Discovery of new methylation markers to improve screening for cervical intraepithelial neoplasia grade 2/3.

BACKGROUND: Assessment of DNA promoter methylation markers in cervical scrapings for the detection of cervical intraepithelial neoplasia (CIN) and cervical cancer is feasible, but finding methylation markers with both high sensitivity as well as high specificity remains a challenge. In this study, we aimed to identify new methylation markers for the detection of high-grade CIN (CIN2/3 or worse, CIN2+) by using innovative genome-wide methylation analysis (MethylCap-seq). We focused on diagnostic performance of methylation markers with high sensitivity and high specificity considering any methylation level as positive. RESULTS: MethylCap-seq of normal cervices and CIN2/3 revealed 176 differentially methylated regions (DMRs) comprising 164 genes. After verification and validation of the 15 best discriminating genes with methylation-specific PCR (MSP), 9 genes showed significant differential methylation in an independent cohort of normal cervices versus CIN2/3 lesions (p < 0.05). For further diagnostic evaluation, these 9 markers were tested with quantitative MSP (QMSP) in cervical scrapings from 2 cohorts: (1) cervical carcinoma versus healthy controls and (2) patients referred from population-based screening with an abnormal Pap smear in whom also HPV status was determined. Methylation levels of 8/9 genes were significantly higher in carcinoma compared to normal scrapings. For all 8 genes, methylation levels increased with the severity of the underlying histological lesion in scrapings from patients referred with an abnormal Pap smear. In addition, the diagnostic performance was investigated, using these 8 new genes and 4 genes (previously identified by our group: C13ORF18, JAM3, EPB41L3, and TERT). In a triage setting (after a positive Pap smear), sensitivity for CIN2+ of the best combination of genes (C13ORF18/JAM3/ANKRD18CP) (74 %) was comparable to hrHPV testing (79 %), while specificity was significantly higher (76 % versus 42 %, p ≤ 0.05). In addition, in hrHPV-positive scrapings, sensitivity and specificity for CIN2+ of this best-performing combination was comparable to the population referred with abnormal Pap smear. CONCLUSIONS: We identified new CIN2/3-specific methylation markers using genome-wide DNA methylation analysis. The diagnostic performance of our new methylation panel shows higher specificity, which should result in prevention of unnecessary colposcopies for women referred with abnormal cytology. In addition, these newly found markers might be applied as a triage test in hrHPV-positive women from population-based screening. The next step before implementation in primary screening programs will be validation in population-based cohorts.

A four-gene methylation marker panel as triage test in high-risk human papillomavirus positive patients.

Cervical neoplasia-specific biomarkers, e.g. DNA methylation markers, with high sensitivity and specificity are urgently needed to improve current population-based screening on (pre)malignant cervical neoplasia. We aimed to identify new cervical neoplasia-specific DNA methylation markers and to design and validate a methylation marker panel for triage of high-risk human papillomavirus (hr-HPV) positive patients. First, high-throughput quantitative methylation-specific PCRs (QMSP) on a novel OpenArray™ platform, representing 424 primers of 213 cancer specific methylated genes, were performed on frozen tissue samples from 84 cervical cancer patients and 106 normal cervices. Second, the top 20 discriminating methylation markers were validated by LightCycler® MSP on frozen tissue from 27 cervical cancer patients and 20 normal cervices and ROCs and test characteristics were assessed. Three new methylation markers were identified (JAM3, EPB41L3 and TERT), which were subsequently combined with C13ORF18 in our four-gene methylation panel. In a third step, our methylation panel detected in cervical scrapings 94% (70/74) of cervical cancers, while in a fourth step 82% (32/39) cervical intraepithelial neoplasia grade 3 or higher (CIN3+) and 65% (44/68) CIN2+ were detected, with 21% positive cases for ≤CIN1 (16/75). Finally, hypothetical scenario analysis showed that primary hr-HPV testing combined with our four-gene methylation panel as a triage test resulted in a higher identification of CIN3 and cervical cancers and a higher percentage of correct referrals compared to hr-HPV testing in combination with conventional cytology. In conclusion, our four-gene methylation panel might provide an alternative triage test after primary hr-HPV testing.

Detection of cervical neoplasia by DNA methylation analysis in cervico-vaginal lavages, a feasibility study.

OBJECTIVE: To explore the feasibility of DNA methylation analysis for the detection of cervical neoplasia in self-obtained cervico-vaginal lavages. METHODS: Lavages collected by a self-sampling device and paired cervical scrapings were obtained from 20 cervical cancer patients and 23 patients referred with an abnormal cervical smear (15 with high-grade cervical intraepithelial neoplasia (CIN2+) and 8 without CIN). All lavages and scrapings were analyzed by liquid based cytology (LBC), Hybrid Capture II (HC-II) for hr-HPV DNA detection and by DNA methylation analysis (JAM3, TERT, EPB41L3 and C13ORF18). Concordance between lavages and scrapings was measured by Cohen's Kappa (k). RESULTS: In lavages and scrapings from cervical cancer patients (n=20), methylation analysis was positive in 19 (95%) and 19 (95%), HC-II in 16 (80%) and 15 (75%) and LBC in 15 (75%) and 19 (95%), respectively. In lavages and scrapings from CIN2+ patients (n=15), methylation analysis was positive in 10 (67%) and 12 (80%), HC-II in 15 (100%) and 15 (100%) and LBC in 11 (73%) and 12 (80%), respectively. Concordance between cervical scrapings and lavages (n=43) was for LBC k=0.522 (p<0.001), hr-HPV testing k=0.551 (p<0.001) and DNA methylation analysis k=0.653 (p<0.001). CONCLUSIONS: DNA methylation analysis in cervico-vaginal lavages obtained by a self-sampling device is feasible and its diagnostic performance appears to be at least comparable to the detection of cervical neoplasia by cytomorphology and hr-HPV. Our pilot study suggests that detection of cervical neoplasia by DNA methylation analysis in cervico-vaginal lavages warrants exploration of its use in large prospective studies.

The ErbB signalling pathway: protein expression and prognostic value in epithelial ovarian cancer.

Ovarian cancer is the most frequent cause of death from gynaecological cancer in the Western world. Current prognostic factors do not allow reliable prediction of response to chemotherapy and survival for individual ovarian cancer patients. Epidermal growth factor receptor (EGFR) and HER-2/neu are frequently expressed in ovarian cancer but their prognostic value remains unclear. In this study, we investigated the expression and prognostic value of EGFR, EGFR variant III (EGFRvIII), HER-2/neu and important downstream signalling components in a large series of epithelial ovarian cancer patients. Immunohistochemical staining of EGFR, pEGFR, EGFRvIII, Her-2/neu, PTEN (phosphatase and tensin homologue deleted on chromosome 10), total and phosphorylated AKT (pAKT) and phosphorylated ERK (pERK) was performed in 232 primary tumours using the tissue microarray platform and related to clinicopathological characteristics and survival. In addition, EGFRvIII expression was determined in 45 tumours by RT-PCR. Our results show that negative PTEN immunostaining was associated with stage I/II disease (P=0.006), non-serous tumour type (P=0.042) and in multivariate analysis with a longer progression-free survival (P=0.015). Negative PTEN staining also predicted improved progression-free survival in patients with grade III or undifferentiated serous carcinomas (P=0.011). Positive pAKT staining was associated with advanced-stage disease (P=0.006). Other proteins were expressed only at low levels, and were not associated with any clinicopathological parameter or survival. None of the tumours were positive for EGFRvIII. In conclusion, our results indicate that tumours showing negative PTEN staining could represent a subgroup of ovarian carcinomas with a relatively favourable prognosis.

Loss of amino acids during hemodialysis: effect of oral essential amino acid supplementation.

The aim of this study was to investigate the effect of amino acid (AA) supplementation on the loss and its time course of free AA, during hemodialysis. 2 uremic patients on regular hemodialysis received an increasing number of essential AA tablets during the dialysis. The loss and its time course of 6 essential and 7 nonessential free AA during dialysis was assayed in the dialysate. The loss per dialysis treatment of the administered essential AA increased almost proportionally with the doses of AA tablets. For the nonessential AA, no definite relationship between doses of AA tablets and losses was observed. At all levels of supplementation, methionine was retained best: 90% of the amount present in the tablets. Threonine showed the lowest retention: 15-55%.