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• W2087093385 abstract "Oncogenic human papillomavirus (HPV) infection is the most important risk factor in cervical carcinogenesis cases; high viral loads, viral integration into the host genome, and gain of the telomerase-related genes, TERT and TERC, are all factors associated with progression to cancer. A recently developed multiparameter HPV 16/18 multiplex ligation-dependent probe amplification (MLPA) assay, which allows the simultaneous assessment of these factors, was applied to a series of 67 normal and (pre)malignant frozen uterine cervical samples, as well as to 91 cytological preparations, to test the ability of the MLPA assay to identify high-risk lesions on the basis of these factors. Validation was performed using quantitative PCR, the PapilloCheck and fluorescence in situ hybridization. Only 5 out of 37 normal tissue samples or low-grade cervical lesions (ie, CIN1 and condyloma) showed either an HPV16 viral load higher than 25 copies per cell, viral integration, and/or gain of one of the telomerase-related genes, whereas for the high-grade cervical lesions, one or more of these risk factors was found in 25 of 30 cases. The HPV MLPA assay showed a sensitivity of 83% and a specificity of 86% in frozen cervical specimens. Furthermore, the feasibility of the MLPA assay was shown for cytological samples, where in 57% of high-grade squamous intraepithelial lesion cases, the high-risk factors were detected using this assay. Oncogenic human papillomavirus (HPV) infection is the most important risk factor in cervical carcinogenesis cases; high viral loads, viral integration into the host genome, and gain of the telomerase-related genes, TERT and TERC, are all factors associated with progression to cancer. A recently developed multiparameter HPV 16/18 multiplex ligation-dependent probe amplification (MLPA) assay, which allows the simultaneous assessment of these factors, was applied to a series of 67 normal and (pre)malignant frozen uterine cervical samples, as well as to 91 cytological preparations, to test the ability of the MLPA assay to identify high-risk lesions on the basis of these factors. Validation was performed using quantitative PCR, the PapilloCheck and fluorescence in situ hybridization. Only 5 out of 37 normal tissue samples or low-grade cervical lesions (ie, CIN1 and condyloma) showed either an HPV16 viral load higher than 25 copies per cell, viral integration, and/or gain of one of the telomerase-related genes, whereas for the high-grade cervical lesions, one or more of these risk factors was found in 25 of 30 cases. The HPV MLPA assay showed a sensitivity of 83% and a specificity of 86% in frozen cervical specimens. Furthermore, the feasibility of the MLPA assay was shown for cytological samples, where in 57% of high-grade squamous intraepithelial lesion cases, the high-risk factors were detected using this assay. Currently preventive screening for (pre)malignant lesions of the uterine cervix is based on the analysis of Papanicolaou stained cytological samples, combined with identification of high risk human papillomavirus (HPV) types.1Jordan J Arbyn M Martin-Hirsch P Schenck U Baldauf JJ Da Silva D Anttila A Nieminen P Prendiville W European guidelines for quality assurance in cervical cancer screening: recommendations for clinical management of abnormal cervical cytology, part 1.Cytopathology. 2008; 19: 342-354Crossref PubMed Scopus (143) Google Scholar, 2Spitzer M Apgar BS Brotzman GL Management of histologic abnormalities of the cervix.Am Fam Physician. 2006; 73: 105-112PubMed Google Scholar Although the specificity of cytology in the detection of high grade lesions is high, its sensitivity can be as low as 50%.3ACOG Practice Bulletin No. 99: management of abnormal cervical cytology and histology.Obstet Gynecol. 2008; 112: 1419-1444Crossref PubMed Scopus (85) Google Scholar, 4Cuzick J Clavel C Petry KU Meijer CJ Hoyer H Ratnam S Szarewski A Birembaut P Kulasingam S Sasieni P Iftner T Overview of the European and North American studies on HPV testing in primary cervical cancer screening.Int J Cancer. 2006; 119: 1095-1101Crossref PubMed Scopus (862) Google Scholar On the contrary, the sensitivity for HPV testing is very high, while its specificity is low.5Rao A Pather S Dalrymple C Mackie A Deans R Carter J The role of HPV testing in patients with possible high-grade cervical cytology.J Obstet Gynaecol Res. 2009; 35: 503-506Crossref PubMed Scopus (5) Google Scholar Obviously, there is a need for a screening approach that is both highly specific and highly sensitive. We recently developed an HPV multiplex ligation-dependent probe amplification (MLPA)-assay6Theelen W Reijans M Simons G Ramaekers FCS Speel EJM Hopman AHN A new multiparameter assay to assess HPV 16/18 viral load and physical status together with gain of telomerase genes in HPV-related cancers.Int J Cancer. 2010; 126: 959-975PubMed Google Scholar which not only identifies HPV16 and 18, but simultaneously assesses viral load and viral integration for these HPV types, as well as gain of the telomerase genes, since these markers were described to be associated with progression to cancer (see below). In our previous study the MLPA-assay was used to determine these parameters in cervical cancer cell lines and a very small subset of clinically derived tissue.6Theelen W Reijans M Simons G Ramaekers FCS Speel EJM Hopman AHN A new multiparameter assay to assess HPV 16/18 viral load and physical status together with gain of telomerase genes in HPV-related cancers.Int J Cancer. 2010; 126: 959-975PubMed Google Scholar Here, the study has been expanded to 67 fresh frozen samples of normal cervix and different stages of CIN, as well as, 91 cytological samples. MLPA is a molecular technique initially developed by Schouten et al7Schouten JP McElgunn CJ Waaijer R Zwijnenburg D Diepvens F Pals G Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification.Nucleic Acids Res. 2002; 30: e57Crossref PubMed Scopus (2092) Google Scholar for quantification of up to 40 genomic targets. For each target a pair of probes is designed. Each probe contains a universal PCR primer sequence and a sequence complementary to the target. When the probes hybridize immediately adjacent to each other they can be ligated and subsequently amplified using universal primers. Because one of the primers is labeled with a fluorescent dye the amplified products can be visualized using capillary electrophoresis. Furthermore, the products can be discerned based on length because of the variable stuffer sequences.8Shen Y Wu BL Designing a simple multiplex ligation-dependent probe amplification (MLPA) assay for rapid detection of copy number variants in the genome.J Genet Genomics. 2009; 36: 257-265Crossref PubMed Scopus (38) Google Scholar We modified the assay as such that a simultaneous quantification of both human and viral targets is possible. Of the 15 to 18 oncogenic HPV-types described9Munoz N Bosch FX de Sanjose S Herrero R Castellsague X Shah KV Snijders PJ Meijer CJ Epidemiologic classification of human papillomavirus types associated with cervical cancer.N Engl J Med. 2003; 348: 518-527Crossref PubMed Scopus (4934) Google Scholar HPV16 and 18 account for 70% of the cervical cancer cases.10Munoz N Castellsague X de Gonzalez AB Gissmann L Chapter 1: HPV in the etiology of human cancer.Vaccine. 2006; 24: 1-10Crossref PubMed Scopus (1083) Google Scholar In addition, it has been described that the viral load for HPV16 increases during cervical carcinogenesis,11Saunier M Monnier-Benoit S Mauny F Dalstein V Briolat J Riethmuller D Kantelip B Schwarz E Mougin C Pretet JL Analysis of human papillomavirus type 16 (HPV16) DNA load and physical state for identification of HPV16-infected women with high-grade lesions or cervical carcinoma.J Clin Microbiol. 2008; 46: 3678-3685Crossref PubMed Scopus (88) Google Scholar while also viral integration into the host genome is described to be associated with progression to cancer.12Klaes R Woerner SM Ridder R Wentzensen N Duerst M Schneider A Lotz B Melsheimer P von Knebel Doeberitz M Detection of high-risk cervical intraepithelial neoplasia and cervical cancer by amplification of transcripts derived from integrated papillomavirus oncogenes.Cancer Res. 1999; 59: 6132-6136PubMed Google Scholar For HPV18 a high viral load is not related to cervical malignancy,13Cheung JL Cheung TH Ng CW Yu MY Wong MC Siu SS Yim SF Chan PK Analysis of human papillomavirus type 18 load and integration status from low-grade cervical lesion to invasive cervical cancer.J Clin Microbiol. 2009; 47: 287-293Crossref PubMed Scopus (29) Google Scholar, 14Hesselink AT Berkhof J Heideman DA Bulkmans NW van Tellingen JE Meijer CJ Snijders PJ High-risk human papillomavirus DNA load in a population-based cervical screening cohort in relation to the detection of high-grade cervical intraepithelial neoplasia and cervical cancer.Int J Cancer. 2009; 124: 381-386Crossref PubMed Scopus (56) Google Scholar, 15Xi LF Koutsky LA Castle PE Wheeler CM Galloway DA Mao C Ho J Kiviat NB Human papillomavirus type 18 DNA load and 2-year cumulative diagnoses of cervical intraepithelial neoplasia grades 2–3.J Natl Cancer Inst. 2009; 101: 153-161Crossref PubMed Scopus (29) Google Scholar but integration into the host genome is seen more often for this HPV type.16Pirami L Giache V Becciolini A Analysis of HPV16, 18, 31, and 35 DNA in pre-invasive and invasive lesions of the uterine cervix.J Clin Pathol. 1997; 50: 600-604Crossref PubMed Scopus (83) Google Scholar, 17Vinokurova S Wentzensen N Kraus I Klaes R Driesch C Melsheimer P Kisseljov F Durst M Schneider A von Knebel Doeberitz M Type-dependent integration frequency of human papillomavirus genomes in cervical lesions.Cancer Res. 2008; 68: 307-313Crossref PubMed Scopus (275) Google Scholar The telomerase genes are included in our MLPA-assay because gain of chromosome 3q, with telomerase RNA component (TERC) as one of the suggested candidate genes18Heselmeyer K Schrock E du Manoir S Blegen H Shah K Steinbeck R Auer G Ried T Gain of chromosome 3q defines the transition from severe dysplasia to invasive carcinoma of the uterine cervix.Proc Natl Acad Sci USA. 1996; 93: 479-484Crossref PubMed Scopus (425) Google Scholar, 19Hopman AHN Theelen W Hommelberg PP Kamps MA Herrington CS Morrison LE Speel EJM Smedts F Ramaekers FCS Genomic integration of oncogenic HPV and gain of the human telomerase gene TERC at 3q26 are strongly associated events in the progression of uterine cervical dysplasia to invasive cancer.J Pathol. 2006; 210: 412-419Crossref PubMed Scopus (111) Google Scholar is frequently identified during carcinogenesis of the cervix. Furthermore, gain of chromosome 5p, containing the telomerase reverse transcriptase (TERT) gene, is also reported to be associated with progression to cancer.20Huang FY Kwok YK Lau ET Tang MH Ng TY Ngan HY Genetic abnormalities and HPV status in cervical and vulvar squamous cell carcinomas.Cancer Genet Cytogenet. 2005; 157: 42-48Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 21Matthews CP Shera KA McDougall JK Genomic changes and HPV type in cervical carcinoma.Proc Soc Exp Biol Med. 2000; 223: 316-321Crossref PubMed Scopus (52) Google Scholar Although all these viral and genomic markers are predominantly detected in high grade lesions they do not identify all these lesions, when assessed separately.11Saunier M Monnier-Benoit S Mauny F Dalstein V Briolat J Riethmuller D Kantelip B Schwarz E Mougin C Pretet JL Analysis of human papillomavirus type 16 (HPV16) DNA load and physical state for identification of HPV16-infected women with high-grade lesions or cervical carcinoma.J Clin Microbiol. 2008; 46: 3678-3685Crossref PubMed Scopus (88) Google Scholar, 19Hopman AHN Theelen W Hommelberg PP Kamps MA Herrington CS Morrison LE Speel EJM Smedts F Ramaekers FCS Genomic integration of oncogenic HPV and gain of the human telomerase gene TERC at 3q26 are strongly associated events in the progression of uterine cervical dysplasia to invasive cancer.J Pathol. 2006; 210: 412-419Crossref PubMed Scopus (111) Google Scholar, 20Huang FY Kwok YK Lau ET Tang MH Ng TY Ngan HY Genetic abnormalities and HPV status in cervical and vulvar squamous cell carcinomas.Cancer Genet Cytogenet. 2005; 157: 42-48Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar As a result the specificity of these individual markers is very high but their sensitivity is low. Through the combination of these markers in the HPV 16/18 MLPA-assay we show that the sensitivity is considerably increased, while the specificity of the assay remains high when applied to frozen cervical specimens. Also we demonstrate the feasibility of the test for cytological preparations to obtain a risk classification based on these MLPA parameters with increasing cytological grading. Sixty-seven frozen cervical specimens including 7 normal ectocervical tissues, 20 normal ectocervical epithelia adjacent to (pre)neoplastic lesions, 10 CIN1 lesions/condylomas, 6 CIN2 lesions, 7 CIN3 lesions, and 17 squamous cell carcinomas were obtained from the Tissue Bank of the University of Liège (Belgium). The samples contained between 30% and 95% (pre)malignant cells. DNA was extracted from each sample by using the NucleoSpin Tissue kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's instructions. The project protocol was approved by the Medical Ethics Committee of the University Hospital of Liège. DNA was also isolated from a series of 84 liquid based cytological samples (Surepath, BD, Franklin Lakes, HJ) from the Department of Pathology (Maastricht University Medical Centre, The Netherlands) which were preselected based on aberrations detected on microscopic inspection. These were also analyzed for HPV triage by GP5+/6+ testing.22van den Brule AJ Pol R Fransen-Daalmeijer N Schouls LM Meijer CJ Snijders PJ GP5+/6+ PCR followed by reverse line blot analysis enables rapid and high-throughput identification of human papillomavirus genotypes.J Clin Microbiol. 2002; 40: 779-787Crossref PubMed Scopus (468) Google Scholar This was also the case for 7 normal samples. DNA was previously isolated using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The MLPA-assay was performed as previously described6Theelen W Reijans M Simons G Ramaekers FCS Speel EJM Hopman AHN A new multiparameter assay to assess HPV 16/18 viral load and physical status together with gain of telomerase genes in HPV-related cancers.Int J Cancer. 2010; 126: 959-975PubMed Google Scholar and illustrated in Figure 1. A multiplex pre-amplification PCR was performed using the Qiagen Multiplex PCR kit. A 20 μl reaction mixture contained Qiagen Master Mix (3 mmol/L MgCl2, dNTPs, and HotstarTaq DNA polymerase), the previously described multiplex primer mix targeting the viral E2, E6, and E7 genes for HPV16 and 18, as well as the human β-globin, MSH2, TERT, and TERC genes6Theelen W Reijans M Simons G Ramaekers FCS Speel EJM Hopman AHN A new multiparameter assay to assess HPV 16/18 viral load and physical status together with gain of telomerase genes in HPV-related cancers.Int J Cancer. 2010; 126: 959-975PubMed Google Scholar (final concentration 20 nmol/L for each forward primer and 200 nmol/L for each reverse primer), and 10 ng of sample DNA. Amplification was performed in a Biometra T1 Thermocycler (Biometra, Göttingen, Germany) as follows: 15 minutes at 95°C, followed by 20 cycles of each 30 seconds at 94°C, 90 seconds at 55°C, and 90 seconds at 72°C, and a final extended elongation step for 10 minutes at 72°C. The pre-amplified product was diluted five times using sterile water, after which 2 μl was mixed with 1.5 μl MLPA-buffer (1.5 M KCl, 300 mmol/L Tris-HCl pH 8.5, 1 mmol/L EDTA; MRC-Holland, Amsterdam, the Netherlands), 1.5 μl of the previously described probe mix containing probes against the described viral and human targets6Theelen W Reijans M Simons G Ramaekers FCS Speel EJM Hopman AHN A new multiparameter assay to assess HPV 16/18 viral load and physical status together with gain of telomerase genes in HPV-related cancers.Int J Cancer. 2010; 126: 959-975PubMed Google Scholar (3 fmol of each synthetic probe oligonucleotide and 1.5 fmol of each M13-derived oligonucleotide in TE buffer) and 3 μl sterile water. After a 5 minutes denaturation step at 98°C in a Biometra T1 Thermocycler with a heated lid, the mixture was incubated for 16 hours at 60°C. For ligation this mixture was diluted to 40 μl with ligation buffer (2.6 mmol/L MgCl2, 5 mmol/L Tris-HCl pH 8.5, 0.013% non-ionic detergents, 0.2 mmol/L nicotinamide adenine dinucleotide) containing 1U heat-stable Ligase-65 enzyme (MRC-Holland, Amsterdam, the Netherlands) and incubated at 54°C for 15 minutes, followed by ligase inactivation at 98°C for 5 minutes. Four μl of this mixture was added to 16 μl of PCR mixture containing dNTPs (2 mmol/L each, Fermentas, St. Leon-Rot, Germany), 1 U Taq-polymerase (MRC-Holland, Amsterdam, the Netherlands), 1× PCR buffer (50 mmol/L KCl, 10 mmol/L Tris-HCl pH 8.5, 1.6 mmol/L MgCl2) and 4 pmol of the two MLPA-PCR primers each, with the forward primer 5′-GTGGCAGGGCGCTACGAACAA-3′ labeled with carboxyfluorescein, and the reverse primer 5′-GGACGCGCCAGCAAGATCCAATCTAGA-3′. Amplification was performed on a Biometra T1 Thermocycler as follows: an initial cycle of 2 minutes at 95°C, followed by 33 cycles of 30 seconds at 94°C, 30 seconds at 60°C, and 1 minute at 72°C, and a final extended elongation step for 10 minutes at 72°C. MLPA buffers and enzymes were obtained from MRC-Holland (Amsterdam, the Netherlands). Amplified carboxyfluorescein-labeled MLPA products were analyzed by electrophoresis on an ABI3730 genetic analyzer (Applied Biosystems, Foster City, CA). One μl of 20× diluted amplified MLPA products was mixed with 8.5 μl of deionized formamide and 0.5 μl of GeneScan-600 LIZ size standard (Applied Biosystems, Foster City, CA) and run in GeneScan mode. All analyses were done according to the manufacturers' instructions. Electropherograms were analyzed by GeneMarker software (Softgenetics, State College, PA), peak height data were exported to Excel files for calculation of ratios and analyzed according to the flow-chart described in Figure 2. HPV positivity was determined based on the presence of both E6 and E7 peaks (Figure 2, step A). E6 and E7 viral loads were estimated by determining the ratio between E6 or E7 and the seven human targets, ie, β-globin (2×), MSH2, TERT (2×), and TERC (2×), and using this ratio in the following specific equations: L = 55.9r11.1, L = 12.5r11.3, L = 16.8r11.3, and L = 43.2r11.4 for 16E6, 16E7, 18E6, and 18E7, respectively (Figure 2, step B). With L being the viral load estimated as HPV copies per human gene copy and r1 being the ratio between target signal intensity and the total signal intensity of all human targets. The average load of E6 and E7 was multiplied by 2 to obtain the viral load in a diploid cell. All fresh frozen samples were analyzed in duplicate and when the viral load in a sample was determined to be less than 1 copy per cell the assay was repeated with a primer and probe mix containing only HPV targets, thus without primers and probes for the human targets, to confirm HPV positivity. When the viral load in a sample was more than 50 copies per cell the sample was diluted with normal human DNA to decrease the viral load and the diluted sample was analyzed again. Due to experimental variability a reference series was included in each experiment. This series consisted of 5 mixtures of plasmids and human DNA, mimicking HPV16 samples with viral loads of 2, 5, 10, 20, and 40 copies per cell of which 30% was integrated. In each experiment the reference series was run in duplicate and the subsequent 16E2.1/E6 or 16E2.2/E6 ratio was plotted against the viral load to determine the viral integration cut-off value for different viral loads. Patient samples with a ratio above the cut-off line were scored as episomal and samples with a ratio below the cut-off line were scored as mixed or integrated (Figure 2, step C). This results in a classification of episomal HPV when less than 30% of the virus in a sample shows integration, mixed (ie, episomal and integrated) HPV when between 30% and 95% of the virus shows integration, and integrated HPV when more than 95% of the virus shows integration. The specificity of the primer and probe mix for HPV16 and 18 was evaluated by tests on HPV16 or 18 plasmid model systems, cell lines containing HPV16 or 18, and clinical samples containing closely related HPV types, ie, HPV31, 33 and 45 (data not shown), as determined with the PapilloCheck (Greiner Bio-One GmbH, Frickenhausen, Germany). Similar to the fresh frozen tissue samples the cytological samples were analyzed in duplicate and reference samples were included in each experiment. For these experiments the reference samples were mixtures of plasmids and human DNA, mimicking an episomal HPV16 and HPV18 sample with a viral load of 2. To determine gain for TERT the ratio between the combined signal intensity of TERTa and TERTc on the one hand and the combined signal intensity of β-globin-a, β-globin-b, and MSH2 on the other hand was calculated. This signal intensity ratio was compared to the ratio as measured for these targets in the normal human DNA sample. When the signal intensity ratio was 1.5× higher than that of the reference sample, TERT was classified as gained. To determine gain for TERC the ratio between the combined signal intensity of TERCa and TERCb on the one hand and the combined signal intensity of β-globin-a, β-globin-b, and MSH2 on the other hand was calculated. This signal intensity ratio was also compared to the ratio as measured for these targets in the normal human DNA sample and classified as gain when it was 1.5× higher than that of the reference sample (Figure 2; step D). All samples were tested for the presence of HPV16, 18, 31, 33 and 45 using a previously described quantitative (qPCR) by Lindh et al23Lindh M Gorander S Andersson E Horal P Mattsby-Balzer I Ryd W Real-time taqman PCR targeting 14 human papilloma virus types.J Clin Virol. 2007; 40: 321-324Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar targeting sequences in the E6/E7 genes, and were scored positive when the threshold cycle (Ct) value was below 35 for HPV16, 18, 31, and 45 and below 30 for HPV33. Furthermore, for the majority of samples viral load and physical status for HPV16 were estimated by the qPCR described by Peitsaro et al24Peitsaro P Johansson B Syrjanen S Integrated human papillomavirus type 16 is frequently found in cervical cancer precursors as demonstrated by a novel quantitative real-time PCR technique.J Clin Microbiol. 2002; 40: 886-891Crossref PubMed Scopus (322) Google Scholar using primers and probes for HPV16 E6 and HPV16 E2 (Biolegio, Malden, The Netherlands) in a 20 μl PCR mixture containing dNTPs (2 mmol/L each, Fermentas, St. Leon-Rot, Germany), 1 U Taq-polymerase (MRC-Holland, Amsterdam, the Netherlands), 1× PCR buffer (50 mmol/L KCl, 10 mmol/L Tris-HCl pH 8.5, 1.6 mmol/L MgCl2), 3 pmol of each primer, 1 pmol of each probe, and 25 ng of sample DNA. The qPCR reactions were performed in a Rotor-Gene 6000 real-time system (Corbett Life Science, Sydney, Australia) as follows: 2 minutes at 95°C, followed by 50 cycles of 30 seconds at 94°C, 90 seconds at 55°C, and 90 seconds at 72°C, and a final extended elongation step for 10 minutes at 72°C. The standard curves were obtained by amplification of a 10-fold dilution series of 5 × 106 to 500 copies of a HPV16 plasmid.6Theelen W Reijans M Simons G Ramaekers FCS Speel EJM Hopman AHN A new multiparameter assay to assess HPV 16/18 viral load and physical status together with gain of telomerase genes in HPV-related cancers.Int J Cancer. 2010; 126: 959-975PubMed Google Scholar The threshold cycle values for both HPV16 E6 and E2 standard curves were plotted against the log of the copy number over the entire range of dilutions and revealed a linear relationship. These standard curves were used to estimate the average HPV copy number in the individual samples based on duplicate measurements. Viral loads were calculated based on the assumption that the samples have a diploid DNA content and a DNA mass of 7.8 pg per diploid cell.25Lee GM Thornthwaite JT Rasch EM Picogram per cell determination of DNA by flow cytofluorometry.Anal Biochem. 1984; 137: 221-226Crossref PubMed Scopus (72) Google Scholar The physical status of the virus was determined on basis of the copy number ratio between E2 and E6. Samples were classified as integrated when no fluorescence was detected for E2 and the ratio was 0, mixed for ratios between 0 and 0.5, and episomal for ratios above 0.5 as previously described.26Kulmala SM Shabalova IP Petrovitchev N Syrjanen KJ Gyllensten UB Johansson BC Syrjanen SM Type-specific persistence of high-risk human papillomavirus infections in the New Independent States of the former Soviet Union Cohort Study.Cancer Epidemiol Biomarkers Prev. 2007; 16: 17-22Crossref PubMed Scopus (41) Google Scholar FISH analysis on a small selection of 4-μm thick, fresh-frozen tissue sections samples was performed as previously described19Hopman AHN Theelen W Hommelberg PP Kamps MA Herrington CS Morrison LE Speel EJM Smedts F Ramaekers FCS Genomic integration of oncogenic HPV and gain of the human telomerase gene TERC at 3q26 are strongly associated events in the progression of uterine cervical dysplasia to invasive cancer.J Pathol. 2006; 210: 412-419Crossref PubMed Scopus (111) Google Scholar using digoxigenin-labeled probes for HPV16 or 18 (PanPath, Budel, The Netherlands) or a Vysis probe set (Vysis, Abbott Molecular, Des Plaines, IL) consisting of: a DNA probe for chromosome 3 centromere labeled with the fluorescent dye Spectrum Green (SG), a 3q26-specific BAC clone containing the TERC gene labeled with Spectrum Orange (SO), and a probe for chromosome 7 centromere labeled with Spectrum Aqua (SA). Images were acquired using a Leica DMRXA microscope (Leica, Wetzlar, Germany) equipped with custom optical filters for 4,6-diamidino-2-phenylindole, SA, SO, and SG (Chroma Technologies, Brattleboro, VT) with a ×40 Plan Apo (NA 1.20) objective. The microscope was connected to a digital black and white CCD camera (Metasystems Image Pro System, Sandhausen, Germany). To determine gain for the TERC target the copy number for chromosome 7 was used as a control for the ploidy of cells. For chromosome 3 and target 3q26 (TERC) the maximum number of signals per nucleus was determined and used as an indicator of copy number, as described previously.19Hopman AHN Theelen W Hommelberg PP Kamps MA Herrington CS Morrison LE Speel EJM Smedts F Ramaekers FCS Genomic integration of oncogenic HPV and gain of the human telomerase gene TERC at 3q26 are strongly associated events in the progression of uterine cervical dysplasia to invasive cancer.J Pathol. 2006; 210: 412-419Crossref PubMed Scopus (111) Google Scholar The PapilloCheck HPV-Screen DNA-chip (Greiner, Frickenhausen, Germany) was used for the qualitative detection and differentiation between 24 types of genital HPV (18 high-risk and 6 low-risk). HPV genotyping was performed as previously described by Jones et al27Jones J Powell NG Tristram A Fiander AN Hibbitts S Comparison of the PapilloCheck DNA micro-array Human Papillomavirus detection assay with Hybrid Capture II and PCR-enzyme immunoassay using the GP5/6+ primer set.J Clin Virol. 2009; 45: 100-104Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar Briefly, for each test at least 40 ng of DNA was used and a 350 bp fragment of the HPV E1 gene was amplified using a multiplex PCR with type-specific primers. An internal PCR control targeting a fragment of the human housekeeping gene ADAT1 was included in each run to avoid false negative results. PCR fragments were fluorescence-labeled with Cy5 and hybridized to specific probes on the PapilloCheck DNA chip. The amplification level was determined by the binding of PCR products to five control spots and their subsequent signal intensity on the chip. Following hybridization and subsequent washing steps, the chip was scanned at excitation wavelengths of 532 and 635 nm. In order for the MLPA-assay to be useful in cervical screening the sensitivity in HPV-typing has to be high. As summarized in Table 1, 52 of the 67 patient samples were found to be positive for HPV16, including 4 of the 7 (57%) normal samples, 14 of the 20 (70%) normal samples adjacent to a lesion, 7 of the 10 (70%) CIN1/condyloma samples, 12 of the 13 (92%) CIN2/3 samples and 15 of the 17 (88%) carcinoma samples. Furthermore, one CIN2/3 and one carcinoma sample were found to be positive for HPV18.Table 1Results of the HPV MLPA-Assay in Frozen Uterine Cervical Tissue Samples, Validated by qPCR, PapilloCheck, and FISHDiag.CaseAge% CellsTypingViral loadPhysical statusTelomerase genes gainHigh risk class. MLPAMLPAqPCRPapilloCheckMLPAqPCR*according to Lindh et al.23 Ct HPV16/18qPCR†according to Peitsaro et al.24 HPV16MLPAqPCR†according to Peitsaro et al.24 E2/E6MLPA TERTMLPA TERCNormal no lesion (n = 7)164−−−−35/37−−−−−−2‡previously described by Theelen et al.647−−−−36/37−−−−−−352−−−−35/−−−−−−−45216−−0.1136/−−epi−−−−57316−−0.1435/40−epi−−−−6381616160.2234/40−epi−−−−7601616162.1034/380.16epiint−−−Normal adj. to lesion (n = 20)875−−−−38/−n.d.−−−−−935−16−−34/−n.d.−−−−−10−−−−−38/38n.d.−−−−−1147−−−−35/−n.d.−−−−−1236−−−−37/−n.d.−−−−−1343−−45−−/−n.d.−−−−−145016−160.00437/−−epi−−−−15341616−0.0534/390.05epimix−−−16−1616160.0733/−0.02epimix−−−17321616580.1332/−0.1epimix−−−18−161616; 590.1834/−0.04epimix−−−19521616; 31n.c.0.1834/390.05epiepi−−−20−1616160.3331/400.33mix (a)§FISH inconclusive.mix−−+21641616n.c.0.4831/−0.22epimix−−−22−161616; 590.5131/380.17epimi" @default.
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• W2087093385 date "2010-10-01" @default.
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• W2087093385 title "Increase in Viral Load, Viral Integration, and Gain of Telomerase Genes during Uterine Cervical Carcinogenesis can be Simultaneously Assessed by the HPV 16/18 MLPA-Assay" @default.
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