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

Abstract

BackgroundDNA 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.MethodsWe 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.ResultsWe 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%.ConclusionsWe 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.

New DNA Methylation Markers and Global DNA Hypomethylation Are Associated with Oral Cancer Development

“…PENK plays a role in cell death and survival, and is downregulated by the proto-oncogenes FOS and JUN in the central nervous system. However, their role in cancer is not well understood (36). …”

Promoter hypermethylation of HS3ST2, SEPTIN9 and SLIT2 combined with FGFR3 mutations as a sensitive/specific urinary assay for diagnosis and surveillance in patients with low or high-risk non-muscle-invasive bladder cancer

“…To quantify the methylation levels in urine samples, where the DNA amount is often limiting, we used the quantitative multiplex methylation-specific PCR (QM-MSP) using the TaqMan MBG probes technology (Life Technologies), a highly sensitive and specific PCR developed previously by our team [18]. QM-MSP, were carried out in a StepOne Plus Real-Time PCR system (Life Technologies).…”

A Study of Hypermethylated Circulating Tumor DNA as a Universal Colorectal Cancer Biomarker

“…For these patients, primitive tumor DNA was analyzed for 25 patients as well as corresponding healthy tissue DNA for 11 of these patients and plasma DNA was analyzed before surgery (n ϭ 49), 5-21 days after surgery (n ϭ 22 from which 18 were drawn at day 5, 1 at day 6, 1 at day 7, 1 at day 8 and 1 at day 21) and during follow-up (6 patients, 28 samples excluding samples before and after surgery). Finally 11 patients with stage II to IV cancer were included (INSERM, CPP-IDF IX-11-019) (see also (30 )). Primitive tumor DNA and corresponding normal tumor DNA were analyzed for these 11 patients.…”