Methylation Liquid Biopsy

About

BACKGROUND TNBC represents 15-20% of all breast cancers which represents almost 460,000 women globally per year. TNBC patients have high risk of early metastasis (mTNBC) and have a poorer prognosis than other types of breast cancer. There are currently few targeted therapies, though many are being developed, and few predictive biomarkers. Monitoring treatment response is key to the successful management of TNBC. Patients are monitored periodically using laboratory tests, imaging studies, and blood biomarkers. Imaging is typically carried out 3 months after treatment initiation and is not able to detect subtle changes in tumor response. This delays the use of more effective treatments and exposes patients to costly, ineffective, and toxic drugs. Breast cancer serum biomarkers like carcinoembryonic antigen (CEA) and CA 15-3 lack specificity and could be abnormally elevated due to a wide variety of causes. Early response assessment is clinically essential for patient survival. Currently there is no single effective treatment for TNBC with patients receiving a wide variety of treatments. Thus, there is urgent need for new methods to rapidly assesses clinical response to treatment so patients could benefit from intensification or timely changes in treatment if needed. TECHNOLOGY OVERVIEW Liquid biopsies are currently being investigated to improve the clinical management of breast cancer. Dying cancer cells lyse and release tumor DNA fragments into the blood stream. This circulating tumor DNA (ctDNA) can be a powerful tool in the management of TNBC. Traditionally, these assays focus on the identification of tumour-specific gene mutations, but they are limited by issues with sensitivity and specificity. DNA methylation is the second generation of liquid biopsies and can offer significant benefits over mutation-based ctDNA testing. Promoter region hypermethylation has been shown to inactivate tumour suppressor genes. These regions of hypermethylation are indicators of carcinogenesis and can be useful biomarkers for ctDNA because of their prevalence and consistency. Queen’s researchers have developed the mDETECT assay for the detection and monitoring of TNBC. mDETECT is a multiplexed PCR assay that uses primers to amplify methylated DNA followed by next generation sequencing (NGS) to analyze the products. Researchers carefully selected tumor-specific methylated regions with high CpG density present in at least 50% of tumor subtypes and absent from normal tissues. In contrast to competitive assays mDETECT requires very low volumes of serum (< 2 ml) and a much smaller number of NGS reads, making it less expensive while retaining high accuracy. The mTNBC assay includes 53 amplicons from 47 regions. This large panel of targets increases the sensitivity of mDETECT ensuring a low level of detection and the CpG sequencing enhances specificity in the presence of large quantities of normal DNA. Serum samples (1.8 mls) collected from mTNBC patients produced an area under the curve (AUC) of 0.97 for tumor detection with 93% sensitivity and 100% specificity. mDETECT outperformed CA15-3, the most commonly used serum marker for metastatic breast cancer. The mDETECT assay is a sensitive, quantitative, and rapid method of assessing tumour burden.