| Objective: Esophageal cancer is one of the top eight common malignant tumors in the world and its distribution appears obvious regionally. In Hebei and Henan Province, the occurrence rate of esophageal cancer is the first one cancer in all of the malignant tumors and it harmed people′s health in this area. The five-year survival rate of early esophageal cancer is more than 90%, whereas that of the total esophageal cancer is less than 10%. So, the key measure to improve the survival rate is early find, early diagnosis and early treatment. However, the diagnosis of early esophageal cancer is difficult because most patients in the early stage have no esophageal symptoms. For this reason, it is crucial to screen possible molecular markers for preventing, diagnosing and treating esophageal cancer and its precancerous lesions on the basis of the occurrence mechanism of this cancer. At present, it is considered that malignancy tumors occurred via two mechanisms: one is genetic mechanism (DNA sequences changed), another is epigenetic mechanism (DNA epigenetic modification and no DNA sequences changed) and DNA methylation is the latter′s main modality. Unlike genetic changes, the status of gene methylation is reversible, which is especially important to the prevention and cure of malignancy tumors. It has been known that the carcinogenesis of the esophagus is associated with aberrant methylation of many genes, but until recently, the status of genes methylation in different stages of esophageal precancerous lesions has not been reported. If we could screen early esophageal cancer even precancerous lesion by using specific molecular markers on the basis of molecular mechanism of esophageal carcinogenesis, the diagnosis rate of early cancer would be improved significantly. P16 and FHIT genes are important tumor suppressor genes in many human cancers. It was suggested that both genes had higher methylation frequencies in tissues of esophageal cancer. In the present study, we designed to detect the status of promoter hypermethylation of p16 and FHIT genes in tissues and plasma of esophageal precancerous lesions, and compared with that of chronic esophatitis and infiltrated esophageal squamous cell cancer (ESCC). Meanwhile, the influence of p16 and FHIT methylation to genes expression was observed by immunohistochemical method. Our study aimed to reveal the role of p16 and FHIT methylation in the occurrence and development of esophageal cancer, and its clinical value in the diagnosis of esophageal precancerous lesions and early cancers. Methods: 1. Esophageal mucosal idion staining in patients of no swallow symptom and pre/postoperative patients of cardia cancer From April 2002 to March 2003, 366 patients of no swallow symptom, 436 preoperative and 32 postoperative patients of cardia cancer were examined by esophageal mucosa iodine staining by Olympus XQ-240 type endoscopy. The position, size, shape and boundary of all visible unstained lesions were recorded accurately. Multispot biopsies were taken from unstained areas. Then, comparison analysis was performed between the endoscopy appearances and histophathological results. 2. Study on p16 and FHIT methylation of tissues of esophageal precancerous lesions From July 2003 to December 2003, 44 cases of esophageal precancerous lesions were acquired by endoscopy, including 22 mild dysplasia, 13 moderate dysplasia and 9 severe dysplasia. In the same period, 14 carcinoma in situ (CIS) and 37 infiltrated carcinoma of esophageal squamous cell also gained by endoscopy. Genomic DNA was isolated from paraffin embedded tissues after histopathological diagnosis. Methylation-specific PCR (MSP) was used to detect 5′-CpG island methylation status of p16 and FHIT genes in esophageal precancerous lesions, CIS and infiltrated ESCC, and comparison analysis was performed in their corresponding normal tissues and 10 chronicesophatitis. 3. The relationship of loss expression and promoter methylation of p16 and FHIT gene in esophageal precancerous lesions All the wax blocks in Part Two of this study were sliced into 30~40 pieces thick 10 μm for isolating tissue DNA, at the same time, 3 pieces thick 4 μm were sliced, and one was stained by HE for phathological morphology observation, two others were prepared for immunohistochemical staining. The expressions of p16 and FHIT proteins were observed in chronic esophatitis, esophageal precancerous lesions, CIS, infiltrated ESCC and their corresponding normal tissues, and the relationship of methylation and protein expression of p16 and FHIT genes were analyzed in this part. 4. p16 and FHIT methylation in plasma Five ml of venous blood of all the patients in Part two of this study was taken immediately after endoscopy. After anticoagulated by sodium citrate, DNA was extracted from 400μl plasma. Then, MSP was used to detect 5′-CpG island methylation status of p16 and FHIT genes in patients of chronic esophatitis, esophageal precancerous lesions, CIS and infiltrated ESCC, and its value in early diagnosis of ESCC was also studied. Results: 1. In 366 patients no swallow symptom,478 abnormal stained lesions were detected and more than 2 abnormal stained lesions in one case were observed in above 1/3 cases. Lesions serious than moderate dysplasia were 28.42%. 2. In 436 preoperative patients of cardia cancer, 85 abnormal stained lesions were observed in 77 (17.66%) cases. Lesions serious than moderate dysplasia were 29(27 cases), which occupied 6.19% in all patients accepting iodine staining. 3. In postoperative group, 23 abnormal stained lesions were observed in 21(65.63%) cases. Lesions serious than moderate dysplasia were 3 (3 cases), which occupied 9.38% in all patients accepting iodine staining. Another 3 cases of Barrett esophagus were diagnosed.4. The results of MSP showed that 53 cases (55.79%) were found p16 methylation in tissues treated by sodium bisulfite of 95 esophageal precancerous lesions and cancers. The frequencies of p16 methylation in mild, moderate and severe atypical hyperplasia, CIS and infiltrated ESCC were 22.73%, 46.15%, 77.78%, 78.57% and 64.86% respectively. In 10 chronic esophagitis, 1 (10%) was found p16 methylation. No statistics difference was found between chronic esophagitis group and mild, moderate dysplasia groups (all P>0.05). Whereas the frequencies of p16 methylation in severe dysplasia, CIS and infiltrated ESCC groups were significantly higher than that in chronic esophagitis group (P<0.05). 5. Forty-nine cases were found FHIT methylation in 95 esophageal lesions. The frequencies of FHIT methylation in mild, moderate and severe atypical hyperplasia, CIS and infiltrated ESCC were 22.73%, 38.46%, 55.56%, 64.29% and 67.57%respectively. No FHIT methylation was found in 10 chronic esophagitis. No statistics difference was found between chronic esophagitis group and mild dysplasia group (P > 0.05). Whereas the frequencies of FHIT methylation in moderate, severe dysplasia, CIS and infiltrated ESCC groups were significantly higher than that in chronic esophagitis group (P<0.05). 6. P16 and FHIT methylation in tissues of esophageal lesions was not correlated with sex, age, family history of upper gastrointestinal cancer (UGIC), smoking and drinking status of the patients (P>0.05). 7. In the corresponding normal tissues of 95 esophageal lesions, 16 cases were detected unsuccessfully because of too little bisulfite-modified DNA. So, in the rested 79 corresponding normal tissues, 5 (6.33%) were found p16 methylation and 3 (3.80%) was found FHIT methylation. Significant difference could be found between esophageal lesions and the corresponding normal tissues (P<0.05). 8. In 105 esophageal lesions and chronic esophagitis, 69 were found p16 and/or FHIT methylation. That is to say, methylation frequency could mount up to 65.71% when the two genes were detected simultaneously. Amongthem, 34 (32.38%) were detected p16 and FHIT methylation at the same time. 9. Immunohistochemical method was used to detect the expression of p16 and FHIT proteins in esophageal lesions and chronic esophagitis. The results showed that the deletion rates of p16 protein expression in mild, moderate and severe atypical hyperplasia and CIS were 18.18%, 30.77%, 66.67% and 71.43% respectively;the deletion rates of FHIT protein expression were 13.64%, 23.08%, 66.67% and 64.29%respectively. In infiltrated ESCC, the deletion rates of p16 and FHIT protein expression were 67.57% and 64.86%. In chronic esophagitis, protein expression deletion was found one in p16 and none in FHIT. No statistics difference was found between chronic esophagitis group and mild, moderate dysplasia groups (all P>0.05). Whereas the deletion rates of p16 and FHIT protein expression in severe dysplasia, CIS and infiltrated ESCC groups were significantly higher than that in chronic esophagitis group (P<0.05). 10. In the corresponding normal tissues of 95 esophageal lesions, protein expression deletion was found 8 (8.42 %) in p16 and 6 (6.32%) in FHIT. Significant difference could be found between esophageal lesions and the corresponding normal tissues. 11. In 53 cases of p16 methylated esophageal lesions, 12 cases (22.64%) showed positive expression and 41 cases (77.36 % ) showed negative expression of p16 protein. While in 42 cases of p16 unmethylated esophageal lesions, 34 cases (80.95%) showed positive expression and 8 cases (19.05%) showed negative expression of p16 protein. Significant difference was shown between them by chi-square test (P<0.05). 12. In 49 cases of FHIT methylated esophageal lesions, 9 cases (18.37%) showed positive expression and 40 cases (81.63 % ) showed negative expression of FHIT protein. While in 46 cases of FHIT unmethylated esophageal lesions, 41 cases (89.13%) showed positive expression and 5 cases (10.87%) showed negative expression of FHIT protein. Significant difference was shown between them by chi-square test (P<0.05). 13. In plasma DNA of 44 esophageal precancerous lesions and 10chronic esophagitis, none p16 and FHIT methylation was detected by MSP. 14. P16 methylation was detected in plasma DNA of 14 cases. Among them, two were diagnosed CIS, and the other 12 cases were diagnosed infiltrated ESCC. That is to say, in 11 CIS and 24 infiltrated ESCC whose tissues existing p16 methylaion, p16 methylaion also detected in the plasma DNA of 18.18% (2/11)CIS and 50.00% (12/24) infiltrated ESCC. No statistic difference was found between the two groups (P>0.05). 15. FHIT methylation was detected in plasma DNA of 16 cases. Among them, two were diagnosed CIS, and the other 14 cases were diagnosed infiltrated ESCC. That is to say, in 9 CIS and 25 infiltrated ESCC whose tissues existing FHIT methylaion, FHIT methylaion also detected in the plasma DNA of 22.22%(2/9) CIS and 56.00%(14/25) infiltrated ESCC. No statistic difference was found between the two groups (P>0.05). 16. In the plasma DNA of 51 esophageal CIS and infiltrated ESCC, 24 cases were found p16 and/or FHIT methylation. That is to say, methylation frequencies could mount up to 47.06% when the two genes were detected simultaneously. Among them, 6 (11.76%) were detected p16 and FHIT methylation at the same time. Conclusions: 1. Iodine staining was very useful to detect esophageal precancerous lesions and obscure early carcinoma. This technology should be treated as the routine examination to patients of no swallow symptom and pre/postoperative cardia carcinoma, so its applicable range was extended. 2. The color and margin after iodine staining were two marked characters for us to judge the damage degrees of the esophageal lesions. The two characters not only could identify the range of the lesions, but also could help to judge the nature of them. 3. Endoscopic doctors should create the awareness of early diagnosis and think highly of iodine staining, which was the key factor to improve the detection rate of esophageal precancerous lesions and early carcinoma. 4. P16 and FHIT methylation existed in the stage of precancerous lesions,and it might be one of the early events in the occurrence of esophageal cancer. The methylation frequency in tissues of esophageal lesions was significantly higher than that in their corresponding normal tissues, which suggested that methylation was an important reason for malignant transformation of esophageal epithelium. P16 and FHIT methylation detected simultaneously could improve the detection rate in esophageal lesions. This method might be treated as the supplement of histophathology for diagnosing esophageal precancerous lesions and early carcinoma. 5. In the stage of esophageal squamous dysplasia, the methylation frequency of the two genes appeared gradually rising tendency with the increasement of degree of the lesions. And the methylation frequency in severe dysplasia, CIS and infiltrated ESCC showed no significant difference, so these lesions should be treated positively. 6. With the increase of histopathologic grade, the expression of p16 and FHIT protein reduced gradually. Loss expression of p16 and FHIT protein was an early event in the occurrence of esophageal precancerous lesions and early carcinoma, and might play an important role in the process of malignant transformation of esophageal epithelium. 7. Loss expression of p16 and FHIT protein was closely related to the promoter methylation of both genes. It suggested that p16 and FHIT genes participated the occurrence and development of esophageal carcinoma by promoter methylation. 8. The status of DNA methylation in plasma could reflect the status in tissue in certain degree. So DNA methylation in plasma might be a candidate molecular biomarker for screening esophageal cancer and for monitoring the reoccurrence of postoperative patients clinically. However, it should be researched deeply if this method was suit for screening esophageal precancerous lesions. 9. P16 and FHIT methylation could be detected in plasma of patients with infiltrated ESCC, even in CIS, which suggested that this method maybe could screen esophageal cancer in the stage of CIS. It was very useful for... |
PDF Full Text Request