Patrick Tan, MD PhD

Professor, Duke-NUS Medical School

Deputy Executive Director, Biomedical Research Council,

Agency for Science, Technology and Research (A*STAR)

30th European Congress of Pathology

Bilbao, Sept 2018

Genomic and Epigenomic Profiles of

Gastrointestinal Cancers

Enterprise | Interest

Patents related to cancer epigenomics

Tay et al., Cancer Research (2003)

Molecular and Clinical Heterogeneity in

Gastric Cancer

Tay et al., (2003) Cancer Research

Today’s Topics

1) What are the regulators of the cancer transcriptional

landscape?

- Epigenomics and Cancer Immunity

2) What are the Key Determinants for Development of

Gastric Cancer?

- Pre-Malignant Condition (Intestinal Metaplasia)

Cancer Gene Expression is Controlled by

Epigenomic Changes

Padmanabhan, Ushijima and Tan (2017)

Nature Reviews Gastro Hepat

Regulatory Elements can be Identified by

Histone Modifications

Modification Regulatory Elements

H3K4Me1 Enhancers

H3K4Me3H3K4Me2H2A.Z

Promoters

H3K27Ac Active Enhancers/ Promoters

H3K27Me3 Inactive Enhancers/ Promoters

H3K36Me3 Transcribed Regions

H3K9Me3 Constitutively Repressed Genes

Example

H3K4Me3

Active Promoter

PoisedPromoter

InactivePromoter

H3K27Me3

Gene Promoters : Critical Integrators of

Regulatory Inputs

Valen and Sandelin (2011) Trends in Genetics

>50% of Human Genes have Multiple Promoters

Davuluri et al (2008) Trends in Genetics

Epigenomic Promoter Profiling Localizes Somatic Promoters

19 N/T pairs + 12 cell lines (H3K4me3, H3K27ac, H3K4me1)

23,000 Promoter Elements (H3K4me3 High, H3K4me1 Low)

UnalteredTumor-Specific Gain

(Somatic Gain)Tumor-Specific Loss

(Somatic Loss)

Somatic Promoters are Correlated with H3K27ac Activity

and Frequently Map to Multi-Isoform Genes

18% of GC Somatic Promoters Comprise

Alternative Promoters

MET – Altered Signaling Shown by N-terminal

Promoter Variants

Muratani et al., 2014 Nat Comm

TCGA (Papillary RCC) 2016 NEJM

Recurrent Alternative Promoters are Predicted to

Cause Loss of N-terminal Immunogenic Peptides

MHC Class I Binding (HLA A, B, C)

Recurrent N-terminal Peptides Stimulate Immune

Responses in Experimental Systems (1)

EpiMAX Immune Profiling (Healthy Donors)

Recurrent N-terminal Peptides Stimulate Immune

Responses in Experimental Systems (2)

GCs with High Alternative Promoter Usage Exhibit

Decreased T-Cell Cytolytic Activity (GZMA, PRF1)*

TCGA – Nature 2014

(RNA-seq)

ACRG – Nat Med 2015

(Nanostring)

* Adjusted for mutation count and

tumor purity

Model - Somatic Promoter Alterations in GC May

Reflect the Impact of Tumor Immunoediting

Summary

• Largest catalogue of epigenome-guided promoter

elements in gastric cancer

• Tumor-specific promoter alterations are widespread

and frequently map to multi-isoform genes

• Alternative promoters can drive expression of novel

pro-oncogenic isoforms (eg RASA3, MET)

• Alternative promoter usage may decrease

tumor antigenicity

• Somatic usage of alternative promoters

may facilitate immune evasion

Qamra et al., (2017) Cancer Discovery

Today’s Topics

1) What are the regulators of the cancer transcriptional

landscape?

- Epigenomics and Cancer Immunity

2) What are the Key Determinants for Development of

Gastric Cancer?

- Pre-Malignant Condition (Intestinal Metaplasia)

Compound Mechanism of action Treatment setting Reference

Cetuximab Anti-EGFR mAB 1st-line metastatic Lordick et al. 2013

Panitumumab Anti-EGFR mAB 1st-line metastatic Waddell et al. 2013

Gefitinib Anti-EGFR TKI 2nd-line metastatic Petty et al. 2017

Trastuzumab Anti-HER2 mAB 2nd-line metastatic Makiyama et al. 2018

Pertuzumab Anti-HER2 mAB 1st-line metastatic Tabernero et al. 2017

Lapatinib Anti-EGFR/HER2 TKI 2nd-line metastatic Satoh et al. 2014

Rilotumumab Anti-HGF mAB 1st-line metastatic Catenacci et al. 2017

Onartuzumab Anti-MET mAB 1st-line metastatic Shah et al. 2017

Napabucasin Anti-STAT3 2nd-line metastatic Shah et al. 2018

Bevacizumab Anti-VEGF mAB Perioperative Cunningham et al. 2017

Bevacizumab Anti-VEGF mAB 1st-line metastatic Ohtsu et al. 2011

Ramucirumab Anti-VEGFR-2 mAB 1st-line metastatic Fuchs et al. 2018

Olaparib PARP inhibitor 2nd-line metastatic Bang et al. 2017

Pembrolizumab PD-1-directed mAB 2nd-line metastatic Shitara et al. Lancet

2018

Advanced GC is Replete with Many Unsuccessful Phase III Trials

Lordick F (in press)

Gastric Cancer and Intra-Patient Heterogeneity

Sundar and Tan (2018) Cancer Discovery

Most Gastric Cancers* Follow a Multi-step

Carcinogenesis Sequence

Yeoh and Tan (2015) Gastroenterology

*Diffuse-type GC does not involve metaplasia

Gastric Cancer Epidemiology Program(GCEP)

Four Singapore Hospitals : NUH, SGH, TTSH, CGH

Funded by National Medical Research Council

GCEP Translational Study (“TransGCEP”)

Selection of High-Risk IM Patients (n=148)

56% moderate/marked IM

All Chinese

All positive for Hp serology

(Previous Infection)

Normal Mucosa

Mild IM (<30%)

IM (≥30% cellularity)

p value (IM vs Normal)

n=43 n=22 n=83

Age (year), mean ±SD

62±7 60±7 62±7 0.17

Race

Chinese 43 (100) 22 (100) 83 (100) --

Gender (%)

Male 22 (51) 12 (55) 42 (51) 1

Female 21 (49) 10 (45) 41 (49)

Smoking (%) 0.048

Current/ Ex-Smoker

9 (21) 4 (18) 32 (39)

Non-smoker 34 (79) 18 (82) 51 (61)

Alcohol consumption (%)

5 (12) 5 (23) 20 (24) 0.1

Family history of GC

in first-degree relative (%)

7 (16) 3 (14) 13 (16) 1

Hp serology positivity (%)

43 (100) 22 (100) 83 (100) --

Chronic gastritis (%) 38 (88) 22 (100) 83 (100) 0.004

Atrophic gastritis (%) 0 (0) 0 (0) 67 (81) --

Low Grade Dysplasia (%)

0 (0) 0 (0) 2 (2) --

EGN (%) 0 (0) 0 (0) 4 (5) --

Endoscopy

surveillance (months), mean ±SD

56±12 58±8 49±18 0.04

DNA Mutations – Point Mutations and Indels

(MAF>4%)

TP53 and ARID1A

Clonal Mutations are Rare

(TP53 – 2%; ARID1A – 3%)

Laser Capture Microdissection

Confirms Mutations in IM Cells

IMs Exhibit Low

Mutation Burdens

Compared to GC

NL

IM

GC

Copy Number Alterations and Telomere Erosion

MYC

NL IM

10% of IMs have sCNAs

Most sCNAs target Chr 8q

IMs have significantly shorter

Telomeres (Genome Instability)

Sequencing Detects More Hp-Infected IMs Compared

to Histology

All 15 have Hp DNA

(100%)33 cases have Hp DNA

(27%)

Genomic Sequencing Can Detect Active

Low-Level Hp Infection

Histology-confirmed HP cases

Show no Hp reads after eradication

(ie Hp DNA is transient)

Giemsa staining confirms Hp infection in

Sequencing positive cases

IMs Exhibit Global DNA Methylation Alterations

and a Subgroup is Hypermethylated

IMs Exhibit Increased

DNA Methylation

Normal Intestinal Metaplasia

IM Hypermethylation is Associated with Genes related

to Cell Fate Commitment and EZH2 Binding

EZH2 Chip-Seq

at IM Regions

Compared to Advanced GC

(CIN, EBV, MSI Subtypes)

IMs do *not* exhibit Intragenic DNA Hypomethyation

Signatures of Advanced GC

Globally, most cancers are

hypomethylated compared to normal tissues

(eg Berman et al 2012 Nat Genet)

Cancer hypomethylation occurs at repetitive and intragenic

regions, contributing to genomic instability

(Sheaffer et al 2016 Cancer Prev Res; Ehlich et al. 2009 Epigenomics)

DNA Hypomethylation is

Exclusively Seen in Advanced GC

Integration with Clinical Outcome

(Regression, Persistence, Progression)

TransGCEP

Samples

LGD/HGD

/EGC

Regression criteria based on Rugge et al (2003)

Progression includes both LGD and HGD, as both have higher GC risk than IM

Factors influencing IM regression and progression

Mutation Burden Telomere Length

DNA Methylation Copy Number Alterations

Regression

Summary Slide: What our Data Supports

Genomic profiling reveals that IMs exhibit low mutational

burdens compared with GCs

In general, TP53 and ARID1A mutations are rare in IM

Some IMs have FBXW7 mutations, chromosome 8q

amplifications, or shortened telomeres

Sequencing detects more IM patients with active H. pylori

infection than histology

(Epi)genomic alterations in IM predict

subsequent disesae progression or

regression

Huang et al., 2018 Cancer Cell

Acknowledgements

Chang Xu

Angie Lay Keng Tan

Minghui Lee

Suting Tay

Kakoli Das

Manjie Xing

Aliya Fatehullah

Syed Muhammad

Fahmy Alkaff

Tony Kiat Hon Lim

Jonathan Lee

Khek Yu Ho

Steven George Rozen

Bin Tean Teh

Nick Barker

Chung King Chia

Christopher Khor

Choon-Jin Ooi

Kwong Ming Fock

Jimmy So

Wee Chian Lim

Khoon Lin Ling

Tiing Leong Ang

Andrew Wong

Khay Guan YeohMing Teh

Kie Kyon Huang

Kalpana Ramnarayanan

Feng Zhu

Supriya Srivastava

Aditi Qamra

Manjie XingNisha Padmanabhan

Jeffrey Kwok

Shenli Zhang

Chang Xu

Yan Shan Leong

Lee-Lim Ai Ping

Qianqao Tang

John Connolly

Kyoung Mee-Kim

Jeeyun Lee

Dennis Kappei

Khay Guan Yeoh