50 Chromosomes

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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). The primers for fragment (Bohringer Mannheim). Between the steps of the amplification of the probe were chosen according to the detection process the slides were washed three times for 5 min sequence of L1.84 published by Devilee et al.16 each in 4 × SSC/Tween 0.05% at room temperature. Without further purification the PCR products were labeled The cells were counterstained with 4′,6-diamidino-2-phe- by nick translation with biotin-16-dUTP or digoxigenin-11- nyl-indole dihydrochloride (DAPI) and mounted in the fluor- ¨ dUTP (Bohringer Mannheim, Mannheim, Germany) according escence antifading agent 1,4-diaza-bicyclo-(2,2,2,)-octane to the instructions of the supplier. The probes specific for (DABCO). Microscopy was performed with a Zeiss Axiophot chromosomes 6 and 17, respectively, were biotinylated; the photomicroscope (Zeiss, Oberkochem, Germany) fitted for probes specifically binding to chromosomes 10 and 18, epifluorescence. Nuclei were photographed with Kodak respectively, were modified with digoxigenin. Ektachrome 400 ASA color film (Kodak, Rochester, NY, USA).

In situ hybridization Nuclei scoring

For double target FISH the probes speciﬁc for chr 6 and 10, Evaluation of the signals in the interphase cells was done and the probes for chr 17 and 18, respectively, were applied according to the criteria published by Hopman et al.18 In the in combination. For each patient one slide was prepared, on control experiments using bone marrow and peripheral blood Screening for hyperdiploidy (Ͼ50) in childhood ALL by FISH J Ritterbach et al 429 Table 2 Hybridization data of normal bone marrow and peripheral blood samples (n = 6)

Probe for chromosome Frequency of spots per nucleus (%)a

01 2 3 4

6 0.05 ± 0.08 4.40 ± 1.61 95.40 ± 1.59 0.12 ± 0.08 0.03 ± 0.05 10 0.23 ± 0.23 4.05 ± 1.18 95.27 ± 1.22 0.40 ± 0.12 0.03 ± 0.08 17 0.03 ± 0.05 4.65 ± 0.50 95.22 ± 0.59 0.05 ± 0.05 0.05 ± 0.05 18 0.00 ± 0.00 3.63 ± 0.86 96.07 ± 1.01 0.08 ± 0.10 0.22 ± 0.26 aGiven are median percentages of the number of spots per nucleus ±s.d.; per sample 1000 nuclei were counted. of healthy donors each probe was tested in single and in dou- one extra copy of the respective chromosome. In 20 patients, ble target hybridization. In the single hybridization experi- three signals for chr 6 were detected, whereas for chr 10 only ments 1000 nuclei were analyzed per slide and probe. For the two hybridization signals were seen. Three patients showed a double target FISHs 200 interphase nuclei were scored per spot combination of two signals for chr 6 and three for 10. slide and probes. Ninety-six children (58.2%) exhibited two signals for chr 6 In the prospective study 100 nuclei per patient were evalu- and 10 in 89.0–99.0% (mean, 94.8%) of the investigated cells, ated on each slide in the series using the probes for chr 6 and thus indicating a diploid karyotype. The remaining children 10, and 200 nuclei per patient on each slide when evaluating showed other spot combinations (Table 3). the probes for chr 6 and 10 (100) and for chr 17 and 18 (100). In 35 (31.0%) of the 113 children of group 2 hybridization signals differing from the values for disomy were found ranging from 4.0 to 91.0% (mean 69.3%). Three signals for all Cytogenetic analysis tested chromosomes were found most often. Seventy-eight patients (69.0%) displayed only two spots in most of their blast Bone marrow (BM) and/or blood (PB) samples were processed cells (88.0–99.0%); mean 94.0%) indicating disomy for the by standard cytogenetic methods.19 Karyotype analyses were respective chromosomes. The different signal combinations done on GTG-banded20 chromosomes; chromosomal aber- for the respective chromosomes found in this group are shown rations were described according to the International System in Table 4 and Figure 1. for Human Cytogenetic Nomenclature (ISCN) (1995).21

Cytogenetic results Flow cytometry Cytogenetic analysis was done for all patients exhibiting sig- For the DNA analysis by ﬂow cytometry, techniques were nals in their nuclei which differed from the two signals for the used as described previously.22 targeted chromosomes. The results are shown in Table 5. In 52 of 67 patients of group 1 (52/165, 31.5%) the cytogenetic analysis revealed a hyperdiploid karyotype with more than 50 Results chromosomes. In two patients (94241, 94422) a translocation t(9;22)(q34;q11) was also present. Patient 94210/A who exhi- FISH experiments – controls bited three signals for chr 6 in 2% of his cells had a hyperdiploid chromosome set with 48 chromosomes, showing tri- In the nuclei prepared from BM and PB of healthy donors somy of chr 14 and chr 21. A further three patients (94009, the probes for chr 6, 10, 17 and 18 showed two signals in 94363, 94375) showed tetraploid clones in their leukemic approximately 95% of counted cells in the single hybridiz- cells, two others (94234, 94115) had a near-haploid karyo- ation experiments (Table 2). Interphase cells with three or four signals for the respective probe were observed in less than 1% + × of the nuclei. Therefore a cut-off value of 2% (mean 3 s.d.) Table 3 Different combinations of ﬂuorescence in situ hybridiz- was chosen for the detection of trisomies by interphase FISH. ation signals found in group 1 In the double hybridization experiments comparable results were obtained. ISH spots ISH spots No. patients chromosome 6 chromosome 10

FISH experiments – patients 002 111 Nuclei of 165 patients were hybridized with the probes spe- 121 2296 ciﬁc for chr 6 and 10 (group 1), whereas the remaining 113 3220 children were examined with DNA probes speciﬁc for chr 6 233 and 10, as well as 17 and 18 in combination, respectively 3336 (group 2). Sixty-seven (40.6%) of the 165 children of group 1 344 showed hybridiation spots in their nuclei that differed from 441 the values for disomy at frequencies ranging from 2.0–93.0% 621 (mean 65.9%). Most of the aberrant nuclei showed three sig- 165 nals for chr 6 and also three spots for chr 10 (n = 36) indicating Screening for hyperdiploidy (Ͼ50) in childhood ALL by FISH J Ritterbach et al 430 The overall frequency based on cytogenetics in our investigated group was: Hyperdiploidy Ͼ50 80 patients = 28.8% Hyperdiploid 47–50 5 patients = 1.8% Hyperhaploidy 2 patients = 0.7% Hypertriploidy 1 patient = 0.4% Near tetraploidy 4 patients = 1.4%

Comparison of DNA index and FISH

Flow cytometric analysis of the cellular DNA contents were Figure 1 Dual-colour FISH analysis on nuclei of leukemic bone performed on cells of 234/278 children (138 of group 1 and marrow cells of patient 95089/A (left panel) and 95107 (right panel). Left panel: Nucleus exhibiting three hybridization signals for chr 6 96 of group 2). 149 patients showed a DNA index 1.00–1.04, (FITC) as well as three signals for chr 10 (rhodamine). Right panel: ie in the diploid range. 138 of them exhibited two signals for Nucleus showing two hybridization spots for chr 17 (FITC) and four all tested chromosomes, whereas 11 children showed discrep- signals for chr 18 (rhodamine); DAPI counterstaining. ant results, which are summarized in Table 6. The DNA indices of 80 patients ranged from 1.04 to 2.00. The FISH signals of 67 children showed agreement with the Table 4 Different combinations of fluorescence in situ hybridiz- DNA measurements. Discrepancies between the measured ation signals found in group 2 DNA index and counted spots in the in situ hybridization experiments were detected in another 12 patients and are ISH spots ISH spots ISH spots ISH spots No. patients summarized in Table 6. One of these children (94115) with a chromosome chromosome chromosome chromosome 6 101718 high DNA index (1.72) showed hybridization spots indicating a hyperhaploid chromosome set (38%), but a hyperdiploid 2 22278karyotype detected by cytogenetics was also present. The 2 123 1FISH experiment of another patient (93348) could not be 2 322 3evaluated. 2 232 2 In five patients DNA indices below the value of 1.00 (0.59– 2 223 20.99) were found; one child (94234) showed good agreement 3 232 1with the hybridization spots indicating a hyperhaploid chro- 3 233 1 2 334 1mosome set, whereas two patients (94038, 95055) exhibited 2 333 1three signals for chr 6 and 10 (3/3) pointing to a hyperdiploid 3 33314karyotype (Table 6). Two patients (93619, 93600) with DNA 3 323 2indices of 0.99 and 0.98 respectively exhibited a normal 3 334 2signal distribution. 3 324 2 To clarify the discrepant results between DNA index 3 322 1Ͼ 3 433 1( 1.04) and hybridization spots we did karyotype analysis 4 644 1subsequently in 10 of 11 patients (Table 6). In seven children 113 (94136, 94137, 94319, 93667, 94012, 94197, 95218) the cytogenetic analysis could not detect the aneuploid cell clone and patient 94324 (1.08) showed a constitutional trisomy of chromosome 21. In one patient (94166) the karyotypic type, and in one child (94236) chromosome numbers in the analysis failed. hypertriploid range were detected. Patient 94728 showed a hyperdiploid karyotype with 51– 53 chromosomes, but neither chr 6 nor 10, 17, or 18 were In two patients (94418, 94247) who showed more than two involved in the trisomic state. This patient would not have signals in 30% of the counted nuclei, the hyperdiploid clone was not found and only cells with a normal karyotype were been detected using the technique of in situ hybridization with observed. The cytogenetic preparations of the bone marrow the probes that we had chosen. of six children were not evaluable because of the poor quality or the absence of metaphases. In group 2, 35 children showed more than two hybridiz- Discussion ation signals for the tested chromosomes. Twenty-eight of them (28/113, 24.8%) exhibited a Ͼ50 karyotype in their According to the cytogenetic findings at diagnosis of ALL, chil- malignant cells. One patient (95089/A) also had a translo- dren can be assigned to different prognostic groups. While cation t(9;22)(q34;q11). Patient 95099 with three signals of for many specific chromosomal translocations with prognostic chr 18 in 6% of the cells showed a hyperdiploid karyotype impact, the PCR technology is successfully applied (eg but without involvement of chr 18. t(9;22)(q34;q11), t(4;11)(q21;q23)),23 the Ͼ50 hyperdiploid Four patients fell within the group of hyperdiploidy with group can either be detected by measurements of the DNA chromosome numbers between 47–50 (95063, 95096, 95168, content of the blast cells or by classical cytogenetic analysis. 95219), while one child exhibited a tetraploid clone in his As cytogenetic investigations of a large number of patients is leukemic cells (95076). The cytogenetic preparations of two a time-consuming technique, it would be of interest to have patients could not be analyzed because of insufficient quality. a quick screening method for the early detection of patients Screening for hyperdiploidy (Ͼ50) in childhood ALL by FISH J Ritterbach et al 431 Table 5 Comparison of FISH signals with cytogenetic results

Patient ID FISH signals Karyotype

6 101718

94234 1 2 ND ND 27,XX,+10,+18,+21 94115 1 1 ND ND 25,XY,+mar/50,XY,+X,+14,+18,+21 No. patients 24 3 3 ND ND Ͼ50 12 3 2 ND ND Ͼ50 334NDNDϾ50 123NDNDϾ50 2 3333 Ͼ50 2 3324 Ͼ50 1 3322 Ͼ50 1 2123 Ͼ50 93331 3 2 ND ND Ͼ50, der(1) 94720 3333 Ͼ50,+i(21)(q10) 94694 3333 Ͼ50,+mar 94719 3333 Ͼ50,der(1)?t(1;2)(p32;q31),der(19)t(1;19)(q23;p13) 95139 3433 Ͼ50,der(1q) 94124 3 3 ND ND Ͼ50,der(1q) 94707 3333 Ͼ50,der(1q) 94329 3 3 ND ND Ͼ50,t(6;7)(q13;q36) 95089/A 3333 Ͼ50,t(9;22)(q34;q11) 94349 3 3 ND ND Ͼ50,trp(1)(q21q42) 94241 3 2 ND ND 46,XY,t(9;22)(q34;q11)/Ͼ50,t(9;22)(q34;q11),+der(22)t(9;22)(q34;q11) 94422 3 2 ND ND 48,XY,add(6)(q25),t(9;22)(q34;q11),+21,+der(22)t(9;22)(q34;q11)[cp6] /51,XY,+5,+6,a7,t(9;22)(q34;q11),+17,+20,+21,+3mar[cp7] 94023 2223 49-53,XY,+X,+4,+18,+21,+21,+mar[cp7] 95099 2223 52,XY,+X,+Y,+4,i(7)(q10),+14,+21,+21 95049 2333 52-53,XY,+X,+4,+10,+14,+17,+18,+21[cp5] 94143 3 3 ND ND 52-55,XX,+X,der(1q),+4,+5,+6,+10,+?14,+?17,+18,+mar1,+mar2,+mar3,mar4[9cp] 94705 3323 53,X,+X,−Y,+4,+6,+10,+14,add(17)(p?),+18,+21,+21 95178 3232 53,XX,+6,+8,+14,+17,+21,+21,+21 93608 3 2 ND ND 53,XY,+X,+4,+6,+14,+17,+18,+21 93326 3 3 ND ND 53,XY,+X,+6,+10,+14,+18,+21,+21 95011/A 3333 54,XY,+X,+4,+6,+der(10)?t(10;14)(q22;q11),+12,−14,+17,+18,+21,+21 93595 3 3 ND n.d 54,XY,+X,+6,+10,+14,+17,+18,+21,+21 95003 3323 54,XY,+X,+6,+10,+14,+18,+21,+21,+mar 95264 3333 54-55,XX,+X,+4,+6,+10,+14,+17,+18,+21,+21[cp6] 95208 3333 54-55,XY,+X,+4,+6,+10,+17,+18,+21,+21,+mar[cp3] 95195 3233 54-55,XY,+X,+4,+6,+16,+17,+18,+21,+21,+mar[cp10] 94663 3334 54-56,XX,+X,+4,+6,+8,+10,+14,+17,+18,+18,+21[cp8] 94364 3 3 ND ND 54-57,XY,+X,+4,+6,+10,+14,+17,+18,+21,+21,+21[cp9] 95031 3334 56,XY,+X,+4,+6,+10,+14,+17,+18,+18,+21,+21 95184 3333 56,XY,+X,der(1)t(1;?),+4,+6,+10,+14,+17,+18,+21,+21,+mar 95113 3333 57,XY,+X,+Y,+4,+5,+6,+10,+14,+17,+18,+21,+21 95171 3333 57-59,XY,+X,+4,+5,+6,+7,+8,+10,+14,+17,+18,+21,+21[cp8] 94286 3 3 ND ND 60-61,XY,+X,+Y,+4,+5,+6,+9,+10,+14,+15,+16,+17,+20,+21,+21,+mar1[cp4] 94387 3 3 ND ND 61-63,XY,+X,+3,+4,+5,+6,+8,+9,+10,+11,+12,+13,+14,+16,+17,+18,+21,+21,+2mar[cp5] 94662 2334 64-65,XX,+X,+1,+2,+3,+4,+7,+?9,+11,+12,+14,+16,+17,+18,+18,+21[cp5] 94236 3 4 ND nd. hypertriploid 94363 3 2 ND ND near tetraploid 94375 4 4 n.d ND near tetraploid 94009 6 2 n.d ND near tetraploid 95076 4644 near tetraploid 95157 2232 45-46 chr., +17 could not be conﬁrmed 95219 2322 45,XX,inc/50,XX,+10,inc/57,XX,+10,inc[1] 94210/A 3 2 ND ND 48,XY,+14,+21 95096 2232 49,XX,+X,+17,+mar 95063 2322 48,XY,+10[inc] 95168 2322 43-45,XY,+10[inc] 94418 3 3 ND ND 46,XY 94247 3 2 ND ND 46,XY

ND, not done; Ͼ50, only counting of chromosomes was possible. Screening for hyperdiploidy (Ͼ50) in childhood ALL by FISH J Ritterbach et al 432 Table 6 Discrepancies of DNA index and FISH results

Patient ID DNA index FISH (number of spots) Karyotype

6101718

(a) Patients with a DNA index of 1.00 94009 1.00 4 (21%) 2 ND ND near tetraploid 94375 1.00 4 (12%) 4 (12%) ND ND near tetraploid 94293 1.00 2 3 (28%) ND ND Ͼ50 93629 1.00 2 3 (27%) ND ND failed 93630 1.00 3 (3%) 2 ND ND failed 94210 1.00 3 (2%) 2 ND ND 48,XY,+14,+21 95219 1.00 2 3 (6%) 2 2 Ͼ50 95168 1.00 2 3 (27%) 2 2 47-50,XY,+10,inc 95157 1.00 2 2 3 (84%) 2 45-46 chr.,+17 could not be conﬁrmed 95096 1.00 2 2 3 (78%) 2 49,XX,+X,+17,+mar 95063 1.00 2 3 (10%) 2 2 47-50 chr., modal 48 chr. (b) Patients with a DNA index ranging from 1.04 to 2.00 and below 1.00 94136 1.21 2 2 ND ND 46,XX (1 metaphase with 50 chr.) 94137 1.07 2 2 ND ND 46,XY 94166 1.20 2 2 ND ND failed 94319 2.00 2 2 ND ND 46 chromosomes 94324 1.08 2 2 ND ND +21c 94411 1.95 2 2 ND ND ND 93667 1.18 2 2 2 2 46,XY and near tetraploid 94012 1.17 2 2 2 2 44-46,X,−X,+mar 94197 1.24 2 2 2 2 46,XY and near tetraploid 94728 1.10 2 2 2 2 51- 53,XY,+X,+9,+14,+21,+21,+mar[cp7] 95218 1.14 2 2 2 2 46,XX,del(17)(q25)/46,XX 94115 1.72 1 1 ND ND 25,XY,+mar/50,XY,+X,+14,+18,+21 94234* 0.59 ± 1.22 1 2 ND ND 27,XX,+10,+18,+21 94038 0.84 3 3 ND ND Ͼ50 95055 0.99 3 3 2 2 Ͼ50

*One metaphase was found with more than 50 chromosomes.

with Ͼ50 chromosomes. The feasibility of applying FISH to a concordant result was found between the DNA index and interphase nuclei to detect numerical abnormalities was the FISH analysis. However, 11 children with a DNA index shown in several investigations.24–27 We started a prospective of 1.00 showed more than two signals for at least one of the FISH study using probes specific for the most frequent trisomic tested chromosomes, and cytogenetics confirmed the FISH chromosomes (6, 10, 17, 18) in order to test this technique as results in eight of them. The trisomic clones of most of these a screening method for the detection of hyperdiploidy Ͼ50 in patients were very small (2–28%) and therefore might be children with ALL. undetectable for flow cytometry. Bone marrow and blood cells of a total of 278 children with On the other hand, another 11 patients with two signals the diagnosis of ALL were hybridized and all but two slides were found with a DNA index above the normal value. Cyto- (0.7%) could be analyzed successfully. genetics could be performed in nine of them and showed only To confirm the hybridization experiments, a cytogenetic one child with a Ͼ50 hyperdiploid karyotype in which, how- analysis was performed for all children showing a spot num- ever, none of the tested chromosomes was trisomic. In another ber differing from the normal values for disomy. The compari- child among normal metaphases one cell with more than 50 son of both techniques showed a high degree of concordance: chromosomes was detected. Hyperdiploidy was also found in in 80 of these 100 children a Ͼ50 hyperdiploid karyotype was one patient with a hyperhaploid karyotype showing only one detected, whereas in five patients hyperdiploidy with 47–50 signal for 6 and two signals for 10. Two cell clones could be chromosomes was found. In the latter group, only one chro- detected by FCM with a DNA index of 0.59 and 1.22. mosome exhibited hybridization signals indicating a trisomic Despite the fact that no cytogenetic data are available for state of the respective chromosome. In addition patients with most of the patients with two signals, the sensitivity of our chromosome numbers in the haploid (n = 2) and in the probe combination is confirmed by the results of flow cytome- triploid/tetraploid (n = 5) range could be identified. The FISH try and by the incidence of hyperdiploidy found in this study results of 10 patients could not be verified by cytogenetics that coincides with previously published data5 and is even due either to a lack of metaphases (n = 8) or to a normal kary- higher than in cytogenetics. otype (n = 2). These two children, however, had a DNA index The aim of this study was to test an alternative fast screening Ͼ1.16, indicating a Ͼ50 hyperdiploid karyotype. method detecting children with a hyperdiploid Ͼ50 kary- If the hybridization experiments showed two signals for otype. Because classical cytogenetics often fail after mailing every tested chromosome, the DNA measurements were and the metaphase quality in this group is often poor, it was counted as a control. DNA indices ranging from 1.00 up to decided to test interphase FISH for screening. As shown by 1.04 were taken as normal. In most of the patients (n = 138), the results, the use of centromeric probes for chromosomes 6, Screening for hyperdiploidy (Ͼ50) in childhood ALL by FISH J Ritterbach et al 433 10, 17 and 18 is sufficient to detect hyperdiploidy Ͼ50 and 10 Devilee P, Thierry RF, Kievits T, Kolluri R, Hopman AHN, Willard can be used for therapy stratification. Adverse prognostic mar- HF, Pearson P, Cornelisse CJ. Detection of chromosome aneuplo- kers like t(9;22) or t(1;19) which can be found in high hyperdi- idy in interphase nuclei from human primary breast tumors using chromosome-specific repetitive DNA probes. Cancer Res 1988; ploid karyotypes1 will be detected by the routinely performed 28 48: 5825–5830. RT-PCR analyses for these rearrangements. The combination 11 Anastasi J, Le Beau MM, Vardiman JW, Fernald AA, Larson RA, of these two screening techniques will give quick results Rowley JD. Detection of trisomy 12 in chronic lymphocytic leuke- usable for therapeutic decisions. Cytogenetics, however, mia by fluorescence in situ hybridization to interphase cells: a should be performed to evaluate the complete karyotype, but simple and sensitive method. Blood 1992; 79: 1796–1801. will not have further therapeutic consequences. As compared 12 Jabs EW, Wolf SF, Migeon BR. Characterization of a cloned DNA with classical cytogenetics the use of interphase FISH using sequence that is present at centromeres of all human autosomes the described probe combination has several advantages: it is and the X chromosome and shows polymorphic variation. Proc Natl Acad Sci USA 1984; 81: 4884–4888. easy to handle, has a high success rate (93%), is able to detect 13 Devilee P, Kievits T, Waye JS, Pearson PL, Willard HF. Chromo- small clones, needs only a small amount of material and is some-specific alpha satellite DNA: isolation and mapping of a less time-consuming. It will, however, not replace other tech- polymorphic alphoid repeat from human chromosome 10. niques, but is a useful alternative to screen for hyperdiploidy Genomics 1988; 3: 1–7. Ͼ50 in childhood ALL. 14 Waye JS, Willard HF. Molecular analysis of a deletion polymor- phism in alpha satellite of human chromosome 17: evidence for homologous unequal crossing-over and subsequent fixation. Nucleic Acids Res 1986; 14: 6915–6927. Acknowledgements 15 Dunham I, Lengauer C, Cremer T, Featherstone T. Rapid gener- ation of chromosome-specific alphoid DNA probes using the poly- merase chain reaction. Hum Genet 1992; 88: 457–462. For excellent technical assistance we are indebted to Astrid 16 Devilee P, Slagboom P, Cornelisse CJ, Pearson PL. Sequence het- Jaeckel regarding fluorescence in situ hybridization and cyto- ¨ erogeneity within the human alphoid repetitive DNA family. genetics, as well as Christina Both, Doris Erb and Thomas Nucleic Acids Res 1986; 14: 2059–2073. Jung. The authors also thank Dr Rosalyn Slater, Rotterdam, 17 Pinkel D, Straume T, Gray JW. Cytogenetic analysis using quanti- The Netherlands, for critically reviewing the manuscript. The tive high-sensitivity fluorescence hybridization. Proc Natl Acad study was granted by the Deutsche Krebshilfe, the Parents’ Sci USA 1986; 83: 2934–2938. 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