Current cervical cancer screening is based on morphological assessment of Pap smears and associated with significant false negative and false positive results. Previously, we have shown that detection of hypermethylated genes in cervical scrapings using quantitative methylation-specific PCR (QMSP) is a promising tool for identification of squamous cell cervical cancer. Aim of the present pilot-study was to evaluate presence of hypermethylated genes in cervical carcinogenesis, both in squamous cell as well as adenocarcinomas. Cervical scrapings were obtained from 30 patients diagnosed with cervical cancer (20 squamous cell carcinomas and 10 adenocarcinomas) and 19 women with histologically normal cervices. The scraped cells were used for determination of promoter hypermethylation by QMSP for 12 genes and for morphological assessment. Overall, CALCA, DAPK, ESR1, TIMP3, APC and RAR-b 2 promoters were significantly more often hypermethylated in cancers than in controls, while adenocarcinomas were more often hypermethylated above the highest control ratio for APC, TIMP3 and RASSF1A promoters. Combining 4 genes (CALCA, DAPK, ESR1 and APC) yielded a sensitivity of 89% (with all adenocarcinomas identified), equal to cytomorphology (89%) and high-risk human papilloma virus (Hr-HPV; 90%). The 4-gene QMSP proved theoretically superior to cytomorphology as well as Hr-HPV in specificity (100% vs. 83 and 68%, respectively), because cytology identified 3 controls as moderate or severe dyskaryosis and 6 controls were positive for Hr-HPV. In conclusions, QMSP of 4 gene promoters combined appears to have comparable sensitivity and potentially better specificity in comparison to ''classic'' cytomorphological assessment and Hr-HPV detection. QMSP holds promise as a new diagnostic tool for both squamous cell carcinoma and adenocarcinoma of the cervix. ' 2006 Wiley-Liss, Inc. Key words: methylation; cervical cancer; QMSP; DAPK; ESR1Cervical cancer is an important cause of death in women worldwide. 1 Cervical carcinogenesis is highly associated with (high-risk) human papilloma virus (HPV) infections. 2 Cytomorphological examination of cervical smears is a widely applied, though not ideal screening method for cervical cancer and its precursors, since the Pap smear has false negative rates of 2-40%, due to a combination of sampling error, processing artifacts and the nature of subjective interpretation. 1,3,4 False-negative cytology can also be found in about 50% of cases when previous negative smears are reviewed from the small proportion of screened women who develop invasive cancer. 4 Moreover, as many as 20% of all Pap smears are interpreted as atypical squamous cells of undetermined significance (ASCUS) or borderline dyskaryotic, leading to increased surveillance and more invasive tests in many of these patients. 3,5 The incidence of squamous cell carcinoma of the cervix has decreased since introduction of nation wide screening programs, compared to a relative increased incidence of adenocarcinoma of the cervix. 6 The efficacy of ...
Purpose: Recently, we reported 13 possible cervical cancer-specific methylated biomarkers identified by pharmacologic unmasking microarray in combination with large-genome computational screening. The aim of the present study was to perform an in-depth analysis of the methylation patterns of these 13 candidate genes in cervical neoplasia and to determine their diagnostic relevance. Experimental Design and Results: Five of the 13 gene promoters (C13ORF18, CCNA1, TFPI2, C1ORF166, and NPTX1) were found to be more frequently methylated in frozen cervical cancer compared with normal cervix specimens. Quantitative methylation analysis for these five markers revealed that both CCNA1 and C13ORF18 were methylated in 68 of 97 cervical scrapings from cervical cancer patients and in only 5 and 3 scrapings, respectively, from 103 healthy controls (P < 0.0005). In cervical scrapings from patients referred with an abnormal Pap smear, CCNA1 and C13ORF18 were methylated in 2 of 43 and 0 of 43 CIN 0 (no cervical intraepithelial neoplasia) and in 1 of 41 and 0 of 41 CIN I, respectively. Furthermore, 8 of 43 CIN II, 22 of 43 CIN III, and 3 of 3 microinvasive cancer patients were positive for both markers. Although sensitivity for CIN II or higher (for both markers 37%) was low, specificity (96% and 100%, respectively) and positive predictive value (92% and 100%, respectively) were high. Conclusion: Methylation of CCNA1 and C13ORF18 in cervical scrapings is strongly associated with CIN II or higher-grade lesions. Therefore, these markers might be used for direct referral to gynecologists for patients with a methylation-positive scraping. (Cancer Epidemiol Biomarkers Prev 2009;18(11):3000-7)
PurposeThe Ki67 proliferation index is a prognostic and predictive marker in breast cancer. Manual scoring is prone to inter- and intra-observer variability. The aims of this study were to clinically validate digital image analysis (DIA) of Ki67 using virtual dual staining (VDS) on whole tissue sections and to assess inter-platform agreement between two independent DIA platforms.MethodsSerial whole tissue sections of 154 consecutive invasive breast carcinomas were stained for Ki67 and cytokeratin 8/18 with immunohistochemistry in a clinical setting. Ki67 proliferation index was determined using two independent DIA platforms, implementing VDS to identify tumor tissue. Manual Ki67 score was determined using a standardized manual counting protocol. Inter-observer agreement between manual and DIA scores and inter-platform agreement between both DIA platforms were determined and calculated using Spearman’s correlation coefficients. Correlations and agreement were assessed with scatterplots and Bland–Altman plots.ResultsSpearman’s correlation coefficients were 0.94 (p < 0.001) for inter-observer agreement between manual counting and platform A, 0.93 (p < 0.001) between manual counting and platform B, and 0.96 (p < 0.001) for inter-platform agreement. Scatterplots and Bland–Altman plots revealed no skewness within specific data ranges. In the few cases with ≥ 10% difference between manual counting and DIA, results by both platforms were similar.ConclusionsDIA using VDS is an accurate method to determine the Ki67 proliferation index in breast cancer, as an alternative to manual scoring of whole sections in clinical practice. Inter-platform agreement between two different DIA platforms was excellent, suggesting vendor-independent clinical implementability.Electronic supplementary materialThe online version of this article (10.1007/s10549-018-4669-2) contains supplementary material, which is available to authorized users.
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