1. Prevention & detection of oral cancer
牙科公共衛生學
Prevention & detection of oral cancer
口腔癌之預防與偵測
陳玉昆副教授: 高雄醫學大學 口腔病理科
~2755

2. 參 考 書 目
Gibbs WW. Untangling the roots of cancer. Sci Am 2003;289:56-65.
What you need to know about cancer. Sci Am 1996 ;289:
Hannen EJM, Riediger D. The quantification of angiogenesis in relation to metastasis in oral cancer: a review. Int. J Oral Maxillofac Surg 2004;33:2-7.
Shieh et al. Role of angiogenic and non-angiogenic mechanisms in oral squamous cell carcinoma: correlation with histologic differentiation and tumor progression. J Oral Pathol Med 2004;33:601-6.
Sharma DC. Betel quid and areca nut are carcinogenic without tobacco. Lancet Oncol 2003;4:587.
Sharma DC. Indian betel quid more carcinogenic than anticipated. Lancet Oncol 2001;2:464.
Braakhuis BJM et al. A genetic progression model of oral cancer: current evidence and clinical implications. J Oral Pathol Med 2004;33:
Braakhuis BJM et al. A Genetic explanation of slaughter’s concept of field cancerization: evidence and clinical implications. Cancer Res 2003;63:
Loktionov A. Common gene polymorphisms, cancer progression and prognosis. Cancer Letters 2004;208 :1-33.
Desmaze C et al. Telomere-driven genomic instability in cancer cells. Cancer Letters 2003;194:
Hiyama E & Hiyama K. Telomerase as tumor marker. Cancer Letters 2003;194:
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:
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:
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.

3. 學 習 目 標 探索癌症之旅 始 點 終 點 Field cancerization 癌化的標準教條 5 3 4 癌細胞的六種超能力
四種癌化理論 6 2
Stages of carcinogenesis
癌症的預防 7
始 點 1
終 點
How cancer
arise

4. 第一站：How Cancer Arises Stochastic Clonal Evolution Model
Interaction between tumor cells
and stromal cells
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. 14

5. Asymmetrical Division
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
Ref. 15

6. Traditional Model of Tumor Formation Mutation Only at the Stem Cell
(a) The 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 of
Tumor Formation
Mature
Definitive
Tissue
Cell
Tumor
Mutation
Mutation Only
at the Stem Cell
(b) Mutation only at the stem cell or progenitor cell level. 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
Ref. 15

7. Cancer Stem Cell Model cancer stem cell mutation Cancer cell Self-
renewing
stem cell
Progenitor
cell
Mature cell
Cancer cell
cancer stem cell
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. 15

8. 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
Resistant to apoptosis
Ref. 15

9. 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 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 to accumulate the necessary number of genetic changes for malignancy to develop

10. A Schematic Diagram Showing Sites of Origins of Putative CSCs in OSCC
Epithelium
Connective tissue
1, CSC might come from epithelial SC or progenitor within basal layer with genetic alterations; 2, muscle-derived SCs; 3, fibroblast-derived SCs; 4, vessel wall-derived SCs; 5, blood-derived SCs; and 6, adipose derived SCs.
Ref. 16

11. Putative Cell Surface Markers of Presumptive CSC
SP-C+CCA+
Tumor Type
Surface Markers
Ref. 16

12. 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
CD44+CD24-
Tumor formed
New tumor formed
Ref. 16

13. A Potential Mechanisms of CSC Formation MUTATION CSC
Progenitors
Self renewal
Stem/progenitor cells
Differentiated cells
Ref. 16

14. B Potential Mechanisms of CSC Formation MULTIPLE GENETIC HITS CSC
Stem/progenitor cells
Ref. 16

15. C D Potential Mechanisms of CSC Formation MULTISTEP DEDIFFERENTIATION
Cancer cell
CSC
FUSION D
Cancer cell
Stem/progenitor cells
Ref. 16

16. DMBA-Induced Hamster Buccal Pouch Model
Hamster buccal-pouch mucosa provides one of the most widely-accepted experimental models for oral carcinogenesis
Carcinogen: DMBA
14-wk
Normal
Ref. 12

17. 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.
Animal Study
Human Study
Ref. 12

18. Isolation and Characterization of Stem Cells from Normal Hamster Buccal Pouch (HBPSC)
Figure 1A-B
Representative sample of the normal hamster buccal pouch tissues revealed no obvious grossly (A; inset) and histological (B, Hematoxylin & eosin stain, 200) changes.
Ref. 13

19. 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

20. Representative sample of the HBPSCs obtained from the normal hamster buccal pouch tissues were spindle-shaped in morphology (200).
Figure 2A-B
Ref. 13

21. Representative sample of the HBPSCs obtained from the normal hamster buccal pouch tissues were able to form colonies, stained with crystal violet (A; B, 100). A B
Figure 3
Ref. 13

22. Cytoplasmic keratin (A, 200) and vimentin (B, 200) stainings were noted for the representative sample of the HBPSCs obtained from the normal hamster buccal pouch tissues. A B
Figure 4A-B
Ref. 13

23. Proliferation rate (# of folds)
Proliferation rates for the HBPSCs obtained from the three normal hamster buccal pouch tissues (p: passage).
Proliferation rate (# of folds)
Pouch 2
Pouch 3
Figure 5
Ref. 13

24. (A) Representative sample of the 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. A N M
GAPDH
PPAR B 50
100
150
200
250
300
350
400 bp
Figure 6A-B
Ref. 13

25. Representative sample of the 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).
Representative sample of the HBPSCs obtained from the normal hamster buccal pouch tissues were able to differentiate towards osteogenic lineage (×200).
Figure 7, inset
Ref. 13

26. Representative sample of the 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
GAPDH
Osteonectin
Nestin
Oct-4
Nanog
Rex-1
100
200
300
400
500
600
700 bp
Figure 9
Ref. 13

27. Representative sample of the 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).
0.9
CD14
% of Max
100
CD 29
93.6
CD 34
1.7
CD 45
1.5
CD 90
85.8
51.3
CD 105
Figure 10
Ref. 13

28. 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.

29. Comparison of Morphology Between Our Isolated Cells & Literature Results
Our isolated cells from DMBA-induced cancer pouch tissue
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. 13, 17

30. Hallmarks of CSCs (1)
Self-renewal, stem cell marker expression, aberrant differentiation, and tumor-initiating potential
OSCC-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)

31. Hallmarks of CSCs (2)
Ref. 18

32. 第二站: Stages of Carcinogenesis
Gentically
altered cell
Hyperlasia
Dysplasia
第二站: Stages of Carcinogenesis
Tumor development
occurs in stages
Oral premalignant lesions
Leukoplakia, Erythroplakia, Oral submucous fibrosis, Verrucous hyperplasia, Erosive lichen planus
Ref. 1
Genetically
altered
cell (CSC)
Hyperplasia
Initiated cell
起始細胞
Dysplasia
基底層完整
基底層完整

33. How Cancer Spreads In situ cancer Invasive cancer Ref. 1 Blood vessel/
lymphatic vessel
Ref. 1
How Cancer Spreads

34. How Cancer Spreads Primary Ref. 1 tumor Normal epithelial cell
Basement membrane
Invasive tumor cell
Blood vessel/
lymphatic channel
Ref. 1
How Cancer Spreads

35. How Cancer Spreads Ref. 1 Secondary tumor site Endothelial/lymphatic
lining
Basement membrane
Metastatic cell
in circulation
Secondary tumor site
Tumor cell
adhering
to capillary
Ref. 1

36. Initiation Phase (Early) 第二站: Further look on stages of carcinogenesis去毒
Ref. 9

37. Initiation Phase (Late)
Ref. 9

38. Promotion Phase (Early)
Mutant clone establishment & appearance
of phenotypically transformed cells
Ref. 9

39. Promotion Phase (Late)
Establishment of phenotypically
transformed cell population (dysplasia)
Ref. 9

40. Progression Phase (Early)
Malignisation
Ref. 9

41. Progression Phase (Middle)
Microinvasion
Ref. 9

42. Advanced invasion and metastasis
Progression Phase (Late)
Advanced invasion and metastasis
Chemotherapy
Ref. 9

43. 第三站: 癌化理論的標準教條 Normal Cell Cycle 崗 哨 G1 arrest Beginning of cycle Cell
Ref. 2
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

44. 標準教條 致癌基因 抑癌基因 Normal Cell Ref. 2 Inhibitory pathways Stimulatory
abnormality
Stimulatory
致癌基因
抑癌基因

45. 下 坡 油 門 全 開 煞 車 失 靈 Aberrant cell cycle — accelerated cars
without brake
Ref. 2
Cell Cycle
下 坡
煞 車 失 靈
油 門 全 開
失 控
Activation of
oncogene
失 控
Inactivation of
tumor suppressor gene

46. Oncogene (1) Ref. 2 Genes for growth factors or their receptors PDGF
Codes for platelet-derived growth factor
Involved in glioma (a brain cancer)
erb-B
Codes for the receptor for epidermal growth factor
Involved 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 receptor
Involved 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

47. Oncogene (2) Ref. 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 clock.
Involved in breast, head and neck cancers
MDM2
Codes for an antagonist of the p53 tumor suppressor protein. Involved in sarcomas and other cancers

48. Tumor Suppressor Gene Ref. 2 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 division.
Involved in pancreatic cancer
NF-1
Codes for a protein that inhibits a stimulatory (Ras) protein.
Involved 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)

49. Tumor Suppressor Gene Ref. 2 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
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

50. A Subway Map for Cancer Pathways
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
癌化理論 → 標準教條：
細胞循環中，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

51. 第四站
Ref. 2
癌化的四個理論
標準教條

52. Ref. 2
修 正 教 條
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
修 正 教 條

53. 早期不穩定理論 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 早期不穩定理論
Ref. 2
早期不穩定理論
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
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

54. 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

55. 隨染色體起舞 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。
Ref. 2
隨染色體起舞
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。

56. Normal & Cancer Chromosomes
Ref. 2 正常
癌 症

57. Field Cancerization (1)
第五站:
Field Cancerization (1)
Ref. 7
Epithelium
Connective tissue
Basal layer with
stem cells
Genetic altered
Patch phase
Field
Expanding
field phase
Precursor lesions
develop within field
Precursor lesions
becomes carcinoma
and new precursor
becomes develop
Carcinoma excised,
field and precursor
lesion remains
Second field tumor
develops from
precursor lesion

58. Field Cancerization (2)
Ref. 8
Histological Proof
Normal
Patch
Carcinoma
Field
Chromosomal
Proof
17p
3p, 9p, 8p, 18q
11q
p arm
q arm
centromere

59. 第六站: 癌細胞的六種魔鬼能力
Ref. 2
癌細胞的第一種魔鬼能力

60. 第六站: 癌細胞的六種魔鬼能力
Ref. 2
癌細胞的第二種魔鬼能力

61. 第六站: 癌細胞的六種魔鬼能力
Ref. 2
癌細胞的第三種魔鬼能力

62. 第六站: 癌細胞的六種魔鬼能力
Ref. 2
癌細胞的第四種魔鬼能力

63. Angiogenesis Factors (1)
Ref. 3
Tumor island

64. Angiogenesis Factors (2)
Ref. 4
Angiogenesis Factors (2)
Newly-formed
vessels
Normal
vessels
Tumor cells

65. Angiogenesis Factors (3)
Ref. 4
Normal
epithelium
Dysplasia
Early localized
tumor
Advanced
invasive
Normal vessel
Newly-formed vessel
Tumor-lined vessel

66. 第六站: 癌細胞的六種魔鬼能力
Ref. 2
癌細胞的第五種魔鬼能力
p arm
q arm
centromere 藍

67. Consequences of teleomere loss
in tumor cells
Ref. 10
Teleomere
(末端粒腺體)
Normal
Teleomere
Mutant
Fusions breakages
Duplication
of 16q: iso16q
Trisomy 16q
Monosomy 16q
Chromosome
instability
Gene
amplification
Chromosome
imbalances

68. Regulation of Teleomeres – Alterative Length of Teleomere
(ALT), Teleomerase or Both
Ref. 10
Growth
Massive Apoptosis
Immortalization
Teleomeres
shorten
Teleomeres
are critically short
Teleomeres
are regulated by
1. ALT
3. Teleomerase & ALT ?
Genomic Instability
2. Telomerase
Repeated cell divisions

69. Immunohistochemical Staining of Teleomerase Reverse Transcriptase (h-TERT)
Ref. 11
Brown color
stained dots
Cancer
tissues

70. 第六站: 癌細胞的六種魔鬼能力
Ref. 2
癌細胞的第六種魔鬼能力

71. Causes and Prevention 第七站 What Causes Cancer? Ref. 1
Chemical-environment
Virus
The top two causes - tobacco and diet-
account for almost two thirds of all cancer
deaths and are amongst most correctable
PAPILLOMA VIRUS is a
significant cause of cancer

72. Most Oral Carcinoma in Taiwan is Associated with Betel Quid
Ref. 12

73. Lancet Oncology 2001; August
Ref. 6
印度檳榔包裝

74. Lancet Oncology 2003; October
Ref. 5
印度的檳榔攤

75. Carcinogens in Work Place (1)
Ref. 1 砷 石棉

76. Carcinogens in Work Place (2)
Ref. 1

77. Genes and Cancer Risk
Ref. 1
基因警察
DNA
修補基因

78. Strategies for Minimizing Cancer Risk
Realistic Goals for Reducing Cancer Mortality
Ref. 1
Food
Strategies
for Minimizing
Cancer Risk
Causes of current
cancer mortality
Realistic population goals
for reduced cancer mortality
Risk factors
Estimated number of deaths in US (thousands per year)
100,000 to 125,000 current deaths
Simple, realistic preventive measures
could save hundreds of thousands of lives
every year in developed countries alone

79. Chemoprevention of Cancer Someday people should be able to
avoid cancer or delay its onset by taking
specially formulated pills or foods
Ref. 1

80. to excessive proliferation genetically damaged cells
Chemoprevention的原理
Ref. 1
Healthy cell
Differentiation cell
Genetic mutations
that can lead
to cancer
Processes that lead
to excessive proliferation
genetically damaged cells
Cancer cell
Damaged cell
(precancer cell)
Programmed death
of altered cells
(Apoptosis)

81. Earlier Detection Advances in Cancer Detection Ref. 1 Chromosome 17
BRCA1
Chromosome 17
A family search for BRCA1 mutation
Earlier Detection
Advances in
Cancer Detection
Tests to look for the presence of a tumor before any symptoms appear may save more lives than new drug therapies do
Ref. 1

82. Some Family Causing Syndromes
Ref. 1
DNA Testing cost
Syndrome
Cancers
Gene

83. Diagnosing Hubert H. Humphrey
Example of Early Detection
Ref. 1
Slide is prepared from
Humphrey’s urine sample taken in 1967
Copies of DNA are
made from PCR
Normal
DNA
Mutant
DNA
美國副總統—韓福瑞
Diagnosing Hubert H. Humphrey
27 Years Later (2004)
DNA is placed on nylon membrane
生物晶片
Mutant DNA
probe
Probe bind to mutant DNA
DNA is purifed and p53 gene is sequenced
Probe for the mutation is constructed
Section of cancer is taken from Humphrey’s bladder surgically removed in 1967
Gel shows
mutation (red arrow) in p53 gene

84. Advances in Tumor Imaging
Mammograms
CT scan
Liver
Hepatoma
Breast
Advances in
Tumor Imaging
New tools yield a three-dimensional view
inside the body and automated advice
on interpreting the anatomical landscape
Ref. 1

85. Advances in Tumor Imaging
Ref. 1
Positron Emission Tomography

86. Immunotherapy/ Stem Cell Therapy for Cancer Orange: Stroma
Ref. 1
Antibodies recognizes specific cells and can be
used to find and selectively destroy tumor cells
Orange: Stroma
Green: Colon cancer cell

87. Fighting Cancer by Attacking
Its Blood Supply
Ref. 1
Inhibition of NOS enzymes by NOS inhibitor
Relatively lower level of NO produced by SCC facilitates angiogenesis & tumor dissemination
NOS inhibitor
NOS inhibitor NO NO
NOS inhibitor
Before therapy
After therapy
NO: nitric oxide
NOS: nitric oxide synthase

88. Summaries 瞭解以下各點： How cancer arise Stages of carcinogenesis 癌化的標準教條
四種癌化理論
Field cancerization
癌細胞的六種超能力
癌症的預防