Abstract
BackgroundThe 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.MethodsGene 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.ResultsSix 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.ConclusionsThe 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.
Highlights
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
RNA quality assessment Prior to microarray hybridization and RT-qPCR analysis, the RNA extracted from the tumor cell lines and the healthy peripheral blood mononuclear cell (PBMC) was checked for quality with the RNA 6000 Nano LabChip Kit run on the Agilent 2100 Bioanalyzer
Gene expression analysis of patients’ blood samples The expression of the same 93 genes was evaluated in blood samples from healthy female volunteers (N = 26) and in peripheral blood samples from patients with breast (N = 52), ovarian (N = 23), cervical and endometrial cancer (25 patients each), using the TaqMan® Low Density Array (TLDA) 96a RTqPCR platform
Summary
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. It has been shown that normal-like breast cancer cells characterized by aggressive behaviour and worse treatment options are not recognized by the CellSearch circulating tumor cell test (Veridex LLC, San Diego, CA), which uses EpCAM for cell isolation [16] This test is the only diagnostic test that is currently approved by the US Food and Drug Administration for the automated detection and enumeration of circulating tumor cells [17]. The analysis of hMAM (human mammaglobin A), the most widely studied marker after CK19 (cytokeratin 19) in breast cancer patients, gene expression identifies patients with nearly 100% specificity at the same sensitivity as CK19 (1 tumor cell in 106 peripheral blood mononuclear cells) [19,20].
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