A multiplex RT-PCR assay for the detection of chimeric transcripts encoded by the risk-stratifying translocations of pediatric acute lymphoblastic leukemia

Abstract

Modern therapy for pediatric acute lymphoblastic leukemia (ALL) is based on the principle of risk stratification. One of the most important laboratory features used to accurately risk stratify patients is the presence of specific chromosomal translocation within the leukemic blasts. In this paper, we describe a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the accurate, sensitive, and rapid identification of chimeric transcripts encoded by the major risk-stratifying translocations of pediatric ALL. This assay will identify both the CML-and ALL-type BCR-ABL transcripts encoded by the t(9;22), all described variants of the E2A-PBX1 transcripts encoded by the t (1;19), the MLL-AF4 transcripts encoded by the t(4;11), and all variants of TEL-AML1 encoded by the t(12;21). In addition, we have developed a reverse dot-blot detection system as an alternative to traditional post-PCR Southern blot analysis. Application of this combined assay to the analysis of 70 leukemic samples and five cell lines resulted in a complete concordance between this multiplex assay and individual PCR reactions. The characteristics of the multiplex assay suggest that its application to routine clinical screening will significantly improve the ability of clinical laboratories to accurate risk stratify pediatric ALL patients.

A simplified flow cytometric assay identifies children with acute lymphoblastic leukemia who have a superior clinical outcome

“…Diagnostic immunophenotyping and chromosomal and genetic analyses were performed by standard techniques. [13][14][15] Initial treatment consisted of methotrexate and/or mercaptopurine, 16 followed 4 days later by a 6-week remission induction therapy with prednisone, vincristine, daunorubicin, asparaginase, and etoposide plus cytarabine. 17 Patients were considered to have lower-risk ALL if they were 1 to 9 years old with a presenting leukocyte count lower than 50 ϫ 10 9 /L or had a DNA index of 1.16 or more.…”

Genes contributing to minimal residual disease in childhood acute lymphoblastic leukemia: prognostic significance of CASP8AP2

“…21,22 The presence of BCR-ABL, E2A-PBX1, and TEL-AML1 fusions or MLL gene rearrangements was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). 23 Among the 288 ALL cases studied (189 to identify genes associated with MRD and 99 to test the clinical significance of the identified genes), 47 were classified as T-lineage ALL and 241 as B-lineage ALL. The latter included 16 cases with BCR-ABL, 22 with E2A-PBX1, 18 with MLL rearrangements, 57 with TEL-AML1, 52 with hyperdiploidy (Ͼ 50 chromosomes), and 76 with other features.…”

Multiplex reverse transcriptase–polymerase chain reaction screening in childhood acute myeloblastic leukemia

“…[7][8][9][10][11][12][13][14][15] This is particularly true for those transcripts found in AML, whereas the clinically most important acute lymphocytic leukemia (ALL)-specific abnormalities have been combined in several types of multiplex assays. 16,17 However, the steadily increasing number of detectable abnormalities makes the conventional screening approaches for single specific fusion transcripts more and more impractical and obsolete.…”