OC 01-02VALIDATION OF HPV DNA ARRAY GENOTYPING ASSAY WITH CERVICAL CANCER SAMPLES

11. Genotyping - Sequencing
A.P. Pesic 1, A.K. Krings 1, M.H. Hempel 2, A.M. Mathewos 3, J.K. Komp 4, D.H. Höfler 5, D.H. Holzinger 5, M.P. Pawlita 5, E.J.K. Kantelhardt 4, A.M.K. Kaufmann 1.
1Gynäkologische Tumorimmunologie, Charité Universitätsmedizin Berlin (Germany), 2AID/GenID Diagnostika, Strassberg (Germany), 3Radiotherapy Center, Addis Ababa University Hospital, Addis Ababa (Ethiopia), 4Institute for Medical Epidemiology, Biostatistics and Informatics, Martin Luther-University Halle, Germany (Germany), 5German Cancer Research Center (DKFZ), Heidelberg (Germany)

Background / Objectives

The purpose of this study was to evaluate the performance of HPV DNA Array for full genotyping of 18 high-risk (16,18,26,31,33,35,39,45,51,52,53,56,58,59,66,68,73,82) and 11 low-risk HPV types (6,11,40,42,44,54,67,69,70,85,97), by using 110 histology confirmed, HPV-DNA positive, cervical cancer samples pre-genotyped by multiplex Luminex-based hybridization assay following BS-GP5+/6+ PCR (BS-GP5+/6+ MPG).


Methods

HPV DNA Array is an E1-based PCR assay. Multiplex PCR is used for amplification of E1 gene sequences of 180bp length. The amplicons are detected and genotyped by reverse hybridization to immobilized DNA probes spotted as triplets in single 96 well-plate wells and read by AID EliSpot reader system and AiDOT software. Assay performance was assessed with 110 cervical cancer samples collected with Delphi screener cervico-vaginal lavage and stored in PreserveCyt. Ethical approval was given by the Institutional Review Board of Addis Ababa University, Ethiopia. Patients were recruited after giving informed consent at Tikur Anbessa teaching hospital, Addis Ababa. HPV detection accuracy was established by comparing HPV DNA Array to BS-GP5+/6+ MPG, as gold standard. BS-GP5+/6+ MPG is L1-based genotyping test. Multiplex PCR is used for amplification of L1 gene sequences of 150bp length.


Results

Concordance of HPV genotype related positivity identified by the two assays was 90%. We observed a complete genotype match in 64.5% (71/110), ≥1 HPV genotype match in 25.5% (28/110), with discordant BS-GP5+/6+ MPG positivity/HPV DNA Array negativity in 7.3% (8/110) and concordant HPV positivity with discrepant types in 2.7% (3/110). The HPV DNA Array showed sensitivity for overall HPV detection of 92.5% with sensitivity for detection of HPV-16: 93% (66/71), HPV-18: 83% (5/6), and HPV-45: 67% (8/12), compared to BS-GP5+/6+ MPG.


Conclusion

In most or all HPV DNA Array false negative HPV-16 (4/5 cases) and HPV-45 samples (4/4 cases), we observed a low HPV copy number in BS-GP5+/6+ MPG. Also in 7 out of 8 discordant samples (BS-GP5+/6+ MPG positive/HPV DNA Array negative), low MPG-Luminex signals were found, arguing for insufficient sample quality.

Overall, HPV DNA Array showed good sensitivity for HPV detection, and due to its technical simplicity and high-throughput potential, this method may be suitable for routine full genotyping in HPV screening.


References