Abstract
IntroductionComparison studies between different analytical methodologies for circulating tumor cells (CTC) detection and molecular characterization are urgently needed, since standardization of assays is essential before their use in clinical practice.MethodsWe compared three different CTC molecular assays. To avoid discrepancies due to pre-analytical errors we used the same cDNAs throughout our study. CTC were isolated using anti-EpCAM and anti-MUC1 coated magnetic beads from 2 × 5 ml of peripheral blood of 254 early and 51 metastatic breast cancer patients and 30 healthy individuals. The same cDNAs were analyzed by: a) singleplex RT-qPCR assay for CK-19; b) multiplex RT-qPCR for CK-19, HER-2, MAGE- A3, and PBGD; and c) a commercially available molecular assay (AdnaTest BreastCancer) for GA733-2, MUC-1, HER-2 and beta-actin.ResultsIn early breast cancer, CK-19 RT-qPCR, multiplex RT-qPCR and the AdnaTest, were positive for the presence of CTC in 14.2%, 22.8% and 16.5% subjects, respectively. The concordance between the AdnaTest and CK-19 RT-qPCR was 72.4% while between the AdnaTest and multiplex RT-qPCR was 64.6%. In patients with overt metastasis, CK-19 RT-qPCR, multiplex RT-qPCR and the AdnaTest were positive in 41.2%, 39.2% and 54.9% patients, respectively. The concordance between the AdnaTest and CK-19 RT-qPCR was 70.6% while between the AdnaTest and multiplex RT-qPCR was 68.6%.ConclusionsAll CTC assays gave similar results in about 70% of cases. Better agreement was found in the metastatic setting, possibly explained by the higher tumor load in this group. Discordances could be attributed to the different gene transcripts used to evaluate CTC positivity. Our results indicate the importance of CTC heterogeneity for their detection by different analytical methodologies.
Highlights
Comparison studies between different analytical methodologies for circulating tumor cells (CTC) detection and molecular characterization are urgently needed, since standardization of assays is essential before their use in clinical practice
In the group of early breast cancer patients, CTC were isolated according to the AdnaTest BreastCancerTM system from 2 × 5 mL peripheral blood and detected in 36/254 patients by cytokeratin 19 (CK-19) RTqPCR (14.2%), while in the group of 51 patients with overt metastasis, CTC were detected in 21 patients (41.2%) (Table 2)
Quality control is an important issue for the clinical use of CTC analysis, and standardization of CTC detection and characterization methodologies are important for the incorporation of CTC into prospective clinical trials testing their clinical utility [26]
Summary
Comparison studies between different analytical methodologies for circulating tumor cells (CTC) detection and molecular characterization are urgently needed, since standardization of assays is essential before their use in clinical practice. Molecular assays are highly sensitive, easy to perform, detect viable CTC, have the advantage of in silico design, and can be automated and subjected to internal and external quality control systems. Another major advantage of molecular assays for CTC detection is the flexibility they offer, especially in a multiplex format, where we can significantly reduce the amount of precious CTC sample, as well as the time and cost of analysis. Few molecular markers provide adequate sensitivity individually, but combinations of markers may offer better sensitivity for CTC detection
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