MRD in ALL

Minimal residual disease in childhood ALL
(patient specific Ig/TCR PCR)
Molecular Biology and Cytometry Course Mol: 2013
Marleen Bakkus
Survival of childhood cancer patients
Major challenges

Treatments are still very complicated with
serious adverse reactions

Late effect in long term survivals can be highly
detrimental
 1/1000 adults is an ex-childhood cancer patient
 The challenge is cure for all but the quality of cure is
also essential
Treatment must be better “fine tuned”
Minimal residual disease
The first step to cure = complete remission (CR)
<5% blasts
Cellular origin of B/T-cell malignancies
leukemia
Myeloid
stem cell
acute
lymphoma
multiple
myeloma
chronic
B-lymphocytes
Stem
cell
precursor cells
lymphoid
stem cell
Bone
marrow
T-lymphocytes
Thymus
Blood
Lymphnode
Bone
marrow
Which genes for MRD?
Unique rearranged DNA
T-cell receptor (TCR)
Immunoglobulin (Ig)
VH
D
JH
germline
N-D-N
VH
JH
rearranged gene
Leader
FR1
CDR1
FR2
CDR2
FR3
CDR3
FR4
More diversity ajunctional regions
VH3-21
Cm
DH3-3 JH4-1
Junctional region
VH3-21(germline)
insertion
TGTATTACTGTGCGAGA
TGTATTACTGT
AGGC
TGTATTACTGTGCG
TATCCGGA
TGTATTACTGTGC
CCGGACTG
TGTATTACTGTGCGAG CTGAGTC
TGTATTACTG
ACATCGA
TGTATTACTGTGCG
CGT
TGTATTACTGTGC
TGTATTACTGTGCGAGA GGCTAG
TGTATTACTGTG
GTCCAG
TGTATTA
CCGGA
DH3-3 (germline)
GTATTACGATTTTTGGAGTGGTTATTATACC
insertion
JH4-1(germline)
ACTACTTTGACTACT
CGATTTTTGGAGTGGTTATTATA
GTCCA
TGACTACT
TTACGATTTTTGGAGTGGTTATTATAC
CGATCG
CTTTGACTACT
TTTTGGAGTGGTTATTATACC
GGT
ACTACTTTGACTACT
TATTACGATTTTTGGAGTGGTTAT
CGTAGCGTA
TTTGACTACT
CGATTTTTGGAGTGGTTATTATA
CGTAG
ACTTTGACTACT
TACGATTTTTGGAGTGGTTATTAT
GGCTAAGG
TGACTACT
TTTGGAGTGGTTAT
CC
ACTTTGACTACT
ATTACGATTTTTGGAGTGGTTATACC
GTCGA
GACTACT
TATTACGATTTTTGGAGTG
CCGTAG
CTACTTTGACTACT
CGATTTTTGGAGTGGTTATTATA
C
ACT
Frequency of TCR and Ig in childhood ALL
B-lineage ALL
T-lineage ALL
IgH
>95%
20-25% (~DH-JH)
Igk
~65% (~Kde)
Igl
15-20%
TCRb
~35%
~90%
TCRg
~55%
~95%
TCRd
~40%
~55%
Vd2-Ja29
~40-45%
Ref.: J.J.M. van Dongen en V.H.J. van der Velden: Detection of minimal residual disease in ALL
PRESENTATION
Blasts > 85%
MRD assesment
Genomic DNA
TCR Gamma
V N J
TCR delta
V N
D
VH-CDR3
V N-D-N J
DIAGNOSIS
PCR
125 bp
Sequencing of the N-region
Clono (N)-specific oligonucleotidic (ASO) probe/primer
Allele-specific
real-time PCR
MRD
PCR
REMISSION
Blasts < 1-5%
Genomic DNA
<0.1%
Target identification
Diagnosis:
 Bone marrow sample
 DNA extraction
 PCR for
 IGH
 IGK
 TCRG
 TCRD
 TCRB (only in T-ALL)
 Sil-Tal (only in T-ALL)
Number of targets in childhood ALL 2010
Aantal targets bij de diagnose en herval stalen ALL kinderen 2010
100%
80%
0 targets
60%
1 target
2 targets
40%
3 of meer targets
20%
0%
B-ALL
T-ALL
B- en T-ALL
0 targets
2%
0%
1%
1 target
12%
7%
11%
2 targets
28%
53%
33%
3 of meer targets
58%
40%
55%
Sequence targets
Heteroduplex analyses
PAGE
heteroduplex
homoduplex


Purify clonal band
Sequence with forward
and reverse primers
Sequence analysis and ASO design
ASO-primer 2
IGHV3-64*05
(-6)



ASO-primer 1
N1
IGHD2-2*01 N2
(-5, -13)
http://www.imgt.org/IMGT_vquest/vquest?...
http://vbase.mrc-cpe.cam.ac.uk/index.php?...
hardcopy
IGHJ1
(-0)
ASO design

OLIGO software

Tm: 53-58°C

Avoid stretches of more than 3 G’s and C’s in the
last 5 bases

Avoid dimer formation
ASO-primers in RQ-PCR
IgH
VH
TCRd
DH
V d2
JH
ASO
n=3
n=6
TCRg
n=1
n=1
Dd3
ASO
Igk
Jg
Vg
ASO
Vk
n=1
n=2
Kde
ASO
ASO
junctional region
Probe
Consensus primer
Ref: Verhagen et al., Leukemia 2000, Van der Velden et al. Leukemia 2002
n=1 n=1
Temperature gradient
60°C
63°C
65°C
Vg9
Patient 10-2
Healthy controls
Temperature gradient
60°C
63°C
65°C
IgH
VH2
Quantification of FU samples

qASO-PCR
Quantitative range: 10-4
Sensitivity: 10-5
FU2
FU1
FU3
FU1
FU2
 FU1: 0,04%
 FU2: positive, non-quantifiable
 FU3: negative
Control gene Albumin
Standard: commercial DNA
European Study Group on MRD detection in ALL
(ESG-MRD-ALL, since 2010 EURO-MRD)
AIMS of ESG-MRD-ALL (initial focus on PCR analysis of Ig/TCR genes):
1. Quality Control Program: 2 times per year: - February / March
- August / September
2. Educational meetings, including evaluation of quality control rounds:
2 times per year: - April / May
- October / November
3. Standardization of MRD techniques
- standardization techniques within each treatment protocol
- guidelines for interpretation of RQ-PCR results
4. Collaborative development and clinical evaluation of new MRD
strategies and new MRD techniques
Ref: VHJ van der Velden, 2007, Leukemia
DCLSG
ALL9
Interfant
99
European Study Group on MRD detection in ALL
(ESG-MRD-ALL; 32 laboratories in 17 countries)
MRCALL 97/99
UKALL-R3
adult ALL
LALA 2000
ESG
MRD
ALL
COALL06-97
EORTCCLG 58951
NOPHO
ALL-2000
Israel
adult ALL
UKALL XII
adult ALL
PETHEMA/
GETH
FRALLE
2000
Europe
BFMAIEOP
ALL-2000 adult
ALL
GMALL
06/99
(pre-)BMT
ALL
Singapore
Stockholm
Glasgow
Copenhague
Petach
Tikwa
Leeds
Sheffield
Singapore
Kiel
London
Amsterdam
Hamburg
Rotterdam Hannover Berlin
Bristol
Lille
Frankfurt
Brussel
Prague
Heidelberg
Paris
Zurich
Vienna
Australia
Padova
Monza
Salamanca
Sydney
EORTC-CLG 58 881
(France, Belgium)
EFS %
100
MRD < 10-2
90
80
70
60
50
VHR
MRD > 10-2
40
30
20
Overall Logrank test: p<0.0001
10
0
(years)
0
O
N
23 120
9
1
12
2
3
4
5
Number of patients at risk :
117
112
105
100
MRD < 10-2
7
5
4
4
MRD > 10-2
Cavé et al., NEJM 1998
EORTC 58 951
by MRD on Day 35
EORTC 58 881
(median follow up>5years)
EFS %
100
EORTC 58 951
(median follow up 2 years)
MRD <
90
EFS %
100
10-2
80
80
70
70
60
60
50
MRD > 10-2
50
MRD > 10-2
40
MRD < 10-2
90
•therapievermindering?
•intensievere therapie?
40
30
30
20
20
Overall Logrank test: p<0.0001
10
0
(years)
0
O N
23 120
9 12
1
2
Overall Logrank test: p<0.0001
10
3
Number of patients at risk :
117
112
105
7
5
4
4
100
4
5
0
(years)
0
10-2
MRD <
MRD > 10-2
O N
37 554
15 41
1
2
3
Number of patients at risk :
343
249
97
19
8
2
4
1
0
5
MRD < 10-2
MRD > 10-2
Treatment scheme ALL in frontline
ASO development
MRD detection is centralised
in one lab for all Belgian ALL
patients
MRD detection
TP0
Induction
TP1
TP2
HD-MTX
+IT
Consolidation
interval
2 years
Decisional threshold (EORTC-CLG guidelines)
10-2
10-3
Late
intensif
Maintenance
New guidelines for MRD monitoring and treatment
CLG-EORTC
MRD guidelines:
MRD to be performed for all patients after IA and after IB
MRD at further timepoints: only if MRD after IB≥10-4
[if MRD after IA ≥10-2 and MRD after IB <10-4  control on a further point is
acceptable]
For VHR patients: MRD to be tested after each block until BMT
Decisions taken from MRD results
Decisional threshold: 10-2 and/or 10-3 after IB/B’)
MRD≥10-2  Patients VLR, AR1, AR2: shift to VHR treatment
 BMT except for the following patients:
1) pts with hyperdiploidy>51chr who reach CR after IA
2) pts with GPR and CR after IA and MRDIB/IB’<10-3
Avoiding AlloSCT
a
d3 fte
5 rR
a
d1 fte
07 r R
d1
35
d1
56
d1
91
d2
17
d2
75
d3
03
d3
64
d4
33
d4
96
d6
22
d3
0
d3
5
SEAL Relapse
1,00E+00
1,00E-01
1,00E-02
1,00E-03
MRD genescan
MRD RQ-ASO
1,00E-04
1,00E-05
1,00E-06
1,00E-07
1,00E-08
3y
QR: 1.10-4, sensitivity: 5.10-5
Conclusion - MRD

Undertreatment could be avoided in normal risk
patients (4/144)

Overtreatment has been avoided in high risk
patients (7/13) and in relapsed patients (5/26)

MRD monitored treatment is a step to
“personalised medicine”
Participating belgium centra
 Gent, UZ Gent (Dr. Y. Benoit)
 Brussel, HUDE Reine Fabiola (Dr. A. Ferster)
 Leuven, Gasthuisberg Leuven (Dr. A. Uyttenbroeck)
 Brussel, UCL Saint Luc (Dr. C. Vermylen)
 Brussel, UZ Brussel (Dr. A. van Damme/Dr. J. van der Werff ten
Bosch)
 Luik, Hopital Citadelle Liege (Dr. M-F. Dresse/ Dr. C. Hoyoux)
 Montegnee, C.H. St. Joseph Esperance (Dr. P. Philippet/ Dr. N.
Francotte)
Molecular-Hematology lab
MRD girls