[CANCER RESEARCH 64, 8101– 8108, November 1, 2004] Absence or Low Expression of Fas-Associated Protein with Death Domain in Acute Myeloid Leukemia Cells Predicts Resistance to Chemotherapy and Poor Outcome Le ´a Tourneur, 1 Ste ´phanie Delluc, 1 Vincent Le ´vy, 2,7 Franc ¸oise Valensi, 3,7 Isabelle Radford-Weiss, 4,7 Ollivier Legrand, 5,7 Jacques Vargaftig, 6,7 Charlotte Boix, 1 Elizabeth A. Macintyre, 3,7 Bruno Varet, 6,7 Gilles Chiocchia, 1 and Agne `s Buzyn 1,6,7 1 De ´partement d’Immunologie, Institut Cochin, Institut National de la Sante ´ et de la Recherche Me ´dicale U 567, Centre National de Recherche Scientifique UMR 8104, Institut Fe ´de ´ratif de Recherche 116, Universite ´ Rene ´ Descartes, Paris V, France; 2 Centre d’Investigations Cliniques et INSERM ERM 321, Ho ˆpital Saint-Louis, Paris, France; 3 Laboratoire Central d’He ´matologie, 4 Laboratoire de Cytoge ´ne ´tique, 5 Service d’He ´matologie Ho ˆtel-Dieu, and 6 Service d’He ´matologie Adultes, Ho ˆpital Necker-Enfants Malades, Paris, France; and 7 Assistance Publique des Ho ˆpitaux de Paris, Paris, France ABSTRACT In acute myeloid leukemia (AML), coexpression of death receptors and ligands of the tumor necrosis factor (TNF) receptor/TNF- superfamily on leukemic cells after chemotherapy is not always accompanied by apoptosis, suggesting that the apoptotic death receptor signaling pathway is disrupted. Because Fas-associated protein with death domain (FADD) is the main adaptor for transmitting the Fas, TNF-related apoptosis-induc- ing ligand receptors, and TNF receptor 1 death signal, expression of FADD was analyzed by Western blot and immunocytochemistry in leu- kemic cells of 70 de novo AML patients treated with the European Organization of Research and Treatment of Cancer AML-10 randomized trial before initiation of induction chemotherapy. Thirty seven percent of patients (17 of 46) with FADD negative/low (FADD /low ) leukemic cells had a primary refractory disease compared with 12% of FADD  patients (3 of 24; P  0.05). FADD /low expression was significantly associated with a worse event-free survival [EFS (P  0.04)] and overall survival (P  0.04). In multivariate analysis, FADD /low protein expression was independently associated with a poor EFS and overall survival (P  0.002 and P  0.026, respectively). Importantly, FADD /low protein expression predicted poor EFS even in patients with standard- or good-risk AML (P  0.009). Thus, we identified low or absent expression of the FADD protein in leukemic cells at diagnosis as a poor independent prognostic factor that can predict worse clinical outcome even for patients with standard- or good-risk AML. INTRODUCTION Chemotherapeutic drugs used for leukemia treatment can kill target cells via several mechanisms, including apoptosis. Apoptosis medi- ated by the Fas and tumor necrosis factor (TNF)-related apoptosis- inducing ligand receptors (TRAIL-Rs) is involved in the antileukemic activity of anthracyclines (1) and etoposide (2), two major cytotoxic drugs for acute myeloid leukemia (AML) therapy. Both were shown to increase TRAIL-R2 [death receptor (DR) 5] expression on human leukemic cell lines and to enhance TRAIL-mediated apoptosis of these cells (3). Moreover, anthracyclines and other drugs up-regulate Fas or Fas ligand (FasL) molecules on tumor cells, which leads to suicide/fratricide death of cancer cells (4, 5). Etoposide can activate Fas clustering and consequently Fas-mediated cell death independ- ently of FasL expression (2). However, in most AML patients, leu- kemic cells in vitro are resistant to Fas-mediated cell death despite expressing the Fas receptor (6), which suggests that the apoptotic Fas signaling pathway is disrupted in AML cells. Because chemotherapeutic drugs can induce apoptosis of leukemic cells, failure in the apoptotic machinery could lead to chemoresistance and may therefore have an impact on clinical outcome (7). In AML, the expression level of molecules involved in the mitochondria- mediated pathway of apoptosis provides important prognostic infor- mation. Although the prognostic value of the expression of bcl-2 antiapoptotic and bax proapoptotic molecules is controversial (8 –10), the bax to bcl-2 ratio is of prognostic value in AML patients (11, 12). The prognostic value of the expression of molecules involved in DR-mediated apoptosis pathways has been less studied. However, it has recently been shown that a defect in the caspase activation pathways correlates with resistance of AML patients to chemotherapy (13), suggesting that the apoptotic signaling pathways induced by DRs are disrupted in chemoresistant AML cells. Engagement of the Fas receptor by three FasL molecules leads to clustering of Fas and consequent recruitment of the Fas-associated protein with death domain (FADD) adaptor protein (14, 15). FADD can in turn activate procaspase 8, which is the initiator of a caspase cascade ultimately resulting in Fas-bearing cell death (16). Although at least two more adaptors for the Fas receptor are known (17, 18), FADD is the main adaptor transmitting the death signal via Fas (19). Moreover, FADD is a key adaptor molecule for numerous DRs because caspase-mediated apoptosis induced by at least TNF receptor 1, DR3, DR4 (TRAIL-R1), and DR5 (TRAIL-R2) needs FADD (20 –24). Until now, the role of FADD in drug-induced apoptosis of leukemic cells has been studied mainly in cell lines. In the U937 human leukemia promonocytic cell line, blockage of the DR signaling pathway by transient expression of FADD antisense or by transfection with a vector encoding a FADD dominant negative mutant has been shown to prevent cytotoxic drug-induced apoptosis of these cells (2). In the present study, we identified the absence or low expression of FADD protein in AML cells at diagnosis as a poor independent prognostic factor. Indeed, complete remission (CR) rate, event-free survival (EFS), and overall survival (OS) were significantly dimin- ished in patients with FADD-negative/low (FADD -/low ) leukemic cells (P = 0.05, P = 0.04, and P = 0.04, respectively). Absence or low expression of FADD protein was independently associated with a poor EFS and a poor OS in multivariate analysis (P = 0.002 and P = 0.026, respectively). Importantly, low or absent expression of FADD protein predicted poor EFS even in patients with standard- or good-risk AML (P = 0.009). In summary, we identified low or absent expression of the FADD adaptor molecule as a new independent marker of poor clinical outcome in AML patients. MATERIALS AND METHODS Patients. Peripheral blood mononuclear cells [PBMCs (n = 36 patients)] or bone marrow (BM) cells (n = 34 patients) of 70 leukemic patients were collected between September 1988 and September 2002 before the initiation of Received 7/2/04; revised 8/23/04; accepted 8/25/04. Grant support: Institut National de la Sante ´ et de la Recherche Me ´dicale. The laboratory is associated with the Ligue contre le cancer, Comite ´ Ile de France. L. Tourneur and S. Delluc are supported by the Fondation de France, Comite ´ Leuce ´mie. The Socie ´te ´ Franc ¸aise de Greffe de Moelle et de The ´rapie Cellulaire ensured the promotion of this study. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Agne `s Buzyn, Service d’He ´matologie Adultes, Ho ˆpital Necker-Enfants Malades, 149 rue de Se `vres, 75015 Paris, France. Phone: 33-1-44495286; Fax: 33-1-44495280; E-mail: agnes.buzyn@nck.ap-hop-paris.fr. ©2004 American Association for Cancer Research. 8101 Research. on December 1, 2015. © 2004 American Association for Cancer cancerres.aacrjournals.org Downloaded from