Int. J. Cancer: 121, 978–983 (2007)
' 2007 Wiley-Liss, Inc.
High rate of centrosome aberrations and correlation with proliferative activity
in patients with untreated B-cell chronic lymphocytic leukemia
Manfred Hensel1*, Martin Zoz1, Christian Giesecke1, Axel Benner2, Kai Neben1, Anna Jauch3, Stephan Stilgenbauer4,
Anthony D. Ho1 and Alwin Kr€amer1,5
1
Department of Internal Medicine V, University of Heidelberg, Heidelberg, Germany
2
Central Unit ‘‘Biostatistics’’, German Cancer Research Center, Heidelberg, Germany
3
Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany
4
Department of Internal Medicine III, University of Ulm, Ulm, Germany
5
Clinical Cooperation Unit Molecular Hematology/Oncology, German Cancer Research Center, University of Heidelberg, Germany
B-cell chronic lymphocytic leukemia (CLL) is characterized by a
high rate of clonal genomic alterations and a low proliferative
activity with cell cycle arrest in G0/G1 phase. Recently, centrosome aberrations have been described as a possible cause of chromosomal instability and aneuploidy in many human malignancies.
To investigate whether centrosome aberrations do occur in CLL
and whether they correlate with common prognostic factors and
disease activity, we examined peripheral blood mononuclear cells
(PBMC) from 70 patients with previously untreated CLL using an
antibody to c-tubulin. All 70 CLL samples displayed significantly
more cells with centrosome aberrations (median: 26.0%, range
11.0–41.5%) as compared to peripheral blood B lymphocytes from
20 age-matched, healthy individuals (median: 2.0%, range 0–6%;
p < 0.001). The extent of centrosome aberrations correlated with
the proliferative activity of the CLL cases as measured by lymphocyte doubling time (p 5 0.02) as well as with time to first treatment
(p 5 0.05). Accordingly, more centrosome aberrations were found
in PHA-stimulated T lymphocytes from healthy individuals as
well as in B cells from surgically removed tonsil tissue of patients
with acute tonsillitis as compared to the peripheral blood B
lymphocytes from the control group. In contrast, no correlation
was observed between centrosome aberrations and immunoglobulin VH gene mutation status or cytogenetically defined risk groups.
These findings suggest that, despite the common observation of
most CLL cells remaining in G0/G1 phase, their centrosome replication process is deregulated and correlates to the proliferative
activity of CLL cells.
' 2007 Wiley-Liss, Inc.
Key words: centrosome aberrations; chronic lymphocytic leukemia;
proliferative activity
Chronic lymphocytic leukemia (CLL) is a heterogeneous disease at the clinical as well as at the molecular and cellular level.1,2
Three decades ago, 2 clinical staging systems, mainly based on
tumor load, have been introduced by Binet et al. 3 and Rai et al.4
Chromosomal changes have been detected in the majority of CLL
samples by interphase cytogenetic analysis using fluorescence
in situ hybridization (FISH).5 Both numerical and structural chromosome aberrations provide information about the clinical course.
17p and 11q deletions, as well as trisomy 12 have been identified
to predict rapid disease progression and short survival times,
whereas 13q deletions, as the sole aberration, are associated with
favorable outcome.5,6 Furthermore, the immunoglobulin VH gene
mutation status has been shown to be an independent prognostic
factor in CLL.7,8 In addition, the lymphocyte doubling time,
reflecting the clinical aggressiveness and proliferative activity, is a
useful parameter with prognostic value and a helpful tool for treatment decisions.9,10 For many years, the proliferative activity of
circulating CLL cells was thought to be low.11 Accordingly, CLL
was thought of as a purely accumulative disease because of a defect of apoptosis in the leukemic clone. Recently, however, it was
demonstrated that the proliferative activity of CLL cells seems to
be much higher as previously appreciated.12 Importantly, proliferation kinetics, as measured by an in vivo isotopic labeling method,
has been shown to correlate with clinical disease progression, but
Publication of the International Union Against Cancer
not with Ig VH gene mutation status, ZAP-70 or cytogenetic
abnormalities.12
As the primary microtubule organizing center of most eukaryotic cells, the centrosome ensures symmetry and bipolarity of
the cell division process, a function that is essential for accurate
chromosome segregation. Centrosome aberrations have recently
been described in both solid tumors and hematological malignancies.13–24 Moreover, it could also be demonstrated that centrosome
aberrations correlate with karyotype abnormalities in solid neoplasias of different origin,13,15,20–22,24–26 as well as in acute and
chronic myeloid leukemias, myelodysplastic syndromes, multiple
myelomas and nonHodgkin’s lymphomas.16,17,27–31 The extent of
centrosome aberrations was higher in aggressive and rapidly
proliferating nonHodgkin’s lymphomas as compared to indolent
lymphomas.17
To investigate whether centrosome aberrations do occur in CLL
and correlate with established prognostic parameters like cytogenetics, VH mutation status and proliferative activity, we examined
peripheral blood mononuclear cells (PBMC) from 70 patients with
untreated CLL and peripheral blood B cells from 20 age-matched
healthy volunteers using an antibody to g-tubulin.24
Materials and methods
Specimen selection
We examined a set of 70 previously untreated patients with
CLL (median age 63.5 years, range 30.1–88.2). All patients were
diagnosed between November 1985 and April 2002. The CLL
diagnosis was based on morphologic and immunophenotypic
features according to the World Health Organization (WHO) classification.32 The last follow-up was in May 2006. Median time
from diagnosis to analysis of centrosome aberrations was 23
months (range, 0–185 months). Median survival was 71 months
(range, 21–231 months). Interphase cytogenetic data were available for 69 patients (99%), obtained by FISH as previously
described by D€
ohner et al.5 VDJ sequencing was performed
according to the ERIC recommendations on IGHV gene mutational status analysis33 and as recently described.34 VH homology
cutoff was 98%. VH gene mutation status was available for
45 patients (64%). Cytogenetic analyses were performed as a
part of study protocols of the German CLL Study Group in 11
patients. For T cell stimulation, PBMC (7 3 106 cells) from 3
healthy donors were cultured in the presence of 1 lg/ml phytoGrant sponsors: German CLL Study Group, the Deutsche Krebshilfe,
the Bundesministerium f€ur Bildung und Forschung.
*Correspondence to: Department of Medicine V, University of
Heidelberg, Im Neuenheimer Feld 410, D-69120 Heidelberg, Germany.
Fax: 149-6221-5633678.
E-mail: [email protected]
Received 8 January 2007; Accepted after revision 5 March 2007
DOI 10.1002/ijc.22752
Published online 6 April 2007 in Wiley InterScience (www.interscience.
wiley.com).
CENTROSOME ABERRATIONS IN CLL
979
TABLE I – PATIENT CHARACTERISTICS (N 5 70)
Age at first diagnosis (years)
Age at PBMC analysis (years)
Male sex
Disease stage at diagnosis
A
B
C
Disease stage at study entry
A
B
C
Duration of disease (month)
Chromosomal aberrations
13q deletion1
12q trisomy
11q deletion
17p deletion
Normal karyotype
VH gene status (available in n 5 45 pts)
Unmutated
Mutated
b 2-microglobulin at study entry (mg/l)
LDH at study entry (U/l)
60.3 (27.9–87.5)
63.5 (30.1–88.2)
47 (67.1%)
55 (80.1%)
11 (16.2%)
2 (2.9%)
52 (74.3%)
15 (21.4%)
3 (4.3%)
23 (0–185)
34 (48.5%)
4 (5.7%)
8 (11.4%)
1 (1.4%)
22 (31.4%)
15 (33.3%)
30 (66.7%)
2.4 (1.0–9.9)
148 (101–534)
1
13q deletion without additional abnormalities of chromosome 11,
12, 17.
hemagglutinin (Sigma, Deisenhofen, Germany) for 3 days. Tonsillectomy specimens from 3 individuals with acute tonsillitis served
as a source of stimulated B lymphocytes. CD20-positive B lymphocytes from peripheral blood of 20 healthy and age-matched
volunteers (median age 60.5 years; range 37–77) and 4 otherwise
healthy patients with monoclonal B-cell lymphocytosis (MBL)
(median age 63.5 years; range 61–66), defined by the presence of
monoclonal lymphocytes with the immunophenotypic characteristics of CLL cells at levels below 5 3 109 l21 in the blood,35,36
were examined as control group. Approval was obtained from the
institutional review board of the University of Heidelberg.
Informed consent was provided according to the Declaration of
Helsinki.
Centrosome staining
Centrosome staining in PBMC was performed as described previously.16 Mononuclear cells were isolated from peripheral blood
by Ficoll gradient centrifugation using Biocoll separating solution
(Biochrom, Berlin, Germany). Around (96.4 6 6.9)% of the
mononuclear cells isolated from peripheral blood of CLL patients
were lymphocytes as calculated from differential blood counts.
Immunophenotyping of the mononuclear cell fraction showed
CD19/CD5 coexpression in (79.7 6 12.9)% of cells, reflecting a
high tumor cell content. Cytospins were stained by using a monoclonal antibody to g-tubulin (Sigma, Deisenhofen, Germany). The
antibody–antigen complexes were detected by incubation for 1 hr
at room temperature with a fluorescein isothiocyanate-conjugated
secondary goat anti-mouse IgG antibody (Convance, Richmond,
CA). Analysis of centrosomal aberrations in B and T lymphocytes
was performed by coimmunostaining with a monoclonal antibody
to g-tubulin (Sigma) and monoclonal antibodies to CD20 or CD3
(Sigma), respectively. Highly cross-absorbed secondary reagents
were purchased from Molecular Probes (Eugene, OR); Alexa-488
and Alexa-594 were used for dual-color detection. Slides were
mounted with phosphate-buffered glycerol (Euroimmun, L€
ubeck,
Germany) and visualized under a fluorescence microscope
(Axioskop; Zeiss, Jena, Germany) by using a 3100 objective. Immunostaining of centrosomes was judged satisfactory when the
characteristic single or paired centrosome pattern was detected in
negative controls. Centrosomes were considered structurally abnormal if they had a diameter of at least twice that of centrosomes
in nonmalignant PBMC and numerically abnormal if they were
FIGURE 1 – Percentage of centrosome aberrations in peripheral
blood mononuclear cells (PBMC) from 70 patients with B-cell chronic
lymphocytic leukemia (CLL), detected by indirect immunofluorescence staining with an anti-g-tubulin antibody, compared to CD20positive B-lymphocytes from 20 age-matched healthy volunteers and
4 individuals with monoclonal B-cell lymphocytosis (MBL). Centrosome aberrations were detectable in 26.0% (range: 11.0–41.5%) of the
PBMC from patients with CLL, 12.2% (range: 8.2–26.8%) of the B
cells from individuals with MBL, and 2.0% (range: 0–6%) of the B
cells from healthy controls (p < 0.001).
present in numbers more than two, as described previously.23 At
least 200 consecutive cells per sample were carefully examined.
Flow cytometry
For two-parameter flow cytometry analysis to assay cell cycle
distribution by DNA content in B or T lymphocytes, cells
were stained with anti-CD20 or anti-CD3 (Becton Dickinson,
Heidelberg, Germany), fixed in ice-cold methanol, stained with
0.1 mg/ml propidium iodide and analyzed by a FACScan flow
cytometer (Becton Dickinson) using Cellquest software.
Statistical analysis
The distribution of the percentage of cells with centrosome
aberrations in controls and CLL patients were compared using the
exact Wilcoxon rank sum test. Differences in the percentage of
cells with centrosome aberrations among cytogenetically defined
subgroups and subgroups defined by IgVH mutation status were
also analyzed by application of the exact Wilcoxon rank sum test.
The correlation between age and the percentage of cells with
centrosome aberrations was estimated by Spearman’s rho. Tests of
changes in the distribution of lymphocyte doubling time depending on the percentage of CLL cells with centrosome aberrations
were done using maximally selected Wilcoxon statistics.37
The primary endpoint of the analysis was treatment-free
interval (TFI), measured from the date of diagnosis until the time
of first therapy, taken as a surrogate for disease progression. The
decision to start treatment was taken according to the NCI
guideline.38 Estimation of TFI and survival distributions were
done by the method of Kaplan and Meier.39 Testing of possible
changes in the TFI distribution with respect to the percentage of
CLL cells with numerical and/or structural centrosome aberrations
was done by maximally selected log-rank statistics.37,40 Depending
on the results of the maximally selected log-rank test, univariable
Cox proportional hazards regression was performed to test the
effect of centrosome aberrations on TFI.41,42 In addition, we used
the ‘‘proportional subdistribution hazards’’ regression model described in Fine and Gray.43 This model directly assesses the effect
of the percentage of cells with centrosome aberrations on the subdistribution of TFI in a competing risks setting with patient’s
980
HENSEL ET AL.
FIGURE 2 – Centrosome aberrations in B-cell CLL. Indirect immunofluorescence staining with an anti-g-tubulin antibody of normal PBMC
with 1 or 2 centrosomes [(a), (b)] and CLL cells with either structural [(c), (d)] or numerical [3 or 4 centrosomes; (e), (f)] centrosome aberrations. As described in Materials and Methods, centrosomes were considered structurally abnormal if they had a diameter at least twice that of
centrosomes from nonmalignant PBMC.
death being the competing risk. To estimate the hazard ratio for a
sensible change in the percentage of centrosome aberrations the
interquartile range (‘‘half sample’’) was used.44
The result of a statistical test was judged as statistically significant at a two-sided p value not larger than 5%. All statistical analyses were performed using SPSS (release 11.0; SPSS, Chicago,
IL) and R, version 2.3.145 using the R packages Design, version
2.0-12, exactRankTests, version 0.8-12, and maxstat, version 0.7-9.
Results
We examined CD20-positive B lymphocytes from 4 patients
with MBL35,36 and PBMC from 70 previously untreated patients
with CLL using indirect immunofluorescence with a monoclonal
antibody to g-tubulin. The analysis was performed at the time of
diagnosis (n 5 21) or during the course of the disease (n 5 49).
Among the 70 CLL patients were 47 males and 23 females with a
median age of 63.5 years (range, 30–88) (Table I). Median
duration of disease, defined as time from diagnosis to analysis of
centrosome aberrations, was 23 months (range, 0–185). Interphase
cytogenetic data were available for 69 patients (99%). Firstly, the
centrosome aberration patterns of the patients with MBL and CLL
were compared to CD20-positive B lymphocytes from peripheral
blood of 20 healthy and age-matched volunteers (median age
60.5 years; range 37–77). All MBL and CLL samples displayed
significantly more cells with centrosome aberrations as compared
to the control individuals. Centrosome aberrations were detectable
in 26.0% (range 11.0–41.5) of the PBMC in CLL, 12.2% (range
8.2–26.8) of the B cells in MBL, but in only 2.0% (range: 0–6%)
of the B lymphocytes in healthy controls (p < 0.001) (Fig. 1). No
correlation between age of the CLL patients and percentage of
cells with centrosome aberrations was found (Spearman’s rho 5
20.08; 95% confidence interval 20.30 to 0.16). Approximately
50% of the centrosomal abnormalities were numerical in nature.
Almost all numerical centrosome alterations were because of cells
harboring 3 or 4 structurally normal centrosomes (Fig. 2).
Next, we sought to evaluate whether centrosomal aberrations do
correlate with established prognostic parameters in patients with
FIGURE 3 – Treatment free interval (TFI) according to the percentage of CLL cells harboring centrosome aberrations (n 5 70 untreated
patients). Q1–4 represent the TFI curve estimates corresponding to
patient groups defined by the quartiles of the distribution of the percentage of CLL cells harboring centrosome aberrations.
CLL. In Figure 3, estimated TFI curves depending on centrosome
aberration patterns are shown. Estimates were computed corresponding to patient groups defined by the quartiles of the distribution of the percentage of CLL cells harboring centrosome
aberrations. Using a univariable Cox proportional hazards regression model, centrosome aberrations were significantly related to
TFI (p 5 0.05). The half sample hazard ratio was 1.57 (95% confidence interval 1.00–2.46). A similar result with a half sample
hazard ratio of 1.58 (95% confidence interval 0.93–2.68) was
obtained using a competing risk regression model with death of
981
CENTROSOME ABERRATIONS IN CLL
TABLE II – CENTROSOME ABERRATIONS IN CHRONIC LYMPHOCYTIC LEUKEMIA ARE NOT CORRELATED TO CYTOGENETIC
RISK PROFILE AND VH-STATUS
Abnormality
Total no.
Median number (range) of cells with abnormal centrosomes (%)
Numerical
1
Normal controls
Healthy individuals with monoclonal B-cell
lymphocytosis (MBL)
CLL patients
Interphase cytogenetics
Favorable risk2
Adverse risk3
VH-status
Unmutated
Mutated
Structural
Total
20
4
0.5 (0.0–4.0)
8.0 (4.1–22.9)
1.0 (0.0–4.0)
4.0 (1.7–6.7)
2.0 (0.0–6.0)
12.2 (8.2–26.8)
56
13
13.5 (2.2–26.5)
14.5 (4.5–25.5)
11.5 (6.0–23.6)
12.0 (5.0–20.9)
26.0 (11.0–39.5)
26.5 (11.5–41.5)
15
30
14.5 (2.2–26.5)
13.5 (4.5–22.0)
11.0 (8.0–20.9)
11.0 (7.0–23.6)
27.0 (20.0–34.5)
25.5 (12.5–39.5)
1
CD20 positive B-lymphocytes from age-matched healthy volunteers were analyzed as controls.–2Favorable risk: normal karyotype or 13q deletion without additional abnormalities of chromosome 11, 12, 17.–3Adverse risk: 11q deletion, 12q trisomy, 17p deletion.
FIGURE 4 – (a) shows the results of the maximally selected Wilcoxon rank sum statistics, performed to test for a potential cut point separating
2 groups with different lymphocyte doubling time distributions. The dashed horizontal line at 3.05 represents the value of the test statistic corresponding to a significance level of 5% and the dashed vertical line indicates the corresponding cutpoint at 29.5%. The corresponding boxplot of
the distribution of lymphocyte doubling time dependent on the percentage of centrosome aberrations is shown in (b) (p 5 0.02).
the patient as competing risk (p 5 0.09). A statistically significant
dependency of overall survival on centrosome aberrations could
not be found (p 5 0.66), most likely explained by the high
percentage of censored survival times (84%).
To test for the hypothesis that centrosome abnormalities are
associated with chromosome aberrations in CLL, we compared
the centrosome aberration patterns with available cytogenetic data
(n 5 69). Patients with 13q deletions (without additional 11q
deletions, 12q trisomy or 17p deletions) and normal karyotypes
were considered having a favorable risk (n 5 56), patients with
11q deletions, 12q trisomy or 17p deletions having an adverse risk
profile (n 5 13). There was no difference in the percentage of
centrosome aberrations in patients with favorable cytogenetic risk
profiles as compared to patients with adverse cytogenetic risk
(Wilcoxon rank sum test: p 5 1.00) (Table II). IgVH mutation status was evaluable in 45 patients. Considering the classical VH
homology cutoff value of 98%, 30 (67%) patients had mutated
and 15 (33%) patients had unmutated VH genes. The incidence of
centrosome abnormalities was comparable in both IgVH sub-
groups (Wilcoxon rank sum test: p 5 0.61) (Table II). In contrast
to cytogenetic risk profile and IgVH mutation status, a significant
negative correlation between the percentage of cells with centrosome aberrations and the proliferative activity of the malignant
lymphocytes, as measured by the lymphocyte doubling time was
found (Spearman’s rho 5 20.22; 95% confidence interval 20.43
to 0.02) (Fig. 4).
To further determine whether the occurrence of centrosome
aberrations in lymphoid cells depends on their proliferative status,
we examined both phytohemagglutinin-stimulated T lymphocytes
from healthy donors and B cells from tonsillectomy specimen of
individuals with acute tonsillitis. Peripheral blood T cells from 3
healthy volunteers were stimulated with PHA or cultured without
stimulation for 3 days and subsequently analyzed by immunofluorescence microscopy. The impact of PHA stimulation on T cell
proliferation was ascertained by FACS analysis after double staining with CD3 and propidium iodide. Centrosome aberrations were
detectable in 0.5% of unstimulated cells as compared to 11%
(median; range 11–18%) of stimulated cells (p 5 0.02). As
982
HENSEL ET AL.
revealed by FACS analysis, 22% (median; range 16–26%) of the
PHA stimulated T cells, but only 1% of the cells without stimulation were in S/G2/M phase of the cell cycle (p 5 0.016). Similarly
and in contrast to peripheral blood B cells of our control group,
5% (median; range 4–7%) of B cells from 3 individuals with acute
tonsilitis harbored centrosomal aberrations with 8–26% of these
cells being in S/G2/M phase of the cell cycle.
Discussion
The findings reported here demonstrate for the first time that
centrosome defects are a common feature of CLL. All CLL
samples displayed significantly more cells with centrosome aberrations as compared to control individuals. Even at the premalignant MBL stage, centrosome amplification can be regularly
detected. When the frequency of centrosome aberrations in CLL
cells was analyzed within cytogenetically defined risk groups, no
correlation of the extent of centrosome abnormalities to the 2
major risk groups was found. In addition, centrosome aberrations
were not associated with the IgVH gene mutation status of the
CLL patients. Instead, centrosomal defects were correlated with
clinical aggressiveness and proliferative activity of CLL, as measured by time to first treatment and lymphocyte doubling time.
Centrosome aberrations have recently been reported in solid
tumors of different origin including brain, breast, lung, colon,
prostate, pancreas, bile duct and head and neck.13,15,20–22,24–26
Hematological malignancies including acute and chronic myeloid
leukemias, multiple myelomas and both Hodgkin- and nonHodgkin lymphomas also display centrosome aberrations at high frequencies.16,17,27–31,46 Moreover, centrosome aberrations have been
shown to occur already in many premalignant lesions, including
preinvasive cancers of prostate, uterine cervix, breast and pancreas
as well as in myelodysplastic syndromes and monoclonal gammopathies of undetermined significance.13,14,21,22,26,30,31 In the
current study, we found that centrosome aberrations are already
present in B lymphocytes from patients with MBL, a lesion considered to represent a premalignant stage of CLL,35,36 suggesting
that centrosome abnormalities do occur early during disease
evolution of CLL, analogous to the above malignancies. This is
further substantiated by the fact that percentage of B lymphocytes
with centrosomal aberrations increases through stages of clonal
lymphocyte expansion. Our finding of an association between centrosome aberrations on one hand and clinical aggressiveness and
proliferative activity of CLL on the other hand is in line with the
finding that aggressive lymphomas harbor more extensive centrosome abnormalities as compared to indolent nonHodgkin lymphomas.17 Within the groups of follicular lymphomas and mantle cell
lymphomas, the number of cells with centrosome aberrations correlated with increased histological grading and ploidy status,
respectively.17 Also, a positive correlation between the amount of
centrosomal abnormalities and the proliferation index/mitotic
index has been found in multiple myelomas, follicular lymphomas, diffuse large B cell lymphomas and mantle cell lymphomas,17 clearly implying that lymphomas with a higher proliferation index display significantly more centrosome aberrations
irrespective of their histological subgroup. In addition, the missing
correlation between centrosome anomalies and karyotype aberrations is also precedented by findings that have recently been
reported for multiple myeloma and Burkitt lymphoma.30,46 Of
note, a case of Burkitt lymphoma with excessive proliferative
activity displayed extensive centrosomal aberrations without
ongoing chromosomal instability.46
CLL is characterized by a low proliferative activity with cell
cycle arrest in G0/G1 phase and a 2n DNA content. However, it
has recently been described that CLL cells from patients with a
poor outcome uniformly have a more extensive proliferative history than those from the subgroup of patients with better outcome,
as judged by telomere length analysis, suggesting that CLL is not
simply an accumulative disease of slowly dividing B lymphocytes
but possibly one of the B cells with extensive proliferative histories.47 Moreover, in vivo measurements of CLL cell kinetics using
a nonradioactive isotopic labeling method demonstrated that CLL
cells often have substantial birth rates that are correlated to disease
activity and progression but not to classical prognostic parameters
like IgVH mutation status and cytogenetic abnormalities.12 Therefore, in analogy to other nonHodgkin-lymphomas, centrosomal
abnormalities of circulating CLL cells might reflect cellular generation emanated from proliferation centers in lymph nodes and
bone marrow of CLL patients. To test for this hypothesis, we
examined in vitro stimulated T lymphocytes from healthy donors
and in vivo stimulated B cells from tonsillectomy specimens of
individuals with acute tonsillitis for changes in centrosome morphology. In accord with the size of the proliferating fractions, as
determined by FACS analysis, the percentage of cells with morphologically abnormal centrosomes was increased among both
stimulated T and B lymphocytes when compared to their unstimulated counterparts. Consistently, we have reported earlier that
quiescent PBMC contain fewer centrosomal aberrations than
activated tonsillar B lymphocytes.17 Importantly, analogous to the
findings reported here, centrosome abnormalities in lymphomas
and multiple myelomas are strongly associated with the mitotic/
proliferation index and the plasma cell labeling index as a markers
for proliferative activity, respectively.17,30
The detailed mechanisms by which centrosome aberrations
develop are still largely unknown. Several oncogenes and tumor
suppressor genes, among the p53 and ATM, have been implicated
in the formation of centrosomal defects in human malignancies.28,48,49 Of note, inactivation of p53 or ATM is a frequent event
in CLL. Both, p53 and ATM abnormalities are associated with a
poor prognosis in this disorder.50 Therefore, it is tempting to speculate that aberrations of the ATM/p53 pathway might be involved
in the generation of centrosomal abnormalities in CLL.
In conclusion, our results indicate that centrosome defects are a
common feature in CLL and suggest that increased proliferation
of CLL cells, which is associated with limited IgVH gene mutations and poor survival, might lead to centrosome amplification
and subsequent chromosomal aberrations. Analogous to multiple
myelomas, acute myeloid leukemias and mantle cell lymphomas,
where the occurrence of centrosome aberrations is associated with
deregulated expression of pericentriolar matrix proteins,30,51,52
gene expression analysis might further improve our understanding
of the causes and consequences of centrosome aberrations in CLL.
Acknowledgement
We thank Mrs. Brigitte Schreiter for excellent technical assistance.
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