Activating FLT3 Mutations Are Detectable in Chronic and Blast Phases of Chronic Myeloproliferative Disorders Other Than Chronic Myeloid Leukemia

Hematopathology / FLT3 IN CHRONIC MYELOPROLIFERATIVE DISORDERS

Pei Lin, MD, Dan Jones, MD, PhD, L. Jeffrey Medeiros, MD, Weina Chen, MD, PhD, Francisco Vega-Vazquez, MD, PhD, and Rajyalakshmi Luthra, PhD

Key Words: FLT3 gene mutations; Chronic myeloproliferative disorders Downloaded from https://academic.oup.com/ajcp/article/126/4/530/1759842 by guest on 30 September 2021

DOI: 10.1309/JT5BE2L1FGG8P8Y6

Abstract FLT3 (CD135) is a member of the class III receptor tyro- FLT3 gene mutations, either internal tandem sine kinase family that is expressed by early hematopoietic duplication (ITD) or D835 point mutations, have been progenitors. It is composed of 5 immunoglobulin-like studied extensively in acute myeloid leukemia and domains in the extracellular region, a single transmembrane myelodysplastic syndrome (MDS). Little is known about sequence, and intracellularly, a short juxtamembrane portion FLT3 mutations in chronic myeloproliferative diseases followed by an interrupted kinase domain.1 (CMPDs) or their relationship with V617F JAK2 Two distinct types of activating mutations of the FLT3 mutations. We analyzed bone marrow samples from 142 gene are most common. An internal tandem duplication (ITD) patients with Philadelphia (Ph) chromosome– CMPD resulting from duplication of the juxtamembrane portion of or CMPD/MDS and from 119 patients with Ph+ the gene occurs in up to 20% of acute myeloid leukemia chronic myeloid leukemia (CML) using a multiplex (AML) and 3% of myelodysplastic syndrome (MDS) cases. A polymerase chain reaction assay. FLT3 mutations, 11 missense point mutation of codon 835, or less commonly ITD and 2 D835, were detected in 13 (9.2%) patients codon 836, involves the activation loop of the FLT3 protein. with CMPD or CMPD/MDS, 7 in blast phase and 6 in These point mutations, collectively referred to here as D835, chronic phase. Analyses for JAK2 mutations in 11 of 13 occur in up to 7% of AML and 3% of MDS cases. Both muta- cases were all negative. By contrast, no FLT3 mutations tions result in constitutive activation of FLT3 kinase by were detected in CML, including 108 chronic and 11 autophosphorylation.1,2 blast phase cases. FLT3 mutations occur in Although FLT3/ITD mutations can induce myeloprolifer- approximately 10% of CMPD and CMPD/MDS but are ative diseases in a transgenic mouse model,3 FLT3 mutations not observed in JAK2+ CMPD or in CML. have been demonstrated mainly in patients with AML or MDS.1,4-6 The mutations are more common in AML with monocytic differentiation, and their presence, especially ITD, is associated with marked leukocytosis and a high risk for relapse. FLT3 mutations also are implicated in leukemic transformation of MDS.1,7 Rare cases of acute lymphoblastic leukemia with FLT3 gene mutations reported are primarily cases associated with MLL gene alterations.8,9 Studies of FLT3 mutations in chronic myeloproliferative diseases (CMPDs) or CMPD/MDS, in blast or chronic phase, are extremely rare. Yokota and colleagues6 studied 20 patients with chronic myelogenous leukemia (CML) and found no mutations. In another study, Pardanani and colleagues10 found

530 Am J Clin Pathol 2006;126:530-533 © American Society for Clinical Pathology 530 DOI: 10.1309/JT5BE2L1FGG8P8Y6 Hematopathology / ORIGINAL ARTICLE no FLT3 mutations in 115 patients with a variety of CMPDs, CA), forward primers for ITD and D835 were labeled with a including 48 with myelofibrosis with myeloid metaplasia, 21 fluorescent dye, 6-carboxyfluorescein (FAM).16 The presence with polycythemia vera, 20 with essential thrombocythemia, of any PCR fragment larger than the wild-type allele was con- 16 with systemic mast cell disease, 4 with hypereosinophilic sidered positive for ITD. D835 PCR products were digested syndrome, 3 with chronic neutrophilic leukemia, and 3 with with the EcoRV restriction enzyme before capillary elec- myelodysplastic syndrome. trophoresis. The wild-type allele cut by this enzyme resulted

Mutation in codon 617 of the JH2 domain of Janus kinase in 2 fragments of 64 and 48 base pairs. In contrast, mutations 2 (JAK2) is the most common molecular abnormality found in at D835 alter the EcoRV recognition site and result in one Philadelphia chromosome (Ph)– CMPD, especially poly- 112-base-pair fragment. The sensitivity of these assays is cythemia vera (70%-80%), essential thrombocythemia (40%- approximately 2%, ie, 1 mutated cell in 50 total cells, as estab- 50%), and chronic idiopathic myelofibrosis (30%-50%), as lished by dilutional studies. Downloaded from https://academic.oup.com/ajcp/article/126/4/530/1759842 by guest on 30 September 2021 well as a small number of myelodysplastic syndromes and To detect V617F JAK2 gene mutations, DNA isolated atypical myeloid disorders (7%).11,12 The relationship from bone marrow aspirate and/or core biopsy specimens was between FLT3/ITD or FLT3/D835 and JAK2 mutation in Ph– amplified using forward primer 5'-TCT TTC TTT GAA GCA CMPD has been rarely explored. GCA AGT ATG AT-3' and biotin-labeled reverse primer 5'- To assess the prevalence of FLT3 mutations in CMPD or ACA AAA ACA GAT GCT CTG AGA AAG G-3'. A single- CMPD/MDS, we reviewed bone marrow aspirate samples stranded biotinylated template strand was prepared for obtained from patients with these diseases and analyzed for sequencing using the Pyrosequencing Vacuum Prep Tool FLT3 mutations by a polymerase chain reaction (PCR) assay (Biotage, Uppsala, Sweden). Pyrosequencing reactions were at our institution. All cases assessed were Ph–, confirmed by performed on a PSQ HS 96 instrument using a JAK2 sequenc- conventional cytogenetics and molecular studies. We also ana- ing primer, 5'-ATT ATG GAG TAT GT-3'.11,12,17 All primers lyzed the corresponding bone marrow samples of 11 patients were obtained from Invitrogen, Carlsbad, CA, and analyses with FLT3 mutation–positive CMPD or CMPD/MDS for the were performed using appropriate negative and positive con- JAK2 V617F mutation. For comparison, we assessed a large trol samples. The sensitivity of detection is approximately number of cases of Ph+ CML. In a subset of cases, we retro- 10%, ie, 1 mutated cell in 10 total cells. spectively analyzed archived, formalin-fixed, paraffin-embed- For archived, formalin-fixed, paraffin-embedded bone ded bone marrow clot samples acquired previously to deter- marrow clot samples, DNA was extracted using the QIAamp mine when the mutations became detectable during the course tissue kit (QIAGEN, Hilden, Germany) according to the man- of disease. ufacturer’s instructions. Subsequent analysis followed the same procedures described for fresh samples. In a few cases, fresh and corresponding archived samples were analyzed and compared to ensure quality. Materials and Methods The clinical history and pathologic diagnosis of each The files of the Molecular Diagnostics Laboratory, patient were reviewed independently and verified by 2 Department of Hematopathology, at our hospital were hematopathologists. Clinical data were obtained by review of searched for all CMPD cases that were analyzed for FLT3 medical records. The peripheral blood smears, Wright- gene mutations from March 1999 to August 2003. The World Giemsa–stained bone marrow aspirate smears, and H&E- Health Organization classification of hematopoietic neo- stained bone marrow biopsy sections of cases with FLT3 plasms was used for diagnosis,13 with unclassifiable cases mutations were reviewed. Conventional cytogenetic analysis showing myeloproliferative and dysplastic features diagnosed was performed on bone marrow aspirate material using meth- as CMPD/MDS. ods previously described.18 A multiplex fluorescent-based PCR method was used to detect ITD and D835 point mutations in FLT3. DNA was isolated from bone marrow aspirate samples by using a standard Results phenol-chloroform extraction method. The presence of ITD was assessed by amplification of the juxtamembrane domain From March 1999 to August 2003, 1,978 consecutive using primers from exons 11 and 12 as described by Kiyoi and bone marrow aspirate samples from 1,167 patients with a vari- Naoe.14 The presence of D835 point mutations was assessed ety of acute and chronic myeloid and lymphoid disorders were by a restriction fragment length polymorphism–mediated analyzed for FLT3 mutations. Among them, 165 samples were assay using primers flanking the mutation site.15 To facilitate obtained from 142 patients with Ph– CMPD or CMPD/MDS, the detection of PCR products by capillary electrophoresis on including chronic (n = 47) or blastic (n = 16) phase of CMPD a 3100 genetic analyzer (Applied Biosystems, Foster City, other than CML, blast (n = 34) or chronic (n = 39) phase of

© American Society for Clinical Pathology Am J Clin Pathol 2006;126:530-533 531 531 DOI: 10.1309/JT5BE2L1FGG8P8Y6 531 Lin et al / FLT3 IN CHRONIC MYELOPROLIFERATIVE DISORDERS chronic myelomonocytic leukemia (CMML), and samples from 5 cases obtained during the chronic phase CMPD/MDS, unclassifiable (n = 6). We also analyzed bone were analyzed for FLT3 mutations, 4 CMPD, unclassifiable marrow samples from 119 patients with CML in chronic or and 1 chronic idiopathic myelofibrosis. The number of sam- blast phase that were confirmed by conventional cytogenetic ples assessed ranged from 1 to 4 in each case, and the blast or molecular studies. Cases of CMML in blast phase were counts ranged from 1% to 8% per sample. FLT3/ITD mutation excluded from final analysis because it is difficult to distin- was detected in 2 CMPD, unclassifiable with 6% of blasts in guish underlying CMML from MDS. the bone marrow aspirate in each. Results were negative in FLT3 mutations were detected in 15 bone marrow aspi- other samples. rate specimens from 13 patients (9.2%) diagnosed with Ph– CMPD or CMPD/MDS. They were ITD in 11 cases and D835 point mutation in 2 cases. FLT3 mutations were not detected Discussion Downloaded from https://academic.oup.com/ajcp/article/126/4/530/1759842 by guest on 30 September 2021 in any of the 119 cases of CML, including 108 in chronic phase and 11 in blast phase. The present study demonstrates that FLT3 mutations Of the 13 patients with FLT3 mutations, 7 were in blast occur in chronic or blast phase of CMPD and CMPD/MDS phase and 6 were in chronic phase of disease. Among the 7 but are not seen in the JAK2-mutated CMPD groups. This patients in blast phase, the diagnoses before the onset of blast finding suggests that events leading to FLT3 mutations may be phase were as follows: CMPD, unclassifiable, 4; poly- distinct from JAK2 mutations. Detectable FLT3 mutations cythemia vera, 1; chronic idiopathic myelofibrosis, 1; and correlated with an increased number of blasts in cases of blast CMPD/MDS, unclassifiable, 1. FLT3 mutation analysis was transformation of CMPD or CMPD/MDS, suggesting that performed at the initial blast phase in 4 patients and during FLT3 mutations may contribute to disease progression, as in persistent blast phase that had lasted for 4, 6, and 7 months, cases of leukemic transformation of MDS.7 However, FLT3 respectively, in 3 other patients. The diagnoses of the 6 mutations also were detected in cases in the chronic phase of patients with chronic phase of disease were as follows: CMPD and CMPD/MDS, suggesting that FLT3 mutations CMML, 4; and CMPD, unclassifiable, 2. Of the 13 patients, 6 alone are insufficient to cause blast transformation. Others had previous chemotherapy. Characteristics of the patients are have found similarly that FLT3 mutations were not detectable detailed further in ❚Table 1❚. in 7 cases of blastic transformation of myeloproliferative dis- To determine whether FLT3 mutations were detectable ease with JAK2 mutation.19 during the chronic phase in CMPD or CMPD/MDS cases with Six patients in the present study group had received FLT3 mutations detected during the blast phase, 9 archived chemotherapy before the detection of FLT3 mutations, raising

❚Table 1❚ Clinicopathologic Features of 13 Patients With Blast or Chronic Phase of Myeloproliferative or Myeloproliferative/Myelodysplastic Disorders Associated With FLT3 Gene Mutations*

Mutations Duration of Archived Samples Diagnosis FLT3 JAK2 Disease (mo) BM Blasts (%) Prior Treatment Assessed for FLT3

CMPD-u, blastic (n = 4) ITD – 22 19 (persistent for 4 mo) Hydroxyurea, idarubicin, 2, both negative cytarabine ITD – 22 23 Hydroxyurea 1 positive† D835 – 9 28 None 1 positive‡ D835 ND 21 27 (persistent for 6 mo) Hydroxyurea, idarubicin, cytarabine 1, negative PV, blastic (n = 1) ITD – 96 88 None — CIMF, blastic (n = 1) ITD – 48 8 (persistent for 7 mo) Hydroxyurea, DCTER 4, all negative CMPD/MDS-u, blastic (n = 1) ITD – 5 82 None — CMML (n = 4) ITD – 2 8 None — ITD – 24 7 Hydroxyurea — ITD – 3 4 None — ITD – 9 3 None — CMPD-u (n = 2) ITD ND 21 3 Cytarabine, IFN-α 1, positive ITD – 48 1 None —

BM, bone marrow; CIMF, chronic idiopathic myelofibrosis; CMML, chronic myelomonocytic leukemia; CMPD, chronic myeloproliferative diseases; CMPD/MDS, chronic myeloproliferative/myelodysplastic disorders; DCTER, dexamethasone, cytarabine, thioguanine, etoposide, and daunorubicin; IFN, interferon; ITD, internal tandem duplication; ND, not done; PV, polycythemia vera; u, unclassifiable; –, negative. * Polymerase chain reaction analysis was performed during the persistent blast phase of diseases in 3 patients. † The archived sample was obtained 12 months prior to the blast phase and had 6% blasts in the aspirate. ‡ The archived sample was obtained 1 month prior to the blast phase and had 6% blasts in the aspirate.

532 Am J Clin Pathol 2006;126:530-533 © American Society for Clinical Pathology 532 DOI: 10.1309/JT5BE2L1FGG8P8Y6 Hematopathology / ORIGINAL ARTICLE the possibility that FLT3 mutations are acquired secondary to 7. Horiike S, Yokota S, Nakao M, et al. Tandem duplications therapy. Alternatively, the neoplastic clone carrying a FLT3 of the FLT3 receptor gene are associated with leukemic transformation of myelodysplasia. Leukemia. 1997;11:1442- mutation may exist before and is resistant to therapy. 1446. Regardless, identification of FLT3 mutations in these patients 8. Taketani T, Taki T, Sugita K, et al. FLT3 mutations in the indicates that some cases of CMPD or CMPD/MDS could be activation loop of tyrosine kinase domain are frequently found potential targets of FLT3 inhibitors.20 in infant ALL with MLL rearrangements and pediatric ALL with hyperdiploidy. Blood. 2004;103:1085-1088. FLT3 mutations were not detected in any of the 119 cases 9. Paietta E, Ferrando AA, Neuberg D, et al. Activating FLT3 of CML, a finding concordant with that reported by Yokota mutations in CD117/KIT+ T-cell acute lymphoblastic and colleagues,6 who found no FLT3 mutations in 20 cases of leukemias. Blood. 2004;104:558-560. CML assessed. The bcl-abl and FLT3 gene mutations seem to 10. Pardanani A, Reeder TL, Kimlinger TK, et al. Flt-3 and c-kit be mutually exclusive. mutation studies in a spectrum of chronic myeloid disorders including systemic mast cell disease. Leuk Res. 2003;27:739- Downloaded from https://academic.oup.com/ajcp/article/126/4/530/1759842 by guest on 30 September 2021 In summary, FLT3 mutations can be identified in chronic 742. and blast phases of CMPD and CMPD/MDS. CMPD cases 11. James C, Ugo V, Le Couedic JP, et al. A unique clonal JAK2 with FLT3 mutations do not carry concurrent JAK2 mutations. mutation leading to constitutive signaling causes FLT3 mutations also are distinctly absent in CML. FLT3 polycythaemia vera. 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