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

Forward Reverse
Fluorescent
Primer Primer
Taqman Probe

Partner 1 Partner 2

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 10 5-106)
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
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

Std.
wild type mutate
RT-
d
PCR

*
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 LIAP 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 LIAP box
Many LAIPS have a low frequency in
normal BM
B: different-from normal approach
(monocytic leukemia
 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
 Answer
A: Positive for MRD

B: Regeneration

C: Indeterminate