Letters to the Editor 216 Analysis of the exon 12 and 14 mutations of the JAK2 in Philadelphia -positive leukemia

Leukemia (2008) 22, 216; doi:10.1038/sj.leu.2404953; In conclusion, we could not identify any JAK2 gene mutations published online 13 September 2007 or deletions among 88 Ph þ CML and Ph þ AL patients.

M Inami1, H Yamaguchi1, S Hasegawa2, Y Mitamura1, 1 2 2 1 1 Previous studies have shown no correlation between transition F Kosaka , A Kobayashi , S Kimura , K Dan and K Inokuchi 1 of disease status for Philadelphia chromosome-positive (Ph þ ) Division of Hematology, Department of Internal Medicine, chronic myelogenous leukemia (CML) and JAK2V617F gene Nippon Medical School, 1-1-5 Sendagi, Bunkyo-Ku, mutation.1,2 Recently, several novel mutations as well as Tokyo, Japan and 2 deletion in exon 12 of the JAK2 gene in JAK2V617F-negative Sekino Clinical Pharmacology Clinic, 3-28-3 Ikebukuro, polycythemia vera3 and JAK2K607N mutation in acute myeloid Toshima-ku, Tokyo, Japan leukemia4 were reported. Because the JAK–STAT pathway plays E-mail: [email protected] an important role in the initiation and progression of Ph þ leukemia,5,6 we analyzed these novel mutations and deletion of the JAK2 gene among Ph þ CML patients and Ph þ acute leukemia (Ph þ AL). We examined 88 CML patients, three patients with Ph þ acute lymphocytic leukemia and two References patients of Ph þ acute biphenotypic leukemia patients. Bone marrow mononuclear cells were obtained from all patients with 1 Jelinek J, Oki Y, Gharibyan V, Bueso-Ramos C, Prchal JT, Verstovsek 4 a written informed consent. S et al. JAK2 mutation 1849G T is rare in acute leukemias but can be found in CMML, Philadelphia chromosome-negative CML, and Among the Ph þ CML cases, 72 patients were in the chronic megakaryocytic leukemia. Blood 2005; 106: 3370–3373. phase, five patients were in the accelerate phase and 11 were in 2 Kronenwett R, Graf T, Neumann F, Pechtel S, Steidl U, Diaz-Blanco E blast crisis (BC). Five Ph þ CML patients had additional et al. Absence of the JAK2 mutation V617F in CD34+ hematopoietic chromosome aberrations and three Ph þ CML patients had stem and progenitor cells from patients with BCR–ABL-positive CML double Ph þ . As for Ph þ CML treatment, 13 had in chronic phase and blast crisis. Leuk Res 2006; 30: 1323–1324. been treated with interferon-a before administration of imatinib 3 Scott LM, Tong W, Levine RL, Scott MA, Beer PA, Stratton MR et al. JAK2 exon 12 mutations in polycythemia vera and idiopathic and six patients were imatinib resistant. erythrocytosis. N Engl J Med 2007; 356: 459–468. V617F We first looked for the JAK2 gene mutation by allelic- 4 Lee JW, Kim YG, Soung YH, Han KJ, Kim SY, Rhim HS et al. The specific (AS-) PCR and direct sequencing as described in a JAK2 V617F mutation in de novo acute myelogenous leukemias. previous report.7 No patients with the JAK2V617F mutation were Oncogene 2006; 25: 1434–1436. identified by AS-PCR. Next, we looked for other mutations in 5 Xie S, Wang Y, Liu J, Sun T, Wilson MB, Smithgall TE et al. exons 12 and 14 of the JAK2 gene by direct sequencing.3,7 Neither Involvement of Jak2 tyrosine phosphorylation in BCR–ABL trans- formation. Oncogene 2001; 20: 6188–6195. mutations nor deletions in exons 12 and 14 of the JAK2 gene were 6 Klejman A, Schreiner SJ, Nieborowska-Skorska M, Slupianek A, detected in these Japanese Ph þ CML and Ph þ AL patients. Wilson M, Smithgall TE et al. The Src family kinase Hck couples The progression of CML to BC requires telomere shortening, BCR/ABL to STAT5 activation in myeloid leukemia cells. EMBO J genomic instability, differentiation arrest and loss of tumor 2002; 21: 5766–5774. suppressor, however, the JAK2 mutations might not be required 7 Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S for these processes. Even so, we think that sequencing of the et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 2005; 365: 1054–1061. entire JAK2 gene and analysis of the possible abnormalities in 8 Shuai K, Halpern J, ten Hoeve J, Rao X, Sawyers CL. Constitutive the JAK–STAT pathway may be necessary because BCR/ABL activation of STAT5 by the BCR–ABL oncogene in chronic directly regulates STAT5.8 myelogenous leukemia. Oncogene 1996; 13: 247–254.

A PDGFRB-positive acute myeloid malignancy with a new t(5;12)(q33;p13.3) involving the ERC1 gene

Leukemia (2008) 22, 216–218; doi:10.1038/sj.leu.2404894; karyotypic level. Here, we describe for the first time a new fusion, published online 9 August 2007 that is ERC1-PDGFRB in a case of acute myeloid leukemia (AML). To detect reliably t(5;12)(q33;p13)/ERC1-PDGFRB by fluores- cence in situ hybridization (FISH) and to differentiate this new Although PDGFRB fuses with different partners in myeloproli- fusion from the classical t(5;12)(q33;p13)/ETV6-PDGFRB,we ferative disorders/myelodysplastic syndromes (MPD/MDS), set up a specific double-color double-fusion assay using the ETV6-PDGFRB is the only fusion gene, which has been RP11-1087E18 clone for the ERC1 gene and the CTD-2601I11 recognized to date when a t(5;12)(q33;p13) is present at the clone for the PDGFRB gene (Figure 1).

Leukemia Letters to the Editor 217 A 36-year-old man was referred to the Division of Hemato- logy, Rome, in November 2006 because of a relapsed AML after an autologous stem cell transplantation (auto-SCT). The previous diagnosis of AML M5a had been made in 2002. The past history revealed a slight leukocytosis (14 Â 109/l) from October 2000. On admission, the peripheral blood count was: hemoglobin, 11.4 g/dl, Platelets 97 Â 109/l, white blood cells, 3.1 Â 109/l (polymorphonuclear cells 12%, lymphocytes 63%, monocytes 11%, Blast 14%). Liver and spleen were enlarged. The bone marrow aspirate was hypercellular with a diffuse infiltration of medium and large blasts with basophilic cytoplasm (71% of nucleated cells). Megakaryocytes were dysplastic. The karyo- type was 46,XY,t(5;12)(q33;p13.3)[9]/92,XXYY,t(5;12)(q33;p13.3) X2[12]. Given the 5q33 breakpoint we performed a double-color metaphase FISH with cosmid 9-4 for the 30-PDGFRB (orange) and cosmid 4-1 (green) for the 50-PDGFRB.1 FISH gave one orange/ green fusion signal on normal five, one orange signal on der(5) and one green signal on der(12), indicating the t(5;12) disrupted PDGFRB. Cosmid 179A6 (50-ETV6) and cosmid 148B6 (30-ETV6) gave two orange/green fusion signals on normal 12 and on der(12), indicating that the 12p13.3 breakpoint mapped telomeric to 1 Figure 1 Double-color double-fusion FISH with clone CTD- ETV6. 2601I11 (orange) and clone RP11-1087E18 (green) on bone marrow FISH with five cosmids for loci/ localized telomeric to sample of relapse: arrows indicate der(5) and der(12). ETV6, that is, cosmid 214C9 for D12S994, cosmid 135H12 for

Figure 2 Nucleotide and sequence of ERC1-PDGFRb.(a) ERC1-PDGFRb mRNA fused in frame, joining nucleotide 3021 (ERC1 exon 15) to nucleotide 1837 (PDGFRb exon 10). (b) ERC1-PDGFRb mRNA fused out-of frame, joining nucleotide 3021 (ERC1 exon 15) to nucleotide 2049 (PDGFRb exon 11). Sequence numbers refer to GenBank accession numbers NM_178040.1 for ERC1 and NM_002609.3 for PDGFRb.

Leukemia Letters to the Editor 218 D12S939, cosmid 139D4 for CACNL1A1, cosmid 152H1 for ERC1-PDGFRB cannot be differentiated from the ETV6-PDGFRB FGF6 and cosmid 147G1 for NOL1 narrowed the 12p13 rearrangement by conventional cytogenetics and that, as breakpoint to a 1 M region, between locus D12S939 and locus expected, this new PDGFRB recombination is sensitive to D12S994.2 We hypothesized that ERC1 (ELKS/RAB6-interacting/ treatment with imatinib. CAST family member 1) was a putative partner for PDGFRB since of all the genes mapping within this DNA segment, only ERC1 contains dimerization domains, which are a common Acknowledgements feature of all PDGFRB partners. FISH with four DNA clones RP11-359B12, RP4-773N5, RP11-1087E18 and RP11-73H11 We thank Dr Geraldine Boyd for assistance in the preparation of encompassing the ERC1 gene from telomere to centromere the manuscript. This study was partly supported by AIRC indicated that the breakpoint fell within clone RP11-1087E18.3 (Associazione Italiana Ricerca sul Cancro), MIUR (Ministero per Nested reverse transcriptase-PCR amplified an ERC1-PDGFRB l’Istruzione, l’Universita` e la Ricerca Scientifica), Fondazione fusion transcript. Patient RNA was extracted with Trizol Cassa di Risparmio and AULL (Associazione Umbra per la lotta (Invitrogen, Carlsbad, CA, USA) from a bone marrow sample contro le leucemie), Perugia and ‘‘Associazione Sergio Luciani’’, and retro-transcribed using the thermoscript RT-PCR system Fabriano, Italy. BC is supported by FIRC (Federazione Italiana (Invitrogen). The first amplification round was performed with Ricerca sul Cancro). Cosmids for PDGFRB, ETV6, and for the short primers ERC1_1756_ex7F (50-GTTGAAGGAGTCCTTGACTG arm of were kindly provided by Dr J Cools and -30) and PDGFRb_2393R (50-TAGATGGGTCCTCCTTTGGTG Dr P Marynen (Centre for Human Genetics, University of Leuven, -30) and the second with primers ERC1_2288_ex11F (50-TCTT Leuven, Belgium); RP11 clones belong to the Roswell Park Cancer CTCTGGCATCCTCAGG-30) and PDGFRBR1 (50-TAAGCATCTT Institute libraries (http://www.chori.org/BACPAC) and were kindly GACGGCCACT-30). The product was cloned in the pGEM-T provided by Dr Mariano Rocchi (DAPEG, University of Bari, Italy). Easy Vector System (Promega, Madison, WI, USA). Blast search The CTD-2601I11 clone belong to the Caltech library and is and sequence analysis showed that nucleotide 3021 (ERC1 exon distributed by Invitrogen. Fondazione Benilde Viotti. 15) was involved in an in-frame fusion with nucleotide 1837 P Gorello1,4, R La Starza1,4, L Brandimarte1, SM Trisolini2, (PDGFRb exon 10) (Figure 2a), and an out-of-frame fusion with V Pierini1, B Crescenzi1, MZ Limongi2, M Nanni2, E Belloni3, nucleotide 2049 (PDGFRb exon 11) (Figure 2b). C Tapinassi3, E Gerbino3, MF Martelli1, R Foa`2, G Meloni2, RT-PCR investigated the reciprocal PDGFRB-ERC1 using PG Pelicci3 and C Mecucci1 1 primers PDGFRb_1121ex5F (50-GTGAACGCAGTGCAGACTG Department of Hematology, University of Perugia, IBiT T-30) and ERC1_ex19_3780R, (50-ATCCAAAGCAGAAAGGCA Foundation, Fondazione IRCCS Biotecnologie nel Trapianto, 0 Perugia, Italy; GA-3 ) followed by nested PCR with primers PDGFRb_ 2 1799ex9F (50-CCGAACATCATCTGGTCTGC-30) and ERC1_ Department of Hematology, University ‘La Sapienza’, Rome, 0 0 Italy and ex19_3689R (5 -AGTGTTTGGTGCCAAGAAGG-3 ). We detected 3European Institute of Oncology and IFOM-IEO Campus, and sequenced a PDGFRB-ERC1 in-frame fusion transcript joining Milan, Italy nucleotide 1836 (PDGFRb exon 9) to nucleotide 3022 (ERC1 exon E-mail: [email protected] 16) (data not shown). 4These authors equally contributed to this work. ERC1 maps at 12p13.3, spans 500 kb, is ubiquitously expressed and is characterized by several coiled-coil domains. Interestingly, ERC1, H4(D10S170) and TPM3 are three PDGFRB References partners in myeloid malignancies that are also involved in human papillary thyroid carcinoma: ERC1 and H4(D10S170) 1 La Starza R, Specchia G, Cuneo A, Beacci D, Nozzoli C, Luciano L fuse with RET as a result of t(10;12)(q21;p13) and of inv(10) et al. The hypereosinophilic sindrome: fluorescence in situ (q11.2q21), producing the ERC1-RET and the H4(D10S170)-RET hybridization detects the del(4)(q12)-FIP1L1/PDGFRA but not autophosphorylated tyrosine kinase, respectively;4,5 TPM3 genomic rearrangements of other tyrosine kinases. Haematologica/ rearranges with the nearby neurotrophic tyrosine kinase THJ 2005; 90: 596–601. 6 2 Kucherlapati R, Marynen P, Turc-Carel C. Report of the Fourth receptor type 1 (NTRK1/1q23) gene. international workshop on human chromosome 12 mapping 1997. The efficacy of imatinib mesylate on PDGFRB-positive Cytogenet Cell Genet 1997; 78: 81–95. chronic myeloproliferative disorders was first reported by 3 http://www.ncbi.nlm.nih.gov/mapview/ (Build 36.2). Accessed Apperley et al.7 in four patients, including one patient with April 2007. aggressive disease with severe and diffuse skin localization who 4 Schwaller J, Anastasiadou E, Cain D, Kutok J, Woijski S, Williams IR failed to respond to corticosteroid, hydroxyurea and interferon et al. H4(D10S170), a gene frequently rearranged in papillarythy- roid carcinoma, is fused to the platelet-derived growth factor a. Since PDGFRB was involved in our patient, imatinib mesylate receptor b in atypical chronic myeloid leukemia with (400 mg/day) was administered for 2 weeks followed by t(5;10)(q33;q22). Blood 2001; 97: 3910–3918. chemotherapy.8 After induction chemotherapy imatinib was 5 Pierotti MA, Santoro M, Jenkins RB, Sozzi G, Bongarzone I, Grieco M restarted. At 30 days from the end of chemotherapy the patient et al. Characterization of an inversion on the long arm of chromosome was in hematological and FISH remission (as assessed with 10 juxtaposing D10S170 and RET and creating the oncogenic sequence genomic clones for PDGFRB). RT-PCR showed persistence of RET/PTC. Proc Natl Acad Sci USA 1992; 89: 1616–1620. 6 Huret JL. TPM3 (Tropomyosin 3). Atlas genet cytogenet oncol the ERC1-PDGFRB transcript, which progressively decreased haematol. August 2001 URL: http://www.infobiogen.fr/services/ under imatinib treatment but did not disappear. In April 2007, chromcancer/genes/TPM3ID225.html. the patient underwent a family unrelated SCT which has been 7 Apperley JF, Gardembas M, Melo JV, Russell-Jones R, Bain BJ, Baxter EJ followed at day þ 20, date of the first marrow monitoring by a et al. Response to imatinib mesylate in patients with chronic disappearance of the ERC1-PDGFRB transcript. myeloproliferative diseases with rearrangements of the platelet-derived b In conclusion we have identified a new partner of growth factor receptor . NEnglJMed2002; 347: 481–487. 8 Cardarelli L, Capria S, Trisolini SM, Cimino G, Diverio D, Guarini A PDGFRB, that is the ERC1 gene which has so far never et al. ID-Ara-C, idarubicin and mylotarg gentuzumab ozogamicin as been involved in hematological malignancies. Based on salvage treatment in advanced AML patients. Bone Marrow the results of this report we wish to highlight that the Transplant 2007; 39 (Suppl 1): S62.

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