MRD detection in AML

Adriano VendittiHematologyFondazione Policlinico Tor VergataRome

Determinants of Treatment Response

RESPONSEOUTCOME

Leukemia

Tumor burdenGrowth potentialDrug resistance

KaryotypeGenetics

Host

AgePharmacogenomics

Therapy

Drug dosageDrug interactions

Assessing MRD:does it worthwhile?

▶ MRD = QoR = CHT-resistance leukemic cells surviving cytotoxic therapy

▶ Normal karyotype is molecularly heterogeneous

► Also patients belonging to the most favorablecategories do have relapse

▶ Cytogenetic/genetic risk-stratification inadequate forpredicting relapse in individual patients

Can MRD detection improve outcomeprediction?

Methods for detecting MRD in AML

▶PCR on chimeric fusion genes mutations gene overexpression

▶Flow cytometry leukemia-associated aberrant phenotypes (LAIP), LAIPs

are absent or very infrequent in NBM

• NPM1

• FLT3

• CEBPA

• MLL-PTD

• RUNX1

Potential molecular targets for MRD

• PML-RARA

• CBFB-MYH11

• RUNX1-RUNX1T1

• MLL-fusion partner

• DEK-NUP214

• WT1

• BAALC

• ERG

• MN1

Fusion genes Mutations Overexpression

25-30% of AMLs

stable at relapse

~ 75% of CN-AML

?stability at relapse

~ 30% ofAMLs

?sensitivity

Minimal residual disease assessed by NPM1specific RQ-PCR (Schnittger, 2009)

Cilloni et al, JCO 2009

The five attributes of modern FC

▶Lightness Its underlying principle is solid, without any intricate

conjecture

▶Quickness AL diagnosis can be provided within 1 h

▶Exactitude Analytical process is highly controlled and data are

quantitatively expressed

▶Visibility Dot plot pattern recognition

▶Multiplicity 6-9 color polichromatic analysis

Del Vecchio et al, Leukemia 2007

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di pasquo e bm postIl2.001 newFSC-Height ->

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di pasquo e bm postIl2.001 newCD3 FITC ->

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di pasquo e bm postIl2.001 newCD3 FITC ->

10 10 10 10 100 1 2 3 4

di pasquo e bm postIl2.001 newCD34 APC ->

10 10 10 10 100 1 2 3 4

di pasquo e bm postIl2.001 newCD34 APC ->

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di pasquo e bm postIl2.001 newCD4 PE ->

Designing MRD studies in AL

Multiple staining at diagnosis

Identification of “leukemia-associated” phenotypes

Definition of a patient’s “immunologic fingerprint”

Immunologic fingerprint used during follow-up

Venditti et al Blood 2000, Venditti et al Leukemia 2003, Buccisano et al Leukemia 2006. Maurillo etVenditti et al Blood 2000, Venditti et al Leukemia 2003, Buccisano et al Leukemia 2006. Maurillo etal JCO 2008, Buccisano et al Blood 2010al JCO 2008, Buccisano et al Blood 2010

Terwijn M et al. ASH 2010

RFS after different courses of CHTand MRD % in BM

MRD% is an independent prognostic factor; use for risk stratification

1st course 2nd course 3rd course

Years

Re

lap

se

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e S

urv

iva

l

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1,0

0 1 2 3 4 5 6 7 8 9 10

MRD+

MRD-

YEARS

Overall S

urvival

0,0

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MRD+ MRD-

Venditti et al, Blood 2000

▶56 pts with de novo AML enrolled in the EORTC/GIMEMAprotocols▶MRD levels ≥ 3.5 ×10-4 cells at the end of consolidationpredicts relapse

Tor Vergata University Experience

Tor Vergata University Experience

Maurillo et al, JCO 2008

Maurillo et al, JCO 2008

Tor Vergata University Experience

RFS OS

Cytogenetics 0.0001 N.S.

MDR1 expression 0.03 N.S.

Post-InductionMRD status N.S. N.S.

Post-ConsolidationMRD status 0.001 0.004

Comprehensive risk-assessment Integration of baseline prognosticators (cytogenetics,genetics) and parameters inherent the quality of response(MRD)

Optimization of post-remission therapy Development of MRD-oriented therapiesMore appropriate use of alloSCT

▶3.5 x 10-4 RLC▶Post-consolidation time-point

Buccisano et al, Blood 2010

ν TVUHν 143 adults with AML in CR (median age 50y, range 18-75; 40 ≥

60y)ν EORTC-GIMEMA AML-10, AML-12, AML-13, AML-17ν MRD+: ≥ 3.5 x 10-4 (0.035%) at post-consolidation

Buccisano et al, Blood 2010

Integrated Risk-Score

Low-Risk High-Risk

Good K / MRD-Int K / MRD-

Adverse KFLT3+Good K / MRD+Int K / MRD+

Integrated Risk-ScoreAlonzo TA et al, abst 761, ASH 2010

Conventional Cytogenetics, Molecular Profiling, andFlow Cytometric Response Data Allow the Creationof a Two-Tiered Risk-Group System for Risk-BasedTherapy Allocation In Childhood AML- a ReportFrom the Children's Oncology Group

MRD monitoring!!!

▶MRD monitoring feasible▶Independent prognostic factor for

Risk of relapseRelapse-free survivalEvent-free survivalOverall survival

MRD monitoring???

▶No agreement on relevant thresholds and checkpoints

Post-induction?Post-consolidation?

▶Is MRD equally relevant in all subsets of AML?▶Will MRD translate in new interventions?▶Can early intervention make a difference?

alloSCT > autoSCT according to MRD status

N=42MRD+ post-cons

N=37MRD- post-cons

Maurillo et al, JCO 2008

B

alloSCT > autoSCT for High-risk AML

auSCT alloSCT Total

L-risk 26 6 32

H-risk 30 17 47

Total 56 23 79

Low-Risk High-RiskGood K / MRD-Int K / MRD-

Adverse KFLT3+Good K / MRD+Int K / MRD+

Buccisano et al, Blood 2010

alloSCT > autoSCT for High-risk AML

0 1 2 3 4 5

Time (yrs)

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Dis

ease F

ree S

urv

ival

P=0.003

AlloSCT (ITT)N=21

AlloSCTN=15

AutoSCTN=53

85%

44%

20%

Buccisano et al, 2010 unpublished

GIMEMA AML1310: a study of risk-adaptedand MRD-directed therapy for adult AML

Low-risk: CBF/Kitwt; NPM1+/FLT3-Int-risk: all othersHigh-risk: Adverse K; FLT3+

Diagnosis

Low-risk

Int-risk

High-risk

MRD-

MRD+

MRD marker

LAIP

Risk stratifCG, molecular

MRD assess

LAIP

FLA-IdasalvageNo CR CR

CRIn

duct

ion

(1 o

r 2 c

ours

es)

Con

solid

atio

n 1

autoSCT

alloSCT

alloSCT:MRD, MUD,UCB, HRD

▶Kinetic of MRD fluctuation between IND andCONS▶Prediction failure for 20-30% MRD neg pts who dorelapse▶PB as alternative source to BM

Points for consideration

100 patients

Buccisano et al, Leukemia 2006

Relationship between MRD level after IND andMRD status after CONS

Maurillo et al, JCO 2008

Excel: Ind – Cons –Good: Ind + Cons –

Poor: Ind + Cons +Ugly: Ind – Cons +

Prediction failure for some MRD neg patients

▶20-30% relapse rate in MRD negative. Whydon’t we predict it?

– Technical reasons: sensitivity/specificity

– Biological reasons: role of LSC

Role of LSC

CD34+CD38-

To find markers and/or properties thatdiscriminate LSC from HSC within theCD34+CD38- compartment

Strategy to discriminate leukemic and normalCC34+ CD38- stem cell

Bakker et al Cancer Res 64: 8443-8450, 2004

AML marker: CLL-1

RFS after different courses of CHTand LSC frequency in BM

After induction:median value of BMRLC: 5.2x10-3 (range 1x10-4-1.64x10-1)median value of PBRLC: 2.85x10-3 (range 1x10-5-1.15x10-1)r=0.84, P<0.001

Maurillo L. et al Haematologica 2007

After consolidation:median value of BMRLC: 4.1x10-3 (range 2x10-5-6.3x10-2)median value of PBRLC: 3.7x10-3(range 1x10-5-1.34x10-1)r=0.82, P<0.001

Peripheral blood MRD after consolidation

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Maurillo L. et al Haematologica 2007

MRD assessment toindividualize therapy of AML

▶ Informs appropriate post-remission therapy

▶ Informs decision regarding transplantation infirst CR

–– Optimizes timing & type of transplantOptimizes timing & type of transplant

▶ Defines need for additional therapy post-transplant

▶ Identifies impending relapse & drives pre-emptive therapy