Baseline identification of clonal V(D)J sequences for DNA-based minimal residual disease detection in multiple myeloma

Abstract

Tracking of clonal immunoglobulin V(D)J rearrangement sequences by next generation sequencing is highly sensitive for minimal residual disease in multiple myeloma. However, previous studies have found variable rates of V(D)J sequence identification at baseline, which could limit tracking. Here, we aimed to define the factors influencing the identification of clonal V(D)J sequences. Bone marrow mononuclear cells from 177 myeloma patients underwent V(D)J sequencing by the LymphoTrack assays (Invivoscribe). As a molecular control for tumor cell content, we sequenced the samples using our in-house myeloma panel myTYPE. V(D)J sequence clonality was identified in 81% of samples overall, as compared with 95% in samples where tumor-derived DNA was detectable by myTYPE. Clonality was detected more frequently in patients with lambda-restricted disease, mainly because of increased detection of kappa gene rearrangements. Finally, we describe how the tumor cell content of bone marrow aspirates decrease gradually in sequential pulls because of hemodilution: From the initial pull used for aspirate smear, to the final pull that is commonly used for research. In conclusion, baseline clonality detection rates of 95% or higher are feasible in multiple myeloma. Optimal performance depends on the use of good quality aspirates and/or subsequent tumor cell enrichment.

Monitoring minimal residual disease in the bone marrow using next generation sequencing

“…For this reason, IGH-tracking remains the backbone of NGS-based MRD tracking. Some assays also include the possibility for light chain tracking, which is particularly beneficial in cases where no clonal IGH sequence can be identified (23,24).…”

“…tumor-specific); but they do not have to be productive. For example, a patient with kapparestricted multiple myeloma will have productive IGH and IGK rearrangements and may also have an unproductive IGH and/or IGK rearrangement, but both the IGL alleles will be in the germline configuration (24,26). Patients with lambda-restricted multiple myeloma will be in the same situation with regards to IGH and IGL but will also have two unproductive IGK rearrangements that can potentially be used for tracking (24).…”

Baseline VDJ clonotype detection using a targeted sequencing NGS assay: allowing for subsequent MRD assessment

“…The commercially available LymphoTrack assays (i.e. IGH Leader, FR1, FR2 and FR3; Invivoscribe Inc, San Diego, CA) and our custom capture next NGS-based panel were used to sequence 74 multiple myeloma samples 1,9,10 . Polymerase chain reaction amplification of the IGH variable region followed by NGS to a target read depth of >20,000 and clonality calling was performed using LymphoTrack as previously described 10 .…”

“…The MiXCR algorithm was used to reconstruct the V(D)J region from short-read data ( Fig. 1) 9,12,13 . The V(D) J clonotypes detected through the targeted NGS panel were then compared to the clonotypes detected in the same samples using the LymphoTrack assay 10 .…”

“…The large number of variations of the V(D)J sequence and the subsequent somatic hypermutation can however negatively affect the annealing of primers and thus decrease the capture rate. The two available commercial assays have overcome this by including multiple probes primers at various distances from the immunoglobulin variable regions (LymphoTrack) or including probes that cover a wide variety of V(D)J combinations including incomplete rearrangements (ClonoSEQ) 9,15 . Using standard short-read sequencing data, the algorithm MiXCR aligns reads mapping to a library of reference germline V, D, J, and C gene sequences to assemble full-length clonotypes.…”