CD155 on Tumor Cells Drives Resistance to Immunotherapy by Inducing the Degradation of the Activating Receptor CD226 in CD8+ T Cells.

The activating receptor CD226 is expressed on lymphocytes, monocytes, and platelets and promotes anti-tumor immunity in pre-clinical models. Here, we examined the role of CD226 in the function of tumor-infiltrating lymphocytes (TILs) and resistance to immunotherapy. In murine tumors, a large proportion of CD8+ TILs had decreased surface expression of CD226 and exhibited features of dysfunction, whereas CD226hi TILs were highly functional. This correlation was seen also in TILs isolated from HNSCC patients. Mutation of CD226 at tyrosine 319 (Y319) led to increased CD226 surface expression, enhanced anti-tumor immunity and improved efficacy of immune checkpoint blockade (ICB). Mechanistically, tumor-derived CD155, the ligand for CD226, initiated phosphorylation of Y319 by Src kinases, thereby enabling ubiquitination of CD226 by CBL-B, internalization, and proteasomal degradation. In pre-treatment samples from melanoma patients, CD226+CD8+ T cells correlated with improved progression-free survival following ICB. Our findings argue for the development of therapies aimed at maintaining the expression of CD226.

Anatomic position determines oncogenic specificity in melanoma.

Oncogenic alterations to DNA are not transforming in all cellular contexts1,2. This may be due to pre-existing transcriptional programmes in the cell of origin. Here we define anatomic position as a major determinant of why cells respond to specific oncogenes. Cutaneous melanoma arises throughout the body, whereas the acral subtype arises on the palms of the hands, soles of the feet or under the nails3. We sequenced the DNA of cutaneous and acral melanomas from a large cohort of human patients and found a specific enrichment for BRAF mutations in cutaneous melanoma and enrichment for CRKL amplifications in acral melanoma. We modelled these changes in transgenic zebrafish models and found that CRKL-driven tumours formed predominantly in the fins of the fish. The fins are the evolutionary precursors to tetrapod limbs, indicating that melanocytes in these acral locations may be uniquely susceptible to CRKL. RNA profiling of these fin and limb melanocytes, when compared with body melanocytes, revealed a positional identity gene programme typified by posterior HOX13 genes. This positional gene programme synergized with CRKL to amplify insulin-like growth factor (IGF) signalling and drive tumours at acral sites. Abrogation of this CRKL-driven programme eliminated the anatomic specificity of acral melanoma. These data suggest that the anatomic position of the cell of origin endows it with a unique transcriptional state that makes it susceptible to only certain oncogenic insults.

Author Correction: Tissue-resident memory CD8+ T cells promote melanoma-immune equilibrium in skin.

Panel j was inadvertently labelled as panel k in the caption to Fig. 4. Similarly, 'Fig. 4k' should have been 'Fig. 4j' in the sentence beginning 'TNF-α-deficient gBT-I cells were…'. In addition, the surname of author Umaimainthan Palendira was misspelled 'Palendria'. These errors have been corrected online.

Tissue-resident memory CD8+ T cells promote melanoma-immune equilibrium in skin.

The immune system can suppress tumour development both by eliminating malignant cells and by preventing the outgrowth and spread of cancer cells that resist eradication1. Clinical and experimental data suggest that the latter mode of control-termed cancer-immune equilibrium1-can be maintained for prolonged periods of time, possibly up to several decades2-4. Although cancers most frequently originate in epithelial layers, the nature and spatiotemporal dynamics of immune responses that maintain cancer-immune equilibrium in these tissue compartments remain unclear. Here, using a mouse model of transplantable cutaneous melanoma5, we show that tissue-resident memory CD8+ T cells (TRM cells) promote a durable melanoma-immune equilibrium that is confined to the epidermal layer of the skin. A proportion of mice (~40%) transplanted with melanoma cells remained free of macroscopic skin lesions long after epicutaneous inoculation, and generation of tumour-specific epidermal CD69+ CD103+ TRM cells correlated with this spontaneous disease control. By contrast, mice deficient in TRM formation were more susceptible to tumour development. Despite being tumour-free at the macroscopic level, mice frequently harboured melanoma cells in the epidermal layer of the skin long after inoculation, and intravital imaging revealed that these cells were dynamically surveyed by TRM cells. Consistent with their role in melanoma surveillance, tumour-specific TRM cells that were generated before melanoma inoculation conferred profound protection from tumour development independently of recirculating T cells. Finally, depletion of TRM cells triggered tumour outgrowth in a proportion (~20%) of mice with occult melanomas, demonstrating that TRM cells can actively suppress cancer progression. Our results show that TRM cells have a fundamental role in the surveillance of subclinical melanomas in the skin by maintaining cancer-immune equilibrium. As such, they provide strong impetus for exploring these cells as targets of future anticancer immunotherapies.

Genomic Profiling of Metastatic Basal cell Carcinoma Reveals Candidate Drivers of Disease and Therapeutic Targets.

Basal cell carcinomas (BCCs) are human beings' most common malignant tumors. Most are easily managed by surgery or topical therapies, and metastasis is rare. Although BCCs can become locally advanced, metastatic BCCs are very uncommon and may be biologically distinct. We assessed the clinicopathologic characteristics of 17 patients with metastatic BCC and pursued whole-exome sequencing of tumor and germline DNA from 8 patients. Genomic profiling revealed aberrant activation of Hedgehog signaling and alterations in GLI transcriptional regulators and Notch and Hippo signaling. Matched local recurrences of primary BCCs and metastases from 3 patients provided evidence of a clonal origin in all cases. Mutations associated with YAP inhibition were found exclusively in 2 hematogenously-spread lung metastases, and metastatic BCCs were enriched for mutations in the YAP/TAZ-binding domain of TEAD genes. Accordingly, YAP/TAZ nuclear localization was associated with metastatic types and Hippo mutations, suggesting an enhanced oncogenic role in hematogenously-spread metastases. Mutations in RET, HGF, and phosphatidylinositol 3­kinase (PI3K)/protein kinase B (AKT) signaling were enriched compared with a cohort of low clinical-risk BCCs. Our results implicate Hippo and PI3K/AKT dysregulation in metastatic progression of BCCs, making these potential therapeutic targets in metastatic disease. The common clonal origin of matched recurrent and metastatic BCCs suggests that molecular profiling can assist in determining the nature/origin of poorly differentiated metastatic tumors of uncertain type. Genes and pathways enriched for mutations in this cohort are candidate drivers of metastasis and can be used to identify patients at high risk of metastasis who may benefit from aggressive local treatment and careful clinical follow-up.

Cross-platform comparison of immune signatures in immunotherapy-treated patients with advanced melanoma using a rank-based scoring approach.

BACKGROUND: Gene expression profiling is increasingly being utilised as a diagnostic, prognostic and predictive tool for managing cancer patients. Single-sample scoring approach has been developed to alleviate instability of signature scores due to variations from sample composition. However, it is a challenge to achieve comparable signature scores across different expressional platforms. METHODS: The pre-treatment biopsies from a total of 158 patients, who have received single-agent anti-PD-1 (n = 84) or anti-PD-1 + anti-CTLA-4 therapy (n = 74), were performed using NanoString PanCancer IO360 Panel. Multiple immune-related signature scores were measured from a single-sample rank-based scoring approach, singscore. We assessed the reproducibility and the performance in reporting immune profile of singscore based on NanoString assay in advance melanoma. To conduct cross-platform analyses, singscores between the immune profiles of NanoString assay and the previous orthogonal whole transcriptome sequencing (WTS) data were compared through linear regression and cross-platform prediction. RESULTS: singscore-derived signature scores reported significantly high scores in responders in multiple PD-1, MHC-1-, CD8 T-cell-, antigen presentation-, cytokine- and chemokine-related signatures. We found that singscore provided stable and reproducible signature scores among the repeats in different batches and cross-sample normalisations. The cross-platform comparisons confirmed that singscores derived via NanoString and WTS were comparable. When singscore of WTS generated by the overlapping genes to the NanoString gene set, the signatures generated highly correlated cross-platform scores (Spearman correlation interquartile range (IQR) [0.88, 0.92] and r2 IQR [0.77, 0.81]) and better prediction on cross-platform response (AUC = 86.3%). The model suggested that Tumour Inflammation Signature (TIS) and Personalised Immunotherapy Platform (PIP) PD-1 are informative signatures for predicting immunotherapy-response outcomes in advanced melanoma patients treated with anti-PD-1-based therapies. CONCLUSIONS: Overall, the outcome of this study confirms that singscore based on NanoString data is a feasible approach to produce reliable signature scores for determining patients' immune profiles and the potential clinical utility in biomarker implementation, as well as to conduct cross-platform comparisons, such as WTS.

Melanomas in children and adolescents: Clinicopathologic features and survival outcomes.

BACKGROUND: Melanomas in the first 2 decades of life are uncommon and poorly understood. OBJECTIVE: To assess clinicopathologic features and survival of children (≤11 years) and adolescents (12-19 years) diagnosed with melanoma. METHODS: A pooled cohort of 514 patients was analyzed (397 Dutch, 117 Australian; 62 children, 452 adolescents). Pathology reports were reevaluated to determine melanoma subtypes. Multivariable Cox models were generated for recurrence-free survival (RFS) and overall survival (OS). RESULTS: Melanoma subtypes were conventional melanoma (superficial spreading, nodular, desmoplastic, and acral lentiginous), spitzoid melanoma, and melanoma associated with a congenital nevus in 428, 78, and 8 patients, respectively. Ten-year RFS was 91.5% (95% confidence interval [CI], 82.4%-100%) in children and 86.4% (95% CI, 82.7%-90.3%) in adolescents (P = .32). Ten-year OS was 100% in children and 92.7% (95% CI, 89.8%-95.8%) in adolescents (P = .09). On multivariable analysis possible only for the adolescent cohort due to the small number of children, ulceration status, and anatomic site were associated with RFS and OS, whereas age, sex, mitotic index, sentinel node status and melanoma subtype were not. Breslow thickness >4 mm was associated with worse RFS. LIMITATIONS: Retrospective study. CONCLUSIONS: Survival rates for children and adolescents with melanomas were high. Ulceration, head or neck location and Breslow thickness >4 mm predicted worse survival in adolescents.

Whole-genome landscapes of major melanoma subtypes.

Melanoma of the skin is a common cancer only in Europeans, whereas it arises in internal body surfaces (mucosal sites) and on the hands and feet (acral sites) in people throughout the world. Here we report analysis of whole-genome sequences from cutaneous, acral and mucosal subtypes of melanoma. The heavily mutated landscape of coding and non-coding mutations in cutaneous melanoma resolved novel signatures of mutagenesis attributable to ultraviolet radiation. However, acral and mucosal melanomas were dominated by structural changes and mutation signatures of unknown aetiology, not previously identified in melanoma. The number of genes affected by recurrent mutations disrupting non-coding sequences was similar to that affected by recurrent mutations to coding sequences. Significantly mutated genes included BRAF, CDKN2A, NRAS and TP53 in cutaneous melanoma, BRAF, NRAS and NF1 in acral melanoma and SF3B1 in mucosal melanoma. Mutations affecting the TERT promoter were the most frequent of all; however, neither they nor ATRX mutations, which correlate with alternative telomere lengthening, were associated with greater telomere length. Most melanomas had potentially actionable mutations, most in components of the mitogen-activated protein kinase and phosphoinositol kinase pathways. The whole-genome mutation landscape of melanoma reveals diverse carcinogenic processes across its subtypes, some unrelated to sun exposure, and extends potential involvement of the non-coding genome in its pathogenesis.

Genetic drivers of non-cutaneous melanomas: Challenges and opportunities in a heterogeneous landscape.

Non-cutaneous melanomas most frequently involve the uveal tract and mucosal membranes, including the conjunctiva. In contrast to cutaneous melanoma, they often present at an advanced clinical stage, are associated with worse clinical outcomes and show poorer responses to immunotherapy. The mutational load within most non-cutaneous melanomas reflects their lower ultraviolet light (UV) exposure. The genetic drivers within non-cutaneous melanomas are heterogeneous. Within ocular melanomas, posterior uveal tract melanomas typically harbour one of two distinct, sets of driver mutations and alterations of clinical and biological significance. In contrast to posterior uveal tract melanomas, anterior uveal tract melanomas of the iris and conjunctival melanomas frequently carry both a higher mutational burden and specific mutations linked with UV exposure. The genetic drivers in iris melanomas more closely resemble those of the posterior uveal tract, whereas conjunctival melanomas harbour similar genetic driver mutations to cutaneous melanomas. Mucosal melanomas occur in sun-shielded sites including sinonasal and oral cavities, nasopharynx, oesophagus, genitalia, anus and rectum, and their mutational landscape is frequently associated with a dominant process of spontaneous deamination and infrequent presence of UV mutation signatures. Genetic drivers of mucosal melanomas are diverse and vary with anatomic location. Further understanding of the causes of already identified recurrent molecular events in non-cutaneous melanomas, identification of additional drivers in specific subtypes, integrative multi-omics analyses and analysis of the tumor immune microenvironment will expand knowledge in this field. Furthermore, such data will likely uncover new therapeutic strategies which will lead to improved clinical outcomes in non-cutaneous melanoma patients.

Multiple eruptive squamoproliferative lesions during anti-PD1 immunotherapy for metastatic melanoma: Pathogenesis, immunohistochemical analysis and treatment.

Treatment with anti-PD1 inhibitors may enhance the risk for developing low grade squamoproliferative skin tumors. Immunohistochemical (IHC) analysis of the immune tumor microenvironment (TME) allows exploration of the pathogenesis and relationship with the PD1/PDL1 axis. Patients with eruptive keratoacanthoma (KA)-like lesions were recruited from the Melanoma Institute Australia, a tertiary referral specialist melanoma treatment center from January 2015 to August 2017. Clinicopathologic evaluation and IHC features of tumor cells (PDL1 expression) and peritumoral microenvironment (PD1, FOXP3, PDL1, CD4:CD8 expressing cells) in 12 eruptive KA-like lesions, were compared with solitary KAs in age and sex matched non-anti-PD1 treated controls. Four patients with repeated episodes of eruptive KA-like and lichenoid lesions developing 2-7 months after commencing pembrolizumab for AJCC stage IV melanoma, were recruited. Eruptive KA-like squamoproliferative lesions occurred in sun exposed sites and in areas of resolving, concomitant or delayed lichenoid reactions. Histologically, the lesions were well-differentiated squamoproliferative lesions resembling infundibulocystic squamous cell carcinoma or KA. IHC of cases and controls revealed low PDL1 expression of both squamous tumor cells and the TME immune cells. The numbers of immunosuppressive FOXP3 positive Tregs and PD1-expressing T-cells were higher in the cases than the controls but the CD4:CD8 ratio (2:1) was similar. The patients best responded to acitretin and were managed surgically if they demonstrated neoplastic features. Accelerated squamoproliferative growth in actinically damaged keratinocytes associated with lichenoid eruptions may be unmasked in patients treated with anti-PD1 immunotherapy potentially contributed to by a local cutaneous immunosuppressed TME.

Expression of NGF/proNGF and Their Receptors TrkA, p75NTR and Sortilin in Melanoma.

There is increasing evidence that nerve growth factor (NGF) and its receptors, the neurotrophic receptor tyrosine kinase 1 (NTRK1/TrkA), the common neurotrophin receptor (NGFR/p75NTR) and the membrane receptor sortilin, participate in cancer growth. In melanoma, there have been some reports suggesting that NGF, TrkA and p75NTR are dysregulated, but the expression of the NGF precursor (proNGF) and its membrane receptor sortilin is unknown. In this study, we investigated the expression of NGF, proNGF, TrkA, p75NTR and sortilin by immunohistochemistry in a series of human tissue samples (n = 100), including non-cancerous nevi (n = 20), primary melanomas (n = 40), lymph node metastases (n = 20) and distant metastases (n = 20). Immunostaining was digitally quantified and revealed NGF and proNGF were expressed in all nevi and primary melanomas, and that the level of expression decreased from primary tumors to melanoma metastases (p = 0.0179 and p < 0.0001, respectively). Interestingly, TrkA protein expression was high in nevi and thin primary tumors but was strongly downregulated in thick primary tumors (p < 0.0001) and metastases (p < 0.0001). While p75NTR and sortilin were both expressed in most nevi and melanomas, there was no significant difference in expression between them. Together, these results pointed to a downregulation of NGF/ProNGF and TrkA in melanoma, and thus did not provide evidence to support the use of anti-proNGF/NGF or anti-TrkA therapies in advanced and metastatic forms of melanoma.

bcGST-an interactive bias-correction method to identify over-represented gene-sets in boutique arrays.

MOTIVATION: Gene annotation and pathway databases such as Gene Ontology and Kyoto Encyclopaedia of Genes and Genomes are important tools in Gene-Set Test (GST) that describe gene biological functions and associated pathways. GST aims to establish an association relationship between a gene-set of interest and an annotation. Importantly, GST tests for over-representation of genes in an annotation term. One implicit assumption of GST is that the gene expression platform captures the complete or a very large proportion of the genome. However, this assumption is neither satisfied for the increasingly popular boutique array nor the custom designed gene expression profiling platform. Specifically, conventional GST is no longer appropriate due to the gene-set selection bias induced during the construction of these platforms. RESULTS: We propose bcGST, a bias-corrected GST by introducing bias-correction terms in the contingency table needed for calculating the Fisher's Exact Test. The adjustment method works by estimating the proportion of genes captured on the array with respect to the genome in order to assist filtration of annotation terms that would otherwise be falsely included or excluded. We illustrate the practicality of bcGST and its stability through multiple differential gene expression analyses in melanoma and the Cancer Genome Atlas cancer studies. AVAILABILITY AND IMPLEMENTATION: The bcGST method is made available as a Shiny web application at http://shiny.maths.usyd.edu.au/bcGST/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The prognostic value of tumor mitotic rate in children and adolescents with cutaneous melanoma: A retrospective cohort study.

BACKGROUND: Mitotic rate is a strong predictor of outcome in adult patients with primary cutaneous melanoma, but for children and adolescent patients this is unknown. OBJECTIVE: We sought to assess the prognostic value of primary tumor mitotic rate in children and adolescents with primary melanoma. METHODS: This was a cohort study of 156 patients who were <20 years of age and who had clinically localized cutaneous melanoma. Patients <12 years of age were classified as children and those 12 to 19 years of age as adolescents. Clinicopathologic and outcome data were collected. Recurrence-free and melanoma-specific survival were calculated. Univariable and multivariable analyses were performed using Cox proportional hazard models. RESULTS: Thirteen of 156 patients (8%) were children. The mitotic rate was ≥1/mm2 in 104 patients (67%) and correlated with increasing Breslow thickness. A positive sentinel node was found in 23 of 61 patients (38%) in whom a sentinel lymph node biopsy specimen was obtained. The median follow-up was 61 months. Five-year melanoma-specific and recurrence-free survival rates were 91% and 84%, respectively. Mitotic rate was a stronger predictor of outcome than tumor thickness and was the only factor independently associated with recurrence-free survival. LIMITATIONS: This research was conducted at a single institution and the sample size was small. CONCLUSION: Mitotic rate is an independent predictor of recurrence-free survival in children and adolescents with clinically localized melanoma.

Telomere sequence content can be used to determine ALT activity in tumours.

The replicative immortality of human cancer cells is achieved by activation of a telomere maintenance mechanism (TMM). To achieve this, cancer cells utilise either the enzyme telomerase, or the Alternative Lengthening of Telomeres (ALT) pathway. These distinct molecular pathways are incompletely understood with respect to activation and propagation, as well as their associations with clinical outcomes. We have identified significant differences in the telomere repeat composition of tumours that use ALT compared to tumours that do not. We then employed a machine learning approach to stratify tumours according to telomere repeat content with an accuracy of 91.6%. Importantly, this classification approach is applicable across all tumour types. Analysis of pathway mutations that were under-represented in ALT tumours, across 1,075 tumour samples, revealed that the autophagy, cell cycle control of chromosomal replication, and transcriptional regulatory network in embryonic stem cells pathways are involved in the survival of ALT tumours. Overall, our approach demonstrates that telomere sequence content can be used to stratify ALT activity in cancers, and begin to define the molecular pathways involved in ALT activation.

Neoadjuvant dabrafenib combined with trametinib for resectable, stage IIIB-C, BRAFV600 mutation-positive melanoma (NeoCombi): a single-arm, open-label, single-centre, phase 2 trial.

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), BRAFV600-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.

RAB27A promotes melanoma cell invasion and metastasis via regulation of pro-invasive exosomes.

Despite recent advances in targeted and immune-based therapies, advanced stage melanoma remains a clinical challenge with a poor prognosis. Understanding the genes and cellular processes that drive progression and metastasis is critical for identifying new therapeutic strategies. Here, we found that the GTPase RAB27A was overexpressed in a subset of melanomas, which correlated with poor patient survival. Loss of RAB27A expression in melanoma cell lines inhibited 3D spheroid invasion and cell motility in vitro, and spontaneous metastasis in vivo. The reduced invasion phenotype was rescued by RAB27A-replete exosomes, but not RAB27A-knockdown exosomes, indicating that RAB27A is responsible for the generation of pro-invasive exosomes. Furthermore, while RAB27A loss did not alter the number of exosomes secreted, it did change exosome size and altered the composition and abundance of exosomal proteins, some of which are known to regulate cancer cell movement. Our data suggest that RAB27A promotes the biogenesis of a distinct pro-invasive exosome population. These findings support RAB27A as a key cancer regulator, as well as a potential prognostic marker and therapeutic target in melanoma.

Whole genome sequencing of melanomas in adolescent and young adults reveals distinct mutation landscapes and the potential role of germline variants in disease susceptibility.

Cutaneous melanoma accounts for at least >10% of all cancers in adolescents and young adults (AYA, 15-30 years of age) in Western countries. To date, little is known about the correlations between germline variants and somatic mutations and mutation signatures in AYA melanoma patients that might explain why they have developed a cancer predominantly affecting those over 65 years of age. We performed genomic analysis of 50 AYA melanoma patients (onset 10-30 years, median 20); 25 underwent whole genome sequencing (WGS) of both tumor and germline DNA, exome data were retrieved from 12 TCGA AYA cases, and targeted DNA sequencing was conducted on 13 cases. The AYA cases were compared with WGS data from 121 adult cutaneous melanomas. Similar to mature adult cutaneous melanomas, AYA melanomas showed a high mutation burden and mutation signatures of ultraviolet radiation (UVR) damage. The frequencies of somatic mutations in BRAF (96%) and PTEN (36%) in the AYA WGS cohort were double the rates observed in adult melanomas (Q < 6.0 × 10-6 and 0.028, respectively). Furthermore, AYA melanomas contained a higher proportion of non-UVR-related mutation signatures than mature adult melanomas as a proportion of total mutation burden (p = 2.0 × 10-4 ). Interestingly, these non-UVR mutation signatures relate to APOBEC or mismatch repair pathways, and germline variants in related genes were observed in some of these cases. We conclude that AYA melanomas harbor some of the same molecular aberrations and mutagenic insults occurring in older adults, but in different proportions. Germline variants that may have conferred disease susceptibility correlated with somatic mutation signatures in a subset of AYA melanomas.

Inter- and intrapatient heterogeneity of indoleamine 2,3-dioxygenase expression in primary and metastatic melanoma cells and the tumour microenvironment.

AIMS: Indoleamine 2,3-dioxygenase (IDO), an immunomodulatory enzyme, facilitates immune escape by tumours and promotes tumour progression. IDO inhibitors with and without additional anti-PD-1 therapy have been evaluated in recent and ongoing melanoma clinical trials, but IDO expression in melanoma tumours, and therefore its potential role as a predictive biomarker remains unknown. This study sought to evaluate IDO expression in immunotherapy-naive metastatic melanoma patients in order to determine patterns of expression in corresponding primary melanomas, locoregional metastases and distant metastases. METHODS AND RESULTS: Here, we evaluated IDO expression using immunohistochemistry in 99 melanoma tumour samples from 43 immunotherapy-naive patients with metastatic melanoma to determine patterns of expression in primary melanomas (n = 29), locoregional metastases (n = 36) and distant metastases (n = 34). Thirty-seven per cent of patients demonstrated tumour IDO expression in at least one specimen. Twelve of 35 patients (34%) with longitudinal specimens (i.e. two or more separate specimens from different disease stages in the same patient) displayed heterogeneous IDO staining between samples. Tumour IDO expression positively correlated with tumour-infiltrating lymphocyte (TIL) score as well as the number of IDO-expressing mononuclear cells in the primary melanoma (P < 0.0001 and P = 0.0011, respectively) and nodal metastases (P = 0.049 and P = 0.037, respectively), but not in distant metastases. Furthermore, tumour IDO expression correlated positively with PD-L1 expression by melanoma cells among all specimens (P = 0.0073). CONCLUSIONS: Therefore, while assessment of tumour IDO expression warrants evaluation in melanoma patient cohorts treated with IDO inhibitors dosed at levels proven to inhibit the target by pharmacodynamic assessment, its utility as a biomarker may be limited by intertumoral heterogeneity.

Combination nivolumab and ipilimumab or nivolumab alone in melanoma brain metastases: a multicentre randomised phase 2 study.

BACKGROUND: Nivolumab monotherapy and combination nivolumab plus ipilimumab increase proportions of patients achieving a response and survival versus ipilimumab in patients with metastatic melanoma; however, efficacy in active brain metastases is unknown. We aimed to establish the efficacy and safety of nivolumab alone or in combination with ipilimumab in patients with active melanoma brain metastases. METHODS: This multicentre open-label randomised phase 2 trial was done at four sites in Australia, in three cohorts of immunotherapy-naive patients aged 18 years or older with melanoma brain metastases. Patients with asymptomatic brain metastases with no previous local brain therapy were randomly assigned using the biased coin minimisation method, stratified by site, in a 30:24 ratio (after a safety run-in of six patients) to cohort A (nivolumab plus ipilimumab) or cohort B (nivolumab). Patients with brain metastases in whom local therapy had failed, or who had neurological symptoms, or leptomeningeal disease were enrolled in non-randomised cohort C (nivolumab). Patients in cohort A received intravenous nivolumab 1 mg/kg combined with ipilimumab 3 mg/kg every 3 weeks for four doses, then nivolumab 3 mg/kg every 2 weeks; patients in cohort B or cohort C received intravenous nivolumab 3 mg/kg every 2 weeks. The primary endpoint was intracranial response from week 12. Primary and safety analyses were done on an intention-to-treat basis in all patients who received at least one dose of the study drug. This trial is registered with ClinicalTrials.gov, number NCT02374242, and is ongoing for the final survival analysis. FINDINGS: Between Nov 4, 2014, and April 21, 2017, 79 patients were enrolled; 36 in cohort A, 27 in cohort B, and 16 in cohort C. One patient in cohort A and two in cohort B were found to be ineligible and excluded from the study before receiving the study drug. At the data cutoff (Aug 28, 2017), with a median follow up of 17 months (IQR 8-25), intracranial responses were achieved by 16 (46%; 95% CI 29-63) of 35 patients in cohort A, five (20%; 7-41) of 25 in cohort B, and one (6%; 0-30) of 16 in cohort C. Intracranial complete responses occurred in six (17%) patients in cohort A, three (12%) in cohort B, and none in cohort C. Treatment-related adverse events occurred in 34 (97%) of 35 patients in cohort A, 17 (68%) of 25 in cohort B, and eight (50%) of 16 in cohort C. Grade 3 or 4 treatment-related adverse events occurred in 19 (54%) patients in cohort A, four (16%) in cohort B, and two (13%) in cohort C. No treatment-related deaths occurred. INTERPRETATION: Nivolumab combined with ipilimumab and nivolumab monotherapy are active in melanoma brain metastases. A high proportion of patients achieved an intracranial response with the combination. Thus, nivolumab combined with ipilimumab should be considered as a first-line therapy for patients with asymptomatic untreated brain metastases. FUNDING: Melanoma Institute Australia and Bristol-Myers Squibb.

HDAC inhibitors restore BRAF-inhibitor sensitivity by altering PI3K and survival signalling in a subset of melanoma.

Mutations in BRAF activate oncogenic MAPK signalling in almost half of cutaneous melanomas. Inhibitors of BRAF (BRAFi) and its target MEK are widely used to treat melanoma patients with BRAF mutations but unfortunately acquired resistance occurs in the majority of patients. Resistance results from mutations or non-genomic changes that either reactivate MAPK signalling or activate other pathways that provide alternate survival and growth signalling. Here, we show the histone deacetylase inhibitor (HDACi) panobinostat overcomes BRAFi resistance in melanoma, but this is dependent on the resistant cells showing a partial response to BRAFi treatment. Using patient- and in vivo-derived melanoma cell lines with acquired BRAFi resistance, we show that combined treatment with the BRAFi encorafenib and HDACi panobinostat in 2D and 3D culture systems synergistically induced caspase-dependent apoptotic cell death. Key changes induced by HDAC inhibition included decreased PI3K pathway activity associated with a reduction in the protein level of a number of receptor tyrosine kinases, and cell line dependent upregulation of pro-apoptotic BIM or NOXA together with reduced expression of anti-apoptotic proteins. Independent of these changes, panobinostat reduced c-Myc and pre-treatment of cells with siRNA against c-Myc reduced BRAFi/HDACi drug-induced cell death. These results suggest that a combination of HDAC and MAPK inhibitors may play a role in treatment of melanoma where the resistance mechanisms are due to activation of MAPK-independent pathways.