Mutated NPM proteins are found in NPMc⁺ leukemic cells of different lineages. (A, left) Epitopes recognized by anti-NPM antibodies. Clones 322 and 376 do not distinguish between wild-type (NPMwt) and mutant NPM (NPMm); clone FC-61991 recognizes NPMwt but not NPMm; the Sil-A and Sil-C antibodies recognize NPMm but not NPMwt. The red square area in NPMm indicates the mutated C-terminal portion of nucleophosmin. (Right) Schematic representation of subcellular distribution of NPMwt, NPMm, and NPMwt/NPMm heterodimer molecules in nuclear (orange) and cytoplasmic (gray) compartments of NPMc⁺ AML. (B, top) Double immunofluorescence staining of paraffin section for myeloperoxidase (Myel: red, cytoplasmic) and nucleophosmin (NPM: green, nuclear plus cytoplasmic) from a AML-M6 (×1000); single arrows indicate NPMc⁺ erythroid blasts and double arrows NPMc⁺ myeloid blasts. (Merge) Myeloid blasts (square box) show cytoplasmic colocalization of myeloperoxidase and NPM (×1000). (B, middle) Three-dimensional reconstruction at confocal microscope of the cell in the square box (arrow) (myeloperoxidase, red; NPM, green; nucleus, blue). (B, bottom) Double immunoenzymatic staining (G/NPM) of AML-M6. Leukemic proerythroblasts (arrows) are double-stained for NPM (clone 376) in blue (nucleus plus cytoplasm) and glycophorin in brown (surface). Myeloid blasts (double arrow) are glycophorin A-negative and show nuclear plus cytoplasmic NPM positivity. APAAP/Immunoperoxidase; no counterstain, ×1000. Multilineage involvement in NPMc⁺ AML (figure indicated as NPM). Nuclear plus cytoplasmic expression of NPM (clone 376) is seen in leukemic myelo-monocytic cells adjacent to bone trabecula (T), occasional strongly positive proerythroblasts (double arrow) and megakaryocytes (arrow); APAAP; hematoxylin counterstain, ×1000. (C, top) Proerythroblasts (arrows) are double-stained for NPM mutant (Sil-C) in blue (cytoplasmic-restricted) and glycophorin in brown (surface). Myeloid blasts (double arrow) are glycophorin-negative and show cytoplasmic-restricted expression of NPM mutant (APAAP/Immunoperoxidase; no counterstain, ×1000). (Middle) Cytoplasmic-restricted expression of mutant NPM protein in myeloblasts, cluster of proerythoblasts (double arrow), and isolated megakaryocyte (arrow). APAAP; hematoxylin counterstain, ×1000. (Bottom) Myeloblasts (arrow) show cytoplasmic-restricted expression of mutant NPM, whereas lymphoid cells (double arrow) are Sil-A negative (APAAP; hematoxylin counterstain, ×1000). (D) Wild-type NPM expression (clone FC-61991) is nuclear plus cytoplasmic (arrow) in leukemic cells of NPMc⁺ AML (left) but nucleus-restricted in NPMc^– AML (right, double arrow) with exception of a mitotic figure (arrow). APAAP; hematoxylin counterstain, ×1000.