Unbiased Next Generation Sequencing-based Approach for Minimal Residual Disease Monitoring in Multiple Myeloma

Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells (MMPC) in the bone marrow. Despite the implementation of high-dose therapy (HDT) and the introduction of proteasome inhibitors and immunomodulatory agents, relapse in MM still occurs at a median of 2 years post-HDT. Quantitative allele-specific oligonucleotide polymerase chain reaction (qASO-PCR) targeting the rearranged immunoglobulin heavy chain locus (IgH) is a well-established monitoring approach for minimal residual disease (MRD). However, for each patient individual PCR primers and assays have to be designed and evaluated, rendering the method unsuitable for standard clinical laboratories since this requires detailed knowledge of immunogenetics and vast experience. Moreover, tumor clones are prone to undergo further mutations, for instance within the CDR3 region, by which the tumor clone – or a subclone – possibly evades detection.

Here we present a targeted next-generation sequencing (NGS) massive-parallel ultra-deep sequencing approach based on consensus and family primer sets that enable the effective and unbiased detection of any rearranged IgH and light chain genes of MMPCs. By using a two-tiered approach of IgH quantification in the first line and kappa and lambda light chain quantification in the second line, we are able to circumvent most drop outs of patients with non-amplifiable IgH sequences. Using one standardized PCR protocol for every patient makes the method applicable to any clinical molecular laboratory. Moreover, it allows quantitative detection of any clonal sequence regardless of changes in the DNA sequence due to ongoing mutations, making this approach more robust and more reliable than qASO-PCR.

The presented approach is highly sensitive for both, IgH and light chain quantification, detecting 1 tumor cell among 330.000 normal B cells out of one PCR reaction (6pg target DNA among 2µg polyclonal background DNA). The sensitivity has been demonstrated in 5 different Myeloma and Burkitt Lymphoma cell lines rearranged for VH1 through VH5 and 2 Myeloma cell lines rearranged for kappa and lambda light chain, respectively. 

As a versatile technique, this approach also allows for MRD screening in other B-cell neoplasia, such as B-CLL, a malignancy, in which ongoing somatic hypermutations can pose an obstacle to qASO-PCR.