Cancer genetics and treatment Cancer genetics and treatment BME1450BME1450

David HoggDavid Hogg

RJ 47 year maleRJ 47 year male

• Presented to family doctor Dec 2003 with Presented to family doctor Dec 2003 with pigmented lesion on scalppigmented lesion on scalp

• Told lesion of no significanceTold lesion of no significance

Melanoma TypesMelanoma Types

Superficial spreading melanomaSuperficial spreading melanoma Nodular melanomaNodular melanoma

Lentigo maligna melanomaLentigo maligna melanoma Acral lentiginous melanomaAcral lentiginous melanoma

RJ 47 year maleRJ 47 year male

• Lesion began to bleed two months lateLesion began to bleed two months late

• Biopsy showed nodular melanomaBiopsy showed nodular melanoma

• Referred to Princess Margaret Hospital Referred to Princess Margaret Hospital melanoma clinic for opinion March 2004melanoma clinic for opinion March 2004

RJ 47 year maleRJ 47 year male

• Sent for wide local excision of melanomaSent for wide local excision of melanoma• Sentinel node biopsy revealed melanoma in 2/6 Sentinel node biopsy revealed melanoma in 2/6

regional lymph nodesregional lymph nodes• Radical neck dissection April 2004: no positive nodesRadical neck dissection April 2004: no positive nodes

RJ 47 year maleRJ 47 year male

• Treated with Interferon-Treated with Interferon-• While on treatment, developed severe While on treatment, developed severe

headaches in July 2004headaches in July 2004

RJ 47 year maleRJ 47 year male

• Treated with radiotherapy to brain plus Treated with radiotherapy to brain plus TemozolomideTemozolomide

• Died of disease August 2004Died of disease August 2004

The art of medicine lies in amusing the patient The art of medicine lies in amusing the patient while the disease runs its course.while the disease runs its course.

Sir William OslerSir William Osler

Cancer – questions and problemsCancer – questions and problems

1.1. What is Cancer?What is Cancer?

2.2. What causes the disease?What causes the disease?

3.3. Why is cancer so difficult to treat?Why is cancer so difficult to treat?

4.4. Where are we going?Where are we going?

Cancer – questions and problemsCancer – questions and problems

1.1. What is Cancer?What is Cancer?

2.2. What causes the disease?What causes the disease?

3.3. Why is cancer so difficult to treat?Why is cancer so difficult to treat?

4.4. Where are we going?Where are we going?

Normal cells - characteristicsNormal cells - characteristics

1.1. Proliferate only when they receive positive Proliferate only when they receive positive growth signals from the environmentgrowth signals from the environment

2.2. Cease growing or die in the absence of such Cease growing or die in the absence of such signalssignals

3.3. Cease growing or die when they receive Cease growing or die when they receive growth-inhibitory or death signalsgrowth-inhibitory or death signals

Normal – characteristics: cont…Normal – characteristics: cont…

4.4. Differentiate or enter senescence (old age) Differentiate or enter senescence (old age) during their lifetime.during their lifetime.

5.5. Respect local anatomic boundaries – will not Respect local anatomic boundaries – will not cross tissue barriers unless specifically told to cross tissue barriers unless specifically told to do so.do so.

6.6. Do not spread to other areas of the body Do not spread to other areas of the body (metastasize).(metastasize).

GrowthGrowthfactorfactor

Growth FactorGrowth Factorreceptorreceptor

Signal transduction Signal transduction pathwayspathways

++

DNA (genes)DNA (genes)

++--

Tumor suppressorTumor suppressor

InhibitoryInhibitoryfactorfactor

GrowthGrowth

Programs/responsesPrograms/responses

No growthNo growth

DeathDeath

DifferentiationDifferentiation

MetastasisMetastasis

InputsInputs

Genetic targetsGenetic targets

Cancer is a genetic disease ICancer is a genetic disease I

• Cancer cells are derived from normal cellsCancer cells are derived from normal cells

• To progress from a normal cell to a To progress from a normal cell to a malignant one, a series of changes in the malignant one, a series of changes in the DNA of the cell must occurDNA of the cell must occur

• These changes alter the behaviour of the cell.These changes alter the behaviour of the cell.

Cancer is a genetic disease IICancer is a genetic disease II

• In almost all cases, a cell must suffer multiple In almost all cases, a cell must suffer multiple genetic ‘hits’ to become fully malignant.genetic ‘hits’ to become fully malignant.

• The number of hits is thought to number about 5-7 The number of hits is thought to number about 5-7 in most types of cancerin most types of cancer

• In many cases, at least some of these genetic lesions In many cases, at least some of these genetic lesions are known, and may differ between different types are known, and may differ between different types of cells (and hence cancer types).of cells (and hence cancer types).

Tumor progression – sequential mutations Tumor progression – sequential mutations

s1s1 s2s2 s3s3 s4s4

Timing of genetic changesTiming of genetic changes

• There is not always a set pattern of genetic There is not always a set pattern of genetic changes as a cell progresses to malignancy.changes as a cell progresses to malignancy.

• In other words, although 5-7 changes must In other words, although 5-7 changes must occur to make a cell fully malignant, the occur to make a cell fully malignant, the order and timing of these alteration may be order and timing of these alteration may be flexible.flexible.

Implications of progressive genetic Implications of progressive genetic alterationsalterations

• Cancers are not cells with “uncontrolled Cancers are not cells with “uncontrolled growth”!growth”!

• Loss of growth control occurs very slowly over Loss of growth control occurs very slowly over several years, in a stepwise fashion.several years, in a stepwise fashion.

• Therefore, defining the genetic changes in a Therefore, defining the genetic changes in a tumor will become as important as histological tumor will become as important as histological study.study.

Melanoma TypesMelanoma Types

Superficial spreading melanomaSuperficial spreading melanoma Nodular melanomaNodular melanoma

Lentigo maligna melanomaLentigo maligna melanoma Acral lentiginous melanomaAcral lentiginous melanoma

Melanoma: a multistage process

Derived in part from Chin Derived in part from Chin et alet al. (1998) Genes Dev. 12: 3467-3481. (1998) Genes Dev. 12: 3467-3481

Normal SkinNormal Skin Benign NevusBenign Nevus Dysplastic Nevus Dysplastic Nevus (DN)(DN)

Radial GrowthRadial GrowthPhase Phase

melanomamelanoma

Vertical GrowthVertical Growth Phase Phase

melanomamelanoma

Timing of genetic lesions in melanomaTiming of genetic lesions in melanoma

APC (FAP)APC (FAP)

Normal colonNormal colon AdenocarcinomaAdenocarcinoma

K-K-rasras MMR MMR

(HNPCC)(HNPCC)

SMAD4SMAD4DCCDCC p53p53

c-src/c-src/Cox2Cox2

Normal melanocyteNormal melanocyte Invasive melanomaInvasive melanoma

DN DN gene?gene?(DNS)(DNS)

Ras? - 10q - Ras? - 10q - 6p6p

CDKN2ACDKN2A(Familial melanoma)(Familial melanoma) p53p53

Cancer – questions and problemsCancer – questions and problems

1.1. What is Cancer?What is Cancer?

2.2. What causes the disease?What causes the disease?

3.3. Why is cancer so difficult to treat?Why is cancer so difficult to treat?

4.4. Where are we going?Where are we going?

What causes genetic changes?What causes genetic changes?

• EnvironmentEnvironment– Radiation, drugs (eg: chemotherapy); infections; Radiation, drugs (eg: chemotherapy); infections;

etcetc

• Genetic backgroundGenetic background– Inherited (germline) mutationsInherited (germline) mutations– Tendency to develop genetic lesions (DNA repair Tendency to develop genetic lesions (DNA repair

deficiencies).deficiencies).

Tumour Formation

Genotype

Somatic MutationsEnvironment

But remember throughout that no external But remember throughout that no external cause is efficient without a predisposition of cause is efficient without a predisposition of the body itself. Otherwise, external causes the body itself. Otherwise, external causes which affect one would affect all...which affect one would affect all...

Galen, 200 A.D.Galen, 200 A.D.

Familial CancerFamilial Cancer

• Now known to occur in many tumor types egs:Now known to occur in many tumor types egs:– Breast (Breast (BRCA1; BRCA2BRCA1; BRCA2))– Colon (Colon (APC; MLH1; MSH2; PMS1; PMS2APC; MLH1; MSH2; PMS1; PMS2))– Melanoma (Melanoma (CDKN2ACDKN2A))– Retinoblastoma (Retinoblastoma (RBRB))– Li-Fraumeni Syndrome (Li-Fraumeni Syndrome (p53p53))

Familial Cancer - CharacteristicsFamilial Cancer - Characteristics

Compared to the corresponding sporadic cancers:Compared to the corresponding sporadic cancers:

1. Age at first presentation is lower1. Age at first presentation is lower

2. Tumors may present at multiple sites in the same 2. Tumors may present at multiple sites in the same tissuetissue

3. Tumors may occur in different tissues3. Tumors may occur in different tissues

4. There may be a family history4. There may be a family history

I:1I:1 I:2I:2

II:2II:2melanomamelanoma

II:3II:3brainbrain

II:4II:4 II:5II:5melanomamelanoma

Dx33Dx332 primaries2 primaries

II:6II:6 II:7II:7melanomamelanoma

II:1II:1multiplemultiple

myelomamyeloma

Melanoma family - example Melanoma family - example

melanomamelanoma

Loss of a functional tumor suppressor geneLoss of a functional tumor suppressor gene

A1A1 A2A2 A1A1 A2*A2* A2*A2*

NormalNormal Mutation ofMutation ofone alleleone allele

Loss of remainingLoss of remainingnormal allelenormal allele

gg ss

I:1I:1 I:2I:2

II:2II:2melanomamelanoma

II:3II:3brainbrain

II:4II:4 II:5II:5melanomamelanoma

Dx33Dx332 primaries2 primaries

II:6II:6 II:7II:7melanomamelanoma

II:1II:1multiplemultiple

myelomamyeloma

Ger

mlin

e D

NA

Ger

mlin

e D

NA

Sort

ed B

M (

mye

lom

a)So

rted

BM

(m

yelo

ma)

Uns

orte

d B

MU

nsor

ted

BM

Wild-typeWild-type

HeteroduplexHeteroduplex

MutantMutant

LOH of the wild-type LOH of the wild-type CDKN2ACDKN2A allele in sorted allele in sorted BM obtained from multiple myeloma patient BM obtained from multiple myeloma patient

melanomamelanoma

Knudson’s Two-Hit ModelKnudson’s Two-Hit Model

SporadicSporadic

Normal gene

Somatic mutation

Somatic mutation

Single TumorsUnilateral

Later-onset

MendelianMendelian

Germline mutation

Multiple TumorsBilateral

Early-onset

Somatic mutation

Familial Cancer - CharacteristicsFamilial Cancer - Characteristics

Compared to the corresponding sporadic cancers:Compared to the corresponding sporadic cancers:

1. Age at first presentation is lower1. Age at first presentation is lower

2. Tumors may present at multiple sites in the same 2. Tumors may present at multiple sites in the same tissuetissue

3. Tumors may occur in different tissues3. Tumors may occur in different tissues

4. There may be a family history4. There may be a family history

Familial MelanomaFamilial Melanoma

• ~ 10% of melanoma patients have at least one affected ~ 10% of melanoma patients have at least one affected family memberfamily member

• Only ~3% of patients have multiple affected relativesOnly ~3% of patients have multiple affected relatives• However, the risk for patients with a predisposition to However, the risk for patients with a predisposition to

melanoma is very high: ~60-80X that of the general melanoma is very high: ~60-80X that of the general (European) population(European) population

Sporadic vs. Familial MelanomaSporadic vs. Familial Melanoma

SporadicSporadic FamilialFamilial

Ave. age at diagnosisAve. age at diagnosis 54 years54 years 35 years35 years

No. of primariesNo. of primaries Usually oneUsually one Often multipleOften multiple

Presence of dysplastic nevi Presence of dysplastic nevi (DN)(DN)

Some casesSome cases Most casesMost cases

Family historyFamily history NoNo2 or more affected 2 or more affected first-degree relativesfirst-degree relatives

Germline mutations in the Germline mutations in the CDKN2ACDKN2A gene that gene that predispose to melanoma - Toronto familiespredispose to melanoma - Toronto families

E1E1 E2E2

E3E3

CDKN2ACDKN2A5’5’ 3’ UTR3’ UTR

deletiondeletion

insertioninsertion

missensemissense

24in24inss

19del19del

6d

el

6d

el

2d

el

2d

el

G-G-34T34T

A5T

A5T

R24P

R24P

M53I

M53I

S56I

S56I

V57I

V57I

D85T

D85T

G122V

G122V

14d

el

14d

el

I49S

I49S

T93P

T93P

V126D

V126D

CDKN2ACDKN2A mutation detection mutation detection rate/number AFMrate/number AFM

0102030405060708090

100

5 or more 4 3 2 multipleprimaries

Number of melanomas in family

% M

uta

tio

ns

fou

nd

Problems in Treating CancerProblems in Treating Cancer

If a cancer depends on at least 5-7 mutations in If a cancer depends on at least 5-7 mutations in different pathways, and these pathways differ from different pathways, and these pathways differ from cell to cell, then how:cell to cell, then how:

1. Do we identify critical pathways in the cell1. Do we identify critical pathways in the cell

2. Determine the critical components of each 2. Determine the critical components of each pathway?pathway?

“The most effective way of destroying a network is to attack its most connected

nodes”

Yuhai Tu, Nature 2000

ToolsTools

1.1. For testing pathways:For testing pathways:

• Cell linesCell lines

• Tumor biopsies from patientsTumor biopsies from patients

2.2. To To knockknock out specific mRNA: out specific mRNA:• siRNAsiRNA

3.3. To affect specific proteins:To affect specific proteins:• DrugsDrugs

4.4. Delivery/attack: ?virusesDelivery/attack: ?viruses

Melanoma treatment with an oncolytic virus, JX-594Melanoma treatment with an oncolytic virus, JX-594 (Mastrangelo (Mastrangelo et al.et al., , Cancer Gene TherapyCancer Gene Therapy, 6: 409, 1999), 6: 409, 1999)