(2012) 26, 2384–2389 & 2012 Macmillan Publishers Limited All rights reserved 0887-6924/12 www.nature.com/leu

ORIGINAL ARTICLE RET fusion are associated with chronic myelomonocytic leukemia and enhance monocytic differentiation

P Ballerini1,7, S Struski2,7, C Cresson3,7, N Prade2, S Toujani4, C Deswarte1, S Dobbelstein2, A Petit1, H Lapillonne1, E-F Gautier3, C Demur2, E Lippert5, P Pages1, V Mansat- De Mas2,3,6, J Donadieu1, F Huguet2, N Dastugue2, C Broccardo3,7, C Perot4,7 and E Delabesse2,3,6,7

Myeloproliferative neoplasms are frequently associated with aberrant constitutive (TK) activity resulting from chimaeric fusion genes or point such as BCR-ABL1 or JAK2 V617F. We report here the cloning and functional characterization of two novel fusion genes BCR-RET and FGFR1OP-RET in chronic myelomonocytic leukemia (CMML) cases generated by two balanced translocations t(10;22)(q11;q11) and t(6;10)(q27;q11), respectively. The two RET fusion genes leading to the aberrant activation of RET, are able to transform hematopoietic cells and skew the hematopoietic differentiation program towards the monocytic/macrophage lineage. The RET fusion genes seem to constitutively mimic the same signaling pathway as RAS mutations frequently involved in CMML. One patient was treated with Sorafenib, a specific inhibitor of the RET TK function, and demonstrated cytological and clinical remissions.

Leukemia (2012) 26, 2384–2389; doi:10.1038/leu.2012.109 Keywords: tyrosine kinase; sorafenib; CMML

INTRODUCTION It is the first report of the involvement of the RET TK in Hematopoietic myeloid malignant diseases are classified in three hematological malignancies. RET, initially identified as an broad categories according to the maturation potential of the in an NIH-3T3 transformation assay,3 encodes a 1114 leukemic cells, from the less mature to the more differentiated: amino-acid that belongs to the TK receptor sub-family. It acute myeloid (AML), myelodysplastic syndromes and binds glial-derived neurotrophic factor (GDNF) ligands associated myeloproliferative neoplasms (MPNs). Chronic myelomonocytic with co-receptors (GDNF-family a co-receptors; GFRa). Upon leukemias (CMML) are characterized by a proliferative disease ligand binding, the receptor dimerizes and its intracellular affecting both myeloid and monocytic lineages and by cytological tyrosine residues are phosphorylated leading to kinase signs of myelodysplasia. The 2008 World Health Organization activation.4 The direct implication of RET in was (WHO) classification has recognized its particularities by evidenced in papillary thyroid carcinoma, where somatic distinguishing CMML from myelodysplastic syndrome and rearrangements result in the fusion of its TK catalytic domain MPNs.1 CMML are defined by persistent peripheral blood with a N-terminal dimerization domain encoded by various monocytosis (41 G/L), absence of the BCR-ABL1 fusion , genes.5 More recently, fusion involving RET merged to KIF5B less than 20% of myeloblasts or monoblasts in the blood have been characterized in 1–2% of colorectal and lung or the bone marrow and evidence of dysplasia in myeloid carcinomas.6,7 Subsequently, point mutations were discovered in lineages. In the absence of dysplasia, acquired cytogenetic the majority of familial or sporadic cases of multiple endocrine abnormalities or persistent monocytosis without classical causes neoplasia syndromes 2A/2B (MEN2A/MEN2B).8 Either RET fusion are alternatives to these criteria. The usual age of presentation of or point mutations enhance the RET TK activity.4 CMML is in the range of 70 years. A closely related form of CMML Conversely, inactivating mutations lead to renal agenesis and is found in childhood and named juvenile myelomonocytic have been found in familial and sporadic cases of Hirschsprung’s leukemia.1 disease, a developmental disorder characterized by the absence of MPNs are frequently associated with a constitutive tyrosine enteric ganglia in the intestinal tract.9 kinase (TK) activity resulting from chimaeric fusion genes or point The two RET fusion genes described here leading to the mutations. Examples of such activations in MPN are BCR-ABL1 in aberrant activation of RET, are able to transform hematopoietic chronic myeloid leukemia or JAK2 V617F in polycythemia vera and cells and skew the hematopoietic differentiation program towards essential thrombocytemia.2 We report here the cloning and the monocytic/macrophage lineage. We also report that the functional characterization of two novel fusion genes BCR-RET patient associated with the BCR-RET fusion protein didn’t respond and FGFR1OP-RET in CMML cases generated by two balanced to but was sensitive to Sorafenib, an inhibitor of TK translocations t(10;22)(q11;q11) and t(6;10)(q27;q11), respectively. activity.

1Department of Hematology, Hoˆ pital Trousseau, Assistance Publique-Hoˆpitaux de Paris (APHP), Paris, France; 2Department of Hematology, Hoˆpital Purpan, Toulouse, France; 3Institut National de la Sante´ et de la Recherche Me´dicale U1037, Centre de Recherche sur le Cancer de Toulouse, Toulouse, France; 4Department of Cytogenetics, Hoˆ pital Saint- Antoine, Assistance Publique-Hoˆpitaux de Paris (APHP), Paris, France; 5Department of Hematology, CHU Bordeaux and INSERM U1035, Universite´ Victor Segalen, Bordeaux, France and 6CHU Toulouse and Universite´ Toulouse III Paul-Sabatier, Toulouse, France. Correspondence: Professor E Delabesse, Department of Hematology, Laboratoire d’He´matologie, Pavillon Lefebvre, Hoˆpital Purpan, Place du Docteur Baylac, TSA40031, Toulouse cedex 9, F-31059 France. E-mail: [email protected] 7These authors contributed equally to this work. Received 13 February 2012; revised 28 March 2012; accepted 30 March 2012; accepted article preview online 19 April 2012; advance online publication, 15 May 2012 RET fusions in chronic myelomonocytic leukemias P Ballerini et al 2385 MATERIALS AND METHODS EGFP-positive). Antibodies used recognized ABL1 (Merck-Calbiochem, Karyotype and fluorescence in situ hybridization (FISH) Darmstadt, Germany, #OP20), AKT (Cell Signaling, Saint Quentin en Yvelines, France, #9272), phospho-AKT (Ser473, Cell Signaling, #9271), b- Karyotypes were established from bone marrow aspiration after 24-h catenin (Becton-Dickinson, Le Pont de Claix, France, #610154), phospho-b- cultures. abnormalities were identified with RHG banding and GTG banding in case 1 and RHG banding in case 2. RET-specific probes catenin (Ser33/Ser37/Thr41, Cell Signaling, #9561), ERK1/2 (Cell Signaling, were prepared by labeling RET-specific fosmids from the WIBR-2 human #9102), diphosphorylated ERK1/2 (Sigma, M8159), STAT3 (Cell Signaling, fosmid library.10 Fosmid G248P84737G5 spanned from the 50/centromeric #9139), phospho-STAT3 (Tyr705, Cell Signaling, #9131), STAT5 (Cell region to RET intron 11 (GRCh37/hg19 assembly location Chr10: Signaling, #9363), phospho-STAT5 (Tyr694, Cell Signaling, #9351) and RET (Cell Signaling, #3223). 43 564 608–43 610 954) and was labeled with Spectrum Orange. Fosmid G248P82944G3 spanned from the end of RET intron 1 to 30/telomeric region of RET (GRCh37/hg19 assembly location Chr10: 43 591 944– 43 636 084) and was labeled with fluorescein isothiocyanate. Morphological and confocal immunofluorescence microscopy Infected BaF/3 cells were centrifuged onto glass coverslips for 3 min at RET RT-PCR 300 r.p.m. in a cytospin centrifuge. Rabbit anti–RET (1/100, Cell Signaling, #3223) or normal rabbit control IgG1 (1/100, eBiosciences, Paris, France) The specific RT-PCR of the FGFR1OP-RET fusion transcript in case 1 was antibodies were added for 3 h at room temperature. Fluorescence images performed using FGFR1OP Ex6-F (50-CATTCTCCACCAAAGTCACCA-30) and were taken using an upright laser scanner confocal microscope (Carl Zeiss RET_R2555 (50-CCTGGCTCCTCTTCACGTAG-30). The reverse fusion transcript LSM710, Le Pecq, France, Â 63 oil immersion objective). RET-FGFR1OP was looked for using RET_F1925 (50-CAACATTTGCCCTCAG- GACT-30) and FGFR1OP_R1206 (50-GCAAGGCACCAATAGAGAGC-30). The specific RT-PCR of the BCR-RET fusion transcript in case 2 was performed using four BCR forward primers (HsBCR_b1A: 50-GAAGTGTTTCAG RESULTS 0 0 0 AAGCTTCTCC-3 ; HsBCR_e1A: 5 -GACTGCAGCTCCAATGAGAAC-3 ; HsBCR_ Recurrent 10q11 chromosomal band translocations occur in CMML e6S2: 50-GACTTACCTGAGCCACCTGGAG-30 and HsBCR_c3A: 50-ACGGCGAG AGCACGGACA-30) and a RET reverse primer (HsRET_X12_R1: 50-CCAAG The rearrangement of the 10q11 chromosomal band was AACCAAGTTCTTCCGAG-30; HsRET_X14_R1: 50-GGAGCCGTATTTGGCGT identified in two independent CMML cases. Case 1 is a 12-week ACTC-30; HsRET_X16_R1: 50-GGGATTCAATTGCCATCCATT-30). infant who presented initially with melena and hepato-spleno- megaly. Blood test revealed a fluctuant hyperleucocytosis RET expression quantification and TK domain resequencing (49–88 G/L) with monocytosis (20–24%), myelemia, , Quantitative RT-PCR of RET was performed according to the EAC (Europe thrombocytopenia (34 G/L) and anemia (Hb 8.8 g/dl). The bone Against Cancer) settings.11 Taqman set primers, located between exons 19 marrow aspiration identified 12% of monocytes and less than 2% and 20, were HsRET_121F1 (50-GGAGACACCGCTGGTGGA-30) and monoblastic cells. A diagnosis of juvenile myelomonocytic HsRET_253R1 (50-AGCTCTCGTGAGTGGTACAGGACT-30). Taqman probe leukemia was established. The karyotype identified a 0 0 was HsRET_204_P1 (5 -TGTCAGACCCGAACTGGCCTGGA-3 ). ABL1 was t(6;10)(q27;q11) balanced translocation (Supplementary used as ubiquitous control gene and quantified using the EAC Taqman Figure 1A). At 2 months of follow-up, a femoral granulocytic set.11 RET quantification was performed in 98 AML, 31 CMML, 2 normal peripheral blood and 3 normal bone marrow samples. RET TK domain was sarcoma occurred with a massive osteolytic infiltration by a resequenced in complementary DNA of samples expressing RET at a RET/ monocytic population with the same translocation ABL1 ratio higher than 0.1 using HsRET_TKDomain_F2 (50-TTCC t(6;10)(q27;q11) associated to additional abnormalities, specially CGGTCAGCTACTCCTCT-30) and HsRET_TKDomain_R2 (50-GCATGTTT a triplication of the derivative : 53,XY, þ 2,6, CTTCCCCTTGTGA-30). t(6;10)(q27;q11), þ der(6)t(6;10)x2, þ 7, þ 7, þ 8, þ 13, þ 19. Case 2 is a 69-year-old male, whose disease was revealed by a Retroviral production splenomegaly of 13 cm associated with a hyperleucocytosis Full-length RET fusion transcripts were amplified using Advantage 2 (63.7 G/L) along with myelemia (35%), monocytosis (3.2 G/L) and Polymerase Mix (Clontech, Saint-Germain-en-Laye, France) with BCR_ thrombocytosis (527 G/L). The bone marrow aspiration revealed a 742U21_SI (50-AGTACTAGTACTCGGCCGCGCCATGGTGGACCC-30) and RET_ hyperplasia with 1% of myeloblast and 4% of promyelocytic cells. 3817L30_BI (50-GGATCCGGATCCAAAAACGAGTCTGCCACCTGGGAACTGA-30) 0 A diagnosis of CMML1 was established. The karyotype identified a for BCR-RET and FGFR1OP_200U25_SI (5 -AGTACTAGTACTCGCGGCTTCGGC t(10;22)(q11;q11) balanced translocation with a loss of chromo- GGTTGTCTTGGAG-30) and RET_3817L30_BI for FGFR1OP-RET andclonedinto the pMSCV-IRES-EGFP (MIE) retroviral vector. MIE-BCR-ABL1P210 was kindly some Y (Supplementary Figure 1B). provided by Ali Turhan (Poitiers, France). Retroviruses were prepared using the packaging cell line Phoenix-Eco. Retroviral supernatant was collected and filtered through a 0.45-mmfilter. The RET TK gene is the target of 10q11 translocations The 6q27 chromosomal band, involved in case 1, is a recurrent Lineage-negative cells transduction breakpoint region in MPN involving FGFR1OP already reported in fusion with the FGFR1 gene.14 FISH analysis confirmed its Bone marrow cells were harvested from 6- to 12-week-old C57BL/6 mice. Lineage-negative (Lin À ) cells were obtained after separation using the involvement in the t(6;10)(q27;q11) translocation (data not shown). Lineage Cell Depletion Kit (Miltenyi Biotec, Paris, France), according to the In the 10q11 chromosomal band, RET gene was thought to be a manufacturer’s instructions. Lin À cells were infected with MIE, MIE-BCR- good candidate, as being the unique gene encoding a TK located in RET, MIE-FGFR1OP-RET or MIE-BCR-ABL1P210 as previously described.12 Serial this region. The breakpoint in the RET gene was confirmed by FISH replating was performed as described by Lavau et al.13 analysis (Supplementary Figure 1C). The FGFR1OP-RET fusion transcript was amplified by RT-PCR (Supplementary Figure 1D). This BaF/3 cell line transformation assay product merged FGFR1OP exon 12 with RET exon 12 MIE-BCR-RET, MIE-FGFR1OP-RET and MIE-BCR-ABL1P210 retroviruses were (Supplementary Figure 1G). The reverse RET-FGFR1OP fusion transduced in the BaF/3 cell line regarding its dependency to IL3 for transcript was not detected in case 1 (data not shown). growth. Three independent infections were performed for each fusion The BCR gene is located in the 22q11 chromosomal band and is gene. Cells were recovered in IL3-containing medium for 48 h and then frequently involved in chronic myeloproliferative disease. FISH plated out in presence or absence of 10 ng/ml of IL3 (Peprotech, Neuilly sur analysis confirmed its involvement in the t(10;22)(q11;q11) Seine, France). Cells were counted daily for 4 days. translocation (data not shown). The breakpoint in RET gene was also confirmed by FISH analysis (Supplementary Figure 1E). The Western blot BCR-RET fusion transcript was amplified by RT-PCR (Supplementary Infected BaF/3 cells cultured with IL3 were sorted using the EGFP Figure 1F) and sequenced. This product merges the BCR exon 4 fluorescence (infected cells cultured in absence of IL3 were already 100% with the same RET exon 12 as in case 1 (Supplementary Figure 1H).

& 2012 Macmillan Publishers Limited Leukemia (2012) 2384 – 2389 RET fusions in chronic myelomonocytic leukemias P Ballerini et al 2386 RET gene is expressed in myeloid neoplasias continuous increase of white blood cells (Figure 1). After identifica- TKs are often activated in hematological malignancies by forced tion of the fusion involving the RET TK gene, the patient was treated dimerization due to the juxtaposition of kinase domain and with Sorafenib (Nexavar, Bayer, Lyon, France). This drug was selected dimerization motifs brought by the fusion partner (such as BCR or according to its in-vitro inhibition of TK activity against RET and its 15,16 FGFR1OP). Furthermore, these fusions often result in an aberrant commercial availability (Supplementary Table 1). Sorafenib was expression of the kinase domain, as the regulatory elements are initially provided at a dose of 800 mg/day, displaying a major clinical brought by the fusion’s 50 partner gene. We then checked the (reduction of the spleen enlargement) and hematological expression of RET in normal peripheral blood and bone marrow, in (normalization of the white blood count) remission (Figure 1). 98 AML and 31 CMML cases by quantitative PCR including case 2. However, no cytogenetic and molecular remissions were achieved Case 2 expressed RET at a high level (RET/ABL1 ratio 0.55) (Figure 1). The dose of Sorafenib was progressively reduced due to compared with the normal counterparts (RET/ABL1 ratio: 0.004 for intolerance (hair loss; weight decrease) to 400 mg/day and later to blood and 0.006 for bone marrow). AML and CMML cases 200 mg/day. The patient is still in complete clinical remission after 27 expressed RET at a lower level than case 2 (RET/ABL1 ratio range: months of treatment. 0.33–0.0001, Supplementary Figure 2). We sequenced the TK Case 1 was rapidly allografted in complete remission. The blood catalytic domain of 6 AML and 7 CMML patients expressing RET at count normalized. He is currently in complete remission with a RET/ABL1 ratio higher than 0.1, but detected no of the chronic GVHD at 3 years of follow-up. TK domain (data not shown). RET fusion genes confer independence of IL3 to the BaF/3 cell line Patient carrying the BCR-RET fusion gene is insensitive to Imatinib BCR-RET and FGFR1OP-RET complementary DNA were expressed in but sensitive to Sorafenib BaF/3 cell line using retroviral vectors and compared with the After the diagnosis of case 2, the white blood cell count increased effect of BCR-ABL1P210 in a transformation assay regarding its rapidly (Figure 1). The patient was initially treated using Imatinib dependency of IL3 for growth. In presence of IL3, no difference of 400 mg per day (Gleevec, Novartis, Rueil-Malmaison, France) growth was observed between mutants compared with the non- during 2 months. This treatment was discontinued owing to the transduced cells or infected cells by the MIE retrovirus (Figure 2). In contrast, withdrawal of IL3 was followed by continuous growth of BaF/3 infected by MIE-BCR-RET and MIE-FGFR1OP-RET retro- viruses in a similar way to MIE-BCR-ABL1P210. As controls, the uninfected BaF/3 cells or infected with the MIE vector died rapidly in absence of IL3 (Figure 2).

RET fusion proteins signaling is different from BCR-ABL1 BCR-RET and FGFR1OP-RET cellular locations were investigated by confocal analysis in BaF/3-transduced cells. Both RET fusion gene products are located in the cytoplasm (Supplementary Figure 3) as BCR-ABL1.17 Canonical pathways involved in transformation were analyzed by western blot analyses. BCR-RET and FGFR1OP-RET enhanced ERK phosphorylation in contrast to BCR-ABL1P210. BCR- RET is specifically associated with the phosphorylations of STAT3 and AKT after withdrawal of IL3. The STAT5 and b-catenin pathways were not modified by any of the RET fusion proteins Figure 1. Clinical response of BCR-RET to Imatinib and Sorafenib. (Supplementary Figure 4). White blood cell count in G/L evolution of case 2 from the initial diagnosis according to treatment: Imatinib at 400 mg/day (I) then Sorafenib at 800 mg/day (S8). The dose of Sorafenib was reduced RET fusion genes skew differentiation to the monocytic/ due to toxicity to 400 mg/day (S4) and 200 mg/day (S2). Number of macrophage lineage mitoses, normal or containing the t(10;22) translocation, are To assess the impact of the two RET fusion genes regarding indicated at each time point where the karyotype was performed. the hematopoietic differentiation, we infected primitive

Figure 2. RET fusion genes render BaF/3 cell line independent to interleukin 3. Number of BaF/3 cells after infection by retroviruses expressing EGFP (MIE), BCR-ABL1P210, BCR-RET or FGFR1OP-RET fusion genes compared with the absence of infection (NT). BaF/3 cells were cultured in presence (left panel) or absence (right panel) of IL3. The mean values of three independent infections for each group are shown. ***Po0.001 (two-way analysis of variance).

Leukemia (2012) 2384 – 2389 & 2012 Macmillan Publishers Limited RET fusions in chronic myelomonocytic leukemias P Ballerini et al 2387

Figure 3. Lineage-negative transduced cells differentiation. Lin À cells were infected by EGFP (MIE), BCR-ABL1P210, BCR-RET or FGFR1OP-RET retroviruses. (a) Proportion of the different types of colonies obtained after plating 104 infected Lin À cells in methylcellulose-containing appropriated . After 7 days, colonies were identified and numbered: BFU-E, burst forming unit-erythroblast; colony-forming unit (CFU)-GEMM colonies containing at least three different types of cells; -M, macrophages; -G, granulocytes; -GM, granulocytes-macrophages. (b) Percentage of butyrate esterase-positive cells in cells retrieved from the methylcellulose 7 days after the first plating. (c) Serial replating of Lin À transduced cells. A fraction of cells was replated every 7 days and colonies were numbered. lineage-negative hematopoietic cells with MIE-BCR-RET, MIE- found in AML where the structure of RET was investigated by FGFR1OP-RET, MIE-BCR-ABL1P210 or MIE retroviruses. RET fusions Southern blot and denaturing gradient gel electrophoresis in 17 skewed hematopoietic differentiation increasing the percentage patients.22 of monocyte/macrophage colonies (CFU-M) in methylcellulose The two RET fusion genes described here merge exon 12 of RET cultures (Figure 3a). Cells were then recovered from the in 30 with BCR exon 4 or FGFR1OP exon 12 in 50. The 50 part of the methylcellulose. Morphology and butyrate esterase staining fusion genes encodes dimerization domains, in a similar way to all revealed that the percentage of monocytes/macrophages cells is the fusion gene partners reported in papillary thyroid, lung and also higher in RET fusion gene-infected cells compared with the colorectal carcinomas associated with RET rearrangements (PTC1 MIE or MIE-BCR-ABL1P210 retroviruses (Figure 3b and [CCDC6], PTC3 [NCOA4], PTC5 [GOLGA5], PTC6 [TRIM24], PTC7 Supplementary Figure 5). [TRIM33], PTC8 [KTN1], PCM1, ELKS and KIF5B5–7). Murine bone marrow cells have a limited ability to grow after All RET fusion genes identified so far therefore contain the several replatings in methylcellulose cultures in presence of entire TK domain of RET and lose its signal sequence, extracellular cytokines.13 In a similar way to BCR-ABL1,18 the two RET fusion ligand-binding and intracellular juxta-membrane domains. The genes do not confer to the infected cells the ability of forming latter domain has a negative effect on the RET signaling.23 Both colonies after the third replating (Figure 3c). BCR and FGFR1OP domains kept in the fusion proteins have dimerization properties.14,24 Consequently, the oncogenic activity of RET fusion proteins could be a combination of a constitutive DISCUSSION dimerization, higher expression and loss of negative regulatory We report here the first involvement of the RET TK in elements. To assess the oncogenicity of FGFR1OP-RET and BCR- hematological malignancies, rearranged by translocation with RET fusion gene products, we performed a BaF/3 transformation two different fusion partners, BCR or FGFR1OP. These events have assay. In a similar way to BCR-ABL1, both RET fusion gene products similar molecular consequences, the fusion of the RET TK domain conferred independence to IL3 to BaF/3 cells. Furthermore, RET with dimerization domains. RET is located in the 10q11 fusion proteins skewed the differentiation of lineage-negative chromosomal band. One CMML case with a t(6;10)(q27;q11), hematopoietic progenitors to the monocytic lineage. Nonetheless, a translocation identical to the case 1, has been reported in a these infected cells were unable to serially replate as BCR-ABL1.18 41-year-old man suffering from an atypical CMML syndrome These results suggested that the RET fusions are able to transform associated with myelofibrosis. The disease rapidly evolved to progenitors already engaged in the myelomonocytic acute monoblastic leukemia (M5a).19 In addition, the 10q11 differentiation. chromosomal band has been implicated in additional cases of RET is physiologically expressed by normal granulocytes, monocytic AML,20,21 suggesting that RET TK activation is a rare but monocytes and macrophages.25 RET is also expressed in AML recurrent event in myelomonocytic leukemias, in a similar way to and CMML as shown by Gattei et al.25 and confirmed in our study, the activation of RET in 1– 2% of lung and colorectal .6,7 We the expression being higher in monocytic and myelomonocytic also investigated the TK domain sequence of RET in 6 AML and 7 AML and the most mature stages. In contrast, its expression is CMML patients expressing RET at the highest levels without absent or weak in the other hematopoietic cell lineages (CD34 þ detecting any point mutation in this region. A similar result was progenitors, eosinophils, B and T cells).25 RET fusion proteins

& 2012 Macmillan Publishers Limited Leukemia (2012) 2384 – 2389 RET fusions in chronic myelomonocytic leukemias P Ballerini et al 2388 activated the phosphorylation of ERK, STAT3 and AKT proteins. 7 Lipson D, Capelletti M, Yelensky R, Otto G, Parker A, Jarosz M et al. Identification of The normal RET protein has been found to activate also ERK and new ALK and RET gene fusions from colorectal and lung cancer biopsies. Nat Med AKT through a phosphorylation of its tyrosine residue 1062 and a 2012; 18: 382–384. subsequent activation of RAS pathway.26–28 Phosphorylation of 8 Marsh DJ, Mulligan LM, Eng C. RET proto-oncogene mutations in multiple the tyrosine 705 of STAT3 and therefore its activation has also endocrine neoplasia type 2 and medullary thyroid carcinoma. Horm Res 1997; 47: been associated with RET fusion genes found in thyroid 168–178. carcinomas and was shown to be independent from JAK TKs.29 9 Skinner MA, Safford SD, Reeves JG, Jackson ME, Freemerman AJ. Renal Case 1 was allografted rapidly after its diagnosis. Case 2 was aplasia in humans is associated with RET mutations. Am J Hum Genet 2008; 82: 344–351. treated initially using Imatinib but the white blood cells count 10 International Sequencing Consortium. Finishing the euchromatic continued to increase suggesting an inefficacity of this TK sequence of the human genome. Nature 2004; 431: 931–945. inhibitor. After identification of the TK involved in the 11 Beillard E, Pallisgaard N, van der Velden VH, Bi W, Dee R, van der Schoot E et al. t(10;22)(q11;q11) rearrangement, the patient’s treatment was Evaluation of candidate control genes for diagnosis and residual disease detec- switched to Sorafenib (Nexavar). Sorafenib was associated with tion in leukemic patients using ’real-time’ quantitative reverse-transcriptase complete hematological and clinical responses without karyotypic polymerase chain reaction (RQ-PCR) - a Europe against cancer program. Leukemia or molecular responses. A strong effect of Sorafenib against the 2003; 17: 2474–2486. 15 12 Quelen C, Lippert E, Struski S, Demur C, Soler G, Prade N et al. Identification of a fusion gene PTC3-RET was demonstrated with an IC50 of 47 nM. A clinical effect of Sorafenib was also demonstrated in a phase II transforming MYB-GATA1 fusion gene in acute basophilic leukemia: a new entity in male infants. Blood 2011; 117: 5719–5722. trial in patients with advanced medullary thyroid carcinomas 13 Lavau C, Szilvassy SJ, Slany R, Cleary ML. Immortalization and leukemic transfor- where patients received Sorafenib 400 mg orally twice daily. Of 16 mation of a myelomonocytic precursor by retrovirally transduced HRX-ENL. patients with sporadic RET mutations, 1 achieved partial response EMBO J 1997; 16: 4226–4237. 30 and 14 had stable disease. 14 Popovici C, Zhang B, Gregoire MJ, Jonveaux P, Lafage-Pochitaloff M, Birnbaum D In conclusion, CMML is a hematopoietic malignancy associated et al. The t(6;8)(q27;p11) translocation in a stem cell myeloproliferative disorder with the frequent activation of the RAS pathway, being mutated in fuses a novel gene, FOP, to fibroblast 1. Blood 1999; 93: 30% of CMML cases.31 The RET fusion genes identified here seem 1381–1389. to constitutively mimic the same signaling pathway than RAS 15 Carlomagno F, Anaganti S, Guida T, Salvatore G, Troncone G, Wilhelm SM et al. mutations.32,33 In addition, the impact of the RET fusion genes on BAY 43-9006 inhibition of oncogenic RET mutants. J Natl Cancer Inst 2006; 98: 326–334. the monocytic lineage questions the role of the normal RET TK 16 Karaman MW, Herrgard S, Treiber DK, Gallant P, Atteridge CE, Campbell BT et al. A activity during the physiological monocytic differentiation. Finally, quantitative analysis of kinase inhibitor selectivity. Nat Biotechnol 2008; 26: Sorafenib has a direct effect over the RET fusion protein occurring 127–132. in CMML. 17 Hantschel O, Wiesner S, Guttler T, Mackereth CD, Rix LL, Mikes Z et al. Structural basis for the cytoskeletal association of Bcr-Abl/c-Abl. Mol Cell 2005; 19: 461–473. 18 Huntly BJ, Shigematsu H, Deguchi K, Lee BH, Mizuno S, Duclos N et al. MOZ-TIF2, CONFLICT OF INTEREST but not BCR-ABL, confers properties of leukemic stem cells to committed murine hematopoietic progenitors. Cancer Cell 2004; 6: 587–596. The authors declare no conflict of interest. 19 Cox MC, Panetta P, Venditti A, Abruzzese E, Del Poeta G, Cantonetti M et al. New reciprocal translocation t(6;10) (q27;q11) associated with idiopathic myelofibrosis and eosinophilia. Leuk Res 2001; 25: 349–351. ACKNOWLEDGEMENTS 20 Ackland SP, Westbrook CA, Diaz MO, Le Beau MM, Rowley JD. Evidence favoring We are grateful for the help provided by Association Laurette-Fugain and Association lineage fidelity in acute nonlymphocytic leukemia: absence of immunoglobulin de Recherche des Maladies du Sang de l’Enfant (ARMHE). CB was supported by grants gene rearrangements in FAB types M4 and M5. Blood 1987; 69: 87–91. from Institut National du Cancer (INCa) and Association pour la Recherche contre le 21 Cuneo A, Ferrant A, Michaux JL, Boogaerts M, Demuynck H, Bosly A et al. Clinical Cancer (ARC). ED, CB and CC were supported by the CITTIL (Cooperacion de review on features and cytogenetic patterns in adult acute myeloid leukemia with investigacion transpirenaica en la terapia innovadora de la leucemia), a POCTEFA lymphoid markers. Leuk Lymphoma 1993; 9: 285–291. grant. We thank Franc¸oise Pflumio, Fre´de´ric Pont and Jean-Jacques Fournie´ for 22 Visser M, Hofstra RM, Stulp RP, Wu Y, Buys CH, Willemze R et al. Absence helpful discussions. 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