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JMD 2003, Vol. 5, No. 4
Copyright © 2003 American Society for Investigative Pathology & Association for Molecular Pathology

Multiplex RT-PCR for the Detection of Leukemia-Associated Translocations

Validation and Application to Routine Molecular Diagnostic Practice

Manuel Salto-Tellez*{dagger}, Suresh G. Shelat{dagger}, Bernice Benoit{dagger}, Hanna Rennert{dagger}, Martin Carroll{ddagger}, Debra G.B. Leonard{dagger}, Peter Nowell{dagger} and Adam Bagg{dagger}

From the Department of Pathology, * National University of Singapore, Singapore; the Department of Pathology and Laboratory Medicine {dagger} and the Department of Medicine, {ddagger} Hematology-Oncology Division, University of Pennsylvania, Philadelphia, Pennsylvania

The aim of this study was to validate the application of a commercially available multiplex reverse transcription polymerase chain reaction (RT-PCR) assay [Hemavision-7 System] for the seven most common leukemia translocations for routine molecular diagnostic hematopathology practice. A total of 98 samples, comprising four groups, were evaluated: Group 1, 16 diagnostic samples molecularly positive by our existing laboratory-developed assays for PML-RAR{alpha}/t (15;17) or BCR-ABL/t (9;22); Group 2, 51 diagnostic samples negative by our laboratory-developed assays for PML-RAR{alpha}/t (15;17) or BCR-ABL/t (9;22); Group 3, 21 prospectively analyzed diagnostic cases, without prior molecular studies; and Group 4, 10 minimal residual disease (MRD) samples. Analysis of the two previously studied cohorts (Groups 1 and 2) confirmed the diagnostic sensitivity and specificity of the multiplex assay with regard to these two translocations. Additionally, however, in the "negative" Group (Group 2) the assay revealed three unanticipated translocations (CBFß-MYH11, BCR-ABL, and MLL-AF4), two of which were confirmed on cytogenetics. Analysis of the prospective cohort demonstrated that the assay was cost-effective and amenable to standard laboratory practice, with an identically sensitive MRD detection rate to that of our laboratory-developed tests. Virtually all of the results obtained were consistent with the phenotype and karyotype by conventional methods. This study illustrates the utility of a kit-based multiplex RT-PCR assay for the molecular diagnosis and monitoring of leukemias and reinforces the complementary roles of molecular testing and cytogenetics in diagnostic hematopathology.




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