Multiplex Reverse Transcription-Polymerase Chain Reaction for Simultaneous Screening of 29 Translocations and Chromosomal Aberrations in Acute Leukemia

Abstract

We have developed a multiplex reverse transcription-polymerase chain reaction (RT-PCR) reaction, which enables us to detect 29 translocations/chromosomal aberrations in patients with acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML). Through the construction and optimization of specific primers for each translocation, we have been able to reduce the set-up to 8 parallel multiplex PCR reactions, thus greatly decreasing the amount of work and reagents. We show the value of our set-up in a retrospective analysis on cryopreserved material from 102 AML and 62 ALL patients. The multiplex RT-PCR detected a hybrid mRNA resulting from a structural chromosomal aberration in 45 of 102 (44%) of the AML and in 28 of 62 (45%) of the pediatric ALL cases. Importantly, in 33% of AML and in 47% of the ALL cases with cytogenetic data, submicroscopic chromosomal aberrations or masked translocations were shown that were not detected in the cytogenetic analysis either for structural reasons or because of an insufficient number of metaphases obtained. This multiplex RT-PCR system, which can handle up to 10 patients with a response time of 2 working days, is thus an important tool that complements cytogenetic analysis in the up-front screening of acute leukemia patients and should provide a rapid and efficient characterization of leukemia cells, even in situations with sparse patient material.

Prospective application of a multiplex reverse transcription‐polymerase chain reaction assay for the detection of balanced translocations in leukaemia: a single‐laboratory study of 390 paediatric and adult patients

“…The second major conclusion from our data pertains to the fact that the incidence of patients positive for fusion transcripts in both the AML (23%) and ALL (32%) subgroups is lower than in our retrospective analysis (Pallisgaard et al, 1998), where the incidence of patients positive for fusion transcripts were determined as 44 and 45% respectively. As the patient materials in the present and in our previous study (Pallisgaard et al, 1998) are not directly comparable, even larger studies are needed to corroborate this observation. Despite this, we feel that the unselected, prospective design of our study with no banking bias coupled with a molecular detection system, argue strongly for a re-evaluation of this important aspect of leukaemia diagnosis.…”

“…Given the high number of balanced translocations described in leukaemia, some of which are very rare, and given that they can be used in detection of minimal residual disease (MRD) (Pallisgaard et al, 1999), we developed a multiplex reverse transcription (RT)-PCR that enables the detection of 27 lesions with nearly 80 fusion gene variants (Pallisgaard et al, 1998). The method was validated on archived material from over 150 AML and acute lymphoblastic leukaemia (ALL) patients, and found to be useful for identifying all translocation positive patients including a series of cytogenetically negative ones.…”

Age‐associated difference in gene expression of paediatric acute myelomonocytic lineage leukaemia (FAB M4 and M5 subtypes) and its correlation with prognosis

“…Morphological diagnosis according to FAB classification, immunophenotype and cytogenetic analysis were performed locally. MLL gene rearrangement was determined by cytogenetic analysis and multiplex reverse transcription polymerase chain reaction (RT-PCR) analysis (Pallisgaard et al, 1998;Salto-Tellez et al, 2003) using the HemaVision kit (DNA Technology, Aarhus, Denmark). Southern blot or fluorescence in situ hybridization analysis was also performed in some patients.…”

Simple multiplex RT‐PCR for identifying common fusion transcripts in childhood acute leukemia

“…However, individual RT-PCR for each fusion transcript is expensive and labor intensive. Pallisgaard et al (1998) has successfully demonstrated eight RT-PCR assays for screening of 29 chromosomal aberrations covering both common and uncommon fusion transcripts for childhood acute leukemia. In this study, we aimed to develop a simple RT-PCR method for the detection of the common and risk-stratifying chimeric transcripts in both childhood ALL and ANLL.…”