| BackgroundGastric cancer(GC)is a common malignant tumor of digestive system.It is currently the third most common cancer and its mortality rate is also ranked third in our country.For East Asians,especially Chinese patients,gastric cancer resection combined with D2 lymph node dissection is the standard treatment for resectable gastric cancer.Even if patients with gastric cancer have undergone radical resection,D2 lymph node dissection and postoperative adjuvant chemotherapy,there is still a local recurrence rate of up to 57%.Since the early symptoms of gastric cancer are not obvious,most patients are already in the locally advanced stage when they see a doctor.How to reduce the postoperative recurrence rate,improve the long-term survival,and find the recurrence and metastasis after surgery as early as possible is a problem that needs to be solved urgently for local advanced gastric cancer(LAGC).The results of perioperative treatment(neoadjuvant chemotherapy/radiotherapy+surgery+adjuvant chemical/radiotherapy)for lower esophagus and esophagogastric junction adenocarcinoma has proved to be better than surgery alone in Western countries.The main advantages of perioperative treatment are that it can reduce tumor staging,increase the R0 resection rate,and the patient's tolerance to preoperative treatment is much better than postoperative treatment,so as to ensure that all patients can receive the expected treatment on time.So far,only a few phase ? studies have proved the safety,tolerability and high pathological complete remission rate of neoadjuvant chemoradiotherapy for gastric cancer,and there are few studies on chemoradiotherapy for LAGC in China.At present,the international multi-center TOPGEAR study,the CRITICS-? study of Netherlands,and the Neo-CRAG study initiated by the Tumor Center of Sun Yat-sen University are actively exploring the neoadjuvant chemoradiotherapy for gastric cancer,and the final study results have not been announced yet.The tumor markers of CEA,CA199,CA724 and CA125 have low sensitivity and specificity in monitoring the recurrence and metastasis of gastric cancer after surgery.As the symptoms of early recurrence and metastasis of gastric cancer are relatively hidden and traditional imagings(such as ultrasound,CT,PET-CT,MRI,etc.)are also difficult to detect early micrometastasis of organs and peritoneal metastasis,which leads to many patients lose the best treatment opportunities.Therefore,we need more accurate biomarkers to predict early recurrence,metastasis and prognosis after gastric cancer surgery.In 1869,Ashworth first proposed the concept of circulating tumor cells(CTCs),which are defined as tumor cells that enter the peripheral blood circulation from solid tumors or metastases due to spontaneous or diagnostic procedures.CTC clusters,or circulating tumor microembolisms(CTM),are formed by the aggregation of several CTCs,which are more aggressive than CTCs,and are considered to be the most important dangerous precursor for tumor metastasis.Because CTC detection has the advantages of non-invasive diagnosis and real-time efficacy monitoring,the consensus of many oncologists at home and abroad and related studies have pointed out that CTC is another non-invasive biomarker after CEA,CA199,CA125,AFP,SCC and other tumor markers.CTC can be used as one of the methods for early screening,treatment response monitoring and prognostic evaluation for liver cancer,breast cancer,colorectal cancer,lung cancer and esophageal cancer.CTC is extremely rare in peripheral blood and about 1 CTC appears in every 106 to 107 peripheral blood nucleated cells.Therefore,this places high requirements on the separation and detection sensitivity of CTC.At present,a few studies of CTC in gastric cancer are mainly based on ISET and the CellSearch system to enrich CTC.However,the positive rate and accuracy of the above-mentioned method of enriching CTC are low.In addition,the CellSearch system has certain restrictions on the detection of CTM,and the detection of CTM can increase the understanding of the recurrence and metastasis of gastric cancer,thereby contributing to the later diagnosis and treatment.Negative enrichment(NE)combined with immunofluorescence CD45 staining and fluorescence in situ hybridization(FISH)(NE-imFISH)have high sensitivity and specificity in CTC enrichment and identification,and can perform the downstream research of CTC,such as molecular analysis.As far as we know,NE-imFISH technology is rarely used in the detection of CTC of gastric cancer,and there is no research to evaluate the predictive effect of CTC on the recurrence,metastasis and prognosis of stage ?-? gastric cancer after radical resection.In the first part of this study,we performed neoadjuvant intensity-modulated radiotherapy and concurrent XELOX chemotherapy and compared it with postoperative chemotherapy alone for operable LAGC.The purpose of this part was to observe the effects of neoadjuvant chemoradiotherapy on the degree of tumor regression,surgical resection rate,tumor stage,postoperative complications,postoperative local recurrence rate,long-term survival and adverse reactions in LAGC patients,so as to choose the most reasonable treatment plan for the diagnosis and treatment of LAGC.In addition,in order to explore the predictive effect of CTC on the recurrence,metastasis and prognosis of gastric cancer after radical resection,we used the NE-imFISH method to detect the CTC before the initial treatment and after operation and dynamically monitored CTC of gastric cancer in the second part of this study.The purpose of this part was to observe the quantitative expression of CTC in different stages of gastric cancer,to study the relationship between CTC and gastric cancer clinicopathological factors and its correlation with the recurrence,metastasis and prognosis of gastric cancer,so as to determine whether CTC can provide diagnostic evidence for the recurrence,metastasis and prognosis of gastric cancer,and ultimately provide guidance for the follow-up of gastric cancer after radical surgery.This study will provide sufficient clinical evidence for the diagnosis and treatment strategy and the prediction of recurrence,metastasis and prognosis of LAGC after radical surgery,so as to provide a reasonable and timely treatment plan for the treatment of LAGC,and ultimately achieve the purpose of prolonging the survival of patients.ObjectiveIn this study,we performed neoadjuvant intensity-modulated radiotherapy and concurrent XELOX chemotherapy for operable LAGC patients.The purpose of the study was to observe the effect of neoadjuvant chemoradiotherapy on the degree of tumor regression,surgical resection rate,tumor staging,postoperative complications rate,postoperative local recurrence rate,long-term survival and adverse reactions,so as to provide guidance for the clinical diagnosis and treatment of LAGC.MethodsFrom January 1 2014 to March 30,2017,71 patients in total with LAGC from Zibo Central Hospital were collected and assigned randomly to treatment group(36 cases)and control group(35 cases).Patients in treatment group were given IMRT(45 Gy/1.8 Gy/f)accompanied by synchronous XELOX of two cycles,followed by surgery,and then postoperative adjuvant XELOX chemotherapy of four cycles was performed.Patients in control group received surgery in advance,and then XELOX chemotherapy of six cycles was given.Patients in control group were given adjuvant IMRT accompanied by synchronous XELOX if there were postoperative radiotherapy indications.All patients were followed up for 3 years.Results1.The effect of neoadjuvant chemoradiotherapy in treatment groupIn the treatment group,5 cases(13.9%)achieved CR,23 cases(63.9%)had PR,4 cases(11.1%)were SD,and 4 cases(11.1%)had PD.The postoperative pathological results showed that 5 cases of TRG 1(13.9%),8 cases of TRG 2(22.2%),10 cases of TRG 3(27.8%),7 cases of TRG 4(19.4%)and.6 cases of TRG5(16.7%)according to the Mandard tumor regression grading system.Of the 32 surgical patients,81.3%of the patients had a decrease in the T stage,and 56.3%of the patients had a decrease in the N stage.Multivariate analysis revealed that independent parameter predicting improved OS and DFS were TRG1-2(HR=0.468,95%CI:0.020-1.103,P=0.017)and TRG1-2(HR=0.468,95%CI:0.001-0.771,P=0.035),respectively.2.Comparison of surgical results between two groupsPatients in treatment group showed a significantly higher R0 resection rate(87.5%vs.62.9%,P=0.021)than patients in control group.3.Comparison of postoperative complications between two groupsThe postoperative complications of two groups mainly manifested as remnant stomach weakness,anastomotic bleeding,anastomotic leakage,anastomotic stenosis,incision infection and intestinal obstruction.The total incidence of postoperative complications in treatment group and control group were 21.9%and 25.7%,respectively.There was no significant difference between two groups(P=0.713).4.Comparison of survival time between two groupsThe 1 year,2 years and 3 years OS rates of treatment group and control group were 86.1%,75.0%,61.1%and 77.1%,65.7%,51.4%,respectively.There was no significant difference in OS between two groups(with Log-rank test,P=0.156).The 1 year,2 years and 3 years DFS rates of treatment group and control group were 80.6%,72.2%,55.6%and 54.3%,48.6%,31.4%,respectively.Patients in treatment group showed a significantly longer DFS than patients in control group(with Log-rank test,P=0.013).Multivariate analysis revealed that independent parameters predicting improved OS including Bormann I(HR=0.114,95%CI:0.018-0.743,P=0.023),pathology with well differentiated(HR=0.187,95%CI:0.046-0.756,P=0.019)and R0 surgery(HR=0.118,95%CI:0.032-0.438,P=0.001).Independent parameters predicting high DFS was R0 surgery(HR=0.151,95%CI:0.047-0.478,P=0.001).5.Comparison of postoperative disease progression patterns between two groups(1)The 3-year local regional recurrence rate of patients in treatment group was 12.5%,which was significantly lower than that in control group of 37.1%(P=0.021).(2)The 1 year,2 years and 3 years local-regional failure free survival(LRFFS)rates of treatment group and control group were 96.9%,93.8%,87.5%and 80.0%,77.1%,62.9%,respectively.Patients in treatment group showed a significantly longer LRFFS than patients in control group(with Log-rank test,P=0.003).(3)The 1 year,2 years and 3 years' distant failure free survival(DFFS)rates of treatment group and control group were 83.3%,77.7%,63.9%and 94.3%,74.3%,62.9%,respectively.Patients in treatment group showed a significantly longer LRFFS than patients in control group(with Log-rank test,P=0.003).There was no significant difference in DFFS between two groups(with Log-rank test,P=0.598).(4)Multivariate analysis revealed that independent parameters predicting improved DFFS including Bormann I(HR=0.102,95%CI:0.012-0.878,P=0.038)and R0 resection(HR=0.118,95%CI:0.030-0.470,P=0.002).No independent parameters affecting LRFFS were found.6.Adverse reactionsThe adverse reactions related to neoadjuvant chemoradiotherapy mainly including nausea,vomiting,diarrhea,leukopenia,anemia,thrombocytopenia and hand-foot syndrome.Most of the adverse reactions are Grade 1-3.Most of the adverse reactions in two groups during adjuvant chemotherapy were Grade 1-3 and no life-threatening Grade 4-5 adverse reactions occurred.There was no significant difference in the incidence of adverse reactions in two groups during adjuvant chemotherapy(P>0.05).Conclusions1.The better of tumor pathological regression(TRG)after neoadjuvant chemoradiotherapy,the better of prognosis of patients.2.Neoadjuvant chemoradiotherapy increased R0 surgical resection rate,reduced the T stage and N stage,and did not increase the incidence of postoperative complications of LAGC.3.Neoadjuvant chemoradiotherapy can reduce the local regional recurrence rate and prolong the 3 years LRFFS of LAGC.4.Neoadjuvant chemoradiotherapy prolong the 3 years DFS of LAGC.5.Multivariate analysis revealed that patients with Bormann I,pathology with well differentiated and R0 resection can get better OS,and patients with R0 resection can get better DFS.6.Neoadjuvant chemoradiotherapy for LAGC is safe,and patients can tolerate adverse reactions.ObjectiveAt present,negative enrichment(NE)combined with immunofluorescence CD45 staining and fluorescence in situ hybridization(FISH)(NE-imFISH)are rarely used in the detection of CTC of gastric cancer,and there is no research to evaluate the predictive effect of CTC on recurrence,metastasis and prognosis of stage ?-?gastric cancer after radical resection.This study mainly used NE-imFISH technology to detect CTC before and after treatment of gastric cancer.The purpose of the study was to observe the quantitative expression of CTC in different stages of gastric cancer before treatment,and to study the relationship between CTC and clinicopathological factors of gastric cancer and its correlation with postoperative recurrence,metastasis and long-term survival of gastric cancer,which to explore whether CTC detection can provide a diagnostic basis for recurrence,metastasis and prognosis of gastric cancer after operation.MethodsA total of 78 patients with gastric cancer who underwent D2 and R0 resection at Zibo Central Hospital from January 5,2015 to March 30,2018 were selected as the main research objects.At the same time,we selected 20 healthy individuals without history of tumors as the control group in order to test the sensitivity and specificity of CTC in the diagnosis of gastric cancer.All patients with gastric cancer will receive CTC testing 1 day before surgery and concurrent radiotherapy and patients who do not require chemotherapy will be receive CTC testing one month after surgery and patients with postoperative chemotherapy will be receive CTC testing one month after adjuvant chemotherapy.After that,all gastric cancer patients were checked CTC every 3 months during the first year,every 4 months in the second year,every 6 months in the third year,and yearly after the fourth year.Healthy people only have one CTC test.Results1.Analysis of CTC detection results in gastric cancer patients and healthy volunteersThe 20 healthy volunteers detected mainly normal diploid cells or white blood cells,of which 5 cases(25%)found CTC(CTC counts were 1,1,1,2,and 3).CTC was detected in 76 patients(97.4%)of 78 patients with gastric cancer before initial treatment,and the median number of CTC was 6(range 1-25)per 3.2ml of blood in 76 patients.The ROC curve was created to determine the best CTC threshold for distinguishing gastric cancer patients from healthy individuals.When cut-off=2,the sensitivity and the specificity of this model for diagnosing gastric cancer were 91.0%and 90.0%,respectively.2.Correlation between the detection of CTC and the clinical and pathological characteristics of gastric cancer patients before treatmentBefore the initial treatment,7 patients with CTC<2 were detected in 78 patients with gastric cancer,including 3 patients(42.9%)at stage I,4 patients at stage ?(57.1%)and 0 patients at stage ?.The number of patients with CTC?2 was 71,6 cases(8.5%)at stage ?,22 cases(31.0%)at stage ? and 43 cases(60.5%)at stage ?.There was significant difference in the composition of preoperative TNM staging of patients with CTC<2 and CTC?2(P<0.001),and the patients with CTC<2 are mainly stage ?,while the patients with CTC?2 are mainly stage ? and ?.The detection rate of CTC?4 in patients with stage ? gastric cancer before surgery was 97.7%,which was significantly higher than stage ?(22.2%)and stage ?(61.5%)(P<0.001),and patients with CTC?5 was 95.3%,which was aslo significantly higher than stage ?(11.1%)and stage II(46.1%)(P<0.001).The 5 patients with CTM were all stage ?.3.Comparison of CTC detection before and after chemoradiotherapy and operation in patients with gastric cancerThe detection rate of CTC?2 after neoadjuvant chemoradiotherapy was 5.3%,which was significantly lower than 89.5%before neoadjuvant chemoradiotherapy among the 19 patients who received neoadjuvant chemoradiotherapy(P<0.001).The detection rate of CTC?2 after surgery was 5.1%,which was significantly lower than 91.5%before surgery among 59 patients who did not receive neoadjuvant chemoradiotherapy(P<0.001).4.Comparison of the recurrence and metastasis rate and the CTC detection rate of gastric cancerThe 1 year,2 years and 3 years imaging recurrence and metastasis rates of 78 patients with gastric cancer were 14.1%,21.8%and 34.6%,respectively.The detection rates of CTC?2 in the first year,second year and third year were 19.2%,29.5%and 41.0%,respectively.The detection rates of CTC?2 in patients with recurrence and metastasis of imaging in the first year,second year,and third year were 90.9%,94.1%,and 96.3%,respectively.5.Survival analysis(1)All patients were followed up for 3 years.The 1 year,2 years and 3 years OS rates and DFS rates of imaging of patients were 91.0%,84.6%,71.8%and 85.9%,78.2%,65.4%,respectively.(2)The 1 year,2 years and 3 years OS rates of patients with CTC?2 and CTC<2 were 81.3%,65.6%,34.3%and 97.8%,97.8%,97.8%,respectively.Patients with CTC<2 showed a significantly longer OS than patients with CTC?2(with Log-rank test,P<0.001).(3)The 1 year,2 years and 3 years DFS rates of imaging of patients with CTC?2 and CTC<2 were 68.8%?50.0%,18.8%and 97.8%,97.8%,97.8%,respectively.Patients with CTC<2 showed a significantly longer DFS than patients with CTC?2(with Log-rank test,P<0.001).(4)The patients with CTC?2 were divided into 2?CTC<5 and CTC?5 groups.The OS of the 2?CTC<5 group was significantly longer than that of the CTC?5 group(with Log-rank test,P<0.001).The DFS of patients with 2?CTC<5 was significantly longer than that of patients with CTC?5 group(with Log-rank test,P<0.001).(5)The OS and DFS of patients with no CTM but CTC?2 were both significantly longer than that of patients with CTM(with Log-rank test,P<0.001).(6)The median DFSCTC of patients with CTC?2 was 18.7 months(5.2-72.1 months),while the median DFS of patients with recurrence and metastasis of imaging was 24.9 months(6.9-73.5 months).The median time to detect CTC?2 was 6.2 months earlier than the median time to of recurrence and metastasis of gastric cancer found by imaging.(7)Multivariate analysis revealed that independent parameters predicting improved DFS including CTC<2(HR=0.085,95%CI:0.010-0.735,0.025)and no CTM was detected after operation(HR=0.007,95%CI:0.001-0.077,P<0.001).Independent parameters predicting high OS were CTC<2(HR=0.042,95%CI:0.005-0.339,P=0.003)and no CTM was detected after operation(HR=0.033,95%CI:0.006-0.178,P<0.001).Conclusions1.The detection of CTC with NE-imFISH technology has high sensitivity and specificity for the diagnosis of gastric cancer.2.The later the staging of gastric cancer before initial treatment,the higher the detection rate of CTC?2 and the number of CTC detections.3.The detection rate of CTC?2 decreased significantly after neoadjuvant chemoradiotherapy and surgery for gastric cancer,indicating that the change of CTC before and after treatment can evaluate the efficacy of treatment.4.With the increase in the detection rate of CTC after operation,the rate of recurrence and metastasis of imaging has also increased year by year.5.The prognosis of patients with CTC?2 detected after operation is poor.The more the number of CTCs detected,especially with CTM,the worse of the prognosis for patients.6.Monitoring the changes of CTC after operation can detect the recurrence and metastasis of gastric cancer earlier than imaging.7.The detection of CTC?2 and CTM after operation are independent influencing factors for the poor prognosis of patients. |
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