Leukemia (1998) 12, 427–433 1998 Stockton Press All rights reserved 0887-6924/98 $12.00 BIOTECHNICAL METHODS SECTION (BTS) BTS Leukemia Detection of hyperdiploid karyotypes (Ͼ50 chromosomes) in childhood acute lymphoblastic leukemia (ALL) using fluorescence in situ hybridization (FISH) J Ritterbach1, W Hiddemann2, JD Beck3, M Schrappe4, G Janka-Schaub5, W-D Ludwig6, J Harbott1 and F Lampert1 ¨ 1Oncogenetic Laboratory, Children’s Hospital, University of Gie␤en; 2Dept of Internal Medicine, University of Gottingen; 3Children’s Hospital, University of Erlangen; 4Children’s Hospital, Medical School of Hannover; 5Children’s Hospital, University of Hamburg; and ¨ 6Robert-Rossle-Clinic, Dept of Hematology, Oncology and Tumor Immunology, Humboldt-University, Berlin, Germany ALL patients with a hyperdiploid karyotype of more than 50 low white blood cell count and common or pre-B cell immu- chromosomes (high hyperdiploidy) carry a better prognosis in nophenotype.6 Moreover patients presenting with more than contrast to patients presenting with other cytogenetic features, and an appropriate less intensive therapy protocol should be 50 chromosomes in their blast cells have a favorable prog- developed for these patients. For this reason it is desirable to nosis compared to children belonging to other karyotypic sub- have a quick screening method identifying those with this type groups.7,8 Karyotyping of patients with ALL, however, is diffi- of hyperdiploidy. We therefore studied the bone marrow and/or cult because of the poor quality of metaphases. Flow blood cells of 278 children with ALL using double target fluor- cytometric measurements of the cellular DNA will identify the escence in situ hybridization (FISH) on interphase. A combi- majority of patients with ploidy variation but provides no nation of DNA probes (repetitive, centromere specific) was applied detecting chromosomes which are most frequently information about the nature of the chromosome aberrations. overrepresented in patients with hyperdiploidy (Ͼ50), at chro- The technique of fluorescence in situ hybridization (FISH) mosomes 6, 10, 17 and 18. All patients showing hybridization using chromosome-specific DNA probes is a powerful tool for signals differing from the normal signal distribution of two the identification of numerical aberrations of the targeted spots for each tested chromosome were analyzed cytogenet- chromosomes in metaphases as well as in interphase cells9,10 ically as well. 102 children (102/278; 36.7%) were found to have and is well documented for several types of malignancy.11 a clone with aberrant FISH results. In 80 patients (80/278, 28.8%) the cytogenetic analysis detected a hyperdiploid The aim of this study was to test the applicability of fluor- karyotype Ͼ50 chromosomes, whereas the remaining patients escent in situ hybridization on interphase as a screening (n = 12) could be related to other ploidy subgroups, ie hyper- method for detecting high hyperdiploid karyotypes in child- diploidy with 47–50 chromosomes, haploidy, triploidy/ hood ALL. A combination of DNA probes was chosen which tetraploidy. Comparison of the FISH results with the measure- hybridize to chromosomes that are most frequently overrep- ments of the DNA content showed good agreement for 88.8% resented in patients with a high hyperdiploid karyotype: chr (208/234) of the investigated patients. The detected rate of 2 28.8% patients with a high hyperdiploid karyotype in our inves- 6, 10, 17, and 18. The results of the FISH experiments were tigated cohort is comparable to the frequency of other studies. compared with the cytogenetic analysis as well as the data of Only one patient was not identified as having a hyperdiploid flow cytometry (FCM). karyotype with our combination of DNA probes. Our results indicate that FISH is a feasible and quick screening method for the detection of hyperdiploid karyotypes (Ͼ50 chromosomes) Patients and methods and other ploidy subgroups. Ͼ Keywords: hyperdiploidy 50; FISH; childhood ALL; cytogenetics Patients Bone marrow and/or blood from 278 children (100 girls, 178 Introduction boys) with ALL at diagnosis was studied prospectively. Of these patients 215 showed a common ALL immunophenotype In childhood acute lymphoblastic leukemia (ALL) hyperdiplo- whereas the remaining 63 were diagnosed as having pre-B- idy with more than 50 chromosomes is found in 25 to 30% ALL. Two patients additionally suffered from Down’s syn- of the patients. The modal chromosome number shows a peak drome. Further clinical data are shown in Table 1. at 55 chromosomes, ranging from 51 to 65, and the chromo- All children were treated according to the protocol of the somes most often involved in trisomies and/or tetrasomies are therapy trials ALL-BFM-90 or CoALL-05-92. Bone marrow chromosomes 4, 6, 10, 14, 17, 18, 20, 21 and the X chromo- and/or blood samples were sent to our laboratory by mail from 1–5 some. The children belonging to this group often present 61 pediatric oncology centers in Germany and Switzerland. with good risk features, such as age between 2 and 10 years, Correspondence: F Lampert, Oncogenetic Laboratory, Children’s Fluorescence in situ hybridization Hospital, University of Gie␤en, Feulgenstr 12, D-35385 Gie␤en, Germany, Fax: 49 641 99 43429 Slide preparation for in situ hybridization on interphase was Received 10 September 1997; accepted 20 November 1997 done with bone marrow/peripheral blood cells remaining Screening for hyperdiploidy (Ͼ50) in childhood ALL by FISH J Ritterbach et al 428 Table 1 Clinical and immunophenotypical data of the patients Ploidy group Age (years) WBC (×109/l) Blasts (%) Immunology c-ALL or Outcome pB-ALL relapse/death Near haploid 1.9–4.9 median, 45.0–72.5 median, 84–97 median, c = 1pB= 1 0/0 3.4 n = 2 58.7 n = 2 90.5 n = 2 Hyperdiploid 1.5–11.4 median, 1.3–133.9 median, 1.99 median, 30 c = 68 pB = 15 3/1 3.8 n = 83 7.6 n = 61 n = 56 Near tripolid 5.8 n = 1pB= 1 0/0 Near tetraploid 2.5–13.2 median, 3.3–18.4 median, 12–90 median, c = 4pB= 1 1/0 4.7 n = 5 5.7 n = 520n = 4 Diploid 1.5–18.4 median, 9.0–375.0 median, 2–96 median, c = 133 pB = 40 4/7 4.8 n = 173 14.9 n = 117 53 n = 114 Hyperdiploid 47–50 2.3–8.6 median, 10.8–216.0 median, 11–95 median, c = 3pB= 2 1/1 4.3 n = 5 14.0 n = 361n = 3 from cytogenetic analyses stored in fixative at −20°C. Cell pel- which either on one area (group 1, 6 and 10) dual-color lets were washed with fresh fixative, resuspended in fixative, fluorescence in situ hybridization was performed, or two and dropped on to cold wet slides. The slides were aged at different sites (group 2, 6, 10, 17 and 18) were hybridized. room temperature for 24 h. Occasionally, if the quantity of Preparations were pretreated with RNAse A (100 ␮g/ml) in cells was insufficient, prepared slides were stored in 70% 2 × SSC, pH 7, for 1 h at 37°C, followed by washes in 2 × SSC ethanol at −20°C until use. Immediately before the experiment and dehydration in a graded ethanol series (70%, 90%, 100%) these slides were dehydrated in 90%, 100% ethanol at at room temperature. 10 ␮l of the hybridization mixture (60% room temperature. formamide, 2 × SSC, 5% dextran sulfate, 500 ␮g/ml of carrier Control experiments were done on PHA-stimulated periph- salmon sperm DNA) containing 2–5 ␮g/ml of each DNA eral blood samples of five healthy donors and on bone mar- probe was applied under a coverslip (24 × 26 mm). row aspirates of three probands without infiltration of their Probe and target DNA were denatured simultaneously for bone marrow, one patient with infectious mononucleosis, one 4 min at 80°C on a heating plate. Hybridization was carried patient with ANLL/M7 in remission. out in a moist chamber at 37°C for 20 h. After hybridization stringent washing was done with 0.1 × SSC at 58°C for 5 min three times, followed by an incubation in 0.1 × SSC at room DNA probes temperature for 5 min and 4 × SSC/Tween 0.05% (pH 7) at room temperature for 5 min. For the detection of chromosomal aneuploidy concerning chromosomes 6, 10, 17 and 18 the following probes, hybridizing to the respective centromeric region of the chro- Probe detection and microscopy mosome, were used: 6: Probe p308, a pBR322 recombinant with a 3.0 kb BAMHI human repetitive sequence (kindly pro- Visualization of the hybridization reaction was performed as vided by Dr EW Jabs).12 10: Palpha 10.1, a pUC9 recombinant described by Pinkel et al.17 Preparations were incubated with with a 0.95 kb RsaI human repetitive sequence (kindly pro- 5% BSA in 4 × SSC/Tween 0.05% for 30 min at 37°Cina vided by Dr P Devilee).13 The inserts of the probes for 6 and moist chamber. The biotinylated probes were detected using 10 were amplified using the appropriate plasmid primers via avidin-conjugated fluorescein isothiocyanate (avidin-FITC; PCR. 17: p17h8, a pSP65 recombinant with a 2.7 kb EcoRI Vector, Burlingame, CA, USA) as the first layer for 50 min. human repetitive sequence (kindly provided by Dr F Amplification of the FITC signals was done with additional Willard).14 Chromosome 17 specific primers were used to layers of biotinylated goat anti-avidin (Vector) and avidin- amplify the probe as described by Dunham et al.15 18: L1.84, FITC. The digoxigenin-labeled probes were simultaneously a pKUN recombinant with a 0.68 kb EcoRI human repetitive detected by the use of an anti-digoxigenin-rhodamine, Fab ¨ sequence (kindly provided by Dr P Devilee).