Comparative evaluation of RNA and DNA approach for HPV diagnosis

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1 Comparative evaluation of RNA and DNA approach for HPV diagnosis Dr. Cristina Lapucci Director of Geneticlab Laboratory and Specialist in Medical Genetics XII International Workshop Of Lower Genital Tract Pathology Rome, 5-7 March 2015

2 Molecular HPV Testing Uses Before new guidelines: HPV test used for follow up in ASCUS or L-SIL patients or in triage patients Following new guidelines: HPV test used as primary screening and the cytology should be done after

3 HPV DNA test VS Cytology Why HPV DNA test is better than cytology? Is more sensitive Is automated Is an earlier marker Critical point of the HPV DNA test: HPV DNA test could be positive in sample that would not develop cancer HPV DNA test detect episomal and integrated DNA To amplify HPV DNA, the region of detection should be intact Possible solution mrna HPV test

4 HPV RNA test VS HPV DNA test - #1 HPV RNA test is sensitive as HPV DNA test* BUT RNA HPV test is more specific than HPV DNA test* because mrna detect only replicating viruses (*) Comparing the performance of six human papillomavirus tests in a screening population. J. Cuzick. British Journal of Cancer (2013). INITIAL INFECTION WITH HPV VIRAL INTEGRATION OF HPV Viral DNA episomal Epithelial DNA HR infection mrna expression of E6 / E7

5 HPV RNA test VS HPV DNA test - #2 HPV RNA test amplify the most conserved E6/E7 sequence Expression of E6/E7 mrna is necessary for progression to cervical disease

6 Comparative evaluation of RNA and DNA approach for HPV diagnosis We analysed two different methods (HPV DNA test and HPV RNA test) with the aim to evaluate the performance of HPV RNA test and if is possible use HPV RNA test as primary screening

7 HPV RNA test with APTIMA assay (Hologic) identifies high-risk HPV infections by targeting E6/E7 mrna. 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 Sensitivity: ~100 copies / reaction represents the next generation in cervical cancer screening

8 The Study 1200 samples DNA (Ab analitica) RNA (APTIMA hologic) NEGATIVE 93,51% NEGATIVE 94,81%

9 HSIL + LSIL Patients SENSITIVITY > 10% HPV RNA Tests HPV DNA Tests Best identification of true positive subjects

10 HSIL samples Positive patients DNA (Ab analitica) RNA (APTIMA hologic) POSITIVE 95,96% POSITIVE 96,97% Best identification of true positive subjects

11 AGC samples AGC patients DNA (Ab analitica) RNA (APTIMA hologic) POSITIVE 0% POSITIVE 50% Poor performance Why? DNA test amplify L1 genomic sequence of HPV mrna test amplify the most conserved E6/E7 sequence

12 Conclusion HPV RNA test could be used as a unique primary screening test HPV DNA typing in combination with HPV RNA test could be performed for follow up of positive patients

13 Thank you for your attention! Dr. Marta Piovan Dr. Andrea Apolloni Dr. Cristina Lapucci Dr. Emilio Vicari Dr. Cinzia Businaro Dr. Lorenzo Dalmonego Dr. Tiziana Bartolotti Dr. Enza Costantino

14 Dr. Cristina Lapucci Director of Geneticlab Laboratory and Specialist in Medical Genetics Geneticlab srl Tel Fax Registered Office: Via Roveredo, 20/B Pordenone (PN) Italy Laboratory: Via Corte Ferrighi, 16/B Noventa Vicentina (VI) Italy Web: