Labec Pharma, S.L HPV OncoTect : Flow cytometry assay based on E6/E7 mrna for the early diagnosis of cervical cancer.

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1 HPV OncoTect : Flow cytometry assay based on E6/E7 mrna for the early diagnosis of cervical cancer.

2 INDEX HPV Cervical cancer Screening tests HPV DNA vs. E6/E7 mrna tests Why to detect E6 and E7? HPV OncoTect HPV OncoTect: Clinical studies HPV OncoTect: Report and use algorithm Introduction of the HPV vaccines HPV OncoTect in primary care Conclusions Annex: media references 2

3 HPV Cervical cancer Screening tests HPV DNA vs. E6/E7 mrna tests Why to detect E6 and E7? HPV OncoTect HPV OncoTect: Clinical studies HPV OncoTect: Report and use algorithm Introduction of the HPV vaccines HPV OncoTect in primary care Conclusions Annex: media references 3

4 HPV HPV or Human Papillomavirus is the principal cause of cervical cancer; it constitutes the sexually transmitted disease with higher incidence. Cervical cancer is the second most common cancer among women worldwide. Each year appears nearly half a million new cases and over women die from this cause. In Spain, although the prevalence is not as high as in other regions, it causes about deaths per year, with an increased tendency. Over 100 HPV types have been identified, of which 15 are known as high risk HPV (HR-HPV) due to their carcinogenic potential. HPV infection has a high prevalence, it has been estimated that around 70% of women are infected by the virus at some point in their lives. 4

5 HPV (cont.) - Circular virus made up by 8000 base pairs. - Naked virus made up by capsid and core (DNA) - High risk oncogenic viral types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82 - Low risk oncogenic viral types: 6, 11, 40, 42, 43, 44, 54, 61, 70, 72 and 81 (leading to condyloma or genital warts) - Sexual transmission - Genome composed by 7 regions of early expression and 2 of late expression. - Regions E6 and E7 are over-expressed in cancer and severe lesions. 5

6 HPV Cervical cancer Screening tests HPV DNA vs. E6/E7 mrna tests Why to detect E6 and E7? HPV OncoTect HPV OncoTect: Clinical studies HPV OncoTect: Report and use algorithm Introduction of the HPV vaccines HPV OncoTect in primary care Conclusions Annex: media references 6

7 HPV and cervical cancer Human papillomavirus (HPV), which infects the skin and genital mucosa, cause benign lesions (warts or condyloma) and also precancerous lesions and cancers, specially cervical cancer. It is in fact a group of over 100 viruses, that differs in the structure of their DNA, which determines its genetic properties, and therefore the different disease that they induce. Those differences allow us to differentiate between those HPV types that cause benign lesions and those that cause cancer and premalignant lesions (16, 18, 33, 45, and others) which are known as high-risk HPV (HR-HPV). Studies performed in the last years have shown presence of HPV infections in more than 99% of the cervical cancers, proving the role of HR-HPV in cervical cancer development. HPV can be spread through direct contact with skin and mucous membranes, such as vulvar, vaginal, anal or oral. There is also a possibility, although remote, of infection by the hands. Therefore, today it is accepted that cervical cancer is a sexually transmitted disease, originated by HPV, the most prevalent STD in sexually active people. 7

8 HPV and cervical cancer (cont.) HPV is a very prevalent virus, in particular during the third decade of life, what has justified up to now the performance of HPV tests as a support to cytology in women over 30 years. CMAJ October 15; 167(8): The development of cervical cancer takes place over about 10 years 1 after HPV infection. It is therefore possible to diagnose and treat patients successfully. The first step of prevention of this cancer is a suitable screening system. [1] Prog Obstet Ginecol. 2006;49 Supl 2:5-62 8

9 Cervical cancer in Spain Incidence of cervical cancer in Spain compared to other cancers in women. Number of new cases of cervical cancer each year in different age groups. WHO/ICO Information Centre on HPV and Cervical Cancer (HPV Information Centre). Summary report on HPV and cervical cancer statistics in Spain [Date accessed]. Available at www. who. int/ hpv centre WHO/ICO Information Centre on HPV and Cervical Cancer 9

10 HPV Cervical cancer Screening tests HPV DNA vs. E6/E7 mrna tests Why to detect E6 and E7? HPV OncoTect HPV OncoTect: Clinical studies HPV OncoTect: Report and use algorithm Introduction of the HPV vaccines HPV OncoTect in primary care Conclusions Annex: media references 10

11 Cervical cancer screening Revision of screening HPV 99,7% Cervical cancer 1 guidelines; HPV, DNA tests. LBC: Liquid cytology. ThinPrep SurePath HPV Vaccines Papanicolaou test 1990s LBC 2000s Walboomers et al., Journal of Pathology 189:12-19,1999 HPV OncoTect 11

12 Minimum lines of screening in Spain Due to the important information now available in the fight against cervical cancer, the introduction of HPV test in clinical practice has been recommended by the SEGO (Spanish Gynecology and Obstetrics Society) in women after 35 years of age in population screening programs, and also in opportunistic and demand screening programs. Population Program: Age: 3 years after the first sexual relationship or at age 25 years Interval: Every 3 years (after a normal cytology 2-3 years) (each year according to risk factors) Test HPV at 35 years of age Opportunistic or demand screening: First control at any age Systematic cytology and colposcopy After 35 years of age: HPV test Consensus Document of the SEGO 2002, amended in

13 HPV Cervical cancer Screening tests HPV DNA vs. E6/E7 mrna tests Why to detect E6 and E7? HPV OncoTect HPV OncoTect: Clinical studies HPV OncoTect: Report and use algorithm Introduction of the HPV vaccines HPV OncoTect in primary care Conclusions Annex: media references 13

14 HPV test: Clinical use Progress in molecular biology has permitted the introduction of new tools that has improved the cervical cancer diagnosis. The introduction of HPV tests in clinical practice has a more relevant role due to the information they provide at clinical level and how they help in the prevention of this disease: Identifies the causal agent: HR-HPV is a necessary cause for cervical cancer The HPV tests are more sensitive than cytology for determining the presence of lesions The sensitivity to detect histological abnormalities confirmed by biopsy ranges from 30-67%, and its specificity between % The correlation between the result of cytology and the diagnosis with biopsy sometimes is not higher than 42% (H- SIL) Helps clear up ambiguous cytologies (ASC-US) Usefulness as a control of cure post-treatment after 6 months Screening HART Tuebingen Hannover Jena France public France private Seattle Canada Combined Cytology sensitivity: NIC2+ Cytology sensitivity 1 Nanda K, McCrory DC, Myers ER, Bastian LA, Hasselblad V, Hickey JD, Matchar DB. Accuracy of the Papanicolaou test in screening for and follow-up of cervical cytologic abnormalities: a systematic review. Ann Intern Med May 16;132(10): Massad LS, Collins YC, Meyer PM. Biopsy correlates of abnormal cervical cytology classified using the Bethesda system. Gynecol Oncol Sep;82(3):

15 HPV test: types There are different types of HPV tests marketed by various commercial companies. The HPV test can generally be classified into two types: HPV DNA Tests: Detect the viral DNA, using as target the capsid genes, L1 and L2 regions of the viral genome. We can differentiate between: - Genotyping tests: they determine the viral type that has infected the patient (for example PCR Roche CLART, HPV2, etc.) - Detection of high or low risk HPV: Does not give us the viral type that infects the woman, but detects if the virus is of high or low risk. (Hybrid Capture, etc.) mrna HPV Tests: Detect the virus mrna; their target are usually the viral oncoproteins E6 and E7, these are over-expressed in severe lesions and cancer. Some tests keep the cell integrity (HPV OncoTect) allowing to determine the percentage of affected cells, others break the cell extracting the totality of the viral RNA (Nuclisene EasyQ). As we shall see, HPV OncoTect has a very superior clinical predictive capability. 15

16 HPV-DNA test: Diagnostic signification In situ hybridization (ISH) Polymerase chain reaction (PCR) Hybrid capture 2 (HCII, Digene/Qiagen) Negative HPV The woman is not infected by HPV It is very difficult to develop cancer or severe lesions High negative predictive value (NPV) (next screening in 3 or 5 years) Positive HPV The woman is infected by HPV This don't means that she will develop cancer, most of the infected women clear the infection spontaneously. Low positive predictive value (PPV) Is low for test of HPV-DNA detection 16

17 HPV-DNA test: Limitations HPV DNA tests detect highly prevalent infections, which 70% disappear within 1 year and a total of 90% within two years (without treatment) 1 Only persistent infections have risk of developing high grade pre-cancerous lesions and cancer. Approximately 25% of women with a high-risk virus will develop a low grade intraepithelial lesion (LSIL) and of those only will progress into a precancerous lesion the 10% 2. [1] Moscicki AB, Schiffman M, Kjaer S, Villa LL. Chapter 5: Updating the natural history of HPV and anogenital cancer. Vaccine 2006;24(3):S42-S51. [2] Prog Obstet Ginecol. 2006;49 Supl 2:

18 HPV mrna test: HPV OncoTect HPV OncoTect New target in the detection of HPV Immunophenotype and fluorescence in situ hybridization combined with flow cytometry Viral integration and mrna of E6 and E7 proteins 18

19 HPV mrna test: Diagnostic signification HPV OncoTect HPV OncoTect negative No oncoproteins expresion Although the virus is present, there is no oncogenic activity High negative predictive value (NPV) HPV OncoTect positive Oncoproteins present at the exocervix level There is unusual cell activity, viral integration and high probability of progression High positive predictive value (PPV) High correlation with progression 19

20 HPV test mrna vs DNA: Diagnostic information HPV-DNA neg HPV OncoTect neg HPV-DNA pos HPV OncoTect neg HPV-DNA pos HPV OncoTect pos There is no HPV in the epithelium There is no production of oncogenic proteins There is an HPV infection There is no production of oncogenic proteins There is an HPV infection There is production of oncogenic proteins Low probabilities of progression to severe lesions and cancer Infection with high probability of progression to severe lesion or cancer 20

21 HPV test mrna vs DNA: Diagnostic information HPV OncoTect NucliSENS EasyQ CLART HPV2 HC2 Molecular target mrna all HPV-HR mrna 5 types of HPV DNA 35 types of HPV DNA 18 types of VPH Method In situ hibridization/ Flow cytometry NASBA/ Real Time PCR/ Clinical Array Hibridization/ microplate capture/amplification signal Diagnostic sensibility 92% (CIN2+) 76,5% (CIN2+) 95% (CIN2+) 90% (CIN2+) Diagnostic especifity 95% (CIN2+) 58,8% (CIN2+) 91% (CIN2+) 82% (CIN2+) Negative predictive value 98% (CIN2+) 90,9% (CIN2+) 99% (CIN2+) 99% (CIN2+) Positive predictive value 87% (CIN2+) 31,7% (CIN2+) 68% (CIN2+) 50% (CIN2+) Conclusion: HC2 and Clarté HPV2 are techniques for detection of HPV DNA and therefore with low positive predictive value when it comes to severe lesions. For this reason, it is required the repetition of the tests and monitoring to observe the evolution of the patient and detect the persistence of the virus. These two tests do not provide, as HPV OncoTect does, information as useful to know the real risk of progression of the lesion. If HPV OncoTect is positive, there is abnormal cell activity, which determines the patient is at high risk of developing cancer. Nuclisens EasyQ is a method for detection of HPV E6/E7 mrna, but only covers 5 of the 15 viral types of HR-HPV that cause cervical cancer. HPV OncoTect detects E6 and E7 of all high-risk viral types. Furthermore Nuclisens EasyQ does not detect over-expression of E6 and E7, it destroys the cell to detect the existence of mrna in the sample, so that, its positive predictive value is not as high as OncoTect HPV, which is an indicator more useful to understand the risk of malignancy. HPV OncoTect maintaining the integrity of the cell, and detect the percentage of cells in which there is over-expression, this means it has a much higher positive predictive value. 21

22 HPV Cervical cancer Screening tests HPV DNA vs. E6/E7 mrna tests Why to detect E6 and E7? HPV OncoTect HPV OncoTect: Clinical studies HPV OncoTect: Report and use algorithm Introduction of the HPV vaccines HPV OncoTect in primary care Conclusions Annex: media references 22

23 Why to detect E6 and E7? As we have seen so far, infection by HPV is necessary but not sufficient for developing severe lesions, and as we have noted, the persistence of the virus in the epithelium is crucial for the clearance or progress of the disease process. This increased capacity is detected by determining the over-expression of HPV E6 and E7 oncoproteins. 15 años 30 años 45 años 23

24 Why to detect E6 and E7? For the development of oncogenic activity it is necessary that the HPV infection persists in the epithelium. This leads to the integration of the viral genome into the host genome, and as a result of this integration, an over-expression of the two oncoproteins E6/E7 takes place. HPV-HR infection PERSISTENT DNA-HPV INTEGRATION in celular genome Celular cycle deregulation: E6/E7 over-expression CANCER Zur Hausen H. Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer May;2(5):

25 Why to detect E6 and E7? E6/E7 oncoproteins synergistically, but through different pathways, cause the disruption of normal cell cycle leading to oncogenic activity. E7 Rb + E2F Triggers the synthesis of proteins necessary for DNA replication Increase of genomic instability Unrestricted cell division (allows pass to S phase) Accumulation of mutations p53 Induce apoptosis of cells with abnormally division phase Loss of cellular growth and replication control E6 Degradation APOPTOSIS Loss of cell cycle control (Chromosomal inestability) CANCER 25

26 Why to detect E6 and E7? The ultimate goal of HPV OncoTect by the detection of E6 and E7 mrna is to avoid those positives for HPV DNA that correspond to lesions that are going to be clarified, considering positive women with real risk of malignancy. Normal cervix Squamous intraepithelial lesion Invasive carcinoma Low grade High grade Cervical intraepithelial neoplasia Viral particles Grade I Grade II Grade III Basement membrane Primary infection in basal cells normally caused by micro-injuries 90% of infections resolve spontaneously without lesion in 2 years (1) 60% of low-grade lesions resolve spontaneously.(1) [ (1) Progress of Gynecology and Obstetrics (SEGO official journal) vol 49 Extraordinary 2, Nov 2006] 26

27 HPV Cervical cancer Screening tests HPV DNA vs. E6/E7 mrna tests Why to detect E6 and E7? HPV OncoTect HPV OncoTect: Clinical studies HPV OncoTect: Report and use algorithm Introduction of the HPV vaccines HPV OncoTect in primary care Conclusions Annex: media references 27

28 HPV OncoTect: Clinical use The detection of HPV E6/E7 mrna as a clinical marker for early detection d of cervical cancer: The detection of HPV E6/E7 mrna indicates the activity of oncogenic HPV and not just the presence of the virus It is the most important clinical indicator for the development of cervical cancer, and can be used as a predictive clinical marker to identify those women at high risk of developing high-grade dysplasic lesions and cancer Due to the integration of HPV DNA and its fragmentation, in many cases the tests based on detection of DNA will give false negative results: 4-25% of the cases 2. According to the SEGO criterion reflected in the revision of its Consensus Paper in 2006, it is defined as the ideal method of screening 3 the one that targets the detection of active transcription of HR-HPV HPV oncogenes or proteins resulting from their expression. (1) HPV HandBook: "Human Papillomavirus and Cervical Cancer", edited by Professor Walter Prendiville and Dr Philip Davies, supports detection of oncogenic activity (page 75 and 76): (2) Sankaranaryanan et al., Accuracy of human papillomavirus testing in primary screening of cervical neoplasia: results from a multicenter study in India. International Journal of Cancer 112: , 2004 (3) Documento de consenso SEGO: La infección por papilomavirus. 28

29 HPV OncoTect: Samples Sampling Samples can be obtained by using liquid cytology, such as ThinPrep Pap Test and SurePath Other options: Labec Pharma provides vials and cervical brushes for sampling The adequacy of the sample can be checked in the analysis phase Analysis of samples Using fluorescence and in situ hybridization of E6 and E7 mrna in exocervical cells from a sample of cytology Quantified cellular level, by flow cytometry HPV OncoTect consists of 7 reagents, kit of 100 tests 29

30 HPV OncoTect: Procedure HPV OncoTect procedure is based on the preservation of cellular integrity, which allows through in situ hybridization and flow cytometry to detect the percentage of exocervical cells that overexpress E6 and E7 Oncoproteins. EXOCERVICAL CELLS with target sequence Fluorochrome coupled with DNA probe complementary to mrna HPV E6, E7 ARNm Cels (%) HPV E6, E7 ARNm+ Cels (%) FIXATION / PERMEABILIZE (R1, R2, R3) ARNm E6/E7 Number Probe 1- FITC --> OLIGONUCLEOTIDES marked with fluorochrome (R5) + HEAT/WASH (R6/R7) ATTAGAAT Flow cytometry 30

31 HPV Cervical cancer Screening tests HPV DNA vs. E6/E7 mrna tests Why to detect E6 and E7? HPV OncoTect HPV OncoTect: Clinical studies HPV OncoTect: Report and use algorithm Introduction of the HPV vaccines HPV OncoTect in primary care Conclusions Annex: media references 31

32 Main clinical studies on HPV OncoTect Clinical Studies: Ameripath Estudio de Validación de HPV OncoTect vs PCR Ameripath Estudio de Validación de HPV OncoTect vs CHII Esoterix: Estudio en Chile: Citología vs HPV OncoTect vs CHII Stanford Study: Expresión de E6 y E7: Correlación expresión E6 y E7 con biopsia (2000 patients) High-throughput cervical cancer screening using intracellular human papillomavirus E6 and E7 mrna quantification by flow-citometry Roberto Narimatsu, MD, et al. Clinical Virology Symposium (April 2008) Quantitative analysis of HPV E6/E7 mrna expression in cells using flow citometry (HPV OncoTect) improves positive predictive value for lesions on biopsy (727 patients) Dan Irwin, Bruce K. Patterson - Stanford University Medical Center HPV in Human Pathology (May 2008) Quantitative analysis of HPV E6/E7 mrna expression in cells using flow citometry (HPV OncoTect) improves positive predictive value for lesions on biopsy (395 pacientes) J.A. López G.Asenjo, Bruce K. Patterson - H Clínico S Carlos, Stanford University Medical Center Eurogin 2008 (November 2008) Flow Cytometry in rapid screening for E6/E7 mrna detection Spathis A.1, Chranioti A.1, Alepaki M.1, Diamantopoulou S.2, Georgoulakis J.1, Tsiodras S.3, Kassanos D.2, Karakitsos P.1 U.O.A, University General Hospital, ATTIKON Combined HPV genotyping and cell-based E6,E7 mrna (HPV OncoTect) detection in cervical cytology maximizes sensitivity and positive predictive value for biopsy-proven CIN2 or above Gerald Coquillard, Bruce K. Patterson Stanford University Medical Center 32

33 Main clinical studies on HPV OncoTect (cont.) HPV in Human Pathology Prague, Czech Republic (May 2008) HPV E6, E7 mrna Expression in Ectocervical Cells (HPV OncoTect) Predicts Disease Progression in Women with Low Grade Intraepithelial Neoplasia (LSIL) Francisco Alameda Quitllet - Hospital Del Mar Quantification of Intracellular HPV E6/E7 mrna Expression Increases the Positive Predictive Value of Cervical Cancer Screening J.A. Lopez G-Asenjo, Bruce K. Patterson - H Clínico U. S Carlos, Stanford University Paper published in Revista de Ginecología y Obstetricia Clínica (2008; 9 (3): ) Presented at the 12th World Congress on the Menopause (May 2008) Relationship between HPV OncoTect and viral genotypes determined by PCR in peri and postmenopausal women González Rodríguez, Henríquez Linares, Sainz de la Cuesta, Ojeda Jabardo, Cantarero García. Gabinete Médico Velázquez. Madrid. Spain Hospital Quirón. Madrid. Spain AEPCC Congress, Spain (November 2008) HPV-OncoTect en pacientes peri y postmenopaúsicas: Implicaciones citológicas y genotípicas (98 patients) González SP 1, Ojeda MD 1, Urzáiz C 2, Albarracín A 1, Peñaloza J 1 (1)U. de Patología Vulvocervical. Gabinete Médico Velázquez. Madrid; (2)Ayuntamiento de Madrid SEGO Congress, Spain (May 2007) Relación entre el resultado del HPV-OncoTect y los hallazgos citológicos en pacientes asintomáticas (327 patients) Juan J. Hernández Aguado, Silvia P. González Rodriguez, M. Dolores Ojeda Jabardo, Marcos Cantarero, Mª Teresa López Gil Gabiente Médico Velázquez, AGE (Agrupación ginecológica española) Relación entre el resultado de HPV OncoTect y los factores de riesgo para la infección cervical por HPV (476 patients) Juan J. Hernández Aguado, Silvia P. González Rodriguez, M. Dolores Ojeda Jabardo, Marcos Cantarero, Mª Teresa López Gil Gabiente Médico Velázquez, AGE (Agrupación ginecológica española) 33

34 Main clinical studies on HPV OncoTect (cont.) QUANTITATIVE ANALYSIS OF HPV E6/E7 mrna EXPRESSION IN CELLS USING FLOW CYTOMETRY (HPV ONCOTECT) IMPROVES POSITIVE PREDICTIVE VALUE FOR LESIONS ON BIOPSY The purpose of this study was to combine HPV DNA screening with HPV E6, E7 mrna detection in an effort to improve the overall performance of cervical cancer screening while HPV/Cervical Cancer Screening Algorithm Using HPV E6, E7 mrna to Improve Positive Predictive Value potentially reducing the number of women requiring colposcopy. Liquid Cytology Specimen based cervical cytology specimens collected in either PreservCyt or Pap HRHPV DNA SurePath were submitted for routing cytology, HPV HRDNA detection by + Hybrid Capture 2 and HPV E6, E7 mrna quantification in cells. Results HPV E6, E7 mrna (OncoTect) from these three analyses were compared to biopsy in 40 cases. The positive + Colposcopy Rescreen (3-6 m) predictive value of HPV E6, E7 mrna quantification in cells was 86% which was greater than HPV DNA alone. The specificity was 96% based on 142 samples with normal cytology. There was a statistically significant difference in the percent of ectocervical cells expressing E6, E7 mrna in women with CIN2, CIN3, or cancer (mean 14.7%) compared to women with normal Colposcopic Biopsy cytology (mean 1.4%) with a P< Cytology NSA Cervicitis/H LGSIL HGSIL SCC WNL 1 2 Methods & Materials ASCUS Liquid based cervical cytology specimens collected in either PreservCyt LSIL (Cytyc, Marlborough MA) or SurePath HSIL (Tripath Imaging, Burlington NC) were WNL (no biopsy) n=378 submitted for routing cytology, HPV HRDNA detection by Hybrid Capture II (Digene, Gaithersberg, MD) and HPV Table 1. Relationship between cytologic and E6, E7 mrna quantification in cells biopsy diagnosis. using HPV OncoTect ASR (Invirion Diagnostics, Oak Brook IL). We analyzed a total of 395 samples including 73 with CIN 2, CIN 3, or squamous cell carcinoma and 322 samples from women with normal cytology. Biopsy was performed based on current standards of care. J.A. G-Asenjo, B Palao, B.K. Patterson Hospital Clinico, Madrid Spain Stanford University Medical Center Stanford University School of Medicine INTRO Results Results Results Conclusions % HPV E6, E7 mrna+ Cells WNL LGSIL HGSIL Figure 2. Relationship between HPV E6, E7 mrna expression and biopsy Figure 1. HPV E6, E7 mrna diagnosis. expression in a patient with normal cervical cytology (top) and in a patient with CIN 3 by biopsy (below) HC2 HPV OncoTect Cytology Diagnosis Biopsy Diagnosis High Levels E6E7 (%) Sensitivity (HGSIL) 94% 91% HSIL HSIL 9.3 HSIL Invasive SCC 14.2 Specificity 73% 97% HSIL NSA 0.32 HSIL HSIL (CIN3) 8.3 PPV (HGSIL) 43% 87% HSIL HSIL (CIN2) 10.4 LSIL NSA 2.1 NPV 98% 98% LSIL HSIL (CIN2) 11.7 LSIL LSIL 1.1 HGSIL n=83 LSIL NSA 2.6 LSIL LSIL 2.5 WNL n=378 LSIL LSIL 3.4 LSIL Cervicitis 1.2 LSIL NSA 1.67 LSIL LSIL low Figure 3. Comparison of HPV OncoTect Table 2. Representative data and Hybrid Capture 2 (HC2) for cervical comparing cytology, biopsy, and E6, cancer screening. CIN 2 or above was E7 mrna expression used as the endpoint in this study The positive predictive value of HPV E6, E7 mrna quantification in cells was 87% which was greater than HPV DNA alone (43%). The specificity was 97% based on 378 samples with normal cytology. There was a statistically significant difference in the percent of ectocervical cells expressing E6, E7 mrna in women with CIN2, CIN3, or cancer (mean 10.7%) compared to women with normal cytology (mean 1.1%) with a P< HPV E6, E7 mrna quantification at the cellular level may be able to reduce the number of colposcopies because of the high positive predictive value for high grade lesions on biopsy 34

35 Main clinical studies on HPV OncoTect (cont.) EUROGIN 2008 Flow Cytometry in rapid screening for E6/E7 mrna detection Spathis A.1, Chranioti A.1, Alepaki M.1, Diamantopoulou S.2, Georgoulakis J.1, Tsiodras S.3, Kassanos D.2, Karakitsos P.1 Dep. Diagnotic Cytopathology, U.O.A, University General Hospital, ATTIKON 3rd Obstetric Gynecology Clinic, U.O.A, University General Hospital, ATTIKON 4th Department of Internal Medicine, U.O.A, University General Hospital, ATTIKON Introduction: For the development of cervical cancer, apart from the simple presence of HPV, the viral oncoproteins E6/E7 have to be over-produced. The aim of this study was to evaluate two different techniques for E6/E7 HPV mrna detection. Materials and Methods: 44 cervical samples in PreserveCyt vials were typed for HPV DNA with ClinicalArrays HPV (Genomica, Spain). Flow cytometric evaluation of E6/E7 mrna of high risk HPV types was performed with HPV Oncotect (Invirion Diagnostics, USA). Endocervical cells were gated according to their FSC-SSC parameters and the cut-off was set at 1,5%. NucliSENS EasyQ HPV (biomerieux, France) was used to amplify E6/E7 mrna from HPV types 16, 18, 31, 33 and 45. Results: 7 samples were negative with both FC and NASBA. 2 samples were negative with FC, but positive with NASBA. 11 samples (5 HgSIL, 3 SCC, 3 AdenoCa) were positive for both methods only if the gating for FC was set according to CAM5.2 and CD16 expression. Another 17 samples (1 WNL, 4 LgSIL, 11 HgSIL, 1 SCC) were positive with both methods. 7 more samples (6 LgSIL, 1 SCC) were only positive in FC, out of witch 5 harbored coinfection of HPV 16 with at least another high risk type The remaining 2 were single HPV16 positive samples. Conclusions: Total agreement of the two methods was at 93,18%. Flow cytometry proved to be more efficient a screening method, since all high risk types can be detected. On the other hand, due to the morphological changes in size and granularity of cells in HgSILs and carcinomas, a three color staining procedure is needed. To conclude with, the two methods seem to be complementary since FC can be easily used as a screening method, while NASBA as a typing one. 35

36 SAMPLE 320 HPV OncoTect Main clinical studies on HPV OncoTect (cont.) COMBINED HPV GENOTYPING AND CELL-BASED E6, E7 mrna (HPV ONCOTECT) DETECTION IN CERVICAL CYTOLOGY SPECIMENS PERFORMED ON A MOLECULAR HYBRID PLATFORM (MOHP) MAXIMIZES SENSITIVITY AND POSITIVE PREDICTIVE VALUE FOR BIOPSY-PROVEN CIN 2 OR ABOVE. Gerald Coquillard, Bruce K. Patterson Stanford University Medical Center Stanford University School of Medicine, Department of Pathology, 3375 Hillview Ave, Palo Alto, CA Abstract Current cervical cancer screening relies on cervical cytologic diagnosis combined with high risk HPV DNA (HRHPV) detection. This screening algorithm identifies women at risk for developing cervical cancer with high sensitivity and high negative predictive value. The positive predictive value (PPV) of HRHPV DNA detection for biopsy proven pre-cancerous lesions, however, is low (15%-25%). In this study, we sought to combine HPV tests with high sensitivity and high positive predictive value to develop a cervical cancer screening test bundle with optimal clinical performance that can be run on the same compact instrument. We collected 100 cytologically normal samples in ThinPrep PreservCyt liquid and 20 samples in ThinPrep PreservCyt liquid that had biopsy proven CIN 2 or above. All samples were analyzed using Hybrid Capture 2 (HC2, Qiagen, Germantown, MD, USA), bead-based HPV Genotyping (Qiagen, Germantown, MD, USA), and HPV OncoTect (Invirion Diagnostics, Oakbrook Il, USA) for cell-based E6, E7 mrna expression. Analysis of bead-based HPV Genotyping and HPV OncoTect were performed on the same MoHP (Invirion Diagnostics, Oakbrook Il, USA). HPV Genotyping had a 95% sensitivity for CIN 2 or above and HPV OncoTect had a 90% PPV for CIN 2 or above. By comparison, HC2 had a 90% sensitivity for CIN 2 or above and a PPV of 50%. Of the 18 cytologically normal samples that were positive by HC2, five were negative by genotyping and four were HPV type 70 a non-high risk genotype. In summary, HPV genotyping and HPV Oncotect can be performed on the same, compact, costeffective instrument and provide superior performance for cervical cancer screening compared to HPV DNA alone. Methods & Materials Results Results Liquid based cervical cytology specimens collected in either PreservCyt (Cytyc, Marlborough MA) or SurePath (Tripath Imaging, Burlington NC) were submitted for routing cytology, HPV HRDNA detection by Hybrid Capture II (Digene, Gaithersberg, MD) HPV E6, E7 mrna quantification in + cells using HPV OncoTect ASR (Invirion Diagnostics, Oak Brook IL) and HPV genotyping using the Qiagen/Genaco HPV Genotyping Assay. Four ml of sample was used for HC2 per manufacturer s recommendations. One ml was used for DNA extraction using the EZ 1 Biorobot (Qiagen, Germantown, MD) then processed for HPV genotype. The HPV genotyping was performed on the MoHP instrument. HPV E6, E7 mrna was performed using the HPV E6, E7 mrna ASR (Invirion Diagnostics, Oakbrook, IL). Briefly, 1 ml of cells in LBP were fixed and permeabilized in PermiFlow (Invirion Diagnostics, Oakbrook, IL) for 1 hour at ambient temperature. Following fixation and permeabilization, the cells were washed once in PBS, ph 7.4, pelleted by centrifugation at 400g, washed again in 2 standard saline citrate (SSC), and pelleted by centrifugation. HPV FISH for E6 and E7 mrna was performed by resuspending the cells in 100 µl hybridization mix (Invirion Diagnostics, Oakbrook, IL)and a cocktail of 5'- and 3'- labeled oligonucleotide probes (HPV OncoTect ASR, Invirion Diagnostics, Oakbrook, IL). Hybridization was performed at 37 C for 30 minutes and was followed by a 5-minute wash in 2 SSC and 0.1% Triton X-100 (ViroTect Hybridization Reagent Kit, Invirion Diagnostics, Oakbrook, IL) and a 15-minute wash in 0.1% SSC and 0.1% Triton X-100. The cells were resuspended in PBS, ph 7.4, with 2% fetal calf serum for analysis on the MoHP (Invirion Diagnostics, Oakbrook, IL). HC2 HPV OncoTect HPV Genotyping Sensitivity (HGSIL) 90% 90% 95% Specificity 82% 98% 91% PPV (HGSIL) 50% 90% 68% NPV 98% 98% 99% Table 1. Representative data comparing Hybrid Capture 2 (HC 2), HPV OncoTect, and HPV Genotyping for detection of biopsy proven CIN 2 or above) WNL HGSIL HPV E6, E7 mrna SAMPLE 320 Figure 1. Cellular HPV E6, E7 mrna (HPV OncoTect/Invirion Diagnostics LLC) on a Molecular Hybrid Platform Figure 2. Multiplex Molecular Detection of HPV Genotype on a Molecular Hybrid Platform. Results Figure 3. Multiplex Molecular Detection of HPV Genotype on a Molecular Hybrid Platform. Reporter fluorescence in normal (HPV-) controls (TOP ROW) and in sample 320 (BOTTOM ROW) Conclusions 36

37 Main clinical studies on HPV OncoTect (cont.) HPV IN HUMAN PATHOLOGY (PRAGUE) Review: Francisco Alameda 1 Bibiana Palao 2 1 Hospital Del Mar 2 Labec Pharma HPV E6, E7 mrna Expression in Ectocervical Cells (HPV OncoTect) Predicts Disease Progression in Women with Low Grade Intraepithelial Neoplasia (LSIL) Current cervical cancer screening relies on cytologic diagnosis combined with HPV DNA detection. This screening algorithm identifies women at risk for developing cervical cancer but does not provide any information on which women with low grade lesions (LSIL) will progress. To address this important deficiency in cervical cancer screening, we prospectively followed women with a cytologic diagnosis of LSIL. Serial samples were frozen in ThinPrep PreservCyt medium at -80o C until analysis. Study subjects were divided into Progressors (n=11) and Non-progressors (n=9) and samples were analyzed at baseline and at the time of progression. Non-progressor samples were analyzed at the same duration as Progressors relative to the baseline sample. To predict which subjects progressed and which subjects did not, we used HPV OncoTect (Invirion Diagnostics, Oakbrook Il, USA), an assay that quantifies on a cell-by-cell basis the overexpression of HPV E6, E7 mrna, on all of the thawed, frozen specimens. Samples from the Progressor group had an average of 5.2% cells that overexpressed HPV E6, E7 mrna relative to HPV negative control cells. Samples from the Non-progressor group averaged 1.6% cells that overexpressed HPV E6, E7 mrna which is below the background cut-off of 2.0% determined on 100 HPV negative cervical cytology samples. The difference in the percentage of cells overexpressing HPV E6, E7 mrna between the Progressor group and the Nonprogressor group was statistically significant (P=0.008). Using a cut-off of 2.0%, HPV OncoTect correctly distinguished all Progressors from Non-progressors. 37

38 Main clinical studies on HPV OncoTect (cont.) HPV E6, E7 mrna EXPRESSION IN ECTOCERVICAL CELLS (HPV ONCOTECT) PREDICTS DISEASE PROGRESSION IN WOMEN WITH LOW GRADE INTRAEPITHELIAL NEOPLASIA (LSIL) Francisco Alameda, Bibiana Pilao, Bruce K. Patterson Hospital Del Mar (Spain), Labec Pharma (Spain) Stanford University Medical Center Current cervical cancer screening relies on cytologic diagnosis combined with HPV DNA detection. This screening algorithm identifies women at risk for developing cervical cancer but does not provide any information on which women with low grade lesions (LSIL) will progress. To address this important deficiency in cervical cancer screening, we prospectively followed women with a cytologic diagnosis of LSIL. Serial samples were frozen in ThinPrep PreservCyt medium at -80o C until analysis. Study subjects were divided into Progressors (n=11) and Nonprogressors (n=9) and samples were analyzed at baseline and at the time of progression. Non-progressor samples were analyzed at the same duration as Progressors relative to the baseline sample. To predict which subjects progressed and which subjects did not, we used HPV OncoTect (Invirion Diagnostics, Oakbrook Il, USA), an assay that quantifies on a cell-by-cell basis the overexpression of HPV E6, E7 mrna, on all of the thawed, frozen specimens. Samples from the Progressor group had an average of 5.2% cells that overexpressed HPV E6, E7 mrna relative to HPV negative control cells. Samples from the Non-progressor group averaged 1.6% cells that overexpressed HPV E6, E7 mrna which is below the background cut-off of 2.0% determined on 100 HPV negative cervical cytology samples. The difference in the percentage of cells overexpressing HPV E6, E7 mrna between the Progressor group and the Non-progressor group was statistically significant (P=0.008). Using a cut-off of 2.0%, HPV OncoTect correctly distinguished all Progressors from Non-progressors. Methods & Materials Results Results Liquid based cervical cytology specimens prospectively collected in PreservCyt (Cytyc, Marlborough MA) were submitted for cytology and an aliquot of cells were frozen in PreservCyt at -80 o C until analysis for HPV E6, E7 mrna expression in cells (HPV OncoTect, Invirion Diagnostics, Oak Brook IL) HPV E6, E7 mrna was performed using the HPV E6, E7 mrna ASR (Invirion Diagnostics, Oakbrook, IL). Briefly, 1 ml of cells in LBP were fixed and permeabilized in PermiFlow (Invirion Diagnostics, Oakbrook, IL) for 1 hour at ambient temperature. Following fixation and permeabilization, the cells were washed once in PBS, ph 7.4, pelleted by centrifugation at 400g, washed again in 2 standard saline citrate (SSC), and pelleted by centrifugation. HPV FISH for E6 and E7 mrna was performed by resuspending the cells in 100 µl hybridization mix (Invirion Diagnostics, Oakbrook, IL) and a cocktail of 5'- and 3'- labeled oligonucleotide probes (HPV OncoTect ASR, Invirion Diagnostics, Oakbrook, IL). Hybridization was performed at 37 C for 30 minutes and was followed by a 5-minute wash in 2 SSC and 0.1% Triton X-100 (ViroTect Hybridization Reagent Kit, Invirion Diagnostics, Oakbrook, IL) and a 15-minute wash in 0.1% SSC and 0.1% Triton X-100. The cells were resuspended in PBS, ph 7.4, with 2% fetal calf serum for analysis on an FC 500 flow cytometer (Beckman-Coulter, Fullerton, CA). Ectocervical cells (red) Non-Progressors Progressors HPV E6, E7 mrna WNL LSIL HSIL or SCC Samples Frozen in ThinPrep Patients Following and Re- Screened at 1.5 years and 3 years (Samples Frozen) % E6, E7 mrna Expression in Ectocervical Cells Figure 1. HPV E6, E7 mrna expression Non-progressors Progressors in frozen ectocervical cells from women with cervical disease (LSIL) that remains the same or regresses (non-progressors) Figure 3. Mean E6, E7 mrna expressing or disease that progresses (LSIL to cells in Progressors (right) and Non- HSIL). progressors (left). Boxes represent the 95% confidence interval. P=0.008 Patients n= Initial Diagnosis HPV E6, E7 mrna+ Cells P Non-Progressors 9 LSIL 1.60% Progressors 11 LSIL 5.20% Normal Control Cells (SeraCare, Milford MA)-1.4% Table 1. Patient statistics Figure 2. Study design schematic Conclusions Samples from the Progressor group had an average of 5.2% cells that overexpressed HPV E6, E7 mrna relative to HPV negative control cells. Samples from the Non-progressor group averaged 1.6% cells that overexpressed HPV E6, E7 mrna which is below the background cut-off of 2.0% determined on 100 HPV negative cervical cytology samples The difference in the percentage of cells overexpressing HPV E6, E7 mrna between the Progressor group and the Non-progressor group was statistically significant (P=0.008). Using a cut-off of 2.0% based on the mean of the Non-progressors plus the 95% confidence interval, HPV OncoTect correctly distinguished 10 out of 11 Progressors from Non-progressors. 38

39 Main clinical studies on HPV OncoTect (cont.) HPV IN HUMAN PATHOLOGY (PRAGUE) Quantification of Intracellular HPV E6/E7 mrna Expression Increases the Positive Predictive Value of Cervical Cancer Screening J.A. Lopez G-Asenjo 1 Bruce K. Patterson 2 1 Hospital Clínico U. San Carlos 2 Stanford University Current methods for HPV screening rely on the detection of L1 DNA from high risk genotypes (HRHPV). These assays have very high negative predictive values (~99%) and as such have been used to triage women to longer screening intervals. The literature has shown that the positive predictive value for precancerous and cancerous lesions is less than 50% for HPV DNA screening. The purpose of this study was to combine HPV DNA screening with HPV E6, E7 mrna detection in an effort to improve the overall performance of cervical cancer screening while potentially reducing the number of women requiring colposcopy. Liquid based cervical cytology specimens collected in either PreservCyt or SurePath were submitted for routing cytology, HPV HRDNA detection by Hybrid Capture 2 and HPV E6, E7 mrna quantification in cells. Results from these three analyses were compared to biopsy in 40 cases. The positive predictive value of HPV E6, E7 mrna quantification in cells was 86% which was greater than HPV DNA alone. The specificity was 96% based on 142 samples with normal cytology. There was a statistically significant difference in the percent of ectocervical cells expressing E6, E7 mrna in women with CIN2, CIN3, or cancer (mean 14.7%) compared to women with normal cytology (mean 1.4%) with a P<

40 Main clinical studies on HPV OncoTect (cont.) RELATIONSHIP BETWEEN HPV-ONCOTEC RESULTS AND VIRAL GENOTYPES DETERMINED BY PCR IN PERI AND POSTMENOPAUSAL WOMEN. González Rodríguez SP1, Henríquez Linares AE2, Sainz de la Cuesta R2, Ojeda Jabardo MD1, Cantarero García R1. 1. Gabinete Médico Velázquez. Madrid. Spain 2. Hospital Quirón. Madrid. Spain OBJECTIVE To establish the correlation between the over-expression of viral oncoproteins E6/E7 and high grade viral genotypes using the HPV-ONCOTECT method. DESIGN AND METHODS A sample of 98 peri/postmenopausal patients over 40 years old was selected among women referred to our Center for the realization of Oncotect Test. The selection was independent of the reasons for the referral (positive HPV test, colposcopic findings, risk factors for cervical premalignant lesions, patients choice, abnormal cytology or biopsy). HPV Oncotect was used to detect the over-expression of E6/E7 oncoproteins, after granting informed consent. A total of 110 tests were performed. Data was analysed using SPSS version 13 Figure 1. Contingency tables RESULTS 35,5 % of the sample was PCR positive to a specific HPV genotype, predominantly a high-risk oncogenic subtype. 34,5% had a positive Oncotect result (> 2.00 % overexpression), predominantly in smokers and high-parity women, and in patients with premalignant lesion in the cytology (Figure 1). We found a statistically significant difference of a positive Oncotect test between high risk and low risk oncogenic subtypes, but not in cases with co-infection with more than 2 genotypes. In several cases we found a positive Oncotect result with a negative viral PCR. This lack of association could be explained by a viral genomic deletions at de the integration phase making the virus undetectable by PCR. CONCLUSION We found significant correlation between different HPV genotypes, clustered as low risk or high risk oncogenic types and positive results in Oncotect, even considering high genotypes dispersion and small sample size. REFERENCES Narimatsu, R. and B.K. Patterson High-throughput cervical cancer screening using intracellular human papillomavirus E6 and E7 mrna quantification by flow-citometry. Am.J. Clin. Pathol.123: Herrington CS. Do HPV-negative cervical carcinomas exist?. J Pathol Sep;189(1):1-3. González Rodríguez SP, Hernández Aguado J.J., Ojeda Jabardo M.D. Relación entre el resultado del HPV-ONCOTECT y el genotipo viral determinado por PCR.In: Proceedings cdrom of the XXIX National Congress of the Spanish Society of Gynaecology & Obstetrics, Granada González Rodríguez SP, Hernández Aguado J.J., Ojeda Jabardo M.D Relación entre el resultado del HPV-ONCOTECT y los hallazgos citológicos en pacientes asintomáticas. PCR.In: Proceedings cd-rom of the XXIX National Congress of the Spanish Society of Gynaecology & Obstetrics, Granada

41 HPV Cervical cancer Screening tests HPV DNA vs. E6/E7 mrna tests Why to detect E6 and E7? HPV OncoTect HPV OncoTect: Clinical studies HPV OncoTect: Report and use algorithm Introduction of the HPV vaccines HPV OncoTect in primary care Conclusions Annex: media references 41

42 HPV OncoTect: reports 42

43 Algorithm for combined use of cytology and HPV OncoTect Cytology neg HPV OT neg Cytology neg HPV OT pos ASCUS / LSIL HPV OT neg ASCUS/ LSIL HPV OT pos HSIL HPV OT pos or neg Screen 3 years Repeat at 6-12 months Repeat at 12 months Colposcopy Biopsy/ Colposcopy Cytology neg HPV neg ASCUS HPV neg > ASCUS HPV neg Cytology HPV pos Screen at 3 years Repeat at 12 months Colposcopy Colposcopy Adapted of Wright et al Obstet Gynecol 103:307 43

44 HPV Cervical cancer Screening tests HPV DNA vs. E6/E7 mrna tests Why to detect E6 and E7? HPV OncoTect HPV OncoTect: Clinical studies HPV OncoTect: Report and use algorithm Introduction of the HPV vaccines HPV OncoTect in primary care Conclusions Annex: media references 44

45 The impact of HPV vaccines GSK and Merck have developed two vaccines against HPV. These vaccines are now being marketed in Spain under the name Gardasil (Merck) and Cervarix (GSK). However, these vaccines are limited to two high risk genotypes - 16 and 18 - representing only 65% of cervical cancer cases (Gardasil also includes two low risk types). Vaccines must not ever leave screening policies The vaccines only protect against two serotypes that cause cervical cancer (HPV 16-18). Only have proven effective in women who have not been in contact with the virus. In the case of Gardasil (quadrivalent) seroconversion is maximum at the age of 13 years, then it decreases with age. Since its introduction is very recent, the first results will not appear until the next 20 years. The screening will still be crucial to monitor the vaccines effectiveness and prevent the impact caused by other serotypes different than 16 and

46 The impact of HPV vaccines (cont.) With the introduction of vaccines and the necessary incidence decreasement of cervical cancer, cytology will diminish its sensitivity and predictive value. Therefore it becomes necessary to reconsider the primary screening algorithm. HPV tests are moving forward in their role as a first line test in the fight against cervical cancer, constituting a research line in a large number of ongoing studies. The algorithm that follows belongs to one of these studies. HPV Negative CONTROL Positive mrna Negative Positive Colposcopy Routine cervical screening with primary HPV testing and cytology triage protocol in a randomised setting. Kotaniemi- Talonen, L. (Finish Cancer Registry) BJC, 93, ,

47 HPV Cervical cancer Screening tests HPV DNA vs. E6/E7 mrna tests Why to detect E6 and E7? HPV OncoTect HPV OncoTect: Clinical studies HPV OncoTect: Report and use algorithm Introduction of the HPV vaccines HPV OncoTect in primary care Conclusions Annex: media references 47

48 What would HPV OncoTect offers in Primary Care? Decongesting of medicine specialized services: suggesting the possible gynaecological check-in in primary care, referring only to the specialist those patients at risk, implementation of HPV OncoTect with cytology will screen those patients who require specialized monitoring. Information highly relevant to the diagnosis and treatment rates of ASC_US cytology (atypia of squamous cells of undetermined significance) and LSIL (low squamous intraepithelial lesion): ASC-US: Cytology is unable to determine if there is a pathology or not, it is necessary to perform an HPV test and monitor the patient, which means repeated tests and visits to the specialist. HPV OncoTect provides more precise information to detect lesion progression, which confers the capability of screening those who should be referred to colposcopy. LSIL: Only 30% of Low-grade lesions are going to progress into severe injury thus requiring monituring and follow-up. HPV OncoTect is able to detect those in which the oncogenic activity is evident and show a high probability of progression. Efficiency in the screening: cytology with HPV Oncotect test would offer more effective information against cervical cancer. Screening every 3 years is now a real possibility in patients with negative cytology and negative HPV test; in this regard HPV OncoTect is the HPV test with greatest positive predictive value, thereby enhancing the safety of diagnosis. Taking as an example the approximately 460,000 cytologies that are done annually in primary care services in the Madrid (Spain) region, and assuming that 4% of them can result ASC-US / LSIL (13,800), statistically we know that only 16% of them would be positive for HPV OncoTect (2,070), which in the case of the other 11,730 women we could reduce the uncertainty and monitoring tests for them, saving time and money. 48

49 Example of deployment OncoTect HPV in primary care Objective: To establish a protocol for active prevention of cervical cancer Protocol: to be tested by HPV OncoTect test women who: AFTER CYTOLOGY After gynecological follow-up submit an abnormal cervical cytology (ASCUS, LSIL and HSIL JOINTLY WITH CYTOLOGY Have submitted identical abnormal cervical cytology (ASCUS, LSIL, HSIL) in one of the last two gynecological revisions. Undertake a review of gynecologic entry (ie, the first review of their gynecological lives or those who are more than 5 years without going to the gynecologist). Being older than 35 years, and not having undergone a test for HPV in the past three years. If both results turn out to be negative, they will not be repeated until three years later (as established by international consensus). Have undergone surgical treatment (biopsy / conization), as control post-treatment 6 months after the operation. 49

50 HPV Cervical cancer Screening tests HPV DNA vs. E6/E7 mrna tests Why to detect E6 and E7? HPV OncoTect HPV OncoTect: Clinical studies HPV OncoTect: Report and use algorithm Introduction of the HPV vaccines HPV OncoTect in primary care Conclusions Annex: media references 50

51 HPV OncoTect: Conclusions HPV OncoTect is the only test capable of detecting oncogenic activity from all HPV types (over-expression expression of E6/E7) Identifies persistent infections from all high-risk HPV types It doesn t t have a limit due to age of the patient The gynaecologist doesn t t need to modify its protocol Prevents unnecessary treatments and extra diagnostics Reduces social alarm produced by HPV infection 51

52 HPV Cervical cancer Screening tests HPV DNA vs. E6/E7 mrna tests Why to detect E6 and E7? HPV OncoTect HPV OncoTect: Clinical studies HPV OncoTect: Report and use algorithm Introduction of the HPV vaccines HPV OncoTect in primary care Conclusions Annex: media references 52