Abstract
Waldenström Macroglobulinemia (WM) is characterized by monoclonal IgM paraprotein and bone marrow (BM) infiltration by lymphoplasmacytic lymphoma. The normal counterpart of WM malignant cell seems to be a post germinal centre IgM B-cell, which tumoral transformation occurs after cessation of somatic mutation (SM) but prior to Class switch recombination (CSR). However, recently has been reported that CSR can be possible “ex-vivo”, since clonotypic transcripts encoding post-switch isotypes have been observed in some WM cells cultured with CD40L/IL-40. However, this process has not been shown to occur “in vivo” until now.
#3754, a 51-year-old woman, was diagnosed in 2001 of WM with a M-IgM spike (46 g/L), anemia (Hb 9·6 g/dL), 76% lymphoplasmacytic monoclonal B-cells in BM and normal cytogenetics. In April 2005, an important IgG increase was observed (23 g/L). Immunofixation demonstrated an IgG-k paraprotein in the mid g-region and a monoclonal IgM-k paraprotein at the b-region corresponding to the two monoclonal peaks detected on serum electrophoresis. After 6-mercaptoethanol treatment, a single band was seen at the line stained with kappa, suggesting the presence of a single clone. Other causes of IgG monoclonal components were excluded considering clinical factors, immunophenotype analyses (San Miguel et al, 2003), quantity of DNA and cell cycle analyses (Ocio et al, 2005). However, the definitive proof for a unique monoclonal population was provided through molecular analysis. A single clonotypic rearrangement was detected by amplifying the complete VDJH fragment at diagnosis moment, according to the protocol describes in Biomed II (Leukemia2003; 17.2257–2317). Method describes from Billadeu et al (Billadeau et al, 1993) was used for isotype identification. So, cDNA monoclonal amplification was observed at tubes corresponding to Cm, Cd and Cg. All monoclonal PCR products were directly sequenced in an automated ABI 377 DNA sequencer. VH, DH & JH segments identification, as well as SH recognition was made using the V-BASE sequence directory alignment program, and the CH regions were compared at BLAST. All sequences obtained showed the same clonotypic CDR3 sequence (VH4-59/JH6) as well as the same SH (10,75%) pattern that monoclonal amplification at diagnosis, indicating the presence of the same clone at that moment (Figure 1).
In conclusion, we report for the first time a WM case in which tumor cells were able to carry out CSR, showing IgG and IgM clonotypic amplification, as well as producing both paraprotein components. This constitutes the first in vivo demonstration that CSR is possible in WM cells, and are able to develop a fully functional isotype class switch recombination not only in vitro but also in vivo.
Figure 1: Deduced amino acid sequence of tumor-derived VDJH gene with the three heavy chain isotypes (A: Cμ, B: Cδ C: Cγ). Sequences indicates the somatic mulation pattern. Comparison for WM are made with the closest germline VH gone; uppercase, replacement (R) mutation; lower case, silent (S) mutation. Each mutation was defined by nuclieotide exchanges in a single codon, with successive mutations leading in some cases to 2 or 3 distinct R or S events. These are shown as aligned amino acid changes at specific sites. Figure 1:. Deduced amino acid sequence of tumor-derived VDJH gene with the three heavy chain isotypes (A: Cμ, B: Cδ C: Cγ). Sequences indicates the somatic mulation pattern. Comparison for WM are made with the closest germline VH gone; uppercase, replacement (R) mutation; lower case, silent (S) mutation. Each mutation was defined by nuclieotide exchanges in a single codon, with successive mutations leading in some cases to 2 or 3 distinct R or S events. These are shown as aligned amino acid changes at specific sites.
Computational identification of 4-carboxyglutamate sites to supplement physiological studies using deep learning