Monitoring changes in circulating tumour cells as a prognostic indicator of overall survival and treatment response in patients with metastatic melanoma.

BACKGROUND: New 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. METHODS: CTCs 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. RESULTS: Baseline 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. CONCLUSIONS: Measuring 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.

The Fallacy of Misplaced Presumption.

One takes one's word that p when a source vouches for p and one accepts the word of that source. If the source is reliable in this case, p is acceptable. The reliability of the source is a measure of its plausibility. If a source has the relevant competence, credibility, authority, that word is acceptable. Likewise, the word may be acceptable if accompanied by a cogent argument, but presumption may be misplaced. One may recognize a presumption for a statement when such recognition is not justified, the positive version of the fallacy. One may refuse to recognize a presumption for a statement when there really is a presumption for the statement, the negative version of the fallacy. The essay proceeds to explore various dimensions of when it is justified to take a source's word for a claim, and when it is justified to reject a claim from a source. The discussion ranges over considerations of sexism and race, cultural differences, and the relationship of presumptions to fallacies. Also considered is the role of trust in taking someone's word and the factors involved in trusting someone.

Evaluation of a multi-marker immunomagnetic enrichment assay for the quantification of circulating melanoma cells.

BACKGROUND: Circulating melanoma cells (CMCs) are thought to be valuable in improving measures of prognosis in melanoma patients and may be a useful marker of residual disease to identify non-metastatic patients requiring adjuvant therapy. We investigated whether immunomagnetic enrichment targeting multiple markers allows more efficient enrichment of CMCs from patient peripheral blood than targeting a single marker. Furthermore, we aimed to determine whether the number of CMCs in patient blood was associated with disease stage. METHODS: We captured CMCs by targeting the melanoma associated markers MCSP and MCAM as well as the melanoma stem cell markers ABCB5 and CD271, both individually and in combination, by immunomagnetic enrichment. CMCs were enriched and quantified from the peripheral blood of 10 non-metastatic and 13 metastatic melanoma patients. RESULTS: Targeting all markers in combination resulted in the enrichment of more CMCs than when any individual marker was targeted (p < 0.001-0.028). Furthermore, when a combination of markers was targeted, a greater number of CMCs were enriched in metastatic patients compared with non-metastatic patients (p = 0.007). CONCLUSIONS: Our results demonstrated that a combination of markers should be targeted for optimal isolation of CMCs. In addition, there are significantly more CMCs in metastatic patients compared with non-metastatic patients and therefore quantification of CMCs may prove to be a useful marker of disease progression.

Immunomagnetic-Enriched Subpopulations of Melanoma Circulating Tumour Cells (CTCs) Exhibit Distinct Transcriptome Profiles.

Cutaneous melanoma circulating tumour cells (CTCs) are phenotypically and molecularly heterogeneous. We profiled the gene expression of CTC subpopulations immunomagnetic-captured by targeting either the melanoma-associated marker, MCSP, or the melanoma-initiating marker, ABCB5. Firstly, the expression of a subset of melanoma genes was investigated by RT-PCR in MCSP-enriched and ABCB5-enriched CTCs isolated from a total of 59 blood draws from 39 melanoma cases. Of these, 6 MCSP- and 6 ABCB5-enriched CTC fractions were further analysed using a genome-wide gene expression microarray. The transcriptional programs of both CTC subtypes included cell survival maintenance, cell proliferation, and migration pathways. ABCB5-enriched CTCs were specifically characterised by up-regulation of genes involved in epithelial to mesenchymal transition (EMT), suggesting an invasive phenotype. These findings underscore the presence of at least two distinct melanoma CTC subpopulations with distinct transcriptional programs, which may have distinct roles in disease progression and response to therapy.

Clinical Application of Circulating Tumor Cells and Circulating Tumor DNA in Uveal Melanoma.

PURPOSE: To evaluate the feasibility of using circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) for the management of uveal melanoma (UM). PATIENTS AND METHODS: Low-coverage whole-genome sequencing was used to determine somatic chromosomal copy number alterations (SCNAs) in primary UM tumors, ctDNA, and whole-genome amplified CTCs. CTCs were immunocaptured using an antimelanoma-associated chondroitin sulfate antibody conjugated to magnetic beads and immunostained for melanoma antigen recognised by T cells 1 (MART1)/glycoprotein 100 (gp100)/S100 calcium-binding protein ß (S100ß). ctDNA was quantified using droplet digital polymerase chain reaction assay for mutations in the GNAQ, GNA11, PLCß4, and CYSLTR2 genes. RESULTS: SCNA analysis of CTCs and ctDNA isolated from a patient with metastatic UM showed good concordance with the enucleated primary tumor. In a cohort of 30 patients with primary UM, CTCs were detected in 58% of patients (one to 37 CTCs per 8 mL of blood), whereas only 26% of patients had detectable ctDNA (1.6 to 29 copies/mL). The presence of CTCs or ctDNA was not associated with tumor size or other prognostic markers. However, the frequent detection of CTCs in patients with early-stage UM supports a model in which CTCs can be used to derive tumor-specific SCNA relevant for prognosis. Monitoring of ctDNA after treatment of the primary tumor allowed detection of metastatic disease earlier than 18F-labeled fluorodeoxyglucose positron emission tomography in two patients. CONCLUSION: The presence of CTCs in localized UM can be used to ascertain prognostic SCNA, whereas ctDNA can be used to monitor patients for early signs of metastatic disease. This study paves the way for the analysis of CTCs and ctDNA as a liquid biopsy that will assist with treatment decisions in patients with UM.

Isolation and detection of circulating tumour cells from metastatic melanoma patients using a slanted spiral microfluidic device.

Circulating Tumour Cells (CTCs) are promising cancer biomarkers. Several methods have been developed to isolate CTCs from blood samples. However, the isolation of melanoma CTCs is very challenging as a result of their extraordinary heterogeneity, which has hindered their biological and clinical study. Thus, methods that isolate CTCs based on their physical properties, rather than surface marker expression, such as microfluidic devices, are greatly needed in melanoma. Here, we assessed the ability of the slanted spiral microfluidic device to isolate melanoma CTCs via label-free enrichment. We demonstrated that this device yields recovery rates of spiked melanoma cells of over 80% and 55%, after one or two rounds of enrichment, respectively. Concurrently, a two to three log reduction of white blood cells was achieved with one or two rounds of enrichment, respectively. We characterised the isolated CTCs using multimarker flow cytometry, immunocytochemistry and gene expression. The results demonstrated that CTCs from metastatic melanoma patients were highly heterogeneous and commonly expressed stem-like markers such as PAX3 and ABCB5. The implementation of the slanted microfluidic device for melanoma CTC isolation enables further understanding of the biology of melanoma metastasis for biomarker development and to inform future treatment approaches.

Detection of BRAF-V600E and V600K in melanoma circulating tumour cells by droplet digital PCR.

OBJECTIVES: Defining the BRAF mutation status in metastatic melanoma patients is critical to selecting patients for therapeutic treatment with targeted therapies. Circulating tumour cells (CTCs) can provide an alternative source of contemporaneous tumour genetic material. However methodologies to analyse the presence of rare mutations in a background of wild-type DNA requires a detailed assessment. Here we evaluate the sensitivity of two technologies for cancer mutation detection and the suitability of whole genome amplified DNA as a template for the detection of BRAF-V600 mutations. DESIGN AND METHODS: Serial dilutions of mutant BRAF-V600E DNA in wild-type DNA were tested using both competitive allele-specific PCR (castPCR) and droplet digital PCR (ddPCR), with and without previous whole genome amplification (WGA). Using immunomagnetic beads, we partially enriched CTCs from blood obtained from metastatic melanoma patients with confirmed BRAF mutation positive tumours and extracted RNA and DNA from the CTCs. We used RT-PCR of RNA to confirm the presence of melanoma cells in the CTC fraction then the DNAs of CTC positive fractions were WGA and tested for BRAF V600E or V600K mutations by ddPCRs. RESULTS: WGA DNA produced lower than expected fractional abundances by castPCR analysis but not by ddPCR. Moreover, ddPCR was found to be 200 times more sensitive than castPCR and in combination with WGA produced the most concordant results, with a limit of detection of 0.0005%. BRAF-V600E or V600K mutated DNA was detected in 77% and 44%, respectively, of enriched CTC fractions from metastatic melanoma patients carrying the corresponding mutations. CONCLUSIONS: Our results demonstrate that using ddPCR in combination with WGA DNA allows the detection with high sensitivity of cancer mutations in partially enriched CTC fractions.