Posted November 1, 2006
Zsolt Jobbagy, MD, PhD
CAP Biochemical and Molecular Genetics Subcommittee
Most patients with chronic myeloid leukemia (CML) exhibit the (9:22) translocation (Philadelphia chromosome). The translated BCR/ABL fusion product is an oncogenic protein with increased ABL tyrosine kinase activity causing neoplastic transformation. Although the diagnosis of CML has been used for a century and a half, only in the last 15 years have new therapeutic modalities significantly impacted the prognosis and quality of life of these patients.1 The therapeutic use of allogeneic stem cell transplantation, interferon-alpha and, most recently, selective tyrosine kinase inhibitors has promoted the development of new techniques for identifying and measuring BCR/ABL transcripts to assess response to therapy.2 Post-therapy follow-up to detect and quantify minimal residual disease (MRD) has become an indispensable component of CML management and prognostication. MRD is commonly defined as persistence of low numbers of leukemic cells despite the absence of histologic evidence and clinical signs and/or symptoms of disease.3
Although classical cytogenetics is still recommended to establish a new diagnosis of CML, its utility for monitoring minimal residual disease is limited by the need for a satisfactory bone marrow biopsy to assess metaphases and its poor sensitivity (need at least 5% abnormal cells for detection) due to the low number of cells examined (20 metaphases). The application of fluorescence in situ hybridization (FISH) can theoretically increase sensitivity tenfold (0.5% detection) by examining 200 white blood cells, but the practical detection level of FISH is 1% or higher.4
Reverse transcriptase-polymerase chain reaction (RT-PCR) amplification is a substantially more sensitive technique for detecting low copy numbers of the BCR/ABL fusion transcript, and the combination of RT-PCR with real-time fluorescence detection allows for reliable quantization. The reported sensitivity for quantitative RT-PCR methods varies, but it typically is expected to detect one leukemia cell in 105 normal white blood cells.5 Higher analytic sensitivity can be obtained using qualitative nested RT-PCR analysis, although the clinical significance of low levels of disease detected by such techniques is not established.
Continuous monitoring of CML MRD using RT-PCR-based quantitative techniques after bone marrow transplantation or during continuous drug therapy allows the assessment of initial response and can alert physicians to potential relapses, even in cases demonstrating cytogenetic remission. This information is invaluable for the adjustment of treatment plans and bears important prognostic significance.6,7 Moreover, it has recently been established that the degree of molecular response at the time of or after achieving complete cytogenetic remission is an independent prognostic factor for progression free survival.8
Despite standardization efforts, such as those within the Europe Against Cancer (EAC) Program in 2003,9 quantitative methods for measuring BCR/ABL transcripts include an array of home-brew and commercial RT-PCR based assays. This variety, along with the absence of an agreed calibration standard, has limited the ability for inter-laboratory correlation of results, although progress has been made establishing baselines and reporting log reductions from those baselines.2,8 Additional progress in these areas, as well as the use of self-contained automated systems for measuring BCR/ABL levels,10 can be anticipated to facilitate more uniform performance and interpretation of CML monitoring over the next several years
- Baccarani M, Saglio G, Goldman J, et al. Evolving concepts in the management of chronic myeloid leukemia. Recommendations from an expert panel on behalf of the European Leukemianet. Blood. 2006; prepublished online May 18.
- Hughes TP, Deininger MW, Hochhaus A, et al. Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for 'harmonizing' current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results. Blood. 2006;108(1):28–37. 3.
- Butturini A, Klein J, Gale RP. Modeling minimal residual disease
(MRD)-testing. Leuk Res. 2003;27(4):293–300.
- Pelz AF, Kroning H, Franke A, et al. High reliability and sensitivity of the BCR/ABL1 D-FISH test for the detection of BCR/ABL rearrangements. Ann Hematol. 2002;81(3):147–153.
- Goldman J. Monitoring minimal residual disease of BCR-ABL-positive chronic myeloid leukemia in the imatinib era. Curr Opin Hematol. 2005;12(1):33–39.
- Mughal TI, Yong A, Szydlo RM, et al. Molecular studies in patients with chronic myeloid leukaemia in remission 5 years after allogeneic stem cell transplant define the risk of subsequent relapse. Br J Haematol. 2001;115(3):569–574.
- Olavarria E, Kanfer E, Szydlo R, et al. Early detection of BCR/ABL transcripts by quantitative RT-PCR predicts outcome after allogeneic stem cell transplantation for chronic myeloid leukemia. Blood. 2001;97(6):1560–1565.
- Press RD, Love Z, Tronnes AA, et al. BCR-ABL mRNA levels at and after the time of a complete cytogenetic response (CCR) predict the duration of CCR in imatinib mesylate-treated patients with CML. Blood. 2006;107(11):4250–4256
- Gabert J, Beillard E, van der Velden VHJ, et al. Standardization and quality control studies of 'real-time' quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia—a Europe Against Cancer program. Leukemia. 2003;17(12):2318–2357.
- Winn-Deen E, Helton B, Van Atta R, et al. Development of an
integrated assay for quantitative real-time detection of BCR-ABL
RNA from peripheral blood. Haematologica. 2006;91(s1):19–20.
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