Comparative Genomic Hybridization As Part of a New Diagnostic Strategy In

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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 chromosomes. 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). Alternatively, 0.5 ml of the isothiocyanate (TRITC)-conjugated rabbit-anti-mouse mono- methanol/acetic acid-fixed cell suspension was resuspended clonal antibodies (Sigma, Vienna, Austria). Biotin-16-dUTP in 0.5 ml pepsin/HCl solution (0.5% pepsin in 0.03 M HCl, was detected with fluorescein-iso-thiocyanate (FITC)-conju- pH 1.5–1.7) and incubated for 10 min at RT. Nuclei were gated avidin D (Vector, Burlingame, CA, USA). Slides were passed through a 30 ␮m nylon mesh and centrifuged at 1200 counterstained with DAPI and embedded in H-1000 mounting r.p.m. for 8 min. The cell pellet was resuspended in 70% etha- medium (Vectashield; Vector). nol. After 2 h at −20°C the material was centrifuged again and Image acquisition, processing and evaluation was perfor- the pellet resuspended in 0.3 ml 0.5% Tween 20 for 10 min. med as described previously using an epifluorescence micro- Cells were stained with 2 ml of a solution that contains 4 g scope (Zeiss, Jena, Germany) equipped with a cooled CCD Na HPO × 2H O, 1 g citric acid and 0.2 mg 4,6-diamino-2- 2 4 2 camera (Photometrics, Tuscon, AZ, USA).27 Fluorescence ratio phenylindole-2 hydrochloride (DAPI) per 100 ml distilled profiles of individual chromosomes were calculated using H O for 1 h. The samples were then analyzed with a PAS III 2 ¨ dedicated software.27 For each case, the mean ratio profiles flow cytometer (Partec, Munster, Germany) using the filter of 10–15 metaphase spreads were analyzed. The threshold combination KG1, BG 38 and UG1 (excitation filter), TK 420 used for the identification of imbalances was defined as the (dichroic mirror) and CG 435 (barrier filter). As a control, fresh 95% confidence interval.27 samples from Ficoll-separated peripheral blood (PB) lympho- cytes of healthy donors were analyzed (0.7–1.00% coefficiency of variation). At least 10 000 nuclei were analyzed Conventional FISH per experiment. For the generation of histograms the ‘Multi- cycle’ cell cycle analysis program (Phoenix Flow System, San Numerical abnormalities were analyzed in interphase nuclei Diego, CA, USA) was used. A sample with one single G0/1 by hybridizing repetitive probes specific for the centromeric CGH of hyperdiploid childhood ALL OA Haas et al 476 regions of chromosomes 6, 8, 10, 17, 18 and X.19 For the abnormalities were observed in only four cases of the hyperdi- numerical evaluation of chromosome 21 a cosmid containing ploid group (patient Nos 3, 4, 5 and 8). Interestingly, in three D21S65 (Oncor, Gaithersburg, MD, USA) and a YAC clone of these instances chromosome 7 was affected. Significantly spanning the BCR region on chromosome 22 were applied.28 more structural changes were present in all four patients of the hypotriploid group. Normal metaphases were only found in one (out of four) of the hypotriploid cases, but were Results detected in seven (out of 10) hyperdiploid cases.

Classical banding analysis Flow cytometry Fourteen cases with childhood ALL were analyzed with classical G- and R-banding techniques (Table 2). According to the We determined the percentage of hyperdiploid cells in the classiﬁcation criteria of the ISCN,25 nine cases (patient Nos analyzed samples by measuring the DNA index by ﬂow cyto- 1, 2 and 4–10) were in the hyperdiploid range with 52–58 metry (Table 1). Each sample analyzed showed one single chromosomes, four cases (patient Nos 11–14) were in the abnormal peak that corresponded to 54–93% of the analyzed hypotriploid range with 63–66 chromosomes and one case cell population (Table 1). Residual diploid cells could be was pseudodiploid with a hyperdiploid DNA index (patient detected in all samples and served as an internal standard. No. 3). As expected, most of the chromosomal changes The single abnormal peak indicated that all abnormal cells of involved were gains of complete chromosomes. Structural a patient harbored the same distinct quantitative chromosomal

Table 2 Summary of cytogenetic and CGH results of 14 hyperdiploid cases of ALL

Patient Cytogenetics CGHa Revisions FISH

1 52,XX,+6,+8,+13,+14,+21,+21 [20] +X,+9,+13,+14,+21,+22 6ϾX, 8Ͼ9, 3 × 22 21Ͼ22 2 46,XX [3] +X,+6,+9,+14,+18, (+19),+21, 2 × 22 (70%) 53,XX,+X,+6,+9,+14,+18,+21,+21 [3] +21,+22 3 × 22 (30%) 3 46,XY [7] +X,+6,+8,+10,+18,+19,+21,+21 3 × X, 3 × 6, 46,XY,del(12)(p12) [3] 3 × 8, 3 × 10, 3 × 18, 4 × 21 4 46,XY [1] +X,+4,+6,−7p,+7q,+10,+11,+17, 4 × 21 (87%) 51– 54,XY,+X,+4,+6,i(7q),+10,+11,+17,+18,+21,add +18,+21,+21,+22 (22q),+mar [cp19] 5 46,XY [2] +X,+4,+6,+8,+10,+14,+17,+18, 56,XY,+X,+4,+6,der(7),+8,+10,+14,+17,+18,+21,+21 [2] +21,+21 6 46,XY [3] +X,+4,+6,+9,+10,+14,+17,+18, 7Ͼ9, 13Ͼ14 4 × 21 (73%) 56,XY,+X,+4,+6,+7,+10,+13,+17,+18,+21,+22 [cp17] +21,+21,+22 7 46,XX [17] +X,+4,+6,+10,+14,+14,+17,+18, 8Ͼ10 4 × 21 (81%) 55,XX,+X,+4,+6,+8,+14,+17,+18,+21,+22 [4] +21,+21,+22 8 46,XY [3] +X,+4,+6,+9,+10,+14,+17,+18, 46,XY,der(7) [3] +21,+21 56,XY,+X,+4,+6,der(7),+9,+10,+14,+17,+18,+21,+21 [5] 9 54,XY,+X,+4,+8,+9,+9,+14,+21,+22 [10] +X,+4,+8,+9,+9,+14,+17,+19,4× 21 (84%) +21,(+21),+22 10 55–58,XY,+X,+4,+4,+5,+6,+8,+10,+14,+17,+18,+21,+21 +X,+4,+4,+5,+6,+8,+10,+14,+17, [cp10] +18,+20,+21,+21 11 63,XY,+X,+Y,dup(1)(q11q44),+4,+5,+6,der(7),+8,+11, +X,+1q,+4,+5,+6,+6p,+8,+8,+10, add(11)(q23),+12,+14,+14,+17,+18,+18,+21,+22, +11,–11qdist,+12,+14,+14,+17, +del(22)(q11)?,+1–2 mar [cp10] +17,+18,+18,+19,+21,+21,+22,+22 12 46,XX[7] +X,+2,+4,+4,+5,+6,+8,+10,+11, 13Ͼ14 63,XX,+X,+2,+del(3)(q13)?,+4,+der(4)t(4;18)(?p15;?q11),+5, +12,+14,+16,+17,+18,+19,+21, +6,+8,+10,+11,+12,+13,+16,+17,−18,+21,+21,+22[cp13]b +21,+22 13 47,XY,+21 [1] +X,(+1),+del(2)(pdist),+3,+4,+5, +del(10)Ͼ+10 4 × 17 65–66,XY,+X,+add(Y)(qter),+add(Y)(qter),+del(2)(p16?),+3, +6,+7,+8,+8,+10,+10,+11,+12,+14, 13Ͻ14 +4,+5,+6,+7,+8,+add(9)(p22?),+10,+del(10)(q24),+11, +14,+17,+17,(+19),+21,+21,+22, +add(11)(pter),+12,+13,+14,+i(17)(q10),+21,+21,+22, +22 +r,+mar [cp24] 14 65,XX,+1,t(1;19)(q23;p13),+t(1;19)(q23;p13),+2,+5,+6,+7,+8, +1,+1,+2,+5,+6,(+6),+7,+8,+9,+10, 2 × 6 (16%) +9,+10,+14,+15,+16,+18,+19,+21,+22,+22,+2 mar[2] +14,+15,+16,(+16),+18,+19,+19,+21, 3 × 6 (38%), +22,+22 4 × 6 (46%)

Discrepancies between the cytogenetic and CGH data are printed in bold and are underlined. Some of the discrepancies could be solved by re-checking the karyotypes (column ‘Revisions’). The results of the conventional FISH experiments are shown in the column to the very right (‘FISH’). aChromosome number in brackets indicate that this particular chromosome was considered to be present only in a fraction of the cells (for explanation see text). bChromosome count of 13 metaphase, complete karyotype from one metaphase only. CGH of hyperdiploid childhood ALL OA Haas et al 477 abnormalities, likely involving the same clone. DNA contents (chromosomes 13, 14 and 15) and G-group (chromosomes 21 of the abnormal clones ranged from 1.09 to 1.42 (median and 22). Thus, in eight instances, the data obtained by CGH 1.19). In 13 out of 14 cases, the DNA index of the abnormal immediately allowed a modification of the cytogenetic kary- clone obtained by flow cytometry corresponded tightly to the otypes (Table 2). In seven cases CGH indicated the gain of DNA index that was calculated from classical banding analy- chromosomes that had not been observed by banding analysis based on the suggestions by Boschman et al29 (Table 3). sis. Subsequent FISH analyses showed that in all instances the The percentage of abnormal cells detected by flow cytome- CGH results were correct (Table 2). Patient 14 was of parti- try concurred with the fraction of blast cells found in BM in cular interest. CGH analysis suggested the presence of two most of the cases from which fresh cells were analyzed. This subpopulations, since both chromosomes 6 and 16 did not finding indicates that all leukemic cells were aneuploid. How- reach the threshold value that indicated ‘pure’ trisomy. DNA ever, compared to the percentage of BM blast cells, flow cyto- index calculations of the two different CGH patterns resulted metry resulted in a significantly smaller fraction of abnormal in DNA indices of 1.39 and 1.44, respectively. The lower cells when the cells were cultured for 24 h. This observation index DNA value showed an excellent correlation with the provides evidence that the hyperdiploid cell populations DNA index obtained by karyotyping, whereas the flow cyto- undergo apoptotosis within a very short period in vitro. metrically measured DNA index showed a better concordance with the higher indexed CGH clone. FISH with a chromosome 6-specific probe revealed interphase cells with two, three or Comparative genomic hybridization (CGH) four copies of chromosome 6, respectively (Table 2).

In concordance with the flow cytometric and cytogenetic data, CGH detected quantitative chromosome abnormalities Discussion in all samples (Table 2). Figure 1 shows a representative example of the mean values of ratio profiles obtained from 10 The accurate determination of the specific karyotype abnor- metaphase spreads of patient 4. The gain of a long arm and malities associated with hyperdiploid ALL is important, the loss of a short arm of chromosome 7 is consistent with the because their detailed definition is a precondition for FISH- presence of an i(7q) chromosome detected by classical band- based minimal residual disease studies.5,30 In addition, ing analysis. In most of the cases whole chromosomes were particular karyotype patterns may have specific prognostic lost or gained. CGH also revealed unbalanced structural implications.2–4,6 Our evaluation of the CGH technique in rearrangements in three cases (Table 2). Figure 2 summarizes hyperdiploid ALL confirms that CGH is a valuable adjunct for all gains and losses observed in the 14 cases analyzed. In the unequivocal assessment of such quantitative changes. The accordance with the cytogenetic literature, we found a non- samples analyzed in our study contained between 75 and random acquisition of chromosomes 4, 6, 10, 14, 17, 18 and 98% (mean 92%) hyperdiploid blast cells. Moreover, the sin- X.6,7 Compared to trisomic chromosomes the shifts of the ratio gle narrow peaks obtained by flow cytometry indicated that profiles of chromosome 21 were much stronger in those 12 the abnormal cell populations were rather homogeneous with cases in which FISH analysis confirmed the presence of tetra- regard to their chromosomal composition. These features somy 21. This observation is in line with the linear correlation guaranteed a reliable detection of copy number changes in detected in cell lines with various numbers of X chromosomes hyperdiploid ALLs by CGH which is superior to that of flow and confirms that to a certain extent it is possible to estimate cytometry and classical cytogenetics. On the other hand, our copy number changes with CGH.21 Of interest, a high pro- findings also show that even a single or few hyperdiploid portion of cases also showed trisomies of chromosomes 5, 8, metaphases can be representative of the clonal changes in 9, 19 and 22 which suggests that trisomies of these chromo- hyperdiploid ALL. somes may be more commonly involved than previously In cytogenetics, the cutoff level for hyperdiploidy has been anticipated by classical banding analyses. set at 50 chromosomes,1,2,4,9,10 whereas in flow cytometry a In some instances (patient Nos 2, 9, 13 and 14) the base line cutoff level of 1.16 has proven to be the best discriminator profile of particular chromosomes was significantly shifted but between favorable and unfavorable groups.8 The quality of the did not reach the upper threshold level determined to classify flow cytometric measurements depends significantly on the ‘trisomy’. These ‘low shift levels’ resulted most likely from the way the cells are prepared and stained as well as on the instru- presence of a trisomy in only a fraction of the cell population. mental outfit for measurement. Estimation of the DNA index In the majority of samples the differences between DNA with a sensitivity of ±5%, which conforms to a difference of indices measured by flow cytometry and DNA indices calcu- ± two to four whole chromosomes, is currently considered as lated from the CGH data according to Boschman et al29 (Table being sufficiently accurate.8,10,13 Our comparison shows that, 3) were marginal (0–3% in 11 out of 14 cases). These results if only the DNA index is used for delineating hyperdiploid suggest that in most cases the same abnormal clone was cases, a significant proportion of those recognized cytogenet- detected by flow cytometry and CGH. ically will be missed. Depending on the sizes of gained chromosomes, even hyperdiploid cases with 52–53 chromosomes can have DNA contents far below 1.16 (Table 1). Comparison of cytogenetic and CGH data At present, FISH analysis with centromere-specific probes is the method of choice for screening numerical chromosome Despite an overall good agreement between the karyotypes abnormalities in interphase cells.15,17,19,20,31 It is a rapid, sim- obtained by cytogenetic banding techniques and CGH, we ple to use and easy to interpret technique. However, a reliable observed at least one karyotype discrepancy per case. Some detection of only the most common numerical deviations in of these discrepancies reflected obvious cytogenetic errors such hyperdiploid cases with conventional FISH requires at mainly caused by a wrong assignment of similar-sized chro- least four to six probes in several hybridization steps.5 Thus, mosomes, in particular chromosomes of the B- (chromosomes compared to conventional FISH screening, CGH clearly pro- 4 and 5), C- (chromosomes X, 6, 7, 8, 9 and 10), D- vides a representative overview of quantitative deviations CGH of hyperdiploid childhood ALL OA Haas et al 478

Figure 1 Mean of the ratio profile calculation of 10 metaphase spreads of patient No. 4. The vertical lines on the right side of the chromosome ideograms represent different ratio values of the fluorescence intensities between the tumor and the reference DNA. The left and right lines define the threshold values at the 95% confidence interval. Gray shaded boxes: regions which due to highly repetitive DNA cannot be interpreted. The overrepresented chromosomes X, 4, 6, 10, 11, 17, 18, 21 and 22 as well as the long arm of chromosome 7 are light gray, whereas the underrepresented short arm of chromosome 7 is dark gray.

which, in addition, is obtainable in only one single hybridiz- exact definition of chromosomal abnormalities associated ation experiment. Considering these facts, CGH is also a cost- with childhood ALL. For the detection of aneuploid cases we efficient technique. On the other hand, CGH also requires use flow cytometry and cytogenetic banding analysis which, cytogenetic expertise and can only be performed with a spe- despite all its disadvantages, still remains the only method that cific image analysis system and the respective software which, can provide a complete picture of both numerical and struc- however, is already contained in most chromosome analy- tural karyotype abnormalities. Aneuploid cases that are ses systems. detected by either of the two methods are then further studied We propose the following strategy for the detection and with CGH. In this diagnostic scenario, multicolor FISH analy- CGH of hyperdiploid childhood ALL OA Haas et al 479

Figure 2 Summary of chromosomal imbalances detected by CGH of 14 cases of childhood hyperdiploid ALL. The vertical line on the left side of the chromosome 7 ideogram represents loss of genetic material in one case, whereas the light gray (hyperdiploid cases) and dark gray lines (hypotriploid cases) on the right side of each chromosome ideogram correspond to a chromosomal gain.

sis can be restricted to the clarification of discrepant results, ing the patients samples and their continuing interest in our ¨ to decipher unusual and/or complex structural chromosomal work. This work was supported by the ‘Fonds zur Forderung abnormalities and to monitor small cell populations. der wissenschaftlichen¨ Forschung’ (grant No. P-10454-MED), the ‘Osterreichische Kinderkrebshilfe’ and the European Com- munity (grant No. CA-CT94-1703). We are indebted to Drs ¨ Acknowledgements Thomas Ried and Evelin Schrock at the NIH, Bethesda, USA, for their help in establishing the CGH technique and their We thank Gertrude Pass, Elisabeth Lang and Eva Winkler for continuous advice. their excellent technical support and all clinicians for provid- CGH of hyperdiploid childhood ALL OA Haas et al 480 Table 3 Relative DNA content of individual chromosomes used lymphoblastic leukaemia (ALL) by flow cytometry: correlation of for the calculations of the DNA content of the abnormal karyotypes FAB classification with DNA stemline and proliferation. Br J and the CGH results (adapted from Boschman et al,29 Table 5) Haematol 1985; 60: 677–686. ¨ 12 Hiddemann W, Harbott J, Haas OA, Budde M, Buchner T, Lam- a pert F. Nachweis von Aberrationen des Karyotyps bei Kindern mit No. DNA content per chromosome relative % ¨ akuten Leukamien: eine vergleichende Analyse von Zytogenetik ¨ und Durchflubzytophotometrie. 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