GENETIC CHANGES IN CANCER AND CANCER TARGETED THERAPY

Syarifuddin Wahid Department of Pathology Faculty of Medicine, Hasanuddin Univ.

Environmental agents (Chemicals, radiation,viruses)

Normal Cell
DNA repair APOPTOSIS DNA damage FAILLURE of DNA repair 1

FAILLURE APOPTOSIS PERMANENT DNA DAMAGE (MUTATION) GENETIC AND PHENOTYPIC CHANGES

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 of DNA Repair Genes DNA mismatch repair Nucleotid excision repair. BRCA1 & BRCA2 Cancer Risk

HNPCC Skin Cancer Breast Ca.

Mutation in the genome of Somatic cells

2 Activation of growth-

promoting oncogenes

Inactivation of gene regulate apoptosis

Inactivation of Tumor suppressor genes Decreased apoptosis

Unregulated cell proliferation

Clonal Expansion

Activation of growthpromoting oncogenes

MUTATION (ONE ALLEL)
INACTIVE PROTOONCOGENE ACTIVE ONCOGENE (CELLULAR ONCOGENE)

Oncogenes
NEU/HER2 H-RAS MYC RET

Lesion
Amplification Point Mutation Translocation Rearranggement

Cancer
Breast,ovary,stomach (Ad Ca) Colon, lung, pancreas Burkitts Lymphoma Thyroid (Ca)

Daerah promoter VEGF manusia

POINT MUTATION ON VEGF GENE -460T>C

+405C>G

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

Normal growth

Viral Gene
Normal production of growth factor

Protooncogene
Carcinogen (chemical, viral and radiation)
Mutation

DNA

Cellular Oncogene Viral Oncogene

Abnodrmal production of growth factor Devective immune survailance

Abnormal Growth NEOPLASM

Inactivation of Tumor suppressor genes and gene regulate apoptosis

MUTATION (BOTH ALLELS , LOH)

Active Genes
RB TP53

DELETION

Inactive Genes
Cancer
Retinoblastoma Sarcoma, lymphoma, Adeno Ca Breast Colorectal (HNPCC)

TS Genes Function
Cell cycle Cell cycle,apoptosis (Guardian of the genome) BRCA1 DNA repair MSH2,MLH1 Mismatch repair

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)

Clonal Expansion

5 angiogenesis
Additional mutation 6

7 Escape from immunity

Tumor Progression Invasion and 8 metastasis MALIGNANT NEOPLASM

Transformed Cells Primary Tumor

Clonal expantion, growth, Diversification, angiogenesis Metastatic subclone INVATION

INTRAVASATION Ecape from imunity Tumor Cell embolus

EXTRAVASATION Metastatic deposit Angiogenesis GROWTH

Normal cell

transformation

Normal Cell

Carcinogen Induced changes

Single Tumor cell

progression

Single tumor cell Tumor Cell variants

30 doublings

1 gm (109cells)

Proliferation of genetically Unstable cells Tumor cells variants heterogeneity

10 doublings

Nonantigenic
Invasive Metastatic

1 kg (1012 cells) Metastases

Requiring fewer growth factor

Metastasis

PRL3 overexpression

Carcinoma

TP53 gene loss

MORPHOLOGIC CHANGES

Late Stage adenoma

Intermediate Stage adenoma

DCC gene loss

RAS gene mutation

Early Stage DNA loses methyl groups adenoma Increased Cell APC gene loss growth Normal Colon cell

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

Normal
HER2 reseptor protein HER2 mRNA

Amplifikasi/Overekspresi

2 1 4
Cytoplasm HER2 DNA Nukleus

Cytoplasmic membrane

1= 2= 3= 4=

jumlah copy gen transkripsi mRNA ekspresi reseptor protein permukaan sel pelepasan reseptor dari bgn extraselluler

VEGF

B CELL DEVELOPMENT
H chain
H & L chain

IgM,IgD

Mature B Stem Cell Pro-B Pre-B Bone Marrow No antigen dependence Activated B Immature B Peripheral lymphoid organ or tissue Self antigen Foreign antigen

CD43+

RAG-1 & RAG-2 expression CD43+ B220 IgM(low) CD19+ CD43+ CD43CD10+ CD20 CD20 CD20 CD20 CD20 CD20

IgM(high) CD20 CD20

IgM(high) CD20 CD20

Tipe targeted therapy

1. Fokus pada molekul yang merupakan 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

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).
2.

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.

Antigen-X

MONOCLONAL ANTIBODY
Anti-X antibodyproducing spleen cells FUSION

Mouse myeloma Cell line

In vitro selection in HAT medium

Clone

cells

Screen supernatant for presence of anti-X antibody Hybridomas producing Monoclonal anti-X antibody

Antigen-X

MONOCLONAL ANTIBODY
Anti-X antibodyproducing spleen cells

FUSION

Mouse myeloma Cell line

Genetic engineering

In vitro selection in HAT medium

Clone cells

MONOCLONAL ANYIBODY-LIKE MOLECULE

Screen supernatant for presence of anti-X antibody Hybridomas producing Monoclonal anti-X antibody

Parent mouse antibody

Genetic engineering
HUMAN/MOUSE CHIMERIC MONOCLONAL ANTIBODY (HUMANIZED MAB)

Idiotype (cdr)

HUMAN/MOUSE CHIMERIC MONOCLONAL ANTIBODY (HUMANIZED MAB)

Antigen Binding Site

Murine Variable region (biru)

H-Chain Fab L-Chain

Human constant region

papain
Constan region (merah)

-s-sHuman constant Fc region

Fc

Gambar 12. Struktur humanized monoclonal antibody.

HUMAN/MOUSE CHIMERIC MONOCLONAL ANTIBODY (HUMANIZED MAB)

Variable region a.amino tikus (murin) (<10%) Constant region a.amino manusia (>90%)

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.

Mismatch repair abnormallities DNA mutation DNA methylation abnormalities

K-ras mutation

DC LOH LOH of other Genes on 18q

P53 mutation And/or LOH

Hyperproliperative epithelium

Adenoma of increasing size and degree of dysplasia

Invasive carcinoma

GASTROINTESTINAL CANCER, 2004.