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(1)VU Research Portal. Matching intended use and type of HPV test in research and clinical practice Geraets, D.T.. 2015. document version Publisher's PDF, also known as Version of record. Link to publication in VU Research Portal. citation for published version (APA) Geraets, D. T. (2015). Matching intended use and type of HPV test in research and clinical practice.. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. E-mail address: vuresearchportal.ub@vu.nl. Download date: 19. Oct. 2021.

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(3) Matching intended use and type of HPV test in research and clinical practice. Daan Geraets. 209130-L-bw-Geraets.

(4) Matching intended use and type of HPV test in research and clinical practice Daan Geraets The studies described in this thesis were financially supported by the Stichting Pathologie Ontwikkeling en Onderzoek (SPOO) Foundation, GlaxoSmithKline Biologicals S.A., the Research and Development fund of the Public Health Service of Amsterdam (project no 2369 and 2371) and AGIS healthcare insurance (RVVZ no 1417000), Spanish public grants from the Instituto de Salud Carlos III (grants numbers FIS PI030240, FIS PI061246, RCESP C03/09, RTICESP C03/10, RTIC RD06/0020/0095, and CIBERESP), Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR 2005SGR 00695 and 2009SGR126), Marató de TV3 Foundation (051530), Sanofi Pasteur MSD, and Merck. Financial support for publication of this thesis was kindly provided by the Stichting Pathologie Ontwikkeling en Onderzoek (SPOO) Foundation. ISBN: 978-94-6259-754-9 Cover design: Bart Geraets Printed by: Ipskamp Drukkers BV Copyright© 2015 by D.T. Geraets, Leiden, the Netherlands All rights reserved. No part of this thesis may be reproduced, stored or transmitted in any form or by any means without prior permission of the author. Cover: picture adapted from Modis et al, EBMO J, 2002. Reproduced with permission.. 209130-L-bw-Geraets.

(5) VRIJE UNIVERSITEIT. Matching intended use and type of HPV test in research and clinical practice. ACADEMISCH PROEFSCHRIFT ter verkrijging van de graad Doctor aan de Vrije Universiteit Amsterdam, op gezag van de rector magnificus prof.dr. F.A. van der Duyn Schouten, in het openbaar te verdedigen ten overstaan van de promotiecommissie van de Faculteit der Geneeskunde op donderdag 3 september 2015 om 11.45 uur in de aula van de universiteit, De Boelelaan 1105. door Daniël Theodoor Geraets geboren te Rotterdam. 209130-L-bw-Geraets.

(6) promotor: prof.dr. C.J.L.M. Meijer copromotor: dr. W.G.V. Quint. 209130-L-bw-Geraets.

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(8) TABLE OF CONTENTS Chapter 1:. General introduction and thesis outline. 9. Part 1: Analytical accuracy of HPV genotyping assays Chapter 2:. The original SPF10 LiPA25 algorithm is more sensitive and suitable for epidemiologic. 43. HPV research than the SPF10 INNO-LiPA Extra Journal of Virological Methods 2015; 215–216: 22–29 Chapter 3:. High genotyping concordance between the digene HPV Genotyping RH Test and the. 53. Reverse Line Blot genotyping assay on GP5+/6+-PCR products Journal of Clinical Virology 2009; S3: S16-S20 Chapter 4:. High-throughput genotyping of High-Risk HPV by the digene HPV Genotyping LQ. 61. Test using GP5+/6+-PCR and xMAP technology Journal of Clinical Virology 2009; S3: S21-S26 Chapter 5:. Universal human papillomavirus genotyping by the digene HPV Genotyping RH and. 69. LQ Tests Journal of Clinical Virology 2011; 50: 276-280 Part 2: Application of HPV genotyping assays in epidemiologic studies Chapter 6:. Cross-sectional study of genital carcinogenic HPV infections in Paramaribo,. 79. Suriname: Prevalence and determinants in an ethnically diverse population of women in a pre-vaccination era Sexually Transmitted Infections 2014; 90 (8): 627-633 Chapter 7:. Human papillomavirus genotype attribution in invasive cervical cancer: a. 89. retrospective cross-sectional worldwide study Lancet Oncology 2010; 11: 1048-1056 Chapter 8:. Detection of rare and possibly carcinogenic human papillomavirus genotypes as. 107. single infections in invasive cervical cancer Journal of Pathology 2012; 228: 534-543 Chapter 9:. Long-term follow-up of HPV16-positive women: Persistence of the same genetic variant and low prevalence of variant co-infections PLoS ONE 2013; 8(11): e80382. 209130-L-bw-Geraets. 121.

(9) Part 3: Application of HPV genotyping assays in clinical studies Chapter 10:. Clinical evaluation of a GP5+/6+-based Luminex assay having full high-risk HPV. 135. genotyping capability and an internal control Journal of Clinical Microbiology 2014; 52 (11): 3996-4002 Chapter 11:. Clinical evaluation of high-risk HPV detection on self-samples using the FTA solid. 147. -state carrier cartridge Journal of Clinical Virology 2013; 57(2):125-129 Chapter 12:. General discussion and conclusions. Chapter 13:. Summary & samenvatting. 171. Addendum:. List of publications. 183. Dankwoord. 185. Curriculum vitae. 187. 209130-L-bw-Geraets. 155.

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(11) CHAPTER 1. General introduction and thesis outline. 9. 209130-L-bw-Geraets.

(12) Chapter 1. 10. 209130-L-bw-Geraets.

(13) General introduction and thesis outline. 1.1. BACKGROUND. 1.1.1 Short introduction Infection of cervical epithelium with a human papillomavirus (HPV) can lead to the development of. adenosquamous. and. neuroendocrine. carcinomas. account for less than 5% of cervical cancers (3). ADC develops from adenocarcinoma in-situ (AIS).. cervical intraepithelial neoplasia (CIN) and ultimately. Precursors. cervical cancer. Technologies that can detect and. intraepithelial neoplasia (CIN) grade 1, 2, and 3. of. SCC. are. classified. as. cervical. distinguish different human papillomaviruses are. (including carcinoma in-situ). All precursor lesions can. important to determine their involvement in disease, to. regress, persist or progress. However, the possibility of. evaluate the efficacy of HPV vaccines, to monitor. regression decreases with increasing CIN grade. The. prevalence of HPV genotypes in immunized cohorts,. risk of progression from CIN3 to invasive cervical. and to identity women at risk for having high-grade. cancer (ICC) is estimated between 30-50% (4-7). The. CIN within cervical cancer screening programs. Over. concept of cervical carcinogenesis is summarized in. 125 commercial HPV tests have been developed, with. Figure 1. differences in design and performance. In this thesis we. CIN2 and CIN3 together are referred to as high-. evaluated the performance of a number of established. grade CIN (HG-CIN). In the USA, women with HG-. and novel technologies for the identification of HPVs in. CIN and AIS are treated to prevent cervical cancer. This. relation to their intended use.. approach. 1.1.2 Cervical cancer Cervical cancer is the fourth most common malignancy. overtreatment,. among women worldwide, with 528,000 new cases and. morphologically. In Europe, small CIN2 localized in a. 266,000 deaths occurring each year (1). Squamous cell. well-visible transformation zone are often followed by. carcinoma (SCC; 80%) and adenocarcinoma (ADC;. periodic colposcopy (one year). CIN1 lesions are. 15%) are the main histological types (2), while. considered low-grade CIN (LG-CIN) for which a wait-. regressive. results. in. since. HG-CIN. a. considerable. currently cannot. degree. progressive be. of and. distinguished. and-see policy is advised.. 11. 209130-L-bw-Geraets.

(14) Chapter 1. 20–30 years. hrHPV SCJ cells Ectocervix or TZ. Productive CIN. Type of hrHPV infection. Transient (latent) infection. Productive (permissive) infection. Morphological appearance. Normal. CIN1/2. Transforming CIN. Cancer. Transforming (non-permissive) infection CIN2/3. Cancer. Infectious viral particles Squamous epithelium Superficial zone. Midzone. Basal layer Basement membrane Dermis. Figure 1: The concept of HPV-mediated cervical carcinogenesis and the according morphological appearance. HPV gains access to the basal cells of squamous epithelium, followed by an ordered expression pattern of viral genes leading to the production and release of new virions. Deregulated expression of E6 and E7 oncoproteins and viral genome integration are associated with malignant transformation, leading to genomic instability, (epi)genetic changes, and ultimately invasive cervical cancer. The outcomes of HPV exposure are represented as a transient infection (no pathology), a productive infection (productive CIN; CIN1 and a subset of CIN2), and a transforming infection (transforming CIN; remaining subset of CIN2 and CIN3). The majority of transforming CIN and cervical cancers are suggested to arise from an hrHPV infection of embryonic squamo-columnar junction (SCJ) cells (adapted from (8) and (9)). 1.1.3 Human papillomaviruses PVs are naked viruses with a circular, double-stranded. open reading frames (ORFs), i.e., E1, E2, E4, E5, E6, E7,. DNA genome of about 7600-8000 base-pairs (bp). expression, replication and survival. The late (L) region. contained in a protein capsid. The genome is divided. encodes the two viral structural proteins, i.e., L1 and L2.. which encode proteins required for viral gene. into three regions, i.e., the long control region (LCR),. By convention, the similarity across the highly. the early (E) coding region, and the late (L) coding. conserved L1 ORF was adapted as the basis for. region. The LCR regulates viral gene expression and. taxonomic classification of PVs (10). A PV “type” is. replication. The E region contains six or more early. defined as a complete PV genome, whose L1 ORF. 12. 209130-L-bw-Geraets.

(15) General introduction and thesis outline. nucleotide sequence is at least 10% different from that. The family of papillomaviridae currently consists. of any other known PV type (11). PV subtypes share 90. of 189 classified PVs distributed over 29 genera (12).. to 98% and variants more than 98% nucleotide. Presently, 170 HPV types have been described and are. sequence identity in L1.. located in the α, β, γ, μ, and ν genera (13). HPVs are. At higher taxonomy levels, PVs have been grouped. strictly epitheliotropic and can infect epithelium and/or. together into species, and species into genera. PV types. mucosal areas of skin, conjunctiva, oropharynx, anus,. within the same genus show less than 60% sequence. penis, vulva, vagina and cervix (11, 14). The β, γ, μ, and. identity to types of other genera (12). Different species. ν genera comprise only cutaneous HPVs, while α genus. within a genus share between 60% and 70% nucleotide. contains both cutaneous and mucosal types divided. identity, while PVs in the same species share between 71% over 13 species. HPVs within these species branch into and 89% identity in L1 ORF nucleotide sequence (10).. three. Genera are denominated by a Greek letter and species. relationship. ancestral. clades. by addition of a number to the letter, e.g., species. papillomaviruses is shown in Figure 2.. between. (15).. The. the. different. phylogenetic human. Alphapapillomavirus 9 (α9) (11).. 13. 209130-L-bw-Geraets.

(16) Chapter 1. Figure 2: Phylogenetic relationship between human papillomaviruses (from (16)).. 14. 209130-L-bw-Geraets.

(17) General introduction and thesis outline. The nomenclature for variant lineages of an HPV. 1.0% difference between complete genomes to define. type was initially based on the observed association. major lineages. Major lineages are named using an. between the variant and the continent of origin, e.g.,. alphanumeric, with the reference genome of each type. European, Asian, African 1&2, North-American, and. always located in the “A” clade. Differences between. Asian-American 1&2 variants of HPV16 (17). Recently,. 0.5–1% are used to designate sublineages (e.g., A1, A2).. a novel classification and nomenclature system has been. The conversion between the novel and initial. proposed (18). This system is based on full-genome. nomenclature for HPV16 is shown in Table 1.. sequence analysis, and uses an approximate cut-off of Table 1: Novel (18) and initial (17) nomenclature of HPV16 variant (sub)lineages. Novel (Burk, 2013). Initial (Ho, 1993). Main lineage. Sublineage. A. A1. European (EUR). A2. European (EUR). A3. Asian (As). B. B1. African 1a (AF1a). B2. African 1b (AF1b). C. C1. African 2a (AF2a). C2. African 2b (AF2b). D. D1. North-American (NA). D2. Asian-American 1 (AA1). D3. Asian-American 2 (AA2). 1.1.4 ervical cancer development HPV and ccervical The large majority of infections with HPVs are rapidly. infections are associated with CIN2, CIN3 and ultimately cervical cancer (Figure 1) (8).. cleared by a cell-mediated immune response, and do. The exact mechanism for deregulation of E6 and E7. not give rise to lesions. About 20% of the remaining. is not well understood. The majority of cervical. persisting HPV infections cause lesions that are. carcinomas contain one or more copies of HPV. considered productive infections. These infections lead. integrated into the host genome. Integration occurs. to the generation of new virions in the upper epithelial. more or less randomly, but viral integration sites often. layers using the host replication machinery under. lie within the ORF of E2, the transcriptional regulator. tightly. E7.. of E6 and E7 (19, 20). The continuous overexpression of. Morphologically, productive infections can appear as. viral oncoproteins E6 and E7 in proliferating basal cells. CIN1 and CIN2, but generally do not persist to progress. of the epithelium leads to genomic instability and. to advanced precursor lesions (CIN3) and cancer. (epi)genetic. (Figure 1).. transformation. Precursor lesions can develop within 2-. controlled. expression. of. E6. and. Only a minority of HPV infections become transforming. Transforming infections are characterized. changes,. and. ultimately. malignant. 3 years after HPV infection (21, 22), while progression from HG-CIN to cancer may take 10-30 years (21, 23).. by deregulated expression of E6 en E7 in the (para)basal cells, i.e., cells with proliferating capacity. Transforming. 15. 209130-L-bw-Geraets.

(18) Chapter 1. 1.2. IMPORTANCE OF HPV GE GENOTYPING NOTYPING TESTS. found in α10, α8, α1, and α13 (“low-risk clade 1”) and α2, α4, α14/15, and α3 (“low-risk clade 2”) (Figure 2). The most recent carcinogenic classification of. 1.2.1 HPV types and disease association HPV genotyping tests have been very important in. mucosal HPV genotypes by the WHO IARC was based. epidemiologic. on the evidence of the epidemiologic risk classification,. studies. towards. HPVs. and. their. association with disease. In a global cervical cancer. supplemented. case-control study, Munoz et al classified fifteen types. evidence when available (26). IARC has classified. as high-risk (hr) (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58,. mucosal types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58,. with. phylogenetic. and. biological. 59, 68, 73, and 82), three as probably high-risk (pr-hr). and 59 as carcinogenic (Class 1), type 68 as probably. (26, 53, and 66), and twelve types as low-risk (lr) (6, 11,. carcinogenic (Class 2A), and types 26, 30, 34, 53, 66, 67,. 40, 42, 43, 44, 54, 61, 70, 72, 81, and CP6108) (Table 2). 69, 70, 73, 82, 85 and 97 as possibly carcinogenic (Class. (24). This observation correlated with the three. 2B). HPV6 and HPV11 were not classifiable as to its. ancestral phylogenetic clades of alpha HPVs (15, 25).. carcinogenicity to humans (Class 3), and the remaining. The hrHPVs and pr-hrHPVs were located in α9, α11, α7,. mucosal HPV types were considered probably not. α5, and α6 (“high-risk clade”), while most lrHPVs were. carcinogenic (Class 4) (Table 2).. Table 2: The epidemiologic risk classification (24) and the WHO IARC carcinogenic classification (26) of mucosal HPV genotypes. WHO IARC, 2009. Munoz, 2003 HighHigh - risk types. Probably highhigh-. LowLow -risk types. risk types Class 1 (carcinogenic) 16, 18, 31, 33, 35, 39, 45, 51, -. Types not included in the study. -. -. 52, 56, 58, 59 Class 2A (probably. 68. -. -. -. 73, 82. 26, 53, 66. 70. 30, 34, 67, 69, 85, 97. -. -. 6, 11. -. Class 4 (probably not -. -. 40, 42, 43, 44, 54, 61,. All other mucosal. 72, 81, CP6108. HPVs. carcinogenic) Class 2B (possibly carcinogenic) Class 3 (not classifiable) carcinogenic) The classification of HPVs that pose a high risk for. lacking. Most of these types are not targeted by. causing cervical cancer is an evolving process,. commonly used HPV genotyping assays. Additional. particularly for the HPV types in Class 2A/B. These. research is required to clarify their causal role and. genotypes have been classified as probably/possibly. clinical importance. A specific definition of the HPVs. carcinogenic mainly based on their close phylogenetic. considered (probably/possibly) high-risk is provided in. relation with established carcinogenic HPVs, but. each individual chapter of this thesis.. epidemiological and biological evidence is virtually. 16. 209130-L-bw-Geraets.

(19) General introduction and thesis outline. Even within the group of established hrHPVs. 100% seroconversion in vaccinees (43). The AS04. (Class 1), there appears to be a difference in. adjuvant system of the bivalent vaccine appears to. carcinogenic potential. Longitudinal studies have. induce a stronger, more sustained antibody response. shown an elevated risk of HG-CIN for HPV16 in. than the conventional adjuvant of the quadrivalent. particular and to a lesser extent for HPV18, compared. vaccine (44), although its effect on duration of. to other high-risk HPV types (27-32). In contrast to. protection is unknown.. other hrHPV genotypes, these two types show a. For both vaccines, HPV genotyping tests targeting. proportional increase in prevalence in women having. a broad range of HPVs were used to demonstrate high. normal cytology, precancer, and ICC (33, 34).. efficacy against several surrogate endpoints, from. Technologies that have the resolution to distinguish. persistent HPV infection to high-grade CIN lesions. intratypic variants were used to investigate differences. (CIN2+) associated with vaccine-targeted HPVs (45).. in risk associations between variants, in particular for. The quadrivalent vaccine has demonstrated strong. those of HPV16, the most carcinogenic HPV. Lineages. protection against genital warts, of which 90% in men. B, C, and D (Non-European) of HPV16 appear to be. and women are caused by vaccine targets HPV6 and 11. more pathogenic in comparison to isolates from the A. (46-48). Long-term follow-up of vaccinated cohorts will. lineage (European) (18). HPV16 lineages B, C and D. determine the duration of protection against vaccine-. persist more frequently (35, 36), are more associated. targeted types (45).. with precancer and specifically CIN3 (35-40), and have. Cross-neutralization of HPV types related to. elevated risks for cancer compared to the HPV16 A. HPV16 and 18 confers cross-protection. Both vaccines. variant lineage. In particular, this increased risk of. provide partial cross-protective efficacy against HPV31. cervical cancer is mostly related to the D (Asian-. and 33 (49, 50). The bivalent vaccine also offers some. American) lineage (40-42).. cross-protection against HPV45 (51, 52).. 1.2.2 HPV vvaccine accine efficacy trials HPV genotyping tests targeting a broad range of HPVs. 1.2.3 Surveillance of HPV type prevalence The bivalent and quadrivalent vaccines have been. were used to demonstrate the efficacy of two HPV. implemented in vaccination programs in various. vaccines.. Biologicals,. countries. Several aspects should be monitored to. Rixensart, Belgium) and Gardasil® (Merck & Co.,. evaluate the effects of an HPV vaccination program, i.e.,. Whitehouse Station, NJ, USA) are two prophylactic. 1) the occurrence of vaccine-preventable disease, 2) the. HPV L1 virus-like particle (VLP) vaccines that have. concentration of naturally occurring HPV type-specific. been licensed to prevent anogenital HPV infections and. antibodies and the prevalence of HPV vaccine-. their associated neoplasia. Cervarix is a bivalent vaccine. associated genotypes, 3) the titers of HPV vaccine type-. protecting primarily against HPV16 and 18, and. associated antibody response, 4) vaccine uptake, and 5). Gardasil is a quadrivalent vaccine that targets besides. vaccine safety. HPV genotyping tests can be used to. HPV16 and 18 also the lrHPV types 6 and 11. Both are. determine the prevalence of HPV genotypes pre- and. three-dose vaccines composed of HPV L1 proteins self-. post-vaccination. This allows monitoring the effects of. assembled. into. produced. in. Cervarix®. (GlaxoSmithKline. virus-like different. particles. expression. (VLPs),. but. nation-wide vaccination on prevalence of HPV. systems. and. genotypes (and possibly type replacement) within an. administered with different adjuvants. In contrast to. immunized cohort.. natural HPV infection, both vaccines induce high titers of neutralizing anti-L1 VLP antibodies, with virtually. 17. 209130-L-bw-Geraets.

(20) Chapter 1. 1.2.4 HPVHPV-based screening In the near future, HPV tests that can detect (and. (BD, Burlington, NC, USA) or ThinPrep PreservCyt. partially identify) a range of hrHPVs (hrHPV DNA. changes of cells are graded according to the degree of. testing) will be implemented in cervical cancer. abnormality. Different cytology classification systems. screening. are used, e.g., the Bethesda 2001 (USA), BSSC (United. programs.. Currently,. cervical. cancer. (Hologic, Marlborough, MA, USA). Morphological. screening programs rely on cytology performed on cells. Kingdom),. scraped from the transformation zone of the cervix and. Papanicolaou. collected in a liquid-based medium, e.g., BD SurePath. classification systems are shown in Table 3.. CISOE-A. (The. Netherlands). (PAP).. The. different. and. cytology. Table 3: Classification systems for cervical cytology (adapted from (53, 54)). BETHESDA Unsatisfactory Negative 2001. for evaluation (NILM). ASC-H Atrophy. ASC-US. HSIL. LSIL. AGC. SCC. AGC favor neoplastic. AIS. AC. Severe Borderline BSCC. Inadequate. Negative. nuclear change. Mild. Moderate. Severe. dyskaryosis dyskaryosis dyskaryosis. dyskaryosis Invasive Glandular neoplasia. CISOE-A. C0. PAP. PAP0. S1, E1-2, O1-2 PAP1. S2-3, O3, E3. S4, E4-5. S5, O4-5. S6, O6, E6. PAP2. PAP3a1. PAP3a2. PAP3b. S7, E7. S8-9, O7-8, E9. PAP4 PAP5. NILM, Negative for intraepithelial lesion or malignancy; ASC-H, atypical squamous cells cannot exclude HSIL; ASCUS, atypical squamous cells of undetermined significance; AGC, atypical glandular cells; LSIL low grade squamous intraepithelial lesion; HSIL, high grade squamous intraepithelial lesion; AIS, endocervical adenocarcinoma in situ; SCC, squamous cell carcinoma; AC, adenocarcinoma; CISOE-A, C composition, I inflammation, S squamous epithelium, O other abnormalities and endometrium, and E endocervical columnar epithelium HrHPV DNA tests are an improved primary cervical cancer screening tool compared to cytology. Randomized controlled trials have shown that hrHPV. 60–70% greater protection against invasive cervical carcinomas compared to cytology (63). In general, these hrHPV DNA tests target a range of. DNA testing detects 30% more CIN2+ and 20% more. 13-14. CIN3+ in women aged 30 years and older (55-62).. identification, or limited only to HPV16 and 18 (partial. Secondly, trials with longitudinal data on CIN3+ in. genotyping). The clinical use of hrHPV genotyping is. subsequent screening rounds (at 3-5 year intervals). currently unknown, but the separate identification of. have shown ~50% lower incidence rates of CIN3+. HPV16 and 18 might be of use in clinical management,. among women with a negative HPV test at baseline. due to their increased risk for cervical cancer compared. compared to those having normal cytology (56, 60-62).. to other hrHPVs.. Moreover,. a. pooled. analysis. of. these. studies. demonstrated that hrHPV-based screening provides. 18. 209130-L-bw-Geraets. hrHPVs. but. do. not. permit. individual.

(21) General introduction and thesis outline. 1.3. DESIGN AND PERFORMANCE PERFORMANCE ASSESSMENT OF HPV TESTS. self-collected by lavage or brush and stored in liquidbased medium, solid carrier cartridge or dryly (68, 69).. 1.3.1 Intended use The design of an HPV DNA test should match its. It is important to realize that the yield and quality of the. intended use. HPV tests designed for epidemiologic. methodology. purposes, e.g., disease association studies, vaccine. processing. Thus, all elements of the diagnostic chain. efficacy trials, and surveillance of HPV prevalence,. determine the outcome of the HPV test used and should. require a high analytical sensitivity and specificity and. be taken into account (68).. the capacity to individually identify a range of HPVs. In a clinical setting, HPV tests should detect hrHPV. 1.3.3 Target selection and amplification Most commercial and in-house tests that have been. infections mainly associated with clinically meaningful. developed for HPV detection and genotyping are based. disease, i.e., CIN2+ lesions (clinical sensitivity), while. on PCR amplification of HPV DNA. DNA PCR-based. limiting the detection of transient HPV infections not. methods will be the focus of this thesis, although some. associated with CIN2+ (clinical specificity). In addition. non-PCR based methods will also be briefly described.. specimen for HPV testing is influenced by the used. for. collection,. storage. and. to primary cervical cancer screening, two other. HPV DNA PCR tests do not characterize the. potential clinical applications of hrHPV testing have. complete HPV genome or the L1 ORF, as is required for. been defined. HrHPV testing can be used as a test-of-. classification of PVs (10). Instead, these assays target. cure for women treated for HG-CIN, and as a triage test. one or more regions within the genome that are. for women with borderline and mild abnormal cytology. sufficient for accurate viral recognition of one or more. (ASC-US/LSIL) (64).. previously classified HPVs. Misrecognition due to. 1.3.2. Clinical specimen, collection and processing A range of clinical specimens have been used for HPV. presence of unclassified types or genetic variants is. analysis, e.g., cervical cells collected by brush and stored. HPV tests, but smaller target regions allow more. in liquid-based medium (cervical swabs) (64), whole. efficient amplification in specimens with poorly. tissue sections (WTS) of freshly frozen or formalin-. preserved DNA, e.g., archival FFPE cervical biopsies.. fixed paraffin-embedded (FFPE) cervical biopsies (65,. The regions in the viral genome targeted by a selection. 66), micro-dissected regions of tissue by laser-capture. of HPV DNA PCR-based assays are shown in Figure 3.. inherently possible when using any HPV DNA assay. The size of viral regions that are amplified vary across. microscopy (LCM) (67), and cervicovaginal specimens. 19. 209130-L-bw-Geraets.

(22) Chapter 1. Figure 3: Schematic representation of primer target regions of different type-specific and broad-spectrum DNA PCRbased assays. The approximate fragment lengths (in nucleotides) are shown in parenthesis. The reference genome of HPV16 was used.. The degree of heterogeneity of the viral genome enables. genotypes present in low concentrations within. two approaches for amplification of viral DNA by PCR. multiple infections, also known as masking (70). Broad-. primers, i.e., broad-spectrum (consensus) primers or. spectrum PCR primers do not necessarily have the same. type-specific (TS) primers. Broad-spectrum primers. analytical sensitivity and specificity for each genotype. target relatively well-conserved genomic sequences,. and amplification efficiency might differ among. enabling the simultaneous amplification of a broad-. individual genotypes. Spiking experiments with plasmid. spectrum of HPV genotypes in a single test using only a. mixtures of different HPV genotypes have shown that a. limited number of primers. By default, these primer sets. competitive effect occurs in mixed infections when one. are not specifically designed for the amplification of. genotype is present in a much lower concentration than. only a single HPV type. Most broad-spectrum PCR. another (PCR competition) (71).. genotyping assays target well-conserved regions in the. As opposed to broad-spectrum PCR, type-specific. L1 ORF. The likelihood that sequence variations occur. (TS) primers target viral sequences that are specific for. at these positions causing false-negativity is relatively. a single genotype. These primers permit highly sensitive. low.. Broad-spectrum. can. be. and specific identification of HPVs. Although TS PCRs. 1). low-. can be designed to target any region in the HPV. stringency PCR conditions to allow some degree of. genome, many TS PCR assays amplify regions in the E6. mismatch acceptance between primers and target. or E7 ORF. These PCR target regions are not. sequence, 2) degenerate primers with nucleotide. interrupted by viral integration, since over-expression. variations at variable base positions, 3) primers with the. of E6 and E7 is required for transforming HPV. non-specific base-analogue inosine at ambiguous base. infections.. accomplished. by. amplification. different. approaches:. positions, and 4) sets of multiple, overlapping primers.. An important advantage of TS PCRs is that they are. A technical limitation of all broad-spectrum PCR-. less prone to underestimate the prevalence of HPV. based assays is the underestimation of the prevalence of. types that have low viral loads in multiple infections.. 20. 209130-L-bw-Geraets.

(23) General introduction and thesis outline. However, TS PCRs also have some limitations. could cause false-negative results (72). In addition, the. Assessing HPV test performance performance and quality assurance The non-clinical performance assessment of HPV tests. performance of a separate type-specific PCR for each. comprises analytical sensitivity (limit-of-detection),. HPV is highly laborious and requires substantial. analytical. quantity of clinical specimen. This issue can be. (reproducibility). addressed by the development of multiplex type-specific. methods). The analytical accuracy of a novel HPV test. (MPTS) PCRs, where multiple TS primer sets are. can be evaluated by comparison with an established. combined in a single PCR reaction for simultaneous. “gold standard” or comparator test, using panels of. amplification of a defined set of HPVs (72).. artificial samples, e.g., plasmids cloned with HPV target. 1.3.4 ReadRead-out method Amplification products generated by broad-spectrum. sequences, and/or clinical specimens for which the HPV. Unknown variations in the primer target sequences. 1.3.5. specificity and. (interference), accuracy. precision. (comparison-of-. test was designed.. and by type-specific PCRs can be detected by. However, an analytically validated HPV test. hybridization to a mix of oligonucleotide probes in a. should not be used in clinical practice without prior. DNA. a. clinical validation, since analytical and clinical accuracy. microtiter well plate. In addition, reverse hybridization. are non-synonymous. The clinical relevance of a novel. (RH) can be done by separate genotype-specific probes. hrHPV test for primary cervical cancer screening has to. immobilized on carriers such as nylon membrane,. be supported by data from longitudinal randomized. nitrocellulose strips, microsphere beads or DNA chips.. controlled trials (RCT). Alternatively, a candidate assay. Read-out can also be performed real-time using. can also be clinically assessed if it shows a clinical. fluorochrome-labelled Taqman probes in a quantitative. sensitivity and specificity that is non-inferior to a. (q)PCR format as opposed to a conventional end-point. reference test that has already been validated in. PCR. Read-out systems of PCR products can be. longitudinal RCT. International guidelines for panel. designed to recognize a range of HPV types. composition and criteria for clinical accuracy and test. simultaneously. reproducibility have been formulated (73).. enzyme. immune-assay. (detection),. (DEIA/EIA). individually. in. (full. genotyping), or as a combination of detection and genotyping (partial genotyping). The range of HPVs targeted by an HPV test should. The laboratories performing hrHPV testing should comply with quality assurance (QA) measures, such as a specific infrastructure for nucleic acid amplification,. be in accordance with its design. Clinically relevant. participation. HPV tests generally target 13 hrHPVs (Class 1 and 2A),. (proficiency panels), and accreditation for clinical. and have often incorporated HPV66 (Class 2B) in. molecular testing (73). Some HPV tests have been. addition. These assays are therefore also referred to as. equipped with an internal control (IC) to aid. hrHPV tests. HPV tests designed for epidemiologic. laboratories in assessing specimen quality and/or. purposes are not necessarily restricted to these hrHPVs. monitoring of sample processing, in order to prevent. and may also include possibly hr- and lrHPVs.. false-negative HPV results. An IC usually comprises a spiked. in. exogenous. external. nucleotide. quality. assessments. sequence. or. an. endogenous target (e.g., the beta-globin housekeeping gene) that is simultaneously extracted and amplified with HPV DNA.. 21. 209130-L-bw-Geraets.

(24) Chapter 1. 1.4. TECHNOLOGIES FOR HPV DETECTION AND GENOTYPING. Cervista has been evaluated in the SHENCCAST II. 1.4.1 Tests for pooled detection of HPVs HPV DNA detection assays provide a qualitative test. colleagues (77). However, concerns have been raised. result for a group of HPVs simultaneously (pooled or. (78), which might be resolved by increasing the assay. grouped detection). These assays generally target 13. threshold for positivity (79, 80).. hrHPVs (Class1/2A) or 14 hrHPVs (Class1/2A and. PCR amplification and and enzyme immuno assays. HPV66 in addition). Tests not intended for a clinical. Most endpoint PCR-based methods utilize consensus. setting can detect additional genotypes, including. primer sets targeting a conserved region of a broad-. lrHPVs (e.g., SPF10 DEIA).. spectrum of viral genomes. Detection of amplimers is. Signal amplification. performed. The Hybrid Capture 2 HPV DNA Test (HC2; Qiagen,. (designated DEIA or EIA) using a cocktail of specific. Hilden, Germany) is the most frequently used HPV test. probes for a defined set of hrHPVs (e.g., GP5+/6+ EIA). in the world. HC2 uses liquid-based chemiluminescent. or a mix of universal probes for a very broad range of. study (76) and clinically validated by Boers and about the relatively low clinical specificity of this assay. in. a. DNA. enzyme. immuno. assay. signal amplification of hybridized target DNA for the. HPVs (e.g., SPF10 DEIA). Presence of HPV is. simultaneous detection of 13 hrHPVs. This assay has no. determined by optical density measurement of labeled. internal control for a human DNA target. The RNA. probes hybridized to single-stranded amplimers in a. probes can cross-hybridize with several other (lr)HPVs. microtiter plate.. (74). The HC2 is intended for primary cervical cancer. The SPF10-PCR-DEIA (Labo Bio-medical Products,. screening, management of women with equivocal. Rijswijk, The Netherlands) was developed around 15. cytology results, and test-of-cure. HC2 has been. years ago (81) and is usually combined in a test. evaluated in longitudinal randomized, controlled. algorithm with the LiPA25 reverse hybridization strip. studies, e.g., NTCC, ARTISTIC, and VUSASCREEN. version 1 (Labo Bio-medical Products; described later). (59-61, 75). HC2 is therefore considered a reference test. (82). This algorithm was designed to have high. for validation of novel hrHPV assays (73).. analytical sensitivity and specificity. The SPF10 primers. The Cervista HPV HR Test (Cervista; Hologic,. are non-degenerated and amplify a 65-bp fragment in. Madison, WI, USA) offers simultaneous detection of. the L1 ORF of a broad-spectrum of at least 69 mucosal. DNA from 14 hrHPVs using signal amplification by. and cutaneous HPV types. Qualitative detection of. Invader chemistry. In a first isothermal reaction, a. HPV is performed in a DEIA with conservative,. probe and an Invader oligonucleotide specifically. universal HPV probes. A control target for the. anneal to HPV DNA to generate an overlapping. housekeeping gene beta-globin can be separately. structure. Enzymes specifically cleave and release the. amplified and detected. The SPF10 PCR system has been. overlapping primary probes. In a second, simultaneous. used in various epidemiologic studies and vaccine trials. isothermal reaction, cleaved flaps combine with a. for the bivalent vaccine, e.g., PATRICIA and CVT (66,. fluorescence resonance energy transfer (FRET) probe,. 83-87). The short region of only 65 bp targeted by SPF10. which generates a fluorescent signal. A region from the. primers is particularly sensitive for amplification in. HIST2H2BE gene is also amplified as an internal. formalin-fixed. control target. The Cervista is indicated for cervical. biopsy specimens, in which DNA is often poorly. cancer risk screening combined with cytology and for. preserved. The sensitivity of the SPF10-PCR-DEIA is too. management of women with equivocal cytology results.. high for application in a clinical setting (88).. 22. 209130-L-bw-Geraets. paraffin-embedded. (FFPE). cervical.

(25) General introduction and thesis outline. The. GP5+/6+-PCR-EIA. has. been. originally. alternative for Amplicor because it has an improved. developed as an in-house test and is now commercially. clinical specificity.. available (EIA kit GP HR; Diassay, Rijswijk, The. 1.4.2. approximately 150 bp in the L1 ORF. Amplification of a. HPV DNA detection tests with partial genotyping A recent generation of HPV tests offers concurrent. broad-spectrum of HPV genotypes using only two. identification of a restricted number of HPVs in. primers is achieved by a relatively low annealing. addition to pooled hrHPV detection (designated as. temperature. A cocktail of probes specific for 14. partial genotyping). Partial genotyping is usually. hrHPVs hybridizes with the GP5+/6+ amplification. limited to HPV16 and 18, with the remaining hrHPVs. products in an EIA format, providing a qualitative. detected as a group. Separate identification of HPV16. result (89). A 313-bp fragment of human DNA is. and 18 might be of use in clinical management, due to. intrinsically co-amplified by the GP5+/6+ primers and. their increased risk for cervical cancer compared to. can function as an internal control (90). A specifically. other hrHPVs.. designed probe can detect this fragment in a separate. Quantitati Quantitative tive PCR amplification. EIA format. The GP5+/6+-PCR-EIA is intended for. In HPV tests with quantitative PCR (qPCR) technology,. primary cervical cancer screening, management of. Taqman probes labeled with a fluorescent dye can. women with equivocal cytology results, and test-of-cure.. hybridize with the amplimer during every annealing. Similarly to HC2, the GP5+/6+-PCR-EIA has been. step. The increase in fluorescence can be monitored. evaluated in longitudinal randomized, controlled. real-time during the exponential increase of amplified. studies, e.g., POBASCAM and SWEDESCREEN (56,. viral target DNA, until limiting reagents, accumulation. 62). The GP5+/6+-PCR-EIA is therefore considered a. of inhibitors or inactivation of the polymerase affect the. reference assay for HPV testing in cervical cancer. PCR efficiency.. screening (73).. fluorescence can be distinguished from the background. Netherlands). The GP5+/6+ primers amplify a region of. The AMPLICOR Human Papillomavirus Test (Amplicor;. Roche. Diagnostics,. Almere,. The first cycle where generated. signal is called the threshold cycle (Ct) or quantification. The. cycle (Cq). The initial concentration of the measured. Netherlands) has broad-spectrum primers that amplify. target is correlated to the Cq value, and can be. a region of 165 bp from L1. Complementary probes can. quantified by including a standard curve dilution series.. detect presence of amplification products of 13 hrHPVs. The Cobas 4800 HPV Test (Cobas) enables the. by measuring the optical density in a microtiter plate. A. qualitative, simultaneous detection of 14 hrHPVs using. beta-globin internal control target is amplified in the. qPCR amplification of a region in L1. This test provides. same PCR reaction and detected in a separate microtiter. individual genotyping of HPV16 and 18, if desired, and. plate. Amplicor has not been clinically validated. a beta-globin internal control target in the same PCR. according to the international criteria (73). In a triage. reaction. The Cobas is intended for cervical cancer risk. population, the clinical sensitivity of Amplicor was only. screening in combination with cytology (co-testing) and. marginally higher compared to HC2, but its clinical. for management of women with equivocal cytology. specificity was significantly lower (91). Two other. results. In recent years, its clinical value has been. studies reported equivalent clinical sensitivity and. supported by the ATHENA trial (94) and by. specificity for Amplicor and HC2 in triage populations. comparison with a reference test (95).. (92, 93). Roche developed and introduced the Cobas. Similar to Cobas, the Abbott RealTime High Risk. 4800 HPV Test (Cobas; Roche Diagnostics) as an. HPV test (Abbott RealTime HR HPV test; Abbott,. 23. 209130-L-bw-Geraets.

(26) Chapter 1. Wiesbaden, Germany) provides qPCR-based detection. efficacy of vaccines, while evidence for the clinical. of 14 hrHPVs, concurrent HPV16/18 genotyping, and a. utility of full genotyping is limited.. beta-globin internal control. A modified GP5+/6+. BroadBroad - spectrum PCR amplification and reverse. primer mix is used for L1-based viral amplification (96).. hybridization. The Abbott RealTime HR HPV test has been clinically. The LiPA25 version 1 (Labo Bio-medical Products) is. validated for HPV-based cervical cancer screening in. used for identification of 25 individual HPV genotypes. women aged 30 years and older in several studies (96-. by reverse hybridization of generated SPF10 amplimers. 98).. with genotype-specific probes immobilized on a reverse The HPV-Risk assay (Self-Screen BV, Amsterdam,. hybridization strip. In a combined test algorithm, SPF10. The Netherlands) is an E7-based broad-spectrum qPCR,. amplimers that are positive by the DEIA (Labo Bio-. which offers detection of 14 hrHPVs and HPV67, with. medical Products) can be used directly for LiPA25,. concurrent HPV16/18 genotyping and sample quality. eliminating the need for a separate PCR reaction (81,. assessment using an endogenous human beta-globin. 82).. internal control target. This assay meets the clinical and. aforementioned epidemiological and vaccine efficacy. reproducibility criteria of the international guidelines. studies.. This. algorithm. has. been. used. in. the. (73) and is also compatible with specimens that were. The INNO-LiPA HPV Genotyping Extra (INNO-. self-collected using lavage- and brush-based devices. LiPA; Innogenetics, Gent, Belgium) is another SPF10-. (99).. based. reverse. hybridization. strip. with. similar. The BD Onclarity HPV Assay (Onclarity; Becton. technology as the LiPA25 version 1 algorithm, but there. Dickinson, Sparks, MD, USA) is an E6/E7-based qPCR,. are significant differences. The INNO-LiPA uses. enabling detection of 14 hrHPVs in three separate. different SPF10 PCR primers and reverse hybridization. reactions. Concurrent genotyping is offered for six. probes than LiPA25, does not have a separate DEIA for. types (i.e., HPV16, 18, 31, 45, 51 and 52), while the. detection of a broad-spectrum of HPV types, and offers. remaining hrHPVs are detected as three separate. a concurrent internal control (HLA-DPB1 gene). The. groups. and. intended use of INNO-LiPA is currently unclear.. HV35/39/68). The test also detects human beta-globin. INNO-LIPA has been used in a clinical evaluation (101),. as an endogenous internal control. The Onclarity has. but has not (yet) been fully clinically validated in a. been clinically validated according to the international. randomized controlled trial or by non-inferiority to a. guidelines (100).. reference test according to defined guidelines (73). In. Signal amplification. addition, it has been used in epidemiologic studies. The Cervista HPV 16/18 Test (Cervista HPV16/18;. (102-104), but the analytical sensitivity of INNO-LiPA. Hologic) is a reflex test for women that tested positive. remains to be investigated.. (i.e.,. HPV33/58,. HPV56/59/66,. by the Cervista HPV HR Test. The Cervista HPV16/18. The LINEAR ARRAY HPV Genotyping Test (LA;. can determine presence of HPV16 and/or 18 using the. Roche Diagnostics) is a reverse hybridization strip with. same Invader chemistry as the Cervista.. immobilized probes for the recognition of 37 anogenital. 1.4.3. HPV (sub)types and an internal control target (beta-. HPV DNA full genotyping tests using broad--spectrum PCR broad. globin) (105). Degenerated PGMY primers are used for. HPV tests with a full genotyping capability for a wide. broad-spectrum amplification of a viral DNA fragment. range of HPV genotypes have great value in estimating. of 450 bp in the L1 ORF (105). LA is intended for the. the epidemiological burden of HPV infections and the. qualitative in vitro test for detection of HPV in clinical. 24. 209130-L-bw-Geraets.

(27) General introduction and thesis outline. specimens. LA has been applied in epidemiological. of human DNA and a control-template is present in the. studies (32, 106), but has not been clinically validated.. PCR master mix to rule out inhibition of amplification.. The Reverse Line Blot (RLB) is an in-house assay. Fluorescently labeled amplification products hybridize. for the identification of 37 HPVs using the same. with specific DNA probes fixed on a DNA chip and are. GP5+/6+ amplification products as the EIA (89, 107).. measured. PapilloCheck has been clinically validated for. Oligonucleotide probes are attached to a nylon. simultaneous detection of 14 hrHPVs according to the. membrane in parallel lines, and PCR products are. requirements for clinical sensitivity and specificity (109).. hybridized perpendicularly using a miniblotter. Hybrids. 1.4.4. RLB has been used in a case-control study by WHO. HPV DNA full genotyping tests usi using ng typetypespecific (q)PCR TypeType-specific PCR amplification. IARC to define the risk-classification of individual. Separate type-specific (TS) PCRs for HPV16 and 18 have. genotypes (24), but also in a screening setting, to. been developed (71) and incorporated into the HPV. identify the respective HPV type(s) in women testing. testing algorithm that was used in the two largest. positive for hrHPV by EIA (108).. efficacy trials of the bivalent vaccine, PATRICIA and. are visualized by a conjugate-substrate reaction. The. Two commercially available alternative assays for. CVT. These assays utilize TS primers targeting short. the genotyping of GP5+/6+ amplimers have been. regions in the E6/E7 ORF (HPV16; 92 bp) and in the L1. recently developed, using the same probes as the RLB. ORF (HPV18; 126 bp) for endpoint PCR amplification,. with minor modifications. The Genotyping Kit HPV GP,. followed by read-out using DEIA technology, as. version 1 (GP5+/6+ strip, Diassay, Rijswijk, The. described before (71). The combination of the L1-based. Netherlands; previously marketed as digene HPV. SPF10 broad-spectrum PCR algorithm followed by TS. Genotyping RH Test) is a strip-based reverse. HPV16/18 PCRs provided a higher analytical accuracy. hybridization assay for identification of 14 hrHPVs and. than both assays alone (71).. 4 probably hrHPVs. This assay was designed as an easy-. Recently, two novel assays for multiplex type-. to-use alternative for RLB and could be used for reflex. specific (MPTS) amplification of regions in E6, were. genotyping following hrHPV positivity by HC2 or EIA.. introduced, i.e., MPTS12 and MPTS123 (Labo Bio-. The LMNX Genotyping Kit HPV GP (GP5+/6+ LMNX,. medical Products). In MPTS12 amplification is. Diassay;. HPV. performed in two separate TS PCRs, and pooled. Genotyping LQ Test) provides identification of the. amplification products are genotyped by strip-based. same HPVs, but read-out is performed using bead-. reverse hybridization targeting 9 hrHPVs, i.e., HPV16,. based xMAP technology on a Luminex platform. This. 18, 31, 33, 35, 45, 52, 58, and 59 (110). MPTS123. platform is more suitable for high-throughput testing,. requires performance and pooling of three separate. enabling the GP5+/6+ LMNX to be used as a reflex. PCRs, and utilizes bead-based xMAP technology for. genotyping test but also as a stand-alone test for hrHPV. identification of 14 hrHPVs and 2 lrHPVs (HPV6 and. detection and concurrent genotyping, if desired.. 11) on a Luminex platform (72). These assays were. previously. marketed. as. digene. The PapilloCheck HPV-Screening (PapilloCheck;. designed to be used in conjunction with the SPF10. Greiner Bio-One GmbH, Frickenhausen, Germany) is a. LiPA25 system and improved the analytical accuracy for. PCR-microarray and allows identification of 24 types,. a broader range of HPVs, similar to the TS16/18 assays.. including 14 hrHPVs. Broad-spectrum primers amplify. This is particularly important when investigating the. a 350 bp region from the E1 ORF. A region in the. cross-protection of the bivalent vaccine against other. human ADAT1 gene is amplified to confirm presence. types than HPV16/18 (86).. 25. 209130-L-bw-Geraets.

(28) Chapter 1. TypeType-specific quantita quantitati uantitative tive PCR amplification. and probe hybridization protection for detection of. TS HPV qPCR assays were internally developed at. E6/E7 mRNA expression (119, 120). Aptima met the. Merck Research Laboratories (MRL) and used for. cross-sectional clinical and reproducibility criteria of. efficacy determination in the Phase III trials of the. the international guidelines for HPV test requirements. quadrivalent vaccine, e.g., the FUTURE I and II (111,. for cervical screening (121). It was noted that these. 112). Merck TS HPV qPCR assays are a set of type-. requirements for cross-sectional equivalence were. specific PCRs for amplification of multiple sequences. formulated for HPV DNA tests and may not necessarily. simultaneously, i.e., in the L1, E6, and E7 ORFs (of. be valid for other molecular markers, e.g., E6/E7 mRNA.. HPV6, 11, 16, 18, 31, 45, 52, and 58) or in the E6 and E7. Longitudinal data are needed to ensure that the long-. ORFs (of HPV33, 35, 39, 51, 56, and 59) (113-115). The. term negative predictive value of this mRNA assay is. triplex or duplex qPCR is performed for each HPV type. similar to those of validated HPV DNA tests and allows. individually. These assays appeared to have a higher. for the same screening intervals (121).. analytical. Genotyping Extra in a direct analytical comparison,. 1.4.6 HPV16 intratypic intratypic variant analysis Sanger sequence analysis of parts of the HPV16 genome,. with a limit of detection (LOD) that was below 50. e.g., E6, L1 and the long control region (LCR) (17, 122). copies/test for all HPVs targeted (114).. was used to identify different HPV16 variants, which. sensitivity. than. INNO-LiPA. HPV. An in-house method comprising a set of E6/E7-. were associated with human population migrations and. based type-specific qPCRs for 13 hrHPVs and 4. continent of origin (18) (Table 1). More recently, a. additional HPVs was developed at the RIATOL. novel HPV16 variant reverse hybridization assay (RHA). laboratory in Antwerpen, Belgium. A separate beta-. was developed and evaluated for simple and accurate. globin qPCR is performed as an endogenous internal. recognition of these HPV16 variant lineages (123). This. control. density. assay uses E6-based PCR amplification of a large, single. sedimentation method has been implemented in the. region (570 bp) for clinical specimens of sufficient. processing of clinical specimens (116). The clinical. quality, or uses primer sets that generate four smaller,. validation of the RIATOL qPCRs was reported by. overlapping regions for samples in which DNA is. Depuydt et al (117).. poorly preserved. The generated amplification products. 1.4.5 HPV mRNA detection tests The detection of hrHPV mRNA encoding viral. encompass. oncoproteins E6 and E7 instead of DNA might allow. oligonucleotide probes in a strip-based RHA. This assay. better distinction between productive (low expression. can differentiate between the four main lineages A. of E6/E7) and transforming (high expression of E6/E7). (European&Asian), B (African 1), C (African 2) and D. infections (118). Overexpression of E6 and E7 is. (North-American&Asian-American),. required for malignant transformation in HPV-related. resolution to distinguish some sublineages, i.e., A1&A2. cancers.. (European) from A3 (Asian) within the main lineage A,. to assess. specimen. quality.. A. The Aptima HPV assay (Aptima; Hologic GenProbe, San Diego, CA, USA) is an HPV assay designed for pooled detection of E6/E7 mRNA from 14 hrHPVs. Aptima is based on target capture after cell lysis, with subsequent transcription-mediated amplification (TMA). 26. 209130-L-bw-Geraets. variant-specific. polymorphisms. (SNPs). that. single are. nucleotide targeted. and. has. by. the. and D1 (North-American) from D2&D3 (AsianAmerican) within the main lineage D. The characteristics of HPV tests described in this chapter are summarized in Table 4..

(29) 209130-L-bw-Geraets HPV16&18. Abbott 14 hrHPVs RealTime HR HPV HPV-Risk assay 15 hrHPVs. GP5+/6+-EIA. Riatol (inhouse) PapilloCheck. None. HPV16&18&3 Three separate BS1&45&51&52e qPCRs 13 hrHPVs and Full TS-qPCRs for each 4 other HPVs type separately 14 hrHPVs and Full BS-PCR and probe 10 other HPVs hybridization 14 hrHPVs None BS-PCR and probe hybridization. 14 hrHPVs. BS-qPCR. HPV16&18. 14 hrHPVs. Cobas. Onclarity. BS-qPCR. HPV16&18. 14 hrHPVs. Aptima. Signal amplification by invader chemistry Transcriptionmediated amplification and probe hybridization BS-qPCR. Nonec. 14 hrHPVs. Cervista. Signal amplification by hybrid capture. None. 13 hrHPVs. HC2. Technology. HPVs detecteda Concurrent genotyping. HPV test. used (if applicable).. Yesf/no. DNA, E6/E7d. DNA, L1, ~150 bp Yesf/no. DNA, E1, ~350 bp Yes/yes. Yes/no. DNA, E6/E7d. DNA, E7, ~150 bp Yes/no. Screening Triage after cytology Test-of-cure Not specified. Clinically validated Not specified. Clinically validated Not specified. Clinically validated Screening Triage after cytology Test-of-cure. Internal control: Ma Main in indicated use endogenous/ exogenous DNA No/n.a. Screening Triage after cytology Test-of-cure DNA Yes/n.a. Screening (with cytology) Triage after cytology mRNA, E6/E7d No/yes Screening (with cytology, among >30 yrs) Triage after cytology (among >21yrs) DNA, L1, ~200 bp Yes/no Screening (with cytology) Triage after cytology DNA, L1, ~150 bp Yes/no Not specified. Clinically validated. Target. POBASCAM SWEDESCREEN. Hesselink, 2010 (109). Depuydt, 2012 (117). Ejegod, 2013 (100). ATHENA Heideman, 2011 (95) Carozzi, 2011 (96) Poljak, 2011 (97) Hesselink, 2013 (98) Hesselink, 2014 (99). 10, 11. -. -. -. -. -. -. Supporting trials and/or This thesis, studies b chapter: NTCC 3, 4 ARTISTIC VUSASCREEN Belinson, 2011 (76) Boers, 2014 (77) Wu, 2010 (119) Monsonego, 2012 (120) Heideman, 2013 (121). and internal control), main indicated use (based on kit manual or literature), supporting trials and/or studies, and the chapters of this thesis in which the assay was. Table 4: Brief overview of a selection of HPV tests, summarizing their technical characteristics (HPVs targeted, concurrent genotyping capability, technology, target,. General introduction and thesis outline. 27.

(30) 28. 209130-L-bw-Geraets. 14 hrHPVs and Full 4 other HPVs 14 hrHPVs and Full 23 other HPVs. 13 hrHPVs. 14 hrHPVs and None at least 55 other HPVs. 14 hrHPVs and Full 11 other HPVs. 14 hrHPVs and Full 14 other HPVs. 13 hrHPVs and Full two other HPVs. GP5+/6+ strip (v1) (RH Test)h PGMY Linear Array. Amplicor. SFP10 DEIA (version 1). SPF10 LiPA25 (version 1). SPF10 INNOLiPA extra. Merck TS qPCRs. None. BS-PCR and probe hybridization. 14 hrHPVs and Full 23 other HPVs. TS-qPCRs for each type separately. BS-PCR and probe hybridization. BS-PCR and probe hybridization. BS-PCR and probe hybridization. BS-PCR and probe hybridization. BS-PCR and probe hybridization BS-PCR and probe hybridization. BS-PCR and probe hybridization. 14 hrHPVs and Full 4 other HPVs. GP5+/6+ LMNX (LQ Test)g GP5+/6+ RLB (in-house). Technology. HPVs detecteda Concurrent genotyping. HPV test. Yes/no. No/no. No/no. DNA, L1&E6&E7 Yesf/no or E6&E7d,i. DNA, L1, 65 bp. DNA, L1, 65 bp. DNA, L1, 65 bp. Coutlee, 2006 (105) Wheeler, 2014 (32) Trabizi, 2014 (106). -. 2. 2, 6, 7, 8, 9, 11. 2, 6, 7, 8, 9, 11. 5. -. Van den Brule, 2002 (107) 3, 4 Munoz, 2003 (24) Rijkaart, 2012 (108) 3, 5. Supporting trials and/or This thesis, studies b chapter: 4, 5, 10, 11. Carozzi, 2007 (92) Mo, 2008 (93) Wentzensen, 2009 (91) Vaccine trials, surveillance Kleter, 1998 (81) and epidemiology PATRICIA CVT Tjalma, 2012 (66) Vaccine trials, surveillance Kleter, 1999 (82) and epidemiology PATRICIA CVT Tjalma, 2012 (66) Not specified. Used in Barzon, 2012 (101) epidemiological studies. Not Dartell, 2013 (102) clinically validated Colon-Lopez, 2014 (103) Kjaer, 2014 (104) Vaccine trials Else, 2011 (114) FUTURE I FUTURE II. Internal control: Ma Main in indicated use endogenous/ exogenous DNA, L1, ~150 bp Yes/no Screening Triage after cytology Test-of-cure DNA, L1, ~150 bp No/no Epidemiology and reflex genotyping after hrHPV positivity by EIA DNA, L1, ~150 bp No/no Reflex genotyping after hrHPV positivity by EIA DNA, L1, ~450 bp Yes/no Not specified. Used in epidemiological and surveillance studies. Not clinically validated DNA, L1, ~165 bp Yesf/no Not specified. Not clinically validated (replaced by Cobas). Target. Chapter 1.

(31) 209130-L-bw-Geraets. HPV16 and 18 Full. HPV16, 18, 31, Full 33, 35, 45, 52, 58, and 59 14 hrHPVs and Full 2 other HPVs. TS16/18. MPTS12. Size of region(s) targeted by primers not specified in kit manual or literature. The LMNX Genotyping kit HPV GP (GP5+/6+ LMNX; Diassay BV) was previously marketed as the digene HPV Genotyping LQ Test (LQ Test, Qiagen).. In case of specimens with poorly preserved DNA, four smaller, overlapping fragments of 104, 109, 65, and 69 bp, respectively, can be amplified.. j. and 59.. A triplex qPCR targets sequences in L1, E6, and E7 for HPV6, 11, 16, 18, 31, 45, 52, and 58; A duplex qPCR targets sequences in E6 and E7 for HPV33, 35, 39, 51, 56,. The Genotyping kit HPV GP, version 1 (GP5+/6+ strip; Diassay BV) was previously marketed as the digene HPV Genotyping RH Test (RH Test, Qiagen).. h. g. Internal control analysis is performed in a separate assay in parallel with HPV analysis.. Onclarity detects additional hrHPVs as three groups, i.e., 33/58, 56/59/66, and 35/39/68.. e. d. i. 9. 2. Cervista offers no concurrent genotyping. Partial genotyping for HPV16 and 18 is possible through reflex testing using a separate assay (Cervista HPV 16/18 Test).. FUTURE I (111); FUTURE II (112).. f. Sanchez, 2011 (123). Van Alewijk, 2013 (72). Supporting trials and/or This thesis, studies b chapter: Van Doorn, 2006 (71) PATRICIA CVT Van Alewijk, 2013 (72) Skinner, 2014 (110). The clinical trials are: NTCC (61); ARTISTIC (60); VUSASCREEN (75); POBASCAM (56); SWEDESCREEN (62); ATHENA (94); PATRICIA (85); CVT (83);. b. hrHPVs refers to aforementioned types plus HPV66 and 67.. c. Target. Internal control: Ma Main in indicated use endogenous/ exogenous TS-PCRs and probe DNA, E6/E7, 92 bp No/no Vaccine trials hybridization and L1, 126 bp separately Two separate TSDNA, E6, 55-139 No/no Vaccine trials PCRs and one probe bp hybridization Three separate TS- DNA, E6, 55-139 No/no Vaccine trials PCRs and one probe bp hybridization TS-PCR and probe DNA, E6, 570 bpj No/no Natural history and hybridization epidemiological studies of HPV16 variant infections. Technology. Unless otherwise specified, 13 hrHPVs refers to types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68; 14 hrHPVs refers to aforementioned types plus HPV66; 15. a. HPV16 variant HPV16 main n.a. RHA variant lineages. MPTS123. HPVs detecteda Concurrent genotyping. HPV test. General introduction and thesis outline. 29.

(32) Chapter 1. 1.5 THESIS OUTLINE In this thesis we aimed to evaluate a number of. In contrast, the GP5+/6+ strip and GP5+/6+. established and novel PCR-based technologies for the. LMNX were evaluated against the Reverse Line Blot. identification. in. assay, an established in-house method for reflex. epidemiologic and clinical studies in accordance with. genotyping following the clinically validated GP5+/6+. of. HPVs,. and. apply. these. their intended use. These novel technologies comprise. PCR, in Chapter 3 and Chapter 4, 4 respectively.. additions and improvements to two established PCR-. Accurate identification of individual hrHPVs by these. based (hr)HPV test algorithms, which are current “gold. assays might be valuable for further risk stratification of. standards” for epidemiologic and for clinical purposes,. women that tested hr HPV positive in a clinical setting.. i.e., the SPF10 LiPA25 (version 1) (Figure 5) and the. In Chapter 5, 5 we investigated if the GP5+/6+ strip and. GP5+/6+ EIA (Figure 6), respectively.. GP5+/6+ LMNX can be used for direct genotyping of. This thesis is divided into three main parts: 1) the. PCR products generated by another hrHPV test, i.e.,. assessment of analytical accuracy of several recently. Amplicor, in addition to the GP5+/6+ amplicons.. developed assays for HPV genotyping (Chapters Chapters 22-5), 2). Part 2: Chapters 6 to 9 describe the applications of. the application of (novel) techniques for HPV. genotyping assays in a research setting, e.g., surveillance. characterization. of prevalence, epidemiology (disease association), and. (Chapters Chapters. 6-9),. in. several. and. 3). avenues the. of. utility. research of. novel. natural history of HPV infections. methodologies for hrHPV detection and self-sample. The SPF10 LiPA25 algorithm was used to determine the. collection within a clinical setting (Chapters Chapters 10 10--11). 11. prevalence of and determinants for hrHPV genotypes in. The findings of this thesis are discussed in the context. Paramaribo, Suriname, providing baseline data prior to. of other studies in the general discussion (Chapter Chapter 12). 12. implementation of an HPV vaccination program in. Part1: Chapters 2 to 5 are technical chapters. These focus. 2013. on evaluating the analytical accuracy of novel HPV. surveillance of HPV prevalence is important for short-. genotyping assays.. term evaluation of the effects of a local HPV. Recently, three novel tests for HPV genotyping have. vaccination program.. been introduced, i.e., INNO-LiPA HPV Genotyping. (Chapter Chapter. Chapter. 7. 6). 6. Pre-. describes. and. the. post-vaccination. HPV. genotype. Extra (INNO-LiPA), the GP5+/6+ strip (previously. distribution established by the SPF10 LiPA25 algorithm. marketed as digene HPV Genotyping RH Test) and the. in a worldwide collection of 10,575 cases of invasive. GP5+/6+ LMNX (previously marketed as digene HPV. cervical carcinoma (ICC). The estimated attribution of. Genotyping LQ Test). Each test was evaluated against. individual HPVs to ICC can provide insight into which. an established genotyping assay that is relevant for its. types should be given priority for assessment of cross-. intended use (‘gold standard’), i.e., analytical or clinical.. protective. The INNO-LiPA was compared to the original SPF10. development of second-generation polyvalent vaccines.. effects. of. current. vaccines. and. for. LiPA25 strip (version 1), the analytically sensitive gold. In the same collection of ICC, we investigated the. standard assay for epidemiologic studies and vaccine. presence of HPV types rarely targeted by current HPV. trials (Chapter Chapter 2). 2 Both assays were evaluated on a. genotyping tests, using a novel sequence methodology. selected panel of cervical swabs and biopsies, the two. (Chapter Chapter 8). 8 The contribution of rare HPVs classified. types of specimens mostly used in epidemiologic HPV. as possibly high-risk (Class 2B) due to their. research.. phylogenetic relation with established hrHPVs (Class 1/2A), is of particular interest. Class 2B HPVs are. 30. 209130-L-bw-Geraets.

(33) General introduction and thesis outline. currently not targets of hrHPV tests intended for. Chapter 10 describes a clinical evaluation of the. cervical cancer screening, but their occurrence as single. GP5+/6+ LMNX using the GP5+/6+ EIA as a clinically. infections in ICC could strengthen the circumstantial. validated comparator hrHPV test. The utilized sample. evidence of a carcinogenic role.. panel was collected from a cervical cancer screening. In Chapter 99, we performed a longitudinal study. setting by an international consortium (VALGENT). towards the natural history of infections with different. and provides a cross-sectional clinical equivalence. variant lineages of HPV16, the most carcinogenic HPV. comparison. A non-inferiority analysis of sensitivity. type. European, African, Asian, North American and. and specificity for detecting women with CIN2+ and. Asian-American HPV16 variants were studied in. CIN3+ allows clinical validation of the GP5+/6+ LMNX. cervical swabs, whole-tissue sections and laser-capture. for screening purposes.. micro-dissected regions from biopsies, using a recently. HrHPV analysis performed on cervicovaginal self-. developed HPV16 variant reverse hybridization assay.. collected specimens seems a suitable alternative for. We aimed to determine the prevalence of the different. liquid-based cervical specimens collected by a physician,. variant lineages, and the dynamics of these infections. particularly for non-responders in cervical cancer. over time.. screening and in low-resource settings. The FTA solid-. Part 3: Chapters 10 and 11 describe the applications of. state carrier cartridge is a novel self-sampling device. genotyping assays in a clinical setting, e.g., for primary. and was compared to physician-collected specimens. cervical cancer screening and combined with a self-. using two different HPV assays, i.e., GP5+/6+ LMNX. sampling device.. 11. and the SPF10 LiPA25 algorithm, in Chapter 11. 31. 209130-L-bw-Geraets.

(34) Chapter 1. Processed clinical specimen. INNO SPF10 PCR. SPF10 PCR (version 1). DEIA (version 1). Chapter 2: Alternative SPF10-based HPV genotyping algorithm. Chapter 7: Global attribution of HPVs to cervical cancer. HPV positive. INNO-LiPA. Chapter 6: Prevaccination surveillance of HPVs in Suriname. LiPA25 (version 1). HPV16 positive. HPV not typed. HPV16 variant RHA. Novel sequence methodology. Chapter 9: Natural history of HPV16 variant infections. Chapter 8: Detection of rare, possibly hrHPVs in cervical cancers. Figure 5: Schematic overview of the SPF10 LiPA25 (version 1) test algorithm that is currently used as the gold standard for HPV detection and genotyping in a research setting. Additional or alternative technologies for HPV characterization that have been recently developed are also shown. The different technologies were evaluated and/or applied in the indicated chapters of this thesis.. 32. 209130-L-bw-Geraets.

(35) General introduction and thesis outline. Processed clinical specimen. GP5+/6+ PCR. Chapter 10: Alternative GP5+/6+-based hrHPV detection read-out. LMNX (LQ Test). EIA. hrHPV positive. Chapter 11: Alternative self-sampling method using FTA solid carrier. Amplicor PCR. Amplicor. hrHPV positive. Reverse Line Blot. LMNX (LQ Test). Strip (RH Test). Chapter 5: Direct Amplicor-based hrHPV genotyping read-out. Chapter 4: Alternative Chapter 3: Alternative GP5+/6+-based hrHPV GP5+/6+-based hrHPV genotyping read-out genotyping read-out. Figure 6: Schematic overview of the GP5+/6+ EIA test algorithm that is currently regarded as one of the clinically validated reference assays for hrHPV detection in a clinical setting. Alternative technologies for hrHPV detection and genotyping as well as a novel method for sample collection are also shown. These novel technologies were evaluated in the indicated chapters of this thesis.. 33. 209130-L-bw-Geraets.

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