Evaluation of Integrated HPV DNA as Individualized Biomarkers for the Detection of Recurrent CIN2/3 during Post-Treatment Surveillance.

PURPOSE: Post-treatment follow-up in women with cervical pre-cancers (CIN3) is mandatory due to relapse in up to 10% of patients. Standard follow-up based on hrHPV-DNA/cytology co-testing has high sensitivity but limited specificity. The aim of our prospective, multicenter, observational study was to test the hypothesis that an individualized viral-cellular-junction test (vcj-PCR) combined with cytology has a lower false positive rate for the prediction of recurrence compared to standard co-testing. METHODS: Pre-surgical cervical swabs served for the identification of HPV16/18 DNA integration sites by next-generation-sequencing (NGS). Samples taken at 6, 12 and 24 months post-surgery were evaluated by cytology, hrHPV-DNA and the patients' individual HPV-integration sites (vcj-PCR on the basis of NGS). RESULTS: Integration sites were detected in 48 of 445 patients (10.8%), 39 of them had valid follow-up data. The false positive rate was 18.2% (95% CI 8.6-34.4%) for standard hrHPV/cytology at six months compared to 12.1% (95% CI 4.8-27.3%) for vcj-PCR/cytology, respectively (McNemar p = 0.50). Six patients developed recurrences (1 CIN2, 5 CIN3) during follow-up. Standard co-testing detected all, whereas vcj-PCR/cytology detected only five patients with recurrences. Data of 269 patients without evidence of HPV16/18 integration were subject to post-hoc analyses. Standard co-testing revealed a false positive rate of 15.7% (95% CI 11.7-20.7%) and predicted ten of fourteen recurrences at six months. CONCLUSIONS: Although highly specific on its own vcj-PCR could not detect all recurrent CIN2/3. Possible reasons for this unexpected result may be multifocal lesions, intratumoral heterogeneity with respect to HPV integration and/or incident CIN.

Combined assessment of 3q26 amplification and promoter methylation in patients with high grade cervical lesions show age specific differences.

A considerable proportion of high grade cervical intraepithelial lesions (CIN2/3) are known to resolve on their own especially among young women. However, since reliable prognostic markers are still lacking, the diagnosis "CIN3" is still an indication for surgery which may result in overtreatment. It is conceivable that a combination of different, ideally independent molecular markers may provide more reliable results. In the present cross-sectional study two established triage markers, 3q26 amplification and a methylation signature, were evaluated in an age-dependent manner. The patient cohort comprised 60 patients with histologically confirmed CIN2/3 in two equally sized age groups (<30 years, ≥30 years). Cervical scrapes were analyzed by interphase fluorescence in situ hybridization for 3q26 amplification and methylation specific PCR (GynTect®) for six different genome regions. Both assays showed a significantly different pattern of test outcome independent of age (P = .001). Moreover, the combination of both assays differed significantly for double positive and double negative cases when comparing the two age groups: In patients <30 years there were clearly less cases with positive methylation signature and amplification of 3q26 as in women ≥30 years (23% vs 63%, Bonferroni adjusted P = .016). Of particular interest is the finding that double negative results were exclusive for the young age group (0% vs 27%, Bonferroni adjusted P = .020). Since regression of CIN2/3 characteristically occurs among young women it is tempting to speculate that a double negative test result could be prognostic for regression of CIN2/3. This will have to be investigated further in a prospective longitudinal intervention study.

Performance of a methylation specific real-time PCR assay as a triage test for HPV-positive women.

BACKGROUND: HPV DNA testing as a primary screening marker is being implemented in several countries. Due to the high HPV prevalence in the screening population, effective triage strategies for HPV-positive cases are required. The aim of this study was to evaluate the performance of a methylation-specific real-time PCR  assay (GynTect®) comprising six marker regions as a triage test. RESULTS: An analytical sensitivity of 0.1 ng genomic DNA corresponding to 15 SiHa cells was achieved. Absolute specificity was observed in the presence of 20 ng unmethylated genomic DNA. In a clinical setting, cervical scrapes of 306 women showing abnormal colposcopy were tested for cytology, HPV positivity, and the GynTect markers ASTN1, DLX1, ITGA4, RXFP3, SOX17, and ZNF671. Of all women, histopathological data were available. The overall sensitivity for GynTect to detect CIN3+ was 67.7% (95% CI 57.3%-77.1%) whereas sensitivity was significantly higher for women of age ≥ 30 years (p = 0.04). All cancer cases (n = 5) were detected by GynTect. The overall false positive rate (= 1-specificity) for women with no CIN was 17.4% (95% CI 12.5-23.1%), with a higher proportion among HPV-positive women (24.0%, 95% CI 16.0-33.6%). In a triage screening setting, where all women underwent HPV testing and the HPV positives in addition GynTect testing, the overall sensitivity would slightly decline but specificity would reach the maximum value of 88.7% (95% CI 83.7-92.6%). CONCLUSION: The GynTect® assay is a robust easy to use assay with high analytical sensitivity and specificity. Moreover, the performance of the assay based on cervical scrapes provides further evidence for the usefulness of methylation markers to detect HPV-positive women with clinically relevant disease.

A promising DNA methylation signature for the triage of high-risk human papillomavirus DNA-positive women.

High-risk human papillomavirus (hrHPV)-DNA testing is frequently performed parallel to cytology for the detection of high-grade dysplasia and cervical cancer particularly in women above 30 years of age. Although highly sensitive, hrHPV testing cannot distinguish between HPV-positive women with or without clinically relevant lesions. However, in principle discrimination is possible on the basis of DNA methylation markers. In order to identify novel DNA regions which allow an effective triage of hrHPV-positive cases, hypermethylated DNA enriched from cervical cancers was compared with that from cervical scrapes of HPV16-positive cases with no evidence for disease by CpG island microarray hybridization. The most promising marker regions were validated by quantitative methylation-specific PCR (qMSP) using DNA from archived cervical tissues and cervical scrapes. The performance of these markers was then determined in an independent set of 217 hrHPV-positive cervical scrapes from outpatients with histopathological verification. A methylation signature comprising the 5' regions of the genes DLX1, ITGA4, RXFP3, SOX17 and ZNF671 specific for CIN3 and cervical cancer (termed CIN3+) was identified and validated. A high detection rate of CIN3+ was obtained if at least 2 of the 5 markers were methylated. In the subsequent cross-sectional study all cervical carcinomas (n = 19) and 56% (13/23) of CIN3 were identified by this algorithm. Only 10% (11/105) of hrHPV-positive women without histological evidence of cervical disease were scored positive by the methylation assay. Of note is that the detection rate of CIN3 differed between age groups. Eight of nine CIN3 were detected among women ≥30 years of age but only five of fourteen among <30 year old group (p = 0.03). The specificity for CIN3+ in the older age group was 76.6% (95% CI 65.6-85.5%). Clinical validation studies are required to determine the usefulness of these novel markers for triage after primary hrHPV testing in a cervical cancer screening setting.

Digital-Direct-RT-PCR: a sensitive and specific method for quantification of CTC in patients with cervical carcinoma.

The detection of circulating tumour cells (CTC) in cancer patients may be useful for therapy monitoring and prediction of relapse. A sensitive assay based on HPV-oncogene transcripts which are highly specific for cervical cancer cells was established. The Digital-Direct-RT-PCR (DD-RT-PCR) combines Ficoll-separation, ThinPrep-fixation and one-step RT-PCR in a low-throughput digital-PCR format enabling the direct analysis and detection of individual CTC without RNA isolation. Experimental samples demonstrated a sensitivity of one HPV-positive cell in 500,000 HPV-negative cells. Spike-in experiments with down to 5 HPV-positive cells per millilitre EDTA-blood resulted in concordant positive results by PCR and immunocytochemistry. Blood samples from 3 of 10 CxCa patients each contained a single HPV-oncogene transcript expressing CTC among 5 to 15*10(5) MNBC. Only 1 of 7 patients with local but 2 of 3 women with systemic disease had CTC. This highly sensitive DD-RT-PCR for the detection of CTC may also be applied to other tumour entities which express tumour-specific transcripts.

An integrative functional genomic and gene expression approach revealed SORBS2 as a putative tumour suppressor gene involved in cervical carcinogenesis.

Human papillomavirus (HPV) types 16 and 18 are known to play a major role in cervical carcinogenesis. However, additional genetic alterations are required for the development and progression of cervical cancer. Our aim was to identify genes which are consistently down-regulated in cervical cancers (CxCa) and which are likely to contribute to malignant transformation. Microarray analyses of RNA from high-grade cervical precancers (CIN2/3) and CxCa were performed to screen for putative tumour suppressor genes (TSG) in predefined regions on chromosomes 4 and 10. Validation of the candidate genes was done by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in 16 normal cervical tissues, 14 CIN2/3 and 16 CxCa. The two most promising genes, SORBS2 and CALML5, were expressed ectopically in various cell lines in order to analyse their functional activity. Reconstitution of SORBS2 expression resulted in a significant reduction in cell proliferation, colony formation and anchorage-independent growth in CaSki, HPKII and HaCaT cells, whereby anchorage-independent growth could only be investigated for CaSki cells. SORBS2 had no effect on cell migration. In contrast, reconstitution of CALML5 expression did not influence the phenotype of all cell lines tested. None of the genes could induce senescence or apoptosis. Our results underline a possible role of SORBS2 as a TSG in cervical carcinogenesis.

Two novel unbalanced whole arm translocations are frequently detected in cervical squamous cell carcinoma.

Chromosomal aberrations are a hallmark of human papillomavirus (HPV)-induced cervical carcinogenesis. The aim of this project was to identify structural chromosomal aberrations which may be characteristic for intraepithelial neoplasias (CIN) and cervical carcinomas (CxCa). Two independent HPV16 immortalized keratinocyte cell lines (HPKIA, HPKII) were used as a cell culture model system for cervical carcinogenesis. Different passages of HPKIA and HPKII were analyzed by multicolor spectral karyotyping. Several chromosomal translocations were identified in HPK cells and were validated by interphase fluorescence in situ hybridization (I-FISH). Three unbalanced whole chromosome arm translocations, der(10;14), der(7;21), and der(7;12), were cell line specific. The presence and frequency of these translocations were then examined by I-FISH in frozen tissue sections from normal cervical epithelia (n=6), CIN2/3 (n=15), and CxCa (n=15). The der(10;14) and der(7;21) were detected in 80% and 53.3% of CIN2/3, and in 60% and 46.7% of CxCa, respectively. The percentage of nuclei with translocations in individual lesions was significantly higher among CxCa. The der(7;12) could only be detected in 27% of CIN2/3. None of the translocations were detected in normal cervical epithelia. The translocated chromosomes may contribute to the clonal expansion of subpopulations in these cases and may thus be of diagnostic relevance.

Quantitative multiplex PCR assay for the detection of the seven clinically most relevant high-risk HPV types.

BACKGROUND: High-risk HPV DNA detection has become a valuable tool for the triage of borderline, questionable and abnormal cytologic findings in cervical carcinoma screening programs. This knowledge is largely based on studies which could only discriminate between low-risk (LR-) and high-risk (HR-) HPV groups. However, it is becoming increasingly clear that HPV genotyping may allow further risk stratification and may offer different treatment options in the future. OBJECTIVES: To establish a fast and cost-effective system not only for genotyping but also for quantification of viral DNA. STUDY DESIGN: Development and validation of a 5' exonuclease fluorescent probe multiplex real-time PCR assay (TaqMan format) for the detection and quantification of the 7 most frequent HR-HPV types (16, 18, 31, 33, 45, 52 and 58) which account for over 87% of cervical carcinomas world-wide. Two PCR reactions are required to detect the designated HPV types. RESULTS: Experiments with plasmid constructs of all 18 HR-HPV DNA showed that the multiplex real-time PCR assay was highly sensitive and specific. Evaluation of DNA extracted from archived cell pellets of cervical scrapes by the multiplex assay and the GP5+/6+-EIA showed identical genotyping for 234 of 261 (89.6%) samples and an almost perfect agreement when considering all typing results (kappa 0.901). Viral load did not correlate with disease progression within the CIN spectrum but significant differences were evident when comparing all CIN with the group lacking CIN (p=0.0028) or with the cancer group (p=0.0001). CONCLUSION: Our multiplex assay will be useful to address questions related to viral persistence at the genotype level, the kinetics of viral load and disease recurrence.

Inner border--a specific and significant colposcopic sign for moderate or severe dysplasia (cervical intraepithelial neoplasia 2 or 3).

OBJECTIVE: Investigation of the correlation between the colposcopic sign inner border and cervical intraepithelial neoplasia (CIN) 2 or 3 and its association with specific human papillomavirus (HPV) types and the age of the patient. STUDY DESIGN: Colpophotographs or cervicograms were taken from 947 women referred due to an abnormal cervical finding. Occurrence of the colposcopic sign inner border was evaluated retrospectively by 2 independent colposcopists. Histologic evaluation was based on punch or cone biopsies. Human papillomavirus testing was done using Hybrid Capture I or a polymerase chain reaction-based HPV test. RESULTS: The prevalence of the colposcopic phenomenon inner border in women with an atypical transformation zone was 7.6% (53/695). In 70% of women with inner border, CIN 2 or 3 was confirmed histologically. The sensitivity of the colposcopic sign inner border for detection of CIN 2 or 3 was 20%, and the specificity was 97%. In patients with inner border, the odds ratio for CIN 2 or 3 was 7.7 (95% CI=4.2-14.3). There was no significant association between inner border and any high-risk HPV type. Cervical intraepithelial neoplasia 2 or 3 associated with inner border was significantly more frequent in patients younger than 35 years. CONCLUSION: Inner border is a rare colposcopic phenomenon but highly specific for CIN 2 or 3 in young women.

The colposcopic feature ridge sign is associated with the presence of cervical intraepithelial neoplasia 2/3 and human papillomavirus 16 in young women.

OBJECTIVE: Interobserver and intraobserver correlation between the colposcopic phenomenon ridge sign and its association with cervical intraepithelial neoplasia (CIN) 2 or 3, with a specific human papillomavirus (HPV) type, and with the age of the patient. STUDY DESIGN: Colpophotographs, cervical smears, and histologic results of punch or cone biopsies of 592 patients were evaluated. Colpophotographs were analyzed retrospectively for the presence or absence of an opaque acetowhite ridge at the squamocolumnar junction (ridge sign) by 3 gynecologists of different experience. RESULTS: Interobserver reliability for colposcopic grading of CIN was between 18.2% and 82.3%. Concerning the ridge sign, interobserver agreement varied between 25.3% and 49.4% according to the observers' experience, and intraobserver reliability varied between 56.4% and 67.5% (Cohen kappa=0.310-0.469). In 83 (14.0%) of 592 patients, a ridge sign was diagnosed by the most experienced investigator. Cervical intraepithelial neoplasia 2 or 3 was confirmed histologically in 53 of these 83 women (63.8%). Sensitivity of ridge sign for detection of CIN 2 or 3 was 33.1%; specificity was 93.1%. Women with ridge sign were significantly younger than women with no ridge sign (p< .001). Ridge sign was associated with the presence of HPV 16 (p< .001). CONCLUSION: Ridge sign is a highly specific marker for CIN 2 or 3 and associated with HPV 16 and young age.

Cumulative 5-year diagnoses of CIN2, CIN3 or cervical cancer after concurrent high-risk HPV and cytology testing in a primary screening setting.

The aim of our study was to assess the cumulative 5-year diagnoses of CIN2, CIN3 or invasive cervical cancer (CIN2+) after concurrent screening by high-risk HPV test and Pap smear in a primary screening setting. Four thousand thirty-four women from Eastern Thuringia/Germany were recruited from 1996 to 1998 for baseline screening that included routine cytology, high-risk HPV testing by consensus primer PCR GP5+/6+ and routine colposcopy. After a median of 59 months 3,153 women participated in final screening using identical methods. Women with abnormal cytology or colposcopy or a positive high-risk HPV test at any time during the study period were recalled for expert colposcopy and histologic verification. CIN2+ was detected in 160 women resulting in a cumulative 5-year proportion of 4.4% (95% CI: 3.7-5.0%). Of 3,702 women who were high-risk HPV negative at baseline, 34 (1.1-95% CI: 0.7-1.4%) had either prevalent CIN2+ or developed CIN2+ within the observation period. HPV/cytology double negatives at baseline were at lowest risk for CIN2+ (1.0-95% CI: 0.7-1.4%) compared to screening positives (16.8-100% depending on combined test results). The 5-year negative predictive value in HPV-/Cyto- women was 99.0% (95% CI: 98.6-99.3%). This suggests that a prolongation of the screening intervals in this group is feasible. However, it should be noted that 1 woman developed a microinvasive carcinoma within the observation period. Moreover, 2 women with prevalent cancer were missed by both tests. The prognostic relevance of concurrent high-risk HPV/cytology screening needs to be verified further by randomized trials.

Detection of disseminated tumor cells in patients with cervical cancer.

PURPOSE: Detection of disseminated tumor cells in a cohort of patients presenting the entire spectrum of invasive cervical cancer. METHODS: Disseminated tumor cells were detected in blood samples taken at different times during surgery or in bone marrow aspirates by a HPV type-specific nested PCR enzyme immunoassay (n-PCR-EIA). A group of 24 patients with HPV-positive cervical cancers representing early and late stages were evaluated, and 15 patients with breast cancer and without HPV-related genital disease served as controls. RESULTS: Disseminated tumor cells were detected in blood samples and/or bone marrow aspirates of 6 of 24 patients. A significant association was found between detectable disseminated tumor cells and recurrent disease ( P=0.013) and between disseminated tumor cells and survival of the patients ( P=0.0054). There was also a clear association between the presence of disseminated tumor cells and tumor size and/or positive lymph node status which, however, was not statistically significant. There was no evidence of increased shedding of tumor cells during surgery. CONCLUSION: Detection of disseminated tumor cells in blood or bone marrow may prove to be of prognostic value, particularly for early-stage cervical cancers.