Development and evaluation of a colorimetric membrane-array method for the detection of circulating tumor cells in the peripheral blood of Taiwanese patients with colorectal cancer

Abstract

Abstract. Early detection is the hallmark of successful cancer treatment. Evidence is accumulating that primary cancers begin shedding neoplastic cells in the circulation at an early stage. To date, a high-sensitivity and high-throughput method for the detection of circulating tumor cells (CTCs) is deficient. In this study, we have developed a high-sensitivity colorimetric membrane-array method to detect CTCs in the peripheral blood of colorectal cancer (CRC) patients as a potential diagnostic tool. Previously, we identified a set of 18 oligonucleotide clones, significantly overexpressed in CRC, which were synthesized and applied to a nylon membrane. Digoxigenin (DIG)-labeled cDNA were amplified by reverse transcriptasepolymerase chain reaction (RT-PCR) from the peripheral blood of 88 Taiwanese CRC patients and 50 healthy subjects, and were then hybridized to the membrane-array. Hybridization signals were detected by color development. Meanwhile, blood samples were analyzed by real-time quantitative PCR (Q-PCR). Subsequently, both methods were compared regarding their correlation, sensitivity and specificity in the detection of CTCs by statistics. The results of membrane-arrays were demonstrated to be closely related to that of Q-PCR (P<0.001). The sensitivity and specificity of membrane-arrays for the detection of CTCs were 94.3% (95% CI, 86.4-102.2%) and 94% (95% CI, 85.9-102.1%), respectively. Moreover, the accuracy of membrane-arrays is higher than that of any one gene by Q-PCR. The detection rate of membrane-arrays was significantly associated with the depth of tumor invasion (P=0.002), the presence of lymph node metastasis (P=0.016), and TNM stage (P=0.005). The preliminary results indicated that the accuracy of membrane-arrays was sufficient to distinguish Taiwanese CRC patients from normal individuals with the advantages of time-saving, cost-effectiveness and high-throughput. Thus, the constructed colorimetric membranearray could be a promising approach for the future detection of CTCs.

Significance of the glycolytic pathway and glycolysis related-genes in tumorigenesis of human colorectal cancers

“…Previously, we have observed that constructed membranearray techniques can be effective and efficient for the confirmation of overexpression of related genes in human cancerous tissues (9)(10)(11). Recently, we have demonstrated that fatty acid metabolism plays an important role in turmorigenesis of human CRCs by microarray-bioinformatics analysis (12).…”

Clinical Application of Automatic Gene Chip Analyzer (WEnCA-Chipball) for Mutant Kras Detection in Peripheral Circulating Cancer Cells of Cancer Patients

“…The traditional techniques such as direct sequencing, polymerase chain reaction and restriction fragment length polymorphism are complicated and can easily be used only in tissue samples, which limits KRAS mutation detection in clinical applications. In order to improve the mutant KRAS detection efficiency, we successfully developed an Activating KRAS Detection Chip and colorimetric membrane array (CLMA) technique capable of detecting KRAS mutation status by screening circulating carcinoma cells in the surrounding bloodstream (Chen et al, 2005;Wang et al, 2006;Chong et al, 2007;Yen et al, 2009;Yang et al, 2010). However, the sensitivity still needs further improvement.…”

“…However, as the technical threshold of chip array remained relatively high, human errors during clinical examinations were commonly seen, and the propagation of associating operations somehow became restricted. The analysis of gene overexpression has led to fundamental progress and clinical advances in the diagnosis of disease (Chen et al, 2005;Wang et al, 2006). The techniques that are commonly used to study gene overexpression include Northern blot, reverse transcriptase polymerase chain reaction (RT-PCR), and real-time PCR (Chong et al, 2007;Yen et al, 2009;Yanget al, 2009).…”

A prospective study comparing multi-gene biomarker chip and serum carcinoembryonic antigen in the postoperative surveillance for patients with stage I–III colorectal cancer

“…The development of recurrent or metastatic lesions was defined as postoperative relapse. CTCs in the peripheral blood were detected using our previously constructed multigene biomarker chip with serial CEA assays at each follow-up [7,[12][13][14][15]. Additional 4 mL samples of peripheral blood were obtained for total RNA isolation.…”

“…Reports have described the detection of circulating tumor cells (CTCs) in the peripheral blood of CRC patients; this method has major prognostic and therapeutic implications [7][8][9][10]. Our recently developed membrane array-based multigene biomarker assay can detect CTCs in the peripheral blood of CRC patients; this is a rational approach for the surveillance of postoperative CRC patients [6,[11][12][13][14][15]. However, a detailed prospective comparative study regarding the diagnostic accuracy of the biomarker chip and serum CEA level detection is required.…”