CD200 As a Prognostic Factor in Acute Myeloid Leukaemia

Letters to the Editor 566 6 Lamballe F, Tapley P, Barbacid M. TrkC encodes multiple 8 Liu Q, Schwaller J, Kutok J, Cain D, Aster JC, Williams IR et al. neurotrophin-3 receptors with distinct biological properties and Signal transduction and transforming properties of the TEL-TRKC substrate speciﬁcities. EMBO J 1993; 12: 3083–3094. fusions associated with t(12;15)(p13;q25) in congenital ﬁbro- 7 Tsoulfas P, Stephens RM, Kaplan DR, Parada LF. TrkC isoforms with sarcoma and acute myelogenous leukemia. EMBO J 2000; 19: inserts in the kinase domain show impaired signaling responses. 1827–1838. J Biol Chem 1996; 271: 5691–5697.

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CD200 as a prognostic factor in acute myeloid leukaemia

Leukemia (2007) 21, 566–568. doi:10.1038/sj.leu.2404559; abnormalities showed that CD200 was signiﬁcantly associated published online 25 January 2007 with worse survival (HR 1.68, 95% CI 1.08–2.62, P ¼ 0.02; Figure 1a and b).

The CD200 gene (aka MOX-2, OX-2) is located on chromosome 3 and encodes a type-1 membrane glycoprotein.1 This protein belongs to the immunoglobulin superfamily and is expressed on many different cell types including T and B lymphocytes and dendritic cells.2 CD200 binds multiple membrane receptor isoforms (CD200R) which have a more tissue restricted expression.3,4 The best characterized isoform, CD200R1, has a longer cytoplasmic tail containing three conserved tyrosine residues that can be phosphorylated.1 These phosphorylated residues interact with signalling adaptor molecules such as Shc, suggesting that the CD200R1 can signal upon binding CD200 ligand providing a more localized response than that provided by cytokines.5 Although CD200(À/À)-deficient mice appear grossly normal and live a normal lifespan, they are susceptible to tissue-specific autoimmunity, suggesting that the function of this protein is to induce immune suppression through the CD200R.6 CD200 also appears to have an alternative role in regulating osteoclast development.7 In leukaemia, CD200 has been shown to be upregulated in B cells from patients with chronic lymphocytic leukemia.8 More recently, CD200 has been shown to be an independent prognostic factor for patients with multiple myeloma.9 In this latter study, expression of CD200 was associated with a bad prognosis. Here we report that in acute myeloid leukaemia (AML), there is a correlation between CD200 expression and the presence of the core binding factor (CBF) associated abnormalities, t(8;21) and inv(16). However, at the same time CD200 expression was linked to worse overall survival in other AML subsets. These data indicate that CD200 is also of prognostic value in AML. Using a cohort of 184 AML trial patients (all processed and diagnosed at Cardiff as part of the Medical Research Council (MRC) AML Trials), complementary RNA was prepared from each sample and hybridized to Affymetrix Human 133A oligonucleotide arrays which allowed the analysis of CD200 gene expression. In approximately 43% of AML patients, CD200 had a ‘present’ Affymetrix call (CD200present) with the remainder giving an absent call (CD200absent). CD200present calls were highly correlated with abnormalities affecting CBF (24/28 vs 56/156, P ¼ 0.0001). There was no evidence of any association Figure 1 CD200 expression in patients with AML. (a) Kaplan– with age at diagnosis (mean ages: present 49 years vs absent 50 Meier plot of the overall survival in non-CBF leukaemia patients present absent years, P ¼ 0.7), WBC (P ¼ 0.3) or sex (P ¼ 0.6), however, larger expressing CD200 (CD200 ) or not (CD200 ). (b) Odds ratio numbers of patients will be required to provide more reliable plots of survival stratified by mutations affecting CBF (t(8;21) and inv(16)). (c) Box and whisker plots depicting microarray data of the evidence and allow for adjustment of clinical and demographic normalized expression of CD200 in AML patients (n ¼ 184) classified w y parameters. Despite the association of CD200 expression with according to FAB criteria. M2 patients without a t(8;21). M2 patients these good risk subtypes, analyses of survival stratified for CBF with a t(8;21). nData outliers.

Leukemia Letters to the Editor 567

Figure 2 Validation of Affymetrix microarray CD200 expression. (a) CD200 gene expression in 20 AML patients was assayed with real-time RT-PCR and normalized to the housekeeping gene S14. The Pearson coefficient of correlation between Affymetrix and real-time RT-PCR is shown. (b) CD200 protein expression was determined by flow cytometric analysis in AML blasts from another 15 AML patients. The upper panels show representative cytometric plots of typical AML blasts (left) expressing CD200 (right). Quadrants delimit background fluorescence of control stained cells. The lower panel shows the Pearson coefficient of correlation between Affymetrix and flow cytometric (mean fluorescence intensity; (MFI)) CD200 values.

We also examined the correlation of level of CD200 suggests that CD200 may have independent prognostic value gene expression with AML subtype (Figure 1c). As might in AML. be expected from the high frequency of CD200present patients Our data analysis was consistent using all available Affymetrix in t(8;21) leukaemias, these patients also significantly probe sets (209582_s_at; 209583_s_at). CD200 expression by overexpressed CD200 by 1.8-fold when compared to FAB-M2 microarray analysis was validated by real-time RT-PCR for 20 patients without this cytogenetic abnormality (P ¼ 0.03). AML patients for which corresponding messenger ribonucleic Induction of CD200 may be a direct consequence of AML1- acid samples were still available (Figure 2a). These data sets ETO expression, because human primary cells transduced were highly correlative (r ¼ 0.763, P ¼ 0.001). We also carried with this fusion gene also overexpress CD200 (A.Tonks, out assessment of CD200 expression at the protein level by flow submitted). Furthermore, patients expressing an inv(16) cytometric analysis using another set of 15 patients for which mutation (generally associated with FAB-M4) also significantly archival material was available. Cells were stained with PE- overexpressed CD200 when compared to M4 patients conjugated anti-CD200 (clone MRCOX-104) in combination without an inv(16) mutation (1.3-fold; P ¼ 0.02). Additionally, with CD45-FITC and CD34-PERCP (all from Becton Dickinson, Cox regression shows that, when adjusted for the type of BD, UK). Again, we found a correlation (r ¼ 0.631; P ¼ 0.01) leukaemia, the level of CD200 expression was significantly between Affymetrix CD200 expression and protein expression associated with worse overall survival (P ¼ 0.04). This data (Figure 2b).

Leukemia Letters to the Editor 568 The mechanism by which CD200 influences survival is recognizes a novel receptor on macrophages implicated in the intriguing. The function of CD200 includes induction of control of their function. Immunity 2000; 13: 233–242. immune suppression through interaction with its receptor, 2 Barclay AN, Wright GJ, Brooke G, Brown MH. CD200 and CD200R,3–6 suggesting a possible role for this molecule in membrane protein interactions in the control of myeloid cells. Trends Immunol 2002; 23: 285–290. influencing immune surveillance. In conclusion, these data 3 Wright GJ, Cherwinski H, Foster-Cuevas M, Brooke G, Puklavec MJ, show for the first time the potential role of CD200 in influencing Bigler M et al. Characterization of the CD200 receptor family in survival in AML and its association with CBF leukaemias. mice and humans and their interactions with CD200. J Immunol 2003; 171: 3034–3046. 4 Gorczynski RM, Chen Z, Lee L, Yu K, Hu J. Anti-CD200R Acknowledgements ameliorates collagen-induced arthritis in mice. Clin Immunol 2002; 104: 256–264. We thank Amanda Gilkes and Megan Musson (Cardiff University) 5 Songyang Z, Margolis B, Chaudhuri M, Shoelson SE, Cantley LC. The phosphotyrosine interaction domain of SHC recognizes for their technical assistance in processing microarray samples. tyrosine-phosphorylated NPXY motif. J Biol Chem 1995; 270: We are grateful to the MRC for access to material from patients 14863–14866. enrolled in the NCRI clinical trials. AT was supported by 6 Hoek RM, Ruuls SR, Murphy CA, Wright GJ, Goddard R, Zurawski Leukaemia Research, UK. SM et al. Down-regulation of the macrophage lineage through interaction with OX2 (CD200). Science 2000; 290: 1768–1771. A Tonks, R Hills, P White, B Rosie, KI Mills, AK Burnett and 7 Lee L, Liu J, Manuel J, Gorczynski RM. A role for the RL Darley immunomodulatory molecules CD200 and CD200R in regulating Department of Haematology, School of Medicine, bone formation. Immunol Lett 2006; 105: 150–158. Cardiff University, Cardiff, UK 8 McWhirter JR, Kretz-Rommel A, Saven A, Maruyama T, Potter KN, E-mail: [email protected] Mockridge CI et al. Antibodies selected from combinatorial libraries block a tumor antigen that plays a key role in immunomodulation. Proc Natl Acad Sci USA 2006; 103: 1041–1046. References 9 Moreaux J, Hose D, Reme T, Jourdan E, Hundemer M, Legouffe E et al. CD200 is a new prognostic factor in Multiple Myeloma. Blood 1 Wright GJ, Puklavec MJ, Willis AC, Hoek RM, Sedgwick JD, Brown First Edition Paper, prepublished online August 31 2006; MH et al. Lymphoid/neuronal cell surface OX2 glycoprotein doi:10.1182/blood-2006-06-029355.

Infusion of allogeneic-related HLA mismatched mesenchymal stem cells for the treatment of incomplete engraftment following autologous haematopoietic stem cell transplantation

Leukemia (2007) 21, 568–570. doi:10.1038/sj.leu.2404550; patient with an end-stage severe aplastic anaemia.8 We now published online 25 January 2007 report on a second patient with bone marrow failure secondary to incomplete engraftment after autologous haematopoietic stem cell transplantation. This patient received infusion of allogeneic MSC. Haematopoiesis recovery was observed with a Primary graft failure is rarely observed after autologous parallel improvement of in vitro clonogenic assay and detection haematopoietic stem cell transplantation, and is usually of allogeneic MSC in recipient bone marrow. associated with a high mortality despite infusion of back up A 40-year-old nulliparous woman with acute myeloid graft and administration of haematopoietic growth factors. leukaemia received an autologous bone marrow transplantation Mesenchymal stem cells (MSC) are part of the bone marrow (ABMT) in complete remission. Conditioning regimen combined microenvironment and support haematopoiesis. In vitro, MSC a 12 Gray fractionated total body irradiation and high dose can differentiate into several tissues1 and possess immunosup- cyclophosphamide (120 mg/kg) followed by infusion of marrow pressive properties.2 After intravenous infusion in mice, MSC are purged by mafosfamide. Primary graft failure occurred despite detected in a wide range of tissues, including bone marrow,3 infusion of back up marrow. Partial recovery on polymorpho- and can enhance engraftment of haematopoietic stem cells.4 nuclear (PMN) and haemoglobin (Hb) was obtained with In human, MSC are still investigational and have been used granulocyte-colony stimulating factor (G-CSF) and erythropoie- mainly in haematopoietic stem cell transplantation. Indeed, tin (EPO) administered three times a week. Thrombocytopenia coinfusion of autologous MSC with autologous peripheral blood remained permanently below 50 Â 109/l. In order to improve haematopoietic stem cells in cancer patients receiving high dose haematopoiesis, infusion of MSC was decided and performed chemotherapy resulted in acceleration of haematopoietic 3 years after ABMT. MSC were isolated from a human leucocyte recovery5 and coinfusion of allogeneic haematopoietic stem antigen mismatched brother bone marrow. Under a compassio- cells with allogeneic MSC was safe.6 More importantly, nate use Osiris Therapeutics (Baltimore, MD, USA) manufac- allogeneic MSC could treat successfully steroid resistant acute tured a patient-speciﬁc product of ex vivo cultured adult human graft-versus-host disease (GVHD).7 MSC. Fifty-six millilitre of bone marrow aspirate was obtained The place of MSC in the treatment of bone marrow failure is under sterile conditions. The culture and expansion of the cells unknown but some in vitro and animal data are in favour to a was performed using 10-stack cell factories (Nalge-Nunc, possible therapeutic role. Indeed, we have previously shown Rochester, NY, USA) and a modiﬁcation of previously published that MSC could improve bone marrow microenvironment in a methods.9 At the end of the culture/expansion period, cells were

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