High-risk Oncogenic Human Papillomavirus Research Articles

Clonal History of Papillomavirus-Induced Dysplasia in the Female Lower Genital Tract

Dysplastic lesions of the vagina or the vulva often occur in women who have a previous history of cervical dysplasia. Most lesions in the female lower genital tract are induced by infections with high-risk oncogenic human papillomaviruses (HR-HPVs), including HPV16 and HPV18. HR-HPV genomes frequently integrate into host cell chromosomes at random sites. We analyzed viral integration sites in multiple metachronous lesions of the lower genital tract from women previously treated for HR-HPV-positive cervical dysplasia or cancer to determine whether the metachronous lesions emerged from a single common preexisting dysplastic cell clone or as consequence of independent HR-HPV infection events in the female lower genital tract. From among 1500 patients with anogenital lesions, seven patients with high-grade vaginal or vulvar lesions and with a previous history of cervical disease (five with prior high-grade cervical dysplasia and two with a history of cervical cancer) were included in this study. Integration sites of HPV16 or HPV18 in vaginal or vulvar lesions were mapped by an adaptor ligation polymerase chain reaction (PCR) method. The sequence information was used to design an integrate-specific PCR assay that was applied to DNA extracted from archival paraffin-embedded material derived from biopsy samples of cervical lesions. Identical HPV DNA integration loci were found in vaginal or vulvar and cervical samples of all lesions available for four of the five patients with a prior history of high-grade cervical dysplasia and for both patients with a history of cervical cancer. These data indicate that high-grade dysplastic lesions in the female lower genital tract may emerge primarily as monoclonal lesions from a transformed cell population derived from the uterine cervix.

The epidemiology of genital human papillomavirus infection

Clinical and subclinical human papillomavirus (HPV) infections are the most common sexually transmitted infections in the world, and most sexually-active individuals are likely to be exposed to HPV infection during their lifetimes. More than 40 genotypes of HPV infect the epithelial lining of the anogenital tract and other mucosal areas of the body; of these, 13–18 types are considered to be high-oncogenic risk HPV types (HR-HPV). Persistent infection with HR-HPVs is now unequivocally established as a necessary cause of cervical cancer and is likely to be responsible for a substantial proportion of other anogenital neoplasms and upper aero-digestive tract cancers. Low oncogenic risk HPV types (LR-HPV) are also responsible for considerable morbidity as the cause of genital warts. Youth and certain sexual characteristics are key risk factors for HPV acquisition and persistence of HPV infection, but other mediating factors include smoking, oral contraceptive (OC) use, other STIs (e.g. chlamydia, herpes simplex virus), chronic inflammation, immunosuppressive conditions including HIV infection, parity, dietary factors, and polymorphisms in the human leukocyte antigen system. Not surprisingly, these factors are also established or candidate cofactors identified in epidemiologic studies of cervical cancer. HPV transmissibility and molecular events in HPV-induced carcinogenesis have been the focus of recent multidisciplinary epidemiologic studies. This shift in research focus coincides with a shift in cancer prevention techniques towards immunization with HPV vaccines and HPV testing of precancerous lesions.

Expression of the high-risk human papillomavirus type 18 and 45 E7 oncoproteins in cervical carcinoma biopsies

E7 proteins are major oncoproteins of high-risk human papillomaviruses (HPVs), which play a key role in cervical carcinogenesis. These proteins have been shown to immortalize primary human cells. Due to the absence of antibodies with suitable sensitivity and specificity, little is known about expression of the E7 oncoproteins in naturally infected tissues. Recently, high-level expression of the E7 protein of HPV-16, the most prevalent oncogenic HPV type, was demonstrated in cervical carcinomas by immunohistochemistry; however, approximately 15 additional high-risk HPV types are known to be associated with cervical carcinoma. It is unknown whether the E7 oncoproteins of HPV-18 and -45, the second and third most prevalent HPV types, are expressed in cervical cancers. Using antibodies against HPV-18 and -45 E7 proteins, it is shown here for the first time that the HPV-18 and -45 E7 proteins can be detected in cervical carcinoma biopsies. Together with anti-HPV-16 E7 antibodies, this could create the possibility of detecting E7 oncoproteins in approximately 80 % of all cervical cancers.

Laminin‐5γ2 chain expression in cervical intraepithelial neoplasia and invasive cervical carcinoma

To analyze the expression of laminin-5 gamma 2 chain, a protein which plays a major role in keratinocyte migration, in cervical intraepithelial neoplasia (CIN), and invasive cervical carcinoma associated with high-risk oncogenic human papillomaviruses (HR-HPVs). The expression of laminin-5 gamma 2 chain protein has been analyzed by immunohistochemistry in 17 cases of low-grade squamous intraepithelial lesions (LSIL-CIN1), 35 high-grade squamous intraepithelial lesions (HSIL-CIN2/3), 18 microinvasive or invasive carcinomas, and three metastatic lymph nodes. All these lesions have been proved to contain HR-HPVs and were also positive for p16 protein which classically is overexpressed at all stages of cervical neoplasia and dysplasia linked with HR-HPVs. 20 cases of normal cervix served as controls. The expression of laminin-5 gamma 2 chain protein was observed in 100% of invasive or microinvasive carcinoma and in their related lymph node metastasis with an immunoreactivity located preferentially at the invasive front of the lesions. All the HSILs (100%) associated with invasive carcinoma were also positive. In contrast, in HSILs without associated invasive component, the expression of the protein has been found in only 34% of cases. In positive HSILs, laminin-5 gamma 2 protein was expressed in basal layers. In LSILs and normal epithelium, no expression of the protein was noted. We conclude the following: (i) the expression of laminin-5 gamma 2 is a late event in cervical carcinogenesis increasing with the grade of dysplastic lesions; (ii) laminin-5 gamma 2 expression facilitates the identification of invasive and microinvasive lesions which could be difficult to diagnose on the basis of routine stains; (iii) laminin-5 gamma 2 expression in HSILs could potentially identify those lesions with a more increased risk of tumor progression.

Requirement of Epidermal Growth Factor Receptor for Hyperplasia Induced by E5, a High-Risk Human Papillomavirus Oncogene

Multicellular organisms rely on complex networks of signaling cascades for development, homeostasis, and responses to the environment. These networks involve diffusible signaling molecules, their receptors, and a variety of downstream effectors. Alterations in the expression or function of any one of these factors can contribute to disease, including cancer. Many viruses have been implicated in cancer, and some of these modulate cellular signal transduction cascades to carry out their life cycles. High-risk human papillomaviruses (HPVs), the causative agents of most cervical and anogenital cancers, encode three oncogenes. One of these, E5, has been postulated to transform cells in tissue culture by modulating growth factor receptors. In this study, we generate and characterize transgenic mice in which the E5 gene of the most common high-risk HPV, HPV16, is targeted to the basal layer of the stratified squamous epithelium. In these mice, E5 alters the growth and differentiation of stratified epithelia and induces epithelial tumors at a high frequency. Through the analysis of these mice, we show a requirement of the epidermal growth factor receptor for the hyperplastic properties of E5.

Genetic abnormalities and HPV status in cervical and vulvar squamous cell carcinomas

Cervical and vulvar cancers are diseases of the female lower genital tract, and high-risk human papillomavirus (HPV) infection is the most important risk factor for the development of both cancers. However, it is clear that additional genetic events are necessary for tumor progression, particularly in HPV-negative cases. We detected the presence of high-risk HPV16 and HPV18 genomes by gene-specific polymerase chain reaction and searched for common genetic imbalances by comparative genomic hybridization (CGH) in 28 cervical and 8 vulvar tumor samples and 7 cancer cell lines. The presence of the HPV genome was detected in 25/28 (89%) cervical tumors and 6/8 (75%) vulvar tumors. CGH of cervical and vulvar tumor samples revealed a consistent pattern of genetic changes in both cancers. Frequent gains were found in 1q, 3q, 5p, and 8q, and less consistent losses were detected in 2q, 3p, 4p, and 11p. Notably, a high-level amplification of 3q was found in 9/28 (32%) cervical tumors and 1/8 (12.5%) vulvar tumors, indicating a pivotal role of gain of 3q in cervical and vulvar carcinogenesis. Furthermore, gains of 5p identified in 9/28 (32%) cervical tumors and 3/8 (37.5%) vulvar tumors were seldom described, particularly in vulvar tumors. Our findings suggest that cervical and vulvar carcinomas bear similar chromosomal alteration hot spots that largely coincide with common genomic lesions during tumor progression, besides the initiation by infection and integration of oncogenic HPV.

Therapy for genital human papillomavirus-related disease

Genital human papillomavirus (HPV) infection is very common, and often sub-clinical and usually resolves without any treatment. Genital warts are caused by HPV 6/11 infection and are one of the commonest clinically recognised disease manifestations of genital HPV. Subjects with genital warts usually perceive them as cosmetically disfiguring, often leading to adverse psychological symptoms, and most subjects with genital warts will present requesting treatment. A wide variety of treatments are available including both provider- and patient-applied therapies. Various individual subject and disease factors mediate appropriate therapy choice. Some of the treatments that are used for genital warts can also be used for some cases of intraepithelial neoplasia caused by high-oncogenic risk HPVs occurring at vulval, anal or penile sites. Specific treatment considerations apply to genital warts in pregnancy and laryngeal papillomatosis and these are also discussed.

HPV infections and cervical cancer prevention. Priorities and new directions. Highlights of EUROGIN 2004 International Expert Meeting, Nice, France, October 21–23, 2004

Cervical cancer is caused by infections with a range of high-risk (oncogenic) Human papillomavirus (HPV) types. It seems likely that practically all (>99%) cervical squamous cell carcinomas are linked with these HPV infections. Cervical cancer has an unequal geographic distribution, with the highest global disease burden confined to the developing countries, where the facilities to combat the disease are clearly insufficient. On the other hand, the declining trends in incidence and mortality rates witnessed in many of the developed countries during the past four decades are mainly attributable to the implementation of organized screening programs based on the use of cervical Pap smear, for example, the Nordic Countries, where an organized screening has resulted in up to 80% reduction in cervical cancer incidence since the early 1960s.

Repression of the human papillomavirus E6 gene initiates p53-dependent, telomerase-independent senescence and apoptosis in HeLa cervical carcinoma cells.

Cervical cancer cells express high-risk human papillomavirus (HPV) E6 and E7 proteins. When both HPV oncogenes are repressed in HeLa cervical carcinoma cells, the dormant p53 and retinoblastoma (Rb) tumor suppressor pathways are activated, and the cells undergo senescence in the absence of apoptosis. When the E6 gene is repressed in cells that continue to express an E7 gene, the p53 pathway, but not the Rb pathway, is activated, and both senescence and apoptosis are triggered. To determine the role of p53 signaling in senescence or apoptosis after repression of HPV oncogenes, we introduced a dominant-negative allele of p53 into HeLa cells. Dominant-negative p53 prevented senescence and apoptosis when E6 alone was repressed but did not inhibit senescence when both E6 and E7 were repressed. To determine whether reduced telomerase activity was involved in senescence or apoptosis after E6 repression, we generated HeLa cells stably expressing an exogenous hTERT gene, which encodes the catalytic subunit of telomerase. Although these cells contained markedly elevated telomerase activity and elongated telomeres, hTERT expression did not prevent senescence and apoptosis when E6 alone was repressed. These results demonstrate that when the Rb tumor suppressor pathway is inactivated by the E7 protein, E6 repression activates p53 signaling, which in turn is required for growth inhibition, senescence, and apoptosis. Thus, sustained inactivation of the p53 pathway by the E6 protein is required for maintenance of the proliferative phenotype of HeLa cervical carcinoma cells.

Taxa de detecção do papilomavírus humano pela captura híbrida II, em mulheres com neoplasia intra-epitelial cervical

OBJETIVO: avaliar a taxa de deteccao do papilomavirus humano (HPV) de alto risco oncogenico em pacientes portadoras de neoplasia intra-epitelial cervical (NIC) e verificar se existe associacao entre a presenca do virus e a carga viral com a gravidade da lesao cervical, assim como qual o melhor ponto de corte para predizer a gravidade desta lesao. METODOS: estudo de corte transversal, no qual foram selecionadas 110 mulheres com citologia e/ou biopsia com diagnostico de NIC. Todas foram submetidas a coleta de nova citologia oncologica, captura hibrida II (CH II), colposcopia e conizacao pela cirurgia de alta frequencia com alca. RESULTADOS: a taxa global de deteccao do HPV de alto risco oncogenico na populacao estudada foi de 77,3%. A avaliacao histopatologica, 81 (73,7%) mulheres apresentavam NIC e, nestas mulheres, a taxa de deteccao do DNA-HPV foi de 87,6%, sendo de 85,9% nas mulheres com NIC 2 ou 3. A CH II apresentou sensibilidade de 87,7%, especificidade de 56%, valor preditivo positivo de 86,6% e valor preditivo negativo de 58,3%, com odds ratio (OR) de 7,76 (2,47 < OR < 25,15) no diagnostico de NIC 2 ou 3. Com ponto de corte de carga viral de 20 pg/mL, selecionado a partir da curva ROC, o valor preditivo positivo da CH II no diagnostico da NIC 2 ou 3, na populacao estudada, foi de 81,3%. CONCLUSOES: a taxa de deteccao do DNA-HPV nas pacientes portadoras de NIC foi de 77,3% e, em mulheres com NIC 2 e 3, foi de 85,9%. O melhor ponto de corte da carga viral para a predicao da lesao cervical de alto grau foi de 20 pg/mL. Acima deste ponto a probabilidade de deteccao do HPV de alto risco oncogenico e maior que 80%.

Prevalence of and risk factors for human papillomavirus infection in adolescent patients attending a general pediatric outpatient department

Human Papilloma Virus (HPV) infection is a key cause of morbidity in adolescent patients. Significant numbers of adolescents receive their health care at pediatric ambulatory facilities (pediatric outpatient departments, private pediatric offices, etc.); however, little is known about the prevalence and risk factors of HPV infections in adolescents seen in these pediatric ambulatory settings. Our study hypothesized that adolescent patients attending a pediatric ambulatory facility would have, compared with other Sexually Transmitted Diseases (STDs), high rates of HPV infection-in particular, significant rates of high risk oncogenic HPV types. In addition, we hypothesized that medical, social, and lifestyle risk factors would accurately predict those teenagers at risk for HPV infection.

Prevalence of and risk factors for human papillomavirus infection in adolescent patients attending a general pediatric outpatient department

Human Papilloma Virus (HPV) infection is a key cause of morbidity in adolescent patients. Significant numbers of adolescents receive their health care at pediatric ambulatory facilities (pediatric outpatient departments, private pediatric offices, etc.); however, little is known about the prevalence and risk factors of HPV infections in adolescents seen in these pediatric ambulatory settings. Our study hypothesized that adolescent patients attending a pediatric ambulatory facility would have, compared with other Sexually Transmitted Diseases (STDs), high rates of HPV infection-in particular, significant rates of high risk oncogenic HPV types. In addition, we hypothesized that medical, social, and lifestyle risk factors would accurately predict those teenagers at risk for HPV infection.

Potential health and economic impact of adding a human papillomavirus vaccine to screening programs.

Recently published results suggest that effective vaccines against cervical cancer-associated human papillomavirus (HPV) may become available within the next decade. To examine the potential health and economic effects of an HPV vaccine in a setting of existing screening. A Markov model was used to estimate the lifetime (age 12-85 years) costs and life expectancy of a hypothetical cohort of women screened for cervical cancer in the United States. Three strategies were compared: (1) vaccination only; (2) conventional cytological screening only; and (3) vaccination followed by screening. Two of the strategies incorporated a vaccine targeted against a defined proportion of high-risk (oncogenic) HPV types. Screening intervals of 1, 2, 3, and 5 years and starting ages for screening of 18, 22, 24, 26, and 30 years were chosen for 2 of the strategies (conventional cytological screening only and vaccination followed by screening). Incremental cost per life-year gained. Vaccination only or adding vaccination to screening conducted every 3 and 5 years was not cost-effective. However, at more frequent screening intervals, strategies combining vaccination and screening were preferred. Vaccination plus biennial screening delayed until age 24 years had the most attractive cost-effectiveness ratio (44 889 dollars) compared with screening only beginning at age 18 years and conducted every 3 years. However, the strategy of vaccination with annual screening beginning at age 18 years had the largest overall reduction in cancer incidence and mortality at a cost of 236 250 dollars per life-year gained compared with vaccination and annual screening beginning at age 22 years. The cost-effectiveness of vaccination plus delayed screening was highly sensitive to age of vaccination, duration of vaccine efficacy, and cost of vaccination. Vaccination for HPV in combination with screening can be a cost-effective health intervention, but it depends on maintaining effectiveness during the ages of peak oncogenic HPV incidence. Identifying the optimal age for vaccination should be a top research priority.

Low-Grade Vulvar and Vaginal Intraepithelial Neoplasia: Correlation of Histologic Features with Human Papillomavirus DNA Detection and MIB-1 Immunostaining

Histologic criteria of low-grade vulvar/vaginal intraepithelial neoplasia (VIN1/VAIN1) are well established; however, a significant interobserver variability in diagnosing VIN1/VAIN1 has been reported. The goal of this study was to evaluate the utility of MIB-1 immunostaining as an adjunct test to increase the diagnostic accuracy in equivocal cases of VIN1/VAIN1. The second goal was to examine the distribution of low- and high–oncogenic risk human papillomaviruses (HPVs) in VIN1/VAIN1 lesions. Consecutive vulvar/vaginal biopsies originally diagnosed as VIN1/VAIN1 (n = 43) or benign (n = 20) were reviewed by two pathologists to obtain a consensus diagnosis. The diagnosis was further confirmed with HPV testing using Short PCR Fragment 10 and Line Probe Assay. MIB-1 immunostaining was performed, and positive staining was defined as a cluster of two or more stained nuclei in the upper two thirds of the epithelial thickness. After verification of the diagnosis using the consensus histologic review and HPV detection as an objective confirmatory test, 31% of cases originally diagnosed as VIN1/VAIN1 were identified as being overdiagnosed. The sensitivity and the specificity of MIB-1 staining for identifying VIN1/VAIN1 were 0.96 and 0.90, respectively. Seventy percent of VIN1 cases were associated with low-risk viral types. In contrast, the majority (84%) of VAIN1 cases were associated with high-risk HPVs. In conclusion, MIB-1 staining is sensitive and specific for identifying VIN1/VAIN1, helpful in verifying the diagnosis in equivocal cases.

Human papillomavirus testing methods.

Testing for human papillomavirus (HPV) relies exclusively on techniques of molecular biology using nucleic acid probes. Tests for HPV using nucleic acid probes have been commercially available since the late 1980s, but early tests were cumbersome, involving the use of nucleic acid probes labeled with radioactive phosphorus (32P). These early HPV tests did not achieve widespread use because they did not detect all oncogenic HPV genotypes. The current commercial HPV detection kit, Digene's Hybrid Capture 2 kit, detects virtually all high-risk oncogenic HPV types, as well as most low-risk nononcogenic HPV genotypes. The Hybrid Capture 2 test format is a proprietary nucleic acid hybridization signal amplification system owned by Digene Corporation. Virtually all test formats for DNA sequence analysis are amenable to applications intended to detect and perhaps quantify the various HPV genotypes. These methods can involve direct hybridization with complementary DNA probes, such as Southern blotting or in situ hybridization, signal amplification, such as the Hybrid Capture 2 method or target nucleic acid amplification, most notably the polymerase chain reaction (PCR). Polymerase chain reaction has been used for HPV detection, genotyping, and viral load determination. General or consensus primer-mediated PCR assays have enabled screening for a broad spectrum of HPV types in clinical specimens using a single PCR reaction. Following amplification using consensus primers, individual HPV genotypes are identified using a variety of methods. Using consensus primers in a test format known as real-time quantitative PCR (RQ-PCR), it is possible to generate viral load (concentration) data from reaction curves generated by monitoring PCR reaction kinetics in real time.

Multiple-primer DNA sequencing method.

A multiple-primer DNA sequencing approach suitable for genotyping, detection and identification of microorganisms and viruses has been developed. In this new method two or more sequencing primers, combined in a pool, are added to a DNA sample of interest. The oligonucleotide that hybridizes to the DNA sample will function as a primer during the subsequent DNA sequencing procedure. This strategy is suited for selective detection and genotyping of relevant microorganisms and samples harboring different DNA targets such as multiple variant/infected samples as well as unspecific amplification products. This method is used here in a model system for detection and typing of high-risk oncogenic human papilloma viruses (HPVs) in samples containing multiple infections/variants or unspecific amplification products. Type-specific sequencing primers were designed for four of the most oncogenic (high-risk) HPV types (HPV-16, HPV-18, HPV-33, and HPV-45). The primers were combined and added to a sample containing a mixture of one high-risk (16, 18, 33, or 45) and one or two low-risk types. The DNA samples were sequenced by the Pyrosequencing technology and the Sanger dideoxy sequencing method. Correct genotyping was achieved in all tested combinations. This multiple-sequencing primer approach also improved the sequence data quality for samples containing unspecific amplification products. The new strategy is highly suitable for diagnostic typing of relevant species/genotypes of microorganisms.

Detecção do DNA do papilomavírus humano após excisão da zona de transformação com alça diatérmica para tratamento de neoplasia intra-epitelial cervical

OBJETIVO: avaliar a presenca do DNA do papilomavirus humano (HPV) de alto risco oncologico antes e quatro meses apos excisao da zona de transformacao com alca diatermica em mulheres com neoplasia intra-epitelial cervical (NIC). METODOS: neste estudo clinico prospectivo foram incluidas 78 mulheres submetidas a excisao da zona de transformacao tratadas no periodo de fevereiro a dezembro de 2001. Todas foram submetidas a colposcopia, citologia oncologica e captura hibrida II (CH II) antes da cirurgia e apos 4±1,25 meses. Para analise estatistica utilizou-se o calculo do odds ratio (OR) com intervalo de confianca de 95% (IC 95%). RESULTADOS: antes da excisao, 67 (86%) mulheres apresentavam CH II positiva para DNA-HPV de alto risco oncologico e destas, apenas 22 (33%) mantiveram a CH II positiva quatro meses apos. A deteccao do DNA-HPV apos o tratamento nao se relacionou com a carga viral previa, presenca de doenca nas margens da peca cirurgica ou idade da mulher. Apos quatro meses, a deteccao do DNA-HPV associou-se significativamente com a presenca de alteracoes citologicas (OR = 4,8; IC 95% = 1,7-13,7), porem nao se relacionou com doenca residual ou recidiva histologica (OR = 6,0; IC 95% = 0,8-52,3). CONCLUSAO: apos o tratamento da NIC, a deteccao do DNA-HPV diminuiu significativamente porem nao se observou relacao com a presenca de doenca residual ou recidiva histologica.

Nucleolin as activator of human papillomavirus type 18 oncogene transcription in cervical cancer.

High risk human papillomaviruses (HPVs) are central to the development of cervical cancer and the deregulated expression of high risk HPV oncogenes is a critical event in this process. Here, we find that the cell protein nucleolin binds in a sequence-specific manner to the HPV18 enhancer. The DNA binding activity of nucleolin is primarily S phase specific, much like the transcription of the E6 and E7 oncoproteins of HPV18 in cervical cancer cells. Antisense inactivation of nucleolin blocks E6 and E7 oncogene transcription and selectively decreases HPV18+ cervical cancer cell growth. Furthermore, nucleolin controls the chromatin structure of the HPV18 enhancer. In contrast, HPV16 oncogene transcription and proliferation rates of HPV16+ SiHa cervical cancer cells are independent of nucleolin activity. Moreover, nucleolin expression is altered in HPV18+ precancerous and cancerous tissue from the cervix uteri. Whereas nucleolin was homogeneously distributed in the nuclei of normal epithelial cells, it showed a speckled nuclear phenotype in HPV18+ carcinomas. Thus, the host cell protein nucleolin is directly linked to HPV18-induced cervical carcinogenesis.

Microtiter Format for Simultaneous Multianalyte Detection and Development of a PCR-Chemiluminescent Enzyme Immunoassay for Typing Human Papillomavirus DNAs

To allow multianalyte binding assays, we have developed a novel polystyrene microtiter plate containing 24 main wells, each divided into 7 subwells. We explored its clinical potential by developing a PCR-chemiluminescent immunoassay (PCR-CLEIA) for simultaneous detection and typing of seven high oncogenic risk human papillomavirus (HPV) DNAs in one well. Seven different oligonucleotide probes, each specific for a high-risk HPV genotype, were separately immobilized in the subwells. Subsequently, a digoxigenin-labeled consensus PCR amplification product was added to the main well. The PCR product hybridized to the immobilized probe corresponding to its genotype and was subsequently detected by use of a peroxidase-labeled anti-digoxigenin antibody and chemiluminescence imaging with an ultrasensitive charge-coupled device camera. Results obtained for 50 cytologic samples were compared with those obtained with a conventional colorimetric PCR-ELISA. The method was specific and allowed detection of 50 genome copies of HPV 16, 18, 33, and 58, and 100 genome copies of HPV 31, 35, and 45. Intra- and interassay CVs for the method were 5.6% and 7.9%, respectively. All results obtained for clinical samples were confirmed by the conventional PCR-ELISA. PCR-CLEIA allows rapid, single-tube simultaneous detection and typing of seven high-risk HPV DNAs with small reagent volumes. The principle appears applicable to the development of other single-tube panels of tests.

Detection of human papillomavirus DNA in malignant lesions from Chinese women with carcinomas of the upper genital tract.

The aim of this study was to determine the prevalence of high-risk oncogenic human papillomaviruses (HPVs) in malignant lesions from Hong Kong Chinese women with carcinomas of the upper genital tract. The presence of high-risk HPVs in 55 cases of endometrial adenocarcinomas and 60 cases of primary epithelial ovarian cancers was detected by polymerase chain reaction (PCR) using consensus primers complementary to late 1 (L1) gene of the genital HPVs. Amplified PCR products were verified and typed by Southern blot analysis using (32)P-labeled DNA probes prepared from cloned HPV-16 and -18 plasmids. To confirm the presence of high-risk HPV types in the tumor tissues, PCR amplification using HPV type 16- and 18-specific primers for part of the E6 gene were also carried out. While HPV-18 was not detected, HPV-16 DNA sequences were identified in 5 (9.1%) of the 55 studied endometrial carcinoma samples. Of the 5 HPV-16-positive cases, there were 4 stage I, and 1 stage II endometrial cancer. In addition, 6 (10%) of the 60 epithelial ovarian carcinomas were positive for high-risk HPVs, which included 5 cases with HPV-16 and 1 case with HPV-18. Clinical staging revealed that 5 of the 6 HPV-positive cases were stage I and the remaining case was stage III ovarian cancer. Histology of the 6 HPV-positive cases showed that there were 1 case of clear-cell adenocarcinoma, 1 case of mucinous cystadenocarcinoma, and 4 cases of mucinous tumor of borderline malignancy. No other HPV types were detected. High-risk HPV was detected in approximately 10% of the tumor samples from women with upper genital tract carcinomas. As compared to the high positive rate of HPV infections in cervical cancer, it appears that HPV infection plays a relatively minor role in the pathogenesis of endometrial and ovarian carcinomas.