GENETIC CHANGES

IN CANCER

AND

CANCER TARGETED THERAPY

Syarifuddin Wahid

Department of Pathology

Faculty of Medicine, Hasanuddin Univ.

Normal Cell

DNA damage DNA repair

Environmental agents

(Chemicals, radiation,viruses)

FAILLURE of DNA repair APOPTOSIS

FAILLURE APOPTOSIS

PERMANENT DNA DAMAGE (MUTATION)

GENETIC AND PHENOTYPIC CHANGES

1

P53 (TP53) Gene

“stress of the cell”

(anoxia, oncogene expression, and

DNA damage)

Activation of P53

cell cycle stop DNA repair

apoptosis

No mutation/carciogenesis

Inherited Mutation of DNA

Repair Genes and Cancer Risk Inherited Mutation Cancer Risk

of DNA Repair Genes

DNA mismatch repair HNPCC

Nucleotid excision Skin Cancer

repair.

BRCA1 & BRCA2 Breast Ca.

Mutation in the genome of

Somatic cells

Activation of growth-

promoting oncogenes

Inactivation of

Tumor suppressor genes

Inactivation of gene

regulate apoptosis

Decreased apoptosis Unregulated cell proliferation

Clonal Expansion

2

3

4

Activation of growth-

promoting oncogenes

INACTIVE

PROTOONCOGENE

MUTATION

(ONE ALLEL) ACTIVE

ONCOGENE

(CELLULAR

ONCOGENE)

Oncogenes Lesion Cancer NEU/HER2 Amplification Breast,ovary,stomach (Ad Ca)

H-RAS Point Mutation Colon, lung, pancreas

MYC Translocation Burkitt’s Lymphoma

RET Rearranggement Thyroid (Ca)

Daerah promoter VEGF manusia

Ket :

a. primer amplikasi dalam

kotak buram, polimorfisme

ditandai dengan angka-angka

dekat dengan tanda panah

(transciption start site). bsaHl

dan BsmFl adalah ensim

pemotong. b Haplotype gen

VEGF, (C) adalah Wild type

promoter

POINT MUTATION

ON VEGF GENE

-460T>C

+405C>G

Protooncogene

Cellular Oncogene

Viral Gene

Carcinogen

(chemical,

viral and

radiation) Viral Oncogene

Normal production of growth factor

Normal growth

Devective immune survailance

Abnodrmal production of growth factor

Abnormal Growth

NEOPLASM

DNA Mutation

Inactivation of

Tumor suppressor genes

and gene regulate apoptosis

Active

Genes

Inactive

Genes

MUTATION

(BOTH ALLELS , LOH)

TS Genes Function Cancer RB Cell cycle Retinoblastoma

TP53 Cell cycle,apoptosis Sarcoma, lymphoma,

(Guardian of the genome) Adeno Ca

BRCA1 DNA repair Breast

MSH2,MLH1 Mismatch repair Colorectal (HNPCC)

DELETION

GROWTH AND

ANTIGROWTH SIGNAL

RETINOBLASTOMA (RB) GENE

RB RB homozygous (Normal)

1st mutation

RB rb heterozygous(N& M)

2nd mutation

homozygous/ loss of

rb rb heterozygosity

(Retinoblastoma)

(LOH)

(LOH)

MALIGNANT NEOPLASM

Clonal Expansion

Tumor Progression

Invasion and

metastasis

angiogenesis

Escape from immunity

Additional mutation

5

6

7

8

Metastatic subclone

Clonal expantion, growth,

Diversification, angiogenesis

Transformed Cells

Primary Tumor

INVATION

INTRAVASATION

EXTRAVASATION

Metastatic deposit

Angiogenesis

GROWTH

Tumor Cell

embolus

Ecape from imunity

transformation

progression

Proliferation

of genetically

Unstable cells

Tumor cells

variants

heterogeneity

Normal cell

Single

Tumor cell

1 gm

(109cells)

1 kg

(1012 cells)

30

doublings

10

doublings

Normal Cell

Carcinogen Induced changes

Tumor Cell

variants

Single tumor cell

Nonantigenic

Invasive

Metastatic

Requiring fewer growth factor

Metastases

Normal

Colon

cell

Increased

Cell

growth

Early

Stage

adenoma

Intermediate

Stage

adenoma

Late

Stage

adenoma

Carcinoma

Metastasis

APC gene loss

DNA loses methyl groups

RAS gene mutation

DCC gene loss

TP53 gene loss

PRL3

overexpression

MORPHOLOGIC

CHANGES

GENETIC CHANGES

MULTISTEPS CHANGES

IN COLON CANCER

CANCER TARGETED THERAPY

Tujuan utama:

Menghambat pertumbuhan dan penyebaran sel tumor dengan cara memblok signal pertumbuhan sel tumor atau menghambat layanan darah ke jaringan tumor.

Sasaran obat

Molekul spesifik yang:

1. Terlibat dlm proses karsinogenesis,

pertumbuhan dan penyebaran

tumor.

2. Terlibat dalam angiogenesis.

3. Menjadi marker dari sel tumor

bersangkutan.

(Molecularly targeted therapy)

Transformasi: Onkogen HER2

1 = jumlah copy gen

2 = transkripsi mRNA

3 = ekspresi reseptor protein permukaan sel

4 = pelepasan reseptor dari bgn extraselluler

Normal Amplifikasi/Overekspresi

Cytoplasm

HER2 reseptor

protein

Cytoplasmic

membrane

Nukleus

HER2 DNA

HER2

mRNA

1

2

3

4

VEGF

Stem Cell Pro-B Pre-B Immature B

Mature B

Bone Marrow

Peripheral lymphoid

organ or tissue

IgM,IgD

No antigen dependence Self antigen Foreign antigen

Activated B

B CELL DEVELOPMENT

RAG-1 & RAG-2 expression

H chain H & L chain

CD43+

CD43+

CD19+

CD10+

CD20

B220

CD43+

CD20

IgM(low)

CD43-

CD20

IgM(high)

CD20

IgM(high)

CD20

CD20 CD20 CD20 CD20 CD20

Tipe targeted therapy

1. Fokus pada molekul yang merupa-kan komponen internal dan fungsi sel. (Menggunakan molekul kecil yang dapat mencapai sel dan meng-hambat

fungsi sel dan akhirnya mati).

- Single transduction inhibitor (Imatinib mesylate, Genefitinib, Laptinib, HER2 tyrosine kinase inhibitor),

- Biologic response Modifier Agent

(Denileukin diftitox), dan

- proteosome inhibitor (Bortezomib).

Tipe targeted therapy

2. Fokus pada molekul di bagian luar sel (membran sel) misalnya reseptor atau molekul membran lainnya.

(Menggunakan antibodi monoklonal)

mis: Anti HER2, Anti CD20

3. Fokus pada molekul (growh factor) yang ada dalam sirkulasi

(Menggunakan antibodi monoklonal)

Mis : Anti VEGF

4. Fokus pada gen (Gene therapies).

JENIS TARGETED THERAPY

1. Menggunakan Small Molecule Drug

(SMD)

2. Menggunakan Monoclonal Antibody

(MAB)

3. Menggunakan Gene Therapy

Cara kerja MAB

Menghambat signal pertumbuhan tumor atau yang memfasilitasi pertumbuhan tumor

• Mengikat growth factor atau molekul lainnya (mis. angiogenesis) yang ada dalam sirkulasi sehingga tidak bisa binding dengan reseptornya.

• Binding dengan reseptor growth factor atau angiogenesis sehingga tidak bisa dicapai oleh growth factor atau molekul angiogenesis.

• Memicu internalisasi reseptor atau pelepasan reseptor dari membran sel.

Cara kerja MAB

Sebagai pencari sel tumor. Mengikat molekul yag ada di permukaan sel tumor

sehingga bahan aktip antikanker (kemoterapi atau radioterapi) yang diikutkan di MAB dapat mencapai dengan tepat sel tumor. (Mengantar obat kemoterapi atau radioaktip mencapai target.

Mengefektifkan sistem imun tubuh membunuh sel tumor (humanized MAB)

Binding dengan protein permukaan (reseptor atau tumor marker) kemudian berinteraksi dengan sistem imun tubuh ang akan membunuh sel target.

Memicu apoptosis sel tumor.

Mouse myeloma

Cell line FUSION

Hybridomas producing

Monoclonal anti-X antibody

Antigen-X

Anti-X antibody-

producing spleen cells

In vitro selection in HAT medium

“Clone” cells

Screen supernatant for presence of anti-X antibody

MONOCLONAL

ANTIBODY

HUMAN/MOUSE CHIMERIC MONOCLONAL ANTIBODY (HUMANIZED MAB)

Genetic engineering

MONOCLONAL

ANYIBODY-LIKE

MOLECULE

Genetic engineering

Mouse myeloma

Cell line FUSION

Hybridomas producing

Monoclonal anti-X antibody

Antigen-X

Screen supernatant for presence of anti-X antibody

“Clone” cells

In vitro selection in HAT medium

Anti-X antibody-

producing spleen cells

MONOCLONAL

ANTIBODY

Parent mouse

antibody

(cdr)

Idiotype

L-Chain

H-Chain

Antigen

Binding Site

Murine

Variable

region

(biru)

Constan

region

(merah)

-s-s-

Fab

papain

Fc

Human constant қ region

Human constant Fc region

HUMAN/MOUSE CHIMERIC

MONOCLONAL ANTIBODY

(HUMANIZED MAB)

Gambar 12. Struktur humanized

monoclonal antibody.

Constant region

a.amino manusia

(>90%)

Variable region

a.amino tikus (murin)

(<10%)

HUMAN/MOUSE

CHIMERIC MONOCLONAL ANTIBODY

(HUMANIZED MAB)

Keuntungan chimeric antibody

• Pembuatan antigen binding site (Fab variable region) yang spesifik pada mencit akan menjamin ikatan efektif dengan target antigen.

• Unsur human pada Fc constan region memungkinkan interaksi lebih efektif dengan human effector mechanisms dari respon imun pasien untuk membunuh sel target.

• Unsur human pada struktur antibodi di atas 90% memperkecil kemugkinan timbulnya reaksi penolakan/allergi/syok dari pasien.

Adenoma of increasing size and

degree of dysplasia

Hyperproliperative

epithelium Invasive carcinoma

P53 mutation

And/or LOH

DC LOH

LOH of other

Genes on 18q

K-ras mutation DNA mutation

DNA methylation

abnormalities

Mismatch repair abnormallities

GASTROINTESTINAL CANCER, 2004.