Monitoring melanoma recurrence with circulating tumor DNA: a proof of concept from three case studies.

BACKGROUND: A significant number of melanoma patients experience recurrence to distant sites, despite having had surgical treatment of the primary lesion, with curative intent. Monitoring of patients for early evidence of disease recurrence would significantly improve management of the disease, allowing timely therapeutic intervention. Circulating tumor DNA (ctDNA) is becoming a well-recognized biomarker for monitoring malignancies and has, in a few studies, been shown to signify disease recurrence earlier than conventional methods. METHODS: We performed a retrospective analysis of plasma ctDNA using droplet digital PCR (ddPCR) in 30 primary melanoma patients with tumors harboring BRAF, NRAS or TERT promoter mutations. Mutant specific ctDNA, measured during clinical disease course, was compared with disease status in patients with confirmed disease recurrence (n = 3) and in those with no evidence of disease recurrence (n = 27). RESULTS: Mutant specific ctDNA was detected in all three patients with disease recurrence at the time of clinically confirmed progression. In one case, plasma ctDNA detection preceded clinical identification of recurrence by an interval of 4 months. CtDNA was not detected in patients who were asymptomatic and had no radiological evidence of recurrence. CONCLUSIONS: This study demonstrates promising results for the use of ctDNA as an informative monitoring tool for melanoma patients having undergone tumor resection of an early stage primary tumor. The clinical utility of ctDNA for monitoring disease recurrence warrants investigation in prospective studies as it may improve patient outcome.

A diagnostic autoantibody signature for primary cutaneous melanoma.

Melanoma is an aggressive form of skin cancer that is curable by surgical excision in the majority of cases, if detected at an early stage. To improve early stage melanoma detection, the development of a highly sensitive diagnostic test is of utmost importance. Here we aimed to identify antibodies to a panel of tumour associated antigens that can differentiate primary melanoma patients and healthy individuals. A total of 245 sera from primary melanoma patients and healthy volunteers were screened against a high-throughput microarray platform containing 1627 functional proteins. Following rigorous statistical analysis, we identified a combination of 10 autoantibody biomarkers that, as a panel, displays a sensitivity of 79%, specificity of 84% and an AUC of 0.828 for primary melanoma detection. This melanoma autoantibody signature may prove valuable for the development of a diagnostic blood test for routine population screening that, when used in conjunction with current melanoma diagnostic techniques, could improve the early diagnosis of this malignancy and ultimately decrease the mortality rate of patients.

Droplet Digital PCR for Mutation Detection in Formalin-Fixed, Paraffin-Embedded Melanoma Tissues: A Comparison with Sanger Sequencing and Pyrosequencing.

The identification of somatic mutations is crucial for guiding therapeutic decisions about personalized melanoma treatment. However, genetic analysis of tumors is usually performed on limited and often low-quality DNA from tumors with low tumor cellularity and high tumor heterogeneity. Different mutation-detection platforms exist, with varying analytical sensitivities. Here we evaluated the detection of common mutations in BRAF, NRAS, and TERT promoter in 40 melanoma FFPE tissues using Droplet Digital (dd)PCR, and compared the results to the detection rates obtained by Sanger sequencing and pyrosequencing. The cellularity of tumors analyzed ranged from 5% to 50% (n = 28) and 50% to 90% (n = 12). Overall, droplet digital (dd)PCR was more sensitive, detecting mutations in 12.5% and 23% of tumors deemed as wild-type by pyrosequencing and Sanger sequencing, respectively. The increased sensitivity of ddPCR was more apparent among tumors with <50% tumor cellularity. Implementation of ddPCR-based assays may facilitate analysis of early-stage tumors and support research into improving outcomes in melanoma patients.

Segmentation of Skin Tumors in High-Frequency 3-D Ultrasound Images.

High-frequency 3-D ultrasound imaging is an informative tool for diagnosis, surgery planning and skin lesion examination. The purpose of this article was to describe a semi-automated segmentation tool providing easy access to the extent, shape and volume of a lesion. We propose an adaptive log-likelihood level-set segmentation procedure using non-parametric estimates of the intensity distribution. The algorithm has a single parameter to control the smoothness of the contour, and we describe how a fixed value yields satisfactory segmentation results with an average Dice coefficient of D = 0.76. The algorithm is implemented on a grid, which increases the speed by a factor of 100 compared with a standard pixelwise segmentation. We compare the method with parametric methods making the hypothesis of Rayleigh or Nakagami distributed signals, and illustrate that our method has greater robustness with similar computational speed. Benchmarks are made on realistic synthetic ultrasound images and a data set of nine clinical 3-D images acquired with a 50-MHz imaging system. The proposed algorithm is suitable for use in a clinical context as a post-processing tool.

Circulating Melanoma Cell Subpopulations: Their Heterogeneity and Differential Responses to Treatment.

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-κß (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.