LAUSR

Dear Editor, Fluorescent in situ hybridization (FISH), metaphase cytogenetics, and single nucleotide polymorphism (SNP) microarray have long been the standard assays for disease diagnostics and subtyping of multiple myeloma. With the advent of next generation sequencing (NGS), our understanding of the genomic landscape in multiple myeloma has been expanded. Recent data show that NGS-based myeloma-targeted assays can identify immunoglobulin heavy chain (IGH) translocations, copy number alterations, as well as somatic mutations. Comprehensive genomic profiling of multiple myeloma is becoming increasingly important for individual risk stratification and therapeutic guidance. Therefore, NGS-based myeloma-targeted assays will gradually replace current standard methods in clinical practice. In addition to initial genomic profiling of multiple myeloma, the depth of treatment response—measured as minimal residual disease (MRD)—is an important prognostic factor. MRD evaluation with multicolor flow cytometry or immunoglobulin V(D)J sequencing to assess depth of treatment response and to inform clinical decisions is becoming standard in many centers across the USA. Recently, we conducted a head-to-head comparison of an NGS-based multiple myeloma-targeted assay (named “myTYPE”) and found that the targeted assay had a high concordance, >99% sensitivity and specificity, for detection IGH translocations and copy number alterations compared to FISH and SNP microarray. In addition, the NGS assay captures somatic mutations in 120 genes that are recurrent and relevant in multiple myeloma. Here, as a follow-up, we assessed whether the targeted NGS assay can detect patient-specific V(D)J clonotypes at diagnosis, something that is required for subsequent longitudinal MRD tracking. 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. 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. The myTYPE assay includes canonical IGH locus was included to capture IGH (14q32) rearrangements. This locus also includes the V(D)J regions of the IGH gene. The MiXCR algorithm was used to reconstruct the V(D)J region from short-read data (Fig. 1). 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. The study was approved by the Institutional Review Board and all patients were seen at Memorial Sloan Kettering Cancer Center and had consented to the tissue acquisition protocol. In 74 samples with a clonal V(D)J sequence detected by Lymphotrack; using myTYPE, the same clonal V(D)J sequence was found in 70 samples (95%). In all 70 samples, the CDR3 sequence detected with myTYPE was identical to the CDR3 sequence reported by LymphoTrack. We went on to examine in detail the four patients where no clonal sequence was detected with