Molecular signatures of gastric cancer an integrated approach to molecular cytogenetics, whole genome copy number and transcriptome profiles

Dissecting simple rearrangements in gastric cancer cell lines 210 9.5 A balanced translocation in IM95 karyotypes and whole genome copy number profiles 10.2 Copy number transitions and

MOLECULAR SIGNATURES OF GASTRIC

CANCER:

AN INTEGRATED APPROACH TO MOLECULAR CYTOGENETICS, WHOLE GENOME COPY NUMBER AND TRANSCRIPTOME PROFILES

LEONG SIEW HONG

Acknowledgements

It is my greatest honor for being given the golden opportunity to be guided and mentored by my most dedicated supervisor Dr Kon Oi Lian Without her continual support and encouragements, this thesis will not come to fruition I thank her deeply for believing in me and to be that guiding star during my darkest days in my scientific pursues I am very grateful to her for always being open to new ideas and allowing me to chart my own course while supporting me in every step of the way She is always there to lend a listening ear and offering me sound advice Her passion and dedication for scientific research had been awe-inspiring and she will always be my role model

This project, although conceived and performed mostly at the National Cancer Center, will not be successful without the support and assistance from many important people I would especially like to acknowledge Dr Tan Soo Yong, Consultant pathologist at SGH As a member of my TAC committee, his feedbacks and advice are very much valued His generosity in sharing his technical expertise and resources had gone a long way in making this study a success I would also like to thank my other advisers and collaborators who had one way on another been tremendously helpful during the course of this work They are: Dr Patrick Tan (my TAC committee chairman, collaborator and adviser), Dr Lai Siang Hui (for histo-pathological assessment of primary GCs), Dr Mac Ho and Dr Tony Lim (in the provision of pre-malignant gastric tissue samples), Dr Nallasivam Palanisamy (who spent many hours relating his molecular cytogenetic experiences)

I am very grateful to Magdalene Koh of the Pathology Department whose tissue sectioning skills are probably the best in Singapore Her technical competency had made a dramatic turnaround in the success of this study I am also very blessed to be a member of a wonderful team at NCC I want to thank the members of my lab: Louise Lee, (for the tremendous bioinformatic support), Ng Wai Har (xenoimplantation of tumor samples), Nelson Chen (for giving me critical feedback in all aspects of my work), Jaichandran (providing his expert advice in some very challenging experiments) and Frank (my new

and enthusiastic member of the lab) To my former colleagues: Mark Tan (my teacher in the initial stages of microarray data analysis), Cheryl Lee (who assisted in cell culture), Serene Lok (always there to hear out my frustrations), Long Yun Chau (now at Harvard), Jaya Visvanathan (Baylor college of Medicine) and Wee Choon Wei (Temasek Life Sciences) You’ve been great fun to be with and have certainly made great impact to my research life You’ve been the best people I have met in all my life and I cherish every moment in the lab because you have made working in the lab very enjoyable I’m also deeply appreciative to Nelson Chen and Louise Lee who had dedicated much time and effort to review this thesis and gave very constructive feedbacks and suggestions Also not forgetting Ng Kia Min who had been a great help in the earlier establishment of the TMA-FISH protocols

I would also like to acknowledge past and current members from Dr Patrick Tan’s lab (Wong Kee Yew, Amit Aggarval, Yu Kun and Tao Jiong) Special thanks to Doris Ma who is always a good friend and forever approachable whenever administrative affairs need to be tackled To the folks at The Wellcome Trust Sanger Institute at Hinxton, Cambridge: Dr Nigel Carter, Ng Bee Ling, Fu Beiyuan, Yang Fengtang, Susan Gribble and Elena Elenor The month-long training had been an eye-opener I treasure the friendship we’ve established and I thank you in everyway I could

I would also specially want to thank Dr Carol Tang (NNI, Singapore) who spurred me on in times of frustration and for being my beacon in the undying passion for scientific discoveries

I would like to thank the Singapore Millennium Foundation for their scholarship support in the program and also their generous sponsorship for the chromosome flow-sorting training at the Sanger Institute

And finally to the most important people in my life: my family I will not have been able to complete this thesis without the continual support of my lovely wife, who stood by me in this arduous journey To my very adorable kids, this thesis is for you too For Dad and Mum, I have fulfilled your dreams and mine too! I love you!!

in oncogenesis

7.2 Recurrent patterns of translocations

8.1 Whole genome copy number analysis

8.3 Global miRNA expression profiling

9 Dissecting simple rearrangements in gastric cancer cell lines 210

9.5 A balanced translocation in IM95

karyotypes and whole genome copy number profiles

10.2 Copy number transitions and translocation

breakpoints

11.2 TMA construction and FISH assay optimization

11.3 18q2 break-apart FISH assay in primary gastric

adenocarcinomas 11.4 18q2 break-apart in non-gastric cancers

12.1 Clinico-pathological features and 18q21q22

breakpoint 12.2 Chromosome 18 aberrations in metaplasia,

dysplasia, early and late stage gastric cancers

13.1 mRNA expression profiling

13.2 Expression of breakpoint genes by

immunohistochemical staining

14 Precise determination of translocation breakpoints by 295 arraypainting

Summary

Gastric cancer (GC) is second only to lung cancer as a leading cause of cancer deaths globally Of the estimated 1 million newly diagnosed patients

curative intent, 60% progress to locoregional and/or distant metastatic

including molecularly targeted agents, are urgently needed for the majority who are beyond surgical cure One strategy for developing new curative treatment is to identify genetic and genomic aberrations that are causal in the initiation and/or progression of specific cancer types Discoveries of druggable

The notable lack of reliable karyotypic data in most solid cancers has

encouraged the view that translocations are rarely associated with epithelial cancer development Fortunately, molecular cytogenetic techniques (e.g comparative genomic hybridization, spectral karyotyping, multicolor

fluorescence in situ hybridization (FISH), high resolution copy number and

tiling arrays, genome annotations and bioinformatics) have made solid tumor cytogenetics more tractable, as demonstrated by recent discoveries of recurrent translocations in human prostate adenocarcinomas

4

and non-small cell lung

Our exploration of this dreaded disease begins with a broad survey of the current state of knowledge in five chapters Chapter 1 reviews the current epidemiological status of GC globally and in Singapore, including a short

discussion of current management and treatment options Chapter 2 explores the interplay of various environmental as well as host and/or bacterial

susceptibility factors that may be associated with GC initiation and

progression In Chapter 3, we examine the African and Asian enigmas –

apparent paradoxes in which high H pylori infectivity coexists with low GC

prevalence As cancer is caused and accompanied by structural and functional genomic alterations, Chapter 4 summarizes and evaluates current

understanding of the molecular pathogenesis of GC In Chapter 5, we review signature chromosomal rearrangements in malignant disorders and particularly note recent discoveries of such translocations in prostate and lung cancers

Chapter 6 details all methods, materials and resources employed in this

project

Data generated in this project appear in Chapters 7 – 14 Our work was

directed at five primary goals:

1) To characterize the chromosomal rearrangements, copy number

alterations, differentially expressed mRNAs and miRNAs in a survey

of 17 GC cell lines as a means of documenting the genomic

complexities of GC These datasets enabled identification of unique

GC signatures based on copy number aberrations and mRNA

expression profiles compared to a well known panel of non-GC cell lines, the NCI-60 panel These results are presented in Chapters 7 and

8

2) To explore the utility of integrating different molecular cytogenetic techniques to fully characterize the fusion breakpoints in a simple rearranged GC cell line, SNU-1, and to propose a translocation

mechanism (Chapter 9)

3) To distinguish “driver” (pathogenic) from “passenger” (collateral) rearrangements Data in Chapters 10 and 11 showed that the novel and recurrent 18q translocation breakpoint in cell lines was also mirrored in

primary GC tumors (but not in non-GC tumors) via a custom-designed

break-apart FISH assay

4) To ascertain clinico-pathological features of 18q breakpoint-positive GCs and to determine chromosome 18 status in pre-malignant lesions (Chapter 12) These include analysis of the expression of two

candidate proteins, Serpin B8 and CD226 antigen by

immunohistochemistry (Chapter 13)

5) To molecularly define junctional DNA of candidate chromosomal fusions, pure fractions of translocated chromosomes were flow-sorted and their breakpoints accurately determined using oligo-based array painting techniques (Chapter 14)

In short, this project highlights the value of integrating a suite of molecular cytogenetic techniques with other genome-wide analyses in discovering

genomic signatures of GC This integrated approach makes solid tumor

cytogenetics more tractable, is generalizable and could help to discover

oncogenic chromosomal rearrangements in other common cancers

References:

1 Garcia, M., et al Global Cancer Facts & Figures 2007

(http://www.cancer.org/docroot/STT/content/STT_1x_Global_Cancer_Facts and_Figures_2007.asp) (2007)

2 Field, K., Michael, M & Leong, T Locally advanced and metastatic gastric cancer: current management and new treatment developments

Drugs 68, 299-317 (2008)

3 Fröhling, S & Döhner, H Chromosomal abnormalities in cancer N

Engl J Med 359, 722-734 (2008)

4 Tomlins, S.A., et al Recurrent fusion of TMPRSS2 and ETS

transcription factor genes in prostate cancer Science 310, 644-648

(2005)

5 Soda, M., et al Identification of the transforming EML4-ALK fusion

gene in non-small-cell lung cancer Nature 448, 561-566 (2007)

6 Mitelman, F., Johansson, B & Mertens, F The impact of

translocations and gene fusions on cancer causation Nat Rev Cancer

7, 233-245 (2007)

List of Tables

Chapter 1

Singapore

seropositivity in major ethnic populations in Singapore

cell lines used in copy number analysis

cell lines used to determine mRNA expression signatures in GCs

Chapter 7

number analysis of 17 GC cell lines

lines

Chapter 8

experimentally validated targets (to date)

Chapter 9

at the deleted cytoband 4q32.3q35.1

Chapter 10

with recurrent breakpoint cytobands

Chapter 11

classification of break-apart status

break-apart status of primary GC tumors cores

break-apart status in primary GC tumors cores

expression correlated with break-apart status

List of Figures

Chapter 1

(GC) among males and females

cancer registries in Asia, Europe and the USA

developing and developed countries

Chapter 4

Hs 746T

in publicly available data using CGH and array CGH

Chapter 5

tumors

Figure 5-2 Schematic representation of possible outcomes of

chromosomal translocations

Chapter 6

section (4 µm)

analysis using anti-human Serpin B8

antibodies against human CD226 antigen, Serpin B2 and SOCS-6

Chapter 7

on metaphase spreads

Figure 7-16 SKYgram illustrating YCC9

Chapter 8

lines using 100K SNP mapping arrays

number gains and losses in 17 GC cell lines

44/60 primary tumors

between 203 primary GCs (training set) versus 17 GC lines and 44 primary GCs (test set)

segregating GC from non-GC cell lines

mRNAs segregating GC from non-GC cell lines

non-GC cell lines

expression signature

hybridization

miRNAs of 17 GC cell lines and 40 primary gastric adenocarcinomas

solution hybridization

Chapter 9

Figure 9-2 Copy number profile of SNU-1 by chromosomal

CGH

number analysis of Chromosome 4

FISH-walking probes at 4q31.1q32.3

number analysis of Chromosome 1q

FISH-walking probes at 1q24.2q25.3

at 1q25.3

FISH-walking probes at 4qter

4q26

at 4q26q32.3

mapping to deleted cytoband 4q32.3q35.1

CGH

Chromosome 1 and 2 in IM95

Chapter 10

analysis and recurrent translocations in 17 GC cell lines

4q32.3 breakpoint in SNU-1

six GC cell lines

probable copy number transition boundaries

Chapter 11

GC cell lines with Chromosome 18 translocation

break-apart probes at chromosome 18

assay

in the "Brute-force" break-apart FISH assay

tissues

translocation and other 18q aberrations in primary GCs

Figure 12-2 Chromosome 18 aberrations in pre-malignant gastric

lesions

Chapter 13

18q aberrant status

by SAM segregated GC cell lines by 18q aberrant status

immunohistochemistry

using an anti-human Serpin B8 monoclonal antibody

of GC tumor cores

using an anti-human antibody for CD226 antigen expression

244K DNA array platform on flow-sorted chromosome spot 3A

Figure 14-10 Array painting copy number plots using Affymetrix

500K SNP mapping array platform on flow-sorted chromosome spot 3A

Figure 14-11 Array painting of chr 18 copy number plots using

Affymetrix 500K SNP mapping array platform on whole genome amplified with Genomiphi

Figure 14-12 Dual-labeled fusion FISH assay validated t(6;18)

fusion in YCC2 metaphase

List of Abbreviations

H pylori Helicobacter pylori

SKY Spectral Karyotyping

Review of the Literature

1 Gastric cancer epidemiology and clinical features

1.1 Epidemiology

1.1.1 Global incidence and mortality

Adenocarcinomas account for close to 95% of all malignant gastric tumors, the remaining being stromal cell tumors, lymphomas, leiomyosarcomas, other rare sarcomas and carcinoids Being by far the most common type of

malignant stomach tumor, gastric adenocarcinomas are generally referred to as gastric cancer (GC) in the research literature, a usage that will be adopted in this thesis GC is a significant global health burden It is the fourth most common cancer after breast, lung and prostate but the second leading cause of cancer mortality (after lung cancer) Globally, an estimated 1 million new

cases are diagnosed each year (Figure 1-1), and more than 800,000 deaths

from GC are recorded

annually1-2

Figure 1-1 Global incidence and mortality of the most common cancers The

bar chart shows estimates of age-specific cancer rates (Adapted from ref 2)

There is great geographical variation in GC incidence The highest rates (> 40 per 100,000) are in East Asia (Japan, China and Korea), South and Central America (Colombia, Costa Rica and El Salvador) North America and most of

(Figure 1-2)

Figure 1-2 Global geographic variation in age-standardized incidence rates of

GC among males (upper panel) and females (lower panel), (Adapted from ref 2)

GC incidence rates are 1.8 to 2.0 times higher in males than in females2 Apart from gender, ethnicity appears to be associated with differences in GC

incidence For example, in the United States, Asians and Pacific Islanders (APIs) and black men have high age-standardized rates (ASR) of 18.6 and 17.4 cases per 100,000, respectively, compared to 10.0 per 100,000 in

Caucasian men (Table 1-1) Similarly, ASR of API and black women are almost twice as high at 10.5 and 8.9 per 100,000, respectively versus 4.7 per

100,000 in Caucasian women Native and Hispanic Americans are also twice

Table 1-1 Age-adjusted incidence and mortality rates of GC by ethnicity

(2001-2005) based on cancer registries in 17 designated SEER (Surveillance, Epidemiology and End Results Program) geographic areas in the USA

(Adapted from ref 3)

The higher ASR

in African-Americans, Hispanics and Asians may be partly due to their

relatively lower socio-economic status, poor access to healthcare and possibly,

an increased exposure to predisposing infections such as Helicobacter pylori

GC incidence rates This striking reduction in GC incidence, especially

among the white population in the US, has been characterized as an

“unplanned triumph” insofar as it was an unintended but welcomed benefit of refrigeration technology, improved food hygiene, increased availability and consumption of fresh vegetables (all of which reduced exposure to putative

gastrointestinal carcinogens), and possibly to lower H pylori infections rates

a lesser extent, in high risk countries such as Japan and China (Figure 1-3)

where current GC incidence rates are still high

Figure 1-3 Stomach cancer incidence rates (1973-1997) obtained from 18

cancer registries in Asia, Europe and the USA (Adapted from ref 7)

The foregoing epidemiologic data does not detail the occurrence of GCs in different anatomic regions within the stomach but rather group all GCs

together regardless of their intra-gastric location The stomach is a relatively large organ which extends from the gastroesophageal junction to the pylorus

It is divided into five regions (named from proximal to distal): cardia, fundus,

body, antrum and pylorus (Figure 1-4)

Figure 1-4 The anatomic divisions of the human stomach (Illustration from

ref 8)

Since the mid-1970s, GCs of the cardia have increased in incidence especially

biologically more aggressive and have a poorer prognosis In high risk Asian nations, reports of increasing proximal GC incidence are mixed Data from

of less than 6% in proximal GCs in Japan was recorded among 843 patients

gastrointestinal endoscopies in a high incidence region of China, Gansu

province, also showed a steady rise in the incidence of proximal GCs (cardia

important to note that GCs of the distal stomach remain predominant in East Asia as reported in these three studies However, as obesity becomes more

prevalent among Asian populations, the rising trend of cardia cancers may be replicated in Asia in the future

GC is the 5

1.1.3 Singapore incidence and mortality

th

Singapore (Figure 1-5) It ranks fourth as a cause of cancer mortality, having

Figure 1-5 Age-standardized cancer incidence and mortality rates of common

cancers in Singapore (Adapted from ref 15)

As in other countries, GC incidence rates are higher in males than females

(1.5:1) (Figure 1-6a) In tandem with declining global rates, Singapore has

also seen a steady decrease in age-standardized incidence rates from 37 per 100,000 persons in the late 1960’s to < 20 per 100,000 among males at the turn of the new millennium GC is more common in older patients, with

incidence rates rising sharply after age 50 (Figure 1-6b) Notwithstanding the

declining incidence, longer life expectancy in Singapore as in other newly

emerging Asian countries, suggests that the clinical burden of GC is unlikely

to diminish greatly in the coming years

Figure 1-6 Age-standardized GC incidence rates in males and females by (a) quinquennia (1968-2002); and (b) age (1998-2002), (Adapted from ref 16)

Singapore is an island nation with a multi-ethnic resident population of 3.6 million comprising 75.2% Chinese, 13.6% Malays, 8.8% Indians and 2.4% Eurasians and other minority groups The remaining 0.88 million are

temporary residents (mainly Asians) Local GC incidence shows a clear ethnic

pattern (Table 1-2) Chinese males and females have the highest incidence

compared to Indians and Malays Males of the latter group have a relative risk (RR < 0.4) of developing GC that is almost three times lower than Chinese males (RR = 1.0) Having attained developed country status, socio-economic conditions and access to healthcare are comparable among Singapore’s three major ethnic groups These highly disparate GC risks thus implicate host

genetic differences or polymorphisms and possible culture-specific practices

as factors that contribute to this interesting ethnicity-associated incidence pattern

Table 1-2 Age-standardized incidence data of GC in the major ethnic groups

in Singapore (1998-2002), (Adapted from ref 16)

As with most other Asian nations, the increase in incidence of cardia tumors is

data are not available to confirm this impression However, crude incidence rates of distally located GCs are reportedly > 3-fold higher than proximal GCs18

1.2 Clinical features, prognosis and treatment

Despite a general global decline in GC incidence, overall mortality rates are still alarming This dismal state of affairs is mainly due to the fact that,

whereas early stage GC has an excellent prognosis, the majority of

symptomatic stomach cancer patients have already progressed to advanced incurable disease at the time of presentation These are usually sporadic (i.e 1.2.1 Lethality of GC

Female incidence (1998-2002)

ASR: age-standardized (to ‘World’ population) rate per 100,000/year

RR: age-adjusted relative risk and 95% confidence interval for Malays and Indians (Chinese as the reference group)

non-familial) tumors that are locally invasive In many cases, regional

(abdominal lymph nodes, peritoneum, liver) and/or distant (lung, bone

marrow) metastases are present at initial diagnosis Early symptoms are nearly always non-specific, and hence more likely to be mistaken for functional dyspepsia, benign gastritis or peptic ulcer disease Alarm symptoms associated with late stage disease include weight loss (62% of patients) and persistent abdominal pain (52%) Other less frequent symptoms are nausea, vomiting, dysphagia, anorexia, early satiety, fatigue, melena, and ulcer-like symptoms Presenting symptoms may also reflect the location and type of tumor For example, patients with cancers of the proximal stomach or gastoesophageal junction are likely to develop dysphagia An advanced distal tumor is liable to cause gastric outlet obstruction Scirrhous-type (linitis plastica) lesions tend to

1.2.2 Prognosis and survival

Prognostic assessment and critical treatment decisions of individual patients

clinicopathological staging systems for GC The Japanese staging system is more detailed and complex as it requires rigorous pathologic assessment of abdominal lymph nodes, considers peritoneal cytology and distinguishes

countries use the simpler staging system jointly developed by the American

categories: 1) Tumor(T): depth and extent of tumor invasion; 2) Node(N):

number of metastatic regional lymph nodes; and 3) Metastases (M): presence

or absence of distant metastases The TNM classification and stage grouping

of stomach cancer is detailed in Table 1-3 Lymph node metastatic status (N)

prognosis and survival of GC by TNM staging is dismal for patients with late

stage tumors (Figure 1-7) For example, 5-year survival rates for stage IIIa,

IIIb and IV patients were 20, 8 and 7%, respectively In the United States,

data from 22 countries and 53 cancer registries reported an overall 5-year

Case control studies in Japan have shown that detection of early gastric cancer

is enhanced through population screening of high-risk individuals and in turn, contributes to overall higher survival rates

27-29

(> 50%, compared to < 21% in Europe) The high cost of such screening programs, however, makes it

Another factor that adversely affects GC prognosis is the high recurrence rate, usually occurring within 2-3 years of surgery, even after R0 resections

(complete resections with histologically negative surgical margins) Adjuvant chemotherapy may be administered to eradicate micrometastases

31

However, most high risk GC patients respond poorly to systemic adjuvant

chemotherapy Clinical trials using treatment regimens based on 5-fluorouracil

in combination with anthracyclines and cisplatin have not markedly improved

Table 1-3 TNM classification and stage grouping (AJCC/UICC), (adapted

from ref 23)

Figure 1-7 Survival statistics by TNM stages (Adapted from ref 21)

1.2.3 Treatment strategies

Surgical resection with curative intent remains the only treatment among current options with proven prospects for long-term survival However even in specialized centers, surgery had managed to improved 5-year survival rates marginally to between 33-47%, depending on surgical skills and patient

chemo-, biologic and radiotherapy are notchemo-, on present evidencechemo-, curative

Nevertheless, there is good evidence to support the use of neoadjuvant

perioperative chemotherapy in patients with locally advanced GC as a means

Accurate pathologic staging of the disease i.e the extent of tumor invasion and metastatic spread to regional lymph nodes, is critical to the assignment of

appropriate treatment strategies Availability of CT (computerized

tomography) and FDG-PET (fluoro-deoxyglucose positron emission

tomography) scans will, for example, enable the T stage and status of local

staging techniques and fitness assessment have led to stage migration and better patient selection This has ironically resulted in fewer than 20% of all

reported post-operative survival has improved by 2- to 3-fold in major centers that practice careful patient selection, many older GC patients are less likely to

meet stringent pre-surgical criteria Table 1-4 summarizes the treatment

options that are generally considered most appropriate for different pathologic stages of GC

Table 1-4 : Treatment strategies with reference to pathological stage of GC

(adapted from ref 21)

In summary, GC is still a significant global health burden especially when multiple factors conspire to make its lethality rather intractable to

improvement These factors are mainly the absence of reliable (sensitive and specific) diagnostic methods for early gastric cancer, advanced disease stage

in a majority of newly diagnosed patients, lack of effective targeted cytotoxic

or cytostatic agents and high local recurrence rates after surgery

References:

1 Parkin, D.M., Bray, F., Ferlay, J & Pisani, P Global cancer statistics,

2002 CA Cancer J Clin 55, 74-108 (2002)

2 Garcia, M., et al Global Cancer Facts & Figures 2007

(http://www.cancer.org/docroot/STT/content/STT_1x_Global_Cancer_Facts_and_Figures_2007.asp) (2007)

3 Ries, L.A.G., et al SEER Cancer Statistics Review, 1975-2005,

(http://seer.cancer.gov/csr/1975_2005/) (2008)

4 Al-Refaie, W.B., et al The impact of ethnicity on the presentation and

prognosis of patients with gastric adenocarcinoma Results from the

National Cancer Data Base Cancer 113, 461-469 (2008)

5 Wiggins, C.L., Becker, T.M., Key, C.R & Samet, J.M Stomach cancer among New Mexico's American Indians, Hispanic whites, and

non-Hispanic whites Cancer Res 49, 1595-1599 (1989)

6 Howson, C.P., Hiyama, T & Wynder, E.L The decline in gastric

cancer: epidemiology of an unplanned triumph Epidemiol Rev 8,

9 Bashash, M., et al Incidence and survival for gastric and esophageal cancer diagnosed in British Columbia, 1990 to 1999 Can J

Gastroenterol 22, 143-148 (2008)

10 Blot,W.J., Devesa, S.S., Kneller, R.W & Fraumeni, J.F Rising

incidence of adenocarcinoma of the esophagus and gastric cardia

12 Lee, J.Y., et al No changing trends in incidence of gastric cardia

cancer in Korea J Korean Med Sci 18, 53-57 (2003)

13 Maeda, H., et al Clinicopathologic features of adenocarcinoma at the gastric cardia: is it different from distal cancer of the stomach? J Am

Coll Surg 206, 306-310 (2008)

14 Zhou, Y., et al A rising trend of gastric cardia cancer in Gansu

Province of China Cancer Lett 269, 18-25 (2008)

15 Lee, H.P., Ling, A., Chow, K.Y & Lubna, A.R in Singapore Cancer

Registry Interim Report in Trends in Cancer Incidence in Singapore 2001-2005, 1-15 (National Registry of Diseases Office NRDO, 2006)

16 Seow, A., Koh, W.P., Chia, K.S., Shi, L.M., Lee, H.P &

Shanmungaratnam, K in Singapore Cancer Registry, Report No 6:

Trends in cancer incidence in Singapore 1968-2002, 1-181 (Singapore

Cancer Registry, 2004)

17 Goh, K.L Changing trends in gastrointestinal disease in the

Asia-Pacific region J Dig Dis 8, 179-185 (2007)

18 Look, M, Gao, F., Low, C.H & Nambiar R Gastric cancer in

Singapore Gastric Cancer 4, 219-222 (2001)

19 Koh, T.J & Wang, T.C in Sleisenger & Fordtran’s Gastrointestinal

and liver disease: pathophysiology, diagnosis, management (eds M

Feldman, LS Friedman & MH Sleisenger) 829-855 (WB Saunders,

2002)

20 Aranha, G.V & Georgen, R Gastric linitis plastica is not a surgical

disease Surgery 106, 758-762 (1989)

21 Clark, C.J., Thirlby, R.C., Picozzi, V., Schembre, D.B., Cummings,

F.P & Lin, E Current problems in surgery: gastric cancer Curr

Probl Surg 43, 566-670 (2006)

22 Japanese Gastric Cancer Association Japanese Classification of

Gastric Carcinoma - 2nd English Edition - Gastric Cancer 1, 10-24

(1998)

23 Greene, F.L., et al in American Joint Committee on Cancer: AJCC

Cancer Staging Manual 6th ed 99-106 (Springer, 2002)

24 Sobin, L.H & Wittekind, C.H in UICC: TNM classification of

malignant tumors 6th ed 65-68 (Wiley, 2002)

25 Siewert, J.R., Böttcher, K., Stein, H.J & Roder, J.D Relevant

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Gastric Cancer Study Ann Surg 228, 449-461 (1998)