J.Gras HPV mRNA Eurogin 2010

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J.Gras HPV mRNA Eurogin 2010

  1. 1. Molecular diagnostics of HPV infection using E6 and E7 mRNA detection: experience in 302 patients Jeremie M. Gras, M.D. Clinical and Molecular laboratory Cliniques Sud Luxembourg, Arlon, Belgium bioMérieux Satellite Symposium Eurogin Congress, Monte-Carlo, 18th February 2010
  2. 2. Molecular diagnostics of HPV infection using E6 and E7 mRNA detection: experience in 302 patients Plan <ul><li>Background </li></ul><ul><li>Patients and methods </li></ul><ul><li>Results </li></ul><ul><li>Discussion </li></ul><ul><li>Conclusion </li></ul>
  3. 3. Molecular diagnostics of HPV infection using E6 and E7 mRNA detection: experience in 302 patients Plan <ul><li>Background </li></ul><ul><li>Patients and methods </li></ul><ul><li>Results </li></ul><ul><li>Discussion </li></ul><ul><li>Conclusion </li></ul>
  4. 4. 1. Background Cervical cancer <ul><li>Cervical cancer is the second most common type of cancer occurring in women </li></ul><ul><li>In 2005, cervical cancer was responsible for more than 250000 deaths </li></ul><ul><li>Human Papillomavirus (HPV) is responsible for 99% of cervical cancer </li></ul><ul><li>Source: WHO </li></ul>
  5. 5. 1. Background HPV mRNA test rationale <ul><li>In the majority of cases, HPV infections regress spontaneously within two years 1 </li></ul><ul><li>Only a small percentage progress to high grade lesions </li></ul><ul><li>E6 and E7 proteins are viral oncogenes that are continuously expressed by malignant phenotypes </li></ul><ul><li>Detection of E6 and E7 mRNA provide more accurate prediction of cancer risk than DNA testing ² </li></ul>1 Schiffman M, Castle PE, Jeronimo J et al. Lancet 2007 ; 370:890-907. 2 Cattani P, Siddu A, D’Onghia S et al. J Clin Microbiol 2009 ; 47(7):2136-41.
  6. 6. 1. Background EasyQ® HPV mRNA test <ul><li>HPV (16,18,31,33,45) E6 and E7 mRNA detection and typing using NASBA principle </li></ul><ul><li>NASBA: Nucleic Acid Sequence Based Amplification ³ </li></ul>3 Compton J. Nature 1991 ; 350(6313): 91-2. Reverse Transcriptase Taq Polymerase Reverse Transcriptase RNAse H T7 polymerase Enzymes + +++ Rapidity Thermal cycles Isothermic (41°C) Conditions RT-PCR NASBA
  7. 7. Molecular diagnostics of HPV infection using E6 and E7 mRNA detection: experience in 302 patients Plan <ul><li>Background </li></ul><ul><li>Patients and methods </li></ul><ul><li>Results </li></ul><ul><li>Discussion </li></ul><ul><li>Conclusion </li></ul>
  8. 8. 2. Patients and Methods Lab description <ul><li>600 files/ day, 24/7 </li></ul><ul><li>40 % in patients; 60 % out patients (including GPs) </li></ul><ul><li>Pre-analytic automation (since May 2009) </li></ul><ul><li>Advanced QC solutions and protocols for clinical chemistry </li></ul><ul><li>New infrastructure for molecular diagnostics (April 2008) </li></ul><ul><li>ISO 15189 accredited for molecular biology (mandatory according to Belgian law) </li></ul>
  9. 9. 2. Patients and Methods ISO 15189 accreditation at Cliniques Sud Luxembourg <ul><li>20 molecular tests have been accredited according to ISO 15189 norm </li></ul><ul><li>Accreditation BELAC N° 370- MED , granted from 23/06/2009 to 22/06/2012 </li></ul><ul><li>HPV mRNA test is an accredited test in CSL Arlon Lab </li></ul>
  10. 10. 2. Patients and Methods Patients <ul><li>Study interval: from September 2008 to May 2009 </li></ul><ul><ul><ul><li>332 HPV mRNA tests performed in 302 patients </li></ul></ul></ul><ul><ul><ul><li>30 tests were repeated for follow up of HPV disease </li></ul></ul></ul><ul><li>Results will focus on the 302 primary HPV tests </li></ul><ul><ul><ul><li>Mean age was 32.7 years old (15.8 to 83 years old) </li></ul></ul></ul><ul><ul><ul><li>Cytology results: </li></ul></ul></ul>
  11. 11. 2. Patients and Methods Methods 1. Cytology first performed, then PreservCyt® vial was transmitted to our lab for preparation (40 mins) 2. Extraction on the easyMAG ® (40 mins) 3. Incubation (40 mins) 4. HPV mRNA amplification and detection using the EasyQ ® test ( NASBA ) (120 mins) TAT for 24 samples: 4 hours
  12. 12. Molecular diagnostics of HPV infection using E6 and E7 mRNA detection: experience in 302 patients Plan <ul><li>Background </li></ul><ul><li>Patients and methods </li></ul><ul><li>Results </li></ul><ul><li>Discussion </li></ul><ul><li>Conclusion </li></ul>
  13. 13. 3. Results Overall Low rate of invalid results
  14. 14. 3. Results ASCUS ASCUS: 66% have a negative HPV test
  15. 15. 3. Results L-SIL L-SIL: 56% have a negative HPV test
  16. 16. 3. Results H-SIL Negative samples have been retested with a DNA method N.B: HPV 61 and 81 are not HR HPVs
  17. 17. 3. Results Unclear cytology (N=48) 10 active infections with HR HPVs
  18. 18. 3. Results Specificity and NPV <ul><li>Calculated by comparison between HPV mRNA test and cytology results </li></ul><ul><li>Specificity: 70.1 % </li></ul><ul><li>Negative Predictive Value: 96.5 % </li></ul><ul><li>Limitations: cervical cytology is a poor gold standard </li></ul>
  19. 19. Molecular diagnostics of HPV infection using E6 and E7 mRNA detection: experience in 302 patients Plan <ul><li>Background </li></ul><ul><li>Patients and methods </li></ul><ul><li>Results </li></ul><ul><li>Discussion </li></ul><ul><li>Conclusion </li></ul>
  20. 20. 4. Discussion Study findings (1/2) <ul><li>EasyQ ® mRNA test gave a low number of invalid results (4%) </li></ul><ul><ul><ul><li>Due to poor sample or bad RNA quality </li></ul></ul></ul><ul><ul><ul><li>Re-testing allowed to find a valid result in most cases </li></ul></ul></ul>a) Invalid results <ul><li>In patients with ASCUS, 66 % of HPV mRNA tests were negative </li></ul><ul><li>In patients with L-SIL, 56 % of HPV mRNA tests were negative </li></ul><ul><li>Overall, negative predictive value was 96.5 % </li></ul>b) Results in ASCUS and L-SIL
  21. 21. 4. Discussion Study findings (2/2) <ul><li>48 samples (17 %) had an unclear cytology screening results </li></ul><ul><li>In several cases, another type of infection was suspected </li></ul><ul><li>HPV mRNA test allowed to find 10 active infections with HR-HPVs </li></ul>c) Interest in patients with unclear cytology results <ul><li>Patients with H-SIL are referred to colposcopy, irrespective of mRNA test result </li></ul><ul><li>H-SIL samples with negative mRNA test were re-tested with a DNA test: in all cases, it was positive for HPV subtypes that technically can not detected by HPV EasyQ ® mRNA test </li></ul>d) Patients with H-SIL
  22. 22. Molecular diagnostics of HPV infection using E6 and E7 mRNA detection: experience in 302 patients Plan <ul><li>Background </li></ul><ul><li>Patients and methods </li></ul><ul><li>Results </li></ul><ul><li>Discussion </li></ul><ul><li>Conclusion </li></ul>
  23. 23. 5. Conclusion <ul><li>Combination of easyMAG ® automated extraction and EasyQ® mRNA test proved to be a fast and reliable method to detect and type HR HPVs in cervical samples </li></ul><ul><li>It detects molecular markers that are closely related to cancer risk </li></ul><ul><li>Integration of HPV mRNA detection in screening algorithms could reduce unneeded additional investigations in many patients with ASCUS or L-SIL </li></ul><ul><li>It allows to detect active HPV infections in patients with unclear cytology results </li></ul>
  24. 24. Thanks Molecular Biology Laboratory, Cliniques Sud Luxembourg ARLON, Belgium Nicolas Hougardy Head Molecular Lab Pierre Goffinet Lab Director Brigitte Léger Nathalie Pinon Bernadette Legère MLTs Andrée Lemaire Françoise Soblet Maggy Conrardy
  25. 25. Thanks And thank YOU for your attention !
  26. 26. Molecular diagnostics of HPV infection using E6 and E7 mRNA detection: experience in 302 patients Jeremie M. Gras, M.D. Clinical and Molecular laboratory Cliniques Sud Luxembourg, Arlon, Belgium bioMérieux Satellite Symposium Eurogin Congress, Monte-Carlo, 18th February 2010

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