Detection Of Circulating Tumor Cells In Breast Cancer Patients And Assessment Of The Prognosis Of Patients In Different CTC Levels

Objective Detect circulating tumor cells with multiparameter flow cytometry proving a high sensitivity and accuracy.Methods To evaluate the sensitivity and accuracy of the method (tumor cells recovery), SKBR-3 BC cells were spiked, respectively, into 7.5ml blood from healthy person. To accurately estimate the number of cells spiked into the blood, control the preparation for the same volume of the cell suspension and cells were counted. The number of Ep-CAM and cytokeratin8,18,19positive cells on FACS CaliberTM was used to calculate the cell recovery. Titration experiments were performed onthree separate occasions and analysis the significantly difference with Mann-Whitney rank sum test.Results Analysis of serial dilutions (0.0001%,0.001%,0.005%,0.05%) of human SKBR-3 tumor cells in normal human blood demonstrated that the lower detection limit for sensitivity of the method was 0.001%, or 10-5, equivalent to ane human cell per 100,000 white blood cells. Recovery and linearity were highly reproducible across three separate experiments, and the number of tumor events recovered could be positively correlated with the number of tumor events expected based on the serial dilutions (y=1.0198x+1E-06, R2=0.997). There was no significantly difference between each group by Mann-Whitney rank sum test (P>0.6).Conclution The technique of detection CTC utilizing multiparameter flow cytometry is practicable and it has high sensitivity, accuracy and repeatbility.Objective Detect the breast cancer CTC with the tranditional reverse transcription polymerase chain reaction (RT-PCR) and compared the sensitivity and specificity between this method and multiparameter flow cytometry.Methods We detect the expression of Ep-CAM in SKBR-3 cell lines (positive control), normal persons (negative control) and 45 breast cancer patients. We compared the expression between advanced breast cancer (ABC) patients and limited breast cancer (LBC) patients. At the same time we detect the CTC in 45 patients and also compared between ABC and LBC.Results We detected the 45 patients with RT-PCR and multiparameter flow cytometry, respectively and the positive rate were 86.67% and 75.56% correspondingly. 18 patients were detected CTC≥5 while 27 patients got CTC<5 with multiparameter flow cytometry. We chose six samples including three typical ABC patients (CTC≥5) and three LBC patients (CTC<5) to detect the expression of Ep-CAM. The results showed hat six samples were found positive for Ep-CAM not only three ABC but also three LBC samples. The LBC positive samples were nearly the same representation with the ABC samples. So, it is hard to distinguish LBC with ABC. However, flow cytometry can identify the two groups clearly and quantifyingly.Conclution When compared with RT-PCR, multiparameter flow cyometry for this research contributed a high specificity although it has a lower sensitivity. However, the technique with multiparameter flow cytometry reduced the false positive rate. Therefore, multiparameter flow cytometry can identify the two groups clearly and quantify ingly.Objective Discussing the clinical value of detection and assessment the prognosis of breast cancer utilizing multiparameter flow cytometry.Methods Forty-five patients with BC and other three normal people were enrolled between September 2006 and June 2008. After we performed detecting CTC among the 45 patients by multiparameter flow cytometry, we analysised patient characteristics such as TNM stage, diameter of tumor, axillary lymph node dissection and metastasis and so on across CTC groups by chi-square as appropriate. Overall survival (OS) was calculated from the date of CTC measurement to the date of death or last follow-up and was estimated using the Kaplan-Meier product limit method and compared across groups using the log-rank test. In addition, a Cox regression analysis for the follow-up was performed in order to get the relative factors on OS.Results Forty-five patients were identified and included in this analysis; 27 (60.0%) patients and 18 (40.0%) patients had CTC<5 and CTC≥5, respectively. Twenty-five (55.6%) patients had metastasis including 10 (22.2%) and 15 (33.3%) patients were no metastasis including 17 (37.8%) and 3 (6.7%) patients had CTC<5 and CTC≥5. Chi-square test analyses showed that there was statistically significantly difference (P= 0.002) in the proportion of patients with CTC<5 and CTC≥5 per 7.5 ml blood between the metastasis group and no metastasis group. There was also statistical difference (P=0.033) in the proportion of patients with CTC<5 and CTC≥5 within different TNM stage. There were no statistically significant difference in age, parimary tumor sites, clinical pathology, diameter of tumor and axillary lymph node dissection (ALND) with CTC<5 and CTC≥5 groups (P>0.05).At the time of last follow-up,17 (37.8%) patients had died including six (13.3%) patients in CTC<5 group and 11 (24.5%) patients in CTC≥5 groups. The median follow-up among all patients was 82.0 weeks (95% confidence interval [CI] 47.70 weeks to 116.305 weeks). The median survival for patients with CTC<5 and CTC≥5 was 95.0 weeks (Std. Deviation,18.67 weeks) and 65.5 weeks (Std. Deviation,30.0 weeks). During the follow-up,11(24.4%) patients got lost contact with seven (15.6%) in CTC<5 and four (8.9%) in CTC≥5 group. Kaplan-Meier was used to analysis the patients survival with Log Rank P=0.004 and Breslow P=0.003, both showed that these two groups had statistically significant difference.In addition, we performed a Cox regression analysis for the follow-up and the significant level is 0.05. The results showed that the variables entered in the model, CTC level (P=0.041), age of the patients (P=0.001), metastasis (P=0.002) retained statistical significance in the multivariate analysis.Conclution 1.There was obvious statistical difference in the proportion of patients with CTC levels within different TNM stage. There was also statistical difference in the proportion of patients with different CTC levels between the metastasis group and no metastasis group. Therefor, it is common to find that the patients who were in high TNM stage and got metastasis could have more chance to be in the CTC≥5 group.2. The population with fewer than 5 CTCs showed significantly higher OS than the group with 5 or more CYCs.3. The status of a patient whether die or not was correlated with three factors: CTC level, age, and metastasis.