Multidimensional Flow Cytometric Analysis of the
Immune System of Multiple Myeloma Patients
Achieving Long Term Disease Control
Roberto J. Pessoa Magalhães, María-Belén Vidriales, Bruno Paiva, Maria-Victoria Mateos, Norma C. Gutierrez,
Ramón García-Sanz, Juan F Blanco, Jose Hernández, Natalia de las Heras, Joaquin Martinez, Elaine Sobral Costa
, Angelo Maiolino, M. Nucci, Javier De La Rubia, Juan-Jose Lahuerta, Alberto Orfao,and Jesús F. San Miguel
Spanish Myeloma Group (GEM),Grupo Castellano-Leones de Gammapatias Monoclonales
Centro de Investigación del Cancer
Serviço de Hematologia
Servicio de Hematologia
Universidade Federal
Universidad
do Rio de Janeiro- UFRJ
de Salamanca-USAL
What Do we Know about MM patients that achieved-Background-
Long Term Disease Control?
Multiple Myeloma was usually considered an incurable disease
However, autotransplant clinical trials data showed that
)
( IFM,SWOG, Arkansas and Pethema/GEM) some patients (6-18%)
9
remain relapse-free for more than 10 years*1...operational cure
g/dl
Long term disease control is not restricted to those who achieved CR*2
(
Asymptomatic
- MGUS-Like signature by GEP*3
phase
- Documented smoldering transformation from MGUS to symptomatic MM*3
Component 6
MGUS
MM
Long Term
Disease
Control
Monoclonal
Remission
Plateau
3
> 5 years
0
*1-Barlogie B, et al. JCO. 2010
*2-Martinez-Lopez J, et al Blood 2011
1st line
1ST therapy
line therapy
Mainteinance
No therapy
*3- Zen FZ, et al Blood 2007
1
What Do we Know about MM patients that achieved-Background-
Long Term Disease Control?
)
What factors are involved in long term disease control
9
g/dl
in Multiple Myeloma?
(
Asymptomatic
phase
Component 6
MGUS
MM
Long Term
Disease
Control
Monoclonal
Remission
Plateau
3
> 5 years
0
*1-Barlogie B, et al. JCO. 2010
*2-Martinez-Lopez J, et al Blood 2011
1st line
1ST therapy
line therapy
Mainteinance
No therapy
*3- Zen FZ, et al Blood 2007
Active Host Immune Suppression in Symptomatic MM-Background-
Dysfunctional*1,2
?
Myeloid-Dcs
Plasmacytoid-Dcs
nio
ratuat
eM
TiMas
iv
ect
Defficient effectors*3-5
Def
CD4 CD4
CD4
NK
NK
B cells: reduced
N-PCs: absent*2
NK
NK
NK
NK
*1-Martin-Ayuso M et al The oncologist 2008;*2-Prat G,
Bone Marrow Stromal Cells
*5-Garcia-sanz R, BJH 1996 ;
et al BJH 2007; *3-Haitakari M ,Hematol-Oncol 2003;
*6-Joshua DA,et al Clin Lymphoma Myeloma
*4Schutt P, Leuk Lymphoma 2006
2008;*7-Dharminder C, Cancer cell 2009
2
-Objectives-
Wheter or not is Long Term Disease Control in MM
Related to an Improved Immunosurveillance ?
AIM:
-To compare the distribution of T, B, NK and DC subsets in PB and BM of
MM achieving long term disease control (LTDC-MM) vs. newly diagnosed
symptomatic MM patients and healthy controls.
Controls and Patients
-Material and Methods-
Inclusion Criteria for Long Term Disease Control MM (LTDC-MM)
· MM patients that after up-front treatment remain in:
· CR
> 5 years...relapse-free
28 patients
· <CR
> 3 years...progression-free
-85% of patients(24) were therapy free for >4 years.The remaining 4 patients were under
Mainteinance therapy (either low dose IFN or Prednisone) but stopped mainteinance > 1 month
before the study
Study Groups
· LTDC-MM
(N=28)
median 9 years(5-20) from diagnosis
· Symptomatic Untreated MM, (MM-s)
(N=23)
· Healthy Adults (HA) > 60 years
(N=10)
· Paired BM and PB samples from each subject
(n= 122 samples)
· Multidimensional Flow Cytometry Studies
3
Targeted cells
8-color Monoclonal Antibody
Panel
T cells
CD4+, CD8+, NK,
CD4/CD45/CD45-RA/CD127/CD8/CD25/CD56/CD3
Activated T cells, T regs
Plasma
Clonal-PCs, Normal-PCs
B cell precursors
CD45/CD38/CD56/CD138/CD19/CyIgK-TCR /CyIg
cells
TCR T cells
Immature, Naïve, Memory, CD20/CD45/SIgG/SIgA/CD19/CD27/SIgM/CD38
Plasmablasts
B cells
Homing, trafficking
CD20/CD45/62L/CXCR4/CD19/CD27/CD10-TCR /CD38
molecules, Clonal-PCs
Plasmacytoid-DCs,
Dendritic
Myeloid-DCs,
HLA-DR/CD45/CD16/CD33/CD34/CD117/CD123/CD14
cells
Circulating Tissue
Macrophages (TiMas)
-Results-
T cell CD4+ and CD8+ subpopulations in PB and BM
Bone Marrow
ells
CD4/CD8 RATIO
c
CD4+
CD8+
HA
HA
* p <.05 vs. LTDC-MM
15%
* p <.05 vs. LTDC-MM
MM-s
3
MM-s
ated
LTDC-MM
LTDC-MM
cleun10%
M
2
**
/B
cells 5%
1
TMBfo
% 0%
0
Peripheral Blood
** p<.001 vs. LTDC-MM
CD4/CD8 RATIO
CD4+
CD8+
5
**
* p <.05 vs. LTDC-MM
HA
HA
* p <.05 vs. LTDC-MM
MM-s
PB1,500
MM-s
4
LTDC-MM
LTDC-MM
lof
MM
3
1,000
cells/T
2
of 500
N.
1
0
0
-LTDC-MM displayed similar numbers of CD8+ cytotoxic cells to
symptomatic MM and both were increased as compared to HA, PB and BM
- More pronounced decrease of the CD4/CD8 T
-
cell ratio vs symptomatic MM -
4
-Results-
Total Natural Killer Cells in PB and BM
Bone marrow
Peripheral blood
4%
* p <.05 vs. LTDC-MM
cells
250
BM
cells
d 3%
PB 200
f
lo
/nucleate
/
-
-
150
2%
CD3
CD3
+
+
100
CD56
CD56
1%
of
BM
N.
50
of%
0
0%
HA
MM-s LTDC-MM
HA
MM-s
LTDC-MM
-LTDC-MM displayed similar numbers of NK cells to symptomatic MM
and both were increased in PB as compared to HA -
- NK dim cells (most cytotoxic compartment) with the same results-
-Results-
T Regulatory Cells in PB and BM
Bone marrow
Peripheral blood
100
1.5
cells
*p<.05 VS LTDC-MM
80
BM
cells
1.0
fPB 60
lo
/
/nucleated
40
cells
0.5
s
Tregs
reg 20
BM
fT
of
.oN
% 0.0
0
HA
MM-s
LTDC-MM
HA
MM-s
LTDC-MM
LTDC-MM showed in BM, decreased number of regulatory T cells vs
Symptomatic MM
* T regulatory cell phenotype: CD4+CD25hi CD127-
5
B cells in PB and BM
-Results-
Bone marrow
Peripheral blood
cells
*p<.005 vs LTDC-MM
BM 6
*p<.005 vs LTDC-MM
cells 300
PBf
lo
4
/nucleated
/ 200
cells
cells
2
100
CD19+
CD19+
of
BM 0
N.
of
0
%
HA
MM-s
LTDC-MM
HA
MM-s
LTDC-MM
LTDC-MM patients show recovered PB and BM B- cell numbers
vs symptomatic MM
-Results-
B cell Differentiation in PB and BM
BM
PB
BM
B-precursors
Immature
Naïve
Memory Plasmablasts
* Normal-Pcs
400
400
* p <.05 vs. LTDC-MM
l
200
HA
200
*
/
cells
MM-s
+
cells
100
LTDC-MM
100
19
+
CD 50
50
CD19
BM
*
PB
of 10
10
of
%
N.
5
5
2
*
2
*
*
0
0
LTDC-MM shows in contrast to symptomatic MM:
- Recovery of B cell precursors and normal PCs in BM
- Recovery of pre-germinal center B cells in PB
*Associated with normalization of CXCR4 expression by PB PCs vs MM-s patients p<.05
6
-Results-
Antigen Presenting Cell Compartment in PB and BM
% of DCs BM
HA MM-s
LTDC-MM
P-DCs
Circulating Tissue
42* 81*
68
Macrophages (TiMas)
Plasmacytoid-DC
57* 17*
32
M-DCs
% of DCs PB
HA MM-s
LTDC-MM
TiMas
Circulating Tissue
69
81*
70
Macrophages (TiMas)
Myeloid-Dcs
22
12*
22
*LTDC-MM showed similar numbers of total
Plasmacytoid-DC
DCs in PB and BM vs MM-s and HA
8.3 5.4
8.3
- PB antigen presenting cells return to normal levels while they remain
altered in BM of LTDC-MM -
*p<.05 VS LTDC-MM
-Results-
Sub-analysis in LTDC-MM
Does the immune system of LTDC-MM patients differs depending on:
I- The presence or not of residual myelomatous cells?
MRD negative vs. MRD positive
II- The duration of long term disease control?
10 yrs vs. 5-10 yrs
7
-Results-
Distribution of normal vs clonal PC in the BM of LTDC-MM (n=28)
- The Plasma Cell compartment of LTDC-MM patients is heterogeneous -
Normal PC
Clonal PC
100% Normal-PCs
1%-95% Clonal-PCs
30% (n=8)
70% (n=20)
Normal BM-profile
Residual clonal PC
MRD-
MGUS-profile
No significant differences were seen in the distribution of T,NK,B and DCs cell populations
between the two subgroups of LTDC-MM , except normal BM PCs were increased in CR pts
-Results-
Distribution of Immune System Cells in LTDC-MM with more than
10 years of Follow-up
*Peripheral
LTDC-MM
Blood
Cell
5 - 10 years (n=21)
10 years (n=7) p-Value
populations
T cells
1102
CD3+
±1042
1756 ±562
p .05
CD4+
443 ±211
747 ±199
p .001
CD8+
604 ±850
940 ±434
p .05
Tregs
35 ±15
45 ±18
NS
B cells
141
CD19+
±94
230 ±109
NS
Immature
8 ±7
11 ±6.8
NS
Naïve
105
p .05
±78
162 ±63
M-DCs
11
p .05
±5.2
19 ±11
*Mean and standard deviation. PB numbers of cells / l
LTDC-MM patients with > 10 years follow up have greater improvement in PB T, B and Dendritic Cells
8
Conclusions
Like symptomatic MM patients, LTDC-MM cases show:
· An expansion of the cytotoxic T and NK cell compartments
In contrast, to symptomatic patients, LTDC-MM cases show:
· A decrease in Tregs
· Normalization of Dcs subsets alterations
· Replenishment of BM precursor-B cells
· An increased B- cell (immature and naïve B-lymphocytes)
production
· Recovery of normal-PC numbers in the BM
Acknowledgments
Servicio de Hematología
Centro de Investigación
Serviço de Hematologia
del Cancer
Universidade Federal do Rio de Janeiro
Hospital Universitario Salamanca
Prof. Alberto Orfao
Prof. Jesús F. San Miguel
Carlos Fernandez Gimenez
Prof. Wolmar Pulcheri
Mª Belén Vídriales
Julia Almeida
Angelo Maiolino
Bruno Paiva
Martin Perez-Andres
Marcio Nucci
Mª Victoria Mateos
Quentin Lecrevisse
Elaine Sobral
Mª Consuelo López-Berges
Norma Gutiérrez
Ramón García-Sanz
Enrique Ocio
José J. Pérez
Gloria Ercilla
Isabel Martin
Investigators and our patients!
Mª Teresa González
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