Esophageal Mucosa Dyeing And Detecting Of P53 And P16 In Improving The Early Diagnostic Rate Of Barrett's Esophagus And Esophageal Adenocarcinoma

Recent years, the incidence of esophageal adenocarcinoma(EAC)increased rapidly , the prognosis of EA is very bad and the survival ratefor 5 years is only 17%. Numerous studies have demonstrated thatBarrett's esophagus(BE) is the precursor of EA and about 30% of BEwill progress to EAC. The esophageal cancer risk for BE patients is30-125 times greater than for the general population. At present , thetherapy of tumors remains difficult in medical studies and clinical worksand after medical and surgical therapy the metaplasia columnar epitheliumcan regress to squamous epithelium. So early detection and therapy of BEmay prevent the progression to invasive adenocarcinoma and improve thepatients' living quality.The definition of BE given by BE Congress of AmericanGastroenterological Association(AGA) at February ,2003 in Chicago is :pathological changes with intestinal metaplasia located betweensquamoucolumnar junction( SCJ) and gastroesophageal junction (GEJ). Itincludes abnormal external appearance of the mucosa of distal ofesophagus at endoscopy and intestinal metaplasia of esophageal mucosaconfirmed by biopsy.Endoscopy incorporating with biopsy and cytologic diagnosis hasbeen considered as the reliable diagnostic approach of BE and itsprogression to invasive malignancy . But in the situation of pathologicalform the variety of the appearance of mucosa especially the existence ofearly tiny focus make it difficult to take a biopsy well and truly just atroutine endoscopy for endoscopic doctors. So in this study we useendoscopic esophageal mucosa dyeing incorporating with biopsy anddetection of relative oncogene to improve the diagnostic rate of BE andEAC.Esophageal mucosa dyeing is a technique that intravital staining ofesophageal epithelia by dyes to make the mucosa lesion to take on aspecial colour at endoscopy. At present the usual staining agents arecompound iodine solution (Lugol's solution), methylene blue, toluidineblue and so on. Toluidine blue is a kind of dye that stain the nucleus of acell, and because the content of DNA in cancer cell is markedly higherthan normal cell there is a clear borderline between tumor epithelium andnormal squamous epithelium after dyeing. Lugol's solution is a kind ofabsorbable dye based on iodine. It has affinity for hepatin ofnon-keratinized squamous epithelium. Squamous epithelium withneoplasia and dysplasia has few hepatins even loss, so it is stainless orslightly staining to iodine solution and there is a great contrast , accordingwhich we can take a biopsy well and truly.The p16 gene located on 9p21 is an important cancer suppressor gene.It encodes an inhibitor of cyclin-dependent kinase 4 (CDK4), which canrestrain the activity of CDK4 specially after combined with CDK4.Consequently cell proliferation and growth are restrained. When the p16gene is damaged by mutation, the protein does not function properly andcells may grow out of control, resulting in cancer.The p53 gene located on short arm of chromosome 17(17p13) isone of the tumorous genes which are studied most widely and deeply. Itinvolves in the negative regulation of cell proliferation. The main functionof p53 protein is restraining cell cycle, inducing apoptosis and acceleratingdifferentiation. When losing the function ,cells divide uncontrollably andform tumors. In addition, the p53 gene lose its function after combinedwith some cancer protein such as E6 protein of HPV 16,SV40 T antigenand adenovirus EIB and mutant P53 not only lose the ability of inhibitingcancer but also can induce cancerization. As P53 gene mutates, ittransforms from cancer suppressor gene into oncogene.30 cases of BE (testing group), 30 cases of reflux esophagitis(RE,comparing group) were selected by endoscopy. Then the tissue specimensof esophageal mucosa which need biopsy and immunohistochemicalstaining was divided into subgroup before byeing and subgroup afterdyeing. All patients were diagnosed according to diagnosis criteria of BECongress of American Gastroenterological Association(AGA) atFebruary ,2003 in Chicago. At routine endoscopy ,we observed theesophageal mucosa of patients with appearance of BE and RE , took 3-5pieces of tissue specimens from where there are hyperaemicspot,hyperplastic grains,partial apophysis, retuse or erosionalfocus,ulcer,and abnormal colour , sprayed 5 to 10 mL of 2% toluidine blueonto the pathological esophageal mucosa, followed by spraying 50 mL tapwater after 1-3minutes, then sprayed 5 to 10 mL of 3% Lugol's solution,followed by spraying tap water and aspirating staining water several timesafter 2-5minutes until there is a clear eye shot, and then we observed andtook 3-5 pieces of tissue specimens of the mucosas which have beenstained by toluidine blue and which are stainless to Lugol's solution. Thetissue samples were fixed in 10% formalin, embedded in paraffin andsectioned. The sections were examined by routine biopsy and then stainedwith immunohistochemistry. Immunohistochemical staining was used todetect the expressions of P16 and P53 in tissue specimens. In routinebiopsy intestinal metaplasia(IM) ,high-grade dysplasia (HGD)andadenocarcinoma (EAC)were considered to be positive .The tissuesamples were considered to be positive for gene expression only whenthere were nuclear staining brown-yellow (P53) or brown (P16). Eachspecimen was observed in ten high-time-fields(×400). Counting thepositive cells, it is positive if the ratio of the positive cells is more than 5%and it is negative if the ratio is less than 5% or no positive cells. PBS wasused as negative control and esophageal section known immunoreactivityto antibodies against P16 and P53 are used as positive control.Result: 1. The detection rates of IM were respectively 70% and93.33% in BE before and after dyeing and there were significantdifferences between them (P<0.05);the detection rates of HGD and EACwere respectively 10% and 36.67% in BE before and after dyeing andthere were significant differences between them (P<0.05). It wasconcluded that Esophageal mucosa dyeing can enhance the veracity ofbiopsy and accordingly the early diagnostic rate of BE and EAC.Compared with RE group ,the detection rates of HGD and EAC of BEgroup is much higher and it confirmed that BE is a progression of EAC. 2.The expression rates of P16 in BE and RE before dyeing were 30%and20%, which was consistent with some previous reports, however, it was56.67% and 40% after dyeing, higher than the rates beforedyeing(especially BE group, P﹤0.05). The expression rates of P53 in BEand RE before dyeing were 23.33%and 16.67%, which was consistentwith some previous reports, however, it was 50% and 40% after dyeing,obviously higher than the rates before dyeing(both groups, P﹤0.05). Itshows there were high expressions of p53 and p16 in esophageal mucosaswith positive dyeing and there were positive relativity between them. Atthe same time, in this result the expresstions of p53 and p16 increasedgradually according to the sequence of reflux esophagitis ,Barrett'sesophagus and esophageal adenocarcinoma , it suggested a positiverelativity and confirmed the above-mentioned progressive sequence atprotein level. 3. After esophageal mucosa dyeing the detection rates ofIM,HGD,EAC and p53, p16 protein all increased greatly, especially in BEgroup , and there was a positive relativity between the detection rates ofp53 ,p16 protein and that of HGD and EAC , so it suggested thatendoscopic esophageal mucosa dyeing incorporating with biopsy anddetection of relative oncogene was an effective method to improve thediagnostic rate of BE and EAC . Endoscopic esophageal mucosa dyeingincorporating with immunohistochemical examination of p53 and p16may be helpful to early diagnosis of BE and EAC and then effectivetherapies can be done , which has very important clinical significance tothe prevention and remedy of EAC.In conclusion, endoscopic esophageal mucosa dyeing and biopsy arethe reliable diagnostic methods of Barrett's esophagus and esophagealadenocarcinoma. Barrett's esophagus is a progression of esophagealadenocarcinoma or the precursor of esophageal adenocarcinoma. To detectthe expression of p53 and p16 is an effective method of improving thediagnostic rate of Barrett's esophagus and esophageal adenocarcinoma.