Incomplete DJH rearrangements as a novel tumor target for minimal residual disease quantitation in multiple myeloma using real-time PCR

Abstract

The hypervariable regions of immunoglobulin heavy-chain (IgH) rearrangements provide a specific tumor marker in multiple myeloma (MM). Recently, real-time PCR assays have been developed in order to quantify the number of tumor cells after treatment. However, these strategies are hampered by the presence of somatic hypermutation (SH) in VDJH rearrangements from multiple myeloma (MM) patients, which causes mismatches between primers and/or probes and the target, leading to a nonaccurate quantification of tumor cells. Our group has recently described a 60% incidence of incomplete DJH rearrangements in MM patients, with no or very low rates of SH. In this study, we compare the efficiency of a real-time PCR approach for the analysis of both complete and incomplete IgH rearrangements in eight MM patients using only three JH consensus probes. We were able to design an allele-specific oligonucleotide for both the complete and incomplete rearrangement in all patients. DJH rearrangements fulfilled the criteria of effectiveness for real-time PCR in all samples (ie no unspecific amplification, detection of less than 10 tumor cells within 10 5 polyclonal background and correlation coefficients of standard curves higher than 0.98). By contrast, only three out of eight VDJH rearrangements fulfilled these criteria. Further analyses showed that the remaining five VDJH rearrangements carried three or more somatic mutations in the probe and primer sites, leading to a dramatic decrease in the melting temperature. These results support the use of incomplete DJH rearrangements instead of complete somatically mutated VDJH rearrangements for investigation of minimal residual disease in multiple myeloma.

Significantly improved PCR-based clonality testing in B-cell malignancies by use of multiple immunoglobulin gene targets. Report of the BIOMED-2 Concerted Action BHM4-CT98-3936

“…These data would therefore confirm that incomplete rearrangements do provide supplementary information, particularly in the presence of SHM. 14,15 Although the three-tube IGH V H -J H multiplex strategy was designed to try and overcome the effects of SHM occurring within the V H region, it is more difficult to overcome mismatches when they occur within the J H segment. The consensus primer used in this study has mismatches to five of the six J H segments.…”

“…14 This type of joining provides an excellent target for PCR-based clonality studies, as the majority of these rearrangements do not show SHM. 15 Amplification of alternative clonal targets has been used to overcome these problems. The use of immunoglobulin light chain gene (IGK and IGL) rearrangements has been applied to clonality testing.…”

Molecular and cytogenetic characterization of expanded B-cell clones from multiclonal versus monoclonal B-cell chronic lymphoproliferative disorders

“…18,30 In parallel, patterns of rearrangement of the immunoglobulin heavy chain variable region genes (IGHV) and immunoglobulin κ (IGKV) and λ (IGLV) light chain genes were analyzed for each FACS-purified B-cell clone 18,[31][32] (see the Online Supplementary Methods section for detailed descriptions). To investigate the level of phylogenetic relationship among IGHV amino acid sequences, a sequence distance tree was built using the neighbor-joining method implemented in the freely available Molecular Evolutionary Genetic Analysis (MEGA) software.…”

Subjects with chronic lymphocytic leukaemia‐like B‐cell clones with stereotyped B‐cell receptors frequently show MDS‐associated phenotypes on myeloid cells

“…In parallel, analysis of the rearrangement patterns of the immunoglobulin heavy chain variable region genes (IGHV) was performed for each FACS-purified B-cell clone (van Dongen et al, 2003;Gonz alez et al, 2003;Henriques et al, 2013Henriques et al, , 2014. Forward and reverse sequences were aligned into a single resolved sequence and then aligned with germline sequences using the ImMunoGeneTics (IMGT) database and tools (http://www.imgt.org/ IMGT_vquest/share/textes/).…”