Preliminary Investigation Of The Clinical Significance Of Detecting Circulating Tumor Cells From Renal Cells Carcinoma Patients Through"Ligand Probe" PCR Based On α-FR

Background:Over the past10years, the incidence of kidney cancer has increased2%-4%; indeed, kidney cancer accounts for approximately2%of all cancers worldwide. Kidney cancer is relatively uncommon in Asia compared with Western countries, but the incidence is increasing more highly in developed Asian nations. As Asian nations develop economically and become more westernized, along with the higher rates of smoking and the growing problem of obesity, the incidence of kidney cancer will continue to approach the incidence of kidney cancer in Western countries. Statistical analysis of kidney cancer incidence in1988-2002base on several cities of China, the results show that kidney cancer incidence rates were4.26per one hundred thousand in1988-1992,5.40per one hundred thousand in1993-1997and6.63per one hundred thousand in1998-2002. Showed an year by year increasing trend. Kidney cancer incidence hold the second in the urinary and male reproductive system tumors of our country.Human renal cell carcinoma (RCC) is thought to arise from the epithelium of the kidney or collecting tubules, which account for80%-90%kidney cancer. Because it high degree of malignancy and lack of sensitivity to radiotherapy and chemotherapy, nearly30%of RCC patients present with metastatic disease at the time of diagnosis despite improvements in medical imageology, So when distant metastases occur, the five years survival less than10%. The serum levels of erythropoietin, ferritin, neuron-specific enolase, and transforming growth factor-P have been measured in an effort to monitor post-nephrectomy patients and/or to predict prognosis; however, more evidence is need to confirm the usefulness of these markers in the clinical management of patients with RCC. In1869, Ashworth was first reported the circulating tumor cells (CTCs), it was those tumor cells shed from the primary tumor into blood circulation. A number of studies have confirmed CTCs can be detection among different stages of the various types of tumors and that the presence of these cells is associated with TNM stage and prognosis, In addition, CTCs have been first time in the recommendations of the American Society of Clinical Oncology (ASCO) as tumor markers for breast cancer in2007. The folate receptor Alpha (a-FR) is a glycoprotein which is attached to the cell surface by a c-terminal glycosyl phosphatidyl inositol anchor, which consists of257amino acids and with an apparent molecular weight of38kDa. It’s expressed in limited quantities on certain normal epithelial cells, but significantly overexpressed in tumors of epithelial lineage, such as75%of kidney cancers. So far, the study of CTCs about RCC is relatively less mainly due to the limitation of current detection technology. Such as RT-PCR、 CellSearch system and Flow Cytometry detection methods were not suited to RCC.Thus, we study the a-FR expression in human renal carcinoma cell line ACHN and7860on the mRNA and protein levels based on summarize literature. And then attempt to using "ligand probe" PCR technology based on a-FR for the quantitative detection of CTCs from RCC patients, and demonstrated the clinical significance of detecting CTCs using "ligand probe" PCR technology. We provides a available CTCs detection technology for RCC patients, no similar reports at domestic and overseas so far.Objection:Discuss the feasibility of "ligand probe" PCR method based on a-FR for quantitative detecting circulating tumor cells from renal cell carcinoma patients, and then demonstrate the preliminary clinical significance of detecting circulating tumor cells through above technology.Materials and Methods:First:(1)Human renal carcinoma cell line ACHN、786-0were cultured with RPMI-1640folic acid-free medium with10%FBS, the logarithmic growth phase cells were harvested with0.25%trypsin-EDTA. While collection of leukocytes in peripheral blood of healthy volunteers as negative control. Three types of cells for extracted total RNA by the acid-guanidinium thiocyanate-phenol chloroform method using a Trizol reagent, total RNA were run on electrophoresis in1.2%agarose gel and stained with ethidium bromide in order to verify the integrity of total RNA. The real-time quantitative PCR for target gene (a-FR) specific amplification was performed by the SYBR Premix Ex TaqTM II Kit, and GAPDH as the reference gene. According to PCR dissolution curves and amplification curves, the comparative method of△△t was used to calculate the relative expression of a-FR mRNA for three types of cells. PCR products were separated by electrophoresis on a1.2%agarose gel and then visualized and photographed under UV light with ethidium bromide staining.(2) Take1×106of three types of cells for extracted total protein through cell protein extraction kit, detection of protein concentration by BCA Protein Assay Kit. Western blot assay was used to analysis the a-FR expression level in those protein samples, GAPDH as an internal reference. The expression difference of a-FR protein was analysis via chemiluminescence detection the hybridization signal.(3) To examine the sensitivity and effectiveness of the "ligand probe" PCR technology in detecting CTCs, validation experiments were conducted with cultured ACHN cells spiked into a whole blood sample obtained from healthy donors. The resuspension cells serially diluted to5different concentrations (1×10-1×105/ul), PBS or10,100,1000,10000, and100000ACHN cells were mixed with3ml of fresh peripheral blood from healthy volunteers and repeated in triplicate. The next step was performed according to standard procedures for detecting cancer cells in blood (vide infra). Simple linear regression analysis was calculated for different dilutions and detection values in parallel in double-blind experiments.Second:(1) Firstly, three ml of fresh whole blood remove the red blood cell by means of using ice cold erythrocyte lysis buffer, the pellet was resuspended and then immunomagnetic beads conjugated with CD45antibodies was used for negative enrichment CTCs; activation solution was added to activate and release the tumor cell a-FR, followed by DNA fragments labeled with Taqman ligand probes specifically conjugated to a-FR were added, then the excessive probes were washed out with washing buffer, finally, eluent was added for elution to the tumor cell-labeled probe.(2) Application ABI Viia7fluorogenic quantitative PCR combined with Taqman probe for specificity amplification of elution DNA fragments. In addition, serial dilutions were made to produce a standard curve using6concentrations in duplicate for the six-point standard substances. The reaction conditions were as follows:stage1(95℃for2min,40℃for30sec,60℃for1min,8℃for5min, and95℃for1min) for1cycle; and stage2(95℃for10sec,35℃for30sec, and72℃for5sec) for40cycles. The fluorescent signal was detected during annealing at35℃in stage2. Least-squares regression analysis was performed by Graphpad Prism5software to calculate the correlation between the logarithm of the initial copy number of the standard substance and the corresponding measured CT value. Based on the standard curve we obtained, the measured raw copy number for each specimen reaction was calculated using the CT value for each PCR interpolated to the external standard curve. For each DNA specimen, the triplicate average raw copy number per well was multiplied by0.144to convert to copies/3ml.Third:Ninety-one peripheral blood specimens were collected from45RCC patients, those specimens were obtained1day before surgery (n=45) and7days after surgery (n=39);7specimens were also obtained during follow-up of3post-operatively patients, we as well as collected pertinent clinical data from RCC patients. Patients with other malignancies were excluded from the RCC group. In addition, there were85peripheral blood specimens obtained from the patients with benign lesions of the kidney (n=35) and healthy volunteer control group (n=50). There were19males and26females among the45RCC patients, with an age range between29and87years (55.58±14.16years). All RCCs were confirmed by pathologic evaluation. Forty-five patients had RCCs, including31clear cell carcinomas,4chromophobe cell carcinomas,8papillary carcinomas, and2collecting duct carcinomas. According to the2010AJCC TNM classification for kidney cancer, there were21stage Ⅰ,14stage Ⅱ,4stage Ⅲ, and6stage Ⅳ RCCs. Thirty-five patients had benign lesions of the kidney (24males and11females), with an age range of22-72years (mean,50.74±14.28years). The benign lesions of the kidney included13patients with kidney stones,7patients with hydronephrosis,7patients with renal cysts,6patients with renal angiomyolipomas, and2patients with renal lipomas. Twenty-seven male and23female healthy volunteers were recruited as the negative control group. Healthy donors had no evidence of an epithelial malignancy or a familial cancer syndrome; however, conventional abdominal CT was not required for negative control subjects. Peripheral blood specimens were obtained by venipuncture; the first2.5ml of blood was discarded to minimize the risk of contamination by dermal epithelial cells. All blood specimens were temporarily stored in a4℃refrigerator after collection and CTCs were detected within5h. The spiking experiments were processed separately from the participant samples to avoid cross-contamination. We analysis the differences of CTCs level among three groups, discuss the correlation of CTCs level in peripheral blood with TNM stage or tumor clinical stage, whether CTCs level changed after surgery.Forth:Statistics:All analyses were performed using SPSS13.0Software (SPSS Inc., Chicago, IL, USA). General linear correlation analysis was performed for the number of spiked tumor cells versus the detection value in the artificial spiking experiment. To determine the threshold for the detection method, we conducted ROC curve analysis. One-way ANOVA was used to analyze different categories. Among RCC patients, one-way ANOVA or t-tests will be used for determine the significance of detection values in different TNM stages, clinical stages, and pre-and post-operative groups. To compare the differences in the two types of surgical methods for treatment of localized RCC patients, the difference between the detection value of the pre-and post-operative groups was analyzed by two independent samples t-tests. The threshold of significance was set at ap<0.05.Results:Fluorogenic Quantitative PCR:The comparative method of△△t was used to calculate the expression levels of a-FR mRNA for renal cancer cell lines, ACHN、786-0was39.77folds and23.12folds than WBC respectively.Western blot:GAPDH was used as the housekeeping gene in western blot study was expressed at relatively constant level in ACHN,786-0and WBC. However, a-FR in human renal carcinoma cell lines ACHN and786-0was excessive expressed, while absence in WBC.Spiking experiments with ACHN RCC cells:The technology of the specific "ligand probe" based on conjunction to a-FR could detect5-5×105cancer cells from3ml of peripheral blood. Simple linear correlation analysis was performed to determine the correlation coefficient between the defined amounts of tumor cells and the detection value (r=0.99,p<0.01).A ROC curve was used for analysis of the detection value of CTCs in all clinical samples, and we determined by the Youden index cut-off values to maximize the sum of the sensitivity and specificity:The area under the ROC curve (AUROC) was maximum when the cut-off value was set at15.4Copies/3ml.CTCs detection value in pre-operation of RCC was significantly higher than healthy controls and renal benign lesions group (p<0.01, p<0.01), as well as significantly correlation with tumor TNM stage and clinical stage(p<0.01).For T1、 T2and non-existence metastasis of RCC patients,radical nephrectomy and nephron sparing surgery have no significant difference in post-operation peripheral blood CTCs levels (p=0.320). Conclusion:The "ligand probe" PCR technology could efficient detection CTCs from peripheral blood for RCC patients. In our study, the technology of CTCs quantitative detection for RCC patients could recover the inadequacy of TNM staging, and it may have some value in evaluate therapy effects and monitoring recurrence.