Human papillomaviruses : role in cervical dysplasia and carcinoma, and use as molecular risk marker for progression

Abstract

The general aim of these studies was to define the role of infection with oncogenic human papillomaviruses (HPV) for development of cervical dysplasia and to obtain early molecular markers for progression to carcinoma.

The results of cytological screening of 500 women in the Stockholm Gynaecologic Health Control were compared with HPV detection by polymerase chain reaction (PCR) and in situ hybridisation (ISH). Most cases with cytological atypia were found to be HPV positive by PCR, and there was a good agreement between PCR and ISH. The oncogenic HPV type 16 was most common (23.4%), followed by the oncogenic types 31 (12.5%) and 18 (10.9%). The general HPV prevalence was 11.1-15.7%, and for atypia the corresponding figure was 2.5%. A 5-year follow-up study of some of these women showed a clearance rate of over 90%.

Cell samples from 476 women being in Stockholm were analysed by PCR to obtain information regarding the frequency of the most common oncogenic and non-oncogenic HPV types found in samples from patients having different grades of dysplasia (cervical intraepithelial neoplasia; CIN I-III). There was an increase in the total frequency of HPV from 69% in "normal" (no present atypia) to 71% in CIN I, 80% in CIN II and 85% in CIN III patients. There was also a shift of the frequencies of HPV types from "normal", having 21% non-oncogenic (HPV6 and 11) and 13% HPV16, to CIN I with 14% non-oncogenic and 18% HPV16, CIN II with 9% non-oncogenic and 21% HPV 16 and CIN III with 1 % non-oncogenic and 46% HPV 16. This underlines the possible role of infection with oncogenic HPV types, and especially HPV 16, for the development of severe dysplasia.

Using multiple PCR primer sets in order to optimise the detection of HPV in invasive cervical carcinomas, we found in a series of 355 cases that 98% were HPV positive.

In order to find early molecular markers for tumour progression, 158 cervical carcinoma cases positive for HPV16 only, were analysed by PCR for possible integration of the viral genome into the cellular chromosomes. Disruption of the viral E I and/or E2 reading frames, indicating such integration, was found in 23 and 29% of the cases, respectively. From these studies, it would seem that a majority of the cases contain episomal copies of HPV16, with intact E1/E2 reading frames. The sites of disruption were mapped in greater detail, and these were almost equally distributed over the 5', middle or 3' end of these reading frames, although, there was some preponderance for disruptions in the 5' end. Patients with carcinomas showing E1/E2 disruptions had a shorter survival time than those without such changes.

A more detailed analysis, using a new technique (rliPCR) with inverse PCR and analysis of flanking human sequences, showed that deletion/integration was in fact present in the majority of cervical carcinomas (80%), but the simultaneous occurrence of episomes had masked their detection with the ordinary PCR technique. However, evidence of integration was absent in all but one of the investigated CIN III lesions.

Mapping of integration of HPV33 in patients classified as CINIII and the cervical carcinoma samples showed that integration was at least as frequent in CIN III as in carcinomas. So, for HPV type 33, in contrast to type 16, integration may be an early event in neoplastic progression.

Integration sites for HPV16 in the cellular chromosomes were mapped in cervical carcinomas by using inverse-PCR together with cloning and fluorescence in situ hybridisation techniques. Such sites were located to chromosomes 1, 3q28, 6p25, 11p13, 13q22 (SiHa cells) and 18q22.