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

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

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

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

5. MRD by PCR: Leukemic Fusion TranscriptsMRD 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 AMLsTogether ~30% of AMLs
• qRT-PCR assaysqRT-PCR assays
• Highly sensitive (1 in 10Highly sensitive (1 in 1055
-10-1066
))
• Normalize to controlNormalize to control
• Absolute copy number vs. degree of reductionAbsolute copy number vs. degree of reduction
• RNA instability, turn around timeRNA instability, turn around time
• Limited applicabilityLimited applicability
• Establish standardized assays and cut-offsEstablish standardized assays and cut-offs

6. Mutation detection –Mutation detection – NPM1NPM1
• NPM1NPM1 mutations in ~30% of overall AML, ~50% of AMLmutations in ~30% of overall AML, ~50% of AML
with normal karyotypewith normal karyotype
• Most are 4 bp insertions, two adjacent sitesMost are 4 bp insertions, two adjacent sites
• Allele-specific primers detect 1 in 10Allele-specific primers detect 1 in 1044
-10-1055
• Post-therapy MRD is prognostic, can monitor kinetics toPost-therapy MRD is prognostic, can monitor kinetics to
predict relapsepredict relapse**
• Other potential markers:Other potential markers: FLT3, MLL-PTD, KIT, DMNT3AFLT3, MLL-PTD, KIT, DMNT3A
*
Schnittger et al. 2009, Blood 114:2220
mutatedwild type
Std.
RT-
PCR

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

8. Approaches
• Must know the patterns ofMust know the patterns of normalnormal andand
recoveringrecovering bone marrowbone marrow
• If available, compare MRD to the originalIf available, compare MRD to the original
phenotypephenotype
• Need detailed description of antigenNeed detailed description of antigen
expression or dot-plotsexpression or dot-plots
• Rely on LIAP to identify leukemic cellsRely on LIAP to identify leukemic cells

9. Criteria for Dx and SensitivityCriteria for Dx and Sensitivity
• LAIP vs “different-from normal” approachLAIP vs “different-from normal” approach
• A: LAIP approach:A: LAIP approach:
• Find aberrant clusters of at least 20 cells,Find aberrant clusters of at least 20 cells,
showing abnormal expression of at least 2showing abnormal expression of at least 2
markers in the LIAP boxmarkers in the LIAP box
• ManyMany “LAIPS” have a low frequency in normal“LAIPS” have a low frequency in normal
BMBM
• B: “different-from normal” approach (monocyticB: “different-from normal” approach (monocytic
leukemialeukemia

10. Sensitivity
• To yield sensitivity of 0.01%, collect at leastTo yield sensitivity of 0.01%, collect at least
200K cells per tube (20/200K = 1 in 10200K cells per tube (20/200K = 1 in 1044
==
0.01%)0.01%)
• Sensitivity may be limited due to backgroundSensitivity may be limited due to background
normal cells, 0.1% or highernormal cells, 0.1% or higher
• * 0.1% is commonly used threshold in the literature* 0.1% is commonly used threshold in the literature

11. Detection of MRD by Flow CytometryDetection of MRD by Flow Cytometry
• Advantages:Advantages:
• Widely applicable (90- 95% of cases)Widely applicable (90- 95% of cases)
• Relatively rapid turn around timeRelatively rapid turn around time
• Disadvantages:Disadvantages:
• Interpretation often challenging, requiresInterpretation often challenging, requires
experienceexperience
• Can be expensiveCan be expensive
• Lack of standardizationLack of standardization

12. Potential challenges
• LAIPs may not cover all leukemic blasts, partialLAIPs may not cover all leukemic blasts, partial
overlap with normaloverlap with normal
• Antigen shift resulting from selection/emergenceAntigen shift resulting from selection/emergence
of subclonesof subclones
• A complete change in LAIPs in about 20% ofA complete change in LAIPs in about 20% of
AML, with 80% having at least one LAIPAML, with 80% having at least one LAIP
similar to the original (Voskova et al)similar to the original (Voskova et al)
• Post therapy hypocellular samplePost therapy hypocellular sample
• Use a comprehensive panel of antibodies toUse a comprehensive panel of antibodies to
establish baselineestablish baseline

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

14. Acknowledgement
• Dr. Jeffrey Jorgensen MD Anderson Cancer Center

15. Answer
• A: Positive for MRD
• B: Regeneration
• C: Indeterminate