Colon Cancer and Hereditary

Cancer Syndromes

Gisela Keller

Institute of Pathology

Technische Universität München

gisela.keller@lrz.tum.de

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.