Molecular Epidemiologic Studies On High Risk Human Papillomavirus In Cervical Cancer Isolated From Sichuan

Cervical cancer is the second most common cancer in women malignanttumor worldwide and only inferior in breast cancer. But more and more manyresearch indicated, the association of cervical cancer and high-risk humanpapillomavirus (HPV) is extremely close, in other words, the infection withHPV is the primary factor which cervical cancer occurs. In recent years, theincidence rate and the mortality rate of cervical cancer were in the stable levelalso had the growth tendency, what is especially anxious is cervical cancerpatients have the obvious youth oriented tendency.Human papillomavirus (HPV) is one kind with the race correlation smallDNA virus which is composed by genome DNA and the capsid, no have anenvelope. The most transmission is the direct sex contact. The carcinogenesisaccording to HPV, divided into low-risk HPV like HPV6, 11 and so on,high-risk HPV like HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 andso on, with cervical cancer correlation, in particular HPV16 and 18.At present, the failures of effective method are performed to controlcervical cancer. HPV vaccine is the first choice for the prevent and therapycervical cancer. In order to design prevention and therapy vaccine, researchershave conducted the multi-level epidemiology studies to the infection withHPV. The studies indicated HPV plays an important role, especially tocervical cancer with high-risk subtypes and between various area,geographical environment, economical condition and population weredifferent. Bosch and Manos collected 1,008 cervical cancers biopsiesspecimens from 22 countries to perform the PCR and found HPV16 and 18 isthe most epidemic subtypes, which is not significant different in variouscountries. But in Iran, S Farjadian (S Farjadian et al;2,003) points out the rateof infection with HPV16 was only 26.7% and HPV18 was not found in thestudy groups. In China, the limited epidemiology investigation demonstratedthat, except HPV16, the high-risk HPV subtypes and the rate of the infectionwas very different between south and north. Therefore, to understand sufficie-ntly the distributions of high-risk HPV genotypes in various areas has animportant reference value to HPV vaccine design.Clinically, genital tract infection with HPV is a long-term process andHPV can exist for many years in cell. Not all of the HPV infection progressdevelop to the cancer. The incidence of cervical cancer is due to interactionamong the viral factor, the host factor and the environment factor. Zebhe(Zebhe et al;1998) suggested that the substitution of T350G in the HPV DNAE6 regions, can strengthen the carcinogenicity of HPV. But it is not supportedby the other researchers. It is a recognition that HPV DNA variation is theimportant attribute which the virus infect procession continues to. The furtherresearch found, at all of the majority cervical cancer tissues, the integration ofHPV DNA and the host cell occurred. At present, people do not have theexplicit conclusion whether there has a relevance between this kind ofintegration and carcinogenicity. But, the thorough understand of the variationand the physical status of HPV DNA in host cells is very importance toexplain the pathogenesis mechanism, design the vaccine, diagnose andtherapy the disease.In China, the HPV material on epidemiology and the studies of HPVinfection characteristic are absent. We investigated 158 cervical cancerpatients from Sichuan area, in which the morbidity of invasive cervical cancerwas moderately high (approximately 22 per 100 000 women). Sichuanprovince has a large population, but actually the epidemiology material onHPV is few.We collected 158 samples cervical cancer tissues, from 2003 to 2004, andinvestigated HPV infection rate in cervical cancer and analyzed thedistribution of 9 high-risk HPV subtypes, of which 7 detected the mutations ofHPVE6 region, including the total of specimens with HPV16 DNA. Further-more, we analyzed the physical state of HPV16 DNA which exists in the hostcell. In order to detect accurately HPV DNA, 158 specimens were subjected toPCR amplication with a pair of primers GP5+/GP6+, E6 type-specific primersand MY09/11 primers with the nested PCR. Three methods supplementedmutually. For the HPV16, we have used E6 type-specific primers to performPCR and next nested PCR. The method of PCR-based on sequencing isaccurate, simple and specific. The genome integration of HPV usually disruptsthe open reading frame (ORF) of E2 gene and results in the lack of E2 genewhich leads to the overexpression of E6 and E7 genes. In this process, E6gene is relatively conservative. E2 and E6 genes fragments were amplificatedin the same tube by multiplex PCR and the PCR products was detecteddifferent quantity through scans, next, calculated E2/E6 ratio. Theoretically,E2/ E6 ratio was equal or exceeded 1 in DNA episomal states and E2/E6 ratiois smaller than 1 in DNA concomitant, E2 fragment is negative in DNAintegrated states. The method for half quota is operated simple and quickly,but not requesting E2 or E6 accurate content. Therefore, the exact instrumentis not requiremented.The infection rate of HPV DNA in Sichuan area. In 158 samples, 5 didnot had a successful to amplificated DNA. 148 specimens were positive forHPV DNA in 153. 5 samples were negative. So, the overall infection rate is96.7% (1.48/153).The distribution of HPV subtypes. In 148 specimens, of 3 could notdetermine HPV subtype. Others 145, the single infection accounts for 77.9%(1.13/145), the mix infection accounts for 22.1% (32/145). The prevalence of9 high risk human papillomavirus (HPV) subtypes (16,18,31,33,35,45,52,58and 59) were studied in 145 isolates with definite subtypes, of which HPV16,58 and 18 were prominent, accounting for 78.6%, 20.0% and 8.3%respectively, followed by45 (4.1%), 52 (4.1%), HPV31 (3.4%), 59 (2.8%)and33 (1.4%). However, no HPV35 was observed in the 145 isolates. Thisdistribution is different with the other area in china, likes Shanxi, Xinjiangand so on.Age and HPV. This group's average age is 48 years old. The incidence rateconcentrates in 32～,39～,48～,56～ and 62～ the age group, accounts forabove 90%. Above 69 years, the incidence rate is only 2%, but it is as high asactually 22% below 32 years old.The mutations of 7 HPV subtypes. In HPV16 E6 ORF region, therewere 14 base substitutions, in the nucleotide position, 94(G→A),168(C→G),173(C→T),176(G→A),178(T→G),188(G→C),203(A→G),240(C→G),276(A→G),335(C→T),350(T→G),442(A→C),464(A→G)and 511(T→C),respectively. The mutations of 94 and511 did not result amino acid changes, but other base variations had all causedamino acid transformation. The base substitutions of 94A, 173T, 203G, 240G,464G and 511C have not been reported previously. What is worth payingattention is the C240G located just right in the P53 degeneration regions.HPV18 E6 were found 2 substitutions(485T→C,549C→A) which did notchange the corresponding amino acid. HPV33 had 6 base substitutions andwas 164T→C,273A→G,364A→C,387A→C,446A→G and 213A→C,respectively. Except A213C, the other mutations had result amino acidchanges. The substitution of 164C has not been reported previously. The basesubstitutions of 482T→C, 549C→A,134C→T,157C→T ,259G→T ,341T→C ,482T→C and 497A→G had been found in HPV45E6 region. Of 482T→C and 549C→A did not result amino acid changes, but 157C→T and 259G→T have all had the change in the amino acid level, other are the silentmutation. The mutations of 134C→T,157C→T ,259G→T and 341T→Chave not been reported previously. HPV52 had altogether found 5substitutions which was 350G→T,356A→G,378A→C,379A→G and 467C→A,respectively. The mutations of G35T and A356G were both silentmutation. Others caused amino acid changes. The mutations of A356G andC467A have not been reported previously. To detect 4 specimens with HPV58and find 2 base substitutions, of which one is 259A→G, another is 388A→C,resulting amino acid change. The A259G has not been reported previously. Wehave found 4 base substitutions that is T102C, C306T, A363C and T402C,respectively, in HPV59 E6 regions. These variants had not all resulted aminoacid change.The physical state of HPV16 DNA. 63 of 114 HPV16positive specimens were concomitant state, 43 cases were episomal and only 8specimens were integrated into the host cells. In clinical stages, the integrationappeared mostly in FIGO III (6/8), 2 specimens in II and no I.In brief, through analyzing 158 patients with cervical cancer, the resultsshowed the infection rate of HPV was 96.7% in Sichuan area and similar tomost other regions. But HPV58 occupied a second high prevalence and wasdifferent with other areas worldwide. In this study, we had novel found somevariants which have not been reported previously, but there substitutionslocated in the P53 degeneration region or T antigen binding points. By theinformation of mutations, we would find the rules of variant in HPV E6 genesand then understand the process of cervical cancer. Furthermore, we will berequested to study these special mutations which are high risk to CC. Analysisof HPV16 variants indicated the E and E derivative strains were main in thisareas. The integrated proportion of HPV16 DNA was only 7% in cervicalcancer tissues and low far previous results (45.5%). It is value that furtherinvestigated. The integration of HPV DNA was usually taken place inhigh clinical stages and as a process sign of cervical cancer.