1 DIAGNÓSTICO Y CLASIFICACIÓN DE LEUCEMIAS AGUDAS CON LOS PANELES EUROFLOW CANCER RESEARCH CENTER, UNIVERSITY & CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of UNIVERSITY HOSPITAL of SALAMANCA (SPAIN) SALAMANCA (SPAIN) Curso Avanzado de Actualización en Oncohematología por Citometria de Flujo, Buenos Aires, 31 de mayo de 2011 DIAGNOSIS OF CLONAL HAEMATOLOGICAL DISORDERS Clinical symptoms Laboratory and signs findings Morphology + cytochemistry Cytogenetics Immunophenotyping Molecular biology/FISH 1. Making the diagnosis Normal ↔ reactive/regenerating ↔ malignant Annually > 300,000 new patients with a hematological malignancy in developed countries 2. Classification of hematopoietic malignancies - relation with prognosis - relevance of risk-group definition in treatment protocols Based on differentiation characteristics and particularly on chromosome aberrations, resulting in fusion gene transcripts or aberrantly (over) expressed genes 3. Evaluation of treatment effectiveness Detection of minimal residual disease (MRD): MRD-based risk-group stratification (treatment reduction or treatment intensification) Annually > 400,000 follow-up samples in leukemia patients (ALL, AML, CML) DIAGNOSTICS IN HEMATO-ONCOLOGY Prepared by JJM van Dongen REQUIRED DEVELOPMENTS IN FLOW CYTOMETRY (status in 2005) Immunobeads – introduce combined cellular/immunobead assays – special immunobead for leukemias Novel antibodies – test new (academic) antibodies for application in intracellular stainings – development of new antibodies against oncoproteins and aberrant signalling pathways Multicolor flow cytometry: 8 color comprehensive panels – inclusion of solid state violet laser – selection of appropriate fluorochromes – compare conjugated antibodies (multiple companies) Development of novel software for complex pattern recognition – combining multiple tubes: calculate data & multivariate analyses – mapping of diagnosis and follow-up leukemia samples against templates of reference “normal/control” samples THE EUROFLOW APPROACH TO LEUKEMIA/LYMPHOMA IMMUNOPHENOTYPING Clinical question Diagnostic screening tube “Diagnostic classification” panel MRD monitoring Evaluation Majority of diseases? Majority of cases? New disease entities? Knowledge 14 Major groups 154 Nosologic entities Experience Reference profiles SET UP OF A FCM LABORATORY FOR LEUKEMIA /LYMPHOMA TYPING: CONVENTIONAL PANEL DESIGN Clinical request/need Purchase a flow cytometer Design of MAb panels (Disease category vs cell lineage oriented) Training Immunophenotypic diagnostic activity started Experience Panel optimization Experience New indications
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DIAGNÓSTICO Y CLASIFICACIÓN DE LEUCEMIAS AGUDAS CON LOS
PANELES EUROFLOW
CANCER RESEARCH CENTER, UNIVERSITY & CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of UNIVERSITY HOSPITAL of SALAMANCA (SPAIN) SALAMANCA (SPAIN)
Curso Avanzado de Actualización en Oncohematología por Citometria de Flujo, Buenos Aires, 31 de mayo de 2011
DIAGNOSIS OF CLONAL HAEMATOLOGICAL DISORDERS
Clinical symptoms Laboratoryand signs findings
Morphology + cytochemistry
Cytogenetics
Immunophenotyping
Molecular biology/FISH
1. Making the diagnosisNormal ↔ reactive/regenerating ↔ malignant
Annually > 300,000 new patients with a hematological malignancy in developed countries
2. Classification of hematopoietic malignancies- relation with prognosis- relevance of risk-group definition in treatment protocols
Based on differentiation characteristics and particularly on chromosome aberrations, resulting in fusion gene transcripts or aberrantly (over) expressed genes
3. Evaluation of treatment effectivenessDetection of minimal residual disease (MRD):
MRD-based risk-group stratification (treatment reduction or treatment intensification)Annually > 400,000 follow-up samples in leukemia patients (ALL, AML, CML)
DIAGNOSTICS IN HEMATO-ONCOLOGY
Prepared by JJM van Dongen
REQUIRED DEVELOPMENTS IN FLOW CYTOMETRY(status in 2005)
Immunobeads– introduce combined cellular/immunobead assays– special immunobead for leukemias
Novel antibodies– test new (academic) antibodies for application in intracellular
stainings– development of new antibodies against oncoproteins and aberrant
signalling pathways
Multicolor flow cytometry: ≥≥≥≥8 color comprehensive panels– inclusion of solid state violet laser– selection of appropriate fluorochromes– compare conjugated antibodies (multiple companies)
Development of novel software for complex pattern recognition– combining multiple tubes: calculate data & multivariate analyses– mapping of diagnosis and follow-up leukemia samples against
templates of reference “normal/control” samples
THE EUROFLOW APPROACH TO LEUKEMIA/LYMPHOMA IMMUNOPHENOTYPING
Clinical question
Diagnostic screening tube
“Diagnostic classification” panel
MRD monitoring
Evaluation
Majority of diseases?
Majority of cases?
New disease entities?
Knowledge
14 Major groups
154 Nosologic entities
Experience
Reference profiles
SET UP OF A FCM LABORATORY FOR LEUKEMIA /LYMPHOMA TYPING: CONVENTIONAL PANEL DESIGN
Clinical request/need
Purchase a flow cytometer
Design of MAb panels(Disease category vscell lineage oriented)
- Davis et al: 2006 Bethesda International Consensusrecommendations on the flow cytometric immunophenotypicanalysis of hematolymphoid neoplasias. Clin Cytometry, 72B, 2007.
- ESCCA (European Society for Clinical Cell Analysis: www.escca.eu)
- European Leukemia Net (www.leukemia-net.org)
- Consenso Latinoamericano (Clin Cytometry, 1998 y 2006)
LEUKEMIA /LYMPHOMA IMMUNOPHENOTYPING: EVALUATION OF ANTIBODY PANELS
Single center panel
Single center experience/evaluation
Experience-based (subjective)
Long time requiredLimited by new:
instruments
techniques
markers
Consensus recommendationsShared experience
<subjectivity
Multicenter evaluation possible
Multicenter panel
Prospective evaluation
Experimentally supported
>objectivity
CONSTRUCTION OF EUROFLOW LEUKEMIA/ LYMPHOMA IMMUNOPHENOTYPING ANTIBODY PANEL
CONSTRUCTION OF EUROFLOW PANELS: MEDICAL INDICATION ORIENTATION/SCREENING & CLASSIFICATION PANELS
Van Dongen et al: EuroFlow antibody panels for standardized n-dimensional flow cytometricimmunophenotyping of normal, reactive and malignant leukocytes. To be published in: Leukemia 2011
ALOT
BCP-ALL T-ALL AML/MDS
4 tubes 4 tubes 4 to 7 tubes
1 tube
3
Step 0: Design strategy
Step 1: Selection of fluorochromes
Step 2: Selection of markers
Step 3: Selection of antibody reagents
Step 4: Selection of antibody combinations
Step 5: Panel constructed
CONSTRUCTION OF EUROFLOW ANTIBODY PANELSALOT (Acute Leukemia Orientation Tube)
Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7
cyCD3 CD45 cyMPO cyCD79a CD34 CD19 CD7 smCD3
� Designed for assessment of the nature of immature blast cell
populations in acute leukemia samples
� Designed to choose appropriate immunophenotypic panel(s)
B-CELL MATURATION IN NORMAL BM NORMAL BM B_CELL MATURATION vsREGENERATION
NORMAL BM B_CELL MATURATION vsREGENERATION
NORMAL BM B_CELL MATURATION vsREGENERATION
10
BCP-ALL VS NORMAL BM B-CELL MATURATION
APS view 1
APS view 2
Case 1 Case 2 Case 3 Case 4
BCP-ALL (CASE 4) VS NORMAL BM B-CELL MATURATION
MULTIPLE BCP-ALL CASES VS NORMAL BM B-CELL MATURATION
Haematogones
BCP-ALL TEL-AML1
BCP-ALL BCR-ABL
BCP-ALL Hyperdiploïdes
BCP-ALL MLL rearranged
BCP-ALL panel
Flow cytometryimmunophenotyping in AML
• Definition of which subpopulations to analyse
Usually blast cells, Less frequently: maturing neutrophils, monocytes, erythroid cells
• Define which markers/combinations of markers
• Define the myeloid lineages involved
• Define the phenotypic alterations to be considered:
Numerical changesFluorescence intensityAsynchronous expression of maturation-associated markers
WHO CLASSIFICATION OF AML*
AML with recurrent genetic abnormalities
AML with myelodysplasia-related changes
AML NOS
Therapy-related myeloid neoplasms
Myeloid sarcoma
Myeloid proliferations related to Down syndrome (DS):- Transient abnormal myelopoiesis- Myeloid leukemia associated with DS
Blastic plasmacytoid dendritic cell neoplasm
* SM-AHNMD
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Multi-tube EuroFlow classification panel for AML/MDS
CD10
CD14
CD71
CD19
APC-H7
AML/
MDS
1
2
3
4
Tube
Diagnosis and
subclassification of AML
and PNH especially focused
on neutrophilic lineage
Diagnosis and
subclassification of AML
and PNH especially
focussed on monocytic
lineage
Diagnosis and
subclassification of AML
especially focused on
erythroid lineage
Aberrant expression of
lymphoid-associated
markers and abnormal
lymphoid maturation
CD11b
IREM2
CD33
CD7
CD117
CD117
CD117
CD117
CD34
CD34
CD34
CD34
CD13
CD64
CD105
CD56
CD16
CD35
CD36
nuTdT
CD45
CD45
CD45
CD45
HLADR
HLADR
HLADR
HLADR
Aim**APCPE-Cy7PerCP-Cy5.5
PEFITCPacific Orange
Pacific Blue
* Further information about the markers and the availability of hybridoma clones is summarized in Appendix A. Backbone markers are indicated in bold; nu= nuclear.
** The described marker combinations might also be applied for disease staging and monitoring of treatment effectiveness (MRD diagnostics)
Responsible scientist: VHJ van der Velden
Multi-tube EuroFlow classification panel for AML/MDS (Part 2)
CD38
CD4
CD9
APC-H7
AML/
MDS
5
6
AML-
M7
7
Tube
Aberrant expression of
markers; detection of stem
cells
Diagnosis and
subclassification of AML
especially focused on
megakaryocytic, basophilic,
mast cell and plasmacytoid
dendritic lineages
Characterization of AML-
M7, mastocytosis
CD22
CD123
CD42b
CD117
CD117
CD117
CD34
CD34
CD34
NG2
CD203c
CD25
CD15
CD42a
and
CD61
CD41
CD45
CD45
CD45
HLADR
HLADR
HLADR
Aim**APCPE-Cy7PerCP-Cy5.5
PEFITCPacific Orange
Pacific Blue
* Further information about the markers and the availability of hybridoma clones is summarized in Appendix A. Backbone markers are indicated in bold;
nu= nuclear.
** The described marker combinations might also be applied for disease staging and monitoring of treatment effectiveness
IN WHAT DO NEOPLASTIC CELLS DIFFER FROM IN WHAT DO NEOPLASTIC CELLS DIFFER FROM NORMAL CELLS ?NORMAL CELLS ?
-- ReflectReflect derailmentderailment ofof proteinprotein expressionexpression((underlyingunderlying geneticgenetic abnormalitiesabnormalities andand//ororchangeschanges in in thethe BM BM microenvironmentmicroenvironment ?)?)
SCF
IL3
IL7
IL9 EPO
IL11TPO
M-CSF
G-CSF
GM-CSF
HematopoyeticStem cell
Lymphoidprecursor
CFU-GMCFU-G
CFU-MCFU-Eo
CFU-Bs
Bone marrow Blood - tissues
Myeloidprecursor
Neutrophil
Monocyte/Macrophage/DC
Eosinophil
Basophil
HEMATOPOIESISHEMATOPOIESIST/NK cellprecursor
B-cellprecursor
Erythrocytes
Platelets
BFU-E
CFU-Mk
pDCprecursor Dendritic cells
Mast cellCFU-MC
WHO CLASSIFICATION OF AML
AML with recurrent genetic abnormalities
AML with myelodysplasia-related changes
AML NOS:- AML with minimal differentiation- AML without maturation- AML with maturation- Acute myelomonocytic leukemia- Acute monoblastic and monocytic leukemia- Acute erythroid leukemia- Acute megakaryoblastic leukemia- Acute basophilic leukemia- Acute panmyelosis with myelofibrosis
Therapy-related myeloid neoplasmsMyeloid sarcomaMyeloid proliferations related to Down syndromeBlastic plasmacytoid dendritic cell neoplasm
IMMUNOPHENOTYPIC IDENTIFICATION OF LINEAGE IMMUNOPHENOTYPIC IDENTIFICATION OF LINEAGE COMMITMENT OF CD34COMMITMENT OF CD34+ + BM CELLSBM CELLS
nTDTnTDT FITCFITC
CyM
PO
CyM
PO
PE
PE
10
010
110
210
310
4
c
10 10 10 10 100 1 2 3 4
TR
AN
SF
OR
ME
D S
SC
TR
AN
SF
OR
ME
D S
SC
CD34 APC
0256
512
768
1024
a
10 10 10 10 100 1 2 3 41024
TRANSFORMED SSCTRANSFORMED SSC
0 256 512 768
bCD
45
PE
RC
PC
D4
5 P
ER
CP
10
010
110
210
310
4
b
Matarraz S et al. Leukemia 2008
Neutrophil precursors
B-cell precursors
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Normal Maturation of CD34+ Neutrophilprecursor cells in the BM
# One ISM-AML case with KIT mutation restricted to mast cells
* Two cases showed coexistence of t(8;21) & D816V KIT mutation in all cellular compartments analyzed
WHO CLASSIFICATION OF AML
AML with recurrent genetic abnormalities
- AML with t(8;21)(q22;q22); RUNX1-RUNX1T1- AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11- APL with t(15;17)(q22;q12); PML-RARA- AML with t(9;11)(p22;q23); MLLT3-MLL- AML with t(6;9)(p23;q34); DEK-NUP214- AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1- AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL1- AML with mutated NPM1- AML with mutated CEBPA
AML with myelodysplasia-related changesAML NOSTherapy-related myeloid neoplasmsMyeloid sarcomaMyeloid proliferations related to Down syndromeBlastic plasmacytoid dendritic cell neoplasm
Diagnosis Genetic Aberrant immunophenotype
lesion
BCP-ALL t(9;22)* CD34hi,CD10+,CD38lo,CD13lo
t(12;21) CD34het,CD10+,CD20-,CD13lo
11q23 CD34+,CD10-,7.1+,CD15+
AML t(15;17) CD34-/+,CD15-/lo,CD2-/lo,CD13het
Inv(16) MPOhi,CD2-/lo
t(8;21) CD19+,CD56+
11q23 CD56+,7.1-/+,CD19-/lo,CD2-/+
AL: GENOTYPIC-PHENOTYPIC ASSOCIATIONS
Ortuño F, Orfao A, Cytometry B, 2004
Bead-based flow cytometric assay for detection of fusion proteins
Dept. of Immunology, Erasmus MC, Rotterdam
Patents: US 6,610,498 B1 (26 August 2003)
US 6,686,165 B2 (3 February 2004)
5´PML
3´RARA
PMLRARA
transcription
mRNA
PML RARA
PML RARA
PML RARA
fusion proteinstranslation
cell lysate
bead
beads coatedwith anti-RARA
antibody
bead
bead
FITC-conjugatedanti-PML antibody
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Results of PML-RARA fusion protein detection using the immunobead assay
10
100
1,000
10,000
100,000
APL AML(non APL)
CML BCP-ALL T-ALL
MF
Iva
lue
500
180
BCR3
BCR2
BCR1
result of
RQ-PCR
negative
n=46 n=1 n=34 n=16n=66
At this moment the technical developments for 7 well-defined
fusion proteins have (virtually) been completed:
● CML: − BCR-ABL : completed RUO kit launched and published