DEK-CAN molecular monitoring of myeloid malignancies could aid therapeutic stratification L Garc ¸on 1,6,7 , M Libura 1,7 , E Delabesse 1 , F Valensi 1 , V Asnafi 1 , C Berger 2 , C Schmitt 3 , T Leblanc 4 , A Buzyn 5 and E Macintyre 1 1 Faculte ´ de Medecine, Universite ´ Paris-Descartes, INSERM EMI U210 and AP-HP He ´matologie-biologique, Ho ˆ pital Necker- Enfants Malades, rue de Se `vres, Paris cedex, France; 2 Pe ´diatrie Oncologie A, CHU Ho ˆ pital Nord, Saint-Etienne, France; 3 Me ´decine Infantile II, Ho ˆ pitaux de Brabois - Ho ˆ pital d’Enfants, Vandoeuvre-les-Nancy, France; 4 He ´matologie Pe ´diatrique, Ho ˆ pital St. Louis, Paris Cedex, France; and 5 He ´matologie Clinique, Ho ˆ pital Necker-Enfants Malades, rue de Se `vres, Paris cedex, France The t(6;9)(p23;q34) is a recurrent chromosomal abnormality observed in 1% of acute myelogenous leukemia (AML), which generates a fusion transcript between DEK and CAN/NUP214 genes. We used a DEK-CAN real-time quantitative (RQ)-PCR strategy to analyze 79 retrospective and prospective samples from 12 patients. Five patients reached DEK-CAN negativity (sensitivity 10 5 ); all underwent early allogeneic hematopoietic stem cell transplantation (median 5.5 months from diagnosis) with some demonstrating molecular positivity at the time of allograft. All four cases in CCR with adequate follow-up (median 18.5 months, range 13–95) demonstrate persistent molecular negativity, whereas all seven patients with persistent DEK-CAN positivity died at a median of 12 months from diagnosis (range 7–27). We conclude that DEK-CAN molecular monitoring by RQ- PCR in t(6;9) malignancies is a useful tool for individual patient management and that molecular negativity is indispensable for survival, but should not be a prerequisite for allografting in this rare, poor prognosis, subset of AML. Leukemia (2005) 19, 1338–1344. doi:10.1038/sj.leu.2403835; published online 23 June 2005 Keywords: acute leukemia; DEK-CAN transcript; minimal residual disease; real-time PCR Introduction The t(6;9)(p23;q34) is a cytogenetic event occurring recurrently in acute myelogenous leukemia (AML) and myelodysplasia (MDS). Since its first description in 1976, 1 more than 70 cases have been reported, allowing the identification of clinical and morphological characteristics: a young age at diagnosis, a predominance of AML-M2 according to the FAB classification, signs of multilineage dysplasia frequently with bone marrow (BM) basophilia, and a very poor prognosis with short disease- free survival (DFS) and overall survival (OS). 2–5 At the molecular level, this translocation generates a chimeric protein, resulting from the fusion between DEK and the 3 0 - terminus of the CAN gene, also known as NUP214. 6 DEK, originally described as a proto-oncogene, is now known to be a major component of metazoan chromatin able to modify the structure of DNA by introducing supercoils. 7,8 Its level was found to be increased in AML BM samples. 9 CAN is a nuclear pore complex protein with multiple FG-peptide sequence motifs and is implicated in nucleocytoplasmic transport. 10,11 The CAN gene is involved in several fusion transcripts described in acute leukemia: not only with DEK in t(6;9)(p23;q34) AML, but also with the SET gene and recently with ABL in T-cell acute lympho- blastic leukemia (ALL). 12,13 The size and structure of the DEK- CAN chimeric protein is the same in all cases, as DNA break- points within both genes are always located in the same intron. 14 Quantitative reverse transcription-polymerase chain reaction (RQ-PCR) is currently used for the detection of minimal residual disease (MRD) in several AMLs. 15 By providing information about the kinetics of clearance of the leukemic clone, reliable MRD quantification can have important predictive value for relapse and can identify groups of patients for whom more intensive treatment can be of benefit. 16 Real-time quantitative (RQ)-PCR can provide useful information, as demonstrated in ALL as well as AML with PML-RARa, CBFb-MYH11, and AML1- ETO transcripts. 17 Few studies of MRD have been performed for DEK-CAN þ AML, with most using only qualitative RT-PCR. 18– 21 A limited number of patients have been analyzed by RQ-PCR, but little is known about the evolution of the DEK-CAN fusion transcript under therapy. 22,23 We therefore decided to quantify DEK-CAN fusion transcript in a series of 12 AML/MDS patients with the t(6;9) translocation and show that molecular evolution under therapy correlates with clinical outcome. Materials and methods Patients and samples We analyzed a group of 10 patients with AML and two with MDS demonstrating a t(6;9)(p23;q34) by conventional cytoge- netics. For all these patients, presence of the DEK-CAN fusion transcript was confirmed by RT-PCR. Patients were treated in one of the seven following French centers: Assistance Publique – Ho ˆ pitaux de Paris de la Pitie ´-Salpe ´trie ` re, Trousseau, Ho ˆ tel-Dieu, Necker-Enfants Malades and Saint-Louis, Paris; Ho ˆ pital Nord, Saint-Etienne; Ho ˆ pital d’Enfants-Brabois, Nancy. All patients were treated and followed in their original institutions between 1996 and 2004. RT-PCR quantification of DEK-CAN fusion transcript at diagnosis or during follow up was performed in the laboratory of Biological Hematology at the Necker-Enfants Malades hospital, after informed consent according to the Declaration of Helsinki. The total number of samples analyzed was 79. For eight patients, diagnosis and follow up samples were analyzed. For the others, only follow up samples at different end points of treatment were available. Analysis of DEK-CAN fusion transcript by RT-PCR and RQ-PCR For cDNA analysis 1 mg of total RNA obtained from BM or peripheral blood (PB) mononuclear cells was reverse transcribed and cDNA quality was assessed relative to the ABL house- keeping gene as described 24 on an ABI PRISMt 7000 (Applied Biosystems, Foster City, CA, USA). Samples with Ct ABL values above 32 (fluorescent threshold 0.1) were considered degraded. Qualitative RT-PCR at diagnosis was performed in the presence of 200 mM dNTP, 2.5 mM MgC l2 and 200 nM of the following primers: DEK-primer C: gCCAAAAgAg AAAAACCTAAA; CAN- primer B: gCAAggATTT ggTgTgAgAT. Real-time quantitative- Received 30 April 2005; accepted 3 May 2005; published online 23 June 2005 Correspondence: Professor E Macintyre, Laboratoire d’He ´matologie, Tour Pasteur, Ho ˆ pital Necker-Enfants Malades, 149-161, rue de Se `vres, 75743 Paris cedex 15, France; Fax: þ 33 1 44 38 17 45; E-mail: elizabeth.macintyre@nck.ap-hop-paris.fr 6 Current address: Laboratoire d’He ´matologie, Ho ˆpital de Bice ˆtre, 78 rue du Ge ´ne ´ral Leclerc, 94275 Le Kremlin-Bice ˆtre, France 7 These two authors have contributed equally to this work Leukemia (2005) 19, 1338–1344 & 2005 Nature Publishing Group All rights reserved 0887-6924/05 $30.00 www.nature.com/leu