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Prepublished online as a Blood First Edition Paper on June 28, 2002; DOI 10.1182/blood-2002-04-1188. This Article Abstract Full Text (PDF) All Versions of this Article: 2002-04-1188v1 101/1/289    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Neben, K. Articles by Krämer, A. Search for Related Content PubMed PubMed Citation Articles by Neben, K. Articles by Krämer, A. Related Collections Neoplasia Brief Reports Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 January 2003, Vol. 101, No. 1, pp. 289-291 NEOPLASIA Brief report Centrosome aberrations in acute myeloid leukemia are correlated with cytogenetic risk profile Kai Neben, Christian Giesecke, Silja Schweizer, Anthony D. Ho, and Alwin Krämer From the Medizinische Klinik und Poliklinik V, Universität Heidelberg, Heidelberg, Germany.     Abstract Top Abstract Introduction Study design Results and discussion References Genetic instability is a common feature in acute myeloid leukemia (AML). Centrosome aberrations have been described as a possible cause of aneuploidy in many human tumors. To investigate whether centrosome aberrations correlate with cytogenetic findings in AML, we examined a set of 51 AML samples by using a centrosome-specific antibody to pericentrin. All 51 AML samples analyzed displayed numerical and structural centrosome aberrations (36.0% ± 16.6%) as compared with peripheral blood mononuclear cells from 21 healthy volunteers (5.2% ± 2.0%; P < .0001). In comparison to AML samples with normal chromosome count, the extent of numerical and structural centrosome aberrations was higher in samples with numerical chromosome changes (50.5% ± 14.2% versus 34.3% ± 12.2%; P < .0001). When the frequency of centrosome aberrations was analyzed within cytogenetically defined risk groups, we found a correlation of the extent of centrosome abnormalities to all 3 risk groups (P = .0015), defined as favorable (22.5% ± 7.3%), intermediate (35.3% ± 13.1%), and adverse (50.3% ± 15.6%). These results indicate that centrosome defects may contribute to the acquisition of chromosome aberrations and thereby to the prognosis in AML. (Blood. 2003;101:289-291) © 2003 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References Genetic instability is a common feature in acute myeloid leukemia (AML). Balanced chromosomal translocations such as t(15;17), t(8;21), and inv16/t(16;16) lead to leukemia-specific fusion transcripts without gain or loss of genetic material, whereas unbalanced chromosome abnormalities result in gains and losses of whole chromosomes or parts thereof.1,2 In AML, both numerical and structural chromosome aberrations have been shown to provide information about the clinical course. In large AML clinical trials, especially numerical chromosome aberrations like losses of chromosomes 5 and 7 as well as complex aberrations were identified as adverse prognostic factors for survival. Defects in chromosome number are thought to occur through missegregation of chromosomes,3 but the mechanism by which this occurs has not been elucidated. There are many potential mitotic targets, which could cause unequal segregation of chromosomes, among those chromosomal, spindle microtubule, and centrosomal defects.4 Centrosome aberrations have been described as a possible cause of numerical chromosome abnormalities in many solid human tumors.5-13 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.4 To investigate whether centrosome aberrations do occur in AML and correlate with cytogenetically defined subgroups, we examined a set of 51 samples with AML by using a centrosome-specific antibody to pericentrin.14     Study design Top Abstract Introduction Study design Results and discussion References Specimen selection We examined a set of 51 patients with AML according to the French-American-British (FAB) classification.15 All patients were diagnosed and treated in our institution between July 2000 and February 2002. Cytogenetic studies were performed after short-term cultures by using standard protocols for G- or R-banding techniques. Karyotype changes were interpreted according to the 1995 International System for Cytogenetic Nomenclature (ISCN) nomenclature.16 Approval was obtained from the institutional review board of the University of Heidelberg for this study. Informed consent was provided according to the Declaration of Helsinki. Centrosome staining For enrichment of mononuclear cells, heparin was added to leukemia samples and centrifuged with Biocoll separating solution (Biochrom, Berlin, Germany). For centrosome immunostaining, cytospins were prepared. The cells were mounted on coated slides and fixed in 20°C acetone for 10 minutes, permeabilized in 0.2% Tween 20 for 5 minutes, blocked in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and 1% human immunoglobulin for 1 hour, followed by standard indirect immunohistochemistry. Briefly, cytospins were stained by using a polyclonal antibody to pericentrin (Convance, Richmond, CA). The slides were washed in PBS 3 times for 5 minutes each. The antibody-antigen complexes were detected by incubation for 1 hour at room temperature with a fluorescein isothiocyanate (FITC)-conjugated secondary goat antimouse immunoglobulin G (IgG) antibody (Convance). The slides were washed in PBS 3 times for 5 minutes each again. Slides were mounted with phosphate-buffered glycerol (Euroimmun, Lübeck, Germany) and visualized under a fluorescence microscope (Axioskop; Zeiss, Jena, Germany) by using a × 100 objective. Calculation of centrosome aberrations 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 at least twice that of centrosomes in nonmalignant control cells and numerically abnormal if they were present in numbers more than 2, as described previously.11 At least 200 consecutive cells per sample were carefully examined. Statistical analysis Differences in the number of cells with centrosome aberrations among cytogenetically defined subgroups were analyzed by the application of the t test for independent samples. All statistical analyses were performed by the statistical software SPSS for Windows, release 6.1.3 (SPSS, Chicago, IL).     Results and discussion Top Abstract Introduction Study design Results and discussion References To investigate whether centrosome aberrations do occur in AML and correlate with cytogenetically defined subgroups, we examined a set of 51 AML samples by using indirect immunofluorescence with an antibody to pericentrin. Our data set consisted of 30 peripheral blood and 21 bone marrow samples obtained from patients with AML at the time of diagnosis (n = 41) or at relapse (n = 10). In particular, there were 27 men and 24 women with a median age of 60 years (range, 18-80 years). A total number of 33 patients were registered as having de novo AML, whereas 18 patients with secondary AML were analyzed. Cytogenetic information was available for 48 of 51 patients. The karyotype classification was similar to that used in other series.1,2 Analogous to the Medical Research CouncilAcute Myeloid Leukemia (MRC AML) 10 trial, all AML patients with a specific abnormality were considered, irrespective of the presence of additional or secondary cytogenetic changes.1 To determine whether blasts from patients with AML harbor abnormal centrosomes, we analyzed 51 patients with AML and compared the centrosome patterns with peripheral blood mononuclear cells from 21 healthy volunteers, including 8 men and 13 women with a median age of 24 years (range, 22-58 years). All 51 AML samples analyzed displayed numerical and structural centrosome aberrations as compared with the control samples (Figure 1). Centrosome abnormalities were detectable in 36.0% ± 16.6% of the blasts in AML but in only 5.2% ± 2.0% of the controls (P < .0001). These findings indicate that centrosome defects are a common feature of AML. Consistently, centrosome aberrations have been previously reported in solid tumors of different origin, including brain, breast, lung, colon, prostate, pancreas, bile duct, and head and neck.5-11 Hematologic malignancies like non-Hodgkin lymphomas and myelodysplastic syndromes also display centrosome aberrations at high frequencies.12-13 View larger version (51K): [in this window] [in a new window]   Figure 1. Centrosome aberrations in acute myeloid leukemia. Indirect immunofluorescence staining of normal bone marrow (A) and acute myeloid leukemia cells (B-C). Cells were immunostained with an antibody to pericentrin, followed by a FITC-conjugated secondary antibody. Original magnification × 1000. To test for the hypothesis that centrosome abnormalities are associated with chromosome aberrations in AML, we compared the centrosome aberration patterns of 48 AML patients with available karyotype information. In comparison to 34 AML patients with normal chromosome count, the extent of numerical and structural centrosome aberrations was higher in 14 AML patients with numerical chromosome changes (50.5% ± 14.2% versus 34.3% ± 12.2%; P < .0001). In line with this finding, studies have provided evidence that centrosome aberrations result in chromosome missegregation and may lead to malignant transformation.17-19 Specifically, centrosome hyperamplification, induced by p53 mutations or Mdm2 overexpression, has been shown to induce aneuploidy.17 In another study, overexpression of tumor-amplified kinase STK15/BTAK induced centrosome amplification, aneuploidy, and malignant transformation.18 In addition, 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 (cdk) 2 in late G1 phase of the cell cycle.20-22 Consequently, the commonly observed abrogation of the p53 and Rb pathways in human malignancies, including AML, will not only facilitate progression toward DNA replication but may also deregulate the centrosome duplication cycle.4,23,24 Because karyotype changes provide prognostic information in AML,1 we correlated centrosome abnormalities to cytogenetically defined risk groups according to the MRC AML 10 trial as shown in Table 1. We found a statistically significant correlation of the extent of centrosome abnormalities to all 3 risk groups (P = .0015), defined as favorable (22.5% ± 7.3%), intermediate (35.3% ± 13.1%), and adverse (50.3% ± 15.6%). This difference was mainly due to structural rather than numerical centrosome aberrations, fitting nicely to the ultrastructural observation that in human breast cancers anaplastic morphology and abnormal mitoses correlate to excess pericentriolar material rather than to an increase in centriole or centrosome numbers.25 The description of p53 and Rb pathway alterations in AML patients with an inferior prognosis and unfavorable cytogenetics further suggests a pathophysiologic link to the induction of centrosome aberrations.23,24                                View this table: [in this window] [in a new window]   Table 1. Centrosome aberrations in acute myeloid leukemia are correlated to cytogenetic risk profile In conclusion, our results indicate that centrosome defects are a common feature of AML and suggest that they may contribute to the acquisition of an increasing karyotypic instability. Because the extent of centrosome abnormalities correlates to cytogenetically defined risk groups in AML, the prognostic importance of centrosome patterns should be studied in prospective trials.     Acknowledgments We thank Mrs Brigitte Schreiter for excellent technical assistance.     Footnotes Submitted April 19, 2002; accepted June 6, 2002. Prepublished online as Blood First Edition Paper, June 28, 2002; DOI 10.1182/blood-2002-04-1188. Supported by the Deutsche José Carreras Leukämie-Stiftung e.V. (DJCLS 2001/NAT-3). The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Alwin Krämer, Institute of Cancer Biology, Department of Cell Cycle and Cancer, Danish Cancer Society, Strandboulevarden 49, 2100 Copenhagen, Denmark; e-mail: ajk{at}cancer.dk.     References Top Abstract Introduction Study design Results and discussion References 1. Grimwade D, Walker H, Oliver F, et al. The importance of diagnostic cytogenetics on outcome in AML: analysis of 1612 patients entered into the MRC AML 10 trial. Blood. 1998;92:2322-2333[Abstract/Free Full Text]. 2. Bloomfield CD, Lawrence D, Byrd JC, et al. Frequency of prolonged remission duration after high-dose cytarabine intensification in acute myeloid leukemia varies by cytogenetic subtype. Cancer Res. 1998;58:4173-4179[Abstract]. 3. Nicklas RB. How cells get the right chromosomes. Science. 1997;275:632-637[Abstract/Free Full Text]. 4. Krämer A, Neben K, Ho AD. Centrosome replication, genomic instability and cancer. Leukemia. 2002;16:767-775[CrossRef][Medline] [Order article via Infotrieve]. 5. Lingle WL, Lutz WH, Ingle JN, Maihle NJ, Salisbury JL. Centrosome hypertrophy in human breast tumors: implications for genomic stability and cell polarity. Proc Natl Acad Sci U S A. 1998;95:2950-2955[Abstract/Free Full Text]. 6. Pihan GA, Purohit A, Wallace J, Malhotra R, Liotta L, Doxsey SJ. Centrosome defects can account for cellular and genetic changes that characterize prostate cancer progression. Cancer Res. 2001;61:2212-2219[Abstract/Free Full Text]. 7. Weber RG, Bridger JM, Benner A, et al. Centrosome amplification as a possible mechanism for numerical chromosome aberrations in cerebral primitive neuroectodermal tumors with TP53 mutations. Cytogenet Cell Genet. 1998;83:266-269[CrossRef][Medline] [Order article via Infotrieve]. 8. Sato N, Mizumoto K, Nakamura M, et al. Centrosome abnormalities in pancreatic ductal carcinoma. Clin Cancer Res. 1999;5:963-970[Abstract/Free Full Text]. 9. Kuo K-K, Sato N, Mizumoto K, et al. Centrosome abnormalities in human carcinomas of the gallbladder and intrahepatic and extrahepatic bile ducts. Hepatology. 2000;31:59-64[Medline] [Order article via Infotrieve]. 10. Gustafson LM, Gleich LL, Fukasawa K, et al. Centrosome hyperamplification in head and neck squamous cell carcinoma: a potential phenotypic marker of tumor aggressiveness. Laryngoscope. 2000;110:1798-1801[Medline] [Order article via Infotrieve]. 11. Pihan GA, Purohit A, Wallace J, et al. Centrosome defects and genetic instability in malignant tumors. Cancer Res. 1998;58:3974-3985[Abstract]. 12. Kearns WG, Sutton JF, Young NS, Liu JM. Centrosome amplification and chromosomal instability in patients with bone marrow failure syndromes [abstract]. Blood. 2001;98:913a. 13. Krämer A, Schweizer S, Neben K, et al. Centrosome aberrations as a possible mechanism for tetraploidization of blastoid mantle cell lymphoma variants [abstract]. Blood. 2001;98:332a. 14. Doxsey SJ, Stein P, Evans L, Calarco PD, Kirschner M. Pericentrin, a highly conserved centrosome protein involved in microtubule organization. Cell. 1994;76:639-650[Medline] [Order article via Infotrieve]. 15. Bennett JM, Catovsky D, Daniel MT, et al. Proposed revised criteria for the classification of acute myeloid leukemia. A report of the French-American-British Cooperative Group. Ann Intern Med. 1985;103:620-625[Medline] [Order article via Infotrieve]. 16. Mitelman F, ed. An International System for Human Cytogenetic Nomenclature. Basel, Switzerland: Karger; 1995. 17. Carroll PE, Okuda M, Horn HF, et al. Centrosome hyperamplification in human cancer: chromosome instability induced by p53 mutation and/or Mdm2 overexpression. Oncogene. 1999;18:1935-1944[CrossRef][Medline] [Order article via Infotrieve]. 18. Zhou H, Kuang J, Zhong L, et al. Tumor amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation. Nat Genet. 1998;20:189-193[CrossRef][Medline] [Order article via Infotrieve]. 19. Marx J. Do centrosome abnormalities lead to cancer? Science. 2001;292:426-429[Free Full Text]. 20. Meraldi P, Lukas J, Fry AM, Bartek J, Nigg EA. Centrosome duplication in mammalian somatic cells requires E2F and Cdk2-cyclin A. Nature Cell Biol. 1999;1:88-93[CrossRef][Medline] [Order article via Infotrieve]. 21. Hinchcliffe EH, Li C, Thompson EA, Maller JL, Sluder G. Requirement of Cdk2-cyclin E activity for repeated centrosome reproduction in Xenopus egg extracts. Science. 1999;283:851-854[Abstract/Free Full Text]. 22. Lacey KR, Jackson PK, Stearns T. Cyclin-dependent kinase control of centrosome duplication. Proc Natl Acad Sci U S A. 1999;96:2817-2822[Abstract/Free Full Text]. 23. Stirewalt DL, Kopecky KJ, Meshinchi S, et al. FLT3, RAS, and TP53 mutations in elderly patients with acute myeloid leukemia. Blood. 2001;97:3589-3595[Abstract/Free Full Text]. 24. Faderl S, Kantarjian HM, Estey E, et al. The prognostic significance of p16INK4a/p14ARF locus deletion and MDM-2 protein expression in adult acute myelogenous leukemia. Cancer. 2000;89:1976-1982[CrossRef][Medline] [Order article via Infotrieve]. 25. Lingle WL, Salisbury JL. Altered centrosome structure is associated with abnormal mitoses in human breast tumors. Am J Pathol. 1999;155:1941-1951[Abstract/Free Full Text]. © 2003 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: B. Rebacz, T. O. Larsen, M. H. Clausen, M. H. Ronnest, H. Loffler, A. D. Ho, and A. Kramer Identification of Griseofulvin as an Inhibitor of Centrosomal Clustering in a Phenotype-Based Screen Cancer Res., July 1, 2007; 67(13): 6342 - 6350. 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[Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) All Versions of this Article: 2002-04-1188v1 101/1/289    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Neben, K. Articles by Krämer, A. Search for Related Content PubMed PubMed Citation Articles by Neben, K. Articles by Krämer, A. Related Collections Neoplasia Brief Reports Social Bookmarking                   What's this?

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