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Preclinical validation of a monochrome real-time multiplex assay for translocations in childhood acute lymphoblastic leukemia.
Clin Cancer Res. 2002 Dec; 8(12):3832-40.CC

Abstract

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.

Authors+Show Affiliations

Research, King Fahad National Centre for Children's Cancer and Research, King Faisal Specialist Hospital and Research Centre, Riyadh, 11211 Saudi Arabia.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Validation Study

Language

eng

PubMed ID

12473597

Citation

Siraj, Abdul K., et al. "Preclinical Validation of a Monochrome Real-time Multiplex Assay for Translocations in Childhood Acute Lymphoblastic Leukemia." Clinical Cancer Research : an Official Journal of the American Association for Cancer Research, vol. 8, no. 12, 2002, pp. 3832-40.
Siraj AK, Ozbek U, Sazawal S, et al. Preclinical validation of a monochrome real-time multiplex assay for translocations in childhood acute lymphoblastic leukemia. Clin Cancer Res. 2002;8(12):3832-40.
Siraj, A. K., Ozbek, U., Sazawal, S., Sirma, S., Timson, G., Al-Nasser, A., Bhargava, M., El Solh, H., Bhatia, K., & Gutiérrez, M. I. (2002). Preclinical validation of a monochrome real-time multiplex assay for translocations in childhood acute lymphoblastic leukemia. Clinical Cancer Research : an Official Journal of the American Association for Cancer Research, 8(12), 3832-40.
Siraj AK, et al. Preclinical Validation of a Monochrome Real-time Multiplex Assay for Translocations in Childhood Acute Lymphoblastic Leukemia. Clin Cancer Res. 2002;8(12):3832-40. PubMed PMID: 12473597.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Preclinical validation of a monochrome real-time multiplex assay for translocations in childhood acute lymphoblastic leukemia. AU - Siraj,Abdul K, AU - Ozbek,Ugur, AU - Sazawal,Sudha, AU - Sirma,Sema, AU - Timson,Georgina, AU - Al-Nasser,Abdallah, AU - Bhargava,Manorama, AU - El Solh,Hassan, AU - Bhatia,Kishor, AU - Gutiérrez,Marina I, PY - 2002/12/11/pubmed PY - 2003/1/22/medline PY - 2002/12/11/entrez SP - 3832 EP - 40 JF - Clinical cancer research : an official journal of the American Association for Cancer Research JO - Clin Cancer Res VL - 8 IS - 12 N2 - 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. SN - 1078-0432 UR - https://www.unboundmedicine.com/medline/citation/12473597/Preclinical_validation_of_a_monochrome_real_time_multiplex_assay_for_translocations_in_childhood_acute_lymphoblastic_leukemia_ L2 - http://clincancerres.aacrjournals.org/cgi/pmidlookup?view=long&pmid=12473597 DB - PRIME DP - Unbound Medicine ER -