Investigation On The Clinical Application Of Gastric Mucosal Marking Targeting Biopsy

PrefaceGastric carcinoma is one of the most prevalent malignancies around the world. In China,its incidence rate and mortality rate rank top among all cancer.However, due to its insidious onset and unspecific manifestations,early detection of cancerous lesions remains a difficult task.Most of the patients with newly diagnosed gastric cancer are in the middle or late stage,the overall 5-year survival rate is only 15-20%.Chronic atrophic gastritis(CAG)with intestinal metaplasia and(or)atypical hyperplasia(intraepithelial neoplasia)are universally recognized important precancerous lesions which are closely related with gastric carcinogenesis.The carcinogenetic rate of CAG in China is 1.2～7.1%,while in other countries,it reaches 8.6～13.8%.Therefore,the intervention and blockage of precancerous lesions are key to gastric cancer prevention.To achieve this goal,several important measures shall be carried out,including①accurate and periodical monitoring of precancerous lesions aiming to the early detection of cancer,and②screening of effective drugs to prevent the progression of precancerous lesions and inversing precancerous lesions to reduce the risk of tumorigenesis.Gastroesophagoscopy and pathological diagnosis of biopsy are the main techniques to monitor development of precancerous lesions clinically.However,how to accurate localize and target gastric mucosa with precancerous lesions for clinical follow-up remains problematic.Currently,ordinary biopsy technique is unable to precisely obtain tissues from the identical location during different follow-up endoscopy examinations.The Marking Targeting Biopsy(MTB),a novel technique originally invented by our lab,employs a specially-designed biopsy forceps to perform biopsy and tissue staining simultaneously.The stained marks are easily visible during follow-up endoscopies.This technique has undergone three technical improvements and repeated preliminary test on animal models.It has been proved to be safe,and the endurance of tattooing marks is long enough for recognition during follow-up endoscopic examinations.This technique and its application have been accredited by engineers from the biological engineering department of Zhejiang University and biological and medical equipment experts.It also achieved national invention patent(mucosal marking targeting biopsy forceps,patency number:ZL 01112558.6).Theoretically,MTB could solve the critical technical problems concerning the precise discrimination of precancerous lesions,but its clinical availability,application regulations and values still necessitate further investigation.The persistent existence of damaging factors and disturbance of defensive capability of gastric mucosa are considered as the main causes for carcinogenesis. Therefore,effective measures to stop inflammation,promote repairing and inverse precancerous lesions have long been pursued.For instance,the eradication therapy of Hp,various gastric mucosal protective agents to eliminate damaging factors,as well as folic acid which is an anti-oxidative agent to stabilize DNA methylization and eliminate mutant cells through apoptotic pathway.Nevertheless,whether these measures are effective remains to be testified in clinical practice.Therefore,in this study,we used MTB technique to follow up patients with precancerous lesions who are treated with blockage medications.We obtained tissue samples from precisely the identical location and compared the development of disease before and after therapy.Through these efforts,we investigated the clinical availability and application of MTB technique,established the procedure regulations, and estimated current blockage therapy for precancerous lesions. Material and MethodsWe collected patients with precancerous lesions by our inclusion and exclusion criteria and established a prospective cohort.After informed consents had been obtained,marking targeting biopsy was performed.1.Marking targeting biopsy:We performed endoscopy on selected patients using disposable mucosal MTB forceps.We chose lesser and greater curvature of antrum, lesser and greater curvature of body,angle and other suspicious lesions to perform injection under the mucosal layer by medical India ink.The concentration of ink is 1:10,and 0.3-0.5ml ink was injected at each point.Biopsy was performed at the end of injection at precisely the same location,tissue samples were sent for pathological analysis.2.Grouping therapy:Patients were classified into Hp positive and Hp negative group. In the positive group,patients were treated with Hp eradication therapy followed by one-week wash-out period.Then patients were categorized into A,B,C three groups,each group were given teprenone(50mg tid)+ folic acid(10mg tid) combined therapy,unspecific medications and no medication respectively.The negative group was directly classified into A,B,C three groups.3.Follow-up,including collection of clinical data,observation of tattooing effect under endoscopy and effect of different regimens.1.Record the duration and local mucosal changes of original tattooing marks at different time points(13week,26w,39w,52w,78w,104w,130w etc)and locations.Perform biopsy at the original location to compare the pathological development of tissue samples before and after treatment.Investigate whether India ink would induce local tissue reaction and influence pathological analysis.2.Observe patients tolerance,adverse effects during endoscopic MTB and associated clinical situations with medications.4.Data analysis:1.Probability statistical analysis was performed on the endurance of tattooing marks,factors which might affect the effect of marking were also analyzed.2.Self-comparison of symptoms and pathological effects was performed Results1.172 patients with precancerous lesions were gradually collected into the prospective cohort from May,2005 to March 2008.Among them,88 were male and 84 were female,the average age was 51.98±10.03(27～78).According to pathological diagnosis,129(75.00%)patients had gastric glands atrophy,149 (86.63%)patients had intestinal metaplasia,33(19.19%)patients had atypical hyperplasia(intraepithelial neoplasia)(including 3 patients with high-grade intraepithelial neoplasia who had already received surgical therapy).2.Follow-up and statistical analysis on precancerous patients monitored by MTB technique:A total of 101 endoscopy examinations were performed,the overall effective rate of marking targeting was as high as 90.48%;at different time points of 13w,26w,39w,52w,78w,104w and 130w,the existence rates of most significant tattooing mark were98.81%,96.43%,92.86%,90.48%,90.48%,90.48%,90.48%.The duration time ranged from 5w to 126w.From the survival curve,the tattooing marks remained stably and clearly visible after 52～130w. Although the duration of tattooing marks at different locations were not consistent, from our observation,the existent rates of tattooing marks at the lesser curvature of antrum and body,angle and special lesions(all were locations gastric cancer preferentially developed)were significantly higher than marks at other locations. These results indicated that MTB technique could satisfy the need for accurately monitoring precancerous lesions.3.Cox regression analysis was made on the persistence of tatooing marks with three factors including performing physicians,treatment medications and pathological states of the patients.Results showed the regression coeffecients were 0.324, -0.252,0.158 respectively(P＞0.05),indicating all the three were not independent influential factors.Through continous updates and improvements for three years,I gained the following experience:①Before injection,ensure the marking ink flows smoothly inside the biopsy forceps;②During injection,the needle should vertically stab into the submucosal layer,push and inject ink until the mucosa swollen slightly;③The best sequence for injection was greater curvature of gastric body-lesser curvature of gastric body-angle-lesser curvature of antrum -greater curvature of antrum,thus could ensure the visibility of all injections;④after injection,rinse mucosa with sterilized water,if the tattooing marks were not clear enough,re-mark immediately;⑤after the whole procedure,inform the patient with subsequent treatment and time for re-examination.4.After MTB,few patients experienced temporary upper gastric pain(commonly resolved within one week).Most patients tolerated MTB well,and no other adverse effect such as fever or melena was observed.Neither endoscopy nor pathological analysis found substantial mucosal inflammatory reaction in tattooing area.Remaining ink could be observed in the interspace of connective tissues,but it did not influence histological and cytological analysis.5.Estimation of the effect of teprenone + folic acid therapy,non-specific therapy and no therapy groups using MTB technique:The results showed no significance among three groups(P＞0.05).The overall effective rates for pathological inversion of precancerous lesions were 56.10%,28.57%,0%,the difference was significant(P＜0.01),the effect of teprenone + folic acid therapy was substantially better than the other two groups.Conclusion(1)Marking targeting biopsy technique is available in the monitoring of precancerous lesions.Its advantages include clear and long-term recognizable tattooing marks, more accurate comparison between biopsy samples,safer and fewer adverse effects.MTB technique is not only able to precisely monitor the development of precancerous lesions,but also beneficial to the estimation of various therapy effects.Therefore,it has very high values in clinical practice.(2)Teprenone combined with folic acid could obviously inverse atrophy,intestinal metaplasia and other precancerous lesions.