Elsevier

Gynecologic Oncology

Volume 92, Issue 3, March 2004, Pages 873-880
Gynecologic Oncology

Physical state and expression of HPV DNA in benign and dysplastic cervical tissue: different levels of viral integration are correlated with lesion grade

https://doi.org/10.1016/j.ygyno.2003.11.035Get rights and content

Abstract

Objective. Human papillomavirus (HPV) infection is the most important event in the malignant transformation of human cervical epithelium. Several high-risk (HR-)HPV subtypes have been identified, which lead to CIN and subsequently to invasive carcinoma. The reason for this phenomenon is still unknown, but it seems to be related to the physical state of HPV DNA.

Methods. Digene HC II test was used to identify HR- and/or low-risk (LR-)HPV infections in cervical swabs of 275 women attending our clinic for routine cytological screening and/or colposcopy because of an abnormal Pap smear comprising low-grade squamous intraepithelial lesions (LGSIL) and high-grade SIL (HGSIL). Specific HR (16, 18, 31, 33, 52b, 58) and LR (6, 11) subtypes were characterized in cervical biopsies of 10 women with benign cellular changes and of 68 women with CIN I–III by the PCR-restriction enzyme method. The physical state of HPV DNA (episomal, mixed and integrated form) was analyzed by bi-dimensional (2D)-gel electrophoresis. In addition, mRNA expression of E6/E7 genes was analyzed by RT-PCR. Furthermore, the relative virus load was determined in nine selected cases. The physical state and transcriptional activity of HPV DNA were then correlated to histopathological results.

Results. LR-HPV infection [27 cases (9.8%)] and HR-HPV infection [121 cases (44%)] of cervical swabs were clearly correlated to the degree of SIL. Further HPV typing in cervical biopsies of 78 women showed that HPV6 and 11 were restricted to benign cellular changes, CIN I and II, whereas HPV16 and 18 were observed predominantly in CIN III/CIS (P = 0.01). No clear distribution pattern was observed for HPV31, 33, 52b and 58. Expression of HPV E6 and E7 transcripts was uniformly correlated with the different physical state of HPV DNA. Analyzing the physical state of these HPV subtypes, HPV6 and 11 could only be detected as an episomal form, independent of SIL grade. In normal epithelium and in CIN I and II, HPV16 and 18 were exclusively found in the episomal form. In CIN III/CIS, 15 of 30 cases of HPV16 (50%) and 16 of 17 cases of HPV18 (94%) were exclusively integrated into the host genome. Like HPV16/18, HPV31, 33, 52b and 58 were also present in the episomal form in normal epithelium and in CIN I and II, but were integrated in 80% of the CIN III/CIS (4/5) cases.

Conclusion. Absent integration of HPV16 DNA in some CIN III/CIS suggests that integration is not always required for progression early dysplastic lesions. In contrast, integration of HPV type 18 and others appears to be of major importance for the transforming efficacy of cervical dysplasia. The applied method represents a sensitive instrument to assess the physical state of HPV and is useful to predict the progression of disease.

Introduction

Specific genotypes of the human papillomavirus (HPV) are known to be the principal cause of cervical squamous intraepithelial lesions (SILs) and cervical cancer [1]. To date, more than 100 different HPV types have been characterized and, based on their potential to induce cervical epithelial malignant transformation, have been classified as low-risk (LR) and high-risk (HR) viruses [2], [3]. The entry of HPV into cervical host cells may be followed by three events: (1) viral DNA is maintained as an episome, thereby establishing a latent infection; (2) conversion of latent into productive infection is associated with the assembly of complete infective virions; (3) viral DNA is integrated into the host genome. It is now generally accepted that the integration of HPV DNA into the host genome and subsequent constitutive expression of the oncoproteins E6 and E7 represent two activation mechanisms for the progression of preinvasive lesions to cervical carcinoma [4], [5]. Integration of viral genomic sequences can cause deletions and/or a disruption of the E2 gene and result in a loss of its function as a regulator of viral gene expression [5]. This event is often followed by up-regulation of E6/E7 gene transcription. E6 and E7 proteins deregulate cell-cycle control through interaction with different cell proteins, for example, tumor suppressor gene products such as p53 and retinoblastoma protein (Rb), thereby initiating the transformation and immortalization of HPV-infected cells [6], [7].

Various studies have demonstrated the presence of integrated HPV16 and 18 genomes in the vast majority of cancers and in cell lines isolated from cervical malignancies [8], [9], [10], [11], [12], [13]. However, the role of the integration of viral DNA into the host genome as a major event leading to malignant transformation of dysplastic cervical epithelium is still discussed, as there have been several studies reporting only the presence of episomal forms of HPV16 DNA in preneoplastic and neoplastic cervical lesions. For example, Matsukura et al. [14] detected exclusively episomal forms of HPV16 DNA in infected cervical epithelium, albeit this form was present in at least 70% of investigated cervical malignancies. Similarly, Fuchs et al. [15] and Das et al. [16] reported the presence of viral HPV16 DNA in up to 30% of cervical carcinomas, and again, viral DNA was exclusively present in the episomal form.

To elucidate the role of HPV6, 11, 16, 18, 31, 33, 52b, 58 DNA integration and mRNA expression patterns in the transformational process of cervical epithelium in benign and dysplastic/preinvasive lesions of the uterine cervix, we used polymerase chain reaction (PCR) and bi-dimensional (2D)-gel electrophoresis to analyze the type-specific integration patterns of viral DNA. In addition, mRNA expression of E6/E7 genes was evaluated by a reverse transcriptase (RT)-PCR and correlated with HPV DNA integration.

We established an RT-PCR test (amplification of papillomavirus oncogene transcripts) that allows discrimination of HPV E6/E7 mRNAs derived from integrated and/or episomal viral genomes. In combination with virus load and Northern blot in certain cases, we defined and confirmed the prevalence of integrate-derived HR-HPV16, 18, 31, 33, 52b and 58 transcripts in cervical swabs from patients with CIN lesions in various stages of progression.

Section snippets

Specimens and patient population

Clinical samples obtained from 275 patients attending our clinic between February 1997 and November 2001 were considered suitable for the study. The ethics committee of the University of Vienna approved this study. Only women who had signed the informed consent form were enrolled. The study population was restricted to women of Caucasian origin undergoing their biennial or triennial routine cytological analysis and/or patients referred to our clinic for colposcopy because of an abnormal

Presence and typing of HPV DNA

To determine the physical state and expression pattern of HPV DNA in preneoplastic and neoplastic cervical lesions, clinical specimens were collected from 275 patients enrolled in the study. Swab specimens were tested for the presence of HPV infection by the HC II test. As shown in Table 1, among the total HPV DNA, the LR- and HR-HPV types were found in 148 patients (53.8%). The HPV detection rate ranged from 27.5% (28 of 102 patients) in women with normal Pap smears (within normal limits, or

Discussion

In the present study, we established a method to detect the physical state and the mRNA expression pattern of viral genomes of several LR- and HR-HPV types in benign and premalignant/preinvasive clinical samples obtained from 78 out of 275 patients. The persistence and progression of dysplastic cervical lesions are influenced by several factors such as lesion severity [21], type of HPV present in the epithelial cells [22], [23], the prevalence and amount of E6/E7 transcripts detected in

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