Minimal Residual Disease analysis in childhood ALL

Minimal Residual Disease Minimal Residual Disease analysis in childhood ALLanalysis in childhood ALL

Dr Jerry HancockDr Jerry HancockScientific Co-ordinator of Scientific Co-ordinator of

UKMRD Laboratory NetworkUKMRD Laboratory Network

Bristol Genetics LabBristol Genetics Lab

SurvivalSurvival in Childhood ALLin Childhood ALLS

urv

ival

Prognostic factors used to direct therapyPrognostic factors used to direct therapy

Fixed factorsFixed factors

Age, sex, white cell count at diagnosis, cytogeneticsAge, sex, white cell count at diagnosis, cytogenetics

Dynamic factorsDynamic factors

response to treatment correlates with prognosisresponse to treatment correlates with prognosis

• slow early response (SER) assessed by microscopy predicts relapseslow early response (SER) assessed by microscopy predicts relapse

• predicts outcome within groups of children with the same fixed risk factors predicts outcome within groups of children with the same fixed risk factors

receiving the same therapyreceiving the same therapy

• BUTBUT the microscope is an insensitive tool for detection of residual leukaemia the microscope is an insensitive tool for detection of residual leukaemia

and most destined to relapse have a rapid responseand most destined to relapse have a rapid response

Intensity of

Therapy

Analysis of outcome for MRC UKALL 97/99 Trial - Analysis of outcome for MRC UKALL 97/99 Trial - Stratification of Treatment based on “clinical risk”Stratification of Treatment based on “clinical risk”

TreatmentTreatment CharacteristicsCharacteristics NN

Arm AArm A<10 yrs AND WCC <10 yrs AND WCC

<50<50AND RERAND RER

6262

Arm BArm B>10 yrs or >10 yrs or WCC >50WCC >50AND RERAND RER

2222

Arm CArm CA or B ANDA or B AND

SER SER Or poor cytosOr poor cytos

1616

Intensity of

Therapy


TreatmentTreatment CharacteristicsCharacteristics NN EFS (%)EFS (%)



6262 8484


2222 7070



1616 7070

Intensity of

Therapy

Only 20% of relapse comes from highest risk groupMany of those cured are over-treated


TreatmentTreatment CharacteristicsCharacteristics NN EFS (%)EFS (%) RelapseRelapse



6262 8484 11 (52%)11 (52%)


2222 7070 6 (28%)6 (28%)



1616 7070 4 (20%)4 (20%)

Minimal Residual DiseaseMinimal Residual Disease

Haematologic remission

1012

1011

107

0

MRD

“cure”

Relapse

MRD Analysis Follow-up

In years

Detection limit of

PCR technique

Detection limit of

cytomorphology

Re

lativ

e f

req

ue

ncy

of

leu

kae

mic

ce

lls

MRD shown to have independent prognostic value in ALLMRD shown to have independent prognostic value in ALL Childhood ALLChildhood ALL

van Dongen van Dongen et al et al 19981998

CavCavé é et al et al 19981998

Coustan-Smith Coustan-Smith et al et al 19981998

Relapsed childhood ALLRelapsed childhood ALL Knechtli Knechtli et alet al 1998 1998

Eckert Eckert et alet al 2001 2001

Goulden Goulden et alet al 2003 2003

Adult ALLAdult ALL Bruggeman Bruggeman et alet al 2006 2006

Raff Raff et alet al 2007 2007

Prognostic value of MRDPrognostic value of MRD

van Dongen van Dongen et alet al, Lancet 1998, Lancet 1998

= 98%

= 20%

= 78%

Quantitative MRD DetectionQuantitative MRD Detection

Three methods currently available :Three methods currently available :

1.1. Flow cytometric immunophenotypingFlow cytometric immunophenotyping Utilises tumour associated aberrant immunophenotypesUtilises tumour associated aberrant immunophenotypes

• E.g. Presence of myeloid markers on leukaemia blastsE.g. Presence of myeloid markers on leukaemia blasts

2.2. Reverse transcriptase (RT) PCRReverse transcriptase (RT) PCR Utilises tumour specific RNA targetsUtilises tumour specific RNA targets

• E.g. Fusion gene transcriptsE.g. Fusion gene transcripts

3.3. Real-time Quantitative (RQ) PCR Real-time Quantitative (RQ) PCR Utilises patient-specific gene rearrangementsUtilises patient-specific gene rearrangements

• E.g. Immunoglobulin and T-cell receptor gene rearrangementsE.g. Immunoglobulin and T-cell receptor gene rearrangements

Utility of method chosen depends upon the aim of the study Utility of method chosen depends upon the aim of the study Important considerations: applicability, stability, sensitivity & quantitationImportant considerations: applicability, stability, sensitivity & quantitation

RQ-PCR methodology is method of choice in most European MRD-based RQ-PCR methodology is method of choice in most European MRD-based clinical trialsclinical trials

RQ-PCR for MRD analysisRQ-PCR for MRD analysis

Methodology identifies Methodology identifies unique Immunoglobulin and/or T-cell unique Immunoglobulin and/or T-cell

receptor gene rearrangements that are clone-specificreceptor gene rearrangements that are clone-specific

98% of patients will have at least one clonal rearrangement98% of patients will have at least one clonal rearrangement

Two patient-specific RQ-PCR assays are designed for each patientTwo patient-specific RQ-PCR assays are designed for each patient

capable of detecting one leukaemic cell in a background of 10,000 capable of detecting one leukaemic cell in a background of 10,000

marrow cellsmarrow cells

• Important to prevent false-negative results due to clonal evolutionImportant to prevent false-negative results due to clonal evolution

80-85% of patients will have two assays quantitative to 1 in 10,00080-85% of patients will have two assays quantitative to 1 in 10,000

Scheme of Investigation Scheme of Investigation - - identification of patient-specific MRD markeridentification of patient-specific MRD marker

PCR analysis of diagnostic DNA

Heteroduplex analysis of PCR products

Purify and sequence clonal rearrangement

Synthesis of 18 - 25 base patient-specific

oligonucleotide

TTGTAGTAGTTACCAGCTGGGCTATGAATACTTCCAGCACTGGG

D region J regionN region

• Identification of V, D and J segment usage

In Patient-specific RQ-PCR

for MRD Analysis

ALL2003 Randomisation AlgorithmALL2003 Randomisation Algorithm

UKMRD UKMRD Laboratory NetworkLaboratory Network

BristolBristol

GlasgowGlasgow

SheffieldSheffield

BartsBarts

Barts & Royal LondonGreat Ormond Street

MiddlesexRoyal Marsden

St GeorgesUniversity CollegeUniversity Hospital

Cambridge

BristolCardiffLeeds Oxford

Southampton

GlasgowAberdeenBelfast Dublin

Dundee EdinburghLiverpool

Newcastle

SheffieldBirmingham

DerbyLeicester

Manchester Nottingham

Norwich

MRD risk groups by RegimenMRD risk groups by Regimen- January 2009- January 2009

CharacteristicsCharacteristics RegisteredRegistered MRD MRD High riskHigh risk

MRDMRDLow riskLow risk

Regimen ARegimen A<10 yrs AND WCC <10 yrs AND WCC


947947 264 (28%)264 (28%) 288 (30%)288 (30%)

Regimen BRegimen B>10 yrs or >10 yrs or WCC >50WCC >50AND RERAND RER

637637 214 (34%)214 (34%) 155 (24%)155 (24%)

TotalTotal 15841584 478 (30%)478 (30%) 443 (28%)443 (28%)

Event Free Survival By TrialEvent Free Survival By Trial

0 1 2 3 4 50

25

50

75

100

PE

RC

EN

T

TIME IN YEARS

74%

80%

88%

At risk:

ALL97 (1997-99) 997 919 865 801 757 732

ALL97-99 (1999-2002) 938 889 849 814 769 745

ALL2003 (2003-) 1828 1368 967 551 201 0

ALL97 (1997-99)

ALL97-99 (1999-2002)

ALL2003 (2003-)

ALL PATIENTS

0 1 2 3 4 5

0

25

50

75

100

PE

RC

EN

T

TIME IN YEARS

16%

8%

At risk:

ALL97-99 (1999-2002) 932 889 848 814 769 744

ALL2003 (2003-) 1819 1368 953 551 201 0

ALL97-99 (1999-2002) ALL2003 (2003-)

No.Patients

No.Events

Obs./Exp.

ALL97-99 (1999-2002) 932 162 1·2

ALL2003 (2003-) 1839 70 0·7

2P = 0·0001

Relapse Risk By TrialRelapse Risk By Trial

Event Free Survival by MRD Risk GroupEvent Free Survival by MRD Risk Group

0 1 2 3 4 5

0

25

50

75

100

PE

RC

EN

T

TIME IN YEARS

At risk:

HIGH 638 451 317 187 68 0LOW 604 467 311 184 89 3

INDETERMINATE 738 566 449 307 161 7

HIGH = 78%

LOW = 95%

INDETERMINATE = 85%

No.Patients

No.Events

Obs./Exp.

HIGH 638 68 1·5LOW 604 12 0·3

INDETERMINATE 738 70 1·2

15-JAN-09 17:51:15

Proposals for ALL 2010Proposals for ALL 2010

All patients on ALL 2020 will have therapy allocated based on MRD

• Treatment on ALL 2003 is currently randomised based on MRD

Including risk groups A, B and C

MRD Analysis done at day 28 and week 11

Sequential analysis of High Risk disease at 2 or 3 extra time-points

Identification of patients with Very High Risk disease

• Novel therapies employed

• BMT?

AcknowledgementsAcknowledgements Members of UKMRD network: - Members of UKMRD network: -

BartsBarts Gary WrightGary WrightMaggie CorboMaggie CorboSheela MedahunsiSheela MedahunsiUlrika JohannsonUlrika JohannsonSusannah AkikiSusannah Akiki

BristolBristol Jerry HancockJerry HancockPaul ArcherPaul ArcherRichard HathwayRichard HathwayKayleigh McDonaghKayleigh McDonaghPaula WaitsPaula WaitsNigel WoodNigel Wood

GlasgowGlasgow Sandra ChudleighSandra ChudleighMary GardinerMary GardinerFrances FeeFrances FeeLinda SmithLinda SmithNicola CraigNicola CraigAnne SproulAnne SproulSteve McKaySteve McKay

SheffieldSheffield Gill WilsonGill WilsonHelen StuartHelen StuartShilpa HaridasShilpa HaridasAmal AfifiAmal AfifiMiranda DurkieMiranda DurkieJane HoldenJane HoldenRichard KirkRichard KirkJames BlackburnJames Blackburn

Clinical Co-ordinator: - Clinical Co-ordinator: -

Nick GouldenNick Goulden

UKMRD Steering Committee:-UKMRD Steering Committee:-

Nick CrossNick Cross

Ajay VoraAjay Vora

Brenda GibsonBrenda Gibson

David GrantDavid Grant

Christine HarrisonChristine Harrison

Finbarr CotterFinbarr Cotter

John MoppettJohn Moppett CLWP and ALL Task ForceCLWP and ALL Task Force ESG-MRD-ALLESG-MRD-ALL:-:-

Jacques Jacques van Dongenvan Dongen

Vincent van Vincent van der Veldender Velden

I-BFM MRD GroupI-BFM MRD Group

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Minimal Residual Disease analysis in childhood ALL