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M inimal R esidual D isease 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 2/3 rd relapse

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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


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

    NPM1-WT & FLT3-ITD

    NPM1-mut & FLT3-ITD

    PML-RARα

    WT1

    RUNX1

    CBFB-MYH11

    Monitor with:

    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 mopost 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 63% 18% 0%

    RR 4.8 NS

    Sensitivity 86.7%

    Specificity 42.3%

    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%.

    • Doubek (ExpHem2009;37:659-672)


    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.

    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

      • 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


    The Leukemia Associated ImmunoPhenotypeVs. 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

      • 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

        • SchuurheisExpert Rev Hem 2010;3:1-5)


    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

    >1%

    • 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 2

        16 to Auto SCT 12 to Allo

    >0.1%

    >0.01%

    <0.01%

    >0.01%

    >0.1%

    >1%

    San Miguel Blood 2001;98;1746-51


    MRD By Flow adds to cytogenetics

    Survival% in Remission

    MRD -

    • Favorable & Intermediate cytogenetics

    • But not to unfavorable or FLT3-ITD

    • FLT3 WT

      • MRD+ adds

    • FLT3-ITD

      • Doesn’t add

    MRD +

    MRD -

    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

    • 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 2ndindution

      • 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 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

    • Rise in MRD presages relapse

      • Follow a molecular marker if available

      • Change in Chimerism:

        • Ratio recipient to donor, especially in CD34+ cells

          • VernerisCurr 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 MarkersYes

    • Definable thresholdsYes, but variable

    • PrognosticYes

    • Action improves Outcome?

      • APL Yes

      • Molecular relapse after AlloYes for 30-40%

      • All othersNot 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


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