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Diagnostic Performance of Plasma DNA Methylation Profiles in Lung Cancer, Pulmonary Fibrosis and COPD.

Wielscher M, Vierlinger K, Kegler U, Ziesche R, Gsur A, Weinhäusel A - EBioMedicine (2015)

Bottom Line: Disease-specific alterations of the cell-free DNA methylation status are frequently found in serum samples and are currently considered to be suitable biomarkers.The results were confirmed using an independent sample set (n = 46) by use of the four top markers discovered in the study (HOXD10, PAX9, PTPRN2, and STAG3) yielding an AUC of 0.85 (95%CI: 0.72-0.95).This technique was capable of distinguishing interrelated complex pulmonary diseases suggesting that multiplexed MSRE enrichment might be useful for simple and reliable diagnosis of diverse multifactorial disease states.

View Article: PubMed Central - PubMed

Affiliation: AIT - Austrian Institute of Technology, Health & Environment Department, Molecular Diagnostics Unit, Muthgasse 11/2, 1190 Vienna, Austria.

ABSTRACT
Disease-specific alterations of the cell-free DNA methylation status are frequently found in serum samples and are currently considered to be suitable biomarkers. Candidate markers were identified by bisulfite conversion-based genome-wide methylation screening of lung tissue from lung cancer, fibrotic ILD, and COPD. cfDNA from 400 μl serum (n = 204) served to test the diagnostic performance of these markers. Following methylation-sensitive restriction enzyme digestion and enrichment of methylated DNA via targeted amplification (multiplexed MSRE enrichment), a total of 96 markers were addressed by highly parallel qPCR. Lung cancer was efficiently separated from non-cancer and controls with a sensitivity of 87.8%, (95%CI: 0.67-0.97) and specificity 90.2%, (95%CI: 0.65-0.98). Cancer was distinguished from ILD with a specificity of 88%, (95%CI: 0.57-1), and COPD from cancer with a specificity of 88% (95%CI: 0.64-0.97). Separation of ILD from COPD and controls was possible with a sensitivity of 63.1% (95%CI: 0.4-0.78) and a specificity of 70% (95%CI: 0.54-0.81). The results were confirmed using an independent sample set (n = 46) by use of the four top markers discovered in the study (HOXD10, PAX9, PTPRN2, and STAG3) yielding an AUC of 0.85 (95%CI: 0.72-0.95). This technique was capable of distinguishing interrelated complex pulmonary diseases suggesting that multiplexed MSRE enrichment might be useful for simple and reliable diagnosis of diverse multifactorial disease states.

No MeSH data available.


Related in: MedlinePlus

Study flow diagram. The discovery was performed on bisulfite converted DNA derived from lung tissue of lung cancer patients, fibrotic ILD, COPD patients and healthy controls. Candidate markers revealed by Illumina 450K arrays were validated on the same sample material via MSRE digestion based qPCR. The best performing assays in this procedure were then used for minimal invasive cfDNA methylation detection. Thus a 4-gene model could be established, which was validated on an independent sample set of 46 patients.
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f0005: Study flow diagram. The discovery was performed on bisulfite converted DNA derived from lung tissue of lung cancer patients, fibrotic ILD, COPD patients and healthy controls. Candidate markers revealed by Illumina 450K arrays were validated on the same sample material via MSRE digestion based qPCR. The best performing assays in this procedure were then used for minimal invasive cfDNA methylation detection. Thus a 4-gene model could be established, which was validated on an independent sample set of 46 patients.

Mentions: The 204 serum samples were randomized and split into three parts each one underwent separate multiplexed MSRE enrichment and Biomark qPCR read out. 46 samples from proof of principal (POP) set were processed separately. Technical replicates of samples as well as negative controls were introduced by the time of cell free DNA (cfDNA) isolation and randomly distributed to the three experimental blocks. Standard curves, which also served as amplification control, as well as additional negative controls, were introduced before multiplexed enrichment. cfDNA was isolated out of 400 μl patients' serum using Roche High pure template preparation kit (Roche) with a modified isolation protocol published previously Wielscher et al., 2011. In a volume of 12 μl cfDNA was digested with four methylsensitive restriction enzymes (MSRE) namely HpaII (ThermoFisher), Hin6I (ThermoFisher), AciI (NewEnglandBiolabs) and HpyCHIV4 (NewEnglandBiolabs). 10 μl of the MSRE digestion reaction was subjected to a multiplexed pre-amplification. Preamplification reaction was performed in a volume of 20 μl using 96 primer pairs per sample. 19 cycles of preamplification were performed. qPCR detection, consisting of 96 single qPCR reactions per sample, was performed on a BioMark™ HD Reader (Fluidigm). For detailed information on all experimental procedures and patients, see study flow figure (Fig. 1) and in supplemental material pp 1–3.


Diagnostic Performance of Plasma DNA Methylation Profiles in Lung Cancer, Pulmonary Fibrosis and COPD.

Wielscher M, Vierlinger K, Kegler U, Ziesche R, Gsur A, Weinhäusel A - EBioMedicine (2015)

Study flow diagram. The discovery was performed on bisulfite converted DNA derived from lung tissue of lung cancer patients, fibrotic ILD, COPD patients and healthy controls. Candidate markers revealed by Illumina 450K arrays were validated on the same sample material via MSRE digestion based qPCR. The best performing assays in this procedure were then used for minimal invasive cfDNA methylation detection. Thus a 4-gene model could be established, which was validated on an independent sample set of 46 patients.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4563135&req=5

f0005: Study flow diagram. The discovery was performed on bisulfite converted DNA derived from lung tissue of lung cancer patients, fibrotic ILD, COPD patients and healthy controls. Candidate markers revealed by Illumina 450K arrays were validated on the same sample material via MSRE digestion based qPCR. The best performing assays in this procedure were then used for minimal invasive cfDNA methylation detection. Thus a 4-gene model could be established, which was validated on an independent sample set of 46 patients.
Mentions: The 204 serum samples were randomized and split into three parts each one underwent separate multiplexed MSRE enrichment and Biomark qPCR read out. 46 samples from proof of principal (POP) set were processed separately. Technical replicates of samples as well as negative controls were introduced by the time of cell free DNA (cfDNA) isolation and randomly distributed to the three experimental blocks. Standard curves, which also served as amplification control, as well as additional negative controls, were introduced before multiplexed enrichment. cfDNA was isolated out of 400 μl patients' serum using Roche High pure template preparation kit (Roche) with a modified isolation protocol published previously Wielscher et al., 2011. In a volume of 12 μl cfDNA was digested with four methylsensitive restriction enzymes (MSRE) namely HpaII (ThermoFisher), Hin6I (ThermoFisher), AciI (NewEnglandBiolabs) and HpyCHIV4 (NewEnglandBiolabs). 10 μl of the MSRE digestion reaction was subjected to a multiplexed pre-amplification. Preamplification reaction was performed in a volume of 20 μl using 96 primer pairs per sample. 19 cycles of preamplification were performed. qPCR detection, consisting of 96 single qPCR reactions per sample, was performed on a BioMark™ HD Reader (Fluidigm). For detailed information on all experimental procedures and patients, see study flow figure (Fig. 1) and in supplemental material pp 1–3.

Bottom Line: Disease-specific alterations of the cell-free DNA methylation status are frequently found in serum samples and are currently considered to be suitable biomarkers.The results were confirmed using an independent sample set (n = 46) by use of the four top markers discovered in the study (HOXD10, PAX9, PTPRN2, and STAG3) yielding an AUC of 0.85 (95%CI: 0.72-0.95).This technique was capable of distinguishing interrelated complex pulmonary diseases suggesting that multiplexed MSRE enrichment might be useful for simple and reliable diagnosis of diverse multifactorial disease states.

View Article: PubMed Central - PubMed

Affiliation: AIT - Austrian Institute of Technology, Health & Environment Department, Molecular Diagnostics Unit, Muthgasse 11/2, 1190 Vienna, Austria.

ABSTRACT
Disease-specific alterations of the cell-free DNA methylation status are frequently found in serum samples and are currently considered to be suitable biomarkers. Candidate markers were identified by bisulfite conversion-based genome-wide methylation screening of lung tissue from lung cancer, fibrotic ILD, and COPD. cfDNA from 400 μl serum (n = 204) served to test the diagnostic performance of these markers. Following methylation-sensitive restriction enzyme digestion and enrichment of methylated DNA via targeted amplification (multiplexed MSRE enrichment), a total of 96 markers were addressed by highly parallel qPCR. Lung cancer was efficiently separated from non-cancer and controls with a sensitivity of 87.8%, (95%CI: 0.67-0.97) and specificity 90.2%, (95%CI: 0.65-0.98). Cancer was distinguished from ILD with a specificity of 88%, (95%CI: 0.57-1), and COPD from cancer with a specificity of 88% (95%CI: 0.64-0.97). Separation of ILD from COPD and controls was possible with a sensitivity of 63.1% (95%CI: 0.4-0.78) and a specificity of 70% (95%CI: 0.54-0.81). The results were confirmed using an independent sample set (n = 46) by use of the four top markers discovered in the study (HOXD10, PAX9, PTPRN2, and STAG3) yielding an AUC of 0.85 (95%CI: 0.72-0.95). This technique was capable of distinguishing interrelated complex pulmonary diseases suggesting that multiplexed MSRE enrichment might be useful for simple and reliable diagnosis of diverse multifactorial disease states.

No MeSH data available.


Related in: MedlinePlus