La malattia residua minima in citometria a flusso: problematiche

La malattia residua minima in citometria a flusso:
problematiche tecniche e impatto clinico
4° UK NEQAS USERS MEETING
Meeting Scientifico
Milano, 27 Ottobre 2011
Giuseppe Gaipa
[email protected]
Outline
• Razionale
• Definizione tecnica e significato in oncoematologia
• Applicazioni e requisiti tecnici
• Flow Cytometry Vs PCR
• MRD by Flow Cytometry: standardizzazione e QC trials
• Impatto clinico (leucemie pediatriche)
• Uso corretto della MRD
LA RISPOSTA ALLA TERAPIA E’ ETEROGENEA
Campana D, Educational Program ASH meeting, 2008
Szczepanski T et al , THE LANCET Oncology 2001
REMISSIONE EMATOLOGICA E MALATTIA
RESIDUA MINIMA: DUE DEFINIZIONI CORRELATE
Diagnosi
Remissione (< 5% blasti)
MALATTIA RESIDUA MINIMA: Livello di malattia al di sotto
del limite di sensibilità dell’indagine morfologica
SIGNIFICATO CLINICO
La malattia residua minima rappresenta un marcatore
surrogato della risposta individuale al trattamento, che
riflette l’effetto combinato di molteplici fattori clinici e
biologici.
Tumor
biology
Drug Response
Genetic
Treatment
environment
Utilizzo in clinica
FASE DELLA TERAPIA
OBIETTIVO
OPZIONE
Induzione della remissione
Risposta precoce
Riduzione/intensificazione
della terapia
Fine induzione
/mantenimento
Previsione/rischio
recidiva
Intensificazione della
Terapia; ricerca donatore
Pre trapianto
Qualità della
Remissione/ efficacia
del purging
Post trapianto
Rischio recidiva
Timing trapianto
Immunosoppressione
/DLI
Requisiti tecnici
• Sensibilità (ideale 10-4 - 10-5)
• Specificita’ (no falsi positivi)
• Stabilità dei marcatori
• Facile standardizzazione
• Quantificazione della MRD
• Riproducibilità tra laboratori (per gli studi multicentrici)
• Rapidita’ (applicabilità in ambito clinico)
Tecniche per lo studio della malattia residua minima
Flow Cytometry
Aberrant Immunphenotypes
RQ-PCR
Junctional regions of Ig/TCR genes
dilution curve
Blast cells
undil.
10-5
CD10
RQ-PCR
10 0
Sensitivity 10-3- 10-5
Applicability 80-90%
10 1
CD58 FITC ->
AIGIBM0302.58.003
RT-PCR chromosome aberration
10 2
CD58
Sensitivity 10-3- 10-4
Applicability > 90%
10 3
10 4
Sensitivity 10-3- 10-6
Applicability 10-50%
Quale tecnica ?
Non esiste una tecnica “migliore” in assoluto.
La scelta puo’ dipendere da molti fattori tra cui:
-Time point dello studio
-Domanda Clinica
-Tipo di protocollo terapeutico
- Expertise del laboratorio
In generale:
Per la identificazione dei pazienti ad alto rischio è preferibile un metodo
con alta specificità, mentre non è richiesta una alta sensibilità.
Per la identificazione dei pazienti ad basso rischio è indispensabile un
metodo con alta sensibilità
RQ-PCR vs Citometria a flusso
RQ-PCR
FLOW
sensitivity
10-4/6
10-4/5
specificity
very high
(clone and patient-specific)
high
(leukemia-associated)
time of response
slow
very fast
costs *
~ 2500 €
350-550 €
standardization
High
High, but operator
dependent
applicability
~ 90
MRD quantification
DNA (log level)
% (of childhood ALL)
* Complete follow-up per patient
>95 % (of childhood ALL)
% or absolute number
of cells
Leukemia-Associated Immunophenotypes
CD20/CD10/CD19/CD34
LLA
LLA
LLA
CD10 PE
LLA
(CD19+ gated)
10 0
10 1
10 2
10 3
CD20 FITC ->
PIENBM2802.20.001
10 4
10 0
10 1
10 2
10 3
10 4
CD20 FITC ->
BISABM0703.20.003
10 0
10 1
10 2
CD20 FITC ->
ROCAMABM0901.20.003
CD10 PE
CD20 FITC
Normal Bone Marrow
10 3
10 0
10 1
10 2
10 3
CD20 FITC ->
PIASBM150903.20.002
CD20 FITC
10 4
10 4
10 0
10 1
10 2
10 3
CD20 FITC ->
GAGIBM2810.20.003
10 4
Cellular Background is crucial
LLA
LLA
LLA
CD10 PE
LLA
10 0
10 1
10 2
10 3
10 4
CD20 FITC ->
PIENBM2802.20.001
10 0
10 1
10 2
10 3
10 4
CD20 FITC ->
BISABM0703.20.003
10 0
10 1
10 2
10 3
10 4
CD20 FITC ->
ROCAMABM0901.20.003
10 2
10 3
10 4
Normal BCP in
T-ALL
Day 78 therapy
Normal BCP
donor
Normal BCP in T-ALL
Day33 therapy
CD10 PE
Normal BCP in T-ALL
Day15 therapy
10 1
CD20 FITC ->
GAGIBM2810.20.003
(CD19+ gated)
CD20 FITC
10 0
10 0
10 1
10 2
CD20 FITC ->
006
10 3
CD20 FITC
10 4
10 0
10 1
10 2
10 3
10 4
CD20 FITC ->
NUGHEDUBM160605.20.003
10 0
10 1
10 2
10 3
CD20 FITC ->
PIASBM150903.20.002
10 4
10 0
10 1
10 2
10 3
CD20 FITC ->
SPALBM2405.20.003
10 4
Phenotypic plasticity in ALL:
drug-induced antigen expression modulation
by steroid-containing induction therapy
PB
BM
Typical but not uniform
patterns (“normalization”):
Diagnosis
CD19-PC5
PB
BM
10 0
10 1
10 2
10 3
10 4
FL2-CD10 PE ->
ZUANBM0512.58.019
up:
10 0
10 1
10 2
10 3
10 4
FL2-CD10 PE ->
ZUANPB0512.20.024
10 0
10 1
10 2
CD11a, CD20, CD45
10 3
10 4
10 0
FL2-CD34 PE ->
MAANBM2703.34.020
10 1
10 2
10 3
10 4
FL2-CD34 PE ->
MAANPB2703.34.026
down: CD10, CD34, TdT
CD99, MyM
Follow-up
~ stable: CD19, CD58
10 0
10 1
10 2
10 3
FL2-CD10 PE ->
ZUANBM1912.58.004
10 4
10 0
10 1
10 2
10 3
FL2-CD10 PE ->
ZUANPB1912.20.008
CD10 PE
10 4
10 0
10 1
10 2
10 3
FL2-CD34 PE ->
MAANBM1004.34.005
10 4
10 0
CD34 PE
Gaipa et al., Leukemia 19: 49 (2005)
10 1
10 2
10 3
FL2-CD34 PE ->
MAANPB0304.34.005
10 4
CD10
diagnosis
10 0
10 1
10 2
10 3
10 4
CD20 FITC ->
D170904.012
10 0
10 1
0.72 %
10 2
10 3
10 4
CD20 FITC ->
D011004.012
10 1
CD20 FITC ->
D230904.016
10 2
CD20
10 1
10 2
10 3
10 4
CD20 FITC ->
D191004.002
day 15
10 0
10 0
10 4
10 0
10 1
CD20 FITC ->
D121004.006
10 2
10 1
10 2
10 3
10 4
CD20 FITC ->
D111104.002
day 33
10 3
10 0
0.8%
10 4
10 0
10 1
CD20 FITC ->
D171104.006
10 2
10 1
10 2
10 3
10 4
10 3
10 4
CD20 FITC ->
D131204.002
day 52
10 3
10 0
day 78
10 3
10 4
10 0
10 1
CD20 FITC ->
D141204.006
10 2
Standardized antibody panel
COLOR
NC-Correlation
1STFITC
2NDPE
SYTO16
CD10orCD7
3RDPE-C 7
Y
CD45
4THAPC
CD19orCD3
BCP- ALL (1 main staining plus 0 – 3 of additionals)
Pro-B ALL
1)
CD20
CD10
CD34
CD19
2)
CD58
CD10
CD34
CD19
3)
CD10
CD34
CD45
CD19
4)
CD10
CD11a
CD45
CD19
1) CD10+CD20
CD38
CD34
CD19
2) CD15 or CD65
CD34
CD45
CD19
T-ALL (1 main staining plus 0 - 1 of additionals)
1)
CD99
CD7
sCD3
CD5
2)
CD99
CD7
sCD3
cyCD3
3)
TdT
CD7
sCD3
cyCD3
QUALITY CONTROL (I) Instrument Setting
Day1
10 0
10 1
10 2
10 3
10 4
CD4 FITC ->
ALMIPB0311.48345.002
• Material: Normal PBMC stained with CD4/CD8/CD3/CD45
Day2
10 0
10 1
10 2
10 3
• Frequency: Daily
10 4
CD4 FITC ->
ALMIPB1011.48345.002
• Parameters: Fluorescence intensity; Compensation
Day3
10 0
10 1
10 2
10 3
10 4
CD8 PE
CD4 FITC ->
ALMIPB0712.48345.006
• Expected results: Mainteining parameters
within established ranges.
Day4
10 0
10 1
10 2
10 3
10 4
CD4 FITC ->
ALMIPB1711.48345.006
CD4 FITC
QUALITY CONTROL (II) Instrument linearity
FL1
FL2
• Material: Calibrite beads (Type IIIa)
• Frequency: two weeks
FL3
FL4
• Parameters: MFI at established PMT voltage
differences in MFI btween beads of the same
color
• Expected results: Mainteining parameters
within established ranges (20 runs in 5 days).
Gating strategy
MRD quantification
• For FCM-MRD measurements, at least 300000 ungated events were
collected and analyzed
• The minimum target sensitivity for quantifying MRD was defined as the
ability to detect 30 clustered MRD events in 3x105 total cellular events
(0.01%)
• However a cluster of at least 10 events with leukemia-associated
immunophenotype and back-gating light scatter was sufficient to
define a sample as “MRD-positive”.
Inter-laboratory sample exchange
(exchange of follow-up samples)
n 63 samples exchanged between the two centers
Center B
positive/negative concordance of MRD-estimates: 90% (κ=0.81).
Center A
Inter-laboratory sample exchange
(exchange of artificial mixture of blasts with normal bone
marrow)
n 164 MRD-values available from 42 submitted samples
observed results
Observed results
100,00
1
107
10,00
ICC 0.98
1,00
0,10
Center 1
5
Center 2
Center 3
0,01
Center 4
5
46
0,00
0,00
0,01
0,10
1,00
10,00
100,00
expected results
Expected results
5 false values each among 113 positive and 51
negative values (sensitivity 95.6%; specificity 90.2%)
STANDARDIZATION AND QUALITY CONTROL:
INTERPRETATION CONCORDANCE ON EXCHANGED FILES
Files n 800
Centers n 4
1
observed results
100,00
1
381
5
10,00
7
1,00
BERLIN
MONZA
PADOVA
VIENNA
24
0,10
0,01
25
356
0,00
0,00
0,01
0,10
1,00
10,00
100,00
expected results
Inter-rater reliability coefficient 0.995
STANDARD OPERATING PROCEDURES
Standardization and Clinical applications of
Flow Cytometric MRD studies in Childhood
Leukemias
The experience of the I-BFM ALL
FLOW-MRD network
AIMS of the I-BFM ALL FLOW-MRD
network
• Make results comparable between study groups
• Foster collaborative research
• Promote FLOW as reliable and standardized tool for
clinical applications
Standardization and Quality Control
Program
Step 1
Step 2
• Rotational personnel exchange (site visits)
• Sample quality monitoring (transport delay in multiple clinical centers)
• Twice yearly group meetings incl. LMD file reviewing
• Preparative quality monitoring
• MoAb standardization; clones and fuorochromes
• Interlaboratory Flow cytometer performances monitoring (IIIa type beads-MESF)
• Standardized MoAb combinations
• Sample Preparation Standardization
Step 3
• Post-acquisition standardization:
- Education by personnel exchange
- Review rounds (incl. Online accessibilty- ftp Server)
- LMD file exchange ring trials
- sample exchange ring trials (spiked or “real“)
MRD QUALITY CONTROL PROGRAM
The UK NEQAS Experience
MRD Quality control - NEQAS
• NEQAS
– 63 Labs registered to date
• Pilot Study – April 2010
– 4 QC rounds per year
• Diagnostic & 2 x FUP samples (B & T Cell ALL)
– Interim October 09- April 2010
• 3 free QC trials to all labs registered
• Resolve technical/logistic issues
• April 2010-2011
• Participants registration costs
• Only 2 trials received
• April 2011-2012
• Participants registration costs
• 4QC rounds due
Courtesy of Jenny Jesson – UK NEQAS
Summary of Neqas Trials
Trial No
Date
Lineag
e
Median
MRD%
No of Labs
% Returns
09021
Nov 2009
B
0.18
30/37
81
09022
Nov 2009
B
0.13
30/37
81
10011
March 2010
B
0.05
40/48
83
10012
March 2010
B
0.03
41/48
85
10021
May 2010
B
0.01
40/56
71
10022
May 2010
B
0.01
39/56
70
10031
Dec 2010
B
0.003
42/56
75
10032
Dec 2010
B
0.001
43/56
77
10041
Dec 2010
B
0
42/56
75
10042
Dec2010
B
0
42/56
75
Courtesy of Jenny Jesson – UK NEQAS
Courtesy of Jenny Jesson – UK NEQAS
NEQAS 0902 - 30 Labs
Courtesy of Jenny Jesson – UK NEQAS
Impatto clinico
(leucemie pediatriche)
Assessment of MRD in ALL is an independent prognostic
indicator and has become a corner-stone for risk stratification
Van Dongen JJM et al.
Lancet 1998,352:1731
Elaine Coustan Smith et al.
The Lancet, vol.351,1998
MRD BASED BFM/AIEOP-ALL INDUCTION THERAPY PROTOCOL
INDUZIONE IB
INDUZIONE IA
MTX IT
MTX IT
L-ASP 5UL/mq
CPM 1 gr./mq
ARA-C:
DAUNO:30mg/mq
75mg/mq/die
VCR:1,5 mg/mq
R
PDN 60 mg/mq die
1
8
PB
PDN
6-MP
DXM 10 mg/mq die
15
BM
22
PB
29
33
BM
36
60mg/mq/die
43
50
57
64
BM
78
BM
PB
MRD
MRD
PCR–based risk assessment:
Low risk: BM+33 and BM+78 negative
High risk: BM+78 > 5 x10-4
Medium: all others
MRD evaluation as an independent
prognostic factor
Conter V et al:Blood. 2010 Apr 22;115(16):3206-14.
Schrappe M et al:Blood. 2011 Aug 25;118(8):2077-84.
AIEOP-BFM ALL 2000 TRIAL – Induction Ia Phase
MTX IT
L-ASP 5UL/mq
DAUNO:30mg/mq
VCR:1,5 mg/mq
R
PDN 60 mg/mq die
1
8
PB
PDN
DXM 10 mg/mq die
15
BM
22
PB
29
33
BM
Flow MRD
Period: December 2000 - December 2004
Patients: 830 childhood with ALL
Centers: 34 Italian Centers
Samples: Bone marrow aspirate on day 15
Clinical impact of day 15 FCM MRD
FCM-MRD based risk groups:
< 0.1% standard risk
< 1%-<10% intermediate risk
> 10 % high risk
1.0
Event free survival
EFS
0.8
0.6
0.4
<0.1
<10%
>=10%
0.2
N. pts
343
382
90
N. events
33
73
44
5 yrs EFS
89.9%(1.7)
79.3%(2.3)
46.1%(5.9)
3
4
0.0
0
1
2
5
YEARS FROM DIAGNOSIS
Basso G et al, Journal of Clinical Oncology 27(31) :5168–5174 (2009)
Day 15 FCM MRD by presenting features:
NCI criteria
A
B
NCI Standard Risk
NCI High Risk
(age 1 to 9 years WBC< 50,000/mL )
(Others )
1.0
1.0
<0.1
<10%
>=10%
0.9
0.8
N. pts
251
264
51
N. rel.
18
38
18
5 yrs Cum. Incidence
8.3%(1.9)
15.5%(2.4)
43.1%(8.6)
0.8
N. pts
92
118
39
N. rel.
6
24
20
5 yrs Cum. Incidence
5.4%(2.4)
22%(4.1)
53.8%(8.4)
0.7
Cum. Incidence
0.7
Cum. Incidence
<0.1
<10%
>=10%
0.9
0.6
0.5
0.4
0.6
0.5
0.4
0.3
0.3
0.2
0.2
0.1
0.1
0.0
0.0
0
1
2
3
YEARS FROM DIAGNOSIS
4
5
0
1
2
3
YEARS FROM DIAGNOSIS
Basso G et al, Journal of Clinical Oncology 27(31) :5168–5174 (2009)
4
5
FLOW-CHART FOR RISK ASSIGNEMENT IN
AIEOP-BFM ALL 2009 TRIAL
- PPR
- No CR d 33
- MLL/AF4 or t(4;11)
- Hypodiploidy
Classical
(non-MRD)
HR criteria
no
PCR MRD
Reliable ?
yes
MRD
SR?
yes
no
no
FCM
MRD
MR?
yes
no
MRD
HR?
yes
PCR
MRD SR
excluded?
MR
HR
no
yes
MRD d15
<0.1%?
no
yes
SR
yes
FCM
MRD d15
≥ 10%?
no
yes
MR
MR
SR
HR
HR
MRD is the best available method to assign
the risk of ALL patients
• There is a close association between the quality of the
molecular remission and the final outcome, independently
on the applied treatments in childhood ALL
• It is still unknown why the exposure to drugs during the early
phases of treatment (induction or consolidation) discloses
different in vivo chemosensitvities which influence the final
treatment outcome
Cazzaniga G. et al Br J Haematol, 2011
MRD as surrogate marker for early
assessment of novel therapies?
• To accelerate approval of novel drugs or 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.
Cazzaniga G. et al Br J Haematol, 2011
MRD as surrogate end point
Activity
early response
MRD
or
Efficacy
or
clinical outcome
EFS-Survival
An end-point for activity
is not necessarily a surrogate for efficacy
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
Conclusions
•
MRD is generally used in ALL to guide post induction or post
consolidation therapy:
- Early time points to decrease treatment
- High MRD levels at late time points for more effective treatments.
•
Either FCM or PCR, or both can be used in MRD-based treatment
protocols depending on the available expertise, resources and specific
protocol design.
•
The combined use of MRD evaluation and the newly available genomic
information 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.
AKNOWLEDGMENTS
Giovanni Cazzaniga
Oscar Maglia
Simona Sala
Simona Songia
Andrea Biondi
Michael N Dworzak
Renate E Panzer-Grümayer
Angela Schumich
M.Tettamanti Research Center, Pediatric Clinic
University of Milan Bicocca, Monza, Italy.
Leonid Karawajew
Richard Ratei
Wolf-Dieter Ludwig
Barbara Buldini
Alessandra Benettello
Barbara Michielotto
Giuseppe Basso
Hemato-Oncology Lab., Pediatric Clinic,
University of Padua, Padua, Italy.
Ester Mejstrikova
Ondrej Hrusak
Pediatric Hematology and Oncology,
Charles University Prague,
Jean- Pierre Bourquin
Division of Pediatric Oncology, University
Children's Hospital Zurich, Switzerland
Children's Cancer Research Institute and
St. Anna Children's Hospital, Vienna, Austria,
Department of Hematology, Oncology,
and Tumor Immunology, Robert-Rössle-Clinic, Charité,
Campus Buch, Berlin, Germany
Andre Schrauder
Martin Schrappe
Pediatrics, University Hospital Schleswig-Holstein,
Campus Kiel, Kiel, Germany
Daniela Silvestri
Maria Grazia Valsecchi
Dept of Clinical and Preventive Medicine,
Università Milano Bicocca, Monza, Italy,
Drorit Luria
Shai Israeli
Sheba Medical Center Tel-Hashomer,
Ramat Gan, Tel Aviv Israel
CLINICAL SIGNIFICANCE OF IMMUNOLOGICAL
DETECTION OF MRD IN CHILDREN WITH ALL
Elaine Coustan Smith et al. The Lancet, vol.351,1998
CLINICAL SIGNIFICANCE OF
MRD IN CHILDREN WITH ALL by PCR
Low-risk group (42.6%; n=55)
Intermediate-risk group
(42.6%; n=55)
High-risk group (14,8%; n=19)
Kaplan-Meierz estimates of the relapse-free survival according
to the residual disease at time points one and two.
Van Dongen JJM et al.; Lancet 1998,352:1731
PATIENTS AND SAMPLES:FILE ANALYSYS
•
Patients selected for files analyses: 31 (randomly chosen dates of
recruitment from early 2002 to late 2003).
•
202 samples from seven time-points of assessment (PB from diagnosis,
days 8, 15, 33; BM days 15, 33, 78) were submitted to all centers for
blinded LMD file interpretation.
•
N° of independent votes on each sample: 800 (four on each sample,one
per center) and compared by the study coordinator, who was not involved
in the original LMD analyses.
FILE ANALYSYS: DATA EVALUATION
Qualitative analyses: the majority votes per sample.
Quantitative concordance: in MRD-positive samples, the median of the
positive values.
Failures were defined as:
i) A negative vote in a sample otherwise qualified positive by most of the
centers, or vice-versa
ii) A positive MRD-level >3x larger or smaller than the median of the
positive values of the sample.
RESULTS (I): INTER-LABORATORY TEST OF
CONCORDANCE IN POSITIVE/NEGATIVE MRD-VOTES
• 106/202 samples were classified as MRD-positive (53%) and 96 as negative.
Per centers agreement on expected vote:
Center 1: 89%
Center 2: 97%
Center 3: 93%
Center 4: 96%
• All four centers agreed on the MRD-status in 76% (153/202) of samples
overall (83 of 106 positive, i.e. 78%; 70 of 96 negative, i.e. 73%).
RESULTS (II): INTER-LABORATORY TEST OF
QUANTITATIVE CONCORDANCE
• Of the106 MRD positive samples correct* level were quoted in:
Center 1: 82%
Center 2: 93%
Center 3: 85%
Center 4: 94%
• All four centers agreed on the correct MRD level in 67% of samples (71/106).
* A positive MRDMRD-level >3x larger or smaller than the median of the positive values
values of the sample
RESULTS (II): INTER-LABORATORY TEST OF
QUANTITATIVE CONCORDANCE
• Of the106 MRD positive samples correct* level were quoted in:
Center 1: 82%
Center 2: 93%
Center 3: 85%
Center 4: 94%
• All four centers agreed on the correct MRD level in 67% of samples (71/106).
* A positive MRDMRD-level >3x larger or smaller than the median of the positive values
values of the sample