Available online at www.annclinlabsci.org Annals of Clinical & Laboratory Science, vol. 41, no. 2, 2011 193

CKIT mutation in therapy-related with MLLT3/MLL chimeric transcript from t(9;11)(p22;q23)

Chae Lim Jung1, Hee-Jin Kim1, Dong-Hwan Kim2, Heejae Huh1, Min-Jung Song1, and Sun-Hee Kim1 Departments of Laboratory Medicine & Genetics1 and Medicine2, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea

Abstract. Gain-of-function mutations of the CKIT have been reported to specifically occur in core-binding factor (CBF) acute myeloid leukemia (AML) with a poor prognostic implication. Here we report a case of therapy-related AML with t(9;11)(p22;q23) who had CKIT mutation. A 48-year-old woman with breast cancer received partial mastectomy followed by 6 cycles of adjuvant chemotherapy and radiation therapy. At 28 months from the diagnosis of breast cancer, she was diagnosed as having AML with blasts 81% in bone marrow. Cytogenetic analysis revealed t(9;11)(p22;q23), and FISH showed 96.5% of MLL break-apart signals. RT-PCR study revealed MLL(11q23)/MLLT3(9p22) chimeric transcript. FLT3-ITD and NPM1 mutations were both negative. Unexpectedly, mutation analyses for CKIT identified D816Y mutation. The patient received induction chemotherapy and achieved complete remission at 1 month. To the best of our knowledge, this is the first report on CKIT mutation in therapy-related AML with MLL rearrangement.

Keywords: CKIT mutation; therapy-related AML; MLL; myeloid neoplasm

Introduction history of chemotherapy or radiotherapy for malignancy. Gene rearrangements such as Acute myeloid leukemia (AML) is a genetically MLL rearrangement and loss of the whole or heterogeneous clonal disorder with cytogenetic part of 5/7 are frequent genetic and molecular alterations. Cytogenetic alterations in TR-MN. aberrations are observed in more than half of cases of AML, and in particular, certain recurrent The CKIT gene (or KIT, the official name aberrations harbor prognostic implications approved by the HUGO [1]. In recent years, the understanding of the committee; MIM# 164920) is located on the molecular pathogenesis of AML has remarkably chromosome band 4q11-12 and encodes a 145- fueled by the identification of recurring point kD transmembrane glycoprotein. It is a member mutations in such as NPM1, FLT3, of the type III family of receptor tyrosine kinase CEBPA, and CKIT [2]. The diagnostic entity (RTK) that is composed of 5 extracellular of therapy-related myeloid neoplasm (TR-MN) immunoglobulin-like domains, a single has been newly introduced in the WHO 2008 transmembrane segment, a juxtamembrane classification [3]. It includes both myelodysplastic (JM) and a split cytoplasmic kinase domain. syndrome and AML in association with previous CKIT plays a role in survival, self renewal and differentiation of hematopoietic stem cells. Address correspondence and reprint requests to Hee-Jun Kim, MD, Gain-of-function mutations in CKIT occur PhD, Assistant Professor, Department of Laboratory Medicine & either in the JM domain or in the kinase domain, Genetics, Samsung, Medical Center, Sungkyunkwan, University causing constitutive activation of the receptor School of Medicine, 50 Ilwon-dong, Gangnam-gu, Seoul, Korea, and thereby contributing to leukemogenesis 135-710; tel +82-2-3410-2710; fax +82-2-3410-2719; e-mail: [4]. Mutations in CKIT have been reported [email protected] 0091-7370/11/0200-193. © 2011 by the Association of Clinical Scientists, Inc. 194 Annals of Clinical & Laboratory Science, vol. 41, no. 2, 2011 in 10-46% of CBF-AML, AML with t(8;21) negative. Unexpectedly, however, the CKIT (q22;q22)/RUNX1-RUNX1T1 rearrangement mutation analysis by direct sequencing of exons or inv(16)(p13.1q22)/t(16;16)(p13.1;q22) or 8, 10, 11, 12, 13, and 17 on the ABI Prism CBFB-MYH11, which accounts for 10-15% of 3100 sequencing analyzer (Applied Biosystems, AML [5-11]. The presence of a CKIT mutation Foster city, CA, USA) identified a heterozygous has been reported to harbor an unfavorable D816Y mutation (c.2446G>T) in exon 17 of prognostic implication in CBF-AML [8-11]. CKIT (Fig. 1C).

In this report, we describe a patient with TR- The patient received induction chemotherapy MN with t(9;11)(p22;q23) who also had with cytarabine and idarubicin and achieved CKIT mutation. This is the first report on a complete remission at 1 month. Currently she case of CKIT mutation in TR-AML with MLL is receiving consolidation chemotherapy with rearrangement. high-dose cytarabine and idarubicin.

Case report

A 48-year-old Korean woman was diagnosed with invasive ductal carcinoma of the breast in February 2008. She underwent partial mastectomy and received 6 cycles of adjuvant chemotherapy (fluorouracil, doxorubicin, and cyclophosphamide) and radiation therapy (dose, 6,050 cGy). After treatment, her condition was stable without evidence of malignancy. Her follow-up course had been uneventful until she presented with upper respiratory infection symptom in Jun 2010, 28 months from the diagnosis of breast cancer. Complete cell counts and peripheral blood examination showed white blood cell count 30.62 x 109/L with 91% blasts, Hemoglobin 8.5 g/dL, and platelet count 21.0 x 109/L. The bone marrow was packed with leukemic blasts (81% of all nucleated cells), which were positive for CD11, CD13, CD33, CD64, CD117, and myeloperoxidase on flow cytomeric analyses.

Cytogenetic analysis on bone marrow aspirate revealed a balanced translocation involving the MLL gene, 46,XX,t(9;11)(p22;q23)[11] (Fig. 1A). FISH study using LSI MLL dual- color, break-apart probe (Abbott Molecular, Des Plaines, IL, USA) showed break-apart MLL signals in 96.5% of interphase cells (Fig. 1B). Reverse transcription polymerase Fig 1.A. Cytogenetic analyses on the bone marrow aspirate in chain reaction (RT-PCR) study confirmed the patient revealed 46,XX,t(9;11)(p22;q23)[11] (arrows). Fig 1.B. Interphase FISH using LSI MLL dual-color, break-apart the presence of MLL(11q23)/MLLT3(9p22) probe revealed MLL gene rearrangement (break-apart signals) in chimeric transcript. Point mutation studies for 96.5% of interphase nuclei. Fig 1.C. Direct sequencing analysis FLT3-ITD and NPM1 mutations were both of the CKIT gene revealed a heterozygous D816Y mutation in exon 17 (2445G>T) (arrow). CKIT mutation in TR-AML with MLL rearrangement 195 Discussion II mutations) work together [4, 7]. Our case suggests that the CKIT mutation can cooperate TR-MN is the most serious long-term with MLL rearrangements in TR-AML, albeit complication in cancer patients. Among the rarely. recurrent cytogenetic abnormalities in TR- AML, MLL gene rearrangement has been As was the case in our patient, CKIT mutations known to be associated with a short latency are most frequently encountered in exon 17 period and prior topoisomerase II inhibitor which encodes the CKIT activation loop in therapy, and presentation as AML without the kinase domain (TKD). Frequent TKD precedent myelodysplastic phase [12]. The mutations include D816V, D816Y, D816H, patient described in this report is in line with and N822K [14], and gain-of-function this observation in that she presented with a mutations of CKIT may serve as a target for full-blown AML with a period of 28 months TK inhibitors [10]. While AML with CKIT after treatment of topoisomerase II inhibitor. D816 mutation are known to be intrinsically The partner gene in our case was the MLLT3 resistant to imatinib, they may respond to other gene on the 9p22 band. The t(9;11)(p22;q23) TK inhibitors such as PKC412 or dasatinib or MLLT3/MLL is the most common MLL [15, 16]. The observation of the occurrence rearrangement in AML and also frequent in of CKIT mutation in non-CBF AML as in TR-AML [3]. our patient demonstrates the potential need for the screening of CKIT mutation in AML Our patient also had heterozygous CKIT without restriction to CBF-AML, potentially D816Y mutation (c.2446G>T) in exon 17, for therapeutic decision-making. along with MLLT3/MLL rearrangement. Since CKIT mutations have been reported mostly in In summary, we reported the first case of CBF-AML, most studies on CKIT mutations CKIT mutation in TR-AML with MLL have been limited to CBF-AML. A few studies rearrangement. This case adds a line of evidence have investigated CKIT mutations in TR-MN. in our understanding of leukemogenic gene Schnittger et al. performed a large-scale study mutations that cooperates with recurrent gene involving ~2,000 unselected patients with AML rearrangements. and reported a frequency of 1.7% of CKIT D816 mutation [11]. Among 125 patients with Acknowledgment TR-AML of the series, CKIT mutation was detected in only one patient who had t(8;21). This study was supported by the Samsung None of 65 patients with MLL rearrangement Medical Center Clinical Research Development had CKIT D816 mutation. Another study on Program grant, #CRS-108-62-3. 140 patients with TR-MN reported 2 cases with CKIT D816V mutation, one of which References had t(8;21) [13]. More recently, a study on 144 patients with newly diagnosed AML reported 1. Baldus CD, Mrózek K, Marcucci G, Bloomfield CD. Clinical outcome of de novo acute myeloid leukaemia patients with CKIT mutations in 5.6% (8/144), including normal cytogenetics is affected by molecular genetic alterations: 4 cases with CBF-AML. The series included a concise review. Br J Haematol 2007;137:387-400. 2 cases with MLL rearrangement, and neither 2. Scholl S, Fricke HJ, Sayer HG, Höffken K. Clinical implications of molecular genetic aberrations in acute myeloid leukemia. J had a CKIT mutation [7]. Cancer Res Clin Oncol 2009;135:491-505. 3. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein According to the two-hit model of H, Thiele J, Vardiman JW. eds. WHO classification of tumours of haematopoietic and lymphoid tissues. 4th ed, IARC Press, leukemogenesis, point mutations of such Lyon, 2008; pp 114-115, 127-129. genes as CKIT and FLT3 (class I mutation) 4. Masson K and Rönnstrand L. Oncogenic signaling from the hematopoietic growth factor receptors c-Kit and Flt3. Cell and fusion transcripts such as CBF-MYH11, Signal 2009;21:1717-1726. RUNX1-RUNX1T1, and PML-RARA (class 5. Beghini A, Ripamonti CB, Cairoli R, Cazzaniga G, Colapietro 196 Annals of Clinical & Laboratory Science, vol. 41, no. 2, 2011 P, Elice F, Nadali G, Grillo G, Haas OA, Biondi A, Morra E, 11. Schnittger S, Kohl TM, Haferlach T, Kern W, Hiddemann W, Larizza L. KIT activating mutations: incidence in adult and Spiekermann K, Schoch C. KIT-D816 mutations in AML1- pediatric acute myeloid leukemia, and identification of an ETO-positive AML are associated with impaired event-free and internal tandem duplication. Haematologica 2004;89:920-925. overall survival. Blood 2006;107:1791-1799. 6. Goemans BF, Zwaan CM, Miller M, Zimmermann M, Harlow 12. Larson RA. Therapy-related myeloid neoplasms. Haematologica A, Meshinchi S, Loonen AH, Hählen K, Reinhardt D, Creutzig 2009;94:454-459. U, Kaspers GJ, Heinrich MC. Mutations in KIT and RAS are 13. Christiansen DH, Andersen MK, Desta F, Pedersen-Bjergaard frequent events in pediatric core-binding factor acute myeloid J. Mutations of genes in the receptor tyrosine kinase (RTK)/ leukemia. Leukemia. 2005;19:1536–1542. RAS-BRAF signal transduction pathway in therapy-related 7. Ishikawa Y, Kiyoi H, Tsujimura A, Miyawaki S, Miyazaki Y, myelodysplasia and acute myeloid leukemia. Leukemia Kuriyama K, Tomonaga M, Naoe T. Comprehensive analysis of 2005;19:2232–2240. cooperative gene mutations between class I and class II in de 14. Sritana N, Auewarakul CU. KIT and FLT3 receptor tyrosine novo acute myeloid leukemia. Eur J Haematol 2009;83:90-98. kinase mutations in acute myeloid leukemia with favorable 8. Cairoli R, Beghini A, Grillo G, Nadali G, Elice F, Ripamonti cytogenetics: two novel mutations and selective occurrence CB, Colapietro P, Nichelatti M, Pezzetti L, Lunghi M, Cuneo in leukemia subtypes and age groups. Exp Mol Pathol A, Viola A, Ferrara F, Lazzarino M, Rodeghiero F, Pizzolo G, 2008;85:227-231. Larizza L, Morra E. Prognostic impact of c-KIT mutations in 15. Growney JD, Clark JJ, Adelsperger J, Stone R, Fabbro D, Griffin core binding factor leukemias: an Italian retrospective study. JD, Gilliland DG. Activation mutations of human c-KIT Blood 2006;107:3463-3468. resistant to imatinib mesylate are sensitive to the tyrosine kinase 9. Boissel N, Leroy H, Brethon B, Philippe N, de Botton S, inhibitor PKC412. Blood 2005;106:721-724. Auvrignon A, Raffoux E, Leblanc T, Thomas X, Hermine O, 16. Schittenhelm MM, Shiraga S, Schroeder A, Corbin AS, Quesnel B, Baruchel A, Leverger G, Dombret H, Preudhomme Griffith D, Lee FY, Bokemeyer C, Deininger MW, Druker C. Incidence and prognostic impact of c-Kit, FLT3, and Ras BJ, Heinrich MC. Dasatinib (BMS-354825), a dual SRC/ gene mutations in core binding factor acute myeloid leukemia ABL kinase inhibitor, inhibits the kinase activity of wild-type, (CBF-AML). Leukemia 2006;20:965-970. juxtamembrane, and activation loop mutant KIT isoforms 10. Paschka P, Marcucci G, Ruppert AS, Mrózek K, Chen H, associated with human malignancies. Cancer Res 2006;66:473- Kittles RA, Vukosavljevic T, Perrotti D, Vardiman JW, Carroll 481. AJ, Kolitz JE, Larson RA, Bloomfield CD. Adverse prognostic significance of KIT mutations in adult acute myeloid leukemia with inv(16) and t(8;21): a Cancer and Leukemia Group B Study. J Clin Oncol 2006;24:3904-3911.