Human DNA decays faster with time than viral dsDNA: an analysis on HPV16 using pathology archive samples spanning 85 years

Abstract

Background: Quality of the nucleic acids extracted from Formalin Fixed Paraffin Embedded (FFPE) samples largely depends on pre-analytic, fixation and storage conditions. We assessed the differential sensitivity of viral and human double stranded DNA (dsDNA) to degradation with storage time. Methods: We randomly selected forty-four HPV16-positive invasive cervical cancer (ICC) FFPE samples collected between 1930 and 1935 and between 2000 and 2004. We evaluated through qPCR the amplification within the same sample of two targets of the HPV16 L1 gene (69 bp, 134 bp) compared with two targets of the human tubulin-β gene (65 bp, 149 bp). Results: Both viral and human, short and long targets were amplified from all samples stored for 15 years. In samples archived for 85 years, we observed a significant decrease in the ability to amplify longer targets and this difference was larger in human than in viral DNA: longer fragments were nine times (CI 95% 2.6-35.2) less likely to be recovered from human DNA compared with 1.6 times (CI 95% 1.1-2.2) for viral DNA. Conclusions: We conclude that human and viral DNA show a differential decay kinetics in FFPE samples. The faster degradation of human DNA should be considered when assessing viral DNA prevalence in long stored samples, as HPV DNA detection remains a key biomarker of viral-associated transformation.