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Preclinical Validation of a Monochrome Real-Time Multiplex Assay for Translocations in Childhood Acute Lymphoblastic Leukemia

Research Centre [A. K. S., G. T., A. A-N., K. B., M. I. G.] and Pediatric Hematology-Oncology [A. A-N., H. E. S.], King Fahad National Centre for Children’s Cancer and Research, King Faisal Specialist Hospital and Research Centre, Riyadh, 11211 Saudi Arabia; Genetics, Institute for Experimental Medicine, Istanbul University, Istanbul, Turkey [U. O., S. Si.]; and Hematology, All India Institute of Medical Sciences, New Delhi, India [S. Sa., M. B.]

Purpose: The purpose is to develop a real-time multiplex reverse transcription-PCR assay for detection and quantification of leukemia-specific chimeric transcripts that identify the genetic subgroups of acute lymphoblastic leukemias (ALLs) proposed by the WHO classification.

Experimental Design: Real-time multiplex assay for t(12;21), t(4;11), and t(1;19) with hypoxanthine phosphoribosyltransferase as internal standard used in tandem with a new real time quantitative-RT-PCR assay for the t(9;22). This new strategy was designed to yield an amplicon from each translocation with a distinct melting peak allowing dependable identification using only Sybr green I, without any need for expensive hybridization probes.

Results: We validated this method with 92 primary ALLs and identified 4 E2A-PBX1, 4 mBCR-ABL and 10 TEL-AML1. When compared with conventional RT-PCRs and Southern blot analyses, 100% concordance was obtained. During the course of these studies, we found marked variations in the levels of the TEL-AML1 transcripts in individual patients. We, therefore, extended the study to accurately and reproducibly determine TEL-AML1 mRNA levels in 47 additional patients with t(12;21). The results indicated that the level of expression of TEL-AML1 varied among individual patients, and it was independent of the WBC count.

Conclusions: Our new real-time multiplex assay can be used for rapid, simple, and reliable classification of pediatric ALL. Its reproducible quantification results should also facilitate studies on minimal residual disease. The observed variation in TEL-AML1 transcript levels is of interest because it could reflect biological and/or clinical heterogeneity in the behavior of these leukemias.

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