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
Research
Clinical Care
Education
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
Proposed model of NKTL pathogenesis
EBV infection
P53 mutations
LMP-1 or other
factors
MYC
activation
Regulate
NF-KB
activation
p53
Deregulation
Induce Survivin
Proliferation
Anti-apoptotic
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, miR363, 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
miR-155
Upregulated
miRNA
miR-378
1000
100
100
10
10
1
1
0.1
Normal
NKTL
0.1
NK Cell Lines
Normal
NKTL
NK Cell lines
Downregulated miRNA
miR-26b
miR-363
10
10
miR-342-5p
10
1
1
1
0.1
0.1
0.1
0.01
0.01
0.01
0.001
0.001
0.001
Normal
NKTL
NK Cell Lines
Normal
NKTL
NK Cell Lines
Normal
NKTL
Nk Cell lines
Selection of high probability predicted target
genes of deregulated miRNA
mirBase
pictar
targetScan
mirtarget2
miRanda
tarBase
Predicted
targets
Intersect with our previous GEP data to
narrow down target genes whose
expression is inversely correlated with
expression of deregulated miRNAs
Final list
Gene expression
profile list (J Pathol. 2011
Mar;223:496-51)
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
Relative expressions
mRNA expression changes upon overexpression of miRNAs by Lentiviral transduction
Mir-101
precursor
control mir-26b
precursor
control
mir-26b
precursor
Relative mRNA levels
Relative mRNA expressions
control
control
BCL2
IGF1
mir-101
precursor
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
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
EZH2 overexpression in majority of NKTL.
6
8
10
12
A
B
P=0.003
P=0.002
Inhibition of EZH2 with DZNep induced cell growth inhibition and apoptosis
in NK malignant cells.
Proposed model of NKTL pathogenesis
EBV infection
JAK3 mutations
P53 mutations
LMP-1 or other
factors
STAT
activation
MYC
activation
Regulate
 miRNA
 EZH2
Proliferation
NF-KB
activation
P53 P53
Deregulation
Deregulation
Induce
Survivin
Anti-apoptotic
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
Ng Siok Bian
Koh Tze Loong
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