July 2016

LGC scientists validate Horizon cell-free DNA reference standards

Using LGC’s expertise in digital PCR (dPCR) and measurement science, LGC worked with Horizon Discovery Group plc (LSE: HZD) to assess the precision of low frequency DNA mutations present in Horizon’s new cell free DNA (cfDNA) Reference Standards.

Performing tumour biopsies to assess disease states and monitor progression is highly invasive. However, as tumours cells break down, they release small amounts of DNA in to the bloodstream of the patient. The ability to monitor the presence of these trace amounts of mutant DNA could enable highly specific diagnoses to be performed based only on a blood test (a so-called ‘liquid biopsy’) rather than an invasive biopsy. In addition, the reaction of the tumour to treatments could be regularly monitored.

One challenge however, is that the frequency of mutant DNA present can as compared to normal DNA be very low (0.1 % and lower) and so there is a need for reference materials to measure the accuracy, sensitivity and specificity of cfDNA assays for tumour detection. Scientists at LGC performed droplet dPCR analysis to validate Horizon’s results and ensure the high quality of two panels of Reference Standards for cfDNA measurements of cancer mutations, as described in a Technical Note published by Horizon.

The first Reference Standard set contains DNA for the most frequently occurring mutation in malignant melanoma (BRAF V600E cfDNA Reference Standard Set).  The second panel contains templates for frequently occurring mutations that occur in lung, colon and breast cancer (EGFR, KRAS, NRAS, PIK3CA, Multiplex I cfDNA Reference Standards). LGC’s dPCR measurements confirmed the accuracy of the specified composition of the mutant compared to the non-mutated gene copy (wild-type sequence) down to 0.1 %. Evaluation of the negative control material, composed of 100% wild-type DNA, also demonstrated their suitability for defining the specificity and limit of detection of assays. 

Independent validation of these Reference Standards, as performed at LGC, adds confidence in their application for assessing the performance of high throughput platforms such as Next Generation Sequencing (NGS) and Quantitative PCR (qPCR) for low level tumour DNA analysis.

Dr. Karin Schmidt, Director Reagent Products, Horizon Discovery said, “Horizon’s Reference Standards offer a source of genetically defined, quantitative, sustainable and independent third party reference material. The validation of our cfDNA standards by as high quality an organisation as LGC confirms their value in supporting the critical role of assay validation and routine performance monitoring for this important new class of assays.”

This project was funded by the UK’s innovation agency, Innovate UK, under the Collaborative Research and Development project ‘Improving Cell and Tissue Analysis for Stratified Medicine’.