Cancer-associated Human Papillomavirus Types Research Articles

Analysis of peripheral blood immune cells after prophylactic immunization with HPV-16/18 ASO4-adjuvanted vaccine.

Persistent infection with oncogenic types of human papillomavirus (HPV) is a causal factor for more than 99% of cervical cancers. Recently, prophylactic vaccines have been developed to prevent infections with cancer-associated HPV types (HPV16 and HPV18). The aim of this study was to analyze the changes in the immune system that occur within four weeks of the first dose of HPV-16/18 ASO4-adjuvanted vaccine. Assessment of the percentages of selected cell populations in peripheral blood of 20 healthy volunteers vaccinated with Cervarix was performed using flow cytometry. The analysis revealed an increase in the proportion of activated B and CD4+ T helper cells and an absence of significant differences in cytotoxic CD8+ T lymphocytes, indicating activation of the humoral response after vaccination, without a significant effect on cellular response. There were no significant changes in the NK cell population, and there was a reduction of the percentage of NKT-like cells, which may result from expiry of the primary response at the time of analysis. The presented results are preliminary, and in the context of the increasing use of the anti-HPV vaccine, it would be worth continuing the study in larger groups of patients and at earlier and later time points in combination with the measurement of specific anti-HPV16 and -HPV18 antibody levels. Such an assessment could therefore contribute not only to better understanding of the exact mechanism of action of the vaccine, but also to defining the immunological parameters that determine its effectiveness.

Expression and characterization of transforming protein E7 from cervical cancer-associated human papillomavirus type 31

E7 protein is a major oncogenic factor of human papillomaviruses (HPVs) that plays a key role in virus-associated human cervical carcinogenesis. To determine the biochemical properties of the E7 protein of high-risk HPV type 31, the gene encoding the protein was cloned into a bacterial vector, pET-32a (+), to allow expression of HPV-31E7 as a thioredoxin (Trx) fusion protein in Escherichia coli BL21 (DE3). The resulting expression level of the fusion protein reached 15 ∼ 20% of the total cell protein and more than 60% of the target proteins were in soluble form upon cultivation for 6 h at 30°C in the presence of 0.5 mM IPTG. The fusion protein Trx-HPV-31E7 was effectively purified by Ni2+-chelating chromatography and analyzed by SDS-PAGE and Western blotting. After release from the fusion protein by enterokinase cleavage and purification to homogeneity, the recombinant HPV-31E7 (rHPV-31E7) was investigated for in vitro interaction with the pocket protein p107, which is known to interact with the amino-terminal portion of the protein. The immunoprecipitation studies revealed strong interactions of rHPV-31E7 protein with p107, suggesting it had binding activities and retained its conformational properties.

Viral RNA Patterns and High Viral Load Reliably Define Oropharynx Carcinomas with Active HPV16 Involvement

Oropharyngeal squamous cell carcinomas (OPSCC) that are associated with human papilloma virus (HPV) infection carry a more favorable prognosis than those that are HPV-negative. However, it remains unclear which biomarker(s) can reliably determine which OPSCC specimens are truly driven by HPV infection. In this study, we analyzed 199 fresh-frozen OPSCC specimens for HPV DNA, viral load, RNA expression patterns typical for cervical carcinomas (CxCaRNA(+)), and the HPV-targeted tumor suppressor protein p16(INK4a) as markers for HPV infection. In this set of specimens, there was a 49% prevalence of DNA for the cancer-associated HPV type 16 (HPV(+)). However, there was only a 16% prevalence of high viral load and only a 20% prevalence of CxCaRNA(+), a marker of HPV16 carcinogenic activity. Among the CxCaRNA(+) tumors, 78% of the specimens exhibited overexpression of p16(INK4a), which also occurred in 14% of the HPV-negative tumors. Using a multivariate survival analysis with HPV negativity as the reference group, CxCaRNA(+) as a single marker conferred the lowest risk of death [HR = 0.28, 95% confidence interval (CI), 0.13-0.61] from oropharyngeal cancer, closely followed by high viral load (HR = 0.32, 95% CI, 0.14-0.73). In contrast, a weaker inverse association was found for OPSCC that were HPV(+) and p16(INK4a) high (HR = 0.55, 95% CI, 0.29-1.08). In summary, our findings argued that viral load or RNA pattern analysis is better suited than p16(INK4a) expression to identify HPV16-driven tumors in OPSCC patient populations.

Human Papillomavirus and Cardiovascular Disease Among U.S. Women in the National Health and Nutrition Examination Survey, 2003 to 2006

The purpose of this study was to examine the association between human papillomavirus (HPV) and cardiovascular disease (CVD) among U.S. women. Oncogenic proteins derived from tumor-associated HPV induce the degradation of tumor suppressor protein p53. Inactivation of p53 is associated with accelerated atherosclerotic process. However, the association between HPV infection with CVD remains unclear. Data were from 2,450 women (age 20 to 59 years) in the National Health and Nutrition Examination Survey, 2003 to 2006. Self-collected vaginal swab specimens were sent for HPV DNA analysis by L1 consensus polymerase chain reaction followed by type-specific hybridization. CVD was ascertained by self-reported diagnosis of myocardial infarction or stroke. A total of 60 females (39 women were HPV DNA positive, whereas 21 were negative) had coronary artery disease. Presence of vaginal HPV DNA was associated with CVD. Odds ratio (OR) of CVD comparing women with presence of vaginal HPV DNA to those without was 2.30 (95% confidence interval [CI]: 1.27 to 4.16) after controlling for demographics, health/sex behaviors, medical comorbidities, cardiovascular risk factors, and management. At the same level of adjustment, OR of CVD comparing women with cancer-associated HPV types to those with negative HPV was 2.86 (95% CI: 1.43 to 5.70). HPV infection, especially cancer-associated oncogenic types, is associated with CVD among women.

TLR9 Expression and Function Is Abolished by the Cervical Cancer-Associated Human Papillomavirus Type 16

Cervical cancer development is linked to the persistent infection by high-risk mucosal human papillomaviruses (HPVs) types. The E6 and E7 major oncoproteins from this dsDNA virus play a key role in the deregulation of the cell cycle, apoptosis, and adaptive immune surveillance. In this study, we show for the first time that HPV type 16 (HPV16), the most carcinogenic type among the high-risk subgroup, interferes with innate immunity by affecting the expression of TLRs. Infection of human primary keratinocytes with HPV16 E6 and E7 recombinant retroviruses inhibits TLR9 transcription and hence functional loss of TLR9-regulated pathways. Similar findings were achieved in HPV16-positive cancer-derived cell lines and primary cervical cancers, demonstrating that this event occurs also in an in vivo context. Interestingly, E6 and E7 from the low-risk HPV type 6 are unable to down-regulate the TLR9 promoter. In addition, E6 and E7 from the high-risk HPV type 18, which are known to persist less competently in the host than HPV16, have reduced efficiency compared with HPV16 in inhibiting TLR9 transcription. Furthermore, a CpG motif derived from the HPV16 E6 DNA sequence activated TLR9, indicating this virus is able to initiate innate responses via the receptor it later down-regulates. This study reveals a novel mechanism used by HPV16 to suppress the host immune response by deregulating the TLR9 transcript, providing evidence that abolishing innate responses may be a crucial step involved in the carcinogenic events mediated by HPVs.

Binding of human papillomavirus 16 E6 to p53 and E6AP is impaired by monoclonal antibodies directed against the second zinc-binding domain of E6

The E6 protein of cancer-associated human papillomavirus type 16 (16E6) binds to p53 and, in association with E6AP, promotes its degradation through the ubiquitin-proteasome pathway. The aim of this work was to develop monoclonal antibodies against 16E6 and to test their effect on the binding of 16E6 to p53 and E6AP, and on the degradation of p53. It was shown that an antibody directed against the N terminus of 16E6 inhibited E6AP-dependent binding to p53 and degradation of p53, whereas two different antibodies directed to the second zinc-binding domain of 16E6 reduced 16E6 E6AP-independent binding to p53 and binding to E6AP but not degradation of p53.

The -4 Phenylalanine Is Required for Substrate Ubiquitination Catalyzed by HECT Ubiquitin Ligases

The reaction cycle of HECT domain ubiquitin ligases consists of three steps: 1) binding of an E2 protein, 2) transfer of ubiquitin from E2 to the HECT domain, and 3) transfer of ubiquitin to the substrate. We report the identification of a determinant that is specifically required for the last step of this cycle, a phenylalanine residue located four amino acids from the C terminus of most HECT domains, referred to here as the -4F. Alteration of this residue in human E6AP and Saccharomyces cerevisae Rsp5p did not affect ubiquitin-thioester formation, but effectively blocked substrate ubiquitination. Alteration of the -4F to alanine with concomitant substitution of a nearby residue to phenylalanine only partially restored Rsp5p activity, indicating that precise spatial placement of this residue is important. C-terminally extended E6AP and Rsp5p proteins were also defective for substrate ubiquitination, providing a likely biochemical understanding of a previously isolated Angelman syndrome-associated mutation of E6AP that alters the stop codon of an otherwise wild-type gene. We propose that the -4F may play a role in orienting ubiquitin when it is tethered to the HECT active site cysteine. This may be necessary to allow for approach of the incoming lysine epsilon-amino group of the substrate.

Expression, purification and immunological characterization of the transforming protein E7, from cervical cancer-associated human papillomavirus type 16.

E7 is the major oncogenic protein produced in cervical cancer-associated human papillomavirus type 16 (HPV16). This protein was expressed in Escherichia coli as a glutathione-S-transferase (GST) fusion protein. E7-enriched inclusion bodies were collected from bacterial lysates, were solubilized in 10 M urea, and the protein was purified using anion exchange column chromatography. After removal of endotoxin with serial Triton X-114 extractions, material of high purity (about 90%) was obtained, which is suitable for use in a human clinical trial. This material was immunogenic, and when used as a vaccine, protected mice against challenge with an HPV16 E7 DNA transfected tumour cell line. Based on this observation, the E7GST fusion protein is currently being used in a human clinical trial of a vaccine against HPV16-induced cervical cancer. This fusion protein could be cleaved with thrombin to remove the GST fusion part and further purified by preparative SDS gel electrophoresis to obtain free E7 with > 98% purity.

Epidemiology of HPV infection among Mexican women with normal cervical cytology.

Cervical cancer is caused by human papillomavirus (HPV) and is the most common cancer among Mexican women, but no population-based studies have reported the prevalence and determinants of HPV infection in Mexico. A population-based study was carried out between 1996 and 1999, based on an age-stratified random sample of 1,340 women with normal cytologic diagnoses from 33 municipalities of Morelos State, Mexico. The prevalence of cervical HPV DNA was determined by reverse line blot strip assay to detect 17 cancer-associated and 10 non-cancer-associated HPV types. Two peaks of HPV DNA prevalence were observed. A first peak of 16.7% was observed in the age group under 25 years. HPV DNA prevalence declined to 3.7% in the age group 35-44 years, then increased progressively to 23% among women 65 years and older. Cancer-associated HPV types were the most common in all age groups; non-cancer-associated HPV types were rare in the young and became more common linearly with age. Twenty-four types of HPV were detected; HPV 16, HPV 53, HPV 31 and HPV 18 were the most common, but none was present in more than 1.7% of subjects. The main determinant of infection with both cancer-associated and non-cancer-associated HPV types was the number of sexual partners in all age groups. Less-educated women were at an increased risk of infection with cancer-associated but not with non-cancer-associated HPV types; low socioeconomic status was associated with detection of non-cancer-associated HPV types. Among young women an increasing number of pregnancies was associated with lower HPV detection and among older women low socioeconomic status was related to increased HPV detection, particularly for the age group 35-54 years. Among women with cancer-associated HPV types, there was a higher intensity of polymerase chain reaction signal in younger than in older age groups (p < 0.001). We present additional evidence for the sexually transmitted nature of HPV infection, regardless of age group and HPV type. We confirm previous findings of a second peak of high-risk HPV infections in postmenopausal women, in this case with a clear predominance of cancer-associated HPV types. In populations with this pattern, which can be related to reactivation of latent HPV infections or high previous exposure in older women, screening with HPV testing can have a reduced specificity among older women if proper cut-off points for HPV positivity are not used. Longitudinal studies of immune responses to HPV infection in different age groups are warranted.

Population-Based Study of Human Papillomavirus Infection and Cervical Neoplasia in Rural Costa Rica

To investigate the association of all types of human papillomavirus (HPV) infection with low-grade squamous intraepithelial neoplasia (LSIL), high-grade squamous intraepithelial neoplasia (HSIL), and cancer, the authors conducted a population-based screening of 9175 randomly selected women in rural Costa Rica. Three thousand twenty-four of these women had suspicious findings at screening and were tested for more than 40 types of HPV. In addition to 12 screening-detected cases of cervical cancer, 28 women from throughout Costa Rica who were diagnosed with cervical cancer during the study period were included in the final analyses. The women in this study were 18 to 94 years old (median age = 39 years). Most were multiparous, and only 11 percent had ever smoked. Sexual activity was begun at an average age of 18 years, and more than half had had only one lifetime sexual partner. Overall 16 percent of the women had some kind of HPV infection. Infection rates ranged from 11 percent of those with normal screening results to 89 percent of those with HSIL and 88 percent of those with a diagnosis of cervical cancer. Women with normal cervical cytology had a low rate (4 percent) of infection with cancer-associated HPV types (HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68). Only 10 percent of women with atypical squamous cells of undetermined significance (ASCUS) had cancer-associated HPV infection, whereas patients with LSIL, HSIL, and cancer were infected at rates of 54 percent, 80 percent, and 79 percent, respectively (Table 1). In women with normal screening results, the rate of infection with any type of HPV was 20 percent for women under 25 years of age. The infection rate fell as low as 5 percent in middle-aged women but increased to 20 percent for women more than 65 years old. More cancer-associated HPV types than non–cancer-associated HPV types were found in women younger than 25 years, but more non–cancer-associated types than cancer-associated types were found in women older than 55 years. However, women with ASCUS had more cancer-associated HPV types than non–cancer-associated HPV types in both age groups. The prevalence of any type HPV infection in women with ASCUS increased in those who were more than 55 years old. The diagnosis of LSIL was most common in younger women (median age = 29 years) and decreased steadily as age increased. The women with HSIL were older (median age = 34 years). The rate of HPV infection for these women reached a peak of 2.0 percent at age 30 years and then began to decrease. However, a second peak of about 2.0 percent developed at 65 years of age. These older women were the least likely to have ever had cervical screening. Three hundred five women with normal screening results were randomly selected to serve as control subjects in this study. Among this group with normal cytologic findings, there were 26 types of HPV present. Only 1.0 percent of the HPV types found were HPV16 or HPV18. Slightly more than 4 percent of the control group had multiple infections that accounted for 39 percent of the HPV types. HPV16 was the most common HPV type in women with LSIL, HSIL, and cancer (16, 45, and 47 percent, respectively). The presence of HPV16 carried a high risk of developing HSIL (odds ratio [OR] = 320, 95% CI = 97–1000) and an even higher risk of developing cancer (OR = 710, 95% CI = 110–4500). Cancer-associated HPV types were seen in approximately 80 percent of all women with HSIL or cancer (and in 50 percent of the women with LSIL). HPV types 16, 58, 51, and 52 were significantly associated with HSIL, with ORs ranging from 1400 (95% CI = 120–16,000) for HPV16 to 20 (95% CI = 3.8–100) for HPV51. HPV types 16 and 18 were significantly associated with the diagnosis of cancer (OR = 470, 95% CI = 68–3300; OR = 120, 95% CI = 6.7–2200, respectively). J Natl Cancer Inst 2000;92:464–474

Population-based study of human papillomavirus infection and cervical neoplasia in rural Costa Rica.

Human papillomavirus (HPV) is the main cause of cervical neoplasia. Because few population-based studies have investigated the prevalence of type-specific infection in relation to cervical disease, we studied a high-risk population, estimating the prevalence of HPV infection and the risk associated with various HPV types. We screened 9175 women in Guanacaste, Costa Rica, to obtain a referent standard final diagnosis, and tested 3024 women for more than 40 types of HPV with a polymerase chain reaction-based system. Among women with normal cytology, HPV infections peaked first in women younger than 25 years, and they peaked again at age 55 years or older with predominantly non-cancer-associated types of HPV and uncharacterized HPV types. Low-grade squamous intraepithelial lesions (LSILs) (n = 189) decreased consistently with age. The prevalence of high-grade squamous intraepithelial lesions (HSILs) (n = 128) peaked first around age 30 years and again at age 65 years or older. Seventy-three percent of LSILs were HPV positive, with HPV16 being the predominant type (16% of positive subjects). HPV was found in 89% of HSILs and 88% of cancers, with HPV16 being strongly predominant (51% and 53% of positive subjects). Virtually all HSILs and cancers had cancer-associated HPV types, with high odds ratios (ORs) and attributable fractions around 80%. Risk for HPV16 was particularly high (OR for HSILs = 320, 95% confidence interval [CI] = 97-1000; OR for cancer = 710, 95% CI = 110-4500). We confirm the early decline of HPV infection with age but note increased prevalence after menopause, which could be related to a second peak of HSILs, an observation that warrants further investigation. At least 80% of HPVs involved in cervical carcinogenesis in this population have been characterized. Polyvalent vaccines including the main cancer-associated HPV types may be able to prevent most cases of cervical disease in this region.

Atypical glandular cells of undetermined significance (AGUS): Cytopathologic features, histopathologic results, and human papillomavirus DNA detection

We intensively reviewed 137 smears initially classified as atypical glandular cells of undetermined significance (AGUS) to refine cytological criteria for evaluating these cases, evaluate histological outcomes, and assess the value of human papillomavirus (HPV) DNA testing in management. Consenting, nonpregnant study participants were identified from a cohort of 46,009 women receiving routine Pap smear screening in a managed care setting. Colposcopy was performed on all women, and at least one histological sample was obtained from each. Review diagnoses were assigned to smears and biopsy specimens by two separate panels of pathologists. DNA testing for cancer-associated HPV types was performed on rinses of cytological samplers after a smear and thin-layer slide had been made. On review, 47 (34%) smears were reclassified as negative, 44 (32%) as AGUS, 30 (22%) as atypical squamous cells of undetermined significance (ASCUS), and 16 (12%) as squamous intraepithelial lesions (SIL). The 19 smears interpreted as high-grade intraepithelial lesions on review included 13 high-grade SIL (HSIL), two HSIL with AGUS, favor neoplastic (endocervical adenocarcinoma in situ [AIS]), and four AGUS, favor neoplastic (AIS). Review histological diagnoses were negative in 105 (77%), squamous or glandular atypia in four (3%), low-grade SIL (LSIL) in nine (7%), HSIL in 12 (9%), AIS in five (4%, including two with concurrent HSIL), and endometrial carcinoma in one (1%). HPV testing identified 11 (92%) of 12 women with histologically confirmed HSIL and all five with AIS (100%). A high-grade intraepithelial lesion or carcinoma is detected in approximately 14% of women with community-based diagnoses of AGUS who are referred for immediate evaluation. Use of refined cytological criteria and HPV DNA testing may permit improved management of women with AGUS.

Targetting of the N-terminal domain of the human papillomavirus type 16 E6 oncoprotein with monomeric ScFvs blocks the E6-mediated degradation of cellular p53.

The E6 protein of cancer-associated human papillomavirus type 16 (HPV16) binds to cellular p53 and promotes its degradation through the ubiquitin pathway. In an attempt to identify the regions of E6 that could be targetted for functional inhibition, we generated monoclonal antibodies to the HPV16 E6 oncoprotein (16E6) and analysed their effect on E6-mediated p53 in vitro degradation. The isolated antibodies recognize the 16E6 oncoprotein expressed in the CaSki carcinoma cell line and strongly inhibit the proteolysis of p53 in vitro by binding specifically to a region of 10 residues located at the N-terminal end of 16E6. The variable regions of these antibodies were cloned and expressed in E. coli as single chain Fvs (scFvs). Purified scFvs were present in monomeric form and totally abolished 16E6-mediated p53 degradation by preventing the formation of E6/p53 protein complexes. Our results demonstrate that monovalent binding of scFvs to the N-terminal end of 16E6 abrogates the biological mechanisms leading to the degradation of p53, and they suggest that this region of 16E6 may be a useful in vivo target for blocking the oncogenic activity of HPV16 E6 protein.

T-helper epitopes identified within the E6 transforming protein of cervical cancer-associated human papillomavirus type 16.

The E6 oncoprotein of human papillomavirus type 16 (HPV16 E6) produced by tumor cells of HPV16-associated cervical carcinoma is poorly immunogenic in patients, but nonetheless is a tumor-specific antigen to which therapeutic vaccine strategies may be directed. To investigate the subunit immunogenicity of E6 protein at the T-helper cell level, we immunized mice with overlapping peptides spanning the entire 158 amino acid sequence. Two peptides recalled a proliferative response in lymph node cells (LNC) from C57BL/6 (H-2b)-immunized mice. One of these peptides also recalled proliferative responses in the context of 5/5 other major histocompatibility complex (MHC) class II haplotypes, indicating a "promiscuous" T-epitope. Minimal consensus motif analysis identified the epitopes as 60VYRDGNPYA68 and 98GYNKPLCDLL107. LNC from mice immunized with T-epitope proliferated in response to challenge with whole E6 protein. Immunization with E6 T-epitopes linked to B-epitopes of HPV16 E7 protein elicited specific antibody indicating that T-cells recognizing the T-epitopes provided cognate "help" for B-cells. LNC from mice co-immunized with E6 T-epitope and the major T-helper epitope of HPV16 E7 (48DRAHYNI54) proliferated comparably when challenged with the peptides individually indicating co-dominance of the two T-epitopes. The findings have implications for incorporation of E6 into a therapeutic vaccine.

High-grade dysplasia in genital warts from two patients infected with the human immunodeficiency virus

Cancer-associated human papillomavirus (HPV) types are detected in genital warts removed from immunosuppressed individuals more commonly than from those occurring in otherwise healthy individuals. The prognosis of genital warts containing cancer-associated HPV types is not known. Because it is assumed that genital warts are benign lesions, they are usually treated by destructive therapies without prior knowledge of histopathology. The aim of the present study was to determine whether genital warts from individuals with or without human immunodeficiency virus (HIV) contain high-risk HPV types or areas of dysplasia. The study design was a nonrandomized analysis of genital warts removed by excision biopsy from 15 HIV-infected patients and 15 HIV-negative patients. The tissue was analyzed for HPV DNA by hybrid capture, and microscopic sections of each biopsy were examined for areas of dysplasia. Genital warts from HIV-infected patients contained cancer-associated ("high risk") HPV types in 9 of 15 cases, including 1 that contained only a high-risk type. High-grade dysplastic abnormalities were present in 2 of the 15 lesions from this group, both of which contained high-risk HPV types. Four genital warts removed from HIV-negative patients contained high-risk HPV types, but none contained dysplastic abnormalities. It is concluded that genital warts from HIV-infected patients often contain high-risk HPV types. Such lesions may exhibit dysplastic changes. The frequency of dysplastic changes in genital warts from HIV-infected patients is not known. Biopsy of genital warts may be indicated prior to additional therapy in HIV-infected patients, and surgical removal should be considered as a preferred treatment option in these patients.

Cancer-associated human papillomavirus types are selectively increased in the cervix of women in the first trimester of pregnancy

OBJECTIVE: Because of incomplete data in the current literature, we sought to determine whether pregnancy is an independent risk for the detection of human papillomavirus infection. STUDY DESIGN: Pregnant patients in their first trimester who are seen for routine care at an obstetrics clinic and nonpregnant patients receiving routine care at a sexually transmitted diseases clinic and a gynecology clinic were recruited. Cervical cells were collected by saline solution lavage, and deoxyribonucleic acid was extracted and tested for the presence of human papillomavirus deoxyribonucleic acid with the hybrid capture assay. This assay detects the deoxyribonucleic acid of five “low cancer risk” and nine “high cancer risk” human papillomavirus types with two separate pools of deoxyribonucleic acid probes. RESULTS: Results from 245 pregnant women, 248 patients from the sexually transmitted diseases clinics, and 246 gynecology clinic patients were analyzed. Human papillomavirus deoxyribonucleic acid was detected in samples from 31% of the pregnant patients compared with 17.7% and 18.6% of the sexually transmitted diseases clinic and gynecology clinic patients, respectively. There was no significant difference in positivity for the “low-risk” human papillomavirus types among the three groups (positivity rates from 8.9% to 12.7%), but the pregnant patients had a significantly higher positivity rate for the “high-risk” human papillomavirus types (24.9% compared with 13.3% and 11.4% for the sexually transmitted diseases and gynecology clinic patients, respectively; p < 0.001). A multiple logistic regression analysis showed that pregnancy was an independent predictor of a positive test result for a “high-risk” human papillomavirus type (odds ratio 1.79, 95% confidence interval 1.11 to 2.89) but not a positive test result for a “low-risk” type. CONCLUSIONS: Because sexual activity was not greater among the pregnant patients, we propose that the increased detection of “high-risk” human papillomavirus types among the pregnant patients represents a selective activation of these viruses by hormonal or immunologic factors associated with pregnancy. (A M J O BSTET G YNECOL 1996;174:1487-93.)

Viral and histopathologic correlates of MN and MIB-1 expression in cervical intraepithelial neoplasia

A recently studied tumor antigen, MN, has been associated with cervical carcinomas and cervical intraepithelial neoplasms (CIN), suggesting that it may serve as a marker for cervical cancer or cancer risk. To determine if expression of the MN antigen paralleled parameters reflecting viral or biological events in precursor epithelium, MN expression was correlated with MIB-1 expression, morphological phenotype, and human papillomavirus (HPV) distribution and type in a series of CINs. Seventy-three percent, 62% and 83% of CIN I, II, and III, respectively, were MN antigen positive. The proportion of neoplastic cells immunoreactive for MN did not correlate with the CIN grade or with HPV types stratified by their association with cancer. Evaluation of serial sections showed no correlation between the frequency of MN antigen staining, the proportion of MIB-1 immunoreactive cells, or the proportion of HPV positive cells detected by in situ hybridization (ISH). CINs associated with prototypical high risk (HPV 16) types exhibited increased immunostaining for the MIB-1 antigen and were more often classified as HSIL in contrast to the other types. Thus, although MN expression previously has been associated strongly with squamous carcinoma, it did not emerge as a specific marker for either cancer-associated HPV types or high grade CIN. CIN I lesions associated with low and high risk HPV types were not distinguished by MIB-1 expression and viral replication. This emphasizes the interrelationship between vegetative viral functions (including viral replication) and morphological phenotype, irrespective of HPV type.

Prevalence and histologic significance of cervical human papillomanvirus DNA detected in women at low and high risk for cervical neoplasia

To determine and compare the prevalence and histologic significance of human papillomavirus (HPV) nucleic acids in cervical specimens from women at low (routine hysterectomy) and high (suspicion of cervical neoplasia) risk for cervical neoplasia. Cervical brushings were taken from the cervices of hysterectomy and conization or loop electrical excision specimens and analyzed for HPV nucleic acids by polymerase chain reaction and restriction fragment length polymorphism analysis. Histopathology was confirmed by review of reports or, for HPV-positive results, re-review of the histopathology. Statistical analysis used Student t test or Fisher exact test. Four hundred seventeen and 43 low- and high-risk cervices, respectively, were studied. Statistically significant differences were observed in the index of HPV positivity between the low- and high-risk groups (1.7 versus 42%, P < .001) and the proportion of HPV being cancer-associated HPV types (14 versus 78%, P = .005). None of the 417 cervices from low-risk women contained HPV 16. In the high-risk group, histologically confirmed cervical intraepithelial neoplasia lesion was statistically more likely to be associated with HPV (59 versus 13%, P = .005). Cervices from routine, low-risk hysterectomies in predominately middle-aged women have an extremely low index of cancer-associated HPVs. Considering the strong association of HPV with histologically proven disease, prospective studies exploring the relationship of cancer-associated HPVs to neoplasia in middle-aged women merit consideration.

Prevalence of Human Papillomavirus in Cervical Cancer: a Worldwide Perspective

Epidemiologic studies have shown that the association of genital human papillomavirus (HPV) with cervical cancer is strong, independent of other risk factors, and consistent in several countries. There are more than 20 different cancer-associated HPV types, but little is known about their geographic variation. Our aim was to determine whether the association between HPV infection and cervical cancer is consistent worldwide and to investigate geographic variation in the distribution of HPV types. More than 1000 specimens from sequential patients with invasive cervical cancer were collected and stored frozen at 32 hospitals in 22 countries. Slides from all patients were submitted for central histologic review to confirm the diagnosis and to assess histologic characteristics. We used polymerase chain reaction-based assays capable of detecting more than 25 different HPV types. A generalized linear Poisson model was fitted to the data on viral type and geographic region to assess geographic heterogeneity. HPV DNA was detected in 93% of the tumors, with no significant variation in HPV positivity among countries. HPV 16 was present in 50% of the specimens, HPV 18 in 14%, HPV 45 in 8%, and HPV 31 in 5%. HPV 16 was the predominant type in all countries except Indonesia, where HPV 18 was more common. There was significant geographic variation in the prevalence of some less common virus types. A clustering of HPV 45 was apparent in western Africa, while HPV 39 and HPV 59 were almost entirely confined to Central and South America. In squamous cell tumors, HPV 16 predominated (51% of such specimens), but HPV 18 predominated in adenocarcinomas (56% of such tumors) and adenosquamous tumors (39% of such tumors). Our results confirm the role of genital HPVs, which are transmitted sexually, as the central etiologic factor in cervical cancer worldwide. They also suggest that most genital HPVs are associated with cancer, at least occasionally. The demonstration that more than 20 different genital HPV types are associated with cervical cancer has important implications for cervical cancer-prevention strategies that include the development of vaccines targeted to genital HPVs.

Association of human leukocyte antigen-B1*03 with cervical cancer in Japanese women aged 35 years and younger

An association of human leukocyte antigen (HLA)-DQw3 alleles with squamous cell carcinoma (SCC) of the cervix has been reported in some European populations, but the significance of HLA-DQw3 has not been examined in other populations to the authors' knowledge. The interaction between HLA-DQw3 and human papillomavirus (HPV) in SCC remains to be clarified. To elucidate the association of HLA-DQ alleles with SCC of the cervix, DNA samples extracted from blood lymphocytes of 23 patients with SCC were amplified by the polymerase chain reaction (PCR) using specific primers for the DQB1 genes, and then, each HLA-DQB1 genotype was defined by digestion with restriction enzymes. Human papillomavirus typing also was performed in all cases by PCR, using specific primers for the E6 regions of cancer-associated HPV types (HPV 16, 18 and 33). Twenty patients (87%) carried a DQB1 gene-encoding HLA-DQw3, compared with 49.4% Japanese control subjects in the International Histocompatibility Workshop panel (P = 0.0003). Human papillomavirus 16 or HPV 18 DNA was detected in 86% of the patients. In 13 of the patients with invasive carcinoma with HPV, a high incidence of not only HLA-DQw3 but also of HLA-DQw1 was observed compared with that in control subjects (P = 0.0019, P = 0.047, respectively). The correlation between DQB1*03 alleles and HPV infection was not statistically significant. The frequency of HLA-DQw3 alleles was higher in the authors' patient group than in the control group, suggesting that the HLA-DQw3 molecules may influence the development of SCC of the cervix in young Japanese women. In the patients with HPV-positive invasive carcinoma, the association with HLA-DQw1 molecules suggested that it also may influence the progression of SCC.