A rapid nested polymerase chain reaction method to detect circulating cancer cells in breast cancer patients using multiple marker genes

Abstract

The aim of the present study was to develop a simple and rapid method for the detection of circulating cancer cells using multiple tumor markers and to investigate the clinical significance of circulating cancer cells in breast cancer patients. A novel rapid nested polymerase chain reaction (PCR) assay, with high sensitivity and specificity, was evaluated, which was considered to be suitable for clinical application. The rapid nested PCR method was used to detect the circulating cancer cells of 142 breast cancer patients, using a panel of marker genes (FAM83A, NPY1R and KRT19), which were identified by the Digital Gene Expression Displayer Tool of the National Cancer Institute-Cancer Genome Anatomy Project. In total, 79.6% of the 142 breast cancer patient blood samples were found to express at least one tumor marker. In addition, the number of positive markers was found to significantly correlate with the disease stage and presence of distant metastasis. Furthermore, positivity for more than one tumor marker appeared to predict a reduced survival time in breast cancer patients.

Keywords: breast cancer; circulating cancer cells; nested polymerase chain reaction; tumor marker.

Figure 1

Evaluation of the rapid nested polymerase chain reaction (PCR). (A) The same detection rate was achieved using the (a) novel rapid and the (b) traditional nested PCR assays. Lanes 1–15, breast cancer patients; lane 16, healthy donor; and lane 17, negative control. (B) Sensitivity of the (a) novel rapid and the (b) traditional nested PCR assays. Lanes 1–7, 10-fold serial dilution of MCF-7 using peripheral blood mononuclear cells obtained from healthy donors (Lane 1, 10²; lane, 2 10³; lane 3, 10⁴; lane 4, 10⁵; lane 5, 10⁶; lane 6, 10⁷; and lane 7, 10⁸ cells); and lane 8, negative control. (C) Amplification using outer and inner primers. Lanes 1 and 2, outer primers only; lanes 3 and 4, outer and inner primers; and lanes 5 and 6, inner primers only.

Figure 2

Analysis of positive detection rates with a multimarker gene panel. The positive detection rate for (A) the three individual marker genes. (B) The positive detection rate increased with the panel of markers.

Figure 3

Kaplan-Meier survival analysis for breast cancer patients, grouped according to the number of tumor markers detected in the peripheral blood.