Repeat tumor biopsies to study genomic changes during therapy are difficult, invasive and data are confounded by tumoral heterogeneity. The analysis of circulating tumor DNA (ctDNA) can provide a non-invasive approach to assess prognosis and the genetic evolution of tumors in response to therapy. Mutation-specific droplet digital PCR was used to measure plasma concentrations of oncogenic BRAF and NRAS variants in 48 patients with advanced metastatic melanoma prior to treatment with targeted therapies (vemurafenib, dabrafenib or dabrafenib/trametinib combination) or immunotherapies (ipilimumab, nivolumab or pembrolizumab). Baseline ctDNA levels were evaluated relative to treatment response and progression-free survival (PFS). Tumor-associated ctDNA was detected in the plasma of 35/48 (73%) patients prior to treatment and lower ctDNA levels at this time point were significantly associated with response to treatment and prolonged PFS, irrespective of therapy type. Levels of ctDNA decreased significantly in patients treated with MAPK inhibitors (p < 0.001) in accordance with response to therapy, but this was not apparent in patients receiving immunotherapies. We show that circulating NRAS mutations, known to confer resistance to BRAF inhibitors, were detected in 3 of 7 (43%) patients progressing on kinase inhibitor therapy. Significantly, ctDNA rebound and circulating mutant NRAS preceded radiological detection of progressive disease. Our data demonstrate that ctDNA is a useful biomarker of response to kinase inhibitor therapy and can be used to monitor tumor evolution and detect the early appearance of resistance effectors.

Metastatic melanoma is a highly heterogeneous tumor; thus, methods to analyze tumor-derived cells circulating in blood should address this diversity. Taking this into account, we analyzed, using multiparametric flow cytometry, the co-expression of the melanoma markers melanoma cell adhesion molecule and melanoma-associated chondroitin sulphate proteoglycan and the tumor-initiating markers ATP-binding cassette sub-family B member 5 (ABCB5), CD271, and receptor activator of NF-κβ (RANK) in individual circulating tumor cells (CTCs) from 40 late-stage (III–IV) and 16 early-stage (I–II) melanoma patients. CTCs were heterogeneous within and between patients, with limited co-expression between the five markers analyzed. Analysis of patient matched blood and metastatic tumors revealed that ABCB5 and RANK subpopulations are more common among CTCs than in the solid tumors, suggesting a preferential selection for these cells in circulation. Pairwise comparison of CTC subpopulations longitudinally before and 6–13 weeks after treatment initiation showed that the percentage of RANK+ CTCs significantly increased in the patients undergoing targeted therapy (N=16, P<0.01). Moreover, the presence of ⩾5 RANK+ CTCs in the blood of patients undergoing targeted therapies was prognostic of shorter progression-free survival (hazards ratio 8.73, 95% confidence interval 1.82–41.75, P<0.01). Taken together, our results provide evidence of the heterogeneity among CTC subpopulations in melanoma and the differential response of these subpopulations to targeted therapy.

Summary Background Multimarker quantitative real-time polymerase chain reaction (qRT-PCR) represents an effective method for detecting circulating tumour cells in the peripheral blood of patients with melanoma. Objectives To investigate whether the phenotype of circulating melanoma cells represents a useful indicator of disease stage, recurrence and treatment efficacy. Methods Peripheral blood was collected from 230 patients with melanoma and 152 healthy controls over a period of 3 years and 9 months. Clinical data and blood samples were collected from patients with primary melanoma (early stages, 0–II, n = 154) and metastatic melanoma (late stages, III–IV, n = 76). Each specimen was examined by qRT-PCR analysis for the expression of five markers: MLANA, ABCB5, TGFβ2, PAX3d and MCAM. Results In total, 212 of the patients with melanoma (92%) expressed markers in their peripheral blood. Two markers, MLANA and ABCB5, had the greatest prognostic value, and were identified as statistically significant among patients who experienced disease recurrence within our study period, being expressed in 45% (MLANA) and 49% (ABCB5) of patients with recurrence (P = 0·001 and P = 0·031, respectively). For patients administered nonsurgical treatments, MCAM expression correlated with poor treatment outcome. Conclusions Circulating tumour cells were detectable at all stages of disease and long after surgical treatment, even when patients were considered disease free. Specifically, expression of ABCB5 and MLANA had significant prognostic value in inferring disease recurrence, while MCAM expression was associated with poor patient outcome after treatment, confirming multimarker qRT-PCR as a potential technique for monitoring disease status.

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Background PD‐1 inhibitors are routinely used for the treatment of advanced melanoma. This study sought to determine whether PD‐L1 expression on circulating tumor cells (CTCs) can serve as a predictive biomarker of clinical benefit and response to treatment with the PD‐1 inhibitor pembrolizumab. Methods Blood samples were collected from patients with metastatic melanoma receiving pembrolizumab, prior to treatment and 6–12 weeks after initiation of therapy. Multiparametric flow cytometry was used to identify CTCs and evaluate the expression of PD‐L1. Results CTCs were detected in 25 of 40 patients (63%). Patients with detectable PD‐L1+ CTCs (14/25, 64%) had significantly longer progression‐free survival (PFS) compared with patients with PD‐L1− CTCs (26.6 months vs. 5.5 months; p = .018). The 12‐month PFS rates were 76% versus 22% in the PD‐L1+ versus PD‐L1− CTCs groups (p = .012), respectively. A multivariate linear regression analysis confirmed that PD‐L1+ CTC is an independent predictive biomarker of PFS (hazard ratio, 0.229; 95% confidence interval, 0.052–1.012; p = .026). Conclusion Our results reveal the potential of CTCs as a noninvasive real‐time biopsy to evaluate PD‐L1 expression in patients with melanoma. PD‐L1 expression on CTCs may be predictive of response to pembrolizumab and longer PFS. Implications for Practice The present data suggest that PD‐L1 expression on circulating tumor cells may predict response to pembrolizumab in advanced melanoma. This needs further validation in a larger trial and, if proven, might be a useful liquid biopsy tool that could be used to stratify patients into groups more likely to respond to immunotherapy, hence leading to health cost savings.

BackgroundNew effective treatments for metastatic melanoma greatly improve survival in a proportion of patients. However biomarkers to identify patients that are more likely to benefit from a particular treatment are needed. We previously reported on a multimarker approach for the detection of heterogenous melanoma circulating tumour cells (CTCs). Here we evaluated the prognostic value of this multimarker quantification of CTCs and investigated whether changes in CTC levels during therapy can be used as a biomarker of treatment response and survival outcomes.MethodsCTCs were captured by targeting the melanoma associated markers MCSP and MCAM as well as the melanoma stem cell markers ABCB5 and CD271. CTCs were quantified in 27 metastatic melanoma patients treated by surgery or with vemurafenib, ipilimumab or dacarbazine. Patients were enrolled prospectively and CTC counts performed at baseline (prior to treatment), during and after treatment.ResultsBaseline CTC numbers were not found to be prognostic of overall survival nor of progression free survival. However, a low baseline CTC number was associated with a rapid response to vemurafenib therapy. A decrease in CTCs after treatment initiation was associated with response to treatment and prolonged overall survival in vemurafenib treated patients.ConclusionsMeasuring changes in CTC numbers during treatment is useful for monitoring therapy response in melanoma patients and for providing prognostic information relating to overall survival. Further studies with larger sample sizes are required to confirm the utility of CTC quantification as a companion diagnostic for metastatic melanoma treatment.

Circulating tumor DNA (ctDNA) may serve as a surrogate to tissue biopsy for noninvasive identification of mutations across multiple genetic loci and for disease monitoring in melanoma. In this study, we compared the mutation profiles of tumor biopsies and plasma ctDNA from metastatic melanoma patients using custom sequencing panels targeting 30 melanoma‐associated genes. Somatic mutations were identified in 20 of 24 melanoma biopsies, and 16 of 20 (70%) matched‐patient plasmas had detectable ctDNA. In a subgroup of seven patients for whom matching tumor tissue and plasma were sequenced, 80% of the mutations found in tumor tissue were also detected in ctDNA. However, TERT promoter mutations were only detected by ddPCR, and promoter mutations were consistently found at lower concentrations than other driver mutations in longitudinal samples. In vitro experiments revealed that mutations in promoter regions of TERT and DPH3 are underrepresented in ctDNA. While the results underscore the utility of using ctDNA as an alternative to tissue biopsy for genetic profiling and surveillance of the disease, our study highlights the underrepresentation of promoter mutations in ctDNA and its potential impact on quantitative liquid biopsy applications.

Please cite this article as: Reid, A., Lymbery, A., Ng, J., Tweedle, S., Ryan, U., Identification of novel and zoonotic Cryptosporidium species in marine fish, Veterinary Parasitology (2008Parasitology ( ), doi:10.1016Parasitology ( /j.vetpar.2009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Page 1 of 21A c c e p t e d M a n u s c r i p t