口腔病理
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口腔病理. Carcinogenesis 癌 化. 陳玉昆副教授 : 高雄醫學大學 口腔病理科 07-3121101~2755 [email protected] References. Gibbs WW. Untangling the roots of cancer. Sci Am 2003;289:56-65. What you need to know about cancer. Sci Am 1996 ;289:28-119.

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口腔病理

Carcinogenesis

癌 化

陳玉昆副教授: 高雄醫學大學 口腔病理科

07-3121101~2755

[email protected]


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References

  • Gibbs WW. Untangling the roots of cancer. Sci Am 2003;289:56-65.

  • What you need to know about cancer. Sci Am 1996 ;289:28-119.

  • Braakhuis BJM et al. A genetic progression model of oral cancer: current evidence and clinical implications. J Oral Pathol Med 2004;33:317-22.

  • Braakhuis BJM et al. A Genetic explanation of slaughter’s concept of field cancerization: evidence and clinical implications. Cancer Res 2003;63:1727-30.

  • Loktionov A. Common gene polymorphisms, cancer progression and prognosis. Cancer Letters 2004;208 :1-33.

  • Kaohsiung Medical University, Oral Pathology Department.

  • Huang AH et al. Isolation and characterization of normal hamster buccal pouch stem/stromal cells – a potential oral cancer stem/stem-like cell model. Oral Oncol 2009;45: e189-e195.

  • Umezawa & Gorham. Dueling models in head and neck tumor formation. Lab Investig 2010; 90:1546-8.

  • Spillane JB, Henderson MA. Cancer stem cells: a review. ANZ J Surg 2007;77:464-8.

  • Zhou ZT, Jiang WW. Cancer stem cell model in oral squamous cell carcinoma. Curr Stem Cell Res Ther 2008;3:17–20.

  • Harper LJ et al. Stem cell patterns in cell lines derived from head and neck squamous cell carcinoma. J Oral Pathol Med 2007;36:594-603.

  • Lim YC et al. Cancer stem cell traits in squamospheres derived from primary head and neck squamous cell carcinomas. Oral Oncol 2011;47:83-91.


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Carcinogenesis(癌化)

綱 要

Field cancerization

癌化的標準理論

5

3

4

四種癌化理論

2

Stages of carcinogenesis

1

How cancer arise - Molecular approach


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(1) How Cancer Arises - Molecular Approach

Stochastic Clonal Evolution Model

Interaction between tumor cells

and stromal cells

Stochastic clonal expansion

Tumor cell

In this model, clonal variants, including stromal cells derived from tumor cells, generate a microenvironment (niche) for tumor cells, and support tumor progression after tumor cells undergo clonal evolution.

Ref. 8


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Mutation Only

at the Stem Cell

Mutation

Stem Cell

Tumor

The cancer stem cell replicates forming an exact copy of itself as well as a continuous supply of heterogeneous tumor cells

Normal Stem Cell

Asymmetrical Division

Stem Cell

EarlyProgenitor

Late Progenitor

Definitive Tissue Line

Stem cells create an exact copy of themselves and an EP cell when they divide. The EP cell then progresses to a late progenitor cell and then to the definitive cell line

Ref. 9


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Mutation Only

at the Stem Cell

Stem Cell

Mutation

Tumor

(b) Mutation only at the stem cell or progenitor cell level. The cancer stem cell replicates forming an exact copy of itself & a continuous supply of heterogeneous tumor cells

Traditional Model of

Tumor Formation

Mature

Definitive

Tissue

Cell

Mutation

Mutation

Mutation

Tumor

Tumor

(a) Traditional model of tumor formation. A series of mutations affect a mature cell, causing it to become malignant. Any cell has the potential to form a tumor

Ref. 9


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Cancer Stem Cell Model (1)

mutation

Self-

renewing

cancer stem cell

mutation

Cancer cell

Self-

renewing

stem cell

Progenitor

cell

Mature cell

Mutation only at the stem cell or progenitor cell level

Ref. 9


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Mutation

Stem Cell

Tumor from an early stem cell

Heterogeneous cancer

Increased metastatic potential

Tumor

Mutation

Early Progenitor

Tumor

Mutation

Late Progenitor

Tumor from a late progenitor cell

Homogenous cancer

Less metastatic potential

Tumor

Definitive Tissue

Line

Cancer Stem Cell Model (2)

In the stem cell model, only the stem cells or their progenitor cells have the ability to form tumors. Tumor characteristics vary depending on which cell undergoes the malignant transformation

Ref. 9


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Cancer Stem Cell Model (3)

  • In hypoxia

    (e.g. within niche)

(b) In increased O2

(e.g. outside niche)

Stem cell in quiescence

Progenitor

or differentiated cell

Ref. 9

Self-renewing stem cell

(normal or cancer)

Stem cells (normal or cancer) reside in a hypoxic niche where self renewal and differentiation activity is balanced. With an increase in oxygen levels, proliferation becomes a dominant feature mediated by an increase in p38 MAPK and p16ink4a. This transiently leads to the expansion of the progenitors, which results in a long-term decrease in the stem cell pool and its eventual exhaustion.

Proliferation Stem cell depletion Exhaustion


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Comparison of Somatic and Cancer Stem Cells

Ref. 9


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Stem cell - Oral Epithelia

  • According to the progression model, the development of most of OSCC takes months or years.

  • As normal human oral epithelia have a rate of renewal estimated to be about 14-24 days, most epithelial cells do not exist long enough to accumulate the genetic changes necessary for the development of an OSCC.

  • The hierarchical stem cell structure present in human oral epithelia indicates that stem cells are the only long-time residents of oral epithelia and, consequently, the only cells able toaccumulate the necessary number of genetic changes for malignancy to develop


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A Schematic Diagram Showing Sites of Origins of Putative CSCs in OSCC

Epithelium

CSC might come from:1. Epithelial SC/progenitor within basal layer with genetic

alterations2. Muscle-derived SCs3. Fibroblast-derived SCs4. Vessel wall-derived SCs5. Blood-derived SCs6. Adipose derived SCs.

Connective tissue

Ref. 10


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Putative Cell Surface Markers of Presumptive CSC

Tumor Type

Surface Markers

SP-C+CCA+

Ref. 10


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Frequencies of CSCs in Various Human Cancers

Human cancer

Recipient mice

Cancer stem cell frequency (%)

Ref. 10


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A minority population of CD44+ cancer cells (<3%/<10% of the cells in head and neck SCC cell line), but not the CD44- cancer cells, generate new tumors in vivo

CD44+CD24- Lineage negative

Tumor formed

CD44+CD24-

CD44+CD24-

New tumor formed

Ref. 10


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Potential Mechanisms of CSC Formation

MUTATION

A

Differentiated cells

Stem/progenitor cells

Progenitors

Self renewal

Self renewal

CSC

(A) Mutation.The cancer stem cells might appear after mutations in specific stem cells or early stem cells progenitors. It is also possible that CSC can be derived from differentiated cells.

Ref. 10


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Potential Mechanisms of CSC Formation

MULTIPLE GENETIC HITS

B

Stem/progenitor cells

CSC

(B) Multiple genetic hits. Progressive genetic alterations drive the transformation of stem/progenitor cells into CSC.

Ref. 10


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MULTISTEP DEDIFFERENTIATION

C

CSC

Cancer cell

Potential Mechanisms of CSC Formation

(C) Multistep de-differentiation. Multistep dedifferentiation of cancer cells might give rise to CSC.

Ref. 10


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Potential Mechanisms of CSC Formation

D

FUSION

CSC

Cancer cell

Stem/progenitor cells

(D) Cell fusion. Cell fusion between cancer cells and stem/progenitor cells might induce CSC.

Ref. 10


Dmba induced hamster buccal pouch model

Carcinogen: DMBA

14-wk

Normal

DMBA-Induced Hamster Buccal Pouch Model

  • Hamster buccal-pouch mucosa provides one of the most widely-accepted experimental models for oral carcinogenesis. (Gimenez-Conti & Slaga 1993)

Ref. 6


Dmba induced hamster buccal pouch model1

DMBA-Induced Hamster Buccal Pouch Model

  • Despite anatomical and histological differences between (hamster) pouch mucosa and human buccal tissue, experimental carcinogenesis protocols for the former induce premalignant changes and carcinomas that are similar to the development of premalignancy and malignancy in human oral mucosa.(Morris 1961)

AnimalStudy

HumanStudy

Ref. 6


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Isolation and Characterization of Stem Cells from Normal Hamster Buccal Pouch (HBPSC)

A

B

Normal hamster buccal pouch tissues revealed no obvious grossly (A; inset) and histological (B, Hematoxylin & eosin stain, 200) changes.

Ref. 7


Minimal criteria of stem cell capacity

Minimal Criteria of Stem Cell Capacity

  • Self-renewal

    ---Colony forming unit (CFU)

    ---Proliferation

    One or more lineages differentiation

    ---Adipogenic differentiation

    ---Osteogenic differentiation

    ---Chondrogenic differentiation

    ---Neurogenic differentiation


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HBPSCs obtained from the normal hamster buccal pouch tissues were spindle-shaped in morphology (200).

Ref. 7


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HBPSCs obtained from the normal hamster buccal pouch tissues were able to form colonies, stained with crystal violet (A; B, 100).

A

B

Ref. 7


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Cytoplasmic keratin (A, 200) and vimentin (B, 200) stainings were noted for the HBPSCs obtained from the normal hamster buccal pouch tissues.

A

B

Ref. 7


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Proliferation rates for the HBPSCs obtained from the three normal hamster buccal pouch tissues (p: passage).

Proliferation rate (# of folds)

Pouch 3

Pouch 2

Ref. 7


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(A) HBPSCs obtained from the normal hamster buccal pouch tissues were able to differentiate towards adipogenic lineage (×200). (B) Expression of PPARγ mRNA (401-bp) upon RT-PCR also indicates adipogenic lineage of HBPSCs obtained from normal hamster buccal pouch tissues; GAPDH (135-bp) was the positive control; H2O was the negative control (N); M: molecular weight marker.

M

N

GAPDH

PPAR

bp

400

350

300

250

200

150

100

A

50

B

Ref. 7


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HBPSCs obtained from the normal hamster buccal pouch tissues were able to differentiate towards chondrogenic lineage (×200); inset: a yellowish chondroid pellet (~3mm in diameter).

HBPSCs obtained from the normal hamster buccal pouch tissues were able to differentiate towards osteogenic lineage (×200).

Ref. 7


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HBPSCs obtained from the normal hamster buccal pouch tissues expressed the differentiation markers (Osteonectin: 323-bp & Nestin: 416-bp) and stem cell markers (Nanog: 364-bp, Rex-1: 232-bp & Oct-4: 717-bp) upon RT-PCR. GAPDH (135-bp) was the positive control; H2O was the negative control (N); M: molecular weight marker.

Osteonectin

GAPDH

Nanog

Nestin

Rex-1

Oct-4

M

N

bp

700

600

500

400

300

200

100

Ref. 7


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100

85.8

% of Max

100

% of Max

0.9

CD 90

CD14

HBPSCs obtained from the normal hamster buccal pouch tissues showed high expression for surface markers: CD29, CD90, and CD105 but very low expression for CD14, CD34, and CD45 (Black/blue line: isotype control, Red line: marker of interest; Max: maximum).

100

100

100

% of Max

% of Max

51.3

93.6

CD 29

CD 105

100

100

% of Max

% of Max

1.5

1.7

CD 34

CD 45

Ref. 7


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DMBA-Induced Hamster Buccal Pouch Model

Isolation of normal HBPSC, we may follow in vitro the sequential changes of the normal HBPSCs during multistep oral carcinogenesis or the alternations of these cells upon irradiation treatment and/or chemotherapy. Hence, the isolated normal HBPSCs, would provide a potential avenue for the future study of CSCs of buccal SCCs.


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Comparison of Morphology Between Our Isolated Cells & Literature Results

Our isolated cells from DMBA-induced cancer pouch tissue

squamospheres

squamospheres

A colony with holoclone characteristics of circular outline and tightly packed cobblestone’ cells (h) is surrounded by cells with a spaced and fusiform paraclone morphology (p). A small colony (m) perhaps corresponds to a meroclone.

Refs. 7, 11


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Hallmarks of CSCs (1)

Self-renewal, stem cell marker expression, aberrant differentiation, and tumor-initiating potentialOSCC-driven squamospheres demonstrated:

  • A number of stem cell markers, such as CK5, OCT4,

    SOX2, nestin, and CD44, Bmi-1, CD133, ALDH1

    (2) Single-dissociated squamosphere cells were able to form

    new squamospheres within 1 week of reseeding

    (3) Serum treatment led HNSCC-driven squamospheres to

    be non-tumorigenic differentiated cancer cells

    (4) Injection of as few as 100 undifferentiated squamosphere

    cells in nude mice gave rise to tumor formation

CSCs is known to be significantly resistant to various chemotherapeutic agents (cisplatin, 5-fluorouracil (FU), paclitaxel, and doxetaxel)- side population cells


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Hallmarks of CSCs (2)

Ref. 12


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(1)小結

1. In stochastic model, clonal variants, including stromal cells derived from tumor cells, generate a microenvironment for tumor cells, and support tumor progression after tumor cells undergo clonal evolution

請注意以下的重點提要

2. CSCs may originate from normalsomatic stem cells, it has been estimated that 3 to 6 genetic events are required totransform a normal human cell into a cancercell

3. Accumulated evidences have identified that CSCs in SCCs of head and neck region including oral cavity function in initiation,maintenance,growth, and metastasisof tumors

Cancer development:

Stochastic clonal evolution model

VS Cancer stem cells model


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Gentically

altered cell

Hyperlasia

Dysplasia

(2)Stages of Carcinogenesis

Tumor development

occurs in stages

Oral potentially malignant disorders (OPMD)

Leukoplakia, Erythroplakia, Oral submucous fibrosis, Verrucous hyperplasia, Erosive lichen planus

Genetically altered cell (CSC):initiated cell (起始細胞)

Hyperplasia

Dysplasia

基底層完整

基底層完整

Ref. 1


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Invasive cancer

In situ cancer

Blood vessel/

lymphatic vessel

How Cancer Spreads

Ref. 1


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Primary

tumor

How Cancer Spreads

Normal

epithelial cell

Basement membrane

Invasive tumor cell

Blood vessel/

lymphatic channel

Ref. 1


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Secondary tumor site

Endothelial/lymphatic

lining

Basement membrane

Tumor cell

adhering

to capillary

Metastatic cell

in circulation

How Cancer Spreads

Ref. 1


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(2)Further look on stages of carcinogenesis

Initiation Phase (Early)

去毒

Ref. 5


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Initiation Phase (Late)

Ref. 5


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Promotion Phase (Early)

Mutant clone establishment & appearance

of phenotypically transformed cells

Ref. 5


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Promotion Phase (Late)

Establishment of phenotypically

transformed cell population (dysplasia)

Ref. 5


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Progression Phase (Early)

Malignisation

Ref. 5


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Progression Phase (Middle)

Microinvasion

Ref. 5


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Progression Phase (Late)

Advanced invasion and metastasis

Chemotherapy

Ref. 5


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Initiation (early, late)

Genetically altered cell (CSC)

Progression (late)

Invasive cancer

Progression (middle)

Microinvasion

Promotion (early)

Hyperplasia

Promotion (late)

Dysplaisa

Progression (early)

In situ cancer

Progression (late)

Metastasis

(2)小結

癌症形成是階段性的vs正常細胞有自衛能力

請注意以下的重點提要

Tumor developmentoccurs instages

Normal cellhas self-defense


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(3) 癌化的標準理論

Normal

Cell Cycle

Beginning

of cycle

Cell

divides

(mitosis)

Cell enlarges

and makes

new proteins

Cell prepares

to divide

Cell rests

G1 arrest

崗 哨

Restriction point: cell

decides whether

to commit itself to

the complete cycle

Cell

replicates

as DNA

Ref. 2


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Inhibitory

pathways

Stimulatory

pathways

Normal Cell

Inhibitory

abnormality

Stimulatory

abnormality

標準理論

致癌基因Oncogene

抑癌基因Tumor suppressor gene

Ref. 2


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油門全開

剎車失靈

下 坡

Cell Cycle

下 坡

下 坡

Aberrant cell cycle —Accelerated car downslope without brake

失 控

Activation of

oncogene

失 控

Inactivation of

tumor suppressor gene

Ref. 2


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Oncogene (1)

Ref. 2


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Oncogene (2)

Ref. 2


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TumorSuppressorGene (1)

Ref. 2


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TumorSuppressorGene (2)

Ref. 2


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基因突變地圖

Ref. 2

在各種癌症中發現超過百種以上的突變基因

癌化的標準理論:

Cell cycle中,正常促進細胞形成基因o過度活化 ,變成致癌基因;而抑制細胞形成基因o發生突變,失去功能X,成為抑癌基因

A Subway Map for Cancer Pathways


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(3)小結

癌化理論 → 標準教條:

細胞循環中,原來正常的腫瘤致癌基因與抑癌基因發生突變而失控;

造成致癌基因過度活化及抑癌基因失去功能

請注意以下的重點提要

Tumor developmentoccurs dueto

formations ofoncogene &tumor suppressor gene


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(4) 癌化的四個理論

標準理論:癌症相關基因被致癌物影響而發生突變,無法製造腫瘤抑制蛋白,並活化致癌蛋白,導致產生癌症

Ref. 2


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修正理論:在癌化前期的細胞基因組當中,累積的隨機突變有顯著的增加,終於影響到癌症相關基因

Ref. 2


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早期不穩定理論:認為細胞分裂的主控基因受致癌物質影響而關閉,造成子代細胞染色體數目異常

Ref. 2

早期不穩定理論

其餘兩個理論專注

在非整倍體所扮演的

角色,也就是染色體

上大規模的變異


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全盤非整倍體理論:非整倍體細胞的基因組非常不穩定,使得癌症基因極易發生突變而形成腫瘤

Ref. 2


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癌症是一種基因的疾病然而癌症的複雜情況,卻不能用簡單的「基因突變」來描述。

最近理論認為,染色體的異常可能才是細胞邁向癌症之路的第一步。

隨染色體起舞

Ref. 2


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Normal & Cancer Chromosomes

Ref. 2

正常

癌 症


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(4)小結

請注意以下的重點提要

癌化的四個理論:(1)致癌基因、抑癌基因;(2)修 正 教 條;(3)早期不穩定理論;(4)全盤非整倍體理論


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Epithelium

Basal layer with

stem cells

Connective tissue

Patch phase

Field

Precursor lesions

develop within field

Carcinoma excised,

field and precursor

lesion remains

(5) Field Cancerization (1)

Ref. 3

Genetic altered

Expanding

field phase

Precursor lesions

becomes carcinoma

and new precursor

becomes develop

Second field tumor

develops from

precursor lesion


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Histological Proof

Chromosomal

Proof

Field

Carcinoma

Normal

Patch

17p

11q

3p, 9p, 8p, 18q

centromere

p arm

q arm

Ref. 4

Field Cancerization (2)


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(5)小結

瞭解Field cancerization的形成:

Normal→Patch→Field→Cancer

瞭解Field cancerization的重要:

腫瘤切除要有足夠的safe margin

請注意以下的重點提要

Formationof field cancerization

Importance of fieldcancerization


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Carcinogenesis(癌化)

Summary(總結)

Field cancerization

癌化的標準理論

5

3

4

四種癌化理論

2

Stages of carcinogenesis

1

How cancer arise - Molecular approach


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