Clinical screening of gene rearrangements in childhood leukemia by using a multiplex polymerase chain reaction-microarray approach.

PURPOSE: Currently, many forms of leukemia are considered potentially curable, with prognosis and clinical outcome strongly dependent on the underlying molecular pathophysiology. A substantial number of leukemia patients harbor nonrandom karyotypic abnormalities that define subgroups with unique biological and clinical features. For detection of these types of gene rearrangements, a combination of multiplex RT-PCR with hybridization on oligonucleotide gel array was presented previously, which identified five chromosomal translocations with fusion variants. In the present study, additional clinically relevant translocations were included in our analysis using a second generation of microarrays. We also expanded significantly on the clinical correlation of our findings. EXPERIMENTAL DESIGN: An oligonucleotide microarray was designed for hybridization with products of a multiplex RT-PCR to identify the following translocations: t(9;22)p190, t(4;11), t(12;21), t(1;19), typical for acute lymphoblastic leukemia; t(9;22)p210 for chronic myeloid leukemia; and t(8;21), t(15;17), inv16, typical for acute myeloblastic leukemia. RESULTS: To demonstrate the potential clinical application of the method, 247 cases of childhood leukemia were screened, and the above-mentioned gene rearrangements were found in 30% of cases. The sensitivity and specificity of the assay is comparable with the RT-PCR technique, so that it can be used to follow minimal residual disease. The feasibility of an additional refinement of the method, on-chip-multiplex PCR, has been successfully demonstrated by identifying a common translocation, t(9;22), in chronic myeloid leukemia. CONCLUSIONS: Our data suggest that the microarray-based assay can be an effective and reliable tool in the clinical screening of leukemia patients for the presence of specific gene rearrangements with important diagnostic and prognostic implications. The method is amenable for automation and high-throughput analysis.

Identification of chromosomal translocations in leukemias by hybridization with oligonucleotide microarrays.

BACKGROUND AND OBJECTIVES: Identification of chromosomal rearrangements is important for a precise risk-stratified diagnosis of hematologic malignancies. As the number of known translocations, specific for different types of leukemia increases, it takes ever more time and increasing amounts of patient's material to screen a single patient with individual polymerase chain reactions (PCR). The aim of this study was to develop a new approach combining specificity with high-throughput sufficient for rapid screening of clinical samples for the presence of numerous translocations. DESIGN AND METHODS: We designed an oligonucleotide microarray and used hybridization with microarrays in combination with multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay for accurate and rapid identification of some major leukemias. The following translocations were used as prototypic: t(9;22) p210 and p190, t(4;11), t(12;21), and t(15;17). This approach was tested on five different cell cultures carrying translocations and on 22 clinical samples from leukemic patients. RESULTS: Distinctive hybridization signals were obtained for all types of chimeric transcripts from cell lines with translocations. Both the type of translocation and the splice variant were determined. The data demonstrated high specificity and reproducibility of the method. Analysis of the 22 clinical samples using the microarray-based approach showed complete agreement with standard PCR analysis. INTERPRETATION AND CONCLUSIONS: Our data suggest that oligonucleotide microarrays can be used as an efficient, alternative approach to the traditional post-PCR Southern blot analysis. The oligonucleotide microarray approach appears suitable for clinical screening of major risk-stratifying translocations in patients with leukemia.