Letters to the Editor 537 Six years after CR and 38 months after the end of the J Ade´laı¨de1,CPe´rot2, V Gelsi-Boyer1,3,4, C Pautas5, 1,3 6 7 1 chemotherapy, the patient presented a relapse with lympha- A Murati , C Copie-Bergman , M Imbert , M Chaffanet , D Birnbaum1 and M-J Mozziconacci1,3 denopathies without central nervous or BM localisation. Cyto- 1 genetic analysis on BM cells after 24 h culture showed a t(8;9) Institut de Cance´rologie de Marseille, De´partement d’Oncologie Mole´culaire, Institut Paoli-Calmettes and U599 translocation as a sole abnormality in 16/29 metaphases. Inserm, Marseille, France; A novel lymph-node biopsy confirmed the relapse of T lympho- 2Laboratoire de Cytoge´ne´tique, Hoˆpital Saint-Antoine, Paris, blastic lymphoma with a TDT þ , CD2 þ , CD3 þ , CD4À, France; CD5 þ , CD7 þ , CD8À, CD10À, CD30À, CD1AÀ, KITÀ 3De´partement de BioPathologie, Institut Paoli-Calmettes, phenotype, and P53 moderate overexpression. A high mitotic Marseille, France; activity was evidenced with MIB1 antibody. The search for an 4Universite´ de la Me´diterrane´e, Marseille, France; 5 association with Epstein–Barr virus was negative with LMP Service d’He´matologie Clinique, Hoˆpital Henri Mondor, Cre´teil, France; antibody. A second CR was obtained after ALL-like induction 6 chemotherapy with the same drugs plus L-asparaginase. After De´partement de Pathologie, Hoˆpital Henri Mondor, Cre´teil, France and three courses of consolidation including high doses aracytine 7Laboratoire d’He´matologie, Hoˆpital Henri Mondor, Cre´teil, and methotrexate, autologous stem cell transplantation was France done in February 2005. The patient is still in CR 7 months after E-mail: [email protected] the graft and the karyotype established on BM in June 2005 was normal on 30 metaphases. Dual-colour fluorescence in situ hybridisation, carried out as described2 on BM metaphasic cells from November 2004, showed the t(8;9)(p22;p24) rearrangement. RT-PCR analysis, carried out as previously described,1–3 demonstrated the PCM1-JAK2 fusion in lymph-node cells of the patient in References September 2004, but not on BM or blood cells in CR. Both PCM1-JAK2 and JAK2-PCM1 transcripts were observed (Figure 1, 1 Reiter A, Walz C, Watmore A, Schoch C, Blau I, Schlegelberger B inset). The breakpoint was localised in JAK2 intron 8 and both et al. The t(8;9)(p22;p24) is a recurrent abnormality in chronic and the JH2 pseudo-kinase domain and tyrosine kinase domain were acute leukaemia that fuses PCM1 to JAK2. Cancer Res 2005; 65: preserved (Figure 1). 2662–2667. The PCM1-JAK2 fusion has been described recently in a pre-B 2 Murati A, Gelsi-Boyer V, Adelaide J, Perot C, Talmant P, Giraudier S ALL.1 We have reported here the first case of this alteration in a et al. PCM1-JAK2 fusion in myeloproliferative disorders and acute erythroid leukemia with t(8;9) translocation. Leukemia 2005; 19: T-cell lymphoma. Our work shows that the spectrum of JAK2 1692–1696. activation may be quite large and include both lymphocyte 3 Bousquet M, Quelen C, De Mas V, Duchayne E, Roquefeuil B, lineages. In the reported case, the presence of the t(8;9) in Delsol G et al. The t(8;9)(p22;p24) translocation in atypical chronic peripheral lymphocytes and in BM cells suggests the targeting of myeloproliferative disorders yields a new PCM1-JAK2 fusion gene. the totipotent stem cells. Oncogene 2005; 24: 7248–7252. 4 Heiss S, Erdel M, Gunsilius E, Nachbaur D, Tzankov A. Myelodys- plastic/myeloproliferative disease with erythropoietic hyperplasia Acknowledgements (erythroid preleukemia) and the unique translocation (8;9)(p23;p24): first description of a case. Hum Pathol 2005; 36: 1148–1151. 5 Tefferi A, Gilliland DG. The JAK2 V617F tyrosine kinase mutation in This work has been supported by Institut Paoli-Calmettes, Inserm myeloproliferative disorders: status report and immediate implica- and grants from Laurette Fugain Association and from Ministries of tions for disease classification and diagnosis. Mayo Clin Proc 2005; Health and Research (Cance´ropoˆle). 80: 947–958.

High incidence of Notch-1 mutations in adult patients with T-cell acute lymphoblastic leukemia

Leukemia (2006) 20, 537–539. doi:10.1038/sj.leu.2404101; Notch-1 producing constitutively activated ICN, has been published online 19 January 2006 described in a number of T-ALL patients,2 and gain-of-function mutations in Notch-1 have recently been reported in 56% of pediatric/adolescent T-ALL patients.3 The mutations affect two ‘hotspot’ regions – those in the highly conserved extracellular The Notch pathway has a crucial role in embryonic develop- heterodimerization domain (HD) are thought to increase Notch-1 ment, determining cell fate of a range of different organs. In the cleavage, whereas truncating mutations of the C-terminal PEST hematopoietic system Notch-1 activation directs T-cell differ- domain are thought to increase the intracytoplasmic half-life of ICN.3 entiation, and in murine models, constitutively activated Although the phenotype of adult T-ALL is similar to its intracellular Notch-1 (ICN) leads to T-cell leukemia.1 In pediatric counterpart, there are significant differences; in humans, there is evidence that Notch-1 dysregulation con- particular it has a poorer prognosis, altered frequency of tributes to the pathogenesis of T-cell acute lymphoblastic cytogenetic abnormalities and oncogene expression.4 We were leukemia (T-ALL). For example, the t(7;9) translocation, whereby interested to know whether some of these differences could be the promoter of the T-cell b-chain is juxtaposed with attributed to the incidence and characteristics of Notch-1

Leukemia Letters to the Editor 538 Table 1 Characteristics and Notch-1 mutational status of 24 adult T-ALL patients

Case Sex Age WCC Mutation Mutation (nucleotide)a Notch-1 Consequence (amino acid) Â 109/l status domain

1 M 19 38 MUT ins2@7473/7474 PEST Y2491X 2 F 17 239 WT FFF 3 M 45 52 MUT G5014A (SNP); ins3@5025/5026 HD-(N);C V1672I (SNP); insG@1675/1676 T7518G (SNP); ins8@7330/7331 PEST P2506P (SNP); fs@2444, STOP@2480 4 M 25 73 MUT T4724C+G4725T; T4757C+G4758T HD-N L1575P; L1586P del1@7390 PEST fs@2464, STOP@2477 5 M 32 8 MUT del3@4852–4854 HD-N delF1618 6 M 37 208 MUT T4790A HD-N L1597H 7 M 39 25 WT FFF 8 M 50 79 WT C5097T (SNP) (HD-C) D1699D (SNP) 9 F 26 81 MUT T4781C HD-N L1594P 10 M 36 39 MUT del3@4776–4778+ins3 HD-N H1592Q+F1593T 11 M 17 2 MUT del2@4780–4781+ins5b HD-N insA@1594/1595 12 M 16 27 MUT C5097T (SNP); C5090A HD-C D1699D (SNP); A1697D 13 M 42 68 MUT ins6@4779/4780b HD-N F1593L+insSP@1593/1594 14 M 18 109 WT FFF 15 M 43 18 WT FFF 16 F 39 65 MUT T4850A HD-N I1617N ins5@7304/7305 PEST insGX@2435/2436 17 M 16 133 WT C5097T (SNP) (HD-C) D1699D (SNP) 18 M 55 41 MUT T4781C HD-N L1594P 19 M 36 8 MUT del3@4852–4854 HD-N delF1618 20 M 21 18 MUT T4724C HD-N L1575P 21 M 42 261 MUT del7@4777–4783+ins10 HD-N delFLR@1593–1595+insHFDG 22 M 35 33 MUT del10@4844À4853+ins7 HD-N delQMIF@1615–1618+insLCR 23 M 23 653 MUT T4757C HD-N L1586P del2@7544–7545 PEST fs@2515, STOP @2518 24 M 20 264 WT C5097T (SNP) (HD-C) D1699D (SNP) WCC ¼ white cell count; MUT ¼ mutant; WT ¼wild type; ins ¼ insertion; del ¼ deletion; SNP ¼ single nucleotide polymorphism; HD-N ¼ N-terminal heterodimerization domain; HD-C ¼ C-terminal heterodimerization domain; fs ¼ frameshift; X ¼ stop codon. aNumbered according to Accession NM_017617. bLow level mutation purified using the fragment collector facility of the WAVEs.

Table 2 Frequency and characteristics of Notch-1 mutations in ALL patients) and there is limited data on Notch-1 mutations in pediatric/adolescent and adult patients with T-ALL these patients. To our knowledge there is just one study published in which five of 14 patients had mutations.5 Two of Mutant in 96 Mutant in pediatric/ 24 adult the three mutations identified in the HD domain had been found adolescent patients in pediatric/adolescent patients, and both PEST domain muta- patientsa tions were novel and led to truncation of the C-terminal tail. We therefore screened presentation DNA samples from 24 adult Mutant in any domain (% of total cohort) 54 (56%) 17 (71%) T-ALL patients entered into the MRC UKALLXII trial for HD only 25 (26%) 12 (50%) mutations in Notch-1 HD domains (both HD-N and HD-C), TAD/PEST only 12 (12%) 1 (4%) HD+TAD/PEST 17 (18%) 4 (17%) transactivation domain (TAD) and PEST domain using denatur- ing high-performance liquid chromatography (DHPLC) (Trans- s HD-N (% of all HD-N mutations) (n ¼ 34) (n ¼ 14) genomic WAVE DNA fragment analysis system). Samples with Leucine to proline 14 (41%) 5 (36%) an abnormal chromatograph were sequenced. Other point mutations 7 (21%) 2 (14%) Overall, 17 patients (71%) had at least one Notch-1 mutation; Insertions and/or deletions 13 (38%) 7 (50%) 12 in the HD domain only, one in the PEST domain only, and HD-C (% of all HD-C mutations) (n ¼ 8) (n ¼ 2) four in both domains (Table 1). Mutations in the HD-N domain Leucine to proline 4 (50%) were the most frequent, affecting over half of the patients (14/24, Other point mutations 4 (50%) 1 (50%) 58%), and tended to cluster in the highly conserved region Insertions and/or deletions 1 (50%) between residues 1593 and 1597. Five had point mutations causing leucine to proline substitutions, which had been TAD/PEST (% of all TAD/PEST mutations) (n ¼ 29) (n ¼ 5) described in the pediatric/adolescent patients,3 but seven had Point mutationsb 10 (34%) Insertions and/or deletionsb 19 (66%) 5 (100%) novel insertions or deletions, four of them complex alterations with contiguous insertions and deletions; all were in-frame. HD ¼ heterodimerization domain; HD-N ¼ N-terminal HD domain; HD- Cloning of products from one patient (#4) showed that the two C C-terminal HD domain; TAD transactivation domain; PEST ¼ ¼ ¼ leucine to proline mutations were on different alleles and PEST domain. aData from Weng et al.3 were therefore either in trans or present in different cells. bAll lead to a truncated protein. Five mutations were detected in the PEST domain; all were novel insertions and/or deletions that led to premature mutations in adult patients, which may impact on signaling stop codons, either through a frameshift or through direct events downstream of Notch-1 and response to therapy. T-ALL insertion of a stop codon, truncating the receptor by 39–119 in adults is relatively infrequent (approximately 20% of adult amino acids.

Leukemia Letters to the Editor 539 Remission DNA samples were available from 15 patients, drift that desensitizes current monitoring techniques in T-ALL.7 nine of whom had a mutation at disease presentation. In each Furthermore, g-secretase inhibitors (GSI) have been shown to case the mutation was no longer detectable in remission, induce cell cycle arrest in vitro in T-ALL cell lines harboring confirming that these were acquired leukemia-specific muta- Notch-1 mutations3 and therefore may offer a rational molecu- tions and not germline polymorphisms. Relapse DNA samples larly targeted approach for these patients who, in the older age were available from four patients (#3, 7, 10 and 17, Table 1). group, have a poor outcome with current therapy. The The two patients with mutations at diagnosis relapsed with the experience gained from clinical trials using GSIs for Alzheimer’s same mutation(s). Neither acquired a different mutation, and the disease, where they inhibit the production of beta amyloid two patients who were wild type at diagnosis did not acquire a peptide from amyloid precursor protein, should expedite trials of mutation at relapse. these agents in T-ALL. The results in our cohort of patients are comparable with those reported in the younger aged patients (Table 2) and emphasise the heterogeneity of mutations that can disrupt Notch-1 function. Of Acknowledgements the 18 different mutations identified, only four have been previously described in the pediatric cohort or LF is supported by the UK Leukemia Research Fund and the Kay T-ALL cell lines.3 The HD domain is responsible for stable Kendall Leukaemia Fund. association of Notch-1 subunits6 and structural integrity in this MR Mansour1, DC Linch1, L Foroni1, AH Goldstone1 region is required to prevent exposure of the site to unregulated 1 proteolytic cleavage. Functional analysis using a Notch-sensitive and RE Gale 1Department of Haematology, Royal Free and University luciferase reporter assay has shown that leucine to proline point 3 College Medical School, 98 Chenies Mews, mutations in the HD domain lead to increased activity. At least London WC1E 6HX, UK 10 of the 13 different HD domain mutations detected in our E-mail: [email protected] cohort are likely to result in an altered conformation, either through insertion of a proline residue, or loss or gain of amino acids. Conversely, the C-terminal PEST domain is an important negative regulatory site required for ICN degradation, and loss of References this region also leads to increased activity in the Notch-sensitive reporter assay, probably through stabilization of ICN.3 All PEST 1 Pear WS, Aster JC, Scott ML, Hasserjian RP, Soffer B, Sklar J et al. domain mutations detected in our patients would lead to a Exclusive development of T cell neoplasms in mice transplanted similarly truncated protein. The probable functional consequence with bone marrow expressing activated Notch alleles. J Exp Med 1996; 183: 2283–2291. of the mutations identified in our study is supported by the 2 Ellisen LW, Bird J, West DC, Soreng AL, Reynolds TC, Smith SD loss of these mutations at remission and reappearance at et al. TAN-1, the human homolog of the notch gene, is relapse, which provides conclusive evidence that they are broken by chromosomal translocations in T lymphoblastic neo- acquired and are specifically associated with the leukemic clone. plasms. Cell 1991; 66: 649–661. The selective expansion of such clones indicates that the 3 Weng AP, Ferrando AA, Lee W, Morris JPt, Silverman LB, mutations are likely to provide some proliferative or survival Sanchez-Irizarry C et al. Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science 2004; 306: advantage to the cell. 269–271. The presence of mutations in our cohort was not restricted 4 Pui CH, Evans WE. Acute lymphoblastic leukemia. N Engl J Med to the young adult patients; 10 of the mutant-positive 1998; 339: 605–615. patients were over 30 years of age at presentation, and median 5 Lee SY, Kumano K, Masuda S, Hangaishi A, Takita J, Nakazaki K age of mutant-positive patients was 35 years compared to 20 et al. Mutations of the Notch1 gene in T-cell acute lymphoblastic years for wild-type patients. This suggests that a similar leukemia: analysis in adults and children. Leukemia 2005; 19: 1841–1843. pathogenic mechanism underlies the development of most 6 Sanchez-Irizarry C, Carpenter AC, Weng AP, Pear WS, Aster JC, cases of T-ALL irrespective of age, and the difference in Blacklow SC. Notch subunit heterodimerization and prevention of curability in the two groups is not related to a differing -independent proteolytic activation depend, respectively, on frequency of Notch mutations. The median presenting white a novel domain and the LNR repeats. Mol Cell Biol 2004; 24: cell count was 58.3 Â 109/l and did not differ significantly 9265–9273. between the two groups (P ¼ 70). 7 Marshall GM, Kwan E, Haber M, Brisco MJ, Sykes PJ, Morley AA et al. Characterization of clonal immunoglobulin heavy chain and I These results suggest that Notch mutations may be good cell receptor gamma gene rearrangements during progression of candidate markers for minimal residual disease detection, childhood acute lymphoblastic leukemia. Leukemia 1995; 9: particularly as they would not be susceptible to the antigenic 1847–1850.

Gain of 6p is an infrequent cause of increased PIM1 expression in B-cell non-Hodgkin’s lymphomas

Leukemia (2006) 20, 539–542. doi:10.1038/sj.leu.2404094; tive genomic hybridization (CGH) allows for the detection of published online 26 January 2006 over- and underrepresented sequences in tumor DNA as compared to normal DNA, and such genomic aberrations have been shown to have prognostic value in several studies of Gains and losses of genomic material are changes that play primary lymphomas. The serine/threonine kinase PIM1 is a central roles in tumor development and progression. Compara- known oncogene that has previously been shown to be

Leukemia