CD200 as a prognostic factor in acute myeloid leukaemia - Nature

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566 6 Lamballe F, Tapley P, Barbacid M. TrkC encodes multiple neurotrophin-3 receptors with distinct biological properties and substrate specificities. EMBO J 1993; 12: 3083–3094. 7 Tsoulfas P, Stephens RM, Kaplan DR, Parada LF. TrkC isoforms with inserts in the kinase domain show impaired signaling responses. J Biol Chem 1996; 271: 5691–5697.

8 Liu Q, Schwaller J, Kutok J, Cain D, Aster JC, Williams IR et al. Signal transduction and transforming properties of the TEL-TRKC fusions associated with t(12;15)(p13;q25) in congenital fibrosarcoma and acute myelogenous leukemia. EMBO J 2000; 19: 1827–1838.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

CD200 as a prognostic factor in acute myeloid leukaemia

Leukemia (2007) 21, 566–568. doi:10.1038/sj.leu.2404559; published online 25 January 2007

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(/)-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 (CD200 present) with the remainder giving an absent call (CD200 absent). CD200 present calls were highly correlated with abnormalities affecting CBF (24/28 vs 56/156, P ¼ 0.0001). There was no evidence of any association with age at diagnosis (mean ages: present 49 years vs absent 50 years, P ¼ 0.7), WBC (P ¼ 0.3) or sex (P ¼ 0.6), however, larger numbers of patients will be required to provide more reliable evidence and allow for adjustment of clinical and demographic parameters. Despite the association of CD200 expression with these good risk subtypes, analyses of survival stratified for CBF Leukemia

abnormalities showed that CD200 was significantly associated with worse survival (HR 1.68, 95% CI 1.08–2.62, P ¼ 0.02; Figure 1a and b).

Figure 1 CD200 expression in patients with AML. (a) Kaplan– Meier plot of the overall survival in non-CBF leukaemia patients expressing CD200 (CD200present) or not (CD200absent). (b) Odds ratio plots of survival stratified by mutations affecting CBF (t(8;21) and inv(16)). (c) Box and whisker plots depicting microarray data of the normalized expression of CD200 in AML patients (n ¼ 184) classified according to FAB criteria. wM2 patients without a t(8;21). yM2 patients with a t(8;21). nData outliers.


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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 gene expression with AML subtype (Figure 1c). As might be expected from the high frequency of CD200present patients in t(8;21) leukaemias, these patients also significantly overexpressed CD200 by 1.8-fold when compared to FAB-M2 patients without this cytogenetic abnormality (P ¼ 0.03). Induction of CD200 may be a direct consequence of AML1ETO expression, because human primary cells transduced with this fusion gene also overexpress CD200 (A.Tonks, submitted). Furthermore, patients expressing an inv(16) mutation (generally associated with FAB-M4) also significantly overexpressed CD200 when compared to M4 patients without an inv(16) mutation (1.3-fold; P ¼ 0.02). Additionally, Cox regression shows that, when adjusted for the type of leukaemia, the level of CD200 expression was significantly associated with worse overall survival (P ¼ 0.04). This data

suggests that CD200 may have independent prognostic value in AML. Our data analysis was consistent using all available Affymetrix probe sets (209582_s_at; 209583_s_at). CD200 expression by microarray analysis was validated by real-time RT-PCR for 20 AML patients for which corresponding messenger ribonucleic acid samples were still available (Figure 2a). These data sets were highly correlative (r ¼ 0.763, P ¼ 0.001). We also carried out assessment of CD200 expression at the protein level by flow cytometric analysis using another set of 15 patients for which archival material was available. Cells were stained with PEconjugated anti-CD200 (clone MRCOX-104) in combination with CD45-FITC and CD34-PERCP (all from Becton Dickinson, BD, UK). Again, we found a correlation (r ¼ 0.631; P ¼ 0.01) between Affymetrix CD200 expression and protein expression (Figure 2b). Leukemia


568 The mechanism by which CD200 influences survival is intriguing. The function of CD200 includes induction of immune suppression through interaction with its receptor, CD200R,3–6 suggesting a possible role for this molecule in influencing immune surveillance. In conclusion, these data show for the first time the potential role of CD200 in influencing survival in AML and its association with CBF leukaemias.

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Acknowledgements We thank Amanda Gilkes and Megan Musson (Cardiff University) for their technical assistance in processing microarray samples. We are grateful to the MRC for access to material from patients enrolled in the NCRI clinical trials. AT was supported by Leukaemia Research, UK.

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A Tonks, R Hills, P White, B Rosie, KI Mills, AK Burnett and RL Darley Department of Haematology, School of Medicine, Cardiff University, Cardiff, UK E-mail: [email protected]

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References 1 Wright GJ, Puklavec MJ, Willis AC, Hoek RM, Sedgwick JD, Brown MH et al. Lymphoid/neuronal cell surface OX2 glycoprotein

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recognizes a novel receptor on macrophages implicated in the control of their function. Immunity 2000; 13: 233–242. Barclay AN, Wright GJ, Brooke G, Brown MH. CD200 and membrane protein interactions in the control of myeloid cells. Trends Immunol 2002; 23: 285–290. Wright GJ, Cherwinski H, Foster-Cuevas M, Brooke G, Puklavec MJ, Bigler M et al. Characterization of the CD200 receptor family in mice and humans and their interactions with CD200. J Immunol 2003; 171: 3034–3046. Gorczynski RM, Chen Z, Lee L, Yu K, Hu J. Anti-CD200R ameliorates collagen-induced arthritis in mice. Clin Immunol 2002; 104: 256–264. Songyang Z, Margolis B, Chaudhuri M, Shoelson SE, Cantley LC. The phosphotyrosine interaction domain of SHC recognizes tyrosine-phosphorylated NPXY motif. J Biol Chem 1995; 270: 14863–14866. Hoek RM, Ruuls SR, Murphy CA, Wright GJ, Goddard R, Zurawski SM et al. Down-regulation of the macrophage lineage through interaction with OX2 (CD200). Science 2000; 290: 1768–1771. Lee L, Liu J, Manuel J, Gorczynski RM. A role for the immunomodulatory molecules CD200 and CD200R in regulating bone formation. Immunol Lett 2006; 105: 150–158. McWhirter JR, Kretz-Rommel A, Saven A, Maruyama T, Potter KN, 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. Moreaux J, Hose D, Reme T, Jourdan E, Hundemer M, Legouffe E et al. CD200 is a new prognostic factor in Multiple Myeloma. Blood First Edition Paper, prepublished online August 31 2006; 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; published online 25 January 2007

Primary graft failure is rarely observed after autologous haematopoietic stem cell transplantation, and is usually associated with a high mortality despite infusion of back up graft and administration of haematopoietic growth factors. Mesenchymal stem cells (MSC) are part of the bone marrow microenvironment and support haematopoiesis. In vitro, MSC can differentiate into several tissues1 and possess immunosuppressive properties.2 After intravenous infusion in mice, MSC are detected in a wide range of tissues, including bone marrow,3 and can enhance engraftment of haematopoietic stem cells.4 In human, MSC are still investigational and have been used mainly in haematopoietic stem cell transplantation. Indeed, coinfusion of autologous MSC with autologous peripheral blood haematopoietic stem cells in cancer patients receiving high dose chemotherapy resulted in acceleration of haematopoietic recovery5 and coinfusion of allogeneic haematopoietic stem cells with allogeneic MSC was safe.6 More importantly, allogeneic MSC could treat successfully steroid resistant acute graft-versus-host disease (GVHD).7 The place of MSC in the treatment of bone marrow failure is unknown but some in vitro and animal data are in favour to a possible therapeutic role. Indeed, we have previously shown that MSC could improve bone marrow microenvironment in a Leukemia

patient with an end-stage severe aplastic anaemia.8 We now 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 parallel improvement of in vitro clonogenic assay and detection of allogeneic MSC in recipient bone marrow. A 40-year-old nulliparous woman with acute myeloid leukaemia received an autologous bone marrow transplantation (ABMT) in complete remission. Conditioning regimen combined a 12 Gray fractionated total body irradiation and high dose cyclophosphamide (120 mg/kg) followed by infusion of marrow purged by mafosfamide. Primary graft failure occurred despite infusion of back up marrow. Partial recovery on polymorphonuclear (PMN) and haemoglobin (Hb) was obtained with granulocyte-colony stimulating factor (G-CSF) and erythropoietin (EPO) administered three times a week. Thrombocytopenia remained permanently below 50 109/l. In order to improve haematopoiesis, infusion of MSC was decided and performed 3 years after ABMT. MSC were isolated from a human leucocyte antigen mismatched brother bone marrow. Under a compassionate use Osiris Therapeutics (Baltimore, MD, USA) manufactured a patient-specific product of ex vivo cultured adult human MSC. Fifty-six millilitre of bone marrow aspirate was obtained under sterile conditions. The culture and expansion of the cells was performed using 10-stack cell factories (Nalge-Nunc, Rochester, NY, USA) and a modification of previously published methods.9 At the end of the culture/expansion period, cells were