery to GBM, leveraging Angiopep-2 peptide-modified, immune-free RBCm and charge conversional components. Improved therapeutic efficacy, higher survival rates, and minimized systemic side effects were achieved in orthotopic U87MG-luc human glioblastoma tumor-bearing nude mice.

Inhibitors targeting the mitogen-activated protein kinase (MAPK) pathway and immune checkpoint molecules have dramatically improved the survival of patients with BRAF V600 -mutant melanoma. For BRAF/RAS wild-type (WT) melanoma patients, however, immune checkpoint inhibitors remain the only effective therapeutic option with 40% of patients responding to PD-1 inhibition. In the present study, a large panel of 10 BRAF V600 -mutant and 13 BRAF/RAS WT melanoma cell lines was analyzed to examine MAPK dependency and explore the potential utility of MAPK inhibitors in this melanoma subtype. We now show that the majority of BRAF/RAS WT melanoma cell lines (8/13) display some degree of sensitivity to trametinib treatment and resistance to trametinib in this melanoma subtype is associated with, but not mediated by NF1 suppression. Although knockdown of NF1 stimulates RAS and CRAF activity, the activation of CRAF by NF1 knockdown is limited by ERK-dependent feedback in BRAF-mutant cells, but not in BRAF/RAS WT melanoma cells. Thus, NF1 is not a dominant regulator of MAPK signaling in BRAF/RAS WT melanoma, and co-targeting multiple MAP kinase nodes provides a therapeutic opportunity for this melanoma subtype., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The use of circulating tumor DNA (ctDNA) to monitor cancer progression and response to therapy has significant potential but there is only limited data on whether this technique can detect the presence of low frequency subclones that may ultimately confer therapy resistance. In this study, we sought to evaluate whether whole-exome sequencing (WES) of ctDNA could accurately profile the mutation landscape of metastatic melanoma. We used WES to identify variants in matched, tumor-derived genomic DNA (gDNA) and plasma-derived ctDNA isolated from a cohort of 10 metastatic cutaneous melanoma patients. WES parameters such as sequencing coverage and total sequencing reads were comparable between gDNA and ctDNA. The mutant allele frequency of common single nucleotide variants was lower in ctDNA, reflecting the lower read depth and minor fraction of ctDNA within the total circulating free DNA pool. There was also variable concordance between gDNA and ctDNA based on the total number and identity of detected variants and this was independent of the tumor biopsy site. Nevertheless, established melanoma driver mutations and several other melanoma-associated mutations were concordant between matched gDNA and ctDNA. This study highlights that WES of ctDNA could capture clinically relevant mutations present in melanoma metastases and that enhanced sequencing sensitivity will be required to identify low frequency mutations., Competing Interests: G.V.L. receives consultant service fees from Amgen, BMS, Array, Pierre-Fabre, Novartis, Merck Sharp and Dohme (MSD) and Roche. A.M.M. is an advisory board member for BMS, MSD, Novartis, Roche and Pierre Fabre. R.F.K. has been on advisory boards for Roche, Amgen, BMS, MSD, Novartis and TEVA and has received honoraria from MSD, BMS and Novartis. M.S.C. is an advisory board member for MSD, BMS, Novartis, Pierre-Fabre, Roche and Amgen. R.A.S. has received honoraria from MSD, Novartis, GSK, BMS, Myriad and NeraCare. J.F.T. has been on advisory boards for GlaxoSmithKline, Provectus Inc, Merck Sharp Dohme and Bristol Myers Squibb, and has received honoraria and travel support from GlaxoSmithKline and Provectus Inc. He has received honoraria for advisory board participation from Merck Sharp Dohme and Bristol Myers Squibb. These companies had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. All remaining authors have declared no conflicts of interest.

Background: Adjuvant dabrafenib plus trametinib therapy improves relapse-free survival in patients with resected stage III melanoma. We aimed to ascertain the proportion of patients who would have a pathological response and a response according to Response Evaluation Criteria in Solid Tumors (RECIST) after neoadjuvant dabrafenib plus trametinib therapy for resectable clinical stage III melanoma., Methods: NeoCombi was a single-arm, open-label, single-centre, phase 2 study done at Melanoma Institute Australia (Sydney, NSW, Australia). Eligible patients were adults (aged ≥18 years) with histologically confirmed, resectable, RECIST-measurable, clinical stage IIIB-C (American Joint Committee on Cancer [AJCC] 7th edition), BRAF V600 -mutant melanoma, and had an Eastern Cooperative Oncology Group performance status of 1 or lower. Patients received 150 mg dabrafenib orally, twice daily, plus 2 mg trametinib orally, once daily, for 52 weeks (12 weeks of neoadjuvant therapy before complete resection of the pre-therapy tumour bed, and 40 weeks of adjuvant therapy thereafter). CT and PET scans were done at baseline and before resection. The primary outcomes were the proportion of patients achieving a complete pathological response and the proportion of patients achieving a response according to RECIST at week 12, analysed as per protocol. This trial is registered with ClinicalTrials.gov, NCT01972347, and follow-up of patients is ongoing., Findings: Between Aug 20, 2014, and April 19, 2017, 40 patients were screened, of whom 35 eligible patients were enrolled, received neoadjuvant dabrafenib plus trametinib, and underwent resection. At the data cutoff (Sept 24, 2018), median follow-up was 27 months (IQR 21-36). At resection, 30 (86%) patients achieved a RECIST response; 16 (46%; 95% CI 29-63) had a complete response and 14 (40%; 24-58) had a partial response. Five patients (14%; 95% CI 5-30) had stable disease, and no patients progressed. After resection and pathological evaluation, all 35 patients achieved a pathological response, of whom 17 (49%; 95% CI 31-66) patients had a complete pathological response and 18 (51%; 95% CI 34-69) had a non-complete pathological response. Treatment-related serious adverse events occurred in six (17%) of 35 patients and grade 3-4 adverse events occurred in ten (29%) patients. No treatment-related deaths were reported., Interpretation: Neoadjuvant dabrafenib plus trametinib therapy could be considered in the management of RECIST-measurable resectable stage III melanoma as it led to a high proportion of patients achieving a complete response according to RECIST and a high proportion of patients achieving a complete pathological response, with no progression during neoadjuvant therapy., Funding: GlaxoSmithKline; Novartis; National Health and Medical Research Council, Australia; and Melanoma Institute Australia., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (©2019 American Association for Cancer Research.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Poly(amidoamine) dendrimer (PAMAM) is well-known for its high efficiency as a drug delivery vehicle. However, the intrinsic cytotoxicity and lack of a detectable signal to facilitate tracking have impeded its practical applications. Herein, we have developed a novel label-free fluorescent and biocompatible PAMAM derivative by simple surface modification of PAMAM using acetaldehyde. The modified PAMAM possessed a strong green fluorescence, which was generated by the C=N bonds of the resulting Schiff Bases via n-π* transition, while the intrinsic cytotoxicity of PAMAM was simultaneously ameliorated. Through further PEGylation, the fluorescent PAMAM demonstrated excellent intracellular tracking in human melanoma SKMEL28 cells. In addition, our PEGylated fluorescent PAMAM derivative achieved enhanced loading and delivery efficiency of the anticancer drug doxorubicin (DOX) compared to the original PAMAM. Importantly, the accelerated kinetics of DOX-encapsulated fluorescent PAMAM nanocomposites in an acidic environment facilitated intracellular drug release, which demonstrated comparable cytotoxicity to that of the free-form doxorubicin hydrochloride (DOX·HCl) against melanoma cells. Overall, our label free fluorescent PAMAM derivative offers a new opportunity of traceable and controlled delivery for DOX and other drugs of potential clinical importance.

Purpose: The combination of a BRAF inhibitor dabrafenib and a MEK inhibitor trametinib (CombiDT) has improved outcomes compared with chemotherapy or BRAF inhibitor monotherapy in advanced BRAF V600E/K melanoma. However, CombiDT causes a high incidence of pyrexia and treatment interruptions. Pharmacokinetic analysis may provide an explanation for the pyrexia., Methods: 34 patients with Stage 3 BRAF V600 melanoma were treated with CombiDT on a clinical trial between August 2014 and June 2017. Plasma concentrations of drugs and metabolites were determined using validated LC-MS assays, in addition to analysis of a panel of cytokines., Results: Pyrexia was experienced by 71% of the patients, with an additional 17% requiring dose interruption related to a pyrexia-like prodrome. Dabrafenib concentrations ranged from 4.0 to 4628 ng/ml and trametinib from 1.0 to 45 ng/ml in 34 patients. N-desmethyl-dabrafenib was the most prevalent metabolite, followed by carboxy- and hydroxy-dabrafenib. No definitive association between pyrexia and AUC or C min of the drugs, or metabolites could be observed. The level of IL-1B at the early during treatment (EDT) (as a % of pre-treatment) was higher in the pyrexia group (median 109% (range 32-681%) than in the no-incidence group [56% (26-79%)] (p = 0.029). Similarly, the level of IL-6 at EDT was higher in the pyrexia group [181% (34-3156%) vs 73% (57-101%)] (p = 0.028)., Conclusions: No apparent associations between pyrexia and exposure to the drugs or metabolites could be observed. Greater elevations in IL-1B and IL-6 were observed in patients with pyrexia during the first week of treatment compared to those without pyrexia.

Purpose: Combination PD-1 and CTLA-4 inhibitor therapy has dramatically improved the survival of patients with advanced melanoma but is also associated with significant immune-related toxicities. This study sought to identify circulating cytokine biomarkers of treatment response and immune-related toxicity., Experimental Design: The expression of 65 cytokines was profiled longitudinally in 98 patients with melanoma treated with PD-1 inhibitors, alone or in combination with anti-CTLA-4, and in an independent validation cohort of 49 patients treated with combination anti-PD-1 and anti-CTLA-4. Cytokine expression was correlated with RECIST response and immune-related toxicity, defined as toxicity that warranted permanent discontinuation of treatment and administration of high-dose steroids., Results: Eleven cytokines were significantly upregulated in patients with severe immune-related toxicities at baseline (PRE) and early during treatment (EDT). The expression of these 11 cytokines was integrated into a single toxicity score, the CYTOX (cytokine toxicity) score, and the predictive utility of this score was confirmed in the discovery and validation cohorts. The AUC for the CYTOX score in the validation cohort was 0.68 at PRE [95% confidence interval (CI), 0.51-0.84; P = 0.037] and 0.70 at EDT (95% CI, 0.55-0.85; P = 0.017) using ROC analysis., Conclusions: The CYTOX score is predictive of severe immune-related toxicity in patients with melanoma treated with combination anti-CTLA-4 and anti-PD-1 immunotherapy. This score, which includes proinflammatory cytokines such as IL1a, IL2, and IFNα2, may help in the early management of severe, potentially life-threatening immune-related toxicity. See related commentary by Johnson and Balko, p. 1452 ., (©2018 American Association for Cancer Research.)

Purpose: BRAF V600E and V600K melanomas have distinct clinicopathologic features, and V600K appear to be less responsive to BRAFi ± MEKi . We investigated mechanisms for this and explored whether genotype affects response to immunotherapy., Experimental Design: Pretreatment formalin-fixed paraffin-embedded tumors from patients treated with BRAFi ± MEKi underwent gene expression profiling and DNA sequencing. Molecular results were validated using The Cancer Genome Atlas (TCGA) data. An independent cohort of V600E/K patients treated with anti-PD-1 immunotherapy was examined., Results: Baseline tissue and clinical outcome with BRAFi ± MEKi were studied in 93 patients (78 V600E, 15 V600K). V600K patients had numerically less tumor regression (median, -31% vs. -52%, P = 0.154) and shorter progression-free survival (PFS; median, 5.7 vs. 7.1 months, P = 0.15) compared with V600E. V600K melanomas had lower expression of the ERK pathway feedback regulator dual-specificity phosphatase 6, confirmed with TCGA data (116 V600E, 17 V600K). Pathway analysis showed V600K had lower expression of ERK and higher expression of PI3K-AKT genes than V600E. Higher mutational load was observed in V600K, with a higher proportion of mutations in PIK3R1 and tumor-suppressor genes. In patients treated with anti-PD-1, V600K ( n = 19) had superior outcomes than V600E ( n = 84), including response rate (53% vs. 29%, P = 0.059), PFS (median, 19 vs. 2.7 months, P = 0.049), and overall survival (20.4 vs. 11.7 months, P = 0.081)., Conclusions: BRAF V600K melanomas appear to benefit less from BRAFi ± MEKi than V600E, potentially due to less reliance on ERK pathway activation and greater use of alternative pathways. In contrast, these melanomas have higher mutational load and respond better to immunotherapy., (©2019 American Association for Cancer Research.)

Cancer immunotherapies provide survival benefits in responding patients, but many patients fail to respond. Identifying the biology of treatment response and resistance are a priority to optimize drug selection and improve patient outcomes. We performed transcriptomic and immune profiling on 158 tumor biopsies from melanoma patients treated with anti-PD-1 monotherapy (n = 63) or combined anti-PD-1 and anti-CTLA-4 (n = 57). These data identified activated T cell signatures and T cell populations in responders to both treatments. Further mass cytometry analysis identified an EOMES + CD69 + CD45RO + effector memory T cell phenotype that was significantly more abundant in responders to combined immunotherapy compared with non-responders (n = 18). The gene expression profile of this population was associated with longer progression-free survival in patients treated with single agent and greater tumor shrinkage in both treatments., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Genetic material derived from tumours is constantly shed into the circulation of cancer patients both in the form of circulating free nucleic acids and within circulating cells or extracellular vesicles. Monitoring cancer-specific genomic alterations, particularly mutant allele frequencies, in circulating nucleic acids allows for a non-invasive liquid biopsy for detecting residual disease and response to therapy. The advent of molecular targeted treatments and immunotherapies with increasing effectiveness requires corresponding effective molecular biology methods for the detection of biomarkers such as circulating nucleic acid to monitor and ultimately personalise therapy. The use of polymerase chain reaction (PCR)-based methods, such as droplet digital PCR, allows for a very sensitive analysis of circulating tumour DNA, but typically only a limited number of gene mutations can be detected in parallel. In contrast, next-generation sequencing allows for parallel analysis of multiple mutations in many genes. The development of targeted next-generation sequencing cancer gene panels optimised for the detection of circulating free DNA now provides both the flexibility of multiple mutation analysis coupled with a sensitivity that approaches or even matches droplet digital PCR. In this review, we discuss the advantages and disadvantages of these current molecular technologies in conjunction with how this field is evolving in the context of melanoma diagnosis, prognosis, and monitoring of response to therapy.

Immunotherapy has changed the landscape of cancer treatment. The introduction of immune checkpoint inhibitors has seen tremendous success in improving overall survival of patients with advanced metastatic cancers and has now become the standard of care for multiple tumor types. However, efficacy of immune checkpoint blockade appears to be limited to immunogenic cancers, and even amongst immune-reactive cancers, response rates are low and variable between patients. Recent data have also demonstrated the rapid emergence of resistance to immune checkpoint inhibitors, with some patients progressing on treatment within one year. Significant research efforts are now directed at identifying predictive biomarkers and mechanisms of resistance to immune checkpoint blockade. These studies are underpinned by comprehensive and detailed profiling of the immune milieu. In this review, we discuss the utility and efficacy of immune cell profiling to uncover biomarkers of response and mechanisms of resistance to immune checkpoint inhibitors.

Analysis of liquid biopsies and the identification of non-invasive biomarkers for the diagnosis and prognosis of solid tumors has grown exponentially over the last few years. This has led to an increasing number of commercial kits optimised for the purification of circulating free (cf) DNA and RNA/miRNA from biofluids such as plasma, serum and urine. To optimise and standardise current practices we sought to evaluate the performance of spin column-based and magnetic bead-based commercial kits. The following commercial cfDNA purification kits were analysed in this study: QIAamp circulating nucleic acid kit (Qiagen, Germany); Plasma/serum cell-free circulating DNA Purification midi kit (Norgen Biotek, Canada); QIAamp minElute ccfDNA mini kit (Qiagen); Maxwell RSC ccfDNA plasma kit (Promega, USA); MagMAX cell-free DNA isolation kit (Applied Biosystems, USA); and NextPrep-Mag cfDNA isolation kit (Bioo Scientific, USA). Extracted DNA from the plasma of healthy individuals, either nonspiked or spiked with DNA fragments or cfDNA, was evaluated for recovery using either a BioRad Experion or ddPCR analysis. This study represents the first to use a comprehensive size distribution of spiked-in DNA fragments to evaluate commercial cfDNA kits. The commonly used spin column-based Qiagen QIAamp circulating nucleic acid kit was found to be the most consistent performing kit across the two evaluation assays employed. The Qiagen QIAamp minElute ccfDNA mini kit represented the best performing magnetic bead-based kit and provides an alternative based on lower cost/sample with a simpler workflow than spin column-based kits., (Copyright © 2018 Elsevier Inc. All rights reserved.)