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Chromosomal integration of high-risk human papillomavirus (HR-HPV) genomes is believed to represent a significant event in the pathogenesis of cervical cancer associated with progression from preneoplastic lesions to invasive carcinomas. This hypothesis is based on experimental data suggesting that integration-dependent disruption of HR-HPV E2 gene functions is important to achieve neoplastic transformation and on clinical data gathered by analyzing lesions induced by human papillomavirus (HPV) 16 and 18 that revealed integrated viral genome copies in the vast majority of cervical cancer cells. However, a substantial fraction of cervical cancers is associated with other HR-HPV types for which virtually no data concerning their integration status have been reported so far. Here, we compared integration frequencies of the five most common oncogenic HPV types (HPV16, 18, 31, 33, and 45) in a series of 835 cervical samples using a specific mRNA-based PCR assay (Amplification of Papillomavirus Oncogene Transcripts). Most precancerous lesions displayed exclusively episomal viral genomes, whereas 62% of the carcinomas had integrated viral genomes. However, the frequency of integrated HR-HPV genomes showed marked differences for individual HR-HPV types. HPV16, 18, and 45 were found substantially more often in the integrated state compared with HPV types 31 and 33. The analysis of the median age of patients with high-grade precancerous lesions and invasive cancers suggests that precancers induced by HPV types 18, 16, and 45 progress to invasive cervical cancer in substantially less time compared with precancers induced by HPV types 31 and 33. These findings suggest that integration of oncogenic HPV genomes in cervical lesions is a consequence rather than the cause of chromosomal instability induced by deregulated HR-HPV E6-E7 oncogene expression. Distinct HR-HPV types apparently provoke chromosomal instability in their host cells to a different extent than is reflected by their integration frequencies in advanced lesions and the time required for CIN 3 lesions to progress to invasive cancer. [Cancer Res 2008;68(1):307–13]

Human papillomavirus (HPV) genomes usually persist as episomal molecules in HPV associated preneoplastic lesions whereas they are frequently integrated into the host cell genome in HPV-related cancers cells. This suggests that malignant conversion of HPV-infected epithelia is linked to recombination of cellular and viral sequences. Due to technical limitations, precise sequence information on viral-cellular junctions were obtained only for few cell lines and primary lesions. In order to facilitate the molecular analysis of genomic HPV integration, we established a ligation-mediated PCR assay for the detection of integrated papillomavirus sequences (DIPS-PCR). DIPS-PCR was initially used to amplify genomic viral-cellular junctions from HPV-associated cervical cancer cell lines (C4-I, C4-II, SW756, and HeLa) and HPV-immortalized keratinocyte lines (HPKIA, HPKII). In addition to junctions already reported in public data bases, various new fusion fragments were identified. Subsequently, 22 different viral-cellular junctions were amplified from 17 cervical carcinomas and 1 vulval intraepithelial neoplasia (VIN III). Sequence analysis of each junction revealed that the viral E1 open reading frame (ORF) was fused to cellular sequences in 20 of 22 (91%) cases. Chromosomal integration loci mapped to chromosomes 1 (2n), 2 (3n), 7 (2n), 8 (3n), 10 (1n), 14 (5n), 16 (1n), 17 (2n), and mitochondrial DNA (1n), suggesting random distribution of chromosomal integration sites. Precise sequence information obtained by DIPS-PCR was further used to monitor the monoclonal origin of 4 cervical cancers, 1 case of recurrent premalignant lesions and 1 lymph node metastasis. Therefore, DIPS-PCR might allow efficient therapy control and prediction of relapse in patients with HPV-associated anogenital cancers.

A cohort of 70 consecutive women at a university hospital colposcopy clinic with untreated CIN I and CIN II (CIN I/II) confirmed by cytology and histology was followed for 1 year in the setting of a prospective trial. In the lesions, the presence of DNA from HPV types was examined by restriction fragment length polymorphism (RFLP) analysis. Aneuploid cell lines were demonstrated by aneuploid histograms generated by high-resolution DNA flow cytometry. HPV type 16 infection and the existence of aneuploid cell lines proved to be significant risk factors for CIN I/II lesions to persist or progress to CIN III in the 1-year follow-up period in the same cohort of patients. The relative risks and 95% confidence intervals (CI) were 1.81 (1.44–2.76) for aneuploid cell lines and 1.74 (1.10–2.76) for HPV type 16 infection in CIN I/II lesions. As a predictive diagnostic test for CIN I/II lesions to persist or progress, the specificity and positive predictive value (PPV) for aneuploid histograms were 100% (CI, 73.5–100%) and 100% (CI, 86.8–100%), respectively. The low sensitivity of 27.3% (CI, 14.9–42.8%) restricted the clinical application of the test, leaving 32 of 44 women with persisting or progressing CINI/II with diploid histograms. HPV type 16 positivity by FRLP had a PPV of 68.4% (CI, 43.5–87.4%) as a prognostic test. Six of 19 HPV 16 infected women showed complete remission of their CIN lesion. A combination of the two tests did not provide any additional information. Cytometry (Comm. Clin. Cytometry) 46:166–171, 2001. © 2001 Wiley-Liss, Inc.

The human tumour susceptibility gene TSG101 has recently been identified on chromosomal locus 11p15.1-15.2 which is frequently affected by genetic alterations in neoplastic lesions of the uterine cervix. Aberrant transcripts of the TSG101 gene have been reported in various tumour entities, including breast, ovarian and prostate cancers, but also in several non-neoplastic tissues. We analysed TSG101 transcription by reverse transcription–polymerase chain reaction (RT–PCR) in a total of 139 clinical samples of cervical tissues and in cervical carcinoma cell lines. Variant transcripts were observed in all cell lines, in 69 of 122 (57%) cervical dysplasia and carcinoma samples and in five of 17 (29%) normal cervical tissues. One specific variant TSG101 transcript ( Δ154-1054 ) was detected with a significantly increased frequency in advanced preneoplastic cervical lesions. However, the relative abundance of variant TSG101 transcripts appeared to be generally low, as only wild-type, but no variant transcripts were detectable in Northern blot analyses of cervical carcinoma cell lines. These data point to a progressive loss of stringent splice control functions or to extended alternative splicing in advanced dysplasia and neoplasia. The relative amounts of variant transcripts do not support a major functional role of TSG101 variants in cervical carcinogenesis.

Proliferation of granulosa cells is inversely related to differentiation and hormone production. The purpose of this study was to evaluate the intrafollicular and serum steroid concentrations and to compare these results to granulosa cell proliferation as measured by DNA flow cytometry. Human granulosa lutein cells in follicular fluid of in-vitro fertilization (IVF) patients were investigated with regard to ploidy, percentage of S-phase cells and proliferation index (PI: percentage of cells in the S- and G2/M-phase). The study was originally designed to indicate an additional marker for the outcome of IVF treatment by DNA flow cytometric measurements of granulosa lutein cells. Follicular fluids of 160 follicles (45 patients) were evaluated; 45.6% (n = 73) of the follicles showed aneuploid granulosa lutein cells and 5.6% (n = 9) of the follicles contained multiploid granulosa cells, defined as at least two aneuploid populations of cells with different DNA indices. A total of 48.8% (n = 78) of the follicles had only diploid cells. Thus50 % of the investigated follicles showed aneuploidy. In all, 73% (33 of 45) of patients had at least one follicle containing aneuploid granulosa lutein cells. The PI of the aneuploid cell populations significantly exceeded that of the diploid cell populations (median: aneuploid: 15.5; diploid: 7.4; P0.0001). The intrafollicular concentrations of testosterone, progesterone and dehydroepiandrosterone sulphate (DHEA-S) were significantly lower in follicles with aneuploid granulosa cell populations. Luteinizing hormone concentration was significantly higher in follicles with aneuploid granulosa cells. Intrafollicular concentrations of oestradiol, follicle stimulating hormone and the serum concentrations of all steroid hormones did not show any significant correlation to ploidy. Although aneuploidy has been reported for oocytes (in approximately 17% of the oocytes), no study, to our knowledge, has observed such a high incidence of aneuploidy in granulosa lutein cells after gonadotrophin stimulation. Except for aneuploidy found in tissues with some characteristics of neoplastic growth (colon adenoma, borderline tumours, endometriosis with atypic cells, etc.), it is unique for non-malignant human cells. The correlation with intrafollicular steroid concentrations points to a possible pathophysiological or physiological relevance of these findings. However, it was impossible to correlate the outcome of IVF with DNA flow cytometry results.

To underline the possibilities and limitations of organ-preserving laser surgery in vulvar intraepithelial neoplasia (VIN) and vaginal intraepithelial neoplasia (ValN), we report on a study running since 1993 in the Department of Gynaecology of the University of Heidelberg. 12 patients with VIN II and III had organ preserving laser surgery. The observation interval was at least 1 year after therapy. All patients underwent a standardised preoperative and postoperative diagnostic procedure in our colposcopy unit. Laser surgery was carried out on patients with VIN in hairless areas. No patient had any previous radiotherapy. Laser surgery was carried out under colposcopic guidance. The depth of vaporisation was 2 mm. A morphometric study was done on the preoperative histology. Summing up, we can say that the colposcopically guided laser surgery was very well tolerated by the patients. It proved efficient as an organ-preserving therapy for VIN II/III an ValN II/III.67% of the VIN II/III patients were free from disease for at least one year after one laser therapy. 25% of the patients had to have two laser therapies to be free from disease for at least one year. 8% of the patients finally had conventional surgery because of VIN recurrence after several laser therapies. There was no development of VIN to invasive cancer in any patient. There were no severe complications. All patients had good cosmetic results. Patients with VIN and CIN seemed to have significantly higher rates of recurrences after laser therapy. There was a high percentage of patients (42%) with positive HPV 16 (detected by PCR). 42% of the patients had DNA aneuploidies. 56% of the ValN II/III patients were free from disease for at least one year after one laser therapy. 21 % of the patients had to have two laser therapies to be free from disease for at least one year. 21% of the patients finally had conventional surgery because of ValN recurrence after several laser therapies. There was no development of ValN to invasive cancer in any patient. There were no severe complications. The majority of recurrences occurred within the first 6 months after therapy. Patients with ValN III, a long history of VaIN, a positive HPV 16 report, and DNA aneuploidies seemed to have higher rates of recurrences after laser therapy. There was a high percentage of patients with positive HPV 16 (50%) and DNA-aneuploidies (42%). 3 Patients with ValN did not have any CIN, VIN or invasive cancer. This stresses the importance of colposcopy in the gynaecological examination procedure for the prevention of cancer.

Background: Dysplastic lesions of the vagina or the vulva often occur in women who have a previous history of cervical dysplasia. Most lesions in the female lower genital tract are induced by infections with high-risk oncogenic human papillomaviruses (HR-HPVs), including HPV16 and HPV18. HR-HPV genomes frequently integrate into host cell chromosomes at random sites. We analyzed viral integration sites in multiple metachronous lesions of the lower genital tract from women previously treated for HR-HPV – positive cervical dysplasia or cancer to determine whether the metachronous lesions emerged from a single common preexisting dysplastic cell clone or as consequence of independent HR-HPV infection events in the female lower genital tract. Methods: From among 1500 patients with anogenital lesions, seven patients with high-grade vaginal or vulvar lesions and with a previous history of cervical disease (fi ve with prior high-grade cervical dysplasia and two with a history of cervical cancer) were included in this study. Integration sites of HPV16 or HPV18 in vaginal or vulvar lesions were mapped by an adaptor ligation polymerase chain reaction (PCR) method. The sequence information was used to design an integrate-specifi c PCR assay that was applied to DNA extracted from archival paraffi n-embedded material derived from biopsy samples of cervical lesions. Results: Identical HPV DNA integration loci were found in vaginal or vulvar and cervical samples of all lesions available for four of the fi ve patients with a prior history of high-grade cervical dysplasia and for both patients with a history of cervical cancer. Conclusions: These data indicate that high-grade dysplastic lesions in the female lower genital tract may emerge primarily as monoclonal lesions from a transformed cell population derived from the uterine cervix. [J Natl Cancer Inst 2005;97:1816 – 21] Dysplastic and neoplastic lesions of the cervix, vagina, vulva, and anus have similar risk factors. It is well accepted that persistent infection with high-risk (i.e., oncogenic) human papillomaviruses (HR-HPVs) are required for the development of most of these lesions ( 1 ) . HR-HPV infections are particularly common in young, sexually active women ( 2 ) . Results of cross-sectional studies have revealed that although up to 40% of such women are infected with an HR-HPV, fewer than 10% of infected women develop persistent HPV infections and only 28% of those who do will develop high-grade intraepithelial dysplasia of the cervix within 5 – 6 years of the initial infection ( 3 – 7 ) . Thus, almost all HR-HPV infections resolve spontaneously without causing clinically relevant dysplastic lesions in the genital tract ( 8 , 9 ) . These epidemiologic features suggest that, although neo plastic trans formation of epithelial cells in the cervix depends on HR-HPV infections, other factors are required to permit pro gression of HR-HPV infections to high-grade lesions and invasive cancer. The clinical manifestations of dysplastic lesions in the various epithelial regions of the female genital tract (cervix, vagina, and vulva) show remarkable differences. For example, the incidence of cervical intraepithelial neoplasia (CIN) is 10-fold higher than the incidence of dysplastic lesions of the vagina or vulva. Also, the incidence of cervical dysplasia and derived cervical carcinomas peaks approximately 10 years earlier than the incidence of vaginal cancer ( 10 , 11 ) . These observations suggest that, despite the high frequency of HPV infection events in the female lower genital tract, transformation of the vaginal or vulvar epithelium occurs substantially less often than that of the cervical epithelium. Most vaginal and vulvar lesions occur in women who have a history of HR-HPV – induced cervical dysplasia or carcinomas ( 12 – 14 ) . These lesions may arise at the same time (synchronous lesions) or up to several years after the initial cervical lesion (metachronous lesions). Several studies ( 15 – 17 ) have demonstrated that women who had had a high-grade cervical lesion or invasive cervical cancer had an increased risk of developing a metachronous cancer within the lower genital tract compared with women in the general population. The existence of both synchronous and metachronous lesions suggests that several independent local infection events induce development of multiclonal dysplastic lesions at distinct anatomic sites. The frequent development of dysplastic lesions in women who have vaginal and vulvar lesions associated with previous cervical disease might refl ect their predisposition for HR-HPV – related lesions compared with the general population. Alternatively, the multicentric lesions might all originate from a distinct epithelial cell

OBJECTIVE To investigate the prevalence of HPV L1 capsid proteins in HPV-infected HSIL and LSIL. STUDY DESIGN Cervical smears from 74 women with cytologically and histologically confirmed LSIL (n = 32) and HSIL (n = 42) were collected prospectively to detect HPV high-risk (hr) types 16, 18, 33, 35, 39, 45, 56 and 58 L1-DNA by standardized L1-consensus primer PCR (MY 09/11) and L1 capsid proteins by immunocytochemistry using monoclonal antibodies T31 (HPV16) and T16 (HPV hr) in a standardized protocol. RESULTS In HSIL and LSIL, L1 DNA was found for HPV hr in 93% and 59% and for HPV16 in 69% and 37% of the specimens, respectively. HPV L1 capsid proteins were detected in HSIL and LSIL for HPV hr in 33% and 44% and for HPV16 in 29% and 31% of the specimens, respectively. Expression of L1 capsid proteins was significantly reduced, by 59.6% for HPV hr L1 DNA-positive HSIL (P < .01) and by 40.4% for HPV 16 L1 DNA-positive HSIL (P < .01). In HPV 16 DNA-positive and HPV hr DNA-positive LSIL, no significant reduction of corresponding L1 capsid protein expression could be demonstrated. CONCLUSION These data suggest a disturbed viral cellular interaction in HPV 16 and HPV hr-infected HSIL, with loss of viral L1 capsid antigen. In this context there is a possible role of T31 and T16 as prognostic markers to predict the prognosis of CIN.

In addition to human papillomavirus (HPV) infection, loss of heterozygosity (LOH) at tumor suppressor gene loci has been frequently observed in cervical cancer. Thus, it may be assumed that detection and characterization of specific LOH profiles in preneoplastic lesions, in addition to HPV typing, might facilitate assessment of progression risk of cervical dysplasia. In this study, the type and frequency of allelic imbalance (allelic loss or allelic reduction) were analyzed in 24 unrelated cervical lesions using 14 polymorphic microsatellite markers at different tumor suppressor gene loci. No allelic loss was observed in four condylomatous lesions, whereas 2 of 13 (15%) CIN I lesions displayed allelic loss at 3p25 and 5q11-13. In high-grade lesions, however, allelic loss occurred in four of six (66%) cases at multiple chromosomal regions (3p14-25, 5p15, 5q11, 5q21, 11p15, and 17q21). Allelic reduction was observed in 4 of 13 (30%) low-grade lesions and 3 of 6 (50%) high-grade lesions. LOH was confined to lesions infected by high-risk HPV types. These data suggest that chromosomal instability is an early event in cervical carcinogenesis. The detection of LOH on multiple chromosome 3p loci in 50% of high-grade lesions suggests that a specific marker panel encompassing this region might enable better assessment of which lesions are likely to regress, persist, or progress.