¹Division of Hematology/Oncology, Cedars-Sinai Research Institute, UCLA School of Medicine, Los Angeles, California, CA 90048, USA

²Institute of Human Genetics, University of Heidelberg, D-69120 Heidelberg, Germany

³Department of Pediatrics IV, Hannover Medical School, Hannover, Germany

⁴Department of Medicine, Kochi Medical School, Kochi 783-8505, Japan

Correspondence to: Seisho Takeuchi, Division of Hematology/Oncology, Cedars-Sinai Research Institute, UCLA School of Medicine, Los Angeles, California, CA 90048, USA

Cytogenetic analysis of childhood acute lymphoblastic leukemia (ALL) identified deletions of chromosome arm 11q. These observations led us to analyse the loss of heterozygosity (LOH) of chromosome arm 11q in 113 primary childhood ALL samples using 14 microsatellite markers. LOH was found in 18 (16%) patients. Detailed examination identified three distinct regions of deletion. The first region is flanked by D11S901 and D11S1391 at 11q22-23 containing the ATM gene. Mutational analysis suggested that the altered gene in this region is not the ATM gene. The second region is flanked by D11S614 and D11S924 at 11q23 containing the MLL gene. The third region is flanked by D11S1356 and D11S614 at 11q23 containing the MLL gene. All the cases with LOH at MLL locus lacked detectable MLL gene rearrangements. In addition, 20 children have been studied both at initial diagnosis and relapse; none of the individuals who relapsed acquired LOH of 11q, suggesting that 11q deletions were infrequently involved in the progression of childhood ALL. Children with 11q LOH had a good response to induction chemotherapy (P=0.015). These data suggest that alterations of putative tumor suppressor genes on 11q are important events in development of childhood ALL. Our map provides important information toward cloning putative tumor suppressor genes associated with childhood ALL.

LOH; ALL; 11q; MLL; ATM

Cytogenetic deletions of the long arm of chromosome 11 were reported in 2 - 5% of patients with childhood ALL (Johansson et al., 1993; Raimondi, 1993; Raimondi et al., 1995). These observations are thought to indicate the presence of a tumor suppressor gene on 11q that is mutated on the remaining allele. However, the frequency and region of the deletions is difficult to estimate by conventional cytogenetic analysis because in part small interstitial deletions are beyond the sensitivity of the technique. We analysed 113 childhood ALL samples for loss of heterozygosity (LOH) of chromosome arm 11q using 14 microsatellite markers.

Primary ALL DNAs were obtained from patients of the ongoing Multicenter Trial ALL-BFM 90 of childhood ALL of the German Berlin - Frankfurt - Münster (BFM) group. The corresponding normal DNAs were obtained from the bone marrow after complete remission were achieved. The LOH analysis was performed as described previously (Takeuchi et al., 1995). The genetic map of chromosome 11 was compiled mainly from the Généthon microsatellite map (Weissenbach et al., 1992; James et al., 1994). Primers were obtained from Research Genetics (Huntsville, AL, USA). Five to 10 g of each DNA was digested with BamHI, separated on 1% agarose gels and transferred to nylon membrane (Hybond-N, Amersham, UK). Blots were hybridized with the ³²P-labeled ps4 probe. This probe will detect the common breakpoints of MLL between exons 5 through 11 with BamHI digests (Raimondi et al., 1995). Hybridized membranes were exposed to films.

Eighteen children (16%) showed LOH at least at one locus on 11q. This is higher than the frequency of cytogenetic deletions of 11q (2 - 5%) in ALL (Johansson et al., 1993; Raimondi et al., 1995). These results show the power of LOH analysis using microsatellite markers. The pattern of LOH in each child is shown in Figure 1. Previous cytogenetic analysis of childhood ALL identified 11q23 as the commonly deleted region (Raimondi et al., 1995). We found that deletions occur in three distinct regions on 11q. The first smallest commonly deleted region is between D11S901 and D11S1391 on 11q22-23 (nos. 32, 110). The ATM gene which is mutated in patients with ataxia telangiectasia is located in this critical region (James et al., 1994; Vorechovsky et al., 1996). Children with ataxia telangiectasia frequently develop ALL, implicating ATM in leukemogenesis (Taylor et al., 1996). However, our mutational analysis suggested that the altered gene in this region is not the ATM gene (Takeuchi et al., 1998).

The second smallest commonly deleted region is between D11S614 and D11S924 on 11q23 (nos. 66, 115). This region contains the MLL gene (James et al., 1994). One individual (no. 58) had a small LOH at D11S1341/D11S976 locus. Since the MLL gene is mapped between D11S1341/D11S976 and D11S1364, this child also has LOH at the MLL locus. The MLL gene has been identified as the usual target of 11q23 translocations. We analysed 14 childhood ALL samples (nos. 5, 12, 29, 30, 35, 45, 46, 58, 66, 74, 100, 115, 138, 149) with LOH at MLL locus for rearrangement of the MLL gene. All 14 samples lacked evidence of a rearranged MLL gene (data not shown). Raimondi et al. reported a similar finding that patients with cytogenetic deletion of 11q23 lacked the MLL gene rearrangements (Raimondi et al., 1995). Taken together, in addition to the translocation, loss of normal function of the MLL gene may be a critical event in leukemogenesis.

DNA samples were obtained at the time of ALL relapse from 20 children (nos. 2, 3, 5, 6, 15, 18, 23, 37, 50, 53, 62, 64, 65, 66, 72, 83, 87, 112, 128, 131) and were analysed for LOH. The following microsatellite loci were used: D11S901, D11S2179, D11S1391, and D11S897 for the first locus; D11S1341, D11S976, D11S614, and D11S924 for the second and third loci. Three children (nos. 5, 66, 83) already had LOH of 11q at their initial diagnosis. Two children (nos. 5, 83) continued to have LOH of 11q at the time of relapse. LOH of sample from individual no. 66 was no longer detectable at the time of relapse. All of the 17 children with a normal 11q at their initial diagnosis also had a normal 11q at the time of their relapse. In total, 19 of 20 children showed the same 11q structure at relapse as at their initial presentation, suggesting that 11q deletions less frequently involved in the progression of childhood ALL.

Clinical information for up to 8 years from their initial diagnosis was available for 110 children examined in this study. LOH of chromosome arm 11q was found in both precursor-B ALLs (15/91; 16%) and T-ALLs (3/19; 16%), suggesting that the tumor suppressor genes in this region are critical to normal B- and T-cell function. No statistically significant associations were found between LOH of 11q and gender, white blood cell counts (WBC) counts at diagnosis, or age. All of the 110 children were treated uniformly, and none with 11q LOH showed a poor response to initial induction chemotherapy. In contrast, 15 of 92 (16%) patients without 11q LOH showed a poor response (P=0.015). The children with LOH of 11q tended to have a lower incidence of relapse over the 8 years of follow-up (22%) as compared to the cohort without the 11q LOH (29%).

Acknowledgements

We are grateful to Jochen Harbott for contributing the cytogenetic data and Wolf Dieter Ludwig for providing the immunophenotypic data. This work was supported in part by National Institutes of Health Grants, the Concern Foundation, Parker Hughes Trust (HP Koeffler), the Deutsche Forschungsgemeinschaft, Deutsche Krebshilfe (CR Bartram), and Grant-in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan (S Takeuchi).

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Figure 1 Summary of detailed LOH map of chromosome arm 11q in childhood acute lymphoblastic leukemia. We performed a detailed LOH analysis of chromosome arm 11q between D11S901 and D11S933 using 113 childhood ALL samples. Eighteen samples which showed LOH at one or more loci are presented. The genetic map of chromosome 11 was mainly compiled from the Généthon microsatellite map. For the markers which are assigned to the same location by linkage analysis, the results obtained from each marker were combined. Status of each chromosome locus is indicated by shading as LOH (black), retention of heterozygosity (white), and not informative (cross-hatched). Patient nos. are listed at the top of each column. The positions of the ATM and MLL genes are indicated