Cell surface MSLN expression in AML. (A) Flow plots from a 5-year-old with MSLN⁺ AML with predominantly CD34⁻/MSLN⁺ leukemia. First plot with CD45/side scatter (SSC) distribution showing leukemic blasts (bright green), normal monocytes (dark green), myeloid cells (blue), and normal lymphocytes (gray). In the second plot, AML is shown in bright green, monocytes are dark green, and CD34⁺ cells are red. The AML is predominantly CD34⁻ but both CD34⁻ and CD34⁺ subset express MSLN. The third plot is gated on CD34⁺ cells, with CD34⁺/MSLN⁺ leukemic blasts in yellow and CD34⁺ normal progenitor cells in red. The fourth, fifth, and sixth plots show normal myeloid cells (blue), normal monocytes (green), and normal lymphocytes (gray), respectively, none of which express MSLN. (B) Flow plots from a 12-year-old with MSLN+/CD34+ AML. First plot with CD45/SSC distribution showing leukemic blasts (red), a few normal monocytes (green), and normal lymphocytes (gray). In the second plot the CD34⁺ blasts demonstrate MSLN expression. Third plot confirms the myeloid nature of CD34⁺/heterogenous CD117⁺ abnormal blasts (red), and normal lymphocytes (gray). The fourth plot confirms myeloid nature of the CD34⁺/CD33⁺ blasts (red), with lymphocytes (gray). (C) Flow plots from a 17-year-old patient with MSLN⁺/CD34⁺ AML. First plot with CD45/SSC distribution showing leukemic blasts (red), and few normal myeloid and normal lymphocytes (orange and gray, respectivly). Second plot shows heterogeneous MSLN expression on abnormal CD34⁺ myeloblasts (red) and normal lymphocytes (gray). The third plot confirms the myeloid nature of CD34⁺/CD117⁺ abnormal blasts (red) and normal lymphocytes (gray). (D) Normal CD34⁺ progenitor cells from an 18-year-old are negative for MSLN expression. The first plot shows CD34⁺ cells with early progenitors with bright CD34⁺/dim CD38⁺ expression in yellow, the second plot shows the characteristic position of these early progenitors (yellow) by CD45 and SSC, and the third plot shows there is no MSLN expression on any normal CD34⁺ cells, either early progenitors (yellow) or uncommitted progenitors (red). APC, allophycocyanin; FITC, fluorescein isothiocyanate; Lymph, lymphocytes; Meso, mesothelin; Mono, monocytes; PE, phycoerythrin; PerCP, peridinin-chlorophyll-protein complex; Prog, progenitors.