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

Abstract

SummaryThe efficacy of four methods to recover DNA from Papanicolaou (Pap)-stained archival cervical smears for optimal detection of human papillomavirus (HPV) DNA by GP5+/bioGP6+ polymerase chain reaction (PCR) was investigated. Two of the methods were based on proteinase K treatment and two based on treatment with guanidinium thiocyanate (GTC). The quality of the DNA as measured by PCR assays amplifying different sizes of the β-globin gene appeared to be superior for the GTC-based assays. Using competitive β-globin PCR assays, one of the GTC-based, assays, provisionally named High Pure PCR Template Preparation (HPPTP) assay, yielded by far the highest quantity of amplifiable DNA. It allowed the recovery of 2.2 × 10 5 to 3 × 10 5 genome equivalents in smears containing 5 × 10 5 to 20 × 10 5 nucleated cells, indicating a mean efficiency of 26% (range of 15-44%). In contrast, the other methods revealed markedly lower efficiencies varying from 1% to 10%. The use of the HPPTP assay as a reliable processing procedure was validated by demonstrating a complete agreement in HPV detection and 93% agreement in HPV typing between 39 archival Pap-stained and paired fresh-frozen cervical smears. This method was applied to 40 archival smears from ten cervical cancer 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 before 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 and diagnosis of cervical cancer was 12.0 ± 2.9 years. All subsequent smears were invariably positive for the same HPV type which was also found in the cervical cancer biopsy. In conclusion, the HPPTP assay provides a reliable and efficient means to extract DNA from Pap-stained archival cervical smears for the detection of HPV DNA by PCR and would be the method of choice for future HPV analysis of archival Pap-stained cervical smears.

Lesiones Preinvasoras De Cuello Uterino: Una Visión Actual

“…La ocurrencia de lesiones intraepiteliales de alto grado (NIE II/III) y de cán-cer cérvico-uterino invasor no puede ser posible sin la existencia previa de una infección persistente por subtipos de alto riesgo de virus papiloma humano (7,8,24) Esta simple, pero revolucionaria aseveración, llevó al desarrollo y evaluación de métodos sensibles y de aplicación clínica de detección del DNA viral (15,(36)(37)(38)(39).…”

“…La tipificación del DNA de virus de alto riesgo es un marcador muy sensible para mujeres en riesgo de neoplasia intraepitelial moderada y severa, así como de cáncer cérvico-uterino, sin embargo, el método tiene una menor especificidad que la citología convencional (37)(38)(39)(40). Su utilización ha sido aprobado como método anexo al tamizaje citológico en mujeres mayores de 30 años (41,42), también para complementar el estudio de citologías atípicas de significado incierto (39,43) y en el seguimiento (curación o persistencia) de lesiones preinvasoras tratadas con métodos ablativos (44).…”

High-risk human papillomavirus clearance in pregnant women: trends for lower clearance during pregnancy with a catch-up postpartum

“…In addition, individual high-risk HPV types were determined. This test has been described previously and has been clinically validated (Jacobs et al, 1997(Jacobs et al, , 2000.…”

The contribution of HPV18 to cervical cancer is underestimated using high-grade CIN as a measure of screening efficiency

“…Representative samples of the formalin-fixed biopsies were processed for PCR analysis (Walboomers et al, 1999;Jacobs et al, 2000). Amplification of a 209 base-pair fragment of the b-globin gene was performed to test the integrity of DNA (Roda Husman et al, 1994).…”