NEOPLASIA

H M Nadjib Dahlan Lubis

Bag Patologi FK USU Medan

NEOPLASIA
- Definitions - Nomenclature - Biology of tumor growth: benign & malignant neoplasms - Differentiation & Anaplasia - Rate of growth - Cancer stem cells & cancer cell liniages - Local invasion - Metastasis - Epidemiology - Molecular basis of cancer - Molecular basis of multistep carcinogenesis - Carcinogenic agents and their cellular interactions - Host defense against tumor- Tumor immunity - Clinical features of tumor

NEOPLASIA / ONKOGENESIS KANKER (Kajian molekular) H Muhd Nadjib Dahlan Lubis

Bag Patologi Anatomi

Fak Kedokteran USU/UISU & RS H Adam Malik Medan

Gen
- Pembawa sifat

- Diturunkan - Penyakit, bakat,

- cara fikir, tingkah laku

Sel
- Sitoplasma
- Inti Kromatin/kromosom DNA: makromol, rantai nukleotid Gen: - simpan & transfer (sepotong kecil DNA)

Onkogen Michael Bishop & Harold Varmus: Protoonkogen penompang pd Acut Transf Retrovirus Onkogen Protoonkogen berobah: - Strutur - Ekspresi V-onk : virus pengtransformasi Menyandi onkoprotein ~ protoonkogen

Produksi tak tgt pd: - factor ptb - signal2 luar lain

Gen pengatur

* Protoonkogen * Antionkogen (Supressor)

* Pengatur apoptosis
* Pemerbaik DNA rusak

Steps of cell proliferation

Growth Factor + Specific Receptor (cell membrane) Activation of Growth Factor activation of signal- transducing protein (inner leaflet of plasma memb) Transmission cytosol nucleus (via second messenger ) Induction & activation of nucl regulatory factors initiate DNA transcription Cell cycle cell division

Cell cycle
Go: quiscent: G1: longest, prepare for division R or Restriction point: attempt to complete the cycle S: replication of DNA G2 M: mitosis, separation of chromosomes & division Checkpoint: regulate the cycle negatively, at 3 stages - G1 S - G2 M - Within mitosis

Product Protein of Oncogens

: PDGF, TGF (EGF) : ret, c-erb B1, c-erb B2 (c-neu)

Growth Factor Growth Factor Receptors

Signal-Transduction Protein : c.ras, c-abl Nuclear transcription protein : myc, myb, jun, fos, rel Cyclin & Cyclin-Dependent Kinases

Mutations of genes that endode GF

- c-sis PDGF (astrocytoma, osteosarcoma) - ras TGF- EGF bFGF (melanoma, not normal melanocyte) bombesin like peptide (Small cell lung ca) - hst-1, int-2 FGF (gastroint, breast)

Growth Factor Receptor

- ret - EGF rec family - c-erb B1 - c-erb B2 (c-neu) - c-erb B3

Activation of GFR:

- Mutation
- Gene rearrangement - Over expression

Mutation - ret MEN type 2A & 2B & Familial med thy ca - c-fms CSF-1 (myeloid leukemia)

Overexpr - c-erb B1 EGF R - lung SCC - urinary bladder ca - g.i.t - astrocytoma

Amplification

- c-erb B2 (c-neu) = 2nd member of EGF rec family) - adenocarcinoma : breast, ovary, lung, sto, sal - c-erb B3 - breast cancer - c-erb B2 sensitive to small amount of GF more aggresive High level of c-erb B2 protein on breast cancer poor prog

Signal Transducing Proteins

- c-ras - c-abl ras inaktif (+ GDP) aktif (+GTP) (guanosine diphosphate) GTPase: aktif inaktif

GAP

: mengaktifkan GTPase (GTPase Activating Protein)

Signal-Tranducing Proteins
- In inner leaflet of plasma memb: - RAS - ABL - GF Inactive (RAS GDP) Active (RAS GTP) activates (down stream regulator of proliferation = RAF-MAP kinase mitogenic cascade) nucleus proliferation - GTPase hydrolyses GTP GDP - GTPase activating protein (GAP= brakes) GTPase - Mutant RAS can GAP, but GTPase activity fails to be augmented - Mutant RAS is trapped in its activated form - Mutation in RAS: - would be mimickened by mutation in GAP - respons to braking action of GAP

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- Disabling mutation of neurofibrin 1 (NF-1) = GAP familial neurofibromatosis type 1 - By point mutation, RAS gene active reveal 3 hot spot around codons 12, 13, & 61 - Amino acids coded by codons 12 & 13 occur in the binding pocket for GTP - Codon 61 for hydrolysis of GTP Convert RAS inactive

ABL
- is dampened by neg regulatory domains - Chr mye leuk, activity is unlease: ABL gene is translocated fuse with breakpoint cluster region (BCR) gene BCR-ABL hybrid RAS-RAF (has potent tyrosine kinase activity) - Therapy w. inhibitor of ABL kinase (STI 571 (Gleevec)) - N ABL localizes in nucleus apoptosis of cells suffers DNA
damage TP53 (BCR-ABL gene can not perform this function b. in cytopl

Cells with BCR-ABL fusion gen is dysregulated

- Inappropriate tyrosine kinase - growth autonomy - apoptosis is impaired

- STI 571: - inhibit growth by neutralizing t kinase activity - apoptosis by nuclear localization of ABL

Nuclear Transcription Factor - MYC, MYB, JUN, FOS, & REL oncogens in t. nucleus - MYC most common - Signal to divide quiescent cells MYC protein - MYC protein DNA transcription of CDK drive cell cell cycle - N: MYC level when cell cycle begin - >< oncogenic version of MYC gene overexpression Dysregulation of MYC gene: - t(8;14) translocation Burkitt lymphoma - amplified bre, col, lung

Cyclin & Cyclin-dependent Kinases

- Cyclin CDK CDK activated phosphorylate target prot - Signals cells D family of cyclins CDK4 & CDK6 active

- Checkpoint is guarded by pRb - CDK phosphorylation of pRb overcomes G1 S hurdle DNA synthetic phase - S G2 cyclin A CDK1 / CDK2 - Early G2: cyclin B CDK1 G2 to M

Karyotypic Changes in Tumors

- Genetic damage - activates oncogens - inactivates tumor suppressor genes - subtle (point mutation) - large enough

- Nonrandom structural abnormality - Balanced translocation - Deletion - Amplification - Whole chromosomes may be gained or lost

Balanced translocation

- most common, esp in hematopoietic neoplasms

- Philadelphia (Ph) chr in chronic myelogenous leukemia: reciprocal & balanced translocation between chr 22 & 9

- Ph chr (-) cases of chr mye leuk: BCR-ABL rearrangement

- 90% Burkitt: transloc between chr 8 & 14 - Follicular B-cell lymphoma: reciprocal transloc chr 14 & 18

Deletions

- more common in nonhematopoietic solid tumor - Retinoblastoma : deletion of chr 13q band 14 - colorectal cancer : deletion of 17p, 5q, dan 18q harbor 3 tumor suppressor genes - Small cell lung ca : deletion of 3p

Amplification

- 2 karyotypic manifestations
1. Homogenously staining regions on single chr Homogenous-staining region (HSR)

2. Double minutes: small paired fragment of chr

- Neuroblastoma : N-MYC - Breast cancer : HER-2

Li-Fraumeni syndrome - inherit 1 mutant p53 allele only 1 hit is needed to inactivate the second, normal allele - >< inherit mutant RB, spectrum of Li-F: varied - >< sporadic, Li-F: younger & multlple primary tumor

Function of p53 in DNA damage - cell cycle arrest - initiation of apoptosis Therapeutic implication

- Radiation & chemotherapy: 2 common modalities - DNA damage & subsequent apoptosis - Tumor retaining normal p53 respons >< tumor w. mutant ALL, terato-ca lung,colorect - Strategi aim: - N p53 activity - selectively killing cells defective in p53

Modulation of MDM2 activity

- modified adenevirus lyse cancer cell that lack p53

p14ARF fuction

- p53 = member of a multigene family - p73 cycle cell arrest, appoptosis - p63 p53 deficiency, compensate

Insensitivity to Growth-Inhibitory Signals

RB gen

- 60% of retinoblastomas are sporadic, remaining: familial

- Knudson (1974): two-hit hypothesis

- 2 hits (mutations) are required Both of the N alleles must be inactivated (2 hits) to

retinoblastoma

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Familial

- children inherit 1 defective copy of RB gens, the other: N - When N RB is lost as result of somatic mutation Ret blast - Required only a single somatic mutation : autosomal dominant

Sporadic

- both N RB alleles are lost

Terminology

- A cell heterozygous at RB locus is not malignant - Cancer develops when: Cell becomes homozygous for t. mutant allele or i.o.w. loses heterozygosity o.t. N RB gen

- Because neop transf is ass.w. loss of both of N copies of RB gene, supressor genrs is called recessive cancer gen

CDK inhibitors(CDKI)

2 families

1. Composed of 3 proteins: CDKN1A (p21), p27, p57

inhibit CDKs broadly 2. 4 members: p15, CDKN2A (p16), p18 & p 19

inhibit selectively cyclin D/CDK4 & cyslin D/CDK6

- Cyclin D overexpressed: bre, eso, liv, subset of lymphoma - Amplification of CDK4 gene melanoma, sarcoma, glioblas - Mutation on cyclin B, cyclin E certain mal neo, but much less frequent than cyclinD/CDK4

p53

- antiproliferative
- apoptosis

- Stress (anoxia, MYC, damaged DBA p53 respons

- N p53 (nonstressed): short half time (20) MDM2 - Stress (assault on DNA): p53 modification release it

from MDM2 half-time

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Protein Product of Tumor-Supressor Genes

- Rb Gene pRb - p53 gene p53 - BRCA-1 & BRCA-2 Genes - Mutation of BRCA-1 gene Ovarian, Prostate, Colon BRCA-2 Male Breast, Ovary, Pro, Pan, Lar

- Molecules that regulate Signal Tranduction - NF-1

- APC gene
- Cell Surface Receptors - TGF-B

- Cadherins
- Other: NF-2 gene, VHL, PTEN, WT-1

Mutation
Inheritance 1 mutant allele predisposes mal only 1 hit is needed to inactivate second (N) allele: Li-Fraumeni synd >< mutant Rb allele, mutant p53: varied >< sporadic, Li-F: younger

? : p53 acts as dimer

Mutant (inactive) p53 protein dimerizes wild-type (normal)

protein & inactive complex (Dominant-negative effect)

mutant-normal dimers Would not 100%: normal-normal dimers still form

Dominant-negatif effect of mutated p53 gene

ability of the protein to dimerize with an inactivate the

normal protein.

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Transduksi NF-1 (Neurofibromin) Transkripsi & siklus sel - Rb pd 13q14 : - Aktif = pRb: bind, seq E2F

- Inaktif = pRb-P G1 S - p53 pd 17p13.l Ca lun, Bre, Col Li-Fraumeni synd: Ca, Sa, La, Brain

Apoptosis
- 3 Huruf mulai b : bcl-x, bax, bag, bad - Ekspresi bcl-2 melindungi Lcy thd apop - p53 pengindus apoptosis - bax >< bcl-2

Pemerbaik DNA
- Manusia dlm lautan carsinogen DNA rusak - Msh-2 manusia pd khr 2 HNPCC

- UV Xer. Pig kanker kulit

- Bloom, Ataxia teleangiectasia, Fanconis an

Biology of Tumor Growth

Kinetics of Tumor Cells Growth - 10u cell, 30 population doubling 109 cells (1 gm): smallest clinically detectable mass) - only 10 further doubling cycle 1012 cells (1 kg): maximal size compatible with life) - Doubling time - Growth fraction: proportion of cells within t tumor population that are in t proliferative pool. - Early (submicr phase): vast of transformed cells are in proli ferative pool. - Grow: leave replicative pool non proliferative pool - By the time t tumor is detectable: most tumor cells are not in repl pool - Even in rapidly growing tumor, Gr. fra: 20%

Concept

- Rate of tumor growth depends on growth fraction & degree of imbalalance between cell production & cell loss

- leukemia & lymphoma, small cell ca of lung

Clinical Implication - High growth susceptible - Low growth resistant

Chemother:

Usahakan Go cycle, debulking

Latent Periode

Biology of Tumor Growth

H M Nadjib Dahlan Lubis Bag Patologi Anatomi Fak Kedokteran USU

Biology of Tumor Growth

Natural history of most malignant tumors

1. Transformation (mal change in t target cell)

2. Growth of transformed cells

3. Local invasion 4. Distant metastases

Formation of tumor by clonal descendants of transformed cell - doubling time intrinsic to t tumor cells - angiogenesis host responses tumor cells / products

Factors tumor growth

- Kinetic - Angiogenesis - Progression & Heterogeneity

Kinetic

- How long does it take clinically over tumor mass ? - original transformed cell (10 u) 30 pop doublings 109 cells ( 1 gm) : smallest clin detectable mass - only 10 further doubling 1012 cells (1 kg = max size

compatible with life)

By t time a solid tumor is clin detected, it has completed a

major portion of its life cycles major impediment in

treatment of cancer

?? Relate to kinetics

- Doubling time - Fraction of tumor cells in the replicative pool - Rate at which cells are shed and lost I t growing lesion

The dividing cells do not complete cell cycle more rapidly than N
equal / longer growth is not commonly associated w. shorte ning of cell cycle time.

Growth fraction = proportion of cells within t tumor pop that are in t proliferative pool

- By the time t tumor is detectable, most cells are not in t replicative pool - Even rapidly growing tumor: GF: 20% - Progressive growth & rate are determined by excess of cell production ove cell loss - High GF more rapid

Concept

- Rate of growth depends on: - GF - Degree of imbalance prod & loss - Leu, Lymphoma, Small cell ca: high GF rapid - Colon, breast : low GF slower

- GF susceptibility to chemotherapy Anticancer drugs act on cells that are in cell cycle tumor 5% of all cells in t replicating pool slow growing refractory to treatm Lymphoma: large pool of div cells melt away with chemoth

Strategy with low GF

1. Shift tumor cells from G0 into t cell cycle

debulking (surgery / radiation) = Combined modality treat

How long ? 1 transformed cell 109 = 90 days (30 pop doubling x cell cycle time of 3 days) After detectable, volume-doubling time (lung & colon) 2-3 months Range: 1 month (childhood cancer) to 1 year (salivary gland)

Cancer Stem Cells & Cancer Cell Lineages

- Progeny of a single cell clonal growth heterogenous population (clinically detectable). - Identifying cancer stem cell (initiate & sustain a tumor : difficult tumor initiating cell = (T-IC): - bre ca (2%) - mye leu (0.1-1%) - T-IC require BMI1 gene: repress t cell cycle inhib: - p161NK4a

- p14ARF
- Cancer stem cells are t initial targets for transformation - CSC = N, have low rate of replication implication for treatment: leave in place t cell capable of generating t tumor tumor can easely recur after treatment

Angiogenesis
- Tumor can not > 1-2 mm d. unless vascularized - Hypoxia p53 apoptosis - Neovasc 1. perfusion: nutrisi & O2 2. end insuline like GF, PDGF, GM-CSF, IL-1 - Tum ass ang fac: VEGF, bFGF sel tumor, inf cell (mac): infiltrate tumor - Anti ang fac: thrombospondin-1: - angiostatin, - endostatin, - vasculostatin - Wild type p53: antiangiogenetic fac - Tumor suppressor gene on 16p inhhibit angiogenesis - Therapy: ang gen inhibitor: endostatin

Mekanisme invasi & metastase

Millions of cells circulation , only a few metastases - tumor are heterogenous in respect to metastases - only certain subclones possess right combination of gene product to complete.

Cascade:
1. Invasi extra cellular matrix 2. Vascular dissemination & homing tumor cell

Invasion of Extracellular Matrix

- Detachement (loosening up) of tumor cells from each other E-cadherin (mol adh) + Catenin (cytosclet) - Attachment to matrix component Fibronectin, laminin, collagen, vitronectin - Degradation of extracell matrix Protease: serine, cystein, matrix metalloprotease (type IV coll) >< TIMP cathepsin D (Ca bre) -Migration of tumor cells - Tum cel derived motility fac - produk pemecahan matrix

Dissemination & Homing

* Dlm pembuluh: lekat homo/heterotypic: pla Lekat ke end bas mem: - adh: - itegrin, laminin rec - CD44 (T lym migrasi) * Tropism: - sel tumor mol adh, ligand pd target - target chemoattractant - unpermissive/unfavorable soil

Vasc dissemination & Homing of tumor cells

- Once in circ, tumor cells are vulnerable to destruction by na-

tural& adaptive imm. Defence.

- Tumor cells aggregate: - among tumor cells (homotypic) - + blood cells, particularly: platelets

- Adhesion moleculs: - integrin, laminin rec, proteolytic enz. - CD44 (T lcy) is used to migrate + hyaluronate (endothelial venules) CD44: - : met colonic cancer

Tumor cells leave the capillaries is related to

- anatomic loc of the primary tumor - natural pathways of drainage do not explain the distribution of metastasis : - prostatic ca bones - bronchogenic ca adrenal,brain - neuroblastoma liver, bones Organ Tropism - tumor cells adhesion mol whose ligands are on t end of target organ

- target organ chemoattractants recruit tumor cells

- target tissueis unpermissive environtment

Molecular Genetics of Metastases

- ? Oncogens / tumor spp genes elicit metastases - No single metastasis gene has been found - However, genes that encode E-caderin, TIMP are considered metastase suppressor genes - to identify: - substractive hybridization of cDNA nm23: - low metastatic potential - 10 x high met ability - highest in bre. tumor: 3/ < nodes

- 2 others: - KAI-1 (chr 11p11-2): suppress met in prostate ca

- KiSS-1 gene (chr 11): mal melanoma

Metastase : Molecular Genetic

* Adakah onkogen & tum supp gen Tidak ada single met. Gen ok sel-sel met memerlukan banyak sifat: - adh rec, - pbt collagenase, - motility fac. * E-cadherin: inh metalloproteinase: met sup gen - nm23 met - KAI-1, KiSS

EBV Epithel & B Lymphocyte EBV + EBV rec (CD21) episome in the nucleus Inf latent : replication (-), cells not killed immortalized >< HPV : EBV : - inactivation of tumor-suppressor genes (-) - viral genes dysregulate: - normal prol - survival signals Viral Genes - LMP-1 bcl-2 >< apoptosis activate growth promoting pathway (mimic activation via Bcell surface mol CD40) N: T cell derived signal (LMP-1 cell growth & cell survival)

- EBNA-2 cyclin D & src family activate transcription of LMP-1

Additional factors must also be involved in Burkitt

1. 2. 3. 4. EBV inf is not limited to geographic locales Burkitt is found EBV inf mononucleosis: self limited EBV genome in only 15-20% of Burkitt (outside Africa) Although EBV immortalizes B cells in vitro, these cell do not tumors when injected into imm.suppressed mice in vivo, there are differences in gene expression in EBV-transformed (but not tumorogenic) B-cell lines & Burkitt
f.i. Tumor cell do not viral-encoded memb prot (target of cytotoxic T cells)

- EBV: - one factor in multistep development - is controlled by imm response on cell membrane

Cofactors - Malaria favor proliferation of cells immortalized by EBV - B-cell do not exp surf ag (recognized by host T cell) relieving mutations: t(8;14) translocation c-myc activation EBNA-1 t(8;14) transloc c-myc activation Over exp of c-myc by itself is not sufficient for mal transformation Mutation N-ras oncogenes Emergence of monoclonal B-cell neoplasm Viral gene expression Ag: be recognized by cytotox T cells

Immunosuppressed patients: - HIV

- Organ transplant recipient
- EBV infected B cells polyclonal exp (lymphoblastoid)

>< B cells in Burkitt, B lbl in imm supr exp cell surf ag

(recognized by T cells)

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Cachexia

* ok nutrit. demand tumor cancer ~ fetus post partum cach (-)

* Anorexia
* x ok Intake - cal. exp ^, BMR ^ * Fat lost = muscle

* TNF-, IL-1, IFN-

Utility of Immunohistochemistry

- Categorization of undifferentiated malignant tumors

- intermediate filaments - Categorization of leukemias and lymphomas - Determination of site of origin of metastatic tumors - prostate specific Ag & thyroglobulin - Detection of molecules that have prognostic or therapeutic significance.

- ERBB2: overexpression poor prognosis

Intermediate Filaments Epithelial Mesenchymal Muscle Glial-astrocytes Neoron (most) Embryonic Keratin Vimentin Desmin Glial Fibrillary Acidic Protein Neurofilament proteins No intermediate filament

Most normal adult cells : only one type of intermediate filament except: - some muscle cells: vimentin & desmin - epithelial cell of salivary & kidney : vimentin & keratin

Tumor Markers - CEA - AFP - PSA - PSMA: prostates ccr

- HCG : testicular tumor

- CA 125 : ovarian tumors - Mutated APC, p53, RAS in stool : colorectal ca. - Mutated p53 & hypermethylated gen in sputum : head & neck - Mutated p53 in urine : bladder ccr

What do protein do ? - Catalyse : enzymes - Signaling: receptor in cell membrane + ligands (extra cell) - Transport & storage - Structure & movement: - collagen (skin, bone,conn tis)

- keratin (hair)
- protein in cytoskleton - Nutrition: casein, ovalbumin - Immunity - Regulation: transcription factor + DNA

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P53 Tidak mengatur sel normal Rem bila DNA rusak Chem. Ion timbun (inti)

menahan G1 HPV, HBV, EBV Mengikat p53 Menghilangkan protektif

Aktifnya onkogen
Struktur - Mutasi noktah - Translokasi khromosom Pengaturan ekspresi - Amplifikasi

Mutasi noktah
ras Menghidrolisa GTP . 90% Adenoca panc, 50% kolon, tiroid

Translokasi khromosom
Penyakit Burkitt La B fol Leu Mcy Khr Gen C-myc Bcl-2 C-abl Khromosom 8q24 18q21 9 Pindah ke 14q band 32 14 22

Amplifikasi - Reduplikasi & Amplifikasi sekuens DNA beratus kopi protoonkogen - Deteksi : - Molekular - Sitogenetik - Imunohistokimia - Bentuk: - Bintik ganda - Homogenous staining reaction - Gen: - N-myc : Neuroblastoma - c-erb B-2 : kanker payudara