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Immunophenotyping of Plasma Cells:
Implications on management
Department of
Cancer Research Centre
Haematology
University Hospital
J.F.
.F Sa
Sa
S n Migue
Mi
l
University of Salamanca, Spain

Faculty disclosure information
Nothing to disclose

BACKGROUND
IMMUNOPHENOTYPING
- Acute Leukemias & Ly
L mphoproliferative
y
disorders:
d
· Mandatory for diagnosis & monitoring
- Multiple Myeloma:
· Restricted
R
to research
· Dif
D ferential
if
diagnosis of unusual cases

IMMUNOPHENOTYPING of PLASMA
CELL
Discrimination between normal vs. clonal PC
- Diff
ti
eren al di
diagnosis of MGUS
MGUS & MM
- Risk of Transformation of MGUS & Smoldering MM
- MRD investigation

Immunophenotype of normal and myelomatous PC
Normal BM
BM
Multiple Myeloma
Antigenic
% cases
Antigenic
% cases
pattern
pattern
CD19
CD19
heterogeneous
homogeneous
100%
93%
( 80% +ve cells)
(-ve)
01
2
3
4
10
10
10
012
10
10
34
10
10
10
10
10
Gate CD 38 ->
CD38 ->
CD45
CD45
homogeneous
homogeneous
94%
83%
(+strong)
(-ve)
10
10
01
10
10
23
10 4
10 0
10
10
10
123
10 4
CD45 ->
CD45 ->
CD56
CD56
heterogeneous
100% homogeneous
57%
( 10% +w
k
ea )
kk)
(
t)
+st
+s rong)
10
10
01
102
103
104
10
10
01
10 2
10 3
10 4
Gate CD 38 ->
CD38 ->
Mateo et al. J Clin Oncol; 2008;26:2737

Immunophenotype of normal and myelomatous PC
Normal BM
Multip
Multi le
p Myeloma
y
py
Antigenic
% cases
Antigenic
% cases
pattern
pattern
CD117
CD1
CD117
CD1
homogeneous
100% homogeneous
30%
( -ve)
(+strong)
10
10
01
102
103
104
10
10
01
10 2
103
104
Gate CD38 ->
CD38 ->
CD28
70%
CD28
(-ve/
30%
homogeneous
25%
30%
weak)
(+strong)
10
10
10
012
103
104
10
10
01
102
103
104
Gate CD38 ->
CD38 ->
CD33
80%
CD33
homogeneous
20%
(-ve/
20%
weak)
eak
(+strong)
10
10
01
102
103
104
10
10
01
102
103
104
Gate CD38 ->
CD38 ->
Mateo et al. J Clin Oncol; 2008;26:2737

Distinction between myelomatous & normal PC*
Se
S l
e e
l ct
e ed
ct
CD
C 38high
3
D 8hi
38
38
pl
pl
p a
l s
a m
s a
m ce
ce
c lls
l
ells
lls
l
n-P
n- C
P
MM-PC
CD19
MM-P
CD19
MM-
CD19
CD45
CD4
CD56
CD5
*92% of MM patients
Mateo G, et al. In: Methods in molecular medicine.
Vol 113. Multiple myeloma: methods and protocols. New Jersey:Humana Press Inc.; 2005: 5-24.

Protocol for immunophenotypic investigation
Double Acquisition
A
Pl
Pane
P
l
ane
f
o MA
Mo
M
b
MA
Double Acquisition
Ab
(quadruple combinations)
FITC
PE
PE/C 5
y
APC
1er
1 STEP
CD38
CD56
CD19
CD45
To
T tal
o
BM cellularity
CD138 CD28
CD33
CD38
CD20 CD117
CD1
CD138
CD38
100
10
01
10
12
10
23
10
344
CD38 FITC ->
FITC: fluoresce
fluoresc i
e n
i isotiocianat
isotiociana e;
e PE: phycoery
phy
t
coery rine; PE/Cy5:
PE/Cy
PE/cyanine
PE/cy
5
anine ;
5 APC: allo-phyco-cy
allo-phy
a
co-cy nine
Identify the phenotypic
aberrancy characteristic of
2nd
2 STEP
malignant PC
Live Gate on
selected fraction*
PA
P TIENT
A
-
TIENT SPECIFIC PROBE
100
10
101
10
102
10
103
10
104
10
In <10% of cases other combination
combination were
Gate CD38 FITC ->
added to
to clarify the
the clonal
clonal nature
nature of the PC:
PC:
cL
cL//cK
cK/CD38/*CD45
/CD38/*CD45--19
19--56
56
* 3,000 gated PC from 106
10 acquired leukocytes
leukocy
eukocy
leukocy

Parameters analyzed
..
..
..
Total cellularity
PC compartment
....
..
.. ........ ..
..
..
....
....
..
..
% myelomatous PC %MM-PC
% normal PC %N-PC
Proportion of N-PC referred to
to
the total-PC
%N-PC/ total-PC
San Miguel et al Blood; 2002; 98:1853-56

IMMUNOPHENOTYPING of PLASMA CELL
Discrimination between normal vs. clonal PC
- Differential diagnosis of MGUS & MM
- Risk of Transformation of Smoldering MM & MGUS
- MRD investigation

Differential diagnosis between MM and
MGUS
MM
MGUS
Clonal
Poly-Clonal
versus
Only 10% of MM patients showed > 5%
>5% poly-PC: 82% MGUS*
poly-
poly PC
The most powerful single criteria for differential diagnosis
1.
Ocqueteau M, Am J Pathol 1998, 152: 1655-65
2.
Updated with 1218 patients (811 MM & 407 MGUS)

IMMUNOPHENOTYPING of PLASMA CELL
Discrimination between normal vs. clonal PC
- Differential diagnosis of MGUS & MM
- Risk of Transformation of MGUS & Smoldering MM
- MRD investigation

MGUS: Flow Cytometry Results in 407
patients*
%T
% ot
To al PC
PC in
in BM
BM*
1.0 (0.1
(0.1-3.7)
% of aPC / BMPC compartment 73 (0-100)
<9
< 5%
95% aPC/B
aPC / MPC
BMPC
334 (82%)
> 95% aPC / BMPC
73 (18%)
* Median
Median (range)
(range)
*Morphology: % BMPC: 4% (1-10%)
Pérez-Persona et al; Blood. 2007: 110; 2586-92

MGUS (403 patients)*
Impact of % aPC/BMPC
aPC/BMPC by FC on PFS
1,0
p=0.0000
5 years
>95% aPC/BMPC
0,8
n= 73 (16 progressions)
0,6
24%
Median 107
<95% aPC/BMPC
gressionor
months
0,4
n= 330 (13 progressions)
progressions)
fPo%
4%
0,2
Median Not
0,0
reached
Follow-up (months)..........52 (>24)
0
24
48
72
96
120
144
168
Progression (%)............. 8%
Months
Pérez-Persona et al; Blood. 2007: 110; 2586-92

MGUS (253 patients)
Multivariate Analysis
Analysis: aPC/BMPC & DNA aneuploidy
1,0
p=0.0014
5 years
>95% aPC/BMPC+
0,8
Aneuploidy
49%
n= 15 (9 progressions
(g
)
0,6
>95% aPC/BMPC or
Aneuploidy
n= 114 (15 progressions)
sion
11%
(p g
s 0,4
No adverse factors
n= 124 (3 progressions)
Progres 0,2
%
2%
0,0
Follow-up (months)
52
......
(>24)
0
24
48
72
96
120
144
168
Months
Progression (%)..........10%
Pérez-Persona et al; Blood. 2007: 110; 2586-92

IMMUNOPHENOTYPING of PLASMA CELL
Discrimination between normal vs. clonal PC
- Differential diagnosis of MGUS & MM
- Risk of Transformation of MGUS & Smoldering MM
- MRD investigation

Risk of Transformation
T
of Smoldering MM
into active MM or Amyloidosis
y
Cumulative probability of progression: 73% at 15 years
- 10% / year during the
the........ 1st 5 years
- 3% / year during the........ next 5 years
- 1% / year during the........ last 10 years
Overal Survival : 60% at 5 years ; 34 % at 10 years
Risk factor for progression:
- MC (>4 g /dl); Heavy chain ( IgA); % PC in BM (>20%)
- Diffuse pattern of BM infiltration.
- Urinary light chain (+ and lambda),
- Reduction of univolved Igs (one or two).
Kyle NEJM 2007; 356:2582-92

Smoldering MM:
Flow Cytometry Results in 93 patients*
%T
% ot
To al PC
PC in
in BM
BM*
2.9 (0.9
(0.9-22.0)
% of
of aPC
aPC / BMPC
BMPC compartment
compartment 97 (35-100)
< 95% aPC / BMPC
37 (40%)
>9
> 5%
95% aPC/B
aPC / MPC
BMPC
56 (60%)
* Median (range)
* Morphology: % PC 14% (4-55%)
Pérez-Persona et al; Blood. 2007: 110; 2586-92

Smoldering Multiple Myeloma
Impact of % aPC/BMPC
aPC/BMPC by
by FC
FC on
on PFS
PFS
1,0
p=0.0000
5 years
0,8
0,
>95% aPC/BMPC
n= 56 (36 progressions)
63%
0,6
ression
Median 40 months
r
<95% aPC/BMPC
Prog
0,4
of
n= 37 (4 progressions)
%
8%
0,2
0,0
Median Not reached
Follow-
Follow up (months)....56
(months)....
(24-107)
0
20
40
60
80
100
120
Progression (%)................42%
Months
Pérez-Persona et al; Blood. 2007: 110; 2586-92

Multivariate analysis for PFS
p
HR
% a PC /BMPC
0.004
4.9
Immunoparesis
0.007
2.6
Pérez-Persona et al; Blood. 2007: 110; 2586-92

Impact of p
pprognostic index on PFS
Immunoparesis
>95% aPC/BMPC
Score (n)
----00 (n=
n=32
32)
+
+//----/+
/+
11(n=
(n=27
27))
+
++
+22(n=
(n=27
27))
Pérez-Persona et al; Blood. 2007: 110; 2586-92

Impact of prognostic index* on TTP in
Sl
Smo d
ldering MM
MM (based on aPC/BMPC by FC & Immunoparesis)
10
1,0
5 years
p= 0.003
Median 23 months
0,8
82%
No adverse factors
n= 32 (3 progr.)
0,6
>95% aPC/BMPC or paresis
rogressionp
n= 27 (12 progr.)
to
0,4
e
Median 73 months
42%
m
>95% aPC/BMPC + paresis
Ti
n= 27 (22 pro
(p gr.
g )
%
0,2
8%
Median not reached
0,0
0
24
48
72
96
120
Months
Pérez-Persona et al; Blood. 2007: 110; 2586-92

Symptomatic Multiple Myeloma
Clonal
10% of MM patients showed > 5% poly-PC
Paiva et al Unpublished data

Symptomatic myeloma patients baseline characteristics
according to the number of N-PC/BMPC (811 patients)
5% N-PC/BMPC
>5% N-PC/BMPC
P
(N=723)
(N=88)
ISS stage I
33%
48%
.02

46%
33%
.01
2M; >3.5 mg/L
Hemoglobin; <100 g/L
44%
20%
<.001
Albumin; <3
<3.5 g/dL
50%
38%
.05
M-component; 3 g/dL
75%
44%
<.001
Immunoparesis
84%
42%
.003
PC by optimal
yp
microscope
p >30% 45%
17%
<.001
High-risk cytogenetics
26%
3%
.006
Any t(4;14), t(14;16) or del(17p)

Symptomatic Multiple Myeloma: Survival according
to the presence or absence of >5% N-PC/BMPC in
bone marrow at
at diagnosis
diagnosis (N=811
(N=81 )
1
PFS
OS
100
p< 0 001
.
100
100
p=
p 0 004
.
80
80
Median:
Not reached
reached
60
60
40
Median:
40
51 months
Median:
20
73 months
20
Median:
39 months
0
0
0
20
40
60
80
100
120
0
25
50
75
100
125
Months
Months
> 5% N-PC/BMPC (n=88)
5% N-PC/BMPC (n=723)

IMMUNOPHENOTYPING of PLASMA CELL
Discrimination between normal vs. clonal PC
- Differential diagnosis of MGUS & MM
- Risk of Transformation of MGUS & Smoldering MM
- MRD investigation

BACKGROUND
· Hig
Hi h-dose
g
chemotherapy
chemothera
and Novel Drug
Dru s
g
Complete remission (CR): 25%-75%
Relapse-free survival (RFS) at 5 year: 40%-70%
· However, patients with MM ultimately relapse
MINIMAL RESIDUAL DISEASE (MRD)
persistence of residual malignant cells

Impact on Survival of MRD by Immnunophenotyping in BM
obtained 3 months after ASCT in CR patients
(
t
nega i
tive Immunofi ti
xa
)
on (n=147)
PFS
OS
100
100
87%
80
80
62%
60
Median:
60
71 months
59% Medians:
40
40
Not reached
30% Median:
37 mo
3onths
20
20
p< 0.001
p= 0.009
0
0
0
25
50
75
100
125
5y
0
20
40
Months
560
80
100
120
140
5y
Months
MRD negative (n=94)
MRD positive (n=53)
Paiva et al; Blood. 2008; 112: 4017-23

Multivariate Analysis
Analysis
PFS
OS
p
risk
p
risk
MRD+ at day +100
+100
0.002
36
3.6
00
0. 2
02
20
2.0
High Risk Cytog*.
0.006
1.79
ns
Age >60y.
ns
0.04
1.6
IFx+ at day +100
ns
ns
*t(4;14), t(4;16), del (17p)
Paiva et al; Blood. 2008; 112: 4017-23

CONCLUSIONS
Immunophenotypic studies have clinical value:
- Differential diagnosis MGUS/MM
- Risk of transformation in MGUS & SMM
SMM
- Investigation
g
of MRD (immunophenot
(p
yp
y ic
p
remission)

Grupo Español de Mieloma (GEM)
Hospitales
Hospitales
Clínico de Barcelona
Gl
General de S
i
egov a
12 Octubre (Madrid)
Cruces (Bilbao)
Clínico de Salamanca
St. Coloma de Gramanet
Clínico de San Carlos (Madrid)
(Barcelona)
Hospital de Badalona
Gregorio Marañon (Madrid)
Clínico de Asturias
Carlos Haya (Málaga)
Fr. Peset (Valenci
(Valenc a
i )
a
H. Tauli
Tauli (Gerona)
Universitario de Canarias
Huesca
Rio Ortega (Valladolid)
Palencia
Cínico de Zaragoza
Alcira (Valencia)
Hospital General de Jerez
H. Del Mar (Barcelona)
Ramón y Cajal (Madrid)
Mahón (Baleares)
Morales Meseguer (
g(Murcia)
Clínico de Málaga
La Fe (Valencia)
Xeral Cies (Vigo)
C.U. de Navarra
Plasencia
Galdakao (Vizcaya)
Cáceres
Clínico de Valladolid
Algeciras
Sant Pau (Barcelona)
Ávila
Arnau Vilanova (Lérida)
Jaén
Universita
Univ
rio
ersita
de
de Santiago
Santiago
S. Pau
Pau i Sta
Sta Tecla
General Universitario de Valencia
(Tarragona)
Universitario de Getafe (Madrid)
General de Guadalajara
Insular de las Palmas
Sagunto (Valencia)
H. de La Princesa (Madrid)
Son Dureta (Mallorca)
Severo Ochoa (Madrid)
Cuenca
Juan XIII (Tarragona
(g
)
Alicante SUS
Toledo
M. Valdecilla (Santander)
Gandía (Valencia)
Albacete
Vall D´Hebrón (Barcelona)
H. Del Bierzo
San Jorge (Huesca)
Fundación Jiménez Díaz
Verge de la Cinta (Tortosa)
(Madrid)
Alarcos (Ciudad Real)
Elda (Alicante)
Mt
Mataró (Md
(Madrid)
id)
VD
V. Del Rosel (C
(Cart
)
agena
Juán Canalejo (Coruña)
Castellón
Ferrol
Mutua Tarrasa
Consorcio Tarrasa
C. Corachán (Barcelona)

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