Letter to the Editor 1458 control. The Mcl-1-specific T-cell clone did not kill this cell line Ms. Bodil K. Jakobsen, Department of Clinical , (Figure 1c). University Hospital, Copenhagen, for HLA-typing of patient The lower rates of relapse in allogeneic transplantation samples. This study was supported by grants from the Danish compared with autologous bone marrow transplantation, the Medical Research Council, The Novo Nordisk Foundation, The striking clinical benefit of donor-lymphocyte infusions as well as Danish Cancer Society, The John and Birthe Meyer Foundation, the finding that human T cells can destroy chemotherapy- and Danish Cancer Research Foundation. resistant cell lines from chronic myeloid and multiple RB Sørensen1, OJ Nielsen2, P thor Straten1 and MH Andersen1 myeloma, have prompted development of immunotherapeutic 1 strategies against hematological cancers.3 Among these ap- Tumor Immunology Group, Institute of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark and proaches, active specific immunization or vaccination is 2Department of Hematology, State University Hospital, emerging as a valuable tool to boost the adaptive immune Copenhagen, Denmark. system against malignant cells. In this regard, the identification E-mail: [email protected] of leukemia-associated antigens is crucial. However, very few antigens are characterized in a conceptual framework in which the biology, microenvironment, and conventional disease management have been taken into consideration. Myeloid cell References factor-1 (Mcl-1) is a death-inhibiting member of the Bcl-2 family that is expressed in early differentiation. Elevated 1 Andersen MH, Becker JC, thor Straten P. The anti-apoptotic member levels of Mcl-1 have been reported for a number of solid and of the Bcl-2 family Mcl-1 is a CTL target in cancer patients. hematopoitic cancers, for example, B-cell chronic lymphocytic Leukemia 2005; 19: 484–485. 4–6 leukemia (B-CLL) and in AML and ALL upon relapse. In B-CLL 2 Andersen MH, Kvistborg P, Becker JC, thor Straten P. Identification patients, higher levels of Mcl-1 are strongly correlated with of an HLA-A1 restricted CTL epitope from Mcl-1. Leukemia 2005; failure to achieve complete remission after single-agent therapy. 19: 1084–1085. In multiple myeloma, Mcl-1 plays an important role in the 3 Mocellin S, Semenzato G, Mandruzzato S, Riccardo RC. Part II: 7 Vaccines for haematological malignant disorders. Lancet Oncol survival of malignant cells. We have reported that spontanous 2004; 5: 727–737. Mcl-1-specific T-cell responses are frequent in cancer patients 4 Zapata JM, Krajewska M, Krajewski S, Huang RP, Takayama S, and that these T-cells are highly cytotoxic against cancer cells. Wang HG et al. Expression of multiple apoptosis-regulatory genes in Hence, Mcl-1 appears to be a very attactrive target for human breast cancer cell lines and primary tumors. Breast Cancer anticancer immunotherapy both in hematopoetic and solid Res Treat 1998; 47: 129–140. cancers. 5 Tang L, Tron VA, Reed JC, Mah KJ, Krajewska M, Li G et al. Expression of apoptosis regulators in cutaneous malignant melano- ma. Clin Cancer Res 1998; 4: 1865–1871. 6 Shangary S, Johnson DE. Recent advances in the development of Acknowledgements anticancer agents targeting cell death inhibitors in the Bcl-2 protein family. Leukemia 2003; 17: 1470–1481. We thank Inge Marie Svane, Herlev University Hospital for 7 Zhang B, Gojo I, Fenton RG. Myeloid cell factor-1 is a critical collection of blood samples. We thank Professor A. Svejgaard and survival factor for multiple myeloma. Blood 2002; 99: 1885–1893.

Development of megakaryoblastic leukaemia in Runx1-Evi1 knock-in chimaeric mouse

Leukemia (2006) 20, 1458–1460. doi:10.1038/sj.leu.2404281; progenitors. Here, we report the development of megakaryo- published online 8 June 2006 blastic leukaemia in Runx1-Evi1 knock-in chimaeric mouse. Runx1-Evi1 knock-in chimaeric mice were created by injecting recombinant TT2 ES cells3 into wild-type blastocyst.4 Chromosomal translocations involving the Runx1 gene create We created six of such chimaeric mice, and five of them showed various chimaeric proteins that are believed to cause human sudden deaths after 7 months of age without any significant leukaemia. Runx1-ETO and Runx1-Evi1, generated as conse- finding in post mortem. Interestingly, one of the chimaeric mice quences of t(8;21) and t(3;21), respectively, share molecular that died at 5 months of age showed marked hepatospleno- structural similarities; they both contain DNA-binding domain megaly. Wright–Giemsa staining of stump preparation from the of Runx1 and transcriptional repression domains from either enlarged spleen demonstrated massive infiltration of large ETO or Evi1. Despite such similarity, the subtypes of leukaemia dysplastic cells, some of which contained multi-lobulated related to each of the chimaeric protein are different; Runx1- nuclei with various size of cytoplasm reminiscent of megakaryo- ETO typically occurs with acute myelocytic leukaemia of M2 blastic leukaemia (Figure 1a). Histology section showed subtype in the French–American–British classification, whereas disrupted gross architecture of the spleen, with white and red Runx1-Evi1 is mostly associated with megakaryoblastic leukae- pulp intermingling (Figure 1c). In the liver, substantial infiltra- mia of M7 subtype or megakaryoblastic crisis in chronic tion of leucocytes was observed around the portal vein myelocytic leukaemia. Experimental animals that express the (Figure 1d). Most of the infiltrating cells consisted of reactive fusion proteins in the hematopoietic cells have been created to , with partial presence of the dysplastic megakaryo- recapitulate the diseases. Both Runx1-ETO transgenic1 and cytic cells observed in the spleen. Multiple fibrin thrombi were conditional knock-in2 mice do not develop leukaemia by itself identified in the portal vein, indicating the occurrence of and require additional genetic aberrations to transform myeloid disseminated intravascular coagulation. These findings all

Leukemia Letter to the Editor 1459

Figure 1 Development of megakaryoblastic leukaemia in Runx1-Evi1 chimaeric mouse. (a) Wright–Giemsa staining of stump preparation from spleen (objective lens (OL), Â 40/0.65; original magnification (OM), Â 400). (b) Electron micrograph of spleen cells (OM, Â 3000). M, myeloid cell; Meg, megakaryocytic cell. (c, d) Hematoxylin–eosin staining of sections from spleen (c) (OL, Â 10/0.40; OM, Â 100) and liver (d) (OL, Â 20/ 0.70; OM, Â 200).

indicate an aggressive form of leukaemia. The electron Health, Labour and Welfare, and Japanese Society for the microscopic analysis of the infiltrating cells in the spleen Promotion of Science. showed 20% of the cells positive for -peroxidase, K Maki1, T Yamagata1, I Yamazaki2, H Oda3 and K Mitani1 substantiating megakaryocytic origin of the leukaemic cells 1 (Figure 1b). Taken together, we conclude that this chimaeric Department of Haematology, Dokkyo Medical University School of Medicine, Tochigi, Japan; mouse developed megakaryoblastic leukaemia. 2Department of Clinical Laboratory and Pathology, The key aspect of our observation is that Runx1-Evi1 protein is Inoue Memorial Hospital, Chiba, Japan and leukaemogenic per se, unlike Runx1-ETO. Such clear difference 3Department of Pathology, Tokyo Women’s Medical in the pathophysiological outcome likely arises from the Evi1 University, Tokyo, Japan portion of Runx1-Evi1 protein. Evi1 is reported to stimulate E-mail: [email protected] activator protein 1 activity,5 repress transforming growth factor- b signaling,6 and inhibit c-Jun N-terminal kinase function.7 Such versatile function of Evi1 may underlie stronger oncogenic capacity of Runx1-Evi1 than Runx1-ETO. Another important References aspect is that the affected lineage in human is conserved in the experimental animal. Our observation indicates strong causal 1 Yuan Y, Zhou L, Miyamoto T, Iwasaki H, Harakawa N, Hether- relationship between the expression of Runx1-Evi1 protein and ington CJ et al. AML1-ETO expression is directly involved in the megakaryoblastic leukaemia. Indeed, the Runx1-Evi1 chimaeric development of acute myeloid leukemia in the presence of gene was isolated from a patient developing megakaryoblastic additional mutations. Proc Natl Acad Sci USA 2001; 98: 10398– 10403. crisis in chronic myelocytic leukaemia that accompanied 2 Higuchi M, O’Brien D, Kumaravelu P, Lenny N, Yeoh EJ, Downing 8 emergence of t(3;21). It is unknown whether Runx1-Evi1 is JR. Expression of a conditional AML1-ETO oncogene bypasses preferentially oncogenic in megakaryoblast, or show exclusive embryonic lethality and establishes a murine model of human maturation block in the megakaryocytic lineage when expressed t(8;21) acute myeloid leukemia. Cancer Cell 2002; 1: 63–74. in early haematopoietic cells. Foetal liver cells from Runx1-Evi1 3 Yagi T, Tokunaga T, Furuta Y, Nada S, Yoshida M, Tsukada T et al. A knock-in heterozygous embryo can give rise to dysplastic novel ES cell line, TT2, with high germline-differentiating potency. 4 Anal Biochem 1993; 214: 70–76. . Such abnormal progenitors may have per- 4 Maki K, Yamagata T, Asai T, Yamazaki I, Oda H, Hirai H et al. Dys- sisted in the adult bone marrow and expanded to cause massive plastic definitive hematopoiesis in AML1/EVI1 knock-in embryos. infiltration of the megakaryoblasts in the liver and spleen. Blood 2005; 106: 2147–2155. 5 Tanaka T, Nishida J, Mitani K, Ogawa S, Yazaki Y, Hirai H. Evi-1 raises AP-1 activity and stimulates c-fos promoter transactivation with dependence on the second zinc finger domain. J Biol Chem Acknowledgements 1994; 269: 24020–24026. 6 Kurokawa M, Mitani K, Irie K, Matsuyama T, Takahashi T, Chiba S This work was supported by Grants-in-Aid from the Ministries in et al. The oncoprotein Evi-1 represses TGF-b signalling by inhibiting Japan of Education, Culture, Sports, Science and Technology, and Smad3. Nature 1998; 394: 92–96.

Leukemia Letter to the Editor 1460 7 Kurokawa M, Mitani K, Yamagata T, Takahashi T, Izutsu K, 8 Mitani K, Ogawa S, Tanaka T, Miyoshi H, Kurokawa M, Mano H Ogawa S et al. The evi-1 oncoprotein inhibits c-Jun N-terminal et al. Generation of the AML1-EVI-1 fusion gene in the t(3;21) kinase and prevents stress-induced cell death. EMBO J 2000; 19: (q26;q22) causes blastic crisis in chronic myelocytic leukemia. 2958–2968. EMBO J 1994; 13: 504–510.

The role of the bax gene polymorphism G(À248)A in chronic lymphocytic leukemia

Leukemia (2006) 20, 1460–1461. doi:10.1038/sj.leu.2404280; Untreated patients only published online 8 June 2006 5 p = 0.04 GA (n = 20) M)

Two recent publications in Leukemia have investigated µ 4 GG (n = 46) the potential role of the bax G(À248)A polymorphism in 1,2 chronic lymphocytic leukemia (CLL), which pertain to our 3 3 4

previous study, and that of Saxena et al. Our single centre values ( 50 study of 203 patients showed that this polymorphism signifi- 2 cantly influenced Bax protein expression, overall survival (P ¼ 0.05) and in particular survival from first treatment 1 (P ¼ 0.012). In contrast to the original paper by Saxena et al.4 we showed no difference in allele frequency between CLL Fludara LD 0 samples and those derived from healthy volunteers, nor an GA GG increased incidence of the polymorphism in advanced stage 2 disease. Unfortunately, Nuckel et al. misquote our work by Figure 1 In vitro sensitivity (LD50) to fludarabine in 66 previously stating that we showed an association between the polymorph- untreated CLL patients defined by bax genotype. ism and advanced stage disease F we did not (P ¼ 0.62). In all 2 other aspects, Nuckel et al. assessed clinical and prognostic 1 parameters that we and Skogsberg et al.1 had previously centre study such as Skogsberg et al. More disconcerting is how the 98 patient ‘subset’ were selected? It is clear from Figure 3b identified as irrelevant with regards to the bax G(À248)A 1 polymorphism. Crucially, they failed to address the pivotal that Skogsberg et al. chose to look at 73 GG patients who had question of the role of the polymorphism on therapeutic died. It is clear that for the GA subgroup they chose dead and response. alive patients. Why did they not just compare the dead GG Although many of the findings from the study of Skogsberg subgroup with the dead GA subgroup? – this would have at least et al.1 (463 patients) and our own are compatible, there are a stopped any, albeit unintentional bias being introduced. How number of major differences. (1) They failed to identify any did the authors choose which of the over 60 live GA subgroup to include in this analysis? significant overall survival difference (median survival 85 1 months versus 102 months; P ¼ 0.21 in 350 patients) or survival Skogsberg et al. stated that they could find no difference in from first treatment (median survival 36 months versus 63 bax RNA expression for the different polymorphic groups. months; P ¼ 0.26 in 98 patients). (2) They could not demonstrate However, they only studied 35 patient samples and failed to a difference in bax RNA expression between the polymorphic state how many of these samples were derived from patients groups. These differences between the two studies are worthy of who had received prior therapy. This is important as it has been shown that previous treatment exposure is a contributory factor further comment. 3–5 Our study was a single centre study in which all the clinical in determining Bax protein expression. In our study, we data was complete, whereas in the study of Skogsberg et al.1 (a measured Bax protein (122 patients) not RNA and found multicentred international collaboration involving seven centres overlapping, but significantly different Bax expression between in three countries), there were some significant omissions of the different polymorphic groups. In new data presented here, clinical data. Survival from treatment data was presented for we show that CLL samples derived from previously untreated À only 98 patients when their study contains 154 Binet B/C patients with the bax G( 248)A polymorphism are more patients. In addition, with a median follow-up of 60 months, a resistant to in vitro fludarabine (Figure 1). However, the large significant number of the 243 stage A patients would also have overlap in fludarabine LD50 values between the polymorphic required therapy and could therefore have been included too. groups indicate that bax genotype is not the only determinant of drug sensitivity in CLL cells. Despite this, their study showed a 27-month difference in 1 survival from therapy between the different polymorphic groups As outlined above, the Skogsberg et al. study has several flaws (36 months versus 63 months; P ¼ 0.26) that is, a trend towards and we look forward to larger prospective studies in CLL and worse survival. We make explicit in our study that virtually all other human malignancies that will hopefully offer definitive the patients were entered into the prevailing Medical Research evidence of the importance of this single-nucleotide polymorph- Council CLL study at the time. This means that the indications to ism in determining treatment response and clinical outcome. commence therapy and disease assessment were standardized C Fegan1,4, J Starczynski2, G Pratt2,3 and C Pepper1 according to internationally accepted criteria and indeed the 1Department of Haematology, Wales School of Medicine, therapies the patients received were not too dissimilar – very Cardiff University, Heath Park, Cardiff, UK; difficult to control for in a retrospective, multinational, multi- 2Department of Haematology, Heart of England NHS Trust,

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