Leukemia (1999) 13 , 1513–1518  1999 Stockton Press All rights reserved 0887-6924/99 $15.00 http:/ / www.stockton-press.co.uk/ leu The immunophenotype of minimally differentiated acute myeloid leukemia (AML-M0): reduced immunogenicity and high frequency of CD34 + /CD38 - leukemic progenitors RT Costello 1,2 , F Mallet 1 , H Chambost 3 , D Sainty 2 , C Arnoulet 2 , J-A Gastaut 2 and D Olive 1,4 1 Immunologie des Tumeurs, 2 De ´partement d’He ´matologie, Institut Paoli-Calmettes, Universite ´ de la Me ´ diterrane ´e; 3 He ´matologie Pe ´diatrique, Ho ˆ pital de la Timone; and 4 Unite ´ INSERM U119, Marseille, France Minimally differentiated acute myeloid leukemia (AML-M0) is a rare FAB subtype (2–3% of AMLs) of poor prognosis. The aim of our study was to characterize AML-M0 expression and regu- lation of adhesion/costimulatory molecule involved in immune recognition, to test blast in vitro immunogenicity, and to deter- mine the percentage of leukemia progenitor cells. Here, we demonstrate that alloimmune recognition of AML-M0 in primary mixed lymphocyte reaction, as evaluated by IL-2 secretion of responding T cells, is reduced in comparison with more differ- entiated subtypes (128  95 pg/ml vs 304  159 pg/ml, P  0.05). These data are in line with low blast cell expression of major histocompatibility complex (MHC) class II DR molecules, and of the CD28 ligand B7-2, which plays an important role in AML immune recognition. Adhesion/costimulatory molecules were up-regulated by leukemic cell stimulation via CD40, and, although less efficiently, by -IFN; both stimuli improved blast cell immunogenicity. We also demonstrate that AML-M0 have a very high percentage (40%  30) of CD34  /CD38  leukemic clonogenic precursors in comparison with more differentiated AMLs (2.5%  2) or non-leukemic CD34  hematopoietic precur- sors (1.8%  0.8). Since the presence of a leukemic cell popu- lation at an early differentiation stage has been identified as a poor prognostic factor, we conclude that the high frequency of CD34  /CD38  blasts in AML-M0 may converge with already identified poor prognosis factors such as chemotherapy resist- ance and cytogenetic abnormalities. The clinical implications of AML-M0 impaired in vitro immunogenicity and a high per- centage of CD34  /CD38  blasts will require comparative analy- sis of additional patients. The increased immunogenicity of blast cells after CD40 triggering provide interesting clues for AML-M0 immunotherapy, that have to be confirmed with an in vivo leukemia model in mice. Keywords: acute myeloid leukemia; AML-M0; leukemia progeni- tors; immune recognition; CD34; CD38 Introduction The French–American–British (FAB) classification 1–3 mini- mally differentiated or M0 acute leukemia (AML-M0) is characterized by undifferentiated morphology by light microscopy, expression of immaturity (CD34 or CD7) and myeloid (CD13 or CD33) markers, 2,4–8 the latter permitting differentiation of AML-M0 from ‘stem cell’ acute leukemia. 9 Low remission rate and frequent relapses contribute to the short survival observed in this subtype. 2,4,10 The reason for this remarkably poor prognosis is not elucidated, but the conver- gence of unfavorable prognostic factors has been proposed. High incidence of cytogenetic abnormalities and expression of multidrug-resistance P-glycoprotein may explain the poor sensitivity of leukemic cells to chemotherapy. 5,10 Escape from immune recognition can contribute to AML prognosis, since Correspondence: D Olive, Immunologie des Tumeurs, Institut Paoli- Calmettes, Universite ´ de la Me ´diterrane ´ e, 232 bd Sainte Marguerite, 13009 Marseille, France; Fax: (33)491 22 36 10 Received 19 April 1999; accepted 8 June 1999 immune response has an important role in the control of ‘minimal residual disease’ as outlined by the allogeneic stem cell transplantation-associated ‘graft-versus-leukemia’ reac- tion. 11–13 Lack of differentiation may contribute to the pres- ence of a large population of leukemic cells capable of sus- taining self-renewal and proliferation, which recently has been demonstrated to be contained in the CD34 + /CD38 + blast subpopulation. 14–19 To gain more insight into AML-M0 physiopathology, we analyzed in vitro alloimmune recognition and adhesion/ constimulatory molecule expression in AML-M0, together with the frequency of CD34 + /CD38 - leukemic progenitors, in comparison with more differentiated blasts from myelo/monocytic leukemias or non-leukemic hematopoietic progenitors from different patients. Materials and methods Patient samples Peripheral blood samples, obtained before specific anti-leu- kemic therapy and after informed consent by the patient, were part of diagnostic procedures. We analyzed nine patients with AML-M0, 23 patients with more differentiated AMLs, and three patients with non-hematopoietic cancers (for the analy- sis of granulocyte colony-stimulating factor (G-CSF)-mobilized hematopoietic progenitors). Diagnosis was established follow- ing FAB classification 1 and specific recommendations for AML-M0 distinction: 2,3,10,20 (1) negativity of light microscopy myeloperoxidase and Sudan black-B or positivity in less than 3% of blasts; (2) negative lymphoid markers (positive terminal deoxynucleotydil transferase or CD7 were accepted); (3) posi- tive CD13 and/or CD33. Slides were independently reviewed by two morphologists (DS and CA). Cell separation Peripheral blood mononucleated cells (PBMCs) from healthy donors and leukemic patients were isolated on Ficoll– Hypaque gradients and viably frozen in liquid nitrogen until use. 21 Regarding leukemic cells, samples with a blast fraction lower than 95% were purified by negative selection using magnetic beads coated with anti-CD3, anti-CD19 and, depending on the phenotype at diagnosis, anti-CD56 and/or anti-CD14 mAbs (Beckman Coulter, Marseille, France). For mixed leukocyte culture against leukemic cells, responding T lymphocytes were purified by sheep erythrocyte rosetting plus overnight adhesion in plastic dishes. 21 Non-leukemic hemato- poietic progenitors were obtained after G-CSF mobilization from patients with non-hematological malignancies. The CD34 + preparation was performed using the MACS column