Preclinical validation of a monochrome real-time multiplex assay for translocations in childhood acute lymphoblastic leukemia.
OBJECTIVE
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.


METHODS
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.
Siraj
Abdul K
AK
Research, King Fahad National Centre for Children's Cancer and Research, King Faisal Specialist Hospital and Research Centre, Riyadh, 11211 Saudi Arabia.
Ozbek
Ugur
U
Sazawal
Sudha
S
Sirma
Sema
S
Timson
Georgina
G
Al-Nasser
Abdallah
A
Bhargava
Manorama
M
El Solh
Hassan
H
Bhatia
Kishor
K
Gutiérrez
Marina I
MI
D015139:Blotting, Southern; D002648:Child; D002675:Child, Preschool; D002878:Chromosomes, Human, Pair 1 / Q000235:genetics; D002880:Chromosomes, Human, Pair 11 / Q000235:genetics; D002881:Chromosomes, Human, Pair 12 / Q000235:genetics; D002888:Chromosomes, Human, Pair 19 / Q000235:genetics; D002891:Chromosomes, Human, Pair 21 / Q000235:genetics; D002894:Chromosomes, Human, Pair 4 / Q000235:genetics; D050676:Core Binding Factor Alpha 2 Subunit; D017931:DNA Primers / Q000737:chemistry; D005260:Female; D016044:Fusion Proteins, bcr-abl / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008297:Male; D051788:Myeloid-Lymphoid Leukemia Protein; D015514:Oncogene Proteins, Fusion / Q000235:genetics / Q000378:metabolism; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000145:classification / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D012334:RNA, Neoplasm / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014178:Translocation, Genetic; D000097764:ETS Translocation Variant 6 Protein
D050676:Core Binding Factor Alpha 2 Subunit; D017931:DNA Primers; D016044:Fusion Proteins, bcr-abl; D018398:Homeodomain Proteins; D051788:Myeloid-Lymphoid Leukemia Protein; D015514:Oncogene Proteins, Fusion; D012333:RNA, Messenger; D012334:RNA, Neoplasm; C113855:MLL-AF4 fusion protein, human; C102421:TEL-AML1 fusion protein; D000097764:ETS Translocation Variant 6 Protein; C089847:E2A-Pbx1 fusion protein