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Cell-Free DNA Assay Could Make Routine Cancer Testing Feasible

By Deborah Borfitz 

January 18, 2023 | Scientists from the National University of Singapore (NUS) have discovered a blood test for monitoring cancer patients that could be done for as little as $35. The new Heatrich-BS assay sequences cell-free DNA (cfDNA) in clinical samples that get heated to isolate cancer-specific signatures, according to Cheow Lih Feng, assistant professor at NUS’s Institute for Health Innovation & Technology and department of biomedical engineering.    

The Heatrich-BS assay—short for heat-enrichment of CpG-rich regions for bisulfite sequencing— represents a potential non-invasive alternative to tissue biopsies that is also more than 20 times cheaper to conduct than other sequencing methods. The focus is on “CpG islands” that have high repetitions of C (cytosine) and G (guanine) nucleotides. While they make up only about 1% of the genome, they’re the spots where cancer-specific signatures tend to be concentrated, Cheow explains. 

As recently reported in Science Advances (DOI: 10.1126/sciadv.abn4030), the Heatrich-BS assay was tested on patients with colorectal cancer and found to highly correlate with the findings of CT scans. Most cancers demonstrate the enrichment of cancer-specific biomarkers in CpG islands, Cheow notes, and would thus benefit from the assay. 

The key breakthrough here is the heating step on clinical samples “to denature the non-informative sections of the genome and enrich for the thermally stable CpG islands,” he says, which is significantly less costly than whole genome bisulfite sequencing (WGBS). That heat would have this effect was an accidental discovery when the team was performing some unrelated experiments.  

In terms of imagined clinical utility, the Heatrich-BS assay may more accurately estimate tumor burden and quantitatively track the response of colorectal cancer patients to treatment and the detection of recurrence, says Cheow. The test may also be useful for doing epigenetic subtyping for purposes of patient stratification in both clinical practice and clinical research. 

Tumors are currently tracked either by measuring protein biomarkers in the blood, a method that lacks sensitivity and specificity, or with CT scans, which are expensive, he says. “An affordable, specific, and sensitive cfDNA assay based on Heatrich-BS can enable clinicians and patients to track cancer progress more closely and facilitate timely interventions to improve treatment outcomes. 

“Increasingly,” Cheow continues, “cancer research is also revealing that different cancer epigenetic subtypes can have different therapeutic outcomes. The ability of Heatrich-BS to infer cancer epigenetic subtype non-invasively from cfDNA can potentially aid doctors in treatment decisions in the future.” 

Breakthrough Moment 

Cheow’s lab has always been interested in developing new methods for epigenetic analysis. One of the earliest assays for DNA methylation used methylation-sensitive restriction enzymes which, if unmethylated, will only cut DNA on specific sequences, he says. But he and his team repurposed this technique to interrogate DNA methylation states in single cells (Science, DOI: 10.1126/science.1240617) and genome-wide (Nucleic Acids Research, DOI: 10.1093/nar/gkz717). 

Decades ago, it was revealed that “while the majority of CpG dinucleotides in the mammalian genome are methylated, there are (G+C)- and CpG dinucleotide-rich regions that are digested into HpaII tiny fragments by methylation-sensitive restriction enzymes,” Cheow says. This led to the recognition of CpG islands, which tend to be highly regulated by DNA methylation to control gene expression. 

“The epigenetic importance of CpG islands and the overrepresentation of CpG dinucleotides in them have inspired methods to enrich them with the use of restriction enzymes,” continues Cheow. “The widely used reduced-representation bisulfite sequencing [RRBS] makes use of restriction enzymes to enrich for CpG-rich regions for epigenetic profiling at a fraction of the cost of [WGBS]. However, RRBS is not suitable for profiling naturally-fragmented DNA samples such as cell-free DNA... [and] DNA methylation analysis of cfDNA with WGBS is too costly to be implemented routinely.” 

The breakthrough moment in Cheow’s lab came when a researcher accidentally heated some fragmented DNA. When the heated samples were sequenced, they were highly enriched in promoter regions where many of the CpG islands reside. 

“The high heat was denaturing most of the double-stranded DNA except for GC-rich DNA [including most CpG islands] that had high thermal stability,” he explains. “We realized that we had a very effective and simple method to enrich for CpG islands in fragmented DNA. We then got together with our colleagues at the National Cancer Center of Singapore which runs the national cancer liquid biopsy program CaLiBRe... to explore the use of this strategy for cost-effective cell-free DNA methylation profiling for cancer detection.” 

Road To Market 

The Heatrich-BS assay now needs to be tested and validated in different types of cancer, says Cheow, and some of that work has already started. “We’d love to see this promising technology being expanded to a larger set of cancers or even for pan-cancer measurement.”  

The utility of Heatrich-BS has so far been demonstrated for monitoring patients with known cancer, he points out. But the CpG islands enriched in the assay also harbor biomarkers for different kinds of cancers, opening the potential for its use as a screening tool for various cancers.  

Discussions are now underway about licensing the technology to a company with “a good track record in developing and launching liquid biopsy products internationally,” Cheow adds.

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