NPM exon 12 mutations are found in NPMc⁺ leukemic cells of different lineages. (A) Patient 1. Bone marrow frozen sections are stained for glycophorin A. The cells selected for analysis are shown before (left) and after laser microdissection (right, empty areas), and are labeled as follows: 1 (small group of glycophorin A-negative myeloid blasts); 2, 3, and 4 (single megakaryocytes); 5 and 6 (small groups of glycophorin A-positive erythroid precursors). APAAP; hematoxylin counterstain, 400×. (B) Sequencing analysis of NPM exon 12 in microdissected erythroid cells from patient 1. The heterozygous insertion of 4 nucleotides generates a shift in the reading frame. The arrow indicates the sequence orientation. (C) Patient 3. (Top, left) Bone marrow paraffin section showing numerous NPMc⁺ megakaryoytes (arrow) and small clusters of NPMc⁺ myeloid blasts; T indicates a bone trabecula. APAAP; hematoxylin counterstain, 200×. (Top, right) Higher magnification from the same case showing NPMc⁺ megakaryocyte (arrow) with marked emperipolesis (double arrows). APAAP; hematoxylin counterstain, 1000×. (Bottom, left) CD61⁺ megakaryocytes selected for laser microdissection are indicated (arrows); CD61⁺ endothelial cells of a blood vessel (asterisk) were used as negative control. APAAP; hematoxylin counterstain, 200×. (Bottom right) Empty areas correspond to microdissected megakaryocytes after laser catapulting. APAAP; hematoxylin counterstain, 200×. (D) Chromatograms of NPM exon 12 sequences obtained from genomic DNA of vessel endothelial cells (control) (top) and megakaryocytes (bottom), microdissected from patient 3. Although a wild-type sequence is detected in the control DNA (vessel), the leukemic megakaryocytes show a heterozygous insertion of 4 nucleotides, creating a shift in the reading frame. The arrow indicates the sequence orientation.