Leukemia (1998) 12, 474–481 1998 Stockton Press All rights reserved 0887-6924/98 $12.00 http://www.stockton-press.co.uk/leu Comparative genomic hybridization as part of a new diagnostic strategy in childhood hyperdiploid acute lymphoblastic leukemia OA Haas1, T Henn1, K Romanakis2, S du Manoir3 and C Lengauer4 1Children’s Cancer Research Institute (CCRI), St Anna Children’s Hospital, Vienna, Austria; 2Institute of Human Genetics, University of Kaiserslautern, Kaiserslautern, Germany; 3National Institutes of Health, Gene Technology Branch, National Center for Human Genome Research, Bethesda, MD, USA; and 4Molecular Genetics Laboratory, Johns Hopkins Oncology Center, Baltimore, MD, USA The detailed definition of karyotype changes associated with the leukemic samples, by a morphologically insufficient qual- hyperdiploid acute lymphoblastic leukemia (ALL) is a precon- ity of abnormal metaphases as well as by a high fraction of dition for their exploitation in minimal residual disease studies with fluorescence in situ hybridization analysis (FISH). In normal metaphases. In addition, it is not clear whether the addition, certain karyotype patterns may have different prog- abnormalities detected in a few analyzable metaphases are nostic implications. We have therefore used comparative gen- representative for the whole leukemic clone. Fluorescence in omic hybridization (CGH) to analyze the quantitative karyotype situ hybridization (FISH) techniques allow the analysis of both abnormalities in 14 cases of hyperdiploid ALL and correlated metaphase spreads and interphase nuclei but have been the results with those obtained by flow cytometry and conven- restricted to the evaluation of selected chromosomes or tional cytogenetic analyses. Despite an overall good agreement 15–20 between the karyotypes obtained by classical banding tech- chromosomal subregions. niques and CGH, we came across at least one karyotype dis- Comparative genomic hybridization (CGH) allows the crepancy per case. Clarification of the discordant findings with detection of chromosomal gains and losses in tissues which fluorescence in situ hybridization (FISH) showed that all stem are not accessible to conventional cytogenetic analysis.21–23 lines had been correctly defined by CGH. In eight cases, how- Genomic DNA from the tissue of interest and normal refer- ever, cytogenetic analyses revealed structural abnormalities ence DNA are differentially labeled and simultaneously that were undetectable by CGH. The other discrepancies were 21,22 mainly due to a cytogenetic misinterpretation of similar sized hybridized to normal human metaphase chromosomes. and shaped chromosomes. Based on these findings we present The copy number changes are elicited by the ratios of tumor a new diagnostic strategy for childhood ALL that includes flow and normal fluorescence intensities along the target meta- cytometry and classical cytogenetics as well as CGH for the phase chromosomes. The major disadvantages of CGH are the analysis of aneuploid cases and FISH to resolve the unavoid- facts that only copy number changes of 5 to 10 megabases able discrepancies. can be detected, and that balanced structural rearrangements Keywords: hyperdiploid ALL; flow cytometry; cytogenetics; fluor- 22 escence in situ hybridization (FISH); comparative genomic hybridiz- are not detectable at all. These drawbacks, however, should ation (CGH) play a minimal role in the analysis of hyperdiploid ALLs, since hyperdiploid ALLs predominantly gain complete chromo- somes. Moreover, the vast majority of their structural changes − Introduction are unbalanced, such as a duplication of 1q, iso and marker chromosomes and should therefore be detectable by CGH. Childhood acute lymphoblastic leukemia (ALL) is a hetero- For this reason we considered CGH an excellent supplement for the diagnostic work-up of childhood ALL. In contrast to geneous group of diseases characterized by distinct patterns 23 of chromosomal abnormalities.1–7 The unequivocal determi- a previous study of CGH in childhood ALL, however, we nation of the karyotype composition could be of great impor- restricted ourselves to aneuploid cases that were selected tance and might eventually be used for the stratification of according to their hyperdiploid DNA content in flow clinical therapy.2–4,6 Hyperdiploidy with more than 50 chro- cytometry. We compared thoroughly the respective results mosomes or a DNA content Ͼ1.16 is one of the strongest with those obtained by cytogenetic analysis, and examined predictors for an extremely low risk of failing chemotherapy, the discrepancies with FISH. regardless of the white blood cell count (WBC).6,8 Such cases are likely to fare well on antimetabolite-based therapy and, therefore, may be spared the toxic effects of more intensive Materials and methods treatment with genotoxic agents.4,6,8 It has been suggested that the prospective outcome of hyperdiploid cases may further be Patients related to the particular karyotype composition. Both trisomy 6 and the combined presence of trisomy 4 and 10 seem to We studied 14 cases of childhood hyperdiploid ALL from indicate an extremely favorable prognosis.9,10 which liquid nitrogen-stored isolated mononuclear bone mar- Several laboratory procedures have been found useful for row (BM) cells were available. The relevant clinical and hem- the analysis of leukemia-associated chromosomal changes. atological characteristics of the patients are summarized in Unfortunately, no technique has been able to reveal a com- Table 1. All patients suffered from B cell precursor ALL. Their plete picture of all genetic alterations. Flow cytometry, blast cells were CD10-, CD19- and HLA-DR-positive. Two although simple and fast, only delineates gross quantitative cases (patients 13 and 14) were also positive for deviations of the DNA content.8,11–14 Classical cytogenetic cytoplasmic IgM. analysis is commonly hampered by a poor in vitro growth of Cytogenetic analysis Correspondence: OA Haas, CCRI, St Anna Children’s Hospital, Kind- erspitalgasse 6, A-1090 Vienna, Austria; Fax: +43 1 40170 481 Unstimulated isolated BM cells were cultured for 24 h. Chro- Received 30 September 1997; accepted 20 November 1997 mosomes were prepared according to standard procedures. CGH of hyperdiploid childhood ALL OA Haas et al 475 Table 1 Hyperdiploid cases of childhood ALL Patient Sex Age at WBC % blasts CG CGH Flow cytometry diagnosis (G/L) DNA index BM PB %abn %CV Mat 1 F 2 11/12 111.0 98 91 1.10 1.10 1.09 54 1.05 ea 2 F 5 2/12 3.0 75 1 1.12 1.13 1.13 38 1.20 ma 3 M 2 4/12 120.0 90 92 1.00 1.13 1.14 93 0.65 e 4 M 7 1/12 2.0 95 17 1.15c 1.18 1.17 57 1.57 ma 5 M 2 4/12 53.1 97 87 1.19c 1.19 1.18 71 2.30 ma 6 M 14 8/12 2.1 91 1 1.19 1.19 1.18 77 2.00 ma 7 F 2 7/12 50.0 93 83 1.17 1.19 1.19 92 2.00 e 8 M 1 5/12 4.5 98 24 1.18c 1.18 1.19 88 1.14 ma 9 M 4 8/12 12.2 86 47 1.16 1.18 1.20 87 1.17 e 10 M 4 10/12 14.0 90 64 1.25 1.26 1.25 54 1.00 ma 11 M 2 10/12 29.1 95 66 ND 1.37 1.36 90 1.00 e 12 F 2 7/12 9.3 89 22 1.39c 1.40 1.37 87 1.40 e 13 M 2 4/12 31.2 95 69 ND 1.41 1.40 58 2.70 eb 14 F 13 5/12 12.6 90 39 1.38c 1.39/1.44d 1.42 80 1.06 ma The DNA contents based on cytogenetic and CGH data were calculated by adding or subtracting the relative DNA contents of the respective gained or lost chromosomes. The relative DNA contents of the individual chromosomes were adapted from the publication by Boschman et al29 (see Table 3). aPB; bPB analyzed with a FACS; cDNA content of marker chromosomes was estimated from their sizes; dtwo clones. F female; M, male; WBC, white blood cell count; bl, blast cells; BM, bone marrow; PB, peripheral blood; Mat, material; e, fresh cells fixed with ethanol; ma, cultured cells fixed with methanol/acetic acid; abn, abnormal; CV, coefficiency of variation; CG, cytogenetic results; CGH, comparative genomic hybridization; ND not done. Slides were G- and /or R- and C-banded with trypsin Giemsa peak located at the diploid level was assigned a DNA content and chromomycin A3, distamycin and DAPI, respectively.24 of 1.0. The presence of additional peaks indicated DNA aneu- Karyotyping was performed on a Genevision 121 chromo- ploidy. A DNA index Ͼ1.0 was considered as a hyperdiploid some analysis system. Although the International System for DNA content. Human Cytogenetic Nomenclature (ISCN) requires the description of hypotriploid karyotype changes in relation to triploidy as the ‘normal range’,25 all descriptions of karyotypes Comparative genomic hybridization and CGH results presented are, for the sake of clarity, based DNA from healthy donors was labeled with digoxigenin-11- on a normal diploid pattern. dUTP (Boehringer Mannheim, Mannheim, Germany) and DNAs from the patients’ bone marrow samples were labeled Flow cytometry with biotin-16-dUTP (Boehringer Mannheim) by nick trans- lation. 500 ng of labeled normal and patient DNA were hybridized together with 20 ␮g of human cot-I DNA and Either freshly isolated, ethanol-fixed or cultured, 10 ␮g salmon sperm DNA to normal metaphase spreads that methanol/acetic acid-fixed cells were prepared as described were pretreated as published previously.26 Digoxigenin-11- previously.14 Fixed cells were resuspended in a solution con- dUTP-labeled probes were detected with mouse-derived taining 3% citric acid and 0.5% Tween 20, and incubated for anti-digoxigenin antibody and tetramethyl-rhodamine- 20 min at room temperature (RT).