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Trisomy 12 in Chronic Lymphocytic Leukemia: An Interphase Cytogenetic Study
By Alejandra Perez Losada, Maija Wessman, Marianne Tiainen, Anton H.N. Hopman, Huntington F. Willard,
Francesc Sole, M. Rosa Caballin, Soledad Woessner, and Sakari Knuutila
Interphase cytogenetics by means of in situ hybridization
with the chromosome 12-specific biotinylated Q satellite
DNA probe pSP 12-1 was used for the study of trisomy 12, the
most common chromosomal abnormality in chronic lymphocytic leukemia. In situ hybridizationwas performedon methanol/acetic acid fixed cells of conventional cytogenetic preparations from eight patients and on morphologically and
immunologically classified cells of cytospin preparations
from seven patients. The results show that trisomy 12 is
more common than assumed on the basis of karyotype
analysis of metaphase chromosomes: 2 of 13 patients with a
normal karyotype in G-banding analysis were shown to have
trisomy 12 by interphase cytogenetics. lmmunophenotyping
of the cells of one patient showed that the trisomy was
restricted to cells with lg light chain clonality. For the
evaluation of the prognostic, therapeutic, and biologic significance of trisomy 12, in situ hybridization should be used in
parallel with karyotype analysis because it allows the study
of all cell populations of both interphase and mitotic cells,
whether neoplastic or normal.
o 1991by The American Society of Hematology.
C
teen of the patients (nos. 1 through 13) had been found to have a
normal karyotype in studies conducted between January 1989 and
February 1990 at the Department of Hematology and Oncology of
the Hospital de la Cruz Roja de Barcelona, Spain. During this
period, this laboratory performed chromosome analyses on a total
of 23 patients with CLL; 16 of them proved to have a normal
karyotype and seven an abnormal one. Of the seven patients with
an abnormal karyotype, one had trisomy 12. One patient (no. 14)
with trisomy 12 was selected as a positive control [47,XX,+12,t(7;
13)(q21;q14)]. In addition, one patient (no. 15) with a structural
aberration of chromosome 12 was included in the study
(46,XY,l2~+,13q-,17q-). This patient most probably had a
three-way balanced translocation between chromosomes 12, 13,
and 17, and thus no additional chromosome 12 material was
present.
Table 1shows the hematologic and clinical characteristics as well
as immunophenotypes of neoplastic cells of the patients studied.
Specimens from six healthy persons (ages 28 to 44 years) known to
have normal karyotype were used as negative controls.
Immunophenotyping of neoplastic cells. Hematologic, cytogenetic, and immunologic analyses were performed on the same
blood samples. In every patient, mononucleated cells from the
peripheral blood were isolated by one-step density-gradientcentrifugation in Ficoll-Paque (Pharmacia Fine Chemicals, Uppsala,
Sweden) and washed twice with phosphate-bufferedsaline. Cytospin
preparations were made and stained by immunoperoxidase or
alkaline phosphatase antialkaline phosphatase technique as de-
HRONIC LYMPHOCYTIC leukemia (CLL) is the
most prevalent form of human leukemia’s* and, with
the rise in the mean age of the population, its incidence is
steadily increasing. In most cases of CLL the neoplastic
cells represent B cells; in less than 5% of the cases the cells
are of T-cell origin.’
Chromosome analyses with banding techniques after
culturing the cells with so-called polyclonal B-cell mitogens
such as Epstein-Barr virus, lipopolysaccarides, protein A,
and pokeweed mitogen have shown clonal chromosome
aberrations in approximately 50% of CLL cases; in the
remaining cases, metaphase cells with normal chromosomes or only interphase cells are seen:,’ This finding might
be explained by the malignant clone being unable to divide
in vitro or by the malignant clone being in fact karyotypically normal. Both of these hypotheses have received
support in the literature.’.’ By studying the chromosomes of
morphologically and immunologically classified cells (MAC
method), we have previously shown that in most of the cases
where karyotype analysis showed a normal karyotype the
metaphase cells studied were normal nonclonal T cells.’ In
some cases they were clonal K-positive or A-positive cells
but still with a normal karyotype. Southern blotting DNA
studies with probes that detect restriction fragment length
polymorphism (RFLP) also support the notion that the
karyotypic study of metaphase cells does not always yield
information about the neoplastic cell pop~lation.’~~
Another
sensitive method is provided by in situ hybridization with
DNA probes detecting repetitive target DNA sequences of
specific chromosomes, which enables one to study numerical chromosome aberrations in interphase nuclei (interphase cytogenetics)?-‘2
Because trisomy 12 is the most common chromosome
abnormality in CLL (more than 30% of cases with clonal
chromosome aberrations) and is suggested to have prognostic v a l ~ e , ~we
, ’ ~have performed an interphase gtogenetic
study on 15 patients with CLL, using a chromosome
12-specific a satellite DNA probe. Our results show that
trisomy 12 is more common than assumed on the basis of
karyotype analyses of metaphase chromosomes and that
trisomy 12 is restricted to cells with light chain clonality.
MATERIALS AND METHODS
Patients. Fifteen patients with CLL were studied. The patients
were selected on the basis of previous cytogenetic findings. ThirBlood, VOI 78,NO3 (August l), 1991 : pp 775-779
From the Department of Medical Genetics, University of Helsinki,
Finland; Departamento de Biologia Animal, Vegetal y Ecologii,
(Unidad Antropologia), Facultad de Ciencias, UniversidadAutbnoma
de Barcelona; Unidad de Hematologia y Oncologla, Hospital de la
C w Roja de Barcelona, Spain; the Department of Molecular Cell
Biology, University of Limburg, Maastricht, The Netherlands; and the
Department of Genetics, Stanford University School of Medicine,
Stanford, CA.
Submitted November 2,1990; acceptedApril I, 1991.
Supported by grants from The Finnish Cancer Sociefy, The Sigrid
Jusklius Foundation, and the Comisibn Interdepartmental de Recerca
i Innovavacid Tecnologica.
Address reprint requests to Sakari Knuutila, PhD, Department of
Medical Genetics, University of Helsinki, Haartmaninkatu 3, 00290
Helsinki, Finland.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore by hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1991 by The American Society of Hematology.
0006-4971I91/7803-OO34$3.00IO
775
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LOSADA ET AL
776
Table 1. Clinical and ImmunologicCharacteristics of the 15 Patients With Chronic LymphocyticLeukemia
Patient
No.
SexJAge
(V)
Therapy Before
Study
1
2
3
4
5
6
7
MI56
MI75
F/66
F/70
MI67
F/75
F/6 1
8
MI63
9
Fi7 1
10
11
MI66
MI68
12
13
F/7 1
Fl71
14
15
W78
MI68
None
None
None
None
None
Chlorambucil
Cyclophosphamide
Vincristine
Prednisone
Cyclophosphamide
Prednisone
Chlorambucil
Prednisone
None
Cyclophosphamide
Adriamycin
Vincristine
Prednisone
None
Chlorambucil
Prednisone
None
None
Leukocyte Count
UN
% of
B Cells
8
63
89/10
81/14
66/18
8913
8018
48/44
75/20
96
86
88
91
96
94
2
29
69/28
4
(109/~)
26
29
Light Chain Clonality
K
A
NS
NS
NS
% of
T Cells
NS
6
19
12
11
7
4
91
62
+
37
55/38
34
+
69
39
49
26/48
8211 1
72
84
19
7
68131
63/30
90
77
+
47
15
84/12
73/24
91
22
+
44
35
44
+
+
+
+
15
21
+
7
50
+
+
6
75
Abbreviations: L, % of lymphocytes; N, % of neutrophils; NS, not studied.
scribed for MAC preparations. The following monoclonal antibodies (MoAbs) were used: Leu14 (anti-CD22; Becton Dickinson,
Erembodegem, Belgium), anti-K and anti-A (Coulter Clone, Coulter
Immunology, Hialeah, FL), and Leu4 (anti-CD3; Becton Dickinson).
Conventional cytogenetic preparations. Whole blood was used
for 3-day cell cultures. One culture was set up in the presence of
pokeweed mitogen (PWM; 100 pg/mL; GIBCO, New York, NY)
and one in the presence of 12-0-tetradecanoylphorbol-13-acetate
(TPA, 2 pg/mL; Sigma, St Louis, MO). To arrest the metaphases,
the cultures were treated with 0.125 pg of deacetylmethylcolchicine (Colcemid; GIBCO). For chromosome preparations, the cells
were subjected to hypotonic treatment with a 0.56% solution of
potassium chloride for 10 minutes. The cells were fixed in methanol/
acetic acid (3:l) for 40 minutes. After centrifugation, the cells were
resuspended in fixative, dropped onto slides, and air-dried. The
preparations were stained by a standard trypsin-Giemsa G-banding procedure (GTG). For patient nos. 4 through 9 and 12 through
15,20 metaphases from PWM-stimulated and 20 metaphases from
TPA-stimulated cultures were analyzed, whereas for patient nos. 1
through 3 and 10 through 11,20 metaphases from TPA-stimulated
cultures only were studied.
MAC preparations of immunologically classifid cells. MAC
preparations were made of noncultured cells. Thus, only interphase cells were analyzed. The MAC (morphology-antibodychromosomes) technique has been described earlier.14-16
Briefly,
mononucleated cells from the peripheral blood were isolated by
one-step density-gradient centrifugation in Ficoll-Paque and washed
twice with phosphate-buffered saline. For immunologic classification, 1 million cells were suspended in 1 mL of RPMI 1640
medium, to which 1 mL of hypotonic solution was added. The
hypotonic solution consisted of 50 mmoliL of glycerol, 5 mmol/L of
potassium chloride, 10 mmol/L of sodium chloride, 0.8 mmol/L of
magnesium chloride, 1mmol/L of calcium chloride, and 10 mmolL
of sucrose. After hypotonic treatment for 5 minutes, the cell
suspension was divided into cytocentrifuge chambers and centrifuged at 4% for 10 minutes. After air-drying ovemight, the cells
were identified by immunoperoxidase or alkaline phosphatase
antialkaline phosphatase technique using the following antibodies:
Leu14 (anti-CD22), anti-K, anti-A, and Leu4 (anti-CD3).
In situ hybridization. The hybridization was performed with a
biotin-labeled, chromosome 12-specific c1 satellite DNA probe
pSP12-1, containing a 340-bp EcoRI fragment." Previous experiments using fluorescence in situ hybridization have shown that this
probe is highly specific for the centromeric region of chromosome
12 and displays essentially no cross-hybridization with other
chromosomes at high stringency." The probe was labeled by
nick-translation using biotin-11-dUTP (Bethesda Research Laboratories, Gaithersburg, MD) or biotin-16-dUTP (Boehringer Mannheim, GmbH, Germany) according to the instructions of the
supplier. Hybridization was performed by a modification of methods described earlier.11.'8'20
Conventional cytogenetic preparations (patient nos. 1 to 8) were
made from methanouacetic acid-fixed cell suspensions (stored at
+4"C for 1 to 6 months) derived from TPA-stimulated cultures.
Fresh slides were incubated in sequential ethanol series (70%,
94%, 99%; 5 minutes each) and air-dried for a few hours. MAC
preparations of immunologically classified cells (patient nos. 9
through 15) were destained by incubation in methanol/acetic acid
tixative for 1 hour at room temperature and air-dried for 1 to 4
hours. The preparations were then, right away or after storage at
-70°C for 0.5 to 2 months, incubated in 0.01 N hydrochloric acid
containing 0.01 to 0.1 mg of pepsin per milliliter at 37°C for 4 to 10
minutes to remove the cytoplasm. To stop the reaction, the slides
were washed in distilled water for 2 to 3 minutes, which was
followed by sequential incubation in ethanol. The slides were
allowed to dry at room temperature for 1 to 4 hours before the
hybridization was performed.
Immediately before hybridization the slides were incubated in a
solution consisting of 60% formamide, 2X standard saline citrate
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TRISOMY 12 IN CLL
777
(2X SSC = 0.3 molL NaCl, 0.03 m o m trisodium citrate, pH 7.0),
and 0.05% polysorbate (Tween) 20 at room temperature for 15
minutes. The hybridization buffer consisted of 50% formamide,
10% (wtivol) dextran sulphate, 2X SSC, and 0.5 mg/mL of herring
sperm DNA. The slides were covered with 0.04 p,g of the
biotinylated chromosome 12-specific DNA probe in 10 )LLof the
hybridization buffer. Denaturation of the probe and cells was
performed at 70°C for 3 minutes. The slides were incubated in a
moist chamber at 42°C for 12 to 16 hours. After hybridization the
slides were washed in three changes of 50% formamide, 2X SSC
(pH 7), and 2X SSC (pH 7) at 42°C for 5 minutes each, followed by
one change in phosphate-bufferedsaline containing 0.05% Tween
20 at room temperature for 10 minutes. For detection of hybridization, the slides were first incubated in normal rabbit serum
(Dakopatts A I S , Glostrup, Denmark), followed by incubation with
mouse antibiotin antibody (Dakopatts) and then with peroxidaseconjugated rabbit antimouse serum (Dakopatts). The color was
developed by incubating the slides in diaminobenzidinetetrahydrochloric acid (DAB; Sigma). The cells were counterstained with
hematoqlin. All analyses of interphase signals were performed
using slides with good cell morphology and complete, nonoverlapping cells, with 200 to 250 cells being analyzed per patient/control.
RESULTS
In situ hybridization was performed on specimens from
15 patients with CLL. In the case of patient nos. 1 through
8, in situ hybridization was performed on fresh conventional cytogenetic preparations. In these patients, previous
chromosome studies had shown a normal karyotype. Table
2 shows the proportions of cells with zero, one, two, or three
signals. In patient nos. 1,2,4, 6, 7, and 8, two hybridization
signals in interphase nuclei were detected in 76% to 98% of
the cells analyzed. No signals or only one signal were seen
in the remaining cells. Patient no. 3 had three signals in 5%
(12 of 250 cells), two signals in 85%, and one or no signals in
10% of the cells studied. In patient no. 5, three signals were
present in 56% (140 of 250 cells), two signals in 34%, and
only one or no signals in 10% of the cells (Fig 1).
In the case of seven patients, in situ hybridization was
performed on MAC preparations. Previous G-banding
analyses had shown a normal karyotype in five of these
patients (nos. 9 through 13); patient no. 14 exhibited
trisomy 12 in 50% of the metaphases analyzed, and patient
no. 15 had a structural aberration of chromosome 12 in 15%
of the metaphases analyzed. Of these seven patients, only
patient no. 14 had cells with three in situ hybridization
signals. In this patient, the frequencies of cells with three
and two (or fewer) were 81% and 19%, respectively, when
counted without regard to cell subset. In this patient in situ
hybridization signals were also analyzed from CD3, K, and A
positive cells. Eighty-seven percent of the A positive B cells
had three signals, whereas in none of the cells positive for K
and T-cell markers three signals were seen (Table 3).
In MAC preparations from the three controls the frequency of two signals was 90% to 93%. The corresponding
figure in conventional preparations from the three other
controls was 73% to 95%. These figures demonstrate that
MAC preparations are equally suitable for in situ hybridization as are conventional cytogenetic preparations. In the six
controls, none of the cells contained three hybridization
signals.
Table 2. In Situ HybridizationSpot Frequencies(%)for Chromosome
12-Specific (ISatellite DNA Probe Among 200 to 250 Cells Analyzed
No. of In Situ Hybridization Spots
Patient No.
0
1
2
3
Conventional cytogenetic preparations
1
2
6
81
98
85
96
34
82
96
76
87
95
73
0
2
3
4
3
1
11
4
2
13
13
1
8
1
6
15
3
13
9
3
14
4
5
0
5
2
1
1
5
1
2
1
1
91
99
90
95
98
15
76
93
92
90
3
4
5
6
7
8
Control 1
Control 2
Control 3
MAC preparations
9
10
11
12
13
14,
15
Control 4
Control 5
Control 6
1
5
3
1
3
19
6
6
9
0
5
0
56
0
0
0
0
0
0
0
0
0
0
0
81
0
0
0
0
Karotypes of patient nos. 1 through 13 were normal. Karyotypes of
patient nos. 14 and 15 were 47,XX,+lZ,t(7;13)(q21;q14) and
46,XY,12p+,l3q-,17q -, respectively.
*Percentages counted without regard to cell subset. See Table 3 for
analysis of immunologically classified cells.
DISCUSSION
The present study shows that the characterization of
human chromosomes in interphase nuclei by in situ hybridization is a powerful method in the study of chromosomal
aneuploidy. Especially in CLL, in which the neoplastic B
cells often fail to proliferate actively despite polyclonal
B-cell activators, the advantages of this technique become
evident. Thus, in a study using in situ hybridization, one can
easily analyze up to 250 cells, which would be very laborious
in conventional karyotype analysis. In our study the possibility to use large numbers of cells provided new information
about the frequency and lineage specificity of trisomy 12 in
CLL.
Our results show that trisomy 12 is more common than
expected on the basis of standard cytogenetic G-banding
analyses. Results by Einhorn et
who used Southern
blotting analysis with probes that detect RFLPs on chromosome 12, corroborate our findings indicating that the
proportion of normal metaphases does not match the
proportion of cells lacking the trisomy. However, these
investigators were unable to find any trisomy 12 patients not
detected in conventional karyotype analysis. In our small
patient material we were able to find two cases of trisomy 12
previously undetected by karyotype analysis. Therefore,
standard chromosome banding analysis of metaphase cells
does not appear to be a sensitive method of detecting
aneuploidy in CLL. Discrepancies between various studies
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778
LOSADA ET AL
Fig 1. Patient no. 5. Interphase cells of methanol/
acetic acid fixed conventional cytogenetic preparations after in situ hybridization with chromosome
12-specific OL satellite DNA probe pSP12-1. Large
arrow indicates a cell with trisomy 12; small arrow
indicates a cell with two in situ hybridizationspots.
concerning the prognostic significance of trisomy 123r6,*’-B
might be explained by this methodologic inadequacy. As in
situ hybridization technique is rapid and relatively easy to
use, more CLL cases should be studied using the chromosome 12-specific DNA probe before one can reliably
estimate the prognostic importance of this trisomy. To fully
elucidate the clinical and biologic significance of trisomy 12,
interphase cytogenetics should be used simultaneously with
metaphase cytogenetics.
In addition to trisomy 12, there are other chromosome
abnormalities, some with prognostic value, eg, of chromosomes 13 and 14, often detected in CLL? Because only a
12-specific probe was used in our interphase cytogenetic
study, we cannot rule out the possibility that the patients
might also have had other chromosomal abnormalities.
Thus, interphase cytogenetics cannot replace karyotype
analysis of metaphase cells. The methods are mmplementary.
None of our healthy controls showed three signals for
chromosome 12 in 200 to 250 cells scored from a predetermined area of the slide. The proportion of control cells with
two signals was 73% to 95%, but never 100%. Whether cells
of zero or one spot represent nullisomi or monosomy 12, or
whether they are due to poor penetration of the probe,
incomplete denaturation of target DNA or possible damage
in DNA during long storage of the cells in fixative is not
known. With chromosome 7 or 9 specific probes, the
frequencies of cells with zero or one hybridization spot has
been reported to be as high as ll%.24s25
Thus, it is evident
Table 3. In S i u Hybridization Spot Frequencies (%)for Chromosome
12-Specifica Satellite DNA Probe Among B and T Lymphocytesof
Patient No. 14
No. of In Situ HybridizationSpots
B lymphocytes
K positive
A positive
T lymphocytes(CD3)
0
1
2
3
3
2
5
3
7
92
8
89
0
87
0
4
At least 200 cells were analyzed in each subset.
that this technique should be used with great caution for
analysis of monosomy of low frequency. Even though the
frequency of the cells with only one signal was as high as
13% to 19% in patient nos. 1,6,8, and 15 and in control no.
3, we do not consider these patients necessarily as cases of
monosomy 12. Similarly, it should be noted that cases with a
very low frequency of trisomic cells may remain undetected
for the reasons mentioned above, and finally, that in patient
nos. 3 and 5, who had three signals in a small proportion of
their cells, trisomy 12 cannot be interpreted to be present
only in some of the neoplastic cells. In patient no. 3 any
definite conclusions are also hindered by the fact that light
chain clonality was not determined, and thus it is possible
that some of the B cells are not clonal neoplastic cells.
Further studies are needed to confirm the existence of
“preneoplastic” cells without the trisomy.
The restriction of trisomy 12 to clonal B cells in patient
no. 14 confirms our earlier findings made by MAC study of
metaphase cells.26 Because only metaphase cells were
analyzed in the previous study, we did not find out whether
nonclonal B cells also had the abnormality. In the case of
patient no. 14, who exhibited A clonality, K positive cells
accounted for less than 1% of all cells. Nevertheless, it was
not difficult to find 200 cells for analysis. None of these cells
contained trisomy 12. The frequency of A positive cells with
two signals was 8%. Whether these “disomic” cells represent normal, nonclonal positive B cells or disomic “preneoplastic cells” cannot be solved because the hybridization is
never 100% effective.
In conclusion, the combination of MAC and in situ
hybridization proved useful in the study of CLL, showing
that standard karyotype analysis fails to show all patients
with trisomy 12, and that trisomy 12 is restricted to cells
with Ig light chain clonality. The combination is superior to
karyotype analysis in the study of trisomy 12 in CLL
because it allows the study of all cell populations of both
interphase and metaphase cells, whether neoplastic, or
normal. No mitogens are required. On the other hand, the
method can be used to study the effect of polyclonal B-cell
activators or B-cell growth factors on the different lympho-
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TRISOMY 12 IN CLL
779
cyte subpopulation^.^^ The method also has the advantage
of enabling the study of the role of chromosome abnormality in the process of malignant transformation. The possibility that part of the neoplastic cell population in CLL might
carry trisomy 12 while some wlls do not presents a chance
to study cancer genes in these different neoplastic cell
populations. Finally, in view of the suggested use of trisomy
12 in CLL as an indication for treatment,’ it is now possible
to monitor the frequency of trisomy 12 in relation to the
clinical response to treatment.
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From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1991 78: 775-779
Trisomy 12 in chronic lymphocytic leukemia: an interphase
cytogenetic study
AP Losada, M Wessman, M Tiainen, AH Hopman, HF Willard, F Sole, MR Caballin, S Woessner
and S Knuutila
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