Detection of Genomic Amplification of the Human Telomerase Gene TERC, a Potential Marker for Triage of Women with HPV-Positive, Abnormal Pap Smears
2009; Elsevier BV; Volume: 175; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2009.090122
ISSN1525-2191
AutoresSonia Andersson, Pavani Sowjanya, Darawalee Wangsa, Anders Hjerpe, Bo Johansson, Gert Auer, Patti E. Gravitt, Catharina Larsson, Keng-Ling Wallin, Thomas Ried, Kerstin Heselmeyer‐Haddad,
Tópico(s)Cancer Genomics and Diagnostics
ResumoThe vast majority of invasive cervical carcinomas harbor additional copies of the chromosome arm 3q, resulting in genomic amplification of the human telomerase gene TERC. Here, we evaluated TERC amplification in routinely collected liquid based cytology (LBC) samples with histologically confirmed diagnoses. A set of 78 LBC samples from a Swedish patient cohort were analyzed with a four-color fluorescence in situ hybridization probe panel that included TERC. Clinical follow-up included additional histological evaluation and Pap smears. Human papillomavirus status was available for all cases. The correlation of cytology, TERC amplification, human papillomavirus typing, and histological diagnosis showed that infection with high-risk human papillomavirus was detected in 64% of the LBC samples with normal histopathology, in 65% of the cervical intraepithelial neoplasia (CIN)1, 95% of the CIN2, 96% of the CIN3 lesions, and all carcinomas. Seven percent of the lesions with normal histopathology were positive for TERC amplification, 24% of the CIN1, 64% of the CIN2, 91% of the CIN3 lesions, and 100% of invasive carcinomas. This demonstrates that detection of genomic amplification of TERC in LBC samples can identify patients with histopathologically confirmed CIN3 or cancer. Indeed, the proportion of TERC-positive cases increases with the severity of dysplasia. Among the markers tested, detection of TERC amplification in cytological samples has the highest combined sensitivity and specificity for discernment of low-grade from high-grade dysplasia and cancer. The vast majority of invasive cervical carcinomas harbor additional copies of the chromosome arm 3q, resulting in genomic amplification of the human telomerase gene TERC. Here, we evaluated TERC amplification in routinely collected liquid based cytology (LBC) samples with histologically confirmed diagnoses. A set of 78 LBC samples from a Swedish patient cohort were analyzed with a four-color fluorescence in situ hybridization probe panel that included TERC. Clinical follow-up included additional histological evaluation and Pap smears. Human papillomavirus status was available for all cases. The correlation of cytology, TERC amplification, human papillomavirus typing, and histological diagnosis showed that infection with high-risk human papillomavirus was detected in 64% of the LBC samples with normal histopathology, in 65% of the cervical intraepithelial neoplasia (CIN)1, 95% of the CIN2, 96% of the CIN3 lesions, and all carcinomas. Seven percent of the lesions with normal histopathology were positive for TERC amplification, 24% of the CIN1, 64% of the CIN2, 91% of the CIN3 lesions, and 100% of invasive carcinomas. This demonstrates that detection of genomic amplification of TERC in LBC samples can identify patients with histopathologically confirmed CIN3 or cancer. Indeed, the proportion of TERC-positive cases increases with the severity of dysplasia. Among the markers tested, detection of TERC amplification in cytological samples has the highest combined sensitivity and specificity for discernment of low-grade from high-grade dysplasia and cancer. Cervical carcinoma is the second most common malignancy among women world-wide.1World Health Organization Comprehensive cervical cancer control: a guide to essential practice. WHO, Geneva, Switzerland2006: 3Google Scholar The introduction of screening programs based on cytological examination of cervical smears resulted in a significant decrease in both incidence and mortality rates.2Koss LG The Papanicolaou test for cervical cancer detection. A triumph and a tragedy.JAMA. 1989; 261: 737-743Crossref PubMed Scopus (660) Google Scholar The most common cervical cancer type is squamous cell cervical carcinoma.3Walboomers JM Jacobs MV Manos MM Bosch FX Kummer JA Shah KV Snijders PJ Peto J Meijer CJ Munoz N Human papillomavirus is a necessary cause of invasive cervical cancer worldwide.J Pathol. 1999; 189: 12-19Crossref PubMed Scopus (6982) Google Scholar Infections with high-risk human papillomavirus (HR-HPV) are almost invariably found in women with neoplastic disease and progression from low-grade to high-grade dysplasia, and invasive disease is very rare in the absence of HR-HPV.4Khan MJ Castle PE Lorincz AT Wacholder S Sherman M Scott DR Rush BB Glass AG Schiffman M The elevated 10-year risk of cervical precancer and cancer in women with human papillomavirus (HPV) type 16 or 18 and the possible utility of type-specific HPV testing in clinical practice.J Natl Cancer Inst. 2005; 97: 1072-1079Crossref PubMed Scopus (886) Google Scholar However, transient HPV infections that do not result in the development of high-grade dysplasia are common in young, sexually active women. The causal relationship between HR-HPV infection and cervical cancer has made the detection of the virus an attractive approach to identify women at risk of developing cervical cancer.5Cuzick J Arbyn M Sankaranarayanan R Tsu V Ronco G Mayrand MH Dillner J Meijer CJ Overview of human papillomavirus-based and other novel options for cervical cancer screening in developed and developing countries.Vaccine. 2008; 26: K29-K41Crossref PubMed Scopus (508) Google Scholar However, it has also become increasingly evident that other factors, in addition to HR-HPV per se, are required for cervical carcinogenesis, since only rarely will women infected with HR-HPV eventually develop cervical cancer. Therefore, biomarkers strongly associated with the propensity of low-grade lesions to progress to high-grade lesions and cancer would be of great value for the individualization of the clinical management of women with abnormal Pap smears.6Gravitt PE Coutlee F Iftner T Sellors JW Quint WG Wheeler CM New technologies in cervical cancer screening.Vaccine. 2008; 26: K42-K52Crossref PubMed Scopus (100) Google Scholar The expression of the HPV protein E7 triggers the activation of the cyclin-dependent kinase inhibitor p16INK4a, which has been explored for its diagnostic potential.7Klaes R Friedrich T Spitkovsky D Ridder R Rudy W Petry U Dallenbach-Hellweg G Schmidt D von Knebel Doeberitz M Overexpression of p16(INK4A) as a specific marker for dysplastic and neoplastic epithelial cells of the cervix uteri.Int J Cancer. 2001; 92: 276-284Crossref PubMed Scopus (872) Google Scholar In normal cells, p16 expression is essentially undetectable by immunocytochemistry. It is possible that such staining will not only improve the chances of detecting premalignant cells, but also aid in the distinction between premalignant and reactive atypia.8Norman I Brismar S Zhu J Gaberi V Hagmar B Hjerpe A Andersson S p16(INK4a) immunocytochemistry in liquid-based cervical cytology: is it feasible for clinical use?.Int J Oncol. 2007; 31: 1339-1343PubMed Google Scholar In addition to infection with HPV and its consequent expression of p16, the majority of cervical carcinomas carry extra copies of the long arm of chromosome 3 and with it genomic amplification of the RNA component of the human telomerase gene (TERC), which resides on cytoband 3q26.9Heselmeyer 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 (424) Google Scholar, 10Heselmeyer K Macville M Schrock E Blegen H Hellstrom AC Shah K Auer G Ried T Advanced-stage cervical carcinomas are defined by a recurrent pattern of chromosomal aberrations revealing high genetic instability and a consistent gain of chromosome arm 3q.Genes Chromosomes Cancer. 1997; 19: 233-240Crossref PubMed Scopus (245) Google Scholar Therefore, genomic amplification of this gene is likely to play a central role in progression from low-grade dysplasia to high-grade cervical intraepithelial neoplasia (CIN) and invasive cancer. In a previous study, we showed that progression is never observed in the absence of genomic amplification, and, inversely, extra copies of this gene are not present in lesions that spontaneously regress.11Heselmeyer-Haddad K Sommerfeld K White NM Chaudhri N Morrison LE Palanisamy N Wang ZY Auer G Steinberg W Ried T Genomic amplification of the human telomerase gene (TERC) in pap smears predicts the development of cervical cancer.Am J Pathol. 2005; 166: 1229-1238Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar The aim of the present study was to validate these findings among women undergoing testing for cervical cancer following an abnormal Pap smear at population-based screening, and to correlate the amplification status of TERC with the histological evaluation of concordant biopsies. Genomic copy numbers of TERC and the oncogene MYC were determined on liquid based cytology (LBC) slides using fluorescence in situ hybridization (FISH), and the findings were systematically compared with HPV status, p16 protein expression, and the results of cytological screening and histopathological diagnoses. We consecutively enrolled 78 women with any grade of cytological abnormalities detected at a population-based screening. The women were referred for extended testing at the Department of Gynecology at the Karolinska University Hospital Huddinge in Stockholm, Sweden. The extended testing took place during 2005, 2 to 6 months after the initial population-based screening. The mean age of the women was 35.3 years (median 33 years, range 23 to 60 years). Enrollment, clinical characterization, and sampling of all women have been previously reported in detail.12Andersson S Hansson B Norman I Gaberi V Mints M Hjerpe A Karlsen F Johansson B Expression of E6/E7 mRNA from 'high risk' human papillomavirus in relation to CIN grade, viral load and p16INK4a.Int J Oncol. 2006; 29: 705-711PubMed Google Scholar From each woman we collected material for a Pap smear and LBC, and performed colposcopy and punch biopsies for histopathological evaluation. In many cases, a subsequent loop electrosurgical excision procedure (LEEP) histopathology and/or Pap smear follow-up were available. Cytological and histopathological samples were re-evaluated by an experienced pathologist (A.H.). Samples were diagnosed in a blinded fashion, ie, the respective assessment was disclosed only after both the cytology and histology samples had been evaluated. Cytological categories were defined according to the Bethesda nomenclature,13Solomon D Davey D Kurman R Moriarty A O'Connor D Prey M Raab S Sherman M Wilbur D Wright Jr, T Young N The 2001 Bethesda System: terminology for reporting results of cervical cytology.JAMA. 2002; 287: 2114-2119Crossref PubMed Scopus (2657) Google Scholar including the subgroups within normal limits (WNL), atypical squamous cells—undetermined significance (ASCUS), low-grade squamous intraepithelial lesions (LSIL), atypical squamous cells—cannot exclude high-grade squamous intraepithelial lesions (HSIL) or high-grade changes (ASC-H), and HSIL. According to Swedish recommendations, cases of koilocytosis without signs of dysplasia are reported as WNL. Therefore, the LSIL group contains samples corresponding to cytological CIN grade 1 (CIN1) only. Histopathological diagnosis followed the World Health Organization criteria14Richart RM A modified terminology for cervical intraepithelial neoplasia.Obstet Gynecol. 1990; 75: 131-133PubMed Google Scholar and included the subclasses benign (WNL), CIN1, CIN2, CIN3, and carcinoma. Women with CIN2 or CIN3 were treated by LEEP. Follow-up data were collected until 2007 (Table 1), including Pap smears in all cases and colposcopy, plus biopsy when indicated. LBC samples were used for all of the HPV analyses, the FISH analyses of TERC, MYC, and HPV, and for the p16 analysis. Additionally, for a subset of cases, 4-μm sections of the biopsies, and/or LEEPs were also hybridized and analyzed. All samples used in the study were obtained with informed consent and permission from the local ethical committee. The flow chart in Figure 1 summarizes the study design.Table 1Comprehensive Table of All Analyses and Clinical Data for All 78 Cases StudiedView Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)*Bold text indicates: TERC positive according to threshold of nine and more nuclei with more than 2 TERC signals.Abbreviations: n.a. (not applicable); n.d. (not determined); bx (biopsy); d (doublet); df (doublet far apart); ds (doublets); diff (diffuse); WNL (within normal limits); ASCUS (atypical squamous cells—undetermined significance); LSIL (low-grade squamous intraepithelial lesions); ASC-H (atypical squamous cells—cannot exclude high-grade squamous intraepithelial lesions (HSIL) or high-grade changes; HSIL (high-grade squamous intraepithelial lesions; (LEEP) loop electrosurgical excision procedure. Open table in a new tab Figure 1Flow chart of the study design.View Large Image Figure ViewerDownload Hi-res image Download (PPT) *Bold text indicates: TERC positive according to threshold of nine and more nuclei with more than 2 TERC signals. Abbreviations: n.a. (not applicable); n.d. (not determined); bx (biopsy); d (doublet); df (doublet far apart); ds (doublets); diff (diffuse); WNL (within normal limits); ASCUS (atypical squamous cells—undetermined significance); LSIL (low-grade squamous intraepithelial lesions); ASC-H (atypical squamous cells—cannot exclude high-grade squamous intraepithelial lesions (HSIL) or high-grade changes; HSIL (high-grade squamous intraepithelial lesions; (LEEP) loop electrosurgical excision procedure. HPV status was evaluated by four different assays: 1) a prototype line blot analysis from Roche; 2) a quantitative real-time PCR method specific for 10 high-risk HPV types (HPV 16, 18/45 group, 31 and 33/35/39/52/58/67 group (Quantovir AB, Uppsala, Sweden); 3) detection of E6/E7 mRNA expression of HPV 16, 18, 31, 33, and 45 HR-HPV with real-time nucleic acid sequence-based amplification assay method (PreTect HPV-Proofer; NorChip AS, Oslo, Norway); and 4) HPV-FISH (see also Figure 1). The Roche line blot assay was performed as described earlier.15Peyton CL Gravitt PE Hunt WC Hundley RS Zhao M Apple RJ Wheeler CM Determinants of genital human papillomavirus detection in a US population.J Infect Dis. 2001; 183: 1554-1564Crossref PubMed Scopus (222) Google Scholar, 16Gravitt PE Peyton CL Alessi TQ Wheeler CM Coutlee F Hildesheim A Schiffman MH Scott DR Apple RJ Improved amplification of genital human papillomaviruses.J Clin Microbiol. 2000; 38: 357-361PubMed Google Scholar, 17Gravitt PE Peyton CL Apple RJ Wheeler CM Genotyping of 27 human papillomavirus types by using L1 consensus PCR products by a single-hybridization, reverse line blot detection method.J Clin Microbiol. 1998; 36: 3020-3027PubMed Google Scholar Details of the method and real-time PCR results obtained with the samples analyzed in this study, have been reported.18Andersson S Safari H Mints M Lewensohn-Fuchs I Gyllensten U Johansson B Type distribution, viral load and integration status of high-risk human papillomaviruses in pre-stages of cervical cancer (CIN).Br J Cancer. 2005; 92: 2195-2200Crossref PubMed Scopus (107) Google Scholar, 19Moberg M Gustavsson I Gyllensten U Real-time PCR-based system for simultaneous quantification of human papillomavirus types associated with high risk of cervical cancer.J Clin Microbiol. 2003; 41: 3221-3228Crossref PubMed Scopus (106) Google Scholar Results from the E6/E7 mRNA with the samples used in this communication have been described in a previous publication.12Andersson S Hansson B Norman I Gaberi V Mints M Hjerpe A Karlsen F Johansson B Expression of E6/E7 mRNA from 'high risk' human papillomavirus in relation to CIN grade, viral load and p16INK4a.Int J Oncol. 2006; 29: 705-711PubMed Google Scholar These previously published data were used only as reference points for performance comparison with the previously unpublished line blot and FISH data (Table 1 and Figure 2).12Andersson S Hansson B Norman I Gaberi V Mints M Hjerpe A Karlsen F Johansson B Expression of E6/E7 mRNA from 'high risk' human papillomavirus in relation to CIN grade, viral load and p16INK4a.Int J Oncol. 2006; 29: 705-711PubMed Google Scholar, 18Andersson S Safari H Mints M Lewensohn-Fuchs I Gyllensten U Johansson B Type distribution, viral load and integration status of high-risk human papillomaviruses in pre-stages of cervical cancer (CIN).Br J Cancer. 2005; 92: 2195-2200Crossref PubMed Scopus (107) Google Scholar, 20Andersson S Wangsa D Flores-Staino C Safari H Mints M Hjerpe A Hagmar B Johansson B Expression of p16(INK4a) in relation to histopathology and viral load of 'high-risk' HPV types in cervical neoplastic lesions.Eur J Cancer. 2006; 42: 2815-2820Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar In addition HPV was detected by FISH (see examples in Figure 3, A–E) as described below. Results from previously published p16 immunocytochemical analyses12Andersson S Hansson B Norman I Gaberi V Mints M Hjerpe A Karlsen F Johansson B Expression of E6/E7 mRNA from 'high risk' human papillomavirus in relation to CIN grade, viral load and p16INK4a.Int J Oncol. 2006; 29: 705-711PubMed Google Scholar are summarized in Table 1. In short, p16 immunoreactivity was determined on LBC slides and scored as negative (−; less than three stained reactive cells per slide) or positive (+, ++, and +++) depending on the staining intensity. Slides for FISH analyses were prepared from samples of LBC specimens according to standard procedures.21Linder J Zahniser D ThinPrep Papanicolaou testing to reduce false-negative cervical cytology.Arch Pathol Lab Med. 1998; 122: 139-144PubMed Google Scholar Four-color FISH analysis was performed on each case using a probe set targeting the centromere of chromosome 7 (CEP7), the TERC locus at chromosome band 3q26 (LSI TERC), the MYC locus at 8q24.2 (LSI MYC), and an HPV probe that contained DNA of the types 16, 18, 30, 45, 51, and 58. The probes were provided by Abbott Molecular, Inc. (Des Plaines, IL) through a Cooperative Research and Development Agreement (CRADA #001039). CEP7 was labeled with Spectrum Aqua, the TERC contig with Spectrum Gold, MYC with Spectrum Red, and the HPV probe was biotin-labeled. The HPV probe was previously shown to detect types 16, 18, 26, 31, 33, 35, 39, 45, 52, 53, 56, 58, 59, 66, and 82,22Sokolova I Algeciras-Schimnich A Song M Sitailo S Policht F Kipp BR Voss JS Halling KC Ruth A King W Underwood D Brainard J Morrison L Chromosomal biomarkers for detection of human papillomavirus associated genomic instability in epithelial cells of cervical cytology specimens.J Mol Diagn. 2007; 9: 604-611Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 23Algeciras-Schimnich A Policht F Sitailo S Song M Morrison L Sokolova I Evaluation of quantity and staining pattern of human papillomavirus (HPV)-infected epithelial cells in thin-layer cervical specimens using optimized HPV-CARD assay.Cancer. 2007; 111: 330-338Crossref PubMed Scopus (15) Google Scholar indicating that the DNA of the HPV types contained in the mix cross-reacts with other types because of sequence homology. There were no samples with HPV 30 or HPV51 infection included in the studies cited.22Sokolova I Algeciras-Schimnich A Song M Sitailo S Policht F Kipp BR Voss JS Halling KC Ruth A King W Underwood D Brainard J Morrison L Chromosomal biomarkers for detection of human papillomavirus associated genomic instability in epithelial cells of cervical cytology specimens.J Mol Diagn. 2007; 9: 604-611Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 23Algeciras-Schimnich A Policht F Sitailo S Song M Morrison L Sokolova I Evaluation of quantity and staining pattern of human papillomavirus (HPV)-infected epithelial cells in thin-layer cervical specimens using optimized HPV-CARD assay.Cancer. 2007; 111: 330-338Crossref PubMed Scopus (15) Google Scholar Hybridization, posthybridization washes, and detection of the HPV probe were performed according to the probe manufacturer's recommendations.22Sokolova I Algeciras-Schimnich A Song M Sitailo S Policht F Kipp BR Voss JS Halling KC Ruth A King W Underwood D Brainard J Morrison L Chromosomal biomarkers for detection of human papillomavirus associated genomic instability in epithelial cells of cervical cytology specimens.J Mol Diagn. 2007; 9: 604-611Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar In brief, the LBC slides were incubated for 2 minutes in 2× standard saline citrate at room temperature followed by pepsin (0.3 mg/ml in 10 mmol/L HCl) at 37°C for 5 to 10 minutes, fixed in 1% neutral-buffered formalin at room temperature for 5 minutes, and then washed in 1× PBS for 5 minutes. Slides were dehydrated in an ethanol series and air-dried. Co-denaturation at 72°C for 2 minutes and overnight hybridization at 37°C were done on a ThermoBrite (Statspin). Slides were then washed in 2× SSC/0.3% Nonidet P-40 for 2 minutes at 48°C and in 2× SSC/0.1% Nonidet P-40 for 1 minute at room temperature. The HPV probe was detected using Alexa Fluor 488 Tyramide Signal Amplification kit number 22 (Invitrogen, Carlsbad, CA) according to the manufacturer's directions. Finally, antifade solution (VectaShield, Vector Laboratories, Burlingame, CA) containing the nuclear counterstain 4,6-diamidino-2-phenylindole was applied and coverslips were added. Image acquisition and analyses were performed using a Leica DM-RXA fluorescence microscope (Leica, Wetzlar, Germany) equipped with custom optical filters for 4,6-diamidino-2-phenylindole, Spectrum Aqua, Spectrum Gold, Spectrum Red, and Spectrum Green (Chroma Technologies, Rockingham, VT) and a ×40 Plan Apo (NA 1.25) objective. Scoring of FISH results was done without knowledge of cytological or histopathological evaluation or any of the other markers. HPV fluorescence patterns and TERC and MYC signals were enumerated by screening and counting the entire slide visually using the Spectrum Green filter for HPV, with the Spectrum Gold filter for TERC, and with the Spectrum Red filter for MYC. All nuclei on a slide were evaluated. On average, 2320 nuclei per slide with a range of 232 to 4996 nuclei were counted. For the HPV probe the results were classified as negative (0), or positive with episomal (1), episomal and integrated (2), or integrated (3) patterns depending on the appearance of the fluorescence signals (see Figure 3 as an example). CEP7, TERC, and MYC were evaluated by signal counts per nucleus. The results were registered as patterns for the probe panel (CEP7-TERC-MYC-HPV) for each nucleus. For example, a nucleus with two signals for CEP7, three signals for TERC, two signals for MYC, accompanied by an HPV-FISH episomal pattern was recorded as a "2321" pattern. Accordingly, cells with normal signal numbers and HPV-FISH negative result were reported as "2220." Their number was recorded on a manual counter and used to establish the total cell count by adding it to the number of nuclei with aberrant patterns. Nuclei with more than two TERC signals and/or more than two MYC signals and/or HPV-FISH positivity were imaged using a CoolSnap camera and multifocus imaging with the Leica CW4000-FISH imaging software for each of the optical filters. This software also allows for registering the imaged nuclei in relocation charts with their positions and their signal patterns. This enabled us to revisit the few cases for which ambiguous signal constellations were observed, and to arrive to a consensus count among three observers (P.S., K.H.-H., and T.R.). The complete FISH results comprised of the total cell count per slide and a list of all of the cells deviating from the FISH pattern of normal cells (see column "Signal patterns observed" in Table 1). All of the other columns provided for the FISH data in Table 1 were calculated from these raw data (eg, number of cells with more than two TERC signals excluding or including cells with four CEP7 and four TERC signals, etc). Cells with four signals for each probe, "444" were categorized as "tetraploid," acknowledging the fact that other chromosomal aneuploidies could exist in these cells. Receiver operating characteristics (ROC) curves were used to establish optimal thresholds and to identify FISH count parameters that best distinguish Thinprep samples with underlying CIN2, CIN3, and cancer histology, from Thinprep samples with underlying WNL and CIN1 histology. ROC curves were generated by plotting the sensitivity for detecting CIN2, CIN3, and cancer versus 1 minus the specificity for detecting WNL and CIN1, obtained at percent cell thresholds ranging from 0% to 10% (0.05% increments). Curves were generated based on the number of tetraploid cells, the number of cells with TERC gain (excluding "tetraploid" cells and cells that have four CEP7 signals and four TERC signals), the number of cells with either tetraploidy or TERC gain (ie, all gains of TERC), the number of cells with MYC gain (excluding "tetraploid" cells and cells that have four CEP7 signals and four MYC signals), and the number of cells with either tetraploidy or MYC gain (ie, all gains of MYC). Curves that come closest to the ideal values of 100% sensitivity and 100% specificity (Figure 4A, top left corner of ROC graph) provide the best combination of sensitivity and specificity (assuming equal importance of each) and optimal thresholds are typically selected from points near the "breaks" in the curves (region closest to top left corner; curve slope near 45°). A better view of the dependence of sensitivity and specificity on threshold can be obtained by plotting the distance from ideal (DFI) versus threshold (Figure 4B). DFI is defined here as the distance from the idealpoint (0,1) on the ROC plot (100% sensitivity, 100% specificity), and is calculated as [(1 − sensitivity)2 + (1 − specificity)2]1/2. DFI is smallest for the best combined sensitivity and specificity (giving equal weight to each) and varies from 0 for thresholds providing 100% sensitivity and 100% specificity, to 1.414 for thresholds providing 0% sensitivity and 0% specificity. A four-color FISH probe set for detection of CEP7, TERC (3q26), MYC (8q24), and HPV was hybridized to 78 cervical LBC samples of a Swedish patient cohort. Examples of the FISH analyses are shown in Figure 3. For all of the patients a biopsy was taken at a colposcopy performed at the same time as the LBC sampling, and for most of the patients a subsequent LEEP histology and Pap smear follow-up were available. When no visible lesion was observed, a biopsy was taken close to the squamo-columnar junction. The LBC samples were also analyzed by line blot and Quantovir for HPV infection, detection of E6/E7 mRNA of HPV 16, 18, 31, 33, and 45 high-risk types of HPV expression with the real-time nucleic acid sequence-based amplification assay and for p16 expression by immunocytochemistry. The study design is presented in Figure 1. Table 1 summarizes the FISH, HPV, and p16 data available for each sample together with the cytologic screening results, the histological diagnoses from biopsy and LEEP, and the clinical follow-up data. The results of the primary Pap smears taken at the population-based screenings at the referral clinics for the 78 women enrolled showed ASCUS in 14 cases, 34 smears had LSIL, 22 had HSIL, and six had ASC-H. In two cases we were unable to retrieve the initial diagnosis. The samples in Table 1 are arranged by the highest grade of histological diagnosis (when biopsy and LEEP histology differed from each other, the higher grade was used). When sorting according to the highest grade histology, 14 of the 78 samples were WNL, 17 samples were CIN1, 22 samples were CIN2, 23 samples were CIN3, and two samples were squamous cell cervical carcinoma, maintaining a good balance among the CIN groups. The number of TERC-positive cases increased with severity of the cervical lesion. Applying a threshold of more than eight TERC-positive cells before a case was considered TERC positive, 7% (one of 14) of WNL cases, 24% (4/17) of CIN1, 64% (14/22) of CIN2, 91% (21/23) of CIN3, and 100% (2/2) of the carcinomas were positive for genomic amplification of TERC. Figure 212Andersson S Hansson B Norman I Gaberi V Mints M Hjerpe A Karlsen F Johansson B Expression of E6/E7 mRNA from 'high risk' human papillomavirus in relation to CIN grade, viral load and p16INK4a.Int J Oncol. 2006; 29: 705-711PubMed Google Scholar, 18Andersson S Safari H Mints M Lewensohn-Fuchs I Gyllensten U Johansson B Type distribution, viral load and integration status of high-risk human papillomaviruses in
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