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

Abstract

Objective Comparison of three different molecular assays for the detection and molecular characterization of circulating tumor cells in breast cancer. Methods The retrospective study compared three different molecular assays to detect CTC in the peripheral blood of 30 healthy individuals and 71 benign breast disease patients and 83 early and 84 metastatic breast cancer patients.All samples were collected at the outpatient, inpatient and physical examination department of Sichuan Provincial People's Hospital from January 2011 to June 2014. The same cDNAs were analyzed by: singleplex RT–qPCR assay for BCL–2, multiplex RT–qPCR for BCL–2, HER–2, HMAM, and a commercially available molecular assay (AdnaTest BreastCancer) for GA733–2, MUC–1, HER–2. The positive of CTC were compared among healthy individuals and benign breast disease patients and breast cancer patients. Chi square test was used to compare the expression of gene markers among the three groups, and the agreement of Kappa test was used to evaluate the method. Results (1) Detection rates of early breast cancer by single RT–qPCR, Adna kits and multiple RT–qPCR were 13.3%, 16.9% and 18.1%, respectively, and the detection of metastatic breast cancer were 31.0%, 42.9% and 35.7%, respectively. There were significant differences in the positive of CTC by three molecular assays between healthy individuals and benign breast disease patients and early breast cancer patients (The test values were 4.235 and 4.301, 5.367 and 5.474, 5.894 and 6.023 respectively, P 0.05). There were significant differences between metastasis breast cancer patients and healthy individuals and benign breast disease patients and early breast cancer patients (The test values were 8.429, 7.553 and 7.061; 10.24, 9.025 and 8.745; 9.658, 8.417 and 8.201 respectively, P<0.05). (2) In early breast cancer: The concordance between AdnaTest and single RT–qPCR was 79.5% while between AdnaTest and multiplex RT–qPCR was 77.1%.No agreement was found among them (The test values were 1.065 and 1.871, P were 0.371 and 0.258). The concordance between single RT–qPCR and multiplex RT–qPCR was 80.7%.No agreement was found between them (The test values was 2.814, P was 0.156). (3) In patients with overt metastasis: The concordance between AdnaTest and single RT–qPCR was 78.6%(The test values was 10.986). While between AdnaTest and multiplex RT–qPCR was 80.9%(The test values was 9.251). Agreements were found among them (P was 0.002 and 0.005 respectively). The concordance between single and multiplex RT–qPCR was 88.1%(The test values was 12.364). Agreement was found between them (P was 0.001). Conclusions No correlations were found among different molecular methods to detect CTC in the early primary breast cancer, but correlations were found in the metastatic breast cancer, suggesting that different rate of CTC caused by the number of CTC and its heterogeneity should be considered to the clinical diagnosis and treatment of breast cancer while molecular method is used.(Chin J Lab Med, 2015, 38: 666–671) Key words: Breast neoplasms; Neoplastic cells, circulating; Tumor markers, biological; Reverse transcriptase polymerase chain reaction