Curso de PatologCurso de Patologíía Moleculara Molecular

““PatologPatologíía Molecular del Canca Molecular del Cancéér de Estr de Estóómagomago””

FFáátima Carneirotima CarneiroIPATIMUP IPATIMUP

& & Medical Faculty/Centro Hospitalar São JoãoMedical Faculty/Centro Hospitalar São João

Porto, PortugalPorto, Portugal

Sporadic cancer

Hereditary cancer

Histo

patholo

gy

Molecular pathology

Genetic

susceptibility

GASTRIC CANCER

“Intestinal” carcinoma Diffuse 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)HER2 (amplification)TPR-MET (rearrangement)c-MET (6.0 Kb mARN)EGFR (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 carcinoma Diffuse carcinoma

• CDH1 gene

E-Cadherin/CDH1 changes

CDH1 mutations(50% - 70%)

Somatic mutations

Diffuse carcinomaDiffuse carcinoma

Absent expression of EAbsent expression of E--cadherincadherin

(inactivation of both alleles in the tumor)(inactivation of both alleles in the tumor)

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

CIN (Chromosomal instability)-Aneuploidy# 3p, 7q, 13q (LOH)# 6q (LOH)# 1q, 5q, 17p (LOH)RAS (overexpression/mutation)HER2 (amplification)TPR-MET (rearrangement)c-MET (6.0 Kb mARN)EGFR (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 carcinoma Diffuse carcinoma

• HER2• EGFR• MSI

Blood born metastases Poor prognosis

HER-2 amplification in intestinal carcinoma

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

HER-2in gastric carcinoma

ToGA TrialHER-2 overexpression in 22% of advanced gastric cancerssignificantly improved survival with trastuzumab

ASCO 2009 (LBA 4509)

HER-2

Prognostic factor YES (56%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

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, 1995

Santos 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

MSIin gastric carcinoma

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

MLH1

methylation

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)

Mixed gastric carcinoma

E-cadherin gene mutations provide a genetic basis for the phenotypic divergence of mixed

gastric carcinomas

CDH1 mutations: 17%

E-cadherin expression

CDH1 mutations: 83%

Machado J et al: Lab Invest 79: 459, 1999

CDH1 mutations

Diffuse component Intestinal component

Laser microdissection

Carvalho B et al: Mixed gastric

carcinomas show similar chromosomal

aberrations in both their diffuse and

glandular componentsCellular Oncology 28:283, 2006

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

More recently confirmed:Zheng HC et al. Mixed-type gastric carcinomas exhibit more aggressive features and indicate the histogenesis of carcinomas. Virchows Archive 452, 2008

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 carcinoma 8490/3 (Signet-ring cell carcinoma and other variants)

Mixed carcinoma 8255/3

WHO – 4th Edition, 2010

PapillaryPapillary TubularTubular

MucinousMucinous

Poorly cohesivePoorly cohesive

MixedMixed

Poorly cohesivePoorly cohesive(signet ring cell)(signet ring cell)

What about the contributions from new high throughput technologies?(WGA,NGS, CGH, expression profiling, etc)

Gut 61:673, 2012

Cancer Res 63:3309, 2003Gastroenterology 141: 476, 2011

Deng N et al, Gut 61:673, 2012

New findings?

� 22 recurrent genomic alterations, both known gastric cancer targets (FGFR2, ERBB2) and genes not previously reported to be amplified in gastric cancer (KLF5, GATA6).

� Genes related to RTK/RAS signaling (FGFR2, KRAS, ERBB2, EGFR and MET are frequently amplified in gastric cancer in a mutually exclusive manner.

� KRAS amplifications associated with adverse prognosis.

Cell lines

G-Intestinal G-Diffuse

Gastroenterology 141: 476, 2011

Tan I et al, Gastroenterology 141:476, 2011

Primary gastric cancers

Tan I et al, Gastroenterol ogy 141:476, 2011

Intrinsic genomic subclasses are prognostic

In vitro chemosensitivity of G-INT and G-DIF cell lines

Intrinsic subtypes of gastric cancer, based on gene expression pattern,

predict survival and response to chemotherapy

New molecular classification of GC?

Sporadic cancer

Hereditary cancer

Histo

patholo

gy

Molecular pathology

Genetic

susceptibility

� 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)

* Most caused by E-cadherin alterations

GASTRIC CARCINOMA

HEREDITARY HEREDITARY –– Germline mutationsGermline mutations

SPORADIC SPORADIC –– Somatic mutationsSomatic mutations

EE--cadherin mutations in diffuse gastric cancercadherin mutations in diffuse gastric cancer

1998/91998/9

EE--cadherin & gastric cancercadherin & gastric cancer

(sporadic and hereditary)(sporadic and hereditary)

20012001

20032003

-- Familial gastric cancer: overview and guidelines for managementFamilial gastric cancer: overview and guidelines for management. (IGCLC). (IGCLC)

-- EE--cadherin expression in gastric cancer cadherin expression in gastric cancer

-- EE--cadherin gene mutations provide a genetic basis for mixed gastricadherin gene mutations provide a genetic basis for mixed gastric carcinomasc carcinomas

-- Second hit ESecond hit E--cadherin gene (cadherin gene (CDH1CDH1) inactivation (promoter ) inactivation (promoter

methylation) in sporadic diffuse gastric carcinoma methylation) in sporadic diffuse gastric carcinoma

-- Functional analyses of EFunctional analyses of E--cadherin cadherin

((CDH1CDH1) germline missense mutations) germline missense mutations

-- Model of development of HDGCModel of development of HDGC20042004

-- Cleft lip/palate and Cleft lip/palate and

CDH1CDH1 mutations in mutations in

families with HDGCfamilies with HDGC

20062006

20052005-- First Portuguese First Portuguese

family with HDGCfamily with HDGC

Int J Surg Pathol Int J Surg Pathol 6: 135, 1998 6: 135, 1998

HistopathologyHistopathology 35: 477, 199935: 477, 1999

Lab Invest Lab Invest 79: 459, 1999 79: 459, 1999

J Med GenetJ Med Genet 36: 873, 199936: 873, 1999N Engl J MedN Engl J Med 344:1904, 2001 344:1904, 2001 OncogeneOncogene 20: 1525, 200120: 1525, 2001Gastroenterol Clin BiolGastroenterol Clin Biol 25: 931, 2001 25: 931, 2001 Hum MutatHum Mutat 19:510, 2002 19:510, 2002 Hum Mol GenetHum Mol Genet 12: 575, 200312: 575, 2003Hum Mol GenetHum Mol Genet 12: 3007, 2003 12: 3007, 2003 Oncogene Oncogene 22:5716, 2003 22:5716, 2003 J PatholJ Pathol 203: 681, 2004203: 681, 2004Virchows Arch Virchows Arch 446: 18, 2005 446: 18, 2005 Clin Cancer ResClin Cancer Res 11:5401, 200511:5401, 2005J Med GenetJ Med Genet 43:138, 200643:138, 2006J Mol Med J Mol Med 84:1023, 2006 84:1023, 2006 Hum Mol GenetHum Mol Genet15:1704, 2006 15:1704, 2006 Hum MutatHum Mutat 28:203, 200728:203, 2007J Pathol J Pathol 211:507, 211:507, 20072007OncogeneOncogene 27: 4255, 2008 27: 4255, 2008 J Clin Pathol J Clin Pathol 61:25, 2008 61:25, 2008 J PatholJ Pathol 216:295, 2008 216:295, 2008 Gastroenterology Gastroenterology 136:2137, 2009136:2137, 2009J Med GenetJ Med Genet 47: 43647: 436, 2010, 2010PLoS One PLoS One 6:e23188 ,6:e23188 ,20112011Cell Mol Life SciCell Mol Life Sci,, 20112011

20072007

-- Novel germline Novel germline CDH1CDH1 mutationsmutations

-- Experimental model in DrosophilaExperimental model in Drosophila

20082008-- NMD mRNA surveillance NMD mRNA surveillance

downregulates aberrantdownregulates aberrant

CDH1CDH1 transcriptstranscripts

-- Second hit of Second hit of

CDH1CDH1 inactivationinactivation

20092009

2010/11/12/132010/11/12/13

-- EE--cadherin repressors in gastric ancercadherin repressors in gastric ancer

-- Prophylactic gastrectomies in asymptomatic carriers of germProphylactic gastrectomies in asymptomatic carriers of germ--line Eline E--

cadherin mutations cadherin mutations

HEREDITARY HEREDITARY –– GermlineGermline mutationsmutations

MLPA analysis and Array CGHMLPA analysis and Array CGH

Deletions Deletions affecting the 5affecting the 5’’--end of end of CDH1CDH1

Deletions Deletions affecting the 3affecting the 3’’--end of end of CDH1CDH1

Large Alu associated Large Alu associated germline deletions of germline deletions of CDH1CDH1in HDGC families: a new mechanism for disruption in HDGC families: a new mechanism for disruption

of Eof E--Cadherin functionCadherin functionOliveira O et al, Hum Mol Genet, 2009Oliveira O et al, Hum Mol Genet, 2009

Germline missense Germline missense CDH1CDH1 mutations in HDGCmutations in HDGC

Splice site mutations

Nonsense mutations

Deletions

and

insertions

Missensemutations

Splice site mutations

Nonsense mutations

Deletions

and

insertions

Missensemutations

Truncating (~80%)Truncating (~80%)

Missense (~20%)Missense (~20%)Functional Assays in CHO cells Functional Assays in CHO cells

(aggregation & collagen invasion (aggregation & collagen invasion

assays)assays)

Missense mMissense mutations affect cellutations affect cell--cell adhesion, cell adhesion, motility and invasionmotility and invasion

T340A, A634V, W409R,T340A, A634V, W409R,V832M, E757KV832M, E757K

A617T, A617T, ……..

Functional IrrelevantFunctional Irrelevant

““neutral variantsneutral variants””

Functional RelevantFunctional Relevant

Adhesion, Motility, Adhesion, Motility, InvasionInvasion

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

CanadaCanada

BrazBrazııll

ChinaChina

USAUSA

FranceFranceGermanyGermanyItalyItaly

NetherlandsNetherlands

New ZealandNew Zealand

South KoreaSouth Korea

JapanJapan

Functional characterization ofFunctional characterization ofCDH1CDH1 missense mutationsmissense mutations(IPATIMUP)(IPATIMUP)

In vivoIn vivo validation of validation of in vitroin vitro assays of assays of CDH1CDH1 missense mutationsmissense mutations

Barber M Barber M et al et al J Pathol 216:295, 2008 J Pathol 216:295, 2008

Translation to Translation to clinical practiceclinical practice

•• Genetic counseling Genetic counseling and testingand testing

•• Early diagnosisEarly diagnosis•• Treatment Treatment (prophylactic (prophylactic gastrectomy)gastrectomy)

UK UK -- Fundus (44.7%), body (40.2%). J Pathol 2008Fundus (44.7%), body (40.2%). J Pathol 2008

USA USA -- 70% in the proximal 1/3 of the stomach. Am J Surg Pathol 200870% in the proximal 1/3 of the stomach. Am J Surg Pathol 2008

New Zealand New Zealand -- Distal stomach and Distal stomach and transitional zone. Gut 2004 transitional zone. Gut 2004

Intramucosal signetIntramucosal signet--ring cell (diffuse) carcinomaring cell (diffuse) carcinoma

In situIn situ (signet ring cell) carcinoma(signet ring cell) carcinoma Pagetoid spread Pagetoid spread of signet ring cells:of signet ring cells:TwoTwo--layer structure: an inner layer composed of benign layer structure: an inner layer composed of benign mucous cells and an outer layer of signet ring cells.mucous cells and an outer layer of signet ring cells.

MucosMucos

aa

MusculaMuscula

ris ris

mucosamucosa

SubmucSubmuc

osaosa

T1aT1aTisTisTNM TNM

stagestage

AA BB

Carneiro F, Charlton A, Huntsman DCarneiro F, Charlton A, Huntsman D4th Edition of WHO book , 20104th Edition of WHO book , 2010

Absent expression of EAbsent expression of E--cadherincadherin

(inactivation of both alleles in the Tis lesions)(inactivation of both alleles in the Tis lesions)

In situIn situ carcinomacarcinoma

Pagetoid spreadPagetoid spread

NonNon--neoplastic mucosaneoplastic mucosawith foveolar hyperplasiawith foveolar hyperplasia

In situIn situ signet ring cellsignet ring cellcarcinomacarcinoma

In situIn situ signet ring cellsignet ring cellcarcinoma and pagetoid spreadcarcinoma and pagetoid spread

CDH1 CDH1 germline mutationgermline mutation

Inactivation of second allele of Inactivation of second allele of CDH1CDH1

??

Pagetoid spread of signetPagetoid spread of signetring cell carcinomaring cell carcinoma

Early invasive (intramucosal)Early invasive (intramucosal)signet ring cell carcinomasignet ring cell carcinoma

CC DD EEAA BB

Development model of HDGCDevelopment model of HDGC

Carneiro F Carneiro F et alet alJ Clin Pathol 61:25, 2008J Clin Pathol 61:25, 2008

The brand new GAPPS syndrome…(A new hereditary gastric cancer syndrome)

Gastric Adenocarcinoma and Proximal Polyposis of the Gastric Adenocarcinoma and Proximal Polyposis of the Stomach (GAPPS): a new autosomal dominant syndrome.Stomach (GAPPS): a new autosomal dominant syndrome.

Worthley et al; Worthley et al; Gut Gut 61:77461:774--779, 2012779, 2012

Familial gastric cancerFamilial gastric cancer

NGS pathology

Thanks for your attentionThanks for your attention

Save the Date

25th European Congress of Pathology

31 August – 4 September 2013in Lisbon, Portugal

www.esp-congress.org