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

• 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?

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

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

PCR

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.

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:

• 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

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)

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

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

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

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

MPFC

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

• 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

• 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

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

Is MRD Prognostic in

AML?

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

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

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• 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

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• 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?

• 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…

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