CANCER RESEARCH CENTRE,CANCER RESEARCH CENTRE,UNIVERSIUNIVERSITYTY AND UNIVERSITY HOSPITAL OFAND UNIVERSITY HOSPITAL OF

SALAMANCA (SALAMANCA (SPAIN)SPAIN)

ACUTE LEUKEMIASACUTE LEUKEMIAS

Sao Paulo, 18th of April 2009

WHO CLASSIFICATION OF WHO CLASSIFICATION OF HAEMATOLOGIC MALIGNANCIESHAEMATOLOGIC MALIGNANCIES

Diagnosis Diagnosis andand classificationclassification ofof clinicobiologicalclinicobiologicalentitiesentities more more thanthan justjust histopathologicalhistopathologicalsubtypessubtypes basedbased onon::

-- HistopathologyHistopathology andand//oror cytopathologycytopathology-- ImmunophenotypeImmunophenotype-- Molecular/Molecular/geneticgenetic findingsfindings-- ClinicalClinical andand haematologichaematologic behaviorbehavior

DIAGNOSIS OF HAEMATOLOGICAL MALIGNANCIESDIAGNOSIS OF HAEMATOLOGICAL MALIGNANCIES

ClinicalClinical symptomssymptoms LaboratoryLaboratoryandand signssigns findingsfindings

MorphologyMorphology + + cytochemistrycytochemistry

CytogeneticsCytogenetics

ImmunophenotypingImmunophenotyping

Molecular Molecular biologybiology/FISH/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

NEW CONTRIBUTIONS IN DIAGNOSINGACUTE LEUKEMIAS

-- EstablishingEstablishing thethe blastblast cellcell gategate..

-- LineageLineage assignmentassignment ofof undifferentiatedundifferentiated//minimallyminimally differentiateddifferentiated ANLLANLL

-- Diagnosis Diagnosis ofof AML AML withwith multilineagemultilineage dysplasiadysplasia

-- RapidRapid identificationidentification ofof relevantrelevant cytogeneticcytogeneticsubgroupssubgroups ofof chronicchronic andand acuteacute leukemiasleukemias

-- StandardizationStandardization ofof multiparametermultiparameter immunoimmuno--phenotypicphenotypic proceduresprocedures

IMMUNOPHENOTYPIC IMMUNOPHENOTYPIC CHARACTERIZATION OF CHARACTERIZATION OF ““NORMALNORMAL”” VS VS

NEOPLASTIC PRECURSORS: NEOPLASTIC PRECURSORS:

THE BLAST CELL GATETHE BLAST CELL GATE

THE BLAST CELL REGION IN NORMAL BM

Mat

urin

gN

eutro

phils Eosinophils

Monocyticcells

MatureLymphocytes

CD34+ B-cellprecursors

HPC

NRBC

A

UNGATED EVENTS

BasophilspDC

CD34- B-cell precursorsCD45-PerCP

SSC

UNGATED BM EVENTS

CD34+/CD117+

HPC

B

SSC

CD34-APC

Mat

urin

gN

eutro

phils Eosinophils

Monocyticcells

MatureLymphocytes

CD34+ B-cellprecursors

HPC

NRBC

A

UNGATED BM EVENTS

BasophilspDC

CD34- B-cell precursors

CD45-PerCP

SSC

CD34 AND THE CD45/SSC BLAST CELL REGION

AntiAnti--CD34CD34

Side

SideSca

tter

Sca

tter

IDENTIFICATION OF PB HPC IDENTIFICATION OF PB HPC

IMMUNOPHENOTYPING IMMUNOPHENOTYPING ofof CD34+ HPCCD34+ HPC((BackgatedBackgated onon CD45CD45dim))

10 10 10 10 100 1 2 3 4

10277.008CD45 PERCPCY5,5 ->CD45-PerCP

NORMAL BONE MARROW

T-SSC

Nucleated red cells

Neutrophil lineage Monocytic lineage

CD34+ HPC

Eosinophils

Lymphocytes

EARLY PHENOTYPE OF CD34+ EARLY PHENOTYPE OF CD34+ MYELOIDMYELOID--COMMITTED HPCCOMMITTED HPC

CELL LINEAGECELL LINEAGE SSCSSC EARLIEST ADDITIONALEARLIEST ADDITIONALMARKERSMARKERS MARKERSMARKERS

ErythroidErythroid stablestable CD36+CD36+ CD105+CD105+

MegakaryocyticMegakaryocytic highhigh CD61+CD61+

NeutrophilNeutrophil highhigh CyMPOCyMPO+,+, CD15/65, CD64CD15/65, CD64

EosinophilEosinophil highhigh CyEPOCyEPO++ CD15/65CD15/65

BasophilBasophil lowlow CD203cCD203c+/+/CD123hiCD123hi

MonocyticMonocytic stablestable CD64+,CD64+, CD35CD35

MastMast cellcell lowlow CD117hiCD117hi, , CyTryp,CD203cCyTryp,CD203c

pDCpDC stablestable CD123hiCD123hi/CD36+/CD36+

IMMUNOPHENOTYPE OF LINEAGE IMMUNOPHENOTYPE OF LINEAGE COMMITTED CD34+HPCCOMMITTED CD34+HPC

CELL LINEAGECELL LINEAGE FREQUENCYFREQUENCY

ErythroidErythroid 15% (5%15% (5%--35%)35%)

MegakaryocyticMegakaryocytic <1%<1%

NeutrophilNeutrophil 31% (1231% (12--39%)39%)

EosinophilEosinophil <1%<1%

BasophilBasophil <1% (<1%<1% (<1%--3%)3%)

MonocyticMonocytic 5% (3%5% (3%--15%)15%)

MastMast cellcell <1% (<1%)<1% (<1%)

pDCpDC 6% (1%6% (1%--15%)15%)

BB--LymphoidLymphoid 23% (<1%23% (<1%--45%)45%)

Total:Total: 81%81%

Neutrophils and Monocytes Follow Different Maturation Pathways

Neutrophil Precursor

Monocyticprecursor

CD35-FITC

CyMPO

-PE

nTDTnTDT FITCFITC

CyMPO

CyMPO

PEPE

100

101

102

103

104

10 10 10 10 100 1 2 3 4

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+

CD34CD34-- BLAST CELLS BLAST CELLS

CD117+/CD34-

Erythroidprecursors

CD34+ HPC

CD117+/CD34-

neutrophilprecursors

Gated CD117+

and/or CD34+ eventsD

CD45-PerCPSSC

CD34+/CD117+

HPC

CD117+/ CD34-

precursors

UNGATED EVENTSC

CD117-PE

SSC

EARLY NEUTROPHIL VS ERYTHROID DIFFERENTIATION

103

102

101

BAND/NEUTROPHIL

METAMYELOCYTMYELOCYTEPROMYELOCYTEMYELOBLAST

CD34

HLA-DR CD117

CD13

CD33

CD11b

CD64

CD65

CD54

CD10

CD35

CD13

PHENOTYPIC CHANGES DURING NORMAL NEUTROPHIL DIFFERENTIATION

MPO

CD15

CD16

MYELOMYELOBLAST CELL CRITERIA:BLAST CELL CRITERIA:

CD34+ and/or CD117+/HLADR+CD34+ and/or CD117+/HLADR+

Neutrophils and Monocytes Follow Different Maturation Pathways

Neutrophil Precursor

Monocyticprecursor

CD35-FITC

CyMPO

-PE

HLADR-FITC

CD117-PE

10 10 10 10 100 1 2 3 4

10944.005CD36 FITC ->

Monocytic differentiation in normal BM

CD36-FITC

CD64-P

E

HLADR CD64 CD36 CD14 IREM2

10 10 10 10 100 1 2 3 4

12784.031MAR FITC ->

MONIREM-2 FITC

CD14-PE

MONOCYTEPROMONOCYTEMONOBLAST

CD117

CD34

LisozymeCD45

CD11b

CD14

HLA-DR

CD36

103

104

102

101

CD64

MPO

CD4

IREM-2

PHENOTYPIC CHANGES DURING MONOCYTIC MATURATION IN NORMAL BM

NEW CONTRIBUTIONS IN DIAGNOSINGACUTE LEUKEMIAS

-- EstablishingEstablishing thethe blastblast cellcell gategate..

-- LineageLineage assignmentassignment ofof undifferentiatedundifferentiated//minimallyminimally differentiateddifferentiated ANLLANLL

-- Diagnosis Diagnosis ofof AML AML withwith multilineagemultilineage dysplasiadysplasia

-- RapidRapid identificationidentification ofof relevantrelevant cytogeneticcytogeneticsubgroupssubgroups ofof chronicchronic andand acuteacute leukemiasleukemias

-- StandardizationStandardization ofof multiparametermultiparameter immunoimmuno--phenotypicphenotypic proceduresprocedures

HEMATOPOIESIS

Stem cell

Lymphoid precursor

Myeloid precursor

T-lymphocytesB-lymphocytesNK-cellsPlasmacytoid DCErythroid cellsMegakaryocytic cellsNeutrophilsMonocytesMonocytic DCEosinophilsBasophilsMast cellsMyeloid DC

IMMUNOPHENOTYPE OF LINEAGE IMMUNOPHENOTYPE OF LINEAGE COMMITTED CD34+HPCCOMMITTED CD34+HPC

CELL LINEAGECELL LINEAGE FREQUENCYFREQUENCY

ErythroidErythroid 15% (5%15% (5%--35%)35%)

MegakaryocyticMegakaryocytic <1%<1%

NeutrophilNeutrophil 31% (1231% (12--39%)39%)

EosinophilEosinophil <1%<1%

BasophilBasophil <1% (<1%<1% (<1%--3%)3%)

MonocyticMonocytic 5% (3%5% (3%--15%)15%)

MastMast cellcell <1% (<1%)<1% (<1%)

pDCpDC 6% (1%6% (1%--15%)15%)

BB--LymphoidLymphoid 23% (<1%23% (<1%--45%)45%)

Total:Total: 81%81%

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+

IDENTIFICATION OF NEWSUBTYPES OF ACUTE LEUKEMIAS

10 10 10 10 100 1 2 3 4

10678.007HLADR FITC ->

10 10 10 10 100 1 2 3 4

10678.007CD45 PerCP/Cy5.5 ->

HLA DR-FITC

CD123-PE

CD45-PerCP Cy5.5

CD34 A

PC

PLASMACYTOID DENDRITIC CELLS

Normal BMNormal BM

10 10 10 10 100 1 2 3 4

10578.011CD 45 PERCPCY5.5 ->

CD45 PerCPCy5.5

10 10 10 10 100 1 2 3 4

10578.007HLADR FITC ->HLA-DR FITC

CD123 PE

T-SSC

CD45-PerCP Cy5.5

AcuteAcute leukemialeukemiaBMBM

88%88%CD4

13%CD5

30%

5%

76%76%

0%

0%

0%

CD7

CD8

CD56

CD94/CD158a

cCD3

CD161

32% CD2

% of positive casesT/NK-cell Ags

CD123hi DC NEOPLASIAS:IMMUNOPHENOTYPE

6%CD20

33%CD22

0%

20%

40%

CD23/CD24

cCD79a

CD79b

0% CD19

% of positive casesB-cell Ags

0%CD14

19%CD15

0%

80%

54%

18%

12%

0%0%

0%0%

CD16

CD32

CD33

CD64

CD65

Lysozyme

MPO

24%CD13

% of positive casesMyeloid Ags

0%CD90

0%CD133

43%

6%

CD117

Tdt

9%CD34

% of positive casesHPC Ags s

10/30 (33%)ALL (T/B)

2/30 (6%)Peripheral NHL (T/B)

9/30 (30%)

1/30 (3%)

DC/NK-cell lymphoma

PCL

8/30 (27%)AML(Mo/M5a)

Morphological diagnosis (n=30)Morphological diagnosis (n=30)

2626 / 4 (87%87% / 13%)Sex (m/f)

64 ± 23 (10 – 91)Age (years)

CD123hi DC NEOPLASIAS:CLINICAL FEATURES

0%

20%

40%

60%

80%

100%CD36

CD14

IREM-2

AML withgranulomonocytic

maturation

AML with monoblastic maturation

AML with monocytic maturation

*

• •

••

*

ACUTE MONOBLASTIC & MONOCYTIC LEUKEMIAS: blast cell maturation patterns

410

10

10

10

01

23

10

10 10 10 10 100 1 2 3 4

IREM-2 FITC

CD14-PE

10

10

10

10

01

23

104

10 10 10 10 100 1 2 3 4

IREM-2 FITC

CD14-PE

ACUTE MONOBLASTIC & MONOCYTIC LEUKEMIAS:

Pattern of blast cell maturation

ACUTE MONOBLASTIC & MONOCYTIC LEUKEMIAS: blast cell maturation patterns

Early pattern of CD14 vs IREM2 expression

CD14+/IREM2- CD14-/IREM2+

% of CD14+ cells 35%+23% 12%+11%

% of IREM2+ cells 8%+13% 28%+17%

AML subtypeMonoblastic/M5a 8/18 2/5Monocytic/M5b 10/18 3/5

Skin/mucosal 0/18 4/5infiltration

NEW CONTRIBUTIONS IN DIAGNOSINGACUTE LEUKEMIAS

-- EstablishingEstablishing thethe blastblast cellcell gategate..

-- LineageLineage assignmentassignment ofof undifferentiatedundifferentiated//minimallyminimally differentiateddifferentiated ANLLANLL

-- Diagnosis Diagnosis ofof AML AML withwith multilineagemultilineage dysplasiadysplasia

-- RapidRapid identificationidentification ofof relevantrelevant cytogeneticcytogeneticsubgroupssubgroups ofof chronicchronic andand acuteacute leukemiasleukemias

-- StandardizationStandardization ofof multiparametermultiparameter immunoimmuno--phenotypicphenotypic proceduresprocedures

WHO CLASSIFICATION (2002 & 2008)

• AML with "recurrent cytogenetic translocations"

• AML with multilineage dysplasia

• Secondary AML

• Other AML (morphological and immunophenotypicclassification

MSSA03.005

CD11b FITC

EP

3

DC

MSSA24.005

CD1 1 b FITC

EP 31DC

CD34 APC (Approaching)

MSSA02.005CD13-PE

CD34

-APC

CD11b-FITC

CD13-PE

CD34-A

PCCD11b-FITC

AML: ALTERED MATURATION OF BM NEUTROPHIL LINEAGE CELLS

NORMAL BM AML BM

Blasts Maturingneutrophils

CD34+ Maturingprecursors neutrophils

“DE NOVO” AML: ABERRANT PHENOTYPES INVOLVING MATURING BM NEUTROPHILS

Aberrant phenotypes (%)Cell lineage

Neutrophils 12/18 (67%)

Monocytic cells 5/18 (27%)

Total 13/18 (72%)

ANALYSIS OF CLONALITY (HUMARA TEST)IN “de novo” AML

#21: neutrophils / HpaII digested

Control cell population(Lymphocytes)/HpaII digested

#21: neutrophils / --

Control cell population(Lymphocytes) / --

“DE NOVO” AML: ABERRANT PHENOTYPES & CLONAL BM HEMATOPOIESIS

% of aberrant phenotypesCell lineage Polyclonal cases Clonal cases

Neutrphils 1/6 (17%) 11/12 (92%)

Monocytes 0/6 (0%) 5/12 (42%)

Total 1/6 (17%) 12/12 (100%)

Case 1 Case 2

KIT MUTATION IN SM: PATTERN OF CELLULAR INVOLVEMENT

Eos Eos

NeutNeut

Mo Mo

LymphsLymphs

NRCNRC

CD34+HPC

CD34+HPC

MastCells

MastCells

KIT mutation

DIAGNOSTIC PATTERNS OF BM CELLULAR INVOLVEMENTSUBTYPES

(% KIT mutated) MC only MC + CD34 Myeloid and/or Germinallymphoid cells

ISM (96%) 49/74 (66%) 7/74 (10%)b 15/74 (20%)a 3/74 (4%)

SMAS (82%) 21/21 (100%) - - -

WDSM (19%) 2/3 1/3 - -

SM- (100%) 8/15 (54%)d 2/15 (13%)d 5/15 (33%)c -AHNMD

ASM (100%) - - 8/11 (73%)c 3/11 (27%)

MCL (83%) 1/2 - 1/2 -

Evolution to AML 0/81 (0%) 0/10 (0%) 0/29 (0%) 4/6 (67%)

KIT MUTATION IN SM: PATTERNS OF BM CELLULAR INVOLVEMENT IN DIFFERENT DIAGNOSTIC SUBTYPES (n=126)

a 4 cases with D816V+ lymphocytes b 2 cases with D816V+ eosinophils c 1 case with D816V+ lymphocytes d 1 case with D816V+ eosinophils

NEW CONTRIBUTIONS IN DIAGNOSINGACUTE LEUKEMIAS

-- EstablishingEstablishing thethe blastblast cellcell gategate..

-- LineageLineage assignmentassignment ofof undifferentiatedundifferentiated//minimallyminimally differentiateddifferentiated ANLLANLL

-- Diagnosis Diagnosis ofof AML AML withwith multilineagemultilineage dysplasiadysplasia

-- RapidRapid identificationidentification ofof relevantrelevant cytogeneticcytogeneticsubgroupssubgroups ofof chronicchronic andand acuteacute leukemiasleukemias

-- StandardizationStandardization ofof multiparametermultiparameter immunoimmuno--phenotypicphenotypic proceduresprocedures

Diagnosis Genetic Aberrant immunophenotypelesion

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,aberrant monocytes

t(8;21) CD19+,CD56+

11q23 CD56+,7.1-/+,CD19-/lo,CD2-/+

AL: GENOTYPIC-PHENOTYPIC ASSOCIATIONS

Bead-based flow cytometric assay for detection of fusion proteins

5' gene A gene 3'B fusion gene

B A

B

A

B A

B

transcription

translation

cell lysate

A

B

A

B

A

B

bead

beads coatedwith anti-Aantibody

bead

bead

AB A B

AB

FITC-conjugatedanti-B antibody

A

Patents: US 6,610,498 B1 (26 August 2003)US 6,686,165 B2 (3 February 2004)

Kindly provided by JJM Van Dongen on behalf of the Euroflow group

Results of the BCR-ABL RUO testing by the EuroFlow laboratories

Samples BCR-ABL PCR assay BCR-ABL immunobead assay* tested negative p190 p210 negative low positive high positive

CML (n=19) 0 1 18 0 16 3

Precursor-B-ALL- childhood (n=50) 49 1 0 49 0 1- adult (n=28) 12 13 3 12 2 14

T-ALL (n=18) 18 0 0 18 0 0

AML (n=27) 27 0 0 27 0 0

Reactive (n=1) 1 0 0 1 0 0

CMPD (n=1) 1 0 0 1 0 0

Mature B-NHL (n=1) 1 0 0 1 0 0

Healthy controls (n=72) NT 72 0 0

*negative: MFI value <135; low positive: MFI value ≥135, but <1,000; high positive: MFI value ≥1,000

BCR-ABL RUO testing by EuroFlowMFI values of the different cell samples (n=217)

135

repeated analysis for confirmationof weak positivitynegativep210p190

result of RQ-PCR

1. High specificity of BCR-ABL RUO− High concordance (100%; 145/145) between BCR-ABL PCR results

and the BCR-ABL RUO results;− Results: - 17/78 precursor-B-ALL were BCR-ABL positive in both

assays (mainly adults)- 19/19 CML were BCR-ABL positive in both assays (with

borderline positivity in one CML case; MFI of 144)- 0/48 of other (acute) leukemias were BCR-ABL positive

2. Mean Fluorescence Intensity (MFI) values− two main groups of positive patient samples were seen:● high level positivity: MFI values ≥ 1,000● lower level positivity: MFI values ≥ 135, but < 1,000

− negative samples were defined as MFI values < 135

3. Different MFI values in precursor-B-ALL and CMLPrecursor-B-ALL: 88% (15/17) high level positivityCML: 84% (16/19) low level positivity (true low expressionor remaining protease activity?)

Results concerning BCR-ABL RUO kitWeerkamp et al, Leukemia, 2009 (in press)

Immunobead flow cytometry with BD™ CBA Flex

beads BCR-ABL t(9;22) fusion protein: Specificity

Black: 697 (t(1;19), neg. control)Blue: TOM-1, BCR-ABL+ (p190)Green: LAMA-84 BCR-ABL+ (p210)Purple: AR230, BCR-ABL+ (p230)

Catching antibody: anti-BCR (clone 3E2C10)Bead: BD-Flex bead (A7)Detection antibody: biotinylated anti-ABL (clone 8E9) + SA-PE

PANELS OF IMMUNOBEADS FOR THE CLASSIFICATION OF ACUTE LEUKAEMIAS

Precursor-B-ALL AML ‘MLL’ T-ALL

BCR-ABL PML-RARA MLL-AF4 CALM-AF10

TEL-AML1 AML1-ETO MLL-AF9 LMO2

E2A-PBX1 CBFB-MYH11 MLL-AF10 HOX11L2

( MLL-AF4) MLL-ENL TAL1

MLL-AF6

AML multiplex tube : PML-RARα t(15;17)

PM L 5D8.16-RARA 9G4.16

0

2000

4000

6000

8000

10000

12000

14000

Sensitivity for detection NB 4 cell line in:

– Negative cell line: less than 10 % (s/n of 21)

– WBC: not tested– PBMC: 10% (s/n 3)

New combination PML 5D8.16 w ith RARA 9G4.16

0

2000

4000

6000

8000

10000

12000

14000

16000

ME1MV 4;

11

K562

697

RCH-ACV

kasu

mi

REH

NB4

MF

I

Assay Cell line patients singleplex multiplexingAML tubePML-RARA + + + to be tested

AML-ETO + + + to be tested CBFß-MYH11 + + + +

Pre-B-ALL

BCR-ABL + + + + E2A-PBX + + + +MLL-AF4 + + + to be tested TEL-AML + + + +

Multiplexen of AML and precursor B-ALL CBA assays

100% K562 100% 697

10% K562/69710% 697/K562

R2 = green = BCR beads

R3 = pink = E2A beads

SIMULTANEOUS DETECTION OF THE BCR-ABL ANDE2A-PBX FUSION PROTEINS

NEW CONTRIBUTIONS IN DIAGNOSINGACUTE LEUKEMIAS

-- EstablishingEstablishing thethe blastblast cellcell gategate..

-- LineageLineage assignmentassignment ofof undifferentiatedundifferentiated//minimallyminimally differentiateddifferentiated ANLLANLL

-- Diagnosis Diagnosis ofof AML AML withwith multilineagemultilineage dysplasiadysplasia

-- RapidRapid identificationidentification ofof relevantrelevant cytogeneticcytogeneticsubgroupssubgroups ofof chronicchronic andand acuteacute leukemiasleukemias

-- StandardizationStandardization ofof multiparametermultiparameter immunoimmuno--phenotypicphenotypic proceduresprocedures

MultiparameterMultiparameter immunophenotypicimmunophenotypicproceduresprocedures: : standardizationstandardization effortsefforts

-- CLSI CLSI ((ClinicalClinical LaboratoryLaboratory StandardsStandards InstituteInstitute):):-- StetlerStetler--StevensonStevenson et al.: et al.: ClinicalClinical flowflow cytometriccytometric analysisanalysisofof neoplasticneoplastic hematolymphoidhematolymphoid cellscells; ; ApprovedApproved guidelineguideline. CLSI . CLSI documentdocument H43H43--A2. CLSI, 2007A2. CLSI, 2007

-- CCSCCS ((ClinicalClinical CytometryCytometry SocietySociety):):

-- DavisDavis et al: 2006 et al: 2006 BethesdaBethesda InternationalInternational ConsensusConsensusrecommendationsrecommendations onon thethe flowflow cytometriccytometric immunophenotypicimmunophenotypicanalysisanalysis ofof hematolymphoidhematolymphoid neoplasias. neoplasias. ClinClin CytometryCytometry, 72B, , 72B, 2007.2007.

-- ESCCAESCCA ((EuropeanEuropean SocietySociety forfor ClinicalClinical CellCell AnalysisAnalysis: : www.escca.euwww.escca.eu))

-- EuropeanEuropean LeukemiaLeukemia NetNet ((www.leukemiawww.leukemia--net.orgnet.org))

Required future developments

Multicolor flow cytometry: >4-6 colors– inclusion of solid state violet laser– compare conjugated antibodies (multiple companies)

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

Development of novel software– novel software for pattern recognition: mapping of

diagnosis and follow-up leukemia samples against templates of “normal/control” samples

T-ALL AMLBCP-ALL

Acute leukemia screening panel

Merge and calculation for the blast cells

Export the blast cell populations & merge them in one data

file

HOW TO BUILD IN A SOFTWARE REFERENCE ACUTE LEUKEMIA SAMPLES ?

B-ALL T-ALL B-ALL T-ALL

GATED B- VS T-LYMPHOID BLAST CELLS:Improving Expert-based classification of blast cell lineage

LINEAGE ASSESSMENT IN ACUTE LEUKAEMIAS (N=158)

BCP-ALL VS T-ALL BCP-ALL VS AML AML VS T-ALL

Information provided about the discriminating parameters:(most informative Mab combinations for diagnosis)

AML with t(4;12) (undifferentiated:CD7++,CD34++, CD3-, CD19-,MPO-, CD117++, CD13/33+)

TM

U/T-M

DEFINITION OF NEW ENTITIES?

All parametersexcept FSC/SSC

All sample aliquots fixed & permeabilizedvs

mixed (surface stainings alone plus fixed & permeabilised)(N=16)

Biological vs technical differences

J AlmeidaJ AlmeidaM AlmeidaM AlmeidaM AnguloM AnguloP BarcenaP BarcenaS BarrenaS BarrenaA A BalanzateguiBalanzateguiJ CiudadJ CiudadA EspinosaA EspinosaJ FloresJ FloresA A GarciaGarcia MonteroMonteroM JaraM JaraQ Q LecrevisseLecrevisseA A LopezLopezMC MC LopezLopez--BergesBergesL L MartinMartin

S S MatarranzMatarranzW NietoW NietoB B PaivaPaivaJ J PerezPerezD PrimoD PrimoA RasilloA RasilloR RivasR Rivas--AmoedoAmoedoA A RodriguezRodriguez--CaballeroCaballeroML ML SanchezSanchezJF San MiguelJF San MiguelJM JM SayaguesSayaguesMD TaberneroMD TaberneroC C TeodosioTeodosioMB MB VidrialesVidriales

GRUPO DE SALAMANCAGRUPO DE SALAMANCA

I I AlaejosAlaejos,, MC MC AlguerAlgueróó,, J Almeida, J Almeida, M Almeida, M Almeida, M Angulo, M Angulo, E Arroyo, E Arroyo, P Barcena, P Barcena, S Barrena, S Barrena, A A BalanzateguiBalanzategui, , A A BortoluciBortoluciA A BrufauBrufau,, C. Bueno, C. Bueno, MD CaballeroMD Caballero C C ChillonChillon,, J Ciudad, J Ciudad, M Corral,M Corral, MC del CaMC del Caññizo, izo, A Duran,A Duran, G Ercilla, G Ercilla, A Espinosa, A Espinosa, E FernE Fernáández,ndez, J Flores, J Flores, M Fuentes, M Fuentes, MA GarcMA Garcíía, a, A GarcA Garcíía, a, R GarcR Garcííaa--Sanz,Sanz, D GonzD Gonzáález,lez, MT GonzMT Gonzáález, lez, M GonzM Gonzáález, lez, N N GutierrezGutierrez,,JM HernJM Hernáándezndez J HernJ Hernáández,ndez, P HernP Hernáández, ndez, M Jara, M Jara, Q Q LecrevisseLecrevisse, , A LA Lóópez, pez, C LC Lóópezpez--BergesBerges, , R R LopezLopez--PerezPerez,, A A MacedoMacedo, , A MartA MartííneznezI MartI Martíín, n, L MartL Martíín, n, M MartM Martíínn--Ayuso,Ayuso, S S MatarranzMatarranz, , G Mateo, G Mateo, MV Mateos,MV Mateos, P MenP Menééndez, ndez, MJ Nieto,MJ Nieto, W Nieto, W Nieto, B B PaivaPaiva, , J PJ Péérez, rez, JA PJA Péérez Simrez Simóón n M PM Péérez Andrrez Andréés, s, D Primo, D Primo, S Quijano, S Quijano, A Rasillo, A Rasillo, O Redondo,O Redondo, A RA Rííos, os, R RivasR Rivas--AmoedoAmoedo, , A RodrA Rodrííguez, guez, I SI Sáánchez,nchez, ML SML Sáánchez, nchez, F F SanguezSanguez--G.G. JF San Miguel, JF San Miguel, M Santiago, M Santiago, JM JM SayagSayagüüeses, , L SuL Suáárez, rez, MD Tabernero, MD Tabernero, C C TeodTeodóósiosio, , JM Vaquero,JM Vaquero,L VL Váázquez, zquez, MB MB VidrialesVidriales,, E E VillaronVillaron

AKNOWLEDGEMENTSAKNOWLEDGEMENTS

I I AbuinAbuin,, M M AlvarezAlvarez--MonMon ML AmigoML Amigo F ArribaF Arriba JL Arroyo,JL Arroyo,M M BarbonBarbon,, A A BarezBarez,, J J BladBladéé,, D Borrego,D Borrego, P Bravo,P Bravo,J Briones,J Briones, MJ MJ CalmuntiaCalmuntia,, M Carnero,M Carnero, F Casanova, F Casanova, C C CerverCerveróó,,E Conde,E Conde, MA Cuadrado,MA Cuadrado, B B DiazDiaz--AgustinAgustin, , J J DiazDiaz--MediavMediav.. A A DurDuráántezntez,,L Escribano,L Escribano, J J FdezFdez--Calvo,Calvo, P P FisacFisac,, L L FlorensaFlorensa,, J J GalendeGalende,,R Galindo,R Galindo, JL JL GarciaGarcia,, J J GarciaGarcia--Conde,Conde, J J GarciaGarcia--LaraLaraññaa,, F Garrido,F Garrido,P Garrido,P Garrido, A A GasconGascon,, L Guerras,L Guerras, I I HerasHeras,, N N HerasHeras,,S Herrero,S Herrero, JJ JJ LahuertaLahuerta,, A LeA Leóón,n, A MartA Martíín,n, M MartM Martíínez,nez,MA MA MontalbanMontalban,, E Montserrat,E Montserrat, JM Moraleda,JM Moraleda, MJ Moro,MJ Moro, JL Navarro,JL Navarro,J J NomdedeuNomdedeu,, R R NuNuññezez,, E Ojeda,E Ojeda, F Ortega, F Ortega, F OrtuF Ortuñño,o,L Palomera,L Palomera, A A PandiellaPandiella,, JA Portero,JA Portero, A Prados,A Prados, A Prieto,A Prieto,F F ProsperProsper,, F Ramos,F Ramos, MJ MJ RodriguezRodriguez,, C C RozmanRozman,, M M RozmanRozman,,A Ruiz,A Ruiz, F RuizF Ruiz--Cabello,Cabello, M Sanz,M Sanz, A A SempereSempere D D SubiraSubira,,B Suquia,B Suquia, JF Tomas,JF Tomas, M Tormo,M Tormo, A UrbanoA Urbano T T VallespiVallespi,,JL Velasco,JL Velasco, V Vicente,V Vicente, N N VillamorVillamor,, J J VillarrubiaVillarrubia,, S S WoessnerWoessner

EuroFlow consortium aims atinnovation in flow cytometry

OBRIGADOOBRIGADO

10 10 10 10 100 1 2 3 4

7938.050CD45 PECY5 ->CD45 PC5CD45 PC5

TR

AN

SFO

RM

ED

SSC

TR

AN

SFO

RM

ED

SSC

10 10 10 10 100 1 2 3 4

7938.050CD45 PECY5 ->CD45 PC5 CD45 PC5

CD

34C

D34

-- PE

P

E

10 10 10 10 100 1 2 3 4

7938.052CD117 APC ->CD117 APCCD117 APC

CD

34C

D34

-- PE

P

E

ACUTE MAST CELL LEUKEMIA

PHENOTYPIC CHANGES DURING NORMAL MAST CELL DIFFERENTIATION

CD34CD203c

FcεεεεRI

CD117

cyTryptase

ACUTE LEUKEMIAS OF MAST CELLS

Phenotypic characteristics:

Associations:

· CD33++, CD7-/+, CD34-/+, tryptase-/+,CD117+/+++, FCRεεεεI-/+, HLADR-/+

· Chronic myeloid leukaemia· Systemic mastocytosis· “De novo” AML: inv(16), AML1/ETO+

Coexistence of KIT mutation

LAL –B Fenotipo y citogenética:

• t(9;22)* Sensitivity 100%, Specificity >90%CD19+ CD10+ CD34++ CD38-/d CD13d

• 11q23♣♣♣♣ Sensitivity 95%, Specificity 85%CD19+ CD10- CD20-CD34+CD15+CD65+ 7.1+

• t(12;21)♦♦♦♦ Sensitivity 86%, Specificity 100%CD19+ CD10+ CD34d CD45-/d DR++

*Leukemia 15:406 ♣♣♣♣ Am J Clin Pathol 111:467 ♦♦♦♦ Leukemia14:1225

IMMUNOPHENOTYPE OF NEOPLASTIC B-CELL PRECURSORS

103

102

101

BAND/NEUTROPHIL

METAMYELOCYTEMYELOCYTEPROMYELOCYTEMYELOBLAST

CD34

HLA-DR CD117

CD13

CD33

CD11b

CD64

CD65

CD54

CD10

CD35

CD13

PHENOTYPIC CHANGES DURING NORMAL NEUTROPHIL MATURATION

MPO

CD15

CD16

ABERRANT PHENOTYPES : THE t(15;17) MODEL

BAND/NEUTROPHIL

METAMYELOCYTEMYELOCYTEPROMYELOCYTE

HLA-DR CD117

CD13

CD33

CD11b

CD64

CD65

CD54

CD10

CD35

CD13MPO

CD15

CD16

10 10 10 10 100 1 2 3 4

11117.003CD 11b FITC ->

10 10 10 10 100 1 2 3 4

9954.007CD11b FITC ->

10 10 10 10 100 1 2 3 4

11117.007CD 15 FITC ->

10 10 10 10 100 1 2 3 4

M3 CD15+ PROMIELOSCD15 FITC ->

CD11b-FITC CD11b-FITC

CD15-FITCCD15-FITC

CD13-PE

CD13-PE

T-SSC

T-SSC

OrfaoOrfao et al, et al, HaematologicaHaematologica, 1999, 1999

CD15

CD11c

MFI values compaired to copy numbers of BCR-ABL (RQ-PCR)

MFI values compaired to ratio of copy numbers of BCR-ABL (RQ-PCR)