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Monitoring of Minimal Residual Disease Principles and Applications. Pei Lin, MD Department of Hematopathology UT M.D. Anderson Cancer Center, Houston, TX. MRD studies in AML: Potential Utility. Definition: Residual disease morphologic complete remission (CR) ( ≤ 5% blasts)
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Monitoring of Minimal Residual Disease Principles and Applications Pei Lin, MD Department of Hematopathology UT M.D. Anderson Cancer Center, Houston, TX
MRD studies in AML: Potential Utility • Definition: Residual disease morphologic complete remission (CR) (≤ 5% blasts) • Time points of testing: post induction, post consolidation, pre-transplant, during CR • Prognostic in most studies • Effectiveness of therapy: quantitative, kinetics • Guidance for risk adjusted therapy • Distinguish early recovery from persistent AML • Predicting early relapse
Methods MRD by PCR Leukemic fusion genes (PML-RARA) Mutations (NPM1) Gene overexpression (WT1) Multiparameter flow cytometry (FCM)
Translocation-specific Quantitative RT-PCR Break-Point Reverse Primer Forward Primer Fluorescent Taqman Probe Amplicon t(8;21) RUNX1-RUNX1T1 Positive Negative
MRD by PCR: Leukemic Fusion Transcripts • Recurrent fusions, e.g. t(15;17), t(8;21), inv(16) • Together ~30% of AMLs • qRT-PCR assays • Highly sensitive (1 in 105-106) • Normalize to control • Absolute copy number vs. degree of reduction • RNA instability, turn around time • Limited applicability • Establish standardized assays and cut-offs
Mutation detection – NPM1 Std. RT-PCR wild type mutated • NPM1 mutations in ~30% of overall AML, ~50% of AML with normal karyotype • Most are 4 bp insertions, two adjacent sites • Allele-specific primers detect 1 in 104-105 • Post-therapy MRD is prognostic, can monitor kinetics to predict relapse* • Other potential markers: FLT3, MLL-PTD, KIT, DMNT3A *Schnittger et al. 2009, Blood 114:2220
Detection of MRD by flow cytometry in AML • Identify aberrant vs. normal myeloid precursors • Leukemia-associated immunophenotypes [LA(I)Ps]: • Aberrant lymphoid antigen (CD19, CD7, CD56…) • Aberrant levels of normally expressed antigens (↓,↑ CD38, CD34…) • Coexpression of early and later antigens (CD34++CD15++) • Altered forward and side scatter
Approaches • Must know the patterns of normal and recovering bone marrow • If available, compare MRD to the original phenotype • Need detailed description of antigen expression or dot-plots • Rely on “LAIP” or deviation form normal to identify leukemic cells
Criteria for Dx and Sensitivity • LAIP vs “different-from normal” approach • A: LAIP approach: • Find aberrant clusters of at least 20 cells, showing abnormal expression of at least 2 markers in the LAIP box • Many “LAIP” have a low frequency in normal BM • B: “different-from normal” approach (monocytic leukemia)
8-color MRD: Baseline study Courtesy of Dr. Jeffrey Jorgensen
BM CD34+: Normal vs. AML MRD Normal AML MRD, 0.1% Courtesy of Dr. Jeffrey Jorgensen
Patient 1 FG, 1-31-2013 FG, 11-9-2012 CD117PE Normal CD34 PE-CY7
Patient 2: 21 days post induction Morphology: 21% of blasts
Post therapy CD34 PE-Cy7 CD64 APC Original
Sensitivity To yield sensitivity of 0.01%, collect at least 200K cells per tube (20/200K = 1 in 104 = 0.01%) Sensitivity may be limited due to background normal cells, 0.1% or higher * 0.1% is commonly used threshold in the literature
Detection of MRD by Flow Cytometry • Advantages: • Widely applicable (90- 95% of cases) • Relatively rapid turn around time • Disadvantages: • Interpretation often challenging, requires experience • Can be expensive • Lack of standardization
Potential challenges • LAIPs may not cover all leukemic blasts, partial overlap with normal • Antigen shift resulting from selection/emergence of subclones • A complete change in LAIPs in about 20% of AML, with 80% having at least one LAIP similar to the original (Voskova et al) • Post therapy hypocellular sample • Use a comprehensive panel of antibodies to establish baseline
Summary • MRD detection by FCM or/and PCR are promising tools to guide therapy and to improve outcomes • Each method has pros and cons • More studies are underway to better incorporate the data into clinical decision making (dose intensification and/or ASCT) • Timing of MRD testing by FCM and the cut off levels for each time point that are significant are being refined
Acknowledgement • Dr. Jeffrey Jorgensen MD Anderson Cancer Center