Minimal residual disease monitoring by quantitative RT-PCR in core binding factor AML allows risk stratification and predicts relapse: results of the United Kingdom MRC AML-15 trial.

The clinical value of serial minimal residual disease (MRD) monitoring in core binding factor (CBF) acute myeloid leukemia (AML) by quantitative RT-PCR was prospectively assessed in 278 patients [163 with t(8;21) and 115 with inv(16)] entered in the United Kingdom MRC AML 15 trial. CBF transcripts were normalized to 10(5) ABL copies. At remission, after course 1 induction chemotherapy, a > 3 log reduction in RUNX1-RUNX1T1 transcripts in BM in t(8;21) patients and a > 10 CBFB-MYH11 copy number in peripheral blood (PB) in inv(16) patients were the most useful prognostic variables for relapse risk on multivariate analysis. MRD levels after consolidation (course 3) were also informative. During follow-up, cut-off MRD thresholds in BM and PB associated with a 100% relapse rate were identified: for t(8;21) patients BM > 500 copies, PB > 100 copies; for inv(16) patients, BM > 50 copies and PB > 10 copies. Rising MRD levels on serial monitoring accurately predicted hematologic relapse. During follow-up, PB sampling was equally informative as BM for MRD detection. We conclude that MRD monitoring by quantitative RT-PCR at specific time points in CBF AML allows identification of patients at high risk of relapse and could now be incorporated in clinical trials to evaluate the role of risk directed/preemptive therapy.

Imaging of multiple mRNA targets using quantum dot based in situ hybridization and spectral deconvolution in clinical biopsies.

Gene expression mapping using microarray analysis has identified useful gene signatures for predicting outcome. However, little of this has been translated into clinically effective diagnostic tools as microarrays require high quality fresh-frozen tissue samples. We describe a methodology of multiplexed in situ hybridization (ISH) using a novel combination of quantum dot (QD)-labeled oligonucleotide probes and spectral imaging analysis in routinely processed, formalin-fixed paraffin embedded human biopsies. The conditions for QD-ISH were optimized using a poly d(T) oligonucleotide in decalcified bone marrow samples. Single and multiplex QD-ISH was performed in samples with acute leukemia and follicular lymphoma using oligonucleotide probes for myeloperoxidase, bcl-2, survivin, and XIAP. Spectral imaging was used for post hybridization tissue analysis, enabling separation of spatially colocalized signals. The method allows quantitative characterization of multiple gene expression using non-bleaching fluorochromes. This is expected to facilitate multiplex in situ transcript detection in routinely processed human clinical tissue.

Monitoring AML1-ETO and CBFbeta-MYH11 transcripts in acute myeloid leukemia.

The core-binding factor (CBF) leukemias comprise acute myeloid leukemia (AML) with t(8;21) and inv(16)/t(16;16), characterized by the presence of the AML1-ETO and CBFbeta-MYH11 fusion genes, respectively. These leukemia-associated genes can now be sensitively and reliably quantified by real-time reverse transcription polymerase chain reaction (RT-PCR) techniques and thus can serve as molecular targets for monitoring residual leukemia. Studies to date suggest that quantitative monitoring of minimal residual disease (MRD) in CBF-positive AML is useful in distinguishing patients at high risk of relapse from those in durable remission. Preliminary results of MRD monitoring by real-time RT-PCR in this subset of AML patients are promising and provide the basis for further evaluation by quantitative analysis in large prospective clinical trials.