The Establishment And Clinical Significance Of A Quantitative Method For Detection Of Circulating Colorectal Tumor Cells In Peripheral Blood

Purpose: To establish a method for detection of circulating colorectal tumor cells by combination of immunomagnetic separation technology with real-time RT-PCR, and investigate the clinical significance of this method.Methods: The colon cancer cells HCT-116 cultured in McCoy`s 5A medium with 10%FBS was harvested, counted, and evenly divided into 4 groups: immunomagnetic enrichment group(GroupA) and non-enriched group (GroupB), another set of negative control group respectively,and serially diluted into 106 to 101. Then the tumor cells mixed with 5 ml whole blood from healthy donor respectively, and peripheral blood mononuclear cells (PBMC)(with tumor cells) was separated by density gradient centrifugation. The PBMC of the study group (GroupA) co-cultured with CD326 monoclonal antibody-coated beads and enriched with immunomagnetic sorting method, while the control group (GroupB) co-cultured with PBS. The resulted cells processed to RNA extraction, and quantitative real-time RT-PCR was performed to assay the expression level of human telomerase reverse transcriptase (hTERT). The experiment was repeated for 10 times. The same experiment was done in 22 newly diagnostic colorectal patients and 22 healthy donors.In which the PBMC was separated from whole blood of the patients by density gradient centrifugation and enriched by immunomagnetic sorting with CD326 monoclonal antibody-coated beads (GroupC). While PBMC from the 22 patients with no enrichment (GroupD), and PBMC from 22 healthy donors with no enrichment (GroupE) as control. The resulted cells processed to RNA extraction, and quantitative real-time RT-PCR was performed to assay the expression level of human telomerase reverse transcriptase (hTERT). The results were correlated with the risk factors of the 22 patients.Results:(1) All PCR amplification curve of the negative control group showed irregular wavy line in the cell experiments, not the typical S-type amplification curves,indicated that all the negative control samples were not showed specific PCR amplification; (2)101-106 cells in each magnitude group of PCR amplification Ct values are shown in Table 5; By relative quantitative comparison of 101-106 cells in each magnitude group hTERT-mRNA expression level, groupA was significantly higher than groupB(P<0.05), Table 6; (3)22 cases of colorectal cancer patients, PCR amplification Ct values of groupC and groupD are in Table 7; By the relative quantitative comparison of 22 cases after the two samples hTERT-mRNA expression level, groupC was significantly higher than groupD(P<0.05), Table8;(4)By relative quantitative comparison of colorectal cancer patients and healthy expression of hTERT-mRNA level, groupC was significantly higher than groupE (P<0.05), Table 9;(5)There are no significant differences in hTERT-mRNA expression between gender,age,size of tumor,location and depth of invasion.(P>0.05). And the hTERT-mRNA expression was significantly related to lymph node metastasis and TNM staging(P<0.05),Table 10.Conclusion:Experimental study established a method for detection of circulating colorectal tumor cells by combination of immunomagnetic separation technology with real-time RT-PCR, by determining the level of hTERT-mRNA expression in PBMC and quantitative analysis, detection sensitivity is better than using RT-PCR detection methods only. Colorectal cancer patients with hTERT-mRNA expression level was significantly related to lymph node metastasis and TNM staging ,it provided new ideas for early detection of clinical colorectal cancer micrometastases.Further study on this research will be valueable with the diagnosis, treatment and prognosis of colorectal cancer.