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Identifying biomarkers to track tumour burden in patient blood

There is a huge need to identify biomarkers to discern which metastatic colorectal cancer patients will benefit from treatment using Regorafenib. Despite being the latest approved drugs for the disease, Regorafenib has limited clinical efficacy and is associated with a number of side effects. However, a lot of patients are treated with Regorafenib as it is the only treatment available for patients who cannot receive anti-EGFR treatment due to K-RAS or BRAF mutation.

Using DNA Methylation to track tumour burden

We have been performing liquid biopsies via methylation biomarkers specifically defined for colorectal cancer: such methylated loci could be used as a universal biomarker to track tumour burden in patients’ blood. Our aim has been to monitor the efficiency of treatment using minimally invasive blood tests without a priori knowledge of the genetic landscape of the tumour.

Using preclinical models (i.e. a large dataset of colorectal cancer cell lines) to compare with normal data, we have identified a subset of methylated markers which are methylated in cancers but not in normal tissue. After such identification in cell lines, we validated our results in-silico using different publicly available dataset (among which the TCGA data) to pinpoint five biomarkers for which we designed locus-specific assays. To do this, we use a derivative of ddPCR called methyl-BEAMing (Li et al., Nat biotechnology 2009), which consist of a dual amplification step with the second PCR using emulsion.

By using these types of assays with the five genes we are able to track the efficacy of conventional treatment (i.e. 5FU based regimen) during stage 4 colorectal cancer. We also assessed these markers in patients involved in a clinical trial with temozolomide, an alkylating agent not usually used in colorectal cancer (Barault et al., Gut 2018).

Identifying universal biomarkers in patients treated with Regorafenib

At the Liquid Biopsies Congress, I will present a follow up to this, looking at universal biomarkers in patients treated with Regorafenib. Given the number of challenges with this drug and lack of companion diagnostics, there is a huge need to identify biomarkers which will highlight which patients will actually benefit from the treatment.

Having previously found an association between circulating methylated DNA and the outcome of colorectal cancer patients who have undergone chemotherapy, the hypothesis was that the five biomarkers we identified could be used to identify cases where treatment with Regorafenib would be beneficial.

Using our assays, we were able to identify non-responder patients. Our assessment of pre and during treatment blood samples confirmed the prognostic value of circulating methylated DNA and its potential as a biomarker which could predict responsiveness to treatment with this drug. We could, therefore, restrict the population of patients who receive this treatment in the early stages of therapy, avoiding overexposure to this type of treatment to those patients who won’t enjoy the future benefits.

Developing an NGS approach for universal biomarkers

The current paper investigates five specific biomarkers, but in the original study published in 2018, there were almost 30 other biomarkers which occur specifically in the tumour. This gives a number of other genes of interest which could also be used as universal biomarkers. Since we are currently using Loci-specific assays, a limitation is the need to restrict the number of genes. However, the next step could be the development of an NGS approach for these types of biomarkers.

The five genes that we identified for locus-specific assays can be used for other types of therapeutics as a potential normaliser for quality checks. These markers could be a surrogate for DNA of tumour origin in the blood and therefore used as a quality check prior to NGS approaches.

 

Ludovic Barault is Senior Research Associate, Candiola Cancer Institute and the University of Torino, Italy. We are looking forward to welcoming him to the Liquid Biopsies Congress: Europe.

 

Presentations at the Liquid Biopsies Congress: Europe will review techniques for single use molecules and selective enrichment. Download the agenda to see what else is on offer.

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