Letters to the Editor 246 7 Life Sciences Division, Department of Statistics, University of 3 Michaud J, Simpson KM, Escher R, Buchet-Poyau K, Beissbarth T, California, Lawrence Berkeley National Laboratory, Carmichael C et al. Integrative analysis of RUNX1 downstream Berkeley, CA, USA and pathways and target genes. BMC Genomics 2008; 31: 363. 8Department of Pediatric Hemato-oncology, Radboud 4 Mikhail FM, Sinha KK, Saunthararajah Y, Nucifora G. Normal and University Nijmegen Medical Center, Nijmegen, transforming functions of RUNX1: a perspective. J Cell Physiol The Netherlands 2006; 207: 582–593. E-mails: [email protected] or 5 Renneville A, Roumier C, Biggio V, Nibourel O, Boissel N, [email protected] Fenaux P et al. Cooperating gene mutations in acute myeloid 9These authors contributed equally to this work. leukemia: a review of the literature. Leukemia 2008; 22: 915–931. 6 Ripperger T, Steinemann D, Go¨hring G, Finke J, Niemeyer CM, Strahm B et al. A novel pedigree with heterozygous germline RUNX1 mutation causing familial MDS-related AML: can these families serve as a multistep model for leukemic transformation? References Leukemia 2009; 23: 1364–1366. 7 Michaud J, Wu F, Osato M, Cottles GM, Yanagida M, Asou N et al. 1 Owen CJ, Toze CL, Koochin A, Forrest DL, Smith CA, Stevens JM In vitro analyses of known and novel RUNX1/AML1 mutations in et al. Five new pedigrees with inherited RUNX1 mutations causing dominant familial platelet disorder with predisposition to acute familial platelet disorder with propensity to myeloid malignancy. myelogenous leukemia: implications for mechanisms of pathogen- Blood 2008; 112: 4639–4645. esis. Blood 2002; 99: 1364–1372. 2 Song WJ, Sullivan MG, Legare RD, Hutchings S, Tan X, Kufrin D 8 Buijs A, Poddighe P, van WR, van SW, Borst E, Verdonck L et al. et al. Haploinsufficiency of CBFA2 causes familial thrombocyto- A novel CBFA2 single-nucleotide mutation in familial platelet penia with propensity to develop acute myelogenous leukaemia. disorder with propensity to develop myeloid malignancies. Blood Nat Genet 1999; 23: 166–175. 2001; 98: 2856–2858. Aberrant telomere structure is characteristic of resistant chronic lymphocytic leukaemia cells Leukemia (2010) 24, 246–251; doi:10.1038/leu.2009.213; and telomerase activity are further prognostic markers as published online 22 October 2009 unfavourable disease was associated with short telomeres4 (reviewed in Van Bockstaele et al.3). This, together with altered expression of several telomeric components,5 indicates that Telomeres are the capping structures of chromosome ends and telomeres are profoundly rearranged in CLL cells. are composed of repeated DNA sequences (B10 kb in adult A subgroup of CLL cells is resistant to DNA damage-induced somatic cells) and a specific set of associated proteins. The apoptosis in vitro (reviewed in Bouley et al.6) and patients regulation of telomere length results from the action of telomere- harbouring these cells inevitably develop the aggressive form of lengthening mechanisms, such as the telomerase complex disease, suggesting that this is a clinically relevant factor. Here, (hTERT, hTR and dyskerin), and of telomere-shortening mechan- we investigated whether specific telomere alterations can isms, such as replication and recombination. Telomerase distinguish CLL cells sensitive (CLL-S) or resistant (CLL-R) to activity is regulated in cis by the shelterin hexa–protein complex DNA damage-induced apoptosis in vitro. A total of 35 patients (TRF1, TRF2, hRAP1, POT1, TIN2 and TPP1) and epigenetic were included in this study (16 CLL-R and 19 CLL-S, clinical and factors.1,2 Proteins involved in DNA replication and repair are biological behaviours were presented in Table 1). Southern blot also associated in telomeric structure and function.2 The analysis showed a significant difference in telomere length shortening of telomere sequences upon cell divisions in most between the two subgroups of patients (Figure 1a). The mean somatic cells results in irreversible cell growth arrest called telomere length was twofold shorter (U-test, Po0.001) in senescence or in apoptosis. A change in telomere function is CLL-R compared with the CLL-S (3552±1151 bp for CLL-R one of the main mechanisms underlying the evolution and and 8010±1668 bp for CLL-S) (Figure 1b). Furthermore, we maintenance of cancer cells. Telomere erosion may be critical observed a positive correlation (R2 ¼ 0.675) between telomere in tumour suppression as it impairs cell proliferation. In restriction fragment length and the in vitro sensitivity to many cancer cells, this is circumvented by reactivating the apoptosis (Figure 1c) that appeared independent of previous expression and/or activity of telomerase or by homologous clinical treatment of patients (Figures 1c and d). The use of the recombination.1,2 Spearman’s rank correlation showed that this association was Chronic lymphocytic leukaemia (CLL) is a B-cell malignancy significant (Po0.0001). These results clearly show that resistant with increased incidence among the elderly. The disease is due CLL cells have shorter telomeres than sensitive CLL cells. to an imbalance between cell death and proliferation resulting The mRNA level of hTERT was assessed by quantitative PCR analysis in an accumulation of malignant cells in the bone marrow and and revealed no significant difference (U-test, P40.05) between peripheral blood (reviewed in Van Bockstaele et al.3). Clinically, the two subgroups of patients (Figure 2a). We also analyzed the the disease varies from indolent, which may progress to/or mRNA level of the negative regulator of the telomerase, PinX1, appear at diagnostic as an aggressive incurable form. The most by quantitative PCR analysis in samples already included in the commonly used biological markers for the disease outcome are analysis for the expression of hTERT. No significant difference the type of chromosomal aberration and the mutational status of (U-test, P40.05) was observed between the two subgroups of immunoglobulin heavy-chain variable region genes (IgVH), cells (Figure 2b). Telomere maintenance and protection mainly often associated with the expression status of Zap70 tyrosine depends on the presence of the six shelterin components and kinase (reviewed in Van Bockstaele et al.3). The telomere length their stoichiometry at chromosome ends is crucial for telomere Leukemia Table 1 Clinical characteristics and outcome of CLL patients Patient’s ID Age Sex Binet’s Treatment Date of treatment Date of sampling IgVH mutation Matutes Chromosomal aberrations Apoptosis stage status score in vitro LLC-S 1 77 m A No January 2007 M 4 del 13q14 monoallelic Sensitive LLC-S 2 77 m A Yes 1994 April 2007 M 4 del 13q14 monoallelic Sensitive LLC-S 3 53 m A Yes 2001 April 2007 M 4 trisomy 12, t(14;17) Sensitive LLC-S 4 89 m A Yes March 2006 December 2006 UM 4 11q- Sensitive LLC-S 5 81 m A No March 2007 M 4 ND Sensitive LLC-S 6 78 m A No December 2006 ND 4 del 13q monoallelic Sensitive LLC-S 7 58 m B No December 2006 M 4 del 13q monoallelic Sensitive LLC-S 8 69 m A No November 2006 M 4 del 13q monoallelic Sensitive LLC-S 9 79 m A No November 2006 ND 4 ND Sensitive LLC-S 10 81 m A No January 2006 ND 5 del 13q monoallelic Sensitive LLC-S 11 63 m A No 2006 M 5 ND Sensitive LLC-S 12 81 f A No May 2007 ND 4 ND Sensitive LLC-S 13 73 f A No November 2006 M 5 ND Sensitive LLC-S 14 63 f A No April 2008 M 5 del 13q14 monoallelic, del 17p monoallelic Sensitive LLC-S 15 78 f A No May 2007 ND 5 ND Sensitive LLC-S 16 85 f A No March 2007 ND 5 ND Sensitive LLC-S 17 74 f A No January 2007 UM 5 trisomy 12, t(14;17 ) Sensitive LLC-S 18 73 f A No December 2006 M 4 ND Sensitive LLC-S 19 63 f A No May 2007 ND 4 ND Sensitive LLC-R 1 78 m A Yes 2002 March 2007 M 5 del 13q14 biallelic Resistant LLC-R 2 56 m C Yes January 2006 October 2006 ND 5 del 13q14 biallelic Resistant Letters to the Editor LLC-R 3 57 m A Yes 1997 January 2006 UM 4 del 13q14 monoallelic Resistant LLC-R 4 72 m A No July 2007 ND 5 Complex karyotype Resistant LLC-R 5 64 m A Yes 2003 January 2007 M 4 del 13q biallelic, del 17p Resistant LLC-R 6 70 m B Yes October 2006 April 2007 ND 4 del 13q14 biallelic, del 17p monoallelic Resistant LLC-R 7 89 m A No July 2007 UM 5 ND Resistant LLC-R 8 75 m ND No October 2007 ND 4 del 13q, del 11q Resistant LLC-R 9 76 m ND No October 2007 ND 5 del 13q, t(14;18) Resistant LLC-R 10 82 m A Yes January 2003 June 2004 M 5 del 13q14 biallelic, del 17p monoallelic Resistant LLC-R 11 68 f A Yes 2005 January 2007 M 5 del 13q14 biallelic, del 17p monoallelic Resistant LLC-R 12 76 f A/B Yes September 2006 April 2007 UM 5 del 13q14monoallelic, del 17p monoallelic Resistant LLC-R 13 60 f A No November 2006 UM 5 del 13q14 biallelic, del 17p monoallelic Resistant LLC-R 14 76 f A Yes January 2006 December 2005 UM 5 ND Resistant LLC-R 15 77 f C Yes 2000 2006 ND 4 ND Resistant LLC-R 16 75 f A Yes September 2007 March 2006 M 5 del 13q14 monoallelic, del 17p monoallelic Resistant Abbreviations: CLL, chronic lymphocytic leukaemia; del, deletion; f, female; IgVH, immunoglobulin heavy-chain variable genes; m, male; M, mutated; ND, not determined; t, translocation; UM, unmutated.