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Intra-clonal heterogeneity in malignant plasma (PC) cells and clonal B cells has been reported in both multiple myeloma (MM) and Waldenstrom macroglobulinemia (WM). Further phenotypic and molecular characterization of inter- and intra-clonal genetic complexity together with humoral immunity will enhance our understanding of disease pathogenesis and identify novel therapeutic strategies. By a large cohort of MM (N = 100) and WM (N = 50) patient samples, we studied evolution of bone marrow patient samples from premalignant stages (MGUS and smoldering MM) to malignant stages (MM and WM) versus age-matched healthy donors (HD) by combining immunophenotypic, molecular, and cytogenetic approaches. We examined phenotypic and molecular features of intra-clonal architecture of malignant PC in MM and clonal B cells in WM together with humoral immunity on a single cell level using mass cytometry (CyTOF). CyTOF is based on rare stable earth elemental isotopes-bound to antibodies to target epitopes on and within cells: up to 40 different markers can simultaneously assess immunophenotyping and analysis of signaling in single cell. To investigate phenotypic profile and molecular signature of intra-clonal heterogeneity of PC in MM, high-dimensional analyses revealed that clonal PC clustered separately from B cells with 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. 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 RARα2. sXBP-1 was significantly upregulated in all MM stages. Furthermore, expression of stem cell markers 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. Both MM and WM are characterized by immune dysfunction; therefore we used CyTOF technology to define the complex immune profile in MM and WM patient samples. Therefore, we examined changes in B cell development, T cell subsets, NK and NKT cells as well as monocytic, granulocytic, erythroid and platelet lineages during evolution of both diseases vs. HD on phenotypic and molecular level. For example, plasmacytoid dendritic cells (pDC) were increased in MM with significantly decreased PD-1 expression on pDC in MM compared to MGUS (P = 0.007). Interestingly, PD-1 and its ligand PD-L1 were variably expressed on B cells (2-9% and 3-27%) and PC (0.5-46% and 3-41%) in MM. A better understanding of inter- and intra-clonal heterogeneity together with host immunity on the phenotypic and molecular/signaling level in MM and WM will provide the framework for identifying and validating novel personalized targeted therapies. Citation Format: Jana Jakubikova, Danka Cholujova, Teru Hideshima, Jacob Laubach, Nikhil C. Munshi, Steven P. Treon, Paul G. Richardson, Efstathios Kastritis, David M. Dorfman, Kenneth C. Anderson. Phenotypic and molecular characterization of inter- and intraclonal heterogeneity in multiple myeloma and Waldenstrom macroglobulinemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2004. doi:10.1158/1538-7445.AM2015-2004