Clinical Applications of BCR-ABL Molecular Testing in Acute Leukemia

Abstract

Recent advances in molecular genetics impact the health care and outcome of patients with acute lymphoblastic leukemia (ALL). BCR-ABL, a common molecular defect in adult ALLIn 1960, Nowell and Hungerford 1 reported the discovery of what came to be known as the Philadelphia chromosome (Ph 1 ) in association with chronic myelogenous leukemia (CML). Their short report entitled "Minute chromosome in human chronic granulocytic leukemias" was the first to demonstrate a leukemia-specific genetic abnormality. In the ensuing decades, the pathology of the Ph 1 has been studied and it is now known to represent an abnormally shortened (derivative) chromosome 22 resulting from translocation with chromosome 9.2 The t(9;22) is found in over 90% of CMLs, in a lesser proportion of acute lymphoblastic leukemias (ALL) or biphenotypic acute leukemias, and in rare cases of de novo acute myelogenous leukemia (AML).The break on chromosome 22q11.2 usually occurs in the major breakpoint cluster region (M-BCR), in the minor breakpoint cluster region (m-BCR), or rarely at other nearby sites. The break on chromosome 9q34 involves the ABL gene, named after Abelson murine leukemia virus where a viral version of the ABL gene was first discovered. The translocation brings the 5Ј end of the BCR gene into juxtaposition with the tyrosine kinase domain of the ABL gene to produce a hybrid gene retaining tyrosine kinase activity. 3Depending on whether the M-BCR or m-bcr breakpoint is involved in the translocation, transcription of the hybrid gene results in chimeric mRNA encoding a 210 kd BCR-ABL fusion protein or a 190 kd BCR-ABL fusion protein, respectively. The reciprocal ABL-BCR translocation also forms a chimeric gene that is capable of being transcribed, but the pathological significance of this reciprocal chimeric gene product is uncertain. Laboratory Tests for t(9;22)Conventional cytogenetics is the recommended test for detecting t(9;22) in newly diagnosed leukemia patients. Chromosome banding analysis has the advantage of high specificity and an ability to detect alternate or additional cytogenetic defects that are valuable in diagnosis and prognosis. However, cytogenetic analysis requires viable marrow cells or more than 10% blasts in the peripheral blood to reliably culture the cells and visualize metaphases. Occasionally fibrosis interferes with marrow aspiration, yielding few analyzable metaphase cells. The number of cells examined determines the sensitivity of karyotyping. A typical examination of 20 cells carries a sensitivity of one in 20, or 5%. Cryptic t(9;22) occurs in about 5% of CML cases and also in a small proportion of ALLs, resulting in false negative karyotypic interpretation in metaphase spreads of cells that actually contain the translocation at the molecular level.

Evaluation of the Cepheid GeneXpert BCR-ABL Assay

“…Generally, the utility of chromosome banding for monitoring minimal residual disease is limited by the need for satisfactory cell culture for visualization of the metaphases. 4 Furthermore, the relatively low number of cells examined (usually 20 metaphases) results in sensitivities similar to those of the routine histological examination of the bone marrow for the presence of leukemic cells (5%). 5 Sensitivity can theoretically be increased by about 10-fold (to 0.5% leukemia cell content detection) by using fluorescence in situ hybridization and examining 200 white blood cells (WBCs); however, the practical sensitivity of interphase fluorescence in situ hybridization is only about 1%.…”

Influence of detection of pretreatment cytogenetic abnormalities on first complete remission and survival in adult acute lymphoblastic leukemia

“…The Philadelphia (Ph) chromosome was the most frequent karyotypic aberration in the high-risk group (35.7% in the high-risk group versus 15.15% in all 33 ALL patients), which is higher than previously reported (15%-30%) [7,13,19,[26][27][28][29][30]. Our observa- CR-complete remission; RD-resistant disease tion pointed for variable immunophenotypes in Ph(+) ALL, without any apparent connection to a specific phenotype.…”

Applications of Real-time Reverse Transcription Polymerase Chain Reaction in Clinical Virology Laboratories for the Diagnosis of Human Diseases

“…This technique is a refinement of the original PCR developed by Kary Mullis and co-workers in the mid 1980's [5,6] . It's potential for high-throughput; together with regular introduction of enhanced or novel chemistries, more reliable instrumentation and improved protocols [7,8] made it the choice of diagnostics. The principle of rRT-PCR assays is as follows, the RNA will be reverse transcriped into cDNA, and it requires a suitable detection system for reporting the presence of PCR products, to monitor the amplification in real-time and the software programs for quantitative analysis [9] .…”