Quantitative Detection Of Human Papillomavirus 16/18 DNA In Laryngeal Carcinomas Using Non-Isotine In Situ Hybridization

HPVs were closely associated with several kinds of human malignant tumors, especially the HPV16/18 types which were called high-risk oncovirus. At present, besides the cervix tumors, there has been an increasing interest in the correlation between IIPVs and the laryngeal carcinomas.Although HPV16/18DNA has been detected in laryngeal carcinoma tissues by some different methods, the accurate mechanism of carcinogenesis of larynx induced by HPV16/18 infection was still unknown. In the present reseach, we quantified HPV16/18 DNA hybridization signals in laryngeal carcinoma using non-isotin in situ hybridization (NISH) and microscopic image analysis technique At the same time the expression levels of PCNA (Proliferating Cell Nuclear Antigen, PCNA) and mutant p53 were also quantitatively examined by immunohistochemical staining and microscopic image analysis. The correlation between the intensity of hybridization signal and staining intensity of PCNA andp53 was analysed statistically. We tried to discuss the quantitative relationship between HPV infection and the grade of proliferative activity and malignance of laryngeal carcinomas1. Materials and MethodsParaffin-embedded tissue sections were obtained from the 63 patients who were operated for laryngeal carcinoma. 42 vocal cord polyps were collected and used as controls. All the cases were pathologically diagnosed.From each specimen, 4 serial tissue sections at 5um thickness were cut for NISH and negative control as well as immunohistochemical staining of PCNA and p53 respectively.1.1 In situ Hybridization (ISH)The slides were baked at 73 癈 overnight. After deparafTinizing, they were digested with freshly diluted 1 X proteinase K, DNase and RNase solution. Hybridization probe was added to slides, followed by denaturing through heating at 95 癈 for 10 minutes in water. The slides were placed in a chamber which was humidified with 50% formamide and incubated at 37癈 to allow hybridization of the probe with the target nucleic acid overnight. After hybridization finished, the slides were blocked with 1 X and 2 X protein block solution for 5 minutes, and combined with the Biotin-Antigen and streptavidine-AP conjugate. The last alkaline phosphatase reaction was developed using BCIP/NBT as chromogenes.1.2 Immunohistochemical MethodAll 63 specimens were deparaffinized in xylene, then rehydrated throughgraded alcohol. Endogenous peroxidase activity was blocked with endogenous peroxidase blocking solution. To reduce non-specific binding, the sections were incubated with 10% non-immunone serum for 5 minutes at room temperature. Followed by incubating with bioinylated second antibody for 10 minutes at room temperature, the p53 or PCNA rat mono-colonal antibody were added to slides for incubating at same condition for 1 hour. At last, the streptavidin alkaline phosphatase and the freshly diluted DAB were sequentially added to slides for visualization.1.3 All the slides were microscopically examined and measured by microimage analysis.2. ResultsThe dark-blue and punctuate distributed positive signals of HPV16/18 hybridization located in nuclei. The positive percentage of HPV16/18 hybridization in laryngeal carcinoma was remarkably higher than it was in vocal cord polyps, as shown in Table 1. The difference between them was statistically significant (x2=l 4.00, p<0 001).Table 1 The percentage of HP VI 6/1 8DN A positive hybridization Tissue sections Cases Positive cases Positive percentageLaryngeal carcinoma 63 23 36.50%Vocal cord polyps 42 2 4.76%The positive percentage, and positive cell numbers as well as squares of hybridization in high, middle and low differentiated laryngeal carcinomas wereshown in Table 2. Both the positive cell numbers and squares in high differentiated cases were higher than they were in low differentiated cases (?257.50,/7<0.001, w=257.00,/x0.001).Table 2. P...