curso de patología molecular “patología molecular del cancér de
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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)
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)
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.
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 (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
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
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
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