Realtime PCR applications in hematology

Anne M. Sproul

The success of treatment of patients with leukemia is judged by several criteria. For the patient and clinician the most desirable outcome is sustained remission from disease and long-term survival. Unfortunately it is unlikely that complete eradication of leukemic cells is ever achieved. Hematological remission is defined as fewer than 5% blast cells in the bone marrow as determined by morphology. Disease levels below this threshold detected by more sensitive methods such as flow cytometry, cytogenetics (including FISH), and PCR are referred to as minimal residual disease (MRD). The sensitivity of PCR has extended the detection limit to one leukemic cell in one million normal cells.

Although qualitative or endpoint PCR has played a major role in the diagnosis and monitoring of leukemia, it is now accepted that quantitative data is more informative. Several large trials including molecular monitoring have shown that some patients still have PCR detectable disease during and at the end of treatment, for example the AML1ETO transcript may persist in AML patients in long-term remission (Jurlander et al., 1996). Whereas these studies have been able to predict poor outcome for some patient groups, e.g. PMLRARA positivity at the end of treatment indicates impending relapse (Lo Coco et al., 1999), it has been more difficult to gain accurate prognostic information from PCR results, e.g. childhood acute lymphoblastic leukemia (ALL) (van Dongen et al., 1998) or chronic myeloid leukemia (CML) (Hochhaus et al., 2000). However by measuring the MRD levels it has been shown that it is possible to stratify risk groups on the basis of MRD level at specific time points in treatment (Biondi et al., 2000).

Real-time PCR is the only truly quantitative method available. It offers other advantages over conventional PCR, for example more rapid turnaround as no post PCR processing is necessary and since closed systems are used the risk of contamination is abolished. These are important in diagnostic laboratories. This chapter refers to real-time PCR using hydrolysis or TaqMan® probes; this reflects the author's personal experience not bias. The same analyses can be performed using hybridization probes and should be regarded as interchangeable. However the reagents referred to have been designed for assays using probes and are not validated for the use of DNA-binding dyes such as SYBR® Green 1.

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