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 (⬎50 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
¨
Oncogenetic Laboratory, Children’s Hospital, University of Gie␤en; 2Dept of Internal Medicine, University of Gottingen; 3Children’s
4
5
Hospital, University of Erlangen; Children’s Hospital, Medical School of Hannover; Children’s Hospital, University of Hamburg; and
¨
6
Robert-Rossle-Clinic, Dept of Hematology, Oncology and Tumor Immunology, Humboldt-University, Berlin, Germany
1
ALL patients with a hyperdiploid karyotype of more than 50
chromosomes (high hyperdiploidy) carry a better prognosis in
contrast to patients presenting with other cytogenetic features,
and an appropriate less intensive therapy protocol should be
developed for these patients. For this reason it is desirable to
have a quick screening method identifying those with this type
of hyperdiploidy. We therefore studied the bone marrow and/or
blood cells of 278 children with ALL using double target fluorescence in situ hybridization (FISH) on interphase. A combination of DNA probes (repetitive, centromere specific) was
applied detecting chromosomes which are most frequently
overrepresented in patients with hyperdiploidy (⬎50), at chromosomes 6, 10, 17 and 18. All patients showing hybridization
signals differing from the normal signal distribution of two
spots for each tested chromosome were analyzed cytogenetically as well. 102 children (102/278; 36.7%) were found to have
a clone with aberrant FISH results. In 80 patients (80/278,
28.8%) the cytogenetic analysis detected a hyperdiploid
karyotype ⬎50 chromosomes, whereas the remaining patients
(n = 12) could be related to other ploidy subgroups, ie hyperdiploidy with 47–50 chromosomes, haploidy, triploidy/
tetraploidy. Comparison of the FISH results with the measurements of the DNA content showed good agreement for 88.8%
(208/234) of the investigated patients. The detected rate of
28.8% patients with a high hyperdiploid karyotype in our investigated cohort is comparable to the frequency of other studies.
Only one patient was not identified as having a hyperdiploid
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 (⬎50 chromosomes)
and other ploidy subgroups.
Keywords: hyperdiploidy ⬎50; FISH; childhood ALL; cytogenetics
Introduction
In childhood acute lymphoblastic leukemia (ALL) hyperdiploidy with more than 50 chromosomes is found in 25 to 30%
of the patients. The modal chromosome number shows a peak
at 55 chromosomes, ranging from 51 to 65, and the chromosomes most often involved in trisomies and/or tetrasomies are
chromosomes 4, 6, 10, 14, 17, 18, 20, 21 and the X chromosome.1–5 The children belonging to this group often present
with good risk features, such as age between 2 and 10 years,
Correspondence: F Lampert, Oncogenetic Laboratory, Children’s
Hospital, University of Gie␤en, Feulgenstr 12, D-35385 Gie␤en,
Germany, Fax: 49 641 99 43429
Received 10 September 1997; accepted 20 November 1997
low white blood cell count and common or pre-B cell immunophenotype.6 Moreover patients presenting with more than
50 chromosomes in their blast cells have a favorable prognosis compared to children belonging to other karyotypic subgroups.7,8 Karyotyping of patients with ALL, however, is difficult because of the poor quality of metaphases. Flow
cytometric measurements of the cellular DNA will identify the
majority of patients with ploidy variation but provides no
information about the nature of the chromosome aberrations.
The technique of fluorescence in situ hybridization (FISH)
using chromosome-specific DNA probes is a powerful tool for
the identification of numerical aberrations of the targeted
chromosomes in metaphases as well as in interphase cells9,10
and is well documented for several types of malignancy.11
The aim of this study was to test the applicability of fluorescent in situ hybridization on interphase as a screening
method for detecting high hyperdiploid karyotypes in childhood ALL. A combination of DNA probes was chosen which
hybridize to chromosomes that are most frequently overrepresented in patients with a high hyperdiploid karyotype: chr
6, 10, 17, and 18.2 The results of the FISH experiments were
compared with the cytogenetic analysis as well as the data of
flow cytometry (FCM).
Patients and methods
Patients
Bone marrow and/or blood from 278 children (100 girls, 178
boys) with ALL at diagnosis was studied prospectively. Of
these patients 215 showed a common ALL immunophenotype
whereas the remaining 63 were diagnosed as having pre-BALL. Two patients additionally suffered from Down’s syndrome. Further clinical data are shown in Table 1.
All children were treated according to the protocol of the
therapy trials ALL-BFM-90 or CoALL-05-92. Bone marrow
and/or blood samples were sent to our laboratory by mail from
61 pediatric oncology centers in Germany and Switzerland.
Fluorescence in situ hybridization
Slide preparation for in situ hybridization on interphase was
done with bone marrow/peripheral blood cells remaining
Screening for hyperdiploidy (⬎50) 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
pB-ALL
Outcome
relapse/death
Near haploid
1.9–4.9 median,
3.4 n = 2
1.5–11.4 median,
3.8 n = 83
5.8 n = 1
2.5–13.2 median,
4.7 n = 5
1.5–18.4 median,
4.8 n = 173
2.3–8.6 median,
4.3 n = 5
45.0–72.5 median,
58.7 n = 2
1.3–133.9 median,
7.6 n = 61
84–97 median,
90.5 n = 2
1.99 median, 30
n = 56
c = 1 pB = 1
0/0
c = 68 pB = 15
3/1
3.3–18.4 median,
5.7 n = 5
9.0–375.0 median,
14.9 n = 117
10.8–216.0 median,
14.0 n = 3
12–90 median,
20 n = 4
2–96 median,
53 n = 114
11–95 median,
61 n = 3
pB = 1
c = 4 pB = 1
0/0
1/0
c = 133 pB = 40
4/7
c = 3 pB = 2
1/1
Hyperdiploid
Near tripolid
Near tetraploid
Diploid
Hyperdiploid 47–50
from cytogenetic analyses stored in fixative at −20°C. Cell pellets were washed with fresh fixative, resuspended in fixative,
and dropped on to cold wet slides. The slides were aged at
room temperature for 24 h. Occasionally, if the quantity of
cells was insufficient, prepared slides were stored in 70%
ethanol at −20°C until use. Immediately before the experiment
these slides were dehydrated in 90%, 100% ethanol at
room temperature.
Control experiments were done on PHA-stimulated peripheral blood samples of five healthy donors and on bone marrow aspirates of three probands without infiltration of their
bone marrow, one patient with infectious mononucleosis, one
patient with ANLL/M7 in remission.
DNA probes
For the detection of chromosomal aneuploidy concerning
chromosomes 6, 10, 17 and 18 the following probes,
hybridizing to the respective centromeric region of the chromosome, were used: 6: Probe p308, a pBR322 recombinant
with a 3.0 kb BAMHI human repetitive sequence (kindly provided by Dr EW Jabs).12 10: Palpha 10.1, a pUC9 recombinant
with a 0.95 kb RsaI human repetitive sequence (kindly provided by Dr P Devilee).13 The inserts of the probes for 6 and
10 were amplified using the appropriate plasmid primers via
PCR. 17: p17h8, a pSP65 recombinant with a 2.7 kb EcoRI
human repetitive sequence (kindly provided by Dr F
Willard).14 Chromosome 17 specific primers were used to
amplify the probe as described by Dunham et al.15 18: L1.84,
a pKUN recombinant with a 0.68 kb EcoRI human repetitive
sequence (kindly provided by Dr P Devilee). The primers for
amplification of the probe were chosen according to the
sequence of L1.84 published by Devilee et al.16
Without further purification the PCR products were labeled
by nick translation with biotin-16-dUTP or digoxigenin-11¨
dUTP (Bohringer Mannheim, Mannheim, Germany) according
to the instructions of the supplier. The probes specific for
chromosomes 6 and 17, respectively, were biotinylated; the
probes specifically binding to chromosomes 10 and 18,
respectively, were modified with digoxigenin.
which either on one area (group 1, 6 and 10) dual-color
fluorescence in situ hybridization was performed, or two
different sites (group 2, 6, 10, 17 and 18) were hybridized.
Preparations were pretreated with RNAse A (100 ␮g/ml) in
2 × SSC, pH 7, for 1 h at 37°C, followed by washes in 2 × SSC
and dehydration in a graded ethanol series (70%, 90%, 100%)
at room temperature. 10 ␮l of the hybridization mixture (60%
formamide, 2 × SSC, 5% dextran sulfate, 500 ␮g/ml of carrier
salmon sperm DNA) containing 2–5 ␮g/ml of each DNA
probe was applied under a coverslip (24 × 26 mm).
Probe and target DNA were denatured simultaneously for
4 min at 80°C on a heating plate. Hybridization was carried
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
temperature for 5 min and 4 × SSC/Tween 0.05% (pH 7) at
room temperature for 5 min.
Probe detection and microscopy
Visualization of the hybridization reaction was performed as
described by Pinkel et al.17 Preparations were incubated with
5% BSA in 4 × SSC/Tween 0.05% for 30 min at 37°C in a
moist chamber. The biotinylated probes were detected using
avidin-conjugated fluorescein isothiocyanate (avidin-FITC;
Vector, Burlingame, CA, USA) as the first layer for 50 min.
Amplification of the FITC signals was done with additional
layers of biotinylated goat anti-avidin (Vector) and avidinFITC. The digoxigenin-labeled probes were simultaneously
detected by the use of an anti-digoxigenin-rhodamine, Fab
¨
fragment (Bohringer Mannheim). Between the steps of the
detection process the slides were washed three times for 5 min
each in 4 × SSC/Tween 0.05% at room temperature.
The cells were counterstained with 4′,6-diamidino-2-phenyl-indole dihydrochloride (DAPI) and mounted in the fluorescence antifading agent 1,4-diaza-bicyclo-(2,2,2,)-octane
(DABCO). Microscopy was performed with a Zeiss Axiophot
photomicroscope (Zeiss, Oberkochem, Germany) fitted for
epifluorescence. Nuclei were photographed with Kodak
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,
and the probes for chr 17 and 18, respectively, were applied
in combination. For each patient one slide was prepared, on
Evaluation of the signals in the interphase cells was done
according to the criteria published by Hopman et al.18 In the
control experiments using bone marrow and peripheral blood
Screening for hyperdiploidy (⬎50) in childhood ALL by FISH
J Ritterbach et al
Table 2
Frequency of spots per nucleus (%)a
Probe for chromosome
6
10
17
18
a
429
Hybridization data of normal bone marrow and peripheral blood samples (n = 6)
0
1
2
3
4
0.05 ± 0.08
0.23 ± 0.23
0.03 ± 0.05
0.00 ± 0.00
4.40 ± 1.61
4.05 ± 1.18
4.65 ± 0.50
3.63 ± 0.86
95.40 ± 1.59
95.27 ± 1.22
95.22 ± 0.59
96.07 ± 1.01
0.12 ± 0.08
0.40 ± 0.12
0.05 ± 0.05
0.08 ± 0.10
0.03 ± 0.05
0.03 ± 0.08
0.05 ± 0.05
0.22 ± 0.26
Given 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 double target hybridization. In the single hybridization experiments 1000 nuclei were analyzed per slide and probe. For the
double target FISHs 200 interphase nuclei were scored per
slide and probes.
In the prospective study 100 nuclei per patient were evaluated on each slide in the series using the probes for chr 6 and
10, and 200 nuclei per patient on each slide when evaluating
the probes for chr 6 and 10 (100) and for chr 17 and 18 (100).
Cytogenetic analysis
Bone marrow (BM) and/or blood (PB) samples were processed
by standard cytogenetic methods.19 Karyotype analyses were
done on GTG-banded20 chromosomes; chromosomal aberrations were described according to the International System
for Human Cytogenetic Nomenclature (ISCN) (1995).21
one extra copy of the respective chromosome. In 20 patients,
three signals for chr 6 were detected, whereas for chr 10 only
two hybridization signals were seen. Three patients showed a
spot combination of two signals for chr 6 and three for 10.
Ninety-six children (58.2%) exhibited two signals for chr 6
and 10 in 89.0–99.0% (mean, 94.8%) of the investigated cells,
thus indicating a diploid karyotype. The remaining children
showed other spot combinations (Table 3).
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
tested chromosomes were found most often. Seventy-eight
patients (69.0%) displayed only two spots in most of their blast
cells (88.0–99.0%); mean 94.0%) indicating disomy for the
respective chromosomes. The different signal combinations
for the respective chromosomes found in this group are shown
in Table 4 and Figure 1.
Cytogenetic results
Flow cytometry
For the DNA analysis by flow cytometry, techniques were
used as described previously.22
Results
FISH experiments – controls
In the nuclei prepared from BM and PB of healthy donors
the probes for chr 6, 10, 17 and 18 showed two signals in
approximately 95% of counted cells in the single hybridization 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.)
was chosen for the detection of trisomies by interphase FISH.
In the double hybridization experiments comparable results
were obtained.
FISH experiments – patients
Nuclei of 165 patients were hybridized with the probes specific for chr 6 and 10 (group 1), whereas the remaining 113
children were examined with DNA probes specific for chr 6
and 10, as well as 17 and 18 in combination, respectively
(group 2). Sixty-seven (40.6%) of the 165 children of group 1
showed hybridiation spots in their nuclei that differed from
the values for disomy at frequencies ranging from 2.0–93.0%
(mean 65.9%). Most of the aberrant nuclei showed three signals for chr 6 and also three spots for chr 10 (n = 36) indicating
Cytogenetic analysis was done for all patients exhibiting signals in their nuclei which differed from the two signals for the
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
chromosomes. In two patients (94241, 94422) a translocation
t(9;22)(q34;q11) was also present. Patient 94210/A who exhibited three signals for chr 6 in 2% of his cells had a hyperdiploid chromosome set with 48 chromosomes, showing trisomy of chr 14 and chr 21. A further three patients (94009,
94363, 94375) showed tetraploid clones in their leukemic
cells, two others (94234, 94115) had a near-haploid karyo-
Table 3
Different combinations of fluorescence in situ hybridization signals found in group 1
ISH spots
chromosome 6
0
1
1
2
3
2
3
3
4
6
ISH spots
chromosome 10
No. patients
0
1
2
2
2
3
3
4
4
2
2
1
1
96
20
3
36
4
1
1
165
Screening for hyperdiploidy (⬎50) in childhood ALL by FISH
J Ritterbach et al
430
The overall frequency based on cytogenetics in our investigated group was:
Hyperdiploidy ⬎50
Hyperdiploid 47–50
Hyperhaploidy
Hypertriploidy
Near tetraploidy
80 patients
5 patients
2 patients
1 patient
4 patients
=
=
=
=
=
28.8%
1.8%
0.7%
0.4%
1.4%
Comparison of DNA index and FISH
Figure 1
Dual-colour FISH analysis on nuclei of leukemic bone
marrow cells of patient 95089/A (left panel) and 95107 (right panel).
Left panel: Nucleus exhibiting three hybridization signals for chr 6
(FITC) as well as three signals for chr 10 (rhodamine). Right panel:
Nucleus showing two hybridization spots for chr 17 (FITC) and four
signals for chr 18 (rhodamine); DAPI counterstaining.
Table 4
Different combinations of fluorescence in situ hybridization signals found in group 2
ISH spots
ISH spots
ISH spots
ISH spots No. patients
chromosome chromosome chromosome chromosome
6
10
17
18
2
2
2
2
2
3
3
2
2
3
3
3
3
3
3
4
2
1
3
2
2
2
2
3
3
3
3
3
3
3
4
6
2
2
2
3
2
3
3
3
3
3
2
3
2
2
3
4
2
3
2
2
3
2
3
4
3
3
3
4
4
2
3
4
78
1
3
2
2
1
1
1
1
14
2
2
2
1
1
1
113
type, and in one child (94236) chromosome numbers in the
hypertriploid range were detected.
In two patients (94418, 94247) who showed more than two
signals in 30% of the counted nuclei, the hyperdiploid clone
was not found and only cells with a normal karyotype were
observed. The cytogenetic preparations of the bone marrow
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 hybridization signals for the tested chromosomes. Twenty-eight of
them (28/113, 24.8%) exhibited a ⬎50 karyotype in their
malignant cells. One patient (95089/A) also had a translocation t(9;22)(q34;q11). Patient 95099 with three signals of
chr 18 in 6% of the cells showed a hyperdiploid karyotype
but without involvement of chr 18.
Four patients fell within the group of hyperdiploidy with
chromosome numbers between 47–50 (95063, 95096, 95168,
95219), while one child exhibited a tetraploid clone in his
leukemic cells (95076). The cytogenetic preparations of two
patients could not be analyzed because of insufficient quality.
Flow cytometric analysis of the cellular DNA contents were
performed on cells of 234/278 children (138 of group 1 and
96 of group 2). 149 patients showed a DNA index 1.00–1.04,
ie in the diploid range. 138 of them exhibited two signals for
all tested chromosomes, whereas 11 children showed discrepant 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
DNA measurements. Discrepancies between the measured
DNA index and counted spots in the in situ hybridization
experiments were detected in another 12 patients and are
summarized in Table 6. One of these children (94115) with a
high DNA index (1.72) showed hybridization spots indicating
a hyperhaploid chromosome set (38%), but a hyperdiploid
karyotype detected by cytogenetics was also present. The
FISH experiment of another patient (93348) could not be
evaluated.
In five patients DNA indices below the value of 1.00 (0.59–
0.99) were found; one child (94234) showed good agreement
with the hybridization spots indicating a hyperhaploid chromosome set, whereas two patients (94038, 95055) exhibited
three signals for chr 6 and 10 (3/3) pointing to a hyperdiploid
karyotype (Table 6). Two patients (93619, 93600) with DNA
indices of 0.99 and 0.98 respectively exhibited a normal
signal distribution.
To clarify the discrepant results between DNA index
(⬎1.04) and hybridization spots we did karyotype analysis
subsequently in 10 of 11 patients (Table 6). In seven children
(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
analysis failed.
Patient 94728 showed a hyperdiploid karyotype with 51–
53 chromosomes, but neither chr 6 nor 10, 17, or 18 were
involved in the trisomic state. This patient would not have
been detected using the technique of in situ hybridization with
the probes that we had chosen.
Discussion
According to the cytogenetic findings at diagnosis of ALL, children can be assigned to different prognostic groups. While
for many specific chromosomal translocations with prognostic
impact, the PCR technology is successfully applied (eg
t(9;22)(q34;q11), t(4;11)(q21;q23)),23 the ⬎50 hyperdiploid
group can either be detected by measurements of the DNA
content of the blast cells or by classical cytogenetic analysis.
As cytogenetic investigations of a large number of patients is
a time-consuming technique, it would be of interest to have
a quick screening method for the early detection of patients
Screening for hyperdiploidy (⬎50) in childhood ALL by FISH
J Ritterbach et al
Table 5
431
Comparison of FISH signals with cytogenetic results
Patient ID
FISH signals
Karyotype
6
10
17
18
94234
94115
1
1
2
1
ND
ND
ND
ND
27,XX,+10,+18,+21
25,XY,+mar/50,XY,+X,+14,+18,+21
No. patients
24
12
3
1
2
2
1
1
93331
94720
94694
94719
95139
94124
94707
94329
95089/A
94349
94241
94422
3
3
3
2
3
3
3
2
3
3
3
3
3
3
3
3
3
3
3
3
3
2
4
3
3
3
3
1
2
3
3
3
4
3
3
3
3
3
2
2
ND
ND
ND
ND
3
2
2
2
ND
3
3
3
3
ND
3
ND
3
ND
ND
ND
ND
ND
ND
ND
3
4
2
3
ND
3
3
3
3
ND
3
ND
3
ND
ND
ND
94023
95099
95049
94143
94705
95178
93608
93326
95011/A
93595
95003
95264
95208
95195
94663
94364
95031
95184
95113
95171
94286
94387
94662
94236
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
3
2
2
3
3
3
2
2
3
3
3
3
3
3
2
3
3
3
3
3
3
3
3
3
4
2
2
3
ND
2
3
ND
ND
3
ND
2
3
3
3
3
ND
3
3
3
3
ND
ND
3
ND
3
3
3
ND
3
2
ND
ND
3
n.d
3
3
3
3
4
ND
4
3
3
3
ND
ND
4
nd.
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50, der(1)
⬎50,+i(21)(q10)
⬎50,+mar
⬎50,der(1)?t(1;2)(p32;q31),der(19)t(1;19)(q23;p13)
⬎50,der(1q)
⬎50,der(1q)
⬎50,der(1q)
⬎50,t(6;7)(q13;q36)
⬎50,t(9;22)(q34;q11)
⬎50,trp(1)(q21q42)
46,XY,t(9;22)(q34;q11)/⬎50,t(9;22)(q34;q11),+der(22)t(9;22)(q34;q11)
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]
49-53,XY,+X,+4,+18,+21,+21,+mar[cp7]
52,XY,+X,+Y,+4,i(7)(q10),+14,+21,+21
52-53,XY,+X,+4,+10,+14,+17,+18,+21[cp5]
52-55,XX,+X,der(1q),+4,+5,+6,+10,+?14,+?17,+18,+mar1,+mar2,+mar3,mar4[9cp]
53,X,+X,−Y,+4,+6,+10,+14,add(17)(p?),+18,+21,+21
53,XX,+6,+8,+14,+17,+21,+21,+21
53,XY,+X,+4,+6,+14,+17,+18,+21
53,XY,+X,+6,+10,+14,+18,+21,+21
54,XY,+X,+4,+6,+der(10)?t(10;14)(q22;q11),+12,−14,+17,+18,+21,+21
54,XY,+X,+6,+10,+14,+17,+18,+21,+21
54,XY,+X,+6,+10,+14,+18,+21,+21,+mar
54-55,XX,+X,+4,+6,+10,+14,+17,+18,+21,+21[cp6]
54-55,XY,+X,+4,+6,+10,+17,+18,+21,+21,+mar[cp3]
54-55,XY,+X,+4,+6,+16,+17,+18,+21,+21,+mar[cp10]
54-56,XX,+X,+4,+6,+8,+10,+14,+17,+18,+18,+21[cp8]
54-57,XY,+X,+4,+6,+10,+14,+17,+18,+21,+21,+21[cp9]
56,XY,+X,+4,+6,+10,+14,+17,+18,+18,+21,+21
56,XY,+X,der(1)t(1;?),+4,+6,+10,+14,+17,+18,+21,+21,+mar
57,XY,+X,+Y,+4,+5,+6,+10,+14,+17,+18,+21,+21
57-59,XY,+X,+4,+5,+6,+7,+8,+10,+14,+17,+18,+21,+21[cp8]
60-61,XY,+X,+Y,+4,+5,+6,+9,+10,+14,+15,+16,+17,+20,+21,+21,+mar1[cp4]
61-63,XY,+X,+3,+4,+5,+6,+8,+9,+10,+11,+12,+13,+14,+16,+17,+18,+21,+21,+2mar[cp5]
64-65,XX,+X,+1,+2,+3,+4,+7,+?9,+11,+12,+14,+16,+17,+18,+18,+21[cp5]
hypertriploid
94363
94375
94009
95076
3
4
6
4
2
4
2
6
ND
n.d
n.d
4
ND
ND
ND
4
near
near
near
near
95157
95219
94210/A
95096
95063
95168
2
2
3
2
2
2
2
3
2
2
3
3
3
2
ND
3
2
2
2
2
ND
2
2
2
45-46 chr., +17 could not be confirmed
45,XX,inc/50,XX,+10,inc/57,XX,+10,inc[1]
48,XY,+14,+21
49,XX,+X,+17,+mar
48,XY,+10[inc]
43-45,XY,+10[inc]
94418
94247
3
3
3
2
ND
ND
ND
ND
46,XY
46,XY
tetraploid
tetraploid
tetraploid
tetraploid
ND, not done; ⬎50, only counting of chromosomes was possible.
Screening for hyperdiploidy (⬎50) in childhood ALL by FISH
J Ritterbach et al
432
Table 6
Patient ID
Discrepancies of DNA index and FISH results
DNA index
FISH (number of spots)
6
Karyotype
10
17
18
(a) Patients with a DNA index of 1.00
94009
1.00
4 (21%)
94375
1.00
4 (12%)
94293
1.00
2
93629
1.00
2
93630
1.00
3 (3%)
94210
1.00
3 (2%)
95219
1.00
2
95168
1.00
2
95157
1.00
2
2
4 (12%)
3 (28%)
3 (27%)
2
2
3 (6%)
3 (27%)
2
ND
ND
ND
ND
ND
ND
2
2
3 (84%)
ND
ND
ND
ND
ND
ND
2
2
2
95096
95063
2
3 (10%)
3 (78%)
2
2
2
1.00
1.00
2
2
near tetraploid
near tetraploid
⬎50
failed
failed
48,XY,+14,+21
⬎50
47-50,XY,+10,inc
45-46 chr.,+17 could not be
confirmed
49,XX,+X,+17,+mar
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
94137
1.07
2
2
ND
94166
1.20
2
2
ND
94319
2.00
2
2
ND
94324
1.08
2
2
ND
94411
1.95
2
2
ND
93667
1.18
2
2
2
94012
1.17
2
2
2
94197
1.24
2
2
2
94728
1.10
2
2
2
ND
ND
ND
ND
ND
ND
2
2
2
2
95218
94115
1.14
1.72
2
1
2
1
2
ND
2
ND
46,XX (1 metaphase with 50 chr.)
46,XY
failed
46 chromosomes
+21c
ND
46,XY and near tetraploid
44-46,X,−X,+mar
46,XY and near tetraploid
5153,XY,+X,+9,+14,+21,+21,+mar[cp7]
46,XX,del(17)(q25)/46,XX
25,XY,+mar/50,XY,+X,+14,+18,+21
94234*
94038
95055
0.59 ± 1.22
0.84
0.99
1
3
3
2
3
3
ND
ND
2
ND
ND
2
27,XX,+10,+18,+21
⬎50
⬎50
*One metaphase was found with more than 50 chromosomes.
with ⬎50 chromosomes. The feasibility of applying FISH to
interphase nuclei to detect numerical abnormalities was
shown in several investigations.24–27 We started a prospective
FISH study using probes specific for the most frequent trisomic
chromosomes (6, 10, 17, 18) in order to test this technique as
a screening method for the detection of hyperdiploidy ⬎50 in
children with ALL.
Bone marrow and blood cells of a total of 278 children with
the diagnosis of ALL were hybridized and all but two slides
(0.7%) could be analyzed successfully.
To confirm the hybridization experiments, a cytogenetic
analysis was performed for all children showing a spot number differing from the normal values for disomy. The comparison of both techniques showed a high degree of concordance:
in 80 of these 100 children a ⬎50 hyperdiploid karyotype was
detected, whereas in five patients hyperdiploidy with 47–50
chromosomes was found. In the latter group, only one chromosome exhibited hybridization signals indicating a trisomic
state of the respective chromosome. In addition patients with
chromosome numbers in the haploid (n = 2) and in the
triploid/tetraploid (n = 5) range could be identified. The FISH
results of 10 patients could not be verified by cytogenetics
due either to a lack of metaphases (n = 8) or to a normal karyotype (n = 2). These two children, however, had a DNA index
⬎1.16, indicating a ⬎50 hyperdiploid karyotype.
If the hybridization experiments showed two signals for
every tested chromosome, the DNA measurements were
counted as a control. DNA indices ranging from 1.00 up to
1.04 were taken as normal. In most of the patients (n = 138),
a concordant result was found between the DNA index and
the FISH analysis. However, 11 children with a DNA index
of 1.00 showed more than two signals for at least one of the
tested chromosomes, and cytogenetics confirmed the FISH
results in eight of them. The trisomic clones of most of these
patients were very small (2–28%) and therefore might be
undetectable for flow cytometry.
On the other hand, another 11 patients with two signals
were found with a DNA index above the normal value. Cytogenetics could be performed in nine of them and showed only
one child with a ⬎50 hyperdiploid karyotype in which, however, none of the tested chromosomes was trisomic. In another
child among normal metaphases one cell with more than 50
chromosomes was detected. Hyperdiploidy was also found in
one patient with a hyperhaploid karyotype showing only one
signal for 6 and two signals for 10. Two cell clones could be
detected by FCM with a DNA index of 0.59 and 1.22.
Despite the fact that no cytogenetic data are available for
most of the patients with two signals, the sensitivity of our
probe combination is confirmed by the results of flow cytometry and by the incidence of hyperdiploidy found in this study
that coincides with previously published data5 and is even
higher than in cytogenetics.
The aim of this study was to test an alternative fast screening
method detecting children with a hyperdiploid ⬎50 karyotype. Because classical cytogenetics often fail after mailing
and the metaphase quality in this group is often poor, it was
decided to test interphase FISH for screening. As shown by
the results, the use of centromeric probes for chromosomes 6,
Screening for hyperdiploidy (⬎50) in childhood ALL by FISH
J Ritterbach et al
10, 17 and 18 is sufficient to detect hyperdiploidy ⬎50 and
can be used for therapy stratification. Adverse prognostic markers like t(9;22) or t(1;19) which can be found in high hyperdiploid karyotypes1 will be detected by the routinely performed
RT-PCR analyses for these rearrangements.28 The combination
of these two screening techniques will give quick results
usable for therapeutic decisions. Cytogenetics, however,
should be performed to evaluate the complete karyotype, but
will not have further therapeutic consequences. As compared
with classical cytogenetics the use of interphase FISH using
the described probe combination has several advantages: it is
easy to handle, has a high success rate (93%), is able to detect
small clones, needs only a small amount of material and is
less time-consuming. It will, however, not replace other techniques, but is a useful alternative to screen for hyperdiploidy
⬎50 in childhood ALL.
Acknowledgements
For excellent technical assistance we are indebted to Astrid
Jaeckel regarding fluorescence in situ hybridization and cyto¨
genetics, as well as Christina Both, Doris Erb and Thomas
Jung. The authors also thank Dr Rosalyn Slater, Rotterdam,
The Netherlands, for critically reviewing the manuscript. The
study was granted by the Deutsche Krebshilfe, the Parents’
Leukemia Research Fund Gie␤en, and the Forschungshilfe
Station Peiper.
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