A systematic comparison of quantitative high-resolution DNA methylation analysis and methylation-specific PCR

Early diagnosis of colorectal cancer (CRC) is of high importance as prognosis depends on tumour stage at the time of diagnosis. Detection of tumour-specific DNA methylation marks in cfDNA has several advantages over other approaches and has great potential for solving diagnostic needs. We report here the identification of DNA methylation biomarkers for CRC and give insights in our methylation-sensitive restriction enzyme coupled qPCR (MSRE-qPCR) system. Targeted microarrays were used to investigate the DNA methylation status of 360 cancer-associated genes. Validation was done by qPCR-based approaches. A focus was on investigating marker performance in cfDNA from 88 patients (44 CRC, 44 controls). Finally, the workflow was scaled-up to perform 180plex analysis on 110 cfDNA samples, to identify a DNA methylation signature for advanced colonic adenomas (AA). A DNA methylation signature (n = 44) was deduced from microarray experiments and confirmed by quantitative methylation-specific PCR (qMSP) and by MSRE-qPCR, providing for six genes’ single areas under the curve (AUC) values of >0.85 (WT1, PENK, SPARC, GDNF, TMEFF2, DCC). A subset of the signatures can be used for patient stratification and therapy monitoring for progressed CRC with liver metastasis using cfDNA. Furthermore, we identified a 35-plex classifier for the identification of AAs with an AUC of 0.80.

Download Full-text

DNA methylation analysis with methylation‐sensitive high‐resolution melting (MS‐HRM) reveals gene panel for glioma characteristics

CNS Neuroscience & Therapeutics ◽

10.1111/cns.13443 ◽

2020 ◽

Vol 26 (12) ◽

pp. 1303-1314

Author(s):

Aleksandra Majchrzak‐Celińska ◽

Emilia Dybska ◽

Anna‐Maria Barciszewska

Keyword(s):

Dna Methylation ◽

High Resolution ◽

High Resolution Melting ◽

Gene Panel ◽

Methylation Analysis ◽

Dna Methylation Analysis

Download Full-text

CADM1, MAL and miR124-2 methylation analysis in cervical scrapes to detect cervical and endometrial cancer

Journal of Clinical Pathology ◽

10.1136/jclinpath-2014-202616 ◽

2014 ◽

Vol 67 (12) ◽

pp. 1067-1071 ◽

Cited By ~ 56

Author(s):

Lise M A De Strooper ◽

Marjolein van Zummeren ◽

Renske D M Steenbergen ◽

Maaike C G Bleeker ◽

Albertus T Hesselink ◽

...

Keyword(s):

Cervical Cancer ◽

Dna Methylation ◽

Endometrial Cancer ◽

Gene Promoter ◽

Intraepithelial Neoplasia ◽

Promoter Hypermethylation ◽

Methylation Analysis ◽

Cervical Intraepithelial Neoplasia Grade ◽

Specific Pcr ◽

Dna Methylation Analysis

AimsGene promoter hypermethylation is recognised as an essential early step in carcinogenesis, indicating important application areas for DNA methylation analysis in early cancer detection. The current study was set out to assess the performance of CADM1, MAL and miR124-2 methylation analysis in cervical scrapes for detection of cervical and endometrial cancer.MethodsA series of cervical scrapes of women with cervical (n=79) or endometrial (n=21) cancer, cervical intraepithelial neoplasia grade 3 (CIN3) (n=16) or CIN2 (n=32), and women without evidence of CIN2 or worse (n=120) were assessed for methylation of CADM1, MAL and miR124-2. Methylation analysis was done by the PreCursor-M assay, a multiplex quantitative methylation-specific PCR.ResultsAll samples of women with cervical cancer (79/79, 100%), independent of the histotype, and 76% (16/21; 95% CI 58.0% to 94.4%) of women with endometrial cancer scored positive for DNA methylation for at least one of the three genes. In women without cancer, methylation frequencies increased significantly with severity of disease from 19.2% (23/120; 95% CI 12.1% to 26.2%) in women without CIN2 or worse to 37.5% (12/32; 95% CI 20.7% to 54.3%) and 68.8% (11/16; 95% CI 46.0% to 91.5%) in women with CIN2 and CIN3, respectively. Overall methylation positivity and the number of methylated genes increased proportionally to the lesion severity.ConclusionsDNA methylation analysis of CADM1, MAL and miR124-2 in cervical scrapes consistently detects cervical cancer and the majority of CIN3 lesions, and has the capacity to broaden its use on cervical scrapes through the detection of a substantial subset of endometrial carcinomas.

Download Full-text