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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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slide1

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)

slide2

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

slide3

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)

slide6

ALOT

1 tube

BCP-ALL

T-ALL

AML/MDS

4 to 7 tubes

4 tubes

4 tubes

slide7

ALOT: B-cell precursor ALL

BM stained with

ALOT 8-color tube

CyCD3

CD7

sCD3

CD19

CyCD79a

CyMPO

CD45

CD34

Responsible scientist: Ludovic Lhermitte

slide8
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)
slide9

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)

slide10

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

slide13

BCP-ALL panel

ALOT

BCP-ALL

slide14

BCP-ALL panel

ALOT

BCP-ALL

slide15

BCP-ALL panel

ALOT

BCP-ALL

slide16

BCP-ALL panel

ALOT

BCP-ALL

Positive Diagnosis

slide17

BCP-ALL panel

ALOT

BCP-ALL

Differential Diagnosis

&

Ambiguous lineage acute leukemia

slide18

BCP-ALL panel

ALOT

BCP-ALL

Maturation stage (EGIL)

slide19

BCP-ALL panel

ALOT

BCP-ALL

Alternative classification

Immunophenotypic features associated with well-defined molecular aberrations

slide20

BCP-ALL panel

ALOT

BCP-ALL

Prognosis markers

slide21

BCP-ALL panel

ALOT

BCP-ALL

LAP markers

slide22

BCP-ALL panel

ALOT

BCP-ALL

slide23

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

slide24

BCP-ALL:DETECTION OF ABERRANT PHENOTYPES

Mix of 3 different regenerating B cell populations (Haematogones)

BCP-ALL blast cells

slide25

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
slide27

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
slide33

PANELS OF IMMUNOBEADS FOR THE CLASSIFICATION OF ACUTE LEUKAEMIAS

Precursor-B-ALL AML ‘MLL’ T-ALL

BCR-ABL PML-RARAMLL-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
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

slide35

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

  • Sensitivity for detection REH cell line in:
    • WBC : 10-50%
    • PBMC : 1-10%
slide36

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%
slide37

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

slide38

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)

slide40

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

slide41

DIAGNOSIS OF CLONAL HAEMATOLOGICAL DISORDERS

Clinical symptoms Laboratory

and signsfindings

Morphology + cytochemistry

Cytogenetics

Immunophenotyping

Molecular biology/FISH

slide42

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

slide43

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

slide45

Clinical question

Screening tube

Diagnostic

panel

MRD

The EuroFlow comprehensive approach

slide46

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

slide47

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

slide48

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

slide49

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

slide50

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

slide51

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

slide52

Panel construction

  • Selection of:
      • Fluorochromes
      • Antibodies
      • Fluorochrome + antibody combinations
      • 8-color combinations
      • Panels
slide53

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
slide54

FLUOROCHROME SELECTION

Initial selection

Further comparisons

*Alternative new additional fluorochromes currently under evaluation

Responsible scientists: T.Kalina, J.Flores

slide55

Panel construction – Antibodies

  • Selection of:
      • Fluorochromes
      • Antibodies
      • Fluorochrome + antibody combinations
      • 8-color combinations
      • Panels
slide56

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 …
slide57

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

slide59

Panel construction

  • Selection of:
      • Fluorochromes
      • Antibodies
      • Fluorochrome + antibody combinations
      • 8-color combinations
      • Panels
slide61

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

slide62

LST – Lymphocytosis screening tube

Able to identify all the sample major populations:

Non-hematopoietic cells

Responsible scientist: J. Flores Montero

slide63

LST – Lymphocytosis screening tube

Able to identify all the sample major populations:

Non-hematopoietic cells

T lymphocytes

Responsible scientist: J. Flores Montero

slide64

LST – Lymphocytosis screening tube

Able to identify all the sample major populations:

Non-hematopoietic cells

T lymphocytes

B lymphocytes

Responsible scientist: J. Flores Montero

slide65

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

slide66

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

slide67

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

slide68

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

slide69

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

slide70

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

slide73

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

slide74

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/CD27CD20/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

slide75

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/CD27CD20/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

slide76

B-CLPD: 4-COLOUR STAINING PANEL

- FITCPEPerCP/Cy5.5 APC

- Control Control CD19 Control - sIgk sIglCD19 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

slide77

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

slide80

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

slide83

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

slide84

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

slide85

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

Version 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 AF700 CD5 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 APCH7 CD81Panel 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

Responsible scientist: S. Böttcher

slide86

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 CD38 Ig 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

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

slide88

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

slide91

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

slide95

B-CLPD: Comparative analysis of “our case”

vs multiple reference groups

Responsible scientists: Sebastian Bottcher

Costa et al, Leukemia, 2010

slide96

B-CLPD:Comparative analysis of “our case”

vs multiple reference groups

Responsible scientist: Sebastian Bottcher

Costa et al, Leukemia, 2010

slide97

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

slide98

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 - - -

slide99

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

slide100

EuroFlow participants

www.euroflow.org

University Institutes / Medical Schools

Erasmus 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 …

slide101

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

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