Letters to the Editor 2564 L Orsi1,2,22, J Rudant1,2,3,22, A Bonaventure1,2, S Goujon-Bellec1,2,3, 21Universite´ Paris 7 Denis Diderot, Paris, France E Corda4,5, T-J Evans6, A Petit7,8, Y Bertrand9, B Nelken10,11, E-mail: [email protected] A Robert12, G Michel13, N Sirvent14, P Chastagner15, 22These authors contributed equally to this work. S Ducassou16, X Rialland17,18,DHe´mon1,2, E Milne19, RJ Scott6, A Baruchel20,21 and J Clavel1,2,3 1INSERM, U1018, Department of Environmental Epidemiology of Cancers, Villejuif, France; REFERENCES 2 Paris-Sud University, UMR-S1018, Department of 1 Lacour B, Guyot-Goubin A, Guissou S, Bellec S, Desandes E, Clavel J. Incidence of Environmental Epidemiology of Cancers, childhood cancer in France: National Children Cancer Registries, 2000–2004. Eur J Research Center in Epidemiology and Population Health, Cancer Prev 2010; 19: 173–181. Villejuif, France; 2 Eden T. Aetiology of childhood leukaemia. Cancer Treat Rev 2010; 36: 286–297. 3French National Registry of Childhood Blood 3 Papaemmanuil E, Hosking FJ, Vijayakrishnan J, Price A, Olver B, Sheridan E et al. Malignancies (RNHE), Villejuif, France; Loci on 7p12.2, 10q21.2 and 14q11.2 are associated with risk of childhood acute 4Fondation Jean Dausset, Centre d’Etude du Polymorphisme lymphoblastic . Nat Genet 2009; 41: 1006–U1073. 4 Trevino LR, Yang WJ, French D, Hunger SP, Carroll WL, Devidas M et al. Germline Humain (CEPH), Paris, France; 5 genomic variants associated with childhood acute lymphoblastic leukemia. Nat Inserm, Univ Paris Diderot, UMR-S-946, Genet 2009; 41: 1001–U1067. Genetic Variation and Human Diseases Unit, 5 Sherborne AL, Hosking FJ, Prasad RB, Kumar R, Koehler R, Vijayakrishnan J et al. Paris, France; Variation in CDKN2A at 9p21.3 influences childhood acute lymphoblastic leukemia 6School of Biomedical Sciences, Faculty of Health, risk. Nat Genet 2010; 42: 492–494. University of Newcastle, Newcastle, 6 Ellinghaus E, Stanulla M, Richter G, Ellinghaus D, Te Kronnie G, Cario G et al. NSW, Australia; Identification of germline susceptibility loci in ETV6-RUNX1-rearranged childhood 7AP-HP, Hoˆpital Armand Trousseau, Paris, France; acute lymphoblastic leukemia. Leukemia 2012; 26: 902–909. 8Universite´ Paris 6 Pierre et Marie Curie, Paris, France; 7 Rudant J, Orsi L, Menegaux F, Petit A, Baruchel A, Bertrand Y et al. Childhood acute leukemia, early common infections, and allergy: the ESCALE study. Am J 9Institut d’He´mato-Oncologie Pe´diatrique, Lyon, France; 10 Epidemiol 2010; 172: 1015–1027. Poˆle Enfant, CHRU, Lille, France; 8 Ehret GB, Munroe PB, Rice KM, Bochud M, Johnson AD, Chasman DI et al. Genetic 11 Universite´ Lille Nord de France, Lille, France; variants in novel pathways influence blood pressure and cardiovascular disease 12 Hoˆpital des Enfants, Toulouse, France; risk. Nature 2011; 478: 103–109. 13AP-HM, Hoˆpital la Timone, Marseille, France; 9 Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MAR, Bender D et al. PLINK: a 14Hoˆpital Arnaud de Villeneuve, Montpellier, France; tool set for whole-genome association and population-based linkage analyses. 15Hoˆpital d’enfants, CHU de Nancy, Vandoeuvre, France; Am J Hum Genet 2007; 81: 559–575. 16Hoˆpital Pellegrin Tripode, Bordeaux, France; 10 Zeng K, Bastos RN, Barr FA, Gruneberg U. Protein phosphatase 6 regulates mitotic 17Hoˆpital Me`re-Enfant, CHU-Nantes, Nantes, France; spindle formation by controlling the T-loop phosphorylation state of Aurora A bound to its activator TPX2. J Cell Biol 2010; 191: 1315–1332. 18CHU d’Angers, Angers, France; 19 11 Healy J, Richer C, Bourgey M, Kritikou EA, Sinnett D. Replication analysis confirms Telethon Institute for Child Health Research, Centre for the association of ARID5B with childhood B-cell acute lymphoblastic leukemia. Child Health Research, University of Western Australia, Haematologica-the Journal 2010; 95: 1608–1611. Crawley, WA, Australia; 12 Mullighan CG, Su X, Zhang J, Radtke I, Phillips LAA, Miller CB et al. Deletion of IKZF1 20AP-HP, Hoˆpital Robert Debre´, Paris, France and and prognosis in acute lymphoblastic leukemia. NEnglJMed2009; 360: 470–480.

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

A microRNA signature specific for hairy cell leukemia and associated with modulation of the MAPK–JNK pathways

Leukemia (2012) 26, 2564–2567; doi:10.1038/leu.2012.149 diagnostic markers and explained some of the peculiar features of leukemic hairy cells.2 In particular, the expression of genes controlling cell adhesion and response to chemokines appeared to be significantly altered in HCL, supporting the aberrant Hairy cell leukemia (HCL) is a chronic B-cell lymphoproliferative adhesion and homing properties of HCL cells.2 No recurrent disorder characterized by marked , and chromosomal alterations have been identified in HCL, while proliferation of tumor cells with hairy appearance that infiltrate the studies based on genome-wide single-nucleotide polymorphism , liver and and circulate at low percentage in analysis confirmed a remarkably stable genome.1,3 More recently, the peripheral blood.1 HCL displays a unique immunophenotype using a whole-exome sequencing approach, the BRAF V600E gene (ANXA1 þ , CD103 þ , CD25 þ and CD11c þ ) that is distinct from mutation was identified as the first genetic hallmark of HCL.4 The that of other B-cell .1 HCL is highly sensitive to purine presence of BRAF-activating mutations in virtually all HCL cases4,5 analogues that induce a response in about 90% of patients.1 strongly suggests that the RAF-MEK-ERK pathway may have a During the past decade, significant progress has been made in critical role in the HCL pathogenesis. the understanding of the HCL pathogenesis.1 Gene-expression A novel layer of biological complexity has been added by the profile analysis identified a unique molecular signature, which discovery of microRNAs (miRNAs) and their implication in cancer. In suggested a derivation from memory B cells, provided novel B cells, miRNAs are expressed in a stage- or transformation-specific

Accepted article preview online 4 June 2012; advance online publication, 29 June 2012

Leukemia (2012) 2546 – 2569 & 2012 Macmillan Publishers Limited Letters to the Editor 2565

Figure 1. HCL display a homogenous miRNA-expression profile. (a) Hierarchical clustering based on miRNA-expression profiles of HCL, SLVL and B-CLL. The analysis is based on the subset of miRNAs displaying at least a two-fold average change in expression levels from the mean across the whole panel. (b) Identification of a HCL-specific miRNA signature by supervised analysis. The Z-score is displayed for each miRNA. fashion, suggesting that they might have developmental and B-cell malignancies (Figure 1a). Similarly, B-CLL tended to cluster pathological roles.6 Although miRNA-expression signatures have together, whereas SLVL did not form a uniform cluster likely been derived for several hematological malignancies, the because of their higher heterogeneity (Figure 1a). contribution of miRNAs to HCL has not yet been analyzed. In In order to identify a HCL-specific miRNA signature, HCLs were order to investigate the potential role of miRNAs in HCL compared with SLVLs, B-CLLs and normal mature B cells at pathogenesis and the possibility of using miRNA-expression different stages of differentiation including naı¨ve, memory and signatures for the differential diagnosis of HCL, miRNA-expression germinal-center B cells. This supervised analysis was performed profiling was performed on peripheral blood-derived CD19 þ Bcells using the Genes@Work software platform8 and identified six from eight HCL patients (all harboring the BRAF V600E gene miRNAs, which were expressed at significantly higher level in HCL mutation4), five patients with splenic with villous compared with normal and other malignant B cells (Figure 1b). (SLVLs; all devoid of the BRAF V600E gene mutation) The HCL-specific signature included the miR-221/miR-222 family, and nine patients with B-cell chronic lymphocytic leukemia (B-CLL; miR-22, miR-24, miR-27a and let-7b. of which five could be tested and turned out to be negative for the MiRNAs exert their function by negatively modulating the BRAF V600E gene mutation) using the Human miRNA microarray kit translation of their targets, and several genes have been already v.1.0 (Agilent Technologies, Santa Clara, CA, USA). For comparison, demonstrated to be directly targeted by the miRNAs identified six each naı¨ve, and memory B-cell samples from here as deregulated in HCL. Interestingly, CDKN1B (p27/Kip1) healthy donors were also included in the study. Unsupervised represents a fully characterized direct target of miR-221/miR-222.9 hierarchical clustering, based on the average linkage method and Low levels of CDKN1B protein expression in HCL have been previ- Pearson’s correlation,7 was used to interrogate the miRNA- ously reported and could not be explained by genetics, decreased expression profiles for their ability of discriminating HCL, SLVL transcription or increased ubiquitin-mediated degradation.10 and B-CLL. The results showed that HCL displayed a unique and Therefore, it has been suggested that low CDKN1B protein levels homogenous phenotype clearly distinct from that of the other in HCL may be due to unknown mechanisms of posttranscriptional

& 2012 Macmillan Publishers Limited Leukemia (2012) 2546 – 2569 Letters to the Editor 2566

Figure 2. MAPK signaling pathway is significantly enriched for targets predicted to be affected by the HCL miRNA signature. A total of 257 targets were predicted by at least four prediction algorithms to be affected by the HCL miRNA signature. The pathway-enrichment analysis was performed on the 257 predicted targets and significant enrichment (upon Bonferroni correction) was detected only for the MAPK signaling pathway. The red label identifies predicted targets. The color reproduction of this figure is available at the Leukemia journal online.

regulation.10 Our data strongly suggest that miR-221/miR-222 overexpression of miRNAs may contribute to make HCL cells more overexpression in HCL represents at least one mechanism, leading resistant to apoptosis. This mechanism may act in concert with the to low CDKN1B protein expression in HCL. constitutive activation of the RAF-MEK-ERK pathway triggered by MiR-24 has been demonstrated to directly target CDKN2A the BRAF V600E mutant. protein (p16),11 a tumor suppressor, largely affected by gene Taken together, the data herein identify a HCL-specific miRNA deletions in human cancer, but not in HCL.3 Overexpression of signature with implications for the pathogenesis and clinical miR-24 in HCL may lead to a negative modulation on CDKN2A management of HCL. In fact, these results reinforce the notion that protein in the absence of lesions affecting the gene locus. The the MAPK pathway has a critical role in the development of HCL. CDKN1A (p21) protein has been recently reported to be regulated This in turn suggests a druggable target because many available by miR-22, whose overexpression in HCL could contribute to make compounds can modulate the altered MAPK–JNK pathway typical HCL cells more resistant to apoptosis.12 of leukemic hairy cells. To gain further insights on the potential role of miRNAs in HCL pathogenesis, we applied five target prediction algorithms CONFLICT OF INTEREST (MiRBase Targets, TargetScan, PicTar, RNA22 and PITA)13 to identify the candidate targets of the six-miRNA HCL-specific signature. BF and ET applied for a patent on the clinical use of the BRAF V600E mutant in HCL. The remaining authors declare no conflict of interest. Targets commonly predicted by at least four of the prediction methods and expressed in B cells were further investigated. Overall, 257 genes were predicted to be targeted by at least one ACKNOWLEDGEMENTS of the HCL-overexpressed miRNAs. The pathway-enrichment analysis BF was supported by an investigator grant from the Associazione Italiana Ricerca sul was performed on the 257 predicted targets using the Database for Cancro and ET was supported by a fellowship (2008/14) from the European Annotation, Visualization and Integrated Discovery.14 The mitogen- Hematology Association. We thank Roberta Pacini and Gianluca Schiavoni for their activated protein kinase (MAPK) pathway appeared as the most technical assistance. significantly enriched in predicted targets of HCL-overexpressed miRNAs, suggesting that miRNA-deregulated expression in HCL Y Kitagawa1,2, M Brahmachary1,3, E Tiacci4, R Dalla-Favera1,5,6,7, may modulate MAPK signaling (Figure 2). Interestingly, most of B Falini4,8 and K Basso1,5,8 the predicted targets belonging to the MAPK signaling pathway 1Institute for Cancer Genetics and H. Irving Comprehensive were involved in the activation of p38 MAPK and Jun N-terminal Cancer Center, Columbia University, kinase (JNK). This pathway has been shown to make HCL cells New York, NY, USA; susceptible to apoptosis, an effect likely triggered by the interac- 2Institute for Integrated Medical Sciences, tion of HCL cells with the microenviroment, in particular with Tokyo Women’s Medical University, the vitronectin-positive cells in the splenic red pulp.15 Of note, Tokyo, Japan; miRNA-mediated repression was not predicted for proteins in 3Joint Centers for Systems Biology, Columbia University, the RAF-MEK-ERK pathway, consistent with a lack of negative New York, NY, USA; modulation of this pathway, which is constitutively active in HCL 4Institute of Hematology, University of Perugia, cells because of BRAF somatic mutation.4 The balance between Perugia, Italy; ERK and JNK/p38 MAPK may have a critical role in HCL, as 5Department of Pathology and Cell Biology, suggested by the observation that active JNK/p38 MAPK makes Columbia University, New York, HCL cells susceptible to apoptosis, whereas they are effectively NY, USA; rescued by ERK activation.15 Therefore, negative modulation of 6Department of Genetics and Development, the pro-apoptotic JNK/p38 MAPK signaling pathways caused by Columbia University, New York, NY, USA and

Leukemia (2012) 2546 – 2569 & 2012 Macmillan Publishers Limited Letters to the Editor 2567 7 Department of Microbiology and Immunology, 7 Eisen MB, Spellman PT, Brown PO, Botstein D. Cluster analysis and display of Columbia University, New York, NY, USA genome-wide expression patterns. Proc Natl Acad Sci USA 1998; 95: 14863–14868. E-mail: [email protected] 8 Califano A, Stolovitzky G, Tu Y. Analysis of gene expression microarrays for 8These two authors contributed equally to this work. phenotype classification. Proc Int Conf Intell Syst Mol Biol 2000; 8: 75–85. 9 le Sage C, Nagel R, Egan DA, Schrier M, Mesman E, Mangiola A et al. Regulation of the p27(Kip1) tumor suppressor by miR-221 and miR-222 promotes cancer cell REFERENCES proliferation. EMBO J 2007; 26: 3699–3708. 1 Tiacci E, Liso A, Piris M, Falini B. Evolving concepts in the pathogenesis of hairy- 10 Chilosi M, Chiarle R, Lestani M, Menestrina F, Montagna L, Ambrosetti A et al. cell leukaemia. Nat Rev Cancer 2006; 6: 437–448. Low expression of p27 and low proliferation index do not correlate in hairy cell 2 Basso K, Liso A, Tiacci E, Benedetti R, Pulsoni A, Foa R et al. Gene expression leukaemia. Br J Haematol 2000; 111: 263–271. profiling of hairy cell leukemia reveals a phenotype related to memory B cells 11 Lal A, Kim HH, Abdelmohsen K, Kuwano Y, Pullmann Jr R, Srikantan S et al. with altered expression of chemokine and adhesion receptors. J Exp Med 2004; p16(INK4a) translation suppressed by miR-24. PLoS One 2008; 3: e1864. 199: 59–68. 12 Tsuchiya N, Izumiya M, Ogata-Kawata H, Okamoto K, Fujiwara Y, Nakai M et al. 3 Forconi F, Poretti G, Kwee I, Sozzi E, Rossi D, Rancoita PM et al. High density Tumor suppressor miR-22 determines -dependent cellular fate through post- genome-wide DNA profiling reveals a remarkably stable profile in hairy cell transcriptional regulation of p21. Cancer Res 2011; 71: 4628–4639. leukaemia. Br J Haematol 2008; 141: 622–630. 13 Saito T, Saetrom P. MicroRNAs—targeting and target prediction. N Biotechnol 4 Tiacci E, Trifonov V, Schiavoni G, Holmes A, Kern W, Martelli MP et al. BRAF 2010; 27: 243–249. mutations in hairy-cell leukemia. N Engl J Med 2011; 364: 2305–2315. 14 Huang da W, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths 5 Tiacci E, Schiavoni G, Forconi F, Santi A, Trentin L, Ambrosetti A et al. Simple toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res genetic diagnosis of hairy cell leukemia by sensitive detection of the BRAF-V600E 2009; 37: 1–13. mutation. Blood 2012; 119: 192–195. 15 Kamiguti AS, Harris RJ, Slupsky JR, Baker PK, Cawley JC, Zuzel M. Regulation of 6 Basso K, Sumazin P, Morozov P, Schneider C, Maute RL, Kitagawa Y et al. hairy-cell survival through constitutive activation of mitogen-activated protein Identification of the human mature miRNome. Immunity 2009; 30: 744–752. kinase pathways. Oncogene 2003; 22: 2272–2284.

Selection of antigen receptors in splenic marginal-zone lymphoma: further support from the analysis of the immunoglobulin light-chain gene repertoire

Leukemia (2012) 26, 2567–2569; doi:10.1038/leu.2012.207 mens or peripheral blood samples as described previously.8–10 Sequences were analyzed using the IMGT databases and the IMGT/V-QUEST tool (version 3.2.17, Universite´ Montpellier 2, CNRS, We recently demonstrated that 430% of the cases with Montpellier, France; www.imgt.org). All sequences, including 41 splenic marginal-zone lymphoma (SMZL) express B-cell sequences previously reported from our groups,9,10 were evaluated receptors (BcRs) that carry a single polymorphic variant of the following the novel strategy detailed in our recent publications.1,8 IGHV1-2 gene (IGHV1-2*04).1 The IGHV1-2*04 BcRs in SMZL are Overall, 113 productive IG LC rearrangements were amplified from characterized by long complementarity-determining region-3 107 cases. Six cases carried two productive IG LC rearrangements (CDR3) and exhibit a low impact of somatic hypermutation along with a single productive HC rearrangement, effectively ruling (SHM), leading to limited changes, which cluster in certain out the possibility of biclonal population. Notably, four of the six positions of the VH domain. Altogether, these features argue for cases with double-productive rearrangements were found to carry selection by antigen in the pathogenesis of SMZL and suggest one each IGKV-IGKJ and IGLV-IGLJ rearrangement, hence raising the heavy chain (HC) dominance in the clonogenic immunoglobulin possibility that an initially expressed IGKV-IGKJ rearrangement was (IG) receptors of SMZL.1 subsequently edited through a process involving inactivation of the That notwithstanding, the possibility that IG light chains (IG LC) IGK locus by a rearrangement involving the kappa-deleting element, might also have an important role in SMZL ontogeny exists and thus, ensuring allelic exclusion.6,8 needs to be investigated. This is supported by studies demonstrat- The IG LC gene repertoire was biased (Supplementary Table 1) ing that IG LCs may be critically implicated in recognition of and with only six genes (IGKV3-20, IGKV4-1, IGKV1-5, IGKV1-8, IGKV1-39, selection by antigen. Examples include: (i) significant LC sequence IGLV2-14), accounting for 72/113 rearrangements (64%). restrictions among estradiol-specific antibodies;2 (ii) impaired In particular, the IGKV3-20 and IGLV2-14 genes predominated by responses to Haemophilus influenzae in individuals lacking a far within IGKV-IGKJ and IGLV-IGLJ rearrangements (20/82 cases, specific allelic variant of the IGKV2D-29 gene; and, (iii) biased IG LC 24%; and 10/31 cases, 32%, respectively). Hence, the IG gene gene usage in autoimmunity (for example, IGKV1-17 and IGLV1-47 in repertoire in SMZL is restricted with regards to not only IG HCs but lupus,3–4 IGKV1-39/1D-39 in Graves disease5). Furthermore, the BcR LCs as well, strongly indicating selection by antigen. specificity may drastically change through genetic processes On the basis of the percentage of IGKV/IGLV gene identity to the affecting LCs, including secondary rearrangements in the context germline (GI), 21/113 sequences (18%) were assigned to a ‘truly of receptor editing6 or distinctive SHM patterns.7 Finally, marked LC unmutated’ subgroup (100% GI), whereas the remaining gene repertoire biases exist in various B-cell lymphomas, strongly sequences (92/113, 82%) exhibited a variable impact of SHM, indicating that LCs can contribute significantly toward shaping ranging from minimal to pronounced (Supplementary Table 2). antigen reactivity of the clonotypic BcRs.8 For statistical comparisons, sequences with 97–99.9% GI were In order to gain insight into the role of IG LCs in SMZL we classified as ‘borderline/minimally mutated’ (n ¼ 61, 54%), whereas explored the IG LC gene repertoire in 107 SMZL cases diagnosed those with o97% GI as ‘significantly mutated’ (n ¼ 31, 28%). as described previously1 and carrying at least one productive LC Rearrangements of most genes did not differ regarding the impact rearrangement. The analysis was performed on spleen speci- of SHM. The IGKV1-5 gene constituted an exception as it was

Accepted article preview online 18 July 2012; advance online publication, 3 August 2012

& 2012 Macmillan Publishers Limited Leukemia (2012) 2546 – 2569