Boštjan J. Kocjan

Which high-risk HPV assays fulfil criteria for use in primary cervical cancer screening?

Several countries are in the process of switching to high-risk human papillomavirus (hrHPV) testing for cervical cancer screening. Given the multitude of available tests, validated assays which assure high-quality screening need to be identified. A systematic review was conducted to answer the question which hrHPV tests fulfil the criteria defined by an international expert team in 2009, based on reproducibility and relative sensitivity and specificity compared to Hybrid Capture-2 or GP5+/6+ PCR-enzyme immunoassay. These latter two hrHPV DNA assays were validated in large randomized trials and cohorts with a follow-up duration of 8 years or more. Eligible studies citing the 2009 guideline were retrieved from Scopus (http://www.scopus.com) and from a meta-analysis assessing the relative accuracy of new hrHPV assays versus the standard comparator tests to detect high-grade cervical intraepithelial neoplasia or cancer in primary screening. The cobas 4800 HPV test and Abbott RealTime High Risk HPV test were consistently validated in two and three studies, respectively, whereas the PapilloCheck HPV-screening test, BD Onclarity HPV assay and the HPV-Risk assay were validated each in one study. Other tests which partially fulfil the 2009 guidelines are the following: Cervista HPV HR Test, GP5+/6+ PCR-LMNX, an in-house E6/E7 RT quantitative PCR and MALDI-TOF (matrix-assisted laser desorption-ionization time-of-flight). The APTIMA HPV assay targeting E6/E7 mRNA of hrHPV was also fully validated. However, the cross-sectional equivalency criteria of the 2009 guidelines were set up for HPV DNA assays. Demonstration of a low risk of CIN3+ after a negative APTIMA test over a longer period is awaited to inform us about its utility in cervical cancer screening at 5-year or longer intervals.

Commercially available assays for multiplex detection of alpha human papillomaviruses

Five main groups of commercial assays for the multiplex detection of alpha human papillomaviruses (HPVs) are currently available. DNA-based screening assays, which test for the presence of 13–14 HPVs without determination of HPV type, have been the standard for HPV detection in the last decade. Assays that combine testing for 14 HPVs and HPV-16 and HPV-18 genotyping are a potential future standard for HPV detection. The clinical value of HPV genotyping assays has still not been finally determined. Recently, one of the mRNA-based assays showed equal clinical sensitivity but higher clinical specificity for CIN2+/CIN3+ in comparison with the validated DNA-based assay. In situ hybridization assays are too laborious and have insufficient clinical sensitivity to be used in routine screening. Automation, price reduction and improvement of clinical specificity are the main goals for the future development of HPV assays.

Commercially available molecular tests for human papillomaviruses (HPV): 2015 update.

Commercial molecular tests for human papillomaviruses (HPV) are invaluable diagnostic tools in cervical carcinoma screening and management of women with cervical precancerous lesions as well as important research tools for epidemiological studies, vaccine development, and implementation and monitoring of vaccination programs. In this third inventory of commercial HPV tests, we identified 193 distinct commercial HPV tests and at least 127 test variants available on the market in 2015, which represents a 54% and 79% increase in the number of distinct HPV tests and variants, respectively, in comparison to our last inventory performed in 2012. Identified HPV tests were provisionally divided into eight main groups and several subgroups. Among the 193 commercial HPV tests, all but two target alpha-HPV types only. Although the number of commercial HPV tests with at least one published study in peer-reviewed literature has increased significantly in the last three years, several published performance evaluations are still not in line with agreed-upon standards in the HPV community. Manufacturers should invest greater effort into evaluating their products and publishing validation/evaluation results in peer-reviewed journals. To achieve this, more clinically oriented external quality-control panels and initiatives are required. For evaluating the analytical performance of the entire range of HPV tests currently on the market, more diverse and reliable external quality-control programs based on international standards for all important HPV types are indispensable. The performance of a wider range of HPV tests must be promptly evaluated on a variety of alternative clinical specimens. In addition, more complete HPV assays containing validated sample-extraction protocols and appropriate internal controls are urgently needed. Provision of a broader range of automated systems allowing large-scale HPV testing as well as the development of reliable, rapid, and affordable molecular point-of-care tests are priorities for the further improvement of HPV tests.

Tumor‐specific and gender‐specific pre‐vaccination distribution of human papillomavirus types 6 and 11 in anogenital warts and laryngeal papillomas: A study on 574 tissue specimens

Anogenital warts and laryngeal papillomas are two most important benign tumors etiologically linked with HPV. In the study, which included both the largest number of laryngeal papilloma tissue specimens (152 specimens from 152 patients) to date and the largest number of prospectively collected and histologically confirmed tissue specimens of anogenital warts obtained from both genders (422 specimens from 315 patients), HPV DNA was detected in 413/422 (97.9%) of anogenital warts and 139/152 (91.4%) of laryngeal papillomas. HPV‐6 and/or HPV‐11 were detected in 291/315 (92.4%) patients with anogenital warts and in 138/152 (90.8%) patients with laryngeal papillomas, indicating that the great majority of both tumors could be prevented with prophylactic quadrivalent vaccine. The HPV‐6 gender‐specific distribution in both anogenital warts and laryngeal papillomas was not statistically significant. In contrast, HPV‐11 was found almost three times more often in males than in females with anogenital warts (16.5% vs. 6.3%; P = 0.008), with a gender neutral HPV‐11 type distribution in laryngeal papillomas. The overall HPV DNA prevalence in anogenital warts was significantly different from that in laryngeal papillomas (97.1% vs. 91.4%; P = 0.01). In the first comparison of the HPV‐6/HPV‐11 type‐specific distribution between patients suffering from anogenital warts and laryngeal papillomas with the same geographic and ethnic background, a significant imbalance in tumor‐specific distribution of HPV‐6 and HPV‐11 was identified: HPV‐6 was statistically more often present in anogenital warts than in laryngeal papillomas (79.0% vs. 59.2%; P = 0.000013), whereas HPV‐11 was statistically more frequent in laryngeal papillomas than in anogenital warts (28.9% vs. 12.4%; P = 0.00003). J. Med. Virol. 84: 1233–1241, 2012.

Detection and differentiation of human papillomavirus genotypes HPV-6 and HPV-11 by FRET-based real-time PCR

A real-time PCR (RT-PCR) assay was developed based on fluorescence resonance energy transfer (FRET) hybridization probe technology, allowing very sensitive and specific detection of HPV-6 and HPV-11, reliable differentiation of HPV-6 and HPV-11, as well as prototypic and non-prototypic HPV-6 genomic variants, in a single PCR reaction. The primers and probe were designed on the basis of multiple alignments of 74 HPV-6 E2 gene sequences and 20 HPV-11 E2 gene sequences. Testing on defined plasmid standards showed that the RT-PCR allowed simple and reliable identification of HPV-6 and HPV-11 using type specific amplification followed by probe-specific post-amplification dissociation analysis. Sensitivity, assessed by probit analysis at a 95% detection level, was 42.9, 43.4, and 25.3 DNA copies per assay for prototypic and non-prototypic HPV-6 variants and HPV-11, respectively. The results obtained by the developed assay on 51 HPV DNA-negative samples and 149 HPV DNA-positive samples, including 81 HPV-6 positive samples (19 prototypic and 62 non-prototypic HPV-6 variants), 28 HPV-11 positive samples, 10 samples of HPV-44 and HPV-74 (the closest relatives of HPV-6 and HPV-11) and 30 samples of 15 other important alpha HPV, showed complete agreement with those obtained with the INNO-LiPA human papillomavirus (HPV) Genotyping Assay and HPV-6 E2 and E6 gene sequencing.

In Inverted Papillomas HPV more likely represents incidental colonization than an etiological factor

Inverted papillomas (IPs) are the most frequent type of sinonasal papillomas. These benign but destructive lesions are known for their high recurrence rate, probably due to incomplete excision. Our aim was to investigate the frequency of human papillomavirus (HPV) infection in patients with IPs and in IPs associated with squamous cell carcinoma (IPsSCC) and to compare it with the frequency of HPV infections in the control group. The influence of HPV infection on the malignant alteration and recurrence rate of IPs was also evaluated. Paraffin-embedded tissue samples from 68 patients with sinonasal IPs and 5 patients with IPsSCC were analyzed in this retrospective study. The control group consisted of 47 patients who had undergone septoplasty or mucotomy of the inferior turbinate. PCR amplification with consensus primer sets was performed to detect alpha-HPVs, and direct sequencing of the PCR products with the same primers was used to determine the HPV genotypes in the samples. We detected HPV DNA in 20 (30.3%) patients with IPs, in 3 (60%) patients with IPsSCC, and in 6 (13%) patients from the control group. The frequency of HPV infection in the study group was statistically significantly higher (p = 0.032) than in the control group. The presence of HPV DNA was not a statistically significant predictor of the recurrence of IPs (p = 0.745) nor was it a statistically significant risk factor for associated SCC (p = 0.32). Since HPV type 11 was the predominant genotype in the IPs, IPsSCC, and in the control cases, we presume that HPV infection may represent incidental colonization rather than being an important etiological factor of IPs.

Comparison of the Abbott RealTime High Risk HPV test and INNO-LiPA HPV Genotyping Extra test for the detection of human papillomaviruses in formalin-fixed, paraffin-embedded cervical cancer specimens

Comparative evaluation of Abbott RealTime and Innogenetics INNO-LiPA on alternately processed formalin-fixed, paraffin-embedded specimens of 31 cervical cancers and 31 uterine myomas showed complete agreement in the detection of 14 assay-common HPV genotypes and partial genotyping of HPV-16 and HPV-18. The tissue preparation protocol was shown to be sample-to-sample contamination safe.

Molecular methods for identification and characterization of novel papillomaviruses.

Papillomaviruses (PV) are a remarkably heterogeneous family of small DNA viruses that infect a wide variety of vertebrate species and are aetiologically linked with the development of various neoplastic changes of the skin and mucosal epithelia. Based on nucleotide similarity, PVs are hierarchically classified into genera, species and types. Novel human PV (HPV) types are given a unique number only after the whole genome has been cloned and deposited with the International HPV Reference Center. As of 9 March 2015, 200 different HPV types, belonging to 49 species, had been recognized by the International HPV Reference Center. In addition, 131 animal PV types identified from 66 different animal species exist. Recent advances in molecular techniques have resulted in an explosive increase in the identification of novel HPV types and novel subgenomic HPV sequences in the last few years. Among PV genera, the γ-PV genus has been growing most rapidly in recent years with 80 completely sequenced HPV types, followed by α-PV and β-PV genera that have 65 and 51 recognized HPV types, respectively. We reviewed in detail the contemporary molecular methods most often used for identification and characterization of novel PV types, including PCR, rolling circle amplification and next-generation sequencing. Furthermore, we present a short overview of 12 and 10 novel HPV types recently identified in Sweden and Slovenia, respectively. Finally, an update on the International Human Papillomavirus Reference Center is provided.

Characterization of human papillomavirus type 120: a novel betapapillomavirus with tropism for multiple anatomical niches

Recent studies indicate that human papillomaviruses (HPVs) from the genera Betapapillomavirus and Gammapapillomavirus are abundant in the human oral cavity. We report the cloning and characterization of a 7304 bp HPV120 genome from the oral cavity that is related most closely to HPV23 (L1 ORF, 83.7 % similarity), clustering it in the genus Betapapillomavirus (β-PV). HPV120 contains five early and two late genes, but no E5 ORF. HPV120 was detected from heterogeneous human biological niches, including the oral cavity, eyebrow hairs, anal canal and penile, vulvar and perianal warts. Characterization of the clinical spectrum of HPV120 infections indicates a broader spectrum of epithelial tropism than appreciated previously for HPV types from the genus β-PV.

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.