胃癌病人外周血微转移的检测及其对术中化疗指导的研究
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摘要
背景：胃癌是最常见恶性肿瘤，其发病率占我国消化道恶性肿瘤第一位，预后较差，即使行广泛淋巴清扫术，五年生存率仍不超过35％。胃癌治疗的最大难题是复发，复发的原因很多，其中最重要的原因之一是：治疗前患者血液中已有目前技术水平不能检测到的微转移。因而，检测并治疗微转移是预防复发的有效途径。CK19、CEA都是研究得较多的胃癌微转移肿瘤标记物，敏感性和特异性较高，但各家实验报告结果不尽相同。对微转移治疗的研究，主要集中在化疗方面，包括术前、术中、术后化疗，但对术中经静脉全身化疗研究较少，对化疗的指征没有统一的认识，本实验采用RT-PCR技术，检测胃癌患者外周血中CK19mRNA、CEAmRNA含量及其变化，指导胃癌患者的术中化疗并观察疗效。
第一章：胃癌病人与外周中血OK19mRNA、CEAmRNA的表达
目的：探讨胃癌病人外周血微转移的检测指标。
方法：取已确诊的胃癌病人92例及正常人20例，取外周血，用RT-PCR法检测外周血CK19mRNA、CEAmRNA表达情况，比较胃癌病人外周血与健康人两项指标的差异，分析胃癌微转移与各临床因素的关系，进行统计学分析。
结果：(1)正常人外周血CK19mRNA、CEAmRNA，全为阴性。胃癌组病人外周血CK19mRNA阳性率46.7％，CEAmRNA阳性率42.4％，两指标总的阳性率(有一项阳性为阳性)为66.3％。(2)胃癌病人外周血OK19mRNA、OEAmRNA联合检测与单独检测之差别。总阳性率为66.3％。与单独检测比较差异有显著性(x~2=23.53，P＜0.01)。(3)微转移与各临床因素的关系：不同病理类型CK19mRNA、CEAmRNA总阳性的表达有差异，(x~2=14.92，P＜0.05)。浸润深度情况不同，外周血CK19mRNA、OEAmRNA的表达有差异，浆膜受累的表达率明显高于未受累者(X~2=13.01，P＜0.01)。病理分期与外周血OK19mRNA、CEAmRNA的表达有关，分期越晚，表达率越高(x~2=13.48，P＜0.05)。淋巴结有转移者CK19mRNA、CEAmRNA的表达率明显高于无淋巴结转移者(x~2=7.14，P＜0.05)。肿块部位与CK19mRNA、CEAmRNA的表达率无关，不同部位的表达率无明显差异(X~2=5.53，P＞0.05)。年龄与CK19mRNA、OEAmRNA的表达无关系(x~2=2.94，P＞0.05)。性别与CK19mRNA、CEAmRNA的表达无关系(x~2=1.23，P＞0.05)。
结论：1采用RT-POR技术检测胃癌病人外周血CK19mRNA、CEAmRNA的表达能较客观地反映胃癌病人外周血微转移的状况。2联合检测OK19mRNA、OEAmRNA能提高微转移的检出率。3胃癌病人外周血微转移与年龄、性别、部位无关，与病理类型、肿瘤浸润深度、临床分期、淋巴结转移情况有关。
第二章：胃癌病人术中化疗方案筛选及化疗体外药敏实验的研究
目的：探讨胃癌术中化疗较合适的方案。
方法：病例选择；第一部分中的病例中取35例，其中女性17例，男性18例，年龄31-74，平均41.6岁，病理类型；高分化腺癌10例，中分化10例，低分化12例，未分化3例。所有病人都未接受抗肿瘤治疗。肿瘤体外药敏实验：采用MTT法：制备20种化疗方案的工作液，分别测定各种方案的抑瘤率。进行统计学分析，选择抑瘤率高者作化疗方案。
结果：20种方案中以5-FU抑瘤率最高(57.36±15.2)，与其他方案比差异有显著性(P＜0.05)
结论：体外药敏实验显示：5-FU对胃癌有较高的抑瘤率，明显优于其他方案，可筛选作为胃癌病人的术中化疗首选方案。
第三章术中化疗对胃癌病人外周血微转移的治疗作用的研究
第一节胃癌病人外周血OK19mRNA、OEAmRNA的表达对术中化疗的指导作用
目的：采用CK19、CEAmRNA的表达来观察术术中化疗对微转移的治疗效果，并探讨术中化疗的效果。
方法：取术前外周血中CK19mRNA、CEAmRNA至少一项呈阳性病人30例和都呈阴性的胃癌病人30例，分成6组，每组10人，按以下分组：1组：CK19mRNA、CEAmRNA都呈阴性，不施术中化疗。2组：CK19mPNA、CEAmPNA都呈阴性，施化疗，方案为术中滴一次5-FU(剂量500mg／m~2)。3组：CK19mRNA、CEArnRNA都呈阴性，施化疗，方案为术前2天及术中各滴一次5-FU(剂量500mg／m~2)。4组：CK19mRNA、CEAmRNA都呈阳性，不施术中化疗。5组：CK19mRNA、CEAmRNA都呈阳性，施化疗，方案为术中滴一次5-FU(剂量同前)。6组：CK19mRNA、CEAmRNACK都呈阳性，施化疗，方案为术前2天及术中各滴一次5-FU(剂量同前)。
每例病人于术程开腹前、胃癌根治术取下标本后、术后3天、术后1周、术后2周采外周静脉血，术中开腹后及取下标本后各采网膜右静脉血一次；术后取癌组织及正常胃组织，用RT-PCR检测CK19mRNA、CEAmRNA表达情况。
结果：癌组织中CK19mRNA、CEAmRNA的表达，所有组病例癌组织总的阳性率为90％-100％，6组之间差异无显著性P=0.824，P＞0.05；正常胃组织CK19mRNA、CEAmRNA的表达情况：只有CK19mRNA有表达，约2／10-1／10例，6组比较，P=0.62，P＞0.05，无显著性差异。外周血CK19mRNA、CEAmRNA总的阳性率各组变化情况：阴性病人手术前后血清CK19mRNA、CEAmRNA的变化各组之间(1组与2，3组比较)无显著性差异(P＞0.05)，阳性病人手术前后血清CK19mRNA、CEAmRNA的变化各组之间(4组与5，6组比较)有显著性差异(P＜0.05)，术中开腹后网膜右静脉血CK19mRNA、CEAmRNA的表达与术前外周血的表达一致，无差异；术中取下标本后网膜右静脉血CK19mRNA、CEAmRNA的表达与术前外周血的表达不一致，原来阴性的病人(1、2、3组)出现一定的阳性率(5／10-6／10)，但三组之间比较差异无显著性(P＞0.05)。
结论：(1)术中化疗能减少胃癌病人外周血中CEAmRNA、CK19mRNA的表达，提示可能对外周血微转移有一定的治疗作用。(2)外周血CEAmRNA、CK19mRNA的表达水平可作为术中化疗的指征参考及疗效观察指标。(3)5-FU术中静滴可作为胃癌病人术中化疗首选方案。
第二节术中化疗对胃癌病人外周血微转移治疗效果的临床观察
目的：分析术中化疗的远期疗效。
方法：对第三部分的病例做长期随访，每例病人都行规范的术后辅助化疗，每组病人以3个月为时间单位计算生存率，绘制生存曲线。然后比较各组生存率的差异。
结果：外周血微转移阴、阳性生存曲线对比(1、4组对比)，x~2=5.24，P＜0.05，差异有显著性。外周血微转移阴性生存率曲线对比：x~2=3.98，P＞0.05，差异无显著性。外周血微转移阳性病人生存曲线的比较x~2=9.47，P＜0.05，3组间的生存率差异有显著性。趋势分析，x~2=8.23，P＜0.05，差异有显著性，说明生存情况好坏依次为：6组、5组、4组。
结论：胃癌病人外周血微转移对疾病的预后有很重要意义，阳性者的术后无病生存期明显短于阴性者。术中化疗对外周血微转移有明显的治疗作用，5-FU静滴化疗有较好的效果。化疗方案还可进一步完善。
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
Backgroung : 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. ( X~2=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 (X~2=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 ( X~2=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 (X~2=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 ( X~2=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 (X~2=5.53, P＞0.05 ) .There was no correlation between the expression of CK19mRNA ,CEAmRNA and the patient's age ( X~2=2.94, P＞0.05)。There was no correlation between the expression of CK19mRNA ,CEAmRNA and the patient's sexual ( X~2=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 vitro
Objective: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 cancer
Objective: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/m_2 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/m_2 iv by drip during operation. Group6: anyone of CK19mRNA、CEAmRNA was positive, chemotherapy was performed as follow: 5-FU 500mg/m~2 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 cancer
Objective: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), X~2 =5.24, P＜0.05。There was no difference between the groups with micrometastases negative(group 1,2,3), X~2=3.98, P＞0.05。There was difference between the groups with micrometastases positive(group 4,5,6), X~2 =9.47, P＜0.05;and the range of survival rates in hign-to-low order was group 6,5,4.(trendy test: X~2 =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.
第一章：胃癌病人与外周中血OK19mRNA、CEAmRNA的表达
目的：探讨胃癌病人外周血微转移的检测指标。
方法：取已确诊的胃癌病人92例及正常人20例，取外周血，用RT-PCR法检测外周血CK19mRNA、CEAmRNA表达情况，比较胃癌病人外周血与健康人两项指标的差异，分析胃癌微转移与各临床因素的关系，进行统计学分析。
结果：(1)正常人外周血CK19mRNA、CEAmRNA，全为阴性。胃癌组病人外周血CK19mRNA阳性率46.7％，CEAmRNA阳性率42.4％，两指标总的阳性率(有一项阳性为阳性)为66.3％。(2)胃癌病人外周血OK19mRNA、OEAmRNA联合检测与单独检测之差别。总阳性率为66.3％。与单独检测比较差异有显著性(x~2=23.53，P＜0.01)。(3)微转移与各临床因素的关系：不同病理类型CK19mRNA、CEAmRNA总阳性的表达有差异，(x~2=14.92，P＜0.05)。浸润深度情况不同，外周血CK19mRNA、OEAmRNA的表达有差异，浆膜受累的表达率明显高于未受累者(X~2=13.01，P＜0.01)。病理分期与外周血OK19mRNA、CEAmRNA的表达有关，分期越晚，表达率越高(x~2=13.48，P＜0.05)。淋巴结有转移者CK19mRNA、CEAmRNA的表达率明显高于无淋巴结转移者(x~2=7.14，P＜0.05)。肿块部位与CK19mRNA、CEAmRNA的表达率无关，不同部位的表达率无明显差异(X~2=5.53，P＞0.05)。年龄与CK19mRNA、OEAmRNA的表达无关系(x~2=2.94，P＞0.05)。性别与CK19mRNA、CEAmRNA的表达无关系(x~2=1.23，P＞0.05)。
结论：1采用RT-POR技术检测胃癌病人外周血CK19mRNA、CEAmRNA的表达能较客观地反映胃癌病人外周血微转移的状况。2联合检测OK19mRNA、OEAmRNA能提高微转移的检出率。3胃癌病人外周血微转移与年龄、性别、部位无关，与病理类型、肿瘤浸润深度、临床分期、淋巴结转移情况有关。
第二章：胃癌病人术中化疗方案筛选及化疗体外药敏实验的研究
目的：探讨胃癌术中化疗较合适的方案。
方法：病例选择；第一部分中的病例中取35例，其中女性17例，男性18例，年龄31-74，平均41.6岁，病理类型；高分化腺癌10例，中分化10例，低分化12例，未分化3例。所有病人都未接受抗肿瘤治疗。肿瘤体外药敏实验：采用MTT法：制备20种化疗方案的工作液，分别测定各种方案的抑瘤率。进行统计学分析，选择抑瘤率高者作化疗方案。
结果：20种方案中以5-FU抑瘤率最高(57.36±15.2)，与其他方案比差异有显著性(P＜0.05)
结论：体外药敏实验显示：5-FU对胃癌有较高的抑瘤率，明显优于其他方案，可筛选作为胃癌病人的术中化疗首选方案。
第三章术中化疗对胃癌病人外周血微转移的治疗作用的研究
第一节胃癌病人外周血OK19mRNA、OEAmRNA的表达对术中化疗的指导作用
目的：采用CK19、CEAmRNA的表达来观察术术中化疗对微转移的治疗效果，并探讨术中化疗的效果。
方法：取术前外周血中CK19mRNA、CEAmRNA至少一项呈阳性病人30例和都呈阴性的胃癌病人30例，分成6组，每组10人，按以下分组：1组：CK19mRNA、CEAmRNA都呈阴性，不施术中化疗。2组：CK19mPNA、CEAmPNA都呈阴性，施化疗，方案为术中滴一次5-FU(剂量500mg／m~2)。3组：CK19mRNA、CEArnRNA都呈阴性，施化疗，方案为术前2天及术中各滴一次5-FU(剂量500mg／m~2)。4组：CK19mRNA、CEAmRNA都呈阳性，不施术中化疗。5组：CK19mRNA、CEAmRNA都呈阳性，施化疗，方案为术中滴一次5-FU(剂量同前)。6组：CK19mRNA、CEAmRNACK都呈阳性，施化疗，方案为术前2天及术中各滴一次5-FU(剂量同前)。
每例病人于术程开腹前、胃癌根治术取下标本后、术后3天、术后1周、术后2周采外周静脉血，术中开腹后及取下标本后各采网膜右静脉血一次；术后取癌组织及正常胃组织，用RT-PCR检测CK19mRNA、CEAmRNA表达情况。
结果：癌组织中CK19mRNA、CEAmRNA的表达，所有组病例癌组织总的阳性率为90％-100％，6组之间差异无显著性P=0.824，P＞0.05；正常胃组织CK19mRNA、CEAmRNA的表达情况：只有CK19mRNA有表达，约2／10-1／10例，6组比较，P=0.62，P＞0.05，无显著性差异。外周血CK19mRNA、CEAmRNA总的阳性率各组变化情况：阴性病人手术前后血清CK19mRNA、CEAmRNA的变化各组之间(1组与2，3组比较)无显著性差异(P＞0.05)，阳性病人手术前后血清CK19mRNA、CEAmRNA的变化各组之间(4组与5，6组比较)有显著性差异(P＜0.05)，术中开腹后网膜右静脉血CK19mRNA、CEAmRNA的表达与术前外周血的表达一致，无差异；术中取下标本后网膜右静脉血CK19mRNA、CEAmRNA的表达与术前外周血的表达不一致，原来阴性的病人(1、2、3组)出现一定的阳性率(5／10-6／10)，但三组之间比较差异无显著性(P＞0.05)。
结论：(1)术中化疗能减少胃癌病人外周血中CEAmRNA、CK19mRNA的表达，提示可能对外周血微转移有一定的治疗作用。(2)外周血CEAmRNA、CK19mRNA的表达水平可作为术中化疗的指征参考及疗效观察指标。(3)5-FU术中静滴可作为胃癌病人术中化疗首选方案。
第二节术中化疗对胃癌病人外周血微转移治疗效果的临床观察
目的：分析术中化疗的远期疗效。
方法：对第三部分的病例做长期随访，每例病人都行规范的术后辅助化疗，每组病人以3个月为时间单位计算生存率，绘制生存曲线。然后比较各组生存率的差异。
结果：外周血微转移阴、阳性生存曲线对比(1、4组对比)，x~2=5.24，P＜0.05，差异有显著性。外周血微转移阴性生存率曲线对比：x~2=3.98，P＞0.05，差异无显著性。外周血微转移阳性病人生存曲线的比较x~2=9.47，P＜0.05，3组间的生存率差异有显著性。趋势分析，x~2=8.23，P＜0.05，差异有显著性，说明生存情况好坏依次为：6组、5组、4组。
结论：胃癌病人外周血微转移对疾病的预后有很重要意义，阳性者的术后无病生存期明显短于阴性者。术中化疗对外周血微转移有明显的治疗作用，5-FU静滴化疗有较好的效果。化疗方案还可进一步完善。
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
Backgroung : 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. ( X~2=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 (X~2=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 ( X~2=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 (X~2=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 ( X~2=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 (X~2=5.53, P＞0.05 ) .There was no correlation between the expression of CK19mRNA ,CEAmRNA and the patient's age ( X~2=2.94, P＞0.05)。There was no correlation between the expression of CK19mRNA ,CEAmRNA and the patient's sexual ( X~2=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 vitro
Objective: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 cancer
Objective: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/m_2 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/m_2 iv by drip during operation. Group6: anyone of CK19mRNA、CEAmRNA was positive, chemotherapy was performed as follow: 5-FU 500mg/m~2 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 cancer
Objective: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), X~2 =5.24, P＜0.05。There was no difference between the groups with micrometastases negative(group 1,2,3), X~2=3.98, P＞0.05。There was difference between the groups with micrometastases positive(group 4,5,6), X~2 =9.47, P＜0.05;and the range of survival rates in hign-to-low order was group 6,5,4.(trendy test: X~2 =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.
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