UTILIDAD DE LA CITOMETRIA DE FLUJO EN EL … · UTILIDAD DE LA CITOMETRIA DE FLUJO EN EL...

1

UTILIDAD DE LA CITOMETRIA DE FLUJO EN EL DIAGNÓSTICO, CLASIFICACIÓN Y

MONITORIZACIÓN DE HEMOPATÍAS MALIGNAS

CANCER RESEARCH CENTER, UNIVERSITY & CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of UNIVERSITY HOSPITAL of SALAMANCA (SPAIN) SALAMANCA (SPAIN)

CursoCurso AvanzadoAvanzado de de ActualizaciActualizacióónn en en OncohematologOncohematologííaa porpor citometriacitometria de de flujoflujoBuenos Aires, 30 de mayo de 2011Buenos Aires, 30 de mayo de 2011

IMMUNOPHENOTYPING OFHAEMATOLOGICAL MALIGNANCIES

- 1953/1994: From the development of the instruments& techniques to the WHO classificationof haematological malignancies.

- 1994/2006: The ability to specifically identifyleukaemic cells: from normal phenotypesto aberrant phenotypic profiles.

- 2006/-: Recent contributions of immuno-phenotyping of haematologicalmalignancies: pointing to the future.

- 1953: The “Coulter” principle and instrument development- 1965/68: Multiparameter and multicolour flow cytometry

Marv van DillaH. Crissman

Joe Gray1970


Wallace Coulter

Wofgang Göhde

- 1953: The “Coulter” principle and instrument development- 1965/68: Multiparameter and multicolour flow cytometry- 1970: FACS: “fluorescence activated cell sorter”.


L. Herzenberg & FACS II (1976)

- 1953: The “Coulter” principle and instrument development- 1965/68: Multiparameter and multicolour flow cytometry- 1970: FACS: “fluorescence activated cell sorter”.

Len & LeeHerzenberg

IMMUNOPHENOTYPING OFHAEMATOLOGICAL MALIGNANCIES FluorescenceFluorescence activatedactivated cellcell sortingsorting

Prepared by A.Salvador

2

¿WHERE CAN I APPLY FLOW CYTOMETRY?

César MILSTEIN

Gunter VALET

Kohler G, Milstein C. Continuous cultures offused cells secreting antibody of pre-definedspecificity. Nature 1975;256:495-497.

- 1953: The “Coulter” principle and instrument development- 1965/68: Multiparameter and multicolour flow cytometry- 1970: FACS: “fluorescence activated cell sorter”.- 1975: Production of monoclonal antibodies


Tabla 2.- Immunological classification

of ALL

Phenotype

B-ALL Ig+

T-ALL SER+

non-T non-B ALL cALLA+

cALLA- 10 10 10 10 100 1 2 3 4

nTDT

CyC

D3

- 1953: The “Coulter” principle and instrument development- 1965/68: Multiparameter and multicolour flow cytometry- 1970: FACS: “fluorescence activated cell sorter”.- 1975: Production of monoclonal antibodies- 1978/80: Immunophenotyping of leukemia cells

Immunophenotypic classification of ALL- 1981: Definition of aberrant marker expression- 1985: Use of Immunophenotyping to classify FAB M7


David MASON


Immunophenotypic classification of ALL- 1981: Definition of aberrant marker expression- 1985: Use of Immunophenotyping to classify FAB M7- 1985: APAAP technique


Loken et al, Blood, 1987 Loken et al, Blood, 1987



Immunophenotypic classification of ALL- 1981: Definition of aberrant marker expression- 1985: Use of Immunophenotyping to classify FAB M7- 1985: APAAP technique- 1986: Benchtop 3-color flow cytometers- 1987: Immunophenotypic analysis of normal hematopoiesis


Immunophenotypic classification of ALL- 1981: Definition of aberrant marker expression- 1985: Use of Immunophenotyping to classify FAB M7- 1985: APAAP technique- 1986: Benchtop 3-color flow cytometers- 1987: Immunophenotypic analysis of normal hematopoiesis- 1988: Second MIC classification of haematological malignancies- 1993: CD45-based blast cell gating/identification- 1994: REAL classification of lymphoid neoplasias- 1997: WHO classification of lymphoid neoplasias


3

FCM IMMUNOPHENOTYPING IN THE 80`S: FCM IMMUNOPHENOTYPING IN THE 80`S: PANELS OF REAGENTS AND TECHNIQUESPANELS OF REAGENTS AND TECHNIQUES

PANELS OF REAGENTS:PANELS OF REAGENTS:

-- PanelsPanels ofof relevantrelevant markersmarkers forfor thethe diagnosticdiagnosticclassificationclassification ofof patientspatients suspectedsuspected ofof::

-- AML, ALLAML, ALL-- BB--CLPD, TCLPD, T--CLPDCLPD

TECHNIQUES:TECHNIQUES:

-- IsolationIsolation ofof MNC MNC -- DifficultDifficult toto distinguishdistinguish normal/normal/leukemicleukemic cellscells

-- IndirectIndirect andand directdirect IF IF –– FewFew fluorochromefluorochrome conjugatedconjugated MAbMAb availableavailable

-- Single Single stainingsstainings –– FewFew fluorochromefluorochrome availableavailable

DIAGNOSIS OF HAEMATOLOGICAL MALIGNANCIESDIAGNOSIS OF HAEMATOLOGICAL MALIGNANCIES

ClinicalClinical symptomssymptoms LaboratoryLaboratoryandand signssigns findingsfindings

MorphologyMorphology + + cytochemistrycytochemistry

CMDCMDMDS MDS ImmunophenotypingImmunophenotyping MRDMRDMGMG

AcuteAcute leukemiasleukemias ChronicChronic lymphoidlymphoidleukemiasleukemias

CLINICAL UTILITY OF IMMUNOPHENOTYPING OF HAEMATOLOGICAL MALIGNANCIES

- Acute leukaemias:- Lineage assignment (myeloid vs lymphoid -B or T-)

- Diagnosis of biphenotypic leukaemias/mixed lymphoid/myeloid- Phenotypic classification of B-cell precursor ALL- Phenotypic classification of T-ALL- Lineage subclassification of AML (e.g: AML with monocytic maturation)

- Chronic lymphoproliferative disorders:- Diagnosis of T & B-cell clonality- Phenotypic classification of T/NK-CLPD- Phenotypic classification of B-cell CLPD

EGIL: DEFINITION OF BAL

Score

c/mCD3

B-Lineage T-Lineage Myeloid lineage

2 cCD79a MPO

cIgM TCR Lisozyme

cCD22

1 CD2, CD5 CD13, CD33

CD20 CD8, CD10 CD117, CDw65

0.5 Tdt, CD24 Tdt, CD7 CD14, CD15

CD1a CD64

Criteria: > 2 points

IMMUNOPHENOTYPIC CLASSIFICATION OF ACUTE LEUKAEMIAS

IMMUNOPHENOTYPIC PATTERNS OF DIFFERENT IMMUNOPHENOTYPIC PATTERNS OF DIFFERENT TYPES OF BTYPES OF B--CLPD CLPD ((Orfao et al, In: “B-CLL”.Humana Press, 2004)

sIgsIg CD5 CD10 CD20 CD5 CD10 CD20 CD11cCD11c CD23 CD24 CD25 CD38 CD43 CD23 CD24 CD25 CD38 CD43 CD79bCD79b CD103 FMC7CD103 FMC7

BB--CLLCLL d d ++ -- d d --/+ /+ ++++ + + + + --/+ /+ ++ d d -- --

PLL + PLL + --/+ /+ -- + + --/+ /+ --/+ + /+ + --/+ /+ --/+ /+ --/+ + /+ + -- ++

HCL + HCL + -- -- ++ ++ ++++ -- --/+ /+ ++++ -- -- + + + + ++

SMZLSMZL + + --/+ /+ -- + + + + -- + + --/+ /+ -- -- + + --/+ +/+ +

LPLLPL + + -- -- + + -- -- + + + + --/+ /+ -- + + -- --/+ /+

MCL + MCL + ++ -- + + --/+ /+ -- + + --/+ /+ -- ++ + + -- --/+/+

FL + FL + -- ++ + + --/+ /+ --/d + /d + --/+ /+ ++ -- + + -- ++

LDBCL + LDBCL + -- -- + + --/+ /+ -- --/+ /+ -- + + -- + + -- ++

BL BL --/+ /+ -- ++ + + -- -- + + -- ++++ --/+ /+ --/+ /+ -- ++

0 256 512 768 1024

10112.001

FSC-Height ->

TR

AN

SF

OR

ME

D S

SC

FSC Height

MNCMNC

10 10 10 10 100 1 2 3 4

EGV47955.003

CD4 APC ->CD4 APC

CD

8 P

erC

P

EGV47955.003

CD3 FITC ->

10 10 10 10 100 1 2 3 4

CD3 APC

TRADITIONAL FCM PHENOTYPING

MNC

MNC

Ficoll

Blood

15%15%

CriteriaCriteria forfor positivitypositivity: >20%: >20%

4

10 10 10 10 100 1 2 3 4

EGV47955.003

CD4 APC ->CD4 APC

CD8

PerC

P

MULTICOLOR FLOW CYTOMETRY vsSINGLE-STAININGS IN ONE TUBE

10 10 10 10 100 1 2 3 4

65295.003CD19 PerCPCY5.5 ->

CD3

APC

CD19 PerCP/Cy5.5


Immunophenotypic classification of ALL- 1981: Definition of aberrant marker expression- 1985: Use of Immunophenotyping to classify FAB M7- 1985: APAAP technique- 1986: Benchtop 3-color flow cytometers- 1987: Immunophenotypic analysis of normal hematopoiesis- 1988: Second MIC classification of haematological malignancies- 1993: CD45-based blast cell gating/identification- 1994: REAL classification of lymphoid neoplasias- 1997: WHO classification of lymphoid neoplasias






FLOW CYTOMETRY:FLOW CYTOMETRY:TYPE OF INFORMATIONTYPE OF INFORMATION

-- IdentificationIdentification ofof cellcell populationspopulations

-- EnumerationEnumeration ofof cellcell numbersnumbers

-- CharacterizationCharacterization ofof cellcell populationspopulations

Identification of different granulocytic subpopulations in childhood BM

E.G. van Lochem et al., Cytometry Part B 2004; 60B: 1-13.

FSC

SS

C

CD11b-APC

CD

13

-PE

CD16-FITC

CD

13-P

E

CD13-PE

SS

C

staining 5CD16/CD13/CD45/CD11b

CD16-FITC

SS

C

CD16-FITC

CD

11

b- A

PC

Myelo/monoblast Promyelocyte MetamyelocyteMyelocyte Neutrophill

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

nTDTnTDT FITCFITC

Cy

MP

OC

yM

PO

PE

PE

10

01

01

10

21

03

10

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

02

56

51

27

68

10

24

a

10 10 10 10 100 1 2 3 41024

TRANSFORMED SSCTRANSFORMED SSC

0 256 512 768

bCD

45

PE

RC

PC

D45

PE

RC

P1

00

10

11

02

10

31

04

b

Matarraz S et al. Leukemia 2008

Neutrophil precursors

B-cell precursors

5

10 10 10 10 100 1 2 3 4

CD38 FITC ->

TR

AN

SF

OR

ME

D S

SC

->

10 10 10 10 100 1 2 3 4

CD38 FITC ->

CD

138 P

erC

P/C

y5 -

>

CD38-FITC

CD38-FITC gated PC

T-S

SC

CD13

8-Pe

rCP/

Cy5.

5

MONOCLONAL GAMMOPATHIES: IDENTIFICATION OF CLONAL PLASMA CELLS

ClonalPC

Normal PC

CD

56

PE

9

1

D

C

CP

A

54

DC

CD19

-Pcp

Cy5

CD56-PE CD45-A

PC

Perez-Andres, J Biol Reg, 2004

DIAGNOSIS OF HAEMATOLOGICAL MALIGNANCIESDIAGNOSIS OF HAEMATOLOGICAL MALIGNANCIES

ClinicalClinical symptomssymptoms LaboratoryLaboratoryandand signssigns findingsfindings

MorphologyMorphology + + cytochemistrycytochemistry

CytogeneticsCytogenetics

ImmunophenotypingImmunophenotyping

Molecular Molecular biologybiology/FISH/FISH

*0.35% of all B-cells & O.O3% of all leucocytes

Clonal B cells

Normal B cells

ImmunophenotypicImmunophenotypic identificationidentification ofof PB PB BB--cellscells withwith a CLLa CLL--likelike phenotypephenotype

Nieto et al, Blood 2009

MINIMAL RESIDUAL DISEASE IN B-CLL

Brugiatelli M et al.(Cancer 1989)

Robertson LE et al.(Blood 1992)

Leonormand B et al.(Leukemia 1994)Cabezudo E et al.(Leukemia, 1997)

García-Vela A et al.(Leukemia, 1999)

Rawstron AC et al.(Blood 2001)

Maloum K et al.(Br J Haematol 2002)

Gupta R et al.(Am J Clin Pathol 2004)

Bottcher S et al.(Leukemia 2004)Moreton P et al.

(J Clin Oncol 2005)Montillo M et al.

(Cancer Invest 2005)

Sensitivity

10-2

10-2

10-3

10-3

10-4

10-4

10-4

10-3

10-4

10-5

10-5

Aberrant criteria

sIgκ+/sIgλ+ ratio

sIgκ+/sIgλ+ ratio

CD19+/CD5+

CD19+/CD5+

CD19+/CD79b+d

CD19+/CD20+d/CD5+/CD79b+d


CD19+/CD5+

CD19+/CD5+/CD43+/CD20+d



Prognostic value

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Not analyzed

Not analyzed

Yes

Yes

METHODS FOR MRD METHODS FOR MRD INVESTIGATIONINVESTIGATION

101000

101011

101022

101033

101044

101055

101066

101077

101088

101099

10101010

10101111

NeoplasticNeoplastic cellscellsSensitivitySensitivitySensitivity MorphologyMorphology, , CytogeneticsCytogenetics, ,

SouthernSouthern--BlotBlot,,

FCM DNA FCM DNA aneuploidyaneuploidy

F.I.S.HF.I.S.H

FlowFlow cytometrycytometry

P.C.RP.C.R..1010--66

1010--55

1010--44

1010--33

1010--22

FCM IMMUNOPHENOTYPING IN THE 90`S: FCM IMMUNOPHENOTYPING IN THE 90`S: PANELS OF REAGENTS AND TECHNIQUESPANELS OF REAGENTS AND TECHNIQUES

PANELS OF REAGENTS:PANELS OF REAGENTS:

-- PanelsPanels ofof informativeinformative combinationscombinations ofof reagentsreagents forfor::

-- AML, ALL, BALAML, ALL, BAL-- MM, WM, MGUSMM, WM, MGUS-- BB--CLPD, TCLPD, T--CLPDCLPD-- MDSMDS

TECHNIQUES:TECHNIQUES:

-- NonNon--NRBC NRBC lysislysis -- DistinctDistinct normal normal vsvs leukemicleukemic phenotypesphenotypes

-- DirectDirect IF IF –– ManyMany fluorochromefluorochrome conjugatedconjugated MAbMAb availableavailable

-- MultipleMultiple stainingsstainings –– IncreasedIncreased numbernumber ofof fluorochromefluorochrome availableavailable

Diagnosis & follow-up ofMRD in acute

leukaemias, CLPD & MM

6

1. Making the diagnosis

Normal ↔ reactive/regenerating ↔ malignantAnnually > 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

JJM van Dongen

What problems are we experiencing?What problems are we experiencing?

-- ManyMany reagentsreagents: : costlycostly andand complexcomplex

-- NeedNeed expertiseexpertise in normal (& in normal (& referencereference) ) cellcell populationspopulations

-- TimeTime consumingconsuming

-- TechnicalTechnical limitationslimitations

-- ManyMany (my) (my) strategiesstrategies toto reachreach a similar a similar resultresult butbutsuboptimalsuboptimal

-- NotNot standardizedstandardized: : reproduciblyreproducibly harmonizedharmonized? ?

-- PartialPartial andand more more limitedlimited clinicalclinical utilityutility thanthan expectedexpected

STANDARDIZATION EFFORTS FORSTANDARDIZATION EFFORTS FORIMMUNOPHENOTYPIC STUDIESIMMUNOPHENOTYPIC STUDIES

-- CLSI CLSI ((ClinicalClinical LaboratoryLaboratory Standards Standards InstituteInstitute):):-- StetlerStetler--Stevenson et al.: Stevenson et al.: ClinicalClinical flowflow cytometriccytometric analysisanalysis ofofneoplasticneoplastic hematolymphoidhematolymphoid cellscells; ; ApprovedApproved guidelineguideline. CLSI . CLSI documentdocument H43H43--A2. CLSI, 2007A2. CLSI, 2007

-- CCSCCS ((ClinicalClinical CytometryCytometry SocietySociety):):-- Davis et al: 2006 Bethesda International Davis et al: 2006 Bethesda International ConsensusConsensus

recommendationsrecommendations onon thethe flowflow cytometriccytometric immunophenotypicimmunophenotypicanalysisanalysis ofof hematolymphoidhematolymphoid neoplasias. neoplasias. ClinClin CytometryCytometry, 72B, , 72B, 2007.2007.

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

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

-- Consenso LatinoamericanoConsenso Latinoamericano ((ClinClin CytometryCytometry, 1998 y 2006), 1998 y 2006)

Standardization in diagnostic flow cytometry

HOWEVER: Standardization according to GLP guidelines demands for much higher levels of standardization

EuroFlow standardization aims at:– usage of comparable flow cytometers (3 lasers and ≥ 8 colors)

– full standardization of instrument settings (e.g. based on standard beads)– standardized laboratory protocols and immunostaining procedures (SOP’s)

– careful selection of optimal antibody clones per marker/CD code– selection of optimal 8-color antibody combinations and fluorochromes– design of combinations of multiple 8-color tubes: estimation and APS view

– new software for fast and easy data analysis with automated pattern recognition– recognition of normal and abnormal leukocyte subsets (complete differentiation

pathways) with the same immunostaining protocols– mapping of new patient samples against large data base of earlier collected

patient samples, analyzed with the same immunostaining protocol

Standardization according to literature generally refers to:– lists of CD codes and markers per disease category

– rarely a specific antibody is recommended and (almost) never a fluorochrome is proposed

CLINICAL APPLICATIONS OF FLOW CYTOMETRY

Microscopy Flow cytometry

Hybridoma technology

Monoclonal antibodies

Fluorochrome-conjugates

From research laboratories to clinical diagnostics

Digital instruments

>4 color flow cytometers

Higher analytical speed

Exponentially growing amount of complexinformation/data

70s-90s

XXI century





7

GUEST EDITORIAL: The Continuing Evolution of Hematology

BEREND HOUWEN

Laboratory Hematology 8:204

© 2002 Carden Jennings Publishing Co., Ltd.

EXCERPT:

The evolution of laboratory hematology is a continual process, as

the papers in the following Special Section demonstrate. These papers

cover a variety of topics related to the practice of laboratory

hematology. Application of extended differential capabilities decreases

the need for routine microscopic intervention for samples with

nucleated red blood cells. Similarly, as careful examination of flagging

performance shows, increased productivity can be achieved by fewer

morphology reviews. In most laboratories, rules for review of

hematology results are applied by the technologists. Many times these

rules are quite informal, but when formalized and embedded in a

computer-based algorithm, they can form a powerful management

tool, as illustrated in one of the papers.

THANK THANK YOUYOU

RECENT CONTRIBUTIONS OF IMMUNO-PHENOTYPING IN THE DIAGNOSIS OF

HAEMATOLOGICAL MALIGNANCIES

-- StandardizationStandardization ofof immunophenotypicimmunophenotypic analysesanalyses

-- DiagnosisDiagnosis ofof clonalclonal multilineagemultilineage diseasedisease

-- RapidRapid screeningscreening ofof relevantrelevant cytogeneticcytogenetic subgroupssubgroups ofofacuteacute leukaemiasleukaemias

-- OntogenicOntogenic characterizationcharacterization ofof CLPDCLPD

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

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

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.

Synchronized light scatter experiments

Normal PB samples processed according to

the standardized EuroFlow sample preparation protocol

7 different normal PB samples acquired in 7 different centers

“Local” settings EuroFlow settings

8

BCLPD panel: classification of an atypical case vs the reference WHO diagnostic groups

Responsible scientist: Sebastian Bottcher

B-CLPD: Comparative analysis of “our case”vs multiple reference groups

Responsible scientists: Sebastian Bottcher

Costa et al, Leukemia, 2010

B-CLPD:Comparative analysis of “our case”vs multiple reference groups

Responsible scientist: Sebastian Bottcher

Costa et al, Leukemia, 2010

REFERENCE DATAFILES: NORMAL vs. CLL B-CELLS

Normal PB (n=8) CLL cases (n=6)

Case number

CD19

-PEC

y7

Normal PB CD19+ B-cells

Case number

CD19

-PEC

y7

CD19+ CLL B-cells

Case number

CD19

-PEC

y7

Normal PB B cells

CLL cases

LAIR1 9.3

CD5 8.6

CD79b 8.2

IgM 8.1

COMPARE A CASE VS NORMAL & CLL B-CELLS

CLL cases (n=6)Normal PB (n=8)

Case number

CD19

-PEC

y7

CD19+ CLL B-cells

SSC

CD19-PECy7CD19-PECy7

SSC

Normal PB B cells

CLL cases

Case number

CD19

PEC

y7

CLLNormal

PB B-cells



-- StandardizationStandardization ofof immunophenotypicimmunophenotypic analysesanalyses

-- DiagnosisDiagnosis ofof clonalclonal multilineagemultilineage diseasedisease

-- RapidRapid screeningscreening ofof relevantrelevant cytogeneticcytogenetic subgroupssubgroups ofofacuteacute leukaemiasleukaemias

-- OntogenicOntogenic characterizationcharacterization ofof CLPDCLPD

9

MAST CELLS

MONOCYTES

LYMPHOCYTES

EOSINOPHILS

NEUTROPHILS

CD34+ HPC

PURIFICATION OF BM MAST CELLS IN MASTOCYTOSIS

Case 1 Case 2

KIT MUTATION IN SM: PATTERN OF CELLULAR INVOLVEMENT

Eos Eos

NeutNeut

Mo Mo

LymphsLymphsNRC

NRC

CD34+HPC

CD34+HPC

MastCells

MastCells

KIT mutation

FREQUENCY OF MULTILINEAL KIT MUTATION IN MASTOCYTOSIS (n=202)

MC & CD34+ HPC

Only MC

Myeloid lineages

Myeloid & lymphoid lineages

% O

F CA

SES

SUBTYPE OF MASTOCYTOSISTeodosio et al, J Alllergy Clin Immunol 2010

SM: PROGNOSTIC FACTORS FOR SM: PROGNOSTIC FACTORS FOR PROGRESSIONPROGRESSION--FREE SURVIVALFREE SURVIVAL

VARIABLE UNIVARIATE MULTIVARIATE

ANALYSIS ANALYSIS

RR (IC 95%) p-value RR (IC 95%) p-value

Age >60 13.2 (1.2-150) 0.03 NS

Cytopenias 16.6 (1.5-179) 0.02 NS

High β2-microglobulin 1.65 (1.2-2.3) 0.003 1.90 (1.2-2.9) 0.003

Germinal KIT mutation 8.01 (1.3-49) 0.02 13.3 (1.4-124) 0.02

Escribano et al, J Allergy Clin Immunol, 2009



-- StandardizationStandardization ofof immunophenotypicimmunophenotypic analysesanalyses ((sessionsession V)V)

-- Diagnosis Diagnosis ofof clonalclonal multilineagemultilineage diseasedisease ((sessionsession I)I)

-- RapidRapid screeningscreening ofof relevantrelevant cytogeneticcytogenetic subgroupssubgroups ofofacuteacute leukaemiasleukaemias ((sessionsession III)III)

-- OntogenicOntogenic characterizationcharacterization ofof CLPD CLPD ((SessionSession III)III)

Percentage of cases

ag

e(y

ea

rs)

0 10 20 30 40 50 60 70 80 90 100

5.7%

5.2%

15.5%

17.8%

21.5%

50%

40

45

50

55

60

65

70

75

80

85

90

95

FrequencyFrequency ofof CLLCLL--likelike MBL MBL cellscells in in healthyhealthy individualsindividuals

MBL: MBL: 80/639 (12.5%)80/639 (12.5%)

10

0% (0/45)0% (0/45)8% (4/45)36% (16/45)0.5Population

(Salamanca)

(6)*

0% (0/10)0% (0/21)18% (4/22)39% (15/38)9Population (Leeds) (8)

3% (1/33)6% (2/33)21% (7/33)58% (19/33)3141Clinic (Leeds)

(8)

3% (4/126)2% (2/126)18% (21/126)44% (56/126)2757Clinic

(Mayo) (12,13)

7% (23/325)18% (58/235)16% (53/325)54%

(178/238)>5,000/uLCLL (17)

17p deletion11q deletionTrisomy 1213q14

deletionMedian

CLL count

Rawstron et al, Cytometry B, 2010 (in press)

CLL vs CLL-LIKE MBL:Genetic features of clonal B-lymphocytes

NoNo CLL-

associated2/7 (29%)0.4%0.5

Population

(Salamanca)

(6)

No4-59/6136/51 (70%)7%1.0Population

(Italy) (4)

Yes3-07, 3-23, 4-3418/20 (90%)80%9Population

(Leeds) (8)

Yes3-07, 4-3412/16 (75%)25%26Familial

(Duke) (24)

Yes3-07, 1-69, 4-34,

3-2384/109 (77%)>95%2757

Clinic (Mayo)

(12,13)

Yes3-07, 3-23, 4-3418/20 (90%)>95%3141Clinic (Leeds)

(8)

-3-07, 1-69, 4-34,

3-23

534/927

(57.6%)>95%>5,000CLL (25)

Similar to CLL?

Predominant

CLL cell IGHV

gene

Cases with

<98% IGHV

homology

CLL-like B-

cells (median

%)

Median

CLL cell

count

Source

CLL vs CLL-LIKE MBL:Biological features of clonal B-lymphocytes

Rawstron et al, Cytometry B, 2010 (in press)

100%

50%

0%10.10.01 5 10 20 40 5030

Whole series

10.10.01 5 10 20 40 5030

40 - 59 years

10.10.01 5 10 20 40 5030

60 - 69 years

100%

50%

0%

100%

50%

0%

100%

50%

0%

≥≥≥≥ 70 years

PB Volume (mL)10.10.01 10 20 40 5030

% ofcases witha CLL-likeclone

5

70%

100% 100%

18%

46%32%

88%

62%36%

100%100%100%

DETECTED FREQUENCY

FREQUENCY OF CLL-like MBL IN HEALTHY ADULTS

100%

50%

0%10.10.01 5 10 20 40 5030

Whole series

10.10.01 5 10 20 40 5030

40 - 59 years

10.10.01 5 10 20 40 5030

60 - 69 years

100%

50%

0%

100%

50%

0%

100%

50%

0%

≥≥≥≥ 70 years

PB Volume (mL)10.10.01 10 20 40 5030

% ofcases witha CLL-likeclone

5

70%

100% 100%

18%

46%32%

88%

62%36%

100%100%100%

CALCULATED FREQUENCYDETECTED FREQUENCY

FREQUENCY OF CLL-like MBL IN HEALTHY ADULTS

Volume of PB analyzed: 1mL

Volume of PB analyzed: 50mL

% of CLL-like cells: 0.02% of the whole B-cell population

ImmunophenotypicImmunophenotypic identificationidentification ofof CLLCLL--likelike clonalclonal BB--cellscells in in healthyhealthy adultsadults

FREQUENCY OF CLLFREQUENCY OF CLL--LIKE MBL IN HEALTHY LIKE MBL IN HEALTHY ADULTS: ANALYSIS OF 50 ADULTS: ANALYSIS OF 50 mLmL OF PB OF PB

Number of positive cases: 8/9

- Frequency: < 0.01%- Count: < 0.12 CLL-like B-cells/µµµµL

0,032 cel/uL0,018 cel/uL

0,016%0,009%

0,49%0,27%

BiclonalK+dL+d

M73CASE 9

0,009 cel/uL0,013%0,14%MonoclonalK+dM88CASE 8


0,02%0,01%

0,18%0,095%

BiclonalK+dL+d

F82CASE 7



NDM87CASE 4


0,008%0,006%

0,11%0,08%

BiclonalK+dL+d

F72CASE 3

0,0018 cel/uL0,0009 cel/uL

0,002%0,001%

0,28%0,14%

BiclonalK+dL+d

F77CASE 2

0,066 cel/uL0,041%0,8%MonoclonalL+dM78CASE 1

ABSOLUTE COUNT

% FROM B-CELLS

% OF WBC (x10-3)

MONOCLONALsIgGENDER AGECASE ID

11

CIC/UNIVERSITY OF CIC/UNIVERSITY OF SALAMANCA SALAMANCA

L Escribano L Escribano I I AlvarezAlvarez TwoseTwoseL Sanchez MuL Sanchez MuññozozI SI Sáánchez Matasnchez MatasA MatitoA Matito

MAST CELL UNIT,MAST CELL UNIT,HOSPITAL VIRGEN del VALLEHOSPITAL VIRGEN del VALLE

TOLEDOTOLEDO

EuroFlow consortium aims at innovation in flow cytometry

www.euroflow.org

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