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N E O P L A S I A

H M Nadjib Dahlan LubisBag Patologi FK USU Medan


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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


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NEOPLASIA / ONKOGENESIS KANKER (Kajian molekular)

H Muhd Nadjib Dahlan Lubis

Bag Patologi Anatomi

Fak Kedokteran USU/UISU & RS H Adam Malik Medan


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G e n

- Pembawa sifat

- Diturunkan - Penyakit, bakat,

- cara fikir, tingkah laku

S e l - Sitoplasma

- Inti Kromatin/kromosomDNA: makromol, rantai nukleotidGen: - simpan & transfer

(sepotong kecil DNA)


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Onkogen

Michael Bishop & Harold Varmus:Protoonkogen penompang pd Acut Transf Retrovirus

Onkogen

Protoonkogen berobah: - Strutur- Ekspresi

V-onk : virus pengtransformasiMenyandi onkoprotein ~ protoonkogen

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


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Gen pengatur

* Protoonkogen

Antionkogen (Supressor)

Pengatur apoptosis

Pemerbaik DNA rusak


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Steps of cell proliferation

Growth Factor + Specific Receptor (cell membrane)

Activation of Growth Factor activation of signal- trans-ducing 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


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Cell cycle

Go: quiscent:G1: longest, prepare for division

R or Restriction point: attempt to complete the cycle

S: replication of DNAG2M: mitosis, separation of chromosomes & division

Checkpoint : regulate the cycle negatively, at 3 stages- G1 S- G2 M- Within mitosis


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Product Protein of Oncogens

Growth Factor : PDGF, TGF (EGF)

Growth Factor Receptors : ret, c-erb B1, c-erb B2 (c-neu)

Signal-Transduction Protein : c.ras, c-abl

Nuclear transcription protein : myc, myb, jun, fos, rel

Cyclin & Cyclin-Dependent Kinases


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Mutations of genes that endode GF

- c-sis PDGF (astrocytoma, osteosarcoma)- ras TGF- EGF

- hst-1, int-2 FGF (gastroint, breast)

bFGF (melanoma, not normal melanocyte)

bombesin like peptide (Small cell lung ca)


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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


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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


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Amplification

- c-erb B2 (c-neu) = 2 nd 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


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Signal Transducing Proteins

- c-ras

- c-abl

ras inaktif (+ GDP) aktif (+GTP)

(guanosine diphosphate)

GTPase: aktif inaktif

GAP : mengaktifkan GTPase (GTPase Activating Protein)


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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 aug-mented

- 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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K0727


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K0728


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K0729


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K0730


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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


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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 DNAdamage TP53 (BCR-ABL gene can not perform this function b. in cytopl


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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


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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 cellcell 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


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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


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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


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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


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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


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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


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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


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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


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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


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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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K70736


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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


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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


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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 19inhibit 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


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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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22

HC

F

P t i P d t f T S G


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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


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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


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? : 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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K0738


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K0739


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K0740


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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


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Apoptosis

- 3 Huruf mulai b : bcl -x, bax, bag, bad

-

Ekspresi bcl-2 melindungi Lcy thd apop

- p53 pengindus apoptosis

- bax >< bcl-2


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Pemerbaik DNA

- Manusia dlm lautan carsinogen DNA rusak

- Msh-2 manusia pd khr 2 HNPCC

- UV Xer. Pig kanker kulit

- Bloom, Ataxia teleangiectasia, Fanconi s an


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Kinetics of Tumor Cells Growth

- 10u cell, 30 population doubling 10 9 cells (1 gm):smallest clinically detectable mass)

- only 10 further doubling cycle 10 12 cells (1 kg):maximal size compatible with life)

- Doubling time

- Growth fraction: proportion of cells within t tumor populationthat are in t proliferative pool.

- Early (submicr phase): vast of transformed cells are in proliferative pool.

- Grow: leave replicative pool non proliferative pool- By the time t tumor is detectable: most tumor cells are notin repl pool

- Even in rapidly growing tumor, Gr. fra: 20%

Concept


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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

Chemother: - High growth susceptible

- Low growth resistant

Usahakan Go cycle, debulking

Latent Periode


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Biology of Tumor Growth

H M Nadjib Dahlan Lubis

Bag Patologi Anatomi Fak Kedokteran USU


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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


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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


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?? 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


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- By the time t tumor is detectable, most cells are not in t repli-

cative pool

- Even rapidly growing tumor: GF: 20%

- Progressive growth & rate are determined by excess of cell

production ove cell loss

- High GF more rapid

C t


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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


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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 10 9

= 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)


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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


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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 matrix2. Vascular dissemination & homing tumor cell


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Invasion of Extracellular Matrix

- Detachement (loosening up) of tumor cells from each otherE-cadherin (mol adh) + Catenin (cytosclet)

- Attachment to matrix componentFibronectin, laminin, collagen, vitronectin

- Degradation of extracell matrixProtease: serine, cystein, matrix metalloprotease(type IV coll)

>< TIMPcathepsin D (Ca bre)

-Migration of tumor cells- Tum cel derived motility fac- produk pemecahan matrix


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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


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Tumor cells leave the capillaries is related to


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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


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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


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EBV Epithel & B Lymphocyte

EBV + EBV rec (CD21) episome in the nucleusInf 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 >< apoptosisactivate growth promoting pathway (mimic activation via B-cell surface mol CD40) N: T cell derived signal

(LMP-1 cell growth & cell survival)

- EBNA-2 cyclin D & src familyactivate transcription of LMP-1

Additi l f t t l b i l d i B kitt


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Additional factors must also be involved in Burkitt

1. EBV inf is not limited to geographic locales Burkitt is found2. EBV inf mononucleosis: self limited3. EBV genome in only 15-20% of Burkitt (outside Africa)4. Although EBV immortalizes B cells in vitro, these cell

do not tumors when injected into imm.suppressed micein vivo, there are differences in gene expression inEBV-transformed (but not tumorogenic) B-cell lines & Burkittf.i. Tumor cell do not viral-encoded memb prot (target ofcytotoxic T cells)

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

Cofactors


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- Malaria favor proliferation of cells immortalized by EBV

- B-cell do not exp surf ag (recognized by host T cell) relievingmutations: 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 neoplasmViral gene expression Ag: be recognized by cytotox T cells


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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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K0750


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K0751


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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-


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Intermediate Filaments


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Most normal adult cells : only one type of intermediate filament

except:

- some muscle cells: vimentin & desmin- epithelial cell of salivary & kidney : vimentin & keratin

Epithelial

MesenchymalMuscleGlial-astrocytesNeoron (most)Embryonic

Keratin

VimentinDesminGlial Fibrillary Acidic ProteinNeurofilament proteinsNo intermediate filament


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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


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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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K0745


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Aktifnya onkogen

Struktur - Mutasi noktah- Translokasi khromosom

Pengaturan ekspresi - Amplifikasi


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Mutasi noktah

r a s Menghidrolisa GTP . 90% Adenoca panc, 50% kolon, tiroid


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Translokasi khromosomPenyakit

Burkitt

La B folLeu Mcy Khr

Gen

C-myc

Bcl-2C-abl

Khromosom

8q24

18q219

Pindah ke

14q band 32

1422


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Amplifikasi

- Reduplikasi & Amplifikasi sekuens DNAberatus kopi protoonkogen

- Deteksi : - Molekular- Sitogenetik

- Imunohistokimia

- Bentuk: - Bintik ganda- Homogenous staining reaction

- Gen: - N-myc : Neuroblastoma- c-erb B-2 : kanker payudara


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