MRD monitoring in Hematological Malignancies: Concepts and Technological Considerations in

Lymphoid malignancies (ALL and MCL)

Elizabeth Macintyre Onco-Hematology laboratory, Necker-Enfants Malades, Paris, France

INSERM 1151 « Normal and Pathological Lymphoid Differentiation »

HST & Taiwanese Society of BMT, 13 April 2019

INDUCTION CONSOLIDATION ( ALLO-SCT)

Hematological CR

Molecular CR

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MAINTENANCE

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Hematologicalrelapse

MRD1 MRD2 MRD3 MRD4 MRD5 MRD6…..

Therapeuticstratification

Pre-emptive Treatment of molecular relapseEarly detection

of relapse

Diagnostic

Minimal Residual Disease: When, How and Why ?

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0.000 10E-6

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NGS?NGF?ddPCR ?

Kinetics vary with disease, and with disease sub-types

MRD sensitivity• Depends on the number of cells or DNA copies analysed and the number of pathological

events to be identified in order to define MRD positivity

• Flow Cytometry (FC)

– 10-30 cells with a pathological phenotype (universal or patient specific)– If 10 cells,

• Analyse 100 000 cells -> Sensitivity 10E-4 or 0.01% (Acquisition 5-10 mins) = Multiparameter FC or MFC• Analyse 10M cells -> Sensitivity 10E-6 ou 0.0001% (Acquisition >45 mins & requires « bulk lysis ») Next Gen Flow

– Flores-Montero J …. Orfao for the EuroFlow initiative, Leukemia. 2017 Jan 20. doi: 10.1038/leu.2017.29.

• DNA– 6.6pg = 1 cell, 6.6µg = 1M cells, 500ng = 75 000 cells– Q-PCR by EuroMRD criteria, 3x500ng = 225 000 cells

• Sensitivity 10E-4 - 10E-5 ou 0.01-0.001% • Depends on backgroud

• There will always be a borderline non-quantifiable gray zone (Poisson distribution)– Positive non-quantifiable (PNQ) ou Positive Below Quantifiable Range (BQR)– Frequency depends on the disease (MCL>ALL)

• It is prudent to take clinical decisions in the Quantifiable Range (QR), at at least 1 log abovethe limit of sensitivity.

– Despite this, the clinical tendency is the opposite ie total negativity.

The perfect MRD target

• Universal

• Optimal, uniform Sensitivity/specificity

• Robust

• RapidCompatible with the clinical impact

• Reproducible within and between centres

• Minimal risque of false positive and false negativeresults

• Price compatible with the benefit provided

• Q-PCR, Flow cytometry, ddPCR and IG/TCR NGS

European Study Group on MRD detection

Chairman: B. SchäferFounding Chairman: J.J.M. van Dongen

Supported by Leukaemia & Lymphoma Research, LeukemiaNet, and EuroClonality

www.EuroMRD.org

March 2018

Gold standard since ≈2000: Design of a clone specific IgH VDJ probe in B lineage ALL

FR1-JH1245FR2-JH1245

Clone Specific anti-junctional PCR primers

(ASO)

TGCAACGACTTCCCCGAGGT

DHVH JH

Clone Specific VDJ or DJ amplification with JH specific Taqman

quantification

D N J

Standard curve and ASO selection based on dilution into pooled PBL from

normal donors

Specificity of clonal vs. non-specific amplification of PBL is variableand determines sensititivity and quantitative range (QR)

Sensitivity = lowest level of detectionQuantitative range = lowest level of robust, reproducible, quantifiable detection

Bruggemann et al. Leukemia, 2010 p251, Van der Velden Leukemia 2007 p604

QR 10-4

Nb PCR Cycles

10-1 10-2 10-3 10-4 10-5

Sensitivity 10-5

Acceptable target

10-1 10-2 10-3 10-4 10-5

Sensibilité 10-4

QR 10-3

Threshold Ct Non-specific

signal in

PBL

Unacceptable target

Threshold Ct

20 pediatric studies(11 249 patients)

16 adult studies (2076 patients)

“Minimal residual disease is a “biomarker” of disease in ALLin the powerful sense that MRD IS the disease”

Cave H et al NEJM 1998, van Dongen JJ et al. Lancet 1998 2017, 20 years on…

THE LEUKEMIC CELL

- morphology

- immuno-phenotyping

- classical and molecular

cytogenetics

- targeted molecular biology

- global approaches: transcriptomics, CGH array,

SNP, WG/WES/RNA sequencing, epigenetics, protéomics

THE PATIENT

- known predisposed

background :

- e.g. Down syndrome

- not known:

- genetic polymorphisms

e.g. drug metabolism

variability

The patient, the disease, their interactions….

Interactions with stroma

Neoangiogenesis

Immune response

MRD is an integrative reflexion of many parameters

Hematologic Malignancies: Regulatory Considerations for Use of Minimal Residual Disease in Development of Drug and Biological Products for Treatment

U.S. Department of Health and Human Services - Food and Drug Administration Center for Drug Evaluation and Research (CDER) Center for Biologics Evaluation and Research (CBER)

For new drugs that have a demonstrated durable CR in patients with relapsed or refractory ALL, FDA has accepted MRD of less than 0.01% as supporting evidence of efficacy. As technologies improve and new clinical findings emerge, the level of MRD needed to support an efficacy claim may change.

First FDA approval of a leukemia drug— Blinatumomab (Blincyto; Amgen)—to include patients who are technically in remission but still have measurable residual disease.

October 2018

MRD AS A SURROGATE MARKER FOR EFFICACY ?

Flow cytometry: the first Q-PCR challenger

❖ 598 MRD samples (children + adultes)

❖ Ig/TCR & 4/5C FCM

❖ QualitativeConcordance : 96% ❖if performed in a standardised fashion

❖ Complementary -> 100% informativity

Garand et al. Leukemia. 2013 Feb;27(2):370-6.

Flow cytometry and IG/TCR quantitative PCR for MRD quantitation in acute lymphoblasticleukemia: a French multicenter prospective study on behalf of the FRALLE, EORTC and GRAALL. 238 pts., 598 samples, 4/5 color FlowGarand et al. Leukemia. 2013 Feb;27(2):370-6.

79 q-PCR BQR:80% MFC negative11% BQR 9% Positive

Droplet Digital PCR : the principle

13MRD & MCLMarion Alcantara

• Droplet generation• ≈ 20000 droplets per sample• ≈ 1 DNA molecule per droplet

• Each droplet is an isolated reaction vessel• In any given droplet, target may or may not be present

• Endpoint assay• Number of droplets with target (positives)• Number of droplets without target (negatives)

Make Droplets PCR Droplets Read Droplets Results

“X” target

copies

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MRD & MCL

Marion Alcantara

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Droplet Digital PCR : advantages

• Absolute quantification of target DNA molecules

• No need for a reference standard curve• Low infiltrated or tissue only samples• Ideallly suited to lymphoma MRD

• High concordance with RQ-PCR• Less Positive Non Quantifiable (PNQ/BQR) samples ?

• Sensitivity and reproducibility• Comparable with RQ-PCR

Drandi et al., J Mol Diagn., 2015Della-Starza et al. BJH 2016

Comparative analysis between RQ‐PCR and digital‐droplet‐PCR of

immunoglobulin/T‐cell receptor gene rearrangements to monitor MRD in (Adult) acute

lymphoblastic leukaemia

Della-Starza et al. BJH, 2016, Volume: 174, Issue: 4, Pages: 541-549

BQR

BQR

35 Q-PCR BQR:50% ddPCR negative25% BQR 25% Positive

ddPCR cf qPCRLess sensitive or more specific ?

ddPCR – an ideal technique for T lymphoblasticLymphoma ?<20% BM blasts and diagnostic material oftentissue only, so ASO Q-PCR and Flow difficult

Pearson r : 0.97

P<0.0001

R squared : 0.94

N=34 samples>diagnostic

MDD

MRD

>blood

BM

effusion

ddPCR corrected for

Albu ddPCR

Clonal quantification of TCR Minimal Disseminated Disease (MDD) & MRDby ddPCR vs. MFC in 34 LB02 pediatric T lymphoblastic lymphomas

Trinquand A, Plesa A. et al., unpublished

10

Next Generation Sequencing Immunogenetics

Anton W. Langerak et al. J Immunol 2017;198:3765-3774

Copyright © 2017 by The American Association of Immunologists, Inc.

Quantification and spikesKnecht H. et al. Leukemia In Press

ALL target identificationBrueggemann M et al. Leukemia In Press

B cell clonality in hematopathologyGroenen P. et al. Leukemia In Press

EuroClonality NGS consortium (France)

A.W. Langerak/ J.J.M. van Dongen (R’dam)

P. Groenen (Nijmegen) - IgK

M. Brüggemann / C. Pott (Kiel) – IgH VDJ

M. Hummel (Berlin) - TCRB

M. Catherwood (Belfast)

F. Davi (Paris) – IgH DJ & IgL

E. Macintyre (Paris) – TCRD (TCRA)

R. Garcia Sanz (Salamanca)

D. Gonzalez (Sutton) K. Stamatopoulos (Thessaloniki)

N. Darzentas (Brno)

G. Cazzaniga (Monza) - TCRG

J. Trka (Prague)

J. Hancock (Bristol)

M. Ladetto (Torino)

M. Lefranc / V. Giudicelli (Montpellier)

M. Giraud M. Salson (Lille) Coordinator: A.W. Langerak

(R’dam)

TCR Beta

Vidjil

Multiplex PCR & GeneScan fragment analysis Multiplex PCR & NGS, representation of top 100 clones

TCR Beta

CDR3 lengthVgenes / clone%

V genes / J genes

NGS Working group

TCR Repertoire in dysimmune states, between clonality and repertoire analysesEuroClonality Amplicon NGS, Vidjil analysis

TCRgamma TCR béta (2 PCR)

Polyclonal

faible clone dans le

polyclonal (diminution

)

Adapter ligation vs one-step NGS PCR: IGH VJ FR3 on PBLs

Arrest Interrogate Bio-informatics

IT-Adapter ligation : IGH VDJ

One-step PCR (PSL): IGH VDJ One-step PCR (PSL): IGH VDJ

IT-adaptor ligation : IGH VDJ

Courtesy B Scheijen, Nijmeigen, NL

Next Generation Sequencing Immunogenetic quantitation of rare events requires an internal calibrator/spike

qPCR vs. (Ion Torrent) Amplicon IG/TR NGS in pediatric ALL.

Better prognostic impact and allows assessment of non-tumor IgH repertoire

The comparison of MRD as measured by qPCR and (Ion Torrent) NGS, the prognostic

significance of NGS, qPCR and FC at various treatment time points and the impact of IgH

repertoire diversity on outcome.

Michaela Kotrova et al. Blood 2015;126:1045-1047

© 2015 by American Society of Hematology

Risk of False positive MRD by qPCR post SCT in pediatric ALLFronkova E, et al. Bone Marrow Transplant 2008; 42: 187–196

Next-generation sequencing indicates false-positive MRD results and better predicts prognosis after SCT in patients with childhood ALLKotrova M et al. Bone Marrow Transplantation volume 52, pages 962–968 (2017)

Oncogenic abnormalities and MRD are independent prognostic factorswhich provide complementary information: GRAALL Adult Ph- ALL

B : Gen : IKZF1 et MLLMRD1 > 10-4MRD1 + distinguishes HR and LR Whatever the oncogenotypeOncogenetic status has no prognosticimpact in MRD- patients

T : Gen : NOTCH1/FBXW7/RAS/PTENMRD1 > 10-4Oncogenetic poor risk defines a poorprognostic group, whatever the MRD status.MRD status has no prognostic impact in oncogenetic low risk patients

Beldjord, Blood 2014

Gen- MRD-Gen+ MRD-Gen- MRD+Gen+ MRD+

Gen- MRD-

Gen+ MRD-

Gen- MRD+

Gen+ MRD+

Integrated use of MRD classification and IKZF1 alt. accurately predicts 79% of relapses in pediatric ALL.

• MRD and IKZF1 in 131 uniformly treated precursor-B-ALL patients • Improvement of risk stratification by combining both?• Results

• strong prognostic significance of MRD classification, independent of IKZF1 alterations• 8 / 11 relapsed cases in the large MRD-M group (n=81; 62%) :IKZF1 alteration+ • integration of both MRD and IKZF1 status

- resulted in a favorable outcome group (n=104; 5 relapses) and a poor outcomegroup (n=27; 19 relapses), - showed a stronger prognostic value than each of the 2 alone (hazard ratio (95%CI): 24.98 (8.29-75.31)).

• MRD and IKZF1 status alone identified only 46 and 54% of the relapses, respectively.•Their integrated use allowed prediction of 79% of all the relapses with 93% specificity.

Wanders E et al., Leukemia 2011

Also true in pediatric T-ALL (MRD, oncogenetics and WBC)

Petit A et al., Blood 2018

O’Connor et al., JCO 2018

MRD should be evaluated indpendently in oncogenic subroupsrequires large patient datasets and sophisticated data management

Blood Cancers

8% of Adult Cancers≈ 50% leukemia, 50% lymphoma

70% Lymphoid, B>>T

45% of Childhood Cancers≈ 70% leukemia, 30% lymphoma

>90% Lymphoid, B>>T

Pediatric

Cancers

Adult

Cancers

MRD in mature lymphoid malignancies

• CLL - Flow• Myeloma - NGS• MCL – Q-PCR, ddPCR, Flow, NGS….• DLBCL, FL and other nodal NHL ….

– Requires development of cfDNA techniques

DNA sources for MRD detection in hematologic malignancies.

Florian Scherer et al. Blood 2017;130:440-452

Q-PCR : the gold standard in MCL MRD

32MRD & MCLMarion Alcantara

• Target• IGH rearrangement

• >90% of patients• t(11;14)

• 25 – 40% of patients• Blood/bone marrow

• Sequencing• Allele-specific primer design• Allele-specific (ASO) RQ-PCR

• Standardized• Sensitive

• Up to 10-5

• Euro-MRD guidelines

• Relative quantification approach• Need a reference standard curve

• Dilutions of the tumor-specific target (diagnostic DNA)

VH4-34(-0)/2N/(-2)DH3-3(-7)/4N/(-5)JH4

ASO

NGS, Vidjil

Too many grey zone values in MCL

PNQ/BQR ≈ 50% of positive results

Retrospective analysis on thawedCryopreserved cells

Being tested prospectivelyin MCLR2 & LYMA101

Kinetics of MRD evolution. Q-PCR (in red) and MFC (in blue) quantification of peripheral blood samples at indicated dates.

Conclusion : Flow cytometry is a more rapid but less sensitive alternative to Q-PCR

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MALNOU MRD

BM MRD

FMC MRD

RCHOP/RDHAP

BEAM (Feb 2006)

4 R

(Aug 2009-May 2011)

R / 3 months 4 R

(Apr 2014-Apr 2016)

R / 3 months

BQR 2/3 &3/3

BQR 1/3

negative

MCL MRD

Digital Droplet PCR Hybrid technologie between Flow and ASO Q-PCR

1.0E-08

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EU-MCL & MCLR2 ddPCR vs Q-PCR

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MCL67 Q-PCR BQR58% ddPCR Neg9% BQR 33% Pos

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R2=0,8772

ALL35 Q-PCR BQR:50% ddPCR Negative25% BQR 25% Positive

High-throughput sequencing

Ladetto et al., Leukemia, 2014

◊ MCL◊ 30 patients

◊ 22 RQ-PCR◊ 26 NGS

◊ 179 samples

INDUCTION CONSOLIDATION ( ALLO-SCT)

Hematological CR

Molecular CR

1012

1010

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mb

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s le

ucé

miq

ues

Temps

108

106

104

102

MAINTENANCE

…

Hematologicalrelapse

MRD1 MRD2 MRD3 MRD4 MRD5 MRD6…..

Therapeuticstratification

Pre-emptive Treatment of molecular relapseEarly detection

of relapse

Diagnostic

Minimal Residual Disease: When, How and Why ?

100%

1% 10E-2

0.01% 10E-4

0.000 10E-6

104

102

Cyt

olo

gyFI

SHFl

ow

Ro

bu

stD

NA

Q-P

CR

RQ

-PC

R

NGS?NGF?ddPCR ?

Kinetics vary with disease, and with disease sub-types

Conclusions

• Q-PCR remains the gold standard for MRD in the EU but not the USA– Flow cytometry can compete if the number of events analysed is increased

to >4M, except in some ALL (and MCL) subtypes– ddPCR is slightly less sensitive than Q-PCR but may be more specific and

merits prospective comparison. • It is very valuable in lymphoma for initial quantification• It is particularly interesting in MCL since there are so many gray zone PNQ/BQR reults

• The relative interests of IG/TR NGS and Q-PCR will be actively evaluatedin the next few years

• The current tendency to require complete MRD negativity encourages the use of techniques with a minimum number of borderline results(ddPCR et NGS cf. qPCR)

• MRD should be evaluated independently in oncogenetic subgroups, atleast in ALL– This requires large patient numbers and encourages international

collaboration

Vahid Asnafi, Nawel Bedjaoui, Jonathan Bond, Chantal Brouzes, Danièle Canioni, Agata Cieslak,

Gaelle Cordonnier, Coralie Derrieux, Charles Dussiaux, Ludovic Lhermitte, Mathieu Simonin, Melania Tesio, Aurore Touzart, Amélie Trinquand, Patrick Villarese and Elizabeth Macintyre

,

M. Alcantara, MH Delfau, M. Callanan, C. Pott, S. Ferrero, O. Hermine……