Abstract
Here we compared clonotype identification by allele-specific oligonucleotide real-time quantitative-PCR (ASO RQ-PCR) and next-generation sequencing (NGS) in 80 multiple myeloma patients. ASO RQ-PCR was applicable in 49/55 (89%) and NGS in 62/78 (80%). Clonotypes identified by both methods were identical in 33/35 (94%). Sensitivity of 10−5 was confirmed in 28/29 (96%) by NGS while sensitivity of RQ-PCR was 10−5 in 7 (24%), 5 × 10−5 in 15 (52%), and 10−4 in 7 (24%). Among 14 samples quantifiable by ASO RQ-PCR, NGS yielded comparable results in 12 (86%). Applicability of NGS can be improved if immunoglobulin heavy-chain incomplete DJ primers are included.
Highlights
Allele-specific oligonucleotide real-time quantitative-PCR (ASO allele-specific oligonucleotide real-time quantitative-PCR (RQ-PCR))-based minimal residual disease (MRD) detection is a well-established approach for treatment monitoring in multiple myeloma (MM) (1, 2)
More recently we reported on a standardized next-generation sequencing (NGS)based protocol for MRD testing with a sensitivity of [10−5] based on the use of triplicates of 1 mg DNA input and 1 million sequencing reads measured by the LymphoTrack-MiSeq platform (5, 6)
In the NGS approach, clonality of diagnostic bone marrow (BM) samples was studied by four PCR assays e.g. LymphoTrack immunoglobulin heavy-chain locus (IGH) complete VDJ, FR1/FR2/FR3 and immunoglobulin k locus (IGK) rearrangements which included both VJ and V-Kappa deleting element (Kdel) rearrangements (5)
Summary
Allele-specific oligonucleotide real-time quantitative-PCR (ASO RQ-PCR)-based minimal residual disease (MRD) detection is a well-established approach for treatment monitoring in multiple myeloma (MM) (1, 2). MM arises from post-germinal center plasma cells, in which hypermutation for enhanced antigen affinity has occurred during antigen-specific B cell responses in the germinal center of lymphoid tissues This renders a relatively low applicability when consensus primers/probes are used for MRD detection by ASO RQ-PCR. We have shown that the use of all patient-specific primers to the V and J genes in combination with a Taqman probe for complementarity-determining region 3 (CDR3) nucleotide sequences, increases the applicability of ASO RQ-PCR to 90% of patients (3, 4) This RQ-PCR-based approach could reach a sensitivity of down to [10−5] by using triplicates of 500 ng DNA for MRD assessment. We compared MRD detection by both ASO RQ-PCR and NGS approaches in a series of 80 MM patients, namely their applicability (rate of successful identification of clonotypes), sensitivity and rate of quantifiable MRD cases
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
