| Objective(1) To compare the laryngoscope classification and pathological diagnosis of vocal cord leukoplakia and explore the relationship between them. To observe the prognosis of the flat and smooth type vocal cord leukoplakis patients, which may provide guidance for treatment or surgical indications in patients with vocal cord leukoplakia; (2) To detect the expression of galectin-3 and autophagy related factors in vocal cord leukoplakia tissues, to analyze the significance of their expression in different categories of vocal cord leukoplakia tissues and possible mechanisms, and to prepare for the following experimental studies.MethodsThe leukoplakia patients were enrolled in our retrospective study, who were inpatients from January 2003 to March 2014 at Fudan University EENT Hospital ENT Department.The laryngoscope were classified as three types:flat and smooth type (Type Ⅰ), bulge and smooth type (Type Ⅱ) and bulge and rough type (Type Ⅲ); the pathological reports were classified as five groups:no dysplasia (Group A); mild dysplasia (Group B); moderate dysplasia (Group C), severe dysplasia (Group D) and canceration (Group E). The numbers of the patients in each group were counted. The follow-up observation interval are at least 160 days, and oberve the prognosis of the flat and smooth type patients.Specimens were taken from the patients who were received operations between April 25 2012 and September 26 2013 at of Fudan University EENT Hospital ENT Department. Specimens were vocal cords polyps, leukoplakia, and laryngeal carcinoma biopsy samples, which for Real-time PCR, Western blot, immunohistochemistry, and electron microscopy experiments. The test indicators were Galectin-3 and autophagy related factors.ResultsIn 1170 patients, a total of 1635 vocal cords, there were 402 (24.6%) were flat and smooth type (Type Ⅰ) vocal cord leukoplakia,609 (37.2%) were bulge and smooth type (Type Ⅱ) and 624 (38.2%) were bulge and rough type (Type Ⅲ) vocal cord leukoplakia.In Type Ⅰ group, the proportion of pathology with no hyperplasia was the largest, it was 76.9%, which was 2.2 and 2.6 times compared with the bulge and smooth, bulge and rough type leukoplakia with no dysplasia. The proportion of Group B, C, D, E in Type I and were 12.7%,2.7%,3.9% and 3.7%.In bulge and smooth type (Type Ⅱ) vocal cord leukoplakia, the proportion of pathology with no hyperplasia was 35.6% that was significantly smaller than the proportion in Type I. The proportion of mild, moderate dysplasia were 35.5% and 9.7%, respectively; the proportion of sereve dysplasia (8.4%) and canceration group (10.8%) were larger than the propotion were in Type I group.In bulge and rough type (Type Ⅲ), the proportion of no, mild and moderate dysplasia were 13.8%,25.3% and 10.1%; severe dysplasia (21.5%) and canceration group (29.3%) were in the largest proportion. The ratio were 2.6 and 5.5 times compared to the ratios of Type Ⅲ Group D and Type II Group D. The ratio of Group E were 2.7 and 7.9 times compared to the ratios of Type III Group E and Type II Group E. The Chi-square test result was significant (pearson Chi-square value was 410.1, p= 0.000) in three rigid laryngoscope classification with different pathology results.A total of 162 flat and smooth type vocal cord leukoplakia patients, they were all received conservative treatment. The follow-up time was 160 days (5.3 months) to 3519 days (9.6 years), median follow-up time was 904 days (2.5 months).100 cases (61.7%) patients were cure (Group A); 48 patients’(29.6%) leukoplakia had no changes (Group B); 11 patients (6.8%) were recrudescence (Group C) and 3 cases (1.9%) were deterioration (Group D).Quantitative PCR detected the expression ratios of galectin-3 in the vocal polyps, vocal cord leukoplakia, larynx carcinoma were 3.28,2.24,0.49. Differences were statistically significant:tpolpy,leukoplakia=2.06,Ppolpy,lcukoplakia=0.05; tpolpy,larynx carcinoma=4.78, Ppolpy, larynx carcinoma=0.00; tleukoplakia, larynx carcinoma-4.47,Pleukoplakia, larynx carcinoma=0.00. And we also detected the expression ratios of lc3b、beclin-1 in the vocal polyps, vocal cord leukoplakia, larynx carcinoma, they were 1.39,2.28,1.06 and 1.86、2.67、1.14. Differences were not statistically significant.We analyzed the gene expression level of galectin-3, Ic3b, beclin-1 in leukoplakia tissues with or with not dysplasia or cancerization, the differences were statistically significant in some results.We analyzed the correlation between galectin-3 and beclin-1 with the results of quantitative PCR in leukoplakia tissues, r=0.36, p-0.02, they had correlation; the correlation between galectin-3 and Ic3b, r=0.08, p=0.62, that means they had no correlation.We used Western Blot to detect the expression of GALECTIN-3 in the vocal polyps, vocal cord leukoplakia, larynx carcinoma tissues; the ratios were 2.30,2.58 and 1.31. The differences were statistical significant in the leukoplakia and larynx carcinoma tissues, tleukoplakia, larynx carcinoma=2.57,pleukoplakia, larynx carcinoma=0.01.We used Western Blot to detect the expression of BECLIN-1 in the vocal polyps, vocal cord leukoplakia, larynx carcinoma tissue; the ratios were 1.81,1.71 and 0.55. The differences were statistical significant in the leukoplakia and larynx carcinoma tissues, tpolpy, larynx carcinoma=2.40,Ppolpy, larynx carcinoma=0.03; tleukoplakia, larynx carcinoma=2.92, Pleukoplakia, larynx carcinoma:=0.01.We analyzed the correlation between GALECTIN-3, BECLIN-1 with the results of Western Blot in leukoplakia tissues, r=0.60, p=0.00, we can consider they had correlation.Immunohistochemical staining showed Galectin-3 is mainly expressed in the cytoplasm and cell membrane, Beclin-1, LC3B expressed mainly in the cytoplasm. TEM scan showed the autophagy body exists in vocal cord leukoplakia tissues.ConclusionThe no or mild dysplasia pathological condition were mainly found in Type I leukoplakia patients (89.6%), which can cure in 61.7% patients by conservative treatment, and the canceration rate was the lowest (3.7%). This type of leukoplakia can be classified as early stage vocal cord leukoplakia, and conservative treatment should be first step. The pathology results in the bulge and smooth type vocal cord leukoplakia were mainly no or mild displasia (71.1%), but the canceration rate was higher (3.7%), that we did not recommend conservative treatment. The pathology results in bulge and rough type (Type III) were mailnly morderate or severe dysplasia or cancerization (60.9%), The canceration rate was the highest (29.3%), which were advanced vocal cord leukoplakia, and surgery were recommended in those patients.Galectin-3 may serve as a marker in distinguishing vocal cord leukoplakia and laryngeal carcinoma; autophagy may occur in vocal cord leukoplakia tissues, the expression of Galectin-3 had some correlation with the expression of autophagy related factor Beclin-1, the interaction location may be in the cytoplasm. |
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