Download - M inimal R esidual D isease in AML
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Minimal Residual Disease in AML
Steven M. Kornblau, M.D.Department of Leukemia
Department of Stem Cell Transplantation and Cellular Therapy
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The MRD Concept• Most patients achieve remission• Most relapse– Cure rate 20-25% overall therefore 2/3rd relapse
• What if we could predict who will eventually relapse ?
• Could we act on this information to benefit the patient?
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Goals for MRD• Residual disease detectable at some time point
– When?– Which marker?
• MRD detection adds prognostic information to presentation features– Should not be something measureable at diagnosis
• A threshold that predicts relapse vs. CCR can be defined– What level is actionable?
• There is a therapeutic response that can be taken– MRD detected more or different therapy– Not detected less therapy needed, less toxicity.
• Serve as a surrogate marker for efficacy?
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Barriers to MRD in AML• Defined marker to follow not always present– Cytogenetics– Mutations– Flow
• Standardized methodology not available• Standardized threshold not defined– Continuous variables measured, dichotomized
endpoints desired.• A therapy that will improve things may not exist.
Co-occurrence is frequent creates added complexity When multiple events are present which do you follow?Are effects: Additive, cancel each other out, synergistic?
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Methods for MRD detection• Multiparameter flow
– limit of detection 1:10-4
– More rapid
• RT-PCR• limit of detection varies by assay, target gene etc.
– Experimentally on cell lines “spiked” 1:10-3 to 10-7 – Practical on patient samples 1:10-4 to 10-5
• Takes longer• Good markers PML-RARα, NPM1, MLL, CEPBA, WT1 EVI1(Mecom) PRAME
• Normal Marrow will give a positive signal for nearly all mutations at some level
•Regenerating marrows are not the same as “Normal” marrows • Literature often incorrectly uses “sensitivity” when they mean the “limit of detection”
or “lowest possible threshold”
Hokland Blood 2011; 117:2577-84
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When to monitor for MRD?• One time– When?
• Immediately post induction • at CR1, at 3 month? ….
• Serially– Starting when? – How often?
– Repeat in ~ 2 weeks• Same or Rising Consider as molecular relapse Act ?• Down or gone, repeat in 2 weeks
How to respond to (conversion ) MRD?
Hokland Blood 2011; 117:2577-84
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PCR
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Problems with using mutations
• Variation in mutation site, insertion site, length• Multiclonality at DX or relapse• Expansion of minor clones• Mutation status can change between DX and relapse
– Mutational shift e.g. FLT3-ITD– Loss or gain of mutations
• Instability can affect usefulness of these markers for MRD
• Use of Next Generation Sequencing to follow clonal evolution over time. Probably u$eful but expen$ive.
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Kinetics of relapse affect frequency of monitoring and source
SLOWER FASTER
NPM1-mut & FLT3-ITD NPM1-WT & FLT3-ITD
PML-RARα
RUNX1CBFB-MYH11
NPM1
WT1
Peripheral Blood Bone MarrowMonitor with:
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• PML/RARα measured by Quantitative RT-PCR is Standard of care– for all or just high risk?
• After induction– ATRA & Anthracycline -useless due to residual apoptotic and differentiated cells– ATO – Any positive is bad
• After consolidation it is clearly bad – Conversion to positive predicts relapse. False positive is rare– Rising PCR at rate of 1 log per month predicts relapse.
• Outcome better if treated after conversion instead of waiting for relapse – ATRA + Chemo era: PETHEMA Leukemia 2007, 21:446-52– Arsenic Era: Grimwade , JCO 2009, 27:3650-8 & Leuk Res 2011;35:3-7– Affects quality of Autograft , if MRD positive don’t use (GIMEMA)– Persistent positive can be salvaged by allograft.
• Sequential monitoring. – Follow the Eur Against Ca program (Leukemia 2003, 14:2318-57)– Marrow better than blood. 1.5 Log more sensitive.– Q 3 month for 36 mo post consolidation
• Economically advantageous $4-11K/QALY
MRD in APL- Take Home
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APL Treated with ATO alone
Chendamarai Blood v119:3143 2012
151 patients treated with ATO single agent.2 step Nested Q-PCR, used BIOMED-1 methods, Quantified by Eur Against Ca protocolsSensitivity 10-3 after 1st round, 10-4 after 2nd round Ct = PML-RARα/ABL * 100 Negative if beyond 4020.5% relapsed, median 15 mo
%+ 100 63% 18% 0%RR 4.8 NSSensitivity 86.7%Specificity 42.3%
Good RiskWBC<5, PLT >20
High RiskWBC >5, or PLT <20
% + after induction 69% 62%
Relapse in Neg 0% 10%
Relapse in Pos 22% 32%
Lead time provided by detection of conversion31 relapses15 > 4 months10 never pos6 not done
False Positive conversion4.6% (8/151)
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How much lead time? Did it matter?– AML N=79, Median age 42.5 (20-67), Standard TX– Frequent monitoring by RQ-PCR
• median 74 samples PER patient!, range 10-237 • but not set schedule over 6-60 months.
– Fusion Gene: N=24, CR=23, Molecular CR (PCR-) in 11/24• Molecular relapse N=33 in 17 patients• 12 Not treated at PCR conversion . 100% relapsed.
– Median lead time was 25.5 days, range 8 to 79 days
• 21 treated at molecular relapse (N=12 patients) Chemo, GO, DLI– CR= 7 molecular PR=7 No response =8 – 4 “cured” 8 Relapse, Median 119 days
– PB and BM- strongly correlated (R= 0.8)– Whole BM and CD34+ and CD34- strong correlation (R=.9)– Pre-emptive therapy salvaged ~33%, delayed relapse 66%.
• Doubek (ExpHem2009;37:659-672)
Fusion N MRD+ Time to Relapse-days
Outcome
TX at MRD TX at Rel
RUNX1 12 8 26 35 60 77 79 6 Alive 1 D 1 A 1 D
CBFB/MYH11 6 3 19 25 1A 1D 1A
MLL 6 6 8 19 21 24 61 3A 1D 2D
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CBFβ AML• False Positive rare in inversion 16 • qRT-PCR -Standardized Europe Against Cancer assay ratio w.r.t. β2M • 53 with inversion 16, age 16-60• 13 samples per patient, blood vs. BM,
– Diagnosis, Induction cycle #1 and #2, – Consolidation #1,2,3,– Follow up 3 6 9 12 15 18 24 36 72 mo
• Marrow more sensitive• Pre TX correlated with % BM blasts, not other clinical features• Kinetics of decline after induction did not correlate with outcomes • After consolidation 59% negative, 2Yr RFS 70% in neg vs. 54% positive
– 14 Positive, 10 relapsed- they never achieved negativity (Median 1190) – 35 negative, 3 relapsed, 2 converted to >10 copies
• Follow Up- 29 Neg at some point, 10 converted, 6 of these relapsed. Lead times were 3, 5 , 6mo for 3, but 3 others at relapse.
Corbaciaglu JCO 28:3724-3729 2010
During Consolidation
Early Follow-up Early Follow-up
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Wilms Tumor 1• Overexpressed in 90% of AML, mutated ~ 10%
– Phase I- tested 9 RQ-PCR protocols in 11 labs, cut 3– Phase II tested 6 in 11 labs, selected best 3– Phase III tested 3 protocols on several standards, picked the best
• Established reference from normals-Often Expressed– 118 PB, 61 BM , 25 G-CSF stim PB
• Tested – Diagnosis 238 PB, 382 BM, 15 with WT1 mutation– After Anthra+ ara-C therapy N=129, 16 repetitively
• Results– Blood = BM at Dx and MRD– Mutant = wild type– High levels in Inv16, FLT3itd, NPM1
Cilloni JCO 2009;27:5195-5201
Magnitude of decline after induction predictive, >2 log
Level after consolidation also predictive
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NPM1• Potentially a great target as 30% mutated • 17 different mutations measured by PCR.
– Type A= 80%, B ,D = 6% each– Limit of detection 1:10,000 to 1:100,000
• 252 NPM1 mutated AML followed 84 relapsed– 47 MRD+ 15-221 days (median 62) before relapse– 15 never MRD- Failure to get 3 log reduction = relapse– 31 MRD never + before relapse– All relapses had the same NPM1 mutation– Sensitivity = 62/93 =66%,– Specificity? Not stated.
• Many time points prognostic• Prognostic after Allo SCT
Schnittger Blood 2009;114:2220-31
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MPFC
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Multiparameter Flow Cytometry• Only 50% have suitable molecular markers for PCR• 80-94% have a flow detectable pattern
– Leukemia Associated ImmunoPhenotype, define at diagnosis– “Different from Normal” define at diagnosis
• Gives a quantitative result– When to assess?– What threshold to use?
• Ranges used from 0.035 to 1%• 0.1% commonly chosen. • No predictive benefit using 0.01% (Leung Blood 2012;120:468-472)
– How many cells to analyses?• Clusters of as few as 20 cells can define MRD
– 200,000 events 1:10:000 = 20 cells
• Recommended to study 1 million as not all blast express the pattern.
• Almost all studies use levels derived retrospectively and lack a validation cohort.
• See Ossenkoppele Br J Haem 2011;153:421-436 for review of literature
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• Must detect the LAIP at the time of DIAGNOSIS to follow later• May be more than one LAIP
• Must follow all of them to pick out minor clones that expand.
• Different from Normal - Use a panel of ab and look for characteristic pattern.– Asynchronous expression very useful. E.g CD34+ and CD123+– Lineage infidelity useful. E.g. CD7 (Lymphoid) expression on AML blasts– Aberrant expression associated with cytogenetic abnormalities
• AML1-ETO : CD19+, CD11a- CD56+/- cCD79a = poor prognosis• CBFβ –MYH11: CD2+• T(15:17) : CD56 in 20%= bad• NPM1: CD13, CD117 CD110 CD123• CEBPA: 7+
• S&S best with 6+ color flow, Abs to LSC & multiple lymphoid Ags • Leukemia Stem Cell frequency
– CD34+ CD38- CD123 CLL-1 CD44 CD47 CD96 & the same aberrant markers.– Low frequency can be a disadvantage
The Leukemia Associated ImmunoPhenotype Vs. the “Different from Normal” approach
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• Background = expression on normal cells– limits both sensitivity & specificity, – Can raise limit of detection to 0.1% to 1%– Background lower in PB vs. BM
• Not all blasts express the aberrant marker– Lowers sensitivity
• Immunophenotype shifts- can be as high as 91%– Most cases have multiple LAIPs reducing the false negative rate.– But looking for the diagnostic pattern will miss newly emergent
patterns
• Flow subject operator expertise– Standardized protocols and automated analyses may help
• Schuurheis Expert Rev Hem 2010;3:1-5)
Multiparameter Flow CytometryPotential Problems
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LSC by FLOW for MRD• CD34+CD38- & – Positive for CD123 CD117 CD25 – Negative for HLA-DR
• Frequency at diagnosis predictive of relapse• Persistence after therapy predictive of relapse• Rarity make BM better than blood• Rarity might decrease sensitivity• Aberrant markers exist– C-type lectin like (CLL-1), not seen on normal
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Is MRD Prognostic in
AML?
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MRD by Flow in Adults AML• 233 consecutive Adults
– Median age 42+ 18– Cytogenetics
• Fav n=49 (Includes 43 APL) Int = 35 Unfav =10 Unk=32
• 3 color + FSC, SSC at diagnosis and remission. – 15K event followed by live gate on larger # of cells – Looked for SAME phenotype as at diagnosis
175 aberrant IP 126 CR with 3+7 x 2 + HDAC+ anthra x 216 to Auto SCT 12 to Allo
# MRD N 3 yr relapse
Median Survival
Low Risk <0.1% 45 14% Not reached
Intermediate Risk >0.1% 64 45% 79 mo
High Risk >1% 17 85% 20 mo
>1%
>0.1%
>0.01%
<0.01%
>1%
>0.1%
>0.01%
San Miguel Blood 2001;98;1746-51
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MRD By Flow adds to cytogenetics
• Favorable & Intermediate cytogenetics
• But not to unfavorable or FLT3-ITD
• FLT3 WT– MRD+ adds
• FLT3-ITD– Doesn’t add
Buccisano Blood 2012;119(2);332-341
MRD -
MRD +
MRD -
MRD +
Survival % in Remission
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Summary of Studies of Prognostic Value of MPFC in AML
Ossenkoppele Gr J Haem 2011:153;421-436
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Does Detecting MRD improve outcomes ?
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MRD in Pedi AML- AML02 Study• Induction 1 with high vs. low dose ara-C + Dauno and etoposide• MRD#1 on day 22, if >1% positive then immediately to induction 2, otherwise wait
for recovery of counts then to induction 2 • Induction 2 ADE +/- Gemtuzumab-ozogamicin• MRD#2 by Flow measurement after 2nd indution
– Low HDAC x 3. Good cyto more often negative– High Allo (n =59). Bad cyto more often positive
• 216 AML enrolled 202 evaluable for MRD #1, 193 for MRD#2• Use of high dose ara-C no effect. Use of GO increased conversion to MRD-
MRD#1 # % Relapse 3yr EFS
Positive (>1) 50 49% 43%
Positive (>0.1) 24 17%
Negative 128 17% 74
MRD#2 # % Relapse 3yr EFS
Positive (>1) 17 65% 36%
Positive (>0.1) 21 49
Negative 155 17% 71%
MRD most powerful in multivariateCBF, 11q23 and FLT3 stay in the model
Triage to ALLO didn’t seem to help the high risk group
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Does early intervention in CR1 help ?• Chemotherapy. – Delayed but didn’t prevent – More trials underway
– Pedi- Clofarabine + ara-C for MRD >0.1%– Adults
» Ceplene + low dose IL2» Anti CD33-gemtuzumab» Dendritic cell vaccination» Azacitidine or Decitabine
• Allo SCT- questionable benefit– Adults
• Walter JCO 2011; 29:1190-97 – Pedi
• Rubnitz. Lancet Oncology 2010;11:534-552• WT1 Jacobson Br J Haem 2009:146:2709-16• Leung Blood 2012; 120:468-72
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MRD Post ALLO SCT• Rise in MRD presages relapse
– Follow a molecular marker if available– Change in Chimerism:
• Ratio recipient to donor, especially in CD34+ cells – Verneris Curr Het Malig Rep 2010;5:157-162
• Rapid dynamics of relapse makes MRD use difficult• Hypomethylating agent can be beneficial
• Platzbecker Leukemia 2012;26:381-89• Azacitadine75mg/m2/day for 7 days. Median 4 (1-11 ) cycles• N=20
– 16 50% increasing donor chimerism , 30% stable– Despite this 13 relapsed Median @ 231 days
– Bolanos-Meade Biol Blood Marrow Transplant 2011;17(5) 754-758• Azacitadine75mg/m2/day for 7 days. • N= 10• Best BM response = CR in 6, 3 progressed, 1 revert to MDS• 2 CR got DLI, 1 developed cGVHD• 4 CR lost all host chimerism 2 with MRD• 1 relapsed• Median survival = 422 Days
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MRD- Meeting the goals?
• Detectable Markers Yes• Definable thresholds Yes, but variable• Prognostic Yes• Action improves Outcome?
– APL Yes–Molecular relapse after Allo Yes for 30-40%– All others Not Yet…
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MRD- what is needed• Standardized assays and cutpoints• Prospective studies to validate– Multicenter– Multiple central labs
• Clinical studies demonstration that action based on MRD improves outcome
• Markers needed for poor prognosis AML• New Therapies that work