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Research. Clinical Care. Education. Molecular markers in NKTL. A/Prof Chng Wee Joo Department of Haematology-Oncology National Cancer Institute of Singapore National University Health System Senior Principle Investigator Cancer Science Institute, Singapore - PowerPoint PPT PresentationTRANSCRIPT
Education
Clinical Care
Research
Molecular markers in NKTL
A/Prof Chng Wee JooDepartment of Haematology-OncologyNational Cancer Institute of SingaporeNational University Health SystemSenior Principle InvestigatorCancer Science Institute, SingaporeNational University of Singapore
Extranodal nasal-type Natural Killer/T-cell lymphoma (NKTL)
• Distinct clinicopathologic entity most commonly affecting Asians and Central and South Americans
• characterized by a clonal proliferation of NK or T cells with a cytotoxic phenotype.
• There is a strong association with Epstein-Barr Virus (EBV), which manifests a type II latency– expression of LMP-1 and EBNA-1, – absence of EBNA-2.
• EBV detected in the neoplastic cells in a clonal episomal form, supporting the role of the virus in tumor pathogenesis
Clinical Spectrum of Extra-nodal Natural Killer / T-cell Lymphoma
Kwong YL. Leukemia 2005; 19:2186
EBV infection
LMP-1 or other factors
MYC activation
P53 mutations
Induce Survivin
Proliferation Anti-apoptotic
Regulate
Proposed model of NKTL pathogenesis
NF-KB activation
p53 Deregulation
Ng SB, et al. J Pathol. 2011 Mar;223(4):496-51
microRNAs: small molecules with a big impact
• MicroRNAs (miRNA) are a class of small,
non-coding RNAs (~20 nts long) that
repress gene expression (in most cases)
– Degrading or repressing mRNAs
– Important class of gene regulators that controls
most biological processes.
– Latest in human: 1527 precursors, 1921 mature
miRNAs (miRbase 19)
• Each miRNA can have hundreds of different
conserved or nonconserved targets
• Samples:– 30 cases of NKTL FFPE– 6 NK cell lines (KHYG-1, NK-92, HANK-1, SNT-8, SNK-6 and
NK-YS)– 3 paired samples of normal NK cells (unstimulated and
stimulated) isolated from buffy coat packs of whole blood samples from blood bank
– 2 cases each of normal skin, intestinal, nasal and lymph node FFPE tissue were also included as control tissue
miRNA are predominantly downregulated in NKTL• miRNA deregulation in NKTL
– In both NK cell lines and FFPE NKTL samples compared to normal NK cells, among the miRNAs showing at least 2-fold and statistically significant difference (p<0.05) in expression:
• 2 upregulated (miR-155 and miR-378)• 39 were down-regulated: miR-342-5p, miR-26b, miR-
363, miR-150 and miR28-5p
– Validation of MEP results• Real-time RT–PCR quantification of miRNAs• Correlation with microRNA transcriptome of NK cell using
sequencing method
quantitative-PCR validation of selected miRNAs consistent with MEP data
Upregulated miRNA
Downregulated miRNA
miR-155
Normal NKTL NK Cell Lines0.1
1
10
100
miR-378
Normal NKTL NK Cell lines0.1
1
10
100
1000
miR-26b
Normal NKTL NK Cell Lines0.001
0.01
0.1
1
10
miR-363
Normal NKTL NK Cell Lines0.001
0.01
0.1
1
10
miR-342-5p
Normal NKTL Nk Cell lines0.001
0.01
0.1
1
10
Selection of high probability predicted target genes of deregulated miRNA
Intersect with our previous GEP data to narrow down target genes whose expression is inversely correlated with expression of deregulated miRNAs
pictarmirBase
targetScan
miRanda
tarBase
mirtarget2
Predicted targets
Gene expression profile list (J Pathol. 2011 Mar;223:496-
51)
Final list
226 target genes of 41 deregulated
miRNA
Validation of miRNA targets: Re-expression of miRNA using lentiviral vector
• Target genes of miR-101, miR-26a, and miR-26b selected (STMN1, BCL2, IGF1, EZH2)
• Lentiviral vectors used to re-express these miRNAs in NKYS cell line
• Results – reduced growth of NKYS– modulated the expression of their predicted target genes– suggesting the potential functional role of the deregulated
miRNAs in the oncogenesis of NKTL
mRNA expression changes upon overexpression of miRNAs by Lentiviral transduction
mir-26b precursor
control control controlmir-26b precursor
Mir-101 precursor
mir-101 precursor
control
BCL2 IGF1
Immunohistochemistry reveals overexpression of target proteins of suppressed miRNAs in NKTL
• IHC for selected target proteins (MUM1/IRF4, BLIMP1, STMN1) of deregulated miRNAs performed on 38 cases of NKTL for validation
BLIMP1 MUM1 STMN1
17/34 (50%) 20/38 (53%) 20/35 (57%)
Mechanism of miRNA deregulation in NKTLRole of MYC•MYC is known to cause extensive repression of miRNA expression (Chang TC, et al. Nat Genet. 2008;40:43-50)•Indeed, in our cohort, tumor samples with increase expression of BLIMP1, MUM1 and STMN1 proteins, regulated by their underexpressed miRNAs, showed higher MYC nuclear expression, consistent with MYC activation
EZH2 overexpression in majority of NKTL.
NK
TL
cell
lines
Nor
mal
NK
ce
lls
NK
TL
Nor
mal
tiss
ue
6
8
10
12
P=0.003 P=0.002
A
B
Inhibition of EZH2 with DZNep induced cell growth inhibition and apoptosis in NK malignant cells.
EBV infection
LMP-1 or other factors
MYC activation
NF-KB activation
P53 mutations
P53 Deregulation
Induce Survivin
Proliferation Anti-apoptotic
Regulate
Proposed model of NKTL pathogenesis
P53 Deregulation
miRNA
EZH2
JAK3 mutations
STAT activation
Future Studies
• MYC-EZH2 in about 50%, JAK3 mutation in about 35% – Do they signify 2 molecular groups of NKTL i.e are these
abnormalities mutually exclusive or overlapping ?– What are the clinical implications if such subtypes exist?– Opportunity to answer these questions as collaborative
projects within the Asian lymphoma study group• STAT3 and p53 mutation• EZH2, MYC and NFKB protein by IHC
Acknowledgement
YAN Junli Viknes Koh Tze Loong
Ng Siok Bian
Acknowledgement and thanks
National University Health System, Singapore Jim Liang-Seah TayBaohong LinChonglei BiJoy TanGaofeng Huang
Queen Mary Hospital, Hong KongYok-Lam Kwong
Tokyo Medical and Dental University, JapanNorio Shimizu
Osaka University Graduate School of Medicine, Japan Katsuyuki Aozasa
Funding from NMRC, NRF, MOE