Genetic Abnormalities And HPV Status In Vulvar Squamous Cell Carcinomas

Vulvar squamous cell carcinoma (VSCC) account for about 80%-90% of women's malignancies. But its etiopathogenisis is still unclear to date. Laboratory and clinical epidemiology studies showed that HPV infection associates closely with development and progression of VSCC. However, current situation of high HPV infection rate and low VSCC incidence indicates that the development of VSCC is a multifactorial polygene involved and multistage process. Chromosomal imbalance is the most important mechanism in development and progression of VSCC. Prognosis of VSCC is relatively poor, and the carcinoma severely do harm to patients' physical and mental health.At present, genetics studies of VSCC is fairly limited. People are not satisfied with isolated studies of structures of oncogenes and suppressor gene anymore. Instead, they will put them in protein and genefamily form associated with vital movement. Therefore we can search for chromosomal alterations, find related oncogenes and suppressor genes, study genic change in development andprogression of VSCC by means of molecular genetics techniques, which are of great significance in discovering rules of development in VSCC and can offer theoretical evidence for gene diagnosis and therapy.Comparative genomic hybridization (CGH),a molecular genetics technique, was established by Kallioniemi on account of differential hybridization and florescence in situ hybridization (FISH) in 1992, which combined FISH and digital image analysis. The technique can detect and fix DNA copy numbers change between genomes in all chromosomal zones.In this study, we detected amplification and loss of genomic DNA copy numbers in VSCC using CGH, to search for and candidate genes in cancerization,and detected HPV-DNA by PCR, levels of MDM2 and P14ARF/P19ARF gene by RT-PCR , expression of MDM2 and P14ARF/P19ARF proteins by Western-blot to analyze the relationship among MDM2, P14ARF/P19ARF, HPV infection and carcinogenesis, development and prognosis of VSCC, which conduce to recognize the biologic characteristic and provide a biologic marker for clinical diagnosis, treatment and prognosis and to deepen comprehension of pathogenesis in VSCC.Materials and MethodsMaterials1.Tumors samples.2.DNA and RNA extraction related agents.3.CGH related agents.4.RT-PCR related agents.5.Western-blot related agents.Methods1. PCRDNA was isolated by phenol/chloroform extraction from VSCC. HPV16/18 -DNA was detected by PCR2. CGHDNA fluorescent probes of VSCC and normal tissue was marked by means of nick-translation, which performed in situ hybridization with normal metaphase chromosome. Afer DAPI staining, hybridization product was photographed in digital image by cold CCD camera through fluorescence microscope . Then QCGH software analysis was used to obtain analysis pictures of each VSCC case.3. Western-blotthe levels of MDM2 and P14ARF proteins were detected by MDM2 and P14ARF special antibody in protein extractive fluid of VSCC.4. RT-PCRTotal RNA was extracted from VSCC tissues using TRIzol reagent, the cDNA synthesized from the mRNA was followed, then amplified PCRproducts of MDM2 and P14ARF/P19ARF, assayed their mRNA the genecopy number. 5.results criterion PCR resultsunder 2% gelose electrophoresis ultraviolet, the results were observed, the target gene appearance was considered as positive result. CGH resultsWe obtained analysis pictures of each case using QCGH software analysis. Reference value was set 1.0 where fluorescence ratio of red and green equals. Threshold was set 1.2(green) and 0.8(red) respectively. Fluorescence ratios exceeding threshold was defined amplification or loss. Western-blot resultsafter sheeting, scanned the film and imputed the results to the computer, the images were analyzed by image analyzing soft-Chemi Image5500v2.03. The value of protein content = the mean of relative gray scale value of the protein band / relative gray scale value of the protein strap in normal vulvar tissue. RT-PCR resultsgel was scanned by UVP gel image analyzing system, and analyzed by image analyzing soft-Chemi Image5500v2.03. Gene copy number = relative gray scale value of sample electrophoresis band / relative gray scale value of 0 -actin electrophoresis band.6.Statistical analysisStatistical analysis was performed using SPSS for windows 11.0 software. Statistical significance was calculated with t-test and chi-squared independence test .The difference was considered significant when the P value was less than 0.05.Results1.HPV in VSCCIn 21 VSCC cases of CGH analysis, 11 was HPV positive and 10 was HPV negative, All the detected HPV were HPV 16. here was no HPV detected in normal vulvar tissue. HPV was detected in 18/42 cases VSCC of PT-PCR and Western-blot, Two of them were HPV18, the others were HPV16.2.CGH analytic resultsMutations of different levels were found in cellular chromosome of each VSCC cases. The averagely abnormal districts were 4.9 in each case, among which loss was more than amlification. But it was no statistical significance. Chromosomal distribution areas of increasing DNA copy number were 3q24— 27 , 8q21 - 23 , 12q13 — 15 and 9p21 - 22 , the frequencies were 42.9%,38.1%,33.3% and 19% respectively. While chromosome distribution areas of decreasing DNA copy number were:4p13-ter,3p24 and 9p21-22, thefrequencies were 52.4%,42.9% and 9.5% respectively. More chromosomal mutations were found in HPV positive cases as compared to HPV negative ones. But it had no statistical significance. In chromosome 3q and 12q regions, frequency of chromosomal which had predominantly statistical differences (p<0.01);Whereas in chromosomal 8q location, frequency of chromosomal amplification was higher in HPV negative group than in HPV positive group, which had predominantly statistical differerces (p<0.01)too. In chromosome 4p and 3p, frequency of chromosomal loss was higher in HPV positive group than in HPV negative group. But the differences were not statistically significant. In chromosome 9p locus, gains were found in HPV positive group but loss in HPV negative group.The differences were not striking.3.The results in Western immumoblottingThe expression of MDM2 protein significantly increased in VSCC than in normal vulvar tissue (P<0.05). The expression of MDM2 gene protein increasing gradually in well, mild and poorly differentiated VSCC. There were significant differences between poorly differentiated tissue and mild/well-differentiated tissue (p<0.05)。P14ARF protein express in VSCC and normal vulvar tissue, there was no significant difference between them. The expression of P14ARF protein was no significant difference among well, mild and poorly differentiated tissue (P> 0.05), the expression of P14ARF protein was not relative with histologicaldifferentiation in VSCC.MDM2 protein expression were not different between HPV positive group and HPV negative group (P>0.05), P14ARF protein expression in HPV positive group was higher than that in HPV negative group, there were statistical significance between them (P<0.05). P14ARF protein expression was not associated with MDM2 protein expression (P>0.05).4.The results in RT-PCR semi-determinalion methodMDM2 gene amplification was not detected in normal vulvar tissue, and in VSCC it was significant higher than that in normal vulvar tissue, there were significant difference between them (P<0.05). MDM2 gene amplification was relative with histological differentiation of carcinoma. It was significant difference between in poorly differentiated and in mild/well differentiated carcinoma (P<0.05).There were p14AFR gene amplification in normal vulvar tissue and VSCC. There was no significant difference between them (P>0.05), which demonstrate P14ARF amplification was not relative with histological differentiation of carcinoma.MDM2 gene amplification was no significant difference between the HPV positive group and the negative group (p>0.05). The positive rate of P14ARF gene amplification was 100% in HPV positive group, and it was 8.3% in HPV negative group, there were statistical significance between the two groups(p<0.05). MDM2 gene amplification was no relative with P14ARF gene amplification (p>0.05).5.The expression of MDM2/P19ARF in mice VIN/VSCC MDM2 mRNA was highly expressed in the VIN1-2, VIN3 and VSCC tissue, the expression of MDM2 had not been found in the vulvar epithelium of the comparison mice, there was no statistical significance between the comparison group and VINs groups (P>0.05). there was statistical significance between comparison group and VSCC group or between VINs groups and VSCC group (P<0.05).P19ARF gene was highly expressed in the comparison group, VIN1-2 and VIN3 groups, but was lower expressed in VSCC group. P19ARF gene was deficient obviously in the VSCC group. There was statistical significance of P19ARF gene expression between VSCC group and comparison group or VINs groups (P<0.05), there was no statistical significance among other groups (P>0.05). In the model group, there was negative relationship between MDM2 and P19ARF (P<0.05)Conclusions1.A lot of nonrandom loci of amplification or loss of DNA copy number were found by CGH in our study. The most prominent amlification was at 3q24-27, in secondary amplification was at 8q21-23 and 12ql3-15; The most...