Research On Detection And Clinical Significance Of CTC In Peripheral Circulation Of Patients With Renal Cell Carcinoma

BackgroundRenal cell carcinoma (RCC) accounts for2%～3%of adult malignant tumor. There were more than twenty hundreds thousands new cases and more than one hundreds thousands mortality cases every year in the world. About1/3of RCC patients appear metastasis when diagnosed and about1/3of limited RCC patients undergo recurrence after surgery. In China, RCC is ranked second in the malignant tumor of urinary system, and its incidence is increasing year by year. Important advances in diagnosis and treatment of RCC will change the natural course of the disease and improve the survival rate of the patients.Tumor cells’ exfoliation, invasion and entry into circulation system is the early stage of tumor metastasis, which provide the possibility to form tumor metastasis lesions. In1869, Ashworth found some cells which were similar to tumor cells in the peripheral blood of a patient suffered advanced carcinoma, and first proposed the concept of the circulating tumor cells (CTC). Circulating tumor cells (CTC) are defined as the tumor cells which detach into circulating system from primary lesion or metastasis lesion. CTC have the tumor specific antigen or genetic characteristics. Detection of circulating tumor cells in the peripheral blood (CTC) and carried on the analysis, and has important clinical application value on the aspects of clear transfer process, auxiliary diagnosis, curative effect monitoring, guiding individual treatment, prognosis. There are more than50kinds of methods for the detection of CTC, Summing up and including detection method based on immune response, fluorescence quantitative PCR method and Detection method based on cell filtration. At present, the mature methods is Cellsearch technology which was developed by Veridex, CellSearch technology is the only method approved by the USA food and Drug Administration (FDA) to detect the CTC. It is a semi-automatic technology, through carrying anti EpCAM antibody of ferromagnetic fluid to enrich the CTC, then CTC were stained by CK and DAPI fluorescent antibody, the last cells were stained by CD45antibody, then CK+/DAPI+/CD45-cells were counted as CTC using automated fluorescence microscopy.Technology of CTC detection includes two steps of enrichment and identification. The enrichment methods are based on specific immune recognition principle and morphology. The limitation of detection method based on immune recognition is, which is expressed only in the epithelial antigen CTC, and does not recognize the occurrence of EMT or CTM, the literature shows that the missing rate will be up to50%. ISET is based on the tumor larger than granulocytes in peripheral circulation, so that tumor cells were isolated from peripheral circulation, which is independent on related antigen antibody reaction and is filtrated directly from peripheral circulation. It will lose less tumor cells and doesn’t destroy the morphological characteristics of tumor cells at the same time, and also improves the sensitivity to a large extent. It has been shown that the detection in breast cancer and lung cancer, ISET technology is higher efficiency than immunomagnetic enrichment method.We would like to detect CTC in peripheral circulation of patients with renal cell carcinoma by CellSearch and ISET respectively, and to compare with the clinical pathological characteristics and previous research results, to try to find a pertinent method for CTC detection in peripheral blood of patients with renal cell carcinoma, and to investigate the clinical significance of CTC detection in peripheral circulation. Materials and Methods1Subjects for study82patients with renal cell carcinoma, who admitted Surgery in Shandong Tumor Hospital from July2013to July2014, were selected as the experimental cases. Of which,63male cases, and19fumale cases.,with age range from33to72years, and with average age of63years. According to the pathological staging,34patients in stage Ⅰ,14patients in stage Ⅱ,13patients in stage Ⅲ,21patients in stage Ⅳ;according to the pathological grading,8cases of patients in stage1,32cases of patients in stage2,27cases of patients in stage3,15cases of patients in stage4. In July2013, we selected20healthy volunteers who were all confirmed health by imaging examination in the tumor hospital of Shandong Province. The experiment has been approval by Shandong Provincial Tumor Hospital Ethical Committee and has got a written approval. Before the experiment, all of the experimental subjects had the consent of experiment, and their family members have signed the informed consent.2Methods2.1Sample collection and processing15.0ml peripheral blood was got one day before surgery and the patients haven’t have a diet before drawing blood. The healthy control group were collected peripheral blood15.0ml without diet. The vein puncture point was taken in the median cubital.7.5ml samples were sent to Shanghai Ding Jing Company, detected by the Celltracks(?)AutoPrep(?)system within96hours;7.5ml samples were detected by the CTC-Biopsy within24hours in the center Laboratory of Shandong Provincial Tumor Hospital.2.3The CellSearch methodUse Johnson company Celltracks(?)utoPrep(?) automatic detection system to test samples. The result analysis (cell interpretation):analyzer, click the Review icon, according to the CTC immunofluorescence staining characteristics, expression CK+DAPI+CD45-blank channel-cells for CTC, on the scan results were determined.2.3The CTC-Biopsy tester (ISET) detection processIn order to the company of Wuhan Youzhiyou CTC-Biopsy tester test sample. According to the method of ISET CTC Hofman VJ reported the interpretation standards:nuclear size variation big (ratio>0.5); the nucleus of greater than24u m; the nucleus is irregular in shape; stains existing3D level; high nucleocytoplasmic ratio, as long as consistent with the above at least4features, can be considered from the morphology of CTC. Cell room physicians interpret the results by pathology.3. The arrangement of materialsThe detection results of registration of the CellSearch method and ISET method, the clinical and pathological features of registration of subjects, a list of records.4. Statistical methodsSPSS19.0statistical analysis software and Excel2003were used in the statistical analysis and charting. Univariate analysis using the four lattice Fisher exact probability test, for a list of Fisher exact probability test and Kendall correlation analysis, Significance was set at P<0.05. Multivariate analysis using Logic regression analysis. Significance was set at P<0.05.Results1The results of CellSearch method and ISET method to detect the healthy subjects peripheral blood20healthy volunteers were detected by the CellSearch method and ISET method, and none of which were found CTCs in the peripheral blood.2Comparison between the results of CTC in peripheral blood of patients with renal cell carcinoma detected by CellSerach system and clinical pathological characteristics 2.1The results of CTC in peripheral blood of patients with renal cell carcinoma using the CellSerach systemIn accepting the detection of82cases of renal clear cell carcinoma patients, there were more than one CTC in18cases of patients’peripheral blood, according to the pathological staging,2cases in stage Ⅰ,1cases in stage Ⅱ,3cases in stage Ⅲ,12cases in stage Ⅳ; according to the pathological grade,2cases in grade1,8cases in grade2,2cases in grade3,6cases in grade4;and in11patients’ peripheral blood more than2CTCs were found, according to the pathological staging,1cases in stage Ⅰ,1cases in stage Ⅱ,1cases in stage Ⅲ,8cases in stage Ⅳ; according to the pathological grade,2cases in grade1,5cases in grade2,1cases in grade3,3cases in grade4..Pathological findings of microvascular invasion in13patients, including6cases of CTC number of1,2patients with CTC number2.22cases of patients with tumor tissue necrosis was found in6cases, the number of cases of CTC1,2CTC number2. With9cases of sarcomatoid patients, including3cases of CTC number of1,1patients with CTC number2. Set system involvement in7patients, including2cases of CTC number of1,0patients with CTC number2.The ECOG score is more than9cases of1patients, including4cases of CTCnumber of1,1patients with CTC number2. Platelet count≥450x109/Lin7patients, including6cases of CTC number of1,2patients with CTC number2.Hemoglobin under normal value18patients, including4cases of CTC number of1,1patients with CTC number2. Neutrophil to lymphocyte ratio was larger than3.6of the16patients, including6cases of CTC number of1,1patients with CTC number2.2.2Study on the correlation between the CTC results of patients with renal cell carcinoma with the clinical pathological featuresSingle factor analysis, according to the Fisher exact test and Kendall correlation analysis, draw the conclusion, cutoff value was1, CTC increased,and the results of clinical staging, microvascular invasion and platelet over450*109/L correlation (P<0.05); cutoff value was2, the results of CTC andlymph node metastasis and pathological staging correlated (P<0.05).Multivariate logistic regression analysis, cutoff value was1,the results of CTCand pathological staging, lymph node metastasis, pathological grade, tumorfound in the necrotic tissue related. Take the cutoff value was2, the results of CTC and pathological staging, pathological grading, sarcomatoid change,neutrophil-to-lymphocyte ratio≥3.6.3. Comparison between the results of CTC in peripheral blood of patients with renal cell carcinoma detected by ISET system and clinical pathological characteristics3.1The results of CTC in peripheral blood of patients with renal cell carcinoma using the ISET system.We have detected82cases of patients with renal clear cell carcinoma,28cases of peripheral blood in patients with CTC positive,4cases of peripheral blood in patients with CTM positive. The pathological stage Ipatients34cases,14cases,13patients in stage Ⅰ, stage Ⅲ patients with stage Ⅳ21patients, according to pathology in8cases of patients with stage1grade,2grade32cases of patients,27cases of patients with stage3,15cases of patients with stage4.82cases of renal cell carcinoma, pathological findings of13patients with microvascular invasion, of which9cases were CTC positive,2cases with CTM positive.22cases of patients with tumortissue necrosis was found, of which9were CTC positive,1cases with CTM positive. With9cases of sarcomatoid patients, including3cases of CTC positive, CTM was not found.7patients with involvement of the collecting system,1of which were positive for CTC, CTM was not found. The ECOG score is more than9cases of1patients, including5cases of CTC positive,CTC was not found. Platelet count≥450x109/L in7patients, of which5cases were CTC positive,1cases with CTM positive. Hemoglobin under normal value18patients, of which6cases were CTC positive,2cases were CTM positive. Neutrophil to lymphocyte ratio was larger than3.6of the16patients, including9cases of CTC positive,2cases with CTM positive. 3.2Correlation of CTC results with the clinical pathological features of patients with renal cell carcinomaSingle factor analysis, according to the Fisher exact test and Kendall correlation analysis, draw the conclusion, ISET detection results of CTC and lymph node metastasis, pathological staging, microvascular invasion and platelet over450X109/L and the neutrophil/lymphocyte is equal to or greater than3.6correlation (P<0.05); CTM only associated with pathological staging(P<0.05). Multivariate logistic regression analysis, the results of CTC and pathological stage, distant metastasis, pathological grading, sarcomatoid, lower than normal hemoglobin related. CTM positive cases were too few, so that to sieve out the influential variables.Conclusions1Detection of peripheral blood CTC patients with renal cell carcinoma by CellSearch was feasible, when cutoff value was1, according to the pathological stage, the detected ratio of Ⅰ stage, Ⅱ stage, Ⅲ stage, Ⅳ stagepositive rates were5.9%(2/34),7.1%(1/14),23.1%(3/13),57.1%(12/21); When cutoff value wes2, according to the pathological stage, the detected ratio of Ⅰ stage, Ⅱstage, Ⅲ stage, IV stagepositive rates were2.9%(1/34),7.1%(1/14),7.7%(1/13),38.1%(12/21);2The sensitivity of CTC detection by CellSearch in peripheral blood of renal cell carcinoma was21.9%(18/82), healthy subjects were not detected in CTC, so the specificity of100%;3The results of CTC in peripheral blood of renal cell carcinoma patients by CellSearch were relevant with pathological staging, lymph node metastasis, pathological classification, tissue necrosis, tumor sarcomatoid change, NLR more than3.6;4Detection of peripheral blood CTC patients with renal cell carcinoma by ISET was feasible, according to the pathological stage, the detected ratio of Ⅰ stage, Ⅱ stage,Ⅲ stage, Ⅳ stagepositive rates were5.9%(2/34),28.6%(4/14),76.9%(10/13),57.1%(12/21); According to the pathological stage, the CTM positive ratio of Ⅰ stage, Ⅱ stage, Ⅲ stage, Ⅳ stage were0(0/34),0(0/14),15.4%(2/13),9.5%(2/21);5The sensitivity of CTC in peripheral blood of renal cell carcinoma patients by ISET detection was34.1%(28/82), healthy subjects were not detected in CTC, so the specificity of100%;6The results of CTC in peripheral blood of renal cell carcinoma patients by ISET were relevant with pathological stage, distant metastasis, pathological grading, sarcomatoid, lower than normal hemoglobin.