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.
Recently several noninvasive methods have been employed to detect circulating tumor cells (CTCs) in cancer patients. In this study, we have developed a highly sensitive, high-throughput colorimetric membrane-array method that was designed to detect a panel of mRNA markers including human telomerase reverse transcriptase (hTERT), , carcinoembryonic antigen (CEA) and mucin 1 (MUC1) mRNA for the presence of CTCs in the peripheral blood of patients with gastric cancer (GC). Digoxigenin-labeled cDNA targets synthesized following total RNA isolation from peripheral blood samples of 64 GC patients and 80 healthy individuals were subjected to membrane-array hybridization. The results showed that membrane array could positively detect 5 cancer cells/ml of peripheral blood in GC cell-dilution experiments. The sensitivity, specificity and diagnostic accuracy for hTERT, CK-19, CEA and MUC1 mRNA ranged from 78.1% to 82.8%, 76.3% to 85% and 81.3% to 83.3%, respectively. Both CEA and MUC1 mRNA expression was correlated significantly with all malignant biological properties of GC, such as macroscopic type, depth of tumor invasion, lymph-node metastasis, TNM stage and coexisting distant metastasis (all p < 0.05). Using these 4 markers in combination, the sensitivity, specificity and diagnostic accuracy of membrane array were raised to 89.1%, 91.3% and 90.3%, respectively. The expression of all 4 mRNA markers was an independent predictor for postoperative recurrence/metastasis. GC patients with the expression of all the 4 mRNA markers showed a poorer survival rate than those without the expression of any 1 mRNA marker (p 5 0.0223). These findings demonstrated that our membrane-array method could detect CTCs in the circulation of GC patients with considerably high sensitivity and specificity. The identification of CTCs in the peripheral blood may be useful in the auxiliary cancer diagnostics or postoperative surveillance of GC patients for recurrence/metastasis. ' 2006 Wiley-Liss, Inc.Key words: circulating tumor cell; membrane array; gastric cancer; molecular diagnosisThe degrees of tumor penetration in the stomach wall and lymph node metastasis have been used for many decades as the 2 major prognostic determinants for gastric cancer (GC) patients. Unfortunately, despite informative staging of patients with GC, some patients with apparently localized disease at diagnosis will subsequently develop recurrent or metastatic diseases. 1-4 One of the major causes is attributed to the presence of disseminated tumor cells shed from the primary carcinoma into circulation, prior to or during surgery. Even in patients with early GC, blood-born metastasis occurs. 2,5,6 Recent attempts to improve staging include sensitive detection of disseminated tumor cells in the blood or lymph nodes by molecular approaches. Evidence increasingly clarifies that primary cancers begin shedding neoplastic cells into the circulation at an early stage. [7][8][9] It is the dissemination of cancer cells into circulation that is widely accepted as the main cause l...
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