B-CELL NEOPLASMS
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B-CELL NEOPLASMS (PRECURSOR AND MATURE). CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN) Multicolor Immunophenotyping: Standardization and Applications March 9-11, 2012 TMH, Mumbay (India). DIAGNOSTICS IN HEMATO-ONCOLOGY. 1.Making the diagnosis

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Cancer research center university university hospital of salamanca spain

B-CELL NEOPLASMS

(PRECURSOR AND MATURE)

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

Multicolor Immunophenotyping: Standardization and Applications

March 9-11, 2012 TMH, Mumbay (India)


Cancer research center university university hospital of salamanca spain

DIAGNOSTICS IN HEMATO-ONCOLOGY

1.Making the diagnosis

Normal  reactive/regenerating  malignant

Annually > 300,000 new patients with a hematological malignancy in developed countries

2.Classification of hematopoietic malignancies

-relation with prognosis

-relevance of risk-group definition in treatment protocols

Based on differentiation characteristics and particularly on chromosome aberrations, resulting in fusion gene transcripts or aberrantly (over) expressed genes

3.Evaluation of treatment effectiveness

Detection of minimal residual disease (MRD):

MRD-based risk-group stratification (treatment reduction or treatment intensification)

Annually > 400,000 follow-up samples in leukemia patients (ALL, AML, CML)

Prepared by JJM van Dongen


Cancer research center university university hospital of salamanca spain

WHO CLASSIFICATION OF LYMPHOID MALIGNANCIES

(2002-2008)

  • B-cell precursor (immature) derived acute

  • lymphoblastic leukemia/lymphoblastic

  • lymphoma

  • - Mature (peripheral) B-cell neoplasms


B cell precursor acute lymphoblastic leukemia lymphoma b all

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)


Cancer research center university university hospital of salamanca spain

ALOT

1 tube

BCP-ALL

T-ALL

AML/MDS

4 to 7 tubes

4 tubes

4 tubes


Cancer research center university university hospital of salamanca spain

ALOT: B-cell precursor ALL

BM stained with

ALOT 8-color tube

CyCD3

CD7

sCD3

CD19

CyCD79a

CyMPO

CD45

CD34

Responsible scientist: Ludovic Lhermitte


Cancer research center university university hospital of salamanca spain

ALOT

ALOT (Acute Leukemia Orientation Tube)

  • Designed for assessment of the nature of immature blast cell populations in acute leukemia samples

  • Designed to choose appropriate immunophenotypic panel(s)


Cancer research center university university hospital of salamanca spain

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)


Cancer research center university university hospital of salamanca spain

CLASSIFICATION OF

PRECURSOR-B ALL & T-ALL

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+)


Bcp all

BCP-ALL

BCP-ALL panel

BCP-ALL


Cancer research center university university hospital of salamanca spain

BCP-ALL panel

BCP-ALL


Cancer research center university university hospital of salamanca spain

BCP-ALL panel

ALOT

BCP-ALL


Cancer research center university university hospital of salamanca spain

BCP-ALL panel

ALOT

BCP-ALL


Cancer research center university university hospital of salamanca spain

BCP-ALL panel

ALOT

BCP-ALL


Cancer research center university university hospital of salamanca spain

BCP-ALL panel

ALOT

BCP-ALL

Positive Diagnosis


Cancer research center university university hospital of salamanca spain

BCP-ALL panel

ALOT

BCP-ALL

Differential Diagnosis

&

Ambiguous lineage acute leukemia


Cancer research center university university hospital of salamanca spain

BCP-ALL panel

ALOT

BCP-ALL

Maturation stage (EGIL)


Cancer research center university university hospital of salamanca spain

BCP-ALL panel

ALOT

BCP-ALL

Alternative classification

Immunophenotypic features associated with well-defined molecular aberrations


Cancer research center university university hospital of salamanca spain

BCP-ALL panel

ALOT

BCP-ALL

Prognosis markers


Cancer research center university university hospital of salamanca spain

BCP-ALL panel

ALOT

BCP-ALL

LAP markers


Cancer research center university university hospital of salamanca spain

BCP-ALL panel

ALOT

BCP-ALL


Cancer research center university university hospital of salamanca spain

BCP-ALL panel

ALOT

BCP-ALL

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


Cancer research center university university hospital of salamanca spain

BCP-ALL:DETECTION OF ABERRANT PHENOTYPES

Mix of 3 different regenerating B cell populations (Haematogones)

BCP-ALL blast cells


Cancer research center university university hospital of salamanca spain

PRECURSOR B-ALL: DNA ANEUPLOIDY

NEOPLASTIC

B-CELLS

NORMAL

B-CELLS

0

256

512

768

1024

RESIDUAL

NON-B-CELLS

FL2-Area ->

RM30243.100

0

256

512

768

1024

FL2-Area ->

RM30243.100

B-CELL PRECURSOR ALL: DNA ANEUPLOIDY


Immunophenotype of neoplastic b cell precursors

IMMUNOPHENOTYPE OF NEOPLASTIC B-CELL PRECURSORS

  • 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


Cancer research center university university hospital of salamanca spain

ADULT PRECURSOR B-ALL:

IMMUNOPHENOTYPE OF BCR/ABL+ CASES

Normal BM

CD34+ B-cells

CD45

CD19

CD19

CD38

10

0

10

1

10

2

10

3

10

4

0

1

2

3

4

10

0

10

1

10

2

10

3

10

4

10

0

10

1

10

2

10

3

10

4

10

10

10

10

10

CD10 CD13 CD34 CD34

CD13 PE ->

CD34 PE ->

CD34 ->

CD10 ->

UVJ39869.004

UVJ39869.002

MO21489004

AP36452.003

DNA diploid

Bcr/abl+ ALL

CD19

CD19

CD45

CD38

10

0

10

1

10

2

10

3

10

4

10

0

10

1

10

2

10

3

10

4

10

0

10

1

10

2

10

3

10

4

10

0

10

1

10

2

10

3

10

4

CD10 CD13 CD34 CD34

CD10 ->

CD13 ->

CD34 ->

CD34 ->

HG30672.002

HG30672.010

HG30672.008

HG30672.006

DNA aneuploid

Bcr/abl+ ALL

CD19

CD19

CD38

CD45

10

0

10

1

10

2

10

3

10

4

10

0

10

1

10

2

10

3

10

4

10

0

10

1

10

2

10

3

10

4

10

0

10

1

10

2

10

3

10

4

CD10 ->

CD13 ->

CD34 ->

CD34 ->

CD10 CD13 CD34 CD34

RFR7690.008

RFR7690.008

RFR7690.007

RFR7690.011


Bead based flow cytometric assay for detection of fusion proteins

Bead-based flow cytometric assay for detection of fusion proteins

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 euroflow mfi values of the different cell samples n 217

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

Weerkamp et al, Leukemia, 2009


Results concerning bcr abl ruo kit

Results concerning BCR-ABL RUO kit

Weerkamp et al, Leukemia, 2009

  • 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

  • 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

  • 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?)


Immunobead flow cytometry with bd cba flex beads bcr abl t 9 22 fusion protein specificity

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

Final Statement about the BCR-ABL RUO testing

  • The main advantages of the immunobead assay are:

  • Not dependent of the breakpoint position in the fusion gene;

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

  • Providing results within several hours;

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

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


Cancer research center university university hospital of salamanca spain

PANELS OF IMMUNOBEADS FOR THE CLASSIFICATION OF ACUTE LEUKAEMIAS

Precursor-B-ALL AML ‘MLL’ T-ALL

BCR-ABLPML-RARAMLL-AF4CALM-AF10

TEL-AML1AML1-ETO MLL-AF9LMO2

E2A-PBX1CBFB-MYH11 MLL-AF10HOX11L2

( MLL-AF4) MLL-ENLTAL1

MLL-AF6


Precursor b all multiplex tube e2a pbx1 t 1 19

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

Sensitivity for detection on cell line <10%

*Exp. Performed on April 27th 2007

Neg. patients

Pos patients


Cancer research center university university hospital of salamanca spain

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

  • Sensitivity for detection REH cell line in:

    • WBC : 10-50%

    • PBMC : 1-10%


Cancer research center university university hospital of salamanca spain

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

Sensitivity for detection MV4;11 line in:

  • 697 cell line: 10%

  • WBC : 10-50% (close to 10%)

  • PBMC: idem 10%


Cancer research center university university hospital of salamanca spain

100% K562

100% 697

R2 = green = BCR beads

R3 = pink = E2A beads

10% 697/K562

10% K562/697

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


Cancer research center university university hospital of salamanca spain

B-CELL NEOPLASMS

(PRECURSOR AND MATURE)

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

Multicolor Immunophenotyping: Standardization and Applications

March 9-11, 2012 TMH, Mumbay (India)


Who mature b cell neoplasms

WHO: Mature B-cell Neoplasms


Cancer research center university university hospital of salamanca spain

B CELL CHRONIC LYMPHOPROLIFERATIVE DISORDERS

Heterogeneous group of diseases typically characterized by a monoclonal expansion of a mature-appearing neoplastic B-lymphocyte

WHO CLASSIFICATION OF B-CLPD

Mature/peripheral B cell chronic lymphoid leukemias:

Chronic lymphocytic leukemia/Small B cell lymphocytic lymphoma

Prolymphocytic leukemia

Hairy cell leukemia

Mature/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 lymphoma

Plasma cell neoplasias:

Multiple myeloma/plasmacytoma


Cancer research center university university hospital of salamanca spain

DIAGNOSIS OF CLONAL HAEMATOLOGICAL DISORDERS

Clinical symptomsLaboratory

and signsfindings

Morphology + cytochemistry

Cytogenetics

Immunophenotyping

Molecular biology/FISH


Cancer research center university university hospital of salamanca spain

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 -/+ - + + - - + - ++ -/+ -/+ - +


Cancer research center university university hospital of salamanca spain

MATUTES et al SCORE FOR B-CLL

MARKER PATTERN SCORE

CD5 positive 1

CD23 positive 1

CD22 dim 1

sIg dim 1

FMC7 negative 1

(CD79b) dim 1

Diagnosis of CLL requires a score > 3(4)

Matutes et al, Leukemia 1994


Who b cell malignancies

WHO: B-cell malignancies

Histology &

cytology

DLBCL

B-PLL

Cytogenetics

MCL

BL

Immunophenotype

CLL

HCL

Clinic

MALT


Cancer research center university university hospital of salamanca spain

Clinical question

Screening tube

Diagnostic

panel

MRD

The EuroFlow comprehensive approach


Cancer research center university university hospital of salamanca spain

Clinical question

Screening tube

Diagnostic

panel

MRD

The EuroFlow comprehensive approach

Suspicion of

Atypical lymphocytes

Monoclonal

High

lymphoma

Splenomegaly

High suspicion of

component

monoclonal

localization in

L

ymphocytosis

acute leukemia

non-IgM,

Unexplained

component

“small cell number”

LN enlargement

e.g. blast cells observed

Bone lesions

cytopenia

non-IgM

samples

e.g. CS

F

,

BM plasmacytosis

vitreous

Sustained

Monoclonal

Unexplained

monocytosis

Eosinophilia

component


Cancer research center university university hospital of salamanca spain

Clinical question

Screening tube

Diagnostic

panel

MRD

The EuroFlow comprehensive approach

Suspicion of

Atypical lymphocytes

Monoclonal

High

lymphoma

Splenomegaly

High suspicion of

component

monoclonal

localization in

L

ymphocytosis

acute leukemia

non-IgM,

Unexplained

component

“small cell number”

LN enlargement

e.g. blast cells observed

Bone lesions

cytopenia

non-IgM

samples

e.g. CS

F

,

BM plasmacytosis

vitreous

Sustained

Monoclonal

Unexplained

monocytosis

Eosinophilia

component

PCS

T

SS

T

ALO

T

LS

T

first tube of PCD

reactive/polyclonal

reactive/polyclonal

clonal/aberrant

other ? B-CLPD

clonal

ALOT – acute leukemia orientation tube

LST–lymphocytosis screening tube

PCD– Plasma cell discrasia screening tube

SST – small sample tube


Cancer research center university university hospital of salamanca spain

Clinical question

Screening tube

Diagnostic

panel

MRD

The EuroFlow comprehensive approach

Suspicion of

Atypical lymphocytes

Monoclonal

High

lymphoma

Splenomegaly

High suspicion of

component

monoclonal

localization in

L

ymphocytosis

acute leukemia

non-IgM,

Unexplained

component

“small cell number”

LN enlargement

e.g. blast cells observed

Bone lesions

cytopenia

non-IgM

samples

e.g. CS

F

,

BM plasmacytosis

vitreous

Sustained

Monoclonal

Unexplained

monocytosis

Eosinophilia

component

PCS

T

SS

T

ALO

T

LS

T

first tube of PCD

reactive/polyclonal

reactive/polyclonal

first

4 tubes

clonal/aberrant

other ? B-CLPD

clonal

first

4 tubes

B-CLPD

B-CLPD

T

-CLPD

NK-CLPD

PCD

BCP-AL

L

T

-AL

L

AML/MDS

complete

limited

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


Cancer research center university university hospital of salamanca spain

Clinical question

Screening tube

Diagnostic

panel

MRD

The EuroFlow comprehensive approach

Suspicion of

Atypical lymphocytes

Monoclonal

High

lymphoma

Splenomegaly

High suspicion of

component

monoclonal

localization in

L

ymphocytosis

acute leukemia

non-IgM,

Unexplained

component

“small cell number”

LN enlargement

e.g. blast cells observed

Bone lesions

cytopenia

non-IgM

samples

e.g. CS

F

,

BM plasmacytosis

vitreous

Sustained

Monoclonal

Unexplained

monocytosis

Eosinophilia

component

PCS

T

SS

T

ALO

T

LS

T

first tube of PCD

reactive/polyclonal

reactive/polyclonal

first

4 tubes

clonal/aberrant

other ? B-CLPD

clonal

first

4 tubes

B-CLPD

B-CLPD

T

-CLPD

NK-CLPD

PCD

BCP-AL

LL

T

-AL

L

AML/MDS

complete

limited

aberrant

+

ab

CL

L

aberrant

CL

L

variou

s

variou

s

variou

s

NK cells

subtype

s

subtype

s

subtype

s

various subtypes

o

f

BCP-AL

L

o

f

T

-AL

L

o

f

AM

L

+

gd

MC

L

aberrant

non-CL

L

of PCD

reactive

MDS

FC

L

reactive

PNH

HC

L

CM

L

other clonal B

CML-BC

other MPD


Cancer research center university university hospital of salamanca spain

THE EUROFLOW APPROACH TO LEUKEMIA/LYMPHOMA IMMUNOPHENOTYPING

Clinical question

Knowledge

Experience

Evaluation

Reference profiles

Diagnostic screening tube

Majority of diseases?

Majority of cases?

New disease entities?

“Diagnostic classification” panel

14 Major groups

154 Nosologic entities

MRD monitoring


Cancer research center university university hospital of salamanca spain

CONSTRUCTION OF EUROFLOW LEUKEMIA/ LYMPHOMA IMMUNOPHENOTYPING ANTIBODY PANEL

Clinical request/need

Proposed strategy

Medical indication

Panel optimization (re-design)

2-8 cycles

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

Panel evaluation

Panel evaluation vsconventional in-use panels

Panel optimization (re-design)

Techniques


Cancer research center university university hospital of salamanca spain

Panel construction

  • Selection of:

    • Fluorochromes

    • Antibodies

    • Fluorochrome + antibody combinations

    • 8-color combinations

    • Panels


Cancer research center university university hospital of salamanca spain

Panel construction

  • Selection of:

    • Fluorochromes

    • Antibodies

    • Fluorochrome + antibody combinations

    • 8-color combinations

    • Panels

  • Reagent stability

  • Brightness

  • Low fluorescence background levels

  • No spectral overlap between fluorochrome emissions


Cancer research center university university hospital of salamanca spain

FLUOROCHROME SELECTION

Initial selection

Further comparisons

*Alternative new additional fluorochromes currently under evaluation

Responsible scientists: T.Kalina, J.Flores


Cancer research center university university hospital of salamanca spain

Panel construction – Antibodies

  • Selection of:

    • Fluorochromes

    • Antibodies

    • Fluorochrome + antibody combinations

    • 8-color combinations

    • Panels


Cancer research center university university hospital of salamanca spain

Panel construction – Antibodies

  • 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 …


Cancer research center university university hospital of salamanca spain

Panel construction – Antibodies

  • 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

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

Responsible scientist: S. Böttcher


Cancer research center university university hospital of salamanca spain

Panel construction

  • Selection of:

    • Fluorochromes

    • Antibodies

    • Fluorochrome + antibody combinations

    • 8-color combinations

    • Panels


Cancer research center university university hospital of salamanca spain

Panel construction – Fluorochrome + antibody BB combinations


Cancer research center university university hospital of salamanca spain

LST – Lymphocytosis screening tube

Suspicion of

Atypical lymphocytes

Monoclonal

High

lymphoma

Splenomegaly

High suspicion of

component

monoclonal

localization in

L

ymphocytosis

acute leukemia

non-IgM,

Unexplained

component

“small cell number”

LN enlargement

e.g. blast cells observed

Bone lesions

cytopenia

F

,

non-IgM

samples

e.g. CS

BM plasmacytosis

vitreous

Sustained

Monoclonal

Unexplained

monocytosis

Eosinophilia

component

PCS

T

SS

T

ALO

T

LS

T

first tube of PCD

Responsible scientist: J. Flores Montero


Cancer research center university university hospital of salamanca spain

LST – Lymphocytosis screening tube

Able to identify all the sample major populations:

Non-hematopoietic cells

Responsible scientist: J. Flores Montero


Cancer research center university university hospital of salamanca spain

LST – Lymphocytosis screening tube

Able to identify all the sample major populations:

Non-hematopoietic cells

T lymphocytes

Responsible scientist: J. Flores Montero


Cancer research center university university hospital of salamanca spain

LST – Lymphocytosis screening tube

Able to identify all the sample major populations:

Non-hematopoietic cells

T lymphocytes

B lymphocytes

Responsible scientist: J. Flores Montero


Cancer research center university university hospital of salamanca spain

LST – Lymphocytosis screening tube

Able to identify all the sample major populations:

Non-hematopoietic cells

T lymphocytes

B lymphocytes

NK cells

Responsible scientist: J. Flores Montero


Cancer research center university university hospital of salamanca spain

LST – Lymphocytosis screening tube

Able to identify all the sample major populations:

Non-hematopoietic cells

T lymphocytes

B lymphocytes

NK cells

Plasma cells

Responsible scientist: J. Flores Montero


Cancer research center university university hospital of salamanca spain

LST – Lymphocytosis screening tube

Able to identify all the sample major populations:

Non-hematopoietic cells

T lymphocytes

(T-cell subpopulations)

B lymphocytes

NK cells

Plasma cells

Responsible scientist: J. Flores Montero


Cancer research center university university hospital of salamanca spain

LST – Lymphocytosis screening tube

Able to identify all the sample major populations:

Non-hematopoietic cells

T lymphocytes

(T-cell subpopulations)

B lymphocytes

(B-cell subsets & Ig light chain restriction)

NK cells

Plasma cells

Responsible scientist: J. Flores Montero


Cancer research center university university hospital of salamanca spain

LST – Lymphocytosis screening tube

Able to identify all the sample major populations:

Non-hematopoietic cells

T lymphocytes

(T-cell subpopulations)

B lymphocytes

(B-cell light chain restriction)

NK cells

Plasma cells

B-NHL panel backbone

Responsible scientist: J. Flores Montero


Cancer research center university university hospital of salamanca spain

Panel construction

  • Selection of:

    • Fluorochromes

    • Antibodies

    • Fluorochrome + antibody combinations

    • 8-color combinations

    • Panels


Characterization markers

Characterization markers

B cell homing

Normal B lymphopoiesis

CD11a, CD11c, CD31, CD49d, CD62L, CXCR5, CCR6, LAIR1

CD10, CD20, CD22 CD24, CD27, CD38 CD39, CD43, CD63 CD81, CD95, CD138 Bcl-2, HLA-DR, IgM

Known to differentiate

CD5, CD23, CD25 FMC7, CD79b, CD103, CD200, sIg

Responsible scientist: Sebastian Bottcher


Characterization markers1

Characterization markers

B cell homing

Normal B lymphopoiesis

CD11a, CD11c, CD31, CD49d, CD62L, CXCR5, CCR6, LAIR1

CD10, CD20, CD22 CD24, CD27, CD38 CD39, CD43, CD63 CD81, CD95, CD138 Bcl-2, HLA-DR, IgM

Known to differentiate

CD5, CD23, CD25 FMC7, CD79b, CD103, CD200,sIg

Responsible scientist: Sebastian Bottcher


Cancer research center university university hospital of salamanca spain

Panel construction – characterization markers

  • 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

X

X

X

X

X

X – univariate analysis

X – multivariate analysis

Responsible scientist: S. Böttcher


Cancer research center university university hospital of salamanca spain

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

Pac

Blue

Pac

Orange

FITC

PE

PerCP-Cy5.5

PECy7

APC

APC-H7

1=

LST

CD20 /CD4

CD45

sIgl

/CD8

sIgK /CD56

CD5

CD19

/TCRgd

CD3

CD38

2

CD20

CD45

CD23

CD10

CD79b

CD19

CD200

CD43

3

CD20

CD45

CD31

LAIR

CD11c

CD19

sIgM

CD81

4

CD20

CD45

CD103

CD95

CD22

CD19

CXCR5

CD49d

5R

CD20

CD45

CD62L

CD39

HLA-DR

CD19

CD27

Responsible scientist: Sebastian Bottcher


Cancer research center university university hospital of salamanca spain

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

Pac

Blue

Pac

Orange

FITC

PE

PerCP-Cy5.5

PECy7

APC

APC-H7

1=

LST

CD20 /CD4

CD45

sIgl

/CD8

sIgK /CD56

CD5

CD19

/TCRgd

CD3

CD38

2

CD20

CD45

CD23

CD10

CD79b

CD19

CD200

CD43

3

CD20

CD45

CD31

LAIR

CD11c

CD19

sIgM

CD81

4

CD20

CD45

CD103

CD95

CD22

CD19

CXCR5

CD49d

5R

CD20

CD45

CD62L

CD39

HLA-DR

CD19

CD27

Responsible scientist: Sebastian Bottcher


Cancer research center university university hospital of salamanca spain

B-CLPD: 4-COLOUR STAINING PANEL

- FITCPEPerCP/Cy5.5 APC

- ControlControlCD19 Control- sIgksIglCD19 CD5

- CD22 CD23 CD19 CD20

- Fmc7CD24CD19 CD34

- CD43CD79bCD19 CD49d

- CyBcl2CD10CD19 CD38

- CD103CD25 CD19 CD11c

- sIgMCD27CD19 CCR6

- CD3CyZap70CD19 CD5


Cancer research center university university hospital of salamanca spain

CLL case 2

CLL case 1

CD19+ CLL B-cells

CD19-PECy7

Case number

REFERENCE DATAFILES FOR CLL B-CELLS

Merge Calculated Data files

CLL case 3

CLL case 4

Responsible scientist: Sebastian Bottcher


Characterization markers cd200

BL

CLL

DLBCL

FL

HCL

LPL

MCL

MZL

Characterization markers:CD200

Responsible scientist: Sebastian Bottcher


Characterization markers lair1 cd305

BL

CLL

DLBCL

FL

HCL

LPL

MCL

MZL

Characterization markers:LAIR1(CD305)

Responsible scientist: Sebastian Bottcher


Cancer research center university university hospital of salamanca spain

CLL immunophenotypic diagnosis

Characterization marker: CD5

HCL

LPL

FL

DLBCL

MZL

MCL

CLL

BL

CD10+

CD10-

Best 5 markers for the DD between CLL and MCL according to EuroFlow analysis

Responsible scientist: S. Böttcher


Pca of total immunophenotype

MCL

CLL

Principal component 2→

2 SD

1 SD

Principal component 1→

PCA of total immunophenotype

Responsible scientist: Sebastian Bottcher


Pca of total immunophenotype1

MCL

CLL

Principal component 2→

2 SD

1 SD

Principal component 1→

PCA of total immunophenotype

Responsible scientist: Sebastian Bottcher


Cancer research center university university hospital of salamanca spain

CLL immunophenotypic diagnosis

Characterization marker: smIgM

HCL

LPL

DLBCL

FL

CLL

BL

MZL

MCL

CD10-

CD10+

Best 5 markers for the DD between CLL and MCL according to EuroFlow analysis

Responsible scientist: S. Böttcher


Cancer research center university university hospital of salamanca spain

CLL immunophenotypic diagnosis

Characterization marker: CD200

DLBCL

MZL

MCL

CLL

BL

HCL

LPL

FL

CD10-

CD10+

Best 5 markers for the DD between CLL and MCL according to EuroFlow analysis

Responsible scientist: S. Böttcher


Cancer research center university university hospital of salamanca spain

Major consecutive development steps in the design of the EuroFlow B-CPLD panel

VersionTubeFluorescence channelPacBPacOFITCPEPECy5PECy7APCAPCCy7

Backbone 11SmIg CD37SmIgCD19CD22CD20

Backbone 21CD20 SmIg CD37SmIgCD19CD22

1CD20CD45IgIgPerCPCy5.5CD19IgMAF700CD5CD222CD20CD45CD103CD10CD5CD19CD43CD22

Panel 13CD20CD45CD81CD79bCD5CD19CD23CD22

4CD20CD45CD31CD63CD5CD19CXCR5CD22

5CD20CD45CD24LAIR1CD5CD19CD11aCD22

6CD20CD45CD38CD25CD138CD19CD11cCD22

1CD20CD45IgIgCD22CD19CD23APCH7CD81Panel 22CD20CD45CD103CD25CD11cCD19IgMCD24

3CD20CD45CD31LAIR1CD5CD19CD43CXCR5

4CD20CD45bcl2CD10CD79bCD19CD38CD49b

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

Responsible scientist: S. Böttcher


Cancer research center university university hospital of salamanca spain

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

VersionTubeFluorescence channelPacBPacOFITCPEPECy5PECy7APCAPCCy7

1CD20CD45IgIgCD22CD19CD23CD81

2CD20CD45CD103CD25CD11cCD19IgM

Panel 33CD20CD45CD31LAIR1CD5CD19CD43

4CD20CD45bcl2CD10CD79bCD19CD38CD49d

5CD20CD45CD24CD95CD19CD200

1CD20CD45IgIgCD22CD19CD23CD81

2CD20CD45CD103CD25CD11cCD19IgMCD43

Panel 43CD20CD45CD31LAIR1CD5CD19CD43CD24

4CD20CD45bcl2CD10CD79bCD19CD83CD49d

5CD20CD45CD24CD95CD19CD200CD31

6CD20CD45CD19CXCR5CD103

1=LSTCD20CD45CD8 +CD56 +CD5CD19 +CD3CD38IgIgTCR2CD20CD45CD23CD10CD79bCD19CD200CD43

Panel 53CD20CD45CD31LAIRCD11cCD19IgMCD81

4CD20CD45CD103CD95CD22CD19CXCR5CD49d

5CD20CD45CD62LCD39HLADRCD19CD27


B clpd diagnostic work flow

Lymphocytosis

Cytopenia

Acute leukemia

LN involvement

Monoclonal

component

Eosinophilia

ALOT

LST

B-CLPD

B-CLPD

limited

broad

CLL

CLL

MCL

non-CLL

FCL

reactive/

HCL

non-aberrant

other clonal B

B-CLPD: Diagnostic work-flow

> 90% pure by BB

Responsible scientists: Juan Flores and Sebastian Bottcher


Cancer research center university university hospital of salamanca spain

HCL

CLL

MCL

CLL

CLL

MZL

CLL

DLBCL

CLL

DLBCL

FL

FL

CLL

CLL

CLL

MCL

MCL

CLL

HCL

CLL

MZL

BCLPD classification panel: modular design

Full panel

Tubes 1 & 2 only

Responsible scientist: Sebastian Bottcher


Pca of total immunophenotype clearly separated diseases

PCA of total immunophenotype: clearly separated diseases

MZL

HCL

MCL

CLL

FL

CLL

Responsible scientist: S. Böttcher


Pca of total immunophenotype2

PCA of total immunophenotype

CD10+

DLBCL

BL

CD10-

DLBCL

MCL

CLL

FL

1 SD separated

2 SD separated

Overlap of 1st SD

Responsible scientist: Sebastian Bottcher


Cancer research center university university hospital of salamanca spain

PCA ofB-CLPD panel

BL vs. DLBCL CD10-

BL vs. MCL

BL vs. CLL

BL vs. HCL

BL vs. FL

BL vs. LPL

BL vs. MZL

BL vs. DLBCL CD10+

BL vs.Normal

Responsible scientist: S. Böttcher

Designed by: Q Lecrevisse


Separation power of different types of bclpd

Separation power of different types of BCLPD

Responsible scientist: S. Böttcher

1 x 1comparison n = 150


Euroflow b clpd panel summary

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

Responsible scientist: Sebastian Bottcher


Expert pathologist agreement with the consensus diagnosis

LPL

MZL

Expert pathologist agreement with the consensus diagnosis

5 expert hematopathologists

~1,400 lymphoma cases

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


Cancer research center university university hospital of salamanca spain

B-CLPD: Comparative analysis of “our case”

vs multiple reference groups

Responsible scientists: Sebastian Bottcher

Costa et al, Leukemia, 2010


Cancer research center university university hospital of salamanca spain

B-CLPD:Comparative analysis of “our case”

vs multiple reference groups

Responsible scientist: Sebastian Bottcher

Costa et al, Leukemia, 2010


Cancer research center university university hospital of salamanca spain

IMMUNOPHENOTYPIC PATTERNS OF DIFFERENT

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


Cancer research center university university hospital of salamanca spain

IMMUNOPHENOTYPIC PATTERNS OF DIFFERENT

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 - - -


Cancer research center university university hospital of salamanca spain

UTILITY OF THE NEW SOFTWARE TOOLS

Standardization of FCM immunophenotyping

  • How to get optimal and comparable measurements?

  • Which are the most appropriate fluorochromes?

  • What is the optimal sample preparation protocol?

REPRODUCIBLE & OBJECTIVE RESULTS


Cancer research center university university hospital of salamanca spain

EuroFlow participants

www.euroflow.org

University Institutes / Medical Schools

Erasmus MC, Rotterdam, NLJ.J.M. van Dongen, V.H.J. van der Velden…

USAL, Salamanca, ESA. Orfao, J. Flores, J. Almeida, Q. Lecrevisse…

IMM, Lisbon, PTP. Lucio, A. Mendonça, A. Parreira a.o…

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

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

UNIVLEEDS, Leeds, GBS. Richards, A.C. Rawstron. P. Evans …

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

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

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

KUL, Leuven, BEN. Boeckx …

HGSA, Porto, PTM. Lima, AH Santos

UFRJ, Rio de Janeiro, BRC. Pedreira, E.S. Costa

Companies (SME’s)

DYNOMICS, Rotterdam, NLE. Dekking, F. Weerkamp …

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


Cancer research center university university hospital of salamanca spain

Contributors - Acknowledgements

Michael Kneba

Monika Brüggemann

Sebastian Böttcher

Stephen Richards

Paul Evans

Matt Cullens

Ruth de Tute

Andy Rawstron

Elizabeth MacIntyre

Vahid Asnafi

Ludovic Lhermitte

Paulo Lucio

Andreia Mendonça

Evan Jensen

Tomek Szczepanski

Lukasz Sedek

  • Ondrej Hrusak

  • Tomas Kalina

  • Ester Mejstrikova

Photo Lukasz Sedek

Jacques van Dongen

Vincent van der Velden

Jeroen Te Marvelde

Alita van der Sluijs

Juan Flores-Montero

Julia Almeida

Quentin Lecrevisse


Cancer research center university university hospital of salamanca spain

THANK YOU


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