Analysis by multiplex PCR of the physical status of human papillomavirus type 16 DNA in cervical cancers

Integration of human papillomavirus (HPV) DNA occurs early in cancer develo pment and is an important event in malignant transformation of cervical can cer. Integration of HPVs preferentially disrupts or deletes the E2 open rea ding frame, which results in the loss of its expression, The preferential d isruption of the E2 gene causes the absence of the E2 gene sequences in the PCR product following integration. Twenty-two carcinomas positive for HPV type 16 (HPV-16) DNA mere first tested far the disruption of the E2 gene by PCR. A specific fragment of the E2 gene was not amplified in 10 cases, sug gesting integration of HPV DNA into the host genome. Next, multiplex PCR fo r the RPV E2 and E6 genes was carried out in the remaining 12 cases. Copy n umbers of both genes should he equivalent in episomal forms, while the E2 g ene copy number will be smaller than that for E6 following the preferential disruption of the E2 gene in concominant forms. Although relative ratios o f HPV E2 to E6 PCR products (E2/E6 ratios) ranged from 1.40 to 2.34 in 10 o f 12 cases, multiplex PCR products from 2 cases displayed extremely low rat ios of 0.69 and 0.61, Southern blot hybridization with an HPV-16 probe reve aled that only in these two cases was both episomal and integrated HPV DNA being carried simultaneously. Thus, multiplex PCR for the E2 and E6 genes o f HPV-16 DNA following PCR for the E2 gene can distinguish the pure episoma l form from a mixed form of episomal and integrated HPV DNA, Clinical appli cation of this technique mill help researchers to understand the implicatio n of the integration of HPV DNA for cervical carcinogenesis and cervical ca ncer progression.