Pilot study of a commercialized human papillomavirus (HPV) genotyping assay: comparison of HPV risk group to cytology and histology

We evaluated a commercialized PCR assay, Linear Array, that detects 37 human papillomavirus (HPV) genotypes, using a sample of liquid cytology specimens (n = 534). We found a strong association of an increasing level of HPV risk (HPV type 16 [HPV16] > HPV18 > other carcinogenic types > noncarcinogenic types > negative specimens) with increasing severities of cytologic interpretations (P(Trend) < 0.0005) and histologic diagnoses (P(Trend) < 0.0005).     

Comparison of the Third Wave Invader human papillomavirus (HPV) assay and the digene HPV hybrid capture 2 assay for detection of high-risk HPV DNA

This study compared the clinical performance of the Digene Hybrid Capture 2 (HC2) assay to that of a prototype Third Wave Invader human papillomavirus (HPV) (IHPV) analyte-specific reagent-based assay for the detection of oncogenic or "high-risk" (HR) HPV DNA using liquid-based cytology specimens. In total, 821 ThinPrep vials were tested using both assays. In accordance with the type-specific probes contained within each test, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for the IHPV assay were 95.9%, 97.6%, 97.5%, and 96.1%, respectively, and those for the HC2 assay were 98.1%, 86.2%, 87.1%, and 97.9%. Overall, the sensitivity and NPV were comparable between the assays, but the IHPV assay demonstrated a better specificity and PPV, since the IHPV assay had fewer false-positive HR HPV results. The incorporation of an internal control to evaluate the cellularity of the test material is an important feature of the IHPV assay and should reduce the risk of false-negative results due to insufficient sample collection rather than the lack of HR HPV DNA. An additional benefit of the IHPV assay was the smaller sample volume required (1 ml versus 4 ml for the HC2 assay).     

Modulation of apoptosis by human papillomavirus (HPV) oncoproteins

Summary. The regulation of host-mediated apoptosis by the E6 and E7 oncoproteins has garnered attention because it is believed to be an important strategy employed by high-risk (HR)-human papillomaviruses (HPVs) to evade immune surveillance. Additionally, the revelation that E5 can protect cells from tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis suggests that it may also play a role in undermining host defense mechanisms. Cellular transformation is an unintended consequence of persistent infection by HR-HPVs, and it is therefore likely that the primary function of E5, E6 and E7 is to regulate cell survival throughout the normal viral life cycle in order to ensure viral replication and promote the spread of progeny. The purpose of this article is to review the literature on the regulation of host-mediated apoptosis by E5, E6 and E7 that describes the mechanisms employed by HR-HPVs to persist in the host and create the conditions necessary for cellular transformation.     

Genomic diversity of human papillomavirus (HPV) Genotype 38

Human papillomavirus (HPV) genotype HPV 38 is a HPV genotype associated with skin cancer and is classified taxonomically in the beta‐PV genus–species 2. At least six genomic variants of HPV 38, including prototype isolate and its subtype FA125, have been characterized so far. In order to investigate further the genomic diversity of HPV 38, a total of 39 HPV 38 positive samples obtained from hairs plucked from pubic, scrotal, perianal or eyebrow regions from 31 immunocompetent healthy male individuals were analyzed. The characterization of genomic variants was based on analysis of L1, E6, and E7 genomic regions. Sequence analysis revealed the presence of a single genomic variant in 35 samples and the presence of at least two different HPV 38 genomic sequences in four samples. A total of nine, nine, and five L1, E6, and E7 genomic variants were identified among 35 isolates, respectively. After combining nucleotide variations in all three genomic regions for a particular isolate, 13 different variants were identified, of which 6 and 7 corresponded to HPV 38 and FA125, respectively. In addition to 5 genomic variants identified previously (prototype isolate, subtype FA125, putative subtype AF091444, isolates U21875 and AF091443), 12 novel genomic variants were characterized. A sixth genomic variant described previously (L38917) was found to be identical with prototype HPV 38 isolate. Taking into account the results of this and previous studies, at least seventeen HPV 38 genomic variants exist today, 12 of which are described for the first time in this study. J. Med. Virol. 81:288–295, 2009. © 2008 Wiley‐Liss, Inc.     

Analysis of centrosome overduplication in correlation to cell division errors in high-risk human papillomavirus (HPV)-associated anal neoplasms

High-risk HPV-associated anal neoplasms are difficult to treat and biomarkers of malignant progression are needed. A hallmark of carcinogenic progression is genomic instability, which is frequently associated with cell division errors and aneuploidy. The HPV-16 E7 oncoprotein has been previously shown to rapidly induce centriole and centrosome overduplication and to cooperate with HPV-16 E6 in the induction of abnormal multipolar mitoses. Based on this function, it has been suggested that HPV-16 E7 may act as a driving force for chromosomal instability. However, a detailed analysis of centrosome overduplication in primary HPV-associated neoplasms has not been performed so far. Here, we determined the frequency of centrosome overduplication in HPV-associated anal lesions using a recently identified marker for mature maternal centrioles, Cep170. We detected centrosome overduplication in a small but significant fraction of cells. Remarkably, centrosome overduplication, but not aberrant centrosome numbers per se or centrosome accumulation, correlated significantly with the presence of cell division errors. In addition, our experiments revealed that in particular pseudo-bipolar mitoses may play a role in the propagation of chromosomal instability in high-risk HPV-associated tumors. These results provide new insights into the role of centrosome aberrations in cell division errors and encourage further studies on centrosome overduplication as a predictive biomarker of malignant progression in HPV-associated anal lesions.     

A prospective study of antibody responses to defined epitopes of human papillomavirus (HPV) type 16 in relationship to genital and anorectal presence of HPV DNA.

The aim of this study was to investigate whether antibody responses against synthetic peptides derived from genital human papillomavirus (HPV) proteins are associated with laboratory-proven genital and anorectal HPV infection. In this study, 158 heterosexual patients (110 women and 48 men) were followed prospectively. At each visit we collected serum samples as well as specimens from several sites in the anogenital area for detection of HPV type 6/11 (HPV-6/11), -16, -18, and -33 DNAs by PCR. Immunoglobulin A (IgA) and IgG responses against disrupted bovine papilloma virions and eight different synthetic peptides derived from HPV-6/11, -16, and -18 were determined for serum samples from the first and the last visits. The subjects attended the Municipal Sexually Transmitted Disease Clinic in Amsterdam, The Netherlands, two to seven times (mean, four times) at approximately 4-month intervals. Women were monitored over a period of 155 person-years, and men were monitored over 65 person-years. The magnitudes of the IgA responses against HPV-16 late protein epitopes L1:13, L1:31, and L2:49 were significantly higher in the sera from the last visit among the currently HPV DNA-positive participants than in HPV DNA-negative persons (P = 0.02). When the persons positive for any HPV type at any time during the follow-up period were compared with those who were negative at all times during the follow-up period, we also found a significant elevation of IgA responses against L1:31 and L2:49 (P = 0.04 and 0.01, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the Digene HC2 assay and the Roche AMPLICOR human papillomavirus (HPV) test for detection of high-risk HPV genotypes in cervical samples

Many different methods with different sensitivity and specificity have been proposed to detect the presence of high-risk human papillomavirus (HR HPV) in cervical samples. The HC2 is one of the most widely used. Recently, a new standardized PCR-based method, the AMPLICOR HPV test, has been introduced. Both assays recognize the same 13 HR HPV genotypes. The performances of these two commercially available assays were compared in 167 consecutive women (for a total of 168 samples) who presented at the Colposcopy Clinic either for a follow-up or for a diagnostic visit. Concordant results were found in 140/168 cervical samples (overall agreement, 83%; Cohen's kappa = 0.63). Twenty-eight samples gave discordant results: 20 were positive with the AMPLICOR HPV test and negative with the HC2 assay, and 8 were negative with the AMPLICOR HPV test and positive with the HC2 assay. The genotyping showed that no HR HPV was detected in the 8 HC2 assay-positive AMPLICOR HPV test-negative samples, while in 8/20 AMPLICOR HPV test-positive HC2 assay-negative samples, an HR HPV genotype was found. The AMPLICOR HPV test scored positive in a significantly higher percentage of subjects with normal Pap smears. All 7 cervical intraepithelial neoplasia grade 3 patients scored positive with the AMPLICOR HPV test, while 2 of them scored negative with HC2. Both tests had positive results in the only patient with squamous cell carcinoma. In conclusion, this study shows that the HC2 assay and the AMPLICOR HPV test give comparable results, with both being suitable for routine use. The differences noted in some cases may suggest a different optimal clinical use.     

Evaluation of a novel multiplex human papillomavirus (HPV) genotyping assay for HPV types in skin warts

Human papillomaviruses (HPV) of the genera alpha, mu, and nu induce benign tumors of the cutaneous epithelia that constitute a significant burden for immunocompromised adults. Currently, no gold standard for genotyping of these HPV types exists. In this study, we describe the prevalence of genus alpha, mu, and nu HPV types in cutaneous warts. We developed a novel multiplex HPV genotyping assay, BSwart-PCR/MPG (BSwart), to type sensitively and specifically 19 cutaneous HPV types frequently found in warts. BSwart-PCR/MPG is based on a multiplex PCR using broad-spectrum primers and subsequent multiplex hybridization to type-specific probes coupled to Luminex beads. In a first application comprising 100 cutaneous warts, the assay was compared to another, recently described genotyping assay, the HSL-PCR/MPG. When a 10-fold dilution series was used, the detection limit was between 10 and 100 HPV genomes per PCR. When comparing the two assays, there was an excellent agreement in detecting dominant HPV types; however, we also obtained evidence for a higher sensitivity of the BSwart assay for multiple infections in these cutaneous warts. Using BSwart, HPV was found in 95% of wart preparations, with HPV1 being most prevalent, followed by types 27, 57, and 2. Both novel BSwart and HSL-PCR/MPG HPV genotyping assays are powerful high-throughput tools that could be used to learn more about the natural history of cutaneous HPV. They would be advantageous to monitor the efficacy of future skin HPV vaccines and to identify novel HPV vaccine candidates.     

Candidate human papillomavirus (HPV) type 27b: nucleotide sequence and heterogeneity with HPV 27

More than 100 human papillomavirus (HPV) types have been reported as tumorigenic agents. HPV types 2 and 27 were identified in benign tumors and classified closely according to the sequence homology. An HPV type 2 related sequence was identified previously in an oral papilloma. However, the correct typing and sequencing was impossible at that time. A candidate HPV was cloned by the long-PCR technique from an oral papilloma and characterized by nucleotide sequence. This virus was basically HPV type 27, but there was some heterogeneity in comparison with the prototype. Most differences were nucleotide-sequence variations. Two of four-nucleotide insertions were frame shift, resulting in longer L1 protein than that of HPV 27.     

Neutralization of human papillomavirus (HPV) pseudovirions: a novel and efficient approach to detect and characterize HPV neutralizing antibodies

The development of vaccines against human papillomaviruses (HPVs) has long been hampered by the inability to grow HPVs in tissue culture and the lack of an efficient neutralization assay. To date, less than 10% of more than 100 different HPV types can be grown in athymic and "SCID" mouse xenograft systems or raft culture systems. Recently, the in vitro generation of HPV pseudovirions and their use in neutralization assays were demonstrated. The major shortcomings of the current approaches to HPV neutralization are the lack of HPV virions for most types for the xenograft methods and the time-consuming and inefficient generation of infective pseudovirions for the latter methods, which precludes their use in large-scale HPV clinical trials or epidemiological studies. We describe here a novel and efficient approach to generating pseudovirions in which HPV virus-like particles (VLPs) are coupled to the beta-lactamase gene as a reporter. We show that it is not necessary to encapsidate the reporter gene constructs into the pseudovirions. Using sera from human volunteers immunized with HPV-11 VLPs expressed in yeast, we demonstrate that our novel neutralization assay compares favorably with the athymic mouse neutralization assay. Furthermore, our assay was used to define neutralizing monoclonal antibodies to HPV-6, which were previously unknown.     

Cervical human papillomavirus (HPV) infection and HPV type 16 antibodies in South African women

There is a high incidence of cervical cancer in South African women. No large studies to assess human papillomavirus virus (HPV) infection or HPV type 16 (HPV-16) exposure have occurred in the region, a requirement for policy making with regards to HPV screening and the introduction of vaccines. Control women (n = 1,003) enrolled in a case control study of hormonal contraceptives and cervical cancer were tested for 27 cervical HPV types by reverse line blot analysis. The seroprevalence of HPV-16 immunoglobulin G (IgG) and IgA antibodies was assessed by a virus-like particle-based enzyme-linked immunoassay of 908 and 904 control women, respectively, and of 474 women with cervical cancer. The cervical HPV prevalence was 26.1%. The HPV-16 IgG seroprevalence was 44.4% and the HPV-16 IgA seroprevalence was 28.7% in control women, and these levels were significantly higher (61.8% and 52.7%, respectively) for women with cervical cancer (odds ratio [OR], 2.1 and 2.8, respectively). The cervical HPV prevalence showed an association with cervical disease, and the HPV-16 IgG prevalence decreased while the HPV-16 IgA prevalence increased with increasing age (P < 0.05). The prevalence of oncogenic HPV types (including HPV-16) decreased with age, whereas nononcogenic HPV types showed limited association with age. Multivariate analysis revealed cervical HPV infection to be associated with herpes simplex virus type 2 infection (OR, 1.7) and increasing years of education (OR, 1.9). HPV-16 IgG antibodies were inversely associated with current smoking status (OR, 0.6), and the presence of HPV-16 IgA antibodies was inversely associated with the use of alcohol (OR, 2.1) and inversely associated with the use of oral contraceptives (OR, 0.6). High levels of exposure to HPV, and particularly HPV-16, were evident in this population. The apparent increase of serum HPV-16 IgA with increasing age requires further investigation.     

Daily self-sampling for high-risk human papillomavirus (HR-HPV) testing

BackgroundSelf-sampling for HPV as part of primary screening is a well-tolerated method for women not attending organized Pap smear screening and could increase coverage of cervical cancer screening.ObjectiveTo investigate if the prevalence of HR-HPV varies from day to day in infected women and if one single sample is reliable for detecting an ongoing infection.Study designThis is a prospective cohort study on 12 premenopausal and 13 postmenopausal women performing daily self-sampling for HR-HPV testing. They were all HR-HPV-positive 1–3 months ago. Postmenopausal women were sampled for 28 days and premenopausal women sampled during bleeding-free days in one menstrual cycle. A possible difference in viral load between the estrogen-dominated proliferative phase and the progesterone-dominated secretory phase was analyzed.Results and conclusionsConsistent results throughout the sampling period were observed for 19 women, with either a daily presence of HPV (14 women) or no HPV at all during the sampling period (5 women). Of 607 samples from 25 women, 596 were consistently positive or negative for HPV during the sampling period and 11 were inconsistent (2%). There was no difference in HPV copy number between the estrogen dominated proliferative or progesterone dominated secretory menstrual cycle phases.The major finding was a high degree of consistency concerning HR-HPV positivity and negativity of HR-HPV in vaginal fluid during a sustained period of daily self-sampling. It does not appear to matter whether the sample is collected in the proliferative or secretory phase.     

Cervical human papillomavirus (HPV) infection in South African women: implications for HPV screening and vaccine strategies

The prevalence of cervical human papillomavirus (HPV) in South African women (n = 1,073) increased from 20.4% (173/848) in women with normal cytology to 41.7% (48/115) in women with atypical squamous cells of undetermined significance, 70.2% (40/57) in women with low-grade squamous intraepithelial lesions, and 83% (44/53) in women with high-grade squamous intraepithelial lesions (HSILs). HPV types 16 and 35 were the dominant types in women with HSILs but not in women in the other categories.     

The association between human leukocyte antigen (HLA)-G polymorphisms and human papillomavirus (HPV) infection in Inuit women of northern Quebec

Background

The human leukocyte antigen (HLA)-G molecules act as negative regulators of the immune response. We analyzed the associations between HLA G polymorphisms and human papillomavirus (HPV) infection and squamous intraepithelial lesions (SIL) in Inuit women from Nunavik, northern Quebec.

Methods

Cervical specimens from a cohort study of 548 Inuit women were tested for HPV DNA. HPV genotypes were classified according to tissue-tropism groupings of alpha-papillomavirus species: alpha group 1 includes low risk (LR) cervical species, group 2 includes high risk (HR) cervical species, and group 3 includes LR vaginal species. HLA-G alleles were typed using direct DNA sequencing.

Results

HLA-G^∗01:01:01 was associated with an increased risk of period prevalent alpha groups 1 (OR = 2.23, 95% CI:1.08–4.59) and 3 (OR = 1.70, 95% CI:1.09–2.65). The homozygous HLA-G^∗01:04:01 genotype was associated with a decreased risk of alpha group 3 infection period prevalence (OR = 1.69 95% CI = 1.07–2.67). No HLA-G alleles were significantly associated with HPV persistence. HLA-G^∗01:01:02, G^∗01:04:01 and G^∗01:06 were associated with high grade (HG)SIL, but the association did not reach statistical significance.

Conclusions

These results suggest that HLA-G polymorphisms play a role in the natural history of HPV infection, likely at the stage of host immune recognition. HLA-G polymorphisms interacted differently with the three alpha papillomavirus groups.     

Prevalence of human papillomavirus (HPV) DNA in larynx and lung carcinomas.

In this retrospective study, we investigated the HPV DNA occurrence in 21 laryngeal and 26 primary lung squamous cell carcinomas. Nonisotopic in situ hybridization (NISH) technique was performed with commercially available digoxigenin-labelled DNA probes for HPV screening. Subtyping for HPV subtypes 6/11, 16/18 and 31/33 was also performed. We observed HPV DNA signals in 10 (47.6%) cases of laryngeal SCC and in only 3 (11.5%) cases of lung SCC. Typing showed signals of HPV 6/11, 16/18 and 31/33 infection in 80%, 40%, 30% of the laryngeal carcinomas, respectively. In the lung, we demonstrated type 16/18 positivity in two and type 6/11 in one of the HPV-positive cases. We found a statistically significant correlation between HPV infection and tumour recurrence (p < 0.035) in laryngeal carcinomas, but not between HPV presence and tumour stage or grade in neither larynx nor lung.     

Validity of vaginal testing in detecting human papillomavirus (HPV) genotypes.

The aim of this study was to assess the validity and usefulness of vaginal scrapes in detecting cervical human papillomavirus (HPV) DNA by the polymerase chain reaction (PCR). The study group comprised 23 women tested positive and 28 women tested negative for cervical HPV DNA by PCR, and confirmed by histopathology. At the time of specimen collection, both vaginal and endocervical scrapes were taken from these women, and tested for HPV DNA by PCR, using MY09/MY11 primer system. HPV genotypes were analyzed by hybridizing PCR products with HPV type-specific biotinylated probes. HPV DNA was detected in both vaginal and cervical scrapes from the HPV-positive, but not from HPV-negative group. In the HPV-positive group, the same HPV type was found in vaginal and endocervical scrapes, giving a positive predictive value of 1.0. The results indicate that HPV types can be detected in vaginal scrapes, and recommend utilization of the less invasive vaginal testing for the routine detection of HPV DNA.     

Prevaccination genomic diversity of human papillomavirus genotype 6 (HPV 6)

Prevaccination genomic diversity of human papillomavirus genotype 6 (HPV 6) was established by sequencing 3798 bp of 77 clinically important HPV 6 isolates obtained from 45 and 32 patients with genital warts and laryngeal papillomas, respectively. By analyzing pooled L1, LCR, E6, E2, and E5 nucleotide data of an individual isolate, a total of 36 different genomic variants were identified, of which six (12 isolates), one (one isolate) and 29 (64 isolates) corresponded to HPV 6b, HPV 6a, and HPV 6vc genetic lineages, respectively. Several novel, potentially important mutations were identified. Non-prototypic HPV 6vc genomic variants were found in the majority of genital warts and laryngeal papillomas included in the study. The presence of serious HPV 6 genome sequence errors was confirmed and novel sequence errors were identified in sequence repositories.     

The human papillomavirus (HPV) vaccine,HPV related diseases and cervical cancer in the post-reproductive years

Prophylactic HPV vaccines have demonstrated high efficacy against a range of HPV related diseases. They have been widely adopted as population health interventions in many jurisdictions and their routine use has been endorsed by the WHO. The development of these vaccines comes after an increased understanding of the natural history and epidemiology of HPV infection and disease in both males and females. Persistent HPV infection with oncogenic types induces malignant transformation in a range of epithelia including the cervix, anogenital regions, the penis and a number of head and neck sites. In relation to HPV disease prevention in the post-reproductive years, most infections occur soon after commencement of sexual activity but new infections do occur throughout the age spectrum. This reduces the likely impact of prophylactic vaccines in this population. The major impact on HPV related disease in this age group will come from advances in screening and early detection of HPV and neoplastic precursors. The most appropriate prevention for any individual man or women in this age group will be an individualised combination of vaccination, screening and early detection depending on the individual's own circumstances.