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ABSTRACT: The contribution of carcinogenic human papillomavirus (HPV) types to the burden of cervical cancer has been well established. However, the role and contribution of phylogenetically related HPV genotypes and rare variants remains uncertain. In a recent, global study of 8,977 HPV-positive invasive cervical carcinomas (ICC), the genotype remained unidentified in 3.7% by the HPV SPF(10) PCR-DEIA-LiPA(25) (version 1) algorithm. The 331 ICC specimens with unknown genotype were analyzed by a novel sequence methodology using multiple, selected, short regions in L1. This demonstrated HPV genotypes that have infrequently or never been detected in ICC, i.e., HPV26, 30, 61, 67, 68, 69, 73 and 82, and rare variants of HPV16, 18, 26, 30, 34, 39, 56, 67, 68, 69, 82 and 91. These are not identified individually by LiPA(25) and only to some extent by other HPV genotyping assays. Most identified genotypes have a close phylogenetic relation with established carcinogenic HPVs and have been classified as possibly carcinogenic by IARC. Except for HPV85, all genotypes in alpha species 5, 6, 7, 9 and 11 were encountered as single infections in ICC. These species of established and possibly carcinogenic HPV types form an evolutionary clade. We have shown that the possibly carcinogenic types were detected only in squamous cell carcinomas, which were often keratinizing and diagnosed at a relatively higher mean age (55.3) than those associated with established carcinogenic types (50.9). The individual frequency of the possibly carcinogenic types in ICC is low, but together they are associated with 2.25% of the 8,338 included ICC with a single HPV type. This fraction is greater than seven of the established carcinogenic types individually. This study provides evidence that possibly carcinogenic HPV types occur as single infections in invasive cervical cancer, strengthening the circumstantial evidence of a carcinogenic role. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
The Journal of Pathology 06/2012; · 6.32 Impact Factor
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Heather Griffin,
Zhonglin Wu,
Rebecca Marnane,
Vincent Dewar, Anco Molijn,
Wim Quint,
Christine Van Hoof,
Frank Struyf,
Brigitte Colau,
David Jenkins,
John Doorbar
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ABSTRACT: High-risk human papillomavirus (HPV) infections are the cause of nearly all cases of cervical cancer. Although the detection of HPV DNA has proved useful in cervical diagnosis, it does not necessarily predict disease presence or severity, and cannot conclusively identify the causative type when multiple HPVs are present. Such limitations may be addressed using complementary approaches such as cytology, laser capture microscopy, and/or the use of infection biomarkers. One such infection biomarker is the HPV E4 protein, which is expressed at high level in cells that are supporting (or have supported) viral genome amplification. Its distribution in lesions has suggested a role in disease staging. Here we have examined whether type-specific E4 antibodies may also allow the identification and/or confirmation of causal HPV-type. To do this, type-specific polyclonal and monoclonal antibodies against three E4 proteins (HPV-16, -18, and -58) were generated and validated by ELISA and western blotting, and by immunohistochemistry (IHC) staining of epithelial rafts containing these individual HPV types. Type-specific detection of HPV and its associated disease was subsequently examined using formalin-fixed paraffin-embedded cervical intra-epithelial neoplasias (CIN, (n = 247)) and normal controls (n = 28). All koilocytotic CIN1 lesions showed type-specific E4 expression of their respective HPV types. Differences were noted amongst E4 expression patterns in CIN3. HPV-18 E4 was not detected in any of the 6 HPV-18 DNA-positive CIN3 lesions examined, whereas in HPV-16 and -58 CIN3, 28/37 (76%) and 5/9 (55.6%) expressed E4 respectively, usually in regions of epithelial differentiation. Our results demonstrate that type-specific E4 antibodies can be used to help establish causality, as may be required when multiple HPV types are detected. The unique characteristics of the E4 biomarker suggest a role in diagnosis and patient management particularly when used in combination.
PLoS ONE 01/2012; 7(12):e49974. · 4.09 Impact Factor
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Wim Quint,
David Jenkins, Anco Molijn,
Linda Struijk,
Miekel van de Sandt,
John Doorbar,
Johann Mols,
Christine Van Hoof,
Karin Hardt,
Frank Struyf,
Brigitte Colau
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ABSTRACT: In 20-40% of cervical intra-epithelial neoplasia (CIN) and in 4-8% of cervical carcinoma tissue specimens, multiple HPV genotypes have been detected. Whole tissue section (WTS) PCR does not determine how the individual types relate causally to complex and multiple CIN. Our objective was to determine whether laser capture micro-dissection (LCM) with HPV PCR genotyping (LCM-PCR) could accurately recover type-specific HPV DNA from epithelial cells in individual areas of CIN and normal epithelium, and whether one or more viruses are present in one lesion. For that, histologically selected samples of CIN and normal epithelium were isolated by LCM and analysed by the SPF(10) PCR/LiPA(25) (version 1) HPV genotyping system for 25 HPV genotypes. HPV genotypes detected in 756 areas of CIN (grade 1, 2 or 3) by LCM-PCR were compared with results obtained by WTS-PCR in 60 cases (74 biopsies). We showed that when a single HPV type is detected by WTS-PCR, that type was almost always (94%; 29/31) recovered by LCM-PCR from CIN. When multiple HPV types were present by WTS-PCR, their distribution within histological sections could be mapped by LCM-PCR. Association of a single HPV type with a discrete area of CIN was found for 93% (372/399) of LCM fragments analysed by PCR. We found colliding CIN lesions associated with separate HPV types and only 62% (61/99) of HPV types detected by WTS-PCR were found in CIN by LCM-PCR. Therefore, the LCM-PCR technique was found very accurate for high-resolution HPV genotyping and for assigning an individual HPV type to an area of CIN. At LCM level, in cervical biopsy sections with multiple HPV infections, the relation between HPV types and CIN lesions is often complex. Almost every HPV type found in CIN by LCM-PCR is associated with a biological separate independent CIN lesion-one virus, one lesion.
The Journal of Pathology 11/2011; 227(1):62-71. · 6.32 Impact Factor
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Wen Chen,
Xun Zhang, Anco Molijn,
David Jenkins,
Ju-Fang Shi,
Wim Quint,
Johannes E Schmidt,
Ping Wang,
Yu-Ling Liu,
Lian-Kun Li, [......],
Jin-Feng Liu,
Qi Zhou,
Ying Hong,
Li Li,
Qing Li,
Hong-Lin Zhou,
Mei-Lu Bian,
Jing Chen,
You-Lin Qiao,
Jennifer S Smith
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ABSTRACT: Prophylactic vaccination against HPV 16 and 18 has the potential for effective prevention of high-grade precancer (cervical intraepithelial neoplasia [CIN)] 2/3) and ICC caused by these viruses (globally 50 and 70%, respectively) when employed in women prior to starting sexual activity. To provide data for decisions on HPV vaccination in China, we determined HPV type-distribution in ICC and CIN 2/3 from women of different regions within China. A multicenter study was conducted by randomized sampling of paraffin blocks of 664 ICC (630 squamous cell carcinoma [SCC]; 34 adenocarcinoma [ADC]), 569 CIN 2/3 cases from seven regions of China. Histological diagnosis was confirmed in 1,233 cases by consensus review. HPV DNA was detected using the SPF10 LiPA25 version 1 assay. HPV prevalence was 97.6% in SCC, 85.3% in adenocarcinoma, and 98.9% in CIN 2/3. HPV 16 (76.7%) and HPV 18 (7.8%) were the most common, together accounting for 84.5% of SCC, followed by HPV 31 (3.2%), HPV 52 (2.2%), and HPV 58 (2.2%). HPV positivity in SCC did not differ notably by region. However, SCC cases from women <or=34 years had higher HPV 16 positivity than women over 50 years, among whom HPV 52, 58, and 39 were more common. HPV 16 and 18 were under-represented, whereas HPV 31, 52, and 58 were over-represented in CIN2/3 compared to SCC. The potential impact of vaccines against oncogenic HPV types 16 and 18 is estimated to be high (84.5%) against total SCC. These data are critical for China's future evaluation of the cost-effectiveness of current cervical cancer vaccines and of HPV-based screening guidelines.
Cancer Causes and Control 09/2009; 20(9):1705-13. · 2.88 Impact Factor
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Stefanie J Klug, Anco Molijn,
Betti Schopp,
Barbara Holz,
Angelika Iftner,
Wim Quint,
Peter J F Snijders,
Karl-Ulrich Petry,
Susanne Krüger Kjaer,
Christian Munk,
Thomas Iftner
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ABSTRACT: Classification of high-risk HPV types for cervical cancer screening depends on epidemiological studies defining HPV type-specific risk. The genotyping tests that are used, are however, not uniform with regard to type-specific detection rates making comparisons between different studies difficult. To overcome the lack of a "gold standard" four tests were evaluated crosswise using 824 cervical smears pretested by HC2. The tests evaluated were the L1-PCR-based assays PGMY09/11 LBA, HPV DNA Chip and SPF LiPA and an E1 consensus PCR followed by cycle sequencing (E1-PCR). A subset of 265 samples was tested in addition with the GP5+/6+ reverse line blot assay. Differences were noted in the sensitivity and range for specific HPV types, e.g. with detection rates for HPV53 ranging from 2.3% to 11.6%. HPV16 was the most prevalent type detected by all tests except for the SPF-10 LiPa, which detected HPV31 more often. Kappa values calculated ranged from poor (k=0.20) to intermediate (k=0.54) for HPV positivity, but were higher for high-risk type positivity (k=0.31-0.61) and best for recognition of HPV16 (k=0.53-0.72). The analytical sensitivity of the tests ranged between 15% and 97% for individual types and specificity was highly dependent on which test system was used as "gold standard" for the analysis. The results of histology were used for calculation of clinical sensitivity and specificity. E1-PCR, PGMY09/11 LBA and SPF-10 LiPA had a high clinical sensitivity (>95%) for the detection of cervical intraepithelial neoplasia 2 or higher, whereas the HPV DNA Chip reached only 84.1%.
Journal of Medical Virology 07/2008; 80(7):1264-74. · 2.82 Impact Factor
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ABSTRACT: The use of a single broad-spectrum human papillomavirus (HPV) DNA-based PCR test may fail to detect lower concentrations of HPV DNA due to competition between different genotypes in mixed infections. To improve HPV detection by PCR, broad-spectrum and type-specific (TS) PCRs were combined, with a focus on HPV-16 and HPV-18. Cervical and cervicovaginal cell samples were obtained from 1,113 healthy women (age range, 15 to 25 years) participating in an HPV-16/HPV-18 candidate vaccine efficacy trial. These samples were tested by a broad-spectrum SPF(10) PCR-DNA enzyme immunoassay, followed by a primer SPF(10)-based line probe assay (SPF(10) LiPA), and HPV-16- and HPV-18-TS PCRs. The results for the majority of the HPV-16/18 SPF(10) LiPA-positive samples were confirmed by TS-PCR (kappa values, 0.775 for HPV-16 and 0.785 for HPV-18). However, TS PCR revealed additional positive samples among those that contained other HPV genotypes due to competition. Conversely, SPF(10) LiPA identified HPV-16 or -18 in samples that remained negative by TS PCR as a result of sampling variation. Analysis of follow-up samples from more than 1,000 women confirmed that the combination of SPF(10)-LiPA with additional HPV-16- and HPV-18-TS PCR diminishes the rate of false-negative diagnosis. The combination of broad-spectrum and TS PCRs resulted in a novel testing algorithm. This combination of assays is more accurate than either method alone, and the novel algorithm offers a highly accurate and effective method for the analysis of HPV infections.
Journal of Clinical Microbiology 10/2006; 44(9):3292-8. · 4.15 Impact Factor
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ABSTRACT: Human papillomaviruses (HPVs) comprise more than 100 genotypes. The mucosal types can be divided into high-risk and low-risk (LR) types depending on the associated disease risk. HPV infection is mainly diagnosed by molecular methods, since reliable serological tools are not available and culture of the virus is not possible. Accurate molecular diagnostic techniques that can be used to inform patient management and follow-up after treatment are now available for detection and identification of HPV. The diagnosis of HPV infections in patients at risk of disease in a clinical setting requires a different approach from that used for epidemiological studies, vaccination trials and natural history studies. This review describes the different molecular methods available for HPV detection and genotyping and their possible clinical utility.
Journal of Clinical Virology 04/2005; 32 Suppl 1:S43-51. · 3.97 Impact Factor
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ABSTRACT: The risk of cervical neoplasia for women with normal Papanicolaou smears was calculated for those whose smears were human papillomavirus (HPV) positive and those whose smears were HPV negative. Data on 347 cases and controls were analyzed in a population-based, nested case-control study. Cases (n = 77) were women who participated in the Utrecht screening program (1976-1984) in the Netherlands and who developed cervical intraepithelial neoplasia 3 or microinvasive or invasive squamous cervical cancer after having a negative smear (1980-1986). Controls (n = 270) were matched on age (+/-5 years) and follow-up period. DNA was isolated from the Papanicolaou smears and was tested for the presence of HPV DNA by using the ultrasensitive broad-spectrum, general short-fragment polymerase chain reaction. HPV was found in 55 (71%) of the baseline smears of the 77 cases and in 31 (11%) of those of the 270 controls. The age-adjusted odds ratios for developing cervical intraepithelial neoplasia or microinvasive or invasive cervical cancer were 19.2 (95 percent confidence interval (CI): 10.3, 35.7) for HPV positivity in general, 5.4 (95% CI: 1.5, 19.5) for infection with low-risk HPV genotypes, 24.0 (95% CI: 12.4, 46.4) for high-risk HPV genotypes, and 104.8 (95% CI: 29.5, 372.7) for HPV type 16.
American Journal of Epidemiology 08/2002; 156(2):158-64. · 5.22 Impact Factor
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ABSTRACT: A comparison of two PCR-based human papillomavirus (HPV) DNA detection and genotyping systems (PGMY LBA and SPF(10) LiPA) was conducted in two laboratories. Both systems are based on broad-spectrum PCR for the detection of HPV DNA, followed by reverse hybridization with type-specific probes. A total of 400 selected cervical scrape specimens in PreservCyt solution (55% normal cytology, 18% atypical squamous cells of unknown significance, 14.8% low-grade squamous intraepithelial lesions [SIL], and 12.5% high-grade SIL) were tested for the presence of HPV DNA. In this selected group of specimens, the overall agreement between the two methods for the detection of any HPV DNA was high (kappa = 0.859). When the 20 common HPV genotypes identified by both methods were considered (HPV types 6, 11, 16, 18, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 56, 58, 59, 66, and 68), compatible genotype-specific results were observed in 96.5% of the samples, even when multiple HPV genotypes were present. However, for some specific HPV genotypes, there were significant differences in HPV detection by the two methods. PGMY LBA detected more HPV type 42 (P = 0.002), HPV type 56 (P = 0.039), and HPV type 59 (P < 0.001), whereas SPF(10) LiPA detected more HPV type 31 (P < 0.001) and HPV type 52 (P = 0.031). For the remaining genotypes, including HPV types 16 and 18, the results obtained by the two methods were not significantly different. In general, both genotyping methods are highly suitable for clinical and epidemiological studies.
Journal of Clinical Microbiology 03/2002; 40(3):979-83. · 4.15 Impact Factor
