1. Pei Lin, MD Department of Hematopathology UT M.D. Anderson Cancer Center, Houston, TX Monitoring of Minimal Residual Disease Principles and Applications

2. MRD studies in AML: Potential Utility Definition: Residual disease morphologic complete remission (CR) (≤ 5% blasts) Definition: Residual disease morphologic complete remission (CR) (≤ 5% blasts) Time points of testing: post induction, post consolidation, pre-transplant, during CR Time points of testing: post induction, post consolidation, pre-transplant, during CR Prognostic in most studies Prognostic in most studies Effectiveness of therapy: quantitative, kinetics Effectiveness of therapy: quantitative, kinetics Guidance for risk adjusted therapy Guidance for risk adjusted therapy Distinguish early recovery from persistent AML Distinguish early recovery from persistent AML Predicting early relapse Predicting early relapse

3. Methods MRD by PCR Leukemic fusion genes (PML-RARA) Mutations (NPM1) Gene overexpression (WT1) Multiparameter flow cytometry (FCM)

4. Partner 1Partner 2 Fluorescent Taqman Probe Amplicon Forward Primer Reverse Primer Break-Point Translocation-specific Quantitative RT-PCR Translocation-specific Quantitative RT-PCR t(8;21) RUNX1-RUNX1T1 PositiveNegative

5. MRD by PCR: Leukemic Fusion Transcripts Recurrent fusions, e.g. t(15;17), t(8;21), inv(16) Recurrent fusions, e.g. t(15;17), t(8;21), inv(16) Together ~30% of AMLs Together ~30% of AMLs qRT-PCR assays qRT-PCR assays Highly sensitive (1 in 10 5 -10 6 ) Highly sensitive (1 in 10 5 -10 6 ) Normalize to control Normalize to control Absolute copy number vs. degree of reduction Absolute copy number vs. degree of reduction RNA instability, turn around time RNA instability, turn around time Limited applicability Limited applicability Establish standardized assays and cut-offs Establish standardized assays and cut-offs

6. Mutation detection – NPM1 NPM1 mutations in ~30% of overall AML, ~50% of AML with normal karyotype NPM1 mutations in ~30% of overall AML, ~50% of AML with normal karyotype Most are 4 bp insertions, two adjacent sites Most are 4 bp insertions, two adjacent sites Allele-specific primers detect 1 in 10 4 -10 5 Allele-specific primers detect 1 in 10 4 -10 5 Post-therapy MRD is prognostic, can monitor kinetics to predict relapse * Post-therapy MRD is prognostic, can monitor kinetics to predict relapse * Other potential markers: FLT3, MLL-PTD, KIT, DMNT3A Other potential markers: FLT3, MLL-PTD, KIT, DMNT3A * Schnittger et al. 2009, Blood 114:2220 mutatedwild type Std. RT- PCR

7. Detection of MRD by flow cytometry in AML Identify aberrant vs. normal myeloid precursors Identify aberrant vs. normal myeloid precursors Leukemia-associated immunophenotypes [LA(I)Ps]: Leukemia-associated immunophenotypes [LA(I)Ps]: Aberrant lymphoid antigen (CD19, CD7, CD56…) Aberrant lymphoid antigen (CD19, CD7, CD56…) Aberrant levels of normally expressed antigens ( ↓,↑ CD38, CD34…) Aberrant levels of normally expressed antigens ( ↓,↑ CD38, CD34…) Coexpression of early and later antigens (CD34++CD15++) Coexpression of early and later antigens (CD34++CD15++) Altered forward and side scatter Altered forward and side scatter

8. Approaches Must know the patterns of normal and recovering bone marrow Must know the patterns of normal and recovering bone marrow If available, compare MRD to the original phenotype If available, compare MRD to the original phenotype Need detailed description of antigen expression or dot-plots Need detailed description of antigen expression or dot-plots Rely on “LAIP” or deviation form normal to identify leukemic cells Rely on “LAIP” or deviation form normal to identify leukemic cells

9. Criteria for Dx and Sensitivity Criteria for Dx and Sensitivity LAIP vs “different-from normal” approach LAIP vs “different-from normal” approach A: LAIP approach: A: LAIP approach: Find aberrant clusters of at least 20 cells, showing abnormal expression of at least 2 markers in the LAIP box 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 Many “LAIP” have a low frequency in normal BM B: “different-from normal” approach (monocytic leukemia) B: “different-from normal” approach (monocytic leukemia)

10. 8-color MRD: Baseline study Courtesy of Dr. Jeffrey Jorgensen

11. BM CD34+: Normal vs. AML MRD Normal AML MRD, 0.1% Courtesy of Dr. Jeffrey Jorgensen

12. Normal BM

13. Normal Bone Marrow

14. Normal BM

16. FG, 1-31-2013 FG, 11-9-2012 CD117PE CD34 PE-CY7 Normal Patient 1

17. Patient 2: 21 days post induction Morphology: 21% of blasts

18. Post therapy CD64 APC CD34 PE-Cy7 Original

19. Sensitivity To yield sensitivity of 0.01%, collect at least 200K cells per tube (20/200K = 1 in 10 4 = 0.01%) To yield sensitivity of 0.01%, collect at least 200K cells per tube (20/200K = 1 in 10 4 = 0.01%) Sensitivity may be limited due to background normal cells, 0.1% or higher Sensitivity may be limited due to background normal cells, 0.1% or higher * 0.1% is commonly used threshold in the literature * 0.1% is commonly used threshold in the literature

20. Detection of MRD by Flow Cytometry Advantages: Advantages: Widely applicable (90- 95% of cases) Widely applicable (90- 95% of cases) Relatively rapid turn around time Relatively rapid turn around time Disadvantages: Disadvantages: Interpretation often challenging, requires experience Interpretation often challenging, requires experience Can be expensive Can be expensive Lack of standardization Lack of standardization

21. Potential challenges LAIPs may not cover all leukemic blasts, partial overlap with normal LAIPs may not cover all leukemic blasts, partial overlap with normal Antigen shift resulting from selection/emergence of subclones 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) 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 Post therapy hypocellular sample Use a comprehensive panel of antibodies to establish baseline Use a comprehensive panel of antibodies to establish baseline

22. Summary MRD detection by FCM or/and PCR are promising tools to guide therapy and to improve outcomes MRD detection by FCM or/and PCR are promising tools to guide therapy and to improve outcomes Each method has pros and cons Each method has pros and cons More studies are underway to better incorporate the data into clinical decision making (dose intensification and/or ASCT) 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 Timing of MRD testing by FCM and the cut off levels for each time point that are significant are being refined

23. Acknowledgement Dr. Jeffrey Jorgensen MD Anderson Cancer Center