1. Minimal Residual Disease analysis in childhood ALL
Dr Jerry Hancock
Scientific Co-ordinator of
UKMRD Laboratory Network
Bristol Genetics Lab

2. Survival in Childhood ALL

3. Prognostic factors used to direct therapy
Fixed factors
Age, sex, white cell count at diagnosis, cytogenetics
Dynamic factors
response to treatment correlates with prognosis
slow early response (SER) assessed by microscopy predicts relapse
predicts outcome within groups of children with the same fixed risk factors receiving the same therapy
BUT the microscope is an insensitive tool for detection of residual leukaemia and most destined to relapse have a rapid response

4. Analysis of outcome for MRC UKALL 97/99 Trial - Stratification of Treatment based on “clinical risk”
Characteristics N
Arm A
<10 yrs AND WCC <50
AND RER 62
Arm B
>10 yrs or
WCC >50 22
Arm C
A or B AND
SER
Or poor cytos 16
Intensity of
Therapy

5. Analysis of outcome for MRC UKALL 97/99 Trial - Stratification of Treatment based on “clinical risk”
Characteristics N
EFS (%)
Arm A
<10 yrs AND WCC <50
AND RER 62 84
Arm B
>10 yrs or
WCC >50 22 70
Arm C
A or B AND
SER
Or poor cytos 16
Intensity of
Therapy

6. Analysis of outcome for MRC UKALL 97/99 Trial - Stratification of Treatment based on “clinical risk”
Characteristics N
EFS (%)
Relapse
Arm A
<10 yrs AND WCC <50
AND RER 62 84
11 (52%)
Arm B
>10 yrs or
WCC >50 22 70
6 (28%)
Arm C
A or B AND
SER
Or poor cytos 16
4 (20%)
Intensity of
Therapy
Only 20% of relapse comes from highest risk group
Many of those cured are over-treated

7. Relative frequency of leukaemic cells
Haematologic
remission
1012
1011
107
MRD
“cure”
Relapse
MRD Analysis
Follow-up
In years
Detection limit of
PCR technique
cytomorphology
Relative frequency of leukaemic cells
Minimal Residual Disease

8. Prognostic value of MRD
MRD shown to have independent prognostic value in ALL
Childhood ALL
van Dongen et al 1998
Cavé et al 1998
Coustan-Smith et al 1998
Relapsed childhood ALL
Knechtli et al 1998
Eckert et al 2001
Goulden et al 2003
Adult ALL
Bruggeman et al 2006
Raff et al 2007

9. van Dongen et al, Lancet 1998
= 98%
= 78%
= 20%

10. Quantitative MRD Detection
Three methods currently available :
Flow cytometric immunophenotyping
Utilises tumour associated aberrant immunophenotypes
E.g. Presence of myeloid markers on leukaemia blasts
Reverse transcriptase (RT) PCR
Utilises tumour specific RNA targets
E.g. Fusion gene transcripts
Real-time Quantitative (RQ) PCR
Utilises patient-specific gene rearrangements
E.g. Immunoglobulin and T-cell receptor gene rearrangements
Utility of method chosen depends upon the aim of the study
Important considerations: applicability, stability, sensitivity & quantitation
RQ-PCR methodology is method of choice in most European MRD-based clinical trials

11. RQ-PCR for MRD analysis
Methodology identifies unique Immunoglobulin and/or T-cell receptor gene rearrangements that are clone-specific
98% of patients will have at least one clonal rearrangement
Two patient-specific RQ-PCR assays are designed for each patient
capable of detecting one leukaemic cell in a background of 10,000 marrow cells
Important to prevent false-negative results due to clonal evolution
80-85% of patients will have two assays quantitative to 1 in 10,000

12. Scheme of Investigation
- identification of patient-specific MRD marker
PCR analysis of diagnostic DNA
Heteroduplex analysis of PCR products
Purify and sequence clonal rearrangement
TTGTAGTAGTTACCAGCTGGGCTATGAATACTTCCAGCACTGGG
D region
J region
N region
Identification of V, D and J segment usage
Synthesis of base patient-specific oligonucleotide
In Patient-specific RQ-PCR for MRD Analysis 8 4

13. ALL2003 Randomisation Algorithm

14. UKMRD Laboratory Network Glasgow Sheffield Bristol Barts Birmingham
Barts & Royal London
Great Ormond Street
Middlesex
Royal Marsden
St Georges
University College
University Hospital
Cambridge
Cardiff
Leeds
Oxford
Southampton
Aberdeen
Belfast
Dublin
Dundee
Edinburgh
Liverpool
Newcastle
Birmingham
Derby
Leicester
Manchester
Nottingham
Norwich

15. MRD risk groups by Regimen - January 2009
Characteristics
Registered
MRD
High risk
Low risk
Regimen A
<10 yrs AND WCC <50
AND RER
947
264 (28%)
288 (30%)
Regimen B
>10 yrs or
WCC >50
637
214 (34%)
155 (24%)
Total
1584
478 (30%)
443 (28%)

16. Event Free Survival By Trial
100
ALL2003 (2003-)
88%
ALL97-99 ( )
80% 75
74%
ALL97 ( )
PERCENT 50 25 1 2 3 4 5
TIME IN YEARS
At risk:
ALL97 ( )
997
919
865
801
757
732
ALL97-99 ( )
938
889
849
814
769
745
ALL2003 (2003-)
1828
1368
967
551
201

17. Relapse Risk By Trial ALL PATIENTS ALL97-99 (1999-2002)
100 75
No.
No.
Obs./
Patients
Events
Exp.
ALL97-99 ( )
ALL97-99 ( )
932
162
1·2
ALL2003 (2003-)
1839 70
0·7
ALL2003 (2003-)
PERCENT 50
2P = 0·0001 25
16% 8% 1 2 3 4 5
TIME IN YEARS
At risk:
ALL97-99 ( )
932
889
848
814
769
744
ALL2003 (2003-)
1819
1368
953
551
201

18. Event Free Survival by MRD Risk Group
LOW = 95%
100
INDETERMINATE = 85% 75
HIGH = 78%
PERCENT 50
No.
No.
Obs./ 25
Patients
Events
Exp.
HIGH
638 68
1·5
LOW
604 12
0·3
INDETERMINATE
738 70
1·2 1 2 3 4 5
TIME IN YEARS
At risk:
HIGH
638
451
317
187 68
LOW
604
467
311
184 89 3
INDETERMINATE
738
566
449
307
161 7
15-JAN-09 17:51:15

19. Proposals 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?

20. Acknowledgements Members of UKMRD network: - Barts Gary Wright
Maggie Corbo
Sheela Medahunsi
Ulrika Johannson
Susannah Akiki
Bristol Jerry Hancock
Paul Archer
Richard Hathway
Kayleigh McDonagh
Paula Waits
Nigel Wood
Glasgow Sandra Chudleigh
Mary Gardiner
Frances Fee
Linda Smith
Nicola Craig
Anne Sproul
Steve McKay
Sheffield Gill Wilson
Helen Stuart
Shilpa Haridas
Amal Afifi
Miranda Durkie
Jane Holden
Richard Kirk
James Blackburn
Clinical Co-ordinator: -
Nick Goulden
UKMRD Steering Committee:-
Nick Cross
Ajay Vora
Brenda Gibson
David Grant
Christine Harrison
Finbarr Cotter
John Moppett
CLWP and ALL Task Force
ESG-MRD-ALL:-
Jacques van Dongen
Vincent van der Velden
I-BFM MRD Group