Early Diagnosis Of Esophageal Cancer Based On Replication-Selective Oncolytic Adenovirus

BackgroundChina is one of the countries with high incidence of esophageal cancer.The morbidity and the mortality of esophageal cancer in China were ranked sixth and fourth separately in malignant tumors in 2012.In recent years,the incidence is rising,and most patients have progressed to the middle or late stages of treatment when their life have been seriously affected.What's worse,the 5-year survival rate is less than 30%.Early lesions such as intramucosal cancer and submucosal superficial cancer are feasible with minimally invasive endoscopic treatment,Which is equivalent to surgery.At the same time,it can reduce surgical trauma and complications.what's more,the patients will recover quickly from surgery.The 5-year survival rate is significantly improved and it is more than 95%.Therefore,it is of great significance to study effective early diagnosis of esophageal cancer.In the 1950 s,esophageal cytology enabled early diagnosis of esophageal cancer.The esophageal mucosal exfoliated cells collected by the netting device were smeared or placed in examination solution,then stained and microscopeed to observe whether there are pathological changes.Previous studies have shown that the positive rate of esophageal exfoliation cytology for the diagnosis of esophageal cancer can reach 80% to 90% in the patients who were suspected esophageal cancer with clinical symptoms.This method also provides conditions for the early diagnosis of esophageal cancer.However,there are some problems such as false positive,false negative and suspicious positive with it.Although many improvements have been made over the years,there are still many problems such as poor sensitivity and specificity.Thus we should do more studies to improve the method based on esophageal exfoliated cells.Adenovirus has many advantages,such as a wide range of hosts,high infection efficiency,no integration into the chromosome,infection of cells in the proliferative phase and non-proliferative phase.Adenoviruses has multiple subtypes,of which adenovirus type 11(Ad11)enters the host cells by binding to CD46 on the surface of the membrane and replicates in the cells.The abnormal expression of CD46 receptor on various tumor cells enables Ad11 more susceptible to tumor cells.Replication-selective oncolytic adenovirus Ad11-5ETel-GFP,which is recombinant by homologous recombination,recognizes CD46 on the surface of tumor cells and then specifically expresses green fluorescent protein(GFP)in these cells.It is easy to identify tumor cells under the fluorescence microscope.What's more,it is sensitive for the virus to infect most tumor cells without replicating in normal cells.This may be a specific,sensitive and economical method to detect tumor cells.ObjectiveAt present,there are not any rapid and simple methods for large-scale screening in the early diagnosis of esophageal cancer.The novel replication-selective oncolytic adenovirus Ad11-5ETel-GFP can specifically replicate and express GFP in tumor cells,which is easy to observe under the fluorescence microscope.In order to find a simple and fast method for early diagnosis of esophageal cancer,we tested its ability to infect esophageal cancer cells and explored the optimal infection conditions,then we applied it to the esophageal exfoliated cells from the patients who were suspected esophageal lesions and explored its ability to detect esophageal cancer.MethodsFirstly,we collected pathological sections of esophageal cancer tissues and adjacent normal tissues from patients with esophageal cancer.Immunohistochemistry was used to detect the expression of CD46 in esophageal cancer tissues and adjacent tissues,then we use Ad11-5ETel-GFP to infect the esophageal cancer cell lines KYSE-70,KYSE-140,KYSE-510,and KYSE-520 respectively.In order to confirm the ability of Ad11-5ETel-GFP to infect esophageal cancer cells and optimize experimental conditions,We used different doses of Ad11-5ETel-GFP and observed the number of positive cells which express GFP at different time.The results above proved that the method we established could detect esophageal cancer cells.Finally,we collected exfoliated cells from patients who were suspected esophageal lesions and needed to be taken biopsy,then the exfoliated cells were infected with Ad11-5ETel-GFP and cultured in the 37°C incubator for a suitable period of time.After that,the cells were collected and fixed,smeared,and stained with DAPI.At last,we counted the number of GFP-positive cells under the fluorescence microscope and compared it with the results of pathological biopsy to evalue its ability to the detection of esophageal cancer.All the data was analyzed by SPSS22.0 statistical software,which was a two-sided test.The level of test was ?=0.05,and P<0.05 was considered statistically significant.Results1.Fisher's exact test was used to compare the expression of CD46 in esophageal cancer tissues and adjacent tissues.The results showed that the expression of CD46 in esophageal cancer tissues was significantly higher than that in adjacent normal tissues.The differences were statistically significant(P<0.05).2.Replication-selective oncolytic adenovirus Ad11-5ETel-GFP could infect esophageal cancer cell lines KYSE-70,KYSE-140,KYSE-510,KYSE-520 and then express GFP.The expression of GFP was positively correlated with infection time and the dose of virus within a certain range.When the virus we used exceeded 2 pfu/cell and the infection time was between 24~48 hours,the detection rate was maintained above 90%.The optimal infection dose was 2 pfu/cell and the optimal infection time was between 24~48 hours.3.A total of 58 esophageal brush specimens were collected from patients with suspected esophageal lesions,including 47 patients with pathologically confirmed esophageal cancer and 11 patients with esophagitis.After infection,GFP expression was positive in 38 patients and negative in 9 patients with esophageal cancer,at the same time it was positive in 2 patients and negative in 9 patients with esophagitis.The detection rate of esophageal cancer was 80.6%.Conclusions1.The expression of CD46 in esophageal cancer tissues is significantly higher than that in adjacent tissues.2.Replication-selective oncolytic adenovirus Ad11-5ETel-GFP can infect esophageal cancer cell lines KYSE-70,KYSE-140,KYSE-510,KYSE-520 and express GFP.The optimal infection dose is 2 pfu/cell and the optimal infection time is within 24~48 hours.3.The detection rate in esophageal exfoliated cells from patients with esophageal cancer is 80.6% by the use of replication-selective oncolytic virus Ad11-5ETel-GFP.There is some significant value in the early diagnosis of esophageal cancer.