Assessment of risk factors and human papillomavirus (HPV) related pathogenetic mechanisms of CIN in HIV-positive and HIV-negative women. Study design and baseline data of the HPV-PathogenISS study

OBJECTIVES: In women with HIV-associated immunosuppression, HPV infections have an increased risk of progression to high-grade cervical intraepithelial neoplasia (CIN). With the HAART-induced prolonged survival and more protracted clinical course of AIDS, progression of CIN to cervical cancer (CC) has become a clinically relevant issue, and the mechanisms responsible for HIV-HPV interactions need further elucidation. The study design and analysis of the baseline data of our new project are presented. MATERIAL AND METHODS: This project is a combination of a prospective cohort study of HIV- and HIV+ women, and a retrospective analysis of CIN lesions and cervical cancer. Up to the present, 244 women have been enrolled (17 HIV+) and subjected to epidemiological interview, colposcopic examination, sampling for HPV testing and typing (PCR, InnoLiPA), and HPV serology. The retrospective series of biopsies were analysed for 13 biomarkers (monitoring key molecular events) using immunohistochemistry and tested for HPV by PCR and TaqMan. RESULTS: HIV- and HIV+ women differ in their exposure status to many of the key epidemiological risk factors of cervical cancer, the most significant ones being number of sexual partners (p = 0.0001), age at onset of sexual activity (p = 0.002), and contraception (yes-no) (p = 0.009). The differences in the baseline clinical observations are less dramatic; HIV-positive women had more frequent HSIL PAP tests (p = 0.040), CIN2 or higher in cervical biopsy (p = 0.049), and external genital warts (p = 0.019). The factors predicting intermediate endpoint markers of cervical cancer, i.e., HSIL PAP smear, ATZ2 in colposcopy, and high-grade CIN in biopsy were analysed in univariate and multivariate regression models. All factors significant in univariate analysis were entered in the multivariate model; HIV-status and Pap smear history maintained their independent predictive power of the HSIL Pap test. The most powerful predictor of ATZ2 colposcopy was HSIL in Pap test. Only the HSIL Pap test and ATZ2 colposcopy remained significant independent predictors of high-grade CIN (p = 0.0001 and p = 0.008, respectively) in the multivariate model. CONCLUSIONS: The three intermediate endpoint markers are closely interrelated, but predicted in part by different covariantes in the causal pathway to cervical cancer. To elucidate whether the increased risk of HIV-positive women to high-grade CIN is due a) to their different exposure status to the risk factors, b) to the direct effects of HIV, or c) to molecular interactions between HIV and HPV, we need to complete these analyses separately in HIV+ and HIV- women.     

Performance of the Roche AMPLICOR human papillomavirus (HPV) test in prediction of cervical intraepithelial neoplasia (CIN) in women with abnormal PAP smear

OBJECTIVES: To assess the performance of a novel PCR-based assay (Roche AMPLICOR HPV test) in detection of cervical pathology as a part of management for abnormal PAP smear (MAPS) and in women participating in cervical cancer screening. STUDY DESIGN: Altogether, 504 women comprising 270 patients referred for colposcopy due to an abnormal Pap smear and another 234 women participating in cervical cancer screening (tested for comparison) were analyzed for oncogenic (HR) Human papillomavirus (HPV) types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 using the Roche AMPLICOR HPV test in cervical samples collected in PreservCyt liquid media. Colposcopic biopsy and/or LEEP cone biopsy was used as the gold standard in the triage group, while liquid-based cytology (LBC) was the reference test in the screening group. RESULTS: The prevalence of HPV was significantly higher in the MAPS group (65.9%) than in the screening group (31.2%) (P = 0.0001). There was a poor concordance between the referral PAP and the current LBC, being only moderate in the screening series, ICC (weighted kappa) = 0.291 (95%CI 0.070-0.459) (P = 0.007), and almost poor in the MAPS Series, with ICC = 0.217 (95%CI 0.04-0.384) (P = 0.023). AMPLICOR HPV positivity increased linearly with the increasing grade of cervical lesions. In detecting high-grade (CIN2-3), colposcopy was the most sensitive test (96.5%), very similar to AMPLICOR (95.2%) (P = 0.731), while LBC with HSIL cutoff was by far the most specific test (99.5%) and showed the highest PPV (96.1%). NPV of colposcopy (97.2%) and AMPLICOR (96.7%) were similar (P = 0.839). Together with abnormal colposcopy and HSIL cytology, the AMPLICOR HPV test is a powerful independent predictor of high-grade CIN2-3, and as such suitable to replace cervical cytology in management of women with abnormal PAP test (MAPS). CONCLUSIONS: The Roche AMPLICOR HPV test is comparable to other HPV tests (HCII, PCR) in detecting CIN in MAPS. However, more data are clearly needed on the performance of AMPLICOR test in management of abnormal PAP and particularly as a screening tool.     

Persistent HPV infection in postmenopausal age women.

OBJECTIVE: Persistence of human papillomavirus (HPV) is associated with an increased risk of developing cervical SIL and cancer in young women. Because this association in older, postmenopausal age women has received little attention, we evaluated persistence of HPV among women in this age group. METHODS: Women (n=105) ages 45-64 were examined annually for 7 years to evaluate HPV in cervical cytologic specimens. PCR, dot blot hybridization and DNA sequencing were used to detect HPV types. RESULTS: The cumulative prevalence of HPV was 34%, and 24% had HPV high-risk oncogenic types which are associated with genital cancers. The most common oncogenic types were HPV-16 (72%) and HPV-31 (16%). The persistence rate of HPV infection was 16%. No specific risk factors were associated with repeat viral positivity. CONCLUSION: Postmenopausal women are infected with persistent oncogenic HPV at a substantial rate, supporting the need for continued screening in postmenopausal women to detect preneoplastic genital lesions.     

Prevalence and genotyping of HPV in cervical cancer among Australian women.

OBJECTIVE: To investigate the association between HPV DNA detection and histological examination of tumor biopsies from women with cervical cancer. METHOD: A total of 186 women with cervical cancer were screened for HPV infection using MY09/MY11 primer based PCR. The status of HPV infection was correlated with histological and demographic characteristics by Fisher's exact test or Chi square test. RESULT: The prevalence of HPV infection in this Australian population was 91.9% (171). Among these 53.8% (100) were HPV type 16, 17.2% (32) HPV type 18, and 21.0% (39) other HPV types. Three significant associations were identified: (1) HPV genotype 18 and adenocarcinomas/adenosquamous carcinomas (P < 0.001); (2) HPV DNA negativity and postmenopausal status (P = 0.02); and (3) HPV DNA positivity and lymphocyte infiltration of tumor tissues (P = 0.03). CONCLUSION: HPV infection is present in the vast majority of cervical cancer patients. However, its presence or genotyping does not seem to have a major influence on the pathogenesis of cervical cancer, such as tumor grade, FIGO staging and metastasis (P > 0.1).     

Human papillomavirus (HPV) DNA in primary cervical cancer and in cancer free pelvic lymph nodes--correlation with clinico-pathological parameters and prognostic significance

OBJECTIVE: To assess whether the presence of human papilloma virus (HPV) DNA and/or several genotypes of HPV DNA in primary cervical cancer and cancer free pelvic lymph nodes are correlated with several clinicopathological parameters of well-defined prognostic significance and whether virological parameters are predictors of long-term survival in cancer patients. PATIENTS AND METHODS: 223 cases of cervical cancer patients included in this retrospective study underwent follow-up evaluation. Survival and cause of death were examined for 204 (91.4%) patients, with a mean follow-up time of 4.4 years. HPV DNA was detected using the high sensitive polymerase chain reaction (PCR) method followed by HPV DNA sequencing for HPV genotyping. These results were correlated with well-defined clinicopathological parameters and survival data. RESULTS: HPV DNA was detected by PCR in 150 of 203 (73.4%) tissue specimens of cervical cancer patients. DNA sequence analysis revealed the presence of HPV 16 (n = 68, 45.3%), HPV 18 (n = 49, 32.6%) and rare HPV types (n = 33, 22.1%). HPV genotypes correlated significantly with histological tumor types, node status, blood vessel invasion and lymph space involvement. The presence of HPV DNA in cervical cancer as well as the genotype of HPV 16 could also be confirmed as significant prognostic factors in the univariate Cox Regression Analysis (RR 2.856, p < 0.003 resp. RR 3.444, p < 0.0001). The presence of HPV DNA in cancer free pelvic lymph nodes was significantly correlated to the concomitant manifestation of pelvic lymph node metastases (RR 3.1, p < 0.0001). In the multivariate analysis, however, HPV DNA in primary tumor and in negative pelvic lymph nodes failed to be of prognostic relevance. Exclusively, HPV 16 appears to impact independently on the overall survival in cervical cancer patients (RR 3.653, p < 0.002). CONCLUSION: The detection of HPV 16 genotype may play an important adjunct role in assessing prognosis of cervical cancer patients. The clinical impact of the presence of HPV DNA in primary tumors and cancer free pelvic lymph nodes remains to be investigated in further studies. The exact mechanisms by which HPV influence the prognosis of cervical cancer patients have to be defined.     

HPV16/18 prevalence in cervical lesions/cancers and p53 genotypes in cervical cancer patients from India

OBJECTIVES: The HPV16/18 code for an oncoprotein-E6, which binds to p53 tumor suppressor protein and degrades the protein via ubiquitination. A common polymorphism of p53 in exon 4 codon 72, resulting in either proline (Pro) or arginine (Arg), affects HPV16/18 E6-mediated degradation of p53 protein in vivo. Hence, in the current study we investigated the prevalence of HPV16/18 in cervical lesions and the distribution of p53 genotypes in cervical cancers and normal healthy women. METHODS: DNA from 337 Indian women with invasive cervical cancers, 164 women with clinically normal cervix, 64 women with low-grade squamous intraepithelial lesions (LSIL), and 5 women with high-grade squamous intraepithelial lesions (HSIL) was examined for the presence of HPV16/18 using consensus primers in a polymerase chain reaction (PCR), and the specific HPV type was identified by Southern hybridization of the PCR product using HPV16/18 type-specific nucleotide sequences as probes. Further, 134 women with cervical cancers and 131 healthy women were used to determine the frequency of p53 genotypes, Pro/Pro, Arg/Arg, and Pro/Arg, using peripheral blood cell DNA to indicate the constitutional genotypes and allele-specific primers, in a PCR-based assay. RESULTS: We observed a prevalence of HPV16/18 in 77% (258/337) of cervical cancer patients, 38% (24/64) of LSILs, 4 of 5 HSILs, and 15.2% (25/164) of normal healthy women. The frequency of distribution of the three genotypes of p53 codon 72 in a subgroup of the HPV16/18-positive cervical cancer patients was Pro/Pro 0.18 and Arg/Arg 0.26, with the heterozygous Pro/Arg 0.56, differing significantly from the genotype frequency in the normal healthy women (chi(2) = 6.928, df = 2, P < 0.05). CONCLUSIONS: A high prevalence of HPV16/18 was observed in the cervical cancers. The prevalence in LSILs confirms HPV16/18 infection as an early event and further indicates a role in progression of lesions. The p53 genotype distribution indicated that women homozygous for Arg genotype were at a 2.4-fold higher risk for developing HPV16/18-associated cervical carcinomas, compared to those showing heterozygous Pro/Arg genotype (odds ratio 2.4, 95% confidence interval 1.89 to 3.04).     

Relationship between HPV typing and abnormality of G1 cell cycle regulators in cervical neoplasm

OBJECTIVE: In tumorigenesis, loss of function of the G1 pathway (p16-CDK4/cyclinD1-pRB pathway (RB pathway) and p14-MDM2-p53 pathway (p53 pathway)) is a theoretically essential event. The simultaneous analysis of all components of the RB and p53 pathway may be able to explain cervical tumorigenesis. However, there are no reports in which all components of the G1 pathway and HPV typing were examined simultaneously in cervical cancer. METHODS: We examined HPV typing and the status of the G1 pathways simultaneously by PCR-SSCP, multiplex PCR, methylation-specific PCR, and immunohistochemical techniques in cervical neoplasia. A total of 105 samples (normal, 10; cervical intraepithelial neoplasm (CIN), 42; invasive cancer (IC), 53) were included. RESULTS: Abnormality of the RB pathway tended to be more frequent in ICs (60.4%) than in CINs (31.0%) (P = 0.069). The primary target was p16 (CIN, 14.3%; IC, 43.4%; P = 0.032). Abnormality of the p53 pathway was detected in ICs (56.6%) and in CINs (40.5%) (P = 0.1494). In particular, strong expression of MDM2 was higher in ICs (32.1%) than in CINs (7.1%) (P = 0.0045). Abnormalities of the RB and p53 pathways were higher in low-risk and negative HPV than in high-risk HPV (81.3% vs 51.4%, P = 0.0657; 81.3% vs 45.9%, P = 0.0328). Seven HPV-negative cases had abnormalities in the RB or p53 pathways. CONCLUSION: In conclusion, abnormality of the G1 pathway may be one of the important mechanisms for carcinogenesis of low-risk and negative HPV cases.     

Human papilloma virus testing in patient follow-up post cone biopsy due to high-grade cervical intraepithelial neoplasia

OBJECTIVE: We evaluated the contribution of the human papilloma virus (HPV) load in planning follow-up and management of women post cone biopsy for high-grade cervical intraepithelial neoplasia (CIN2-3). METHODS: Ninety-six suitable women were followed-up by Pap smears: two consecutive abnormal smears dictated referral for colposcopy-directed biopsy. Before colposcopy, HPV tests determined high-risk HPV DNA type and load (Hybrid Capture System type I). Patients histologically diagnosed with CIN1 or CIN2-3 underwent repeat conization or hysterectomy for residual disease. HPV load was compared to cytology for the detection of residual disease. RESULTS: At follow-up, 20/89 (22.4%) studied women had positive cytology reports of either low- (n = 11) or high-grade (n = 9) squamous intraepithelial lesion (SIL). Colposcopic biopsies diagnosed 9 CIN1 and 8 CIN2-3 cases. Residual disease was corroborated in 16/17 (94.1%) women and the status was readjusted based on cone biopsy/hysterectomy: CIN2-3 in 9 and CIN1 in 7. The positive prediction values for CIN2-3 residual disease with high-grade SIL, CIN2-3 on colposcopic punch biopsy, and high HPV load were 89, 100, and 100%, respectively. For CIN1 residual disease with low-grade SIL, CIN1 on colposcopic punch biopsy, and low and borderline HPV load, they were 54.5, 77.7, and 100%. The HPV load was a more accurate predictor for CIN1 or CIN2-3 on the cervical specimen in cases with low-grade SIL or CIN1 on colposcopic biopsy. CONCLUSIONS: Evaluating HPV loads after a positive cytology report may assist in triaging women post conization biopsy for CIN2-3 to appropriate treatment. Its high positive predictive value, specificity, and sensitivity for CIN1 and CIN2-3 and supplementary information could be especially pertinent for clinical management of low-grade SIL cases.     

HPV oligonucleotide microarray-based detection of HPV genotypes in cervical neoplastic lesions

BACKGROUND: In this study we examined the use of a new-human papillomavirus (HPV) detection method, the HPV oligonucleotide microarray system (Biomedlab Co., Korea), which we compared with the well-established HPV DNA detection system (Hybrid Capture II; HC-II, Digene Co.). This new method prompted us to develop a new HPV genotyping technique, using the oligonucleotide microarray, to detect the generic and type-specific sequence of HPV types. In particular, we undertook the evaluation of the clinical efficacy of the HPV oligonucleotide microarray for detecting HPV in cervical neoplastic lesions. METHODS: One hundred forty patients were involved and classified into three groups according to their histopathologic diagnoses: Group I (nonspecific chronic cervicitis; n = 61), Group II (low-grade squamous intraepithelial lesion (SIL); koilocytosis, and mild dysplasia; n = 39), and Group III (high-grade SIL; moderate, severe dysplasia and in situ carcinoma; n = 40). Cytological diagnoses were based on the Bethesda System and cervical samples were analyzed by the two methods. The HPV oligonucleotide microarray detected 15 types of high-risk HPV (HPV-16/-18/-31/-33/-35/-39/-45/-51/-52/-56/-58/-59/-66/-68/-69) and 7 types of low-risk HPV (HPV-6/-11/-34/-40/-42/-43/-44). RESULTS: In 105 of the 140 cervical samples (75%), HPV DNAs were examined using the HC-II method. HPV detection rates using the HPV microarray agreed with those of HC-II. One HC-II-positive, but HPV microarray-negative, case occurred in the low-grade SIL (Group II) and was later confirmed negative for HPV. The other HPV microarray-positive but HC-II-negative case was found to be HPV-18 by PCR. Low-risk types of HPV were detected in 3 of 39 low-grade SIL cases (Group II) using the HPV microarray. HPV-16 was the most frequent type (32.1%) in all specimens tested, and was significantly more frequent in low-grade or high-grade intraepithelial lesions (Groups II or III) than in normal controls (Group I) (P < 0.05). HPV-58 was the second most common type (17.5%) in Group III. The HPV microarray was found to have advantages in terms of identifying the HPV genotypes and cases of multiple HPV infection. Double HPV infections were detected in 12 cases and triple HPV infections in 7 cases. Two cases were positive for four types of HPV (HPV-16/18/33/35, HPV-16/18/58/68). The sensitivity of HPV testing (HC-II; 94.9%, HPV microarray; 93.7%) for identifying patients with squamous intraepithelial lesion was significantly better than the sensitivity of cytology (77.1%, P < 0.05). On using multiple logistic regression analysis to estimate the relative risk of SIL versus HPV type, HPV-16-positive cases were found to have a 7.5-fold risk of SIL (95% CI = 3.28-16.51; P < 0.01). HPV-33 and HPV-58 were found to be significantly related to high-grade SILs (P < 0.01). CONCLUSIONS: Our results suggest that the HPV oligonucleotide microarray is highly comparable to HC-II for detecting HPV in cervical specimens. The HPV oligonucleotide microarray provides useful information on viral genotype and multiple HPV infections in HPV-related cervical lesions. Genetic information on HPV in cervical specimens might be a particular benefit of the new procedure in the management of cervical neoplastic lesions     

Lack of association between HLA DQB1 alleles with HPV infection and histology findings in cervical cancer in Australian women

Chen S, Tabrizi SN, O'Sullivan H, Fairley CK, Quinn MA, Garland SM. Lack of association between HLA DQB1 alleles with HPV infection and histology findings in cervical cancer in Australian women. Int J Gynecol Cancer 1999; 9: 220–224.
The association of human leukocyte antigen (HLA) types with cervical cancer and human papillomavirus (HPV) infection is controversial. In this study we examined 186 Australian women who had biopsy-confirmed cervical cancer and detected HLA DQB1 alleles and HPV DNA, using the polymerase chain reaction (PCR) amplification and sequence specific oligonucleotides probe (SSOP) hybridization methods. We analyzed the frequencies of 11 DQB1 alleles according to HPV DNA status (HPV positivity and HPV genotyping) and histology (tumor type, staging, grades, lymphocyte infiltration and nodal status). No significant differences among these 11 HLA DQB1 alleles were found with respect to HPV status and histology.     

hWAPL基因多态性增加HPV16/18阳性宫颈癌患者的发病危险性

目的:检测宫颈癌患者外周血人半翼基因(hWAPL)的单核苷酸多态性(SNP)及宫颈癌组织中的HPV DNA感染型别,探讨宫颈癌的发病易感性。方法:应用基质辅助激光解吸附电离飞行时间质谱检测技术检测150例宫颈癌患者(病例组)和150健康妇女(对照组)外周血hWAPL基因的12个标签SNP(tagSNP)位点的多态性;采用基因芯片方法对宫颈脱落细胞进行HPV DNA分型检测。结果:与对照组比较,病例组hWAPL基因的SNP位点rs11595882 T(P=0.001,OR=2.481)、rs10887621 C(P=0.040,OR=1.610)、rs11202058 G(P=0.043,OR=1.479)的危险等位基因频率明显高于对照组(P0.05)。病例组hWAPL基因SNP rs7083506(CC+CT)(P=0.011,OR=3.273)、rs11595882(TT+TC)(P=0.002,OR=2.510)、rs7918136(TT+TA)(P=0.011,OR=3.273)、rs11202058的(GG+GA)(P=0.011,OR=3.273)危险等位基因型的频率显著高于对照组(P0.05)。HPV16/18阳性的宫颈癌患者的rs11595882和rs11202058位点的危险等位基因频率和等位基因型频率显著高于HPV16/18阳性的正常人群(P值均小于0.01)。结论:hWAPL基因的多态性改变是宫颈癌发生的危险因素,尤其对合并高危型HPV16/18阳性的患者尤为显著。     

Prevalence and predictors of squamous intraepithelial lesions of the cervix in HIV-infected women in Lusaka, Zambia

OBJECTIVES: HIV-infected women living in resource-constrained nations like Zambia are now accessing antiretroviral therapy and thus may live long enough for HPV-induced cervical cancer to manifest and progress. We evaluated the prevalence and predictors of cervical squamous intraepithelial lesions (SIL) among HIV-infected women in Zambia. METHODS: We screened 150 consecutive, non-pregnant HIV-infected women accessing HIV/AIDS care services in Lusaka, Zambia. We collected cervical specimens for cytological analysis by liquid-based monolayer cytology (ThinPrep Pap Test) and HPV typing using the Roche Linear Array PCR assay. RESULTS: The median age of study participants was 36 years (range 23-49 years) and their median CD4+ count was 165/microL (range 7-942). The prevalence of SIL on cytology was 76% (114/150), of which 23.3% (35/150) women had low-grade SIL, 32.6% (49/150) had high-grade SIL, and 20% (30/150) had lesions suspicious for squamous cell carcinoma (SCC). High-risk HPV types were present in 85.3% (128/150) women. On univariate analyses, age of the participant, CD4+ cell count, and presence of any high-risk HPV type were significantly associated with the presence of severely abnormal cytological lesions (i.e., high-grade SIL and lesions suspicious for SCC). Multivariable logistic regression modeling suggested the presence of any high-risk HPV type as an independent predictor of severely abnormal cytology (adjusted OR: 12.4, 95% CI 2.62-58.1, p=0.02). CONCLUSIONS: The high prevalence of abnormal squamous cytology in our study is one of the highest reported in any population worldwide. Screening of HIV-infected women in resource-constrained settings like Zambia should be implemented to prevent development of HPV-induced SCC.     

Low incidence of HPV DNA in sera of pretreatment cervical cancer patients

OBJECTIVE: The aim of this study was to investigate the feasibility of using DNA in the circulation as a diagnostic tool for cervical cancer. METHODS: We used PCR followed by Southern hybridization to detect human papillomavirus (HPV) DNA in serum samples taken from patients of cervical cancer before treatment. RESULTS: A total of 60 samples were analyzed. In a set of 40 samples, without knowledge of the HPV DNA status in the corresponding cervical carcinomas, we could detect 8 (20%) positive samples, of which 7 (17.5%) were HPV 16 and 1 (2.5%) was HPV 18. In another set of 20 samples, known to be HPV 16 infected in the corresponding cervical carcinomas, we detected only 4 (20%) HPV-16-positive samples. The occurrence of HPV DNA in sera of cervical cancer patients seems sporadic. CONCLUSION: The low incidence indicates that serum HPV DNA has limited application in the diagnosis of cervical cancer.     

Factors predicting human papillomavirus clearance in cervical intraepithelial neoplasia lesions treated by conization

OBJECTIVE: The objective was to identify the factors, if any, that may predict long-term results of CIN treatment and HPV clearance/persistence after locally excisional therapy. METHODS: A series of 252 women with CIN lesions treated by conization were subjected to sequential HPV detection by repeated PCR during the prospective posttreatment follow-up. Factors predicting viral clearance during the follow-up (10.26 months) were elaborated using univariate and multivariate statistical techniques applied on epidemiological, clinical and biological data of the lesions. RESULTS: Sensitivity of the PAP test in detecting high-grade lesions was 93.9%, and specificity 27.3%. Odds ratio for having CIN 3/Stage IA1 squamous cervical cancer in the cone with HSIL PAP test was 5.69; 77.8 and 22.2% residual disease were found among PCR-positive and -negative cases, respectively. HPV DNA was negative in 74/252 (29.8%) samples at the first PCR. Multivariate logistic regression analysis showed that HPV 16 was an independent explanatory factor for high-grade CIN (P = 0.0001). HPV clearance increased to 63.5% at completion of the follow-up, corresponding to the monthly clearance rate of 5.27%. In Kaplan-Meier analysis, the highly significant (P = 0.0001) predictors of HPV clearance/persistence were age, lesion grade in the biopsy, lesion grade in the cone, volume of the cone, length of active sexual life, and involvement of endocervical margin (P = 0.0013). In chi-square tests, high-risk HPV type (P = 0.001) was such a predictor. In multivariate (Cox) model, the significant independent predictors of HPV clearance were involved endocervical margin (P = 0.001), lesion grade in the cone (P = 0.004), high-grade lesion in the colposcopic biopsy (P = 0.023), age (P = 0.029), and HSIL in PAP smear (P = 0.029). CONCLUSIONS: These data suggest that posttreatment follow-up should include both the PAP test and HPV detection techniques for early detection of any patients at increased risk for disease recurrence and progression, because of persistent oncogenic HPV types.     

子宫颈癌微卫星不稳定性和HPV感染的研究

目的　探讨微卫星不稳定性 (microsatelliteinstability ,MI)及人乳头瘤病毒 (HPV)感染与宫颈癌的相关性。方法　 2 2例宫颈浸润性鳞癌石蜡标本 ,选取 3、 5、 6号染色体上的 3个微卫星位点D3S1 2 89、D5S4 0 6、D6S2 77进行MI分析 ;选用HPV 1 6 / 1 8型特异引物进行HPV检测 ;应用免疫组化法检测Ki6 7蛋白的表达。结果　在宫颈癌标本中 3个位点均未发现MI和杂合性缺失 (LOH)的改变 ;HPV1 6 / 1 8检出率为 77 3% ,Ki6 7指数明显高于对照组。结论　 3个微卫星位点未见MI和LOH的改变 ,与国外报道不同 ,可能与种族差异有关。HPV分型及Ki6 7蛋白表达的检测有助于宫颈病变的评价     

A comparative analysis of human papillomavirus types 16 and 18 and expression of p53 gene and Ki-67 in cervical,vaginal, and vulvar carcinomas

This study aimed to investigate the correlation between HPV positivity, p53 overexpression, and cell proliferative activity in cervical, vaginal, and vulvar squamous cell carcinoma. METHODS: Sixteen vaginal and 31 vulvar squamous cell carcinomas were examined retrospectively for overexpression of p53 gene and Ki67 antigen by immunohistochemistry and for the presence of HPV types 16 and 18 DNA using a polymerase chain reaction (PCR) method. The results were compared with those obtained from 40 cervical squamous cell carcinomas. RESULTS: HPV type 16 or 18 DNA was detected in 21 (52.8%) of 40 cases of cervical carcinomas and p53 overexpression in one (2.5%), while HPV DNA sequences were detected in seven (43.7%) of 16 cases of vaginal carcinoma and p53 overexpression in three (18.7%). With regard to vulvar carcinoma, HPV was harbored in four (12.8%) of 31 cases and p53 overexpression in 19 (61.2%). These results indicated statistically significant inverse correlations between HPV positivity and p53 overexpression (R = -0.999, P < 0.0001). Overexpression of Ki-67 was detected in 28 (70.0%) of 40, 12 (75.0%) of 16, and 21 (67.7%) of 31, cervical, vaginal, and vulvar carcinomas, respectively. There was no significant difference among the three groups. CONCLUSIONS: In cervical carcinoma, HPV types 16 and 18 might play a common causal role, and in vulvar carcinoma, p53 gene mutations might be a main causal factor for carcinogenesis. Vaginal carcinoma, on the other hand, is considered to have transitional characteristics between cervical and vulvar carcinoma.     

Viral DNA load, physical status and E2/E6 ratio as markers to grade HPV16 positive women for high-grade cervical lesions

OBJECTIVES: Cervical intraepithelial neoplasias (CIN) associated with high-risk (HR) human papillomavirus infection, in addition to HR-HPV typing need other viral marker testing to distinguish a subset of lesions with clinical relevant infections. This study has evaluated the significance of viral markers, such as viral load, physical status and E2/E6 ratio, to stratify HPV16 infected women at a single point in time for grade of cervical lesions. METHODS: One hundred sixty-six cytological specimens were selected from women with low (n=72) and high (n=94) grade squamous intraepithelial lesions (SIL), and positive to HPV16. All the 72 LSIL were CINI, 83 of the 94 HSIL were CINII/III and 11 SCC (Squamous Cervical Carcinoma). Cytological specimens were analysed by two different SYBR Green Real-time PCR assays (RT-PCR). Specific primers for both E2 and E6 viral genes and GAPDH cellular gene were designed to determine viral load, physical status and E2/E6 ratio. RESULTS: The viral load was significantly higher in HSIL than in LSIL. In CINI episomal DNA was prevalent (72.2%), mixed forms (episomal and integrated) were 27.8%, suggestive of an early integration of viral DNA into cellular genome, no pure integrated forms were detected. However in CINII/III mixed DNA forms were prevalent (73.5%). In SCC pure integrated DNA was prevalent (81.8%) in absence of episomal forms. E2/E6 ratio decreased significantly from CINI to CINII/III and SCC with a linear trend. The logistic regression analysis showed that viral load higher than 1.38x10(6) genome copies per 300 ng of total DNA associated with E2/E6 ratio lower than 0.90 was highly significant in differentiating CINII/III versus CINI, while the only E2/E6 value lower than 0.17 was significant in differentiating SCC from CINI. CONCLUSIONS: Viral load higher than 1.38x10(6) genome copies per 300 ng of total DNA and E2/E6 ratio values allow HPV16 infected women with high grade cervical intraepithelial lesions to be recognized.     

Human papillomavirus (HPV) DNA detection in self-collected urine

OBJECTIVE: Non-invasive sampling of human genitals to identify high-risk individuals with subclinical oncogenic HPV infection remains a challenge. The study was designed to see if self-collected urine can be used as a simple, non-invasive sampling for screening HPV, particularly for screening/monitoring general population or young adolescents or infants, if they are to be immunized by HPV vaccines. METHOD: Self-collected urine samples from 100 sexually unexposed college going girls and cervical scrapes from 104 normal healthy sexually active married women were used in this study. Additionally, a group of 55 women were recruited for collecting first urine and later scraped cervical cells to validate urine sampling by directly comparing HPV positivity between the two types of biological specimens. A dry 'paper smear' method for specimen collection and a simple single tube protocol was employed for PCR detection of HPV infection. RESULTS: Out of 100 sexually inexperienced college going girls, only 6 (6%) were positive for HPV infection as revealed by L1 consensus primer and 4 (4%) of them were positive for HPV 16 but none was found positive for HPV 18 DNA. Out of 104 sexually active married women who were cytologically reported as negative by Pap test, 11 (10.5%) were found HPV positive and 7 (6.7%) of them had infection of high-risk HPV type 16. Both urine and later cervical scrapes from a group of 55 women collected as dry 'paper smear' showed perfect matching positivity for HPV between urine and cervical scrape. CONCLUSIONS: The use of urine coupled with its dry collection as 'paper smear' facilitating their easy transport, storage and direct PCR detection of HPV DNA opens up an alternative non-invasive approach for population screening of HPV infection, at least in such cases as children and infants in whom invasive samples are difficult to obtain.     

A population-based study of cervical carcinoma and HPV infection in Latvia

OBJECTIVES: We wished to quantify the population-based importance of cervical carcinoma risk factors in Latvia. METHODS: Totally, 223 of 224 eligible cases of incident invasive cervical carcinoma were enrolled during July 1998-February 2001 in Latvia. An age-matched sample of 300 healthy control women was selected from the Latvian population registry and 239 of these women (79%) were enrolled. A demographic and life-style questionnaire was completed, cervical brush samples were analyzed for human papillomavirus (HPV) DNA by PCR and serum samples for HPV antibodies. RESULTS: Risk factors for cervical cancer in multivariate analysis were HPV type 16 or 18 DNA positivity (OR = 32.4; CI 95% 16.5-63.6) and living in the capital (OR = 2.4; CI 95% 1.2-4.7). Oral contraceptive use was not a risk factor (OR = 0.4; CI 95% 0.2-1.1). A strong protective effect was found for having had more than three Pap smears in the last 5 years (OR = 0.07 CI 95% 0.03-0.19). CONCLUSIONS: Inadequate population coverage of Pap smears, in spite of excessive smear usage, caused 28.4% of cervical cancers in age groups eligible for screening. HPV type 16 infection was the most important risk factor for cervical cancer in Latvia, with a population-attributable risk percent for all ages of 58.5%.