Inter and Intra-Clonal Heterogeneity in Multiple Myeloma and Waldenstrom Macroglobulinemia

Introduction: Intra-clonal heterogeneity in malignant plasma (PC) cells and B-cells has recently been reported in both multiple myeloma (MM) and Waldenstrom macroglobulinemia (WM). Further phenotypic and molecular characterization of inter- and intra-clonal genetic complexity will enhance our understanding of disease pathogenesis and identify novel therapeutic strategies.

Methods: In this study, we compared normal and malignant PC maturation-associated B-cell subsets using bone marrow samples from individuals with monoclonal gammopathy of undetermined significance (MGUS), smoldering MM (SMM), newly diagnosed MM, and relapsed/refractory MM versus age-matched healthy donors (HD). We also similarly analyzed WM. In addition to corrupted B-cell lineage, we examined phenotypic and molecular features of intra-clonal architecture (complexity) of malignant PC in MM and clonal B-cells in WM on a single cell level using time-of-flight mass cytometry (CyTOF) technology. CyTOF technology is based on rare stable earth elemental isotopes-bound to antibodies to target epitopes on and within cells: up to 40 different markers on a single cell can simultaneously assess including phenotype, transcription factors, regulatory signaling molecules and enzymes, as well as activation of signaling molecules. The resulting high-dimensional data were analyzed by SPADE, viSNE and Wanderlust software.

Results: Our high-dimensional data of clustered analyses showed significantly decreased CD19+CD27- patient cells in MM with cytogenetic abnormalities (cytog+) including del(13q), t(4;14), t(14;16), t(3;14), +1q or t(11;14) versus patient cells in MM without any cytogenetic abnormalities (cytog-; P=0.013). In contrast, there was a significant increase of transitional B cells (CD19+CD27-IgM+CD10-IgDlow) in patients with MM cytog+ vs. MM cytog- (P=0.028). A significant increase of mature (naïve) B cells (CD19+CD27-IgM+CD10-IgD+) was also detected in MM cytog+ versus MM cytog- patients (P=0.013), but not in WM cytog- vs. WM cytog+ (46XY, -Y, +18q, +6p, 14q). Clonal PC (CD19-CD38++CD138+CD45-/dim; either cyk or cyl +) were significantly upregulated in MM cytog+ compared to MM cytog- (P=0.021) by CyTOF analyses. To investigate phenotypic profiles and molecular signature of intra-clonal heterogeneity of PC in MM, high-dimensional analyses by SPADE and viSNE revealed that clonal PC clustered separately from B cells by, virtue of high CD319 and CD47 expression; variable expression of CD52, CD56, CD81, CD44, CD200; and low expression of CD28, CD117, CD338, CD325, and CD243. For example, adhesion CD56 and anti-adhesion CD52 molecules were significantly increased in MM cytog+ compared to MM cytog-. Clonal PC highly expressed IFR4 and Notch1; variously expressed FGFR3, sXBP-1, KLF4 and c-Myc; and only minimally expressed Bcl-6, WHSC-1 (MMSET) and RARa2. sXBP-1 was significantly upregulated in all MM stages compared to HD. Furthermore, expression of stem cell markers including Sox-2, Oct3/4 and Nestin was detected only at low level in clonal PC, except for higher expression of Nanog. In WM, clonal B cells expressed Bcl-6 (4-36%) and MYD88 (2-27%) by CyTOF analyses. Finally, cluster analyses by SPADE and viSNE allows for detection of phenotypic and molecular changes not only in clonal populations but also at distinct B-lineage maturation stages, such as expression of Pax-5 and Bcl-2 on early B cell progenitors. This data represents a cohort of MM (N=35) and WM (N=15) patients; a significantly larger data set of MM (N=100) and WM (N=50) will be presented.

Conclusion: This study characterizes the molecular and phenotypic profile associated with inter- and intra-clonal heterogeneity in MM and WM. It not only enhances our understanding of disease pathogenesis, but may allow for individualized targeted therapy.