DIAGN ÓSTICO Y CLASIFICACI ÓN DE DIAGNOSIS OF CLONAL ... · DIAGN ÓSTICO Y CLASIFICACI ÓN DE...

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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)

Training

Immunophenotypicdiagnostic activity

startedExperience

Panel optimization

Experience

New indications

2

STANDARDIZATION EFFORTS FORIMMUNOPHENOTYPIC STUDIES

- CLSI (Clinical Laboratory Standards Institute):- Stetler-Stevenson et al.: Clinical flow cytometric analysis ofneoplastic hematolymphoid cells; Approved guideline. CLSI document H43-A2. CLSI, 2007

- CCS (Clinical Cytometry Society):

- 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

Clinical request/need

Medical indication

Design of MAb panels (Medical

indication-oriented) & immuno-phenotyping strategy

TechniquesPanel evaluation vsconventional in-use

panels

Panel optimization (re-design)

Panel evaluation

Proposed strategy

Panel optimization (re-design)

2-8 cycles

other MPD

Monoclonal

component

Monoclonal

component

non-IgM

ALOT LST PCD SST

BCP-ALL T-ALL AML/MDS B-CLPDlimited

B-CLPDbroad T-CLPD NK-CLPD

Sustainedmonocytosis

Eosinophilia

reactive/polyclonal

other B-CLPD

reactive/

non-aberrant

CLL

non-CLL

CLL

MCL

FCL

HCL

other clonal B

reactive

aberrant +αβ

aberrant +γδ

reactive

aberrant

NK cellsvarious subtypes

of BCP-ALL

various subtypes

of T-ALL

various subtypes

of AML

MDS

PNH

CML

CML-BC

Acute leukemia CytopeniaLymphocytosis

LN involvement

Suspect small cell samples

(e.g. CSF, FNA, vitreous)

clonal

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)

Acute Leukemia Orientation Tube (AL0T)

Target Antigen Fluorochrome conjugateGating markers

(first level)Gating Markers (second level) Immaturity markers Lineage markers

cyMPO FITC X My

cyCD79a PE X B, T

CD34 PerCP Cy5.5 X X -

CD19 PE CY7 X B, My

CD7 APC X X T, My

smCD3 APC H7 X T

cyCD3 Pacific Blue X T

CD45 PO X X -

AL

OT

ALOT: B-cell precursor ALL

BM stained withALOT 8-color tube

CyCD3CD7sCD3CD19

CyCD79aCyMPOCD45CD34

Responsible scientist: Ludovic Lhermitte

BCP-ALL


BCP-ALL T-ALL


BCP-ALL T-ALL AML


4

BCP-ALL T-ALL AML

Single « virtual » merged

tube/data file


BCP-ALL T-ALL AML


BCP-ALL T-ALL AML


BCP-ALL T-ALL AML


BCP-ALL T-ALL AML


ALOT (Acute Leukemia Orientation Tube)


5

ALOT: IMMUNOPHENOTYPIC CLASSIFICATION OF BLASTS

T-ALL vs BCP-ALLBCP-ALL vs AMLT-ALL vs AML

T-ALL vs BCP-ALLBCP-ALL vs AMLT-ALL vs AML

BLASTSBLASTS

BLASTS

BLASTS

BLASTS BLASTS

Development of immunostainings protocols – 8-color combinations

Monoclonalcomponent

Monoclonal

componentnon-IgM

ALOT LST PCD

Sustainedmonocytosis

Eosinophilia

Acute leukemia CytopeniaLymphocytosis

LN involvement

SST

Suspect small cell samples

(e.g. CSF, FNA, vitreous)

Pac Blue Pac Orange FITC PEPerCP

Cy5.5PE Cy7 APC APC H7

cyCD3 CD45 cyMPO cyCD79a CD34 CD19 CD7 CD3

Objectives:

•Assessment of the nature of immature blast cell populations in acute leukemia samples (B, T

versus non-lymphoid or mixed phenotype);

•Orientation towards the most appropriate complementary antibody panel(s): BCP-ALL, T-ALL,

and/or AML/MDS







BCP-ALL panel:

T-ALL panel:






CD20 CD45 CD58 CD66c CD34 CD19 CD10 CD38

Kappa CD45 Cyµµµµ CD33 CD34 CD19IgM

CD117Lambda

CD9 CD45 TdT CD13 CD34 CD19 CD22 CD24

CD21 CD45CD15

CDw65NG2 CD34 CD19 CD123 CD81




cyCD3 CD45 TdT CD99 CD5 CD10 CD1a CD3

cyCD3 CD45 CD2 CD117 CD4 CD8 CD7 CD3

cyCD3 CD45 TCRγδγδγδγδ TCRαβαβαβαβ CD33 CD56 cyTCRββββ CD3

cyCD3 CD45 CD44 CD13 HLA Dr CD45RA CD123 CD3

BCP-ALL panel:

T-ALL panel:



T-ALL panel


cyCD3 CD45 TdT CD99 CD5 CD10 CD1a smCD3

cyCD3 CD45 CD2 CD117 CD4 CD8 CD7 smCD3

cyCD3 CD45 TCRγδγδγδγδ TCRαβαβαβαβ CD33 CD56 cyTCR ββββF1 smCD3

cyCD3 CD45 CD44 CD13 HLADR CD45RA CD123 smCD3

T-ALL panel








6

T-ALL panel







T-ALL panel







Positive Diagnosis

T-ALL panel







Differential Diagnosis

&

Ambiguous lineage acute leukemia

T-ALL panel







Classical classification

« Maturation stage »

(EGIL)

T-ALL panel







Alternative T-ALL classification

Maturation stage

T-ALL panel







Alternative T-ALL classification

Maturation stage

&

Well-defined molecular abberations

7

T-ALL panel







LAP Markers

T-ALL panel







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

29 parameters

13 parameters

Colorful dots = normal maturation stage

White dots = T-ALL samples

13 parameters

Colorful dots = normal maturation stage

White dots = T-ALL samples

BCP-ALL panel



smIgK CD45 cyIgM CD33 CD34 CD19

smIgM

+CD117 smIgL


CD21 CD45

CD15

+CDw65 NG2 CD34 CD19 CD123 CD81

BC

P-A

LL

8

BCP-ALL panel




smIgM

+CD117 smIgL


CD21 CD45

CD15



cyCD3 CD45 cyMPO cyCD79a CD34 CD19 CD7 smCD3ALO

TB

CP

-ALL

BCP-ALL panel





smIgM

+CD117 smIgL


CD21 CD45

CD15


Positive Diagnosis

ALO

TB

CP

-ALL

BCP-ALL panel





smIgM

+CD117 smIgL


CD21 CD45

CD15


Differential Diagnosis

&

Ambiguous lineage acute leukemia

ALO

TB

CP

-ALL

BCP-ALL panel





smIgM

+CD117 smIgL


CD21 CD45

CD15


Maturation stage (EGIL)

ALO

TB

CP

-ALL

BCP-ALL panel





smIgM

+CD117 smIgL


CD21 CD45

CD15


Alternative classification

Immunophenotypic features associated with well-

defined molecular aberrations

ALO

TB

CP

-ALL

BCP-ALL panel





smIgM

+CD117 smIgL


CD21 CD45

CD15


Prognosis markers

ALO

TB

CP

-ALL

9

BCP-ALL panel





smIgM

+CD117 smIgL


CD21 CD45

CD15


LAP markers

ALO

TB

CP

-ALL

BCP-ALL panel





smIgM

+CD117 smIgL


CD21 CD45

CD15


LS CD45 CD19 CD34 cyCD3 cyMPO cyCD79a CD7 smCD3 CD20 CD58 CD66C CD10 CD38 smIgK cyIgM CD33 smIgM+CD117 smIgL CD9 TdT CD13 CD22 CD24 CD21 CD15+65 NG2 CD123 CD81

31 parameters

ALO

TB

CP

-ALL

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

11

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


and PNH especially

focussed on monocytic

lineage

Diagnosis and


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


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

(MRD diagnostics)

Responsible scientist: VHJ van der Velden

IMMUNOPHENOTYPIC CHARACTERIZATIONIMMUNOPHENOTYPIC CHARACTERIZATIONOF MDSOF MDS

HOW SIMILAR ARE NEOPLASTIC CELLS TO NORMAL HOW SIMILAR ARE NEOPLASTIC CELLS TO NORMAL CELLS ?CELLS ?

-- ReflectReflect cellcell lineagelineage andand maturationmaturation stagestage..

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 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

12

Normal Maturation of CD34+ Neutrophilprecursor cells in the BM

STAGE I STAGE II STAGE III

CD34+ CD34+ CD34lo

CD117+ CD117+ CD117+HLADR+ HLADR+ HLADRlo

MPO+/++ MPO++ MPO++CD15/65- CD15/65+ CD15/65++CD64- CD64- CD64+ 10 10 10 10 100 1 2 3 4

10277.008

CD45 PERCPCY5,5 ->

CD45-PerCP

T-S

SC

Normal BM

HEMATOPOIETIC MATURATION IN NORMAL BM

CD36 FITC

Monocytic (MPO-/DR+)

Erythroid/DC

10

010

110

210

3

CD64

PE

10 10 10 10 100 1 2 3 4

Gated CD34+ BM cells

Normal Maturation of Erythroid Cells in the BM

STAGE I STAGE II STAGE III STAGE IV

CD34+ CD34+ CD34lo CD34-CD117+ CD117+ CD117+ CD117lo

HLADR+ HLADRlo HLADRlo HLADR-CD36lo/+ CD36+ CD36+ CD36+CD105- CD105+ CD105+ CD105lo/-

CD71lo CD71lo CD71hi CD71hi

CD45lo CD45lo CD45lo CD45-10 10 10 10 100 1 2 3 4

HLAHLA--DRDR FITCFITCC

D1

17

CD

11

7P

EP

E

h

10

010

110

210

310

4

10

4

HLADR FITC

10 10 10 10 100 1 2 3 4

CD

12

3 P

E

e

Basophil

pDC

10

010

110

210

3

CD36 FITC

Monocytic

Erythroid

10

010

110

210

3

CD

64

PE

f

10 10 10 10 100 1 2 3 4

Matarraz S et al. Leukemia 2008

IMMUNOPHENOTYPIC IDENTIFICATION OF LINEAGE IMMUNOPHENOTYPIC IDENTIFICATION OF LINEAGE COMMITMENT OF CD34COMMITMENT OF CD34+ + BM CELLSBM CELLS

Mast cells

IMMUNOPHENOTYPE OF CD34+ IMMUNOPHENOTYPE OF CD34+ MYELOIDMYELOID--COMMITTED HPCCOMMITTED HPC

CELL LINEAGECELL LINEAGE SSCSSC IMMUNOPHENOTYPEIMMUNOPHENOTYPE

ErythroidErythroid stablestable CD36+CD36+, , CD64CD64--, , CD45loCD45lo, , CD105+CD105+

MegakaryocyticMegakaryocytic highhigh CD61+,CD61+, CD45loCD45lo

NeutrophilNeutrophil highhigh CyMPOCyMPO+,+, CD13hiCD13hi

EosinophilEosinophil highhigh CyMPOCyMPO--, CD15/65+, , CD15/65+, CyEPOCyEPO++

BasophilBasophil lowlow CD123hiCD123hi, , HLADRloHLADRlo, , CD117loCD117loCD45hiCD45hi, , CD203cCD203c++

MonocyticMonocytic stablestable CyMPOCyMPO--, CD64+,, CD64+, DR+, DR+, CD117loCD117lo

MastMast cellcell lowlow CD117hiCD117hi, , HLADRloHLADRlo, , CD45hiCD45hi

pDCpDC stablestable CD123hiCD123hi, , HLADRhiHLADRhi, , CD36+CD36+

UNGATED BM EVENTS

CD34+

HPC

B

SSC

CD34-APC

Mat

urin

g

Neu

trop

hils

Eosinophils

Monocytic

cells

Mature

Lymphocytes

CD34+ B-cell

precursors

HPC

NRBC

A

UNGATED BM EVENTS

Basophils

pDC

CD34- B-cell precursors

CD45-PerCP

SSC

EARLY MYELOID PRECURSORS

CD117+/

CD34-

Erythroid

precursors

CD34+ HPC

CD117+/CD34-

neutrophil

precursors

Gated CD117+

and/or CD34+ eventsD

CD45-PerCP

SSC

CD34+/CD117+

HPC

CD117+/ CD34-

precursors

UNGATED EVENTSC

CD117-PE

SSC

13

10 10 10 10 100 1 2 3 4

10277.008

CD45 PERCPCY5,5 ->

CD45-PerCP

T-S

SC

Normal BM

HEMATOPOIETIC MATURATION IN NORMAL BM

CD36 FITC

Monocytic (MPO-/DR+)

Erythroid/DC

10

010

110

210

3

CD64

PE

10 10 10 10 100 1 2 3 4

Gated CD34+ BM cells

10 10 10 10 100 1 2 3 4

10944.005

CD36 FITC ->

MONOCYTIC MATURATION IN NORMAL BM

CD36-FITC

CD64

-PE

HLADR CD64 CD36 CD14 IREM2

IREM-2 APC

CD14

-APC

H7

CD14-APCH7

FLAER

-FIT

C

10 10 10 10 100 1 2 3 4

10678.007

HLADR FITC ->

10 10 10 10 100 1 2 3 4

10678.007

CD45 PerCP/Cy5.5 ->

HLA DR-FITC

CD12

3-PE

CD45-PerCP Cy5.5

CD34

APC

PLASMACYTOID DENDRITIC CELLS

Normal BMNormal BM

10 10 10 10 100 1 2 3 4

10578.011

CD 45 PERCPCY5.5 ->CD45 PerCPCy5.5

10 10 10 10 100 1 2 3 4

10578.007

HLADR FITC ->HLA-DR FITC

CD12

3 PE

T-S

SC

CD45-PerCP Cy5.5

AcuteAcute leukemialeukemiaBMBM

10 10 10 10 100 1 2 3 4

7938.050

CD45 PECY5 ->CD45 PC5CD45 PC5

TR

AN

SF

OR

ME

D S

SC

TR

AN

SF

OR

ME

D S

SC

10 10 10 10 100 1 2 3 4

7938.050

CD45 PECY5 ->CD45 PC5 CD45 PC5

CD

34

CD

34-- P

E

P

E

10 10 10 10 100 1 2 3 4

7938.052

CD117 APC ->CD117 APCCD117 APC

CD

34

CD

34-- P

E

P

E

ACUTE MAST CELL LEUKEMIA

PHENOTYPIC CHANGES DURING NORMAL MAST CELL DIFFERENTIATION

CD34CD203c

FcεεεεRI

CD117

cyTryptase

WHO CLASSIFICATION OF AML*



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

10 10 10 10 100 1 2 3 4

12651.031

CD45 PERCPCY5.5 ->

MON

10 10 10 10 100 1 2 3 4

12844.014 copia

CD45 PERCPCY5.5 ->

10 10 10 10 100 1 2 3 4

12704.008

CD56 PE ->

10 10 10 10 100 1 2 3 4

10277.014

CD56 PE ->

MON

10 10 10 10 100 1 2 3 4

12844.014 copia

MAR FITC ->

10 10 10 10 100 1 2 3 4

12784.031

MAR FITC ->

MON

CD45-PerCP

CD45-PerCP

IREM2-FITC

IREM2-FITC

CD14

-PE

T-S

SC

T-S

SC

CD14

-PE

Normal BM

MDS BM

MONOCYTIC LINEAGE IN MDS: ABERRANT PHENOTYPES

CD56-PE

CD56-PE

T-S

SC

T-S

SC

14

N-DIMENSIONAL NEUTROPHIL MATURATION IN NORMAL BM

Myeloblasts Promyelocytes

MyelocytesMeta-

myelocytes

Bands

Neutrophils

N-DIMENSIONAL NEUTROPHIL MATURATION IN NORMAL BM

CD38

APC

CD15

FITC

CD10

APC

CD45

PO

CD16

FITC

CD11

7PE

CD11

bAPC

HLA

DRP

E

CD13

PECD

34Pe

rCP

Maturation stage of neutrophil precursors in normal BM

N-DIMENSIONAL NEUTROPHIL MATURATION IN NORMAL BM VS AML

CD15 26.6

CD117 19.4

SSC 19.2

FSC 19.0

CD11b 6.0

Maturation stage of AML blasts

Aberrant phenotype of AML blasts

Aberrant markers

Neutrophil maturation in normal BM: definition of maturation stages based on principal component analysis (PCA) 0f 10 parameters

BM 1 BM3BM2

BM4 BM5

All

BM

BM6 BM8BM7

Antigen expression patterns during monocyticmaturation in normal BM

BM 1 BM3BM2

BM 4 BM5

All

BM

BM 6 BM8BM7

Monocytic maturation in normal BM: definition of maturation stages based on principal component analysis (PCA) of data on 10 parameters

15

Erythroid maturation in normal BM: definition of maturation stages based on principal component analysis (PCA) of data on 10 parameters

BM 1 BM3BM2

BM 4 BM5

All

BM

BM 6BM8BM7 RAEB-1

AML/MDS vs normal BM: myelomonocytic maturation

Normal BM

CD34+ HPC:

Neutrophil

Immature

Pre-B

Neutrophil

Immature

CyMPO-PE

CyMPO-PE HLADR-FITC

HLADR-FITC

CD11

7-PE

CD11

7-PE

“DE NOVO” AML: ABERRANT PHENOTYPES & CLONAL HEMATOPOIESIS (n=68)

Phenotype Polyclonal AML# Clonal AML*

N=12 N=49

Normal phenotype 58% 2%

<2 altered lineages 83% 8%

N. of altered lineages 0,7 2,7

Total 12/61 (20%) 49/61 (80%)

Fernández et al, 2011 (in preparation)

# 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 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

16

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

● precursor-B-ALL: − BCR-ABL : completed

− TEL-AML : completed

− E2A-PBX1 : completed

− MLL-AF4 : completed

● AML: − AML1-ETO : completed

− CBFB-MYH11: completed

− PML-RARA : completed prototype testing completed

Core-factor tubeMultiplex tube: prototype completed

Precursor-B-ALL tubeMultiplex tube close to completion

MUCHASGRACIAS

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