Letters to the Editor 1052 the secondary malignancies and cardiovascular events, may not 2 Aoudjhane M, Labopin M, Gorin NC, Shimoni A, Ruutu T, Kolb HJ directly relate to SCT itself. et al. Comparative outcome of reduced intensity and Long-term follow-up should also consider issues of quality of myeloablative conditioning regimen in HLA identical sibling life (QOL). This initial analysis shows that chronic graft-versus- allogeneic haematopoietic stem cell transplantation for patients older than 50 years of age with acute myeloblastic leukaemia: a host disease rates and the duration of required immune retrospective survey from the Acute Leukemia Working Party suppression were lower after RIC. Chronic graft-versus-host (ALWP) of the European group for Blood and Marrow Transplanta- disease is a dominant factor determining QOL, suggesting better tion (EBMT). Leukemia 2005; 19: 2304–2312. long-term QOL with RIC; however, formal QOL assessment is 3 Martino R, Iacobelli S, Brand R, Jansen T, van Biezen A, required to determine this question. Finke J et al. Retrospective comparison of reduced-intensity The major limitation of this study is the nonrandomized conditioning and conventional high-dose conditioning for allo- geneic hematopoietic stem cell transplantation using HLA- comparison and the selection biases in allocating patients to one identical sibling donors in myelodysplastic syndromes. Blood of the regimens. However, because patients were selected for 2006; 108: 836–846. RIC based on high risk for non-relapse mortality, and because 4 Ringde´n O, Labopin M, Ehninger G, Niederwieser D, Olsson R, OS was similar among these patients given RIC and eligible Basara N et al. Reduced intensity conditioning compared with patients given MAC, it is possible that results in standard risk myeloablative conditioning using unrelated donor transplants in patients may be similar. Randomized studies, for patients in CR patients with acute myeloid leukemia. J Clin Oncol 2009; 27: 4570– 4577. (preferentially CR1) with long-term follow-up and assessment of 5 Shimoni A, Hardan I, Shem-Tov N, Yeshurun M, Yerushalmi R, late complication and QOL, will be needed to determine the Avigdor A et al. Allogeneic hematopoietic stem-cell transplantation best approach in this setting. MAC and possibly the new in AML and MDS using myeloablative versus reduced-intensity reduced toxicity myeloablative regimens are still the best conditioning: the role of dose intensity. Leukemia 2006; 20: 322– approach in patients with active disease. 328. 6 Socie´ G, Stone JV, Wingard JR, Weisdorf D, Henslee-Downey PJ, Bredeson C et al. Long-term survival and late deaths after allogeneic Conflict of interest bone marrow transplantation. Late Effects Working Committee of the International Bone Marrow Transplant Registry. N Engl J Med 1999; 341: 14–21. The authors declare no conflict of interest. 7 Mohty M, de Lavallade H, El-Cheikh J, Ladaique P, Faucher C, Fu¨rst S et al. Reduced intensity conditioning allogeneic stem cell A Shimoni, I Hardan, N Shem-Tov, R Yerushalmi transplantation for patients with acute myeloid leukemia: long term and A Nagler results of a ‘donor’ versus ‘no donor’ comparison. Leukemia 2009; The Division of Hematology and Bone Marrow 23: 194–196. Transplantation, Chaim Sheba Medical Center, 8 Valca´rcel D, Martino R, Caballero D, Martin J, Ferra C, Nieto JB Tel-Hashomer, Israel et al. Sustained remissions of high-risk acute myeloid leukemia and E-mail: [email protected] myelodysplastic syndrome after reduced-intensity conditioning allogeneic hematopoietic transplantation: chronic graft-versus-host disease is the strongest factor improving survival. J Clin Oncol 2008; 26: 577–584. References 9 Hegenbart U, Niederwieser D, Sandmaier BM, Maris MB, Shizuru JA, Greinix H et al. Treatment for acute myelogenous leukemia 1 Blaise D, Vey N, Faucher C, Mohty M. Current status of reduced- by low-dose, total-body, irradiation-based conditioning and hemato- intensity-conditioning allogeneic stem cell transplantation for acute poietic cell transplantation from related and unrelated donors. myeloid leukemia. Haematologica 2007; 92: 533–541. J Clin Oncol 2006; 24: 444–453.

KANK1, a candidate tumor suppressor , is fused to PDGFRB in an imatinib-responsive myeloid neoplasm with severe thrombocythemia

Leukemia (2010) 24, 1052–1055; doi:10.1038/leu.2010.13 of myeloproliferative neoplasm, most likely essential thrombo- published online 18 February 2010 cythemia. There was no detectable BCR–ABL transcript or JAK2 V617F mutation, which is present in about 50% of patients with essential thrombocythemia. The karyotype performed on bone The majority of myeloproliferative neoplasms are associated marrow cells showed 46,XY,t(5;9)(q31B32;p22B?24.3) in 16 with activating mutations in tyrosine kinase .1 of 18 metaphases (Figure 1a). Treatment with hydroxyurea was About 20 different fusion products of the PDGFRB gene, which started, but elicited no response. After two-and-a-half months of encodes platelet-derived growth factor receptor-b (PDGFRb), treatment, the platelet count had increased to 1400 Â 109/l and have been described in patients with myeloid neoplasms the neutrophil and eosinophil counts to 13 Â 109/l and associated with eosinophilia.1,2 Myelodysplastic features 0.9 Â 109/l, respectively. The patient experienced a transient and thrombocytopenia are often associated with PDGFRB ischemic attack that was attributed to the thrombocythemia. translocations.3 Fluorescence in situ hybridization analysis established the Here, we describe a new acquired t(5;9) chromosomal disruption of the PDGFRB locus. As this tyrosine kinase receptor translocation in a 67-year-old male patient who presented with is highly sensitive to imatinib mesylate,2,3 the patient was thrombocythemia (platelets, 904 Â 109/l) without prominent treated with a low dose of this drug (100 mg/day). At 4 months eosinophilia or neutrophilia. Bone marrow smears showed an after the initiation of this treatment, platelet and leukocyte increased number of polymorphic megakaryocytes without any counts were back to normal levels (Figure 1b). After 12 months blast cells or dysplastic features. Morphology was suggestive of follow-up, the patient remains in complete hematological

Leukemia Letters to the Editor 1053

Figure 1 The t(5;9) translocation implicates KANK1 as a new fusion partner to PDGFRB in a patient with imatinib-sensitive thrombocythemia. (a) G-banded partial karyotype of the patient showing the t(5;9). (b) Evaluation of the patient’s platelet counts (continuous line) and white blood cell counts (dashed line) as a function of time. Arrows indicate when the treatments were initiated (HU, hydroxyurea). (c) The RNA and protein sequences of the KPb breakpoint are shown. (d)KPb expression was analyzed by nested RT-PCR in cDNA derived from bone marrow samples from the patient with the t(5;9)-positive myeloid neoplasm (lane 2), or from patients with chronic myelogenous leukemia (lanes 3 and 4) or with acute lymphoblastic leukemia (lane 5). (e) A schematic representation of KANK1, PDGFRb and KANK1–PDGFRb (KPb) is shown. cc, coiled-coil domain; A, domain; octagons in PDGFRb represent Ig-like domain; TM, transmembrane domain; and Kinase, kinase domain. The position of the breakpoint is indicated by arrowheads. Localization of the two primer sets used in nested PCR (d) is represented by arrows.

remission. The response to low-dose imatinib pointed to could be cultured for several weeks in the absence of IL-3 (data constitutive activation of PDGFRb as the cause of thrombo- not shown), indicating that KPb is able to transform hemato- cythemia in this patient. poietic cells. To evaluate KPb sensitivity to imatinib mesylate, Using rapid amplification of cDNA ends PCR, we identified Ba/F3–KPb cells were exposed to increasing concentrations of the KANK1 gene (also called ANKRD15) on 9 as the drug, which inhibited cell growth in the absence of IL-3 the fusion partner of PDGFRB (Figure 1c). The fusion was (Figure 2b). The estimated IC50 for KPb was around 7 nM, which confirmed by fluorescence in situ hybridization using specific corresponded to the reported values for ETV6–PDGFRb and KANK1 probes (Supplementary Figure S1) and by nested PCR FIP1L1–PDGFRa inhibition, and is about two orders of (Figure 1d), as described in Supplementary Materials and magnitude lower than the concentration required to inhibit methods. The translocation fuses exon 2 of KANK1 BCR–ABL.2 (ENST00000382293, Ensembl database, up to nucleotide Autophosphorylation of tyrosine residues in the intracellular 3020) to exon 9 of PDGFRB (ENST00000261799, from part of receptor tyrosine kinases is a critical step to achieve nucleotide 1714). The junction with exon 9 of PDGFRB is their full activation.2 After immunoprecipitation of PDGFRb- unusual and was mentioned only once regarding an containing proteins, we showed that KPb was constitutively ETV6–PDGFRB fusion (unpublished data in Curtis et al4). In tyrosine phosphorylated in Ba/F3 cells (Figure 2c). As expected, line with several previously reported PDGFRb hybrids, KANK1– this was not observed in the presence of imatinib, suggesting PDGFRb (KPb) contains three coiled-coil domains of KANK1, that KPb phosphorylated itself. STAT transcription factors are which may mediate the oligomerization of the hybrid protein, downstream targets of the PDGF receptors.2 Aberrant activation as shown for wild-type KANK1.5 It also includes the fifth of STAT5 has an important role in hematopoietic cell immunoglobulin (Ig)-like extracellular domain, the transmem- transformation by many tyrosine kinase oncogenes, including brane domain and the kinase domain of PDGFRb (Figure 1e). ETV6–PDGFRB.2 By western blot with specific anti-phospho-site The KANK1 gene was identified as a potential tumor antibodies, we showed that STAT5, as well as the related factor suppressor gene at 9p24, whose expression was lost in renal STAT3, were phosphorylated in the presence of KPb in an cell carcinoma tissues and cell lines. The 9p chromosomal imatinib-dependent manner (Figure 2d). region, including KANK1, is also deleted in various cancers, The uncommon breakpoint within PDGFRB found in KPb such as in acute lymphocytic leukemia and in other diseases.5 leads to the addition of the fifth Ig-like domain of the PDGFRb The concomitant disruption of a tumor suppressor and activation extracellular part to the hybrid protein. In the wild-type of a receptor tyrosine kinase by translocation has been suggested receptor, this domain is not implicated in ligand binding, which in other cases, such as ETV6–PDGFRB. is mediated by the first three Ig-like domains.2,6 Although the To assess the transforming activity of KPb, we generated function of this domain has not been fully defined, it was lentiviral vectors expressing wild-type KANK1 or KPb and suggested that in c-KIT, another member of the PDGFR family, transduced the murine IL-3-dependent hematopoietic cell line homotypic interactions between the fourth and to a lesser extent Ba/F3. The KPb protein contains the first 741 residues of KANK1 between the fifth Ig-like domains allow the intracellular part to fused to the last 692 residues of PDGFRb. In the absence of IL-3, achieve the exact position needed for the receptor activation.6 only KPb-expressing cells proliferated, as shown in a [3H] Therefore, we investigated whether the fifth Ig-like domain thymidine incorporation assay (Figure 2a). Ba/F3–KPb cells could have a role in the activation of KPb. We generated a

Leukemia Letters to the Editor 1054

Figure 2 KPb is an activated oncogene that stimulates cell proliferation. Ba/F3 cells were transduced with KPb, wild-type KANK1 or the empty vector control. (a, b) Cells were grown in absence of IL-3, and cell proliferation was measured by a [3H]thymidine incorporation assay as described in Material and methods. Cells proliferated to a similar extent in the presence of IL-3 (data not shown). (b) The effect of increasing imatinib concentrations was tested on proliferation of Ba/F3 cells stably expressing KPb.(c) Ba/F3 cells were treated for 4 h with imatinib (0.5 mM) as indicated. Cells were lysed and KPb was immunoprecipitated with an antibody against PDGFRb. Immunoprecipitates were separated by SDS–PAGE and blotted with an antibody against phosphorylated tyrosines (pTyr). As a control, the membrane was reprobed with an anti-PDGFR antibody. The bands shown are derived from the same blot and the same X-ray film. (d) Cells were left in cytokine-free medium for 4 h with imatinib (0.1 mM) as indicated. Cell lysates were immunoblotted with antibodies against phosphorylated or total STAT5 and STAT3. IL-3 was added for 10 min before lysis of Ba/F3.

mutant devoid of this domain (DIg5) in which the PDGFRb in familial cerebral palsy. It is therefore possible that the sequence starts with exon 11, as in most of the described hybrids remaining wild-type KANK1 allele is not expressed in the (Supplementary Figure S2a). The DIg5 mutant stimulated Ba/F3 patient cells. cell proliferation to a similar extent compared with KPb, The patient presented thrombocythemia, but no prominent suggesting no key function of this Ig-like domain in KPb eosinophilia, in sharp contrast with the reported clinical features activation (Supplementary Figure S2b). Altogether, this indicated of myeloid neoplasms associated with PDGFRB rearrangements, that the breakpoint in PDGFRB can be located within intron 8 or which often include thrombocytopenia.1,3 Therefore, we spec- intron 10 with little impact on the activation of the protein. The ulate that KANK1 may also have a role in the KPb-associated higher frequency of alterations in intron 10 may be related to the thrombocythemia. This is in line with a previous report by size of this intron (3142 base pairs versus 1316 for intron 8). Kralovics et al.,8 indicating that KANK1 expression was We found no evidence of the recurrence of KANK1 decreased in polycythemia vera and essential thrombocythemia. rearrangement in myeloid neoplasms. In our database of Furthermore, KANK1 was shown to inhibit cell migration and B30 000 samples of patients with hematological malignancies, actin polymerization through the modulation of Rho GTPases.5 we found eight cases of myeloid malignancies with unexplained As Rho GTPases and the actin cytoskeleton have important 9p terminal cytogenetic alterations and tested them with functions during megakaryopoiesis, KANK1 might regulate fluorescence in situ hybridization probes specific for the KANK1 thrombocyte production. locus. No additional case of KANK1 disruption was found. In previous reports of treatment of PDGFRb-associated Future studies will have to search for other types of alterations in myeloid neoplasms with imatinib mesylate, a daily dose of the KANK1 gene in such diseases, like deletions, mutations and 400 mg was administered to most patients (range, 200–800 mg), epigenetic changes. Remarkably, Lerer et al.7 suggested that in line with common practice in BCR–ABL-positive chronic KANK1 is expressed only from one allele, and is even imprinted myelogenous leukemia.3 In FIP1L1–PDGFRa-positive

Leukemia Letters to the Editor 1055 myeloproliferative neoplasm, a dose of 100 mg/day was shown to 2Centre for Human Genetics, Cliniques universitaires Saint- 2 a b Luc, Universite´ catholique de Louvain, Brussels, Belgium and be effective. As PDGFR and have similar sensitivity to imatinib, 3 a low dose is likely to be effective in the treatment of PDGFRB- Hematology Unit, Cliniques universitaires Saint-Luc, associated myeloid neoplasms, as suggested by the present report, Universite´ catholique de Louvain, Brussels, Belgium E-mail: [email protected] with potential benefits in terms of costs and side effects. 4These authors equally contributed to this work. In conclusion, we identified a new t(5;9) translocation product between PDGFRB and a novel partner, KANK1,ina patient with thrombocythemia who responded well to low-dose imatinib. References

1 Tefferi A, Vardiman JW. Classification and diagnosis of myelopro- Conflict of interest liferative neoplasms: the 2008 World Health Organization criteria and point-of-care diagnostic algorithms. Leukemia 2008; 22: The authors declare no conflict of interest. 14–22. 2 Jones AV, Cross NC. Oncogenic derivatives of platelet-derived growth factor receptors. Cell Mol Life Sci 2004; 61: 2912–2923. 3 David M, Cross NC, Burgstaller S, Chase A, Curtis C, Dang R et al. Acknowledgements Durable responses to imatinib in patients with PDGFRB fusion gene-positive and BCR-ABL-negative chronic myeloproliferative This work was supported by grants from the Salus Sanguinis disorders. Blood 2007; 109: 61–64. foundation, the Bekales foundation and Action de Recherches 4 Curtis CE, Grand FH, Waghorn K, Sahoo TP, George J, Cross NC. Concerte´es (Communaute´ Franc¸aise de Belgique). SM is the A novel ETV6-PDGFRB fusion transcript missed by standard recipient of a fellowship from the Maurange foundation (managed screening in a patient with an imatinib responsive chronic myeloproliferative disease. Leukemia 2007; 21: 1839–1841. by the Roi Baudouin foundation), FD is a FRS-FNRS research 5 Kakinuma N, Zhu Y, Wang Y, Roy BC, Kiyama R. Kank proteins: fellow and FT is supported by a scholarship from Ope´ration structure, functions and diseases. Cell Mol Life Sci 2009; 66: Te´le´vie. We wish to thank Jean-Luc Vaerman for providing 2651–2659. samples and Catherine Marbehant for excellent technical assis- 6 Yang Y, Yuzawa S, Schlessinger J. Contacts between membrane tance. We are grateful to Thomas Michiels for generous donations proximal regions of the PDGF receptor ectodomain are required for of reagents and to Laurent Knoops and Stefan Constantinescu for receptor activation but not for receptor dimerization. Proc Natl Acad Sci USA 2008; 105: 7681–7686. very helpful discussions. This study was approved by the ethics 7 Lerer I, Sagi M, Meiner V, Cohen T, Zlotogora J, Abeliovich D. committee of the medical faculty (ref # F/2005/02). Informed Deletion of the ANKRD15 gene at 9p24.3 causes parent-of-origin- consent was obtained. dependent inheritance of familial cerebral palsy. Hum Mol Genet 1,4 2,4 3 1 2005; 14: 3911–3920. S Medves , FP Duhoux , A Ferrant , F Toffalini , 8 Kralovics R, Teo SS, Buser AS, Brutsche M, Tiedt R, Tichelli A et al. 2 2 2,4 1,4 G Ameye , J-M Libouton , HA Poirel and J-B Demoulin Altered gene expression in myeloproliferative disorders correlates 1 de Duve Institute, Universite´ catholique de Louvain, with activation of signaling by the V617F mutation of Jak2. Blood Brussels, Belgium; 2005; 106: 3374–3376.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

Human leukocyte antigen (HLA) A1-B8-DR3 (8.1) haplotype, tumor necrosis factor (TNF) G-308A, and risk of non-Hodgkin lymphoma

Leukemia (2010) 24, 1055–1058; doi:10.1038/leu.2010.17; be in linkage disequilibrium. TNF is located on chromosome published online 11 February 2010 6p21.3 among the Class III genes of the major histocompatibility complex, 250 kb centromeric to the HLA-B locus and 850 kb telomeric to the Class II HLA-DR locus. It is well documented Large-scale consortial efforts now provide convincing evidence that Caucasian populations carry the 8.1 ancestral haplotype that the pro-inflammatory cytokine tumor necrosis factor (TNF) (AH) that includes the TNF-308A allele (HLA-A1-B8-TNF-308A- promoter polymorphism (TNF G-308A), which is thought to DR3-DQ2).3 Notably, the 8.1 AH is implicated in the risk of increase TNF-a protein expression resulting in inflammation, is numerous autoimmune conditions, including those associated associated with increased risk of non-Hodgkin lymphoma (NHL) with NHL (for example, systemic lupus erythematosus and and specifically with the NHL subtype, diffuse large B-cell Sjogren’s syndrome).3–5 It remains unknown, however, whether lymphoma (DLBCL) among Caucasians.1 The largest effort to the association reported for TNF-308A is due to or independent date from the International Lymphoma Epidemiology Consor- from HLA alleles and/or haplotypes. tium (InterLymph) comprising 7999 incidence NHL cases and Here, we present data from 555 controls and 610 cases from a 8452 controls report that TNF-308A carriers have a 1.25-fold US population-based case–control study of NHL where HLA (per allele) increased risk for DLBCL and a 1.35-fold increased Class I and Class II alleles were evaluated in the context of TNF risk for marginal zone lymphoma.2 with regard to their role in risk for NHL and NHL subtypes. A limitation to the published association studies, however, is As has been previously described,6 the multi-center National the inability to delineate the association between TNF and NHL Cancer InstituteFSurveillance, Epidemiology and End Results from human leukocyte antigen (HLA) alleles that are known to (NCI-SEER) NHL case–control study population comprised 1321

Leukemia