monitoring of minimal residual disease principles and applications
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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) - PowerPoint PPT PresentationTRANSCRIPT
Pei Lin, MDPei Lin, MD
Department of Hematopathology Department of Hematopathology
UT M.D. Anderson Cancer Center, Houston, TXUT M.D. Anderson Cancer Center, Houston, TX
Monitoring of Minimal Residual Disease Monitoring of Minimal Residual Disease Principles and ApplicationsPrinciples and Applications
MRD studies in AML: Potential MRD studies in AML: Potential UtilityUtility
• Definition: Residual disease morphologic Definition: Residual disease morphologic complete remission (CR) (complete remission (CR) (≤ ≤ 5% blasts)5% blasts)
• Time points of testing: post induction, post Time points of testing: post induction, post consolidation, pre-transplant, during CR consolidation, 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
Methods
• MRD by PCR
• Leukemic fusion genes (PML-RARA)
• Mutations (NPM1)
• Gene overexpression (WT1)
• Multiparameter flow cytometry (FCM)
Partner 1 Partner 2
FluorescentTaqman Probe
Amplicon
Forward Primer
Reverse Primer
Break-Point
Translocation-specific Quantitative RT-Translocation-specific Quantitative RT-PCRPCR
t(8;21)RUNX1-RUNX1T1 Positive Negative
MRD by PCR: Leukemic Fusion MRD by PCR: Leukemic Fusion TranscriptsTranscripts
• 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 assays qRT-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
Mutation detection – Mutation detection – NPM1NPM1• NPM1 NPM1 mutations in ~30% of overall AML, ~50% of mutations in ~30% of overall AML, ~50% of
AML with normal karyotypeAML with 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 Post-therapy MRD is prognostic, can monitor kinetics to predict relapseto predict relapse**
• Other potential markers: Other potential markers: FLT3, MLL-PTD, KIT, FLT3, MLL-PTD, KIT, DMNT3ADMNT3A
*Schnittger et al. 2009, Blood 114:2220
mutated
wild typeStd.RT-PCR
Detection of MRD by Detection of MRD by flow cytometry in AMLflow cytometry in AML
• Identify aberrant vs. normal myeloid precursorsIdentify aberrant vs. normal myeloid precursors
• Leukemia-associated immunophenotypes Leukemia-associated immunophenotypes [[LA(I)PsLA(I)Ps]]::
• Aberrant lymphoid antigen (CD19, CD7, CD56…)Aberrant lymphoid antigen (CD19, CD7, CD56…)
• Aberrant levels of normally expressed antigens Aberrant levels of normally expressed antigens ((↓,↑ ↓,↑ CD38, CD34…)CD38, CD34…)
• Coexpression of early and later antigens Coexpression of early and later antigens (CD34++CD15++)(CD34++CD15++)
• Altered forward and side scatterAltered forward and side scatter
Approaches• Must know the patterns of Must know the patterns of normalnormal and and
recoveringrecovering bone marrow bone marrow
• If available, compare MRD to the original If available, compare MRD to the original phenotypephenotype
• Need detailed description of antigen Need detailed description of antigen expression or dot-plotsexpression or dot-plots
• Rely on “LAIP” or deviation form normal Rely on “LAIP” or deviation form normal to identify leukemic cellsto identify leukemic cells
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 2 showing abnormal expression of at least 2 markers in the LAIP boxmarkers in the LAIP box
• Many Many ““LAIPLAIP” ” have a low frequency in have a low frequency in normal BMnormal BM
• B: “different-from normal” approach B: “different-from normal” approach (monocytic leukemia)(monocytic leukemia)
8-color MRD: Baseline study
Courtesy of Dr. Jeffrey Jorgensen
BM CD34+: Normal vs. AML MRDN
orm
al
AM
L M
RD
, 0.1
%
Courtesy of Dr. Jeffrey Jorgensen
Normal BM
CD45 V500-A
SS
C-A
-10210
210
310
410
5
0
65536
131072
196608
262144
CD45dim gate
7.5%
CD34 PerCP-Cy5-5-AS
SC
-A-10
210
310
410
5
0
65536
131072
196608
262144
0.8%
CD34-Percp
CD34 PerCP-Cy5-5-A
CD
38
FIT
C-A
-102
103
104
105
-102
102
103
104
105
CD64 PE-Cy7-A
CD
34
Pe
rCP
-Cy5
-5-A
-102
103
104
105
-102
102
103
104
105
CD14 APC-H7-AC
D6
4 P
E-C
y7
-A
-102
103
104
105
-102
102
103
104
105
CD33 PE-AC
D3
4 P
erC
P-C
y5
-5-A
-102
103
104
105
-102
102
103
104
105
CD13 APC-A
CD
33
PE
-A
-102
103
104
105
-102
102
103
104
105
CD64 PE-Cy7-A
CD
38
FIT
C-A
-102
103
104
105
-102
102
103
104
105
Normal Bone Marrow
HLA-DR V450-A
CD
34
Pe
rCP
-Cy5
-5-A
-102
103
104
105
-102
102
103
104
105
CD123 APC-A
CD
34
Pe
rCP
-Cy5
-5-A
-102
103
104
105
-102
102
103
104
105
HLA-DR V450-A
CD
12
3 A
PC
-A
-102
103
104
105
-102
102
103
104
105
CD2 FITC-AC
D3
4 P
erC
P-C
y5
-5-A
-102
103
104
105
-102
102
103
104
105
HLA-DR V450-A
CD
11
7 P
E-A
-102
103
104
105
-102
102
103
104
105
CD117 PE-A
CD
34
Pe
rCP
-Cy5
-5-A
-102
103
104
105
-102
102
103
104
105
Normal BM
CD5 FITC-A
CD
38
AP
C-A
-102
103
104
105
-102
102
103
104
105
CD19 PE-Cy7-AC
D3
8 A
PC
-A-102 103 104 105
-102
102
103
104
105
CD7 PE-A
CD
38
AP
C-A
-102 103 104 105
-102
102
103
104
105
CD56 V450-AC
D3
8 A
PC
-A-10
210
310
410
5
-102
102
103
104
105
CD19 PE-Cy7-A
CD
34
Pe
rCP
-Cy5
-5-A
-102
103
104
105
-102
102
103
104
105
%
CD19 PE-Cy7-A
SS
C-A
-102
103
104
105
0
65536
131072
196608
262144
0.8%
CD19-PeCy7 gate
Normal BM
FG, 1-31-2013
CD45 V500-A
SS
C-A
-102
102
103
104
105
-400
65236
130872
196508
262144
CD45dim gate6.7%
CD33 PE-A
CD
34 P
E-C
y7-A
-102
103
104
105
-102
102
103
104
105
CD34 PE-Cy7-A
CD
11
7 P
E-A
-10210
210
310
410
5
-102
101
103
104
105
%
CD34 PE-Cy7-A
CD
117 P
E-A
-102
103
104
105
-102
102
103
104
105
CD45 V500-A
SS
C-A
-102
102
103
104
105
-400
65236
130872
196508
262144
CD45dim gate57.7%
CD33 PE-A
CD
34
PE
-Cy7
-A
-10210
210
310
410
5
-102
101
103
104
105
%
FG, 11-9-2012
CD33 PE-A
CD
34
Pe
rCP
-Cy5
-5-A
-102
103
104
105
-102
102
103
104
105
CD
117P
ECD34 PE-CY7
Normal
Patient 1
Patient 2: 21 days post induction
CD45 V500-A
SS
C-A
-102
102
103
104
105
-400
65236
130872
196508
262144CD45dim gate58.5%
CD34 PE-Cy7-AS
SC
-A-10
210
210
310
410
5-400
65236
130872
196508
262144CD34-PEcy711.2%
Morphology: 21% of blasts
Post therapy
CD56 V450-A
CD
34
Pe
rCP
-Cy5
-5-A
-10210
210
310
410
5
-102
100
103
104
105
CD64 PE-Cy7-A
CD
34 P
erC
P-C
y5-5
-A
-102
102
103
104
105
-102
100
103
104
105
1.5%
0.3% 16.8%
CD34 PE-Cy7-A
CD
38
Pe
rCP
-Cy5
-5-A
-10210
210
310
410
5
-102
101
103
104
105
0.0%0.4%
28.1%71.5%
CD34 PerCP-Cy5-5-A
CD
38 F
ITC
-A
-102
102
103
104
105
-102
100
103
104
105
12.3%
1.1% 3.6%
CD56 PerCP-Cy5-5-A
CD
11
7 P
E-A
-10210
210
310
410
5
-102
101
103
104
105
CD64 APC C
D34
PE
-Cy7
Original
Sensitivity
• To yield sensitivity of 0.01%, collect at To yield sensitivity of 0.01%, collect at least 200K cells per tube (20/200K = 1 least 200K cells per tube (20/200K = 1 in 10in 104 4 = 0.01%)= 0.01%)
• Sensitivity may be limited due to Sensitivity may be limited due to background normal cells, 0.1% or higher background normal cells, 0.1% or higher
• * 0.1% is commonly used threshold in the * 0.1% is commonly used threshold in the literatureliterature
Detection of MRD by Flow Detection of MRD by Flow CytometryCytometry
• 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, requires Interpretation often challenging, requires experienceexperience
• Can be expensiveCan be expensive
• Lack of standardization Lack of standardization
Potential challenges• LAIPs may not cover all leukemic blasts, partial LAIPs may not cover all leukemic blasts, partial
overlap with normaloverlap with normal
• Antigen shift resulting from Antigen shift resulting from selection/emergence of subclones selection/emergence of subclones
• A complete change in LAIPs in about 20% of A complete change in LAIPs in about 20% of AML, with 80% having at least one LAIP AML, 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 to Use a comprehensive panel of antibodies to establish baselineestablish baseline
Summary
• MRD detection by FCM or/and PCR are promising MRD detection by FCM or/and PCR are promising tools to guide therapy and to improve outcomestools to guide therapy and to improve outcomes
• Each method has pros and consEach method has pros and cons
• More studies are underway to better incorporate More studies are underway to better incorporate the data into clinical decision making (dose the data into clinical decision making (dose intensification and/or ASCT)intensification and/or ASCT)
• Timing of MRD testing by FCM and the cut off Timing of MRD testing by FCM and the cut off levels for each time point that are significant are levels for each time point that are significant are being refinedbeing refined
Acknowledgement
• Dr. Jeffrey Jorgensen MD Anderson Cancer Center