According to authors of a study published in the American Journal of Hematology, molecular assays for the detection of minimal residual disease (MRD), based on sequencing the third complementarity-determining regions (CDR3) of immunoglobulin-variable genes for heavy and light chains, are suitable for routine clinical use in the setting of multiple myeloma.

DNA-based next-generation sequencing (NGS) of gene rearrangements associated with  immunoglobulin heavy (IGH) and light (IGK/IGL) chains is being increasingly used to detect the presence of MRD — low-level disease not identified through conventional assessments — in patients with multiple myeloma. One approach focuses on the CDR3 sequences of the heavy and light chains of tumor-specific immunoglobulins, as it represents the most diverse section of the immunoglobulin V(D)J region, for long-term tracking of MRD. 

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While the prognostic implications of the presence of MRD are widely accepted, more evidence supporting the long-term stability of tumor-specific sequences identified at baseline is needed before the use of NGS for the detection of MRD over time can be incorporated into routine clinical practice for patients with multiple myeloma. Furthermore, more information is needed with respect to whether rearrangement sequences in the V-J regions of tumor-specific immunoglobulin light chains can also be used for longitudinal MRD detection, since IGH clones are not detectable in all patients with the disease. 

In this study, standard RNA sequencing was performed on bone marrow specimens from an in-house cohort of 30 patients with multiple myeloma and 10 healthy individuals.  In addition, previously unpublished, as well as publically available, RNA sequencing data were accessed. In total, a RNA-sequencing data set of heavy and light chain CDR3 regions was obtained from 905 samples (893 plasma cell myeloma samples and 12 normal bone marrow samples) from 841 individuals. 

Notably, 1 or more clonal CDR3 sequences was detected in baseline samples of all patients with multiple myeloma. While clonal CDR3 light chain sequences were identified in 100% of samples, clonal CDR3 heavy chains (IGH) sequences were found in 85% of these samples. 

Another important finding of this study was that while samples from healthy controls showed polyclonal CDR3 sequences, a median of 81% of the myeloma samples were dominated by a single dominant CDR3 clone. 

In addition, although subclones with evolved CDR3 sequences were observed in 3.2% of samples at baseline, no evidence of clonal evolution of dominant CDR3 sequences was detected following analyses of 101 specimens collected at different time points for 45 patients with multiple myeloma. For each of the patients tested, the dominant clonal heavy and light chain CDR3 sequence remained unchanged over time, supporting the stability of these clones.

Furthermore, greater than 99% of heavy chain CDR3 sequences were determined to be unique (ie, not present in any other independent samples searched, including samples from healthy controls), although a somewhat lower probability for uniqueness was observed for light chain CDR3 sequences. Regarding the latter observation, it was determined that the likelihood of CDR3 light chain sequence uniqueness increased with the number of somatic mutations and insertions. Based on these findings, light chain clones with more than 1 somatic mutation or a V-J insertion length exceeding a specified median value were deemed appropriate for use in MRD tracking.  

These findings from RNA-based sequencing were validated by comparison with results obtained using standard DNA amplicon-based sequencing, with a higher sensitivity observed for the former assay. 

Although the study authors did not consider an RNA-based sequencing assay as feasible for MRD testing in a clinical setting, based on the findings from this study, they concluded that “going forward, molecular MRD assays enable reliable long-term tracking of clonal heavy and light chain sequences with applicability approaching 100%, which renders it an optimal strategy for routine clinical use.”

Reference

Rustad EH, Misund K, Bernard E, et al. Stability and uniqueness of clonal immunoglobulin CDR3 sequences for MRD tracking inmultiple myeloma [published online October 1, 2019]. Am J Hematol. doi: 10.1002/ajh.25641