Towards Diagnostic ctDNA Testing in Multiple Myeloma: How does Automated Magnetic Bead-Based Cell-Free DNA Extraction Compare to the Leading Silica Membrane-Based Manual Extraction?

Abstract

ctDNA in Multiple How does Automated Magnetic Bead-Based Cell-Free DNA Extraction Compare to the Leading Silica Membrane-Based Manual Abstract Introduction : Diagnosis and monitoring of haematological malignancies rely heavily on bone marrow biopsies. Less invasive peripheral blood biopsies are an emerging alternative, involving isolation of cell-free DNA (cfDNA) and circulating tumour DNA (ctDNA) as a source of tumour information. To date, the Qiagen QIAamp Circulating Nucleic Acid kit is considered the gold standard for cfDNA isolation. However, it is time-consuming, labour intensive and relatively costly. To move ctDNA analysis into a diagnostic setting, standardisation by partially automating cfDNA extraction is highly desirable. This study revisited cfDNA extraction in multiple myeloma, a haematological cancer potentially shedding relatively high amounts of ctDNA. Methods: Four plasma samples from three myeloma patients carrying the NRASQ61R mutation and six healthy controls were used in this study. cfDNA was isolated from plasma with the manual method, Qiagen QIAamp Circulating Nucleic Acid kit and the automated Promega RSC Maxwell ccfDNA kit. The cfDNA yields were assessed and ctDNA (NRAS) detection compared by droplet digital PCR (ddPCR). Results: Although the Promega kit was more convenient, ctDNA detection was more sensitive with the Qiagen kit. Indeed, ddPCR successfully detected low NRAS mutant load in Qiagen extracts while only high NRAS mutant load was confidently detected in Promega extracts. Conclusion: The Promega kit is easier to use and more economical, but the Qiagen kit yielded significantly higher amounts of cfDNA. Moreover, critically low patient ctDNA concentration was only detectable using the Qiagen kit. This supports the superiority of the Qiagen kit for monitoring minimal residual disease.