IMPORTANCIA DE PARAMETROS CLINICO-BIOLOGICOS en la ESTRATIFICACION del TRATAMIENTO en LMA

IMMUNOPHENOTYPE OF MALIGNANT CLONE
Implications of management
Department of Haematology
Cancer Research Centre
University Hospital
J.F. San Miguel
University of Salamanca, Spain

BACKGROUND
IMMUNOPHENOTYPING
- Acute Leukemias & Lymphoproliferative disorders:
· Mandatory for diagnosis & monitoring
- Multiple Myeloma:
· Restricted to research
· Differential diagnosis of unusual cases

IMMUNOPHENOTYPING of PLASMA CELL in
MULTIPLE MYELOMA
Characterization of antigenic profile of PC
- Prognostic influence of specific antigens
- Correlation protein expression & gene markers
- MRD investigation in MM

Prognostic influence of antigenic profile in Myeloma
Controversial results (CD10, CD20, Myeloid...)
- Technical pitfalls
. Differences in the Clone of MoAb & Flurochromes
. Single vs multiparametric labelling
. Criteria for definition of positivity
- Treatment heterogeneity*
* Uniformely treated with GEM2000 protocol
protocol (VBCMP/VBAD (x6) followed
(VBCMP/VBAD (x6) followed by ASCT)
627 patients; median OS: 62 months; RFS: 36 months
627 patients; median OS: 62 months; RFS: 36 months

Antigenic profile of plasma cells
Multiparametric flow-cytometric approach
Panel of MoAbs
(double acquisition)
(quadruple combinations)
1st
1st STEP
2nd
2nd STEP
Total cellu
Total cellu larity
Specific selection of PCs*
FITC
PE
PE/Cy5
APC
CD38
CD56
CD19
CD45
C
CD138
CD28
CD33
CD38
S
C
S
S

ED S
D
M
E
CD20
CD117
CD138
CD38
R
M
O
R
O
F
S
N
TRANSF
100 101 102 103 104
A
TRANSF
R
10
100
0
10
101
1
10
102
2
10
103
3
10
104
4
T
FITC: fluorescein isothiocyanate; PE: phycoeritrin;
CD38 FITC ->
PE/Cy5: PE/cyanine-5; APC: alophycocyanine
Gate CD38 FITC ->
* 3,000 CP obtained from 106
6 acquired leukocytes

Patterns of antigenic expression in PC of MM
Patterns of expression
n= 627 patients
-ve
-
+weak
+
++bright
++
-/+het
-/+
CD38
-
80%
20%
-
CD138
2%*
25%
63%
-
CD19
94%
-
1%
5%
CD20
83%
6%
7%
4%
CD28
60%
11%
20%
9%
CD33
80%
4%
7%
9%
CD45
70%
10%
10%
10%
CD117
65%
14%
16%
5%
CD56
22%
10%
58%
10%
* Usually
Usually associ
a
ated
ssociated with apoptotic
poptotic cells
cells

Prognostic influence of PC antigenic expression (I)
CD56
CD117
-- CD117 + (n=195)
-- CD56 +
CD56 (n=272)
1,0
-- CD117 +
1,0
-- CD117 -ve (n=388)
-- CD56 ve
CD56 ve (n=355)
,9
-- CD117 -ve (n=388
,8
,8
38 m
,7
,6
38 m
PFS
,6
PFS
41 m
,4
,5
33 m
,4
31 m
,2
,3
,2
0,0
p=0.05
,1
p=0.01
0,0
0
6
12
18
24
30
36
42
48
54
60
0
6
12
18
24
30
36
42
48
54
60
66
72
-- CD56 +
CD56 (n=272)
-- CD117 +
(n=195)
1,0
-- CD56 ve
CD56 ve (n=355)
1.0
-- CD117 -ve
-ve (n=388)
,8
.8
OS
NR
,6
NR
.6
,4
59 m
58 m
.4
,2
.2
0,0
p=0.03
p=0.1
0.0
0
6
12
18
24
30
36
42
48
54
60
0
6
12
18
24
30
36
42
48
54
60
66
72
Months from diagnosis
Months from diagnosis

Prognostic influence of PC antigenic expression (II)
CD19
CD28
1,0
-- CD19 +
CD19 (n=47)
-- CD28 + (n=235)
1,0
-- CD28 + (n=235
,9
-- CD19 -ve
CD19 -ve (n=580)
,9
-- CD28 -ve
CD28 -ve (n=368)
,8
,8
,7
,7
,6
,6
36m
37 m
,5
,5
PFS ,4
28 m
,4
31 m
,3
,3
,2
,2
,1
p=0.2
,1
p=0.05
0,0
p=0.0
0
6
12
18
24
30
36
42
48
54
60
0,0 0 6 12 18 24 30 36 42 48 54 60
1,0
-- CD28 + (n=235)
-- CD19 +
CD19 (n=47)
1,0
-- CD28 + (n=235
,9
,9
-- CD28 -ve
CD28 -ve (n=368)
-- CD19 -ve
CD19 -ve (n=580)
,8
,8
,7
62 m
,7
66m
,6
,6
OS ,5
,5
54 m
,4
54 m
,4
,3
,3
,2
,2
,1
p=0.06
,1
p=0.1
0,0
0,0
0
6
12
18
24
30
36
42
48
54
60
0
6
12
18
24
30
36
42
48
54
60
Months from diagnosis
Months from diagnosis

Prognostic influence of PC antigenic expression (III)
PFS (median)
p
OS (median)
p
CD20 -
n= 473
n= 473
37 m 0.5
65 m
0.7
+ n= 100
+ n= 100
33 m
62 m
CD33 -
n= 463
n= 463
36 m
0.2 62 m 0.3
+ n= 118
+ n= 118
32 m 58 m
CD45 -
n= 514
n= 514
36 m 0.8 59
36 m 0.8
m
59 m
0.7
+ n=100
+ n=100
37 m
62 m

Prognostic influence of PC antigenic expression (V)
PFS (median)
p
OS (median)
p
CD20 -
n= 473 37 m 0.5
65 m
0.7
+ n= 100
33 m
62 m
CD33 -
n= 463 36 m
0.2 62 m 0.3
+ n= 118
32 m 58 m
CD45 -
n= 444 36 m 0.8 59 m 0.7
+ n=141
37 m
62 m
PFS
OS
-ve
37 m
1.0
1.0
-ve
37
1.0
1.0
+weak
35 m
.9
.9
+weak
35
.9
.9
.8
.8
++bright 45 m
.8
.8
.7
.7
-/+ het
36 m
CD45
.7
.7
.6
.6
.6
.6
.5
.5
.5
.5
-ve
58 m
.4
.4
-ve
58
.4
.4
+weak
NR
.3
.3
+weak
.3
.3
.2
.2
++bright
NR
p=0.6
.2
.2
.1
.1
-/+ het
50 m
p=0.5
.1
.1
-/+ het
50 m
0.0
0.0
0.0
0.0
66
66
60
60
54
54
48
48
42
42
36
36
30
30
24
24
18
18
12
12
6
6
0
0
66
66
60
60
54
54
48
48
42
42
36
36
30
30
24
24
18
18
12
12
6
6
0
0

Prognostic influence of phenotypic profiles
Prognostic influence of phenotypic profiles
Prognostic influence of phenotypic profiles
Prognostic influence of phenoty
Prognostic influence of phenotypic profiles
Prognostic influence of phenot
CD56 & CD117
CD56 & CD28
CD117 & CD28
ve
+ve
+v
+ve

1,0
-- CD56
D56
5 ve
ve
D56
CD
CD
CD117
117 -ve
-ve
117-v
-ve
117 (n
( =
n 303)
=
-- CD117 +veCD28-ve (n=277)
1,0
-- CD56
D56
5 ve
D56
CD28+ve
28+v (n
(n
( =87
=
)
87)
1,0
-- CD117 +ve
CD117 CD28-ve
CD28-ve(n
=303
=
)
303
1,0
-- CD56
D56 ve CD28+ve (n
(n =87
=
)
87)
1,0
(n=303)
-- CD117 +veCD28-ve (n
,9
-- ot
o h
t er
h s
er (n=
( 14
n= 0)
14
,9
-- ot
o h
t er
h s
er (n=
( 372)
n=
,9
-- oth
t ers
er (n=140)
14
n= 0)
140)
-- others (n=37
n= 2)
37
-- oth
th ers (n=87)
=87)
=87)
8
,8
,8
,8
,7
,7
,7
,6
31 m
31
,6
,6
31 m
31
37 m
37
NR
,5
,5
,5
,4
,4
,4
PFS
41 m
41
29 m
29
33 m
33 m
33 m
33
,3
,3
,3
,2
,2
,2
p=0.009
p=0.0
p=0. 0
0
0.0 5
0
,1
p=0.
p=0 0
0
0.0 9
0
p=0.0
p=0.0
p=
05
0
p 0.001
,1
p=0.009
,1
p=0.005
,1
,1
p=0.00
=
1
0.00
0,0
0,0
0,0
0
6
12 18 24 30 36 42 48 54 60 66 72
0
0
6
12 18 24 30 36 42 48 54 60 66 72
6
12 18 24 30 36 42 48 54 60 66 72
Month
Mo
s
Month fr
nths
s
nths om
fro
fr
dia
m
om di g
a
dia nos
g
i
gnos
nos
gnoss
is
i
Mont
Mon hs
Months fr
hs om
fr
dia
om d
om i
om d a
i gnos
g
dia no
n s is
i
gnoss
i
Months
Months
Mo
fr
Months om
o
fr m
o
dia
dia
om
g
dia no
n s
gnos i
s s
is
Months from diagnosis
i
1,0
1.0
1,0
,9
.9
,9
,8
.8
,8
,7
NR
.7
NR
,7
NR
OS ,6
.6
,6
OS ,5
.5
,5
NR
42 m
42
NR
,4
.4
,4
p=0.002
p=0.002
0.002
p=0.00
0.002
p=0.0
0.002
p=0.
,3
.3
,3
,2
.2
,2
p=0
p= .
p=0 0
. 1
0.01
0
0.01
p=0.00
0.0 2
0
p=0
p= .
p=0 0
. 4
0.04
0
0.04
,1
.1
,1
0,0
0.0
0,0
0
6
12 18 24 30
36 42 48 54 60 66 72
0
6
12 18 24 30 36 42 48 54 60 66 72
0
6
12 18 24 30 36 42 48 54 60 66 72
Mont
Mon hs
Months fr
hs om
fr
dia
om
omdia
om
gnos
g
dia nos
g
is
i
gnoss
i
Months
Months
Mo
fr
Months om
fro
fr m
fro dia
di
om a
di g
dia nos
gnos
g
is
nos
Months
Months
Mo
fr
Months om
fro
fr m
fro dia
di
om a
di g
dia no
n s
gnos i
s s
is
i

Prognostic influence of phenotypic profiles
CD56 & CD117
CD56 & CD28
1.0
1.0
CD56+CD117+ n= 130 45 m
.9
CD5
CD5 6+CD
6+CD 28
28 - n= 1116
= 1116 41
41 m
m
CD56+CD117+ n= 130 45 m
.9
+/- or +/-
n=267 36 m
+/+ or -/-
n=266
=266
36 m
+/- or +/-
n=267 36 m
.8
.8
CD56-CD117-
n=186
31 m
CD5
CD5 6-CD
6-CD 28
28 + n=116 2
=116
9
29
m
m
CD56-CD117-
n=186
31 m
.7
.7
.6
.6
.5
PFS .5
.4
.4
.3
.3
.2
.2
p=0.001
p=0.01
.1
.1
0.0
Months from diagnosis
0.0
0
6
12
18
24
30
36
42
48
54
60
66
72
0
6
12
18
24
30
36
42
48
54
60
66
72
Months from diagnosis
Months from diagnosis
Months from diagnosis
CD28 & CD117
CD2
CD2 8-CD
8-CD 117
117 + n
+ n = 142
45 m
1.0
+/- or +/-
n=327 37 m
.9
CD2
CD2 8+CD
8+CD 117
117 -
n=114
=114
29 m
.8
.7
.6
.5
PFS .4
.3
.2
.1
p=0.0005
0.0
0
6
12
18
24
30
36
42
48
54
60
66
72

Multivariate analysis for RFS and OS
RFS
OS
P
P
% BMPC by FCM >20%
0.01
NS
% BMPC in S-phase 2.5%
0.03
0.004
Non-hyperdiploidy DNA
0.05
NS
ISS
0.003
0.0001
Calcium 11 mg/dl
0.005
NS
Platelets 130 · 109
0 /L
0.017
0.04
CD56- CD117-
0.004
0.02

Correlation between chromosomal abnormalities &
DNA content
Hyperdiploid
Non-hyperdiploid
P
Del(13q)
19% (32/166)
45% (87/194) .001
IGH translocations:
Total
10% (15/142)
49% (87/175)
.001
t(11;14)
4% (6/142)
20% (36/175)
.05
no t(11;14) 6% (9/142)
29% (51/175)
Mateo et al; Clin. Can. Res, 2005

Correlation between antigen expression & DNA content
Hyperdiploid (414) Non-hyperdiploid (455)
P
CD20 +
28% (40/142)
72% (102/142) .0001
CD28 +
39% (123/319)
61% (196/319)
.0001
CD33 +
63% (103/164)
37% (61/164)
.0001
CD56 -
35% (118/340)
65% (134/201)
.0001
CD117 -
36% (192/526)
64% (334/526) .0001
Number of cases: 869
Mateo et al; Clin. Can. Res, 2005

Correlation between
antigenic expression & chromosomal abnormalities
Del 13 p
t(11;14) p Other IGH
CD20 -
33% (36/73) NS
8% (19/244)
.0001 20%
.0001 20% (49/244)
+ 33% (92/277)
32% (21/66) 14% (9/66)
CD56 -
35% (51/144)
NS
24% (31/129)
.001 20% (26/129)
+ 30% (69/266)
6% (11/197) 19% (36/197)
CD117 -
37%
37% (85/232)
.002 16%
.002 16% (34/211)
22% (46/211)
+ 26% (31/121)
6% (6/101)
.001 13% (13/101)
FISH analysis (n=410)
Mateo et al; Clin. Can. Res, 2005

Incidence of Aberrant Phenotypes in PC from MM
N= 627 patients
80%
80
57%
60
92%
50
40
25%
30%
30
20%
21%
20
13%
10
0
CD38
CD56
CD28 CD33
CD117
sIg
CD20
Infra-
Over- expression
expression
Asynchronous expression
expression

II. Antigen over-expression
Normal BM
Multiple Myeloma
Antigenic
Antigenic pattern
pattern
% cases
Antigenic pattern
% cases
CD56
CD56
heterogeneous
heterogeneous
homogeneous
100%
57%
( 10% +weak)
(+strong)
0
1
2
3
4
10 0
101
102
103
104
10
10
10
10
10
Gate CD 38 ->
CD38 ->
CD28
CD28
homogeneous
70%/30%
25%
(-ve/w
(-ve/w eak)
eak)
(+strong)
100
101
102
103
104
10 0
101
102
103
104
Gate CD38 ->
CD38 ->
CD33
CD33
homogeneous
80%/20%
20%
(-ve/w
(-ve/w eak)
eak)
(+strong)
0
1
2
3
4
10 0
10 1
102
103
104
10
10
10
10
10
Gate CD38 ->
CD38 ->

Additional useful markers
Normal BM
Multiple Myeloma
Antigenic
Antigenic pattern
pattern
% cases
Antigenic
Antigenic pattern
pattern
% cases
CD19
CD19
heterogeneous
heterogeneous
homogeneous
100%
93%
( 80% +ve cells)
(-ve)
0
1
2
3
4
10 0
10 1
10 2
10 3
10 4
10
10
10
10
10
Gate CD 38 ->
CD38 ->
CD45
CD45
homogeneous
homogeneous
(+strong)
94%
(-ve)
83%
10 0
10 1
10 2
10 3
10 4
10 0
10 1
10 2
10 3
10 4
CD45 ->
CD45 ->

Distinction between myelomatous & normal PC
Selected
Select
e ed
Selected CD38high
CD38hi
CD38
plas
pl
ma
plas
c
ma e
c lls
asma cells
lls
n-PC
n-PC
MM-PC
CD19
MM-P
CD19
CD45
CD56

PROTOCOL for MRD
Double Acquisition
Panel of MoAb
(quadruple combinations)
FITC
PE
PE/Cy5
APC
1er
1 STEP
CD38
CD56
CD19
CD45
Total BM cellularity
Total BM cellularity
CD138
CD28
CD33
CD38
TRANSFORMED SSC
100
101
102
103
104
TRANSFORMED SSC
CD20
CD117
CD138
CD38
CD38 FITC ->
FITC: fluorescein isotiocianate; PE: phycoerytrine; PE/Cy5:
PE/cyanine5; APC: allo-phyco-cyanine
C
S
S

D
2nd
2
STEP
ME
Identify the phenotypic aberrancy
R
O
Live Gate on selected
F
characteristic of malignant PC
S
fraction*
on*
N
A
R
PATIENT-SPECIFIC PROBE
10
10 0
0
10
10 1
1
10
10 2
2
10
10 3
3
10
10 4
4
T
Gate CD38 FITC ->
* 3,000 gated PC from 106
3,000 gated PC from 106 acquired leukocytes

MRD: Sensitivity of Immunophenotyping
Dilutional Experiments
1:1
1:10
1:100
15%
1.4%
0.13%
MM- PC
MM- PC
MM- PC
5
6

-
>
5
6

-
>
5
6

-
>
D
D
D
C
C
C
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
CD38 ->
Gate CD38 ->
Gate CD38 ->
1:1000
1:10000
0.015%
0.0014%
MM- PC . .
MM- PC
. ...
. ... . .
.. .. . ..
. ..... .
..... .
...
.
. .. .. .
. . ... . .
.
. .
..
. ... ... ........ ..
->
. . ....
. .
.
6
56 ->
.
. .. ..
.
.. .. .
... .. ..
D5
. . ..
Sensitivity
. ..... .. .
Sensitiv
C
.. .. ........
CD
. ... . ..
. .. ..
. . .
. .
. ..
.
. ... .. .
.. ....
.
.
. .
. ..
. .
.
.
.
level: 10-4
level: 10
100
101
102
103
104
100
101
102
10 3
104
Gate CD38 ->
Gate CD38 ->

MRD: parameters analyzed
.
.
.
Total cellularity
PC compartment
..
.
. .... .
.
.
.. ..
. .
% myelomatous PC %MM-PC
PC %MM-PC
% normal PC %N-PC
% normal PC %N-PC
Proportion of N-PC referred to the
total-PC
%N-PC/ total-PC

Spanish multi-centre protocol GEM (2000-2004)
Diagnosis
VBCMP/VBAD (x4)
Non-responding
Stem cell collection
VBCMP/VBAD (x2)
Double-Trx
MRD
investigation
ASCT (1º)
Complete remission
Partial response
3m post-ASCT
(negative electrophoresis)*
(positive electrophoresis)
ASCT (2º)
Mini-ALO-Trx
Maintenance
Relapse
* Immunofixation either ve or +ve

Correlation between immunophenotyping & electrophoretic responses
at three months post-ASCT (n=200)
Partial response
Complete remission
EF-positive
IFx-positive
IFx-negative
p
MRD evaluation
n=74 cases
n=27 cases (21%)
n=99 cases (79%)
MM-PC
0.76 ± 0.9 #
0.76 ± 0.9
0.28 ± 0.4
0.1 ± 0.3
0.006
MRDnegative cases
8% #
8%
MRDnegative cases
33%
64%
0.003
% n-PC/TPC
42 ± 33 #
42 ± 33
69 ± 34
86 ± 25
0.008
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
MRD-ve: <0.01% residual MM PC

Changes in PC compartment following ASCT
20
14
10
1
0.8
0,3
0,25
0,21
0,2
n-PC
%
0,15
0,14
0,1
0,05
0,035
MM-PC
0
0,03
0,007
0
100
80
80
77
60
%
40
% n-PC / TPC
20
0
4
0
VBCMP/
Diagnosis
3m post-Trx
12m post-Trx
VBAD (x6)
Results expressed as mean

Changes in PC distribution following ASCT
Diagnosis
Post-Trx
N-PC
MM-PC
MM-PC
CD19
CD19
CD45
CD45
CD56
CD56
Gate CD38

Impact on RFS of MRD (by immunophenotyping) in BM
obtained 3 months post-ASCT in 200 patients
%MM-PC
1,0
p=0.0001
,9
,8
,7
NR
-- <0.01% MM-PC
,6
-- 0.01% to 1% MM-PC
,5
al
--
40m
1% MM-PC
v
iv
--
v
iv
,4
,3
e
e
sur
23m
e-fr
,2
a
ps
,1
Rel
0,0
0
6
12
18
24
30
36
42
48
54
60
66
72
78
Months from immunophenotypical analysis (3 months post-ASCT)

Impact on RFS of MRD (by immunophenotyping) in BM
obtained 3 months post-ASCT in 99 patients
post-ASCT in 99 patients in CR (IFx-)
%MM-PC
%N-PC / total PC
1,1
1,0
p=0,02
p=0,01
1,0
,9
,9
,8
,8
NR
,7
NR
,7
,6
,6
al ,5
36m
,5
v
iv
32m
,4
,4
e
e
sur
,3
,3
e-fr
,2
,2
a
ps
Rel ,1
,1
0,0
0,0
0
6
12
18
24
30
36
42
48
54
60
66
0
6
12
18
24
30
36
42
48
54
60
66
Months from immunophenotypical analysis (3 months post-ASCT)
-- <0.01% MM-PC (MRD ve)
-- 75 % N-PC/total PC
-- 0.01% MM-PC
-- < 75 % N-PC/total PC

Impact on RFS of immunophenotyping
at 3 months post-ASCT in 200 patients
%MM-PC
1,0
p=0.0001
,9
,8
,7
NR
,6
-- <0.01% MM-PC (n=82)
,5
al
v
iv
-- 0.01% to 1% MM-PC (n=100)
v
iv
40m
-- 0.01% to 1% MM-PC
,4
-- 1% MM-PC (n=18)
,3
e
e
sur
23m
e-fr
,2
a
ps
,1
Rel
0,0
0
6
12
18
24
30
36
42
48
54
60
66
72
78
Months from immunophenotypical analysis (3 months post-ASCT)

Impact on RFS of immunophenotyping
at 3 months post-ASCT in 99 CR (IF-) patients
%MM-PC
%N-PC / total PC
1,1
1,0
p=0,02
p=0,01
1,0
,9
,9
,8
,8
NR
,7
NR
,7
,6
,6
al ,5
36m
,5
v
iv
32m
,4
,4
e
e
sur
,3
,3
e-fr
,2
,2
a
ps
Rel ,1
,1
0,0
0,0
0
6
12
18
24
30
36
42
48
54
60
66
0
6
12
18
24
30
36
42
48
54
60
66
Months from immunophenotypical analysis (3 months post-ASCT)
-- <0.01% MM-PC (n=66)
-- 75 % N-PC/total PC (n=80)
-- 0.01% MM-PC (n=33)
-- < 75 % N-PC/total PC (n=19)

CONCLUSIONS
Inmunophenotypic studies have clinical value:
- Prognostic influence of antigen expression
- Correlation genetic changes/protein profile
- Investigation of MRD (immunophenotypic remission)

Grupo Español de Mieloma (GEM/Pethema)
Hospitales
Hospitales
Clínico de Barcelona
General de Segovia
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 (Valencia)
H. 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 (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
Universitario de Santiago
S. Pau i Sta Tecla (Tarragona)
General Universitario de Valencia
General de Guadalajara
Universitario de Getafe (Madrid)
Sagunto (Valencia)
Insular de las Palmas
Son Dureta (Mallorca)
H. de La Princesa (Madrid)
Cuenca
Severo Ochoa (Madrid)
Alicante SUS
Juan XIII (Tarragona)
M. Valdecilla (Santander)
Toledo
Albacete
Gandía (Valencia)
H. Del Bierzo
Vall D´Hebrón (Barcelona)
Fundación Jiménez Díaz (Madrid)
San Jorge (Huesca)
Elda (Alicante)
Verge de la Cinta (Tortosa)
V. Del Rosel (Cartagena)
Alarcos (Ciudad Real)
Castellón
Salamanca´Group:
Mataró (Madrid)
Mutua Tarrasa
Juán Canalejo (Coruña)
Consorcio Tarrasa
G.Mateo, N.Gutierrez, R.Lopez, M.Mateos,
Ferrol
C. Corachán (Barcelona)
R.Garcia-Sanz, A.Orfao