Molecular understanding of Peripheral T-cell Lymphoma

Javeed IqbalDepartment of Pathology and Microbiology

James O. Armitage Center for Leukemia and Lymphoma ResearchUniversity of Nebraska Medical Center, Omaha, NE

ICKSH 201960th Annual meeting, Seoul, Korea

I have no personal or financial interests to declare:

I have no financial support from an industry source at the current presentation.

대한혈액학회 Korean Society of Hematology

COI disclosureName of author ：Javeed Iqbal

2

University of Nebraska Medical Center

PTC

LCurrent Challenges

diagnosis

Treatment

Molecular investigation

-heterogeneity- 30-50% of cases are PTCL, not otherwise specified (PTCL-NOS)

Most PTCL entities show poor responses to conventional chemotherapy

Lack of authentic cell lines and genetically relevant animal models for major PTCL

entities

Lymphoma Classification

Iqbal et al. Blood Reviews, 2017

Morphology/Clinical

+ Immunohistochemistry

+ Cytogenetics, FISH

+ Molecular Biology + Gene Expression Profiles (GEP)

+ Next Generation Sequencing (NSG)

Classification project-1999Nebraska

Transcriptomics

GenomicsPTCL entities ~19 ~25 ~30

1998 20011956 1974 1982 1988

Rap

papo

rt

Luke

& C

ollin

sK

iel c

lass

ifica

tion

Kie

l cl

assi

ficat

ion/

upd

ate

Wor

king

Fo

rmul

atio

n

W.H.O ClassificationR

EAL

C

lass

ifica

tion

NC

I

2008 2016

.

CLP DN1 DN4 DP

CD8

CD4

CD8

γδ

TFH

TH1

T REG

HSC

CD4

ThymusPeriphery

Bone Marrow

Cytotoxic T

TBX21

AITLATLL

IFN-γ

IL4IL5IL13

IL21CXCL13IL10

TGF-β

STAT5Helper T

Cortex Medulla

CD4+CD8+

DN2 DN3

CD44+CD25-

CD44+CD25+

CD44-CD25+

CD44-CD25-

Pre-TCRβDependent

TCRαRearrangements

TCRβRearrangements

CD4-CD8-

NOTCHIL7

γδ

TH2GATA3

MHCClass I

MHCClass II

CD24

Mature T cell development and activation

Complexity of T-cell immunobiology, numerous subsets and functional plasticity makes meaningful disease classification challenging

HSC: Hematopoietic stem cells CLP: common lymphoid progenitor

β-Selection phase Repertoire selection phase

Iqbal & deLeval Agressive Lymphomas; series: Hem. Malignancies, 2019

W.H.O. classification of mature T/NK-cell neoplasms (2016 revised version)

CutaneousExtranodalNodal Leukemic

Primary Cutaneous CD30+ T-cell Disorders

Primary Cutaneous Gamma/Delta TCL

Extra-nodal NK/T-cell lymphoma, nasal type

ENK/TCL

Enteropathy-associated T-cell lymphomaEATL

Hepatosplenic T-cell lymphoma HSTL

Subcutaneous Panniculitis-like TCL

Adult T-cell leukemia/lymphomaATLL

Aggressive NK-Cell Leukemia

T-cell Prolymphocytic Leukemia

T-cell Large Granular Lymphocytic Leukemia

Mycosis Fungoides

Sézary Syndrome

Primary Cutaneous CD30+ lymphoproliferative disorders

Primary Cutaneous γδ TCL

Peripheral T-cell Lymphoma, Not Otherwise Specified

PTCL-NOS

Anaplastic large-cell lymphoma, ALK(+)ALCL

Angioimmunoblastic T-cell lymphoma

AITL

Follicular PTCL

Anaplastic large-cell lymphoma, ALK(-) ALCL

Monomorphic epitheliotropic intestinal T-cell lymphoma

Breast implant-associated anaplastic large cell lymphoma*

Indolent T-cell lymhpoproliferativedisorder of the gastrointestinal tract*

Primary cutaneous anaplastic large cell lymphomaLymphomatoid papulosisPrimary cutaneous γδ T-cell lymphomaPrimary cutaneous CD8+ aggressive epidermotropiccytotoxic T-cell lymphoma* Primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder*Hydroa vacciniforme-like lymphoproliferative disorderSevere mosquito bite allergy

Chronic lymphoproliferative disorder of NK cells*Systemic EBV-positive T-cell lymphoma of childhood*Chronic active EBV infection of T- and NK-cell type, systemic formAdult T-cell leukemia/lymphoma

Nodal PTCL with TFH phenotype

Adapted from Swerdlow et al. Blood 2016

University of Nebraska Medical Center

No major improvement in clinical outcome since last three decades in PTCL

OS of PTCL-NOS/AITL

PTCL-NOS30%

AITL22%

ALK-ALCL7%

ENKTCL12%

ALK+ ALCL8%

ATLL11%

HSTL2%

EATL5%

Others3%

Overall frequency of PTCL subtypes

Park et al. Int J Hematol 2014Adams et.al. J Clin Oncol. 2016Vose et.al. J Clin Oncol. 2008

36% France

20% Asia

38%USA

Adam

set

al.

J C

lin O

ncol

. 201

6La

uren

tet

al.

J C

lin O

ncol

.201

7

Park

et.

alIn

tJ H

em 2

014

Iqbal & deLeval Agressive Lymphomas; series: Hem. Malignancies, 2019

Evolution of molecular analysis techniques

Cytogenetics Gene expression profiling

Chromosomal copy number

profiling

Next generation sequencing

(RNA-seq; DNA-seq)

We are beginning to use sophisticated techniques to identify patient-and tumor-related differences that increase response rates and decrease toxicity of lymphoma therapy

-Genomic defined personalized medicine

University of Nebraska Medical Center

Molecular diagnosisAITLALCLPTCLNOS ( novel subgroups)

Pathobiology and target characterizationGenetic/epigenetic driverFunctional consequences

Rationalize/Justify the new clinical investigations

Genomic Signatures in PTCL

Genetic or molecular abnormalities defining the biology of the major PTCL subtypes

University of Nebraska Medical Center

Unsupervised hierarchical clustering of PTCL cases and normal T-cells

Iqbal et al. Blood 2010

Major PTCL entities form tight clusters with cases of PTCL-NOS and other rare entities interspersed.

Gene expression-based molecular predictors of the major subgroups of PTCL

More than half of the PTCL-NOS cases were not molecularly classified

AITL

ATLL

ALK+ALCL

Iqbal et.al Blood 2010

Molecular characteristics of AITL gene signature

CD21

CXCL13 PD1

FDCAngiogenesis

CD20

CD4

Gene signatures define crucial features in pathobiology & oncogenic pathways

Cytokine/chemokine signature

B-cell receptor signalingGerminal center–related genes

CD20CD79aIGH

CXCL13CXCR5CXCL8

T- activationTFH cell markersICOS

CTLA4PD1BTLA

FDC markersCR1 CR2 (CD21),CD23,CLU CD200

NF-κB activation pathwayIL6/STAT3 activation pathwayTGF-β signalingVEGF signalingIL12 signalingTFH gene signatures

Oncogenic pathways

P <0.05/ FDR<0.2

AITL

Iqbal & deLeval Aggressive Lymphomas; series: Hem. Malignancies, 2019

University of Nebraska Medical Center

TP53

upr

egul

ated

ta

rget

sig

natu

re

Mon

ocyt

ic s

igna

ture

B-c

ells

sig

natu

re

AITL

sur

viva

l m

odel

sco

re

H&E CD3

CD20

CD3

CD20

H&E

Development of prognostic models in AITL82

AIT

L

CD16+ mDC

CD141+mDC

82 A

ITL

CD

141+

mD

C

CD

16+

mD

C

Tumor microenvironment significantly influences AITL

prognosisIqbal et al. Blood. 2014

Refinement of prognostic signatures

2019

Genomic aberrations in AITLFr

eque

ncy

(%)

Gai

nLo

ss

Genomic copy number analysis using Affymetrix platform

Heavican et al. Blood 2019

Mutation spectrum in AITL

- Epigenetic dysregulation

- TCR signaling cascade

-1-0.8-0.6-0.4-0.2

00.20.40.60.8

1 TET2 VAF Equal TET2 VAF HIgh

RH

OA/

IDH

2

TET

2

WES RNA-seq

Major aberration

~145 AITL

IDH2R172 mutations defines a unique AITL subgroup

Chao et.al ( unpublished)Rohr et.al (unpublished)Chao et al. Blood 2015

P=NS

prop

ortio

n

• IDH2 mutants show significant hypermethylation in proximal promoter region compared to wild type and normal tonsil

• Histone modifications associated with IDH2 mutation

Aberrant CD28 signaling in AITL

Exon 2Exons 4, 3 Exon 1CD28 ICOS

Direction of transcription

Rohr et al. Leukemia 2016

CD80>>>>CD86

APC

CD28 mRNA expression

CD28 mutation spectrum in AITL

Developing genetically faithful murine models

TET2 70-80%IDH2R172 20-30%

Human AITL

Murine model

Cre-CD4+;Tet2FL/FL

Overall survival

Long term deficiency of Tet2 in CD4 + T cell leads to T-cell lymphoma with TFH immunophenotype

Heavican et.al, Unpublished

-Tet2-DNMT3A

AITL

IDH2R172

++ Chr5/21

GC

reac

tion

Epigenetic changes

Genomic CN changes

Integrative genomic analysis in AITL

Pathogenetic Evolution

Heavican et al. Blood 2019

RHOAG17V

University of Nebraska Medical Center

—AITL molecular signature reflected TFH cellular origin and suggested role of tumor microenvironment in disease pathobiology

—IDH2 & TET2 mutations co-occur suggesting unique cooperation in T-cell lymphomagenesis

—IDH2 mutations defines a unique subset of AITL patients

—Genes involved in TCR signaling and T-cell differentiation are predominantly hyper methylated in IDH2 mutant cases

—Genetically relevant murine models may lead to better understanding of lymphoma biology

Summary-I

University of Nebraska Medical Center

III-Delineating molecular subgroups within PTCL-NOS

Refinement of molecular diagnostic signatures

Unique molecular signatures were identified for major PTCL entities

Blood. 2014 May 8;123(19):2915-23.Lymphoma and Leukemia Molecular Profiling Project (LLMPP) initiative Iqbal et al. Blood 2014

HST

CL

NKCL / γδ-PTCL NK- / γδ T-cell lines CT(αβ)-PTCL T-cell

lines

Identification of γδ-PTCL from PTCL-NOS

H &E

TCR-γ

Markers StatusCD3ε 7/7 (+)CD2 2/4 (+)CD5 1/7 (+)CD7 1/1 (+)CD8 3/5 (+)CD4 1/6 (+)CD56 3/4 (+)TIA1 4/4 (+) Granzyme B 2/3 (+)TCR-beta 5/5 (-)EBER-1 3/5 (-)

OS

of γδ

PTC

L

γδ-PTCL have similar gene expression signature as NKCL but distinct from CT(αβ)- PTCL & HSTCL

Iqbal et al. Leukemia. 2011

STAT3 and STAT5B mutations identified in NK or γδ-T cell derived lymphomas

-Stat3 and Stat5B are often mutated at the SH2 domain in NK and γδ-T cell lymphomas-In vitro data analysis showed sensitivity of this mutations to JAK1/2 inhibition

Kucuk et.al Nat Commun. 2015

Evaluation of pathological vs molecular diagnosis

Out of 152 PTCL-NOS cases, a subset of cases were classified as unique PTCL entities

HTLV1

IDH2R172 mutation

IHC[CD30/TIA/GZB)

IHC(TCR-γ)

Validation of the molecular classification

One-third of PTCL-NOS cases were not molecularly classified into WHO

recognized PTCL entities

GATA3Unclassifiable

TBX21

37 0.920 1.4149 2.08

nmedian OS

(years)

Time (years)

Prop

ortio

n (O

S)

p=0.01

(B)

PTCL-NOS is subdivided into two major subgroups

(A)

Prob

abili

ty

(LO

OC

V)

TBX21GATA3

Probability in TBX21 subgroupProbability in GATA3 subgroup

TBX21 Unclassifiable GATA3

Iqbal et al. Blood 2014

APC

CD4CD

3γC

D3ε

TCRα

/βJAK2

TYK2

IL-12R

JAK1

JAK2

IFNγR

IL12

IFNγ

MH

CII

STAT1

STAT4TBX21EOMES

Cellular immunityInflammation

NOTCH3

DLL

1/4

APC

CD4CD

3γC

D3ε

TCRα

/β

JAK1

JAK3

IL-2R

JAK1

JAK3

IL4R

IL2

IL4

MH

CII

STAT6

STAT5GATA3C-MAF

Humoral immunityAb production

NOTCH 1/2

Jagg

ed1/

2

TH1/2 differentiation schematic program

TH2TH1

MacrophagesCD8+T-cellsNK cells

B-cellsIgE, IgG1, IgG3

IL2INFγ

IL4IL10IL13

Distinct Copy Number Aberrations and potential target genes associated with molecular PTCL subgroups

Heavican et al. Blood 2019BC

L11B

PTCL-GATA3 (CN gain)PTCL-GATA3 (CN loss)PTCL-TBX21CN gain/loss

0

40

40

n=61

Freq

uenc

y (%

)

REL

CD28 TP

63,TP

RG1

TRRA

P

MYC

ATM

PLCG

1STAT

3

ITPR

3

CDKN

2A

FAS,

PTE

N

FOXO

1

TP53

JAK3

IBTK

PRDM

1ZC

3H12

D, L

ATS1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 20 22

Unique mutation profiles in molecular PTCL subgroups

Heavican et al. Blood 2019

C57BL/6x129

Trp53R172H+/-;Cd4-Cre+/-

Ptenfl/+;Cd4-Cre+/-

Ptenfl/fl;Cd4-Cre+/-

Trp53R172H+/-;Ptenfl/+;Cd4-Cre+/-

Trp53R172H+/-;Ptenfl/fl;Cd4-Cre+/-

Generation and validation of the murine models

* * * * * **

* p53 mutation

Human PTCL n= 36

Significant differences in overall survival between genotypes

WT, n=38

Trp53R172H+/-;Cd4-Cre+/-, n=14 (Trp53+/-)

Ptenfl/+;Cd4-Cre+/-, n=20 (Pten+/-)

Ptenfl/fl;Cd4-Cre+/-, n=24 (Pten-/-)

Trp53R172H+/-;Ptenfl/+;Cd4-Cre+/-, n=19 (Trp53+/-;Pten+/-)

Trp53R172H+/-;Ptenfl/fl;Cd4-Cre+/-, n=9 (Trp53+/-;Pten-/-)

- Pten loss accelerates tumorigenesis in Trp53 (functional KO) mice & cooperates with Trp53 loss in tumorigenesis- No tumor observed in Pten heterozygous mice

University of Nebraska Medical Center

Genetic analysis within PTCL-TBX21 subgroup

Dendrogram for clustering PTCL-NOS cases using centered correlation and complete linkage

CT-PTCL

Cor

rela

tion

Other PTCL-U Other PTCL-U

(A) Hierarchal clustering (C) GSEA analysisIFNγ responsive genes CD8+ T-cell gene signature

P<0.01 P<0.005

(E) Granzyme B expression by immunohistochemistry in CT-PTCL

H & E Granzyme B

Prop

ortio

n

YearsYears

(D) Survival of the CT-PTCL group

CT-PTCLPTCL-NOS

p=0.05

OS

Prop

ortio

n

CT-PTCLPTCL-NOS

p=0.06

EFS

Hours after stimulation

(B) Expression of the CT-PTCL signature in normal CD8+ T-cells stimulated with anti-CD3, anti-CD28 and IL12 for various time intervals (hours)

CD8+ T-cell 0 2 8 24 48

Identification of cytotoxic αβ-PTCL group from PTCL-NOS

Iqbal et.al Blood 2010International peripheral T-cell lymphoma Project

H&E CD3 TIA1

(B)

TIA1H&E CD3

(C)

Cytotoxic

Pan-B

plasma-cell

Immunoglobulin

Cytotoxic- plasma cell signature

sign

atur

e

p=0.2 p=0.05

Quartile

(D)

Prop

ortio

n

Time (years) Time (years)

Q1Q2Q3Q4

Q4Q1+Q2+Q3

OS in TBX21 subgroup OS in TBX21 subgroupQ1 Q2 Q3 Q4

Tumor microenvironment influences prognosis in PTCL-TBX21 subgroup

Blood. 2014 May 8;123(19):2915-23.Iqbal et al. Blood 2014

PTCL-TBX21 AITL0

25

50

%R

882

Mut

atio

nsAITL

PTC

L-N

OS

n = 39% (69/176)

DNMT3A

Distinct DNMT3A mutational spectrum in PTCL-TBX21

R882

R882

DNMT3A dimer interface

R887

R887

DNA

GATA3 n = 27% (6/22)TBX21 n = 40% (12/30)TFHlike n = 10% (1/10)

R882

Unpublished

0 5 10 15 200

50

100

Years

Perc

ent s

urvi

val

DNMT3AΔWT

p = 0.04

0 5 1 0 1 5 2 00

5 0

1 0 0

Y e a rs

Per

cen

t su

rviv

al

DNMT3AΔWT

p = 0.01

0 5 1 0 1 5 2 00

5 0

1 0 0

Y e a rs

Per

cen

t su

rviv

al DNMT3AΔ

WT

p< 0.001

0 2 4 60

5 0

1 0 0

Y e a rs

Per

cen

t su

rviv

al

DNMT3AΔWT PTCL-GATA3

p = 0.9

0 5 1 0 1 5 2 00

5 0

1 0 0

Y e a rs

Pe

rce

nt

su

rviv

al DNMT3AΔ

WT

p = 0.09

0 5 1 0 1 5 2 00

5 0

1 0 0

Y e a rsP

erc

en

t s

urv

iva

l

p = 0.003

WT DNMT3AΔ

DNMT3AR882

Prognostic significance of DNMT3A mutations

n=44

n=83 n=83

n=37n=7

n=31

n=14

n=12

n=9n=13

n=4

n=30

n=52

PTCLs (excluding ALCLs) AITL

PTCL-NOS PTCL-TBX21 PTCL-GATA3

Unpublished

n=14 n=9

Activated CD8+ T cells

N.E.S. = 1.62p < 0.01q = 0.12

PTCL-TBX21

DNMT3A∇ mutations define CD8+ T-cell subgroup in PTCL-TBX21

WT DNMT3AΔ GSEA

DNMT3A mutationCytotoxic SignatureActivated CD8+T cell signatureCD4/CD8 by IHC

DNMT3A∆ WT Total Signal*0.00

0.10

0.20

0.30

0.40

CD8

Sign

atur

e

Cibersort analysisBioinformatics programs

H&E

H&E

CD4

CD4

CD8

CD8

DN

MT3

A W

TD

NM

T3A∇

Unpublished

University of Nebraska Medical Center

Summary-III

‒ PTCL-NOS can be subdivided into two molecular subgroups with distinct molecular pathobiology and cellular-origin

‒ Molecular subgroups of PTCL-NOS evolve using different oncogenic pathways

‒ Tp53 role in T-cell differentiation in PTCL-GATA3 subgroup is further warranted.

‒ DNMT3A mutation likely defines a CD8= T cell cytotoxic subgroup in PTCL-TBX21

University of Nebraska Medical Center

Speculations of cell-of-origin for PTCL subsets

Adap

ted

from

O'S

hea

and

Paul

. 201

0. S

cien

ce

AITL

PTCL-NOS/GATA3 subgroup

PTCL-NOS/TBX21 subgroup

ALCL (ALK+) ALCL (ALK-)?

ATLL

γδT γδPTCL

GEP and immunophenotypic findings corroborate these speculations

de Leval & Gaulard Blood 2014

PTCL entities are genetically distinct

Heavican et al. Blood 2019

University of Nebraska Medical Center

The complexity of PTCL can finally be addressed with the integration of global genomic analyses, which demonstrated that molecularly

defined PTCL subgroups have diverse genetic features and arise by distinct genetic pathways

Graphical summary

-Tet2-DNMT3A

AITL

IDH2R172

++ Chr5/21

GC

reac

tion

14q (BCL11B)+Chr 3 gain

Epigenetic Changes

Genomic copy number changes

PTCL-GATA3PTCL-TBX21

CD

KN2A

-TP5

3PT

EN-P

I3K

AMPK-mTOR

-Tet1/2-DNMT3A

RHOAG17V

University of Nebraska Medical Center

Fresh frozen RNA using Affymetrix platform (Blood, 2014)

-Total RNA isolation-Refined gene signature-normalization and model correction-Molecular diagnosis

Refined PTCL signature on nCounterTM

Assay development

Dia

gnos

tic

sign

atur

e ge

nes

Normalization genes

HG-U133 (Affymetrix platform)

FFPE RNA using NanoString platform

PTCL subtyping assay development for clinical setting

Validation of diagnostic model for PTCL classification

2019-Total RNA isolation-Refined gene signature-normalization and model correction-Molecular diagnosis

TCRα

TCRβ

CD3δ

CD3ε

CD3ε

CD3γ

CD3ζ

CD3ζ

CD28

CD28

CD4/

CD8

MHC

MHC

Ag

JAK/STAT

Cyto

kine

Rece

ptor

Cyto

kine

Rece

ptor

Cytokine

SOS GRB2

LAT

SLP7

6

LCK

ZAP-70

PI3K

AKT

mTOR

VAV NCK

GADSITK

PLCγ1

IP3

PIP2

RAS

DAG

MALT1 Bcl10 CARMA1

PKCθ

IKKβ IKKγ

IKKαTAK1

MKK7

JNK2

NFκBIκB

FOS JUN NFκB NFAT

IκB

Degraded

RAF

MEK1/2

ERK1/2

Calc

ium

Chan

nel

Ca2+

Ca2+

CaM

Calcineurin

CaMKIV

CREB

NFAT

NFATIP3R

IntracellularCa2+ Store

MKK3/6 MKK4/7

p38 JNK

MEKK1

NFκB

Rac/cdc42PIP3

PTCL-NOS GATA3: constitutively active

PI3K and mTOR pathways

PTCL-NOS TBX21: constitutively active

NFκB and STAT3 pathways

ALK-ALCL: enriched mTOR pathway signatures

ALK-ALCL: constitutively active JAK/STAT3 pathway

NK/T-cell lymphoma: activation of NFκB and JAK/STAT3 pathways

HSTL: high frequency STAT3 mutation

Molecularly defined AITL: oncogenic pathways NFκB, TGFβ and IL-6

signaling identified

TemsirolimusEverolimus

Bortezomib

Ruxolitinib

STAT3

Integrating new genomic information for targeted therapy in PCTL

Iqbal et al. Blood Rev. 2016

NCI Rare Tumor Initiative (Refractory TCGA-like approach)

• Fresh Frozen PTCL cases with available germline • Other sites (anticipated) in Asia for rare PTCLs

Prio

rity

list • PTCL-NOS

• AITL • ALCL • ATLL • NCKL • rare one

Projected numbers ~500 PTCLs

Comprehensive genomic characterization of PTCL entities

Acknowledgements

Iqbal LaboratoryAlyssa BouskaTayla HeavicanTyler HerekWaseem LoneJiayu YuSneha R.

Chan LaboratoryWing C. ChanTimothy McKeithanYuping Liu

Pileri LaboratoryStefano PileriMaria Antonella Laginestra

National Cancer Center SingaporeSoon Thye LimChoon Kiat Ong

Weill Cornell Medicine/ Cornell UniversityGiorgio Inghirami

Fu LaboratoryKai FuAndy Bi

Catalina AmadorTimothy GreinerKai FuJulia VoseJames ArmitageMartin BastLynette Smith

University of Nebraska Medical Center, OMAHA (USA)

Dennis WeisenburgerCity of Hope Medical Center (USA)

N.C.I-NIH (USA)Lou StaudtGeorge WrightElaine Jaffe

International PTCL Project

LLMPP consortium members

Funding Support

• Clinical and Translation Research Program• Eppley Cancer Center Support• Nebraska Medical Center Research Support

• SPORE• UH2/UH3• STTR• PO1

Acknowledgements

UNMC