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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
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
Difference between normal and malignant PC
Plasmocytes malins
Plasmocytes normaux
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
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
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%)
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+
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
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?
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
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
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
“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”
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
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
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)
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
Sarasquete ME et al; Haematologica; 2005
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.
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
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
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
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
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.
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.
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-
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+
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
- 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
• 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
END
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