colon cancer and hereditary cancer syndromes filecolon of a 30 year old fap patient . familiar...
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Colon Cancer and Hereditary
Cancer Syndromes
Gisela Keller
Institute of Pathology
Technische Universität München
Colon Cancer and Hereditary Cancer
Syndromes
• epidemiology
• models of colorectal carcinogenesis
• tumor suppressor genes and oncogenes
• molecular classification
Globocan 2012, IACR: International Agency for Research on Cancer
http://globocan.iarc.fr
World: Estimated incidence and mortality, both sexes
Colorectal Cancer in Germany
• 62430 new cases /year 2010
• Second (women) and third (men) most common cancer
Robert-Koch Institut 2013
Genetic Model of Colon Carcinogenesis
(Fearon-Vogelstein Model)
• alterations in 4 to 6 genes
• alterations in a preferential sequence
• accumulation is important
• activation of oncogenes and inactivation of tumor
suppressor genes
Colorectal Cancer and Age
from: Varmus und Weinberg, Gene und Krebs, Spektrum der Wissenschaft 1992
Colorectal Cancer and the Number of Alterations as
Rate Limiting Events
from: Varmus und Weinberg, Gene und Krebs,
Spektrum der Wissenschaft 1992
one
event
2 events
4-6 events
• benign epithelial neoplasia
• exophytic growth pattern
• atypic nuclei (enlarged, hyperchromatic spindle-
shaped, ovoid)
normal
epithelia
tubular
adenoma
Adenomas
modified from
www.sgpgi.ac.in/path/seminars/Image6.gi
adenoma carcinoma
Adenoma Carcinoma
Accumulation of Genetic Alterations in
the Adenoma-Carcinoma-Sequence
Fearon ER and Vogelstein B: A Genetic Model for Colorectal Tumorigenesis. Cell 61:759-767, 1990
normal
epithelia
hyperprolifera-
tive epithelia
adenoma
carcinoma
APC
LOH 5q
Genetic Alteration in the
Adenoma-Carcinoma-Sequence
APC Gene (adenomatous polyposis coli)
• chromosome 5q21
• multiple functions - cell adhesion, migration
- chromosome segregation, genetic stability
- Wnt-signaling
Reya and Clevers (2005), Nature 434:843-850
Colon Epithelium APC and Wnt-Signaling
Elisabeth Pennisi (1998), Science 281: 1438-1441
APC Gene (adenomatous polyposis coli)
• chromosome 5q21
• multiple functions - cell adhesion, migration
- chromosome segregation, genetic stability
- Wnt-signaling
• tumor suppressor gene
- somatic mutations: 40-70% of adenomas and carcinomas
- germline mutations: FAP-syndrome (familial adenomatous
polyposis coli)
Tumor Suppressor Genes
• suppress cell proliferation
• multiple functions
– signal transduction
– cell cycle regulation
– apoptosis
– DNA-repair
• loss of function important
Two Hit Model of Inactivation of Tumor
Suppressor Genes (Knudson 1971)
1. mutation
somatic
2. mutation
somatic
sporadic tumor
1. mutation
germline
2. mutation
somatic
hereditary tumor
colon of
a 30 year old
FAP patient
Familiar Adenomatous Polyposis Coli (FAP)
• germline mutations in the APC gene
• autosomal dominant inherited tumor disease
• > 100 adenomas in the colon
• high cancer risk
• early age on onset
• frequency: 1% of colorectal carcinomas
normale
epithelia
hyperprolifera-
tive epithelia
adenoma
carcinoma
APC
LOH 5q
K-ras
Genetic Alterations in the
Adenoma-Carcinoma-Sequence
Oncogene
• activated proto-
oncogene
• promote cell
proliferation
K-ras (Kirsten rat sarcoma viral oncogene homolog)
• somatic mutations
- in 40-50% of carcinomas and adenomas (> 1cm)
• G-protein
• signal transduction (receptor tyrosine kinase, e.g. EGFR = epidermal growth factor receptor)
Function and Activation of K-ras
Signal transduction
Gschwind et al. 5 :361-369, 2004.
Oncoprotein
• K-ras – mutations in 40-50% of carcinomas and adenomas ( >1cm)
– G-protein
– signal transduction
• c-myc
– overexpressed in up to 70% of carcinomas
– transcription factor
• EGFR – overexpressed in 30-80% of carcinomas
– receptor for the growth factor EGF
Oncogenes in Colorectal Tumors
Receptor Tyrosine Kinases as Therapeutic
Targets
Gschwind et al. 5 :361-369, 2004.
Antibodies against EGFR:
Cetuximab
Treatment of
metastatic colon carcinomas
K-ras Mutation and Therapy Response
Copyright © American Society of Clinical Oncology
89 patients with metast. CRC
therapy with Cetuximab
Lievre, A. et al. JCO, 26:374-379 2008
K-ras
K-ras
nonmutated
mutated nonmutated
muta-
ted
progression
free survival
overall
survival
Gschwind et al.
Nature Rev Cancer 5 :361-369, 2004.
Randomized study
(n=394)
• Cetuximab + best supportive
care
versus
• best supportive care alone
Result
K-ras mutations: 42.3%
Median OS:
9.5 versus 4.8 Monate
„This is really a sea change in practice“
Last month, an expert panel said that patients with advanced
colorectal cancer should not be treated with cetuximab or
panitumumab if their tumors have mutations in the K-Ras oncogene.
.....The European regulatory agency has already changed the
cetuximab label, restricting ist use to patients with wild-type K-
Ras.....
NEWS
K-Ras Mutations Are Changing Practice in Advanced Colo-
rectal Cancer
Caroline McNeil
Journal of the National Cancer Institute Advance Access
originally published online on November 25, 2008
Mutationsanalytik TUM-Pathologie
0
50
100
150
200
250
300
350
400
450
500
550
600
650
2007 2008 2009 2010 2011 2012 2013
KRAS Analysen
Jahr
normale
epithelia
hyperprolifera-
tive epithelia
adenoma
carcinoma
APC
LOH 5q
DCC
SMAD 2/4
LOH 18q
K-ras
Genetic Alterations in the
Adenoma-Carcinoma-Sequence
DCC (deleted in colorectal cancer)
SMAD2/4 (mother against decapentaplegic homolog)
• tumor suppressor gene, chromosome 18q21
– LOH 18q: 50% of late ademonas 70% of carcinomas
• DCC
– mutations: 6% of carcinomas
– reduced expression: 30% of carcinomas
– cell adhesion, migration
• SMAD2/4
– mutations:14% of carcinomas
– TGF-b signaling
normal
epithelia
hyperprolifera-
tive epithelia
adenoma
carcinoma
APC
LOH 5q
SMAD2/4
LOH 18q
K-ras TP53
LOH 17p
Genetic Alterations in the
Adenoma-Carcinoma-Sequence
• tumor suppressor, chromosome 17p13
– somatic mutations: 50% of carcinomas
– LOH: 75% of carcinoma
• protein of 393 amino acids, MW 53kdal
• multiple functions
– cell cycle control (G1-S „checkpoint“)
– DNA repair
– apoptosis
TP53
Genetic Model of Colon Carcinogenesis
(Fearon-Vogelstein Model)
• alterations in 4 to 6 genes
• alterations in a preferential sequence
• accumulation is important
• activation of oncogenes and inactivation of tumor
suppressor genes
Science. 2007 Oct 11; [Epub ahead of print]
• sequencing of 18191 genes in 11 tumors
• number of mutations / tumor
- colon: 77
- breast: 101
• Identification of 280 CAN (cancer)-genes
• Number of mutated CAN-genes / tumor
- colon: 15
- mamma: 14
The Genomic Landscapes of Colon and
Breast Cancer: Mutations
• only few CAN-genes are commonly mutated
• large number of genes are mutated at low frequency
large tumor heterogeneity
Fig. 3 Cancer genome landscapes
Wood LD et al., Science 2007, Oct 11
K-ras
APC
TP53
Few gene mountains, many gene hills
normal
epithelia
hyperprolifera-
tive epithelia
adenoma
carcinoma
APC
LOH 5q
SMAD2/4
LOH 18q
K-ras TP53
LOH 17p
Genetic Alterations in the
Adenoma-Carcinoma-Sequence
chromosomal instability
Molecular Classification of Colorectal
Carcinomas
• chromosomale instability (CIN)
Molecular Classification of Colorectal
Carcinomas
• chromosomale instability (CIN)
• microsatellite instability (MSI)
Microsatellites
• short repetitive DNA-sequences
• widely distributed in the genome
• function unknown
• highly polymorphic
CA 15 - 30
CAGTAA 15 - 30
replication
deletion insertion
replication replication
Mismatch-
repair proteins
MSH2 / MSH6
MLH1 / PMS2
Microsatellite instability
Microsatellite Instability
BAT 25
BAT 26
Normal
Tumor
Tumor
Normal
Molecular Classification of Colorectal
Carcinomas
• chromosomal instability (CIN)
• microsatellite instability (MSI)
-MSI-H: > 2/5 markers unstable
-MSI-L: 1/5 marker unstable
-MSS: stable
Microsatellite Instability (MSI) and DNA -
Mismatch Repair Genes
• MSI - additional alleles in the tumor
• defects in mismatch repair genes – MLH1, MSH2, MSH6, PMS2
• mutations in genes with repetitive sequences in the coding region – TGF-b-receptor type II, IGFII-receptor, axin2, bAX
• MSI-H in colorectal carcinomas - sporadic: 15 - 20%
- hereditary (HNPCC): 80 - 90%
Lynch-Syndrome (HNPCC - hereditary nonpolyposis colorectal cancer)
• germline mutations in DNA-mismatch repair genes
(MLH1, MSH2, MSH6, PMS2)
• tumor spectrum
– colon
– extracolonic (endometrium, stomach, small intestine, urothel carcinomas)
• syn-, metachrone carcinomas
• early age of onset
• frequency: 2-3% of colorectal carcinomas
MSH2 MSH6
PMS2 MLH1
Immunohistochemistry: MMR - Proteins
Germline mutation: MLH1, c.511G>T, p. Glu171Stop
Colon-Ca
ED: 32J
Colon-Ca,
Endometrium-Ca
ED: 35J
Colon-Ca,
DD-Ca
ED: 30J
Pedigree of an HNPCC-Family
Molecular Classification of Colorectal
Carcinomas
• chromosomal instability (CIN)
• microsatellite instability (MSI)
-MSI-H: > 2/5 markers unstable
-MSI-L: 1/5 marker unstable
-MSS: stable
• epigenetic instability
CpG island methylator phenotype (CIMP)
-CIMP-H: >30% methylated genes
-CIMP-L: <30% methylated genes
• covalent modification of cytosine
(CpG sites)
• function:
- gene expression
- chromosomal stability
• 70% of CpGs are methylated
• CpG islands in promoter regions are not methylated,
transcription possible
Epigenetic Alteration: DNA-Methylation
promoter hypermethylation
silencing of transcription
Colorectal Carcinogenesis
• classical adenoma-carcinoma-sequence
• serrated carcinogenesis
Serrated Morphology of Epithelial
Crypts
Serrated Carcinogenesis
• activation of the MAPK-signaling
-BRAF
-KRAS
• inhibition of apopotosis
serrated morphology
• DNA methylation
CpG island methylator phenotype (CIMP) -CIMP-H: >30%
-CIMP-L: <30% methylated genes
sessile
serrated
adenoma
CIMP-H
carcinoma
MSI-H normal
epithelia BRAF
methyl. MLH1
mut. TGbRII,
IGFR2
Sessile serrated carcinogenesis
partial methyl.
MLH1, methyl. MGMT
mut. p53
carcinoma
MSI-L, MSS
HP?
normal
epithelia
traditional
serrated
adenoma
(TSA) /
villous
adenoma
carcinoma
MSI-L, MSS
KRAS
Traditional serrated carcinogeneis – mixed type
methyl.
MGMT
CIMP-L
HP?
CIN
mut. p53, APC
LOH 3p, 18q
CIMP-H
carcinoma
MSI-H normal
epithelia BRAF
methyl. MLH1
mut. TGbRII,
IGFR2
Sessile serrated carcinogenesis
partial methyl.
MLH1, methyl. MGMT
mut. p53
carcinoma
MSI-L, MSS
HP? sessile
serrated
adenoma
sessile
serrated
adenoma
carcinoma
MSI-H
Clinical Characteristics
carcinoma
MSI-L, MSS
traditional
serrated
adenoma/
villous
adenoma
carcinoma
MSI-L, MSS
Traditional serrated carcinogenesis
proximal localization
sex: F>M
distale localization
sex: M>F
Sessile serrated carcinogenesis
high-risk-subtype
5 year survival
<30%
low-risk-subtype
5 year survival
>70%
Molecular Classification of Colorectal
Carcinomas
CIN
CIMP- H
MSI-H
57%
12%
20%
3%
8%
CIMP-L
CIMP-negativ
APC, K-ras,
p53 mutation
„serrated
pathway“
„classical adenoma
carcinoma pathway“
from: Jass JR, Histopathol 2007, 50:113-130
K-ras, p53-
mutation
MLH1-methyl.
BRAF-mutation
Nature. 2012 Jul 18;487(7407):330-7.
Comprehensive molecular characterization of
human colon and rectal cancer.
Cancer Genome Atlas Network.
Collaborators (326)
Genomweite Analysen von 276 Karzinomen
Exom-Sequenzierung
DNA-Kopien-Anzahl
Promoter-Methylierung
mRNA-Expression
microRNA-Expression
The Cancer Genome Atlas Network Nature 487, 330-337 (2012) doi:10.1038/nature11252
Mutation frequencies in human CRC.
The Cancer Genome Atlas Network Nature 487, 330-337 (2012) doi:10.1038/nature11252
Diversity and frequency of genetic changes leading to
deregulation of signalling pathways in CRC.