Correspondence 2250 Table 1 and clinical details of the eight patients with dup(21)

Patient no. Sex/age WBC EFS OS AML1 Form of (years) x109/l (months) (months) signals/cell dup(21)

2848 M/5 2 57 62+ 5 LA 46,XY,dup(21)(q?) 3382 M/11 6 16 39 5 SM 46,XY,i(9)(q10),del(11)(q2?1),der(21)dup(21)(q?) 3767 F/10 1 32 35+ 4–6 M 46,XX,dup(21)(q?) 4414 M/7 9 8 8 5+ M 45,XY,t(6;19)(p21;p13),der(7)t(7;15)(p1;q1),del(11)(p13), -15,del(16)(q2),ider(21)(q10)dup(21)(q?) 4780 M/10 80 13 + 13+ 5 SA 45,XY,À11,del(12)(p1?2),der(20)t(11;20)(q?;q?),dup(21)(q?) 5655 F/8 55 8+ 8+ 4–6 M 46,XX,del(1)(q4?),del(6)(q1?5),del(7)(q2?2q3?1),dup(21)(q ?) 5674 M/6 55 5+ 5+ 6+ SA 47,XY,+X,dup(21)(q?) 5898 M/5 31 NK NK 4–6 M 47,XY,+X,del(16)(q13),i(17)(q10),ider(21)(q10)dup(21)(q?)/ 47,idem,add(7)(p1) EFS, event-free survival; OS, overall survival; LA, large acrocentric; SA, small acrocentric; M, metacentric; SM, small acrocentric.

Dr John Crolla and his team, Wessex Regional Genetics References Laboratory, Salisbury, for growing and preparation of the AML1- specific cosmids; the Clinical Trial Service Unit (CTSU), Oxford, 1 Harewood L, Robinson H, Harris R, Jabbar Al-Obaidi MS, Jalali GR, for clinical and survival data. We are grateful to the following Martineau M et al. Amplification of AML1 on a duplicated UKCCG cytogenetic laboratories for providing cytogenetic and 21 in acute lymphoblastic : a study of 20 FISH data as well as fixed cell suspensions: West Midlands cases. Leukemia 2003; 17: 547–553. 2 Jabbar Al-Obaidi MS, Martineau M, Bennett CF, Franklin IM, Regional Genetics Services, Birmingham Women’s Hospital; Goldstone AH, Harewood L et al. ETV6/AML1 fusion by FISH Haematology Cytogenetics Laboratory, University Hospital of in adult acute lymphoblastic leukemia. Leukemia 2002; 16: Wales, Cardiff; SE Scotland Cytogenetics Services, Western 669–674. General Hospital, Edinburgh; LRF Centre for Childhood Leuke- 3 Mikhail FM, Serry KA, Hatem N, Mourad ZI, Farawela HM, El mia, Great Ormond Street Hospital, London; Cytogenetics Kaffash DM et al. AML1 gene over-expression in childhood acute Department, St James Hospital, Leeds; Merseyside & Cheshire lymphoblastic leukemia. Leukemia 2002; 16: 658–668. 4 Morel F, Herry A, Le Bris M-J, Douet-Guilbert N, Le Calvez G, Genetics Laboratory, Liverpool Women’s Hospital, Liverpool; Marion V et al. AML1 amplification in a case of childhood Oncology Cytogenetics Service, Christie Hospital, Manchester; acute lymphoblastic leukemia. Cancer Genet Cytogenet 2002; North Trent Cytogenetics Service, Sheffield Children’s Hospital 137: 142–145. NHS Trust, Sheffield. 5 Alvarez Y, Coll MD, Bastida P, Ortega JJ, Caballin MR. AML1 amplification in a child with acute lymphoblastic leukemia. Cancer HM Robinson1 1Leukaemia Research Fund Cytogenetics Group, Genet Cytogenet 2003; 140: 58–61. ZJ Broadfield1 Cancer Sciences Division, University of KL Cheung1 Southampton, Southampton, UK; L Harewood1 2Clinical Trial Service Unit, Radcliffe Infirmary, 1 Oxford, UK; RL Harris 3 GR Jalali1 Paediatric Oncology, John Radcliffe Hospital, M Martineau1 Oxford, UK AV Moorman1 KE Taylor1 S Richards2 C Mitchell3 CJ Harrison1

Comprehensive analysis of gene alterations in acute megakaryoblastic leukemia of Down’s syndrome

Leukemia (2003) 17, 2250–2252. doi:10.1038/sj.leu.2403121 of AMkL. TMD is developed in about 10% of DS infants and Published online 21 August 2003 resolves spontaneously in most cases. However, 20% of TMD TO THE EDITOR cases develop AMkL within 3 years, suggesting that additional genetic alterations might occur during the progression from TMD Down’s syndrome (DS) children have the 10- to 20-fold increased to AMkL. Recently, somatic of the GATA1 gene were risk of developing acute leukemia compared with non-DS reportedly associated with AMkL of DS children.1 In addition, it children. (AML) in DS children shows was reported that GATA1 mutations were present in the TMD unique characteristics as to be a predominance of the mega- blasts, and that the identical GATA1 was found in karyoblastic leukemia (AMkL) and a preceding history of the sequential samples collected from a patient during TMD and transient myeloproliferative disorder (TMD), whose blasts are subsequent AMkL.2–5 These results suggested that GATA1 morphologically and phenotypically indistinguishable from those mutations are an early event in DS leukemogenesis and contribute

Leukemia Correspondence 2251 Table 1 Mutations of GATA1 and genes in AMkL and TMD patients with DS

UPN Age (years) Sex GATA1 mutation p53 mutation

AMkL-1 1 year 2 months M 22 del 1 bp None AMkL-2 1 year 10 months M 90–91 ins 1 bp None AMkL-3 1 year 8 months F 31–32 ins 1 bp None AMkL-4 2 year 1 months F 104–105 ins 1 bp ex6 199Gly-Arg AMkL-5 2 year 8 months F 193–194 ins 20 bp None AMkL-6 1 year 11 months F 24–37 del 14 bp None AMkL-7 1 year 9 months M 128C-T (37Gly-stop) None AMkL-8 NA F 53 T-C (12Ser-Pro) None AMkL-9 1 year 11 months M 208 del 1 bp None AMkL-10 1 year 9 months F 297–298 ins 22 bp None AMkL-11 1 year 9 months F 202–213 del 12 bp and ins 5 bp None AMkL-12 1 year 9 months M 94C-T (Ser-Phe) None AMkL-13 2 years F 62–63 ins 1 bp ex6 200Asn del 1 bp (frame shift) AMkL-14 1 year 7 months M 62–63 ins 1 bp None TMD-1 5 day F 107T-C (30Ser-Pro) None TMD-2 7 day F 22G-A (no ) None TMD-3 10 day M 22G-A (no start codon) None TMD-4 3 day M 29 C-T (4Leu-Ser) ex5 163Tyr-His Mutations of GATA1 and p53 genes in AMkL and TMD patients with DS are shown. GATA1 mutations were found in all AMkL and TMD cases. All GATA1 mutations were clustered within 2.p53 mutations were found in two of the 14 AMkL cases and one of the four TMD cases. No FLT3, N-RAS and c-KIT mutation was found in all cases. NA, not applicable.

to both pathogenesis of TMD and AMkL, while the acquisition of Mutations of GATA1 were found in all AMkL and TMD cases additional genetic alterations might be necessary for the progres- and clustered within the first coding exon (exon 2) consistent sion from TMD to AMkL. To date, a lot of genetic alterations, with previous reports (Table 1). Each of the mutations from 11 which are involved in the pathogenesis of leukemia, have been cases was an insertion or a deletion, which altered the reading accumulated. Especially, mutations of FLT3, N-RAS, p53 and cKIT frame of GATA1 resulting in the introduction of a premature genes are frequently found in AML, and likely to be associated stop codon. Five mutations were missense mutations; one with the disease progression. To examine whether these mutations mutation resulted in a stop codon and two mutations resulted in are involved in the progression of TMD to AMkL, we comprehen- a loss of the first initiation codon. To evaluate additional sively analyzed these mutations in AMkL and TMD of DS. gene mutations in DS AMkL, we analyzed mutations in FLT3, N- The study population included 14 AMkL and four TMD cases RAS, p53 and cKIT genes. p53 mutations were found in two of of DS. The diagnosis of AMkL and TMD was based on 14 AMkL and one of four TMD cases, although no FLT3, N-RAS morphology, histopathology, the expression of leukocyte and cKIT mutation was found in all AMkL and TMD cases differentiation antigens and/or the French–American–British (Table 1). classification. The diagnosis of DS was confirmed by the In this study, we comprehensively analyzed the gene conventional cytogenetic analysis. samples from mutations, which were commonly found in AML, although we the patients with AMkL and TMD were subjected to the Ficoll– could not define the specific gene mutations related to the Hypaque (Pharmacia LKB, Uppsala, Sweden) density-gradient evolution from TMD to AMkL of DS. Since cKIT mutations in centrifugation and cryopreserved in liquid nitrogen before use AML were restrictedly found in core-binding factor , after obtaining informed consent from all parents of the patients. no mutations in AMkL were well acceptable.9 Mutations of FLT3 High-molecular weight DNA was extracted from the samples by and N-RAS are so far the most frequent gene alterations the standard method. For screening of GATA1 mutations, we reported to be involved in AML. An incidence of FLT3 amplified genomic DNA corresponding to exon 2 of GATA1 by mutations is apparently associated with the age of patients polymerase chain reaction (PCR) using the primers: forward with AML,7 although its association of N-RAS mutations primer, 50-GGATTTCTGTGTCTGAGGACC-30 and reverse pri- has not been demonstrated. FLT3/ITD has been found in mer, 50-CAAGACAGCCACTCAATGGAG-30. Amplified pro- approximately 20% of adult patients, but in 34% of patients ducts were cloned into pGEM-T Easy vector (Promega, over 55 years of age. In contrast, it has been found in only Madison, WI, USA) and sequenced on a DNA sequencer (310; approximately 10% of childhood patients. Furthermore, it Applied Biosystems, Foster City, CA, USA) using a BigDye has been found in only one of 23 (4.3%) infant AML patients terminator cycle sequencing kit (Applied Biosystems). FLT3 gene under 1 year of age. The association of FLT3/D835Mt with mutations of internal tandem duplication of the juxtamembrane aging remains unclear, while our study showed that it was domain (FLT3/ITD) and in the activation loop (FLT3/D835Mt), found in 44 of 713 (6.2%) adult patients and four of 100 (4%) N-RAS gene mutations of codons 12, 13 and 61 and p53 gene childhood patients with AML. These results indicated that the mutations of 5–8 were examined as previously reported absence of FLT3 mutations in TMD and AMkL of DS is likely to and were confirmed by the sequencing procedure.6–8 To detect be related to the lower age of patients. It was reported that p53 mutations at D816 within the activation loop of the cKIT gene, mutations might be involved in the evolution from TMD to we amplified exon 17 of the cKIT gene by genomic PCR using AMkL, because two of three AMkL harbored p53 mutations, but the forward primer, 50-CCTCCAACCTAATAGTGTA-30 and the all seven TMD did not.10 However, we could not demonstrate reverse primer, 50-TCCTTTAACCACATAATTACAATCAT-30, and the relationship between the evolution of AMkL and p53 amplified products were directly sequenced. mutations.

Leukemia Correspondence 2252 Approximately 60% of AML cases reportedly harbored References mutations in FLT3, N-RAS, p53 or cKIT gene, while it remains unclear which kind of genetic events are involved 1 Wechsler J, Greene M, McDevitt MA et al. Acquired mutations in in the disease progression in the remaining cases. In addition, GATA1 in the megakaryoblastic leukemia of . Nat it remains unclear why GATA1 mutations uniquely occur in Genet 2002; 32: 148–152. DS children. Further studies are required to elucidate this 2 Mundschau G, Gurbuxani S, Gamis AS, Greene ME, Arceci RJ, Crispino JD. Mutagenesis of GATA1 is an initiating event in Down issue, although the same etiology might be involved in the syndrome leukemogenesis. Blood 2003; 101: 4298–4300. acquisition of GATA1 mutations and the evolution of AMkL 3 Hitzler JK, Cheung J, Li Y, Scherer SW, Zipursky A. GATA1 from TMD. mutations in transient leukemia and acute megakaryoblastic leukemia of Down syndrome. Blood 2003; 101: 4301–4304. 4 Groet J, McElwaine S, Spinelli M et al. Acquired mutations in GATA1 in neonates with Down’s syndrome with transient myeloid disorder. Lancet 2003; 361: 1617–1620. 5 Rainis L, Bercovich D, Strehl S et al. Mutations in exon 2 of GATA1 are early events in megakaryocytic malignancies associated with Acknowledgements trisomy 21. Blood 2003; 102: 981–986. 6 Nakano Y, Naoe T, Kiyoi H et al. Prognostic value of p53 gene This work was supported by Grants-in-Aid from the Japanese mutations and the product expression in de novo acute myeloid Ministry of Health, Labour and Welfare and the Ministry of leukemia. Eur J Haematol 2000; 65: 23–31. Education, Culture, Sports, Science and Technology. 7 Kiyoi H, Naoe T. FLT3 in human hematologic malignancies. Leukemia Lymphoma 2002; 43: 1541–1547. Y Hirose1,5 1Department of Infectious Diseases, Nagoya 8 Kiyoi H, Naoe T, Nakano Y et al. Prognostic implication of FLT3 K Kudo2,5 University School of Medicine, Nagoya, Japan; and N-RAS gene mutations in acute myeloid leukemia. Blood 1999; H Kiyoi1 2Department of Pediatrics, Nagoya University 93: 3074–3080. Y Hayashi3 Graduate School of Medicine, Nagoya, Japan; 9 Boissan M, Feger F, Guillosson JJ, Arock M. c-Kit and c-kit mutations 3 T Naoe4 Department of Pediatrics, Graduate School of in mastocytosis and other hematological diseases. S Kojima2 Medicine, University of Tokyo, Tokyo, Japan; J Leukocyte Biol 2000; 67: 135–148. 4Department of Hematology, Nagoya University 10 Malkin D, Brown EJ, Zipursky A. The role of p53 in Graduate School of Medicine, Nagoya, Japan differentiation and the megakaryocytic leukemias of Down syndrome. Cancer Genet Cytogenet 2000; 116: 1–5.

T-cell signaling and costimulatory molecules in B-chronic lymphocytic leukemia (B-CLL): an increased abnormal expression by advancing stage

Leukemia (2003) 17, 2252–2254. doi:10.1038/sj.leu.2403100 interaction induced tumor rejection and immunity to tumor rechallenge in a T-cell-dependent manner. The CD28 molecule TO THE EDITOR is constitutively expressed on T cells, while CTLA-4 is primarily localized intracellular and only transiently is expressed on the B-cell chronic lymphocytic leukemia (B-CLL) is a clonal surface upon activation. lymphoproliferative disorder with a highly variable clinical The aim of the present study was to analyze the expression of course and prognosis, which is associated with immune CD54, CD28 and CTLA-4 molecules, as well as the CD3z chain dysregulation, especially in the progressive phase. The leukemic in T cells of B-CLL patients and relate to the stage of the disease. may be regulated by inhibitory or growth-promoting B-CLL patients with progressive (n ¼ 7) and nonprogressive signals by autologous T cells. These cells have been shown to (n ¼ 11) disease entered the study. The criteria for diagnosis and 5 exhibit functional abnormalities related to disease activity.1,2 disease progression have been described elsewhere. Age- The adhesion molecule CD54 is constitutively expressed on T matched healthy donors (n ¼ 11) were used as controls. T cells cells and upregulated during immune activation. The CD3z were enriched from peripheral blood mononuclear cells by chain connects the T-cell (TCR) to the intracellular nylon wool separation. Direct and indirect surface and signaling machinery leading to T-cell activation.3 For an intracellular immunofluorescence staining were performed. efficient T-cell activation engagement of costimulatory receptors Cells were analyzed by flow cytometry using a FACScan and is required as well. A balance between the CD28- and CTLA-4- data were processed by the CellQuest software. For comparison mediated regulatory signals seems to be indispensable. Upon of independent variables, the Kruskal–Wallis and Mann–Whit- TCR stimulation accompanied by CD28/B7 interaction, cyto- ney U-test were applied. The Wilcoxon signed-rank test was kine production and upregulation of cell surface molecules used for pairwise variables. Spearman’s rank correlation leading to T-cell activation and proliferation occur. Lack of measured the correlation between marker expression and CD28 costimulation induces either T-cell apoptosis or anergy.3 disease stage. All statistics were performed using the Stat View Reduced IL-2 production by T cells, IL-2 receptor down- software. þ þ regulation and G1-phase arrest is induced when CTLA-4 binds A higher frequency of CD4 /CD54 T cells was found in to the B7 family receptors. CTLA-4-deficient mice died from a patients with progressive disease compared to controls (Po0.01) þ lymphoproliferative disease, characterized by multiple organ (Figure 1). In the CD8 subset, the frequency of CD54 cells was infiltration and T-cell activation.4 Blocking of the CTLA-4/B7 higher than within the CD4 subset in patients with nonpro- gressive disease and in controls (Po0.05 and 0.01) (Table 1). 5These authors equally contributed to this work Moreover an increased frequency of CD4 þ /CD54 þ T cells was

Leukemia