Morphologic and molecular findings in a case of CALR-mutated essential thrombocytemia that evolved into a CALR-mutated BCR-ABL⁺ chronic myeloid leukemia. (A-C) Bone marrow biopsy in 2011. (A) Normocellular BM with increased amount of large hyperlobated “staghorn” megakaryocytes (PAS stain). (B-C) Higher magnification showing the loose clusters of megakaryocytes with mainly staghorn forms (B: Giemsa stain; C: periodic acid Schiff stain). (D-F) BM biopsy in 2014. (D) Hypercellular BM with increased myeloid:erythroid ratio and increased amount of small hypolobated dwarf megakaryocytes (PAS stain). Insert: Interphase fluorescent in situ hybridization analysis using BCR-ABL dual color, dual fusion translocation probe (Zytotomed, Zytolight BCR-ABL) shows one red signal (red arrow), one green signal (green arrow), and two red/green fusion signals (yellow arrow) indicative of a t(9;22)(q34;q11). (E) Higher magnification showing the atypical small hypolobated megakaryocytes in a background of left-shifted granulopoiesis (periodic acid Schiff stain). (F) CD61 (Dako; Glostrup, Denmark) staining highlights the small hypolobated megakaryocytes. (G) Graphic depiction of the peripheral blood counts from January 2011 to January 2015. The patient was treated originally with interferon 135 µg. In October 2014, he was switched to nilotinib 150 mg (2-0-2). The top table shows the quantitative reverse transcription polymerase chain reaction (qRT-PCR) results of the BCR-ABL fusion transcript in international standard (IS). The bottom table shows the next-generation sequencing results of the allele burden of CALR mutant. (H) Fragment length analysis of CALR exon 9 hotspot region shows concurrent amplification of the wild-type allele resulting in a 257-bp fragment and a mutated allele of 262 bp in both BM biopsies. (I) Sanger sequencing of the CALR exon 9 hotspot region confirmed an identical frameshift mutation in both biopsies (c.1154_1155insTTGTC, p.K385fs*47). Fluorescent in situ hybridization images were acquired with a ×100/1.40 oil immersion objective in a Zeiss Axio fluorescence microscope (Zeiss) equipped with the appropriate filters sets and an Axio CAM MRm camera (Zeiss) and were documented and processed by using the Axio Vision Rel 4.8 software (Zeiss). Immunohistochemical analysis was performed on an automated immunostainer (Ventana Medical Systems, Tucson, AZ), following the manufacturer's protocols. Fragment analysis of CALR exon 9 hotspot region was performed by using Phusion Hot Start DNA polymerase (Finnzymes) with adequate amplification conditions and D4-fluorescent dye primer modification (Sigma-Aldrich).²  The products were separated by capillary electrophoresis on the GenomeLab GeXP Genetic Analysis System and analyzed with GenomeLab GeXP 10.2 software (Beckman Coulter, Krefeld, Germany). Sequencing of CALR exon 9 hotspot region was performed by using M13-tailed primers (forward: 5′-CTGGTCCTGGTCCTGATGTC-3′; reverse: 5′-GGGGACATCTTCCTCCTCAT-3′) and Phusion Hot Start DNA polymerase with adequate amplification conditions, followed by dye terminator cycle sequencing (Quick Start Master Mix) using M13 primers and capillary electrophoresis on the GenomeLab GeXP Genetic Analysis System 10.2 software. Next-generation sequencing was applied for mutational screening of CALR (exon 9). By using a 2-step PCR, design amplicons were generated that included sequencing adaptors for Roche 454 sequencing and an individual multiplex identifier (MID tag) to allow multiplexing. After amplicon pooling, the library was purified (QIAquick PCR purification kit; Qiagen, Hilden, Germany) followed by agencourt AMPure XP (Beckman Coulter) and quantified by using the Quant-iT PicoGreen Kit (Invitrogen, Carlsbad, CA). Following the emulsion-based PCR amplification (GS Junior emPCR kit), clonally amplified beads were enriched according to the manufacturer’s recommendations and quantified on a CASY cell counter (Roche). The 454 sequencing data were generated on a GS Junior using the GS Junior Titanium Sequencing Kit (Roche). The expected coverage was ×1000. Data analysis was performed by using JSI Sequence Pilot, SEQNext (JSI Medical Systems GmbH, Kippenheim, Germany). Hb, hemoglobin.