CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN) Multicolor...
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CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN) Multicolor Immunophenotyping: Standardization and Applications March 9-11, 2012 TMH, Mumbay (India) PLASMA CELLS (MORMAL AND NEOPLASTIC)
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Transcript of CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN) Multicolor...
CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN)
Multicolor Immunophenotyping: Standardization and ApplicationsMarch 9-11, 2012 TMH, Mumbay (India)
PLASMA CELLS(MORMAL AND NEOPLASTIC)
TherapyTreatment compliance
In vivo drug kinetics
Tumor micro-environment
Tumor cell features
MRD MONITORING IN HAEMATOLOGICAL MALIGNANCIES
100
101
102
103
104
105
106
107
108
109
1010
1011
N.
of
tum
or
cells
10-6
10-5
10-4
10-3
10-2
Resistance
Complete remission
Immunological CR
Molecular CRSen
sit
ivi
tyS
en
sit
ivi
ty
- CML- APL- Childhood ALL- …
Therapeutic decisions
Morphology, Cytogenetics
Southern-Blot,
FCM DNA aneuploidyF.I.S.H
Flow cytometry
P.C.R.
MRD TECHNIQUES FOR HAEMATOPOIETIC MALIGNANCIES
FCM immunophenotyping PCR/RT-PCR analyses (sensitivity) (sensitivity)
Disease category LAIP sIgk/sIgl Junctional Reg Chromosomal
or TCRVb Ig/TCR genes aberrations(10-3-10-4) (10-2-10-3) (10-3-10-6) (10-4-10-6)
Precursor B-ALLChildren 80-90% NA 95% 40-50%Adults 70-80% NA 90% 35-45%
T-ALLChildren >95% 30-35% >95% 10-25%Adults >95% ? 90% 5-10%
Chronic B-cell leukemias <5% >95% >95% 10-25% Chronic T-cell leukemias 5-10% 60-65% 95% <5% B-cell lymphomas <5% >95% 70-80% 25-30% T-cell lymphomas 20-25% 50-60% 95% 10-15%
AML 70-90% NA 10% 10-30% CML NA NA NA >95%
From: Szczepanski, Orfao et al, Lancet Oncol, 2001; 2: 409-417
BACKGROUNDBACKGROUND
IMMUNOPHENOTYPING IMMUNOPHENOTYPING
- Acute Leukemias & Lymphoproliferative disorders:
• Mandatory for diagnosis & monitoring
- Multiple Myeloma:
• Restricted to research
• Differential diagnosis of unusual cases
- Acute Leukemias & Lymphoproliferative disorders:
• Mandatory for diagnosis & monitoring
- Multiple Myeloma:
• Restricted to research
• Differential diagnosis of unusual cases
Plasma cell quantification (BM infiltration)
Plasma cell quantification (BM infiltration)
• Morphological PC count :
• Morphological PC count :
- area of BM smear- infiltration pattern
- area of BM smear- infiltration pattern
Variability
• Immunophenotyping:
• Immunophenotyping:
- precise identification by CD38/CD138
- precise identification by CD38/CD138
10 10 10 10 100 1 2 3 4
CD38 FITC ->
CD
138
PerC
P/C
y5 -
>
Co-expression of CD38/CD138Co-expression of CD38/CD138
10 10 10 10 100 1 2 3 4CD38 FITC ->
TR
AN
SFO
RM
ED
SS
C -
>
High-intensityHigh-intensity
10 10 10 10 100 1 2 3 4
CD138 PerCP/Cy5->
TR
AN
SFO
RM
ED
SS
C -
>Specific expressionSpecific expression
++ ==
- but…..diluted sample lower numbers
• Correlation between Immunophenotyping & Morphology:
• Correlation between Immunophenotyping & Morphology:
• Prognostic influence of the number of BMPC:
• Prognostic influence of the number of BMPC:
Morphology
Years from diagnosis
20181614121086420
Pro
po
rtio
n o
f p
ati
en
ts a
live
1.0
.8
.6
.4
.2
0.0
PC 20%n= 111
(46 months)
PC >20%n= 501 (28 months)
p=0.0002
Morphology
Years from diagnosis
20181614121086420
Pro
po
rtio
n o
f p
ati
en
ts a
live
1.0
.8
.6
.4
.2
0.0
PC 20%n= 111
(46 months)
PC >20%n= 501 (28 months)
p=0.0002
Immunophenotyping
Years from diagnosis
14121086420
Pro
po
rtio
n o
f p
atie
nts
aliv
e
1.0
.8
.6
.4
.2
0.0
CP >10% n= 253 (21 months)
PC 10%n= 145 (53 months)
p<0.0001
Immunophenotyping
Years from diagnosis
14121086420
Pro
po
rtio
n o
f p
atie
nts
aliv
e
1.0
.8
.6
.4
.2
0.0
CP >10% n= 253 (21 months)
PC 10%n= 145 (53 months)
p<0.0001
0 25 50 75 100
Proportion of plasma cell by morphology
0
25
50
75
100
Pro
po
rtio
n o
f p
lasm
a ce
ll b
y fl
ow
cyt
om
etry
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R2= 0,39
0 25 50 75 100
Proportion of plasma cell by morphology
0
25
50
75
100
Pro
po
rtio
n o
f p
lasm
a ce
ll b
y fl
ow
cyt
om
etry
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R2= 0,4
• High-dose chemotherapy and Novel Drugs Complete remission (CR): 25%-75% Relapse-free survival (RFS) at 5 year: 40%-70%
• High-dose chemotherapy and Novel Drugs Complete remission (CR): 25%-75% Relapse-free survival (RFS) at 5 year: 40%-70%
BACKGROUNDBACKGROUND
• However, patients with MM ultimately relapse
MINIMAL RESIDUAL DISEASE (MRD)
• However, patients with MM ultimately relapse
MINIMAL RESIDUAL DISEASE (MRD)persistence of residual malignant cellspersistence of residual malignant cells
MM PCMM PC Normal PCNormal PCvsvs
Are myelomatosous PC different from normal PC?
Are myelomatosous PC different from normal PC?Ocqueteau, Am J Pathol, 1996; San Miguel et al, Blood, 2002
MM vs Normal BM plasma cells
10 10 10 10 100 1 2 3 4
JC67634.001CD38 ->
14%
10 10 10 10 100 1 2 3 4
JC67634.001CD38 ->
10 10 10 10 100 1 2 3 4
JC67634.001CD38 ->
10 10 10 10 100 1 2 3 4
JC67634.001CD45 ->
10 10 10 10 100 1 2 3 4
PA67603.001CD38 ->
3%
10 10 10 10 100 1 2 3 4
PA67603.002CD38 ->
10 10 10 10 100 1 2 3 4
PA67603.002CD38 ->
10 10 10 10 100 1 2 3 4
PA67603.002CD45 ->
Abnormal plasma cells
Normal plasma cells
MM PLASMA CELL
CyIg+
PC-associated Ags:
CD38++/+++....100%
CD138 +.......98%
B-cell-associated Ags:
CD19+..........3-8% CD20+..........2-25% CD22+..........20-30% CD10+..........6-20% HLA-DR+het….. 10% CD23+.......... 0% FMC7+......... 0%
HPC-associated Ags:
CD34+..........0%
CD117 +......27%
Adhesion molecules:
CD56+/++.....60-70%b1/b2 integrins 98%CD54….……….50-70%
Co-stimulatory Ags:
CD28+/++....... 30-40%
CD40 +....... 100%
CD81,CD27-/lo.40-50%
CD52……………….10-
50%
Myeloid-associated Ags:
CD13+......... 28%CD33 +/++..... 24%
Pan-leuc. Ag:
CD45+...20-40%
Rawstron et al, Haematologica 2008
00
1010
2020
3030
4040
5050
6060
7070
8080
9090
100100
CD19CD19 CD38CD38 CD45CD45 CD56CD56 CD28CD28 CD33CD33 CD117CD117 CD20CD20
96%96%
80%80%
73%73%
60%60%
36%36%
18%18%
32%32%
17%17%
Asynchronous expressionAsynchronous expressionInfra-expressionInfra-expression
Mateo et al. J Clin Oncol; 2008;26:2737
Over-expressionOver-expression
92%
Incidence of aberrant phenotypes in PC from MMIncidence of aberrant phenotypes in PC from MM
MOST USEFUL ANTIGENS FOR THE DETECTION OF ABERRANT PC IN MM
Antigen Expression % MM with Requirement forNormal Altered altered expression
MRD studies
CD19 + (>70%) - 95% EssentialCD56 - (>85%) ++ 75% EssentialCD20 - (100%) + 10% PreferredCD117 - (100%) + 30% PreferredCD28 -/dim (100%) ++ 15% RecommendedCD81 + -/dim N.A. RecommendedCD27 ++ -/dim 40-50% Recommended
N.A.: not analyzed/not reported.Rawstron et al, EMN consensus, Haematologica, 2008
Immmunophenotype of normal vs clonal PC
10000
8000
6000
4000
2000
0Clonal PCNormal PC
10000
8000
6000
4000
2000
0Clonal PCNormal PC
4000
3000
2000
1000
0Clonal PCNormal PC Clonal PCNormal PC
3000
2000
1000
0
CD38
HLA-I 2-microglobulin
CD40
% o
f p
osit
ive
PC
Mean
FL I
nte
nsit
y
p=0.21 p=0.005
60
50
40
30
20
10
0Clonal PCNormal PC
CD95 p=0.72
p0.001
80
60
40
20
0Clonal PCNormal PC
CD56p0.001
120
100
80
60
40
20
0Clonal PCNormal PC
CD86p0.001
p=0.002
Clonal PCNormal PC
100
80
60
40
20
0
CD126p0.001
Perez-Andres et al, Leukemia, 2005; Perez-Andres et al, Int J Cancer, 2009
MGUS vs MM: IMMUNOPHENOTYPIC PANELS
N.of PB AMCA FITC PE PerCPCy5.5 APC PE-Cy7 APCH7colors PO
3 CD38 CD56 CD19
4 CD38 CD56 CD19 CD45
6 CD38 CD56 CD45 cyIgk CD19 cyIgl
8 CD45 CD138 CD38 CD56 CD117 cyIgk CD19 cyIg l
Characterization markers
CD10, CD20, CD22 CD24, CD27, CD38 CD39, CD43, CD63
CD81, CD95, CD138 Bcl-2, HLA-DR, CyIg
Normal B lymphopoiesis
CD11a, CD11c, CD31, CD49d, CD62L, CXCR5, CCR6, CD303
B cell homing
Known to differentiate
CD13, CD15, CD28, CD33, CD56, CD45,
CD117, b2M
7 informative markers
NORMAL vs NEOPLASTIC PC: IMMUNOPHENOTYPIC PANELS
N.of PB HV500 FITC PE PerCPCy5.5 APC PE-Cy7 Alexa700colors HV450 PO APC-H7
3 CD38 CD56 CD19
4 CD38 CD56 CD19 CD45
6 cyIgL cyIgk CD19 CD45 CD56 CD38
CD138 CD117 CD19 CD45 CD56 CD38
8 CD45 CD138 cyIgL cyIgk CD138 CD117 CD56 CD38
CONSTRUCTION OF EuroFlow MRD PANELS: MMM
M #
1M
M #
2N
orm
al B
M #
1N
orm
al B
M #
2
SS
C
SS
C
SS
C
SS
C
CD38-FITC
CD38-FITC
CD38-FITC
CD38-FITC
SS
C
SS
C
SS
C
SS
C
CD38-FITC
CD38-FITC
CD38-FITC
CD38-FITC
Identify PC Select PC
Merge PC(n-cases)
Principal component analysis(n=12 markers)
CD19 19.83
CD56 19.02
CD81 12.29
CD45 11.47
CD27 9.95
CD117 9.34
CD38 5.18
MOST INFORMATIVE MARKERS
Most informative markers
EuroFlow PANEL: Plasma cell dyscrasias
Abnormal PC detection /classification in MGUS & MM (APS view)
Normal PCs
Abnormal PCs
CD19:PE Cy7-A LOGICAL 12.16
CD45:PacB-A LOGICAL 11.81
Responsible scientist: J.Flores
PCD panel: Backbone markers
PacBlue
PacOrange
FITC PE PerCP-Cy5.5
PECy7 APC APC-H7
1 CD45 CD138 CD38 CD19
2 CD45 CD138 CD38 CD19
Responsible scientists: Juan Flores
PCD panel: Backbone markers
PacBlue
PacOrange
FITC PE PerCP-Cy5.5
PECy7 APC APC-H7
1 CD45 CD138 CD38 CD19
2 CD45 CD138 CD38 CD19
Responsible scientists: Juan Flores
PacBlue
PacOrange
FITC PE PerCP-Cy5.5
PECy7 APC APC-H7
1 CD45 CD138 CD38 CD56 CD27 CD19 CyIgk CyIgl
2 CD45 CD138 CD38 CD28 b2M CD19 CD117 CD81
10 10 10 10 100 1 2 3 4
CD38 FITC ->
TR
AN
SFO
RM
ED
SS
C -
>
10 10 10 10 100 1 2 3 4
CD38 FITC ->
CD
138
PerC
P/C
y5 -
>
CD38-FITC
CD38-FITC gated PC
T-SS
C
C
D138-P
erC
P/C
y5.5
MONOCLONAL GAMMOPATHIES: IDENTIFICATION OF CLONAL PLASMA CELLS
Clonal PC
Normal PC
CD
56
PE
9
1
D
C
CP
A
54
DC
CD
19-P
cp
Cy5
CD56-PE CD45-APC
Perez-Andres, J Biol Reg, 2004
MRD TECHNIQUES FOR HAEMATOPOIETIC MALIGNANCIES
FCM immunophenotyping PCR/RT-PCR analyses (sensitivity) (sensitivity)
Disease category LAIP sIgk/sIgl Junctional Reg Chromosomal
or TCRVb Ig/TCR genes aberrations(10-3-10-4) (10-2-10-3) (10-3-10-6) (10-4-10-6)
Precursor B-ALLChildren >90% NA 95% 40-50%Adults >95% NA 90% 35-45%
T-ALLChildren >95% 30-35% >95% 10-25%Adults >95% ? 90% 5-10%
Chronic B-cell leukemias >95% >95% >95% 10-25% Chronic T-cell leukemias 70-80% 60-65% 95% <5% B-cell lymphomas 90% >95% 70-80% 25-30% T-cell lymphomas 75-90% 50-60% 95% 10-15% Multiple myeloma >90% >90% 70-80% NT
AML 70-90% NA 10% 30-40%* CML NA NA NA >95%
* Increased through the usage of additional molecular markers (e.g.: WT1, NMP1 & FLT3 mutations
BM plasma cells in MGUS
10 10 10 10 100 1 2 3 4
JR67635.001CD38 ->
0,35%
10 10 10 10 100 1 2 3 4
JR67635.002CD38 ->
10 10 10 10 100 1 2 3 4
JR67635.002CD38 ->
50%
50%
0 256 512 768 1024
JR67635.002FSC-Height ->
10 10 10 10 100 1 2 3 4
JR67635.002CD38 ->
10 10 10 10 100 1 2 3 4
JR67635.002CD45 ->
Differential diagnosisDifferential diagnosis
Risk of MGUS transformation2
Cases with predominantly (>95%) CD19- ve PC.... High risk (26% transformed in 31 months)
Risk of MGUS transformation2
Cases with predominantly (>95%) CD19- ve PC.... High risk (26% transformed in 31 months)
2. Rawstron A, Blood 2003, 102, 36 a (Abstr.116)2. Rawstron A, Blood 2003, 102, 36 a (Abstr.116)
MGUSMGUSMMMM
Only 20% of MM patients showed poly-PC
and constantly <5% (median: 0.25%)1
Only 20% of MM patients showed poly-PC
and constantly <5% (median: 0.25%)1
The most powerful single criteria for differential diagnosis (even in stage I MM)The most powerful single criteria for differential diagnosis (even in stage I MM)
versus
versus
>5% poly-PC: 98% MGUS
ClonalClonal Poly-ClonalPoly-Clonal
1. Ocqueteau M, Am J Pathol 1998, 152: 16551. Ocqueteau M, Am J Pathol 1998, 152: 1655
Prognostic influence of phenotypic profilesPrognostic influence of phenotypic profiles
CD56 & CD28CD56 & CD28
Months from diagnosisMonths from diagnosis726660544842363024181260
1.0
.9
.8
.7
.6
.5
.4
.3
.2
.1
0.0
p=0.01p=0.01
CD56+CD28- n= 1116 41 m+/+ or -/- n=266 36 mCD56-CD28+ n=116 29 m
CD56+CD28- n= 1116 41 m+/+ or -/- n=266 36 mCD56-CD28+ n=116 29 m
Months from diagnosisMonths from diagnosis
CD56 & CD117CD56 & CD117
726660544842363024181260
1.0
.9
.8
.7
.6
.5
.4
.3
.2
.1
0.0
CD56+CD117+ n= 130 45 m+/- or +/- n=267 36 mCD56-CD117- n=186 31 m
CD56+CD117+ n= 130 45 m+/- or +/- n=267 36 mCD56-CD117- n=186 31 m
p=0.001p=0.001
Months from diagnosisMonths from diagnosis
CD28 & CD117CD28 & CD117
726660544842363024181260
1.0
.9
.8
.7
.6
.5
.4
.3
.2
.1
0.0
CD28-CD117+ n= 142 45 m+/- or +/- n=327 37 mCD28+CD117- n=114 29 m
CD28-CD117+ n= 142 45 m+/- or +/- n=327 37 mCD28+CD117- n=114 29 m
p=0.0005p=0.0005
PFSPFS
PFS
• Kyle & Alexanian 1980a.
• Estimated incidence: 15% of newly diagnosed MMb.
• Estimated Risk of progression: 10% per yearc vs. 1% on
MGUS
a Kyle 1980, Alexanian 1980; bRajkumar 05; cKyle 05
IntroductionSmoldering Multiple Myeloma
Proportion of aPC referred to the total-PC (aPC/BMPC)
Proportion of aPC referred to the total-PC (aPC/BMPC)
2nd step
PC compartment
2nd step
PC compartment
..
..
..
....
....
..
..
..
....
..
..
.. .. .... .. ..
% aPC/BMPC% aPC/BMPC
% nPC/BMPC% nPC/BMPC
1st stepTotal cellularity
1st stepTotal cellularity
PC analysis in BM by FCPC analysis in BM by FC
% PC within BM cellularity% PC within BM cellularity
% Total PC in BM* 2.8 (0.9-22.0)
% of aPC / BMPC compartment 97 (35-100)
< 95% aPC / BMPC 36 (40%)
> 95% aPC / BMPC 53 (60%)
Flow Cytometry Results
* Median (range)
Impact of % aPC/BMPC by FC on Progression Free Survival
<95% aPC/BMPCn= 36 (4 progressions)
>95% aPC/BMPCn= 53 (34 progressions)
0 20 40 60 80 100 120
Months
0,0
0,2
0,4
0,6
0,8
1,0
% o
f P
rogr
essi
on F
ree
Surv
ival
p=0.0000
Median Not reached
Median 40 months
92%37%
5 years
Multivariate analysis for PFS
p HR
% a PC /BMPC 0.004 4.9
Immunoparesis 0.007 2.6
Impact of prognostic index on PFS
Immunoparesis >95% aPC/BMPC Score (n)
- - 0 (n=32)
+ / - -/+ 1 (n=27)
+ + 2 (n=27)
Impact of prognostic index on PFS
>95% aPC/BMPC or paresisn= 27 (12 progressions)
>95% aPC/BMPC + paresisn= 27 (22 progressions)
No adverse factorsn= 32 (3 progressions)
0 20 40 60 80 100 120
Months
0,0
0,2
0,4
0,6
0,8
1,0
% o
f P
rogr
essi
on F
ree
Su
rviv
al
Median not reached
Median 75 months
Median 20 monthsp= 0.003
91%
58%
18%
5 years
MM: IMMUNOPHENOTYPIC IDENTIFICATION OF NEOPLASTIC
PLASMA CELLS IN REMISSION BM
MM: Diagnostic vs remission BM
MM: Diagnostic vs remission BM
MRD/remissionMRD/remissionDiagnosisDiagnosis
MM patients show few poly-PC constantly
<5% (median: 0.25%)1
MM patients show few poly-PC constantly
<5% (median: 0.25%)1
versus
versus
ClonalClonalPoly-ClonalPoly-Clonal
FLOW MRD IN MM: Why, when and how?
- Does response to therapy impact on long-term patient outcome?
- Does flow-based MRD improve prognostic stratification of myeloma patients?
- Is flow-based MRD a well suited technique for MRD assessment in MM?
- Can flow-based MRD techniques be used in routine diagnostic labs?
In the ASCT setting, there is a large body of
evidence showing an association between
optimal response (CR/VGPR) and long-term
outcome (PFS and OS)
In the ASCT setting, there is a large body of
evidence showing an association between
optimal response (CR/VGPR) and long-term
outcome (PFS and OS)
• 10 prospective trials (2991 patients): All showed a positive correlation (statistically significant in 8) . Similar findings in 5/8 retrospective trials
(Van de Velde, Hematologica 2007, 92, 1399)
Impact of CR in the ASCT setting
- Significant correlation between maximal response and outcome prospective studies (<0.00001) & rétrospective studies (< 0.00001)
Is it the same CR & VGPR ??
0 12 24 36 48 60 72 84 96
Months from diagnosis
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
Cu
mu
lati
ve P
rop
ort
ion
Su
rviv
ing
CR vs nCR CR vs PR nCR vs PR
P=0.01P<10-6
P=0.04
0 12 24 36 48 60 72 84 96
Months from diagnosis
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
Cu
mu
lati
ve P
rop
ort
ion
Eve
nt
Fre
e S
urv
ivin
g
CR vs nCR or PR nCR vs PR
P<10-5
P=0.07
CR, n=278 nCR, n=124 PR, n=280 PD, n=25
EFS OS
Lahuerta et al. JCO 2008;26:5775–5782
CR and nCR are not the same: “depth of response”PETHEMA-GEM 2000: Outcome according to post-transplant response
CR nCR PR PD
Medians EFS, months 61 40 34 13
Medians OS, months NR NR 61 15
CR correlates with long-term PFS and OS in elderly patients treated with novel
agents
Gay et al. Blood 2011
PFS OS
P<0.001 P<0.001
CR
VGPR
PR
CR
VGPR
PR
• Retrospective analysis: 3 randomized European trials of GIMEMA and HOVON groups (n=1175)
• First-line treatment
MP (n=332), MPT (n=332), VMP (n=257), VMPT-VT (n=254)
• Significant benefit also seen when analysis is restricted to patients >75 years old
Depth of response
MR
PR
VGPR/ nCR
CR
sCR
Molecular/Flow CR
Treatment initiation
Progression
TTP
MRD investigation in MM : molecular & Immnunophenotypic tools
Which level of response should be measured?Depth of response is related to TTP
G EM 2000 (n=510*)
GEM 2000 (n=510*)D iagnosis
Diagnosis
VBM CP/
VBAD
VBMCP/
VBAD
G EM 2005<65y (n=369*)
GEM 2005<65y (n=369*)D iagnosis
Diagnosis
Thalidom ide/D exam ethasone
(TD )
(n=121)
Thalidomide/Dexamethasone
(TD)(n=121)
VBM CP/VBAD (x4)
Bortezom ib (x2)(n=126)
VBMCP/VBAD (x4)
Bortezomib (x2)(n=126)
ASC T
ASCT3m post-ASC T
3m post-ASCT
M R D
investigation
MRDinvestigation
M RD
investigation
MRDinvestigation
6 cyclesBortezom ib/
Thalidom ide/D exam ethasone
(VTD) (n=122)
Bortezomib/Thalidomide/
Dexamethasone(VTD) (n=122)
ASC T
ASCT3m post-ASC T
3m post-ASCT
M R D
investigation
MRDinvestigation
M RD
investigation
MRDinvestigation
6 alterning cycles
G EM 2005>65y (n=246*)
GEM 2005>65y (n=246*)
D iagnosis
Diagnosis
(n=206)
(n=206)
(n=157)
(n=157)
* Patients ach ieving C R or VG PR after treatm ent w ithout M RD investigation w ere excluded from the ITT ana lysis
* Patients achieving CR or VGPR after treatment without MRD investigation were excluded from the ITT analysis
6 cycles
Bortezom ib/ M elpha lan/ P rednisone (VM P) (n=121)
Bortezomib/ Melphalan/ Prednisone (VMP) (n=121)
Bortezom ib/ Tha lidom ide/ P rednisone (VTP) (n=125)
Bortezomib/ Thalidomide/ Prednisone (VTP) (n=125)M RD
investigation
MRDinvestigation
(n=102)
(n=102)
Analysis of immunophenotypic response (IR) by MFC in 619 myeloma patients included in three consecutive Spanish trials
(n=295)
(n=295)(n=222)
(n=222)
EF + n=108
EF + n=108
#MM-PC#MM-PC
# % N-PC/BMPC# % N-PC/BMPC
0.76 0.76
MRD evaluationMRD evaluation
86% 86% *MRD+ cases*MRD+ cases
44 44
#Results expressed as medians#Results expressed as medians
Complete remissionComplete remissionPartial ResponsePartial Response
IFx - n=147 (79%)
IFx - n=147 (79%)
0.10.1 <.001<.001
<.001<.001
36%36%
8585
<.001<.001
IFx +n=40 (21%)
IFx +n=40 (21%)
62%62%
0.210.21
7373
PP
*≥0.001% MM-PC
Correlation between immunophenotyping & electrophoretic responses at
three months post-ASCT (GEM 2000 trial, n=295)
Paiva et al; Blood. 2008, 112: 4017-4023
GEM2000 & GEM2005: Impact on survival of achieving an Immunophenotypic CR after HDT/ASCT
independent of the induction regimen
OSPFS
P <.001
GEM2000
GEM2005 (<65y)
P <.001
P =.802
120100806040200
100
80
60
40
20
0
P =.640
P =.091
P =.132
1251007550250
100
80
60
40
20
0
Paiva et al. Blood 2010. 116; abstr 1910
M-component positiveM-component negative
------- Follow-upP Progression
DeathTreatment interruption
1 IgG ----------------------------------------------------------------------2 B-J ------------------------------------------------------------------------------------------3 IgG -----------------------------------------------------------------------------------------------4 IgA ------------------------------------------------------------------------------------------------------5 IgG ---------------------------------------------------------------------------------------------------------------6 B-J ------------------------------------------------------------------------------------------ P -------------------------------7 IgG ----------------------------------------------------------------------------------------------------------------------------------
8 B-J -------------------------------- P ----------------------------9 IgG -------------------------------------------------------------------10 B-J --------------------------------------------------------------------11 IgG -----------------------------------------------------------------------12 IgA ------------------------------------------- P ------------------------------13 IgA ------------------------------------------- P ---------------------------------14 IgG --------------------------------------------------------------------------------15 IgA ---------------------------------------------------------------------------------16 IgG --------------------------------------------------------------------------------------------17 IgG ------------------------------------------------------------------------------- P -------18 IgG ------------------------------------------------------------------------------------------19 IgA --------------------------------------------------------------------- P --------------------20 IgG ------------------------------------------------------------------------------------------------------21 IgA ---------------------------------------------------------------------------------------------------------------22 IgA ---------------------------------------------------------------------------------------------------- P -----------23 IgA -------------------------------------------------------------------------------------------------------------------------24 IgA ---------------------------------------------------------------------------------------------------- P -------------------------25 IgA ------------------------------------------------------------------------------------------- P ----------------------------------------26 IgG -------------------------------------------------------------------------------------------------------------------------------------------27 IgA -------------------------------------------------------------------------------------------------------------------------------------------
IR / non-CR
non-IR / CR
1 2 3 4 5 6 7 8 9 10 11 12 // 16 // 20 // 24 // 28 // 32 // 36 // 40 // 44 // 48
Post-Induction Maintenance (months)
Patient no.
Kinetics of response: conventional CR vs. immunophenotypic response (IR)
7/7 (100%) patients turned IFx-
10/20 (50%) patients turned IFx+
Paiva et al, JCO, 2011
0 25 50 75 100 125
0
20
40
60
80
100
MRD- IFE-71 m (n= 94)
MRD- IFE+65 m (n=31)
MRD+ IFE- 37 m (n=53)
p= 0.002
At 5 years: 59% 49% 24% 17%
MRD+ IFE+ 37 m (n=117)
Months
GEM2000: Impact on survival of achieving an immunophenotypic CR vs. conventional CR after
HDT/ASCT
PFS
GEM 2000 trial: Multivariate Analysis
p risk p risk
MRD+ at day +100 0.002 3.6 0.02 2.0
High Risk Cytog*. 0.006 1.79 ns
Age >60y. ns 0.04 1.6
IF+ at dey +100 ns ns
PFS OS
Paiva et al; Blood. 2008 t(4;14), t(4;16), del (17p)
GEM 2000+2005: Immunophenotypic response & FISH for the prediction of early relapse in CR patients after HDT/ASCT (n=241)
MRD negative + Standard risk FISH (n=58)MRD positive OR High-risk FISH (n=45)MRD positive + High-risk FISH (n=7)
OSPFS
P <.001
Months
P <.001
Months
Medians: NRMedian: 97m
Median: 43m
80%
93%
120100806040200
100
80
60
40
20
0
0%
Median: 17m
@ 1y after ASCT
120100806040200
100
80
60
40
20
0
Median: 64m
Median: 35m
GEM 2005(>65y): Impact on survival of the depth of response after induction therapy (n=102)
Immunophenotypic response (n=31) “Stringent CR” (n=11) CR (n=9) PR (≥70% reduction) (n=51)
60
50
40
30
20
10
0
100
80
60
40
20
0
Months
P <.001
PFS
Months
P =.353
OS
6050403020100
100
80
60
40
20
0
Updated results from the MRC myeloma IX trialUpdated results from the MRC myeloma IX trial
Owen et al. IMW Paris 2011 abstr O-09
• 711 intensively treated patients (CVAD or CTD and HDM)
• at 3 months post-HDM: 66% remained MRD+ve
• highly predictive of outcome (PFS; p=0.0001)
• increased MRD-ve rates with consolidation and maintenance prolongation of PFS
• 510 non-transplant eligible patients (CTDa or MP)
• only 8% became MRD- but a significantly improved PFS was demonstrated (p=0.028)
• Immunophenotypic CR predicted outcome in CR (IFx -) patients and both standard and high-risk cytogenetic groups
MM: Flow cytometry immunophenotyping vs. molecular monitoring of MRD ?
Molecular techniques Flow cytometry
Speed 2-3 days (up to weeks) fast: 1-2 hours
Target DNA or RNA protein/cells (RNA is an instable target) (“end-product”)
Applicability 70-75% >95%
Sensitivity 10-5-10-6 10-4-10-5
Multiplexing technically demanding relatively easy(even 25 to 100 tests per tube)
Accuracy semi-quantitative quantitative
Focus all cells in sample any subpopulation(or: prior purification) (FACSorted for further analyses)
Facilities special laboratories needed only standard lab needed(pre-PCR lab, PCR lab, etc) (+ flow cytometer)
Modified from J.J.M. van Dongen
HOW TO SIMPLIFY & OPTIMIZE
FLOW-BASED MRD STRATEGIES
- Improve the design of MRD panels for a greater efficiency and higher reproducibility.
- Construct reference data files for normal and neoplastic cells (e.g.: per disease category)
- Multi-n-dimensional comparison of normal vs neoplastic cell populations (e.g.: at diagnosis and follow-up):
- Automated PCA-guided approach for homogeneous cell populations(e.g. lymphoid)
- Maturation tools for heterogeneous cell populations(e.g. myeloid)
CONSTRUCTION OF EuroFlow MRD PANELS: MMM
M #
1M
M #
2N
orm
al B
M #
1N
orm
al B
M #
2
SS
C
SS
C
SS
C
SS
C
CD38-FITC
CD38-FITC
CD38-FITC
CD38-FITC
SS
C
SS
C
SS
C
SS
C
CD38-FITC
CD38-FITC
CD38-FITC
CD38-FITC
Identify PC Select PC
Merge PC(n-cases)
Principal component analysis(n=14 markers/parameters)
CD19 19.83
CD56 19.02
CD81 12.29
CD45 11.47
CD27 9.95
CD117 9.34
CD38 5.18
MOST INFORMATIVE MARKERS
Automated classification of normal vs aberrant plasma cells in MM
Diagnosis Post-transplantPre-Transplant
m-PC (22%)
n-PC (0.12%)n-PC (0.03%)+m-PC (0.8%)
Comparative View Comparative ViewComparative View
FLOW-BASED MRD IN MM: Conclusions
- Response to therapy impacts on long-term patient outcome both in the transplant and non-transplant settings
- Flow-based MRD improves prognostic stratification of myeloma patients particualrly among those cases who reach CR
- Flow-based MRD is a well suited technique for MRD assessment in MM
- Full standardized and automated flow-based MRD approaches for MM have been developped
Shall flow-based MRD be introduced in the treatment-decision algorithms of new
(randomized) trials ?
CIRCULATING NEOPLASTIC PLASMA CELLS
MGUS SMM MM P.-value
% of cases with PB M-PC 7 (21%) 18 (69%) 43 (75%) <.001
N. of M-PC/mL 0.3 0.2 4.0 <.001% of PB M-PC 0.004 0.02 0.09 .002
Paiva et al, Leukemia (under revision); ASH 2010 (abstract #617)
MINIMAL NUMBER OF PC REQUIRED TO BE ANALYZED IN A MRD ASSAY FOR MM
N. of tests in the MRD assay
N. of total nucleated cells/test
N. of events/test to define a PC population
1 1,000,000 100
2 500,000 50
3 333,334 34
4 250,000 25
5 200,000 20
Rawstron, Orfao et al, Haematologica, 2008
Grupo Español de Mieloma (GEM)Hospitales
Clínico de Barcelona12 Octubre (Madrid)Clínico de Salamanca
Clínico de San Carlos (Madrid)Hospital de Badalona
Clínico de AsturiasFr. Peset (Valencia)
Universitario de CanariasRio Ortega (Valladolid)
Cínico de ZaragozaHospital General de JerezRamón y Cajal (Madrid)
Morales Meseguer (Murcia)La Fe (Valencia)C.U. de Navarra
Galdakao (Vizcaya)Clínico de ValladolidSant Pau (Barcelona)
Arnau Vilanova (Lérida)Universitario de Santiago
General Universitario de ValenciaUniversitario de Getafe (Madrid)
Insular de las PalmasH. de La Princesa (Madrid)
Severo Ochoa (Madrid)Juan XIII (Tarragona)
ToledoGandía (Valencia)
Vall D´Hebrón (Barcelona)San Jorge (Huesca)
Verge de la Cinta (Tortosa)Alarcos (Ciudad Real)
Mataró (Madrid)Juán Canalejo (Coruña)
Ferrol
HospitalesGeneral de Segovia
Cruces (Bilbao) St. Coloma de Gramanet
(Barcelona)Gregorio Marañon (Madrid)
Carlos Haya (Málaga)H. Tauli (Gerona)
HuescaPalencia
Alcira (Valencia)H. Del Mar (Barcelona)
Mahón (Baleares)Clínico de MálagaXeral Cies (Vigo)
PlasenciaCáceres
AlgecirasÁvilaJaén
S. Pau i Sta Tecla (Tarragona)General de Guadalajara
Sagunto (Valencia)Son Dureta (Mallorca)
CuencaAlicante SUS
M. Valdecilla (Santander)Albacete
H. Del BierzoFundación Jiménez Díaz (Madrid)
Elda (Alicante)V. Del Rosel (Cartagena)
CastellónMutua Tarrasa
Consorcio TarrasaC. Corachán (Barcelona)
G.Mateo, M. Perez-Andres, N.Gutierrez, R.Lopez, M.Mateos, R.Garcia-Sanz, J.Almeida, J.San Miguel
Salamanca´Group:
Cytometry Service & Department Medicine, CICancer, University Salamanca-CSIC, Salamanca, SpainMartín Perez-AndresLeandro ThiagoAlberto Orfao
Wilheminenhospital, Wien, Austria Niklas Zojer
University Medical Center, Groningen, Netherlands
Nico A Bos
Serv. Hematology, Hosp. Univ. Salamanca, Salamanca, Spain Bruno PaivaJesus F San Miguel
Vrije Universiteit, Brussel, Belgium
Karin Vanderkerken
Aalborg Hospital Science & Innovation Center AHSIC Aarhus University Hospital, DenmarkHans E Johnsen
University of Heidelberg, Heidelberg, Germany
Dirk Hose
INSERM, U847, CHU Montpellier, Institute of Research in Biotherapy, Université Montpellier1 Montpellier, FranceBernard KleinAnouk Caraux
School of Medicine, Southampton, UKSurinder S Sahota
Erasmus University Medical Center, Rotterdam, NetherlandsPieter Sonneveld
SERVICIO DE CITOMETRIA, DEPARTAMENTO DE MEDICINA Y CENTRO DE INVESTIGACIÓN DEL CÁNCER (IBMCC) UNIVERSIDAD
DE SALAMANCA
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