01517–02–2004 (to SB); KT was supported by a fellowship from the Fondazione Italiana Ricerca sul Cancro (FIRC). S Fabris 1,5 , D Ronchetti 1,2,5 , L Agnelli 1 , L Baldini 2 , F Morabito 3 , S Bicciato 4 , D Basso 4 , K Todoerti 1,2 , L Lombardi 1 , G Lambertenghi-Deliliers 2 and A Neri 1,2 1 Centro di Genetica Molecolare ed Espressione Genica, Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milan, Italy; 2 Dipartimento di Scienze Mediche, Universita ` degli Studi di Milano, Milan, Italy; 3 U.O. Ematologia, A.O. ‘Annunziata’, Cosenza, Italy and 4 Dipartimento dei Processi Chimici dell’Ingegneria, Universita ` degli Studi, Padua, Italy E-mail: neri.a@policlinico.mi.it 5 The first two authors contributed equally to this work References 1 Fonseca R, Barlogie B, Bataille R, Bastard C, Bergsagel PL, Chesi M et al. Genetics and cytogenetics of multiple myeloma: a workshop report. Cancer Res 2004; 64: 1546–1558. 2 Hanamura I, Stewart JP, Huang Y, Zhan F, Santra M, Sawyer JR et al. Frequent gain of chromosome band 1q21 in plasma-cell dyscrasias detected by fluorescence in situ hybridization: incidence increases from MGUS to relapsed myeloma and is related to prognosis and disease progression following tandem stem-cell transplantation. Blood 2006; 108: 1724–1732. 3 Agnelli L, Bicciato S, Mattioli M, Fabris S, Intini D, Verdelli D et al. Molecular classification of multiple myeloma: a distinct transcriptional profile characterizes patients expressing CCND1 and negative for 14q32 translocations. J Clin Oncol 2005; 23: 7296–7306. 4 Callegaro A, Basso D, Bicciato S. A locally adaptive statistical procedure (LAP) to identify differentially expressed chromosomal regions. Bioinformatics 2006; 22: 2658–2666. 5 Zhan F, Huang Y, Colla S, Stewart JP, Hanamura I, Gupta S et al. The molecular classification of multiple myeloma. 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Expression of Polycomb-group (PcG) protein BMI-1 predicts prognosis in patients with acute myeloid leukemia Leukemia (2007) 21, 1116–1122. doi:10.1038/sj.leu.2404623; published online 22 March 2007 BMI-1, a member of the Polycomb group of gene family, is essential for the self-renewal of hematopoietic, neural and cancerous stem cells. 1 BMI-1 is highly expressed in malignan- cies like B cell non-Hodgkin lymphoma, Hodgkin’s lymphoma, colorectal carcinoma, liver carcinoma, non-small cell lung cancer, breast carcinoma and medulloblastoma. Interestingly, the study of BMi-1 knockout mice revealed that BMi-1 is indispensable for the self-renewal of both leukemic and normal hematopoietic stem cells. 2 In addition to its vital role in stem cell self-renewal, BMI-1 regulates the proliferative output of primitive hematopoietic progenitors in a critical manner. 1 We reported previously BMI-1 as a marker of disease progression in cases of myelodysplastic syndrome (MDS). 3 Therefore, we assumed that BMI-1 expression could be used to predict the prognosis of patients with acute myeloid leukemia (AML) and thus could serve as a tool to tailor therapy for this disease. This is the first study, in which flow cytometry was used to determine the expression of BMI-1 in AML blasts of human subjects. We studied bone marrow (BM) samples (except two from peripheral blood (PB) and granulocytic sarcoma patient from the lymph node) obtained from 64 patients with newly diagnosed AML (Supplementary Tables 1 and 2). According to the Helsinki declaration, informed consent was obtained from the patients and donors (used as control), and an institutional review board approved the study. AML M3 patients were excluded from this study, due to their different disease biology and different treatment strategy. All cases of de novo AML were diagnosed as per the FAB criteria. Granulocytic sarcoma was diagnosed using lymph node biopsy. Secondary AML (MDS-AML or t-AML) was diagnosed according to the revised recommendations of the international working group on AML. 4 Complete remission (CR), partial remission , overall survival (OS), relapse-free survival (RFS) and remission duration (RD) were defined as per recommendations of the same group. 4 BM blasts p5%, absolute neutrophil count X1000 ml and platelet count X100 000 ml were the criteria for CR. Cytogenetic remission or molecular remission was not used to define CR. Flow cytometry with appropriate antibody was performed to confirm the diagnosis and morphologic leukemia-free state. Survival was measured from the date of diagnosis to the date of death or last follow-up. Figure 1 (A) BMI-1 expression in AML cell lines by immunoblot analysis. Immunoblot analysis demonstrated BMI-1 expression in five AML cell lines: KG-1 (3), Mono-7 (4), HEL (5), HL-60 (6) and U-937 (7). WI-38 (1) and IL-2-stimulated peripheral blood T cells (2) expressed much less BMI-1 protein. (B) BrdU incorporation by acute promonocytic leukemic cells, U-937: Cell cycle analysis was performed with the staining pattern of 7-AAD and BrdU in (b–d). In (a), M1, M2 and M3 represent BMI-1 negative cells, cells with lower intensity of BMI-1 and higher intensity of BMI- 1, respectively. Dotted line indicates isotype control. BMI-1-negative U-937 cells were collected in (b), cells with lower intensity of BMI-1 were shown in (c) and cells with higher intensity of BMI-1 were depicted in (d). R4, R5, R6 and R7 are indicative of G0/G1 phase, S phase, G2/M phase and apoptotic cells, respectively. (C) BMI-1 expression in AML patients. Patient 16, a case of CD34 þ AML M1 had higher BMI-1 expression (94.57%). Patient 3, a case of CD34 þ AML M0 had the lower expression (21.88%). Patient 21, a case of CD34 – AML M2 had the lower expression (6.91%) comparable to control CD34 þ cells as described in Materials and method. (D) BMI-1 expression (%) in FAB subtypes and secondary AML. Control CD34 þ cells and leukemic blasts were examined for BMI-1 expression. ‘FF’ indicates mean value. Letters to the Editor 1116 Leukemia