Abstract
Cytogenetic abnormalities identified by conventional cytogenetics (CC) have important prognostic and therapeutic roles in myelodysplastic syndromes (MDS). Fluorescence in situ hybridization (FISH) complements CC since it is able to evaluate large numbers of interphase and metaphase nuclei. The question has been raised as to whether interphase FISH in addition to CC is able to imprive the level of detection of del(5q) and del(20q) in MDS. This study performed interphase FISH with 5q and 20q probes in a series of 158 MDS patients with a normal karyotype. No hidden del(5q) or del(20q) was detected. A review of the literature identified 20 patients (1.96%) of 1018 patients (including the current series) and 3 (0.91%) of 331 patients to have a del(5q) or del(20q). Therefore, interphase FISH adds little, if any, improvement to the probability of detecting these deletions. However, interphase FISH is recommended in patients with no cell growth or when fewer than 20 metaphases are available for CC analysis.
Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal diseases of pluripotent hematopoietic stem cells or progenitor cells. They are characterized by ineffective hematopoiesis, increased apoptosis, blood cytopenias and a propensity to evolve to acute myeloblastic leukemia (1, 2).
Clonal cytogenetic abnormalities are observed in 40-60% of patients with de novo MDS, but in 80% of those with therapy-related MDS (secondary MDS) (3). The most common chromosomal abnormalities include 5q-, -7/7q-, +8, 11q-, 12p-, 13q-, 17p-, 20q-, and +21 (4). Conventional cytogenetic (CC) studies identify these clonal abnormalities in the majority of the MDS patients. However, some patients might have an apparently normal karyotype in CC because of poor bone marrow sample, lack of dividing neoplastic cells, or ill-defined chromosomes in addition to those who have a truly normal-appearing karyotype (5). Fluorescence in situ hybridization (FISH) on metaphase cells using a panel of different probes helps to improve the probability of identifying an abnormality that might have been missed by CC (5-7). Metaphase FISH also relies on the presence of dividing neoplastic cells, which is not always the case as for CC.
The question has been raised as to whether interphase FISH with a panel of probes targeting the most common chromosomal abnormalities might be a valuable alternative when a normal karyotype or no karyotype is obtained by CC (8-10). This reports details the Authors' experience in 158 patients with a normal karyotype and reviews the relevant literature.
Patients and Methods
Patients. A series of 158 consecutive MDS patients referred to the cytogenetic laboratory of the Brest University Hospital between January 1995 and December 2007 was selected because a normal karyotype was found in all 20 metaphases analyzed in conventional cytogenetics. The patients were categorized by their bone marrow morphology according to the French-American-British (FAB) classification (11, 12) and reclassified according to World Health Organization (WHO) criteria (13).
Conventional cytogenetics. Cytogenetic analysis was performed on bone marrow cells of the patients at the time of the diagnosis. Bone marrow cultures were synchronized for 17 hours by fluorodeoxyuridine (FudR 10−7 M), before being released by thymidine (10−5 M) for six hours. They were then exposed to colcemide and harvested according to standard procedures. The chromosomes were R-banded and the karyotypes described according to ISCN (2009) (14).
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.
Results of interphase FISH in MDS patients associated with a normal karyotype.
Fluorescent in situ hybridization. FISH studies were performed on 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 interphase nuclei using locus-specific identifier (LSI) for bands 5q31 (EGR1 probe labeled in red; Abbott, Rungis, France), 5q33-34 (CSF1R probe labeled in red; Abbott), and LSI for band 20q12 (D20S108 labeled in red; Abbott). LSI 5p15.2 (450-kb probe extending from D5S721 to D5S23 labeled in green; Abbott) and bacterial artificial chromosome clone RP4-610C12 (20q11.1 band) were used as controls. Twenty metaphase cells and 100 interphase nuclei were analyzed for each patient.
Results
Table I shows the distribution of the 158 MDS patients with a normal karyotype in conventional cytogenetics according the 2008 revision of the WHO classification of myeloid neoplasms.
Sequential FISH analyses revealed two green signals (control signals) and two red signals (corresponding to the EGR1 or CSF1R and D20S108 probes) in the metaphases and nuclei analyzed from all 158 patients.
Discussion
Detection of chromosome abnormalities is useful in the stratification of patients into prognostic subgroups (15-17). The presence of a normal karyotype is associated with a better prognosis in MDS patients. However, the lack of chromosome abnormalities in CC does not always mean that the karyotype is normal. Indeed, results obtained using banding techniques can be hampered by efficacy of bone marrow culture, lack of dividing neoplastic cells, spreading of metaphases, bias of metaphase analysis and quality of chromosomes. Furthermore, low mosaicism can be overlooked when 20 to 25 metaphases are examined, which is common practice in cytogenetic laboratories.
Since Jenkins et al. and Kibbelaar et al. reported additional numerical imbalances of chromosomes 7 and 8 in MDS detected by FISH but not by CC, interphase FISH has been proposed as a complementary technique to detect partial deletions of the long arm of chromosomes 5 and 20 when a normal karyotype has been found in conventional cytogenetics (9, 10).
No submicroscopic deletion of the long arm of chromosomes 5 and 20 was found among the 158 MDS patients with a normal karyotype examined by interphase FISH. Several studies have investigated the usefulness of interphase FISH in patients with a normal karyotype (Table II). A total of 1018 patients (including this series) was analyzed using the EGR1 (5q31) probe for the majority of them; 20 patients (1.96%) were found to have a deletion of the EGR1 probe. Three of the 331 patients (0.91%) studied by interphase FISH using the D20S108 (20q12) probe had a deletion. These ‘negative’ results also confirm that 5q and 20q deletions are usually so large that they can be detected by conventional cytogenetics (18-20).
In conclusion, the negative results obtained by interphase FISH to detect hidden 5q and 20q deletions suggest that conventional cytogenetic analysis identifies the great majority of these deletions in MDS patients. In rare cases, it may even be superior to interphase FISH if the deletions occur outside the probe loci used for FISH. Therefore, CC, associated or not with metaphase FISH, is a superior method of evaluating chromosomes 5 and 20 abnormalities in MDS compared to interphase FISH alone. However, interphase FISH is recommended in cases with no cell growth or when fewer than 20 metaphases are available for CC analysis.
- Received January 7, 2011.
- Revision received February 22, 2011.
- Accepted February 22, 2011.
- Copyright© 2011 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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