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Interphase FISH Does Not Improve the Detection of DEL(5Q) and DEL(20Q) in Myelodysplastic Syndromes

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Interphase FISH Does Not Improve the Detection of DEL(5Q) and DEL(20Q) in Myelodysplastic Syndromes ANTICANCER RESEARCH 31: 1007-1010 (2011) Interphase FISH Does Not Improve the Detection of DEL(5q) and DEL(20q) in Myelodysplastic Syndromes NATHALIE DOUET-GUILBERT1,2,3, ANGÈLE HERRY1, MARIE-JOSÉE LE BRIS2, NADIA GUÉGANIC1, CLÉMENT BOVO1, FRÉDÉRIC MOREL1,2,3 and MARC DE BRAEKELEER1,2,3 1Laboratoire d’Histologie, Embryologie et Cytogénétique, Faculté de Médecine et des Sciences de la Santé, Université de Bretagne Occidentale, Brest, France; 2Service de Cytogénétique, Cytologie et Biologie de la Reproduction, Hôpital Morvan, CHU Brest, Brest, France; 3Institut National de la Santé et de la Recherche Médicale (INSERM) U613, Brest, France Abstract. Cytogenetic abnormalities identified by conventional 11q-, 12p-, 13q-, 17p-, 20q-, and +21 (4). Conventional cytogenetics (CC) have important prognostic and therapeutic cytogenetic (CC) studies identify these clonal abnormalities in roles in myelodysplastic syndromes (MDS). Fluorescence in situ the majority of the MDS patients. However, some patients hybridization (FISH) complements CC since it is able to might have an apparently normal karyotype in CC because of evaluate large numbers of interphase and metaphase nuclei. The poor bone marrow sample, lack of dividing neoplastic cells, question has been raised as to whether interphase FISH in or ill-defined chromosomes in addition to those who have a addition to CC is able to imprive the level of detection of del(5q) truly normal-appearing karyotype (5). Fluorescence in situ and del(20q) in MDS. This study performed interphase FISH hybridization (FISH) on metaphase cells using a panel of with 5q and 20q probes in a series of 158 MDS patients with a different probes helps to improve the probability of identifying normal karyotype. No hidden del(5q) or del(20q) was detected. an abnormality that might have been missed by CC (5-7). A review of the literature identified 20 patients (1.96%) of 1018 Metaphase FISH also relies on the presence of dividing patients (including the current series) and 3 (0.91%) of 331 neoplastic cells, which is not always the case as for CC. patients to have a del(5q) or del(20q). Therefore, interphase The question has been raised as to whether interphase FISH adds little, if any, improvement to the probability of FISH with a panel of probes targeting the most common detecting these deletions. However, interphase FISH is chromosomal abnormalities might be a valuable alternative recommended in patients with no cell growth or when fewer when a normal karyotype or no karyotype is obtained by CC than 20 metaphases are available for CC analysis. (8-10). This reports details the Authors’ experience in 158 patients with a normal karyotype and reviews the relevant Myelodysplastic syndromes (MDS) are a heterogeneous group literature. of clonal diseases of pluripotent hematopoietic stem cells or progenitor cells. They are characterized by ineffective Patients and Methods hematopoiesis, increased apoptosis, blood cytopenias and a propensity to evolve to acute myeloblastic leukemia (1, 2). Patients. A series of 158 consecutive MDS patients referred to the Clonal cytogenetic abnormalities are observed in 40-60% cytogenetic laboratory of the Brest University Hospital between January 1995 and December 2007 was selected because a normal of patients with de novo MDS, but in 80% of those with karyotype was found in all 20 metaphases analyzed in conventional therapy-related MDS (secondary MDS) (3). The most cytogenetics. The patients were categorized by their bone marrow common chromosomal abnormalities include 5q-, -7/7q-, +8, morphology according to the French-American-British (FAB) classification (11, 12) and reclassified according to World Health Organization (WHO) criteria (13). Correspondence to: Professor Marc De Braekeleer, Laboratoire de Conventional cytogenetics. Cytogenetic analysis was performed on Cytogénétique, Faculté de Médecine et des Sciences de la Santé, bone marrow cells of the patients at the time of the diagnosis. Bone Université de Bretagne Occidentale, 22, avenue Camille Desmoulins, marrow cultures were synchronized for 17 hours by fluoro- CS 93837, F-29238 Brest cedex 3, France. Tel: +33 298016476, Fax: deoxyuridine (FudR 10–7 M), before being released by thymidine +33 298018189, e-mail: [email protected] (10–5 M) for six hours. They were then exposed to colcemide and harvested according to standard procedures. The chromosomes Key Words: Interphase FISH, MDS, normal karyotype, del(5q), were R-banded and the karyotypes described according to ISCN del(20q). (2009) (14). 0250-7005/2011 $2.00+.40 1007 ANTICANCER RESEARCH 31: 1007-1010 (2011) Table I. Classification of the 158 MDS patients with a normal karyotype in conventional cytogenetics according to the 2008 revision of the WHO classification of myeloid neoplasms. Disease according to the 2008 revised WHO classification Number of patients Refractory cytopenia with unilineage dysplasia (RCUD) 39 Refractory cytopenia with ring sideroblasts (RARS) 17 Refractory cytopenia with multilineage dysplasia (RCMD) 40 Refractory anemia with excess blasts-1 (RAEB-1) 20 Refractory anemia with excess blasts-2 (RAEB-2) 17 Myelodysplastic syndrome, unclassified 12 Myelodysplastic/myeloproliferative neoplasm, unclassifiable 3 Chronic myelomonocytic leukemia 10 Table II. Results of interphase FISH in MDS patients associated with a normal karyotype. Reference Tissue Number of patients del(5q) del(20q) Probe Number Probe Number (21) BM 27 EGR1 (5q31) 0 (8) BM 101 EGR1 (5q31) 5 (5) BM 32 EGR1 (5q31) 0 D20S108 (20q12) 0 (22) 22 CSF1R (5q33) 1 (23) BM 30 EGR1 (5q31) 0 D20S108 (20q12) 0 (24) BM 57 EGR1 (5q31) 2 D20S108 (20q12) 3 (25) BM/PB 55 EGR1 (5q31) 0 (26) BM 21 EGR1 (5q31) 3 (27) BM 324 EGR1 (5q31) 9 (28) BM 54 EGR1 (5q31) 0 D20S108 (20q12) 0 (29) BM 137 EGR1 (5q31) 0 This report BM 158 EGR1 (5q31) 0 D20S108 (20q12) 0 BM: Bone marrow; PB: peripheral blood. Fluorescent in situ hybridization. FISH studies were performed on Discussion the same fixed material as the conventional cytogenetic analyses. Cell preparations were stored in fixative at –20˚C until use. For each patient, FISH techniques were performed on metaphase cells and Detection of chromosome abnormalities is useful in the interphase nuclei using locus-specific identifier (LSI) for bands stratification of patients into prognostic subgroups (15-17). 5q31 (EGR1 probe labeled in red; Abbott, Rungis, France), 5q33- The presence of a normal karyotype is associated with a 34 (CSF1R probe labeled in red; Abbott), and LSI for band 20q12 better prognosis in MDS patients. However, the lack of (D20S108 labeled in red; Abbott). LSI 5p15.2 (450-kb probe chromosome abnormalities in CC does not always mean that extending from D5S721 to D5S23 labeled in green; Abbott) and the karyotype is normal. Indeed, results obtained using bacterial artificial chromosome clone RP4-610C12 (20q11.1 band) were used as controls. Twenty metaphase cells and 100 interphase banding techniques can be hampered by efficacy of bone nuclei were analyzed for each patient. marrow culture, lack of dividing neoplastic cells, spreading of metaphases, bias of metaphase analysis and quality of Results chromosomes. Furthermore, low mosaicism can be overlooked when 20 to 25 metaphases are examined, which Table I shows the distribution of the 158 MDS patients with a is common practice in cytogenetic laboratories. normal karyotype in conventional cytogenetics according the Since Jenkins et al. and Kibbelaar et al. reported additional 2008 revision of the WHO classification of myeloid neoplasms. numerical imbalances of chromosomes 7 and 8 in MDS detected Sequential FISH analyses revealed two green signals by FISH but not by CC, interphase FISH has been proposed as (control signals) and two red signals (corresponding to the a complementary technique to detect partial deletions of the long EGR1 or CSF1R and D20S108 probes) in the metaphases arm of chromosomes 5 and 20 when a normal karyotype has and nuclei analyzed from all 158 patients. been found in conventional cytogenetics (9, 10). 1008 Douet-Guilbert et al: Interphase FISH in MDS No submicroscopic deletion of the long arm of detecting occult chromosome lesions in myelodysplastic chromosomes 5 and 20 was found among the 158 MDS syndromes with normal karyotype. Leukemia 15: 1841-1847, 2001. patients with a normal karyotype examined by interphase 9 Jenkins RB, Le Beau MM, Kraker WJ, Borell TJ, Stalboerger FISH. Several studies have investigated the usefulness of PG, Davis EM, Penland L, Fernald A, Espinosa R and Schaid DJ: Fluorescence in situ hybridization: a sensitive method for interphase FISH in patients with a normal karyotype (Table trisomy 8 detection in bone marrow specimens. Blood 79: 3307- II). A total of 1018 patients (including this series) was 3315, 1992. analyzed using the EGR1 (5q31) probe for the majority of 10 Kibbelaar RE, Mulder JW, Dreef EJ, van Kamp H, Fibbe WE, them; 20 patients (1.96%) were found to have a deletion of Wessels JW, Beverstock GC, Haak HL and Kluin PM: Detection the EGR1 probe. Three of the 331 patients (0.91%) studied of monosomy 7 and trisomy 8 in myeloid neoplasia: a by interphase FISH using the D20S108 (20q12) probe had a comparison of banding and fluorescence in situ hybridization. deletion. These ‘negative’ results also confirm that 5q and Blood 82: 904-913, 1993. 11 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, 20q deletions are usually so large that they can be detected Gralnick HR and Sultan C: Proposals for the classification of the by conventional cytogenetics (18-20). myelodysplastic syndromes. Br J Haematol 51: 189-199, 1982. In conclusion, the negative results obtained by interphase 12 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, FISH to detect hidden 5q and 20q deletions suggest that Gralnick HR and Sultan C: Proposed revised criteria for the conventional cytogenetic analysis identifies the great majority classification of acute myeloid leukemia. A report of the French- of these deletions in MDS patients. In rare cases, it may even American-British Cooperative Group.
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