Assessment of a six gene panel for the molecular detection of circulating tumor cells in the blood of female cancer patients

Abstract

Background: The presence of circulating tumor cells (CTC) in the peripheral blood of cancer patients has been described for various solid tumors and their clinical relevance has been shown. CTC detection based on the analysis of epithelial antigens might be hampered by the genetic heterogeneity of the primary tumor and loss of epithelial antigens. Therefore, we aimed to identify new gene markers for the PCR-based detection of CTC in female cancer patients.

Methods: Gene expression of 38 cancer cell lines (breast, ovarian, cervical and endometrial) and of 10 peripheral blood mononuclear cell (PBMC) samples from healthy female donors was measured using microarray technology (Applied Biosystems). Differentially expressed genes were identified using the maxT test and the 50% one-sided trimmed maxT-test. Confirmatory RT-qPCR was performed for 380 gene targets using the AB TaqMan® Low Density Arrays. Then, 93 gene targets were analyzed using the same RT-qPCR platform in tumor tissues of 126 patients with primary breast, ovarian or endometrial cancer. Finally, blood samples from 26 healthy women and from 125 patients (primary breast, ovarian, cervical, or endometrial cancer, and advanced breast cancer) were analyzed following OncoQuick enrichment and RNA pre-amplification. Likewise, hMAM and EpCAM gene expression was analyzed in the blood of breast and ovarian cancer patients. For each gene, a cut-off threshold value was set at three standard deviations from the mean expression level of the healthy controls to identify potential markers for CTC detection.

Results: Six genes were over-expressed in blood samples from 81% of patients with advanced and 29% of patients with primary breast cancer. EpCAM gene expression was detected in 19% and 5% of patients, respectively, whereas hMAM gene expression was observed in the advanced group (39%) only. Multimarker analysis using the new six gene panel positively identified 44% of the cervical, 64% of the endometrial and 19% of the ovarian cancer patients.

Conclusions: The panel of six genes was found superior to EpCAM and hMAM for the detection of circulating tumor cells in the blood of breast cancer, and they may serve as potential markers for CTC derived from endometrial, cervical, and ovarian cancers.

Figure 1

Graphical scheme of the experimental plan. Following a step down strategy, six genes from initially 27.686 genes were identified as new gene markers for the RT-qPCR based detection of circulating tumor cells (CTC). In microarray analysis, we compared expression profiles of PBMC isolated by Ficoll gradient centrifugation from healthy individuals and various established cancer cell lines. In microarray validation, we compared expression profiles of PBMC isolated by Oncoquick from healthy individuals and cell lines. cDNA was amplified according to a published protocol [25]. For the experimental analysis of patients samples, we used Oncoquick only. cDNA was amplified using the TargetAmp™1-Round aRNA Amplification Kit.

Figure 2

Sensitivity of RT-qPCR using TLDA platform. Expression levels of 93 candidate genes were analyzed using cDNA generated from total RNA isolated from peripheral blood samples from a healthy female donor and the same blood spiked with 4, 40 and 400 T47-D tumor cells after cell enrichment. RNA was pre-amplified using the TargetAmp™1-Round aRNA Amplification Kit. Average C_t values obtained from RT-qPCR amplification of CCNE2, DKFZp762E1312, EMP2, MAL2, PPIC, and SLC6A8 transcripts using the TLDA format are shown. MAL2 and CCNE2 gene expression was below the detection limit of RT-qPCR in the unspiked blood. The detection sensitivity of the respective marker gene was estimated to be 40 and 400 tumor cells per 15 ml whole blood.

Figure 3

RT-qPCR analysis of marker gene expression in peripheral blood. Gene expression was analyzed in blood samples taken from patients (triangles) with recurrent breast cancer (A), and in blood samples taken at first diagnosis from breast (B), endometrial (C), cervical (D) and ovarian (E) cancer patients. Blood from healthy females (circles) served as a control group. Mononuclear cells were enriched with the Oncoquick density gradient. RT-qPCR was performed following a RNA pre-amplification step. Average C_t values obtained from duplicates were normalized to GAPDH gene expression. Cut-off threshold values calculated from the mean average normalized gene expression in healthy female blood as indicated by horizontal lines for the respective gene markers (DKFZp762E1312 1.39, SLC6A8 2.92, PPIC 3.61, EMP2 6.84, MAL2 14.61, CCNE2 16.83).