Alberto Orfao
Juan Flores-Montero
Julia Almeida
Quentin Lecrevisse
Jacques van Dongen
Vincent van der Velden
Jeroen Te Marvelde
Alita van der Sluijs
Michael Kneba
Monika Brüggemann
Sebastian Böttcher
Tomek Szczepanski
Lukasz Sedek
Stephen Richards
Paul Evans
Matt Cullens
Ruth de Tute
Paulo Lucio
Andreia Mendonça
Ondrej Hrusak
Tomas Kalina
Ester Mejstrikova
Ludovic Lhermitte
Elizabeth MacIntyre
Vahid Asnafi
Anne-Sophie Bedin
Euroflow
EuroFlow 8-color immunostaining protocols for
diagnosis and classification
of haematological malignancies
demonstrated on pediatric perspective
Euroflow
•
•
Technical aspects of flow cytometry = standardization of measurement and
technical set up across labs and instruments
Design of 8-color flow cytometry panels for diagnosis and management of
haematological malignancies
–  selection of markers and the most optimal fluorochromes
–  design and testing of software with novel features enabling analyzis of
polychromatic data
•
Design of database of uniformly measured samples together with
molecular information
Novel concepts of data interpretation present in the Infinicyt
software
•
•
•
•
Generation of „virtual“ (concatenated) merged files for direct and simultaneous
analysis of all samples of interest
Calculation of parameters present in different tubes using „backbone“ markers
Automated interpretation and sample comparison based on bioinformatic tools
implemented in the software (APS, principal component analysis)
Reference images based of normal samples enabling direct comparison with the
sample of interest
Panels overview
ALOT
(acute leukemia orientation tube)
1 tube
BCP-ALL
T-ALL
AML/MDS
4 tubes
4 tubes
4 to 7 tubes
ALOT
Pac Blue
Pac Orange
FITC
PE
PerCP Cy5.5
PE Cy7
APC
APC H7
cyCD3
CD45
cyMPO
cyCD79a
CD34
CD19
CD7
smCD3
§  Designed for lineage orientation and to choose optimal
secondary panel
slide designed by L. Lhermitte, ALL panel coordinator
ALOT (acute leukemia orientation tube)
patient 2 years old MDS RAEB, complex karyotype, FAB M7
mature B cells
B cell
precursors
T cells
granulocytes
blasts
Principal component analysis
•  Mathematical procedure that transforms a number of possibly correlated
variables into a smaller number of uncorrelated variables called principal
components
•  The first principal component accounts for as much of the variability in the
data as possible
•  Populations defined by PCA not dependent on experience of the cytometrist
APS view of single ALOT tube
2D
3D
Comparison of individual disease categories
Extraction of blasts from individual fcs files
BCP-ALL
T-ALL
AML
Single « virtual » merged tube/
data file
slide designed by L. Lhermitte, ALL panel coordinator
ALOT (Acute Leukemia Orientation Tube)
BCP-ALL cases (n=89)
T-ALL cases (n=27)
AML cases (n=36)
slide designed by L. Lhermitte, ALL panel coordinator
BCP-ALL
ALOT
BCP-ALL panel
Pac Blue Pac Orange
FITC
PE
PerCP Cy5.5 PE Cy7
APC
APC H7
cyCD3
CD45
cyMPO
cyCD79a
CD34
CD19
CD7
smCD3
CD20
CD45
CD58
CD66c
CD34
CD19
CD10
CD38
smIgK
CD45
cyIgM
CD33
CD34
CD19
smIgM
+CD117
smIgL
CD9
CD45
TdT
CD13
CD34
CD19
CD22
CD24
CD21
CD45
CD15
+CDw65
NG2
CD34
CD19
CD123
CD81
T-ALL panel
T ALLpanel
Pac Blue Pac Orange
FITC
PE
PerCP Cy5.5 PE Cy7
APC
APC H7
cyCD3
CD45
TdT
CD99
CD5
CD10
CD1a
smCD3
cyCD3
CD45
CD2
CD117
CD4
CD8
CD7
smCD3
cyCD3
CD45
TCR gd
TCR a b
CD33
CD56
cyTCR b F1
smCD3
cyCD3
CD45
CD44
CD13
HLADR
CD45RA
CD123
smCD3
slide designed by L. Lhermitte, ALL panel coordinator
Calculated file
based on
backbone markers
BCP ALL 31
parameters
T ALL 29
parameters
Aims:
•  Diagnosis
•  Maturation stage
•  Biphenotypic
leukemia
identification
•  Correlation with
known molecular
aberrations
•  Prognosis
Differential diagnosis of BCP ALL (not only in
childhood)
•
•
•
•
reactive conditions (i.e. viral infection)
„smouldering“ leukemia presenting as aplastic anemia
AML
B NHL infiltrating bone marrow (predominating subtype in
childhood Burkitt lymphoma, less frequent lymphoblastic
lymphoma, other subtypes are rare)
•  non hematopoietic tumor infiltrating bone marrow (i.e.
neuroblastoma – in combination with increased percentage of
hematogones might mimick BCP ALL)
•  hemophagocytic lymphohistiocytosis
Differential diagnosis of T ALL (not only in childhood)
•  T NHL (% of blasts in bone marrow plays a role)
•  early T cell ALL close category to biphenotypic leukemia or AML
•  leukemised peripheral T cell lymphoma or anaplastic large cell
lymphoma
•  mediastinal biopsy – recognize thymic cells
Genotypic subgroups BCP ALL typical
characteristics
•  tel-aml1: CD66cneg, CD10pos, CD33 and/or CD13 might be positive
•  bcr-abl:CD66cpos, CD10pos, CD38 heterogenous, myeloid markers
might be positive
•  e2a-pbx: CD10pos,CD9pos,intra-IgMpos
•  hyperdiploidy: CD66cpos, other myeloid markers typically negative
•  mll gen rearrangement: CD10neg,CD20neg,NG2pos, CD15pos,
CD65pos
Kalina et al., BMC Cancer, 2005
Hrusak, Porwit, Leukemia 2002
Newly defined subgroups potentially relevant:
- BCR-ABL like (defined by gene expression profilling) : 50% have
rearranged CRLF2 (recently incorporated into pediatric flow
panels), activation of JAK-STAT pathway
- ERG deletions – unique gene expression profile (Yeoh et al.
2002), aberrant CD2, loss of CD19 during the early phase of the
treatment
- IKZF1 alterations, PAX-5 deletion difficult to predict by flow
TUBE 1
CD20:
CD58:
CD66c:
CD10:
CD38:
CD20
CD45
CD58
CD66c
CD34
CD19
CD10
CD38
differentiation marker, potential therapeutic target
identified in expression studies as MRD marker
frequently aberrantly expressed (hyperddiploid, BCR-ABLpos )
differentiation marker
differentiation marker
gate CD19
non malignant bone marrow
CD10 APC
CD10 APC
cALL
proB ALL
(mll-af4pos)
CD20 PB
CD20 PB
PCA1: CD10, CD34,
CD66c
Distinct profile of MLL rearranged BCP cases
PCA2: CD66c, CD9, CD10
Maturation tool (can be also defined directly
on APS view)
Any marker of interest
can be analyzed in the
context of normal cells
Comparison of BCP ALL and normal samples
tube 1: CD58-CD10-CD34-CD45-CD19-CD20-CD38
3 BCP diagnostic sample
3 T ALL during induction treatment
(typically only mature B cells are
present)
3 regenerating samples
Subgroups T ALL typical characteristics
•  compared to BCP ALL less typical immunophenotypic
profiles corresponding to genetic subgroups are known so
far
•  cortical T – good prognosis especially in adults (CD1apos )
•  early T cell precursor – unfavorable subtype in pediatric
and adult cohorts (CD1aneg, CD8neg, CD5weak, stem cell or
myeloid marker OR gene expression profile associated
with ETP, no Auer rods (Coustan-Smith et al. Lancet Oncology 2009)
T-ALL panel
Pac Blue Pac Orange
FITC
PE
PerCP Cy5.5
PE Cy7
APC
APC H7
cyCD3
CD45
TdT
CD99
CD5
CD10
CD1a
smCD3
cyCD3
CD45
CD2
CD117
CD4
CD8
CD7
smCD3
cyCD3
CD45
TCR gd
TCR a b
CD33
CD56
cyTCR b F1
smCD3
cyCD3
CD45
CD44
CD13
HLADR
CD45RA
CD123
smCD3
T ALL classification panel – example ETP ALL
CyCD3
CyCD3
CyCD3
CyCD3
CD45
CD45
CD45
CD45
NuTdT
CD2
TCR γδ
CD44
CD99
CD117
TCRαβ
CD13
„classic“ T
ALLs
CD5
CD4
CD33
DR
CD10
CD8
CD56
CD45RA
CD1a
CD7
CyTCRb
CD123
SmCD3
SmCD3
SmCD3
SmCD3
cyMPO
CD5
All cases fulfilling CD5 only partly positive
(1 of the criterion of Coustan-Smith et al.)
All cases intra-CD3pos
Variable expression of myeloid markers
¾ of cases TCR genes in germ line
configuration
¾ biphenotypic according EGIL classification
cyCD3
T-ALL panel
identification of early T cell ALL (ETP) and biphenotypic cases
LS CD45 CD19 CD34 cyCD3 cyMPO cyCD79a CD7 smCD3 TdT CD99 CD5 C10 CD1a CD2 CD117 CD4 CD8 TCRγδ TCRαβ CD33 CD56 cyTCR ΒF1 CD44 CD13 HADR CD45RA CD123
„classic“ T ALLs
All cases fulfilling CD5 only partly positive
(1 of the criterion of Coustan-Smith et al.)
All cases intra-CD3pos
Variable expression of myeloid markers
¾ of cases TCR genes in germ line
configuration
¾ biphenotypic according EGIL classification
slide designed by A.Orfao
slide designed by A.Orfao
B NHL rationale
•  responsible scientist Sebastian Bottcher
Classifying mature B-NHL by
flow
•  State of the art
•  Recent developments
•  Standardized 8-color panel for B-NHL
–  Univariate analyses
–  Multivariate analyses
–  Retest Reliability
–  Example cases
CD5 -
CD5 +
CD10 -
MZL
HCL
DLBCL
FL
MCL
CLL
MCL
PLL
LPL
MZL
DLBCL
CD10 +
FL
DLBCL
BL
HCL
FL
DLBCL
MCL
BL
Blood 2008;111:3941-67
CD5 -
CD5 +
CD10 -
MZL
HCL
DLBCL
FL
MCL
CLL
MCL
PLL
LPL
MZL
DLBCL
CD10 +
FL
DLBCL
BL
HCL
FL
DLBCL
MCL
BL
Blood 2008;111:3941-67
Blood 2008;111:3941-67
Current status:
immunophenotypic diagnosis
Points
0
1
SmIg
strong
dim
CD5
negative
positive
CD23
negative
positive
FMC7
positive
negative
CD22
strong
dim
CD79b
strong
dim
≥ 4 points: CLL; < 4 points: other B-NHL
Matutes et al., Leukemia, 1994
Current status:
immunophenotypic diagnosis
Points
0
1
SmIg
strong
dim
CD5
negative
positive
CD23
negative
positive
FMC7
positive
negative
CD22
strong
dim
CD79b
strong
dim
≥ 4 points: CLL; < 4 points: other B-NHL
Matutes et al., Leukemia, 1994
EuroFlow B-NHL panel
Pac
Blue
Pac
FITC
Orange
PE
PerCPCy5.5
PECy7
APC
APCH7
1=
LST
CD20 CD45
/ CD4
λ/CD8
κ/CD56
CD5
CD19 /
TCRγ/δ
CD3
CD38
2
CD20 CD45
CD23
CD10
CD79b
CD19
CD200
CD43
3
CD20 CD45
CD31
LAIR
CD11c
CD19
IgM
CD81
4
CD20 CD45
CD103
CD95
CD22
CD19
CXCR5
CD49d
5
CD20 CD45
CD62L
CD39
HLA-DR
CD19
CD27
van Dongen et al., Leukemia, 2012
Conclusions
•  Fully technically standardized panel, allowing
for direct inter-lab directly comparisons
•  Database with built up together with robust
diagnostic information.
•  Innovative flow cytometry data interpretation
strategy (1st commercial software which
implemented real bioinformatic tool):
- reduced subjectivity
- automated pattern-guided data analysis
www.euroflow.org
EuroFlow participants
University Institutes / Medical Schools
Erasmus MC, Rotterdam, NL J.J.M. van Dongen, V.H.J. van der Velden
USAL, Salamanca, ES
A. Orfao, J. Flores, J. Almeida, Q. Lecrevisse a.o.
IMM, Lisbon, PT
P. Lucio, A. Mendonça, A. Parreira a.o.
UNIKIEL, Kiel, DE
M. Kneba, S. Böttcher, M. Ritgen, M. Brüggemann a.o.
AP-HP, Paris, FR
E. Macintyre, L. Lhermitte, V. Asnafi a.o.
UNIVLEEDS, Leeds, GB
S. Richards, A.C. Rawstron. P. Evans a.o.
DPH/O, Prague, CZ
O. Hrusak, T. Kalina, E. Mejstrikova a.o.
SAM, Zabrze, PL
T. Szczepanski, L. Sedek a.o.
DCOG, The Hague, NL
E. Sonneveld, A. van der Sluijs-Gelling a.o.
KUL, Leuven, BE
N. Boeckx a.o.
HGSA, Porto, PT
M. Lima, AH Santos
UFRJ, Rio de Janeiro, BR
C. Pedreira, E.S. Costa
Companies (SME’s)
DYNOMICS, Rotterdam, NL
E. Dekking, F. Weerkamp a.o.
CYTOGNOS, Salamanca, ES M. Martin, J. Bensadon, J. Hernandez, M. Muñoz a.o.