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Immunophenotype in multiple
myeloma and MRD detection
Thomas Matthes
Flow Cytometry Laboratory
Hematology and Clinical Pathology
Hôpitaux Universitaires de Genève
Luzern, 30th march 2012
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Copyright ©2008 Ferrata Storti FoundationRawstron, A. C. et al. Haematologica 2008;93:431-438
List of most useful antigens for the detection of
aberrant plasma cells in multiple myeloma
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Difference between normal and malignant PC
Plasmocytes malins
Plasmocytes normaux
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Incidence of phenotypically aberrant MM-PC
0
10
20
30
40
50
60
70
80
90
100
CD19 CD38 CD45 CD56 CD28 CD33 CD117 CD20
96%
80%
73%
60%
36%
18%
32%
17%
Asynchronous expressionInfra-expression Over-expression
Mateo G, et al. J Clin Oncol; 2008;26:2737
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8-colour EuroFlow panels for
PC dyscrasias
Tube PB PO FITC PEPerCP-
Cy5.5PE-Cy7 APC APC-H7
Baseline CD45 CD138 CD38 CD56 β2micro CD19 cyIgκ cyIgλ
Baseline CD45 CD138 CD38 CD28 CD27 CD19 CD117 CD81
MRD CD45 CD138 CD38 CD56 CD27 CD19 CD117 CD81
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DD: MM vs MGUS
The most powerful single criteria for differential diagnosis !
Ocqueteau M, Am J Pathol 1998, 152: 1655
MGUSMM
versus
> 5% nPC: 98% MGUS
PCa: Clonal PCn: Poly-Clonal
Presence of nPC only in 20% of MM patients; always <5% (median: 0.25%)
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Mateo et al. J Clin Oncol; 2008;26:2737
Prognostic influence of CD28 & CD117 expression
on PC
p= 0.01
38% n=128
29% n=362
21% n=149
Months from diagnosis847260483624120
Pro
gres
sio
n f
ree
surv
ival
1,0
0,8
0,6
0,4
0,2
0,0
PFS
p= 0.0001
OS
Months from diagnosis847260483624120
Ove
rall
surv
ival
1,0
0,8
0,6
0,4
0,2
0,0
72% n=128
57% n=362
43% n=149
CD117+ CD28- CD117- CD28CD117+ CD28+-
CD117- CD28+
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TherapyTreatment
compliance
In vivo drug
kinetics
Tumour micro-
environment
Tumour cell
features
100
101
102
103
104
105
106
107
108
109
1010
1011
No
. o
f tu
mo
ur
ce
lls
10-6
10-5
10-4
10-3
10-2
Resistance
Complete remission
Immunophenotypic CR
Molecular CRSe
ns
itiv
ity
- CML
- APL
- Childhood ALL
- …
Therapeutic
decisions
Morphology, cytogenetics
Southern-blot,
FCM DNA aneuploidy
F.I.S.H
Flow cytometry
P.C.R.
Minimal residual disease (MRD)
Modified from J.J.M. van Dongen
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Depth of response
MR
PR
VGPR/ nCR
CR
sCR
Molecular/flow CR
Treatment initiation
Progression
Time
Depth of response is related to TTP
Which level of response should be
measured in MM?
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Complete response (CR):
negative IF in serum and urine
< 5% PC in BM
disappearance of soft tissue plasmocytoma
stringent CR: CR and
normal FLC ratio 0.26 – 1.65
absence of clonal PC in BM by IHC or Flow (2-4 colors)
molecular CR: CR and
negative ASO-PCR in BM (sensitivitiy 10-5 )
Flow CR: CR and
absence of phenotypically aberrant clonal PC in BM
106 cells analyzed; 4≥ colors; sensitivity 10-4
IMWG uniform response criteria:2 consecutive measurements are required (BM only once)
Rajkumar V et al, 2011
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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=0.01
P<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
of
Even
t Fr
ee S
urv
ivo
rs
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 JJ, 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
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CR correlates with long-term PFS and OS in
elderly patients treated with novel agents
Gay F et al. Blood;117:3025-31.
• 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
OSPFS
p<0.001
CR
VGPR
PR
Months
Pro
bab
ility
of
pro
gre
ssio
n-f
ree
surv
ival
0 24 48 72
1.0
0.8
0.6
0.2
0.4
0.0p<0.001
CR
VGPR
PR
Months
Ove
rall
pro
bab
ility
of
surv
ival
0 24 48 72
1.0
0.8
0.6
0.2
0.4
0.0
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“Achieving CR is an important prognostic factor at all stages of
treatment , including before and after ASCT, with first-line treatment
in the non-transplant setting, and in the relapsed setting.”
Chanan-Khan AA and Giralt S, JCO, 2010
“CR constitutes a surrogate for OS and a clinically relevant
end-point in MM studies”
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Do FLC measurements add additional value to IF ?
52 patients in CR after ASCT or AlloT (45 patients) or conventional
chemotherapy:
CR with negative IF
FLC was negative in 51 patients (98%)
- in 13 patients presence of oligoclonal IF: FLC negative
Kröger N et al.; 2010
Conclusion: FLC allows faster detection of remission or relapse
than IF, but is not more sensitive than IF
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MRD evaluation by PCR (Qualitative & Semi-q) in Multiple Myeloma patients: prognostic value
Author Context Sensitivity N MRD Status PFS OS
Corradini QL ASO-PCR 10-6 29 20 positive 55% NRJCO 1999 Auto/Allo 9 negative 78%
López-Pérez et al QL cons-PCR 10-3-10-4 27 12 positive 20 m* 20%*Leukemia 2000 Auto, apheresis 11 negative 40 m 86%
Martinelli QL ASO-PCR 10–6 44 32 positive 65%* NRJCO 2000 Auto/Allo 12 negative 93%
Corradini QL ASO-PCR 10-6 48 16 positive 0% NRBlood 2003 Allo 19 mixed 33%
13 negative 100%
Ladetto et al, QL Nested-PCR 10-6 39 33 positive 66% NRJCO, 2010 VTP Post-Auto 6 negative 100%
Terragna et al, QL Nested-PCR NR 67 27 positive NR VTD: 67% negativeASH 2010 VTD vs. TD post-auto 60 negative NR TD: 52%
negative
López-Pérez et al Semi-QT FL-PCR 10-3-10-4 23 14 positive 19 m* 28%*Leukemia 2000 Auto, apheresis 13 negative 39 m 81%
Bakkus Semi-QT PCR LDM 10-6 59 38 >0.015% 16 m* NRBJH 2004 Auto 22 <0.015% 64 m
Martínez-Sánchez et al Semi-QT FL-PCR 10-3-10-4 53 25 positive 28%* 68%BJH 2008 28 negative 68% 86%
QL: Qualitative; QT: quantitative; NR: Not reported
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Advantages Disadvantages
• Time consuming
• Labour consuming
• Applicability: ~75%
• Specificity: 100%
• Sensitivity: 10-6 (10-5 - 10-4)
Sarasquete ME, et al. Haematologica. 2005;90:1365-1372.Davies FE, et al. Best Pract Res Clin Haematol. 2002;15:197-222.
Fenk R, et al. Haematologica. 2004;89:557-66.Lioznov M, et al. Bone Marrow Transplant. 2008;41:913-916.
Real-time PCR of VDJ rearrangements for
MRD investigation in MM(Allelic-Specific Oligonucleotide ASO PCR)
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Comparison of molecular and Flow CR
32MM patients after ASCT: CR 3 months after transplant
BM analysis:
- PCR (Allele-specific oligonucleotide real-time quantitative PCR)
in 75% of patients;
- Cytometry (4-color flow)
in 90% of patients
for 24 patients both measurements were possible
Sarasquete ME et al; Haematologica; 2005
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Sarasquete ME et al; Haematologica; 2005
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Correlation of MM-PC detected by RQ-PCR vs. MFC
MRD by ASO-RT-PCR (% of tumour cells)
MR
D b
yfl
ow
cyto
met
ry(%
of
tum
ou
rce
lls)
0 1.00.1-0.01
1
0,1
0.001
0.01
5.0
R=0.853P < .00001
Puig et al. Haematologica 2011;96 (suppl 1):[abstr O-09]. Presented at IMW 2011 Paris.
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Disadvantages
• Applicability: >90%
• “Single-cell” analysis
• Sensitivity: 10-4
• Speed
• Simplicity
• Only PC compartment
• No specific tumor antigens
• Limited number of proteins studied
• Heterogeneous BM infiltration
• Final product analysis: protein (antigen)
Advantages
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Immunophenotypic CR*: absence of phenotypically
aberrant PC at a sensitivity level of 10-4
Normal PC
Myelomatous PC
Normal PC Normal PC
Myelomatous PC
Baseline Follow-up
MRD + MRD -
San Miguel et al Blood; 2002; 99:1853
*Rajkumar et al (IMWG). Blood 2011 117: 4691-4695
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GEM2000: Impact on survival of achieving an immunophenotypic CR after HDT/ASCT (n=295)
PFS OS
MRD negative (n=125) 42%MRD positive (n=170) 58%
0 25 50 75 100 125
0
20
40
60
80
100
p< 0.0001
Median: 71m
Median: 37m
Median: NA
p= 0.002
Median: 89m
0 20 40 60 80 100 120 140
0
20
40
60
80
100
22%
60%
60%
82%
Paiva B ; Blood; 2008
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MRC myeloma IX: Impact on survival of achieving an immunophenotypic response after HDT/ASCT
(n=526)
Rawstron A, et al. Haematologica 2011;96 (suppl 1):[abstr O-09]. Presented at IMW 2011 Paris.
–MRD– –MRD+
p-value for logrank test = 0.0280
Overall survival by 100-day outcomefor intensive pathway patients
Pro
po
rtio
n o
f p
atie
nts
ev
en
t fr
ee
OS
1.0
0.8
0.6
0.2
0.4
0.0
0.9
0.7
0.5
0.1
0.3
0 24 48 726 12 18 30 36 42 54 60 66
p-value for logrank test = 0.0001
Progression-free survival by 100-day outcomefor intensive pathway patients
Pro
po
rtio
n o
f p
atie
nts
ev
en
t fr
ee
PFS
1.0
0.8
0.6
0.2
0.4
0.0
0.9
0.7
0.5
0.1
0.3
0 24 48 726 12 18 30 36 42 54 60 66
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MRC myeloma IX trial: What about consolidation
and maintenance therapy after HDT/ASCT?
Progression-free survival by 100-day outcome assessed in all patients by MRD/maintenance;T: thalidomide; NM: no maintenance
p-value for logrank test = 0.0053
Pro
po
rtio
n e
ven
t fr
ee
1.0
0.8
0.6
0.2
0.4
0.0
0.9
0.7
0.5
0.1
0.3
0 24 48 726 12 18 30 36 42 54 60 66
T; MRD– T; MRD+ NM; MRD– NM; MRD+
Rawstron A, et al. Haematologica 2011;96 (suppl 1):[abstr O-09]. Presented at IMW 2011 Paris.
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6050403020100
100
80
60
40
20
0
Months
P =0.001
PFS
Immunophenotypic CR 90% at 3y
“Stringent CR” 38% at 3y
Conventional CR 57% at 3y
PR (≥70% reduction) 28% at 3y
GEM2005>65y: impact of depth of response on survival
Paiva et al; J Clin Oncol. 2011;29(12):1627-33.
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M-component positive
M-component negative------- Follow-up
P Progression
Death
Treatment interruption
GEM2005>65y: Kinetics of response: conventional CR vs. immunophenotypic CR
Paiva B, et al. J Clin Oncol. 2011;29:1627-1633
1 IgG ----------------------------------------------------------------------
2 B-J ------------------------------------------------------------------------------------------
3 IgG -----------------------------------------------------------------------------------------------
4 IgA ------------------------------------------------------------------------------------------------------
5 IgG ---------------------------------------------------------------------------------------------------------------
6 B-J ------------------------------------------------------------------------------------------ P -------------------------------
7 IgG ----------------------------------------------------------------------------------------------------------------------------------
Flow- / IFE+
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.
7/7
(100%)
patients
turned
IFx-
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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 -------------------------------------------------------------------------------------------------------------------------------------------
GEM2005>65y: Kinetics of response: conventional CR vs. immunophenotypic CR
Paiva B, et al. J Clin Oncol. 2011;29:1627-1633
Flow- /
IFE+
Flow+
/ IFE-
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.
7/7
(100%)
patients
turned
IFx-
10/20
(50%)
patients
turned
IFx+
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FISH cytogenetics and immunophenotypic CR for the prediction of
early relapse of patients in CR after HDT/ASCT
TTP after day+100 HDT/ASCT
P <.001P <.001
Median: NR
Median: 83m
Median: 28m
80%
94%
Standard-risk FISH + MRD negative (n=58)
High-risk FISH OR MRD positive (n=45)
High-risk FISH + MRD positive (n=7)
0%
Median: 6m
1y
Median: 47m
Median: 21m
1009080706050403020100
100
80
60
40
20
0
42%
89%
2.5y
76%
5y
OS after day+100 HDT/ASCT
100806040200
100
80
60
40
20
0
Number of daysNumber of days
Paiva et al; Blood. 2012 119: 687-691
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- 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 at follow-up):
- Automated PCA-guided approach for homogenous cell populations
(e.g. lymphoid)
- Maturation tools for heterogenous cell populations
(e.g. myeloid)
How to optimize MRD detection by cytometry
Sensitivity of 10-6
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• Malignant PC exhibit a characteristic phenotype, which allows
their recognition and distinction form normal PC in practically all
patients
• Depth or response matters: the better the quality of response, the
longer the survival (CR, sCR,molecular CR, IP CR)
• MRD techniques can contribute to the monitoring of the efficacy of
intensification and maintenance therapies in order to avoid under-
or over-treatment
• IP CR is clinically relevant in MM: clear discrimination between
groups of patients with different PFS and OS
• Multiparameter flow cytometry (8-10 colors) could be considered
as the method of choice for MRD monitoring in MM
SUMMARY
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