Petros Karakitsos MD, PhD. Professor, Director Department of Cytopathology Medical School, University of Athens University General Hospital ATTIKON

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1 Petros Karakitsos MD, PhD Professor, Director Department of Cytopathology Medical School, University of Athens University General Hospital ATTIKON

2 Human Papilloma Virus (HPV)

3 Αναδιπλασιασμός των HPV στο πολύστιβο πλακώδες επιθήλιο ρατινοποιημένη Virus release Κοκκιώδης E4 L2 L1 Virus assembly/ Virus release Genome amplification Ακανθωτή Βασική στιβάδ α E6, E7 E1, E2, E4, E5 Viral DNA Genome maintenance/ Cell proliferation Genome maintenance p97 p670 p670 Πολύστιβο πλακώδες κερατινοποιημένο επιθήλιο Πολύστιβο πλακώδες επιθήλιο Doorbar J. Clin Sci (Lond) 2006; 110:

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5 Αυτοκάθαρση HPV λοίμωξης σε γυναίκες Λοιμώξεις μεhpv 16 και18 καθώς και με άλλους ογκογόνους τύπους είναι πολύ συχνότερα εμμένουσες σε σχέση με λοιμώξεις από ογκογόνους τύπους HPV 1 4 Σε γυναίκες ηλικίας15 25 έτων, >80% of HPV λοιμώξεων είναι ποροδικές 5 Σε γυναίκες μεγαλύτερης ηλικίας οι HPV λοιμώξεις φαίνεται ότι είναι εμμένουσες 6 Εμμένουσες λοιμώξεις από ογκογόνους τύπους HPV θεωρούνται αναγκαίος παράγοντας για ανάπτυξη ΚΤΜ Study (Country) n Average follow-up, years Median duration of infection, months Type 16 Type 18 Type 6 Ho, 1998 (USA) Muňoz, 2004 (Colombia) Richardson, 2003 (Canada) Woodman, 2001 (UK) Ho GY, et al. N Engl J Med 1998; 338: ; 2. Muňoz N. J Infect Dis 2004; 190: ; 3. Richardson H. Cancer Epidemiol Biomarkers Prevent 2003; 12: ; 4. Woodman CB. Lancet 2001; 357: Londesborough P. Int J Cancer (Pred Oncol) 1996; 69: ; 6. Castle PE, et al. J Infect Dis 2005; 19:

6 HΡV ένας διαφορετικός ιός A Sophisticated Immune Evasion Mechanism 1-4 Φτωχή έκθεση στα αντιγονοπαρουσιαστικά κύτταρα (APC). Δεν επάγει ικανοποιητική ανοσιακή απάντηση Χρησιμοποιεί το γονιδίωμα του κυττάρου ξενιστή μέσω του κυτταρικού κύκλο απελευθερώσει Καθόλου φλεγμονή, νέους ιούςκαμία 1-4 έλξη των κυττάρων Δεν του προκαλεί ανοσιακού κυτταρικό συστήματος θάνατο 1-4 Εισέρχεται στο επιθηλιακό κύτταρο της βασικής στοιβάδας και ενσωματώνεται Τοπική Ανοσοκαταστολή στο DNA Πολλαπλασιάζεται στο κύτταρο και παραμένει εξ ολοκλήρου ενδοεπιθηλιακά. 1-4 Τοπική Όχι Λοίμωξη Ιαιμία 1-4 (πχ Ηπατίτιδα) Λοίμωξη επιθηλιακών κυττάρων λόγω εκδορών Stanley M. Vaccine 2006; 24: S106-13, 2.Tindle, Nat Rev Cancer 2002; 2, 59, 3.Stanley M. Vaccine 2006; 24: S16-22, 4. Stanley M. HPV Today 2007; 11: 1-16

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8 Potential Clinical Application of HPV related biomarkers Primary screening test Triage of women prior to referral to colposcopy Triage of women after colposcopy Triage of women with LG colposcopy

9 D. Elgui de Oliveira / Cancer Letters 247 (2007) Aneuploidy

10 Μεταβολή της έκφρασης του γονιδιώματος του HPV κατά τη διάρκεια της εξέλιξης σε ΚΤΜ

11 Infection cycle of HUMAN PAPILLOMAVIRUS (HPV) (simplified model) HPV infection of the cell nucleus Latent infection Of no clinical value Active infection Of clinical value Integration of viral DNA into the human genome Cell death Release of the virus Malignant cells virus Capsid protein Cell differentiation Viral DNA Replication of the viral DNA + Production of the capsid and shard protein Initiation of the proliferation of the virus Nucleus with chromosomal DNA Nucleus with chromosomal and viral DNA, in absence of capsid protein

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15 Novel kinetochore proteins in cervical pre-cancer

16 HPA: Hybridization Protection Assay, NASBA: Nucleic Acid Based Amplification, TMA: Transcritpion Mediated Amplification *Results are from studies in ASC-US samples 1 Szarewski, A. et al. Comparison of predictors for high-grade cervical intraepithelial neoplasia in women with abnormal smears. Cancer Epidemiol Biomarkers Prev 17, (2008). 2 Castle, P. E. et al. Performance of carcinogenic human papillomavirus (HPV) testing and HPV16 or HPV18 genotyping for cervical cancer screening of women aged 25 years and older: a subanalysis of the ATHENA study. Lancet Oncol 12, (2011). 3 Stoler, M. H. et al. High-risk human papillomavirus testing in women with ASC-US cytology: results from the ATHENA HPV study. Am J Clin Pathol 135, (2011). 4 Johnson, L. R. et al. A comparison of two methods to determine the presence of high-risk HPV cervical infections. Am J Clin Pathol 130, (2008). 5 Stevens, M. P. et al. Comparison of the Digene Hybrid Capture 2 assay and Roche AMPLICOR and LINEAR ARRAY human papillomavirus (HPV) tests in detecting high-risk HPV genotypes in specimens from women with previous abnormal Pap smear results. J Clin Microbiol 45, (2007). 6 Gravitt, P. E., Schiffman, M., Solomon, D., Wheeler, C. M. & Castle, P. E. A comparison of linear array and hybrid capture 2 for detection of carcinogenic human papillomavirus and cervical precancer in ASCUS-LSIL triage study. Cancer Epidemiol Biomarkers Prev 17, (2008). 7 Pista, A., Verdasca, N. & Oliveira, A. Clinical performance of the CLART human papillomavirus 2 assay compared with the hybrid capture 2 test. J Med Virol 83, (2011). 8 Chranioti, A. et al. Performance evaluation of manual and automated (MagNA pure) nucleic acid isolation in HPV detection and genotyping using Roche Linear Array HPV Test. Infect Dis Obstet Gynecol 2011, (2011). 9 Molden, T., Kraus, I., Karlsen, F., Skomedal, H. & Hagmar, B. Human papillomavirus E6/E7 mrna expression in women younger than 30 years of age. Gynecol Oncol 100, (2006). 10 Keegan, H. et al. Comparison of HPV detection technologies: Hybrid capture 2, PreTect HPV-Proofer and analysis of HPV DNA viral load in HPV16, HPV18 and HPV33 E6/E7 mrna positive specimens. J Virol Methods 155, (2009). 11 Coquillard, G., Palao, B. & Patterson, B. K. Quantification of intracellular HPV E6/E7 mrna expression increases the specificity and positive predictive value of cervical cancer screening compared to HPV DNA. Gynecol Oncol 120, (2011). 12 Kottaridi, C. et al. Clinical Performance of Human Papillomavirus E6, E7 mrna Flow Cytometric Assay Compared to Human Papillomavirus DNA Typing. Anal Quant Cytol Histol 33, (2011). 13 Castle, P. E. et al. A cross-sectional study of a prototype carcinogenic human papillomavirus E6/E7 messenger RNA assay for detection of cervical precancer and cancer. Clin Cancer Res 13, (2007).

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22 Background: Human papillomavirus (HPV) testing is more sensitive than cytology for detecting high-grade cervical intraepithelial neoplasia (CIN). We evaluated the performance of high-risk HPV (hrhpv) testing in routine screening. Methods: In all, women (29 61) enrolled in our population-based cohort study were offered both cytology and hrhpv testing. High-risk HPV-positive women with normal cytology and an age-matched subcohort of hrhpv-negative women with normal cytology were invited for repeat testing after 1 and/or 2 years and were referred for colposcopy if they presented with abnormal cytology and/or a positive hrhpv test. The hrhpv-positive women with borderline or mild dyskaryosis (BMD) and all women with moderate dyskaryosis or worse (>BMD) were directly referred for colposcopy. Women with BMD and an hrhpv-negative test were advised to repeat cytology at 6 and 18 months and were referred for colposcopy if the repeat cytology test was abnormal. The main outcome measure was CIN grade 3 or worse (CIN3+). Results were adjusted for non-attendance at repeat testing. Results: The hrhpv-positive women with abnormal cytology had a CIN3+ risk of 42.2% (95% confidence interval (CI): ), whereas the hrhpv-positive women with normal cytology had a much lower risk of 5.22% (95% CI: ). In hrhpv-positive women with normal cytology, an additional cytology step after 1 year reduced the CIN3+ risk to only 1.6% (95% CI: ) if the repeat test was normal. The CIN3+ risk in women with hrhpv-positive normal cytology was higher among women invited for the first time (29 33 years of age) (9.1%; 95% CI: ) than among older women (3.0%; 95% CI: ). Conclusion: Primary hrhpv screening with cytology triage in women aged >30 years is an effective way to stratify women on CIN3+ risk and seems a feasible alternative to cytological screening. Repeat cytology after 1 year for hrhpv-positive women with normal cytology is however necessary before returning women to routine screening. HPV DNA testing in population-based cervical screening (VUSA-Screen study): results and implications D C Rijkaart, J Berkhof, F J van Kemenade, V M H Coupe, L Rozendaal, D A M Heideman, R H M Verheijen, S Bulk, W Verweij, P J F Snijders and C J L M Meijer British Journal of Cancer 106, (28 February 2012)

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24 HPV TPPT HPV+TPPT Sensitivity Specificity NPV TPPT Direct HPV TPPT= ThinPrep Pap Test Ferreccio et al. Cancer Epidemiol Biomarkers Prev. 2003;12:

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26 Potential Clinical Application of HPV related biomarkers Primary screening test Triage of women prior to referral to colposcopy Triage of women after colposcopy Triage of women with LG colposcopy

27 Tests Flow p16 NASBA HPV Best combination Assess various biomarkers (single tests or combinations) in LSIL in order to identify: possible CIN2+ colposcopy referral

28 HPV DNA test ASCUS: triage Arbyn, JNCI 2004a Arbyn, JNCI 2004b LG SIL: triage X Arbyn, Gynecol Oncol 2005 Kyrgiou, Cancer Treatment Reviews 2006

29 New recommendations for the use of HPV-tests in Denmark GUIDELINES FOR HPV E6/E7 mrna TESTING IN NORWAY

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31 LSIL Sensitivity hrhpv DNA Negative Positive Specificity - HPV 16 or - hrhpv and P16 Negative Positive Routine screening Surveillance Colposcopy Tsoumpou et. Al. Gynecologic Oncology 121 (2011) Valasoulis at. al. Eur. J. Gynecol. Oncol : , 2011

32 A. Spathis et.al. Plos one Volume 7,Issue 11, e49205

33 Suggested work flow Flowchart of the rules of a model produced by automated text processing K. Wagholikar et. al. (2012)

34 Meta analysis of meta analyses

35 A Clinical Decision Support System Patient s Data (examinations results, medical history, etc) CDSS Support Information (diagnostic or therapeutic recommendations, predictions, etc) Data Transfor mation (medical data to mathema tical data) Artificial Intelligence Models Statistical Pattern Recognition Models (Bayesian, SVM, etc) Artificial Neural Networks Genetic Algorithms Fuzzy Logic Data Interpret ation (number s to medical informati on)

36 A Clinical Decision Support System for Cervical Cancer Screening and Triage Patient s Data (Pap test, HPV DNA test, NASBA, Flow, p16) WNL ASCUS LSIL HSIL CA Support Information (Histology) Pap test=lsil HPV DNA type=16 NASBA type=16 Flow=neg. P16=neg. Data Transfor mation AI Model Knowledge Database Data Interpre tation Histology Prediction (probabilities) Normal=4% CIN 1= 60% CIN 2/3 =30% Ca = 6%

37 Performance Comparison Cytology Sensitivity= 78.23% Specificity= 94.04% PPV= 80.99% NPV= 93.01% FPR= 5.96% FNR= 21.77% OA= 90.17% CART Sensitivity= 85.12% Specificity= 92.58% PPV= 66.88% NPV= 97.25% FPR= 7.42% FNR= 14.88% OA= 91.46% LVQ Sensitivity= 85.16% Specificity= 98.01% PPV= 85.71% NPV= 97.92% FPR= 1.99% FNR= 14.84% OA= 96.42% PNN Sensitivity= 92.52% Specificity= 96.25% PPV= 88.89% NPV= 97.54% FPR= 3.75% FNR= 7.48% OA= 95.33%

38 CLART HPV: 45 NASBA: 45 HPV OncoTect : Positive Pap Test HPV DNA Type NASBA Type Flow P16 AIS Positive Negative Normal CIN I CIN II/III Ca 1.1% 5.9% 8% 85%

39 Algorithms

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42 The Problem with False Negatives How severe is the disease? When will the patient be tested again? Problem of False Positives The trade off between sensitivity and specificity can best be visualized by constructing a ROC curve How rapidly does the disease progress? In our society, there is a tendency to overweight on sensitivity In the case of cytology the cutoffs are defined by the various TBS categories (ASCUS+, LSIL+, etc.) A lower cut-off causes a higher sensitivity at the expense of a lower specificity Increased Detection is not necessarily the same as Increased Sensitivity; Detection is only good if it is real

43 Efficiency= Optimal resources allocation in order to achieve goals Efficacy= Selection of appropriate goals and achievement of goals Technology Tests Doing things right Doing the right things