Novel Insights into the Molecular Pathogenesis of T-cell Lymphomas
Kojo S. J. Elenitoba-Johnson MD Peter Nowell MD Professor Director, Division of Precision and Computational Diagnostics Department of Pathology and Laboratory Medicine University of Pennsylvania
Society of Hematopathology Workshop Molecular Genetics of Hematopoietic Neoplasms Chicago, Sept 9, 2017 Disclosure
GENOMENON: Co-Founder Outline Background
• WHO classification of mature T cell lymphoma
Recent updates in genetics of T cell lymphoma
• PTCL/AITL • CTCL/SS • ALCL • Pathobiologic and therapeutic implications
• Conclusions Outline Background
• WHO classification of mature T cell lymphoma
Recent updates in genetics of T cell lymphoma
• PTCL/AITL • CTCL/SS • ALCL • Pathobiologic and therapeutic implications
• Conclusions 2016 World Health Organization
Mature T-cell neoplasms
Cutaneous Extranodal Nodal Leukemic
Mycosis fungoides (MF) NK/TCL nasal type Peripheral TCL-NOS Adult T-cell leukemia/lymphoma Sézary syndrome Enteropathy- Anaplastic large Cell associated TCL lymphoma (ALK +) T-cell prolymphocytic Primary cutaneous CD30+ T- leukemia cell Disorders Monomorphic epitheliotropic intestinal T- Anaplastic Large Cell Primary cutaneous ALCL cell lymphoma* lymphoma (ALK -) T-cell large granular lymphocytic leukemia Breast implant- Primary cutaneous Hepatosplenic TCL γδ TCL associated ALCL* Chronic Subcutaneous lymphoproliferative Primary cutaneous CD8+ Angioimmunoblastic disorder of NK cells* aggressive epidermotropic Panniculitis-Like TCL TCL cytotoxic TCL* Systemic EBV+ T-cell Follicular T cell Aggressive NK-cell Primary cutaneous acral lymphoproliferative lymphoma* leukemia CD8+ TCL* disorder of childhood Nodal peripheral T cell Primary cutaneous CD4+ lymphoma with TFH *Provisional entity. small/medium T cell lymphoproliferative disorder* phenotype*
Hydroa vacciniforme-like Relative frequencies of mature T-cell lymphomas
International T-Cell Lymphoma Project
1. Vose JM et al. J Clin Oncol. 2008;26(25):4124-4130. Subclassification of PTCL and Survival
International T-Cell Lymphoma Project. J Clin Oncol. 2008;26(25):4124-4130. Molecular diagnostic signatures of TCL subgroups
Iqbal J et al., Blood Rev 2016
8 Diagnostic and biologic gray zones in PTCL
• Morphologic, immunophenotypic and genetic overlap
AITL
PTCL-NOS Markers to consider:
ALCL, ALK-negative
Herrera AF et al. Cancer. 2014;120(13):1993-1999. Herrera AF et al. Cancer. 2014;120(13):1993-1999. Outline Background
• WHO classification of mature T cell lymphoma
Recent updates in genetics of T cell lymphoma
• PTCL/AITL • CTCL/SS • ALCL • Pathobiologic and therapeutic implications
• Conclusions Peripheral T-cell Lymphoma/ Angioimmunoblastic T-cell Lymphoma Mutational landscape of PTCL and AITL
12 Cell-type specific mutations in AITL
13 Nguyen and Chiba, Blood Cancer J 2017
Angioimmunoblastic T-cell Lymphoma
Cortes JR and Palomero T. Current Opinions in Hematology 2016 Summary
Mutations in RHOA 70% of AITL; 20%PTCL
Loss of function of TET2 and DNMT3A frequent
IDH2 R172H is highly enriched in AITL and associates with TET2 mutations
Mutations targeting the TCR signaling pathway (CD28, Fyn) contribute to PTCL/AITL pathogenesis
Current model for pathogenesis of AITL suggests initial TET2/DNMT3A mutation followed by RHOA or IDH mutation with lineage commitment to TFH cells
16 Summary Targeted therapy for T-cell lymphoma
Cortes JR and Palomero T. Current Opinions in Hematology 2016 Cutaneous T-cell Lymphoma
Mycosis Fungoides/Sezary Syndrome SÉZARY SYNDROME
• Malignancy of post-thymic T-cells characterized by triad of erythroderma, lymphadenopathy, pruritus
• Complex karyotypes without recurrent structural variants
19 Whole Genome Sequencing Confirms Complexity of Sézary Syndrome Genomes
20 Kiel M, et al. Nat Commun 2015 Exome Sequencing Identifies Regions of Recurrent Aneuploidy in 74 Sézary Patients • 1p36.11 TP53 • 3p21.31 • 9p21.3 • 10p11.22 • 10q23.31 RB1 • 10q21.2 • 13q14.2 • 17p • 19p PTEN CDKN2A 21 Loss of Function of ARID1A in Sézary Genomes (25/62; 40%)
22 ARID1a inactivation detected by immunocytochemistry Loss of Function of TET1/2 in Sézary Genomes (38/62; 61%)
24 Epigenetic Modifiers are Targeted by Aneuploidy and Mutation in Sézary Genomes
25 Choi J et al Nat Genet. 2015 Sep;47(9):1011-9 Wang L et al Nat Genet. 2015 Dec;47(12):1426-34 da Silva Almeida AC et al Nat Genet. 2015 Dec;47(12):1465-70 Ungewickell A et al Nat Genet. 2015 Sep;47(9):1056-60. CD80/86 TCR/CD3 ~36% TNF ~18% PDCD1 FAS-L R2 R2 CTLA4-CD28 * CD4 TGFβ R2 IL-2 IL2R εδ ε γ RII RI P Smad 2
FAS Y Lck JAK1/3 LAT GRB2 PP DDDD DD P ~11% ITK PI3K P P TRADD TRADD TAB2 P P ~9% ZAP70 RIP STAT3 STAT5 TRAF2 TRAF3 PIP2 PIP3 P
PLCγ1 P kinase P Smad 3 TAK1 ~45% P ~36% PTEN ZEB1 TAB1 Apoptosis ~25% IP3 DAG ~15% A20 PKCθ CARMA1 ++ RAS AKT BCL10 Loss of Function [CA ] MALT1 Genotoxic stress Gain of Function Calcineurin ~12% IKK ~30% Growth Arrest MAPK AP-1 NFκB ATM Cell Proliferation NFAT c-FOS c-JUN NFκB
~25% ~67% Cell cycle MYC MLL ~26% ~25% P progression RB1 DNMT EZH2 PRC BMI1 HDAC CDK4 CCND ~40% H3K27 P ~50% CDK6 CCND Me2/3 X p16 STAT3 TET1/2 E2F CDKN2A/B ~40% ~47% P STAT5 H3K4 p19 MDM2 KDM6B Me2/3 p53 p21 X ~92% RB1 ARID, SMAR ~50% ~40% TRX MLL, SETD NCOR ARID1A Cell cycle arrest Summary
• Genomic complexity
• Somatic mutations in chromatin remodelers and epigenetic modifiers (ARID1A, TET2, DNMT3A)
• Mutations in oncogenic drivers coexist in neoplastic cells (JAK1/3, STAT3/5)
• Mutations in tumor suppressors (p53, p16, PTEN)
28 Anaplastic Large Cell Lymphoma Rearrangements in ALK- ALCL
73 ALK- ALCL • 22/73 (30%) DUSP22 translocated • 6/73 (8%) TP63 translocated • Mutually exclusive • 45 were ALK/DUSP22/TP63 triple negative
Parillia Castellar ER et al Blood 2014 Primary cutaneous CD30-positive T-cell lymphoproliferative disorders
● 2nd most common group of cutaneous T-cell lymphomas (CTCL): 30% of cases
● 2 different types:
H&E CD30 ► Primary cutaneous anaplastic large cell lymphoma (c-ALCL)
► Lymphomatoid papulosis (LyP)
● No consistent genetic abnormalities
31
NPM1 TYK2
• Nucleophosmin 1 • Non-receptor tyrosine protein kinase • Multiple functions particularly in • First member of JAK family nucleolus associating with • Signal transduction by interferons ribonucleolar proteins and interleukins
33 Translocations involving TYK2 were found in clinical samples by FISH
17.2% of CD30+ cutaneous LPDs
25 3/15 20 2/14 15
0/151 10 % occurrence% 5
0 Lymphomatoid Primary Other mature T- papulosis cutaneous cell lymphoma anaplastic large cell lymphoma 34 Tyk2 mediates multiple important inflammatory cytokine responses
N- FERM SH2 pseudokinase kinase -C 1 1187
IL-23 p19 p40 subunit subunit IL-12 p40 subunit INFα/β p35 subunit
Jak2 Tyk2 Tyk2 Jak1
Jak2 P Tyk2 STAT 1-5
35 Expression of pSTAT5 in NPM1-TYK2+ lymphoma
pSTAT5
CD30+LPD CD30+LPD NPM1-TYK2 - NPM1-TYK2 + Ectopic expression of NPM1-TYK2 protein in HEK293FT cells activates STAT proteins
37 In vivo modeling of NPM1-TYK2 fusion
hNPM1-TYK2
Elenitoba-Johnson Lab Unpublished 38 Knockdown of TYK2 in MyLa cells decreases cell proliferation
39 Targeting NPM1-TYK2 fusion protein in MyLa cells decreases proliferation
100
IC50 = 130 nM 50 Relative growth Relative
Elenitoba-Johnson Lab, 0 Unpublished 0 1 2 3 4 Log 10 (JAK inhibitor I, nM) 40 NDI-031301 inhibits downstream signaling pathways of NPM1-TYK2 and PCM1-JAK2
MyLa Mac-1
30 min 1 h 3 h 6 h 30 min 1 h 3 h 6 h
NID-031301 (3 µM) - + - + - + - + - + - + - + - + p-NPM-TYK2 (Y1054/1055) p-PCM1-JAK2 (Y1007/1008)
NPM-TYK2 PCM1-JAK2
p-STAT1 (Y701)
STAT1
p-STAT4 (Y693)
STAT4
p-STAT3 (Y705) p-STAT3 (Y705)
STAT3 STAT3
p-STAT5 (Y694) p-STAT5 (Y694)
STAT5 STAT5
β-actin β-actin
Elenitoba-Johnson Lab,Unpublished NDI-031301 inhibits TYK2
● Proliferation assay (72h)
IC50 Cell line (μM) 150 HH NR
MyLa (NPM1-TYK2) 3.407 100 Hut-78 (JAK1 and JAK3 3.050 HH mutation)
MyLa
Hut-78 H9 (JAK1 and JAK3 mutation) 3.337 50 H9 M ac-1 Mac1 (PCM1-JAK2) 3.516 MJ Relative growth (% of control) of (% growth Relative
MJ 2.824 0 0 10 100 1,000 10,000 NR: not reached
NDI-031301 (nM) Summary
● Recurrent TYK2 translocations in cutaneous T-cell lymphomas
● NPM1 as one of the fusion partners
● NPM1-TYK2 translocation results in constitutive activation of TYK2 kinase
● NPM1-TYK2 fusion protein: oncogenic effects through STATs activation
● Evidence for causation and sufficiency in vivo is pending
● TYK2 may be a novel therapeutic target in a subset of CD30+ lymphoproliferative disorders
Velusamy et al. BLOOD 2015
43 Deregulated JAK/STAT signaling in ALCL, ALK -ve
Crescenzo R, et al. Cancer Cell April 2015 Anaplastic large cell lymphoma
Crescenzo R, et al. Cancer45 Cell April 2015 Subclassification of ALK- ALCL
ALK- ALCL
DUSP22 Rearrangement TP63 Rearrangement TYK2 Rearrangement VAV Rearrangement Functional Proteogenomics of ALCL Glycoproteomics for identification of biomarkers and therapeutic targets
• Glycosylation is a common post translational modification
• Glycoproteins are expressed on cell surface and secreted
• Most CD markers recognize glycoproteins Nature Medicine 2009, 15: 828
48 Glycoproteomic Profiling By Solid Phase Extraction of Glyoproteins
Peptide N Glycosidase F
∆0.98406 amu
PNGase F
Hydrogen atom = 1.00782580
LC-MS/MS analysis 49 N-glycoproteomic profiles classify lymphoid neoplasia - 1 - 4 299 1106P 519 - 1 - 12 - 11 - M2 - B1 - 1 - 2A - 1 - Ly2 - Ly1 - 78 - 786 - 92 - 92MI - 518 - 218 - 18 - 318 - 266 HH Hut DEL SUD - HL Mac Mac Karpas SR SUP JURKAT YT NK NK U KMS KMS 8226 RPMI FL DEV Karpas NCEB Granta Rec FL OCI SUD - HL BJA - B Ramos OCI Raji FL FL KMH - 2 Med L1236 L428 T/NK-cell B-cell
ALCL NK MM MCL BL/tFL CHL MF SS ALCL, ALK+ ALCL, ALK- T-ALL NK MM NLPHL tFL/GCB MCL PMBL DLBCL BL CHL A distinct cytokine signature is characteristic of ALK+ ALCL - 1 - 4 299 1106P 519 - 1 - 12 - 11 - M2 - B1 - 1 - 2A - 1 - Ly2 - Ly1 - 78 - 786 - 92 - 92MI - 518 - 218 - 18 - 318 - 266 HH Hut Mac Mac DEL SUD - HL Karpas SR SUP JURKAT YT NK NK U KMS KMS 8226 RPMI FL DEV Karpas NCEB Granta Rec FL OCI SUD - HL BJA - B Ramos OCI Raji FL FL KMH - 2 Med L1236 L428 T/NK-cell B-cell ALCL,ALK+ IL2Rα IL31Rβ
● IL2Rα (CD25)
● IL31Rβ (Oncostatin M receptor)
Potential novel biomarkers IL31Rβ is selectively expressed in ALK+ALCL
● Cell lines ● 56 primary biopsies of ALCL
ALCL, ALK+
1 ALK-, - Tonsil IL31Rβ- DHL 92MI - - DEL SR786 SupM2 MAC1 MAC2A Jurkat HT1080 Karpas 299 Karpas SU RAJI L428 KMH2 HH HUT78 YT NK BJAB HT1080
IL31Rb
GAPDH
ALK-, ALK+, IL31Rβ+ IL31Rβ+
DEL Karpas 299 SR786 SU-DHL-1 8.75 2.16 2.46 4.76
IL31Rβ + IL31Rβ -
ALK + 21 0 SupM2 MAC2A Jurkat 13.7 1.02 1.02 ALK - 14 21 Χ2 = 20.16
Count p<0.001
IL31Rβ CRISPR-Cas9 sgRNA genome-wide vulnerability
14, 250 Lentiviral sgRNAs Vector Designs: sgRNA sgRNA expression: U6, U6-Tet, H1 or H1-Tet Markers: GFP, RFP, PuroR, Promoters: UbiC, EF1a, CMV
PCR & Cloning Packaged Pooled Lentiviral sgRNA Library Barcoded Pooled Plasmid sgRNA Library Target Cell Transduction Barcoded Quantitative sgRNA Identification of 80-90% of Amplification Enriched or Sequences Depleted sgRNA Within 1 Order of Frequency Corresponding to Transduced Gene Targets Magnitude Barcode Representation Target Cells Cytokine receptor pathways are exquisite vulnerability targets in ALK+ALCL
IL6-STAT3 IL2-STAT5
Markov Chain Monte Carlo Simulation IL31Rβ contributes to oncogenesis in ALK+ALCL
p <0.01 80 Scramble shRNA shRNA 60 β
40 WT shRNA Scramble IL31R β
IL31R Percentage Percentage
IL31Rβ shRNA of tumor growth 20 of tumor growth tumor of
0 Scramble IL31Rβ β-actin shRNA shRNA
IL31Rβ knockdown abrogates tumor growth in ALK+ALCL, xenotransplants
Rolland D et al., PNAS 2017 56 Summary
Anaplastic large cell lymphoma
Cytokine/receptor – JAK- STAT signaling plays a prominent role in ALCL pathogenesis
Opportunities for targeted vulnerabilities
57 Outline • Background
– WHO classification of mature T cell lymphoma
• Recent updates in genetics of T cell lymphoma
– PTCL/AITL – CTCL/SS – ALCL
• Pathobiologic and therapeutic implications
• Conclusions Recurrent large structural alterations in T-cell neoplasms
Disease entity Structural Abnormality Frequency
ALK+ ALCL NPM1-ALK 84%
Various-ALK 16%
T-PLL TRA-TCL1A 70-80%
TRA-MTCP1 10%
HSTL i(7q)(q10) >80%
EATL 9q gains ~70%
16q12.1 loss ~30%
ALK- ALCL, c-ALCL IRF4/DUSP22 translocations 25%
TYK2/ NPM1-TYK2 17%/4%
VAV rearrangements 4%
PCM-JAK2 rare
PTCL P53-related genes 6%
F-PTCL ITK-SYK 18-38% 59 AITL ITK SYK rare Recurrent somatic gene mutations in T-cell neoplasms Gene Disease entity Frequency JAK1/JAK3 T-PLL 40% NKTCL/EATL 20-35% STAT3 T-LGLL 27-40% Leukemias JAK/STAT GD HSTCL/EATL 9% CLPD-NK 30% ALK-ALCL STAT5B T-PLL 36% GD HSTCL/EATL 36% T-LGLL rare CD28 AITL, MF/SS 11%, rare Co-stimulatory TCR signaling FYN AITL; PTCL, NOS 3% ,rare PKCG1 AITL, MF/SS, PTCL, 12%,20%,15% NOS PRKCB NKTCL 33% NF-κB CARD11 MF/SS, NKTCL 15%,24% Lymphomas TNFRSF1B MF/SS, NKTCL 6%, rare TET2 AITL, F-PTCL 33-47%, 58% Epigenetic IDH2 AITL ~25% DNMT3A AITL; PTCL, NOS 11% overall SETD2 EATL I/II 32%
Other RHOA AITL; PTCL, NOS 60 67%; 18% CCR4 MF/SS 7% Impact on therapeutic decisions
Lymphoma Genetic features Therapeutic relevance
ALCL ALK ALK inhibitor cALCL TYK2 TYK2 inhibitor AITL ITK/SYK SYK inhibitor T-PLL/ENKTCL JAK3 JAK3 inhibitor T-PLL STAT5B STAT5 inhibitor T-LGLL, NK-LPD STAT3 STAT3 inhibitor
AITL TET2, IDH2, DNMT3A Epigenetic modulators PTCL, NOS DNMT3A Epigenetic modulators F-PTCL TET2 Epigenetic modulators
61 Conclusions
• Genetic basis of T-cell lymphomas reveals complexity within each entity
• Profound implications for taxonomy
• Biologic subcategories and prognosis
• Targeted therapeutics Acknowledgements
Department of Pathology, Department of Internal Medicine, University of Pennsylvania University of Michigan Megan S. Lim Ryan A. Wilcox Delphine Rolland Department of Hematopathology, Ozlem Onder Department of Pathology, MD Anderson Cancer Center Roberto N. Miranda University of Michigan Thirunavukkarasu Velusamy L. Jeffrey Medeiros Mark J. Kiel Department of Pathology, Anagh A. Sahasrabuddhe Seoul National University Hospital Catherine A. Dixon Nathanael G. Bailey (Univ of Pittsburgh) Yoon K. Jeon Bryan L. Betz Department of Pathology, Noah A. Brown Henry Ford Health System Alexandra C. Hristov Kedar V. Inamdar 63 QUESTIONS?
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