IFN-γ Signaling Sensitizes Melanoma Cells to BH3 Mimetics.

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.

Musculoskeletal immune-related adverse events with the use of checkpoint inhibitors in malignancy.

BACKGROUND: Immunotherapy has revolutionised the treatment of many malignancies. Along with their success, there have been inflammatory and immune-related adverse events (irAE). There is a paucity of literature describing the Australian experience of rheumatic irAE. AIMS: To describe and characterise the Royal North Shore Hospital and Westmead Hospital cohort with rheumatic irAE. METHODS: This case series reports on 17 patients with advanced cancer treated at two sites in Sydney, Australia who were referred for rheumatological evaluation from 2013 to 2016. Data were collected retrospectively and inspected for clinical signs, duration of immunotherapy prior to onset of symptoms, management strategies and cancer outcomes. RESULTS: Patients presented with arthralgias, myalgias, periarticular and systemic symptoms. Onset of rheumatological symptoms was variable, with a median of 4 months (range 0.2-24) for monotherapy and 5.05 months (range 0.2-6.9) for combination. The predominant findings were of tenosynovitis (23%) and large joint involvement (65%). All patients were seronegative for RF and anti-CCP. Most patients responded well to non-steroidal anti-inflammatory drugs or low-dose prednisone (59%) and remained on immunotherapy (77%). The majority (76%) of patients experienced concomitant irAE in other organ systems. Sixty-five percent of patients had complete response of their malignancy to immunotherapy. CONCLUSION: Rheumatic irAE are heterogenous clinical entities, which require further evaluation into classification, patient susceptibility and response. From our study, there was no clear clinical pattern. The present case series supports that rheumatic irAE may be associated with tumour response. However, there is still limited experience in rheumatic irAE management and outcomes.

First-in-human, phase I/IIa study of CRLX301, a nanoparticle drug conjugate containing docetaxel, in patients with advanced or metastatic solid malignancies.

Background This was a phase I/IIa study to investigate the tolerability, efficacy and pharmacokinetics (PK)/ pharmacodynamics (PD) of CRLX301, CDP-based nanoparticle formulation of docetaxel. Methods The study was conducted in two parts. In part 1, dose-escalation using a standard 3 + 3 design was performed in two dosing schedules (every week (QW) and every 3 weeks (Q3W)). Part 2 was comprised of a dose expansion at 75 mg/m2 Q3W. PK studies were performed on both dosing schedules. Results Forty-two patients were recruited onto the study with a median age of 64(range 38-76); median number of prior systemic therapies was 5(range 0-10). Grade 3/4 treatment-related toxicities included: neutropenia (21.4 %), infusion related reaction (11.9 %), anemia (7.1 %), fatigue (4.8 %), diarrhea (4.8 %), and peripheral neuropathy (4.8 %). The maximum tolerated dose was 75 mg/m2 given on the Q3W schedule and was not determined on the QW schedule. In this heavily pre-treated population, four patients (12.9 %) achieved stable disease (SD) ≥ 4 months and 2 patients (6.5 %) achieved partial response (PR) for a clinical benefit rate (CBR) of 19.4 % (6/31 patients). The PRs were seen in prostate and breast adenocarcinoma (one each). CRLX301 exhibited some PK advantages over docetaxel including higher retention of drug in plasma, slower clearance and controlled slow release of docetaxel from the carrier. Conclusions In this heavily pretreated patient population, the safety profile was acceptable for CRLX301 therapy. There was some evidence of preliminary tumor efficacy, but further work is necessary to find the optimal dose and schedule of this formulation.Clinicaltrials.gov trial registration number: NCT02380677 (Date of registration: March 2, 2015).

Management of melanoma brain metastases: Evidence-based clinical practice guidelines by Cancer Council Australia.

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

Selective Oral MEK1/2 Inhibitor Pimasertib: A Phase I Trial in Patients with Advanced Solid Tumors.

BACKGROUND: The Ras/Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (Ras/Raf/MEK/ERK) signaling cascade is frequently constitutively activated in human cancers. Pimasertib is a selective and potent adenosine triphosphate non-competitive MEK1/2 inhibitor. OBJECTIVE: Our objectives were to describe the results of a phase I, first-in-human, dose-escalation trial of pimasertib that investigated the maximum tolerated dose, recommended phase II dose, and safety, as well as other endpoints. PATIENTS AND METHODS: Four dosing schedules of pimasertib (once daily [qd], 5 days on, 2 days off; qd, 15 days on, 6 days off; continuous qd; continuous twice daily [bid]) were evaluated in patients with advanced solid tumors. Each treatment cycle lasted 21 days. The primary objective was to determine the maximum tolerated dose based on dose-limiting toxicities (DLTs) evaluated during cycle 1, and the recommended phase II dose (RP2D). Secondary objectives included safety, pharmacokinetics, pharmacodynamics, and antitumor activity. RESULTS: Overall, 180 patients received pimasertib (dose range 1-255 mg/day). DLTs were mainly observed at doses ≥ 120 mg/day and included skin rash/acneiform dermatitis and ocular events, such as serous retinal detachment. The most common drug-related adverse events were consistent with class effects, including diarrhea, skin disorders, ocular disorders, asthenia/fatigue, and peripheral edema. The median time to maximum pimasertib concentration was 1.5 h across dosing schedules, and the apparent terminal half-life was 5 h across qd dosing schedules. Pimasertib decreased ERK phosphorylation within 2 h of administration, which was maintained for up to 8 h at higher doses and prolonged with bid dosing. CONCLUSIONS: Based on the safety profile and efficacy signals, a continuous bid regimen was the preferred dosing schedule and the RP2D was defined as 60 mg bid. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00982865.

Selective Oral MEK1/2 Inhibitor Pimasertib in Metastatic Melanoma: Antitumor Activity in a Phase I, Dose-Escalation Trial.

BACKGROUND: Pimasertib is a selective, potent mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor. OBJECTIVES: The aim of this study was to describe the efficacy, safety, and pharmacodynamics of pimasertib at pharmacologically active doses in a cohort of patients with locally advanced/metastatic melanoma from a first-in-human study of pimasertib. METHODS: This was a phase I, open-label, two-part, dose-escalation study. Part 1 was conducted in patients with solid tumors and identified the maximum tolerated dose, while Part 2 was restricted to patients with advanced/metastatic melanoma. Endpoints included safety, pharmacodynamics, and antitumor activity. We present data for patients with melanoma only from both parts of the study. RESULTS: In total, 93 patients with melanoma received pimasertib, 89 of whom received pharmacologically active doses (28-255 mg/day) across four dose regimens in the two parts of the study. The objective response rate was 12.4% (11/89): complete response (n = 1) and partial response (PR; n = 10). Six patients responded for > 24 weeks. Nine of the 11 responders had tumors with B-Raf Proto-Oncogene, Serine/Threonine Kinase (BRAF; n = 6) and/or NRAS Proto-Oncogene, GTPase (NRAS; n = 3) mutations. Forty-six patients had stable disease (SD). In patients with ocular melanoma (n = 13), best overall response was PR (n = 1), SD (n = 11), and disease progression (n = 1). Phosphorylated extracellular signal-regulated kinase (pERK) levels were substantially reduced within 2 h of treatment and inhibition was sustained with continuous twice-daily dosing. Treatment-related, recurrent, grade 3 or higher adverse events were reported in eight patients, including diarrhea, and skin and ocular events. CONCLUSION: Results from this phase I study indicate that pimasertib has clinical activity in patients with locally advanced/metastatic melanoma, particularly BRAF- and NRAS-mutated tumors, at clinically relevant doses associated with pERK inhibition in peripheral blood mononuclear cells. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00982865.

Tumor MHC Expression Guides First-Line Immunotherapy Selection in Melanoma.

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.

Design and Testing of a Custom Melanoma Next Generation Sequencing Panel for Analysis of Circulating Tumor DNA.

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.

Long-term Follow-up of Standard-Dose Pembrolizumab Plus Reduced-Dose Ipilimumab in Patients with Advanced Melanoma: KEYNOTE-029 Part 1B.

PURPOSE: Combination therapy with reduced-dose programmed death 1 inhibitor plus standard-dose cytotoxic T-lymphocyte-associated antigen 4 inhibitor demonstrated efficacy, but substantial toxicity, in melanoma. We present long-term results of part 1B of KEYNOTE-029, which assessed safety and efficacy of standard-dose pembrolizumab plus reduced-dose ipilimumab in advanced melanoma. PATIENTS AND METHODS: Part 1B was an expansion cohort of the open-label, phase Ib portion of KEYNOTE-029. Eligible patients had advanced melanoma and no previous immune checkpoint inhibitor therapy. Patients received pembrolizumab 2 mg/kg (amended to 200 mg) every 3 weeks plus ipilimumab 1 mg/kg every 3 weeks (four cycles), then pembrolizumab alone for up to 2 years. Primary end point was safety; secondary end points included objective response rate (ORR), progression-free survival (PFS), duration of response (DOR), and overall survival (OS). RESULTS: A total of 153 patients received at least one dose of pembrolizumab plus ipilimumab. At a median follow-up of 36.8 months, 71.9% had received four doses of ipilimumab and 30.7% had completed 2 years of pembrolizumab; 26.1% completed both treatments. Treatment-related adverse events occurred in 96.1% (47.1% grade 3/4; no deaths), leading to discontinuation of one or both study drugs in 35.9%. ORR was 62.1% with 42 (27.5%) complete and 53 (34.6%) partial responses. Median DOR was not reached; 36-month ongoing response rate was 84.2%. Median PFS and OS were not reached; 36-month rates were 59.1% and 73.4%, respectively. CONCLUSIONS: Standard-dose pembrolizumab plus reduced-dose ipilimumab demonstrated robust antitumor activity, durable response, and favorable long-term survival with manageable toxicity.

Clinicians' attitudes and perceived barriers and facilitators to cancer treatment clinical practice guideline adherence: a systematic review of qualitative and quantitative literature.

BACKGROUND: Clinical Practice Guidelines (CPGs) synthesize the best available evidence to guide clinician and patient decision making. There are a multitude of barriers and facilitators to clinicians adhering to CPGs; however, little is known about active cancer treatment CPG adherence specifically. This systematic review sought to identify clinician attitudes, and perceived barriers and facilitators to active cancer treatment CPG adherence. METHODS: A systematic search was undertaken of five databases; Ovid Medline, PsychInfo, Embase, Scopus, CINAHL, and PROQUEST. The retrieved abstracts were screened for eligibility against inclusion criteria, and a full text review was conducted of all eligible studies. Data were extracted, and a quality assessment was conducted of all included studies. The qualitative papers were thematically analyzed. Attitudes, barriers, and facilitating factors extracted from the quantitative papers were categorized within the qualitative thematic framework. RESULTS: The search resulted in the identification of 9676 titles. After duplicates were removed, abstracts screened, and full texts reviewed, 15 studies were included. Four themes were identified which related to negative clinician attitudes and barriers to active cancer treatment CPG adherence: (1) concern over CPG content and currency of CPGs; (2) concern about the evidence underpinning CPGs; (3) clinician uncertainty and negative perceptions of CPGs; and (4) organizational and patient factors. The review also identified four themes related to positive attitudes and facilitators to active cancer treatment CPG adherence: (5) CPG accessibility and ease of use; (6) endorsement and dissemination of CPGs and adequate access to treatment facilities and resources; (7) awareness of CPGs and belief in their relevance; and (8) belief that CPGs support decision making, improve patient care, reduce clinical variation, and reduce costs. CONCLUSION: These results highlight that adherence to active cancer treatment CPG recommendations by oncology clinicians is influenced by multiple factors such as attitudes, practices, and access to resources. The review has also revealed many similarities and differences in the factors associated with general CPG, and active cancer treatment CPG, adherence. These findings will inform tailored implementation strategies to increase adherence to cancer treatment CPGs. TRIAL REGISTRATION: PROSPERO (2019) CRD42019125748.

Longitudinal Monitoring of ctDNA in Patients with Melanoma and Brain Metastases Treated with Immune Checkpoint Inhibitors.

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.

Transcriptional downregulation of MHC class I and melanoma de- differentiation in resistance to PD-1 inhibition.

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 MITFlow/AXLhigh de-differentiated phenotype and cancer-associated fibroblast signatures. We demonstrate that TGFß drives the treatment resistant phenotype (MITFlow/AXLhigh) 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.

Clinicians' attitudes to oncology clinical practice guidelines and the barriers and facilitators to adherence: a mixed methods study protocol.

INTRODUCTION: Clinical practice guidelines (CPGs) are designed to reduce inappropriate clinical variation and improve the quality of care. Barriers to CPGs include a lack of awareness of CPGs, access to them, time pressures and concerns regarding the evidence underpinning CPG development, implementation and dissemination. The objectives of this study are to assess clinicians' attitudes to CPGs for cancer treatment and the perceived barriers to and facilitators of CPG adherence in order to inform the implementation of cancer treatment CPGs. METHODS AND ANALYSIS: A mixed methods study will be conducted using a three-phase, sequential design, with each phase informing the next. In phase 1, a qualitative study using recorded interviews will investigate clinicians' attitudes to CPGs for cancer treatment and perceptions of barriers and facilitators to CPG adherence (n=30); interview transcripts will be analysed thematically. In phase 2, a survey will quantify the frequency of attitudes, barriers and facilitators identified in phase 1, in a broader clinical sample (n=200). In phase 3, a workshop forum will be held to facilitate discussions examining the implications of phase 1 and 2 findings for cancer CPG implementation strategies (n=40) leading to recommendations for improvements to practice. The workshop discussion will be recorded, and the transcript will be analysed thematically. ETHICS AND DISSEMINATION: This study has received ethics approval in New South Wales, Australia (2019/ETH11722, #52019568810127). Study findings will be published in peer-reviewed journals and will form part of a doctoral thesis and be presented at national and international conferences.

Adjuvant dabrafenib plus trametinib versus placebo in patients with resected, BRAFV600-mutant, stage III melanoma (COMBI-AD): exploratory biomarker analyses from a randomised, phase 3 trial.

BACKGROUND: Adjuvant dabrafenib plus trametinib reduced the risk of relapse versus placebo in patients with resected, BRAFV600-mutant, stage III melanoma in the phase 3 COMBI-AD trial. This prespecified exploratory biomarker analysis aimed to evaluate potential prognostic or predictive factors and mechanisms of resistance to adjuvant targeted therapy. METHODS: COMBI-AD is a randomised, double-blind, placebo-controlled, phase 3 trial comparing dabrafenib 150 mg orally twice daily plus trametinib 2 mg orally once daily versus two matched placebos. Study participants were at least 18 years of age and underwent complete resection of stage IIIA (lymph node metastases >1 mm), IIIB, or IIIC cutaneous melanoma, per American Joint Committee on Cancer 7th edition criteria, with a BRAFV600E or BRAFV600K mutation. Patients were randomly assigned (1:1) to the two treatment groups by an interactive voice response system, stratified by mutation type and disease stage. Patients, physicians, and the investigators who analysed data were masked to treatment allocation. The primary outcome was relapse-free survival, defined as the time from randomisation to disease recurrence or death from any cause. Biomarker assessment was a prespecified exploratory outcome of the trial. We assessed intrinsic tumour genomic features by use of next-generation DNA sequencing and characteristics of the tumour microenvironment by use of a NanoString RNA assay, which might provide prognostic and predictive information. This trial is registered with ClinicalTrials.gov, number NCT01682083, and is ongoing but no longer recruiting participants. FINDINGS: Between Jan 31, 2013, and Dec 11, 2014, 870 patients were enrolled in the trial. Median follow-up at data cutoff (April 30, 2018) was 44 months (IQR 38-49) in the dabrafenib plus trametinib group and 42 months (21-49) in the placebo group. Intrinsic tumour genomic features were assessed in 368 patients (DNA sequencing set) and tumour microenvironment characteristics were assessed in 507 patients (NanoString biomarker set). MAPK pathway genomic alterations at baseline did not affect treatment benefit or clinical outcome. An IFNγ gene expression signature higher than the median was prognostic for prolonged relapse-free survival in both treatment groups. Tumour mutational burden was independently prognostic for relapse-free survival in the placebo group (high TMB, top third; hazard ratio [HR] 0·56, 95% CI 0·37-0·85, p=0·0056), but not in the dabrafenib plus trametinib group (0·83, 95% CI 0·53-1·32, p=0·44). Patients with tumour mutational burden in the lower two terciles seem to derive a substantial long-term relapse-free survival benefit from targeted therapy (HR [versus placebo] 0·49, 95% CI 0·35-0·68, p<0·0001). However, patients with high tumour mutational burden seem to have a less pronounced benefit with targeted therapy (HR [versus placebo] 0·75, 95% CI 0·44-1·26, p=0·27), especially if they had an IFNγ signature lower than the median (HR 0·88 [95% CI 0·40-1·93], p=0·74). INTERPRETATION: Tumour mutational burden alone or in combination with IFNγ gene expression signature or other markers for an adaptive immune response might be of relevance for identifying patients with stage III melanoma who might derive clinical benefit from targeted therapy. Further validation in prospective clinical trials is warranted. FUNDING: Novartis Pharmaceuticals.

Mitogen-activated protein kinase dependency in BRAF/RAS wild-type melanoma: A rationale for combination inhibitors.

Inhibitors targeting the mitogen-activated protein kinase (MAPK) pathway and immune checkpoint molecules have dramatically improved the survival of patients with BRAFV600 -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 BRAFV600 -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.

Efficacy of novel immunotherapy regimens in patients with metastatic melanoma with germline CDKN2A mutations.

BACKGROUND: Inherited CDKN2A mutation is a strong risk factor for cutaneous melanoma. Moreover, carriers have been found to have poor melanoma-specific survival. In this study, responses to novel immunotherapy agents in CDKN2A mutation carriers with metastatic melanoma were evaluated. METHODS: CDKN2A mutation carriers that have developed metastatic melanoma and undergone immunotherapy treatments were identified among carriers enrolled in follow-up studies for familial melanoma. The carriers' responses were compared with responses reported in phase III clinical trials for CTLA-4 and PD-1 inhibitors. From publicly available data sets, melanomas with somatic CDKN2A mutation were analysed for association with tumour mutational load. RESULTS: Eleven of 19 carriers (58%) responded to the therapy, a significantly higher frequency than observed in clinical trials (p=0.03, binomial test against an expected rate of 37%). Further, 6 of the 19 carriers (32%) had complete response, a significantly higher frequency than observed in clinical trials (p=0.01, binomial test against an expected rate of 7%). In 118 melanomas with somatic CDKN2A mutations, significantly higher total numbers of mutations were observed compared with 761 melanomas without CDKN2A mutation (Wilcoxon test, p<0.001). CONCLUSION: Patients with CDKN2A mutated melanoma may have improved immunotherapy responses due to increased tumour mutational load, resulting in more neoantigens and stronger antitumorous immune responses.

Long-term outcomes in patients with BRAF V600-mutant metastatic melanoma receiving dabrafenib monotherapy: Analysis from phase 2 and 3 clinical trials.

BACKGROUND: Previous analyses of BREAK-2 and BREAK-3 showed that durable outcomes lasting ≥3 years are achievable with dabrafenib in some patients with BRAF V600-mutant metastatic melanoma (MM); however, additional follow-up is needed to fully characterise the long-term impact of dabrafenib in these patients. METHODS: BREAK-2 was a single-arm phase 2 study evaluating dabrafenib in treatment-naive or previously treated BRAF V600E/K-mutant MM. BREAK-3, a randomised (3:1) phase 3 study, assessed dabrafenib versus dacarbazine in previously untreated unresectable or metastatic BRAF V600E-mutant melanoma. Five-year analyses were performed. RESULTS: All BREAK-2 patients (N = 92 [V600E, n = 76; V600K, n = 16]) discontinued treatment by the data cutoff. Median follow-up was 13.0 months. In BRAF V600E patients, 5-year progression-free survival (PFS) and overall survival (OS) were 11% and 20%, respectively. Subsequent immunotherapy was received by 22% of patients. In BREAK-3, median follow-up was 17.0 and 12.0 months in the dabrafenib (n = 187) and dacarbazine (n = 63) arms, respectively. Thirty-seven patients (59%) receiving dacarbazine crossed over to dabrafenib following disease progression as per protocol. Five-year PFS was 12% in the dabrafenib arm; all dacarbazine-arm patients progressed or were censored by 5 years. Dabrafenib improved PFS versus dacarbazine, regardless of baseline lactate dehydrogenase levels. Five-year OS rates were 24% and 22% in the dabrafenib and dacarbazine arms, respectively. Subsequent therapy in each arm included anti-CTLA-4 (dabrafenib [24%] and dacarbazine [24%]) and/or anti-PD-1 (8% and 2%) treatment. No new safety signals were observed. CONCLUSIONS AND RELEVANCE: These data, representing extended follow-up for dabrafenib monotherapy, demonstrate that durable benefit lasting ≥5 years is achievable in a subset of patients. TRIAL REGISTRATION: ClinicalTrials.gov (BREAK-2, NCT01153763; BREAK-3, NCT01227889).

Analysis of the Whole-Exome Sequencing of Tumor and Circulating Tumor DNA in Metastatic Melanoma.

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.

Targeted Therapy in Advanced Melanoma With Rare BRAF Mutations.

PURPOSE: BRAF/MEK inhibition is a standard of care for patients with BRAF V600E/K-mutated metastatic melanoma. For patients with less frequent BRAF mutations, however, efficacy data are limited. METHODS: In the current study, 103 patients with metastatic melanoma with rare, activating non-V600E/K BRAF mutations that were treated with either a BRAF inhibitor (BRAFi), MEK inhibitor (MEKi), or the combination were included. BRAF mutation, patient and disease characteristics, response, and survival data were analyzed. RESULTS: Fifty-eight patient tumors (56%) harbored a non-E/K V600 mutation, 38 (37%) a non-V600 mutation, and seven had both V600E and a rare BRAF mutation (7%). The most frequent mutations were V600R (43%; 44 of 103), L597P/Q/R/S (15%; 15 of 103), and K601E (11%; 11 of 103). Most patients had stage IV disease and 42% had elevated lactate dehydrogenase at BRAFi/MEKi initiation. Most patients received combined BRAFi/MEKi (58%) or BRAFi monotherapy (37%). Of the 58 patients with V600 mutations, overall response rate to BRAFi monotherapy and combination BRAFi/MEKi was 27% (six of 22) and 56% (20 of 36), respectively, whereas median progression-free survival (PFS) was 3.7 months and 8.0 months, respectively (P = .002). Of the 38 patients with non-V600 mutations, overall response rate was 0% (zero of 15) to BRAFi, 40% (two of five) to MEKi, and 28% (five of 18) to combination treatment, with a median PFS of 1.8 months versus 3.7 months versus 3.3 months, respectively. Multivariable analyses revealed superior survival (PFS and overall survival) with combination over monotherapy in rare V600 and non-V600 mutated melanoma. CONCLUSION: Patients with rare BRAF mutations can respond to targeted therapy, however, efficacy seems to be lower compared with V600E mutated melanoma. Combination BRAFi/MEKi seems to be the best regimen for both V600 and non-V600 mutations. Yet interpretation should be done with care because of the heterogeneity of patients with small sample sizes for some of the reported mutations.

Pneumocystis jirovecii in a patient on dose-dense chemotherapy for early breast cancer.

A 70-year-old woman underwent adjuvant chemotherapy with dose-dense doxorubicin and cyclophosphamide for early breast cancer. After her fourth cycle of chemotherapy, she developed severe fatigue and cough with rapid-onset hypoxic respiratory failure. Investigations demonstrated extensive bilateral consolidation with positive bronchial washings for Pneumocystis jirovecii by polymerase chain reaction (PCR). Despite high-dose trimethoprim-sulfamethoxazole, she progressed to multi-organ failure and succumbed. Pneumocystis jirovecii pneumonia (PJP) has traditionally rarely occurred in women on adjuvant breast cancer chemotherapy but may pose a more serious risk in dose-dense regimes due to higher concurrent exposure to anti-emetic corticosteroids. Clinicians are alerted to the need for vigilance of this rare complication and for rationalization of dexamethasone dosage to mitigate this risk, particularly in the era of modern triple-agent anti-emetic regimens.