Figure 1
Figure 1. JAK2VG17F allele frequency and bone marrow histology. (A) The percentage of mutated JAK2 alleles increased from 5% (prior to imatinib therapy) to 23% (after 23 months of imatinib therapy), while the initial BCR-ABL e19a2 fusion transcript became undetectable after 11 months of treatment. The dotted line indicates the JAK2 wild-type status of 125 control volunteers (87 bone marrow biopsies from patients with Ph+ CML and peripheral blood from 38 healthy blood donors). For the Pyrosequencer assay (Biotage, Uppsala, Sweden), 25 ng of formalin-fixed and paraffin-embedded (FFPE) bone marrow biopsy–derived DNA was used to generate a 102-bp JAK2 polymerase chain reaction (PCR) product (JAK2 forward, 5′-TATGATGAGCAAGCTTTCTCACAAG-3′; JAK2 reverse, 5′-AGAAAGGCATTAGAAAGCCTGTAGTT-3′; GenBank accession no. AL161450) and a 130-bp MPL product (MPL forward, 5′-ATCTCCTTGGTGACCGCTCTG-3′; MPL reverse, 5′-TGGTCCACCGCCAGTCTG-3′; GenBank accession no. U68161). The Pyrosequencer detects the pyrophosphate release per wild-type G or mutated T incorporation at JAK2 position 1849 or at MPL position 1544, respectively, by a connected enzyme cascade with a luciferase-induced light signal. The light signal intensity is proportional to the amount of G or T and allows a quantification of the mutated T allele (positive/negative controls: JAK2 mutant cell line HEL, MPL mutated patient sample, and JAK2/MPL wild-type cell line HL-60). (B) Pretherapy bone marrow trephine revealed increased cellularity with a prominent promyelocytic proliferation and typical micromegakaryocytes (Giemsa stain; original magnification, ×1000). (C) Posttherapy bone marrow trephine showed predominant megakaryocytic proliferation with large and bizarre megakaryocytes arranged in clusters (Giemsa stain; original magnification, ×1000). (D) Megakaryocytic proliferation was associated with a marked increase of argyrophilic fibers (silver stain; original magnification, ×1000). Images in panels B-D were produced with a BX51 microscope equipped with a 40×/0.8 Vplan/Apo objective, a DP50 digital camera, and DP3.1 software, all from Olympus (Hamburg, Germany).

JAK2VG17F allele frequency and bone marrow histology. (A) The percentage of mutated JAK2 alleles increased from 5% (prior to imatinib therapy) to 23% (after 23 months of imatinib therapy), while the initial BCR-ABL e19a2 fusion transcript became undetectable after 11 months of treatment. The dotted line indicates the JAK2 wild-type status of 125 control volunteers (87 bone marrow biopsies from patients with Ph+ CML and peripheral blood from 38 healthy blood donors). For the Pyrosequencer assay (Biotage, Uppsala, Sweden), 25 ng of formalin-fixed and paraffin-embedded (FFPE) bone marrow biopsy–derived DNA was used to generate a 102-bp JAK2 polymerase chain reaction (PCR) product (JAK2 forward, 5′-TATGATGAGCAAGCTTTCTCACAAG-3′; JAK2 reverse, 5′-AGAAAGGCATTAGAAAGCCTGTAGTT-3′; GenBank accession no. AL161450) and a 130-bp MPL product (MPL forward, 5′-ATCTCCTTGGTGACCGCTCTG-3′; MPL reverse, 5′-TGGTCCACCGCCAGTCTG-3′; GenBank accession no. U68161). The Pyrosequencer detects the pyrophosphate release per wild-type G or mutated T incorporation at JAK2 position 1849 or at MPL position 1544, respectively, by a connected enzyme cascade with a luciferase-induced light signal. The light signal intensity is proportional to the amount of G or T and allows a quantification of the mutated T allele (positive/negative controls: JAK2 mutant cell line HEL, MPL mutated patient sample, and JAK2/MPL wild-type cell line HL-60). (B) Pretherapy bone marrow trephine revealed increased cellularity with a prominent promyelocytic proliferation and typical micromegakaryocytes (Giemsa stain; original magnification, ×1000). (C) Posttherapy bone marrow trephine showed predominant megakaryocytic proliferation with large and bizarre megakaryocytes arranged in clusters (Giemsa stain; original magnification, ×1000). (D) Megakaryocytic proliferation was associated with a marked increase of argyrophilic fibers (silver stain; original magnification, ×1000). Images in panels B-D were produced with a BX51 microscope equipped with a 40×/0.8 Vplan/Apo objective, a DP50 digital camera, and DP3.1 software, all from Olympus (Hamburg, Germany).

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