Molecular diagnosis and monitoring of CML

The traditional method of diagnosing CML and monitoring disease status is the cytogenetic analysis of bone marrow-derived metaphases for the presence of the Philadelphia chromosome. However, cytogenetics is negative in approximately 10% of patients at diagnosis and at least half of these patients will have a BCR-ABL fusion detectable by either fluorescence in situ hybridization (FISH) or RT-PCR. The level of sensitivity of cytogenetics is about 1%, which means that in a patient in apparent cytogenetic remission, more than 1010 leukemic cells may still be present. The most sensitive method for the detection of residual leukemic cells is RT-PCR, which can detect one cell in a background of 105 to 106 cells. This means that, even with a negative result, a large number of malignant cells may still be present and could contribute to disease relapse.

FISH analysis relies on the co-localization of large genomic probes specific to the BCR and ABL genes. FISH has several advantages over conventional cytogenetics. It can be performed on metaphase or interphase cells and on peripheral blood. Comparison of marrow and blood samples by FISH analysis shows high concordance. One potential problem with FISH is the random co-localization of the signals from the BCR and ABL probes, such that 8-10% of normal cells will appear positive. This has been circumvented, in part, by D-FISH (double FISH), which uses probes that span the breakpoint region. However, at diagnosis, when most cells are BCR-ABL-posi-tive, FISH is a highly accurate diagnostic test as false-negative results are a rarity and false positives are not a concern when more than 90% of cells are positive.

RT-PCR can be used to amplify the region around the splice junction between BCR and ABL. The high sensitivity of this technique makes it ideal for the detection of minimal residual disease. RT-PCR and, more recently, quantitative RT-PCR have been used to detect residual CML cells following allogeneic stem cell transplantation. These studies have shown that BCR-ABL transcripts can be detected for several months after transplantation. Whether persistence of BCR-ABL transcripts beyond 6 months is predictive of relapse has not been resolved, but a rising level of BCR-ABL transcripts by quantitative RT-PCR after transplantation is highly predictive of relapse. As with FISH in peripheral blood and marrow, RT-PCR the values show a high level of concordance. Both false-positive and false-negative results are possible with RT-PCR and rigorous controls are required to detect these events. False negatives can be due to poor-quality RNA or failure of the reaction, while false positives can be due to contamination. As the majority of patients treated with imatinib will be cytogenetically and FISH-negative, PCR monitoring has been increasingly incorporated into monitoring strategies. Studies of the levels of molecular response to imatinib are being conducted to determine if this correlates with outcome.

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