presentación de powerpointmonocytic lineage infidelity in cmml vs amml matarraz s, cytometry b...
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23/10/2017
1
Immunophenotype of dysplastic and
leukemic monocytic cells
Dr. Sergio Matarraz
Cytometry Service (NUCLEUS).
Department of Medicine
Centro de Investigación del Cáncer
IBSAL-Universidad de Salamanca/CSIC
Identification of early-monocytic commitment is
relatively often tough in AML
Promonocytes
NSE- monocytic leukemias
Poor prognosis of Monocytic aberrancies in low-risk MDS
Metze K, Cell Immunol 2016
Tra
nsfo
rm
ed
SS
C
CD34
Cell lineage Phenotype
Frequency
Normal BM
(%)
Neutrophil CyMPO+, CD13hi 31 (12-39)
B-Lymphoid nTdT+, cyCD79a+, CD19+ 23 (<1-45)
Erythroid CD36+, CD64-, CD45dim, CD105+ 15 (5-35)
pDC CD123hi, HLA-DRhi, CD36+ 6 (1-15)
Monocytic CyMPO-, CD64+, HLA-DR+,
CD117dim
5 (3-15)
Basophil CD123hi, HLA-DRdim, CD117dim,
CD45hi, CD203c+
<1 (<1-3)
Megakaryocytic CD61+, CD45dim <1
Eosinophil CyMPO-, CD15/65+, CyPEo+ <1
Mast cell CD117hi, HLA-DRdim, CD45hi <1
Monocytic differentiation in normal BM
Monocytic lineage
Erythroid lin.
CD36
CD
64
Gated CD34+ BM cells
CD64: high-affinity IgG receptor FcgRI
(van der Poel et al, J. Immunol 2011)Matarraz et al, Leukemia 2008
Matarraz S et al. Cytometry B 2015
Monocytic precursors(e.g. monoblasts)
MatureMonocytes
Promonocytes
HLA-DR (PacB)
CD
117
(P
EC
y7
)
CD
64 (P
E)
MatureMonocytes
CD14 (APCH7)
CD
35 (F
IT
C)
CD300e (IREM2) (APC)
MatureMonocytes
CD
14 (A
PC
H7
)
CD
36 (F
IT
C)
CD14 (APCH7)
MatureMonocytes
CD34+
Monocytic differentiation in normal BM
CD34+ CD34+
Promonocytes
CD14 (APCH7)
CD
14
(A
PC
-C
y7)
CD312 (APC)
CD300e+
CD34+
CD
86
(P
E-C
y7)
CD300e+
CD34+
CD
62L
(B
V 6
50
)
CD34+
CD
38
(B
V-6
05
)
CD300e+
Manuscript in preparation
CD14 (APC-Cy7)
Other markers of Monocytic maturation
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Monocyte subsets in BM and PB
CD300e (APCCy7)
CD
14
(P
erC
PC
y5
.5
)
CD14 (PerCPCy5.5)
CD
16
(B
V7
85
)
Passlick B, Blood 1989Ziegler-Heitbrock L, Blood 2010Hofer TP, Blood 2015
iMo ncMo SLAN- ncMo SLAN+
SLAN is a modification of PSGL-1: switch in trafficking potential(skin, lymphoid, non-lymphoid tissues…?),
Classical Monocytic cellsiMo
ncMo SLAN-
ncMo SLAN+
iMo and ncMo downregulate fuctional monocytic markers
CD
62
L(B
V6
50
)
CD
86
(P
E-C
y7
)
CD
38
(B
V6
05
)
CD312 (APC)
CD
14
(A
PC
-C
y7
)
CD
36
(F
IT
C)
CD300e (APCCy7)
CD14 (APC-Cy7)
CD300e+
BM monocytic
and CD34+ HPC
Dysplastic and Leukemic
Monocytic Differentiation
Monocytic alterations in MDSAbnormal cell distribution Frequency
Maturation blockades 56%
Abnormal antigen expression patterns
Abnormal granularity (SSC) 30%
Abnormal CD45 23%
Abnormal distribution of immature/mature cells 47%
Abnormal CD33 3%
Abnormal HLA-DR 10%
Abnormal CD11b/HLA-DR pattern 10%-29%
Asynchronous antigen expression
Expression of CD34 12%
Abnormal CD14 20%
Abnormal CD13 39%
Abnormal CD36 31%
Abnormal CD64 23%
Abnormal CD15 33%
Expression of lineage infidelity markers
Lineage infidelity CD2 9%
Lineage infidelity CD5 2%
Lineage infidelity CD7 3%
Lineage infidelity CD19 2%
Overexpression of CD56 15%
Stetler-Stevenson, Blood 2001
Ogata, Blood 2002
Wells, Blood 2003
Malcovati, Leukemia 2005
Benesch, Hematology 2007
Matarraz, Leukemia 2008
Stachursky, Leuk Res 2008
Van de Loosdrecht, Blood 2008
Subirá, Transl Res 2008
Kern, Cancer 2010
Matarraz, Cytometry 2010
Kern, Leuk Lymph 2011
Westers, Leukemia 2012
Matarraz, Cytometry B 2015
Harrington, Am J Clin Pathol 2016
Normal BM
CD
13 (P
E)
Maturation blockades and asynchronisms
MDS-MLD
CD11b (APC)
MDS-EB2
CD
35 (FIT
C)
MatureMonocytes
Normal BM
MDS Maturation blockades and abnormal expression
CD14 (APCH7)
CD
35
(F
IT
C)
MDS-MLD MDS-EB2
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CD
35 (FIT
C)
MatureMonocytes
Normal BM
CD14 (APCH7)
CD
35
(F
IT
C)
Monocytic AML Monoblastic AML
AMML blockade, asynchrony and abnormal expression Aberrant CD300e vs. CD14 pattern
Normal BM
CD300e (APC)
CD
14 (A
PC
H7)
5%
MatureMonocytes
MatureMonocytes
35-55%
MDS
AML
(pattern inversion)
5%+
CD
64
(P
E)
CD
14
(A
PC
H7)
60%
CD300e: identification of mature monocytes
15%+
40%+
45%-
55%+
MDS 2
CD300e (APC)
CD14 (APCH7)
18%
30%
42%
48%+
MDS 1 MDS 3
Dysplastic and Leukemic
Monocytic Differentiation
Less frequent Markers
CD
62
L(B
V 6
50)
CD
38
(B
V-6
05
)
CD300e+ CD300e+
CD34+
CD34+
CD
36
(F
IT
C)
CD
86
(P
E-C
y7
)
CD14 (APC-Cy7)
Monocytic differentiation in CMML
CD300e (APCCy7)
Normal level Normal level
blockade
CD
14
(A
PC
-C
y7
)
CD312 (APC)
CD300e (APCCy7)
CMML AML
CD300e and CD312 in CMML
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CD300e (APCCy7) CD14 (APC-Cy7)
CD
62
L(B
V 6
50)
CD300e+
CD34+
CD34+C
D3
8 (B
V-6
05
)
CD
86
(P
E-C
y7
)
CD
36
(F
IT
C)
Monocytic differentiation in Monocytic leukemia
CD
14
(A
PC
-C
y7
)
CD312 (APC)
CD300e (APC)
Monocytic differentiation in Monocytic leukemia
CD300e (APCCy7)
CD
62
L(B
V 6
50)
CD300e+
CD34+
CD34+
CD
38
(B
V-6
05
)
Monocytic differentiation in Monoblastic Leukemia
CD
86
(P
E-C
y7
)
CD14 (APC-Cy7)
CD
36
(F
IT
C)
CD64++
CD
14
(A
PC
-C
y7
)
CD312 (APC)
CD300e (APC)
Monocytic differentiation in Monoblastic Leukemia
CD14 (APC-Cy7) C
D6
4 (F
IT
C-C
y7
)
CD
62
L(B
V 6
50)
CD
38
(B
V-6
05
)
CD300e+
CD34+
CD34+
CD14 (APC-Cy7)
CD
86
(P
E-C
y7
)
CD
36
(F
IT
C)
Monocytic differentiation in Monocytic/DC leukemia
CD300e (APCCy7)
Next-generation flow cytometry for
evaluation of monocytic maturation
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Monocytic alterations in MDSAbnormal cell distribution Frequency
Maturation blockades 56%
Abnormal antigen expression patterns
Abnormal granularity (SSC) 30%
Abnormal CD45 23%
Abnormal distribution of immature/mature cells 47%
Abnormal CD33 3%
Abnormal HLA-DR 10%
Abnormal CD11b/HLA-DR pattern 10%-29%
Asynchronous antigen expression
Expression of CD34 12%
Abnormal CD14 20%
Abnormal CD13 39%
Abnormal CD36 31%
Abnormal CD64 23%
Abnormal CD15 33%
Expression of lineage infidelity markers
Lineage infidelity CD2 9%
Lineage infidelity CD5 2%
Lineage infidelity CD7 3%
Lineage infidelity CD19 2%
Overexpression of CD56 15%
Stetler-Stevenson, Blood 2001
Ogata, Blood 2002
Wells, Blood 2003
Malcovati, Leukemia 2005
Benesch, Hematology 2007
Matarraz, Leukemia 2008
Stachursky, Leuk Res 2008
Van de Loosdrecht, Blood 2008
Subirá, Transl Res 2008
Kern, Cancer 2010
Matarraz, Cytometry 2010
Kern, Leuk Lymph 2011
Westers, Leukemia 2012
Matarraz, Cytometry B 2015
Harrington, Am J Clin Pathol 2016
Vs. NormalMatarraz S et al. Cytometry B 2015
Routine diagnostic is mainly based on experience
Monocytic precursors(e.g. monoblasts)
MatureMonocytes
Promonocytes
HLA-DR (PacB)
CD
11
7 (P
EC
y7)
CD
64
(P
E)
CD14 (APCH7)
MatureMonocytes
CD14 (APCH7)
CD
35
(F
IT
C)
CD300e (IREM2) (APC)
MatureMonocytes
CD
14
(A
PC
H7
)
CD
36
(F
IT
C)
CD14 (APCH7)
MatureMonocytes
CD34+
CD64 (APC)
CD
62
L (B
V 6
50
)
Promonocytes+
MatureMonocytes
10-dimensional normal monocytic maturation
CD34+CD64lo
CD34-CD64het
CD64hiCD14-
CD64hiCD14het
CD14hiCD300-
iMoncMo SLAN-
ncMo SLAN+
CD14hiCD300+
BM
PB ncMo<5%
Maturation stageFrequency(from all monocytic cells)
Bone marrow
CD34+CD64lo 1% (0.03-2%)
CD34-CD64het 2% (0.5-3%)
CD64hiCD14- 6% (3.4-8%)
CD64hiCD14het 11% (5-14%)
CD14hiCD300- 25% (17-43%)
CD14hiCD300+ 57% (48-65%)
Peripheral blood
iMO 46%
ncMO SLAN- 22%
ncMO SLAN+ 30%
CD35/CD14/CD64/CD300e/CD34/CD45/CD117/HLA-DR/FSC/SSC
13-color Normal BM monocytic maturation
CLASSICAL MONOCYTIC STAGES
Normal BM samplesMonocytic database
NGF for interpretation of cell maturation
Responsible scientists: S. Matarraz, Q. Lecrevisse, J. Verde
CD35/CD14/CD64/CD300e/CD34/CD45/CD117/HLA-DR
NGF: database for monocytic lineage
Normalized maturation
CD36/CD14/CD64/CD300e/CD34/CD45/CD62L/CD16/SLAN/CD117/HLA-DR/CD33
Monocytic cells
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Normal BM
CD
13 (P
E)
Maturation blockades and abnormal expressionMDS 1
CD11b (APC)
MDS 2
Normal BM
CD
13 (P
E)
Maturation blockades and abnormal expressionMDS 1
CD11b (APC)
25SD
-10SD
15SD
-20SD
Monocytic cells (stages)
MDS 2
Immaturity
Blockade
asynchronism
CD
35 (FIT
C)
MatureMonocytes
CD14 (APCH7)
Maturation blockades and abnormal expression
MDS 36SD
-8SD
MDS 3
One blockade
Two blockades
CD300e (APC)
MatureMonocytes
CD
14 (A
PC
H7)
35-55%
NGF in AMML monitoring
Cell distribution and
phenotype CMMLMonoblastic
leukemiaMonocytic leukemia
↑ % CD34+ 0% 20% 85%
↓ CyMPO 40% 90% 70%
↓ %CD36+ cells 20% 90% 0%
↓ %CD11b+ cells 70% 100% 28%
↓ %CD15+ cells - 70% 0%
↓ %CD35+ cells 10% 90% 0%
↓ %CD14+ cells 30% 100% 0%
↓ %CD300e+ cells 10% 100% 10%
Mo
no
cy
tic
m
atu
ratio
n
Altered monocytic patterns in CMML vs AMML
Matarraz S, Cytometry B 2015
Aberrant phenotype CMMLMonoblastic
leukemiaMonocytic leukemia
CD34+ 0% 45% 43%
CD16 50% 0% 60%
CD19 0% 55% 43%
CD7 20% 45% 15%
NuTdT 0% 0% 20%
CD56* 70% 70% 80%
NG2 (7.1) 0% 70% 60%
Monocytic lineage infidelity in CMML vs AMML
Matarraz S, Cytometry B 2015
23/10/2017
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Aim
Phenotype of AMML blast cells at baseline and follow-up
Phenotype of monocytic cells at complete remisson (CR)
Prediction of response to treatment
ICUS (n=45)
Database for Monocytic lineage evaluation
Responsible scientists: S. Matarraz, Q. Lecrevisse, J. Verde
CD35/CD14/CD64/CD300e/CD34/CD45/CD117/HLA-DR
Normal BM (n=74)
AMML samples BM
N=166
Diagnostic CR No CR Relapse Total
MRD+ MRD-
36 8 82 17 23 166
Flow: >2-5 million cells for MRD studies
Treatment: Idarrubicin/Cytarabine 3+7 (PETHEMA guidelines)
Median follow-up: 7.3 months
Phenotypic evaluation of Monocytic lineage
Merged follow-up BM samples in AMML
Diagnostic
MRD 1: relapse
MRD+ 3-4: CR
MRD 2: CR MRD-
15 SD
-15 SD
Pe
rc
en
ta
ge
of eve
nts
Monocytic cells (stages)
Diagnostic
Relapses
Pe
rc
en
ta
ge
of eve
nts
Monocytic cells (stages)
Merged follow-up BM samples in AMML
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Phenotype of AMML blasts and Monocytic Cells at diagnostic and
follow-up BM
RC
800
600
400
200
0
48
42
6343
62
134
7641
11773
39
13
AMML
DIAGNOSTICNo RC RELAPSE
Pos induction
MONOCYTIC CELLS
p<0.001
AMML
Blasts
AMML
Blasts
All Cases Cases at CR400
300
200
100
0
48
42
43
76117
73
49
112
p=0.06
EMR- EMR+
Post Induction
MONOCYTIC CELLSPost Induction
MONOCYTIC CELLS
CR No CR MRD- MRD+
N. of S
tandard D
eviations
(S
D)
N. of S
tandard D
eviations
(S
D)
*99.5% CI
Prediction of relapse according to monocytic cell phenotype
CR CR
PRE-RELAPSE
BM
Monocytic cells
p<0.001
RELAPSE
800
600
400
200
0
48
42
6343
62
417349
112
39
13
DIAGNOSTIC
Response to Induction according leukemic cell phenotype
NO CR CR
Response to Induction
Phenotypic alterations at diagnosis
800
600
400
200
0
88
p=0.01
N. o
f S
ta
nd
ard
D
eviations
(S
D)
250
200
150
100
50
0
112
65
NO RESPONSE
Response to Consolidation
COMPLETE REMISSIONDISEASE RELAPSE
Response to consolidation vs monocytic phenotype
Phenotype of Monocytic cells at CR
N. of S
tandard D
eviations
(S
D)
P<0.001
Disease-free survival according to MONOCYTIC phenotype
P=0.002
>50SD
<50 SD
2520151050
100%
80%
60%
40%
20%
0%
AMML at CR AMML at CR and MRD-
Time to relapse (months)
P=0.004100%
80%
60%
40%
20%
0%
2520151050
>50 SD
<50 SD
Conclusions I
• An appropriated combination of markers is required for an accurate analysis (and monitoring) of monocytic differentiation.
• The use of reference flow cytometry databases provides an objective and
accurate evaluation of the degree of deviation from normal of and stage of blockade BM (monocytic) cells in MDS
• Other molecules require investigation for highest sensitivity of detection of alteredmonocytic cells in MDS and other myeloid neoplasms
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Conclusions II
A trend to a higher number and degree of phenotypic alterations are detected in blast from relapsing patients
A high degree of impairment of monocytic maturation (dysplasia vs. MRD+?)
in regenerating hematopoiesis may help prediction for disease relapse
The use of NGF tolos provides a more objective monitoring of monocyticalterations in AMML and likely other AML subtypes: we´re on it!
Further studies in larger series are required to validate the potential of NGF
tools for evaluation of prognostic risk of AML patients
Acknoledgements
H.U. Central de Asturias: Enrique Colado.
CIC Salamanca: Alberto Orfao, Pilar Leoz, Quentin Lecrevisse,
Juan Flores, Alejandro Berkovits, Susana Barrena, Antonio López,
Juana Ciudad, Miriam Santos, Miriam Fierro, Rosana Rivas, Laura
Gutierrez, Daniela Damasceno.
H.U. Getafe/Fundación Jiménez Díaz: Ana Yeguas Bermejo.
Erasmus Medical Center, Rotterdam: Vincent van der Velden.
Leiden University Medical Center: Jacques van Dongen.ESCCA 2017