Original articles
The application of comparative genomic hybridization as an additional tool in the chromosome analysis of acute myeloid leukemia and myelodysplastic syndromes

https://doi.org/10.1016/S0165-4608(00)00386-1Get rights and content

Abstract

In acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) there are frequently complex karyotypes with multiple structurally altered chromosomes, many of which are marker chromosomes of unknown origin. The aim of this study was to apply comparative genomic hybridization (CGH) to cases of AML or MDS in transformation submitted for routine cytogenetic analysis to investigate whether this approach would yield any further information and, if possible, to predict which cases would benefit from CGH analysis. Nineteen cases with AML or MDS in transformation were analyzed. CGH revealed nine cases with gains or losses of chromosomal material. In six of these cases the chromosomal location of this material was not apparent from cytogenetic analysis especially when multiple markers were present. By using fluorescence in situ hybridization (FISH) with specific libraries for the chromosome regions that showed discordance between CGH and conventional cytogenetics, we were able to identify the chromosome location of material within the karyotype. In this group of six patients, four cases of an unbalanced translocation involving regions of chromosomes 5 and 17 were characterized. Three of these cases had additional abnormalities, including two cases with regions of amplification in which oncogenes are located (MYC, MLL) and one case with a dic(7;21)(p10;p10). In all six cases it was possible to characterize complex chromosomal aberrations such as derivative chromosomes, marker chromosomes, and ring chromosomes. This study demonstrates that CGH can detect true gain and loss of critical chromosome regions more accurately than conventional karyotyping in cases with very complex karyotypes, and can thus prove useful in predicting prognosis and pinpointing areas of the genome that require further study. Also, CGH can be a useful technique to identify the origin of marker chromosomes, and it can assist in choice of probes for confirmatory FISH, when there is no clue provided from the analysis of G-banded chromosomes.

Introduction

Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS)-related AMLs are a very heterogeneous group of hematological diseases that show a diverse spectrum of chromosomal aberrations, molecular abnormalities, and immunophenotypes. Acute myeloid leukemia may be induced by disruption of the normal structure and function of genes that control the balance of proliferation and differentiation in hematopoietic precursors [1].

In patients with AML, about 20–40% of patients never achieve remission following standard induction chemotherapy. Only 30–40% of patients who achieve remission will eventually become long-term survivors despite treatment with intensification chemotherapy, bone marrow transplantation, or both 2, 3. Therefore, it is greatly beneficial to allocate patients to different risk groups to prevent disease relapse and to improve long-term survival rates. Many parameters can influence prognosis, including age, FAB type, clinical variables, especially presentation white cell count, and the expression of certain immunophenotypic surface markers, but appropriate analysis of chromosomal abnormalities is one of the most valuable prognostic indicators in AML. Chromosomal abnormalities have been reported in 50–95% of patients with primary AML in different studies 2, 4, 5. Those AML patients with specific balanced rearrangements such as t(15;17), t(8;21), or inv(16) have a relatively good prognosis. Those AML patients with complex karyotypes, often including several marker chromosomes, especially patients with monosomy 5 or del(5q), monosomy 7, or abnormalities of 3q, have been regarded as a poor prognostic group 2, 4, 6, 7, 8, 9.

Many cases of AML and MDS have structurally altered chromosomes, as a part of a complex karyotype, which cannot be fully identified by cytogenetic analysis alone. Identification of chromosomal regions involved in rearrangements and net gains and losses of chromosomal copy numbers could be of considerable clinical importance. Fluorescence in situ hybridization (FISH) can provide some information but requires the correct selection of specific probes, and only a few chromosomes or chromosomal regions can be examined in a single experiment in the practical routine laboratory setting.

Comparative genomic hybridization (CGH) was developed to proved an adjunct to FISH and to overcome these drawbacks, as there is no requirement for test material metaphase preparations [10]. It allows more rapid comprehensive assessment of chromosomal gains and losses in a tumor genome in order to predict prognosis and to choose appropriate treatment protocols in AML patients with complex karyotypes that include marker chromosomes.

Nineteen cases with AML or MDS transforation were studied by CGH and FISH to obtain any further information, especially in cases with complex karyotypes studied by conventional cytogenetics.

Section snippets

Patients samples

Nineteen AML or AML/MDS transformation bone marrow samples were analyzed. These were samples in which sufficient material remained after cultures had been set up for routine cytogenetic analysis. Karyotyping and CGH were done independently.

Preparation for CGH experiments

Metaphase spreads were prepared according to standard protocols from phytohemagglutinin-stimulated, methotrexate-synchronized peripheral blood lymphocytes from a karyotypically normal male and female. Test DNA from fresh bone marrow samples and control DNA

Conventional karyotyping and CGH results

In 7 of the 19 patients, normal results were observed both by cytogenetics and CGH. Cytogenetic analysis of the remaining cases revealed complex karyotypes with multiple chromosome aberrations in eight cases, and two cases showed a balanced t(15;17); two cases failed to yield a result. The CGH analysis of these two failed cases (cases 8 and 17) showed a normal result in case 8 and showed multiple gains and losses of specific chromosome regions in case 17. Analysis by CGH could not detect any

Discussion

In hematologic malignancies, exact analysis of certain chromosomal rearrangements—such as unbalanced translocations, marker chromosomes, or ring chromosomes—is a critical step in identifying the genes or chromosomes that are crucially related to the pathogenesis of a disease and are important in predicting the prognosis of the patients. The presence of marker chromosomes or complex chromosome aberrations is a major cause of incomplete G-banding analysis.

Several methods are used to investigate

Acknowledgements

We are grateful to Dr. Cumming, Dr. Fitzsimmons, Dr. Parker, Dr. Shahriari, and Dr. Watson for providing patient samples.

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