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Clinical Cancer Research Vol. 10, 3059-3063, May 1, 2004
© 2004 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

DNA Aneuploidy and Integration of Human Papillomavirus Type 16 E6/E7 Oncogenes in Intraepithelial Neoplasia and Invasive Squamous Cell Carcinoma of the Cervix Uteri

Peter Melsheimer1, Svetlana Vinokurova2, Nicolas Wentzensen2, Gunther Bastert1 and Magnus von Knebel Doeberitz2

1 Department of Obstetrics, Gynecology and Gynecological Oncology, Medical School, and 2 Institute of Molecular Pathology, Department of Pathology, University of Heidelberg, Heidelberg, Germany


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Increasingly deregulated expression of the E6-E7 oncogenes of high-risk human papillomaviruses (HR-HPVs) has been identified as the major transforming factor in the pathogenesis of cervical dysplasia and derived cancers. The expression of these genes in epithelial stem cells first results in chromosomal instability and induces chromosomal aneuploidy. It is speculated that this subsequently favors integration of HR-HPV genomes into cellular chromosomes. This in turn leads to expression of viral cellular fusion transcripts and further enhanced expression of the E6-E7 oncoproteins. Chromosomal instability and aneuploidization thus seems to precede and favor integration of HR-HPV genomes.

Experimental Design: To prove this sequential concept, we analyzed here the sequence of events of DNA aneuploidization and integration in a series of HPV-16-positive cervical dysplastic lesions and carcinomas. Eighty-five punch biopsies of HPV-16-positive cervical lesions (20 CIN1/2, 50 CIN3, and 15 CxCa) were analyzed for DNA ploidy by DNA flow cytometry and for integration of HPV E6/E7 oncogenes using the amplification of papillomavirus oncogene transcripts assay, a reverse transcription-PCR method to detect integrate-derived human papillomavirus oncogene transcripts.

Results: DNA aneuploidy and viral genome integration were both associated with increasing dysplasia (P < 0.001, {chi}2 test for trend). In addition, DNA aneuploidy was associated with increased viral integration (P < 0.01, Fisher’s exact test). Nineteen of 20 (95%) lesions with integrated viral genomes had aneuploid cell lines; however, only 19 of 32 (59%) lesions with aneuploid cell lines had integrated viral genomes.

Conclusions: These data support the hypothesis that aneuploidization precedes integration of HR-HPV genomes in the progression of cervical dysplasia. Accordingly, deregulated viral oncogene expression appears to result first in chromosomal instability and aneuploidization and is subsequently followed by integration of HR-HPV genomes in the affected cell clones.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Genetic instability is a hallmark of most malignant tumors. It occurs early in the development of precancers and enables them to develop a growing reservoir of proliferating cells that stepwise acquire mutations that permit unrestricted growth of cancer cells (1) . In most cancers, genetic instability is achieved by alterations of the mitotic spindle apparatus that usually are caused by multipolar mitoses and an increasing disequilibrium in distribution of the chromosomes. Because of the severe numerical and structural changes of the chromosomes, this type of genetic instability is referred to as chromosomal instability (2) . Because of the acquired increasing disturbances of cell cycle regulation, resistance to apoptosis, and vascularization, these cells might expand locally and create an increasing reservoir of cells with an abnormal structure and number of their chromosomes. The structural and numerical chromosomal changes result in an increasing shift of the overall DNA content of the cells, a phenomenon commonly referred to as aneuploidy. In cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (CxCa), DNA ploidy estimation has been established as a prognostic factor that allows to estimated the relative progression risk into more advanced lesions (3, 4, 5) .

Persistent infection with high-risk types of the human papillomavirus (HPVs) are the major risk factor for the development of cervical cancer (6) . Two viral genes, E6 and E7, are continuously expressed in high-risk types of the HPV-transformed cells, and their expression is required to induce and maintain the neoplastic phenotype of cervical cancer cells (7) . The E6-E7 gene products cooperate in disturbing cell cycle regulation, preventing apoptosis (8 , 9) , and inducing multiple mitotic aberrations, chromosomal instability, and nuclear aneuploidy (10 , 11) . Thus, cell clones that express the viral E6 and E7 oncogenes undergo chromosomal instability and rapidly develop aneuploidy.

Cervical cancer emerges from a series of histologically well-defined precursor lesions, referred to as CIN grades 1–3. The majority of low-grade dysplastic lesions regress spontaneously, and only a small percentage of lesions progress to severe dysplasia and finally cervical cancer (12 , 13) . The current view of cervical carcinogenesis suggests that in the initial events, high-risk types of the HPV-infected epithelial stem cells undergo specific changes that overcome the tight transcriptional control of viral gene expression in normal nontransformed epithelial stem cells (14) . Inactivation of these cellular control functions permits deregulated transcription of the viral oncogenes E6 and E7 from episomal HPV genomes that in turn confers chromosomal instability and provokes an increasing aneuploidization of the respective cells (11 , 15) . The increasing structural changes support the integration of foreign extra-chromosomal HPV genomes into chromosomes of the host cell. Some of the integrated genome fragments are transcribed, resulting in hybrid mRNA molecules that encompass viral sequences encoding the E6 and E7 gene products at the 5'-end and heterogeneous cellular sequences at the 3'-end (16) . HPV DNA integration seems to be randomly distributed over the human genome with a preference for unstable chromosomal areas (17) . Upon integration, intrinsic viral repressors of gene expression are lost (18) , and cellular regulatory factors can additionally enhance HPV oncogene expression (19) . Cotranscription of viral genes with cellular sequences contributes to the stability and thereby also transforming activity of the respective transcripts (20 , 21) . Thus, cells with integrated HPV genomes that express integrate-derived papillomavirus oncogene transcripts (iPOTs) seem to gain strong growth advantages and are preferentially selected for clonal neoplastic outgrowth (22) . This concept implies that aneuploidization occurs before integration of the viral genome in a sequential pathogenetic model. In recent in vitro studies, this sequential series of events has been demonstrated for high-risk types of the HPV-transformed keratinocytes (23 , 24) . To test whether this model is also valid for naturally occurring lesions, we have investigated the time course of aneuploidization and viral genome integration during the progression of cervical precancerous lesions.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Sampling and Preparation.
Eighty-five tissues samples, which were obtained from patients with HPV-16-positive CIN1/2 (n = 20), CIN3 (n = 50), and CxCa (n = 15) at the Colposcopy Clinic of the Women’s Hospital of the University of Heidelberg, were included in the study. They were part of colposcopically guided punch biopsies, which were taken for diagnostic purpose after written consent from suspicious lesions of the cervix uteri. One part was used for preparation of H&E-stained slides. The remaining part was shock frozen in liquid nitrogen and stored at –70°C. Fresh frozen sections were prepared before cytometric DNA analysis, HPV analysis and amplification of papillomavirus oncogene transcripts-PCR to confirm that tissues indeed contained the expected dysplastic lesions.

Flow Cytometric DNA Analysis.
To determine the cellular DNA content, a standard protocol was applied (25) . The tissues were minced and incubated in a detergent solution (0.1 M citric acid and 0.5% Tween 20; Serva, Heidelberg, Germany) with gentle shaking for 20 min at room temperature. After adding six volumes of a staining solution (0.4 M disodium hydrogen phosphate and 5 µM 4',6-diamidino-2-phenylindole; Partec, Münster, Germany), this mixture was stored for 24 h at room temperature before additional analysis. A PAS II flow cytometer (Partec) was used to analyze the samples. Lymphocytes were used to calibrate the system. The maximum permitted coefficient of variation for calibration was 2%. At least 10,000 cells were counted in each sample. DNA histogram cell cycle analysis was performed as described before (26) , using Multicycle software (Phoenix Flow System, San Diego, CA). The G0-G1, S, and G2-M phases of the cell cycle were calculated. Cases were regarded as acceptable for analysis if the coefficient of variation of the G0-G1 peak was <7.0. Lesions were classified as diploid, tetraploid, or aneuploid (Fig. 1)Citation . If two distinct G0-G1 peaks were present with a DNA index of >1.15 (each containing >10% of total cell population), the histogram was considered aneuploid. By convention, the first G0-G1 peak (on the far left) represented the diploid peak. Diploid and tetraploid lesions were classified as nonaneuploid. Histogram patterns were termed diploid if there was a single G0-G1 peak. A histogram was classified as tetraploid only if a large peak in G2-M (DNA index of 1.80–2.2) made up >20% of the total curve and was also associated with a corresponding 8 N peak.



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  		Fig. 1. Aneuploid DNA histograms of two CIN3 lesions with integrated human papillomavirus type 16 E6/E7 oncogenes. Histograms were generated by flow cytometry from fresh frozen punch biopsy tissues after cell enucleation and staining with the fluorescent dye 4',6-diamidino-2-phenylindole according to a standardized protocol. A, aneuploid DNA histogram demonstrating two clones with a proportion of aneuploid cells and a DNA index of 53.1 and 1.13%, respectively. B, aneuploid DNA histogram demonstrating three clones with a proportion of aneuploid cells and DNA indices of 15.4%/1.66 and 41.1%/1.88, respectively. By convention, the first peak (on the far left) represents the diploid peak.

 
HPV Analysis.
RNA from frozen tissues was isolated using a RNA isolation kit (ToTALLY RNA kit; Ambion, Inc., Austin, TX) as recommended by the supplier. DNA was extracted using an additional protocol for simultaneous extraction of RNA and DNA (Technical Bulletin 161; Ambion, Inc.).

Typing of HPV DNA was performed as previously described, using a standardized general primer mediated PCR system (GP5+/GP6+) with the colorimetric enzyme immunoassay read out system (27) .

For amplification of papillomavirus oncogene transcripts, total RNA (1 µg) from HPV-16-positive probes was reverse transcribed and amplified using a protocol, as reported in detail previously (16) . The final PCR products were electrophoresed in 1.2% agarose gels, blotted onto nylon membranes (Hybond N+, Amersham Life Science, Buckinghamshire, United Kingdom), and hybridized with a HPV-16 E7-specific probe. Labeling and detection of the probes was performed with the enhanced chemiluminescence oligo-labeling and detection kit (Amersham Pharmacia Biotech, Freiburg, Germany) according to the manufacturer’s instructions. Amplimeres, which displayed a different size than the major E7-E1^E4 episomal transcript (~1050 bp in length for HPV-16), were suspected to be derived from integrated HPV genomes (Fig. 2)Citation . To confirm specificity, PCR products of interest were excised from the gel, extracted using the Qiagen Gel Extraction kit (Qiagen, Hilden, Germany), and subjected to direct DNA sequence analysis. Sequence data were analyzed and compared with public data bases using the BLAST analysis program (National Center for Biotechnology Information).



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  		Fig. 2. Amplification of papillomavirus oncogene transcripts (APOT) in three human papillomavirus type16-positive CIN3 samples. The PCR products of ~1050 bp represent the abundant episomal transcripts (ePOTs). Amplimeres with a different size represent integrate-type transcripts (iPOTs). Lane 1: CIN3 with positive detection of both ePOTs and iPOTs. Lane 2: CIN3 with positive detection of iPOTs. Lane 3: CIN3 with positive detection of ePOTs.

 
Statistical Analysis.
For statistical analysis, we used the SPSS software, version 10 (SPSS, Munich, Germany). Comparisons yielding P of <0.05 were considered to be statistically significant. Applied statistical tests were two sided.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Prevalence of DNA Aneuploidy and Transcription of Integrated HPV-16 E6/E7 Oncogenes.
Eighty-five HPV-16 positive lesions, including 20 CIN1/2, 50 CIN3, and 15 invasive squamous cell carcinomas, were analyzed in this study. The age of patients from whom the samples were obtained was 33.1 years (median) with 95% confidence interval of 30.9–34.9 years and range from 23 to 85 years. DNA aneuploidy was detected by flow cytometry analysis in 32 of 85 cervical biopsies (Table 1)Citation . The percentage of aneuploid cell clones ranged from 20% in the low grade lesions (CIN 1) to 32% in the high grade lesions (CIN2/3) and to 80% in the invasive cervical carcinomas. Prevalence of DNA aneuploidy was thus significantly linked to degree of neoplastic progression (P = 0.001, {chi}2 test for trend). However, prevalence of DNA aneuploidy was not influenced by other factors as, for example, women’s age.


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  		Table 1 Prevalence of DNA aneuploidy and transcripts of integrated human papillomavirus type 16 (HPV)-E6/E7 oncogenes in HPV-16-positive CIN1/2, CIN3, and cervical squamous cell carcinoma (CxCa) tissue samples

 
Transcripts derived from integrated HPV-16 E6/E7 oncogenes were detected in 20 of 80 samples (Table 1)Citation . Here again, a close correlation between the grade of the lesions and the presence of iPOTs was observed. In the group of low-grade lesions (CIN1), only 5% expressed iPOTs, whereas in high-grade lesions (CIN 2/3), 14% expressed iPOTs. In cervical carcinomas, the fraction of iPOT-expressing lesions raised to 80%. Thus, the prevalence of iPOTs was clearly linked to the degree of dysplasia (P = 0.001, {chi}2 test for trend). Here again, the prevalence of iPOTs was not influenced by women’s age (P = 0.58, Wilcoxon test for unpaired samples).

Association between DNA Aneuploidy and Integration of HPV-16 E6/E7 Oncogenes.
Because both parameters, aneu-ploidy, and prevalence of iPOTs were associated with increasing grades of dysplasia of the HPV-16-positive lesions analyzed here, the question arose whether the iPOTs occur preferentially in aneuploid lesions, suggesting that aneuploidy favors integration of HPV genomes, or whether expression of iPOTs might be found preferentially in high-grade lesions that might not be in all cases aneuploid, suggesting that in this scenario integration of papillomavirus genomes might precede and subsequently favors aneuploidy. To clarify this question, we compared the prevalence of DNA aneuploidy and iPOTs in the series of HPV-16-positive lesions. Nineteen of 20 (95%) of lesions that expressed iPOTs were aneuploid, whereas, however, only 19 of 32 (59%) of the aneuploid lesions expressed iPOTs (P < 0.001, Fisher’s exact test; Table 2Citation ). These data clearly demonstrate that almost all lesions with integrated papillomavirus genomes were aneuploid, whereas only a fraction of the aneuploid lesions expressed iPOTs.


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  		Table 2 Frequency table of DNA aneuploidy and integration of viral E6/E7 oncogenes detected in 85 human papillomavirus (HPV) type 16-positive cervical biopsies (20 CIN1/2, 50 CIN3, and 15 invasive squamous cell carcinomas)a

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Invasive carcinomas of the uterine cervix develop gradually through well-characterized precursor lesions. Usually only very few of the precancerous lesions progress to invasive carcinomas, whereas most lesions either persist or spontaneously regress within a couple of months (12 , 13) . Thus, the clonal selection process in cervical carcinogenesis seems to be rather inefficient, enabling only few cell clones to grow out as invasive carcinomas. These slow dynamics suggest that a random trial and error process continuously generates new slightly modified cell populations, of which, most die off, leaving only a minor fraction the chance to survive and expand. The genetic basis for these continuous selection events is the generation of increased genomic plasticity in cells, leading to the necessary secondary alterations such as increased proliferation, resistance to apoptosis, increased angiogenesis, invasion through basal membranes, and other features typical for neoplastic growth. In the case of cervical cancer, two papillomavirus oncogenes, E6 and E7, were shown to induce and maintain the genomic instability (15) . The perturbation of the mitotic apparatus in replicating cells that express HPV oncogenes leads to the formation of multiple centrosomes that cause multipolar mitosis with severe numerical and structural aberrations of the chromosomes, a hallmark of aneuploidy. Aneuploidy is a significant progression marker in cervical precancer, suggesting that aneuploid cell clones are more likely to survive and progress to more advanced dysplasia, as compared with euploid cell clones (4) . Similarly, integration of high-risk HPV genomes and the expression of iPOTs emerged as strong progression factor (16 , 28) . Several lines of experimental evidence demonstrated that integration of the viral genomes results in transcriptional deregulation because of cis-acting elements, the stabilization of the mRNA, resulting in an relative increase of viral oncogene products, and thereby also in increased transforming potential of the iPOTs if compared with the less stable episome-derived papillomavirus oncogene products (ePOTs; Refs. 19, 20, 21 ). Because iPOTs were shown to have a significantly higher transforming potential as compared with ePOTs, the question arose of whether chromosomal instability is induced by integrated HPV rather than by episomal viruses. However, expression of ePOTs might be sufficient to induce chromosomal instability. This might provoke repeated chromosomal breakage and fusion events during that fragments of the episomal viral genomes might be integrated into the chromosomal DNA sequences. After aneuploidy induction by ePOTs, HPV integration occurs, leading to the formation of cells expressing iPOTs with an increased neoplastic potential. These iPOT-expressing cells should more rapidly progress to invasive carcinomas, a feature that has been demonstrated by many independent studies (16 , 28 , 29) . The latter hypothesis is supported by a recent study (24) that showed that in vitro ePOTs are fully sufficient to induce chromosomal instability.

To test which of these two hypothesis holds true in the natural history of cervical dysplasia, we have compared the frequency of aneuploid cell clones and cells that already express iPOTs in cervical lesions at different dysplastic stages. Because significantly more lesions were aneuploid even if they did not express iPOTs, whereas almost all lesions that expressed iPOTS were aneuploid, it appears that aneuploidy precedes the outgrowth of cell clones that express iPOTs. Thus, these data are in good agreement with the in vitro observation made by Duensing et al. (24) that expression of ePOTs is fully sufficient to induce chromosomal instability. However, chromosomal instability seems to support integration events of the HPV genomes that in turn permit the expression of iPOTs. The apparently rapid progression of lesions that express iPOTs additionally supports the concept that iPOTs confer a significantly stronger transforming activity and rapidly push the selection of carcinoma cells. Aneuploidy testing in cervical lesions has been successfully applied to identify high-grade lesions with higher progression tendency. In a refined diagnostic setting, HPV integration analysis can indicate progressing lesions with even higher specificity and might be used as a patient-specific tumor-, metastasis-, and recurrency marker.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Magnus von Knebel Doeberitz, Institute of Molecular Pathology, University of Heidelberg, Im Neuenheimer Feld 220, D-69120 Heidelberg, Germany. Phone: 49-6221-56-24-76; Fax: 49-6221-56-50-81; E-mail: knebel{at}med.uni-heidelberg.de

Received 11/12/03; revised 1/ 6/04; accepted 1/16/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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