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

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

Multicolor Immunophenotyping: Standardization and ApplicationsMarch 9-11, 2012 TMH, Mumbay (India)

B-CELL NEOPLASMS (PRECURSOR AND MATURE)

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

- B-cell precursor (immature) derived acute lymphoblastic leukemia/lymphoblastic

lymphoma

- Mature (peripheral) B-cell neoplasms

WHO CLASSIFICATION OF LYMPHOID MALIGNANCIES

(2002-2008)

B-cell precursor acute lymphoblastic leukemia/lymphoma

(B-ALL)• B Lymphoblastic Leukemia/Lymphoma, not

otherwise specified

• B lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities

– BCP-ALL/LL with t(9:22) (q34;q11.2); BCR/ABL

– BCP-ALL/LL with t(v;11q23); MLL rearranged

– BCP-ALL/LL with t(12;21) (p13;q22); TEL/AML1 (ETV6-RUNX1)

– BCP-ALL/LL with hyperdiploidy

– BCP-ALL/LL with hypodiploidy (Hypodiploid ALL)

– BCP-ALL with t(5;14)(q31;q32)(IL3-IGH)

– BCP-ALL with t(1;19)(Q23;P13.3); (E2A-PBX1; TCF3/PBX1)

ALOT

BCP-ALL T-ALL AML/MDS

4 tubes 4 tubes 4 to 7 tubes

1 tube

ALOT: B-cell precursor ALL

BM stained with ALOT 8-color tube

CyCD3CD7sCD3CD19

CyCD79aCyMPOCD45CD34

Responsible scientist: Ludovic Lhermitte

ALOT (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 MycyCD79a PE X B, T

CD34 PerCP Cy5.5 X X -CD19 PE CY7 X B, MyCD7 APC X X T, My

smCD3 APC H7 X TcyCD3 Pacific Blue X TCD45 PO X X -

ALOT

EuroFlow ALOT: assessment of blast cell lineage

Van Dongen et al: EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes. Leukemia 2012 (under revision)

Precursor-B ALL:

- B I (CD19+/cCD79a+/CD10-)- B II (CD10+/Ig-)- B III (cIgm+)- B IV (sIg+)

T-ALL:

- T I (CD7+,cCD3+)- T II(CD2+ &/or CD5+ &/or CD8+- T III (CD1a+)- T IV (CD1a-,CD3+)

CLASSIFICATION OF PRECURSOR-B ALL & T-ALL

BCP-ALL panel


CD20 CD45 CD58 CD66c CD34 CD19 CD10 CD38

smIgK CD45 cyIgM CD33 CD34 CD19smIgM

+CD117 smIgL

CD9 CD45 TdT CD13 CD34 CD19 CD22 CD24

CD21 CD45CD15

+CDw65 NG2 CD34 CD19 CD123 CD81

BCP-ALL

BC

P-A

LL

BCP-ALL panel




+CD117 smIgL


CD21 CD45CD15


BC

P-A

LL

BCP-ALL panel




+CD117 smIgL


CD21 CD45CD15



cyCD3 CD45 cyMPO cyCD79a CD34 CD19 CD7 smCD3ALO

TB

CP

-ALL

BCP-ALL panel





+CD117 smIgL


CD21 CD45CD15


ALO

TB

CP

-ALL

BCP-ALL panel





+CD117 smIgL


CD21 CD45CD15


Positive Diagnosis

ALO

TB

CP

-ALL

BCP-ALL panel





+CD117 smIgL


CD21 CD45CD15


Differential Diagnosis&

Ambiguous lineage acute leukemia

ALO

TB

CP

-ALL

BCP-ALL panel





+CD117 smIgL


CD21 CD45CD15


Maturation stage (EGIL)

ALO

TB

CP

-ALL

BCP-ALL panel





+CD117 smIgL


CD21 CD45CD15


Alternative classification

Immunophenotypic features associated with well-defined molecular aberrations

ALO

TB

CP

-ALL

BCP-ALL panel





+CD117 smIgL


CD21 CD45CD15


Prognosis markers

ALO

TB

CP

-ALL

BCP-ALL panel





+CD117 smIgL


CD21 CD45CD15


LAP markers

ALO

TB

CP

-ALL

BCP-ALL panel





+CD117 smIgL


CD21 CD45CD15


markers present in the virtual merged

and calculated tube Panel

orie

ntatio

n

Positiv

e dia

gnosis

Differ

entia

l dia

gnosis

Ambig

uous lin

eage

acute

leuke

mia

scr

eenin

g

Mat

uratio

n of a

rrest

sta

ging

Prognosi

s

Phenoty

pic fe

ature

s as

soci

ated

with c

hrom

osam

al a

bnormal

ity

MRD L

AP det

ectio

n

ALOT X X X XBCP-ALL - Tube 1 X X X X XBCP-ALL - Tube 2 X X X X XBCP-ALL - Tube 3 X X X X XBCP-ALL - Tube 4 X X X X X

ALO

TB

CP

-ALL

BCP-ALL panel





+CD117 smIgL


CD21 CD45CD15


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

BCP-ALL:DETECTION OF ABERRANT PHENOTYPES

Mix of 3 different regenerating B cell populations (Haematogones)

BCP-ALL blast cells

0 256 512 768 1024

RM30243.100FL2-Area ->

PRECURSOR B-ALL: DNA ANEUPLOIDY

NORMALB-CELLS

NEOPLASTIC

B-CELLS

RESIDUAL NON-B-CELLS RM30243.100

FL2-Area ->0 256 512 768 1024

B-CELL PRECURSOR ALL: DNA ANEUPLOIDY

BCP-ALL Phenotype vs cytogenetics:

• 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

Leukemia 14:1225

IMMUNOPHENOTYPE OF NEOPLASTIC B-CELL

PRECURSORS

10 10 10 10 100 1 2 3 4

HG30672.002CD10 ->

10 10 10 10 100 1 2 3 4

HG30672.006CD34 ->

10 10 10 10 100 1 2 3 4

HG30672.008CD34 ->

10 10 10 10 100 1 2 3 4

HG30672.010CD13 ->

10 10 10 10 100 1 2 3 4

RFR7690.007CD34 ->

10 10 10 10 100 1 2 3 4

RFR7690.008CD10 ->

10 10 10 10 100 1 2 3 4

RFR7690.008CD13 ->

10 10 10 10 100 1 2 3 4

RFR7690.011CD34 ->

10 10 10 10 100 1 2 3 4

UVJ39869.002CD34 PE ->

10 10 10 10 100 1 2 3 4

UVJ39869.004CD13 PE ->

10 10 10 10 100 1 2 3 4

MO21489004CD34 ->

10 10 10 10 100 1 2 3 4

AP36452.003CD10 ->

ADULT PRECURSOR B-ALL: IMMUNOPHENOTYPE OF BCR/ABL+ CASES

Normal BM CD34+ B-cells

DNA diploid Bcr/abl+ ALL

DNA aneuploid Bcr/abl+ ALL

CD10 CD13 CD34 CD34

CD10 CD13 CD34 CD34

CD10 CD13 CD34 CD34

CD

19

CD

19

CD

19

CD

45

CD

45

CD

45

CD

38

CD

38

CD

38

CD

19

CD

19

CD

19

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

transcrip tion

translation

cell lysate

A

B

A

B

A

B

bead

beads coatedw ith 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

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

135

repeated analysis for confirm ationof w eak positiv itynegativep210

p190

result o f R Q -PC R

Weerkamp et al, Leukemia, 2009

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

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

Final Statement about the BCR-ABL RUO testing

The main advantages of the immunobead assay are: 1. Not dependent of the breakpoint position in the fusion gene;

2. No need for special laboratory facilities other then a routine flow cytometer;

3. Providing results within several hours;

4. The possibility to run in parallel to routine immunophenotyping: no extra technician time needed !!;

5. Allowing multiplexing with differently-labeled beads, that can detect different fusion proteins within the same disease category

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

Precursor B-ALL multiplex tube: E2A-PBX1 t(1;19)

Sensitivity for detection on cell line <10%

Specificity E2A-PBX1 (Dec 12, 2008)

0

200

400

600

800

1000

1200

1400

1600

1800

2000

RS(4,11) K562 697 RCH-ACV Kasumi NB4 REH

cell line

MF

I

0

100

200

300

400

500

600

700

800

med

ian

sign

al

*Exp. Performed on April 27th 2007Neg. patientsPos patients

Specificity TEL-AML (July 30th 2008)

0

10000

20000

30000

40000

50000

60000

70000

ME1 RS4,11 K562 697 RCH-ACV

Kasumi NB4 REH

cell line

MF

I

Sensitivity for detection REH cell line in:– WBC : 10-50%– PBMC : 1-10%

TEL-AML in patients

0

10000

20000

30000

40000

50000

60000

70000

dono

r 1

dono

r 2

dono

r 3

dono

r 4

dono

r 5

TE

L-A

ML

patie

nt 1

TE

L-A

ML

patie

nt 2

TE

L-A

ML

patie

nt 3

TE

L-A

ML

patie

nt 4

TE

L-A

ML

patie

nt 5

B-A

LL c

ontr

ol 1

B-A

LL c

ontr

ol 2

B-A

LL c

ontr

ol 3

B-A

LL c

ontr

ol 4

B-A

LL c

ontr

ol 5

healthy control TEL-AML precursor B-ALL w ithouttranslocation

MF

I

Precursor B-ALL multiplex tube: TEL-AML1 t(12;21)

Sensitivity for detection MV4;11 line in:– 697 cell line: 10%– WBC : 10-50% (close to 10%)– PBMC: idem 10%

Specificity MLL-AF4

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

ME1

RS(

4;11

)

MV(

4;11

)

ALL-

PO

K562 69

7

RC

H-A

CV

Kasu

mi

NB4

REH

MFI

patients in MLL-AF4

0

1000

2000

3000

4000

5000

6000

7000

8000

do

no

r

do

no

r

do

no

r

do

no

r

do

no

r

ML

L-

ML

L-

ML

L-

ML

L-

ML

L-

B-A

LL

B-A

LL

B-A

LL

B-A

LL

B-A

LL

MLL-AF4 precursor B-ALL control

MF

I

Precursor B-ALL multiplex tube: MLL-AF4 t(4;11)

100% K562 100% 697

10% K562/697

10% 697/K562

R2 = green = BCR beads

R3 = pink = E2A beads

SIMULTANEOUS DETECTION OF THE BCR-ABL AND E2A-PBX FUSION PROTEINS

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

Multicolor Immunophenotyping: Standardization and ApplicationsMarch 9-11, 2012 TMH, Mumbay (India)

B-CELL NEOPLASMS (PRECURSOR AND MATURE)

WHO: Mature B-cell Neoplasms

B CELL CHRONIC LYMPHOPROLIFERATIVE DISORDERSHeterogeneous group of diseases typically characterized by

a monoclonal expansion of a mature-appearing neoplastic B-lymphocyte

Mature/peripheral B cell chronic lymphoid leukemias: Chronic lymphocytic leukemia/Small B cell lymphocytic lymphoma Prolymphocytic leukemia Hairy cell leukemiaMature/pheripheral B-cell lymphomas: Lymphoplasmacytic lymphoma Splenic marginal zone lymphoma Extranodal marginal zone lymphoma (MALT-type) Nodal marginal zone lymphoma Follicular lymphoma Mantle cell lymphoma Diffuse large B-cell lymphoma Burkitt lymphomaPlasma cell neoplasias: Multiple myeloma/plasmacytoma

WHO CLASSIFICATION OF B-CLPD

DIAGNOSIS OF CLONAL HAEMATOLOGICAL DISORDERS

Clinical symptoms Laboratory and signs findings

Morphology + cytochemistry

Cytogenetics

Immunophenotyping

Molecular biology/FISH

IMMUNOPHENOTYPIC PATTERNS OF DIFFERENT

TYPES OF B-CLPD (Orfao et al, In: “B-CLL”.Humana Press, 2004) sIg CD5 CD10 CD20 CD11c CD23 CD24 CD25 CD38 CD43 CD79b CD103 FMC7

B-CLL d + - d -/+ ++ + + -/+ + d - -

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

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

SMZL + -/+ - + + - + -/+ - - + -/+ +

LPL + - - + - - + + -/+ - + - -/+

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

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

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

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

MATUTES et al SCORE FOR B-CLL

MARKER PATTERN SCORE

CD5 positive 1CD23 positive 1CD22 dim 1sIg dim 1FMC7 negative 1(CD79b) dim 1

Diagnosis of CLL requires a score > 3 (4)

Matutes et al, Leukemia 1994

WHO: B-cell malignancies

Histology &cytology

DLBCLB-PLL

Cytogenetics

MCLBL

Immunophenotype

CLLHCL

Clinic

MALT

The EuroFlow comprehensive approach

Clinical question

Screening tube

Diagnostic panel

MRD


Monoclonalcomponent

Monoclonalcomponent

non-IgM,Bone lesions

BM plasmacytosisSustained

monocytosisUnexplainedEosinophilia

High suspicion ofacute leukemia

e.g. blast cells observedUnexplained

cytopenia

Atypical lymphocytesSplenomegalyLymphocytosis

LN enlargement

Highmonoclonalcomponent

non-IgM

Suspicion oflymphoma

localization in“small cell number”samples e.g. CSF,

vitreous

Clinical question

Screening tube

Diagnostic panel

MRD

Monoclonalcomponent

Monoclonalcomponent


BM plasmacytosis

ALOT LST PCSTfirst tube of PCD

SST

Sustainedmonocytosis

UnexplainedEosinophilia

reactive/polyclonal

other ? B-CLPD



cytopenia


LN enlargement

clonal

reactive/polyclonal

clonal/aberrant


non-IgM



vitreous

ALOT – acute leukemia orientation tubeLST – lymphocytosis screening tubePCD – Plasma cell discrasia screening tubeSST – small sample tube

Clinical question

Screening tube

Diagnostic panel

MRD


Monoclonalcomponent

Monoclonalcomponent


BM plasmacytosis


SST

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



reactive/polyclonal

other ? B-CLPD



cytopenia


LN enlargement

clonal

reactive/polyclonal

clonal/aberrant

first4 tubes

PCD


non-IgM



vitreous

first4 tubes

B-CLPDcomplete

Comprehensive network of panels aiming the diagnosis and characterization of the major WHO

entities

Clinical question

Screening tube

Diagnostic panel

MRD


Clinical question

Screening tube

Diagnostic panel

MRD


Monoclonalcomponent

Monoclonalcomponent


BM plasmacytosis


SST

BCP-ALLL T-ALL AML/MDS B-CLPDlimited T-CLPD NK-CLPD



reactive/polyclonal

other ? B-CLPD

CLL

non-CLL

CLL

MCL

FCL

HCL

other clonal B

reactive

aberrantab+

reactive

aberrantNK cellsvarious

subtypesofBCP-ALL

MDS

PNH

CML

CML-BC

other MPD



cytopenia


LN enlargement

clonal

reactive/polyclonal

clonal/aberrant

first4 tubes

PCD

various subtypesof PCD


non-IgM



vitreous

first4 tubes

varioussubtypesof T-ALL

varioussubtypesofAML

B-CLPDcomplete

aberrantgd+

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 LEUKEMIA/ LYMPHOMA IMMUNOPHENOTYPING ANTIBODY

PANELClinical

request/need

Medical indication

Design of MAb panels (Medical indication-oriented) & immuno-phenotyping

strategy

TechniquesPanel evaluation vs conventional in-use

panels

Panel optimization (re-design)

Panel evaluatio

n

Proposed

strategy

Panel optimization (re-design)

2-8 cycles

Panel construction

Selection of:

• Fluorochromes• Antibodies• Fluorochrome + antibody combinations• 8-color combinations• Panels

Selection of:


Reagent stability

Brightness

Low fluorescence background levels

No spectral overlap between fluorochrome emissions

Panel construction

Initial selection

FL Chann

elLaser

Commonly available

fluorochromes

1 Violet Pacific Blue

HORIZON V450

2* Violet AmCyan

Pacific OrangeHORIZON V500

3 BlueFITC

Alexa Fluor 488

4 Blue PE

5 Blue PE-TxRed

6 BluePerCP Cy5.5

PerCP

7 Blue PE Cy7

8 Red APC

Alexa Fluor 647

9 RedAPC Cy7APC H7

Alexa Fluor 700

Further comparisons

FL Chann

elLaser

Commonly available

fluorochromes

1 Violet Pacific Blue

HORIZON V450

2 Violet AmCyan*

Pacific OrangeHORIZON V500

3 BlueFITC

Alexa Fluor 488

4 Blue PE

5 Blue PE-TxRed

6 BluePerCP Cy5.5

PerCP

7 Blue PE Cy7

8 Red APC

Alexa Fluor 647

9 RedAPC Cy7APC H7

Alexa Fluor 700

FLUOROCHROME SELECTION

*Alternative new additional fluorochromes currently under evaluation

Responsible scientists: T.Kalina, J.Flores

Panel construction – Antibodies

Selection of:


Backbone antibodies

The same antibodies in every tube of the panel

Essential for merge-calculation function

Characterization antibodies

- Lineage assessment, - Differentiation lineage

markers,- Maturation stage, - Aberrant markers,- Relation to cytogenetic

abnormality, - LAP, - MRD …


Backbone antibodies

The same Ab in every tube of the panel

Essential for merge-calculation function

Backbone markers:

Should identify all cells belonging to the target lineage, either normal or malignant

Backbone candidates for B-CLPD: CD19? CD20? CD22? CD37?

- Aberrant underexpression of CD19 and/or CD20 frequently observed- κ/CD37/λ/CD19/CD22/CD20 tested in 69 B-NHL cases

Responsible scientist: S. Böttcher


Panel Construction: selection of backbone B cell markers


Conclusion: CD37 & CD22 redundant, as CD20 PacB plus CD19 PE-Cy7 were sufficient to identify all malignant B cells in all cases

Panel construction

Selection of:


Panel Pac Blue

Pac Orange FITC PE

PerCP

Cy5.5

PE Cy7 APC APC

H7

BCP-ALL CD45 CD34 CD19

T-ALL cyCD3 CD45 CD3

AML/MDS HLADR CD45 CD34 CD11

7

B-CLPD CD20 CD45 CD19

T-CLPD CD4 CD45 CD3 CD8

NK-CLPD CD45 CD3 CD56 CD19

PCD CD45 CD138 CD38 CD19

Panel construction – Fluorochrome + antibody BB combinations

Monoclonalcomponent

Monoclonalcomponent


BM plasmacytosis


SST





cytopenia


LN enlargement


non-IgM



vitreous

Pac Blue

Pac Orang

eFITC PE

PerCP Cy5.5

PE Cy7 APCAPC H7

CD20CD4

CD45Lambd

aCD8

KappaCD56

CD5CD19

TCR gdCD3 CD38

LST – Lymphocytosis screening tube

Responsible scientist: J. Flores Montero

Pac Blue

Pac Orang

eFITC PE

PerCP Cy5.5

PE Cy7 APCAPC H7

CD20CD4

CD45Lambd

aCD8

Kappa

CD56CD5

CD19TCR gδ

CD3 CD38


Able to identify all the sample major populations:

Non-hematopoietic cells


Pac Blue

Pac Orang

eFITC PE

PerCP Cy5.5

PE Cy7 APCAPC H7

CD20CD4

CD45Lambd

aCD8

KappaCD56

CD5CD19

TCR gδCD3 CD38




T lymphocytes


Pac Blue

Pac Orang

eFITC PE

PerCP Cy5.5

PE Cy7 APCAPC H7

CD20CD4

CD45Lambd

aCD8

KappaCD56

CD5CD19TCR gδ

CD3 CD38




T lymphocytes

B lymphocytes


Pac Blue

Pac Orang

eFITC PE

PerCP Cy5.5

PE Cy7 APCAPC H7

CD20CD4

CD45Lambd

aCD8

Kappa

CD56CD5

CD19TCR gδ

CD3 CD38




T lymphocytes

B lymphocytes

NK cells


Pac Blue

Pac Orang

eFITC PE

PerCP Cy5.5

PE Cy7 APCAPC H7

CD20CD4

CD45Lambd

aCD8

KappaCD56

CD5CD19

TCR gδCD3 CD38




T lymphocytes

B lymphocytes

NK cells

Plasma cells


Pac Blue

Pac Orang

eFITC PE

PerCP Cy5.5

PE Cy7 APCAPC H7

CD20

CD4CD45

Lambda

CD8

Kappa

CD56CD5

CD19

TCR gδ

CD3 CD38




T lymphocytes

B lymphocytes

NK cells

Plasma cells

(T-cell subpopulations)


Pac Blue

Pac Orang

eFITC PE

PerCP Cy5.5

PE Cy7 APCAPC H7

CD20CD4

CD45Lambd

aCD8

KappaCD56

CD5CD19

TCR gδCD3 CD38




T lymphocytes

B lymphocytes

NK cells

Plasma cells


(B-cell subsets & Ig light chain restriction)


Pac Blue

Pac Orang

eFITC PE

PerCP Cy5.5

PE Cy7 APCAPC H7

CD20CD4

CD45Lambd

aCD8

KappaCD56

CD5CD19TCR gδ

CD3 CD38




T lymphocytes

B lymphocytes

NK cells

Plasma cells


(B-cell light chain restriction)

B-NHL panel backbone


Panel construction

Selection of:


Characterization markers

CD10, CD20, CD22 CD24, CD27, CD38 CD39, CD43, CD63

CD81, CD95, CD138 Bcl-2, HLA-

DR, IgM

Normal B lymphopoiesi

s

CD11a, CD11c, CD31, CD49d, CD62L, CXCR5,

CCR6, LAIR1

B cell homing

Known to differentiate

CD5, CD23, CD25 FMC7, CD79b,

CD103, CD200, sIg

Responsible scientist: Sebastian Bottcher

Characterization markers

CD10, CD20, CD22 CD24, CD27,

CD38 CD39, CD43, CD63 CD81, CD95, CD138 Bcl-2,

HLA-DR, IgM

Normal B lymphopoiesi

s

CD11a, CD11c, CD31, CD49d, CD62L, CXCR5,

CCR6, LAIR1

B cell homing

Known to differentiate

CD5, CD23, CD25 FMC7, CD79b,

CD103, CD200,sIg


Tested markers (n=66):

• Backbone markers (e.g. CD19, CD20, CD22, CD37, CD45).• Lineage assignment and maturation stage (e.g. Bcl-2,

HLA-DR, IgM, CD10, CD43, CD24, CD27, CD38, CD39, CD63, CD81, CD95, CD138).

• Disease specific (e.g. CD5, CD23, CD25, CD79b, CD103, CD200).

• Integrins and chemokine receptors (e.g. CD11a, CD11c, CD31, CD49d CD62L, CXCR5, LAIR1).

150 cases of B-Lymphoproliferative disorders tested; aim:

• Improve differential classification of B-NHL• Avoid markers with redundant information

FINAL: 4 tube 8-color panel (20 antibodies)

X

XX

X – univariate analysis

X – multivariate analysis

X

XX

Panel construction – characterization markers


LST + BCLPD classification panel

CD49dCXCR5CD19CD22CD95CD103CD45CD204

CD27CD19HLA-DRCD39CD62LCD45CD205R

CD81sIgMCD19CD11cLAIRCD31CD45CD203

CD43CD200CD19CD79bCD10CD23 CD45CD202

CD38CD3CD19/

TCRgd

CD5sIgK /CD56

sIgl/CD8

CD45CD20 /CD4

1=LST

APC-H7APCPECy7PerCP-Cy5.5

PEFITCPacOrang

e

PacBlue

CD20/CD4/CD45/sIgl/sIgK/CD8/CD56/CD5/CD19/CD38/CD23/CD10/CD79b/CD200/CD43/CD31/LAIR1/CD11c/sIgM/CD81/CD103/CD95/CD22/CXCR5/CD49d/CD62L/CD39/HLA-DR/

CD19/CD27

30-colors flow cytometry !


CD20/CD4/CD45/sIgl/sIgK/CD8/CD56/CD5/CD19/CD38/CD23/CD10/CD79b/CD200/CD43/CD31/LAIR1/CD11c/sIgM/CD81/CD103/CD95/CD22/CXCR5/CD49d/CD62L/CD39/HLA-DR/

CD19/CD27

30-colors flow cytometry !

LST + BCLPD classification panel

CD49dCXCR5CD19CD22CD95CD103CD45CD204

CD27CD19HLA-DRCD39CD62LCD45CD205R

CD81sIgMCD19CD11cLAIRCD31CD45CD203

CD43CD200CD19CD79bCD10CD23 CD45CD202

CD38CD3CD19/

TCRgd

CD5sIgK /CD56

sIgl/CD8

CD45CD20 /CD4

1=LST

APC-H7APCPECy7PerCP-Cy5.5

PEFITCPacOrang

e

PacBlue


B-CLPD: 4-COLOUR STAINING PANEL

- FITC PE PerCP/Cy5.5 APC

- Control Control CD19 Control - sIgk sIgl CD19 CD5 - CD22 CD23 CD19 CD20

- Fmc7 CD24 CD19 CD34- CD43 CD79b CD19

CD49d- CyBcl2 CD10 CD19 CD38- CD103 CD25 CD19 CD11c- sIgM CD27 CD19 CCR6- CD3 CyZap70 CD19

CD5

REFERENCE DATAFILES FOR CLL B-CELLS

CD19+ CLL B-cells

Case number

C

D1

9-P

EC

y7

CLL case 2

CLL case 3

CLL case 4

Merge Calculated Data files


CLL case 1

Characterization markers:

CD200


MZLMCLHCL LPLFLCLL DLBCLBL

Characterization markers:

LAIR1(CD305)

MZLMCLHCL LPLFLCLL DLBCLBL


Characterization marker: CD5

MZL MCLHCL LPL FL CLL DLBCLBL

Parameter Significance

1 IgM 14.02

2 CD200 13.76

3 CD79b 11.94

4 CD23 8.57

5 CD38 7.73

CLL immunophenotypic diagnosis

CD10+ CD10-

Best 5 markers for the DD between CLL and

MCL according to EuroFlow analysis


PCA of total immunophenotype

MCL

CLL

Principal component 1 →

Pri

nci

pal co

mpon

en

t 2

→

1 SD 2 SD



MCL

CLL

Principal component 1 →

Pri

nci

pal co

mp

on

en

t 2

→

1 SD 2 SD


PC1

1 IgM 14.09

2 CD200 14.06

3 CD79b 13.39

4 CD23 8.60

5 CD20 6.43

…

MZL MCLHCL LPL FL CLL DLBCLBLCD10+ CD10-


1 IgM 14.02

2 CD200 13.76

3 CD79b 11.94

4 CD23 8.57

5 CD38 7.73




Characterization marker: smIgM


MZL MCLHCL LPL FL CLL DLBCLBLCD10+ CD10-


1 IgM 14.02

2 CD200 13.76

3 CD79b 11.94

4 CD23 8.57

5 CD38 7.73




Characterization marker: CD200


Major consecutive development steps in the design of the EuroFlow B-CPLD panelVersion Tube Fluorescence channel

PacB PacO FITC PE PECy5 PECy7 APC APCCy7

Backbone 1 1 SmIg CD37 SmIg CD19 CD22 CD20

Backbone 2 1 CD20 SmIg CD37 SmIg CD19 CD22

1 CD20 CD45 Ig Ig PerCPCy5.5 CD19 IgM AF700CD5 CD22

2 CD20 CD45 CD103 CD10 CD5 CD19 CD43 CD22

Panel 1 3 CD20 CD45 CD81 CD79b CD5 CD19 CD23 CD22

4 CD20 CD45 CD31 CD63 CD5 CD19 CXCR5 CD22

5 CD20 CD45 CD24 LAIR1 CD5 CD19 CD11a CD22

6 CD20 CD45 CD38 CD25 CD138 CD19 CD11c CD22

1 CD20 CD45 Ig Ig CD22 CD19 CD23 APCH7CD81

Panel 2 2 CD20 CD45 CD103 CD25 CD11c CD19 IgM CD24

3 CD20 CD45 CD31 LAIR1 CD5 CD19 CD43 CXCR5

4 CD20 CD45 bcl2 CD10 CD79b CD19 CD38 CD49b

PacB: Pacific Blue; FITC: Fluorescein isothiocyanate; PE: Phycoerythrin; Cy5: cyanin5; Cy7: Cyanin7; APC: Allophycocyanin; PerCP: Peridinin-chlorophyll-protein; AF700: Alexa Fluor 700; H7: Hilite7


Major consecutive development steps in the design of the EuroFlow B-CPLD panel (continued)Version Tube Fluorescence channel

PacB PacO FITC PE PECy5 PECy7 APC APCCy7

1 CD20 CD45 Ig Ig CD22 CD19 CD23 CD81

2 CD20 CD45 CD103 CD25 CD11c CD19 IgM

Panel 3 3 CD20 CD45 CD31 LAIR1 CD5 CD19 CD43

4 CD20 CD45 bcl2 CD10 CD79b CD19 CD38 CD49d

5 CD20 CD45 CD24 CD95 CD19 CD200

1 CD20 CD45 Ig Ig CD22 CD19 CD23 CD81

2 CD20 CD45 CD103 CD25 CD11c CD19 IgM CD43

Panel 4 3 CD20 CD45 CD31 LAIR1 CD5 CD19 CD43 CD24

4 CD20 CD45 bcl2 CD10 CD79b CD19 CD83 CD49d

5 CD20 CD45 CD24 CD95 CD19 CD200 CD31

6 CD20 CD45 CD19 CXCR5 CD103

1=LST CD20 CD45 CD8 + CD56 + CD5 CD19 + CD3 CD38Ig Ig TCR

2 CD20 CD45 CD23 CD10 CD79b CD19 CD200 CD43

Panel 5 3 CD20 CD45 CD31 LAIR CD11c CD19 IgM CD81

4 CD20 CD45 CD103 CD95 CD22 CD19 CXCR5 CD49d

5 CD20 CD45 CD62L CD39 HLADR CD19 CD27

B-CLPD: Diagnostic work-flow

Monoclonalcomponent

ALOT LST

B-CLPDlimited

B-CLPDbroad

Eosinophilia

reactive/non-aberrant

CLL

non-CLL

CLL

MCL

FCL

HCL

other clonal B

Acute leukemia CytopeniaLymphocytosis

LN involvement

> 90% pure by BB

Responsible scientists: Juan Flores and Sebastian Bottcher

CLL MCL

MCLCLL MCL

Full

pane

lTu

bes

1 &

2 o

nly

BCLPD classification panel: modular design

CLL HCL

CLL HCL

CLL MZL

CLL MZL

CLL FL

CLL FL

CLL DLBCL

CLL DLBCL


MZL HCL MCL CLL FL CLL

PCA of total immunophenotype: clearly separated diseases


PC1

1 CD38 9.87

2 CD10 8.92

3 IgM 8.48

4 CD200 8.33

5 CD81 7.37

…

PC1

1 IgM 14.09

2 CD200 14.06

3 CD79b 13.39

4 CD23 8.60

5 CD20 6.43

…

MCL CLLCD10+DLBCL BL

2 SD separated 1 SD separated



FL CD10-DLBCL

Overlap of 1st SD

PCA of B-CLPD panel

Responsible scientist: S. Böttcher Designed by: Q Lecrevisse

BL vs. DLBCL CD10-

BL vs. DLBCL CD10+

BL vs. CLL

BL vs. FL

BL vs. HCL

BL vs. LPL

BL vs. MCL

BL vs. MZL

BL vs.Normal

Separation power of different types of BCLPD

CLL DLBCL CD10+

DLBCLCD10-

FL HCL LPL MCL MZL

BL

CLL

DLBCL CD10 +

DLBCL CD10 -

FL

HCL

LPL

MCL

1 x 1comparison n = 150

2 SD separated

1 SD separated

Overlap of 1st SD


EuroFlow B-CLPD panel: summary

• B-CLPD panel allows unequivocal classification of most mature B-cell malignancies according to WHO

• Most differential diagnoses achieved (n=32/36) efficiently except for the following 1 vs 1 comparisons:

FL vs DLBC MZL vs LPL LPL vs DLBCL MZL vs DLBCL


Expert pathologist agreement with the consensus diagnosis

The NHL Classification Project, Bood 1997;89:3909-3918 Kindly provided by Raul Braylan

MZL

LPL

5 expert hematopathologists~1,400 lymphoma cases

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


Costa et al, Leukemia, 2010


TYPES OF B-CLPD CD5 CD19 CD38 CD20 CD23 CD10 CD79b CD43 CD11c IGM CD103 CD22

B-CLL + lo hi lo + lo lo

MCL +

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

MZL lo -/+

LPL lo -/+

FL lo +

LDBCL + -/+

LDBCL -/+ -/+

BL hi lo + + hi


TYPES OF B-CLPD CD200 CD31 CD305 CD81 CD95 CXCR5 CD49d CD62L CD39 CD27

B-CLL hi hi -/+ lo -

MCL lo -/+ -

HCL hi hi lo + -

MZL lo

LPL

FL lo lo lo - -

LDBCL lo

LDBCL + +

BL lo lo hi - - -

• How to get optimal and

comparable measurements?

• Which are the most

appropriate fluorochromes?

• What is the optimal sample

preparation protocol?

REPRODUCIBLE & OBJECTIVE RESULTS

UTILITY OF THE NEW SOFTWARE TOOLSStandardization of FCM immunophenotyping

EuroFlow participantsUniversity Institutes / Medical SchoolsErasmus MC, Rotterdam, NL J.J.M. van Dongen, V.H.J. van der Velden…

USAL, Salamanca, ES A. Orfao, J. Flores, J. Almeida, Q. Lecrevisse…

IMM, Lisbon, PT P. Lucio, A. Mendonça, A. Parreira a.o…

UNIKIEL, Kiel, DE M. Kneba, S. Böttcher, M. Ritgen, M. Brüggemann …

AP-HP, Paris, FR E. Macintyre, L. Lhermitte, V. Asnafi …

UNIVLEEDS, Leeds, GB S. Richards, A.C. Rawstron. P. Evans …

DPH/O, Prague, CZ O. Hrusak, T. Kalina, E. Mesjstrikova …

SAM, Zabrze, PL T. Szczepanski, L. Sedek …

DCOG, The Hague, NL E. Sonneveld, A. van der Sluijs-Gelling ...

KUL, Leuven, BE N. Boeckx …

HGSA, Porto, PT M. Lima, AH Santos

UFRJ, Rio de Janeiro, BR C. Pedreira, E.S. Costa

Companies (SME’s)DYNOMICS, Rotterdam, NL E. Dekking, F. Weerkamp …

CYTOGNOS, Salamanca, ES M. Martin, J. Bensadon, J. Hernandez, M. Muñoz …

www.euroflow.org

Contributors - Acknowledgements

Elizabeth MacIntyreVahid AsnafiLudovic Lhermitte

Juan Flores-MonteroJulia AlmeidaQuentin Lecrevisse

Jacques van DongenVincent van der VeldenJeroen Te MarveldeAlita van der Sluijs

Michael KnebaMonika BrüggemannSebastian BöttcherStephen RichardsPaul EvansMatt CullensRuth de TuteAndy Rawstron

Tomek SzczepanskiLukasz Sedek

Paulo LucioAndreia MendonçaEvan Jensen

Ondrej HrusakTomas KalinaEster Mejstrikova

Photo Lukasz Sedek

THANK YOU

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

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