alk t-lymphoproliferative diseases alk23/10/2017 1 escca 2017, thessaloniki, sep 24-27, 2017 paula...

23/10/2017

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ESCCA 2017, Thessaloniki, Sep 24-27, 2017Paula C. Fernandez, MD/PhDInstitute for Laboratory Medicine, Kantonsspital Aarau, Switzerland

T-Lymphoproliferative Diseases

Mature T-cell Leukemias (leukemic/disseminated)

• T-cell prolymphocytic leukemia (T-PLL)• T-cell LGL leukemia (T-LGL)• Chronic LPD of NK cells*• Aggressive NK-cell leukemia• # Systemic EBV+ T-cell lymphoma of childhood• Adult T-cell lymphoma/leukemia (HTLV1+) (ATLL)

• Peripheral T-cell lymphoma, NOS (PTCL-NOS)• # Nodal peripheral T-cell lymphoma w. TFH phenotype *• Angioimmunoblastic T-cell lymphoma (AITL)• # Follicular T-cell lymphoma*• Anaplastic large cell lymphoma, ALK+ (ALK+ALCL)• # Anaplastic large cell lymphoma, ALK- (ALK-ACLCL)

Nodal T-cell Lymphomas

*Provisional entities# change from 2008

• Sézary syndrome (SS)• Mycosis fungoides (MF)• Primary cutaneous CD30+ T-LPD

• Lymphomatoid papulosis• Primary cuteanous ALCL

• Primary cutaneous gd T-cell lymphoma• # Primary cutaneous aggressive epidermotropic

cytotoxic T-cell lymphoma*• # Primary cutaneous acral CD8+ T-cell lymphoma*• # Primary cutaneous CD4+ small/medium LPD*• # Hydroa-vacciniforme-like LPD

Cutaneous T-cell Lymphomas (extranodal)

Mature T- and NK-cell NeoplasmsWHO classification 2016

LPD: lymphoproliferative disorder; TFH: T follicular helper

Extranodal T-cell Lymphomas

• Extranodal NK/T-cell lymphoma, nasal type• Enteropathy-associated T-cell lymphoma (former

EATL type I)• # Monomorphic epitheliotropic intestinal

lymphoma (former EATL type II, now MEITL)• # Indolent LPD of GI-tract*• Hepatosplenic T-cell lymphoma (HSTL)• # Breast-implant-associated anaplastic large cell

lymphoma*• Subcutaneous panniculitis-like T-cell lymphoma

(SPTCL)

• Pediatric-type FL

• Duodenal-type FL

• In situ follicular/mantle cell neoplasia

• EBV+ mucocutaneous ulcer

• *Indolent T/NK‐cell proliferations (GIT, Skin)

• *Breast implant-associated ALCL

• Primary cutaneous CD4+ small/medium T-cell LPD

• * Primary cutaneous acral CD8+ T-cell lymphoma

WHO 2016 update: recognition of indolent “Lymphoma/LPD“

*provisional entities

Indolent T/NK-cell proliferations

Early lymphoid lesions: conceptual, diagnostic and clinical challenges, Haematologica 2014;99(9)

• Interface between benign and malignant

• Include clonal proliferations, cells may carry molecular hallmarks of malignant counterpart

• No autonomous proliferation, limited potential for progression -> conservative treatment

Disease Genetic/molecular alteration

Frequency Effect

T-PLL TRA/D-TCL1 fusionTRA/D-MTCP1 fusion

JAK1/JAK3/STAT5Bmut.ATM deletion/mutation

75%1

75%70%

Aberrant TCL1 expression and activation of AKT pathwayAberrant MTCP1 expression and activation of AKT pathwayConstitutive activationImpaired DNA damage response

T-LGL STAT3 mutationsSTAT5B mutations

30-40%Rare

Constitutive activationConstitutive activation

CLPD-NK STAT3 mutations 25-30% Constitutive activation

ATLL JAK3mutationCCR4 mutation

10%25%

Constitutive activationPI3K/AKT activation

SS TCF3 deletionTP53 deletion

ARID1A mutations

70%75%40%

Enhanced cell-cycle progressionImpaired DNA damage responseChromatin remodeling

Recurrent genetic alterations in mature T/NK-cell leukemias

Gaulard&Levald, SeminHematol, 2014Bailey &Elenitoba-Johnson, Curr. Hematol. Malig. Rep 2015

T-PLL: T-cell prolymphocytic leukemia; T-LGL: T-cell large granular lymphocyte leukemia; CLPD-NK: chroniclymphoproliferative disorders of NK cells; ATLL: Adult T-cell lymphoma / leukemia; SS Sèzary Syndrome

slide by J. Almeida, Salamanca,Spain

Disease Genetic/molecular alteration

ALK+ALCL t(2;5)(p23;q35) (NPM-ALK)t(1;2) (TPM3-ALK)t (2;3) (TFG-ALK)Inv2 (ATIC-ALK)

t(2;22) (CTLC-ALK)

ALK-ALCL DUSP22-IRF4 locus on 6p25.3* (30%)P63 on 3q28* (8%)

AITL(and TFH-like PTCL-NOS)

RHOA mutationsTET2, IDH2 and DNMT3A mutations

PTCL-NOS “GATA3” (↑GATA3, CCR4, IL18RA, CXCR7 and IK)

“TBX21” (↑TBX21, EOMES, CXCR3, IL2RB, CCL3 and IFNg)

HTCL Isochromosome 7q (majority of cases)

* 5y-OS of 90% for DUSP22+ALK-ALCL vs 85% for ALK+ALCL (vs. 17% for P63+ cases)

Gaulard&Levald, SeminHematol, 2014Hapgood&Savage, Hematol. Oncol. 2015slide by J. Almeida, Salamanca,Spain

Recurrent genetic alterations in mature T/NK-cell leukemias

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• Lower incidence (than B-cell disorders)• Diverse and heterogenic group of disorders• Classification is driven by pathology/clinics• Even within one entity: immunophenotypic heterogeneity• Immunophenotypic overlaps with normal, reactive• Clonality assessment cumbersome• Few entities with recurrent genetic aberrations for confirmation

Detection and classification of T-cell neoplasmsis a challenge…

as a consequence:

• Knowledge of normal immunophenotypic pattern is essential• Knowledge of typical phenotypes of T/NK-NHLs is important• Diagnosis cannot be established solely based on immunophenotype

Surrogate information

• Clinical indication for sending material?• Lymphocytosis• Morphologically atypical lymphocytes• Organomegaly (spleen/liver)• Lymphnode enlargement• Effusions• Skin lesions, erythrodermia• Eosinophilia*• Fever*, wasting* (B-symptoms), rashes*• Auto-immune phenomena*: cytopenia, hemolytic anemia• B-cell deregulation*: hyper(hypo)gammaglobulinemia

• Age, gender, ethnicity

• EBV, HTLV status

* Paraneoplastic manifestations (cytokine production, cytotoxic activity)

CD8 - FITC

CD

4 -

PEC

y5

WHO DISEASE ENTITIES

clinic

histology & cytology

genetic &molecular

immunophenotype

How to recognize and classify T NHLs with immunophenotyping?

• Recognizing abnormal and the differentiation from activated phenotypes

• Maturation stage?

• Expression of classification antigens? -> CD30, cyTCL1, CD279

-> altered expression (up/downregulation)-> abnormal patterns-> in combination with scatter characteristics

TSCM: stemcell memory

TCM: central memory

TEM:effector memory

TEff:effector

Post-thymic: T cell differentiation

TRM: resident memory

Progressive differentiation

Mahnke et al. Eur J Immunol 2013

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Cell Origin of T-cell Lymphoma

PLL

De Leval&Gaulard, ASH Educational Book, 2008

Antigens investigated in T-cell malignancies

• «pan T-cell»: CD3, CD4, CD8, CD7, CD2, CD5, TCRab, TCRgd and CD45

• co-stimulatory antigens: CD28, CD27, CD26

• maturation-related: CD45RA, CD45RO

• homing receptors: CCR7 (CD197)

• activation-related: CD38, HLA-DR, CD25, CD11b, CD11c

• NK-associated: CD16, CD94

• cytotoxicity-related: CD56, CD57, Granzyme, Perforin

• classification markers: CD30 (ALCL), CD10 (AITL), cyTCL1 (PLL), CD279/PD-1 (AITL)

T-cell lymphoma cases: Panel used

Screening PacB PacO FITC PEPerCP-Cy5.5

PECY7 APC APCH7

LymphoidScreeningTube: LST

CD4CD20

CD45 CD8Anti-sIgl

CD56Anti-sIgk

CD5 Anti-TCRgd

CD19

smCD3 CD38

TCLPDtube

PacB PacO FITC PEPerCPCy5.5

PECY7 APCAPCH7

AIM

1 CD4 CD45 CD7 CD26 smCD3 CD2 CD28 CD8 Identification Sézary Sy.

2 CD4 CD45 CD27 CCR7 smCD3 CD45RO CD45RA CD8 Maturation stage

3 CD4 CD45 CD5 CD25 smCD3 HLADR cyTCL1 CD8 T-PLL

4 CD4 CD45 CD57 CD30 smCD3 CD11c CD8 Cytotoxic phenotype & ALCL

5 CD4 CD45 cyPerf cyGrz smCD3 CD16 CD94 CD8 Cytotoxic phenotype & T-LGL

6 CD4 CD45 CD279 smCD3 CD8 AITL

T-PLL: T-cell prolymphocytic leukemia, ALCL: Anaplastic large cell lymphoma, T-LGL: T-cell large granular lymphocytic leukemia, AITL: Angioimmunoblastic T-cell lymphomaCy: cytoplasmic, Perf: Perforin, Grz: Granzyme

Analysis and Gating (Software Infinicyt)

1) Exclusion of doublets and debris:

2) Select Lymphocytes:

-> double check in individual plots(e.g. SSC/CD3) that all events selected)

3) Lymphocytes: select B, T

B-cellsT-cells

NK-cells


4) check B-cells

B-cells Plasma cells? B-progenitors?

Kappa/lambda should overlapp for CD19 and CD20


5) check NK-cells

Include: CD56+CD8-/+NK-cells Include only CD4-

NK are CD3-CD5-CD38+

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6) check T-cellsTCRg/d pos cells All other T-cells:

Normal T-cellsubpopulations: CD4+CD8+(dim)CD4+(dim)CD8+CD4-CD8-

Normal Patterns of activation: CD5 and CD38

naive recently activated late activated/ terminally differentiated

Reactive T cells in a 10y oldwith EBV infection

reactive

CD

3

CD5

normal

CD4/CD8 ratio 0.2 scatter CD38+

Pictograms from M. Lima, Porto, Portugal

Screening: what to look for

No

rmal

PB

T-C

LPD

PB

Ratio: 0.9

Ratio: 0.2

large granularlymphocyte leukemia(LGL)

Example:

Green: normal T-cellsGrey: B- and NK-cellRed: abnormal T-cells

Screening: what to look for

No

rmal

PB

Ratio: 0.9

T-C

LPD

PB

Ratio: 930

Prolymphocytic leukemia (PLL)Example:

Green: normal T-cellsGrey: B- and NK-cellRed: abnormal T-cells


TCLPDtube


PECY7 APCAPCH7

AIM








Normal Patterns of activation

naive Recently activated Late activated/ Terminally differentiated

Romero P, J Immunol 2007; 178

CD4

CD8


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T-CLPD tube 1

No

rmal

PB

T-C

LPD

PB

Example: Sézary Syndrome, pathognomonic: CD7-, CD26-

Green: normal T-cellsRed: abnormal T-cellsGrey: B- and NK-cell

T-CLPD tube 1

No

rmal

PB

T-C

LPD

PB

PLLExample:

Green: normal T-cellsRed: abnormal T-cellsGrey: B- and NK-cell

T-CLPD tube 1

No

rmal

PB

T-C

LPD

PB

LGLExample:

Green: normal T-cellsRed: abnormal T-cellsGrey: B- and NK-cell T-cell lymphoma cases: Panel used

TCLPDtube


PECY7 APCAPCH7

AIM








Maturation stages

by J. Almeida, Salamanca, Spain

Different possible combinations: shown CCR7/CD27/CD45RA/CD45RO




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No

rmal

PB

T-C

LPD

PB

LGLExample:

T-CLPD tube 2Green: normal T-cellsRed: abnormal T-cellsGrey: B- and NK-cell

T-CLPD tube 2

No

rmal

PB

T-C

LPD

PB

PLLExample:


TCLPDtube


PECY7 APCAPCH7

AIM











www.control-T.de

TCL1: T-cell lymphoma breakpoint 1

Over-expression of this oncogene is due to translocation in proximityof TCR-regulatory elements-> classification marker for PLL

T-CLPD tube 3

No

rmal

PB

T-C

LPD

PB

T-C

LPD

PB

PLL with inv14q(q11;q32)TCL1

PLL with t(X;14)(q28;q11)MTCP1 (homologue of TCL1)

CD25

HLA

DR

ATLL

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TCLPDtube


PECY7 APCAPCH7

AIM










CD30 expression in T-NHL

www.scienceofCD30.com

T-CLPD tube 4

No

rmal

PB

T-C

LPD

PB

T-C

LPD

PBAnaplastic

Large CellLymphoma(ALCL)

LGL


TCLPDtube


PECY7 APCAPCH7

AIM








Normal Patterns

late activated/ terminally differentiated

recently activatednaive

Romero P, J Immunol 2007; 178Pictograms by M. Lima, Porto, Portugal

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T-CLPD tube 5

No

rmal

PB

T-C

LPD

PB

T-C

LPD

PB

PLL

LGL



TCLPDtube


PECY7 APCAPCH7

AIM







by J. Almeida, Salamanca, Spain

T-CLPD tube 6

No

rmal

PB

T-C

LPD

PB

AITL

case from J. Almeida, Salamanca, Spain

Mature T-cell Leukemias (leukemic/disseminated)

• T-cell prolymphocytic leukemia• T-cell LGL leukemia (T-LGL)• Chronic LPD of NK cells*• Aggressive NK-cell leukemia• # Systemic EBV+ T-cell lymphoma of childhood• Adult T-cell lymphoma/leukemia

• Peripheral T-cell lymphoma, NOS • # Nodal peripheral T-cell lymphoma w. TFH phenotype *• Angioimmunoblastic T-cell lymphoma• # Follicular T-cell lymphoma*• Anaplastic large cell lymphoma, ALK+ • # Anaplastic large cell lymphoma, ALK-

Nodal T-cell Lymphomas

*Provisional entities# change from 2008

• Sézary syndrome • Mycosis fungoides• Primary cutaneous CD30+ T-LPD and subtypes• Primary cutaneous gd T-cell lymphoma• # Primary cutaneous aggressive epidermotropic

cytotoxic T-cell lymphoma*• # Primary cutaneous acral CD8+ T-cell lymphoma*• # Primary cutaneous CD4+ small/medium LPD*• # Hydroa-vacciniforme-like LPD

Cutaneous T-cell Lymphomas (extranodal)

Mature T- and NK-cell NeoplasmsWHO classification 2016

LPD: lymphoproliferative disorder, TFH: T follicular helper

Extranodal T-cell Lymphomas

• Extranodal NK/T-cell lymphoma, nasal type• Enteropathy-associated T-cell lymphoma • # Monomorphic epitheliotropic intestinal

lymphoma• # Indolent LPD of GI-tract*• Hepatosplenic T-cell lymphoma• Subcutaneous panniculitis-like T-cell lymphoma• # Breast-implant-associated anaplastic large cell

lymphoma*

cytotoxic non-cytotoxic variable

phenotype

Strategy for immunophenotypic classification of T-cell neoplasms

based on «normal counterparts» -> identify, quantify and characterize abnormal T- or NK-cell population

• cell type (CD4+, CD8+, CD4+CD8+, CD4-CD8-, TCRab, TCRgd)• establish clonality criteria: aberrant immunophenotypes, TCRVb usage• maturation stage (naive, memory, effector)• functional properties (expression of NK/cytotoxicity-related antigens)• activation status (resting/activated)• sample type/clinics -> localization (leukemic, nodal, extranodal)

What are the most relevant aberrant immunophenotypic features andare they compatible with any disease entity?

your case match? DD?WHO entities

leukemic/nodal/extranodalnon-cytotoxic/cytotoxic

diagnosis will be based on clinics, histopathology,morphology, geneticsand immunophenotype

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Thank you foryour attention

thanks to:Margarida Lima, Porto, PortugalJulia Almeida, Salamanca, Spain

alk t-lymphoproliferative diseases alk23/10/2017 1 escca 2017, thessaloniki, sep 24-27, 2017 paula...

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