Study Of HLA-DR Antigen And Alleles In Etiology Of Cervical Cancer

introductionInfection with high-risk human papillomaviruses is established as the requisition for cervical carcinogenesis. Less than 1% of infected women cannot spontaneously clear HPV and develop into cervical cancer. Patients infected with HIV or operated for kidney transplantation easily encounter persistent infection with HPV. Those facts suggest that immune response to virus may be very important to cervical carcinogenesis.Human leukocyte antigen molecules are responsible for the presentation of foreign antigens to the immune system; They play a central role in the immune recognition and subsequent clearance of virally-infected cells. Since HLA genes are highly polymorphic, the products encoded by HLA genes have different determinant groups and their affinity capacity of binding with antigenic peptide is different. Hence, polymorphic HLA genes are likely associated with risk of cervical cancer.To investigate the role of local immune state in cervix in cervical cancernogenesis, Imrnunohistochemistry staining methods were used to detect the expressions of HLA-DR antigen, CD4 molecule and HPV16/18E6 protein in mormal cevix, CIN and cervical cancer. We also use sequence specific primer-polymerase chain reaction to screen alleles related with susceptibility of cervical cancer.Materials and methods一,sequence specific primer-polymerase chain reaction(PCR-SSP)Sample: 34 cases of relatively normal cervix and 53 cases of cervical cancer were obtained from the department of gynaecology and obstetrics of shengjing hospital affiliated to china medical university. All samples were confirmed after operation.DNA extraction: Ten consective 5μm-thicked sections for one sample were deparaffinized with dimethy benzene, and DNA were extracted by using phenol-choroform methold.HPV detection: Consensus primers GP5+/GP6+ were used to amplify a 150 bp long fragment in the conserved region of the HPV L1 gene so as to detect the presence of HPV。The PCR reaction consisted of an initial denaturation step (94℃, 5 min), followed by 35 cycles of denaturation (94℃, 35 s), annealing (40℃, 50 s), and extension (72℃, 45 s). A final extension step (72℃, 5 min) terminated the reaction. Amplification products were then analyzed by electrophoresis in a GeneFinder-stained 2.5% agarose gel at 80 V for 40 min, and visualized under UV light.HLA-DRB1 genotyping: HLA genotyping was done using PCR methodology with sequence specific primers as described by Olerup and Zetterquist. The PCR reaction consisted of an initial denaturation step (94℃, 5 min), followed by 36 cycles of denaturation (94℃, 35 s), annealing (60℃, 45 s), and extension (72℃, 35 s). A final extension step (72℃, 5 min) terminated the reaction. Every sample was performed PCR reaction for 6 times for HLA-DRB1*1001,*11,*15,*01,*13,*0901 and PCR products were analyzed as above.二,immunohistochemistrySample: 8 cases of relatively normal cervix, 15 cases of CIN, and 33 cases of cervical cancer were collected from the department of gynaecology and obstetrics of shengjing hospital affiliated to china medical university. All patients had no radiotherapy or chemotherapy before operation and all specimen were confirmed after operation.Method: Immunohistochemical staining was performed by streptavidin peroxidase technique. Tissue sections (4μm) were deparaffinized and rehydrated. Heat-induced epitope retrieval in microwave oven was for HLA-DR and CD4, while trypsin digested epitope retrieval for HPV16/18E6 protein. And then the slides were treated with 0.3% hydrogen peroxide to quench endogenous peroxidase activity. Non-specific binding was blocked with goat serum. Slides were then incubated with the monoclonal antibodies mouse anti-human HLA-DR, CD4 and HPV16/18 E6 at 4℃to stay overnight, respectively. After samples were developed with the peroxidase SP kit, the slides were briefly counterstained with Harris' hematoxylin , dehydrated, air-dried and mounted with Histoclad and examined under an Olympus BX40 microscope. Every slide was assessed according to staining intensity and rates of positive cells.Statistical analysis: The software SPSS 11.5 was applied for statistical analysis. T test and Pearson chi-square test were used to compare cases and controls.resultsHPV DNA positive rate of cervical cancer (75%, 40/53 )was much moe than that of control group (24%, 8/34) and difference reached to significance (P < 0.05) .The allele frequencies of HLA-DRB 1*1001, * 11, *15 in cervical cancer group and control group were 35.85%, 20.59%; 30.19%, 14.71%; 43.40%, 26.47%, respectly. The allele frequencies in cervical cancer group were much higher than those of control group. While opposite results were observed for HLA-DRBl*01, *13 allele (18.87% vs 47.06%, P < 0.05; 50.94% vs 82.35%, P < 0.05 ), and among those who were positive for HPV DNA , *13 allele frequency was significately lower in cervical cancer group than control group (40.00% vs 87.50%, P < 0.05).The rates of positive expression for HLA-DR antigen in normal cervix , CIN and cervical cancer were 37.5%,73.3%,81.8%, respectively., with significant differences (P < 0.05 ); The expression rates for CD4 were 62.5%,40.0%,36.4%, resctively, which tended to decrease progressively, but failed to achieve statistical significantces(P > 0.05 ). While the rates of positive expression for HPV16/18E6 protein were 37.5%,60.0%,75.8%. respectively, both showed significant differences (P < 0.05 ). The expression of HLA-DR antigen and HPV16/18E6 protein in cervical cancer tissue was different as clinical stages,degrees of differentiation and lymphoid node metabasis, without significant differences (P > 0.05).conclusion1. The allele frequency for HPV.HLA-DRB1 in cervical cancer patients and healthy persons was significantly different. A positive association with cervical cancer was observed for HLA-DRB1*1001, *11, *15 alleles, which may be predisposing genes; and a negative association for HLA-DRB1*01,*13, which may have protective effect . HLA-DRB1*13 may be can hold back the carcinogenesis role of HPV.2. As an exogenous antigen, HPV16/18 E6 protein excited tumor cells to overexpress HLA-DR antigen. Such tumor cells acted as antigen presenting cells and enchanced immune response.3. These tumor cells could not effectively present HPV antigen without enough CD4 T cells. Hence, the key of immunotherapy for cervical cancer was to adjust elements of immune response and to coordinate their function with each other.