Eur J Haematol 2002: 69: 309–314 Copyright  Blackwell Munksgaard 2002

Printed in UK. All rights reserved

EUROPEAN
JOURNAL OF HAEMATOLOGY
ISSN 0902-4441

Cytogenetic, FISH, and molecular studies

in a case of B-cell chronic lymphocytic
leukemia with karyotypic evolution
Chena C, Cerretini R, Noriega MF, Narbaitz M, Scolnik M, Palacios
M, Neme D, Bruno S, Slavutsky I. Cytogenetic, FISH, and molecular
studies in a case of B-cell chronic lymphocytic leukemia with karyotypic
evolution.
Eur J Haematol 2002: 69: 309–314.  Blackwell Munksgaard 2002.
Abstract: We report the clinical, cytogenetic, fluorescence in situ
hybridization (FISH) and molecular findings in a 54-yr-old male patient
diagnosed with B-cell chronic lymphocytic leukemia (B-CLL), who
showed progression to a diffuse large B-cell lymphoma (Richter’s syn-
drome). Genetic studies were performed at diagnosis and during the
Richter’s transformation (RT). A clonal karyotype with two dicentric
chromosomes, psu dic(12,21)(q24;q10) and dic(17,18)(p11.2;p11.2), was
found. Both rearrangements were confirmed by FISH. Molecular
cytogenetics analysis using p53 probe showed monoallelic loss of this
tumor suppressor gene in 43.8% and 77.3% of cells for the first and the
second studies, respectively). In both studies, deletions of D13S319
(18% and 12% of cells) and D13S25 loci (13% and 12% of cells) at
13q14 were found. Polymerase chain reaction analysis showed the
MBR/JH rearrangement of the bcl-2 gene. FISH studies using LSI bcl-2/
IgH probe allowed quantifying the clonal cell population with this
rearrangement (4% and 6.6% of cells at diagnosis and RT, respectively).
To our knowledge, this is the first case with a psu dic(12,21) described in
B-CLL. The low percentage of cells with the 13q14 deletion and bcl-2/
IgH rearrangement suggests that they were secondary events that
resulted from clonal evolution. Our patient had a short survival
(9 months) and a clear lack of response to several therapeutic agents,
confirming the association of p53 gene deletion and karyotypic evolu-
tion with disease progression.
Christian Chena1, Roxana
Cerretini1,5, Mara Fernanda
Noriega1, Marina Narbaitz2,
Mariano Scolnik3, Mara Fernanda
Palacios3, Daniela Neme4, Salvador
Bruno4, Irma Slavutsky1
1
Department of Genetics, 2Division of Pathology,
3
Department of Oncological Immunology, 4Division of
Oncohematology, Instituto de Investigaciones
Hematolgicas `Mariano R. Castex', Academia Nacional
de Medicina, Buenos Aires; 5Centro Nacional de
Gen,tica M,dica, Buenos Aires, Argentina
Key words: chronic lymphocytic leukemia; Richter's
syndrome; bcl-2 gene; p53 deletion; karyotypic
evolution; FISH analysis
Correspondence: Lic. Christian Chena, Departamento de
Gen,tica, Instituto de Investigaciones Hematolgicas
`Mariano R. Castex', Academia Nacional de Medicina,
Pacheco de Melo 3081, 1425 Buenos Aires, Argentina
Tel: + (5411) 4805–5759/8803 ext 241/291
Fax: + (5411) 4803–9475
e-mail: cpchena@hematologia.anm.edu.ar
Accepted for publication 31 October 2002

Clonal chromosome abnormalities can be detected
in approximately 50% of patients with chronic
lymphocytic leukemia (CLL). Different studies
found trisomy 12 to be the most frequent chromo-
some anomaly, followed by structural aberrations
of the long arm of chromosomes 13 and 14 (1, 2).
However, the introduction of fluorescence in situ
hybridization (FISH) studies has demonstrated that
the most common chromosome abnormalities in
CLL are 13q14 deletions, followed by deletion in
11q22-q23, trisomy 12, monoallelic loss of 17p13,
where p53 tumor supressor gene is located, and
deletion in 6q21 (2).
The most frequent translocation in non-Hodg-
kin’s lymphoma (NHL) of the B-cell type is
t(14,18)(q32.3;q21.3) which is found in follicular,
diffuse large-cell lymphomas, and occasionally in
other histological subtypes of NHL (3). Mole-
cular studies of this translocation have disclosed
a juxtaposition of the bcl-2 gene with the Ig
heavy-chain gene locus. On chromosome 18, the
breakpoints primarily occur at two loci, the
major breakpoint region (MBR) and the minor
cluster region (mcr), in approximately 60% and
30% of cases, respectively. On the contrary,
cytogenetically detectable t(14,18) has been rarely
reported in CLL patients (1, 2). The molecular
analysis of this pathology has detected preferen-
tially, although not exclusively, variant (vcr)
bcl-2 rearrangements involving the 5¢ flanking
region of the bcl-2 gene with the Ig light-chain
genes (4).

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Chena et al.

About of 13% of CLL patients evolve into a

diffuse large B-cell lymphoma, a process known as
Richter’s syndrome (RS). This evolution is usually
associated with a rapidly progressive clinical course
and poor outcome (5). We report a case of CLL
with histologic transformation to RS, refractory to
treatment, in which dicentric chromosomes, p53
and 13q14 deletions, and the MBR/JH molecular
rearrangement of the bcl-2 gene were found.

Case report
A 54-yr-old man was referred to our Institute in
October 2000 with generalized non-bulky lymph-
adenopathies and hepatosplenomegaly. He had no
systemic symptoms. The peripheral blood count
was: WBC 82 · 109 L)1 (90% lymphocytes, 3%
prolymphocytes), Hb 10.4 g dL)1, and platelet
count 144 · 109 L)1. LDH was raised to 551 IU
L)1 (normal range 140–280 IU L)1) and b2-micro-
globulin to 10 lg mL)1 (normal value < 3 lg
mL)1). A direct Coombs test was positive. In
addition BUN was 34 mg dL)1, creatinine 1 mg
dL)1, GOT 23 IU L)1, and GPT 19 IU L)1, Total
bilirubin was 0.6 mg dL)1. Serum protein electro-
phoresis showed two oligoclonal bands (0.10 and
0.18 g dL)1).
The bone marrow (BM) aspirate showed 70%
lymphocytes with typical CLL morphology. The
histopathological study of the BM revealed an
infiltration of 70% small CD20-positive B-lympho- Fig. 1. (a) Section of a lymph node showing large cells with
cytes with diffuse pattern. A flow cytometry study round nuclei and a central nucleolus, prolymphcytes, and a
few small lymphocytes (Giemsa stain, 400·). (b) Immuno-
showed expression of pan-B antigen (CD19, histochemistry study showing large cells expressing nuclear
CD20, CD22) with coexpression of CD5 and Ki 67 antigen (clon Mib1, 400·).
CD23. The population was monoclonal as seen by
k-chain restriction. The immunophenotypic pattern
achieved five points for B-CLL in the scoring lymphadenophathies. He was started on an ESHAP
system proposed by Matutes et al. (6). A diagnosis combination chemotherapy (etoposide, cisplatin,
of B-CLL, RAI stage III, was made. The patient cytosine arabinoside). The patient had a short
started with CHOP chemotherapy (cyclophospha- partial remission. However, the disease progressed
mide, doxorubicin, vincristine, and prednisone). after the second cycle and the decision of treatment
Right after the second CHOP cycle, an axillary with hyper-CVAD was taken. Following the first
lymphadenophathy that had progressed to phase of this regimen the patient died due to septic
13 · 8 cm in size was found. The histopathological shock, 9 months after the diagnosis of CLL .
study of a lymph node biopsy showed a pattern of
small lymphocytic lymphoma/CLL in transforma- Cytogenetic, FISH, and molecular studies
tion to a diffuse large B-cell lymphoma (Richter’s
syndrome). The immunohistochemistry, using the Cytogenetic studies were performed at diagnosis
Ki67 antigen, disclosed 50% of proliferating cells and during Richter’s transformation (RT). BM
(Fig. 1). Flow cytometry analysis showed two cells were cultured in F-10 medium supplemented
different sized B-cell populations with identical with 15% fetal calf serum by direct methods and
immunophenotypes, and similar to those found in with stimulation of different mitogens (Table 1).
the initial sample. G- and C-banding techniques were used. Karyo-
Subsequently, the patient received fludarabine typic abnormalities were described according to the
(25 mg m)2 d)1 · 3 d), cyclophosphamide (1 g m)2d)1), ISCN (7).
and dexamethasone (40 mg d)1 · 3 d) with fur- For FISH analysis, #17 and #18 whole chromo-
ther progression of disease to bulky generalized some painting probes (CAMBIO, Cambridge,

310
 Karyotypic evolution in CLL

Table 1. Cytogenetic results on BM cells of a patient with CLL

Clinical stage Culture Karyotype

Diagnosis 24 h-wM 44, XY, psu-dic(12;21)(q24;q10),

dic(17;18)(p11.2;p11.2)[6]/46, XY[16]
RT 96 h-PWM 44, XY, psu-dic(12;21)(q24;q10),
dic(17;18)(p11.2;p11.2)[2]/46, XY[13]
96 h-LPS 44, XY, psu-dic(12;21)(q24;q10),
dic(17;18)(p11.2;p11.2)[7]/46, XY[11]
72 h-PHA 46, XY[15]

wM, without mitogen; PWM, pokeweed mitogen; LPS, lipopolysaccharide; PHA,

phytohemagglutinin; RT, Richter's transformation.

Fig. 2. G-banded karyotype of a bone marrow cells show-

UK), chromosomes #12, #17, and #18 -specific
a-satellite DNA probes (VYSIS, Downer Grove,
IL), and the locus-specific DNA probes RB-1 for
retinoblastoma gene, D13S319 and LSI D13S25 at
13q14 band, p53 at 17p13 (Spectrum Orange), and
IgH Spectrum Green/BCL2 Spectrum Orange
(VYSIS, Downer Grove, IL) were used. FISH
was performed according to manufacturer’s proto-
cols. Hybridization signals were analyzed on the
cytogenetic preparations from the patient and
controls in 400 interphase nuclei.
DNA extraction was performed by conventional
means. A nested polymerase chain reaction (PCR)
was done in a 50 lL final volume using 1.5 lg of
DNA, 0.1 lm oligonucleotide primers, 20 lm of all
dNTPs, 2.5 lm Cl2Mg, buffer 1X (10 mm Tris–
HCl, 50 mm KCl) and 5 U lL)1 Taq polymerase.
The outer and the inner primers described by
Gribben et al. (8) for the MBR and the JH
consensus region were used. For the first stage,
the PCR protocol consisted of 25 cycles that
included: denaturation at 94 C for 1 min, anneal-
ing at 55 C for 1 min, extension at 72 C for
1 min, and tailing at 72 C for 10 min The second
stage of PCR was reamplification of 5 lL of the
first PCR product, and consisted of 30 cycles:
denaturation at 94 C for 30 s, annealing at 53 C
for 1 min, extension at 72 C for 30 s, and tailing at
72 C for 10 min. PCR products were analyzed by
electrophoresis in 2% agarose gel with ethidium
bromide and visualized under UV light. Positive
products give a 180–250 bp specific signal.
ing dic(17;18)(p11.2;p11.2) and psu-dic(12;21)(q24;q10).
Phytohemagglutinin (PHA)-stimulated culture
showed only normal metaphases (Table 1). Both
rearrangements were confirmed by FISH using
painting probes (Fig. 3). The C-banding technique
revealed the presence of the inactivated centromere
of chromosome 21, indicating the pseudo-dicentric
nature of der(12): psu-dic(12,21)(q24;q10). FISH
using l-satellite DNA probes of chromosomes 17
and 18 showed a signal of each probe on der(17,18),
confirming the dicentric condition of this chromo-
some: ish dic(17,18)(p11.2;p11.2)(wcp17+wcp18+
D17Z1+D18Z1+).
Table 2 shows the molecular cytogenetic analy-
sis. FISH using p53 probe showed monoallelic
deletion of this gene, revealing the presence of
43.8% and 77.3% of clonal cells for the first and
second studies, respectively. In both studies, dele-
tions of D13S319 and D13S25 loci at the 13q14.3
band with diploid RB-1 were found. Trisomy 12
was not observed.

Results
At diagnosis, cytogenetic analysis showed 6/22
unstimulated BM cells with the karyotype 44, XY,
der(12)t(12,21)(q24;q11), der(17,18)(q10;q10) (Fig. 2).
Only one cell with 45, XY, der(17,18)(q10;q10)
was also observed, suggesting clonal evolution.
The second study, performed during RT, showed
the same clone in 2/15 and 7/18 metaphases Fig. 3. Metaphase hybridized with chromosome 12 and 21
from pokeweed mitogen (PWM)- and lipopolysac- painting probes showing normal chromosomes 12 (red) and
charide (LPS)-stimulated BM cells, respectively. 21 (green) and psu-dic(12; 21)(q24;q10) (arrow).

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Chena et al.

Table 2. FISH analysis in a patient with CLL

Clinical stage FISH (%)

+12 RB-1 D13S319 D13S25 p53 bcl-2/JH

Diagnosis 1.0 10.0 18.0 13.2 43.8 4.0
RT 0.7 6.6 11.7 11.6 77.3 6.6

RT, Richter's transformation.

Cut-off for positivity (mean of normal control + 3SD) was 2.8%, 12.8%, 10.1%,
7.5%, and 5.4% for + 12 and monoallelic deletions of RB-1, D13S319, D13S25, and
p53, respectively. For LSI IgH/BCL2 dual-color probe, the presence of two fusion
signals (yellow), one orange, and one green signal indicated the occurrence of the
translocation t(14;18).

A nested PCR for the MBR/JH rearrangement of

the bcl-2 general showed a positive 200 bp-specific
band (Fig. 4). FISH analysis using bcl-2/IgH probe
allowed us to quantify the clonal cell population
with t(14,18), showing that a small subset of cells
had this rearrangement (4% and 6.6% of cells for
the first and second studies, respectively) (Table 2)
(Fig. 5). Translocation t(14,18) was not detected by
conventional cytogenetics.
Discussion
In this report we present a B-CLL patient with
dicentric chromosomes, monosomy of the p53
gene, a 13q14 deletion, the MBR/JH rearrangement
of the bcl-2 gene, and karyotypic evolution, who
had a poor clinical course with RT and short
survival. As for dic(17,18), only eight B-CLL
patients with this anomaly have been reported in
the literature: four cases described by Döhner et al.
(9), three patients referred by Callet-Bauchu et al.
(10), and one case recently reported by Espinet
et al. (11), four of them as a sole anomaly.
Moreover, two reports have described unbalanced
whole-arm translocations der(17,18)(q10;q10), as a
part of a complex karyotype in which the probable
Fig. 4. Agarosa gel electrophoresis of PCR products
showing the MBR/JH rearrangement of the bcl-2 gene. Lane
1, molecular weight marker; lane 2, positive control; lane 3,
negative control; lane 4, patient.
dicentric condition was not evaluated (12, 13).
These findings would suggest this abnormality as a
recurrent alteration in B-CLL patients. In the first
study of our case, the presence of one cell with
dic(17,18) as a sole anomaly would suggest it as the
first event leading to CLL that was followed by
stepwise clonal evolution in the leukemic cells
leading to lymphoma. Thus, an origin of lymphoma
from leukemic cells through progressive cytogenetic
and molecular cytogenetic changes is proposed.
Loss of the short arm of chromosome 17, where
the p53 tumor suppressor gene localizes, has been
observed in 4% of cytogenetically evaluable CLL
patients. However, FISH studies have shown that

Fig. 5. Interphase nuclei hybridized with IgH Spectrum Green/BCL2 Spectrum Orange (VYSIS) probe showing: translo-
cation (two fusion yellow, one red, and one green signal) and no translocation (two green and two red signals).

312

monoallelic deletions of this gene occur in 9–15%
of cases (2, 9). Different reports have shown that
p53 gene deletions have strong implications in the
clinical course of the disease, with significantly
shorter median survival, advanced clinical stage,
and resistance to chemotherapy. In addition, p53
gene abnormalities occur in about 60% of cases
with Richter’s syndrome (2, 9). Our case, with p53
deletion originating in a dic(17,18), showed pro-
gression of the disease with a Richter’s transfor-
mation and a clear lack of response to several
therapeutic agents (alkylating agents, anthracyclin,
and purine analogs). In cases reported by Döhner
et al. (9), FISH and SSCP analysis revealed a
biallelic inactivation of the p53 gene. Our case
showed a monoallelic loss of p53 as determined by
FISH. Because molecular analysis of p53 was not
performed, it is unknown whether the remaining
allele was also inactivated.
Structural aberrations of the long arm of
chromosome 12 have been observed in about 5%
of CLL cases (1) and in approximately 15% of
patients with Richter’s syndrome (14). To our
knowledge this is the first case with a psu-
dic(12,21) described in B-CLL. As is known,
dicentric chromosomes have been found with
different incidences in malignant cells, according
to the type of tumor analyzed. The significance of
this abnormality depends on whether they con-
tribute to overall gain or loss, or if the translo-
cation results in the formation of a fusion gene
(15).
In our case, molecular studies showed the MBR/
JH bcl-2 gene rearrangement and FISH analysis
demonstrated the presence of this anomaly in a
small subpopulation of the leukemic cells, indica-
ting that this finding would be a secondary event.
Although the overexpression of bcl-2 gene is a
frequent phenomenon in B-CLL cases, the rear-
rangement of this gene has been observed in a
minor subset of patients with this pathology.
Among them, only a few CLL patients with the
MBR/JH breakpoint have been reported (16–18),
vcr rearrangements being the most frequently
found (4, 19). Besides, our results agree with those
previously reported by Merup et al. (16), who
suggested that bcl-2 rearrangements in B-CLL
might be a secondary phenomenon that occurs late
during disease progression.
Finally, our patient showed the 13q14 deletion
involving D13S319 and D13S25 loci, with diploid
RB-1. Once more, these findings support the idea
that a still unknown tumor suppressor gene, named
DBM (deleted in B-cell malignancies), is located at
13q14, telomeric to RB-1 (20, 21). In addition, the
low percentage of cells with the 13q14 deletion
suggests that it was a secondary genetic event that
Karyotypic evolution in CLL
resulted from karyotypic evolution. Although
B-CLL has been considered a genetically stable
disease, different studies (22–24) would indicate
that clonal evolution is not uncommon in these
patients, and would be associated with clinical
disease progression.
In conclusion, a new case of dic(17;18), with
other multiple genetic alterations and clonal evolu-
tion, has been presented. The poor clinical course
with Richter’s transformation and the short survi-
val observed in this patient support the association
of p53 gene deletion and karyotypic evolution with
disease progression.
Acknowledgements
This work was supported by grants from the National Research
Council (CONICET), the National Agency of Scientific and
Technical Promotion (ANPCyT), Fundación ‘Alberto
J Roemmers’, and Fundación Acción Oncohematológica.
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