Circulating tumor DNA predicts survival in patients with resected high risk stage II/III melanoma

Background: Patients with high-risk stage II/III resected melanoma commonly develop distant metastases. At present, we cannot differentiate between patients who will recur or those who are cured by surgery. We investigated if circulating tumor DNA (ctDNA) can predict relapse and survival in patients with resected melanoma. Patients and methods: We carried out droplet digital polymerase chain reaction to detect BRAF and NRAS mutations in plasma taken after surgery from 161 stage II/III high-risk melanoma patients enrolled in the AVAST-M adjuvant trial. Results: Mutant BRAF or NRAS ctDNA was detected (≥1 copy of mutant ctDNA) in 15/132 (11%) BRAF mutant patient samples and 4/29 (14%) NRAS mutant patient samples. Patients with detectable ctDNA had a decreased disease-free interval [DFI; hazard ratio (HR) 3.12; 95% confidence interval (CI) 1.79–5.47; P < 0.0001] and distant metastasis-free interval (DMFI; HR 3.22; 95% CI 1.80–5.79; P < 0.0001) versus those with undetectable ctDNA. Detectable ctDNA remained a significant predictor after adjustment for performance status and disease stage (DFI: HR 3.26, 95% CI 1.83–5.83, P < 0.0001; DMFI: HR 3.45, 95% CI 1.88–6.34, P < 0.0001). Five-year overall survival rate for patients with detectable ctDNA was 33% (95% CI 14%–55%) versus 65% (95% CI 56%–72%) for those with undetectable ctDNA. Overall survival was significantly worse for patients with detectable ctDNA (HR 2.63; 95% CI 1.40–4.96); P = 0.003) and remained significant after adjustment for performance status (HR 2.50, 95% CI 1.32–4.74, P = 0.005). Conclusion: ctDNA predicts for relapse and survival in high-risk resected melanoma and could aid selection of patients for adjuvant therapy

Plasma total cell-free DNA (cfDNA) is a surrogate biomarker for tumour burden and a prognostic biomarker for survival in metastatic melanoma patients

Introduction Tumour burden is a prognostic biomarker in metastatic melanoma. However, tumour burden is difficult to measure and there are currently no reliable surrogate biomarkers to easily and reliably determine it. The aim of this study was to assess the potential of plasma total cell free DNA as biomarker of tumour burden and prognosis in metastatic melanoma patients. Materials and methods A prospective biomarker cohort study for total plasma circulating cell-free DNA (cfDNA) concentration was performed in 43 metastatic melanoma patients. For 38 patients, paired blood collections and scan assessments were available before treatment and at first response evaluation. Tumour burden was calculated as the sum of volumes from three-dimensional radiological measurements of all metastatic lesions in individual patients. Results Baseline cfDNA concentration correlated with pre-treatment tumour burden (ρ = 0.52, P < 0.001). Baseline cfDNA levels correlated significantly with hazard of death and overall survival, and a cut off value of 89 pg/μl identified two distinct prognostic groups (HR = 2.22 for high cfDNA, P = 0.004). Patients with cfDNA ≥89 pg/μl had shorter OS (10.0 versus 22.7 months, P = 0.009; HR = 2.22 for high cfDNA, P = 0.004) and the significance was maintained when compared with lactic dehydrogenase (LDH) in a multivariate analysis. We also found a correlation between the changes of cfDNA and treatment-related changes in tumour burden (ρ = 0.49, P = 0.002). In addition, the ratio between baseline cfDNA and tumour burden was prognostic (HR = 2.7 for cfDNA/tumour volume ≥8 pg/(μl*cm3), P = 0.024). Conclusions We have demonstrated that cfDNA is a surrogate marker of tumour burden in metastatic melanoma patients, and that it is prognostic for overall survival.Fil: Valpione, S.. University of Manchester; Reino Unido. Christie NHS Foundation Trust; Reino UnidoFil: Gremel, G.. University of Manchester; Reino UnidoFil: Mundra, P.. University of Manchester; Reino UnidoFil: Middlehurst, P.. University of Manchester; Reino UnidoFil: Galvani, E.. Christie NHS Foundation Trust; Reino Unido. University of Manchester; Reino UnidoFil: Girotti, Maria Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. University of Manchester; Reino UnidoFil: Lee, R.J.. University of Manchester; Reino UnidoFil: Garner, G.. University of Manchester; Reino UnidoFil: Dhomen, N.. University of Manchester; Reino UnidoFil: Lorigan, P.C.. Christie NHS Foundation Trust; Reino UnidoFil: Marais, R.. University of Manchester; Reino Unid

Brain microenvironment-driven resistance to immune and targeted therapies in acral melanoma.

BACKGROUND: Combination treatments targeting the MEK-ERK pathway and checkpoint inhibitors have improved overall survival in melanoma. Resistance to treatment especially in the brain remains challenging, and rare disease subtypes such as acral melanoma are not typically included in trials. Here we present analyses from longitudinal sampling of a patient with metastatic acral melanoma that became resistant to successive immune and targeted therapies. METHODS: We performed whole-exome sequencing and RNA sequencing on an acral melanoma that progressed on successive immune (nivolumab) and targeted (dabrafenib) therapy in the brain to identify resistance mechanisms. In addition, we performed growth inhibition assays, reverse phase protein arrays and immunoblotting on patient-derived cell lines using dabrafenib in the presence or absence of cerebrospinal fluid (CSF) in vitro. Patient-derived xenografts were also developed to analyse response to dabrafenib. RESULTS: Immune escape following checkpoint blockade was not due to loss of tumour cell recognition by the immune system or low neoantigen burden, but was associated with distinct changes in the microenvironment. Similarly, resistance to targeted therapy was not associated with acquired mutations but upregulation of the AKT/phospho-inositide 3-kinase pathway in the presence of CSF. CONCLUSION: Heterogeneous tumour interactions within the brain microenvironment enable progression on immune and targeted therapies and should be targeted in salvage treatments

Distinct subclonal tumour responses to therapy revealed by circulating cell-free DNA.

BACKGROUND: The application of precision medicine in oncology requires in-depth characterisation of a patient's tumours and the dynamics of their responses to treatment. PATIENTS AND METHODS: We used next-generation sequencing of circulating cell-free DNA (cfDNA) to monitor the response of a KIT p.L576P-mutant metastatic vaginal mucosal melanoma to sequential targeted, immuno- and chemotherapy. RESULTS: Despite a KIT mutation, the response to imatinib was mixed. Unfortunately, tumours were not accessible for molecular analysis. To study the mechanism underlying the mixed clinical response, we carried out whole-exome sequencing and targeted longitudinal analysis of cfDNA. This revealed two tumour subclones; one with a KIT mutation that responded to imatinib and a second KIT-wild-type subclone that did not respond to imatinib. Notably, the subclones also responded differently to immunotherapy. However, both subclones responded to carboplatin/paclitaxel, and although the KIT-wild-type subclone progressed after chemotherapy, it responded to subsequent re-administration of paclitaxel. CONCLUSION: We show that cfDNA can reveal tumour evolution and subclonal responses to therapy even when biopsies are not available

B-RAF Mutant Alleles Associated with Langerhans Cell Histiocytosis, a Granulomatous Pediatric Disease

Langerhans cell histiocytosis (LCH) features inflammatory granuloma characterised by the presence of CD1a+ dendritic cells or 'LCH cells'. Badalian-Very et al. recently reported the presence of a canonical (V600E)B-RAF mutation in 57% of paraffin-embedded biopsies from LCH granuloma. Here we confirm their findings and report the identification of two novel B-RAF mutations detected in LCH patients.Mutations of B-RAF were observed in granuloma samples from 11 out of 16 patients using 'next generation' pyrosequencing. In 9 cases the mutation identified was (V600E)B-RAF. In 2 cases novel polymorphisms were identified. A somatic (600DLAT)B-RAF insertion mimicked the structural and functional consequences of the (V600E)B-RAF mutant. It destabilized the inactive conformation of the B-RAF kinase and resulted in increased ERK activation in 293 T cells. The (600DLAT)B-RAF and (V600E)B-RAF mutations were found enriched in DNA and mRNA from the CD1a+ fraction of granuloma. They were absent from the blood and monocytes of 58 LCH patients, with a lower threshold of sequencing sensitivity of 1%-2% relative mutation abundance. A novel germ line (T599A)B-RAF mutant allele was detected in one patient, at a relative mutation abundance close to 50% in the LCH granuloma, blood monocytes and lymphocytes. However, (T599A)B-RAF did not destabilize the inactive conformation of the B-RAF kinase, and did not induce increased ERK phosphorylation or C-RAF transactivation.Our data confirmed presence of the (V600E)B-RAF mutation in LCH granuloma of some patients, and identify two novel B-RAF mutations. They indicate that (V600E)B-RAF and (600DLAT)B-RAF mutations are somatic mutants enriched in LCH CD1a(+) cells and absent from the patient blood. Further studies are needed to assess the functional consequences of the germ-line (T599A)B-RAF allele

Genomic Evaluation of Multiparametric Magnetic Resonance Imaging-visible and -nonvisible Lesions in Clinically Localised Prostate Cancer

Background The prostate cancer (PCa) diagnostic pathway is undergoing a radical change with the introduction of multiparametric magnetic resonance imaging (mpMRI), genomic testing, and different prostate biopsy techniques. It has been proposed that these tests should be used in a sequential manner to optimise risk stratification. Objective To characterise the genomic, epigenomic, and transcriptomic features of mpMRI-visible and -nonvisible PCa in clinically localised disease. Design, setting, and participants Multicore analysis of fresh prostate tissue sampled immediately after radical prostatectomy was performed for intermediate- to high-risk PCa. Intervention Low-pass whole-genome, exome, methylation, and transcriptome profiling of patient tissue cores taken from microscopically benign and cancerous areas in the same prostate. Circulating free and germline DNA was assessed from the blood of five patients. Outcome measurement and statistical analysis Correlations between preoperative mpMRI and genomic characteristics of tumour and benign prostate samples were assessed. Gene profiles for individual tumour cores were correlated with existing genomic classifiers currently used for prognostication. Results and limitations A total of 43 prostate cores (22 tumour and 21 benign) were profiled from six whole prostate glands. Of the 22 tumour cores, 16 were tumours visible and six were tumours nonvisible on mpMRI. Intratumour genomic, epigenomic, and transcriptomic heterogeneity was found within mpMRI-visible lesions. This could potentially lead to misclassification of patients using signatures based on copy number or RNA expression. Moreover, three of the six cores obtained from mpMRI-nonvisible tumours harboured one or more genetic alterations commonly observed in metastatic castration-resistant PCa. No circulating free DNA alterations were found. Limitations include the small cohort size and lack of follow-up. Conclusions Our study supports the continued use of systematic prostate sampling in addition to mpMRI, as avoidance of systematic biopsies in patients with negative mpMRI may mean that clinically significant tumours harbouring genetic alterations commonly seen in metastatic PCa are missed. Furthermore, there is inconsistency in individual genomics when genomic classifiers are applied. Patient summary Our study shows that tumour heterogeneity within prostate tumours visible on multiparametric magnetic resonance imaging (mpMRI) can lead to misclassification of patients if only one core is used for genomic analysis. In addition, some cancers that were missed by mpMRI had genomic aberrations that are commonly seen in advanced metastatic prostate cancer. Avoiding biopsies in mpMRI-negative cases may mean that such potentially lethal cancers are missed

Targeting BRAF for patients with melanoma

The prognosis of patients with metastatic melanoma is poor and not influenced by systemic therapy with cytotoxic drugs. New targeted agents directed against the RAS-RAF-MEK-ERK pathway show promising activity in early clinical development and particular interest is focused on selective inhibitors of mutant BRAF, which is present in one half of the cases of metastatic melanoma. The majority of patients on early trials of these drugs develop secondary resistance and subsequent disease progression and it is, therefore, critical to understand the underlying escape mechanisms leading to resistance

Phase II study of the farnesyltransferase inhibitor R115777 in advanced melanoma (CALGB 500104)

BACKGROUND: Multiple farnesylated proteins are involved in signal transduction in cancer. Farnesyltransferase inhibitors (FTIs) have been developed as a strategy to inhibit the function of these proteins. As FTIs inhibit proliferation of melanoma cell lines, we undertook a study to assess the impact of a FTI in advanced melanoma. As farnesylated proteins are also important for T cell activation, measurement of effects on T cell function was also pursued. METHODS: A 3-stage trial design was developed with a maximum of 40 patients and early stopping if there were no responders in the first 14, or fewer than 2 responders in the first 28 patients. Eligibility included performance status of 0–1, no prior chemotherapy, at most 1 prior immunotherapy, no brain metastases, and presence of at least 2 cutaneous lesions amenable to biopsy. R115777 was administered twice per day for 21 days of a 28-day cycle. Patients were evaluated every 2 cycles by RECIST. Blood and tumor were analyzed pre-treatment and during week 7. RESULTS: Fourteen patients were enrolled. Two patients had grade 3 toxicities, which included myelosuppression, nausea/vomiting, elevated BUN, and anorexia. There were no clinical responses. All patients analyzed showed potent inhibition of FT activity (85-98%) in tumor tissue; inhibition of phosphorylated ERK and Akt was also observed. T cells showed evidence of FT inhibition and diminished IFN-γ production. CONCLUSIONS: Despite potent target inhibition, R115777 showed no evidence of clinical activity in this cohort of melanoma patients. Inhibition of T cell function by FTIs has potential clinical implications. Clinicaltrials.gov number NCT0006012