Circulating tumour DNA analysis demonstrates spatial mutational heterogeneity that coincides with disease relapse in myeloma.

Mutational characterisation in multiple myeloma (MM) currently relies on bone marrow (BM) biopsy, which fails to capture the putative spatial and genetic heterogeneity of this multifocal disease. Analysis of plasma (PL)-derived circulating free tumour DNA (ctDNA) as an adjunct to BM biopsy, for mutational characterisation and tracking disease progression, was evaluated. Paired BM MM cell DNA and ctDNA from 33 relapsed/refractory (RR) and 15 newly diagnosed (ND) patients were analysed for KRAS, NRAS, BRAF and TP53 mutations using the OnTarget Mutation Detection (OMD) platform. OMD detected 128 mutations (PL=31, BM=59, both=38) indicating the presence of PL mutations (54%). A higher frequency of PL-only mutations was detected in RR patients than ND (27.2% vs 6.6%, respectively), authenticating the existence of spatial and genetic heterogeneity in advanced disease. Activating RAS mutations were more highly prevalent than previously described with 69% harboring at least one RAS mutation. Sequential ctDNA quantitation with droplet digital PCR through longitudinal PL tracking of specific clones in seven patients demonstrated changes in fractional abundance of certain clones reflective of the disease status. We conclude that ctDNA analysis as an adjunct to BM biopsy represents a noninvasive and holistic strategy for improved mutational characterisation and therapeutic monitoring of MM.

A method for parallel, automated, thermal cycling of submicroliter samples.

A large fraction of the cost of DNA sequencing and other DNA-analysis processes results from the reagent costs incurred during cycle sequencing or PCR. In particular, the high cost of the enzymes and dyes used in these processes often results in thermal cycling costs exceeding $0.50 per sample. In the case of high-throughput DNA sequencing, this is a significant and unnecessary expense. Improved detection efficiency of new sequencing instrumentation allows the reaction volumes for cycle sequencing to be scaled down to one-tenth of presently used volumes, resulting in at least a 10-fold decrease in the cost of this process. However, commercially available thermal cyclers and automated reaction setup devices have inherent design limitations which make handling volumes of <1 microL extremely difficult. In this paper, we describe a method for thermal cycling aimed at reliable, automated cycling of submicroliter reaction volumes.