Minimal Residual Disease in AML
Steven M. Kornblau, M.D.
Department of Leukemia
Department of Stem Cell Transplantation and
Cellular Therapy
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?
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?
Barriers to MRD in AML
• Defined marker to follow not always present
– Cytogenetics
– Mutations
– Flow
Co-occurrence is frequent creates added complexity
When multiple events are present which do you follow?
Are effects: Additive, cancel each other out, synergistic?
• Standardized methodology not available
• Standardized threshold not defined
– Continuous variables measured, dichotomized
endpoints desired.
• A therapy that will improve things may not exist.
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.
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Hokland Blood 2011; 117:2577-84
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”
When to monitor for MRD?
• One time
– When?
• Immediately post induction
• at CR1, at 3 month? ….
• Serially
– Starting when?
– How often?
How to respond to (conversion ) MRD?
– Repeat in ~ 2 weeks
• Same or Rising  Consider as molecular relapse Act ?
• Down or gone, repeat in 2 weeks
Hokland Blood 2011; 117:2577-84
Problems with using mutations
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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.
Kinetics of relapse affect frequency of
monitoring and source
SLOWER
FASTER
NPM1
NPM1-WT & FLT3-ITD
NPM1-mut & FLT3-ITD
PML-RARα
WT1
CBFB-MYH11
Monitor with:
RUNX1
Peripheral Blood
Bone Marrow
MRD in APL- Take Home
• 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
APL
Treated
with
ATO
alone
151 patients treated with ATO single agent.
2 step Nested Q-PCR, used BIOMED-1 methods, Quantified by Eur Against Ca protocols
Sensitivity 10-3 after 1st round, 10-4 after 2nd round
Ct = PML-RARα/ABL * 100 Negative if beyond 40
20.5% relapsed, median 15 mo
%+
100
RR
Sensitivity
Specificity
63% 18%
4.8
NS
86.7%
42.3%
0%
Good
Risk
WBC<5,
PLT >20
High Risk
WBC >5,
or PLT <20
% + after induction
69%
62%
Relapse in Neg
0%
10%
Relapse in Pos
22%
32%
Lead time provided by
detection of conversion
31 relapses
15 > 4 months
10 never pos
6 not done
False Positive conversion
4.6% (8/151)
Chendamarai Blood v119:3143 2012
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%.
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
1A 1D
CBFB/MYH11
6
3
19 25
1A 1D
1A
MLL
6
6
8 19 21 24 61
3A 1D
2D
• Doubek (ExpHem2009;37:659-672)
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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
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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.
During Consolidation
Early Follow-up
Early Follow-up
Corbaciaglu JCO 28:3724-3729 2010
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
Magnitude of decline after
induction predictive, >2 log
Level after consolidation also
predictive
Cilloni JCO 2009;27:5195-5201
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
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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
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
The Leukemia Associated ImmunoPhenotype
Vs. the “Different from Normal” approach
• 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
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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
Multiparameter Flow Cytometry
Potential Problems
• 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)
LSC by FLOW for MRD
• CD34+CD38- &
– Positive for CD123 CD117 CD25
– Negative for HLA-DR
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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
Is MRD
Prognostic in
AML?
MRD by Flow in Adults AML
• 233 consecutive Adults
>1%
– Median age 42+ 18
– Cytogenetics
>0.1%
• Fav n=49 (Includes 43 APL) Int = 35 Unfav =10 Unk=32
>0.01%
• 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 2
16 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
<0.01%
>0.01%
>0.1%
>1%
San Miguel Blood 2001;98;1746-51
MRD By Flow adds to cytogenetics
Survival
• Favorable &
Intermediate
cytogenetics
• But not to
unfavorable or
FLT3-ITD
• FLT3 WT
– MRD+ adds
% in Remission
MRD -
MRD +
MRD -
• FLT3-ITD
– Doesn’t add
MRD +
Buccisano Blood 2012;119(2);332-341
Summary of Studies of Prognostic
Value of MPFC in AML
Ossenkoppele Gr J Haem 2011:153;421-436
Does Detecting
MRD improve
outcomes ?
MRD in Pedi AML- AML02 Study
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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
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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 multivariate
CBF, 11q23 and FLT3 stay in the
model
Triage to ALLO didn’t seem to help
the high risk group
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
MRD Post ALLO SCT
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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
MRD- Meeting the goals?
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Detectable Markers
Definable thresholds
Prognostic
Action improves Outcome?
– APL
– Molecular relapse after Allo
– All others
Yes
Yes, but variable
Yes
Yes
Yes for 30-40%
Not Yet…
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