The Detection And Clinical Significance Of Different Phenotypes Of Circulating Tumor Cells

Background:Breast cancer is the most common malignant tumor in women.As a systemic diseases,recurrence and metastasis can occur even in the early stage,which are the prime causes of patients’ deaths.Circulating tumor cells(CTCs)who exsit in the peripheral blood of patients are key factor influencing the prognosis. Epithelial-mesenchymal transition(EMT),whereby epithelial tumor cells are thought to lose their apical-basal polarization and cell-cell contacts,permitting invasion through adjacent tissue as spindle-shaped mesenchymal tumor cells with a motile cytoskeleton play a important role in the occur, survival and form a new metastasis site. There are three kinds of phenotypes we can detect in the peripheral blood by the marker difference, they are mixed epithelial–mesenchymal CTCs, mesenchymal CTCs and epithelial CTCs. There is also some CTCs(three or more) get together and become a cluster of CTCs, which is called circulating tumor microemboli(CTM).It’s studied that mesenchymal CTCs and CTM tend to be make recurrence and metastasis.But the relationship of different phenotypes of CTCs between pathological features of breast cancer patients are still unclear.With these questions we performed this preliminary study and tried to get answer.Objective: To study the relationship of different phenotypes of CTCs in the peripheral blood and compare the CTCs level and the count of different phenotypes of CTCs with clinicopathological characteristics of primary tumor and molecular subtypes of breast cancer patients.Method:Samples of 10 m L peripheral blood from 71 cases of patients with invasive breast cancer and 10 cases of patients with breast fibroadenoma for the first time were removed erythrocytes by red blood cell lysis buffer at the first,and then depletion of CD45+ leukocytes by immunomagnetic bead separation,after that isolate CTCs by membrance, then at last RNA–in situ hybridization for analysis the CTCs.The expression of ER,PR,HER-2,Ki-67 was detected by immumohistochemistry.The relationship of the CTCs level and the percents of different phenotypes of CTCs in the peripheral blood between the percents of CTCs with different phenotypes and clinicopathological characteristics of primary tumor and molecular subtypes of breast cancer patients were analyzed.Results:1 The positive rate of total CTCs is 85.9%(61/71) while no CTC was detected was detected in all patients with breast fibroadenoma.The median of three phenotypes of CTCs detected was epithelial 1,mesenchymal 2,mixed 2,only one patient had detected a CTM that consists of three mesenchymal CTCs.2 Of all the 71 breast cancer patients, 25 cases was stageⅠand stage Ⅱ,18 cases was stage Ⅲ,2 cases was stage Ⅳ, while 26 cases was recurrent and metastatic. The detecting rates of different stages were 76%(16/25),94.44%(17/18),100%(28/28).The median number of stageⅠand stage Ⅱwere epithelial 1,mesenchymal 0.5,mixed 1,that of stage Ⅲwere epithelial 1,mesenchymal 3,mixed 2 while that of recurrent/metastatic were epithelial 1,mesenchymal 3,mixed 5.5.There is significant difference in the level of CTCs between different stages(P<0.001).So were the count of mesenchymal CTCs and mixed epithelial–mesenchymal CTCs between different stages(P*=0.002,P**<0.001).The CTCs level of patients with recurrent/metastatic breast cancer was significantly higher(P*<0.001) than that of patients with operable breast cancer,and so were the count of mesenchymal CTCs and mixed epithelial–mesenchymal CTCs(P**=0.006,P***<0.001).3 The immounstaining of the patients showed that the cases of ER+ patients was 47 while ER- was 24.There was no significant difference in the level of CTCs between the patients of ER+ and that of ER.PR high expression(≥20%+) cases was 35,PR low expression(<20%+or-) cases was 36,HER-2+ cases was 24,HER-2- cases was 47,Ki-67 high expressed(≥14%) cases was 60,Ki-67 low expressed(<14%) cases was 11.There was significant difference in the level of CTCs between the patients with ER+ and that with ER-(P=0.007),while there was significant difference in the level of CTCs between the patients with PR high expression and that with PR high expression(P=0.002).There was no significant difference in the level of CTCs between the patients with HER-2+ and that with HER-2-(P=0.115) and so was the Ki-67 high expression and low expression(P=0.477).4 The percents of four molecular subtype was Luminal A11.27%,Luminal B 54.93%,HER-2 15.50%,triple negative breast cancer 18.30%.There was significant difference in the CTCs level between different molecular subtypes of breast cancer(P=0.049).There was significant difference in the count of mesenchymal phenotype of CTCs between different molecular(P=0.002).The level of CTCs and the count of mesenchymal phenotype of CTCs in patients with HER-2 and TNBC subtype was significantly higher than patients with Lumial A and Luminl B.Conclusion:1 The method we establishe to detect the CTCs,which combines membrance with dual-colorimetric RNA–in situ hybridization can detecte all kinds of CTCs besides CTM,and analysis different phenotypes of CTCs. It has been proved have extremely high sensitivity and specific, and it’s useful for clinical practice.2 There is significant correlation in CTCs level between different stages of breast cancer, high stage as well as recurrent/metastatic breast cancer has high CTCs level and high count of mesenchymal CTCs.3 There is significant difference in the CTCs level between different molecular subtypes of breast cancer. The level of CTCs and the count of mesenchymal phenotype of CTCs in patients with HER-2 and TNBC is significantly higher than patients with Lumial A and Luminl B.