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

Abstract

BACKGROUND Circulating tumor DNA (ctDNA) has emerged as a good candidate for tracking tumor dynamics in different cancer types, potentially avoiding repeated tumor biopsies. Many different genes can be mutated within a tumor, complicating procedures for tumor monitoring, even with highly sensitive next-generation sequencing (NGS) strategies. Droplet-based digital PCR (dPCR) is a highly sensitive and quantitative procedure, allowing detection of very low amounts of circulating tumor genetic material, but can be limited in the total number of target loci monitored. METHODS We analyzed hypermethylation of 3 genes, by use of droplet-based dPCR in different stages of colorectal cancer (CRC), to identify universal markers for tumor follow-up. RESULTS Hypermethylation of WIF1 (WNT inhibitory factor 1) and NPY (neuropeptide Y) genes was significantly higher in tumor tissue compared to normal tissue, independently of tumor stage. All tumor tissues appeared positive for one of the 2 markers. Methylated ctDNA (MetctDNA) was detected in 80% of metastatic CRC and 45% of localized CRC. For samples with detectable mutations in ctDNA, MetctDNA and mutant ctDNA (MutctDNA) fractions were correlated. During follow-up of different stage CRC patients, MetctDNA changes allowed monitoring of tumor evolution. CONCLUSIONS These results indicate that MetctDNA could be used as a universal surrogate marker for tumor follow-up in CRC patients, and monitoring MetctDNA by droplet-based dPCR could avoid the need for monitoring mutations.

Colorectal cancer early methylation alterations affect the crosstalk between cell and surrounding environment, tracing a biomarker signature specific for this tumor

“…Tumor fragments of DNA in the blood stream can be used as a surrogate sample to tissue biopsy. Cell free circulating DNA is a mixture of nucleic acids from normal (mainly from leukocytes) and from tumor tissues (ctDNA) Liquid biopsy test using genetic or epigenetic alterations in plasma DNA has been proposed for early diagnosis and early detection of relapse …”

“…Cell free circulating DNA is a mixture of nucleic acids from normal (mainly from leukocytes) and from tumor tissues (ctDNA) 21 Liquid biopsy test using genetic or epigenetic alterations in plasma DNA has been proposed for early diagnosis 6 and early detection of relapse. [22][23][24] Methylation of GRIA4, SLC8A1 and SYN3 was assessed in circulating DNA of CRC patients. Assays were successful in at least one replicate for 43, 45 and 41 cases for GRIA4, SLC8A1 and SYN3, respectively.…”

Methylation analysis of Gasdermin E shows great promise as a biomarker for colorectal cancer

“…To date, a handful of "proof of concept" studies have emerged, outlining the basis of such methods in breast and colorectal cancers. [47][48][49] Given that colorectal cancer initially develops from colon polyps, and that epigenetic modifications are believed to be precursors of cancer formation, 5 another step would be testing GSDME methylation in polyps to assess its potential as an early biomarker for colorectal cancer. Considering the models' robustness and the low number of predictor probes needed, the answer might ultimately lie in the use of GSDME methylation in liquid biopsies as a sensitive, minimally-invasive and cost-effective detection method for colorectal cancer.…”

Germline and somatic mutations in the MTOR gene in focal cortical dysplasia and epilepsy

“…Custom LNA double-dye probes designed to detect both mutant alleles c.6644C>T (p.Ser2215Phe) and c.6644C>A (p.Ser2215Tyr) and wild-type were purchased from Eurogentec. RainDrop Digital PCR system (Raindance Technologies Billerica) was used as previously described 23 with the following modifications: dPCR was performed with Taqman Universal Master Mix (life Technologies), 125 nM final concentration of both probes and 250 nM final concentration of primers.…”