PTCL-NOS: Gene expression profiling Javeed Iqbal Department of Pathology and Microbiology James O. Armitage center for Leukemia and Lymphoma Research University of Nebraska Medical Center Omaha,NE T- cell Lymphomas: we are close to the finalization”
History of Lymphoma Classification Kiel classification Luke & Collins classification/u Classification Formulation REAL Rappaport NCI Working W.H.O pdate Kiel Classification 1982 1988 1998 2001 2016 2008 1956 1974 Nebraska Morphology/ Clinical Classification project-1999 + Immunohistochemistry + Cytogenetics, FISH + Molecular Biology + Gene Expression Profiles Transcriptomics + Next Generation Sequencing Genomics PTCL entities ~19 ~25 ~30
Gene Signatures in PTCL • The genomic characteristics of each tumor through expression of a unique set of genes is known as “GENE SIGNATURE Molecular diagnosis Pathobiology and target characterization Rationalize/Justify the new clinical investigations University of Nebraska Medical Center
Mature T cell development and activation γδ γδ CD24 Medulla IFN- γ Cortex MHC T H 1 Class II TBX21 HSC Pre- TCRβ TCRα TCR β T H 2 IL4 CD4 CD4 Dependent Rearrangements Rearrangements IL5 GATA3 NOTCH IL13 Helper T STAT5 IL7 T FH DN1 DN2 DN3 DN4 DP T REG BCL6 CLP IL21 FOXP3 CD44+ CD44- CD44+ CD44- CD4+ CXCL13 IL10 CD25- CD25+ CD25+ CD25- CD8+ TGF- β CD4 - CD8 - CD8 CD8 Cytotoxic T MHC Class I Periphery Bone Marrow Thymus -selection phase repertoire selection phase HSC: Hematopoietic stem cells CLP: common lymphoid progenitor Complexity of T-cell immunobiology, numerous subsets and functional plasticity makes disease classification challenging
W.H.O. classification of mature T/NK-cell neoplasms (2016 revised version) Nodal Extra-nodal Cutaneous Leukemic Peripheral T-cell Lymphoma, Not Otherwise Specified PTCL-NOS Angioimmunoblastic T-cell lymphoma AITL Nodal PTCL with T FH phenotype Follicular PTCL Anaplastic large-cell lymphoma, ALK(+)ALCL Anaplastic large-cell lymphoma, ALK(-) ALCL adapted from Swerdlow SH et al. Blood 2016 127:2375-2390)
Overall frequency of PTCL subtypes Others 3% EATL HSTL 5% 2% ATLL PTCL-NOS Scott V. Adams et.al 11% J Clin Oncol. 2016 30% 38% USA ALK+ ALCL 8% Int J Hem 2014 Park et. al 20% ENKTCL AITL Asia Laurent et.al J Clin Oncol. 2017 12% 22% 36% France ALK-ALCL 7% Park, S. & Ko, Y.H. Int J Hematol (2014) Scott V. Adams et.al J Clin Oncol. 2016; 34: 963 – 971 Vose et.al J Clin Oncol. 2008, 26:4124 – 4130
Major PTCL subtypes have inferior clinical outcome Diagnosis Adult T-cell leuk/lymph (ATLL) Anaplastic large cell lymphoma, ALK- 1.0 Anaplastic large cell lymphoma, ALK+ 0.9 Angioimmunoblastic T-cell lymphoma 0.8 Peripheral T-cell lymphoma, NOS 0.7 Proportion Generic NK-cell 0.6 0.5 0.4 0.3 0.2 0.1 Test: p<0.001 0.0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 - Xu et.al PLOS One 2014 “ No Survival Improvement for PTCL/AITL patients over the Past Two Decades: A Population- Based Study of 1207 Cases”
No major improvement in clinical outcome since last three decades in PTCL OS of PTCL-NOS/AITL University of Nebraska Medical Center
High-through put technologies dissecting distinct molecular and prognostic subgroups Transcriptomics Gene expression profiling: (mRNA expression) Genomic DNA hybridization (DNA copy number variations) Genomics Next generation sequencing Dissecting out Genetic Evolution of molecular subgroups Patient-centered Delivery to patient care Laboratory discoveries Research /communities
Unsupervised hierarchical clustering of PTCL cases and normal T cells Major entities of PTCL form tight clusters with cases of PTCL-NOS and other rare entities interspersed. University of Nebraska Medical Center Blood 2010;115:1026-1036
Gene expression-based molecular predictors of the major subgroups of PTCL AITL ATLL ALK+ALCL More than half of the PTCL-NOS cases were not molecularly classified International PTCL Project Blood 2010;115:1026-1036
Identification of cytotoxic ( ) PTCL group from PTCL- NOS (A) Hierarchal clustering (C) GSEA analysis IFN responsive genes CD8+ T-cell gene signature Correlation P<0.01 P<0.005 Other Other PTCL-U CT-PTCL PTCL-U Dendrogram for clustering PTCL-NOS cases using centered correlation and complete linkage (D) Survival of the CT-PTCL group (B) Expression of the CT-PTCL signature in normal CD8+ T-cells OS EFS Proportion stimulated with anti-CD3, anti-CD28 and Proportion IL12 for various time intervals (hours) CT-PTCL CT-PTCL PTCL-NOS PTCL-NOS CD8+ T-cell p=0.06 0 2 8 24 48 p=0.05 Years Years (E) Granzyme B expression by immunohistochemistry in CT-PTCL Hours after stimulation H & E Granzyme B International peripheral T-cell lymphoma Project Blood 2010;115:1026-1036
Identification of -PTCL from PTCL-NOS -PTCL have similar gene expression signature as NKCL but distinct from CT(α )- PTCL and HSTCL HSTCL NK- / T-cell NKCL / -PTCL T-cell CT( α )-PTCL lines lines Markers Status CD3 7/7 (+) CD2 2/4 (+) CD5 1/7 (+) CD7 1/1 (+) H &E CD8 3/5 (+) OS of PTCL CD4 1/6 (+) CD56 3/4 (+) TIA1 4/4 (+) Granzyme B 2/3 (+) TCR-beta 5/5 (-) TCR- EBER-1 3/5 (-) Leukemia. 2011 Feb;25(2):348-58.
Refinement of molecular diagnostic signatures Unique molecular signatures were identified for major PTCL entities Lymphoma and Leukemia Molecular Profiling Project (LLMPP) initiative Blood. 2014 May 8;123(19):2915-23. Blood 2014
Robust molecular signature for ALK(-)ALCL Gene signature/pathway enrichment summary in ALK(-)ALCL Re-classified from PTCL-NOS ALK(+) ALCL ALK(-) ALCL PTCL-NOS ALK ALK ALK(-) ALCL is molecularly distinct from PTCL-NOS and ALK(+)ALCL Blood. 2014 May 8;123(19):2915-23.
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 Nat Commun. 2015 Jan 14;6:6025.
Evaluation of pathological vs molecular diagnosis of 152 PTCL-NOS cases, a subset of cases were classified into unique PTCL entities AITL Infrequent n=117 14% 20% IDH2 mutation 33% 1 of 17 IDH2 mutation PTCL-NOS n=152 Blood. 2014 May 8;123(19):2915-23.
One-third of PTCL-NOS cases were not molecularly classified into WHO recognized PTCL entities n=251 n=121 ₋ AITL ₋ ATLL 32% ₋ ALK(-)ALCL PTCL-NOS ₋ ALK(+)ALCL ₋ NKCL ₋ -PTCL
Unsupervised clustering of PTCL-NOS showed at least 3 major clusters (a) (b) (mRNA) Gata3 Cluster 2 cluster3 Cluster1 TBX21 TBX21 CXCR3 (mRNA) STAT1 Ig signature EOMES (c) GATA3 proportion CCR4 121 PTCL-NOS Years
PTCL-NOS can be further divided two major subgroups (A) (B) Probability (LOOCV) Probability in TBX21 subgroup Probability in GATA3 subgroup median OS (years) n 37 0.9 GATA3 Proportion (OS) Unclassifiable 20 1.41 TBX21 49 2.08 p=0.01 TBX21 GATA3 Time (years) TBX21 Unclassifiable GATA3
GEP identified distinct oncogenic pathways INF- gene NF- B target gene Plasma cell gene signature signature signature GATA3 TBX21 IHC validation -catenin oncogenic Proliferation MYC targets signature PI3Kinase regulated (a) Myc induced gene targets (up) Ribosomal transcripts (mTOR) Proliferation related
Tumor microenvironment significantly influences the prognosis in PTCL-TBX21 subgroup (D) OS in TBX21 subgroup OS in TBX21 subgroup Q1 Q2 Q3 Q4 Q1+Q2+Q3 Quartile Q4 Q1 Q2 Q3 Proportion Q4 Cytotoxic signature Pan-B plasma-cell p=0.2 p=0.05 Immunoglobulin Time (years) Time (years) (B) TIA1 H&E CD3 (C) H&E CD3 TIA1 Blood. 2014 May 8;123(19):2915-23.
T H 1/2 differentiation ProgramSchematic APC Jagged1/2 APC MHCII DLL1/4 MHCII T H 2 T H 1 TCR / CD3 CD3 TCR / CD3 CD3 CD4 NOTCH 1/2 CD4 NOTCH3 JAK1 IL-2R JAK2 IL-12R IL2 STAT5 GATA3 Humoral immunity IL12 Cellular immunity JAK3 TBX21 STAT4 C-MAF TYK2 Abs production EOMES Inflammation STAT6 STAT1 JAK1 IL4R IFN R JAK1 IL4 IFN JAK3 JAK2
Schematic of the Gene Expression and Dosage Integration algorithm University of Nebraska Medical Center Proc Natl Acad Sci U S A. 2008 Sep 9; 105(36): 13520 – 13525.
• Major Subgroups within PTCL-NOS characterized by distinct genomic abnormalities PTCL-TBX21
CN Abnormalities Associating with Overall Survival CDKN2A TP53 MYC
Novel PTCL subgroups have distinct Chromosomal Abnormalities
• Unique Mutation Profiles in molecular subgroups
Distinct spectrum of DNMT3A mutations PTCL-TBX21 AITL University of Nebraska Medical Center
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