The Study On Micrometastases In The Peripheral Blood Of Patients With Gastric Cancer: Detection Of Circulating Cancer Cells And Its Significance For Guiding Intraoperative Chemotherapy

The study on micrometastases in the peripheral blood of patients with gastric cancer :Detection of circulating cancer cells and its significance for guiding intraoperative chemotherapyBackgroung : Gastric cancer is the most common malignat tumour, the incidence of this disease heads all other digestive system malignant tumours in our country. The prognosis of gastric cancer is worse. Even if the wide lymphadenectomy is performed, the survival rate of 5 years is not over 35%. The most difficult problem of therapy is recurrence.There are many reasons for recurrence, one of the most important reasons is that there are undetectable micrometastases in the blood of patients before primary treatment. So it is a effective way for prevention of recurrence to detect and treat the micrometastases .CK19 and CEA are the markers studied by many people ,which are with better specific and sensitivity ,but the experiment results are different from each other. Most of studies in therapy for micrometastases are adjuvant chemotherapy , including preoperative ,intraoperative ,and postoperative chemotherapy. There are relatively little studies in intraoperative chemotherapy via vein, and no identical indications of chemotherapy. In this experiment , by RT-PCR technique ,we detected serum content of CK19mRNA and CEAmRNA in peripheral blood of patients with gastric cancer, then indicated the intraoperative chemotherapy with the detecting results and followed up these patients. Charpter one: Difference of CK19mRNA and CEACmRNA in peripheral blood between normal people and patients with gastric cancer.Objective:To find a suitable index for the detection of micrometastases in the peripherial blood of patients with gastric cancer.Methods: A total of 92 patients who had gastric cancer and 20 health people were taken peripheral blood ,the serum content of CK19mRNA and CEAmRNA were detected by RT-PCR technique,and the results were analysed on statistical principle. The links between clinical factors and micrometastases were investigated. Results(1) CK19mRNA,CEAmRNA in health people's peripheral blood all were negative.The positive rate of CK19mRNA of patients was 46.7%, the positive rate of CEAmRNA was 42.44%,the general positive rate(anyone was positive) was 66.3 %. (2) The difference between single and combined detection of CK19mRNA,CEAmRNA in peripheral blood of gastric cancer patients :The positive rate of combined detections was 66.3% ,There was significant difference between single detection and combined detection. ( X²=23.53, P＜0.01 )。(3) The relationships between micrometastases and clinical factors:The positive rates of cmbined detection of CK19mRNA,CEAmRNA were different among those patients with different pathological types (X²=14.92, P＜0.05) ;With the differential depth of invasion,there were differences in expressions of CK19mRNA,CEAmRNA in peripheral blood :the expression in those men with infiltrated serosa was significantly higher those without infiltrated serosa ( X²=13.01, P＜0.01 ) .The expression of CK19mRNA,CEAmRNA in peripheral blood correlated with pathological stage:The later the stage was ,The higher the rate of expression was (X²=13.48, P＜0.05 ) the expression of CK19mRNA,CEAmRNA in those men with metastatic lymph nodes was significantly higher than those without metastatic lymph nodes ( X²=7.14, P＜0.05 ) . The expression of CK19mRNA ,CEAmRNA was not related to the position of tumours:there was not significant difference in expression among those with differential position of tumours (X²=5.53, P＞0.05 ) .There was no correlation between the expression of CK19mRNA ,CEAmRNA and the patient's age ( X²=2.94, P＞0.05)。There was no correlation between the expression of CK19mRNA ,CEAmRNA and the patient's sexual ( X²=1.23, P＞0.05 ) .Conculusions: (1) Detecting expression of CK19mRNA,CEAmRNA in peripheral blood by RT-PCR technology can relatively accurately diagnose the micrometastases in peripheral blood of gastric cancer. (2) Combined detection of CK19mRNA,CEAmRNA can raise the positive rates of micrometastases. (3) The micromestastases in peripheral blood of gastric cancer doesn't correlate with the patient's age ,sexual and the position of tumours, but correlate with pathological types ,the depth of invasion, clinical stage,the metastases of lymphnod.Charpter two:Screening the scheme for introperative chemotherapy of gastric cancer by chemosensitivity testing in vitroObjective:To select a suitable scheme for intraoperative chemotherapy of gastric cancer.Methods:35 patients of the cases in part one were includedin this study, There were 17 women and 17 men.The mean age of the patients was 41.6 years(range:31-74 years).The pathological types of these patients includede well-differentiated adenocarcinoma(10cases), moderately-differentiated adenocarcinoma(10 cases),poor-differentiated adenocarcinoma (12 cases)and no-differentiated adenocarcinoma(3 cases).All patients had underwent no therapy for tumour before their hospitalization.The chemosensitivity testing used MTT assay,20 kinds of testing solution were prepared to examine the sensitivity of gastric cancer to these drugs.The inhibitory rate of tumour cells was examined on each scheme and analysed with statistics .The scheme whose inhibitory. rate of tumour cells was higher was selected.Results:Of 20 schemes,the inhibitory rate of 5-FU is the highest (57.36±15.2) . It is significant different to other scheme (P＜0.05)Conclusion: In chemosensitivity testing in vitro,it was showed that 5-FU was of higher inhibitory rate and signigicant superior to other schemes.5-FU can be selected as the intraoperatrive chemotherapy scheme of ganstric cancer. Charpter three: The effects of intraoperative chemotherapy on the mieromestastases in peripheral blood of patients with gastric cancer.Part one :The guiding significance of CK19mRNA and CEACmRNA in peripheral blood of patients with gastric cancerObjective:To observe the effect of intraoperative chemotherapy on the micrometastases by CK19mRNA,CEAmRNA;and investigate the index of intraoperative chemotherapy.Methods: A total of 60 patients were divided into 6 groups of 10 cases, among those patients were 30 people whose anyone of CK19mRNA,CEAmRNA was positive and 30 people whose CK19rnRNA , CEAmRNA all were negative. The groups were as follows: Group1: CK19mRNA,CEAmRNA all were negative ,no intraoperative chemotherapy was performed. Group2: CK19mRNA,CEAmRNA all were negative, intraoperative chemotherapy was performed. Group3: CK19mRNA,CEAmRNA all were negative , intraoperative chemotherapy was performed as follow: 5-FU 500mg/m₂ iv by drip during operation and two days before operation. Group4: anyone of CK19mRNA,CEAmRNA was positive,no intraoperative chemotherapy was performed. Group5: anyone of CK19mRNA,CEAmRNA was positive , intraoperative chemotherapy was performed as follow: 5-FU 500mg/m₂ iv by drip during operation. Group6: anyone of CK19mRNA,CEAmRNA was positive, chemotherapy was performed as follow: 5-FU 500mg/m² iv by drip during operation and two days before operation.Every case was taken peripheral blood at preoperation, postoperation,three days after operation, a week and two weeks after operation.Intraoperative blood was taken out from the righrt gastroepiploic vein atfter abdominal being opened and the specimen being taken off. The cancer tissue and normal tissue of every case were taken after operation. CK19mRNA,CEAmRNA in above samples were checked by RT-PCR technique.Results: The general positive rate(anyone was positive) in cancer tissue of all cases was 9/10-10/10, There was no difference among 6 groups( P=0.824, P＞0.05).The expression of CK19 mRNA, CEAmRNA in normal tissue was that only CK19mRNA expressed in some cases (1/10-2/10). There was no difference among 6 groups( P=0.62, P＞0.05).The change curve of micrometastases in peripheral blood showed: there were not significant differences between group 1 ,2, 3 whose CK19mRNA,CEAmRNA all were negative (P＞0.05). There were significant differences between group 4,5,6 whose anyone of CK19mRNA,CEAmRNA was positive (P＜0.05). The expression of CK19mRNA,CEAmRNA in blood from right gastroepiploic vein after the abdominal being opened was consistent with the expression at preoperation.The expression of CK19mRNA,CEAmRNA in blood from right gastroepiploic vein after the specimen being taken off were not consistent with the expression at preoperation:the micromestastases of some case whose micromestastases were negative at preoeration(group1,2,3) became positive(5/10-6/10),but there was no difference between group 1,2,3(P＞0.05).Conclusions: (1)The intraoperative chemotherapy is effective on the therapy of micrometastases,it can decrease the micrometastases in peripheral blood of the patients with gastric cancer. (2) CEAmRNA and CK19mRNA in peripheral blood can be taken as the indication of intraoperative chemotherapy and a symbol of curative-effect.(3) 5-FU by intravenous injection can be selected as the intraoperative chemotherapy scheme of gastric cancer, which has higher effective rate ,but minor toxicity, and more scheme may be selected according to patient's concrete condition.Part two:The clinical study on the effects of intraoperative chemotherapy on the mieromestastases in peripheral blood of patients with gastric cancerObjective:To analyse the long-term effects of intraoperative chemotherapy on micromestastases in peripheral blood of patients with gastric cancer.Methods:The patients of part three were followed up,and each case underwent standard postoperative chemotherapy. The relapse-free survival rates of every group were counted 3 moths by 3 months.The curves of survival were depicted,and the differences among these groups were investigated.Results:The curves of relapse-free survival were analysed: There was significant difference between the patients with micrometastases positive and those with micrometastases negative (group 1 and group 4), X² =5.24, P＜0.05。There was no difference between the groups with micrometastases negative(group 1,2,3), X²=3.98, P＞0.05。There was difference between the groups with micrometastases positive(group 4,5,6), X² =9.47, P＜0.05;and the range of survival rates in hign-to-low order was group 6,5,4.(trendy test: X² =8.23, P＜0.05, the difference was significant.Conclusions:The micromestastases in peripheral blood of patients with gastric cancer is very important to the prognosis of patients ,the relapse-free survival of patients with positive were significant shorter than those with negative.The intraoperative chemotherapy is significant effective on micrometastase in peripheral blood.5-FU by intravenous for intraoperative chemotherapy is of better effects,but the scheme will be improved further.