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Leukemia (2003) 17, 2207–2213 & 2003 Nature Publishing Group All rights reserved 0887-6924/03 $25.00 www.nature.com/leu

Centrosome aberrations as a possible mechanism for chromosomal instability in non-Hodgkin’s lymphoma A Kra¨mer1, S Schweizer1, K Neben2, C Giesecke1, J Kalla3, T Katzenberger3, A Benner4, HK Mu¨ller-Hermelink3, AD Ho1 and G Ott3 1 Medizinische Klinik und Poliklinik V, Universita¨t Heidelberg, Heidelberg, Germany; 2Abteilung Molekulare Genetik, Deutsches Krebsforschungszentrum, Heidelberg, Germany; 3Institut fu¨r Pathologie, Universita¨t Wu¨rzburg, Wu¨rzburg, Germany; and 4Central Unit Biostatistics, Deutsches Krebsforschungszentrum, Heidelberg, Germany

Recently, centrosome aberrations have been described as a possible cause of aneuploidy in many solid tumors. To investigate whether centrosome aberrations occur in nonHodgkin’s lymphoma (NHL) and correlate with histologic subtype, karyotype, and other biological disease features, we examined 24 follicular lymphomas (FL), 18 diffuse large-B-cell lymphomas (DLCL), 33 mantle cell lymphomas (MCL), and 17 extranodal marginal zone B-cell lymphomas (MZBCL), using antibodies to centrosomal proteins. All 92 NHL displayed numerical and structural centrosome aberrations as compared to nonmalignant lymphoid tissue. Centrosome abnormalities were detectable in 32.3% of the cells in NHL, but in only 5.5% of lymphoid cells from 30 control individuals (Po0.0001). Indolent FL and MZBCL contained only 25.8 and 28.8% cells with abnormal centrosomes. In contrast, aggressive DLCL and MCL harbored centrosome aberrations in 41.8 and 35.0% of the cells, respectively (Po0.0001). Centrosomal aberrations correlated to lymphoma grade, mitotic, and proliferation indices, but not to the p53 labeling index. Importantly, diploid MCL contained 31.2% cells with abnormal centrosomes, while tetraploid samples harbored centrosome aberrations in 55.6% of the cells (Po0.0001). These results indicate that centrosome defects are common in NHL and suggest that they may contribute to the acquisition of chromosomal instability typically seen in NHL. Leukemia (2003) 17, 2207–2213. doi:10.1038/sj.leu.2403142 Published online 18 September 2003 Keywords: centrosome aberrations; non-Hodgkin’s lymphoma; chromosomal instability; mantle cell lymphoma; follicular lymphoma; diffuse large B-cell lymphoma

Introduction Faithful segregation of chromosomes into daughter cells is essential for maintaining the genetic stability of most organisms. Chromosome segregation is mediated by the mitotic spindle.1–3 Although it is not completely understood how spindles are assembled, the centrosome appears to play an important role in the process.2,4 In a manner highly reminiscent of DNA replication, the centrosome needs to be duplicated exactly once per cell cycle. Recently, it has been demonstrated that centrosome duplication in somatic cells is controlled by the phosphorylation status of the retinoblastoma (Rb) protein, release of the transcription factor E2F from Rb binding, and subsequent activation of cyclin-dependent kinase 2 (cdk2) in late G1 phase.5–7 Consequently, the commonly observed abrogation of the Rb pathway in human malignancies will not only facilitate progression towards DNA replication but may also deregulate the centrosome duplication cycle.8 Genetic instability is a common feature of malignant neoplasias. It is frequently characterized by numerical chromoCorrespondence: A Kra¨mer, Department of Cell Cycle and Cancer, Institute of Cancer Biology, Danish Cancer Society, Denmark; Fax: þ 45-3525-7721 Received 23 June 2003; accepted 14 August 2003; Published online 18 September 2003

some abnormalities, a condition known as aneuploidy. Furthermore, recent results demonstrate that aneuploid cells exhibit continuous changes in chromosome number throughout their lifetimes, suggesting that this chromosome instability may contribute to tumor progression.9 Defects in chromosome number are thought to occur through missegregation of chromosomes,1,9 but the mechanism by which this occurs has not been elucidated. However, many recent studies have provided evidence that centrosome aberrations result in chromosome missegregation and may lead to malignant transformation.10–12 Specifically, centrosome amplification, induced by p53 mutations or Mdm2 overexpression, has been shown to induce aneuploidy.10 Also, abrogation of Rb function by HPV16 E7 protein expression as well as overexpression of both centrosomal matrix proteins like pericentrin and centrosome-associated kinases and phosphatases like STK15/Aurora A and Cdc14A, induced centrosome amplification, aneuploidy, and malignant transformation.11,13–15 Furthermore, recent reports describe centrosome aberrations in human solid tumors of different origin, including brain, breast, lung, colon, pancreatic, biliary tract, and head and neck tumors.13,16–22 Acute myeloid leukemias23 and myelodysplastic syndromes (Kearns et al. Blood 2001; 98: 913a, abstract) also display centrosome aberrations at high frequencies. In almost all patients with non-Hodgkin’s lymphoma (NHL), acquired chromosome abnormalities are present. Some entities are characterized by highly specific primary translocations, such as follicular lymphoma (FL) and mantle cell lymphoma (MCL). These aberrations are thought to be necessary for tumor initiation, and their molecular consequences may be essentially different, implying resistance to apoptosis, conferred by the t(14;18)(q32;q21) chromosome translocation in FL or cell cycle dysregulation by the translocation t(11;14)(q13;q32) in MCL. In addition to typical balanced translocations, at least 75% of NHL display numerical aberrations.24,25 Whereas 75% of FL harbor numerical chromosome aberrations, gains or losses of whole chromosomes can be found in almost 100% of diffuse large-Bcell lymphoma (DLCL). In MCL, a blastoid variant with a striking tendency to harbor chromosome numbers in the tetraploid range has been identified, a phenomenon that is rare in other B-cell neoplasms.26 Secondary structural and numerical chromosome abnormalities have been shown to provide information about the clinical course of individual patients and the risk of transformation of FL to DLCL.27 To investigate whether centrosome abnormalities contribute to chromosomal instability in NHL and to elucidate the possible biological consequences, we have investigated centrosome aberrations in 24 FL, 18 DLCL, 33 MCL, and 17 extranodal marginal zone B-cell lymphomas (MZBCL) using indirect immunofluorescence analysis of centrosomal antigens, and correlated the results to histological subtypes and karyotype.

Centrosome aberrations in non-Hodgkin’s lymphoma A Kra¨mer et al

2208 Materials and methods

Specimen selection All cases of NHL had been diagnosed and categorized according to the principles of the WHO classification.28 A total of 92 cases, including 24 FL (16 FL grades 1 and 2; eight FL grade 3a), 18 DLCL, 19 diploid MCL, 12 tetraploid MCL, two MCL with unknown ploidy status, and 17 MZBCL were selected for the present study. Formalin-fixed and paraffin-embedded material was stained for hematoxylin and eosin, Giemsa, periodic acid Schiff, and Gomori’s silver impregnation. In all cases, immunophenotyping was performed for diagnostic purposes on paraffin sections using B- and T-cell markers. Peripheral blood mononuclear cells (PBMC) from 21 healthy volunteers, including eight males and 13 females with a median age of 24 years (range 22–58 years) as well as tonsillectomy specimes from nine individuals with tonsillitis served as negative controls.

Mitotic, proliferation, and p53 labeling indices The mitotic index was assessed in lymphoma cases by counting mitoses in 10 randomly selected high-power fields ( 40) in Giemsa-stained slides. Proliferation indices were estimated by two independent observers in areas with the highest reactivity for the MIB1 antibody (Dianova, Hamburg, Germany), detecting the Ki67 antigen, on paraffin slides in steps of 10%. p53 labeling indices were recorded as the percentage of positive tumor nuclei after staining with the DO-7 antibody (Dako, Hamburg, Germany).

Cytogenetic studies Classical cytogenetic studies were performed in 76 cases. Unstimulated 10-ml cell cultures were set up in RPMI 1640 medium containing 1–2 106 cells/ml culture medium and allowed to grow overnight. Metaphase preparation involved 20 min of exposure to colchicin, hypotonic shock in 0.075 mol/l KCl, and fixation in methanol/acetic glacial acid (3:1). Metaphases were stained using a trypsin-Giemsa standard technique, and the results were evaluated according to the ISCN.29 The number of structural aberrations in a given case was counted as the sum of structural aberrations in the karyotype, implying that the translocations between two chromosomes were counted as one aberration, as were deletions or additions occurring in one chromosome. Numerical aberrations were counted as one aberration, and supernumerary chromosomes with structural aberrations were counted as two aberrations.

Fluorescence in situ hybridization (FISH) and DNA flow cytometry In MCL cases without classical cytogenetics available, ploidy was determined using either FISH or DNA flow cytometry as previously described.26

many).30 In addition, a randomly chosen part of the slides was stained with a monoclonal antibody to g-tubulin (n ¼ 10; Sigma, Deisenhofen, Germany;17,18,22) or a polyclonal antibody to pericentrin (n ¼ 18; Convance, Richmond, CA, USA;23,31). Nonmalignant lymphoid cells from 30 individuals served as negative controls. For centrosome immunostaining, cryosections (B5 mM thick) of lymphoma tissue mounted on coated slides were fixed in 201C acetone for 10 min, permeabilized in 0.2% Tween-20 for 5 min, blocked in phosphate-buffered saline (PBS) containing 1% BSA and 1% human immunoglobulin for 1 h, followed by standard indirect immunohistochemistry. Briefly, cryosections were stained using antibodies to pericentrin, g-tubulin, or human autoantibodies against centrosomes. The slides were washed in PBS three times for 5 min each. The antibody–antigen complexes were detected by incubation for 1 h at room temperature with FITC-conjugated secondary antibodies, including goat anti-mouse IgG (Convance, Richmond, CA, USA) and goat anti-human IgG (Euroimmun, Lu¨beck, Germany). The slides were washed in PBS three times for 5 min each again. Nuclear DNA was counterstained with propidium iodine and Evans blue. Slides were mounted with phosphatebuffered glycerol (Euroimmun, Lu¨beck, Germany) and visualized under a fluorescence microscope (Axioskop, Zeiss, Jena, Germany) using a 100 objective. Raw digital immunofluorescence images from each preparation were recorded electronically on a digital camera with a Nikon chip (Coolpix 990, Nikon, Tokyo, Japan).

Calculation of centrosome aberrations Immunostaining of centrosomes was judged satisfactory when the characteristic single or paired centrosome pattern was detected in non-neoplastic cells adjacent to the tumor. Centrosomes were considered abnormal if they had a diameter at least twice that of centrosomes in normal control cells, if they were present in numbers 42, and if they were structurally abnormal, as described previously.13 At least 200 cells per sample were carefully examined. Centrosome aberration percentages reflect the number of cells with numerical and/or structural centrosome abnormalities per 100 cells counted. Cells with both aberration types were counted only once.

Statistical analysis Pairwise comparisons of differences in the location of the distributions of the percentage of cells with centrosome aberrations among nonmalignant controls and NHL as well as between NHL subtypes were carried out using the Mann– Whitney test. Always, two-sided P-values were computed. An effect was considered as statistically significant at a P-value p0.05. A generalization of Spearman’s r was computed to detect nonmonotonic associations between two variables.32 The statistical analyses were performed using the statistical software packages SPSS, release 6.1.3 (SPSS Inc.) and S-Plus, version 3.4 (Insightful Corp.).

Results

Centrosome staining Cytogenetic, FISH, and DNA flow cytometry studies Cryosections from 92 NHL, including 24 FL, 18 DLCL, 33 MCL, and 17 MZBCL were examined by immunostaining with autoantibodies against centrosomes (Euroimmun, Lu¨beck, GerLeukemia

Classical cytogenetic data were available for 76 of the 92 NHL samples examined. All cases except for one MZBCL displayed


2209 clonal chromosome aberrations. One out of 24 FL, five out of 18 DLCL, and 12 out of 33 MCL harbored a near tetraploid karyotype. The cytogenetic hallmark of FL, the t(14;18)(q32;q21) chromosome translocation, was detected in 19 of 24 (79%) FL cases; 30 of 33 MCL harbored the t(11;14)(q13;q32) translocation. For three MCL samples no data on the t(11;14)(q13;q32) status were available. A chromosome translocation t(11;18)(q21;q21) was present in five of 17 (29%) MZBCL. In four MCL cases, ploidy as well as t(11;14)(q13;q32) status and in nine MZBCL t(11;18)(q21;q21) status was determined by FISH. DNA flow cytometry revealed ploidy in three additional MCL.

Centrosome aberrations in NHL Controls: A median of 5.2% (range 1.0–8.4%) PBMC and 8.6% (range 3.5–14.5%) of the cells from the tonsillectomy specimens displayed centrosome abnormalities, resulting in the detection of centrosome aberrations in a total median of 5.5% (range 1.0–14.5%) nonmalignant lymphoid cells from 30 control individuals (Table 1). Malignant lymphomas: All 92 NHL samples analyzed displayed more cells with numerical and structural centrosome aberrations as compared to the controls (Figure 1). Centrosome abnormalities were detectable in 32.3% (range 15.1–68.6%) of the NHL cells, but in only 5.5% (range 1.0–14.5%) of the control cells (Po0.0001, Figure 2). Whereas FL and MZBCL contained 25.8% (range 15.1–34.7%) and 28.8% (range 16.4– 34.7%) cells with abnormal centrosomes, aggressive DLCL and MCL harbored centrosome aberrations in 41.8% (range 35.4–54.5%) and 35.0% (range 21.8–68.6%) of the cells, respectively (Po0.0001). Differences between nonmalignant controls and NHL as well as between indolent and aggressive NHL subtypes were statistically highly significant. When the FL were analyzed with respect to their grading, FL grades 1 and 2 exhibited centrosome aberrations in 23.1% (range 15.1–32.7%) of their cells (n ¼ 16). In contrast, FL grade 3a contained 30.0% (range 25.2–34.7%) cells with abnormal centrosomes (n ¼ 8,

Table 1

Figure 1 Centrosome aberrations in NHL. (a) Indirect immunofluorescence staining of normal bone marrow. The arrowhead indicates a pair of normal centrosomes. (b–d) Indirect immunofluorescence staining of NHL cells. In (b) the arrowheads indicate structurally abnormal centrosomal material of increased size. In (d) a cell with predominantly numerical centrosome aberrations is shown. Cells were immunostained with an antibody to pericentrin, followed by a FITC-conjugated secondary antibody.

P ¼ 0.003). As shown in Figure 3, a positive correlation between centrosome aberrations and mitotic/proliferation indices was found for FL and DLCL as well as for near diploid and near tetraploid MCL, when analyzed separately. In contrast, neither for FL nor for DLCL or near diploid and near tetraploid MCL, a statistically significant correlation between centrosome aberra-

Centrosome aberrations in different subtypes of B-cell NHL

NHL subtype

Total no.

Median number (range) of cells with abnormal centrosomes in % Numerical

Structural

Total

Controls PBMC Tonsils

30 21 9

0.0 0.5 0.0

(0.0–2.5) (0.0–2.5) (0.0–0.3)

5.1 4.5 8.6

(1.0–14.5) (1.0–7.0) (3.5–14.5)

5.5 5.2 8.6

(1.0–14.5) (1.0–8.4) (3.5–14.5)

FL Grade 1/2 Grade 3a

24 16 8

2.3 2.3 2.3

(0.0–5.7) (0.0–5.7) (0.5–4.5)

23.3 20.4 27.0

(14.2–34.1) (14.2–30.0) (21.7–34.1)

25.8 23.1 30.0

(15.1–34.7) (15.1–32.7) (25.2–34.7)

DLCL

18

1.0

(0.0–5.9)

40.5

(35.0–52.5)

41.8

(35.4–54.5)

MCL Near diploid Near tetraploid Unknown

33 19 12 2

1.4 0.9 3.6 0.5

(0.0–14.5) (0.0–7.5) (0.0–14.5) (0.4–0.5)

33.7 28.9 50.2 36.8

(21.3–59.9) (21.3–36.3) (33.5–59.9) (33.9–39.7)

35.0 31.2 55.6 37.3

(21.8–68.6) (21.8–43.9) (43.5–68.6) (34.4–40.1)

MZBCL

17

0.4

(0.0–2.7)

27.8

(16.4–34.3)

28.8

(16.4–34.7)

NHL (total)

92

1.2

(0.0–14.5)

30.3

(14.2–59.9)

32.3

(15.1–68.6)

NHL, non-Hodgkin’s lymphoma; PBMC, peripheral blood mononuclear cells; FL, follicular lymphoma; DLCL, diffuse large-B-cell lymphoma; MCL, mantle cell lymphoma; MZBCL, marginal zone B-cell lymphoma. Leukemia


2210 tion level and p53 labeling index was found. p53 overexpression with more than 50% of nuclei showing a distinct staining was found in 0 of 24 FL (0%), five of 18 DLCL (28%), three of 17 near diploid MCL (18%), and two of nine near tetraploid MCL (22%). The majority of centrosome aberrations were structural rather than numerical in nature. Whereas numerical centrosome aberrations were detectable in a median proportion of 1.2% (range 0.0–14.5%) of the cells in NHL, structural centrosome abnormalities were found in 30.3% (range 14.2–59.9%) of the NHL cells. Essentially the same results were obtained using antibodies against g-tubulin (n ¼ 10) or pericentrin (n ¼ 18) in a randomly chosen subgroup of the samples (data not shown).

Relationship between centrosome aberrations and cytogenetic abnormalities Figure 2 Centrosome aberrations in different subtypes of B-cell NHL. The differences between controls and lymphoma entities, indolent and aggressive lymphomas as well as near diploid and near tetraploid MCL are shown as a box plot and were statistically evaluated with the Mann–Whitney U-test. FL, follicular lymphoma; DLCL, diffuse large-B-cell lymphoma; MCL, mantle cell lymphoma; MZBCL, marginal zone B-cell lymphoma.

As centrosomes play a role in the maintenance of genomic stability through control of mitotic chromosome segregation, we asked whether there is a dependency of the ploidy status on the percentage of cells with abnormal centrosomes. We found that the extent of centrosome aberrations in near tetraploid MCL was significantly greater than in near diploid MCL with median proportions of 55.6% (range 43.5–68.8%) vs 31.2% (range

Figure 3 Correlation between centrosome aberrations and proliferation index/mitotic index in NHL. Centrosome abnormalities correlate positively with the proliferation index (a and b) and mitotic index (c and d) in FL and DLCL (a and c) as well as in near diploid and near tetraploid MCL when analyzed separately (b and d). We computed a generalization of Spearman’s r to also detect nonmonotonic associations between 2 variables. (a) r2 ¼ 0.57, Po0.001; (b) r2 ¼ 0.02, P ¼ 0.89 for near diploid MCL; r2 ¼ 0.30, P ¼ 0.20 for near tetraploid MCL; (c) r2 ¼ 0.41, Po0.001; and (d) r2 ¼ 0.20, P ¼ 0.20 for near diploid MCL; r2 ¼ 0.38, P ¼ 0.12 for near tetraploid MCL. Leukemia


2211 21.8–43.9%) of the cells carrying centrosome aberrations (Po0.0001, Table 1, Figure 2). A significant correlation between abnormal centrosomes and ploidy was also found when only numerical centrosome aberrations were taken into account with a median of 3.6% (range 0.0–14.5%) vs 0.9% (range 0.0–7.5%) cells harboring numerical centrosome aberrations in near tetraploid and near diploid MCL, respectively (P ¼ 0.005). In contrast, the extent of centrosome aberrations was not different between near diploid and near tetraploid samples in FL (25.8%, range 15.1–34.7 vs 22.3%) and DLCL (41.8%, range 35.4– 54.5% vs 40.4%, range 37.3–42.1%), respectively.

Discussion Genetic instability is now widely recognized as an essential factor in the evolution of cancer.9,33,34 In the vast majority of malignancies, this instability appears to involve gains and losses of whole chromosomes or large parts thereof, leading to aneuploidy.9,34,35 The mechanisms regulating the fidelity of mitotic chromosome separation in mammalian cells are therefore of great interest. Centrosome aberrations have recently been implicated in the induction of aneuploidy in different malignancies by malsegregation of chromosomes during anaphase of mitosis.8,13,16–23 As centrosomes play a role in the maintenance of genomic stability through control of mitotic chromosome segregation, we were interested in the analysis of a functional dependency between the occurrence and extent of centrosome aberrations and the ploidy status in NHL. The results presented here demonstrate for the first time that centrosomes are structurally and numerically abnormal in all four B-cell NHL subtypes analyzed. The frequency of cells harboring centrosome aberrations correlates with the histological lymphoma subtype. Aggressive lymphomas (DLCL, MCL) harbor more extensive centrosome abnormalities as compared to indolent lymphomas (FL, MZBCL). In keeping with this finding, a positive correlation between the amount of centrosome aberrations and the proliferation index/mitotic index was found in FL, DLCL, and MCL, clearly implying that lymphomas with a higher proliferation index display significantly more centrosome aberrations irrespective of the histological subgroup. It is interesting to compare these data to the control group, where quiescent PBMC harbored less aberrations than activated tonsillar B cells. Within the group of FL, the number of cells with centrosome aberrations correlates with increased histological grading. For MCL, the frequency of cells harboring centrosome aberrations correlates with ploidy, with near tetraploid MCL having far more extensive centrosome abnormalities as compared to near diploid MCL. Of note, no such correlation was found for FL and DLCL. The association between centrosome abnormalities and clinical aggressiveness of lymphoma subtypes described here is in line with recent reports of expanding centrosome aberrations concomitant with tumor progression in prostate, pancreatic, breast, as well as head and neck cancer.18,21,22,36–38 Also, our finding of increased amounts of centrosome abnormalities in grade 3a FL (reminiscent of the amount of centrosome aberrations found in DLCL) as compared to grade 1/2 FL, corresponds well to recent reports describing that centrosome abnormalities correlate with loss of tissue differentiation in prostate and breast cancer.17,18,36,37 Rb and p53 pathway alterations in NHL with increased growth fraction, higher grade transformation, chromosomal instability, and inferior prognosis might, at least in part, account for this finding.24,26,39–44

The detection of predominantly structural instead of numeric centrosome aberrations in the lymphoma samples analyzed might reflect the clustering of multiple centrosomes into larger aggregates, thereby reducing the number of individual microtubule-organizing centers to a manageable number per cell, possibly capable of establishing a bipolar mitotic spindle. Recent in vitro data from Borel et al,45 who describe clustering of multiple centrosomes at single spindle poles with subsequent mitotic cells forming bipolar spindles in p53 or Rb-deficient cell lines, support this concept. In contrast to carcinomas of breast and prostate, where numerical centrosome aberrations are primarily associated with aneuploidy,36,37 structural centrosome abnormalities in NHL might initially contribute to increased proliferation rather than to abnormal mitoses. Aggressive cancers at many sites frequently have chromosome numbers in the triploid–tetraploid range, with numerous structural rearrangements. Therefore, Shackney et al45 have proposed a conceptual model for the evolution of solid tumors in which doubling of the complete karyotype leads to an unstable state that is followed by the loss of whole chromosomes and structural rearrangements. Tetraploidization with subsequent chromosome loss from tetraploidy has also been described recently in NHL.46 Blastoid subtypes of MCL have a striking tendency to harbor chromosome numbers in the tetraploid range, a feature possibly being related to the inappropriate expression of cyclin D1.26 The centrosome duplication cycle is controlled by the phosphorylation status of the Rb protein, release of the transcription factor E2F from Rb binding, and subsequent activation of cdk 2 in late G1 phase of the cell cycle.5–7 Notably, aside from cyclin D1 and cdk4, the deregulated expression of E2F and cdk2 has been described in MCL.47 Also, cyclin D1 overexpression and p53 as well as p16INK4A deletion are especially associated with blastoid variants of MCL.39–41 The scenario described above would therefore fit with the concept that MCL is a lymphoid tumor in which both primary and secondary genetic events cause not only progressive cell cycle destabilization but also increasing centrosome aberrations, finally leading to mitotic failure and doubling of the whole karyotype.24,26,39–41 High centrosome aberration levels might further explain why tetraploidy in MCL is not stable, but generally progresses to an aneuploid state. Although the number of near tetraploid FL and DLCL cases examined is low, in FL and DLCL no association between centrosome aberrations and ploidy status was found. The specificity of this finding for MCL further corroborates our model, since cell cycle deregulatory events leading to centrosome amplification and the tendency to tetraploidization clearly separates MCL from other types of Bcell NHLs.26 The detailed mechanisms by which centrosome aberrations develop in malignant tumors are still largely unknown. In addition to cdk2 activity, other genes such as the tumor suppressors p5310,48–52 and BRCA1,53 and centrosome-associated kinases like breast tumor-amplified kinase STK15/BTAK/ Aurora A,11,51 have been described to induce centrosome abnormalities. Alternative to centrosome hyper-replication due to deregulated cdk2 activity in the late G1 phase, mitotic failure with concomitant tetraploidization and doubling of centrosome numbers has been proposed to be a cause rather than a consequence of centrosome aberrations, especially in p53deficient malignancies.51,52 Since p53 mutations do occur in blastoid MCL, cell division defects as a source of centrosome abnormalities cannot completely be ruled out. However, our finding of a missing association between the p53 labeling index and centrosome aberration levels in MCL, FL, and DLCL argues Leukemia


2212 against a major role for abrogation of p53 function as a cause of centrosome abnormalities in NHL. An association between the amount of centrosome aberrations and chromosomal instability has recently also been described for some solid tumors as well as for acute myeloid leukemia,16–18,23,36,37 fitting nicely to our observations in NHL. Since secondary chromosome aberrations are believed to be related to lymphoma transformation,24,54 and the number of chromosomal alterations rather than the distribution of single aberration types seems to be an important factor in assessing prognosis in NHL,27,55 increasingly aberrant centrosomes might be causally related to lymphoma progression. In conclusion, our results indicate that centrosome defects are a common feature of NHL and suggest that they may contribute to the aquisition of an increasing chromosomal instability. Since the extent of centrosome abnormalities is associated with histologically and cytogenetically defined NHL subtypes as well as the level of chromosomal instability, the prognostic importance of centrosome aberration patterns should be studied in prospective trials. Gene expression analysis of genes relevant to centrosome structure, function, and replication might further improve our understanding of the causes and consequences of centrosome aberrations in NHL.

Acknowledgements We thank Mrs Brigitte Schreiter, Mrs Christel Kohaut, and Mrs Heike Bru¨ckner for excellent technical assistance as well as an anonymous reviewer for thoughtful comments. This work was supported by the Deutsche Jose´ Carreras Leuka¨mie-Stiftung eV. (DJCLS 2001/NAT-3), the Deutsche Forschungsgemeinschaft (KR 1981/1-1), and the European Commission ‘Growth program, research project molecular and biological risk factors in mantle cell lymphoma’, Contract Number QLG1-CT-2000-00687. AK is a Heisenberg scholar of the Deutsche Forschungsgemeinschaft.

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