Upgraded standardized minimal residual disease detection by next-generation sequencing in multiple myeloma

Abstract Minimal residual disease (MRD) is one of the most powerful prognostic factors in multiple myeloma. Therefore, standardization and easy operation of MRD testing is 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 (1 μg DNA input/replicate) for one million sequencing reads by LymphoTrack-MiSeq platform. To improve the technique, here 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 14/19 (78%) patients, with a significant (P = 0.04) impact on progression-free survival based on high- vs low- MRD levels. Reproducibility of detection was confirmed by the extremely small inter-run 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 was verified and the prognostic impact of NGS -based MRD in myeloma confirmed.