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Tetraploidy and 5q deletion in myelodysplastic syndrome: A case report
Cancer Genetics and Cytogenetics 183 (2008) 64e68
Short communication
Tetraploidy and 5q deletion in myelodysplastic syndrome: A case report Iya Znoykoa, Robert K. Stuartb, Tara Ellinghama, Jennifer Wintersc, Daynna J. Wolffa, Denise I. Quigleya,* a
Department of Pathology and Laboratory Medicine, bDepartment of Hematology and Oncology, Medical University of South Carolina, 165 Ashley Ave, Ste. 309, Charleston, SC 29425 c Pathology Sciences Medical Group, Sentara Laboratory Services, Norfolk, VA 23510 Received 2 January 2008; accepted 31 January 2008
Abstract
Tetraploidy is a very rare cytogenetic abnormality in myelocytic malignancies, and its significance is unclear to date. We report here on a 68-year-old male diagnosed with myelodysplastic syndrome/ refractory anemia with excess blasts (MDS/RAEB). Cytogenetic analysis of his bone marrow biopsy at initial clinical presentation and in subsequent studies revealed the presence of two abnormal clones, 92,XXYYand 92,XXYY,del(5)(q13q33). Interphase fluorescence in situ hybridization analysis of abnormal cells confirmed interstitial deletion in 5q, demonstrated predominance of the tetraploid clone and persistent presence of the tetraploid clone with 5q deletion. The patient was not responsive to Revlimid (lenalidomide) treatment, which is routinely used in patients with 5qe syndrome. However, a subsequent course of therapy with the methyl-transferase inhibitor decitabine resulted in clinical and cytogenetic remission. Our data suggest that the unique complex abnormality of tetraploidy and 5q deletion described here for the first time in MDS is characterized by distinct disease etiology, the mechanism of which could involve epigenetic inactivation of gene expression via methylation. Ó2008 Elsevier Inc. All rights reserved. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Myelodysplastic syndromes (MDS) are a group of hematopoietic stem cell disorders characterized by ineffective hematopoiesis and peripheral blood cytopenias [1]. They are categorized into high-risk (O10% marrow blasts) and low-risk (!10%) groups [1]. High-risk MDS bears considerable resemblance to acute myeloid leukemia (AML) and is more likely to evolve into AML [1]. Approximately 50e60% of patients with de novo MDS and more than 85% of individuals with secondary MDS (after cytotoxic therapy) show nonrandom chromosomal aberrations that may involve isolated or multiple abnormalities [2]. Therefore, analysis of recurrent cytogenetic abnormalities in MDS is widely used for diagnosis and for determining prognosis and management. Deletion of the long arm of chromosome 5 [del(5q)] is the most common chromosomal abnormality in MDS, occurring at a frequency of 10e15% [3,4]. Del(5q) also occurs in AML [4] and several other cancers [5e7]. While the deleted * Corresponding author. Tel.: 843-792-1181; fax: 843-792-1248. E-mail address: [email protected] (D.I. Quigley). 0165-4608/08/$ e see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2008.01.022
region of chromosome 5 can vary greatly in size between individual patients, two regions have been identified that correspond to subtypes of MDS. A 4-megabase interval within 5q31 has been defined for patients with more aggressive MDS, including secondary MDS, while a more telomeric region is deleted in the patients with del(5q) syndrome[8]. The commonly deleted region (CDR) or critical region has been defined as 5q31~q33 [8,9] and contains multiple genes involved in cellular growth, hematopoiesis, cell cycle control, cell adhesion, and tumor suppression [2,10,11]. Massive hyperdiploidy (O50 chromosomes) and tetraploidy (4n) are rare cytogenetic abnormalities in myelocytic malignancies. A handful of MDS cases with massive hyperdiploidy have been published to date [12e18], whereas there is only one report, to the best of our knowledge, of two MDS patients with tetraploidy [15]. The combination of tetraploidy and del(5q) has not been reported previously in MDS. Hyperdiploidy has been associated with poor prognosis in MDS [12,14e16], but in light of the small numbers of reported cases, its significance is unclear. In this report, we describe the clinical, cytogenetic, and molecular cytogenetic findings in a case of high-risk MDS with the unique finding of tetraploidy and 5q deletion.
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65
2. Materials and methods 2.1. Case report The patient was a 68-year-old generally healthy man referred by his local primary care physician due to decreased hemoglobin and white cell count. Analysis of his blood and bone marrow aspirate demonstrated macrocytic anemia, severe neutropenia, megaloblastic changes in the red cell line, and occasional uninuclear morphology of megakaryocytes. Excess of blasts evaluated by differential cell count and flow cytometry with myeloid markers was 13 and 19%, respectively. Though the patient demonstrated clinical features of the 5qe syndrome (macrocytic anemia, normal to elevated platelet count, and neutropenia), he did not respond to treatment with Revlimid (lenalidomide), which was administered daily for 12 weeks. Moreover, during this period, his platelet count decreased from 350,000 to 118,000, and the patient developed AML, with a repeat bone marrow examination showing 35% myeloblasts marking for myelomonocytic lineage. However, subsequent treatment with two cycles of decitabine therapy resulted in marked improvement in the patient’s condition, with resolution of severe neutropenia and decrease in blast count to 2 and 3% (estimated by differential cell count and flow cytometry, respectively). Finally, the patient achieved clinical and cytogenetic remission. 2.2. Cytogenetics and fluorescence in situ hybridization (FISH) analysis Bone marrow was cultured in RPMI 1640 media (Invitrogen, Carlsbad, CA) and Chang BMC media (Irvine Scientific, Santa Ana, CA), harvested, and slides were prepared according to standard laboratory methods. Metaphase cells were imaged and karyotypes were generated using the Cytovision System version 3.6 (Applied Imaging, Santa Clara, CA). Cytogenetic abnormalities were described according to the International System of Human Cytogenetic Nomenclature (ISCN) 2005 [19]. FISH analysis for del(5q) was performed using probes EGR1 (5q31), CSF1R (5q33~q34), and control probes D5S23 and D5S721 (5p15.2) on slides prepared from fixed cell pellets according to the manufacturer’s recommended procedure (Abbott Molecular/Vysis, Des Plains, IL). 3. Results Cytogenetic analysis of the bone marrow aspirate revealed the presence of cells with normal karyotypes as well as two abnormal cell lines: a tetraploid clone and a clone with tetraploidy and del(5q) (Fig. 1; Table 1). It is noteworthy that the abnormal mitotic cell population at presentation and subsequent cytogenetic tests at 1 and 3 months were dominated by the tetraploid cell line with 5q deletion. However, at the 5-month follow-up (when AML was diagnosed), while the tetraploid/del (5q) clone was persistent, the majority of
Figure 1. Representative chromosomes 5 showing tetraploidy and deletion of one 5q (arrow).
abnormal dividing cells were represented by the tetraploid clone without 5q deletion (Table 1). Later, the patient was started on decitabine therapy, and by the next cytogenetic evaluation at 10 months, no abnormal cells were observed by routine cytogenetic analysis; the patient had reached cytogenetic remission (Table 1). To detect the presence of residual disease, we performed interphase FISH. We used two probes specific for the 5q CDR, namely CSF1R and EGR1 (see Materials and methods). Both probes have shown similar results (data not shown) and proved to be equally suitable for the identification of the 5q deletion in our case. According to the FISH results, tumor burden at 1 month was 12.4%, and it increased twofold by the fifth month (Table 2), when the patient evolved to AML (see Materials and methods). However, after decitabine treatment, at the 10-month follow-up, the number of abnormal cells had fallen to 3.5%. This value correlates well with the number of blasts measured by differential cell count and flow cytometry (2 and 3%, respectively, see Materials and methods). Interestingly, during the whole observation period, despite of the overall number of abnormal cells, the ratio between the two clones (tetraploid and tetraploid with 5q deletion) remained the same, 2:1 (Table 2). 4. Discussion Chromosomal abnormalities in neoplastic marrow cells often correlate closely with specific clinical and biologic characteristics of the disease and serve as a tool to predict the clinical outcome and develop effective therapeutic approaches. In this paper, we describe the successful treatment of a myelodysplastic syndrome-refractory anemia with excess blasts patient with the unique finding of tetraploidy and 5q deletion. Tetraploidy is a very rare abnormality in hematologic malignancies, especially in MDS. In fact, we are aware Table 1 Results of routine cytogenetic studies Date
46,XY
92,XXYY
92,XXYY,del(5)(q13q33)
At presentation 1 month 3 months 5 months 10 months
18 5 17 15 20
e e e 4 e
2 2 3 1 e
I. Znoyko et al. / Cancer Genetics and Cytogenetics 183 (2008) 64e68
66 Table 2 Result from FISH studies Representation of cells
Date
Distribution of abnormal cells
No. normal No. abnormal cells cells Tetraploid (%) (%) (%)
1 month 199 (87.6) 5 months 319 (74.0) 10 months 248 (96.5)
33 (12.4) 112 (26.0) 9 (3.5)
23 (69.7) 143a (66.2) 6 (67.7)
Tetraploid with deletion 5q (%) 10 (30.3) 73 a (33.8) 3 (33.3)
a Additional abnormal cells were scored, so the number of abnormal cells represented here is greater than that indicated in the ‘‘No. abnormal cell’’ column.
of only one publication concerning tetraploidy in MDS patients [15]. A large-scale study performed in Japan involving 979 patients with different types of hematologic malignancies revealed two cases of tetraploidy in MDS patients [15]. Little more information is available about hyperdiploidy (O50 chromosomes) in MDS [12e18]. In the majority of these reports, hyperdiploidy is associated with poor outcome [12,15,16,20], and increase in the percentage of hyperdiploid cells paralleled the increase in percentage of blasts in the bone marrow [13]. The 5q deletion is also associated with poor prognosis in high-risk MDS patients [10]. Therefore, the combination of tetraploidy and 5q deletion would suggest an unfavorable prognosis for our high-risk MDS patient. Fortunately, due to an optimized treatment course, our patient has achieved clinical and cytogenetic remission. Cytogenetic and FISH analysis of the bone marrow biopsy from the patient reported here has revealed two abnormal cell lines: tetraploid clone and tetraploid clone with 5q deletion. Since only one chromosome 5 shows deletion of the q arm, we conclude that development of tetraploidy preceded 5q deletion in clonal evolution. Furthermore, although the tetraploid clone without deletion was the predominant clone by FISH (Table 2), the mitotic cell population (analyzed cytogenetically) at initial clinical presentation, and then at 1- and 3-month follow-ups, was dominated by tetraploid cells with del(5q) (Table 1). These findings suggest that the clone bearing both abnormalities has a higher mitotic rate and is therefore better represented during routine chromosome analysis. However, more frequent cell divisions of the evolved clone do not affect its overall share among abnormal cells (Table 2), possibly due to its increased apoptotic index. Since the patient demonstrated clinical features of the 5qe syndrome (macrocytic anemia, normal to elevated platelet count, and neutropenia), he was initially treated with the immunomodulatory drug lenalidomide (Revlimid), which is particularly effective in patients with 5qe syndrome [21] and was recently approved by US Food and Drug Administration for MDS treatment [1]. There are not enough data on the therapeutic effect of lenalidomide in patients with high-risk MDS [21e23], but it is the drug of choice for
treatment of low-risk MDS with del(5q), where it has a dramatic therapeutic effect [21,22]. Lenalidomide was developed by modification of the first-generation immunomodulatory drug thalidomide in a drug discovery program. The initial phase I/II clinical study showed that treatment with lenalidomide abrogated red cell transfusion need in 83% of low-risk MDS patients with del(5q) and that cytogenetic remissions were frequently observed [22]. A large phase II follow-up study of 148 patients recently confirmed these promising results, showing erythroid responses and transfusion independency within a median of 4.6 weeks in 76 and 67% of the patients, respectively, and complete cytogenetic remission in 45% of the patients [21]. The molecular basis of this remarkable drug response is unknown, but according to the recent study, it could result from the ability of lenalidomide to inhibit growth of del(5q) erythroid progenitors and up-regulation of the potential tumor-suppressor genes SPARC and Activin A [2]. Intriguingly, SPARC and RPS14, a component of 40s ribosomal subunit, are among the genes showing haploinsufficiency in 5qe syndrome [24,25] and could represent target genes for lenamidolidamide treatment [2]. Our patient was not responsive to treatment with lenamidolide, administered daily for 12 weeks, though in a clinical trial the median time of the response was 4.6 weeks and 90% patients responded within 12 weeks [26]. In one study, lenalidomide worked equally well for patients with isolated versus complex karyotypes as long as del(5q) was present [21]. There are several possible reasons for the nonresponsiveness of our patient to lenamidolide treatment. First, clinical trials were performed in low-risk MDS patients in which the del(5q) is associated with a favorable outcome [10,11]. In high-risk MDS (as in our case), however, a del(5q) is associated with a poor prognosis [10]. Second, though the mechanism of lenamidolide effect in 5q syndrome is not determined, one of the published theories implies that haploinsufficiency for 5q is critical [2]. If that is the case, the presence of three copies of a normal chromosome 5 in the tetraploid clone of our patient could provide for a compensatory effect. Finally and most importantly, according to the accepted point of view, the cell of origin in 5qe syndrome is a pluripotent hematopoietic stem cell and 5q deletions represent an early event in MDS pathogenesis [27,28]. In our case, however, development of tetraploidy apparently preceded 5q deletion in clonal evolution, thus suggesting a different disease etiology than 5qe syndrome. Since the patient did not respond to lenamidolide treatment and his disease had progressed to AML, subsequent treatment with the hypomethylating agent decitabine was initiated. Decitabine has been shown to be effective and well tolerated for the treatment of high-risk MDS, for which treatment options were previously scarce [29]. In clinical trials, decitabine treatment reduced red blood cell transfusion requirement, improved quality of life, and produced statistically significant delays in time to AML and death [29]. However, the drug produced the rather low
I. Znoyko et al. / Cancer Genetics and Cytogenetics 183 (2008) 64e68
clinical remission (CR) rate of 9% [29]. Fortunately, our patient has shown remarkable response to treatment with decitabine and eventually achieved clinical and cytogenetic remission. The effect of decitabine, an S-phaseespecific inhibitor of DNA methyltransferase, relies on its ability to decrease levels of methylation, which is responsible for abnormal silencing of many genes, including tumor suppressor genes in neoplasia. Therefore, a possible mechanism of disease underlying the observed cytogenetic abnormalities, tetraploidy and 5q deletion, may involve epigenetic inactivation of gene expression via methylation. This finding is of particular interest in light of the recently published data involving the model of a-catenin therapy [11]. The study demonstrated that silencing of key tumor-suppressor genes within the 5q CDR results from the loss of one of the alleles via deletion and epigenetic inactivation of the remaining allele. In vitro experiments indicate that inactivation of the remaining allele is caused by promoter methylation and histone deacetylation and can be reversed by using inhibitors of methyltranferase and histone deacetylase [11]. These data suggest that combinational treatment with methyltransferase inhibitors (such as decitabine or azacitidine) and histone deacetylase inhibitors (such as valproic or suberoylanilide hydroxamic acid), which are being studied in clinical trials now [1,29], might specifically benefit the subgroup of individuals with del(5q) who are not responsive to treatment with immunomodulatory drugs.
References [1] Estey E. Acute myeloid leukemia and myelodysplastic syndromes in older patients. J Clin Oncol 2007;25:1908e15. [2] Pellagatti A, Jadersten M, Forsblom AM, Cattan H, Christensson B, Emanuelsson EK, Merup M, Nilsson L, Samuelsson J, Sander B, Wainscoat JS, Boultwood J, Hellstrom-Lindberg E. Lenalidomide inhibits the malignant clone and up-regulates the SPARC gene mapping to the commonly deleted region in 5q- syndrome patients. Proc Natl Acad Sci USA 2007;104:11406e11. [3] Sole F, Espinet B, Sanz GF, Cervera J, Calasanz MJ, Luno E, Prieto F, Granada I, Hernandez JM, Cigudosa JC, Diez JL, Bureo E, Marques ML, Arranz E, Rios R, Martinez Climent JA, Vallespi T, Florensa L, Woessner S. Incidence, characterization and prognostic significance of chromosomal abnormalities in 640 patients with primary myelodysplastic syndromes. Grupo Cooperativo Espan˜ol de Citogenetica Hematologica. Br J Haematol 2000;108: 346e56. [4] Mauritzson N, Albin M, Rylander L, Billstrom R, Ahlgren T, Mikoczy Z, Bjork J, Stromberg U, Nilsson PG, Mitelman F, Hagmar L, Johansson B. Pooled analysis of clinical and cytogenetic features in treatment-related and de novo adult acute myeloid leukemia and myelodysplastic syndromes based on a consecutive series of 761 patients analyzed 1976e1993 and on 5098 unselected cases reported in the literature 1974e2001. Leukemia 2002;16:2366e78. [5] Miura I, Graziano SL, Cheng JQ, Doyle LA, Testa JR. Chromosome alterations in human small cell lung cancer: frequent involvement of 5q. Cancer Res 1992;52:1322e8. [6] Hahn SA, Seymour AB, Hoque AT, Schutte M, da Costa LT, Redston MS, Caldas C, Weinstein CL, Fischer A, Yeo CJ, et al. Allelotype of pancreatic adenocarcinoma using xenograft enrichment. Cancer Res 1995;55:4670e5.
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[7] Verma RS, Manikal M, Conte RA, Godec CJ. Chromosomal basis of adenocarcinoma of the prostate. Cancer Invest 1999;17:441e7. [8] Jaju RJ, Boultwood J, Oliver FJ, Kostrzewa M, Fidler C, Parker N, McPherson JD, Morris SW, Muller U, Wainscoat JS, Kearney L. Molecular cytogenetic delineation of the critical deleted region in the 5q- syndrome. Genes Chromosomes Cancer 1998;22:251e6. [9] Boultwood J, Fidler C, Strickson AJ, Watkins F, Gama S, Kearney L, Tosi S, Kasprzyk A, Cheng JF, Jaju RJ, Wainscoat JS. Narrowing and genomic annotation of the commonly deleted region of the 5qsyndrome. Blood 2002;99:4638e41. [10] Giagounidis AA, Germing U, Aul C. Biological and prognostic significance of chromosome 5q deletions in myeloid malignancies. Clin Cancer Res 2006;12:5e10. [11] Liu TX, Becker MW, Jelinek J, Wu WS, Deng M, Mikhalkevich N, Hsu K, Bloomfield CD, Stone RM, DeAngelo DJ, Galinsky IA, Issa JP, Clarke MF, Look AT. Chromosome 5q deletion and epigenetic suppression of the gene encoding alpha-catenin (CTNNA1) in myeloid cell transformation. Nature Med 2007;13:78e83. [12] Iyer RV, Sait SN, Matsui S, Block AW, Barcos M, Slack JL, Wetzler M, Baer MR. Massive hyperdiploidy and tetraploidy in acute myelocytic leukemia and myelodysplastic syndrome. Cancer Genet Cytogenet 2004;148:29e34. [13] Borgstrom GH, Vuopio P, De la Chapelle A. Polyploidy of the bone marrow. Scand J Haematol 1976;17:123e31. [14] Manley R, Cochrane J, Patton WN. Polyploidy in myelodysplastic syndrome: a case report. Cancer Genet Cytogenet 1998;106: 170e2. [15] Watanabe A, Inokuchi K, Yamaguchi H, Mizuki T, Tanosaki S, Shimada T, Dan K. Near-triploidy and near-tetraploidy in hematological malignancies and mutation of the p53 gene. Clin Lab Haematol 2004;26:25e30. [16] Tanaka M, Takeuchi H, Kaku K, Oka Y. Myelodysplastic syndrome associated with hypotriploidy. Acta Haematol 1996;95: 148e50. [17] Acar H, Caliskan UU, Kaynak M, Yildirim MS, Largaespada DA. Hyperdiploid karyotype in a childhood MDS patient. Clin Lab Haematol 2001;23:255e8. [18] Wong WY, Wu SQ. Pentasomy 8 in pediatric myelodysplastic syndrome. Cancer Genet Cytogenet 2000;121:220e2. [19] Shaffer LG, Tommerup N. An International System for Human Cytogenetic Nomenclature. Basel: S. Karger, 2005. [20] Stamatoullas A, Callat MP, Marreiros S, Tilly H, Bastard C. Unusual complex hyperdiploid karyotypes in myelodysplastic syndromes. Cancer Genet Cytogenet 2006;170:129e32. [21] List A, Dewald G, Bennett J, Giagounidis A, Raza A, Feldman E, Powell B, Greenberg P, Thomas D, Stone R, Reeder C, Wride K, Patin J, Schmidt M, Zeldis J, Knight R. Lenalidomide in the myelodysplastic syndrome with chromosome 5q deletion. N Engl J Med 2006;355:1456e65. [22] List A, Kurtin S, Roe DJ, Buresh A, Mahadevan D, Fuchs D, Rimsza L, Heaton R, Knight R, Zeldis JB. Efficacy of lenalidomide in myelodysplastic syndromes. N Engl J Med 2005;352: 549e57. [23] Raza A, Reeves JA, Feldman EJ, Dewald GW, Bennett JM, Deeg HJ, Dreisbach L, Schiffer CA, Stone RM, Greenberg PL, Curtin PT, Klimek VM, Shammo JM, Thomas D, Knight RD, Schmidt M, Wride K, Zeldis JB, List AF. Phase II study of lenalidomide in transfusion-dependent, low- and intermediate-1-risk myelodysplastic syndromes with karyotypes other than deletion 5q. Blood 2007. [24] Boultwood J, Pellagatti A, Cattan H, Lawrie CH, Giagounidis A, Malcovati L, Della Porta MG, Jadersten M, Killick S, Fidler C, Cazzola M, Hellstrom-Lindberg E, Wainscoat JS. Gene expression profiling of CD34þ cells in patients with the 5q- syndrome. Br J Haematol 2007;139:578e89. [25] Ebert BL, Pretz JL, Bosco J, Chang CY, Tamayo P, Galili N, Raza A, Root D, Attar E, Ellis SR, Golub TR. Identification of RPS14 as the
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5q- syndrome gene by RNA interference screen. Blood 2007; 110(11):1. [26] Galili N, Cerny J, Raza A. Current treatment options: impact of cytogenetics on the course of myelodysplasia. Curr Treatmt Opt Oncol 2007;8:117e28. [27] Jaju RJ, Jones M, Boultwood J, Kelly S, Mason DY, Wainscoat JS, Kearney L. Combined immunophenotyping and FISH identifies the involvement of B-cells in 5q- syndrome. Genes Chromosomes Cancer 2000;29:276e80.
[28] Nilsson L, Astrand-Grundstrom I, Arvidsson I, Jacobsson B, Hellstrom-Lindberg E, Hast R, Jacobsen SE. Isolation and characterization of hematopoietic progenitor/stem cells in 5q-deleted myelodysplastic syndromes: evidence for involvement at the hematopoietic stem cell level. Blood 2000;96:2012e21. [29] Plimack ER, Kantarjian HM, Issa JP. Decitabine and its role in the treatment of hematopoietic malignancies. Leukemia Lymphoma 2007;48:1472e81.
Short communication
Tetraploidy and 5q deletion in myelodysplastic syndrome: A case report Iya Znoykoa, Robert K. Stuartb, Tara Ellinghama, Jennifer Wintersc, Daynna J. Wolffa, Denise I. Quigleya,* a
Department of Pathology and Laboratory Medicine, bDepartment of Hematology and Oncology, Medical University of South Carolina, 165 Ashley Ave, Ste. 309, Charleston, SC 29425 c Pathology Sciences Medical Group, Sentara Laboratory Services, Norfolk, VA 23510 Received 2 January 2008; accepted 31 January 2008
Abstract
Tetraploidy is a very rare cytogenetic abnormality in myelocytic malignancies, and its significance is unclear to date. We report here on a 68-year-old male diagnosed with myelodysplastic syndrome/ refractory anemia with excess blasts (MDS/RAEB). Cytogenetic analysis of his bone marrow biopsy at initial clinical presentation and in subsequent studies revealed the presence of two abnormal clones, 92,XXYYand 92,XXYY,del(5)(q13q33). Interphase fluorescence in situ hybridization analysis of abnormal cells confirmed interstitial deletion in 5q, demonstrated predominance of the tetraploid clone and persistent presence of the tetraploid clone with 5q deletion. The patient was not responsive to Revlimid (lenalidomide) treatment, which is routinely used in patients with 5qe syndrome. However, a subsequent course of therapy with the methyl-transferase inhibitor decitabine resulted in clinical and cytogenetic remission. Our data suggest that the unique complex abnormality of tetraploidy and 5q deletion described here for the first time in MDS is characterized by distinct disease etiology, the mechanism of which could involve epigenetic inactivation of gene expression via methylation. Ó2008 Elsevier Inc. All rights reserved. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Myelodysplastic syndromes (MDS) are a group of hematopoietic stem cell disorders characterized by ineffective hematopoiesis and peripheral blood cytopenias [1]. They are categorized into high-risk (O10% marrow blasts) and low-risk (!10%) groups [1]. High-risk MDS bears considerable resemblance to acute myeloid leukemia (AML) and is more likely to evolve into AML [1]. Approximately 50e60% of patients with de novo MDS and more than 85% of individuals with secondary MDS (after cytotoxic therapy) show nonrandom chromosomal aberrations that may involve isolated or multiple abnormalities [2]. Therefore, analysis of recurrent cytogenetic abnormalities in MDS is widely used for diagnosis and for determining prognosis and management. Deletion of the long arm of chromosome 5 [del(5q)] is the most common chromosomal abnormality in MDS, occurring at a frequency of 10e15% [3,4]. Del(5q) also occurs in AML [4] and several other cancers [5e7]. While the deleted * Corresponding author. Tel.: 843-792-1181; fax: 843-792-1248. E-mail address: [email protected] (D.I. Quigley). 0165-4608/08/$ e see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2008.01.022
region of chromosome 5 can vary greatly in size between individual patients, two regions have been identified that correspond to subtypes of MDS. A 4-megabase interval within 5q31 has been defined for patients with more aggressive MDS, including secondary MDS, while a more telomeric region is deleted in the patients with del(5q) syndrome[8]. The commonly deleted region (CDR) or critical region has been defined as 5q31~q33 [8,9] and contains multiple genes involved in cellular growth, hematopoiesis, cell cycle control, cell adhesion, and tumor suppression [2,10,11]. Massive hyperdiploidy (O50 chromosomes) and tetraploidy (4n) are rare cytogenetic abnormalities in myelocytic malignancies. A handful of MDS cases with massive hyperdiploidy have been published to date [12e18], whereas there is only one report, to the best of our knowledge, of two MDS patients with tetraploidy [15]. The combination of tetraploidy and del(5q) has not been reported previously in MDS. Hyperdiploidy has been associated with poor prognosis in MDS [12,14e16], but in light of the small numbers of reported cases, its significance is unclear. In this report, we describe the clinical, cytogenetic, and molecular cytogenetic findings in a case of high-risk MDS with the unique finding of tetraploidy and 5q deletion.
I. Znoyko et al. / Cancer Genetics and Cytogenetics 183 (2008) 64e68
65
2. Materials and methods 2.1. Case report The patient was a 68-year-old generally healthy man referred by his local primary care physician due to decreased hemoglobin and white cell count. Analysis of his blood and bone marrow aspirate demonstrated macrocytic anemia, severe neutropenia, megaloblastic changes in the red cell line, and occasional uninuclear morphology of megakaryocytes. Excess of blasts evaluated by differential cell count and flow cytometry with myeloid markers was 13 and 19%, respectively. Though the patient demonstrated clinical features of the 5qe syndrome (macrocytic anemia, normal to elevated platelet count, and neutropenia), he did not respond to treatment with Revlimid (lenalidomide), which was administered daily for 12 weeks. Moreover, during this period, his platelet count decreased from 350,000 to 118,000, and the patient developed AML, with a repeat bone marrow examination showing 35% myeloblasts marking for myelomonocytic lineage. However, subsequent treatment with two cycles of decitabine therapy resulted in marked improvement in the patient’s condition, with resolution of severe neutropenia and decrease in blast count to 2 and 3% (estimated by differential cell count and flow cytometry, respectively). Finally, the patient achieved clinical and cytogenetic remission. 2.2. Cytogenetics and fluorescence in situ hybridization (FISH) analysis Bone marrow was cultured in RPMI 1640 media (Invitrogen, Carlsbad, CA) and Chang BMC media (Irvine Scientific, Santa Ana, CA), harvested, and slides were prepared according to standard laboratory methods. Metaphase cells were imaged and karyotypes were generated using the Cytovision System version 3.6 (Applied Imaging, Santa Clara, CA). Cytogenetic abnormalities were described according to the International System of Human Cytogenetic Nomenclature (ISCN) 2005 [19]. FISH analysis for del(5q) was performed using probes EGR1 (5q31), CSF1R (5q33~q34), and control probes D5S23 and D5S721 (5p15.2) on slides prepared from fixed cell pellets according to the manufacturer’s recommended procedure (Abbott Molecular/Vysis, Des Plains, IL). 3. Results Cytogenetic analysis of the bone marrow aspirate revealed the presence of cells with normal karyotypes as well as two abnormal cell lines: a tetraploid clone and a clone with tetraploidy and del(5q) (Fig. 1; Table 1). It is noteworthy that the abnormal mitotic cell population at presentation and subsequent cytogenetic tests at 1 and 3 months were dominated by the tetraploid cell line with 5q deletion. However, at the 5-month follow-up (when AML was diagnosed), while the tetraploid/del (5q) clone was persistent, the majority of
Figure 1. Representative chromosomes 5 showing tetraploidy and deletion of one 5q (arrow).
abnormal dividing cells were represented by the tetraploid clone without 5q deletion (Table 1). Later, the patient was started on decitabine therapy, and by the next cytogenetic evaluation at 10 months, no abnormal cells were observed by routine cytogenetic analysis; the patient had reached cytogenetic remission (Table 1). To detect the presence of residual disease, we performed interphase FISH. We used two probes specific for the 5q CDR, namely CSF1R and EGR1 (see Materials and methods). Both probes have shown similar results (data not shown) and proved to be equally suitable for the identification of the 5q deletion in our case. According to the FISH results, tumor burden at 1 month was 12.4%, and it increased twofold by the fifth month (Table 2), when the patient evolved to AML (see Materials and methods). However, after decitabine treatment, at the 10-month follow-up, the number of abnormal cells had fallen to 3.5%. This value correlates well with the number of blasts measured by differential cell count and flow cytometry (2 and 3%, respectively, see Materials and methods). Interestingly, during the whole observation period, despite of the overall number of abnormal cells, the ratio between the two clones (tetraploid and tetraploid with 5q deletion) remained the same, 2:1 (Table 2). 4. Discussion Chromosomal abnormalities in neoplastic marrow cells often correlate closely with specific clinical and biologic characteristics of the disease and serve as a tool to predict the clinical outcome and develop effective therapeutic approaches. In this paper, we describe the successful treatment of a myelodysplastic syndrome-refractory anemia with excess blasts patient with the unique finding of tetraploidy and 5q deletion. Tetraploidy is a very rare abnormality in hematologic malignancies, especially in MDS. In fact, we are aware Table 1 Results of routine cytogenetic studies Date
46,XY
92,XXYY
92,XXYY,del(5)(q13q33)
At presentation 1 month 3 months 5 months 10 months
18 5 17 15 20
e e e 4 e
2 2 3 1 e
I. Znoyko et al. / Cancer Genetics and Cytogenetics 183 (2008) 64e68
66 Table 2 Result from FISH studies Representation of cells
Date
Distribution of abnormal cells
No. normal No. abnormal cells cells Tetraploid (%) (%) (%)
1 month 199 (87.6) 5 months 319 (74.0) 10 months 248 (96.5)
33 (12.4) 112 (26.0) 9 (3.5)
23 (69.7) 143a (66.2) 6 (67.7)
Tetraploid with deletion 5q (%) 10 (30.3) 73 a (33.8) 3 (33.3)
a Additional abnormal cells were scored, so the number of abnormal cells represented here is greater than that indicated in the ‘‘No. abnormal cell’’ column.
of only one publication concerning tetraploidy in MDS patients [15]. A large-scale study performed in Japan involving 979 patients with different types of hematologic malignancies revealed two cases of tetraploidy in MDS patients [15]. Little more information is available about hyperdiploidy (O50 chromosomes) in MDS [12e18]. In the majority of these reports, hyperdiploidy is associated with poor outcome [12,15,16,20], and increase in the percentage of hyperdiploid cells paralleled the increase in percentage of blasts in the bone marrow [13]. The 5q deletion is also associated with poor prognosis in high-risk MDS patients [10]. Therefore, the combination of tetraploidy and 5q deletion would suggest an unfavorable prognosis for our high-risk MDS patient. Fortunately, due to an optimized treatment course, our patient has achieved clinical and cytogenetic remission. Cytogenetic and FISH analysis of the bone marrow biopsy from the patient reported here has revealed two abnormal cell lines: tetraploid clone and tetraploid clone with 5q deletion. Since only one chromosome 5 shows deletion of the q arm, we conclude that development of tetraploidy preceded 5q deletion in clonal evolution. Furthermore, although the tetraploid clone without deletion was the predominant clone by FISH (Table 2), the mitotic cell population (analyzed cytogenetically) at initial clinical presentation, and then at 1- and 3-month follow-ups, was dominated by tetraploid cells with del(5q) (Table 1). These findings suggest that the clone bearing both abnormalities has a higher mitotic rate and is therefore better represented during routine chromosome analysis. However, more frequent cell divisions of the evolved clone do not affect its overall share among abnormal cells (Table 2), possibly due to its increased apoptotic index. Since the patient demonstrated clinical features of the 5qe syndrome (macrocytic anemia, normal to elevated platelet count, and neutropenia), he was initially treated with the immunomodulatory drug lenalidomide (Revlimid), which is particularly effective in patients with 5qe syndrome [21] and was recently approved by US Food and Drug Administration for MDS treatment [1]. There are not enough data on the therapeutic effect of lenalidomide in patients with high-risk MDS [21e23], but it is the drug of choice for
treatment of low-risk MDS with del(5q), where it has a dramatic therapeutic effect [21,22]. Lenalidomide was developed by modification of the first-generation immunomodulatory drug thalidomide in a drug discovery program. The initial phase I/II clinical study showed that treatment with lenalidomide abrogated red cell transfusion need in 83% of low-risk MDS patients with del(5q) and that cytogenetic remissions were frequently observed [22]. A large phase II follow-up study of 148 patients recently confirmed these promising results, showing erythroid responses and transfusion independency within a median of 4.6 weeks in 76 and 67% of the patients, respectively, and complete cytogenetic remission in 45% of the patients [21]. The molecular basis of this remarkable drug response is unknown, but according to the recent study, it could result from the ability of lenalidomide to inhibit growth of del(5q) erythroid progenitors and up-regulation of the potential tumor-suppressor genes SPARC and Activin A [2]. Intriguingly, SPARC and RPS14, a component of 40s ribosomal subunit, are among the genes showing haploinsufficiency in 5qe syndrome [24,25] and could represent target genes for lenamidolidamide treatment [2]. Our patient was not responsive to treatment with lenamidolide, administered daily for 12 weeks, though in a clinical trial the median time of the response was 4.6 weeks and 90% patients responded within 12 weeks [26]. In one study, lenalidomide worked equally well for patients with isolated versus complex karyotypes as long as del(5q) was present [21]. There are several possible reasons for the nonresponsiveness of our patient to lenamidolide treatment. First, clinical trials were performed in low-risk MDS patients in which the del(5q) is associated with a favorable outcome [10,11]. In high-risk MDS (as in our case), however, a del(5q) is associated with a poor prognosis [10]. Second, though the mechanism of lenamidolide effect in 5q syndrome is not determined, one of the published theories implies that haploinsufficiency for 5q is critical [2]. If that is the case, the presence of three copies of a normal chromosome 5 in the tetraploid clone of our patient could provide for a compensatory effect. Finally and most importantly, according to the accepted point of view, the cell of origin in 5qe syndrome is a pluripotent hematopoietic stem cell and 5q deletions represent an early event in MDS pathogenesis [27,28]. In our case, however, development of tetraploidy apparently preceded 5q deletion in clonal evolution, thus suggesting a different disease etiology than 5qe syndrome. Since the patient did not respond to lenamidolide treatment and his disease had progressed to AML, subsequent treatment with the hypomethylating agent decitabine was initiated. Decitabine has been shown to be effective and well tolerated for the treatment of high-risk MDS, for which treatment options were previously scarce [29]. In clinical trials, decitabine treatment reduced red blood cell transfusion requirement, improved quality of life, and produced statistically significant delays in time to AML and death [29]. However, the drug produced the rather low
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clinical remission (CR) rate of 9% [29]. Fortunately, our patient has shown remarkable response to treatment with decitabine and eventually achieved clinical and cytogenetic remission. The effect of decitabine, an S-phaseespecific inhibitor of DNA methyltransferase, relies on its ability to decrease levels of methylation, which is responsible for abnormal silencing of many genes, including tumor suppressor genes in neoplasia. Therefore, a possible mechanism of disease underlying the observed cytogenetic abnormalities, tetraploidy and 5q deletion, may involve epigenetic inactivation of gene expression via methylation. This finding is of particular interest in light of the recently published data involving the model of a-catenin therapy [11]. The study demonstrated that silencing of key tumor-suppressor genes within the 5q CDR results from the loss of one of the alleles via deletion and epigenetic inactivation of the remaining allele. In vitro experiments indicate that inactivation of the remaining allele is caused by promoter methylation and histone deacetylation and can be reversed by using inhibitors of methyltranferase and histone deacetylase [11]. These data suggest that combinational treatment with methyltransferase inhibitors (such as decitabine or azacitidine) and histone deacetylase inhibitors (such as valproic or suberoylanilide hydroxamic acid), which are being studied in clinical trials now [1,29], might specifically benefit the subgroup of individuals with del(5q) who are not responsive to treatment with immunomodulatory drugs.
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