Semiquantitative analysis of human papillomavirus DNA in cervical intraepithelial neoplasia by a differential polymerase chain reaction

Abstract

A differential polymerase chain reaction (PCR), which takes account of both the human papillomavirus (HPV) type and the amount of viral DNA, was applied to 100 cervical intraepithelial neoplasias (CIN) in order to improve the characterisation of the lesions. One hundred women with cytological smears suggestive of CIN I-III and a colposcopically guided punch biopsy were studied. DNA was phenol-extracted and a consensus PCR with L1 primers and a type-specific PCR for HPV-16 and-18 E6 were performed in parallel. The different sensitivity of the PCR methods allowed a semiquantitative analysis of viral DNA. HPV analysis was possible in all 100 very small cervical punch biopsies. Of the lesions 79% were HPVpositive. The percentage of HPV-16-positive lesions increased significantly (P 0.001) with the degree of severity of CIN (CIN I: 46%, CIN II: 63%, CIN III: 85%). The percentage of CIN with a high viral load increased also with the grade of CIN (CIN I: 8%, CIN II: 33%, CIN III: 44%). Cytologically positive lesions (Pap IIID or IVa) had significantly more frequently (p 0.02) a high viral load: 39% vs. 10% of cytologically false negative lesions. In conclusion, type-specific PCR had a very high sensitivity in the detection of HPV-DNA also in cytologically false negative CIN. The addition of the less sensitive consensusPCR allowed a semiquantitative analysis of the viral copy number. The higher amount of viral DNA in cytologically positive lesions, which may correspond to a higher rate of proliferation, reflects a possible role of the viral load in the progression of CIN. In clinical practice, differential HPVPCR could help to improve the management of CIN.