Clonal architecture and evolutionary history of Waldenström's macroglobulinemia at the single-cell level

Abstract

To provide insight into the subclonal architecture and co-dependency patterns of the alterations in Waldenström's macroglobulinemia (WM), we performed single-cell mutational and protein profiling of eight patients. A custom panel was designed to screen for mutations and copy number alterations at the single-cell level in samples taken from patients at diagnosis (n=5) or at disease progression (n=3). Results showed that in asymptomatic WM at diagnosis, MYD88L265P was the predominant clonal alteration; other events, if present, were secondary and subclonal to MYD88L265P. In symptomatic WM, clonal diversity was more evident, uncovering combinations of alterations that synergized to promote clonal expansion and dominance. At disease progression, a dominant clone was observed, sometimes accompanied by other less complex minor clones, which could be consistent with a clonal selection process. Clonal diversity was also reduced, probably due to the effect of treatment. Finally, we combined protein expression with mutational analysis to map somatic genotype with the immunophenotype. Our findings provide a comprehensive view of the clonality of tumor populations in WM and how clonal complexity can evolve and impact disease progression.

Keywords: Disease mechanisms; Genomic alterations; Protein expression; Single-cell; Waldenström's macroglobulinemia.

Fig. 1.

Clonal architecture of different disease stages of Waldenström's macroglobulinemia (WM) at the single-cell level. (A) Presence of wild-type (WT; green) versus heterozygous MYD88^L265P (red) and deletion of 6q (outlined in blue) in an asymptomatic WM patient at diagnosis (MW1). (B) Presence of WT (green) versus heterozygous MYD88^L265P (red) and deletion of 6q (outlined in blue) in an asymptomatic WM patient at diagnosis (MW2). (C) Distribution of somatic variants (MYD88^L265P and CXCR4^S344*) and copy number alterations (deletion of 6q, deletion of 17p and amplification of 3q) in a patient with symptomatic WM at diagnosis (MW5). (D) Distribution of somatic variants (MYD88^L265P, heterozygous and homozygous, and CXCR4^S338*) in one symptomatic WM patient at disease progression (MW6). (E) Distribution of somatic variants (heterozygous MYD88^L265P) and copy number alterations (deletion of 6q and amplification of 3q) in one symptomatic WM patient at disease progression (MW7). (F) Presence of MYD88^L265P, deletion of 6q and deletion of TRAF3 in one patient with symptomatic WM at the time of disease progression (MW8). In all panels, rows represent the individual cells and columns represent the regions covered by the panel amplicons. Color scale indicates the number of normalized reads for copy number alterations.

Fig. 2.

Copy number alterations in selected regulatory regions of 6q in one patient with asymptomatic WM. Rows represent the individual cells and columns represent the amplicons of 6q region. Yellow-blue color scale indicates the copy number alterations. The number of deleted amplicons/genes differed between cells, with TNFAIP3 the most frequently deleted gene. CNV, copy-number variation; HET, heterozygous; LP, lymphoplasmacytes; PC, plasma cells; SNV, single-nucleotide variant; WT, wild type.