Received: 15 November 2018 | Revised: 22 January 2019 | Accepted: 22 April 2019 DOI: 10.1002/ccr3.2208 CASE REPORT Ultrastructural, cytogenetic, and molecular findings in mast cell leukemia: Case report Anna Bosch‐Vilaseca1 | Anna Monter‐Rovira1 | Sabina Cisa‐Wieczorek1,2 | Guadalupe Oñate 1 | Elena Bussaglia 1 | Maite Carricondo 1 | Ángel Remacha 1 | Clara Martínez 1 | Marta Pratcorona 1 | María Laura Blanco 1 | Josep F. Nomdedéu1 1 Laboratory of Hematology, Hematology Department, Hospital de la Santa Creu Abstract i Sant Pau. Autonomous University of We report a de novo aleukemic form of MCL with a complex monosomic karyo- Barcelona (UAB), Barcelona, Spain type with LOH for multiple chromosomes and TP53 mutation. Additionally, whereas 2 INSERM, Université Aix Marseille, D816V KIT was not found, the c‐Kit transmembrane domain p.M541L variant was Marseille, France detected which is the most common SNP of KIT gene in humans with controversial Correspondence pathogenic role. In these cases, it is crucial to perform a rapid broad molecular study Josep F. Nomdedéu, Laboratory of for an accurate diagnosis which could help to initiate targeted therapy. Hematology, Hematology Department, Hospital de la Santa Creu i Sant Pau, Mas Casanovas, 90, 08035 Barcelona, Spain. KEYWORDS Email: jnomdedeu@santpau.cat acute leukemia, genomics, molecular biology, systemic mastocytosis Mast cell leukemia (MCL) is an extremely rare and aggres- harbor c‐Kit tyrosine kinase domain mutations, most fre- sive form of systemic mastocytosis (SM),1 accounting for quently D816V,4 without recurrent chromosomal aberrations <1% of all adult SM, with ≥20% atypical and immature mast detected to date.5 Therapeutic approaches are limited and, in cells (MC) in bone marrow. There are two known forms of general, prognosis of MCL is poor with less than one‐year presentation for MCL, the classic case in which MCs ac- survival in most patients.3 count for ≥10% of the peripheral white blood cells and the We report the case of an 86‐year‐old Caucasian male more frequent aleukemic variant with less than 10% MCs referred to the emergency care with upper gastrointestinal in peripheral blood. De novo MCL should be distinguished bleeding due to esophageal ulcers (LA grade D esophagitis), from secondary MCL, evolving from other advanced SM. which were successfully treated by endoscopy and with a Diagnostic criteria and classification of SM have been up- proton‐pump inhibitor, and concomitant severe acute throm- dated in the 2017 World Health Organization classification.2 bocytopenia, that prevented from taking a biopsy of the le- Most cases of MCL have markedly high levels of serum sion. Initially, the thrombocytopenia was medically treated tryptase, peripheral cytopenia, and/or leukocytosis with eo- as immune‐mediated without response. In addition, grade sinophilia. Patients can develop any of the typical clinical 2 normocytic anemia (Hb 80‐99 g/L) persisted in the fol- features of SM due to organ impairment or MC activation, lowing blood tests, with no renal or hepatic failure and nor- although skin lesions are not usually detectable. Atypical mal lactate dehydrogenase, a normal leukocyte total count MCs show an abnormal antigen expression, characteristically (5.1 × 109/L [3.8‐11.0]) with 71% neutrophil, 5% myelocyte, CD2 and/or CD25 with the expression of concurrent imma- and 1% of atypical MCs and high levels of serum tryptase turity markers.1-3 Furthermore, most cases of aggressive SM (501 µ/L [<15]). Bosch‐Vilaseca and Monter‐Rovira contributed equally to this work. This is an open access article under the terms of the Creat​ive Commo​ns Attri​butio​n‐NonCo​mmerc​ial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. © 2019 The Authors. Clinical Case Reports published by John Wiley & Sons Ltd. Clin Case Rep. 2019;7:1395–1398.  wileyonlinelibrary.com/journal/ccr3 | 1395 1396 |    BOSCH‐VILASECA et al. Bone marrow was diffusely infiltrated by 60% of large cells were positive for c‐Kit (CD117) and CD2 by immuno- atypical cells with loose chromatin and centric and round histochemical staining. Myeloperoxidase was negative, chlo- nuclei, atypical mast cell type I (MC I), and occasionally roacetate esterase weakly positive, and toluidine blue stain even larger ones with polylobed nuclei (promastocytes or revealed metachromatic granulated blast‐like cells (Figure atypical MC II) distributed in multiple aggregates.6 Most of 1C,D). Electronic microscopy disclosed three distinct types the atypical MCs exhibited a characteristic wide cytoplasm of MCs (immature, mature, and activated MCs) characterized with azurophilic granules and multiple heavy inclusions or by its cytoplasmic granules which appear during their matu- bizarre‐looking vacuoles, not stained with May‐Grünwald‐ ration (Figure 1E‐G). Giemsa (MGG) (Figure 1A,B). Dysplastic features were The immunophenotype of the neoplastic cells assessed absent in the peripheral blood and the bone marrow. These by multiparametric flow cytometry was as follows: CD117, (A) (B) (C) (D) (H) (E) (F) (G) F I G U R E 1 (A‐F) Morphology and immunohistochemistry. A, BM infiltration by atypical MCs type I and type II (arrow). B, Amplified bilobed MC type II or promastocyte (arrow). C, c‐Kit (CD117) immunohistochemical staining. D, Toluidine blue staining showing metachromatic MCs (arrows). (E‐G) MCs ultrastructure at different maturation stages. E, Immature MC with irregular agranulated cytoplasm, mitochondria, and smooth ER. F, Mature MC with a lot of microvilli on cell surface and granules that vary in size, shape, and electron density. G, Activated MC granules clustered near to the exocytosis sites. H, Sanger sequencing of TP53 exon 8 revealed the presence of a G→A base change resulting in a missense mutation G266R. BM, bone marrow; ER, endoplasmic reticulum; GR, granule; MC, mast cell; MT, mitochondria; SG, secretory granule BOSCH‐VILASECA et al.    | 1397 TABLE 1 (A) List of LOH as determined using CytoScan 750K rapid clinical deterioration, midostaurin was started, before Array; (B) Allelic variants identified by NGS obtaining the final molecular studies results. Despite this, the A clinical status worsened and the patient died one month after diagnosis. Chrom. Cytoband/Location Start (Mb)a End (Mb)a To sum up, we report an aleukemic form of MCL case 3 3p21.31‐p21.2 47 002 139 50 797 870 with a complex monosomic karyotype. We also identified 5 5q34‐q35.2 167 244 759 176 110 659 LOH regions of chromosomes 3, 5, 7, 9, and 10.8 Of note, 7 7p21.2‐p15.3 14 753 209 22 086 697 our patient harbored a TP53 mutation which has already 7 7q11.21 62 569 501 66 530 404 been defined as an early transforming event in hematologic malignancies.9 As it has been reported, our patient lacked 7 7q31.31 117 485 006 120 694 229 the D816V KIT mutation; however, a c‐Kit transmembrane 9 9q31.1‐q31.2 107 580 656 111 256 573 domain p.M541L variant was detected and it could modify 10 10q22.1‐q22.2 73 731 650 77 131 019 the phenotype of a neoplastic myeloid progenitor trans- B formed by TP53 mutation. In fact, this KIT allelic vari- ant in exon 10 is the most common SNP found in the KIT Gene AA variant cDNA variant VAF gene in humans and its pathogenic role is controversial.7 KIT p.M541L c.1621A>C 0.57 The contributing effects of the other variants remain to be TP53 p.G266R c.796G>A 0.075 elucidated.5,10,11 GATA2 p.A164T c.490G>A 0.54 Further MCL cases should be analyzed with the newly EZH2 p.D185H c.553G>A 0.48 available techniques before drawing more precise conclu- ANKRD26 p.Q20R c.59A>G 0.56 sions about the genomic landscape of this infrequent disease. Abbreviations: AA, aminoacid; Chrom, chromosome; VAF, variant allele In these cases, it is crucial to perform a rapid broad molecular frequency. study for an accurate diagnosis which could help to initiate a Nucleotide position of the origin and the end of the aberration in the reference targeted therapy. genome (hg19). CD2, CD25, CD34, HLA‐DR, and CD123 were positive, AKNOWLEDGM ENTS whereas CD45, MPO, and TdT were negative. Conventional cytogenetic studies revealed a com- This work was supported in part by grants AGAUR 2014 plex monosomic karyotype: 35,XY,‐1,‐3,‐5,add(5)(q32) SGR 383 from Generalitat de Catalunya and Instituto de ,‐7,‐10,‐11,‐12,‐13,‐15,‐16,‐17,‐18,+mar1[2]/35,XY,i(1) Salud Carlos III FIS 16/00940, Ministerio de Economía y (q10),‐3,‐5,add(5)(q32),‐7,‐10,‐11,‐12,‐13,‐15,‐16,‐17,‐18[ Competitividad, Spain. 2]/46,XY[16] (Figure S1). DNA arrays performed with an Affymetrix platform and Cytoscan 750K confirmed these CONFLICT OF INTEREST findings along with the detection of loss of heterozygosity regions (LOH) (Table 1A). The authors declare no conflict of interest. Mutation screening with conventional PCR techniques, as previously described, ruled out mutations in KIT D816V, AUTHOR CONTRIBUTIONS FLT3, NPM1, CEBPA, MLL, IDH1, IDH2, as well as the re- arrengements CBFB‐MYH11 and RUNX1‐RUNX1T1. AB‐V and AM‐R: conceived and designed the analysis, col- An amplicon‐based targeted next‐generation sequencing lected the data, performed the analysis, and wrote the paper; (NGS) myeloid panel of 42 genes (Haloplex TM, Agilent SC‐W: contributed data or analysis tools (electronic micros- Technologies) routinely used in our institution based on copy study); EB and MC: contributed data or analysis tools published studies of Bullinger et al group detected a KIT (molecular analysis); GO, ÁR, CM, MP, and MLB: aided in M541L variant in exon 10 (transmembrane domain)7 and a interpreting the results and worked on the manuscript. JFN: TP53 missense pathogenic mutation that was confirmed by conceived the study and were in charge of overall direction Sanger sequencing (Figure 1H). Other concurrent allelic and planning. All authors discussed the results and com- variants of GATA2, EZH2, and ANKRD26 genes were de- mented on the manuscript. tected (Table 1B). All these variants have previously been described and reported as polymorphisms of uncertain patho- ORCID genic significance. Therefore, the patient was diagnosed with de novo aleu- Anna Bosch‐Vilaseca https://orcid. kemic MCL, according to the 2017 WHO criteria. Due to a org/0000-0002-9147-3692 1398 |    BOSCH‐VILASECA et al. R E F E R E NC E S blasts detected by single nucleotide polymorphism microarray‐ based whole‐genome scanning. Genes Chromosomes Cancer. 1. Georgin‐Lavialle S, Lhermitte L, Dubreuil P, Chandesris 2016;55(4):389‐396. MO, Hermine O, Damaj G. Mast cell leukemia. Blood. 9. Desai P, Mencia‐Trinchant N, Savenkov O, et al. Somatic muta- 2013;121(8):1285‐1295. tions precede acute myeloid leukemia years before diagnosis. Nat 2. Swerdlow SH, Campo E, Harris NL, et al. (Eds). WHO Med. 2018;24(7):1015‐1023. Classification of Tumours of Haematopoietic and Lymphoid 10. Hirsch P, Zhang Y, Tang R, et al. Genetic hierarchy and temporal Tissues, Revised 4th edn. 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