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Neurofilament Heavy polypeptide CpG island methylation associates with prognosis of renal cell carcinoma and prediction of antivascular endothelial growth factor therapy response.

Dubrowinskaja N, Gebauer K, Peters I, Hennenlotter J, Abbas M, Scherer R, Tezval H, Merseburger AS, Stenzl A, Grünwald V, Kuczyk MA, Serth J - Cancer Med (2014)

Bottom Line: The NEFH CGI methylation demonstrated a tumor-specific increase (P < 0.001), association with advanced disease (P < 0.001), and distant metastasis (P = 0.005).Median OS following targeted therapy was 29.8 months for patients with low methylation versus 9.8 months for the group with high methylation (P = 0.028).We identified NEFH methylation as a candidate epigenetic marker for prognosis of RCC patients as well as prediction of anti-vascular endothelial growth factor-based therapy response.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Hannover Medical School, 30625, Hannover, Germany.

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(A) Neurofilament Heavy polypeptid (NEFH)–CpG island (CGI) organization and location of the qMSP assay relative to the transcription start site (TSS) and exon 1 and 2. CpG sites are indicated by vertical lines. (B) qMSP data for control measurements in duplicate of fully methylated DNA (1), unmethylated DNA (2), unconverted DNA (3) and the blank sample (4). (C) Measurement of assay linearity and efficiency using a twofold dilution series of fully methylated in non-methylated control DNA. (D) Relative methylation levels determined by qMSP for control samples, normal primary cells and various cancer cell lines representing human cancers as specified.
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fig01: (A) Neurofilament Heavy polypeptid (NEFH)–CpG island (CGI) organization and location of the qMSP assay relative to the transcription start site (TSS) and exon 1 and 2. CpG sites are indicated by vertical lines. (B) qMSP data for control measurements in duplicate of fully methylated DNA (1), unmethylated DNA (2), unconverted DNA (3) and the blank sample (4). (C) Measurement of assay linearity and efficiency using a twofold dilution series of fully methylated in non-methylated control DNA. (D) Relative methylation levels determined by qMSP for control samples, normal primary cells and various cancer cell lines representing human cancers as specified.

Mentions: The analysis of converted methylated (M), converted non-methylated (U), and non-converted DNA control samples demonstrated that the NEFH qMSP specifically detects M–DNA while U and non-converted samples remained undeterminable both in the QC1 control reaction as well as in the NEFH-specific PCR (Ct > 45 cycles, Figure 1B). A log2 dilution series adjusted for a constant total amount of converted DNA showed good efficiency and high linearity of the qMSP assay (Figure 1C). Using the slope of the qMSP calibration line we calculated a PCR efficiency of 0.95. Measurement of methylation in cell lines used as substitutes for frequent human cancers as well as normal primary cells and control DNAs demonstrated no methylation neither in normal primary cells of renal or prostatic origin nor in mock controls (Figure 1D). Low methylation was detected for normal mammary primary cells. Breast and urothelial cancer cell lines overall demonstrated highest relative methylation values while >25% relative methylation was detected in two of six RCC and one of three prostate cancer cell lines (Figure 1D).


Neurofilament Heavy polypeptide CpG island methylation associates with prognosis of renal cell carcinoma and prediction of antivascular endothelial growth factor therapy response.

Dubrowinskaja N, Gebauer K, Peters I, Hennenlotter J, Abbas M, Scherer R, Tezval H, Merseburger AS, Stenzl A, Grünwald V, Kuczyk MA, Serth J - Cancer Med (2014)

(A) Neurofilament Heavy polypeptid (NEFH)–CpG island (CGI) organization and location of the qMSP assay relative to the transcription start site (TSS) and exon 1 and 2. CpG sites are indicated by vertical lines. (B) qMSP data for control measurements in duplicate of fully methylated DNA (1), unmethylated DNA (2), unconverted DNA (3) and the blank sample (4). (C) Measurement of assay linearity and efficiency using a twofold dilution series of fully methylated in non-methylated control DNA. (D) Relative methylation levels determined by qMSP for control samples, normal primary cells and various cancer cell lines representing human cancers as specified.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3987080&req=5

fig01: (A) Neurofilament Heavy polypeptid (NEFH)–CpG island (CGI) organization and location of the qMSP assay relative to the transcription start site (TSS) and exon 1 and 2. CpG sites are indicated by vertical lines. (B) qMSP data for control measurements in duplicate of fully methylated DNA (1), unmethylated DNA (2), unconverted DNA (3) and the blank sample (4). (C) Measurement of assay linearity and efficiency using a twofold dilution series of fully methylated in non-methylated control DNA. (D) Relative methylation levels determined by qMSP for control samples, normal primary cells and various cancer cell lines representing human cancers as specified.
Mentions: The analysis of converted methylated (M), converted non-methylated (U), and non-converted DNA control samples demonstrated that the NEFH qMSP specifically detects M–DNA while U and non-converted samples remained undeterminable both in the QC1 control reaction as well as in the NEFH-specific PCR (Ct > 45 cycles, Figure 1B). A log2 dilution series adjusted for a constant total amount of converted DNA showed good efficiency and high linearity of the qMSP assay (Figure 1C). Using the slope of the qMSP calibration line we calculated a PCR efficiency of 0.95. Measurement of methylation in cell lines used as substitutes for frequent human cancers as well as normal primary cells and control DNAs demonstrated no methylation neither in normal primary cells of renal or prostatic origin nor in mock controls (Figure 1D). Low methylation was detected for normal mammary primary cells. Breast and urothelial cancer cell lines overall demonstrated highest relative methylation values while >25% relative methylation was detected in two of six RCC and one of three prostate cancer cell lines (Figure 1D).

Bottom Line: The NEFH CGI methylation demonstrated a tumor-specific increase (P < 0.001), association with advanced disease (P < 0.001), and distant metastasis (P = 0.005).Median OS following targeted therapy was 29.8 months for patients with low methylation versus 9.8 months for the group with high methylation (P = 0.028).We identified NEFH methylation as a candidate epigenetic marker for prognosis of RCC patients as well as prediction of anti-vascular endothelial growth factor-based therapy response.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Hannover Medical School, 30625, Hannover, Germany.

Show MeSH
Related in: MedlinePlus