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MethylDetect Looks to Leverage High-Resolution Melt Tech for Oncology Assay Development

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NEW YORK (GenomeWeb) – On the heels of a study presented late last year demonstrating its high-resolution melt (HRM)-based assay technology for the detection of aberrant methylation of the MLH1 gene, Danish firm MethylDetect is hoping to position itself as a tool supplier for the burgeoning field of epigenetic diagnostics development.

Apart from the study's specific findings in relation to MLH1 and Lynch syndrome screening,  the data — which were shared at the UK's NCRI Cancer Conference last November — provide an important validation of the firm's research-use-only technology in clinical diagnostics development, said MethylDetect Cofounder and CEO Tomasz Wojdacz.

The company is now hoping that the validation data can help it ink partnerships with academia and industry to demonstrate wide applicability of its HRM technology in screening of a variety of biomarkers.

Interest in epigenetic biomarkers and their translation into clinical tests continues to strengthen and diversify. In addition to the possibility for specific, qualitative assays like those that MethylDetect is supporting, investigators are also exploring how measuring patterns of methylation across the genome might help detect or inform treatment strategies for cancer and other diseases.

For gene, or loci-specific assays, commercial tests have largely used PCR platforms thus far. Commercial assays include those from MDx Health that employs a proprietary methylation-specific PCR approach, and one from Epigenomics that uses real-time PCR with a fluorescent hydrolysis probe for the methylation-specific detection of the Septin 9 DNA target.

Wojdacz initially created the methylation-sensitive HRM method at the heart of MethylDetect's commercial offering as an academic researcher, publishing on the technique in 2007 in Nucleic Acids Research.

In essence, he said, the technique relies on the fact that PCR amplification of methylated DNA results in a cytosine-rich PCR product while non-methylated loci yield a tyrosine-rich product. These PCR products display different melting profiles when subjected to HRM.

More specifically, PCR products amplified from non-methylated loci will have a relatively low melting temperature and melt earlier in a temperature gradient than the PCR products amplified from the methylated version of the same locus.

According to Wojdacz the company's proprietary primer design aids analysis of difficult samples like liquid biopsies and FFPE tissue, where PCR bias might otherwise mask the signal of low-concentration methylated alleles of interest amidst a higher background of non-methylated alleles.

"I was working in methylation from the very beginning and I was having trouble detecting methylation that I knew was there in samples with the methods we were using, so this started with me trying to fix that," Wojdacz said.

The solutions developed and patented as part of Wojdacz's academic work provide MethylDetect with a system for creating sensitive, rapid, and cheap assay kits compared to other available protocols.

Wojdacz said that customers who have chosen to work with MethylDetect products — like the Sarah Cannon group, which collaborated with the company on the study presented at the NCRI conference — have praised the fact that assays can be performed with run-of-the-mill PCR equipment, include controls for validation, and feature primers that allow them to work with difficult sample types.

MethylDetect's business model includes offering a wide variety of pre-defined low-cost assays, as well as bespoke assay creation services, which apply the firm's probe design to create a sensitive test for specific loci of interest, as in the Sarah Cannon MLH1 effort.

Wojdacz said that the company is also working with fellow Danish company Ampliqon to expand its offering to more of a sample-to-answer product, though that effort has only just started.

In the study presented at the NCRI meeting, investigators led by Sarah Cannon researcher Diana Pelka set out to develop an assay to detect MLH1 promoter hypermethylation, something that is a component of the UK's NICE-mandated Lynch syndrome pre-screen pathway, but for which there does not yet exist a CE-IVD solution.

Lynch syndrome is associated with germline mutations in genes which encode proteins that repair errors in the normal replication of DNA, resulting in what is called mismatch repair deficiency (dMMR).

According to the Sarah Cannon authors, although Lynch syndrome is the hallmark of dMMR, mismatch repair can also arise somatically, for example via methylation-mediated silencing of the MLH1 gene in some colorectal cancers.

As a result, the UK's advisory body NICE recently recommended that analysis of MLH1 promoter methylation (and testing for BRAF V600E mutations, which have been shown to be associated with MLH1 hypermethylation) be used in the pre-screening of patients who are suspected to have Lynch syndrome. In that way, physicians can pick out these sporadic, somatic dMMR cases, avoiding unnecessary further genetic testing for an inherited disorder like Lynch syndrome.

Pelka and colleagues validated a methylation-specific HRM assay for MLH1 promoter analysis using MethylDetect's primers and controls, coupled with bisulfite conversion and other reagents from Qiagen.

The group used paired tumor and normal FFPE samples from 12 colorectal cancer cases, 4 prostate cancers, 2 endometrial tumors, 1 bladder cancer, and 1 cancer of unknown origin — all with varying statuses as to microsatellite instability, MMR, BRAF mutations, and Lynch syndrome.

According to the authors, all 11 microsatellite-stable samples scored as MLH1 unmethylated, as did the four known and suspected Lynch syndrome samples. The six MSI-high samples (including one of two with a deleterious BRAF mutation) scored as hypermethylated.

In all, the assay scored all samples as expected, except for the other BRAFVal600Glu sample, though this was from a patient with suspected Lynch syndrome, and thus may not represent a true miss for the assay.

Wojdacz said that MethylDetect is now beginning similar collaborations with others groups in the same vein as the Sarah Cannon effort, including both research and commercial groups. He said that the company is working, for example, with a US firm on research into methylation-based liquid biopsy testing, though he did not name this partner.

The firm is backed by venture capital, having completed a seed funding round last year supported by Danish firm Borean Innovation. Wojdacz did not disclose how much was raised, but said the company is now approaching the close of another funding round.

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