Molecular Pathologyof Gastric Cancer

Fátima CarneiroIPATIMUP & FMUP/HSJ

Porto, Portugal

Twenty europeans countries with the highest incidence of gastric cancer

Portugal

Types and sub-types of gastric carcinoma

•Adenocarcinoma•Adenosquamous carcinoma•Anaplastic carcinoma•Blue cell carcinoma•Carcinoma with lymphoid stroma•Carcinoma simplex•Desmoplastic carcinoma•Differentiated carcinoma•Diffuse carcinoma•Expanding carcinoma•Glandular carcinoma•Infiltrative carcinoma•Intestinal carcinoma•Intestinal cell type carcinoma•Linitis plastica•Lymphoepithelial carcinoma•Lymphoepithelioma-like carcinoma•Mixed carcinoma•Medullary carcinoma withlymphoid stroma

•Mucinous adenocarcino-ma•Mucoid carcinoma•Mucous cell type carcinoma•Nonglandular carcinoma•Papillary adenocarcinoma•Parietal cell carcinoma•Poorly-differentiated adenocarcinoma•Pyloric-cardiac cell carcinoma•Scirrhous carcinoma•Signet-ring cell carcinoma•Small cell carcinoma•Solid carcinoma•Solid medullary type•Squamous cell carcinoma•Tubular adenocarcinoma•Types I, II, III and ]IVof Goseki et al.•Unclassified carcinoma•Undifferentiated carcinoma

•Localization •Histotype

Ancient times 1800 Present day

Cancer of distal stomach (non-cardia)

Cancer of proximal stomach (cardia)

Incide

nce

Changing epidemiology

Morphologic heterogeneity

METAGENOMICS

The stomach displays a diverse microbiota when H. pylori is absent or low in abundance

Resident or transient populations of ingested microbes??

Stomach H. pylori -

Stomach H. pylori +

Andersson et al., PLoS ONE 2008

• Helicobacter pylori infection• Diet• Smoking

Environment(Epi)Genetic alterations

Gastriccancer

Gene-environment interaction

Risk of gastric cancer developmentH. pylori virulent genotypes 15 to 17IL-1 gene polymorphism 3.3H. pylori virulence & IL-1B polymorphism 87

Machado et al. Gastroenterology 121: 823, 2001 Figueiredo et al, JNCI 94: 1680, 2002

• Polymorphisms: Mucin genes; Pro-inflammatory genes

• Mutations in “low” or “high”penetrant genes

Familial aggregation

Sporadic cancer

Hereditary cancer

Morphology

Molecular pathology

Intestinal (glandular) carcinoma Diffuse (isolated cell-type) carcinoma

• Elderly patients, mainly males• Decreasing incidence everywhere• Blood-born metastases

• Young patients, mainly females• Familial/hereditary conditioning• Dissemination to the peritoneum

CIN (Chromosomal instability)-Aneuploidy# 3p, 7q, 13q (LOH)# 6q (LOH)# 1q, 5q, 17p (LOH)RAS (overexpression/mutation)ERBB-2 (amplification)TPR-MET (rearrangement)c-MET (6.0 Kb mARN)EGF (overexpression)TGF-a (overexpression)

p 53 (LOH, mutation)APC (LOH, mutation)DCC (LOH)p 16 (hypermethylation)

MSI (Microsatellite Instability)

S-Tn, T, S-TCDw75, S-LexLaminin, collagen IVu-PA, Cat-D NM 23 (loss)CD 44 (variants)

Diploidy# 3p, 7q, 13q (LOH)# 6q (LOH)

c-MET (6.0 Kb mRNA)

p 53 (LOH, mutation)

TGF-ß (overexpression)

K-SAM (amplification)E-cadherin (loss/mutation)

S-Tn, T, S-TCDw75, S-Lex

NM 23 (loss)CD 44 (variants)

Intestinal (glandular) carcinoma Diffuse (isolated cell) carcinoma

ERBB-2

PT NM LMMucosa

ERBB-2 amplification in intestinal carcinoma

David L, Seruca R, Nesland J, Soares P, Sansonetty F, Holm R, Borresen AL,Sobrinho-Simões M: c-erbB-2 expression in primary gastric carcinomas and their metastases. Mod Pathol 5:384, 1992

Blood born metastases Poor prognosis

ERBB-2 amplification in intestinal carcinoma

David L et al; Mod Pathol 5:384, 1992Barros-Silva J et al; Br J Cancer 100: 487,2009

ERBB-2In gastric carcinoma

ToGA TrialERBB2 overexpression in 22% of advanced gastric cancersSignificantly improved survival with trastuzumab

ASCO 2009 (LBA 4509)

ERBB-2

Prognostic factor YES (56%)(about 40 studies) NO (44%)

a

3’-UTR EGFR A13 REPEAT

20/63 (31.7%)

EGFR mutations in sporadic gastric carcinoma

3’-UTR EGFR A13 REPEAT

EGFR mutations 2/77 cases

EGFR amplification 4/30 cases

15-30% of the cases

Peltomäki P et al. Cancer Res 53:5853, 1993Seruca R et al. Int J Cancer 64:32, 1995Santos NR et al. Gastroenterology 110:38, 1996

Microsatellite Instability (MSI) in gastric cancer

Survival of patients

Multivariate analysis• Staging (pTNM) (p< 0.0008)• Venous invasion (p= 0.004)• Histological classification (p=0.08)• Microsatellite instability (p=0.04)

Seruca R et al. Int J Cancer 64: 32, 1995Santos N et al. Gastroenterology 110: 38, 1996 Oliveira C et al. Am J Pathol 153: 1211, 1998

MSI is a molecular marker of good prognosis

in sporadic gastric cancer

Univariate analysis

Unmethylated MethylatedMSI-H 25% 75%MSS 100% -

MLH

1 met

hylation

hMLH1 promoter hypermethylation is the main mechanism underlying MSI in SGC

Mismatch Repair (MMR) gene mutations are rare in sporadic gastric carcinomas

(Do not explain most of the MSI cases)

MSI status

K-RAS Exon 1

BRAF Exon 15

MSI-H 10/36 (28%) 0/38 (0%)

MSS 0/46 (0%) 1/139 (0.72%)

p=0.0001 NP

KRAS and BRAF in sporadic gastric carcinoma

Oliveira et al, Oncogene 22: 9192, 2003Brennetot C et al, Gastroenterology 125, 2003

V600E

S-Le x

Orcein staining

Venous invasion High expression of S-Lex

Absent 5.6%Present 94.4%

p=0.0025

Amado et al. Gastroenterology 114: 462, 1998

High S-Lex expression is a marker of poor prognosisin sporadic gastric cancer

Histotype cases (n) CD44v6 overexpression (%)

Intestinal 14 7 (50.0)

Diffuse 13 10 (76.9)

Mixed 16 10 (62.5)

TOTAL 43 27 (62.8)

CD44v6 in sporadic gastric carcinoma

Normal mucosa

IM (complete)

Dysplasia (low grade)

Hyperplastic polyp

IM (incomplete)

Dysplasia (high grade)

CD44v6 in precursor lesions of gastric carcinoma

What about diffuse carcinoma?

CIN & Aneuploidy# 3p, 7q, 13q (LOH)# 6q (LOH)# 1q, 5q, 17p (LOH)RAS (overexpression/mutation)ERBB-2 (amplification)TPR-MET (rearrangement)c-MET (6.0 Kb mARN)EGF (overexpression)TGF-a (overexpression)

p 53 (LOH, mutation)APC (LOH, mutation)DCC (LOH)p 16 (hypermethylation)

MSI

S-Tn, T, S-TCDw75, S-Lex

Laminin, collagen IVu-PA, Cat-D NM 23 (loss)CD 44 (variants)

Diploidy# 3p, 7q, 13q (LOH)# 6q (LOH)

c-MET (6.0 Kb mRNA)

p 53 (LOH, mutation)

TGF-ß (overexpression)

K-SAM (amplification)E-cadherin (loss/mutation)

S-Tn, T, S-TCDw75, S-Lex

NM 23 (loss)CD 44 (variants)

E-cadherin gene (CDH1) plays the major role

Intestinal (glandular) carcinoma Diffuse (isolated cell) carcinoma

E-Cadherin changes in diffuse carcinoma

CDH1 mutations(50% - 70%)

Somatic mutations

E-cadherin gene alterations in sporadic gastric carcinoma

Machado JC et al. Oncogene 20:1525, 2001

MutationLOH

Promoter methylation

“2nd HIT”“1st HIT”

met unmet

Inactivation of CDH1 in sporadic gastric carcinoma

METHYLATION

NO ALTERATION

LOH+MUTATIONS

Inactivation of CDH1 in sporadic gastric carcinoma

What about the mixed type of gastric carcinoma?

Mixed gastric carcinomas show similar chromosomal aberrations in both their diffuse and glandular components

Diffuse component

Intestinal component

Carvalho B et al. Cellular Oncology 28:283, 2006

What about the mixed type of gastric carcinoma?

E‐cadherin

E-cadherin mutations in mixed gastric carcinomas

”Intestinal” component 17%”Diffuse” component 83%

Machado JC et al: E-cadherin gene mutations provide a genetic basis for the phenotypic divergence of mixed gastric carcinomas

Lab Invest 79: 459, 1999

Histological type

Glandular (n=89)Isolated cell (n=14)Solid (n=28)Mixed (n=82)

5-year survival rate

52%67%48%16%

Carneiro F et al. Pathol Res Pract 191: 571, 1995

Morphology Molecular pathology

4-1-02 - ICD-O Code

Adenocarcinoma 8140/3 Papillary adenocarcinoma 8260/3 Tubular adenocarcinoma 8211/3 Mucinous adenocarcinoma 8480/3 Signet-ring cell carcinoma 8490/3

WHO – 3rd Edition

Signet ring cell ca? Mixed ca?

Some shortcomings:

4-1 Gastric carcinoma Gregory Y. Lauwers Fátima Carneiro David Y. Graham Maria-Paula Curado Silvia Franceschi Elizabeth Montgomery Masae Tatematsu Takenori Hattori

4-1-02 - ICD-O Code Adenocarcinoma 8140/3 Papillary adenocarcinoma 8260/3 Tubular adenocarcinoma 8211/3 Mucinous adenocarcinoma 8480/3 Poorly cohesive carcinomas (including signet ring cell 8490/3 carcinoma and other variants)Mixed carcinoma 8255/3

WHO – 4th Edition, 2010

Major histological

types

• pTNM• Venous invasion

• Sialyl-Lex

• ERBB-2• E-cadherin

• Microsatellite instability

Poor prognosis

Good prognosis

Normal mucosa: neg

Intestinal metaplasia & dysplasia: pos

Sporadic gastric carcinoma

MSIIntestinal GC, older age

MLH1 methylation

Good prognostic biomarker

Oncogenic Mutations

KRAS negative

BRAF negative

EGFR positive

Biomarker for targeted therapy (EGFR inhibitors?)

CD44v6

New diagnostic biomarker

E-cadherin

Poor prognostic biomarkerLOH+ Mut

Nodal metastization, Advanced stage

Sporadic cancer

Hereditary cancer(1-3%)

Morphology

Molecular pathology

Gastric cancer in hereditary cancer syndromes

Syndromes Genetic alterations

• Lynch syndrome (HNPCC) MMR• Li-Fraumeni syndrome TP53• Peutz-Jeghers syndrome STK1• Familial adenomatous polyposis APC

Sporadic (90%)

Familial Aggregation (10%)Familial Gastric Cancer (FGC)Familial Intestinal Gastric Cancer (FIGC)Familial Diffuse Gastric Cancer (FDGC)

Hereditary (1%)Hereditary Diffuse Gastric Cancer (HDGC)

GASTRIC CARCINOMA

Maori kindred

Guilford P et al. Nature 392:402,1998

E-cadherin gene(CDH1) germline

mutations

Hereditary DiffuseGastric Cancer

1999

E-cadherin changes in familial gastric cancer

2001

2003

Familial gastric cancer: overview and guidelines for management.

Prophylactic gastrectomies in asymptomatic carriers of germ-line E-cadherin mutations

Functional analyses of E-cadherin (CDH1)germline missense mutations

Model of development of HDGC2004

Cleft lip/palate and CDH1 mutations in families with HDGC

2006

2005 Report of the first Portuguese family with HDGC J Med Genet 36: 873, 1999

N Engl J Med 344:1904, 2001 Gastroenterol Clin Biol 25: 931, 2001 Hum Mutat 19:510, 2002 Hum Mol Genet 12: 575, 2003Hum Mol Genet 12: 3007, 2003 Oncogene 22:5716, 2003 J Pathol 203: 681, 2004Virchows Arch 446: 18, 2005 Clin Cancer Res 11:5401, 2005J Med Genet 43:138, 2006J Mol Med 84:1023, 2006 Hum Mol Genet15:1704, 2006 Hum Mutat 28:203, 2007Oncogene 27: 4255, 2008 J Clin Pathol 61:25, 2008 J Pathol 216:295, 2008 Gastroenterology 136:2137, 2009J Med Genet 47: 436, 2010

2007Novel germline CDH1 mutations

Experimental model in Drosophila

2008NMD mRNA surveillance downregulates aberrantCDH1 transcripts

Second hit of CDH1 inactivation

2009

2010

CLINICALHISTORY

HISTOLOGICALCLASSIFICATION

MOLECULAR CLASSIFICATION

IDENTIFICATION OF FAMILIAL CASES

The story of hereditary diffuse gastric carcinoma

HEREDITARY – Germline mutations

SPORADIC – Somatic mutations

E-cadherin mutations in diffuse gastric cancer

HEREDITARY – Germline mutations

Missense mutations (20%)

Functional Assays in CHO cells (aggregation & collagen

invasion assays)

Missense mutations affect cell-cell adhesion, motility and invasion

T340A, A634V, W409R,V832M, E757K

A617T, ….

Functional Irrelevant

“so-called variants”

Functional Relevant

Adhesion, Motility, Invasion

Suriano G et al. Hum Mol Genet 12:3007, 2003

Oliveira C, 2010

Oliveira C, 2010

Oliveira C, 2010

Familial gastric cancer: overview and guidelines for management

(International Gastric Cancer Linkage Consortium)

Carriers of germline E-cadherin truncating mutations

Caldas C, Carneiro F, Lynch H et alEur J Genet 36: 873, 1999

Intensive screeningProphylactic gastrectomy

Carneiro F et al: J Pathol 203:68, 2004

In 9 prophylactic gastrectomies from North America

(Huntsman et al, 2001 and Chun et al, 2001),

all exhibited foci of intramucosal diffuse carcinoma

Hereditary diffuse gastric cancer (HDGC)

E-cadherin germline mutations in HDGC

G62V

T118RP172R L214P

G239R

D244GA298T

T340A

W409RI415L

P429S

V487A

A592T

T599S

A617T

A634V R732Q P799R

V832M

TRUNCATING

MISSENSE

In vivo validation of CDH1 germline missense mutations

Suriano G et alJ Mol Med 84:1023, 2006

In vivo validation of in vitro assays of CDH1 missense mutations

Barber M et al J Pathol 216:295, 2008

Barber M et al J Pathol 216:295, 2008

The highest number of foci was found in the fundus (44.7%) followed by the body (40.2%).

Rogers W et alAm J Surg Pathol 32:799, 2008

70% of the total signet ring cell foci were locted within the proximal 1/3 of the stomach

Gastrectomies in New Zealand in CDH1 carriers

Charlton A et al: Hereditary diffuse gastric cancer: predominance of multiple foci of signet ring cell carcinoma in distal stomach and transitional zone. Gut 53;814-820, 2004

Total gastrectomies in asymptomatic CDH1 mutation carriers (n=96)(prophylactic gastrectomies & gastrectomies for early carcinoma)

• Intramucosal SRC carcinoma 87 (91%)

• Total gastrectomies studied under research protocol 73• Number of cases with intramucosal SRC carcinoma 70* (96%)

* 2/3 cases negative for SRC intramucosal carcinoma displayed in situ carcinoma

Intramucosal signet ring cell (diffuse) carcinoma

In situ (signet ring cell) carcinoma

Pagetoid spread of signet ring cells:

Two-layer structure: an inner layer composed of benign mucous cells

and an outer layer of signet ring cells.

Pagetoid spread of signet ring cells and early invasion of lamina propria

In situ (signet ring cell) carcinoma Pagetoid spread of signet ring cells:Two-layer structure: an inner layer composed of benign

mucous cells and an outer layer of signet ring cells.

Mucosa

Muscularis mucosa

Submucosa

T1aTisTNM stage

A B

Carneiro F, Charlton A, Huntsman D4th Edition of WHO book , 2010

Non-neoplastic mucosawith foveolar hyperplasia

In situ signet ring cellcarcinoma

In situ signet ring cellcarcinoma and pagetoid spread

CDH1 germline mutation

Inactivation of second allele of CDH1

?

Pagetoid spread of signetring cell carcinoma

Early invasive (intramucosal)signet ring cell carcinoma

C D EA B

Development model of HDGC

Carneiro F et alJ Clin Pathol 61:25, 2008

CLINICALHISTORY

HISTOLOGICALCLASSIFICATION

MOLECULAR CLASSIFICATION

First Portuguese HDGC Family (Porto)

Second Portuguese HDGC Family (Coimbra)

Genetics, Pathology and Clinics of Familial Gastric Cancer

The story of Hereditary Diffuse Gastric Cancer (HDGC)

Methylation

DNA demethylating agents

Gene structure alterations

Partners of E-cadherin signalling

2nd HIT of CDH1 silencing?

ModulatorsE-cadherin expression

Carla Oliveira Gianpaolo SurianoJosé Carlos MachadoCéu FigueiredoPaulo FerreiraRita MateusRachid KaramHerculano MoreiraManuel Cardoso de OliveiraFátima CarneiroRaquel SerucaManuel Sobrinho-Simões

Paul PharoahCarlos Caldas

Department of Oncology, University of Cambridge UK

David HuntsmanDavid Owen

Thomas Smyrk

J. van KriekenJ de Bruin

Hospitais da Universidade de Coimbra

Augusta CiprianoMário Rui Silva

VU University Medical CenterBeatriz CarvalhoGerrit Meijer

Academic Medical CenterJohan OfferhausAnya Milne

THANKS FOR YOUR ATTENTION

First Portuguese Family with HDGC (Porto)

Second Portuguese Family with HDGC (Coimbra)

Oliveira et al: Virchows Arch 446: 181-184, 2005