1. Education
Clinical Care
Research
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
National University of Singapore

2. 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

3. Clinical Spectrum of Extra-nodal Natural Killer / T-cell Lymphoma
Kwong YL. Leukemia 2005; 19:2186

4. 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

5. 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

6. • 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

7. 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

8. quantitative-PCR validation of selected miRNAs
consistent with MEP data
Upregulated
miRNA
Downregulated miRNA
miR-155
Normal NKTL NK Cell Lines
0.1
1
10
100
miR-378
Normal NKTL NK Cell lines
0.1
1
10
100
1000
miR-26b
Normal NKTL NK Cell Lines
0.001
0.01
0.1
1
10
miR-363
Normal NKTL NK Cell Lines
0.001
0.01
0.1
1
10
miR-342-5p
Normal NKTL Nk Cell lines
0.001
0.01
0.1
1
10

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
pictar
mirBase
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

11. 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

12. 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

13. 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%)

15. Mechanism of miRNA deregulation in
NKTL
Role 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

17. EZH2 overexpression in majority of NKTL.
NKTLcelllines
NormalNKcells
NKTL
Normaltissue
681012
P=0.003 P=0.002
A
B

18. Inhibition of EZH2 with DZNep induced cell growth inhibition and
apoptosis in NK malignant cells.

19. 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

20. 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

21. Acknowledgement
YAN Junli Viknes Koh Tze Loong
Ng Siok Bian

22. Acknowledgement and thanks
National University Health
System, Singapore
Jim Liang-Seah Tay
Baohong Lin
Chonglei Bi
Joy Tan
Gaofeng Huang
Queen Mary Hospital, Hong Kong
Yok-Lam Kwong
Tokyo Medical and Dental University,
Japan
Norio Shimizu
Osaka University Graduate School of
Medicine, Japan
Katsuyuki Aozasa
Funding from NMRC, NRF, MOE