ORIGINAL ARTICLE Incidence of additional genetic changes in the TEL and AML1 genes in DCOG and COALL-treated t(12;21)-positive pediatric ALL, and their relation with drug sensitivity and clinical outcome WAG Stams 1 , HB Beverloo 2 , ML den Boer 1 , RX de Menezes 1,3 , RL Stigter 1 , E van Drunen 2 , NL Ramakers-van-Woerden 4 , AH Loonen 4 , ER van Wering 5 , GE Janka-Schaub 6 and R Pieters 1,5 1 Erasmus MC, Sophia Children’s Hospital, Erasmus University Medical Center Rotterdam, Division of Pediatric Oncology/ Hematology, Rotterdam, The Netherlands; 2 Erasmus MC, Erasmus University Medical Center Rotterdam, Department of Clinical Genetics, Rotterdam, The Netherlands; 3 Centre for Human and Clinical Genetics, Leiden University Medical Centre, Leiden, The Netherlands; 4 VU Medical Center, Amsterdam, The Netherlands; 5 Dutch Childhood Oncology Group, The Hague, The Netherlands and 6 COALL study group, University Children’s Hospital, Hamburg, Germany Clinical heterogeneity within t(12;21) or TEL/AML1-positive ALL (25% of childhood common/preB ALL) indicates that additional genetic changes might contribute to outcome. We studied the relation between additional genetic changes in TEL(ETV6) and AML1(RUNX1) (FISH), drug sensitivity (MTT assay) and clinical outcome in 143 DCOG and COALL-treated t(12;21)-positive ALL patients. Additional genetic changes in TEL and AML1 were present in 83% of the patients, and consisted of (partial) deletion of the second TEL gene (70%), an extra AML1 gene (23%) or an extra der(21)t(12;21) (10%). More than one additional change was observed in 20%. Disease-free survival (pDFS) of DCOG patients without additional genetic changes (4 years pDFS7s.e. 53717%) and of those with an extra der(21)t(12;21) (60722%) is poorer than that of compared to patients with other additional genetic changes in TEL or AML1 (7976%; P-trend ¼ 0.02). This was mainly due to the occurrence of early relapses within 2.5 years after the first diagnosis. Similar observations were found in the COALL cohort, albeit not significant owing to limited follow-up. Multivariate analysis including age, WBC and genetic abnormalities in TEL and/or AML1 showed that especially, in vitro resistance to predniso- lone (hazard ratio 5.78, 95% CI 1.45–23.0; P ¼ 0.01) is an independent prognostic factor in DCOG- and COALL-treated t(12;21)-positive ALL. Leukemia (2006) 20, 410–416. doi:10.1038/sj.leu.2404083; published online 19 January 2006 Keywords: childhood ALL; TEL-AML1; genetic abnormalities; clinical outcome; drug resistance Introduction The t(12;21)(p13;q22) occurs in B25% of childhood acute lymphoblastic leukemia (ALL), and is restricted to precursor B- cell lineage leukemia. The t(12;21) involves fusion of the TEL(ETV6) gene at chromosome 12p13, with the AML1(RUNX1) gene at chromosome 21q22. This translocation fuses the 5 0 terminus of the TEL gene (residues 1–336) in frame, with almost the entire coding sequence (residues 21–480) of the AML1 gene. The breakpoint most often occurs in intron 5 of TEL, and intron 1 of AML1. A frequent translocation variant results in fusion between intron 5 of TEL and intron 2 of AML1. Both TEL and AML1 are frequent targets of chromosomal translocations in a variety of myeloid and lymphoid leukemias. 1,2 TEL contains an N-terminal pointed (PNT) dimerization domain that mediates homodimerization. 3,4 The C-terminal DNA-binding domain homologous to all Ets proteins, recognizes a purine-rich GGAA/T core motif within promoters and enhancers of various genes. 5 TEL has a role in both angiogenesis and hematopoiesis. 6 AML1 encodes a transcription factor that binds the enhancer core sequence TGT/cGGT through its N-terminal Runt homol- ogy domain (RHD). 7 The DNA-binding affinity of AML1 is increased by heterodimerization through the RHD with the core binding factor (CBF) b protein, forming the CBF. Transcriptional activation occurs through the C-terminal trans- activation (TA) domain. CBF is essential for definitive hemato- poiesis of all lineages. 1 The translocation fusion product TEL/AML1 contains the PNT domain of TEL and the RHD, and TA domain of AML1. Several studies have investigated the prognostic value of t(12;21) positive ALL (reviewed in Loh and Rubnitz 8 ). In general, t(12;21)-positive ALL is associated with a good prognosis. However, conflicting data on the percentage of patients entering relapse and the proportion of t(12;21)-positive cases at relapse have been reported. 8 Overall, the reported prognostic relevance of t(12;21) seems to depend on the intensity of the treatment protocol. A possible explanation for this finding can be ascribed to the fact that t(12;21)-positive ALL cells are in vitro more sensitive to L-Asparaginase (L-Asp) than t(12;21) negative ALL cells. 9,10 However, within the t(12;21)-positive ALL group, large interindividual differences in cellular in vitro sensitivity to L-Asp were found. The clinical heterogeneity in response to therapy as well as the large heterogeneity in in vitro sensitivity to L-Asp suggests that additional genetic changes might be important for the differences in drug sensitivity and clinical outcome. Approximately, 70% of t(12;21)-positive ALL cases also show loss of the second TEL allele. 11,12 Trisomy 21 and duplication of the der(21)t(12;21) are also found in t(12;21)-positive patients, with the latter apparently being more common in relapsed cases. 13–16 However, the number of patients screened in these studies is limited. The prognostic significance of additional genetic abnormalities in TEL and AML1 is therefore unknown. We retrospectively studied the incidence of additional genetic changes in the TEL and AML1 genes, and their relationship with drug sensitivity and clinical outcome in 143 t(12;21)-positive children with ALL. Received 20 July 2004; revised 17 October 2005; accepted 10 November 2005; published online 19 January 2006 Correspondence: Dr ML den Boer, Erasmus MC - Sophia Children’s Hospital, Division of Pediatric Oncology/Hematology, Dr Molewater- plein 60, 3015 GJ Rotterdam, The Netherlands. E-mail: m.l.denboer@erasmusmc.nl Leukemia (2006) 20, 410–416 & 2006 Nature Publishing Group All rights reserved 0887-6924/06 $30.00 www.nature.com/leu