口腔病理

陳玉昆副教授 : 高雄醫學大學 口腔病理科 07-3121101~2755 yukkwa@kmu.edu.tw

Carcinogenesis癌 化

References1. Gibbs WW. Untangling the roots of cancer. Sci Am 2003;289:56-65.2. What you need to know about cancer. Sci Am 1996 ;289:28-119.3. Braakhuis BJM et al. A genetic progression model of oral cancer: current evidence and

clinical implications. J Oral Pathol Med 2004;33:317-22.4. Braakhuis BJM et al. A Genetic explanation of slaughter’s concept of field

cancerization: evidence and clinical implications. Cancer Res 2003;63:1727-30.5. Loktionov A. Common gene polymorphisms, cancer progression and prognosis. Cancer

Letters 2004;208 :1-33.6. Kaohsiung Medical University, Oral Pathology Department.7. 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.

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

9. Spillane JB, Henderson MA. Cancer stem cells: a review. ANZ J Surg 2007;77:464-8.10. Zhou ZT, Jiang WW. Cancer stem cell model in oral squamous cell carcinoma. Curr

Stem Cell Res Ther 2008;3:17–20.11. Harper LJ et al. Stem cell patterns in cell lines derived from head and neck squamous

cell carcinoma. J Oral Pathol Med 2007;36:594-603.12. 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.

1. Gibbs WW. Untangling the roots of cancer. Sci Am 2003;289:56-65.2. What you need to know about cancer. Sci Am 1996 ;289:28-119.3. Braakhuis BJM et al. A genetic progression model of oral cancer: current evidence and

clinical implications. J Oral Pathol Med 2004;33:317-22.4. Braakhuis BJM et al. A Genetic explanation of slaughter’s concept of field

cancerization: evidence and clinical implications. Cancer Res 2003;63:1727-30.5. Loktionov A. Common gene polymorphisms, cancer progression and prognosis. Cancer

Letters 2004;208 :1-33.6. Kaohsiung Medical University, Oral Pathology Department.7. 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.

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

9. Spillane JB, Henderson MA. Cancer stem cells: a review. ANZ J Surg 2007;77:464-8.10. Zhou ZT, Jiang WW. Cancer stem cell model in oral squamous cell carcinoma. Curr

Stem Cell Res Ther 2008;3:17–20.11. Harper LJ et al. Stem cell patterns in cell lines derived from head and neck squamous

cell carcinoma. J Oral Pathol Med 2007;36:594-603.12. 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.

Carcinogenesis( 癌化 )

How cancer arise - Molecular approach

Stages of carcinogenesis

癌化的標準理論

四種癌化理論

Field cancerization

綱 要

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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.

Stochastic Clonal Evolution Model

(1) How Cancer Arises - Molecular Approach

Ref. 8

Stochastic clonal expansionInteraction between tumor cellsand stromal cells

Tumor cell

Definitive Tissue Line

Early Progenitor

Late Progenitor

Stem Cell

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

Normal Stem Cell

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

Tumor

Stem Cell

Mutation Onlyat the Stem Cell

Mutation

Ref. 9

Asymmetrical Division

(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

Traditional Model ofTumor Formation

MatureDefinitiveTissueCell

TumorTumor

Mutation

Mutation

Mutation

Ref. 9

(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

Tumor

Stem CellMutation

Mutation Onlyat the Stem Cell

Cancer Stem Cell Model (1)

mutation

mutationSelf-renewingstem cell

Progenitorcell

Mature cell

Cancer cell

Self-renewingcancer stem cell

Ref. 9

Mutation only at the stem cell or progenitor cell level

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

Tumor from an early stem cellHeterogeneous cancerIncreased metastatic potential

Tumor from a late progenitor cellHomogenous cancerLess metastatic potential

Tumor

Tumor

Early Progenitor

Late Progenitor

Definitive TissueLine

Stem CellMutation

Mutation

Mutation

Tumor

Ref. 9

Cancer Stem Cell Model (3)

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.

(a) In hypoxia(e.g. within niche)

(b) In increased O2

(e.g. outside niche)

Stem cell in quiescence Proliferation Stem cell depletion Exhaustion

Self-renewing stem cell(normal or cancer)

Progenitoror differentiated cell Ref. 9

Comparison of Somatic and Cancer Stem Cells

Somatic Stem Cell Cancer Stem Cell

Self renew, highly regulated Self-renew, poorly regulated

Differentiate, produces mature tissue

Differentiate, produces tumor

Migrate to distant tissues Metastasize to distant sites

Long lifespan Long lifespan

Resistant to apoptosis Resistant to apoptosis

Ref. 9

• 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 to accumulate the necessary number of genetic changes for malignancy to develop

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.

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

Ep

ith

eliu

mC

on

nec

tive

tis

sue

Ref. 10

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.

Putative Cell Surface Markers of Presumptive CSC

SP-C+CCA+

Tumor Type Surface Markers

Ref. 10

Frequencies of CSCs in Various Human Cancers

Human cancer Recipient mice Cancer stem cell frequency (%)

Ref. 10

CD44+CD24- Lineage negative

CD44+CD24-

CD44+CD24-Tumor formed

New tumor formed

Ref. 10

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

Potential Mechanisms of CSC Formation

CSC

MUTATIONA

Progenitors

Self renewal

Self renewal

Stem/progenitor cellsDifferentiated cells

(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

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

CSC

MULTIPLE GENETIC HITSB

Stem/progenitor cells

Potential Mechanisms of CSC Formation

Ref. 10

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

Potential Mechanisms of CSC Formation

Ref. 10

CSC

C

Cancer cell

MULTISTEP DEDIFFERENTIATION

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

CSC

FUSIOND

Cancer cell

Stem/progenitor cells

Potential Mechanisms of CSC Formation

Ref. 10

DMBA-Induced Hamster Buccal Pouch Model

14-wk

Normal

Carcinogen: DMBA

• 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 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)

AnimalAnimal StudyStudy

HumanHuman StudyStudy

Ref. 6

A B

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

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

• Self-renewal---Colony forming unit (CFU)---Proliferation

One or more lineages differentiation---Adipogenic differentiation---Osteogenic differentiation---Chondrogenic differentiation---Neurogenic differentiation

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

Ref. 7

A

B

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

Ref. 7

A B

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

Ref. 7

Prol

ifera

tion

rate

(# o

f fol

ds)

Pouch 2 Pouch 3

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

Ref. 7

A

NM GAPDH PPAR

B

50

100

150

200250300350400

bp

(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.

Ref. 7

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).

Ref. 7

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

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.

M N GA

PD

H

Os

teo

ne

cti

n

Ne

sti

n

Oc

t-4

Na

no

g

Re

x-1

100

200

300

400500600700

bp

Ref. 7

0.9

CD14 %

of

Max

100

CD 29

% o

f M

ax

93.6

100

CD 34

% o

f M

ax

100

1.7

CD 45

1.5

% o

f M

ax

100

CD 90

85.8

% o

f M

ax

100

51.3

CD 105

100

% o

f M

ax

100

Ref. 7

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).

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.

DMBA-Induced Hamster Buccal Pouch Model

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.

Comparison of Morphology Between Our Isolated Cells & Literature Results

Our isolated cells from DMBA-induced cancer pouch tissue

Refs. 7, 11

squamospheressquamospheres

squamospheressquamospheres

Self-renewal, stem cell marker expression, aberrant differentiation, and tumor-initiating potentialOSCC-driven squamospheres demonstrated:(1) 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

Hallmarks of CSCs (1)

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

Hallmarks of CSCs (2)

Ref. 12

(1) 小 結

Cancer development:Stochastic clonal evolution modelVS Cancer stem cells model

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

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

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

請注意以下的重點提要

Genticallyaltered cell

HyperlasiaDysplasia

Tumor developmentoccurs in stages

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

Hyperplasia

Dysplasia

Oral potentially malignant disorders (OPMD)Leukoplakia, Erythroplakia, Oral submucous fibrosis, Verrucous hyperplasia, Erosive lichen planus

基底層完整

基底層完整

(2) Stages of Carcinogenesis

Ref. 1

In situ cancer

Invasive cancer

Blood vessel/lymphatic vessel

Ref. 1

How Cancer Spreads

Primary tumor

Normal epithelial cell

Basement membrane

Invasive tumor cellBlood vessel/lymphatic channel

How Cancer Spreads

Ref. 1

Endothelial/lymphaticlining

Basement membrane

Metastatic cellin circulation

Secondary tumor site

Tumor celladheringto capillary

Ref. 1

How Cancer Spreads

Initiation Phase (Early) (2) Further look on stages of carcinogenesis

去毒

Ref. 5

Initiation Phase (Late) Ref. 5

Promotion Phase (Early) Mutant clone establishment & appearance

of phenotypically transformed cells

Ref. 5

Promotion Phase (Late) Establishment of phenotypically

transformed cell population (dysplasia)

Ref. 5

Progression Phase (Early)

Malignisation

Ref. 5

Progression Phase (Middle)

Microinvasion

Ref. 5

Progression Phase (Late) Advanced invasion and metastasis

ChemotherapyRef. 5

(2) 小 結

Tumor development occurs in stagesNormal cell has self-defense

癌症形成是階段性的 vs 正常細胞有自衛能力Initiation (early, late)

Genetically altered cell (CSC)

Promotion (early)Hyperplasia

Promotion (late)Dysplaisa

Progression (early)In situ cancer

Progression (middle)Microinvasion

Progression (late)Invasive cancer

Progression (late)Metastasis

請注意以下的重點提要

NormalCell CycleCell enlargesand makesnew proteins

Beginningof cycleCell

divides(mitosis)

Cell preparesto divide

Cell replicatesas DNA

Cell rests

Restriction point: celldecides whetherto commit itself tothe complete cycle

崗 哨

(3) 癌化的標準理論

G1 arrest

Ref. 2

InhibitorypathwaysNormal Cell

Inhibitoryabnormality

Stimulatoryabnormality

Stimulatorypathways

標準理論

致癌基因Oncogene

抑癌基因Tumor suppressor gene Ref. 2

Activation ofoncogene

Inactivation of tumor suppressor gene

Cell Cycle

失 控失 控

下 坡

下 坡

下 坡

剎車失靈

油門全開

Aberrant cell cycle —Accelerated car downslope without brake

Ref. 2

Oncogene (1) Genes for growth factors or their receptors

PDGF Codes for platelet-derived growth factorInvolved in glioma (a brain cancer)

erb-B Codes for the receptor for epidermal growth factorInvolved in glioblastoma (a brain cancer) and breast cancer

erb-B2 Also called HER-2 or neu. Codes for a growth factor receptor involved in breast, salivary gland and ovarian cancers

RET Codes for a growth factor receptorInvolved in thyroid cancer

Genes for growth factors or their receptors

Ki-ras Involved in lung, ovarian, colon and pancreatic cancers

N-ras Involved in leukemia

Ref. 2

Oncogene (2) Genes for growth factors or their receptors

c-myc Involved in leukemia and breast, stomach and lung cancers

N-myc Involved in neuroblastoma (a nerve cell cancer) and glioblastoma

L-myc Involved in lung cancer

Genes for growth factors or their receptors

Bcl-2 Codes for a protein that normally blocks cell suicide.Involved in follicular B cell lymphoma

Bcl-1 Also called PRAD1. Codes for cyclin D1, a stimulatory component of the cell cycle clockInvolved in breast, head and neck cancers

MDM2 Codes for an antagonist of the p53 tumor suppressor protein. Involved in sarcomas and other cancers

Ref. 2

Tumor Suppressor Gene (1)Genes for proteins in the cytoplasm

APC Involved in colon and stomach cancers

DPC4 Codes for a relay molecule in a signaling pathway that inhibits cell divisionInvolved in pancreatic cancer

NF-1 Codes for a protein that inhibits a stimulatory (Ras) proteinInvolved in neurofibroma and pheochromocytoma (cancers of the peripheral nervous system) and myeloid leukemia

NF-2 Involved in meningioma and ependymoma (brain cancers) and schwannoma (affecting the wrapping around peripheral nerves)

Ref. 2

Tumor Suppressor Gene (2)

Genes for proteins whose cellular locations is not yet clear

BRCA1 Involved in breast and ovarian cancers

BRCA2 Involved in breast cancer

VHL Involved in renal cell cancer

Genes for proteins in the nucleus

MTS1 Codes for the p16 protein, a braking component of the cell cycle clock Involved in a wide range of cancers

RB Codes for the pRB protein, a master brake of the cell cycle. Involved in retinoblastoma and bone, bladder, small cell lung and breast cancer

p53 Codes for p53 protein, which can halt cell division and induce abnormal cells to kill themselves. Involved in a wide range of cancers

WT1 Involved in Wilms’ tumor of the kidney

Ref. 2

基因突變地圖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

Ref. 2

(3) 小 結

Tumor development occurs due toformations of oncogene & tumor

suppressor gene

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

(4) 癌化的四個理論

Ref. 2

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

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

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

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 Ref. 2

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

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

正常 癌 症

Ref. 2Normal & Cancer Chromosomes

(4) 小 結

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

請注意以下的重點提要

(5) Field Cancerization (1)

Patch phase

Precursor lesionsdevelop within field

Carcinoma excised,field and precursorlesion remains

Expandingfield phase

Field

Second field tumordevelops fromprecursor lesion

Precursor lesionsbecomes carcinomaand new precursorbecomes develop

Epithelium

Connective tissue Basal layer withstem cells

Genetic altered

Ref. 3

Field Cancerization (2)

Carcinoma

11q

FieldPatch

Histological Proof

Chromosomal Proof

p armq arm

centromere

Normal

17p 3p, 9p, 8p, 18q

Ref. 4

(5) 小 結

Formation of field cancerizationImportance of field cancerization

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

Carcinogenesis( 癌化 )

How cancer arise - Molecular approach

Stages of carcinogenesis

癌化的標準理論

四種癌化理論

Field cancerization

Summary( 總結 )

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