DIAGNOSTIC AND
PROGNOSTIC TOOLS IN
HAEMATOLOGICAL
MALIGNANCIES IN 2010
(PART II)
Flow cytometry
Immunophenotyping
• Identification of molecular protein
characteristics of abnormal cells
• Labelling with fluorescent
monospecific monoclonal antibodies
• Multiparametric flow cytometry
The objectives of
immunophenotyping in
hematological disorders
• Lineage assignment
– Lymphoid acute/chronic
– Myeloid acute/chronic
– Erythroid
– Megakaryocytic
• Classification /Scoring
• Detection of MPAL
• Maturation anomalies
• Identification of Minimal Residual
Disease patterns
Before....
Now.....
Harrogate 7th HLDA : 250 CD
Adelaide 2004 8th HLDA : 339 CD
Barcelona 2010 : >360 CD
Immunophenotype of
lymphoid cells
• Well established maturation sequence
• Staged and controlled to prepare long lived
antigen-specific cells
• Identified by studying leukemias
• Validated on normal cells
• Numerous similarities between B and T cells:
– Lineage committment
– Gene rearrangements for antigen recetpors
– Selection
B lineage associated
markers
CD22
or
CD19
BCR
CD79
CD21
ITAM
ITAM
ITAM
ITAM
ITIM
ITAM
ITIM
ITAM
ITIM
ITIM
CD23
COOH
CD20
CD24
P
NH2
BONE MARROW
Stem Cell
Pro-B Cell
Early-B Cell
Pre-B Cell
Naive B cell
H0L0
H0L0
TISSUES
ACTIVATION &
CLONAL
PROLIFERATION
Immunocyte
Immunoblast
HxL0 HxL0 HRL0 HRLR
CD34 cCD79 cCD79 cCD79 cCD79 sCD79
DR
cCD22 CD22 CD22 CD22 CD22
CD19 CD19 CD19 CD19 CD19
CD21 CD20 CD20 CD20
CD10 CD21 CD21 CD21
CD10 cµ
sµ/sd
Memory
B cells
Plasma cells
Classification of B- ALL (EGIL)
cCD79/CD
19/c or
sCD22
CD10
c-µ
sIg
B-I (pro-B)
+
-
-
-
B-II
(common)
B-III (pre-B)
+
+
-
-
+
+
+
-
B-IV (mature)
+
+
+
+
T lineage associated
markers
TCR
CD3g d e
ITAMITAMITAM
CD5
ezz/zh
CD2
ITAMITAMITAM
ITAMITAM
ITAMITAM
CD4
CD1
CD8
CD7
BONE
MARROW
Corticothymocyte
Stem cell
Pro-T cell
TISSUES
THYMUS
Medullary
Thymocyte
sCD3
CD7
CD2
CD5
CD8
Naïve T cell
ACTIVATION
& CLONAL
PROLIFERATION
Immunocyte
Immunoblast
g0d0
a0b0
CD34
DR
g0d0
a0b0
cCD3
CD7
CD2
CD5
gXdX
a0b0
cCD3
CD7
CD2
CD5
CD1
gXdX
aXbX
cCD3
CD7
CD2
CD5
CD4
CD8
gXdX
aRbR
sCD3
CD7
CD2
CD5
CD4
Memory
T cells
Effector T
cells
Classification of T-ALL (EGIL)
cCD3
CD7
CD2
CD5
CD8
CD1a+
sCD3+/
CD1a-
T-I (pro-T)
+
+
-
-
-
T-II (pre-T)
+
+
+
-
-
T-III (cortical
T)
T-IV (mature
T)
+
+
+
+
-
+
+
+
-
+
Lymphoproliferative disorders
MARGINAL
ZONE
NODAL MALT
T LYMPHOMAS
SEZARY)
LARGE CELLS ANAPLASTIC
PERIPHERAL
ANGIOCENTRIC
INTESTINAL
HCL
MANTLE ZONE
BURKITT
INTESTINAL OR
LUNG MALT
Follicular,
mantle zone,
marginal zone
SLVL
FOLLICULAR
WALDENSTROM
CENTROBLASTIC or
IMMUNOBLASTIC
Large cell Lymphomas
Circulating marginal zone cells
MYELOMA
Matutes’ scoring of CLL
POSITIVE
NEGATIVE
OR WEAK
CD5
1
0
CD23
1
0
sIg
0
1
FMC7
0
1
CD22/CD79b
0
1
Comparative
immunophenotypes
SMZL/SLVL
HCL
HCLv
CD19
CD22
CD5
CD23
sIg
CD43
FMC7
CD10
CD103
CD11c
CD25
CD79b
CD20
IgM
IgM, IgG..
IgG
CLL
MCL
FL
Immunophenotype of
myeloid cells
• Known maturation sequence
• Massive production of cells with no
specificity
• Early and late differentiation markers
• Important lineage promiscuity
Myeloid lineage markers
CD117
MPO
CD13
CD33
COOH
E E
CD35
NH2
CD14
P
L
L
L
L
L
L
L
L
L
L
K
CD11b
Mg
Mg
Mg
CD15
CD65
CD36
3-fucosyl-N-acetyl-lactosam
céramide dodecasaccharid
DR
BONE
MARRROW
PERIPHERAL
BLOOD
TISSUES
CD4
CD19
CD11B
CD14
CD36
CD34
DR
Stem cell
CD117
CD13
CD33
MPO
CD7
Immature precursors
CD15
CD65
CD16
CD32
CD64
Differentiated cells
Evolution immunohénotypique
au cours de la maturation granuleuse (B Husson)
Myeloblast
CFU-GM
Promyelocyte
Myelocyte
Metamyelocyte
Band
Granulocyte
CD15
CD66
HLA-DR
CD11b
CD16
CD117
CD34
CD33
CD13
CD13
CD11c
CD11c
CD33
CD15
CD11b
CD66
CD16
Detection of BAL :
EGIL’scoring system
• NOT to be used for lineage assignment
• Scoring based on the lineage specificity of
critical differentiation antigens
• Calculation of each lineage “score”
• In BAL, at least two lineages have scores
HIGHER than 2
• In “variant” AL coexpression with a score <2
is not rare
Detection of BAL :
EGIL ’scoring system
BLINEAGE
T-LINEAGE
MYELOID
LINEAGE
2 points
CD79
cµ
cCD22
CD3
TCR
MPO
(lysozyme)
1 point
CD19
CD10
CD20
CD2
CD5
CD8
CD10
CD13
CD33
CDw65
CD117
0.5 point
TdT
CD24
TdT
CD7
CD1a
CD14
CD15
CD64
WHO’s new classification
Extensive
immunophenotype
AUL
Acute
undifferentiated
leukemia
With t(9;22)
Others
MPAL
Incl NK
Mixed Phenotype
Acute leukemia
With
t(n;11q23)
NOS
WHO’s new criteria
of MPAL
• Myeloid lineage
– MPO
– Or strong monocytic engagement
• NSE
• CD14, CD11c, CD36, CD64, Lysozyme
• B-lineage
– Bright CD19 + another B marker
– Low CD19 + two other B markers
• T-lineage : cCD3 (strong, PE or APC)
One word on MDS
Proposition de score
Wells, 2003
0
1
2
3
Working conference 2008
M Loken, A van de Loosdrecht, K Ogata,
A Orfao, D Wells
• Classical blasts  enumeration
– DR+/11b– CD34
– CD117
• Anomalies of the CD13/CD16
pathway
• Abnormal expression anormale of
DR on monocytes
Stachurski, 2008
•
Blasts
–
–
–
•
Granulocytes
–
–
•
CD34, CD117
Abnormal CD2, CD5, CD7, CD56
Increased CD117
Deganulation
Abnormal expression of CD33, CD13, CD11b, CD16, CD15, CD64, CD10, CD14,
DR
Monocytes
–
Anomalies of CD33, CD13, CD11b, CD15, CD64, CD14, DR
In practice, for
hematological malignancies
• Depending on
– Previous clinical and morphological
information
– Sample volume
– Monoclonal antibodies availability
Consensual European Panel,
European LeukemiaNet (2005)
For quick orientation or paucicellular samples
• cCD3, MPO, cCD79a, TdT
• CD7, CD2, CD10, CD19, CD22 (s or c), sIg,
CD13, CD33, CD34
• CD45 for gating purposes
Sublineage classification and definition of clinical
entities (also with adapted gating strategy)
• DR, CD1a, CD4, CD5, CD8, CD3 (m), IgM (c),
CD14, CD117, CD56, CD65, CD41 or CD61,
RBC marker such as glycophorin A
Complementary panel of useful referenced
markers
Acute leukemia Diagnosis Panel
Other useful markers (>20)
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
MPO/LF (lactoferrin) (c): (i) Identification of late neutrophil granulocyte
compartment (Lactoferrin positive) (25,52,54); (ii) for refined
detection/quantification of MPO+ early myeloid cells in combination with CD14
(25,49)
LZ (lysozyme) (c): (i) for myeloblastic leukaemia; (ii) to discriminate pDCs and
myeloid cells (53); (iii) to positively identify early monocytic cells (48)
K/L : (i) on surface for clonality, (ii) in the cytoplasm for rare B-IV cases
CD11b, CD11c : negative in APL (14)
CD15 : for myeloblastic leukemia (42)
CD16 : to discriminate mature PMNs (9)
CD35/36 : for GEIL´s AML classification (11), for RBC after excluding monocytes
(10)
CD58 : to distinguish between normal regenerating B cells and B-cell blasts (59)
CD64 : for AML (9)
CD68 (c): (i) for AML (bright) and subset of B-ALL (weak) (53); (ii) for positive
identification of normal pDCs (bright) (55)
CD71 : for cell proliferation/activation and/or RBC (22,41)
CD86 : prognostic factor in AML (34)
CD99 : to differentiate between blasts and non blastic T-cells (19)
CD123 : IL-3 R, for pDC and AML, some NK (24)
TCR chains for T-ALL, c and/or s (50)
Therapeutic targets: CD20 (40), CD52 (40), CD45 (39), CD33 (31), CD123 (3), CD87
(46), CD44 (20), uPAR(CD87)/uPACD116 (1)
Chronic lymphoproliferative diseases.
Mandatory panel (20 Abs)
• Samples : peripheral blood, bone marrow, LN
suspensions… (Fine needle aspiration for primary
screeening to avoid unnecessary biopsies)
• Gating markers
CD19, CD3, CD56*
• B oriented panel gated on CD19
CD5, CD20, CD23, CD103, CD10, K, L, Ig, CD25, CD79b, CD38
• T oriented panel gated on CD3 or other T-lineage marker
CD2, CD3, CD4, CD5, CD8, CD7,
Lymphoproliferative diseases:
additional useful markers (< 15)
• B lineage
– CD2, CD7, CD123, FMC7, CD138, DR, CD24*, CD43
– (G, A, M, D), CD81*
– Cytoplasmic : Bcl2, Zap70 (relative to internal control)
• T lineage
– TCRs*, CD30, CD10
• NK panel excluding CD19 and CD3 cells/ CD56
– CD57, CD16, CD94, perforin, granzyme B
*CD81/CD22 useful for CLLfollow-up based on dim co-expression
level
*V-beta panel and imunophenotype
*Absence of CD24 on marginal zone and HCL
• Note : CD52 if alemtuzumab considered
Chemosensitivity
• Minimal residual disease
• Quick assessment of response to
therapy
Why look for minimal residual
disease?
The founders
San Miguel, 1997
Campana, 1999
Kern, 2004
MRD in ALL
MRD B-II ALL
CD34
1.5 x 10-2
CD19
MRD in AML
CD117
MRD - AML
CD34
2.1 10-3
AML peripheral blast cells
decrease
D0
SSC
SSC
A
a
CD45
CD16
CD16
e
D
CD45
5.3%
Blasts
100
g
CD11b
h
CD11b
CD16
CD11b
SSC
d
CD16
SSC
CD14
b
C
B
c
FSC
f
E
CD45
3.9%
Blasts
73.7
b
CD11b
e
D2
CD16
CD16
D1
CD11b
1.1%
Blasts
CD11b
CD16
g
0.5%
0.2%
Blasts
9.4
f
CD11b
D4
D3
21.2
d
CD11b
CD16
c
CD16
CD11b
CD16
CD16
CD16
a
CD11b
Blasts
3.6
h
CD11b
Blast slope
100
90
80
70
DFS
60
50
Slope < -25
40
Slope
30
-25 < > -15
Slope > -15
20
10
0
0
200
400
600
800
Time (Days)
1000
1200
1400
Conclusions
•
•
•
•
•
Consensual approach
Rapid and informative diagnostic tool
Precise definition of the disease at diagnosis
Therapeutic indications
Aberrant immunophenotypes useful for
follow up
• Search for new markers:
– New monoclonals
– Microarrays