View
5
Download
0
Category
Preview:
Citation preview
Nuovi markers bio-molecolari nelle Sindromi Linfoproliferative
La Leucemia Acuta Linfoblastica
Genova, 11 Novembre 2014 Alterazioni molecolari in onco-ematologia:
dalla diagnosi allo studio della Malattia Residua Minima (MRD)
Estimated frequency of specific genotypes in childhood ALL
Hyperdiploid >50, 25,0%
TEL-AML1, 20,0%
E2A-PBX1, 4,0% MLL-AF4, 2,0%
BCR-ABL1, 2,0% BCR-ABL1-like; 9.0% CRLF2, 4,0%
IKZF1, 12,0%
ERG, 3,0%
dic(9;20), 2,0% iAMP21, 2,0%
E2A-HLF, 0,5% Hypodiploid; 0.5% Other MLL-R, 4,0%
Other BCP-ALL, 4,5%
TAL1, 7,0%
TLX3, 2,3% LYL1, 1,4%
TLX1, 3,0% MLL-ENL, 3,0%
ETP; 2.0% Others T-ALL; 1.7%
Modified from Pui et al, Blood. 2012;120:1165
T-ALL
BCP-ALL
Molecular genetic subsets of ALL are age-dependent
<1 1 2 3 4 5 6 7 8 9 10 11 12 13 14
years
Annual incidence rate of childhood ALL
per 106 population
t(9;22) or BCR/ABL: 33% (SE 9%), N= 27 t(4;11) or MLL/AF4: 30% (SE 10%), N= 23
t(1;19) or E2A/PBX1: 5-J.-EFS 93% (SE 6%), N= 15 t(12;21) orTEL/AML1: 95% (SE 3%), N= 44 >50 chromosomes (hyperdipl.): 86% (SE 3%), N=198 normal karyotype: 78% (SE 3%), N=244
years 0 1 2 3 4 5 6 7 8 9 10 11 12
0
20
40
60
80
100
pEFS
(%)
Prognosis of ALL according to genetics ALL-BFM 90
ALL Study (year) 70 76 79 81 83 86 90 95 2000 2009
WBC x x x
Total leukemic cell mass (RF) x x x x
Age x x x Sex x CNS x x x x Thymic tumor x x x
Immunophenotype (T vs non-T) sP sP x x x x
No CR to phase Ia x x x x x x x
Prednisone response day 8 x x x x x
MRD x x
t(9;22) BCR-ABL x x x EsPhALL
t(4;11) MLL-AF4 x x x t(12;21) ETV6-RUNX1 x Ploidy x
Childhood ALL trials: criteria for risk group stratification
p-Su
rviv
al (%
)
0
20
40
60
80
100
Childhood ALL: The Treatment Dilemma
Treatment intensity (number of drugs combined, dose intensity)
0
10
20
30
40
50
p-M
orbi
dity
(sev
ere)
(%)
p-M
orta
lity
(trea
tmen
t rel
taed
) (%
)
0 10
Status at the end of the 1990's
The questions:
• How to identify those children (20-25%) who ultimately relapse with disease that is highly refractory to current therapy.
• How to identify those children (25%, or more?) who are likely „over-treated“ and may well be cured using less intensive regimens resulting in fewer toxicities and long term side effects.
Improved overall survival in childhood acute lymphoblastic leukemia
Hunger SP, Pediatr Blood Cancer 2005;45(7):876–80
MRD as “surrogate” marker to assess heterogeneity in response to treatment
MRD detection in Acute Lymphoblastic Leukemia:
Fusion transcripts resulting from chromosomal translocations Immunoglobulin (IG) and T cell Receptor (TR) gene rearrangements Multiparametric Flow cytometry
Variability of the V(D)J region of IG/TR gene rearrangements as patient and clone-specific target for MRD detection
VH 5 ' 3 ' JH
VH primer ASO primer/probe
Junctional (“N”) regions
JH primer
Ig H
DH
Patient Marker Junction Junctional sequence Sensitivity
AC15 Vd2-Dd3 -20 / 9 /-13 TGAAGGGTCTT TCGGGCCCC CACAGTGCTAC 4AN40 Vd2-Dd3 -5 /14 / 0 TGAAGGGTCTTACTACTGTGCCTGTG CACCTGACGTACTT ACTGGGGGATACGCACAGTGCTAC 4AX21 Vd2-Dd3 -1 / 2 / 0 TGAAGGGTCTTACTACTGTGCCTGTGACAC GA ACTGGGGGATACGCACAGTGCTAC 4AA34 Vd2-Dd3 -3 /14 / 0 TGAAGGGTCTTACTACTGTGCCTGTGAC CTTTCACCCTTTTT ACTGGGGGATACGCACAGTGCTAC 5
Initial semi-quantitative ‘dot blot’ analysis by 32P-labelled ASO probes
Germline fluorescent probe
Forward junctional primer
Reverse primer
R Q V N J
MRD detection by monitoring Ig/TcR rerrangements
10-1 10-2 10-3 10-4 PB undil
Advanced RQ-PCR MRD detection in childhood ALL in the AIEOP-BFM 2000 study
• Germline TaqMan probes;
• Design of specific primers for each patient specific target;
• Sensitivity testing of all the primers/probe combinations; • Sensitivity testing by modifying annealing temperature;
• MRD detection experiment;
• Parallel albumin analysis on each sample;
• Final data calculation (normalization).
It strongly depends on the therapeutic time point assessed:
• Early time points • the applicability of MRD could be higher as much as earlier is
the prognostic time point.
• Late time points • the persistence of residual blasts beyond 4 to 6 months or the
re-emergence of residual disease, even at the level of 1x10-4, predicts clinical relapse. However, the clinical usefulness of late MRD determination is limited.
Clinical impact of MRD
I-BFM-SG ALL-MRD-Study: Update Outcome by MRD detection levels at w5 (tp 1) and w12 (tp 2)
MRD at tp. 1+2 neg.: 0.98, SE=.02 (N= 55, 1 event) MRD pos but < 10-3 at tp. 2: 0.76, SE=.06 (N= 55, 14 events) MRD at tp. 1+2 >=10-3: 0.16, SE=.08 (N= 19, 16 events)
years
p: 1-2: .0003; 1-3: .0001; 2-3: .0001 mrd
_upd
2.ka
m 0
2JA
N03
P
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
0 1 2 3 4 5 6 7 8 9 10 11
Basis for risk stratification in trial ALL-BFM 2000
Low risk group
Intermediate risk group
High risk group
0
M
10 12
II
MR - 1
MR - 2
R2
H R 1'
H R 2'
H R 3'
III
BM sampling
12Gy*
* presymptomatic cranial irradiation (18[24] Gy for CNS pos. pts only) # selected indications for allo-BMT (in all strata of HR) ° SR: 2 molecular marker with a sensitivity of =<10-4 available (obligatory)
MRD Timepoints
1 2 (3) (4) (5)
II R1
SR°: • no HR criteria • MRD neg. at tps. 1+2
HR: MRD level at tp. 2 >=10-3 HR:
PRED-PR t(9;22) t(4;11) NR d33
20
12 Gy* only T-ALL
SR - 2
SR - 1
BFM
B M T#
III 12 Gy* only T-ALL
30/7/2000
22
1b
MR: • no HR criteria • no SR-criteria
III III III
III
II
29 26
II 18Gy*
12Gy*
II AIEOP
IA-D+
IA-P+
IB R
1a
G-CSF
104 W. 52 + IA-D: Protocol I, Phase A with DEXA IA-P: Protocol I, Phase A with PRED
10 weeks interim maintenance with 6-MP / MTX
H R 1'
H R 2'
H R 3'
R3
HR - 1
HR - 2
H R 1'
H R 2'
H R 3'
6-MP/MTX 4 Wks.
6-MP/MTX 4 Wks.
MRD as surrogate marker for risk-based
patient stratification
AIEOP-BFM ALL 2000 protocol
MR: n = 1495
n=1495
MRD-MR n = 1708 (51%)
HR: n = 270
n=270
MRD-HR n = 270 (8%)
MRD classification feasible n = 3265 (77%)
Final risk group assignment in AIEOP-BFM ALL 2000 with and without MRD results (n=4239)
MRD classification not feasible n = 974 (23%)
SR: n = 1241
n=1241
MRD-SR n = 1287 (40%)
n=213 n=46
n = 424
n = 843 n = 131
n = 2338 n = 660 Final clinical risk groups (all eligible pts = 4239)
29% 55% 16%
AIEOP-BFM ALL 2000 protocol: Outcome results
311 deaths 89.4%(0.7)5 yrs Prob.
SURVIVAL524 events 79.4%(1.1)
5 yrs Prob.
EFS
AIEOP-BFM 2000
4239 patients
CORS/Hannover - Apr 2006
Prob
abili
ty
0.0
0.2
0.4
0.6
0.8
1.0
YEARS FROM DIAGNOSIS0 1 2 3 4 5
1241N. pts
50N. events
91.4%(1.5)5 yrs EFS
SR 20002338
N. pts
281
N. events
78.8%(1.6)
5 yrs EFS
MR 2000660
N. pts
193
N. events
59.5%(3.2)
5 yrs EFS
HR 2000
AIEOP-BFM 2000by final risk
4239 patients
CORS/Hannover - Apr 2006
EFS
0.0
0.2
0.4
0.6
0.8
1.0
YEARS FROM DIAGNOSIS0 1 2 3 4 5
n=29%
55%
16%
by Final Risk n=4239
All BCP and T-ALL n=4239
1290N. pts
52N. events
91.5%(1.5)5 yrs EFS
SR1705
N. pts
200
N. events
75.7%(2.3)
5 yrs EFS
MR270
N. pts
114
N. events
36.8%(6.6)
5 yrs EFS
HR
AIEOP-BFM 2000by MRD
3265 patients
CORS/Hannover - Apr 2006
EFS
0.0
0.2
0.4
0.6
0.8
1.0
YEARS FROM DIAGNOSIS0 1 2 3 4 5
n=40%
51%
8%
by MRD n=3265
Definition of risk groups in ALL-BFM 95: SR: PRED-GR; WBC <20,000, and age 2-5y MR: PRED-GR; WBC >= 20,000, or age <1y, or >= 6y HR: PRED-PR, or induction failure, or Ph+ ALL
2552 15% 53% 32% All
215 (100%) 61% 29% 9% HR
1306 (100%) 16% 55% 29% MR
1031 (100%) 5% 55% 40% SR MRD risk criteria
ALL 2000
All HR MR SR
ALL-BFM 95 risk criteria
MRD risk groups of ALL-AIEOP BFM 2000 in comparison to risk groups according to ALL-BFM 95 criteria
(eligible patients classifiable by MRD)
Relapses in BCP-ALL by MRD risk groups AIEOP-BFM ALL 2000
1348 61 6.0%(0.8) 7.2%(1.2)1647 266 21.0%(1.2) 22.3%(1.4)189 60 34.9%(3.8) 38.5%(5.0)
SRIRHR
N.pts N. rel. 5-yrs CI 7-yrs CI
p-value<0.001
Cum
. Inc
iden
ce
0.0
0.2
0.4
0.6
0.8
1.0
Years from diagnosis0 1 2 3 4 5 6 7
Conter V et al Blood 2010; 115: 3206
Relapses in T-ALL by MRD risk groups AIEOP-BFM ALL 2000
75 5 7.6%(3.3)292 51 17.6%(2.2)97 36 37.7%(5.0)
SRIRHR
N.pts N. rel. 7-yrs CI
p-value: overall<0.001; SR vs MR=0.02; MR vs HR<0.001
Cum
. Inc
iden
ce
0.0
0.2
0.4
0.6
0.8
1.0
Years from diagnosis0 1 2 3 4 5 6 7
Schrappe et al. Blood 2011; 118: 2077
401N. pts
13N. events
93.5%(1.9)5 yrs EFS
SR264
N. pts
27
N. events
67.5%(8.2)
5 yrs EFS
MR8
N. pts
3
N. events
58.3%(18.6)
5 yrs EFS
HR
AIEOP-BFM 2000TEL/AML pos - by MRD
673 patients
CORS/Hannover - Apr 2006
EFS
0.0
0.2
0.4
0.6
0.8
1.0
YEARS FROM DIAGNOSIS0 1 2 3 4 5
MRD-SR 1.0, SE=.00 (N= 6, no event)MRD-MR .50, SE=.15 (N=16, 6 events)MRD-HR .23, SE=.12 (N=18, 11 events)
years
Log-Rank p = .016
dsm
c040
5.ta
b 18
MA
Y05
P
0.00.10.20.30.40.50.60.70.80.91.0
0 1 2 3 4
Pts enrolledSep/Jul 00–Oct 04(Status April 05)
AIEOP + ALL-BFM 2000, EFS MRD Risk Groups, BCR/ABL pos
MRD results in genetically defined subgroups
BCR/ABL pos BCP-ALL (Pre-TKI)
t(12;21) n=673 pts
Summary of MRD results in childhood ALL
• The long term EFS results of the retrospective MRD study confirmed the prognostic value of MRD monitoring at two early time points.
• The cooperative AIEOP-BFM ALL2000 protocol was the first large multicentric trial using early MRD monitoring as a major prognostic factor for risk stratification in childhood ALL.
– feasibility of early MRD monitoring in a large proportion of patients (~80%)
– MRD confirmed its high prognostic value in all the biological subgroups of patients.
– Definition of a large SR group with excellent outcome
• MRD-SR MRD negative at TP 1 and TP 2 with at least one, preferably more than one PCR-MRD marker with a sensitivity of at least 10-4
• MRD-MR MRD positive at TP 1 and/or TP 2 and MRD at TP 2 <10-3 with at least one PCR-MRD marker
‚Slow early responders‘ (SER) MRD >10-3 at TP 1 and still positive at TP 2 by PCR-MRD
• MRD-HR PCR-MRD >10-3 at TP 2 FCM-MRD >10% at d15 Hypodiploidy <44
• PCR-MRD not stratified : FCM-MRD at d15: <0,1% SR
>0,1% and <10% MR
AIEOP-BFM ALL 2009: new MRD Risk Criteria
MRD stratification PCR : 94.0% FCM : 5.6% Overall: 99.6%
N=1290, 52 events
AIEOP-BFM ALL2000 Outcome by number of markers
Pts enrolled Sep/Jul 00–Sept 05 (Status April 06)
years
P
0.0 0.1
0.2 0.3
0.4 0.5 0.6 0.7
0.8 0.9 1.0
0 1 2 3 4 5 6
.91, SE=.01
MRD-SR 2000 (at least 2 sensitive markers,
TP1 + TP2 negative)
.96, SE=.01
years
P
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
0 1 2 3 4 5 6
N=300, 11 events
MRD-MR 2000 (only 1 sensitive marker,
TP1 + TP2 negative)
Outcome of Childhood ALL by Flow Cytometric Measurement of Residual Disease on Day 15 Bone Marrow
Basso G. JCO 2009
TP1 LOW+, TP2 NEG 0.83, SE=0.03 (N=305, 38 events) TP1 LOW+, TP2 POS 0.79, SE=0.05 (N= 97, 16 events) TP1 HIGH+, TP2 NEG 0.73, SE=0.05 (N=106, 28 events) TP1 HIGH+, TP2 POS 0.40, SE=0.07 (N= 89, 40 events)
years
Log-Rank p = <.0001
aieo
p_bf
m.ta
b 30
APR
09
P
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
0 1 2 3 4 5 6 7 8
AIEOP + ALL-BFM 2000 EFS (6 years) BCP-ALL, non-HR
Patient enrollment AIEOP: 09/00 – 07/06 BFM: 07/00 – 06/06 (Status June 08)
SER, slow early responers
DI >1.6 .86, SE=.05 (N=59, 8 events) DI >1.16-<1.6 .87, SE=.01 (N=769, 99 events) DI >.81 -<1.16 .77, SE=.01 (N=3405, 680 events) DI <.81 .49, SE=.10 (N=24, 12 events)
ALL-BFM 90-2000 Age > 1 year EFS (5 years) DNA index
years
ehes
t090
5.ta
b 09
NO
V05
P
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
DNA Index<0.8 or < 44 chromosomes
DNA Index ≥1.16 or >50 chromosomes
IB M IAD
HR
T/non-‐HR
pB#/non-‐HR M
II
IB
AIEOP-BFM ALL 2009 pCRT 12 Gy if age > 2 yrs* / in selected subgroups no CRT + 6x IT MTX
IA’
IA
IA
R1
Immunology unknown or pB-‐ALL + TEL/AML1 neg + FCM-‐MRD d15 >0.1%
TEL/AML1 pos and/or FCM-‐MRD d15 <0.1%
Prot. IA with 2 DNR doses (day 8 and 15)
Prot. IA with 4 DNR doses (day 8, 15, 22 and 29)
# or immunophenotype unknown * in paSents with CNS disease (CNS 3) tCRT with 12 Gy/18 Gy (dose age-‐adapted))
MR II
II R2
II SR
PEG-‐L-‐ASP 2500 IU/m2 every 2 weeks, over 20 weeks in total
PEG-‐L-‐ASP 4 x 2500 IU/m2 over 4 weeks
IB+
IB
IA
53 104 w 12 1 22 31 43 20 10
IACPM
RHR
T-‐ALL
pB-‐ALL#
HR 1‘
HR 2‘
HR 3‘ III III III
pCRT 12 Gy if age > 2 yrs* / in selected subgroups no CRT + 6x IT MTX
„NRd33 only“ FCM d15 >10%
„MRD-‐MR SER“ „MRD-‐HR only“
SCT
DNX-‐FLA + SCT
IA’ IA
European Study Group on MRD detection in ALL (ex ‘ESG-MRD-ALL’; 57 laboratories in 23 countries)
− Standardization − Guidelines − Quality Control Rounds − Education − Developments
EuroClonality NGS consortium
A. Langerak / J. van Dongen (Rdam)
P. Groenen (Nijmegen)
M. Brüggemann / C. Pott (Kiel)
M. Hummel (Berlin)
M. Catherwood (Belfast)
F. Davi (Paris)
E. Macintyre (Paris)
R. Garcia Sanz (Salamanca)
K. Stamatopoulos (Thessaloniki)
N. Darzentas (Brno)
G. Cazzaniga (Monza) J.Hancock (Bristol) J.Trka (Prague) M.Ladetto (Turin)
M. Lefranc / V. Giudicelli (Montpellier) Coordination : A.W. Langerak
3. RQ-PCR design and sensitivity testing
4. MRD analysis of follow-up samples
2. MRD PCR target identification
1. DNA preparation
a. PCR-heteroduplex analysis
b. Sequencing of clonal rearrangements
c. Sequence interpretation
d. Selection of MRD-PCR targets
a. BM sampling at diagnosis (≥ 5ml)
b. MNC-density gradient separation (1x107 cells)
c. Genomic DNA extraction (≥ 10µg)
a. Design of allele-specific oligonucleotide primers
b. RQ-PCR analysis of dilution series of diagnostic sample
c. RQ-PCR data interpretation
a. RQ-PCR analysis of follow-up samples (control gene)
b. RQ-PCR analysis of follow-up samples (Ig/TCR targets)
c. RQ-PCR data interpretation
d. Calculation of MRD level
( 2-3 days )
( 1-2 weeks )
( 1-2 weeks )
( 1-2 weeks )
3. RQ-PCR design and sensitivity testing
4. MRD analysis of follow-up samples
2. MRD PCR target identification
1. DNA preparation
a. PCR-heteroduplex analysis
b. Sequencing of clonal rearrangements
c. Sequence interpretation
d. Selection of MRD-PCR targets
a. BM sampling at diagnosis (≥ 5ml)
b. MNC-density gradient separation (1x107 cells)
c. Genomic DNA extraction (≥ 10µg)
a. Design of allele-specific oligonucleotide primers
b. RQ-PCR analysis of dilution series of diagnostic sample
c. RQ-PCR data interpretation
a. RQ-PCR analysis of follow-up samples (control gene)
b. RQ-PCR analysis of follow-up samples (Ig/TCR targets)
c. RQ-PCR data interpretation
d. Calculation of MRD level
( 2-3 days )
( 1-2 weeks )
( 1-2 weeks )
( 1-2 weeks )
Flow diagram of RQ-PCR MRD diagnostics :
IG/TR sequencing
Bioinformatic analysis (data interpretation and
calculation of MRD levels)
Adult- ALL , Raff et al., Blood 2007
Early detection of relapse
17/28
5/77
13/15
Pediatric- ALL , Paganin et al., JCO 2014
Early detection of relapse
Major points still to be addressed: • treatment context;
• prospective validation;
• multivariate analysis.
How to integrate new novel genetic markers with MRD-defined subgroups?
Gene Expression Profiling SNParrays (copy number analysis)
Next Generation Sequencing
Novel genomic alterations with prognostic or therapeutic relevance
Gene/Subgroup Alteration Frequency Function Prognostic or therapeutic relevance
PAX5 Focal deletions, translocations, sequence mutations 30% of BCP-ALL Transcription factor required for B-
lymphoid development not related to outcome
IKZF1 Focal deletions or sequence mutations
15% of pediatric BCP-ALL cases Transcription factor required for
lymphoid development poor outcome 70-80% BCR-ABL1 ALL 35% of high-risk BCR-
ABL1negative ALL
JAK1/2
Pseudokinase and kinase domain mutations 20%-35% Down syndrome ALL
Constitutive JAK-STAT activation May be responsive to JAK inhibitors
10% high-riskBCR-ABL1negative ALL
CRLF2 IGH@-CRLF2 or P2RY8-CRLF2, resulting in overexpression
5%-15% pediatric and adult BCP-ALL, Associated with mutant JAK in up
to 50% of cases poor outcome >50% Down syndrome-ALL,
15% pediatric high-risk ALL Associated with IKZF1 alteration and JAK mutations
IL7RA Small ins/del and sequence mutations
10% pediatric T-ALL; <1% in BCP-ALL
Constitutive activation of the IL7 receptor; associated to CRLF2 rearrangements in BCP-ALL.
prognosis not known; may be responsive to JAK/STAT
inhibitors
SH2B3 Deletion and sequence mutations very rare in BCP-ALL, associated to BCR-ABL1-like
negative regulator of JAK2 signaling
May be responsive to JAK inhibitors
CREBBP Focal deletion and sequence mutations
20% of relapsed BCP-ALL; commonly acquired at relapse
Mutations result in impaired histone acetylation and
transcriptional regulation
Associated with glucocorticoid resistance
TP53 Deletions and sequence mutations
3% of BCP-ALL, commonly acquired at relapse
Loss of function or dominant negative poor outcome
BCR-ABL1-like GEP/CNA similar to BCR-ABL1 10% of BCP-ALL Activating cytokine receptor and kinase signaling
May be responsive to TKI or to JAK inhibitors
ETP T-ALL/myeloid immature immunophenotype 6-12% of T-ALL stem cell disease May be responsive to high-dose
cytarabine or epigenetic therapy
Adapted from ref.1. BCP-ALL= B-cell precursor Acute Lymphoblastic Leukemia, GEP=Gene Expression Profile; CNA=Copy Number Abnormalities; TKI=Tyrosine Kinase Inhibitor.
Mullighan C, Nature 2008;453:110
Mullighan C, N Engl J Med 2009;360:470
Impact of Ikaros deletions in childhood ALL
Validation Cohort, BCP, Ph- ALL (N=237) Validation Cohort, BCP ALL (N=258) Original Cohort (N=221)
Impact of Ikaros deletions in childhood ALL
DCOG protocol. Waanders E, Leukemia 2011;25:254
MRD / IKZF1 based stratification in non-HR
MRD-IR by IKZF1 status
MRD-LR/IR and IKZF1 wt
MRD-LR/IR and IKZF1 del
IKZF1 wt
IKZF1 mut
DCOG-ALL-11 treatment protocol
M IA,IB IV MP/MTX
M MP/MTX+Dex/VCR
M HR1 MP/MTX
SR
MR
HR II
SCT
Risk group stratification: - MRD - prednisone response - CR day 33 - t(4;11)
IA,IB
IA,IB
HR2 HR3 HR1 HR2 HR3
Down/TELAML1: no anthr
IKZFdel: anthracycl
Other: anthracycl
MP/MTX+Dex/VCR
MP/MTX+Dex/VCR MP/MTX
Courtesy of Rob Pieters, DCOG
Treatment adapted on genomic lesions
Palmi C, Haematologica 2013;98:1226
overall
Intermediate Risk
Impact of Ikaros deletions in childhood ALL
n % n %SR 109 30,6% 8 2,2%IR 222 62,4% 42 11,8%HR 25 7,0% 4 1,1%
No YesIKZF1 deletionsFinal
stratification
Relapses in IKZF1 deleted: 0/8 SR vs 10/42 IR (23.8%) and 3/4 HR
EFS and CIR by CRLF2 expression in BCP-ALL Intermediate risk patients
P=0.04 P<0.0001
P=0.24(solo rel) P=0.03(solo rel)
214N. pts
39N. rel.
17.6%(2.6)5 yrs Cum. Incidence
NEG15
N. pts
9
N. rel.
61.1%(12.9)
5 yrs Cum. Incidence
POS
pB AIEOP 2000Intermediate Risk - by CRLF2 deletion
229 patients
CORS - Nov 2010
Cum
. Incid
ence
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
YEARS FROM DIAGNOSIS0 1 2 3 4 5
10N. pts
1N. events
90%(9.5)5 yrs EFS
NEG10
N. pts
6
N. events
37.5%(16.1)
5 yrs EFS
POS
pB AIEOP 2000CRLF2>=20 - by CRLF2 deletion
20 patients
CORS - Nov 2010
EFS
0.0
0.2
0.4
0.6
0.8
1.0
YEARS FROM DIAGNOSIS0 1 2 3 4 5
A new genomic subgroup : ‘Ph-like ALL’
Prognostic impact of Ph-like ALL
Mullighan C et al 2009; Loh M et al Blood 2013;121: 485-488
Den Boer ML et al 2009;
DFS
EFS
Cazzaniga G, ASH 2013 n. 353, Monday 9, 11:30 AM
p-value<0.001
1.0
0.8
0.6
0.4
0.2
0.0
EFS
0 1 2 3 4 5
BCP-ALL Total
Ph-like clustering
Case No. 235 11 (4.7%)
Age (mean) 5.7 10.3
WBC count 35,389 157,506
4-year EFS 83.20% 66.70%
Kiyokawa N, ASH 2013 n. 352, Monday 9, 11:15 AM
Kinase alterations in US Ph-like (detectable by multiplex RT-PCR)
Adapted from Mullighan, ASH 2013
Gene Drug n° of partners n° of cases partner genes
ABL1 Dasatinib 6 13 ETV6, NUP214, RCSD1, ZMIZ1, RANBP2, SNX2
ABL2 Dasatinib 3 7 RCSD1, ZC3HAV1, PAG1
CSF1R Dasatinib 1 4 SSBP2
PDGFRB Dasatinib 3 8 EBF1, ZEB2, TNIPI
CRLF2 JAK2 inhibitor 2 IGH, P2RY8
JAK2 JAK2 inhibitor 10 19 BCR, ETV6, PAX5, SSBP2, EBF1, others
EPOR JAK2 inhibitor 2 9 IGH, IGK
EBF1-PDGFRB (Ph-like) responds to TKI
Weston BW et al, J Clin Oncol. 2013;31:e413-6
Lengline E et al, Haematologica. 2013;98:e146-8
days
Conclusions
• Childhood ALL has become a paradigm of success in modern oncology. Still treatment failure rate is 15-20%.
• Response (MRD) oriented risk stratification cannot yet be fully replaced by genetic markers at time of diagnosis.
• New risk groups are being defined but their characterization is still not standardized.
• Need to minimize long-term health complications in the large population of leukemia survivors.
• Need for new drugs, new statistical methods, and transnational collaborations across large and well-characterized patient populations.
Acknowledgments Lilia Corral Simona Songia Tiziana Villa Eugenia Mella Valentina Carrino Giuseppe Gaipa Oscar Maglia Simona Sala Laura Levati Vincenzo Rossi Andrea Biondi Valentino Conter Giuseppe Masera Daniela Silvestri Maria Grazia Valsecchi Fondazione Tettamanti
Emanuela Giarin
Maddalena Paganin Katia Polato
Barbara Buldini
Barbara Michelotto
Giuseppe Basso
Clinica Ped. Univ. Padova
Grants by Fondazione Cariplo, AIRC and Comitato M.L.Verga
BFM Germany BFM Austria BFM Suisse
AIEOP Center
• Immunophenotyping • Cytomorphology (diagnosis) • Evaluation of response (d8) • Preparation of DNA (d0, d33, w12) • Banking of Cells and DNA • MRD of local patients
Padova (centralized)
If material is insufficient: phone call for repuncture
Molecular genetics: • Translocations (RNA) • MRD:
• Identification of MRD targets • Testing of specifity and sensitivity
• MRD quantification of follow up time points
• Interpretation of results
• DNA of diagnosis • DNA of follow up time points
Genetics and MRD result for final risk group assignment
Flow diagram of diagnostic management
Monza
Definition of MRD in AIEOP-BFM ALL 2000
• SR: MRD negative on day 33 (post-induction,TP1), and at day 78 (before "Prot. M" = TP2), if measured with at least 2 markers with a sensitivity of at least 10-4.
• MR: no "SR-MRD" or "HR-MRD" criteria are met.
• HR: MRD at time point 2 (d 78) is positive at ≥10-3.
How to integrate MRD in the design of the new childhood ALL
treatment protocol ?
1) FCM-MRD @d15 >10% ! HR 2) Stratification of patients who are not stratifiable by
PCR-MRD (and without classical HR criteria):
FCM-MRD @d15 <0,1% ! SR >0,1% and <10% ! MR
-> Stratification in >95% of patients
Role of FCM-MRD d15 for the stratification in AIEOP-BFM 2009
How to integrate MRD in the design of the new ALL treatment protocol ?
• MRD detection, currently the best available individual
risk assessment method, was available for stratification in almost all patients (combined use of PCR and FCM)
• R1: Treatment reduction for low-risk pts but controlled by MRD
• R2: Targeted and monitored intensification for all pts with intermediate relapse risk
• R3: Early targeted and monitored intensification in high risk pts
Perspectives
• MRD is generally used in ALL to guide post induction or post consolidation therapy.
• Whilst it is unlikely that MRD studies could be completely replaced by novel risk factors, the combined use of MRD evaluation and the newly available genomic information on leukaemia presenting features, like Ikaros or CRLF2 gene deletions will further improve risk assignment of ALL patients.
• However, clinically relevant improvements in ALL treatment can only result if MRD-based stratification is paralleled by the finding of the appropriate therapeutic strategy.
Monitoraggio molecolare della malattia minima residua nelle leucemie acute pediatriche:
standards o ricerca ?
Prerequisites of a reliable technique to detect MRD
a. Sensitivity of at least 10-4, although it depends on the clinical question;
b. Specificity, to prevent false-positive results c. being quantifiable within a large dynamic range; d. stability over-time of leukaemia-specific markers, to prevent false-
negative results, particularly in long-term studies; e. reproducibility between laboratories (essential for multicenter
trials); f. careful standardization and quality control checks; g. rapid availability of results (in time for clinical usefulness)
Problems and pitfalls of MRD-PCR detection via Ig/TCR genes
1. Time consuming, labor intensive and expensive • Target identification, selection and testing: 3-4 weeks • RQ-PCR analysis of follow-up samples:1-2 weeks
2. Extensive knowledge and experience needed: • Structure of Ig/TCR genes and rearrangement processes • Ig/TCR gene rearrangement patterns in ALL (precursor-B-
ALL ↔ T-ALL; children ↔ adults)
3. International comparability of MRD-PCR results • Between MRD-PCR centers of same treatment protocol • Between treatment protocols
→ Standardization, guidelines and quality control
European Study Group on MRD detection in ALL (EuroMRD)
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
Basis for accreditation of laboratory diagnostics
EuroMRD collaborative history
1993
Start IG/TcR analysis in I-
BFM
Quality Control within the EuroMRD-ALL
Positive 1.00E-05 1.00E-04 1.00E-03 1.00E-02
Mea
n
QR: 5x10-4
RQ-PCR analysis of a follow-up sample using provided primers/probe set
→ MRD level differs less than 2-fold between various laboratories
MRD-PCR laboratories 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
QR 21 Task 1: QR, S, MRD1 and MRD2
0
10
20
30
40
50
60
70
80
90
100
case 1 case 2 case 3 case 4 case 5 case 6 case 7 case 8 case 9 case 10
Perc
ent o
f lab
s wi
th s
ame
resu
lt
QRSMRD 1MRD 2
Inter-laboratory concordance on QR, sensitivity, MRD1 and MRD2
Subclonal architecture in ALL
Linear
Moderate Complex
Anderson, Nature 2011 Notta, Nature 2011
Darwinian clonal evolution of a cancer
Evolutionary speciation, from Charles Darwin's
(1837) Greaves, M. Cancer stem cells: back to Darwin? Semin. Cancer Biol. 20, 65–70 (2010).
Changes in clonal architecture in ALL
Clonal relationship of diagnosis and relapse samples in ALL
Mullighan, C. G. et al. Science 322, 1377–1380 (2008).
Perspectives : Clinical use of MRD
1. Identification of subgroup of patients with different kinetic of early tumor reduction
2. Identification of patients with different outcome within genetically homogeneous subgroups
3. Identification of impending relapse
4. ‘Molecular relapse’ ?
Prospective MRD monitoring in chilhood ALL for molecular relapse detection ?
• Systematic controls for MRD on all patients have relatively low chances (in children) to identify “MRD relapses”
• MRD monitoring may be quite stressful (for patients/families and labs)
• HR subgroups could be more suitable for a strategy of MRD monitoring
• A second sample for confirmation of MRD positivity should be required
MRD as surrogate marker for early assessment of novel therapies?
" The pressure to accelerate approval of novel drugs or the attempt to shorten the time to trial results has generated a growing interest on the use of end-points on activity (response) as surrogate end-points for efficacy (survival or event free survival).
" However, deciding that efficacy of treatment can be assessed in terms of molecular response requires that MRD levels are properly validated as a surrogate end-point.
An end-‐point for acSvity is not necessarily a surrogate for efficacy
AcSvity or Efficacy
MRD as surrogate end point
‘… It has to be pointed out that surrogate markers cannot serve as final proof of clinical efficacy or long term benefit. If they are intended to be the basis for regulatory review and approval then, unless they are properly validated, there should be a predetermined plan to supplement such studies with further evidence to support clinical benefit, safety and risk/benefit assessment.’ EmeaCHMP/EWP/83561/2005
MRD EFS-‐Survival
early response or clinical outcome
Conclusions ‘standards or research’?
" ‘Standard’ • Indepenent prognostic value at end-induction • Standarized method • Quality control rounds
" ‘Research’ • MRD depends on treatment and time points -> no clear value
outside clinical protocols • Clonal background and evolution • Combination of MRD and new genetic biomarkers • Validation as a surrogate marker of efficacy • Future use of next generation sequencing (development and
validation) • Biology of different response (MRD) ?
Molecular MRD monitoring
in chilhood AML
(…) AML is lagging behind acute lymphoblastic leukemia with respect to the implementation of MRD criteria for guidance during therapy. AML is particularly disadvantaged compared with ALL in that approximately half of AML patients lack a molecular target suitable for MRD monitoring.
Elisabeth Paietta. Minimal residual disease in acute myeloid leukemia: coming of age. ASH 2012 - Eucational
Molecular targets for MRD quantification in AML
# Fusion transcripts - t(8;21)(q22;q22) / AML1-ETO - inv(16) or t(16;16)(p13q22) / CBFβ-MYH11 15% of cases RNA, non-linear correlation with cells Pre-leukemia/SC monitoring
# Mutations (allele specific primers)
ex. NPM1, FLT3-ITD low mutation rate in chilren unstable targets
# Overexpression
ex. WT1, EVI1, PRAME
WT1 expression monitoring in chilhoo AML
• Various methodologies to quantify WT1 expression • Aim: To standardize WT1 mRNA quantitation the
European Study Group on WT1 Expression in Childhood AML was established including centers from Germany, Italy and Czech Republic
WT1 Quality controls
Leukemia 2009, 23 (8), 1472-1479 - Standardization of WT1 mRNA quantitation
WT1 expression in AML and healthy BM
2 log 20%
PB
Acknowledgments Lilia Corral Simona Songia Tiziana Villa Eugenia Mella Valentina Carrino Giuseppe Gaipa Oscar Maglia Simona Sala Laura Levati Vincenzo Rossi Andrea Biondi Valentino Conter Giuseppe Masera Daniela Silvestri Maria Grazia Valsecchi Fondazione Tettamanti
Emanuela Giarin
Maddalena Paganin Katia Polato
Barbara Buldini
Barbara Michelotto
Giuseppe Basso
Clinica Ped. Univ. Padova
Grants by Fondazione Cariplo, AIRC and Comitato M.L.Verga
BFM Germany BFM Austria BFM Suisse
Surrogacy requires that the effect of the intervention on the ‘candidate’surrogate predicts its effect on
true clinical outcome
Prentice’s definition
Treatment Surrogate Clinical
X
Surrogate endpoint needs to be validated It is treatment (class) specific
“Surrogate scenario”
40% 10%
4 24
28
40% 10%
6 16
22
% failures 4 yrs
n. of failed
Same rate of failures in responders/non responders
Modest effect on clinical outcome
Standard treatment 100 pts
40 60
YES NO
Experimental treatment 100 pts
60 40
YES NO
High ac`vity
Response
MG Valsecchi, CORS Monza
Main objective of the consortium is to develop, standardize, and validate IG / TR NGS tools for:
i.) clonality assessment; ii.) MRD analysis; iii.) repertoire analysis / somatic mutation analysis
Platform-independent assay design Scientifically independent group, no exclusive interactions with commercial partners in the field of NGS (whenever relevant and useful, collaboration will be initiated, for example for optimal dissemination of developed assays / tools)
EuroClonality-NGS consortium
EuroClonality NGS consortium
A. Langerak / J. van Dongen (Rdam)
P. Groenen (Nijmegen)
M. Brüggemann / C. Pott (Kiel)
M. Hummel (Berlin)
M. Catherwood (Belfast)
F. Davi (Paris)
E. Macintyre (Paris)
R. Garcia Sanz (Salamanca)
K. Stamatopoulos (Thessaloniki)
N. Darzentas (Brno)
G. Cazzaniga (Monza)
M. Lefranc / V. Giudicelli (Montpellier) Coordination : A.W. Langerak
Phase 1 Technical WorkPackages
1 Development of IG-TR PCR-based NGS assays (all teams)
2 Bioinformatics pipeline (N. Darzentas)
3 Capture-based NGS strategies (D Gonzalez)
Phase 2 Application WorkPackages
4 NGS-based clonality assessment (P. Groenen / M. Hummel)
5 NGS-based MRD assessment (M. Brüggemann / C. Pott)
6 NGS-based Ig repertoire analysis (F. Davi / K. Stamatopoulos)
7 NGS-based TR repertoire analysis (A. Langerak / E. Macintyre)
EuroClonality-NGS consortium
Position IGH
V-J
IGH
D-J
IGK
V-J / V-Kde
IGL
V-J
TRB
V-J / D-J
TRG
V-J
TRD
V-J / D-D/J
“FR3”
(clonality,
MRD)
~200 bp
Kiel
Salam.
Paris-P
Thess
Nijmegen
Rotterdam
Belfast
NOT Berlin
Kiel
Monza
Paris-N
London
Paris-N Monza
London
“leader”
(repertoire) Paris-P n.a.
(phase 2?)
t.b.d.
(phase 2?)
t.b.d. NOT NOT NOT
EuroClonality-NGS – PCR-based assays
454 GS Flex, Junior (Roche) HiSeq, MiSeq (Illumina)
Ion Torrent (Life)
Platform independence
• wet lab work • 'raw' data from the NGS machine
• data preparation, including error correction
• meta-‐analysis, e.g. IMGT/HighV-‐QUEST Nikos Darzentas Vojta Bystry Jana Silhava Bioinformatics Analysis Team -‐ BAT -‐ bat.infspire.org Central European Institute of Technology -‐ CEITEC Masaryk University -‐ MU Brno, Czech Republic
EuroClonality-NGS – Bioinformatic pipeline
basic major steps 00. everything is logged, in different formats 01. parameter choice, can be defaults, can be organised in 'scenarios' 02. reading files, counting sequences, handling data 03. if applicable: joining paired-‐end reads 04. if applicable: across-‐samples comparison and info gathering 05. primer / adapter cutting 06. quality trimming, filtering, masking 07. FASTQ to FASTA 08. dereplicating 09. error correction 10. final filtering steps 11. junction analysis, summaries, matrices, visualisations 12. wrapping up -‐ minilogging, compressing/removing files
EuroClonality-NGS – Bioinformatic pipeline
Nikos Darzentas
error correction error corrections are attempted through a module that can be repeated as many times as requested or required there is actually an 'auto' mode that goes on (within limits) until it reaches a plateau of small number of corrections error correction decisions can be different, for many reasons: -‐ for different sequence regions, e.g. V, junction, J etc. -‐ for different receptors, i.e. IG and TR -‐ for different samples, runs, machines, protocols critically, we can learn on-‐the-‐fly from a sample or sequence region (e.g. V region in TR, which should be in germline-‐configuration) to apply in another region (e.g. junction) or other samples there can also be multiple individual factors affecting the decisions, e.g. strand biases
EuroClonality-NGS – Bioinformatic pipeline
Nikos Darzentas
cluster 19 10 20 30 40 50 60 70 80 90 100 110 120 . | . | . | . | . | . | . | . | . | . | . | . | . gagctctgtgaccgccgcggacacggctgtgtattacTGTTCGAGCAGCTTAGGTGTGGCAGTNGCTGGTACGGGTTCGGACTACTTTGAGCAATGGagccagggaaccngggtcaccgtctcctcag g t g n g -‐ t g a n |after corrections -‐ g -‐ g t -‐ g -‐ g -‐ g -‐ t g a -‐ g
EuroClonality-NGS – Bioinformatic pipeline
Nikos Darzentas
Exome Capture – TruSeq (Illumina) DNA $ Fragmentation ~200 bp $ Library Prep $ Hybridisation to biotin-DNA baits $ Streptavidin pull down $ Amplify/index $ Sequence
EuroClonality-NGS – Capture-based assays
BCL2 IGH
FFPE
FF
EuroClonality-NGS – Capture-based analysis
David Gonzalez ! to be extended to V(D)J rearrangements (IG-TR)
Recommended