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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