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Acute Leukemia
11/06/2015
Why MRD?
J.J.M van Dongen, et al. Blood. 2015 Jun 25; 125(26): 39964009.
Hazard ratio
Dar 29 MRD>0.01%
4.31
<0.001
2.25
<0.001
Trisomies 4 and 10
.570
<0.001
1.51
.018
TEL-AML1
.778
.151
Day8 M1 marrow
1.034
.789
COG: AML
MRD at the end of therapy
Disadvantages:
Not standardized
Subjective interpretation
Pattern Recognition
Two Strategies:
Leukemia-Associated immunophenotype (LAP)
Deviation from normal maturation
- Advantages
- Conceptually simple and objective
- Reduced reagent expense for follow up
- Disadvantages
- Requires pre-treatment sample to define LAP
- Requires immunophenotypic stability
- Any event in pre-defined gate regarded as MRD
- Background noise (nonspecific staining)
Diagnostic BM,
Day 29 BM:
MRD: 0.09%
Immunophenotypic Stability
Immunophenotypic Stability
ALL
- 30 consecutive patients with MRD detectable
- All had some change in immunophenotype
- CD10 and CD34 down-modulation, CD19, CD20 and CD45RA
up-modulation, CD58 stable
- Associated with use of steroid in induction therapy
Immunophenotypic Stability
T-ALL
- Disadvantages:
Requires detailed immunophenotypic knowledge (expert)
Subjective
More time consuming
http://www.cytometry.org/newsletters/eICCS-2-1/article3.php
End of Induction:
MRD: 0.5%
ALL
>95% of pediatric ALL
>90-95% of adult ALL
AML
85% of pediatric AML
80-95% of adult AML
Timing
Induction nadir (day 14)
- Reduced background populations
- Hypoplastic with many apoptotic cells
End of induction
- ALL - Few immature B cells
- AML - Active marrow regeneration, increased precursors
End of consolidation
- ALL - Larger number of immature B cells
- AML - Normal marrow populations
Quantitation
Analytical Sensitivity
ALL: 0.01%, AML: 0.1%
Determinants:
Degree of immunophenotypic aberrancy
Number and immunophenotype of background populations
Denominator
Total nucleated cells
- Most comparable to morphology
- DNA binding dye often used (Syto16, Draq5, etc.)
Incomplete RBC lysis, platelet aggregates
White cells
- CD45 positive cells + neoplasm
- Variable CD45 on early NRBCs
- Overestimation with erythroid hyperplasia
Mononuclear cells
- Exclude granulocytic lineage (high side scatter)
- Most comparable to Ficoll-prepared samples
Early MRD literature used Ficoll
Hemodilution
Bone marrow is a semi-solid tissue
- Absolute cell concentration has little meaning
Sources of Variability
Identification (false positive or negative)
- Insufficiently informative reagents
- Improper assay validation
- Immunophenotypic shift
- Inexperienced interpreters
Quantitation
- Too few events acquired
- Denominator effects (2 fold)
- Sample degeneration
- Hemodilution
Reproducibility
Central reference lab system
- East= Dr. Borowitz, John Hopkins University
- West= Dr. Wood, University of Washington
% of cases 50
MRD >.01%
40
30
20
10
0
JHU(n=2282)
UW(n=1947)
d29
d15*
d8 blood
Standardization/COG decentralization
Not standardized
NCI funding mechanism changed
- No longer funds reference lab testing in support of clinical trials
- When: June 2016
Decentralization:
- COG B-ALL Flow MRD Testing Approval Process
Calculation
Tube1/2(MRD/B cells) x Tube3(B cells/Mononuclear cells)
Molecular tests
RT-PCR of fusion transcript or mutations
Ryan J., et al. MRD detection in childhood ALL patients at multiple time-points reveals
high levels of concordance between molecular and immunophenotypic approaches. Br
J Haematol 2009 144: 107-115
Pre- and day 29 post-treatment B lymphoblast frequencies by HTS versus mpFC pretreatment
and day 29 post-treatment, clonal B lymphoblasts were identified by HTS (red) or by mpFC
(blue; N = 91).
Conclusions
Minimal residual disease (MRD) is strongly associated with poor
outcome
MRD detection is a very important step in current risk-stratified
leukemia treatment
Two sensitive detection methods: Flow cytometry and molecular
Flow cytometry is the standard method for MRD detection in COG
protocols in USA
Flow cytometry has the advantages of fast, cost effective, sensitive
and applicable to wide range of leukemia
Immunophenotype change is common
Accurate FCM detection needs consistent FCM techniques,
informative immunophenotype and knowledge of normal patterns of
antigen expression
NGS, more sensitive MRD detection method is in development