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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. - PowerPoint PPT Presentation
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DIAGNOSTIC AND PROGNOSTIC TOOLS IN
HAEMATOLOGICALMALIGNANCIES 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....
Before....
Now.....
Now.....
Harrogate 7th HLDA : 250 CDAdelaide 2004 8th HLDA : 339 CDBarcelona 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
CD19
ITAMITAM
CD22
or
ITIMITIMITIMITIM
ITIMITIMITIMITIM
ITAMITAM
CD20
P
CD24
CD21
NH2
COOH
CD23
BCRCD79
ITAM
ITAM
ITAM
ITAM
H0L0
Stem Cell
CD34 DR
H0L0
Pro-B Cell
cCD79 cCD22CD19
HxL0
Early-B Cell
cCD79CD22CD19 CD21 CD10
HxL0
cCD79 CD22CD19CD20CD21CD10
HRL0
Pre-B Cell
cCD79CD22CD19CD20CD21cµ
HRLR
Naive B cell
sCD79CD22CD19CD20CD21sµ/s
BONE MARROW
ACTIVATION &CLONAL PROLIFERATION
TISSUES
Plasma cells
Memory B cells
Immunocyte
Immunoblast
Classification of B- ALL (EGIL)
cCD79/
CD19/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
CD1
CD2
TCR
CD3
ITAMITAM
ITAMITAM
ITAMITAM
ITAMITAM
ITAMITAM
ITAMITAM
ITAMITAMITAMITAMITAMITAM ITAMITAM
CD4
CD5
CD8
CD7
BONE MARROW
Stem cell
CD34 DR
Pro-T cell
cCD3CD7CD2CD5
THYMUS
cCD3CD7CD2 CD5CD1
Cortico-thymocyte
XX
cCD3 CD7CD2CD5CD4CD8
XXXX
Medullary Thymocyte
sCD3CD7CD2CD5CD4
sCD3CD7CD2CD5CD8
XXRR
Naïve T cell
ACTIVATION& CLONAL PROLIFERATION
TISSUES
Immunocyte
Immunoblast
MemoryT cells
Effector T cells
Classification of T-ALL (EGIL)
cCD3
CD7
CD2CD5CD8
CD1a+
sCD3+/CD1a-
-
+ -
T-I (pro-T) + + - - -
T-II (pre-T) + + + -
T-III (cortical T)
+ + +
T-IV (mature T)
+ + + - +
Lymphoproliferative disorders
MANTLE ZONE
FOLLICULAR
CENTROBLASTIC orIMMUNOBLASTIC
Large cell Lymphomas
BURKITT
T LYMPHOMASSEZARY)LARGE CELLS ANAPLASTIC PERIPHERALANGIOCENTRICINTESTINAL
MARGINAL ZONE NODAL MALTHCL
INTESTINAL OR LUNG MALTFollicular, mantle zone, marginal zone
SLVLCirculating marginal zone cells
WALDENSTROM
MYELOMA
POSITIVE NEGATIVE OR WEAK
CD5 1 0
CD23 1 0
sIg 0 1
FMC7 0 1
CD22/CD79b 0 1
Matutes’ scoring of CLL
Comparative immunophenotypes
SMZL/SLVL
HCL HCLv CLL MCL FL
CD19
CD22
CD5
CD23
sIg IgM IgM, IgG..
IgG
CD43
FMC7
CD10
CD103
CD11c
CD25
CD79b
CD20
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
CD13
EE
NH2
COOH
EE
CD14
LL
L
L
L
LL
L
L
L
P
CD33
CD11b
Mg
Mg
Mg
MPO
KK
CD117
CD15CD65
3-fucosyl-N-acetyl-lactosamine
céramide dodecasaccharide
CD36
CD35
Immature precursors
Differentiated cells
CD34DR
CD117CD13CD33MPOCD7
DRDR
CD4CD4
CD19CD19
CD11BCD11B
CD14CD14
CD36CD36
CD15CD15
CD65CD65
CD16CD16
CD32CD32
CD64CD64
DRDR
CD4CD4
CD19CD19
CD11BCD11B
CD14CD14
CD36CD36
CD15CD15
CD65CD65
CD16CD16
CD32CD32
CD64CD64
Stem cell
BONEMARRROW
PERIPHERALBLOODTISSUES
CFU-GM Myeloblast Promyelocyte MyelocyteMetamyelocyte
Band Granulocyte
HLA-DR
CD34
CD117
CD33
CD13
CD11c
CD15
CD66
CD15
CD66
CD11b
CD11b
CD11c
CD33
CD13
CD16
CD16
Evolution immunohénotypique au cours de la maturation granuleuse (B Husson)
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
B-
LINEAGE
T-LINEAGE
MYELOIDLINEAGE
2 points CD79cµ
cCD22
CD3TCR
MPO(lysozyme)
1 point CD19CD10CD20
CD2CD5CD8
CD10
CD13CD33
CDw65CD117
0.5 point TdTCD24
TdTCD7
CD1a
CD14CD15CD64
WHO’s new classification
Extensive immunophenotype
AULAcute
undifferentiated leukemia
MPALMixed
Phenotype Acute leukemia
OthersIncl NK
With t(9;22) 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 2008M 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– CD34, CD117– Abnormal CD2, CD5, CD7, CD56– Increased CD117
• Granulocytes– 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 purposesSublineage 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 PanelOther 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 CD19CD5, 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
Campana, 1999San Miguel, 1997
Kern, 2004
MRD in ALL
MRD B-II ALL
CD19
CD
34
1.5 x 10-2
MRD in AML
2.1 10-3
CD34
CD
11
7
MRD - AML
AML peripheral blast cells decrease
SS
C
CD45b
SS
C
CD14c
BS
SC
CD16d
CS
SC
FSCa
A
CD45
CD
11b
e
D
CD11b
CD
16
g CD11b
CD
16
5.3%
100
h
Blasts
CD45
CD
16
f
E
D0
D1
CD11b
CD
16
a
CD11b
CD
16
3.9%
73.7
b
Blasts
D2
CD11b
CD
16
c
CD11b
CD
16
1.1%
21.2
d
Blasts
D3
CD11b
CD
160.5%
9.4
f
Blasts
CD11b
CD
16
e CD11b
CD
16
g
D4
CD11b
CD
16
0.2%
3.6
h
Blasts
Blast slope
0 200 400 600 800 1000 1200 1400
100
90
80
70
60
50
40
30
20
10
0
Time (Days)
DF
S
Slope < -25
Slope -25 < > -15
Slope > -15
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
