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Aberrant methylation of NPY, PENK, and WIF1 as a promising marker for blood-based diagnosis of colorectal cancer.

Roperch JP, Incitti R, Forbin S, Bard F, Mansour H, Mesli F, Baumgaertner I, Brunetti F, Sobhani I - BMC Cancer (2013)

Bottom Line: We assessed the results by receiver operating characteristic curve (ROC), using cumulative methylation index (CMI) as variable threshold.We optimized a QM-MSP for simultaneously quantifying their methylation levels.Our assay can be an effective blood test for patients where CRC risk is present but difficult to assess (e.g. mild symptoms with no CRC family history) and who would therefore not necessarily choose to go for further examination.

View Article: PubMed Central - HTML - PubMed

Affiliation: Profilome SAS, Paris Biotech 24 rue du Faubourg St Jacques, Paris 75014, France. jp.roperch@profilome-sas.com.

ABSTRACT

Background: DNA methylation is a well-known epigenetic mechanism involved in epigenetic gene regulation. Several genes were reported hypermethylated in CRC, althought no gene marker was proven to be individually of sufficient sensitivity or specificity in routine clinical practice. Here, we identified novel epigenetic markers and assessed their combined use for diagnostic accuracy.

Methods: We used methylation arrays on samples from several effluents to characterize methylation profiles in CRC samples and controls, as established by colonoscopy and pathology findings, and selected two differentially methylated candidate epigenetic genes (NPY, PENK). To this gene panel we added WIF, on the basis of being reported in literature as silenced by promoter hypermethylation in several cancers, including CRC. We measured their methylation degrees by quantitative multiplex-methylation specific PCR (QM-MSP) on 15 paired carcinomas and adjacent non-cancerous colorectal tissues and we subsequently performed a clinical validation on two different series of 266 serums, subdivided in 32 CRC, 26 polyps, 47 other cancers and 161 with normal colonoscopy. We assessed the results by receiver operating characteristic curve (ROC), using cumulative methylation index (CMI) as variable threshold.

Results: We obtained CRC detection on tissues with both sensitivity and specificity of 100%. On serum CRC samples, we obtained sensitivity/specificity values of, e.g., 87%/80%, 78%/90% and 59%/95%, and negative predictive value/positive predictive value figures of 97%/47%, 95%/61% and 92%/70%. On serum samples from other cancers we obtained sensitivity/specificity of, e.g, 89%/25%, 43%/80% and 28%/91%.

Conclusions: We showed the potential of NPY, PENK, and WIF1 as combined epigenetic markers for CRC diagnosis, both in tissue and serum and tested their use as serum biomarkers in other cancers. We optimized a QM-MSP for simultaneously quantifying their methylation levels. Our assay can be an effective blood test for patients where CRC risk is present but difficult to assess (e.g. mild symptoms with no CRC family history) and who would therefore not necessarily choose to go for further examination. This panel of markers, if validated, can also be a cost effective screening tool for the detection of asymptomatic cancer patients for colonoscopy.

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Efficiency of quantitative simplex (QS) and multiplex (QM) methylation-specific PCR. The diagrams illustrate comparison of both methods, namely A: quantitative multiplex methylation-specific PCR (QM-MSP) and B: quantitative singleplex methylation-specific PCR (QS-MSP). The reactions have been performed in duplicate. We used a mixture of primers and hydrolysis methylated probe specific to only amplify methylated alleles of Albumin, WIF1, PENK, and NPY genes along with a titration of human genomic DNA at various concentrations ranging from 10 up to 0.01 ng/well. On each dilution, the cycle threshold (Ct) was determined for standard DNA. Nearly identical Ct values for each DNA dilution indicate uniform primer performance over 3 logs. The slope of −3.32 (100% efficiency) reflects a 2-fold amplification of DNA per cycle corresponding to a high efficiency. The correlation coefficient R2 of 0.99 shows a high degree of linearity over the entire range.
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Figure 2: Efficiency of quantitative simplex (QS) and multiplex (QM) methylation-specific PCR. The diagrams illustrate comparison of both methods, namely A: quantitative multiplex methylation-specific PCR (QM-MSP) and B: quantitative singleplex methylation-specific PCR (QS-MSP). The reactions have been performed in duplicate. We used a mixture of primers and hydrolysis methylated probe specific to only amplify methylated alleles of Albumin, WIF1, PENK, and NPY genes along with a titration of human genomic DNA at various concentrations ranging from 10 up to 0.01 ng/well. On each dilution, the cycle threshold (Ct) was determined for standard DNA. Nearly identical Ct values for each DNA dilution indicate uniform primer performance over 3 logs. The slope of −3.32 (100% efficiency) reflects a 2-fold amplification of DNA per cycle corresponding to a high efficiency. The correlation coefficient R2 of 0.99 shows a high degree of linearity over the entire range.

Mentions: We evaluated the performance of two different PCR-based assays, quantitative singleplex-MSP (QS-MSP) and quantitative multiplex-MSP (QM-MSP), in order to quantify the methylation levels of NPY, PENK, and WIF1. For co-amplifying two methylation-specific DNA targets in real-time, we used the associations of Fam/Vic and Ned/Vic fluorophore probes as each probe presents a strong individual spectral intensities with limited overlapping absorption spectra. We compared QS-MSP and QM-MSP to determine which assay agreed best with the detection thresholds (Ct) on a serial dilution experiment from 10 ng to 10 pg of methylated DNA. Both QS-MSP and QM-MSP gave similar cycle threshold (Ct) values for each dilution point (data not shown) with similar high amplification efficiency (Figure 2A, 2B).


Aberrant methylation of NPY, PENK, and WIF1 as a promising marker for blood-based diagnosis of colorectal cancer.

Roperch JP, Incitti R, Forbin S, Bard F, Mansour H, Mesli F, Baumgaertner I, Brunetti F, Sobhani I - BMC Cancer (2013)

Efficiency of quantitative simplex (QS) and multiplex (QM) methylation-specific PCR. The diagrams illustrate comparison of both methods, namely A: quantitative multiplex methylation-specific PCR (QM-MSP) and B: quantitative singleplex methylation-specific PCR (QS-MSP). The reactions have been performed in duplicate. We used a mixture of primers and hydrolysis methylated probe specific to only amplify methylated alleles of Albumin, WIF1, PENK, and NPY genes along with a titration of human genomic DNA at various concentrations ranging from 10 up to 0.01 ng/well. On each dilution, the cycle threshold (Ct) was determined for standard DNA. Nearly identical Ct values for each DNA dilution indicate uniform primer performance over 3 logs. The slope of −3.32 (100% efficiency) reflects a 2-fold amplification of DNA per cycle corresponding to a high efficiency. The correlation coefficient R2 of 0.99 shows a high degree of linearity over the entire range.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219483&req=5

Figure 2: Efficiency of quantitative simplex (QS) and multiplex (QM) methylation-specific PCR. The diagrams illustrate comparison of both methods, namely A: quantitative multiplex methylation-specific PCR (QM-MSP) and B: quantitative singleplex methylation-specific PCR (QS-MSP). The reactions have been performed in duplicate. We used a mixture of primers and hydrolysis methylated probe specific to only amplify methylated alleles of Albumin, WIF1, PENK, and NPY genes along with a titration of human genomic DNA at various concentrations ranging from 10 up to 0.01 ng/well. On each dilution, the cycle threshold (Ct) was determined for standard DNA. Nearly identical Ct values for each DNA dilution indicate uniform primer performance over 3 logs. The slope of −3.32 (100% efficiency) reflects a 2-fold amplification of DNA per cycle corresponding to a high efficiency. The correlation coefficient R2 of 0.99 shows a high degree of linearity over the entire range.
Mentions: We evaluated the performance of two different PCR-based assays, quantitative singleplex-MSP (QS-MSP) and quantitative multiplex-MSP (QM-MSP), in order to quantify the methylation levels of NPY, PENK, and WIF1. For co-amplifying two methylation-specific DNA targets in real-time, we used the associations of Fam/Vic and Ned/Vic fluorophore probes as each probe presents a strong individual spectral intensities with limited overlapping absorption spectra. We compared QS-MSP and QM-MSP to determine which assay agreed best with the detection thresholds (Ct) on a serial dilution experiment from 10 ng to 10 pg of methylated DNA. Both QS-MSP and QM-MSP gave similar cycle threshold (Ct) values for each dilution point (data not shown) with similar high amplification efficiency (Figure 2A, 2B).

Bottom Line: We assessed the results by receiver operating characteristic curve (ROC), using cumulative methylation index (CMI) as variable threshold.We optimized a QM-MSP for simultaneously quantifying their methylation levels.Our assay can be an effective blood test for patients where CRC risk is present but difficult to assess (e.g. mild symptoms with no CRC family history) and who would therefore not necessarily choose to go for further examination.

View Article: PubMed Central - HTML - PubMed

Affiliation: Profilome SAS, Paris Biotech 24 rue du Faubourg St Jacques, Paris 75014, France. jp.roperch@profilome-sas.com.

ABSTRACT

Background: DNA methylation is a well-known epigenetic mechanism involved in epigenetic gene regulation. Several genes were reported hypermethylated in CRC, althought no gene marker was proven to be individually of sufficient sensitivity or specificity in routine clinical practice. Here, we identified novel epigenetic markers and assessed their combined use for diagnostic accuracy.

Methods: We used methylation arrays on samples from several effluents to characterize methylation profiles in CRC samples and controls, as established by colonoscopy and pathology findings, and selected two differentially methylated candidate epigenetic genes (NPY, PENK). To this gene panel we added WIF, on the basis of being reported in literature as silenced by promoter hypermethylation in several cancers, including CRC. We measured their methylation degrees by quantitative multiplex-methylation specific PCR (QM-MSP) on 15 paired carcinomas and adjacent non-cancerous colorectal tissues and we subsequently performed a clinical validation on two different series of 266 serums, subdivided in 32 CRC, 26 polyps, 47 other cancers and 161 with normal colonoscopy. We assessed the results by receiver operating characteristic curve (ROC), using cumulative methylation index (CMI) as variable threshold.

Results: We obtained CRC detection on tissues with both sensitivity and specificity of 100%. On serum CRC samples, we obtained sensitivity/specificity values of, e.g., 87%/80%, 78%/90% and 59%/95%, and negative predictive value/positive predictive value figures of 97%/47%, 95%/61% and 92%/70%. On serum samples from other cancers we obtained sensitivity/specificity of, e.g, 89%/25%, 43%/80% and 28%/91%.

Conclusions: We showed the potential of NPY, PENK, and WIF1 as combined epigenetic markers for CRC diagnosis, both in tissue and serum and tested their use as serum biomarkers in other cancers. We optimized a QM-MSP for simultaneously quantifying their methylation levels. Our assay can be an effective blood test for patients where CRC risk is present but difficult to assess (e.g. mild symptoms with no CRC family history) and who would therefore not necessarily choose to go for further examination. This panel of markers, if validated, can also be a cost effective screening tool for the detection of asymptomatic cancer patients for colonoscopy.

Show MeSH
Related in: MedlinePlus