A simplified and reliable HPV testing of archival Papanicolaou-stained cervical smears: application to cervical smears from cancer patients starting with cytologically normal smears

The efficacy of four methods to recover DNA from Papanicolaou (Pap)-stained archival cervical smears for optimal detection of human papillomavirus (HP V) DNA by GP5+/bioGP6+ polymerase chain reaction (PCR) was investigated. Tw o of the methods were based on proteinase K treatment and two based on trea tment with guanidinium thiocyanate (GTC). The quality of the DNA as measure d by PCR assays amplifying different sizes of the beta-globin gene appeared to be superior for the GTC-based assays. Using competitive beta-globin PGR assays, one of the GTC-based, assays, provisionally named High Pure PCR Te mplate Preparation (HPPTP) assay, yielded by far the highest quantity of am plifiable DNA. It allowed the recovery of 2.2 x 10(5) to 3 x 10(5) genome e quivalents in smears containing 5 x 10(5) to 20 x 10(5) nucleated cells, in dicating a mean efficiency of 26% (range of 15-44%). In contrast, the other methods revealed markedly lower efficiencies varying from 1% to 10%. The u se of the HPPTP assay as a reliable processing procedure was validated by d emonstrating a complete agreement in HPV detection and 93% agreement in HPV typing between 39 archival Pap-stained and paired fresh-frozen cervical sm ears. This method was applied to 40 archival smears from ten cervical cance r patients (selected from a group of 200 patients) which had a history of 3 -6 smears with the first smear being Pap 1 or 2 taken at least 5 years befo re cancer was diagnosed. The average time period between the first Pap 1/2 smear that contained the same HPV type as in the corresponding carcinoma an d diagnosis of cervical cancer was 12.0 +/- 2.9 years. All subsequent smear s were invariably positive for the same HPV type which was also found in th e cervical cancer biopsy. In conclusion, the HPPTP assay provides a reliabl e and efficient means to extract DNA from Pap-stained archival cervical sme ars for the detection of HPV DNA by PCR and would be the method of choice f or future HPV analysis of archival Pap-stained cervical smears. (C) 2000 Ca ncer Research Campaign.