minimal residual disease analysis in childhood all dr jerry hancock scientific co-ordinator of ukmrd...
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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
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 EFS (%)EFS (%)
Arm AArm A<10 yrs AND WCC <10 yrs AND WCC
<50<50AND RERAND RER
6262 8484
Arm BArm B>10 yrs or >10 yrs or WCC >50WCC >50AND RERAND RER
2222 7070
Arm CArm CA or B ANDA or B AND
SER SER Or poor cytosOr poor cytos
1616 7070
Intensity of
Therapy
Only 20% of relapse comes from highest risk groupMany of those cured are over-treated
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 EFS (%)EFS (%) RelapseRelapse
Arm AArm A<10 yrs AND WCC <10 yrs AND WCC
<50<50AND RERAND RER
6262 8484 11 (52%)11 (52%)
Arm BArm B>10 yrs or >10 yrs or WCC >50WCC >50AND RERAND RER
2222 7070 6 (28%)6 (28%)
Arm CArm CA or B ANDA or B AND
SER SER Or poor cytosOr poor cytos
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
<50<50AND RERAND RER
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