Upgraded Standardized Minimal Residual Disease Detection by Next-Generation Sequencing in Multiple Myeloma

Minimal residual disease (MRD) is one of the most powerful prognostic factors in multiple myeloma. Therefore, standardization and easy operation of MRD testing are crucial. Previously, we validated the sensitivity of 10^-5 with spike in of plasmid controls for a standardized next-generation sequencing (NGS) approach based on triplicate measurements of bone marrow by LymphoTrack-MiSeq platform. To improve the technique, we replaced spike-in plasmid controls by genomic DNA from myeloma cells. A spike-in control of 0.001% was consistently detected in all 19 samples tested, confirming a uniform sensitivity of 10^-5 of this upgraded protocol. MRD was detected in 14 of 19 patients (78%), with a significant (P = 0.04) impact on progression-free survival based on high versus low MRD levels. Reproducibility of detection was confirmed by the extremely small interrun variation tested in three patients. In nine patients, MRD was tested in parallel by allele-specific oligonucleotide real-time quantitative PCR. NGS showed an improved sensitivity and provided quantification of MRD for cases assigned positive but not quantifiable by real-time quantitative PCR, obviating the need of patient-specific probes/primers. In summary, the use of genomic DNA as spike-in control simplifies NGS detection of MRD while preserving the sensitivity of 10^-5. Validity and reproducibility of the standardized procedure were verified, and the prognostic impact of NGS-based MRD in myeloma was confirmed.