Molecular markers detecting circulating melanoma cells by reverse transcription polymerase chain reaction: methodological pitfalls and clinical relevance

Abstract

Herein, we expound the theory of circulating melanoma cells (CMCs) and their detection with reverse transcription polymerase chain reaction as a molecular staging approach. We discuss the molecular markers that have been used for CMC detection focusing on the use of these markers for multiplex detection analysis. Finally, we comment on the contradictory data of CMC detection studies in the literature and we propose possible solutions which may contribute to the clinical significance of CMC detection in patient management.

Circulating Serologic and Molecular Biomarkers in Malignant Melanoma

“…However, this approach has several limitations, including problems with interlaboratory reproducibility, such as variability of PCR sensitivities, selection of primers, and different PCR protocols. 71 There are also factors inherent to the tumor biology because false-negative results may occur secondary to the heterogeneity in expression of melanoma antigens, especially if a single marker is used, 88 Many markers are also expressed by normal melanocytes, resulting in false-positive results. 71 Moreover, each melanoma marker determination reflects disease status at the time the sample was collected.…”

“…71 There are also factors inherent to the tumor biology because false-negative results may occur secondary to the heterogeneity in expression of melanoma antigens, especially if a single marker is used, 88 Many markers are also expressed by normal melanocytes, resulting in false-positive results. 71 Moreover, each melanoma marker determination reflects disease status at the time the sample was collected. Therefore, sequential sampling may be important to accommodate for intermittent shedding, but the ideal schedule of testing remains to be determined.…”

“…Therefore, sequential sampling may be important to accommodate for intermittent shedding, but the ideal schedule of testing remains to be determined. 71 Given these limitations, this approach has not been validated for general use to date, although research is ongoing.…”

Detecting Minimal Residual Disease in Neuroblastoma: Still a Ways to Go

“…The widespread use of RQ-PCR analysis for detection of disease-specific prognostic markers in leukemia patients provides an excellent example of the usefulness of this technique in diagnostics and treatment control. The rationale for use of RQ-PCR is far less apparent in investigations of disease-associated mRNA expressed by circulating tumor cells in patients with solid malignancies such as colorectal cancer, lung cancer, and melanoma (11)(12)(13), and at the present time RQ-PCR detection of MRD in neuroblastoma appears to fall in this category. However, further studies using this technology invariably may lead to new findings, as seen in the work of Stutterheim et al Stutterheim et al (4 ) are important because these investigators found all markers except PHOX2B to show at least some expression in CD34ϩ samples.…”

“…Differences in the cell lines used may account for some of the differences in results, because in various neuroblastoma cell lines the mean number of specific target transcripts per cell may vary widely (8 ). Recently Nezos et al (11 ) reviewed methodological pitfalls that may explain differences in results between studies using RQ-PCR in investigations of circulating tumor cells.…”