Hospital

Chronic lymphoproliferative disorders

Large group of disorders characterized byproliferation and accumulation of morphologic andimmunophenotypic mature lymphocytes in diverselocations

Classification is initially based on lymphoid lineageassessment

B-cell

CD19+

CD20+

Igs+

T- & NK- cells

CD3+

CD7+

CD4+/CD8+

CD3-

CD7+

CD56+

CD16+

Consequently build upon cytomorphologic, anatomopathologic, immunophenotypic,genetic, epidemiologic and clinical features

Chronic lymphoproliferative disordersCLL/small lymphocytic lymphomaB-cell prolymphocytic leukaemiaSplenic marginal zone lymphomaHairy cell leukaemiaSplenic B-cell lymphoma/leukaemia, unclassifiableLymphoplasmacytic lymphomaIgM MGUSHeavy chain diseasesPlasma cell neoplasmsExtranodal marginal zone lymphoma of MALT tissuesNodal marginal zone lymphomaFollicular lymphomaPaediatric-type follicular lymphomaLarge B-cell lymphoma with IRF4 rearrangementPrimary cutaneous follicle centre lymphomaMantle cell lymphomaDiffuse large B-cell lymphoma (DLBCL), NOST-cell/histiocyte-rich large B-cell lymphomaPrimary diffuse large B-cell lymphoma of the CNSPrimary cutaneous diffuse large B-cell lymphoma, leg typeEBV-positive diffuse large B-cell lymphoma, NOSEBV-positive mucocutaneous ulcerDLBCL associated with chronic inflammationLymphomatoid granulomatosisPrimary mediastinal (thymic) large B-cell lymphomaIntravascular large B-cell lymphomaALK-positive large B-cell lymphomaPlasmablastic lymphomaPrimary effusion lymphomaHHV8-associated lymphoproliferative disordersBurkitt lymphomaBurkitt-like lymphoma with 11q aberrationHigh-grade B-cell lymphomaB-cell lymphoma, unclassifiable,

with features intermediate between DLBCL and classic Hodgkin lymphoma

T-cell prolymphocytic leukaemiaT-cell large granular lymphocytic leukaemiaChronic lymphoproliferative disorder of NK cellsAggressive NK-cell leukaemiaEBV-positive T-cell and NK-cell LPDs of childhood

Systemic EBV+ T-cell lymphoma of childhoodChronic active EBV infection of T- and NK-cell type, systemic formHydroa vacciniforme-like lymphoproliferative disorderSevere mosquito bite allergy

Adult T-cell leukaemia/lymphomaExtranodal NK/T-cell lymphoma, nasal typeIntestinal T-cell lymphoma

Enteropathy-associated T-cell lymphomaMonomorphic epitheliotropic intestinal T-cell lymphomaIntestinal T-cell lymphoma, NOSIndolent T-cell lymphoproliferative disorder of the GIT

Hepatosplenic T-cell lymphomaSubcutaneous panniculitis-like T-cell lymphomaMycosis fungoidesSézary syndromePrimary cutaneous CD30-positive T-cell LPDs

Lymphomatoid papulosisPrimary cutaneous anaplastic large cell lymphoma

Primary cutaneous peripheral T-cell lymphomas, rare subtypesPrimary cutaneous gamma delta T-cell lymphomaPrimary cutaneous CD8-positive aggressive epidermotropic cytotoxic T-cell lymphomaPrimary cutaneous acral CD8-positive T-cell lymphomaPrimary cutaneous CD4+ small/medium T-cell LPD

Peripheral T-cell lymphoma, NOSAngioimmunoblastic T-cell lymphoma and other nodal lymphomas of T follicular helper (TFH) cell origin

Angioimmunoblastic T-cell lymphomaFollicular T-cell lymphomaNodal peripheral T-cell lymphoma with TFH phenotype

Anaplastic large cell lymphoma, ALK-positiveAnaplastic large cell lymphoma, ALK-negativeBreast implant-associated anaplastic large cell lymphoma

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Chronic lymphoproliferative disordersCLL/small lymphocytic lymphomaB-cell prolymphocytic leukaemiaSplenic marginal zone lymphomaHairy cell leukaemiaSplenic B-cell lymphoma/leukaemia, unclassifiableLymphoplasmacytic lymphomaIgM MGUSHeavy chain diseasesPlasma cell neoplasmsExtranodal marginal zone lymphoma of MALT tissuesNodal marginal zone lymphomaFollicular lymphomaPaediatric-type follicular lymphomaLarge B-cell lymphoma with IRF4 rearrangementPrimary cutaneous follicle centre lymphomaMantle cell lymphomaDiffuse large B-cell lymphoma (DLBCL), NOST-cell/histiocyte-rich large B-cell lymphomaPrimary diffuse large B-cell lymphoma of the CNSPrimary cutaneous diffuse large B-cell lymphoma, leg typeEBV-positive diffuse large B-cell lymphoma, NOSEBV-positive mucocutaneous ulcerDLBCL associated with chronic inflammationLymphomatoid granulomatosisPrimary mediastinal (thymic) large B-cell lymphomaIntravascular large B-cell lymphomaALK-positive large B-cell lymphomaPlasmablastic lymphomaPrimary effusion lymphomaHHV8-associated lymphoproliferative disordersBurkitt lymphomaBurkitt-like lymphoma with 11q aberrationHigh-grade B-cell lymphomaB-cell lymphoma, unclassifiable,

with features intermediate between DLBCL and classic Hodgkin lymphoma

T-cell prolymphocytic leukaemiaT-cell large granular lymphocytic leukaemiaChronic lymphoproliferative disorder of NK cellsAggressive NK-cell leukaemiaEBV-positive T-cell and NK-cell LPDs of childhood

Systemic EBV+ T-cell lymphoma of childhoodChronic active EBV infection of T- and NK-cell type, systemic formHydroa vacciniforme-like lymphoproliferative disorderSevere mosquito bite allergy

Adult T-cell leukaemia/lymphomaExtranodal NK/T-cell lymphoma, nasal typeIntestinal T-cell lymphoma

Enteropathy-associated T-cell lymphomaMonomorphic epitheliotropic intestinal T-cell lymphomaIntestinal T-cell lymphoma, NOSIndolent T-cell lymphoproliferative disorder of the GIT

Hepatosplenic T-cell lymphomaSubcutaneous panniculitis-like T-cell lymphomaMycosis fungoidesSézary syndromePrimary cutaneous CD30-positive T-cell LPDs

Lymphomatoid papulosisPrimary cutaneous anaplastic large cell lymphoma

Primary cutaneous peripheral T-cell lymphomas, rare subtypesPrimary cutaneous gamma delta T-cell lymphomaPrimary cutaneous CD8-positive aggressive epidermotropic cytotoxic T-cell lymphomaPrimary cutaneous acral CD8-positive T-cell lymphomaPrimary cutaneous CD4+ small/medium T-cell LPD

Peripheral T-cell lymphoma, NOSAngioimmunoblastic T-cell lymphoma and other nodal lymphomas of T follicular helper (TFH) cell origin

Angioimmunoblastic T-cell lymphomaFollicular T-cell lymphomaNodal peripheral T-cell lymphoma with TFH phenotype

Anaplastic large cell lymphoma, ALK-positiveAnaplastic large cell lymphoma, ALK-negativeBreast implant-associated anaplastic large cell lymphoma

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Epidemiology:≈ 7-8% of human tumors≈ 40% of hematologic tumors≈ 300.000 new cases / year in western countries

Risk factors:• Altered immunity• Infections• Toxic / environmental exposure

MFC in CLPD

1. Diagnostic screening- Normal reactive/regenerating clonal

2. Classification - Definition of biologic and clinical entities

- Risk group definition

- Prognostic stratification

3. Assessment of response to treatment -Residual disease detection

-Risk group definitions

-Prognostic stratification

Increased T cell population

Normal / residual

T cells

Normal / residual T cells

T-PLL

EuroFlow strategy in CLPD

van Dongen et al, Leukemia 2012

EuroFlow strategy in CLPD

van Dongen et al, Leukemia 2012

LST

PacB PacO FITC PEPerCpCy5.5

PE Cy7

APCAPCH7

LSTCD4

CD20CD45

CD8sIgl

CD56sIgk

CD5CD19TCRgd

CD3 CD38

EuroFlow strategy in CLPD

van Dongen et al, Leukemia 2012

LST

PacB PacO FITC PEPerCpCy5.5

PE Cy7

APCAPCH7

LSTCD4CD20

CD45CD8sIgl

CD56sIgk

CD5CD19TCRgd

CD3 CD38

• Identification of targeted populations: mature B-, T- & NK-cells

• Subsetting into major subpopulations

• Detection of abnormal cell populations requiring further evaluation

• Altered (distribution or absolute cell) concentration

• Aberrant immunophenotypic pattern

EuroFlow strategy in CLPD

van Dongen et al, Leukemia 2012

LST validation

B-cells CD8hi T-cells NK-cellsCD4+ T-cells

Principal component analysis

Principal component 1

Pri

nci

pal

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mp

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Lymphoid cells abnormality N

Immunophenotypic profile (n=227) 227/233 (97,4%)

Altered numers /distribution (n=172) 172/233 (73,8%)

Total (n=233) 233/233 (100%)

Conventional vs reference data base interpretation

EuroFlow strategy in CLPD

van Dongen et al, Leukemia 2012

LST validation

B-cells CD8hi T-cells NK-cellsCD4+ T-cells

Principal component analysis

Principal component 1

Pri

nci

pal

co

mp

on

en

t 2

Lymphoid cells abnormality N

Immunophenotypic profile (n=227) 227/233 (97,4%)

Altered numers /distribution (n=172) 172/233 (73,8%)

Total (n=233) 233/233 (100%)

Conventional vs reference data base interpretation

EuroFlow strategy in CLPD

van Dongen et al, Leukemia 2012

LST: complementarity with classification panels

PacB PacO FITC PEPerCpCy5.5

PE Cy7

APCAPCH7

LST=1CD4CD20

CD45CD8sIgl

CD56sIgk

CD5CD19TCRgd

CD3 CD38

2 CD20 CD45 CD23 CD10 CD79b CD19 CD200 CD43

3 CD20 CD45 CD31 CD305 CD11c CD19 sIgM CD81

4 CD20 CD45 CD103 CD95 CD22 CD19 CD185 CD49d

5 CD20 CD45 CD62L CD39 HLADR CD19 CD27Eur

oFlow B

-CLPD

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+

EuroFlow LST AGI data base

LST data base(s) requirements:

• Mature aberrant lymphocytes can

infiltrate various tissues

• Peripheral blood

• Bone marrow

• Lymph node

• Other fluids

• Wide range of patients’ age:

• Normal <-> reactive <-> clonal

• Boarder variability in:

• Lymphoid cell (sub) populations presence

and distribution

EuroFlow LST PB data base construction

Flores-Montero, Journal of Immunological Methods 2019

Key steps:

1. Selection / Staining and acquisition of

normal-reactive bone marrow samples

2. Inspection of technical quality

3. Analysis and identification of all cell

populations in the sample

4. Samples incorporation to the data base

5. Identification of biological and/or technical

outliers

6. Prospective validation

LST sample selected (n=119)

QC check for technical and

biologic variables (n=73)

Samples included in

the data base (n=46)

EuroFlow LST data base construction

Flores-Montero, Journal of Immunological Methods 2019

Key steps:

1. Selection / Staining and acquisition of

normal-reactive bone marrow samples

2. Inspection of technical quality

3. Analysis and identification of all cell

populations in the sample

4. Samples incorporation to the data base

5. Identification of biological and/or technical

outliers

6. Prospective validation

Gating strategy for normal PB populations

EuroFlow LST data base construction

Flores-Montero, Journal of Immunological Methods 2019

Key steps:

1. Selection / Staining and acquisition of

normal-reactive bone marrow samples

2. Inspection of technical quality

3. Analysis and identification of all cell

populations in the sample

4. Samples incorporation to the data base

5. Identification of biological and/or technical

outliers

6. Prospective validation

EuroFlow LST data base construction

Flores-Montero, Journal of Immunological Methods 2019

Key steps:

1. Selection / Staining and acquisition of

normal-reactive bone marrow samples

2. Inspection of technical quality

3. Analysis and identification of all cell

populations in the sample

4. Samples incorporation to the data base

5. Identification of biological and/or technical

outliers

6. Prospective validation

Tumor cell populations

Normal (residual) cell populations

% c

ells

MA

% c

ells

MA

% cells AGI

% cells AGI

EuroFlow automated analysis LST data bases

Automated gaiting & identification process

Clustering

Classification

Output

Evaluation of CHKs

Linkage to classification data bases

Reporting

Automated analysis LST in BM sample

File selection

Automated analysis LST in BM sample

Data base selection

Automated analysis LST in BM sample

AGI output

10%

Automated analysis LST in BM sample

After evaluation of CHKs

Automated analysis LST in BM sample

Linkage to classification data bases

Automated analysis LST in BM sample

Summary

Automated analysis LST in BM sample

Summary

Clonal B-cell comparison withreference population of distinctdisease categories (Follicularlymphoma-based view)

Automated analysis LST in PB sample

Report

Automated analysis LST in PB sample

Report

EuroFlow Automated analysis in CLPD (LST)

Concluding remarks:

Automated analysis is a robust and accurate tool to support

analists in the diagnosis and classification of CLPD which

contributes to the systematic, more standardized and fast

analysis and reporting.