Upgraded Standardized Minimal Residual Disease Detection By Next Generation Sequencing In Multiple Myeloma

Abstract

Background We have previously validated the sensitivity of 10-5 using spike-in plasmid controls in a standardized experimental design based on triplicate of bone marrow DNA that acquired one million sequencing reads by next generation sequencing (NGS) in each replicate of 1 μg DNA input using the LymphoTrack-MiSeq platform. Herein, we attempted to simplify operation by the use of spike-in controls with genomic instead of plasmid DNA in an additional 19 myeloma patients, which was compared with minimal residual disease (MRD) data derived from allele-specific oligonucleotide (ASO) real-time quantitative (RQ)- PCR. Methods To simplify operation of the spike-in controls, we replaced spike-in plasmid controls of immunoglobulin heavy chain sequences with genomic DNA from CD138 sorted cells of MM patients with clonal IGH/IGK rearrangement with highly diverse sequence for two reasons. First, diluting plasmid from stored concentration to appropriate concentration for use (generally from 109 to 6 copies per microliter) is too tedious and susceptible to possible pipeting error. Secondly, the concentration of spike-in controls is more accurate with the use of gDNA since the cell numbers of both the spike-in controls and MRD samples are measured by real-time PCR. On the other hand, calculation of number of copies of plasmid is based on the assumption that the average weight of a base pair is 650 Daltons. MRD assessment was based on the use of triplicates of 1 μg DNA input and a sequencing depth of 1 million sequencing reads per replicate as previously. The number of cells contained in 1μg of each sample was measured by the real time PCR standard curve method using plasmids, in which the albumin gene is cloned. Two spike-in controls generated from genomic DNA of myeloma cells were added to each replicate, one at the concentration of 10-5 for validation of the sensitivity of 10-5, and the other at 10-4 for obtaining an amplification factor, i.e. percentage of tumor alleles per sequence read. The MRD level in each replicate was calculated from the corresponding reads of the myeloma-specific sequence and the amplification factor. The final MRD level of a sample was defined as the mean MRD levels of the triplicates. Results The spike-in control of 0.001% concentration was consistently detected in all 19 follow up samples tested, confirming a uniform Abstracts 17th International Myeloma Workshop, September 12-15, 2019 | 275 sensitivity of 10-5 of this standardized experimental protocol. Moreover, this standardized NGS approach demonstrated MRD positivity in 9/13 (69%) patients achieving complete response. Furthermore, NGS showed an improved sensitivity and provided quantification of MRD for cases assigned 'positive but not quantifiable' by ASO RQ-PCR, without the use of patient-specific probes/primers. Conclusion The use of genomic DNA has simplified verification of the sensitivity of 10-5, and operation of the standardized LymphoTrack-MiSeq-based MRD detection, hence an effective tool for MRD monitoring in MM.