Germline variants of homology-directed repair or mismatch repair genes in cervical cancer.

While cervical cancer is associated with a persistent human papillomavirus (HPV) infection, the progression to cancer is influenced by genomic risk factors that have remained largely obscure. Pathogenic variants in genes of the homology-directed repair (HDR) or mismatch repair (MMR) are known to predispose to diverse tumour entities including breast and ovarian cancer (HDR) or colon and endometrial cancer (MMR). We here investigate the spectrum of HDR and MMR germline variants in cervical cancer, with particular focus on the HPV status and histological subgroups. We performed targeted next-generation sequencing for 5 MMR genes and 12 HDR genes on 728 German patients with cervical dysplasia or invasive cancer. In total, 4% of our patients carried a pathogenic germline variant, based on ClinVar classifications and additional ESM1b and AlphaMissense predictions. These included 15 patients with truncating variants in HDR genes (BARD1, BRCA1, BRCA2, BRIP1, FANCM, RAD51D and SLX4). MMR-related gene variants were less prevalent and mainly of the missense type. While MMR-related gene variants tended to associate with adenocarcinomas, HDR gene variants were commonly observed in squamous cancers. While one patient with HPV-negative cancer carried a pathogenic MMR gene variant (in MSH6), the HDR germline variants were found in patients with HPV-positive cancers and tended to associate with HPV18. Taken together, our study supports a potentially risk-modifying role of MMR and HDR germline variants in cervical cancer but no association with HPV-negative status. These variants may be exploitable in future therapeutic managements.

Genome-wide association study and functional follow-up identify 14q12 as a candidate risk locus for cervical cancer.

Cervical cancer is among the leading causes of cancer-related death in females worldwide. Infection by human papillomavirus (HPV) is an established risk factor for cancer development. However, genetic factors contributing to disease risk remain largely unknown. We report on a genome-wide association study (GWAS) on 375 German cervical cancer patients and 866 healthy controls, followed by a replication study comprising 658 patients with invasive cervical cancer, 1361 with cervical dysplasia and 841 healthy controls. Functional validation was performed for the top GWAS variant on chromosome 14q12 (rs225902, close to PRKD1). After bioinformatic annotation and in silico predictions, we performed transcript analysis in a cervical tissue series of 317 samples and demonstrate rs225902 as an expression quantitative trait locus (eQTL) for FOXG1 and two tightly co-regulated long non-coding RNAs at this genomic region, CTD-2251F13 (lnc-PRKD1-1) and CTD-2503I6 (lnc-FOXG1-6). We also show allele-specific effects of the 14q12 variants via luciferase assays. We propose a combined effect of genotype, HPV status and gene expression at this locus on cervical cancer progression. Taken together, this work uncovers a potential candidate locus with regulatory functions and contributes to the understanding of genetic susceptibility to cervical cancer.

Diagnostic cervical excision in patients with HPV positivity, cytological abnormalities- and preoperative cervical stenosis.

AIM: There is currently no protocol for classifying patients with HPV persistence and preoperative stenosis of the cervical canal. This has a significant impact on cytology results, colposcopy results and the possibility of obtaining reliable cervical histology outcomes. Our analysis clearly shows that colposcopy and cytology underestimate the histological results in patients with limited visibility due to the presence of a type 3 transformation zone (TZ). Our analysis revealed a significant discrepancy between the colposcopy and cytology results and the histological outcomes. Insufficient colposcopy led to the underdiagnosis of dysplastic lesions in patients with a type 3 TZ and cervical stenosis. In the case of repeated cytological abnormalities and inadequate colposcopy examination, it is crucial to perform a diagnostic conization to exclude high-grade dysplastic changes and cervical carcinoma. METHODS: We conducted a retrospective analysis of 1,021 conizations performed in tertiary care hospital in Wolfsburg, Germany between 2014 and 2020. Of these surgical procedures, 89 were diagnostic conizations. In our analysis, we defined diagnostic conization as a procedure performed when there is HPV persistence and repeated cytologic abnormalities in combination with a type 3 TZ, and when it is not possible to retrieve a relevant cervical histology sample. RESULTS: In this period, 8.7% of all conizations were diagnostic excisions. We found histological abnormalities in 48 of 89 patients (53.9%). The histological examination of the excised cone revealed high-grade cervical intraepithelial neoplasia (CIN/HSIL) in 9 patients (10.1%) and CIN 2+ (HSIL) in 23 out of the 89 patients (25.8%). Two cases of early-stage cervical carcinoma (FIGO IA1 and FIGO IA2) were confirmed (2.3%). CONCLUSION: Patients with cervical stenosis, high-risk HPV persistence and repeated cytological abnormalities are at high risk of undetected high-grade cervical dysplasia. Histologic confirmation must be ensured in this patient consultation and this can be achieved by performing diagnostic excisions.

Quality assurance using quality indicators for prevention and early detection of cervical cancer in certified gynaecological dysplasia units and consultancies.

PURPOSE: Cervical cancer is the fourth most common cancer in women worldwide. A successful screening concept for cervical cancer reduces the incidence and mortality of cervical cancer. Quality indicators (QIs) derived from the screening guidelines for cervical cancer and used by the certified dysplasia units and dysplasia consultancies are evaluated in this paper. The aim of this paper is to present the current data from the annual reports of these units and consultancies. METHODS: The results of the basic data and indicators for the audit year 2022 in the gynaecological dysplasia consultancies and units are presented. In 2022, 84 dysplasia consultancies and 42 units were audited. 40 units and 84 consultancies are included in the annual report. QI outcomes for patients treated in certified dysplasia units and dysplasia consultancies are analysed. Median, overall proportion, and standard deviation were calculated for each QI. RESULTS: The indicator year 2021 was analysed, which was audited in 2022 and evaluated in 2023. A total of nine QIs were analysed. Most target goals were met by the 84 certified dysplasia consultancies and by the 40 dysplasia units. The QIs evaluated are implemented to a very high degree. The targets for the three QIs were achieved by both the dysplasia consultancies and the units in at least 95% of the certified centres (QI 1: 100%, QI 2: 95%, QI 3: 100%; QI 1: 100%, QI 2: 97%, QI 3: 100%, respectively). The presentation of patients to the tumour board by the consultancies/units is working; the units are attending the tumour board more regularly than in previous years. Where the target was not met, the auditors issued deviations or reduced the duration of the certificate. The cases are discussed intensively in the sense of an individual case analysis and with the determination of measures on-site. CONCLUSIONS: The targets for the various indicators were largely met by the dysplasia consultancies and units in the 2022 audit year. The certification of gynaecological dysplasia consultancies/units which have to cooperate with certified gynaecological cancer centres, has for the first time ensured the continuity of healthcare from prevention and early diagnosis to treatment of gynaecological cancers.

Evaluation of FAM19A4/miR124-2 methylation performance in the management of CIN3 diagnosed pregnant women.

Pregnant women diagnosed with CIN3 have high regression rates after delivery. Biomarkers are needed to only identify pregnant women with progressive CIN requiring treatment to reduce overreferral and overtreatment. In our study we evaluated the performance of the FAM19A4/miR124-2 methylation test for molecular triage on FFPE samples of CIN3+-diagnosed pregnant women with known clinical course over time as well in a cross-sectional setting. In this German multicenter retrospective study biopsy material was collected from pregnant women diagnosed with cervical cancer (n = 16), with CIN3 that progressed to cancer during pregnancy (n = 7), with CIN3 that regressed to CIN1 or less within 6 months after delivery (n = 41), without CIN (n = 16), CIN3 covering 3-4 quadrants (n = 14) and randomly selected CIN3 (n = 41). FAM19A4/miR124-2 methylation analysis was performed blinded on first diagnosis. All pregnant women with cervical cancer and with CIN3 progressing to cancer tested positive for FAM19A4/miR124-2 methylation (100%, 22/22). In the regressing CIN3 group 47.5% and in the group without CIN 21.6% tested methylation positive. High-volume CIN3 and random selected CIN3 were methylation-positive in 91.7% and 82.1%, respectively. Methylation levels were significantly higher in progressive CIN3 and cancer compared to the controls (P < .0005). The likelihood ratio of a negative methylation test (LR-) for progressive CIN3+ was 0 (95% CI: 0-0.208). A negative FAM19A4/miR124-2 methylation test can rule out progressive CIN disease in pregnant women diagnosed with CIN3. This can help the clinician by managing these pregnant women with conservative follow-up until after delivery.

Association of genomic variants at PAX8 and PBX2 with cervical cancer risk.

Cervical malignancy is triggered by human papillomavirus infection but the risk for cervical cancer has a hereditary component. From a recent Genome Wide Association Study meta-analysis, 2q14.1 (PAX8) and 6p21.32 (PBX2) have been proposed as novel cervical cancer susceptibility loci. We investigated the two main signals at these loci in an independent case-control series of 2578 cases with cervical dysplasia or carcinoma and 1483 healthy females. We find significant associations for both variants, rs10175462 at PAX8 and rs2856437 at PBX2, with overall cervical disease (rs10175462: odds ratio [OR] 0.82, 95% confidence interval [CI] 0.74-0.91, P = 2.4 × 10-4 ; rs2856437: OR 1.52, 95% CI 1.14-2.02, P = .004). Both variants showed evidence of association with invasive squamous cervical cancer (rs10175462: OR 0.80, 95% CI 0.68-0.94, P = .006; rs2856437: OR 1.56, 95% CI 1.03-2.36, P = .036) and with high-grade dysplasia (rs10175462: OR 0.79, 95%CI 0.70-0.90, P = 1.9 × 10-4 ; rs2856437: OR 1.58, 95% CI 1.15-2.17, P = .005). A combined analysis of high-grade dysplasia and invasive cervical cancer also showed significant associations for both variants (rs10175462: OR 0.81, 95% CI 0.73-0.91, P = 2.4 × 10-4 ; rs2856437: OR 1.57, 95% CI 1.18-2.10, P = .002). No association was detected for rs2856437 with low-grade dysplasia, while rs10175462 showed weak evidence of association (P = .05). RNA analyses in cervical samples revealed that PAX8 transcripts were upregulated in HPV-positive lesions (P = .008) but this was not observed in the presence of the protective minor allele of rs10175462. The rs10175462 genotype also correlated with reduced levels of the lncRNA PAX8-AS1 (P < .001). Taken together, our results extend the evidence for a link between genomic risk variants at the HLA region (PBX2) with cervical disease and support PAX8 as the first consistent non-HLA cervical cancer susceptibility locus.

[Vaccination against human papillomavirus]. / Impfung gegen humane Papillomviren.

Vaccination against human papillomavirus (HPV), which has been proven to be highly effective and safe, is recommended as part of standard vaccination by the German Standing Committee on Vaccination (STIKO) for 9­ to 14-year-old girls and boys. Up to 90% of cervical cancer and its precancerous lesions can be prevented with timely vaccination (before first intercourse). In addition, the effectiveness extends to the primary prevention of HPV-associated neoplasms of the vulva, vagina, anus, penis and oropharynx. The HPV vaccination is the focus of the global initiative of the WHO calling on German health policymakers to significantly increase the immunization coverage of the German population, which is currently only 45-60%. Due to the high immunogenicity and the convincing long-term effects, the goals of eliminating cervical cancer and significantly reducing other HPV-associated cancers are theoretically achievable.

Association of genomic variants at the human leukocyte antigen locus with cervical cancer risk, HPV status and gene expression levels.

The human leukocyte antigen (HLA) locus on chromosome 6 has been reported to be associated with cervical cancer. We investigated two independent single-nucleotide polymorphisms in a large case-control series of cervical dysplasia and carcinoma that has been newly established by the German Cervigen Consortium, comprising a total of 2481 cases and 1556 healthy females. We find significant associations for both variants, rs9272117 at HLA-DQA1 and rs2844511 at MICA and HCP5, with cervical disease. Both variants showed evidence of association with invasive cervical cancer (rs9272117: OR 0.89, 95% CI 0.79-0.99, P = .036; rs2844511: OR 1.17, 95% CI 1.04-1.31, P = .008) and with high-grade dysplasia (rs9272117: OR 0.78, 95% CI 0.70-0.87, P = 7.1 × 10-6 ; rs2844511: OR 1.13, 95% CI 1.01-1.26, P = .035), as well as in a combined analysis of both groups (rs9272117: OR 0.83, 95% CI 0.75-0.91, P = 6.9 × 10-5 ; rs2844511: OR 1.14, 95% CI 1.04-1.26, P = .005). Variant rs2844511, but not rs9272117, also showed modest evidence of association with low-grade dysplasia (OR 1.26, 95% CI 1.04-1.54, P = .019). In case-only analyses, rs2844511 tended to predict HPV status (P = .044) and rs9272117 tended to associate with HPV16 (P = .022). RNA studies in cervical samples showed a significant correlation in the transcript levels of MICA, HCP5 and HLA-DQA1, suggesting extensive co-regulation. All three genes were upregulated in HPV16-positive samples. In stratified analyses, rs9272117 was associated with HLA-DQA1 levels, specifically in HPV-positive samples, while rs2844511 was associated with MICA and HCP5 levels. The risk allele of rs2844511 was required for correlations between MICA or HCP5 with HLA-DQA1. Altogether, our results support 6p21.32-33 as the first consistent cervical cancer susceptibility locus and provide evidence for a link between genetic risk variants, HPV16 status and transcript levels of HLA-DQA1, HCP5 and MICA, which may contribute to tumor immune evasion.

Reproductive capacity and recurrence of disease after surgery for moderate and severe endometriosis - a retrospective single center analysis.

BACKGROUND: Endometriosis can be associated with considerable pain and sterility. After surgical excision of moderate or severe endometriosis lesions, the rate of recurrence reaches up to 67%. The objective of this retrospective study was to establish the recurrence and pregnancy rates following surgical resection of stage III/IV endometriosis lesions. Indications for operation were endometriosis symptoms, sonographic findings and/or infertility. METHODS: A total of 456 patients who underwent stage III/IV endometriosis surgery between 2004 and 2014 were sent a questionnaire relating to their postoperative medical treatment, pregnancies, relief of symptoms and recurrence. Responses of 206 patients (45.2%) and their clinical data were analysed for this study. RESULTS: A total of 66.5% (N = 137) of patients had stage III disease, and 33.5% (N = 69) had stage IV disease. The average age was 37 years (17-59). A total of 63.1% (N = 130) of surgeries were performed by laparoscopy, 21.8% (N = 45) were performed by laparotomy and 15% (N = 31) were performed by conversion. Complete resection of endometriosis lesions was achieved in 90.8% of patients (N = 187). After surgery, 48.5% (N = 100) of the women did not receive hormonal treatment; the main reason was the desire for children in 53%. Complete or partial relief in complaints was achieved in 93.2% (N = 192). The rate of recurrence was 21.8% (N = 45). The statistically significant factors that was associated with a higher risk to develop recurrence was an age < 35 (p < 0.005). After surgery, 65.8% (79/120) of patients who wished to have children became pregnant. There was a statistically significant association among a higher postoperative pregnancy rate and age < 35 (p < 0.003) in multivariate logistic regression analysis and laparoscopic surgical access in univariate logistic regression analysis (p < 0.01). CONCLUSION: We assessed the high percentage of complete or partial relief of symptoms of 93.2%, the high postoperative pregnancy rate of 65.8% and the low rate of recurrence of 21.8% compared to international literature to be very encouraging for women suffering from moderate and severe endometriosis. Though laparoscopy is considered the 'gold standard'of endometriosis surgery, laparotomy still may be indicated in patients with extensive endometriosis especially to preserve reproductive function.

Peritoneal contamination with ICG-stained cervical secretion as surrogate for potential cervical cancer tumor cell dissemination: A proof-of-principle study for laparoscopic hysterectomy.

INTRODUCTION: Intracorporal colpotomy during radical hysterectomy for cervical cancer is discussed to be a risk factor for peritoneal dissemination of tumor cells. It might lead to increased recurrence rates after laparoscopic radical hysterectomy compared with abdominal hysterectomy, as shown by the recent LACC study. Data on the frequency or mechanisms of peritoneal contamination are missing. We aimed to analyze peritoneal contamination of cervical secretion during intracorporal colpotomy with a novel indocyaningreen (ICG)-based technique. MATERIAL AND METHODS: In this prospective proof-of-principle study, patients undergoing routine laparoscopic or robot-assisted hysterectomy were selected. ICG was specifically applied to the cervical surface and routine surgery was performed. During colpotomy, pictures under white and fluorescence light were taken to evaluate frequency of contamination. RESULTS: By using cervically applied ICG we were able to visualize directly peritoneal contamination with cervical secretion during intracorporal colpotomy. We detected peritoneal contamination in 9/12 (75%) patients undergoing routine laparoscopic hysterectomy. Contamination of laparoscopic instruments occurred in 60% of the patients. When contamination occurred, it was routinely detectable during all steps of colpotomy. There were no adverse effects during surgery. CONCLUSIONS: Peritoneal contamination with cervical secretion frequently occurs during intracorporal colpotomy. This novel technique represents a promising tool for feasible and direct visualization of peritoneal contamination during colpotomy. The technique may be easily implemented in further studies on laparoscopic and abdominal hysterectomy and serve as a quality assessment tool for surgeons and surgical techniques.

Application of sentinel lymph node dissection in gynecological cancers: results of a survey among German hospitals.

PURPOSE: Evaluating the application of the sentinel lymph node dissection (SLND) in gynecological cancers among German hospitals. METHODS: Between March and June 2016 an online questionnaire on SLND in gynecologic cancers was sent by email to all German gynecologic cancer centers, all university hospitals and general hospitals for which an email address was available. The survey contained 61 questions regarding the SLND in vulvar, cervical, endometrial and ovarian cancer. RESULTS: In total, 63 clinics, including 13 (20.6%) university hospitals, 28 (44.4%) hospitals offering maximum care and 22 (34.9%) general hospitals, responded to the questionnaire. Most clinics (46/63, 73%) performed SLND in vulvar cancer with a median amount of 7.8 (range 1-43) SLND per year. 56.5% of the clinics included patients according to the German national guidelines and performed ultrastaging of negative SLN. Furthermore, 18/63 (28.5%) of the responding clinics applied SLND in cervical cancer including 7 (77.8%) centers which conducted isolated SLND without radical pelvic lymph node dissection (LND). Preoperative imaging with planar lymphoscintigraphy (LSG) was applied in 12/18 (66.7%) of the clinics. SLND in endometrial cancer was reported by 4/63 (6.4%) hospitals. Three of them (75%) regularly performed a subsequent radical pelvic LND. One clinic (1.5%) reported SLND in ovarian cancer in combination with radical LND. CONCLUSION: Especially in vulvar and cervical cancer, isolated SLND appears to be partially implemented in the routine surgical treatment. However, this survey illustrates a wide heterogeneity regarding inclusion criteria and application of the SLND approach.

Excellent analytical and clinical performance of a dry self-sampling device for human papillomavirus detection in an urban Chinese referral population.

AIM: To evaluate the analytical and clinical effectiveness of cervicovaginal self-sampling with a dry sampling device (Evalyn Brush) for high-risk human papillomavirus (hr-HPV) testing and detection of cervical disease. METHODS: The study population consisted of 101 patients from a large gynecological outpatient clinic in Shanghai referred for abnormal cervical screening results and 101 women without cervical lesions. Self-samples obtained in the clinic and physician-collected cervical specimens (reference) were stored at -20 °C for 16-18 weeks and then transferred to 20 ml of ThinPrep medium and tested for hr-HPV using a multiplex real time polymerase chain reaction assay. All women had a colposcopic examination with a Pap smear and directed or random biopsies. RESULTS: High risk-HPV was detected in 92 patients (45.5%) with the self-collected cervicovaginal specimens and in 93 (46.0%) with the physician-collected cervical specimens, resulting in an agreement of 97.5% and a Kappa of 0.95 (95% confidence interval 0.91-0.99). Among all of the included women, 46 (22.8%) had cervical intraepithelial neoplasia grade 3 or worse (cervical intraepithelial neoplasia 3+). Hr-HPV was found in 43 of these patients (93.5%) with self-sampling and in 44 (95.7%) with the physician-collected specimens. CONCLUSIONS: Self-collected dry cervicovaginal samples transferred to ThinPrep medium and tested for hr-HPV using a clinically validated polymerase chain reaction assay showed very good agreement with physician-collected cervical specimens and a very high hr-HPV positivity rate for cervical intraepithelial neoplasia 3 +.

Clinical presentation, treatment and outcome of vaginal intraepithelial neoplasia.

PURPOSE: To characterize the clinical presentation and outcome of patients with vaginal intraepithelial neoplasia (VAIN). METHODS: Medical records of 65 women with VAIN treated between 2005 and 2012 at the colposcopy clinic of a German university hospital were retrospectively evaluated for VAIN grade, HPV status, VAIN localization, treatment method and relapse rate. Follow-up data were available for 53 patients (82 %). RESULTS: Mean age was 61 years (range 32-89 years). Most lesions (55 %) were found in the upper vaginal third; 42 % were multifocal. Multifocal VAIN was more frequently HPV positive than single lesions (p = 0.059). Of all women with known HPV status, 83 % were high-risk (HR) HPV positive and 32 % had a simultaneous CIN earlier or at the same time as the VAIN, mostly CIN 3 (66 %). Two-thirds had a hysterectomy in the past, often because of high-grade CIN. Most cases of VAIN were treated with CO2 laser vaporization. A relapse of the disease could be confirmed for 57 %. HR-HPV positive VAIN was significantly more likely to relapse than HR-HPV negative VAIN (p = 0.005). There were three cases of vaginal cancer with surrounding VAIN 3 or vaginal cancer diagnosed after primary treatment of VAIN 3 and one case of vaginal cancer during follow-up 22 months after the first laser vaporization. All of these cases were HR-HPV positive. CONCLUSIONS: VAIN has a high relapse rate and a high progression rate to invasive cancer especially if HR-HPV positive. Therefore, adequate follow-up examinations are mandatory.

Detection of E6-AP as a potential therapeutic target in cervical specimen of HPV-infected women.

PURPOSE: Silencing of HPV oncogenes or their human host client proteins using topically applied small interfering RNA (siRNA) may be an attractive nonsurgical strategy for CIN treatment. An exploratory clinical investigation was designed to evaluate E6-AP mRNA expression levels in different stages of cervical intraepithelial neoplasia and during the menstrual cycle. METHODS: In 38 premenopausal women aged 18-45 years referred to colposcopy clinic, analysis of serum hormones, cervical smears for cytology and HPV DNA, cervical biopsy, p16 immunohistochemistry and E6-AP mRNA expression levels in cervical smears and biopsies were performed. The intra-subject variability in E6-AP mRNA expression of vaginal smears was assessed and compared to cervical biopsy specimens. RESULTS: RNA of sufficient quantity and quality was available for E6-AP expression analysis from 97 % of the collected cervical smears and from 56 % of the collected biopsy samples. The normalized RNA levels from cervical smears were approximately tenfold higher compared to biopsies. There was little influence by the phase of the menstrual cycle or by CIN stage. Real-time PCR showed that the expression level of E6-AP is in a range (<28 C t) that would allow for detection of at least 100-fold modulation by a therapeutic agent (based on an assay LOD of C t = 36). CONCLUSIONS: Our findings suggest a potential therapeutic approach using E6-AP siRNA as a specific molecular target in cervical intraepithelial neoplasia.

Quality of ChatGPT-Generated Therapy Recommendations for Breast Cancer Treatment in Gynecology.

Introduction: Artificial intelligence (AI) is revolutionizing medical workflows, with self-learning systems like ChatGPT showing promise in therapy recommendations. Our study evaluated ChatGPT's performance in suggesting treatments for 30 breast cancer cases. AI's role in healthcare is expanding, particularly with tools like ChatGPT becoming accessible. However, understanding its limitations is vital for safe implementation. Material and Methods: We used 30 breast cancer cases from our medical board, assessing ChatGPT's suggestions. The input was standardized, incorporating relevant patient details and treatment options. ChatGPT's output was evaluated by oncologists based on a given questionnaire. Results: Treatment recommendations by ChatGPT were overall rated sufficient with minor limitations by the oncologists. The HER2 treatment category was the best-rated therapy option, with the most accurate recommendations. Primary cases received more accurate recommendations, especially regarding chemotherapy. Conclusions: While ChatGPT demonstrated potential, difficulties were shown in intricate cases and postoperative scenarios. Challenges arose in offering chronological treatment sequences and partially lacked precision. Refining inputs, addressing ethical intricacies, and ensuring chronological treatment suggestions are essential. Ongoing research is vital to improving AI's accuracy, balancing AI-driven suggestions with expert insights and ensuring safe and reliable AI integration into patient care.

Validation and functional follow-up of cervical cancer risk variants at the HLA locus.

Cervical cancer is the fourth most common cancer in females. Genome-wide association studies (GWASs) have proposed cervical cancer susceptibility variants at the HLA locus on chromosome 6p21. To corroborate these findings and investigate their functional impact in cervical tissues and cell lines, we genotyped nine variants from cervical cancer GWASs (rs17190106, rs535777, rs1056429, rs2763979, rs143954678, rs113937848, rs3117027, rs3130214, and rs9477610) in a German hospital-based series of 1122 invasive cervical cancers, 1408 dysplasias, and 1196 healthy controls. rs17190106, rs1056429 and rs143954678/rs113937848 associated with cervical malignancies overall, while rs17190106 and rs535777 associated specifically with invasive cancer (OR = 0.69, 95% CI = 0.55-0.86, p = 0.001) or adenocarcinomas (OR = 1.63, 95%CI = 1.17-2.27, p = 0.004), respectively. We tested these and one previously genotyped GWAS variant, rs9272117, for potential eQTL effects on 36 gene transcripts at the HLA locus in 280 cervical epithelial tissues. The strongest eQTL pairs were rs9272117 and HLA-DRB6 (p = 1.9x10E-5), rs1056429 and HLA-DRB5 (p = 2.5x10E-4), and rs535777 and HLA-DRB1 (p = 2.7x10E-4). We also identified transcripts that were specifically upregulated (DDX39B, HCP5, HLA-B, LTB, NFKBIL1) or downregulated (HLA-C, HLA-DPB2) in HPV+ or HPV16+ samples. In comparison, treating cervical epithelial cells with proinflammatory cytokine γ-IFN led to a dose-dependent induction of HCP5, HLA-B, HLA-C, HLA-DQB1, HLA-DRB1, HLA-DRB6, and repression of HSPA1L. Taken together, these results identify relevant genes from both the MHC class I and II regions that are inflammation-responsive in cervical epithelium and associate with HPV (HCP5, HLA-B, HLA-C) and/or with genomic cervical cancer risk variants (HLA-DRB1, HLA-DRB6). They may thus constitute important contributors to the immune escape of precancerous cells after HPV-infection.

Enzyme-linked immunosorbent assay for p16(INK4a) - a new triage test for the detection of cervical intraepithelial neoplasia?

OBJECTIVE: To evaluate enzyme-linked immunosorbent assay (ELISA) for cyclin-dependent kinase inhibitor 2A protein (p16(INK4a) ) on self-collected cervicovaginal lavage samples as an additional triage test to identify women with high-grade cervical intraepithelial neoplasia (CIN). DESIGN: Retrospective feasibility, sensitivity and specificity study. SETTING: University Medical School, Germany. SAMPLE: One hundred and fifty-two patients from the colposcopy clinic were included. METHODS: All women used a cervico-vaginal lavage device (Delphi Screener) for self-sampling and had gynecological examinations with Pap smears, cervical smears in ThinPrep PreservCyt solution and Cervatec medium for human papillomavirus (HPV) testing (Qiagen Hybrid Capture 2) and colposcopic examinations with biopsies if abnormalities were detected (72 women; 51%). All cytological samples were examined by p16(INK4a) ELISA. MAIN OUTCOME MEASURES: Sensitivity and specificity of p16(INK4a) ELISA for high-grade CIN. RESULTS: Complete data were available for 140 women. Among these, 62 women (46%) presented with an atypical Pap smear and 65 (46.4%) were high-risk HPV positive in the reference smear sample. Seventeen women (12%) had CIN 3+. Twenty-seven (19%) physician-collected samples were p16(INK4a) ELISA positive. In contrast, p16(INK4a) ELISA turned out to be positive in only one (1%) vaginal lavage sample. CONCLUSIONS: Our study shows that self-sampling with cervicovaginal lavage followed by p16(INK4a) ELISA is not suitable for the detection of high-grade CIN.

Evaluation of CIN2/3 Lesion Regression in GynTect® DNA Methylation-Marker-Negative Patients in a Longitudinal Study.

Cervical intraepithelial neoplasia (CIN) grade 2/3 has a high spontaneous regression rate, especially among women ≤29 years of age. To reduce overtreatment, reliable prognostic biomarkers would be helpful. The main aim of this study was to analyze the negative predictive value of the methylation marker panel GynTect® for lesion regression. In this prospective, multicenter, longitudinal observational proof-of-concept study, women aged ≤29 years with histologically confirmed CIN2 (n = 24) or CIN3 (n = 36) were closely monitored without treatment for up to 24 or 12 months, respectively. The outcome was either regression, persistence, or progression of the lesion. For each patient, a single baseline sample (V0) for cytology, hrHPV detection and methylation analysis was taken. In a primary analysis, the negative predictive value (NPV) of a GynTect®-negative test result at V0 for regression was determined. We tested the null hypothesis NPV ≤ 70% against the alternative hypothesis NPV ≥ 90%. Twelve of the eighteen GynTect®-negative CIN2 patients showed regression (NPV = 67%, 90% CI 44-85%, p = 0.53). Of the 27 GynTect®-negative CIN3 lesions, 15 regressed (NPV = 56%, 90% CI 38-72%, p = 0.92). Although the majority of GynTect®-negative lesions regressed, the postulated NPV of ≥90% was not observed. Thus, the clinical relevance for an implementation of the GynTect® assay for patients undergoing watchful waiting remains questionable. Further studies with longer observation periods should be undertaken.

Association of two genomic variants with HPV type-specific risk of cervical cancer.

PROBLEM: Human papillomavirus infection is integral to developing invasive cervical cancer in the majority of patients. In a recent genome-wide association study, rs9357152 and rs4243652 have been associated with seropositivity for HPV16 or HPV18, respectively. It is unknown whether these variants also associate with cervical cancer triggered by either HPV16 or HPV18. METHODS: We investigate whether the two HPV susceptibility variants show association with type-specific cervical cancer in a genetic case-control study with cases stratified by HPV16 or HPV18, respectively. We further tested whether rs9357152 modulates gene expression of any of 36 genes at the human leukocyte antigen locus in 256 cervical tissues. RESULTS: rs9357152 was associated with invasive HPV16-positive cervical cancer (OR 1.33, 95%CI 1.03-1.70, p = 0.03), and rs4243652 was associated with HPV18-positive adenocarcinomas (OR 2.96, 95%CI 1.18-7.41, p = 0.02). These associations remained borderline significant after testing against different sets of controls. rs9357152 was found to be an eQTL for HLA-DRB1 in HPV-positive cervical tissues (pANOVA = 0.0009), with the risk allele lowering mRNA levels. CONCLUSIONS: We find evidence that HPV seropositivity variants at chromosome 6 and 14 may modulate type-specific cervical cancer risk. rs9357152 may exert its effect through regulating HLA-DRB1 induction in the presence of HPV. In regard of multiple testing, these results need to be confirmed in larger studies.

Evaluation of a new multiplex real-time polymerase chain reaction assay for the detection of human papillomavirus infections in a referral population.

OBJECTIVES: Human papillomavirus (HPV) testing is an important part of cervical cancer screening and management of women with atypical screening results. This study was conducted to evaluate the analytical and clinical performance of the Abbott RealTime High-Risk HPV assay (RealTime) in a referral population, in comparison to the Qiagen Hybrid Capture 2 High-Risk HPV DNA Test (hc2). METHODS: RealTime is a new polymerase chain reaction assay that detects 14 high-risk HPV genotypes with simultaneous differentiation between HPV 16 and HPV 18. Five hundred forty-five routine cervical smear samples (ThinPrep) from women who were referred to 2 German colposcopy clinics were included in the study. All samples were tested with both assays for the detection of high-risk HPV DNA. Specimens with repeatedly discordant results were genotyped by Linear Array (Roche) and in-house polymerase chain reaction assays. RESULTS: Both assays showed excellent overall agreement (92.8%; κ = 0.86) on 545 samples. Analytical sensitivity of RealTime was comparable to that of hc2 (97.6% vs 95.1%, P = 0.189), whereas RealTime demonstrated significantly higher analytical specificity compared with hc2 (100% vs 93.1%, P < 0.0001). RealTime showed no cross-reactivity with untargeted HPV genotypes in this study. The clinical performance of the assays was evaluated based on histology results available from 319 women (90 nonpathological, 73 cervical intraepithelial neoplasia [CIN] 1, 75 CIN 2, 74 CIN 3, and 7 invasive cancers). High-risk HPV detection rates observed in women with CIN 1, CIN 2+, and CIN 3+ diagnosis, respectively, were comparable for both assays: 47.9%, 92.3%, and 97.5% (RealTime) and 47.9%, 92.3%, and 93.8% (hc2). Detection of HPV 16/18 with RealTime was highly correlated with severity of dysplasia: less than CIN 2, 30.5%; CIN 2+, 59.0%; CIN 3+, 71.6%. CONCLUSIONS: These results support the use of RealTime for routine detection of HPV infections in a referral population.