advances in immunophenotyping of multiple myeloma … na... · advances in immunophenotyping of...
TRANSCRIPT
ADVANCES IN
IMMUNOPHENOTYPING OF
MULTIPLE MYELOMA
ADVANCES IN
IMMUNOPHENOTYPING OF
MULTIPLE MYELOMA
Servicio de Citometria y Servicio de Hematología
Universidad y Hospital Universitario de Salamanca
Servicio de Citometria y Servicio de Hematología
Universidad y Hospital Universitario de Salamanca
Centro de Investigación del
Cáncer
Centro de Investigación del
Cáncer
Sao Paulo, 18th of April, 2009
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
MM PLASMA CELLMM PLASMA CELL
cIg+
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%ββββ1/β/β/β/β2 integrins . 98%
Co-stimulatory Ags:
CD28+/++....... 30-40%
CD40 +....... 100%
CD27 -/dim .... 40-50%
Myeloid-associated Ags:
CD13+......... 28%CD33 +/++..... 24%
Pan-leuc. Ag:
CD45+...20-40%
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 patternVariability
• 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
Per
CP
/Cy5
->
Co-expression of CD38/CD138Co-expression of CD38/CD138
10 10 10 10 100 1 2 3 4CD38 FITC ->
TR
AN
SF
OR
ME
D S
SC
->
High-intensityHigh-intensity
10 10 10 10 100 1 2 3 4
CD138 PerCP/Cy5->
TR
AN
SF
OR
ME
D S
SC
->
Specific expressionSpecific expression
++ ==
- but…..diluted sample →→→→ lower numbers
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
10 10 10 10 100 1 2 3 4
CD38 FITC ->
TR
AN
SF
OR
ME
D S
SC
->
10 10 10 10 100 1 2 3 4
CD38 FITC ->
CD
138
Per
CP
/Cy5
->
CD38-FITC
CD38-FITC gated PC
T-SSC
CD138-Pe
rCP/Cy5.5
MONOCLONAL GAMMOPATHIES: IDENTIFICATION OF CLONAL PLASMA CELLS
Clonal PC
Normal PC
CD
56
PE
9
1
D
C
CP
A
54
DC
CD19-PcpC
y5
CD56-PE CD45-APC
Perez-Andres, J Biol Reg, 2004
Frequency of Aberrant PhenotypesN=195 N=195 patientspatients
001010202030304040505060608080
CD56CD56 CD28CD28 CD33CD33 CD117CD117 CD20CD20
Asynchronous expression
52%52%
25%25%
16%16% 15%15%
11%11%
92%
CD38CD38
80%80%
Over- expressionInfra-expression
sIgsIg
21%21%
Almeida et al, Br J Haematol, 1999
RESULTS: RESULTS: ImmmunophenotypeImmmunophenotype ofof
normalnormal vsvs clonalclonal PCPC
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
CD38CD38 HLAHLA--IIαααααααα ββββββββ22--microglobulinmicroglobulin CD40CD40
%
% ofofpositive PC
positive PC
Mean FL
Mean FL In
tensity
Intensity
p=0.21p=0.21 p=0.005p=0.005
60
50
40
30
20
10
0Clonal PCNormal PC
CD95CD95
p=0.72p=0.72
pp<<<<<<<<0.0010.001
80
60
40
20
0Clonal PCNormal PC
CD56CD56
pp<<<<<<<<0.0010.001120
100
80
60
40
20
0Clonal PCNormal PC
CD86CD86
pp<<<<<<<<0.0010.001
p=p=0.0020.002
Clonal PCNormal PC
100
80
60
40
20
0
CD126CD126
pp<<<<<<<<0.0010.001
MOST USEFUL ANTIGENS FOR THE DETECTION OF ABERRANT PC IN MM
Antigen Expression % MM with Requirement for
Normal Altered altered expression MRD studies
CD19 + (>70%) - 95% Essential
CD56 - (>85%) ++ 75% Essential
CD20 - (100%) + 10% Preferred
CD117 - (100%) + 30% Preferred
CD28 -/dim (100%) ++ 15% Recommended
CD81 + -/dim N.A. Recommended
CD27 ++ -/dim 40-50% Recommended
N.A.: not analyzed/not reported.
Rawstron et al, EMN consensus, Haematologica, 2008
CLINICAL APPLICATIONS OF CLINICAL APPLICATIONS OF IMMUNOPHENOTYPING OF IMMUNOPHENOTYPING OF NEOPLASTIC PLASMA CELLSNEOPLASTIC PLASMA CELLS
Diagnostic classification
Prognostic evaluation
Treatment monitoring
CLINICAL APPLICATIONS OF CLINICAL APPLICATIONS OF IMMUNOPHENOTYPING OF IMMUNOPHENOTYPING OF NEOPLASTIC PLASMA CELLSNEOPLASTIC PLASMA CELLS
Diagnostic classification(differential diagnosis between MGUS vs MM)
Prognostic evaluation
Treatment monitoring
MM PC MGUS PCvsvs
Are myelomatosous PC different
from MGUS PC ?
ANTIGEN CD38*
CD19
CD138
CD9
CD13
CD33
CD40
CD56*
ANTIGEN CD38*
CD19
CD138
CD9
CD13
CD33
CD40
CD56*
MGUS: POLYCLONAL
PC (n=76)
100
100
100
100
17
3
100
0
MGUS: POLYCLONAL
PC (n=76)
100
100
100
100
17
3
100
0
CONTROLS' PC (n= 10)
100
100
100
100
20
7
100
0
CONTROLS' PC (n= 10)
100
100
100
100
20
7
100
0
Results expressed as positive cases (> 20% positive PC) *Strong reactivity Other Ags explored were constantly negative
INMUNOPHENOTYPE OF POLYCLONAL PC IN MGUS VS CONTROLS
INMUNOPHENOTYPE OF POLYCLONAL PC IN MGUS VS CONTROLS
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
%
of
cas
es
1 2 3 1 2 3 4 1 2
MGUS MM PCLN. of pathologic PC clones
% ofca
ses
Rasillo et al, Cancer, 2003
INTRATUMOURAL GENETIC HETEROGENEITY IN MM vs OTHER MONOCLONAL GAMMOPATHIES
RESULTS: RESULTS: ImmunophenotypeImmunophenotype ofof clonalclonal PCPC in in
MGUSMGUS vsvs MMMM andand PCLPCL
10000
8000
6000
4000
2000
0PCLMMMGUS
10000
8000
6000
4000
2000
0PCLMMMGUS
3000
2000
1000
0PCLMMMGUS
CD86CD86
CD38CD38 HLAHLA--IIαααααααα ββββββββ22--microglobulinmicroglobulin CD40CD40
%
% ofofpositive PC
positive PC
Mean FL
Mean FL In
tensity
Intensity
pp<<<<<<<<0.0010.001 p=p=0.0080.008
100
80
60
40
20
PCLMMMGUS0
CD126CD126
pp=0.64=0.64
4000
3000
2000
1000
0PCLMMMGUS
p=p=0.020.02
CD95CD9560
50
40
30
20
10
0PCLMMMGUS
p=p=0.0060.006120
100
80
60
40
20
0PCLMMMGUS
p=p=0.150.15
p=p=0.010.01
80
60
40
20
0PCLMMMGUS
CD56CD56
P=P=0.560.56 *
*
*
*
*
Perez-Andres et al, Leukemia, 2005; Int J Cancer, 2008
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 ->
AbnormalAbnormal plasma plasma cellscells
Normal plasma Normal plasma cellscells
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 transformation 2
Cases with predominantly (>95%) CD19- ve PC.... High risk (26% transformed in 31 months)
Risk of MGUS transformation 2
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)
versusversus
>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
MGUS MGUS vsvs MM: MM: IMMUNOPHENOTYPIC PANELSIMMUNOPHENOTYPIC PANELS
N.of PB AMCA FITC PE PerCPCy5.5 APC PE-Cy7 Alexa700colors PO APC-Cy7
3 CD38 CD56 CD19
4 CD38 CD56 CD19 CD45
6 cyIgL cyIgk CD19 CD45 CD56 CD38
8 CD19 CD45 cyIgL cyIgk CD138 CD28 CD56 CD38
CLINICAL APPLICATIONS OF CLINICAL APPLICATIONS OF IMMUNOPHENOTYPING OF IMMUNOPHENOTYPING OF NEOPLASTIC PLASMA CELLSNEOPLASTIC PLASMA CELLS
Diagnostic classification
Prognostic evaluation-Enumeration of myelomatous PC-Patterns of antigen expression
-Risk of progression of smoldering MM
Treatment monitoring
• Correlation between Immunophenotyping & Morphology:• Correlation between Immunophenotyping & Morphology:
0 25 50 75 100
Proportion of plasma cell by morphology
0
25
50
75
100
Pro
po
rtio
no
fp
lasm
a ce
llb
y fl
ow
cyto
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R2= 0,4
• 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
port
ion
of p
atie
nts
aliv
e
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
port
ion
of p
atie
nts
aliv
e
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
port
ion
of 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
port
ion
of 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
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R2= 0,39
0 25 50 75 100
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CLINICAL APPLICATIONS OF CLINICAL APPLICATIONS OF IMMUNOPHENOTYPING OF IMMUNOPHENOTYPING OF NEOPLASTIC PLASMA CELLSNEOPLASTIC PLASMA CELLS
Diagnostic classification
Prognostic evaluation-Enumeration of myelomatous PC-Patterns of antigen expression
-Risk of progression of smoldering MM
Treatment monitoring
CD56CD56
PF
SP
FS
48484242363630302424181812126600
1,01,0
,8,8
,6,6
,4,4
,2,2
0,00,0
60605454
— CD56 + (n=272)— CD56 –ve (n=355)
— CD56 + (n=272)— CD56 –ve (n=355)
p=0.05p=0.05
38 m38 m
33 m33 m
Months from diagnosisMonths from diagnosis
OS
OS
48484242363630302424181812126600
1,01,0
,8,8
,6,6
,4,4
,2,2
0,00,0
60605454
NRNR
59 m59 m
p=0.03p=0.03
— CD56 + (n=272)— CD56 –ve (n=355)
— CD56 + (n=272)— CD56 –ve (n=355)
CD117CD117
726660544842363024181260
1,0
,9
,8
,7
,6
,5
,4
,3
,2
,10,0
— CD117 + (n=195)— CD117 -ve (n=388)
— CD117 + (n=195)— CD117 -ve (n=388)
31 m31 m
41 m41 m
p=0.01p=0.01
1.0
.8
.6
.4
.2
0.0726660544842363024181260
Months from diagnosisMonths from diagnosis
— CD117 +(n=195)— CD117 -ve (n=388)
— CD117 +(n=195)— CD117 -ve (n=388)
58 m58 m
NRNR
p=0.1p=0.1
Prognostic influence of PC antigenic expression (I)Prognostic influence of PC antigenic expression (I)
Mateos et al, JCO, 2008
Prognostic influence of PC antigenic expression (II)Prognostic influence of PC antigenic expression (II)
CD19CD19
Months from diagnosisMonths from diagnosis
PF
SP
FS
4842363024181260 6054
36m36m
28 m28 m
p=0.2p=0.2
— CD19 + (n=47)— CD19 -ve (n=580)
— CD19 + (n=47)— CD19 -ve (n=580)
1,0
,9
,8
,7
,6
,5
,4
,3
,2
,10,0
OS
OS
60544842363024181260
1,0
,9
,8
,7
,6
,5
,4
,3
,2
,10,0
— CD19 + (n=47)
— CD19 -ve (n=580)
— CD19 + (n=47)
— CD19 -ve (n=580)
62 m62 m
54 m54 m
p=0.06p=0.06
CD28CD28
605448423630241812600
1,01,0
,9,9
,8,8
,7,7
,6,6
,5,5
,4,4
,3,3
,2,2
,1,1
0,00,0
— CD28 + (n=235)— CD28 -ve (n=368)
— CD28 + (n=235)— CD28 -ve (n=368)
31 m31 m
37 m37 m
p=0.05p=0.05
544842363024181260
1,01,0
,9,9
,8,8
,7,7
,6,6
,5,5
,4,4
,3,3
,2,2
,1,1
0,00,0
— CD28 + (n=235)— CD28 -ve (n=368)
— CD28 + (n=235)— CD28 -ve (n=368)
54 m54 m
66m66m
p=0.1p=0.1
60
Months from diagnosisMonths from diagnosisMateos et al, JCO, 2008
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
Mateos et al, J Clin Oncol, 2008
Non-hyperdiploidy DNA Non-hyperdiploidy DNA
% BMPC by FCM >20%% BMPC by FCM >20%
% BMPC in S-phase ≥≥≥≥2.5%% BMPC in S-phase ≥≥≥≥2.5%
P P
Multivariate analysis for RFS and OS
RFSRFS OSOSPP
0.010.01 NSNS
Platelets ≤≤≤≤ 130 · 109/LPlatelets ≤≤≤≤ 130 · 109/L
0.030.03 0.0040.004
0.050.05 NSNS
ISS stage 3ISS stage 3 0.0030.003 0.00010.0001
0.0170.017 0.040.04
Calcium ≥≥≥≥11 mg/dlCalcium ≥≥≥≥11 mg/dl 0.0050.005 NSNS
CLINICAL APPLICATIONS OF CLINICAL APPLICATIONS OF IMMUNOPHENOTYPING OF IMMUNOPHENOTYPING OF NEOPLASTIC PLASMA CELLSNEOPLASTIC PLASMA CELLS
Diagnostic classification
Prognostic evaluation-Enumeration of myelomatous PC-Patterns of antigen expression
-Risk of progression of smoldering MM
Treatment monitoring
• 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
BMPC + High MC (>3 IgG or >2g/dl IgA)
> 10% of BMPC + MC
>10% BMPC + High MC
Predictive factorsAlexanian IgABlood; 1980 BJ >200 mg/24 h Arch Intern Med, 1988 MC >3 g/dl
Facon MC >3 g/dlAm J Hematol, 1995 Hb <12 g/dl
PC >25%
Weber MC >3g/dlB J Haematol; 1997 IgA
BJ >50mg/ 24 h Pathological MRI
Cesana IgAJ Clin Oncol; 2002 BJ +Haematologica; 2004 % BMPC
Rosiñol Evolving type*B J Haematol; 2003 IgA
*SMM with constant increase of MC
Parameters Associated with Disease Progression
N 89
Diagnosis jan96- sep04
Age* (range) 69 (43-88)
Gender (M/F) 44/45
Follow-up*(range) 50 (24-107)
Progression (%) 38 (42)
Characteristics of the Series
Patients (%)
BMPC + High MC (>3 IgG or >2g/dl IgA) 21 (24)
>10% of BMPC + MC 45 (50)
>10% BMPC + High MC 23 (26)
*median (in months)
Perez-Persona et al, Blood, 2007
% 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)
Proportion of aPC referred to the total-PC (aPC/BMPC)
Proportion of aPCreferred 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 withinBM cellularity% PC withinBM cellularity
Impact of % aPC/BMPC by FC onProgression 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
fPro
gres
sion
Fre
e S
urvi
val
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
ImmunoparesisImmunoparesis >95% >95% aPCaPC/BMPC/BMPC ScoreScore(n)(n)
-- -- 0 (n=32)0 (n=32)
+ / + / -- --/+/+ 1 (n=27)1 (n=27)
++ ++ 2 (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
fPro
gres
sion
Fre
e S
urvi
val
Median not reached
Median 75 months
Median 20 monthsp= 0.003
91%
58%
18%
5 years
CLINICAL APPLICATIONS OF CLINICAL APPLICATIONS OF IMMUNOPHENOTYPING OF IMMUNOPHENOTYPING OF NEOPLASTIC PLASMA CELLSNEOPLASTIC PLASMA CELLS
Diagnostic classification
Prognostic evaluation
Treatment monitoring
Changes in PC distribution following ASCT
MM-PCMM-PC
CD56
CD45
CD19
DiagnosisDiagnosis
Gate CD38Gate CD38
N-PCN-PC
MM-PCMM-PC
CD56CD45
CD19
Post-TrxPost-Trx
MINIMAL NUMBER OF PC REQUIRED TO BE ANALYZED IN A MRD ASSAY FOR MM
20200,0005
25250,0004
34333,3343
50500,0002
1001,000,0001
N. of events/test to define a PC population
N. of total nucleated cells/test
N. of tests in the MRD assay
Comparison between ASCT & Chemotherapy(changes in PC compartment)
Comparison between ASCT & Chemotherapy(changes in PC compartment)
Results expressed as median (range)
MRD -ve : <10 -5 MM-PC with only normal PC being detected
Results expressed as median (range)
MRD -ve : <10 -5 MM-PC with only normal PC being detected
pp
MM-PCMM-PC
n-PCn-PC
% n-PC/TPC% n-PC/TPC
MRD –ve casesMRD –ve cases
ASCT3 m post-Trx
n=47
ASCT3 m post-Trx
n=47
0,04 (0-3,2)0,04 (0-3,2)
0,21 (0-1,6)0,21 (0-1,6)
86 (0-100)86 (0-100)
36%36% 0,040,04
0,010,01
0,250,25
0,010,010,17 (0-3,7)0,17 (0-3,7)
0,12 (0-0,9)0,12 (0-0,9)
35 (0-100)35 (0-100)
15%15%
Chemo.After 12 cycles
n=40
Chemo.After 12 cycles
n=40
San Miguel et al, Blood, 2002
Spanish multi-centre protocol GEM (2000-2004)Spanish multi-centre protocol GEM (2000-2004)
DiagnosisDiagnosis
VBCMP/VBAD (x4)VBCMP/VBAD (x4)
RelapseRelapse
VBCMP/VBAD (x2)VBCMP/VBAD (x2)
Stem cell collectionStem cell collection
Complete remission(negative electrophoresis)*
Complete remission(negative electrophoresis)*
MaintenanceMaintenance
Partial response(positive electrophoresis)
Partial response(positive electrophoresis)
ASCT (2º)ASCT (2º) Mini-ALO-TrxMini-ALO-Trx
* Immunofixation either –ve or +ve * Immunofixation either –ve or +ve
ASCT (1º)ASCT (1º)
3m post-ASCT3m post-ASCT
Non-respondingNon-responding
Double-TrxDouble-Trx
MRDinvestigation
Correlation between immunophenotyping & electrophore tic responses
at three months post-ASCT (n=200)
Correlation between immunophenotyping & electrophore tic responses
at three months post-ASCT (n=200)
EF-positive n=74 cases
EF-positive n=74 cases
MM-PCMM-PC
% n-PC/TPC% n-PC/TPC
0.76 ± 0.9 #0.76 ± 0.9 #
MRD evaluationMRD evaluation
8% #8% #MRDnegative casesMRDnegative cases
42 ± 33 #42 ± 33 #
Results expressed as mean ± SD
# p significance between EF-positive (PR) and CR IFE- positive cases: p=0.005; p= 0.0001; p=0.0001
Results expressed as mean ± SD
# p significance between EF-positive (PR) and CR IFE- positive cases: p=0.005; p= 0.0001; p=0.0001
Complete remissionComplete remissionPartial responsePartial response
IFx-negativen=99 cases (79%)
IFx-negativen=99 cases (79%)
0.1 ± 0.30.1 ± 0.3 0.0060.006
0.0080.008
64%64%
86 ± 2586 ± 25
0.0030.003
IFx-positiven=27 cases (21%)
IFx-positiven=27 cases (21%)
33%33%
0.28 ± 0.40.28 ± 0.4
69 ± 3469 ± 34
pp
78726660544842363024181260
1,0
,9
,8
,7
,6
,5
,4
,3
,2
,1
0,0
RFS of MM: Impact of immunophenotypingat 3 months post-ASCT (n=200 patients)
RFS of MM: Impact of immunophenotypingat 3 months post-ASCT (n=200 patients)
Rel
apse
-fre
e su
rviv
alR
elap
se-f
ree
surv
ival
— <0.01% MM-PC— <0.01% MM-PC
— ≥≥≥≥ 1% MM-PC— ≥≥≥≥ 1% MM-PC
Months from immunophenotypical analysis (3 months post-ASCT)Months from immunophenotypical analysis (3 months post-ASCT)
%MM-PC%MM-PC
p=0.0001p=0.0001
40m40m
23m23m
— 0.01% to 1% MM-PC — 0.01% to 1% MM-PC
NRNR
Paiva et al, Blood, 2008
RFS: Impact of immunophenotypingat 3 months post-ASCT in 99 CR (IF-) patients
RFS: Impact of immunophenotypingat 3 months post-ASCT in 99 CR (IF-) patients
6660544842363024181260
1,1
1,0
,9
,8
,7
,6
,5
,4
,3
,2
,1
0,0
%MM-PC%MM-PC
p=0,02p=0,02
NRNR
36m36m
p=0,01p=0,01
NRNR
32m32m
60544842363024181260
1,0
,9
,8
,7
,6
,5
,4
,3
,2
,1
0,066
Rel
apse
-fre
e su
rviv
alR
elap
se-f
ree
surv
ival
Months from immunophenotypical analysis (3 months post-ASCT)Months from immunophenotypical analysis (3 months post-ASCT)
— ≥≥≥≥ 75 % N-PC/total PC— ≥≥≥≥ 75 % N-PC/total PC
%N-PC / total PC%N-PC / total PC
— <0.01% MM-PC— <0.01% MM-PC
— ≥≥≥≥ 0.01% MM-PC— ≥≥≥≥ 0.01% MM-PC — < 75 % N-PC/total PC— < 75 % N-PC/total PC
MRD IN MM: CORRELATION BETWEEN RQ-PCR AND FCM
00
0,20,2
0,40,4
0,60,6
0,80,8
11
1,21,2
1,41,4
1,61,6
1,81,8
11 22 33 44 55 66 77 88 99 1010 1111 1212 1313 1414 1515 1616 1717 1818 1919 2020 2121 2222
PatientsPatients
%
% T
umou
rT
umou
r cel
lsce
lls
%RQ%RQ--PCRPCR%FCM%FCM
R = 0.861
2323 2424
0,860,38221
0,971,14422
1,61,39924
1,311,27923
0,80,21920
0,6861.6119
0,260,11318
0,250,43517
0,0650,07716
0,0470,0115
0,0140,06214
00,10613
00,08212
00,02411
00,00310
00,0029
00.00098
007
006
005
004
003
002
001
%FCM%RQ-PCRCases
�
�
x
�xx
x
Sarrasquete et al, Haematologica, 2005
MRD IN MULTIPLE MYELOMA: APPLICABILITY OF FCM VS RQ -PCR
PatientsPatients in CR (N=53)in CR (N=53)
CCases ases withwith aberrantaberrant phenotypesphenotypes (N=48)(N=48)
ProblProbl emsems associatedassociated withwith thethe samplesample (N=16) (N=16)
VeryVery lowlow infiltrationinfiltration (N=9) infiltraci(N=9) infiltraci óón muy bajan muy baja
DegradedDegraded DNA (N=4) DNA (N=4)
InsufficientInsufficient DNA (N=3)DNA (N=3)
Problems Problems associassoci atedated withwith thethe methodmethod ((RQRQ--PCR)PCR) (N=(N= 8)8)
No No clonalclonal rearrangementrearrangement detecteddetected (N=3) (N=3)
VeryVery short Nshort N --regionregion (N=3)(N=3)
Cases Cases withwith mutationsmutations in in thethe targettarget sequencesequence (N=2)(N=2)
APPLICABILITY OF FCM: 90%
APPLICABILITY OF RQ-PCR: 75%Sarrasquete et al, Haematologica, 2005
Consensus medical indications of multiparameter flow cytometry
immunophenotyping in the study of MM and other MG_____________________________________________________________________
Clinical utility Useful flow cytometry parameters_____________________________________________________________________
Differential diagnosis of MM vs MGUS % aberrant BMPC from total BMPC
Identification of aberrant phenotypes Decreased expression (CD19, CD27, CD38,
CD45 CD81)
Overexpression (CD28, CD33, CD56)
Asynchronous expression (CD20, CD117,
sIg)
Diagnosis of unusual cases PC-associated markers (e.g. CD138, cIg, CD38hi)
& aberrant PC markers (see above)
Patient monitoring % of aberrant BMPC/totalBM cells
% normal BMPC/ all BMPC
_____________________________________________________________________ MM: multiple myeloma; MGUS: monoclonal gammopathy of undetermined significance; BM: bone marrow; PC: plasma cells.
Rawstron et al, EMN consensus, Haematologica, 2008
CONCLUSIONS
Inmunophenotypic studies have high clinical value:
- Differential diagnosis MGUS/MM
- Prognostic influence of FCM number of myelomatous PC
- Prognostic influence of the patterns of antigen expression
- Prediction of the risk of transformation in SMM
- Investigation of MRD (immunophenotypic remission)
Grupo Español de Mieloma (GEM)Hospitales
Clínico de Barcelona12 Octubre (Madrid)Clínico de Salamanca
Clínico de San Carlos (Madrid)Hospital de BadalonaClínico 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:
OBRIGADO