Detection And Clinical Significance Of Circulating Tumor Cells In Peripheral Blood Of Colorectal Cancer Patients Using Cellular Fluorescence Amplification Catalyzed By T7 RNA Polymerase Technique

BackgroundColorectal cancer is one of the most common malignant gastrointestinal diseases in many countries. Curative operation combined with chemotherapy and radiotherapy is the main treatment at present. But in recent decades, the 5-year survival rate has not improved obviously. The most common causes of death are local recurrence and metastasis after radical surgery for colorectal cancer patients. Studies suggested that tumor cells in peripheral blood and bone marrow and lymph nodes were the sources of local recurrence and metastasis and a vital predictor for prognosis and systematic therapy, so it was of clinical important to detect them. At earlier times, general cytology methods were used to detect the tumor cells in peripheral blood and bone marrow, however, these methods needed a large amount of tumor cells for detecting, so the sensitivity was very low. With rapid progress of molecular biology and cell biology and birth of PCR and RT-PCR as well as emergence of many kinds of tumor markers, it becomes possible to detect single circulating tumor cell. In recent years, RT-PCR and other relevant methods have played a predominant role in detecting m RNA in circulating tumor cells of colorectal cancer. These methods have a much higher sensitivity than ever. However, the results from different research groups were very different, and the conclusions they made were even diametrically opposed. Therefore, it is necessary to develop a new method to detect circulating tumor cell of colorectal cancer. In 2006, Zhang and his colleagues developed a new way to detect serum proteins with high sensitivity and specificity, which was called fluorescence amplification catalyzed by T7 RNA polymerase technique (FACTT). We use FACTT to detect single circulating tumor cell in the peripheral blood of colorectal cancer patients called Cellular FACTT and investigate the relationship between the rate of circulating tumor cell and clinical pathological factors and its clinical importance.MethodPartâ… To construct a model in vitro of circulating tumor cell in peripheral blood of colorectal cancer. The tumor cells are detected by RT-PCR and Cellular FACTT using CK20 and CEA as tumor cell markers respectively to determine the sensitivity and specificity.Partâ…¡We used Cellular FACTT to detect the clinical specimen of clinical patients. CEA and CA199 and CA242 were determined by ELISA. The expression of P53,nm23 H-1 in colorectal cancer tumor tissue were examined by immunochemistry. The relationship between the detection rate of circulating tumor cells in peripheral blood and clinical pathological factors was analyzed. For those negative patients prior to surgery were detected again 24h after surgery. Some patients who received chemotherapy after surgery were also examined after chemotherapy.Results1. Cellular fluorescence amplification catalyzed by T7 RNA polymerase technique (Cellular FACTT) was developed. Using CEA as marker to detect the tumor cell in model of colorectal cancer circulating tumor cell Cellular FACTT has a sensitivity of 5 tumor cells from 107 nucleated cells and a specificity of 96.7%.2. Cellular FACTT was used to detect the circulating tumor cell in the peripheral blood of 68 colorectal cancer patients prior to surgery, 51 cases (75%) were positive.3. The detection rate of circulating tumor cell in the peripheral blood of 68 colorectal cancer was closely related with TNM stage, tumor cell differentiation and metastasis status.4. The positive incidence of CEA, CA242 and CA199 in the peripheral blood of colorectal cancer patients was 35.3%,26.5% and 32.4%. Patients of CEA, CA242 and CA199 positive were 83.3%,72.2% and 77.3% positive for circulating tumor cell. The detection rate of circulating tumor cell in the CEA negative group is still very high.5. 17 patients who were negative prior to surgery were detected 24h after operation, 13 were positive (76.5%). 6. 12 positive patients who received chemotherapy early after operation were detected after chemotherapy, 4 were positive (33.3%).7. The positive incidences of P53, nm23 H-1 in colorectal cancer tissue examined by immunochemistry were 57.4%and 50%. The detection rate of circulating tumor cell in the peripheral blood was related with nm23 H-1 expression.Conclusion1. Cellular FACTT is a new technique which combines the specificity of antibody with the sensitivity of T7 RNA polymerase. It has a sensitivity of detecting 5 HT29 cells from 107 nucleated cells.2. The detection rate of circulating tumor cell in the peripheral blood by Cellular FACTT was related with TNM stage, tumor cell differentiation and metastasis status..3. The expression of nm23 H-1 was related with detection of circulating tumor cell in the peripheral blood.4. Cellular FACTT has a very high detection rate even in the CEA, CA19-9 and CA242 negative group.5. Circulating tumor cells were detected by Cellular FACTT even after early chemotherapy following colorectal cancer radical surgery.6. Circulating tumor cells detected by Cellular FACTT can be beneficial for predicting clinical outcome and guiding treatment.