Comparison of three molecular assays for the detection and molecular characterization of circulating tumor cells in breast cancer

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.

A Comparison of Three Methods for the Detection of Circulating Tumor Cells in Patients with Early and Metastatic Breast Cancer

“…The complexity of comparison between different assays regarding CTC prevalence is nicely depicted in the report by Strati et al evaluating three molecular assays for CTC detection; a singleplex RT-qPCR assay for CK-19, a multiplex RT-qPCR for CK-19, HER-2, MAGE- A3, and PBGD and the AdnaTest Breast Cancer [37]. Importantly, in that study, pro-analytical and important analytical variables were kept the same in order to obtain a more precise comparison.…”

Biomarker Associations with Efficacy of Abiraterone Acetate and Exemestane in Postmenopausal Patients with Estrogen Receptor–Positive Metastatic Breast Cancer

“…The methods used in these analyses also did not allow for multiplex testing, which would have enabled multiple biomarkers to be measured simultaneously (16). This may affect data accuracy and appearance of discordance, as only a few individual molecular markers provide adequate sensitivity for CTC detection.…”

Hydrogen Peroxide-Responsive Nanoprobe Assists Circulating Tumor Cell Identification and Colorectal Cancer Diagnosis

“…6,7 After enrichment, thus far, identification and molecular analysis of CTCs majorly rely on the genetic and surface antigen analysis, such as genomic DNA sequencing, 8 determination of DNA and RNA methylation, 9 expression of CK19, HER2, MAGE-A3, PBGD on cell membrane, etc. 10,11 Although these methods show many merits, their drawbacks cannot be ignored. For instance, in genetic analysis, direct DNA sequencing of rare CTC is difficult unless DNA amplification is performed.…”