Download Oral cancer

Oral cancer: Insights into
etiology and molecular basis
and implications for
prevention and treatment
Nikolaos G. Nikitakis, MD, DDS, PhD
Diplomate, American Board of Oral and Maxillofacial
Pathology
Associate Professor, Dept. of Oral Pathology and
Medicine, University of Athens, Greece
Lecture Outline
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The problem
Molecular carcinogenesis
Methods for studying
molecular markers and
targets
Investigated molecular
markers and targets in
oral cancer
Limitations and challenges
Conclusions
Oral Cancer Epidemiology

The sixth most common cancer
worldwide
MEN
ASR: 5.2/100,000/year
WOMEN
ASR: 2.5/100,000/year
Globocan 2008: Incidence of Oral Cancer
Oral cancer
Changing trends
in oral cancer epidemiology

Decrease in the overall incidence of
oral and oropharyngeal cancer in the
last decades
– Decreases in smoking habit
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Increase in the incidence of cancers in
specific anatomic subsites and in
younger individuals
– ?Role of HPV infection
The problem
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
Galen, 130-201 AC
Despite significant progress in the
fields of surgery, radiotherapy and
chemotherapy, oral cancer
prognosis remains dismal
Clinical and histopathological
parameters are not always
accurate predictors
Available therapeutic modalities are
associated with significant side
effects and are not always effective
The problem

To accurately determine
diagnosis and prognosis e.g.
– the likelihood of malignant
transformation (MT) in oral PMD
– the possibility of lymph node
metastasis (LNM) in oral cancer

Hippocrates, 460-370 BC
To select the most appropriate
treatment
– Higher efficacy with less side
effects
Malignant transformation
in oral PMD
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A subset of oral PMD
(e.g. leukoplakia and
erythroplakia) will
progress into invasive
cancer
Clinical and
histopathological
features alone (e.g.
degree of epithelial
dysplasia) cannot
reliably predict which
lesions will progress
Lymph node metastasis
of oral cancer

Metastasis of oral cancer to
regional lymph nodes: the
single most important adverse
prognostic factor and indicator
of survival
– Detection of LNM reduces the 5year survival rates by about 50%

Preoperative nodal status
assessment is crucial for the
selection of the optimal
therapeutic approach
The use of molecular
markers and targets

Advanced
knowledge of the
molecular basis of
cancer has
prompted the
investigation of a
number of
biomarkers as
possible biomarkers
and targets
Molecular basis of
carcinogenesis

The various types of
cancer represent
different diseases
characterized by
specific molecular
alterations
The Hallmarks of Cancer
Hanahan D, Weinberg RA. Cell 2000:57–70
The hallmarks of cancer and the novel molecular events
in OSCC-related inflammation
Wu R et al. J DENT RES 2010;90:561-572
Copyright © by International & American Associations for Dental Research
Oncogenes
and Tumor Suppressor Genes

Various genes and corresponding protein
products have been found to participate in
the regulation of cell proliferation, survival
and spreading of cancer cells
Oncogenes: promoters of tumor growth
 Τumor suppressor genes: inhibitors of tumor
growth

Molecular alterations
and monoclonality
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Carcinogenesis relies on the accumulation
of molecular alterations
Tumor formation is based on the
proliferation of a cellular clone derived
from a specific precursor cell with critical
molecular alterations
The progress
of carcinogenesis


The various carcinogens act by
inducing multiple alterations in critical
molecules
Theory of biphasic carcinogenesis
–

Action of initiators and promoters
Theory of multistep carcinogenesis
“ In the survival of favoured individuals
and races, during the constantlyrecurring struggle for existence, we
see a powerful and ever-acting form of
selection. ”
Charles Darwin, 1859
Multistep Carcinogenesis
Colon Adenocarcinoma
Vogelstein et al.
Multistep Oral Carcinogenesis
Normal
Hyperplasia
EGFR
9p21 del
p16/p14
Trisomy 7
Telomerase
Dysplasia
3p del
17p13
(p53 mut)
Tetraploidy
SCC
CIS
11q13
13q21
8p del
Cyclin D1
Aneuploidy
18q del
10q23
3p26
pTEN
Califano, Sidransky et al.
Field cancerization
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Presence of one or more mucosal areas
harboring cancer-associated genetic or
epigenetic alterations
Provides an explanation for the high
frequency of recurrences and second
primary tumors in a large mucosal area
Has been associated with
– Diffuse exposure to carcinogens
– Lateral expansion and migration of a monoclone
of genetically altered cells
Braakhuis et al. Nat Rev Cancer. 2011;11:9-22.
Methods for studying
molecular markers
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Protein assays
–
–
–
–
Immunohistochemistry
ELISA
Western blot
Proteomics
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Two dimensional gel
electrophoresis
Mass spectrometry
mRNA assays
– RT-PCR (Real Time)
– Serial analysis of gene
expression (SAGE)
– RNA protection assays (RPAS)
– Northern blot
– Genomics

cDNA Microarrays
Methods for studying
molecular markers

DNA & Chromosomes
– PCR
– Southern blot
– Fluorescence in situ hybridization – FISH
(multiplex FISH)
– Flow cytometry
– Comparative genomic hybridization (CGH)
– Spectral karyotyping (SKY) and cytogenetics
– Gene sequencing for mutational analysis
– Single nucleotide polymorphism (SNP)
– LOH analysis
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Restriction fragment length polymorphism analysis
Microsatellite analysis
Adjuvant techniques
– Laser capture microdissection
– Tissue microarrays
Schema of omics technologies, their corresponding analysis targets,
and assessment methods
Wu R et al. J DENT RES 2010;90:561-572
Copyright © by International & American Associations for Dental Research
Investigated molecular
markers and targets
in oral PMDs

Cytogenetics
– Loss of heterozygocity (LOH)
– Aneuploidy

Cell cycle regulation
Loss of Heterozygosity
(LOH)
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Allelic imbalance: One copy of a
polymorphic marker may be lost (LOH)
or amplified (allelic gain)
LOH
– loss of normal function of one allele of a
gene (in which the other allele was
already inactivated)
– results in absence of a functional TSG in
the lost region
Loss of Heterozygosity
(LOH) in oral PMDs

LOH at 3p, 9p21, 17p13 and 8p22-24
and other loci harboring important
TSG (such as p16 and p53)
Association with MT rate
– Mao et al. 1996: 37% of patients with
LOH at 3p and 9p progressed to cancer
(vs. 6% of patients without LOH)
– Partridge et al. 2000: Up to 75% risk of
tumor development if ΑΙ at 2 or more key
microsatellite markers
Loss of Heterozygosity
(LOH) in oral PMDs

Association with MT rate
– Lee et al. 2000: Original cohort of 70
patients
– Zhang et al. 2012: prospective cohort of
296 patients with mild/moderate
dysplasia
High-risk lesions (3p and/or 9p LOH): 22.6fold increase risk of progression
 Using additional markers (4q and 17p): 65.4fold increase for the high-risk group

Aneuploidy
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Abnormal number of
chromosomes Imbalanced DNA content
Percentage of
aneuploidy in oral
cancer: 50-60% (up to
90%)
Aneuploidy in Oral PMDs
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Percentage of aneuploidy in oral PMDs:
20-45% (up to 83%)
Correlation with histological degree of
dysplasia
Aneuploid lesions may transform to
malignancy at a higher rate
Torres-Rendon et al. Oral Oncol. 2009;468-73
Aneuploid
Aneuploid
Diploid
Klanrit et al. Oral Oncol. 2007;43:310-6
Cell Cycle Regulation
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Cyclins and CDKs
Rb family
p53 family and CDKIs
Cdc2
Cell division
Cyclin B
M
Cell prepares
to divide
Cell grows
p18,p19
C
C
INK4
p15,p16
Cdk4 /6
Cdc2
G1
G2
Cyclin D1/2/3
CIP/KIP
p21, p27
Cyclin A
Cdk2
Cyclin A
C
p57
Cdk2
Cyclin E
DNA replication
S
Cyclin D1 in oral PMDs
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Kovesi et al. J Oral Pathol Med
2006: Correlation of Cyclin D1
expression with clinical severity
Cyclin D1
– regulates cell cycle
progress through G1
phase
– overexpression in oral
PMDs
– correlation with degree
of dysplasia
– specific polymorphisms
are associated with oral
PMDs
Retinoblastoma (Rb) and p16
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Unphosphorylated Rb binds to
and inhibits the transcriptional
factor E2F
Formation of cyclic D-CDK4
complex results in
phosphorylation and
inactivation of Rb
p16: inhibits cyclin D-CDK4
complex
E2F
Rb
E2F
Active
Inactive
P
P
Rb
E2F DP
S Phase Genes
Retinoblastoma (Rb) and p16 in
oral PMDs
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pRb loss is associated with transition form
hyperplasia to dysplasia
p16: positive or negative correlation with
degree of dysplasia (surrogate HPV marker)
Total Rb
Un-p-Rb
p53

Guardian of the
genome
– Regulation of cell
cycle, apoptosis,
differentiation and
DNA repair

The most frequently
inactivated TSG in
cancer
p53 and cancer

Kussie et al, Science 274:948, 1996
Mechanisms of
inactivation
– Acquired mutations in both
gene alleles
– Hereditary p53 mutation
– Interaction with HR-HPV
E6 protein
– MDM2 overexpression and
p53 degradation
p53 mutation prevalence in human tumors
IARC Database, R8
p53 in oral PMDs
Non
MT

MT

Cruz et al. J Pathol. 1998;184:360-8
Increased IH
detection due to
stabilizing mutations
and evasion of
degradation
Correlation with
degree of dysplasia
and risk of MT
remains controversial
Cell cycle dysregulation
by HR-HPV
Leemans et al. Nat Rev Cancer. 2011;11:9-22
Modes of HPV infection
von Knebel Doeberitz and Vinokurova Archives Med Res. 2009
Biomarkers of
neck metastasis

Numerous studies have
investigated the role of
molecules that directly or
indirectly participate in
the metastatic cascade
– Invasion of extracellular
matrix
– Vascular dissemination and
homing of tumor cells
– Angiogenesis and
lymphangiogenesis
– Miscellaneous
Molecular Markers
associated with Invasion

Adhesion molecules
–
–
–
–
Cadherins and Catenins
Integrins
CD44
Ep-CAM
Lim et al. Clin Cancer Res 2004: Patients with low
expression of E-cadherin should be considered a
high-risk group for late cervical metastasis
Molecular Markers
associated with Invasion
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Proteolytic enzymes
– Matrix metalloproteinases (MMPs)

Collagen IV, Tissue inhibitors of metalloproteinases (TIMPs)
– Cathepsins (-B, -D, –L)
Katayama et al. Clin Cancer Res 2004:
Expressions of MMP-9 and TIMP-2 have
predictive value for tumor metastases
Molecular Markers associated
with Angiogenesis and
Lymphangiogenesis
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Angiogenesis: formation of
new vessels
Prerequisite for the growth
of tumor cells in a hypoxic
environment
Angiogenesis and
lymphangiogenesis provide
passageways to the
circulation that allow
metastatic spread
Regulators of angiogenesis:
– A long list of promoting and
inhibiting factors, that exhibit
various mechanisms of action
Molecular Markers associated
with Angiogenesis and
Lymphangiogenesis

Microvessel density (MVD)
– Methology for quantification of angiogenesis and
correlation with LNM remain controversial
Guttman et al. Oral Oncology 2004:
Lymph node metastasis was positively
correlated with the microvessel count
using the CD-34 marker
Anti-CD34
Molecular Markers associated
with Angiogenesis and
Lymphangiogenesis

Sauter et al. Clin Cancer Res 1999: Vascular
endothelial growth factor is a marker of
tumor invasion and metastasis in squamous
cell carcinomas of the head and neck
Vascular endothelial
growth factor (VEGF)
family members:
– VEGF-A, -B, -C, -D, -E and
PlGF
– Bind to VEGF receptors on
endothelial cells promoting
migration, proliferation and
vascular permeability
– Play a prominent role in
angiogenesis and
lymphangiogenesis
Molecular Targets
in Oral Cancer

Aberrant molecular events in oral
cancer can serve as potential specific
and effective therapeutic targets
– Preclinical models

Cell lines, Animal models
– Clinical trials

Phase I, II and III
Molecular Targets
in Oral Cancer

In recent years, novel molecular targeted
therapies have been applied in the
treatment of oral cancer
– Cetuximab (mAb against EGFR)
– Erlotinib (TKI against EGFR)
– Dual or pan-HER TKIs
– Angiogenesis inhibitors (e.g. bevacizumab)
– Other inhibitors (against IGF-1R, MET,
PI3K/AKT/mTOR)
Molecular Targets in Oral
Cancer
Le Tourneau and Siu, Curr Opinion Oncol 2008;20:256-63
Cell Signaling
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Dysregulations in cell signaling pathways are
frequent and crucial events in carcinogenesis
Many signaling pathways are involved in oral
oncogenesis
–
–
–
–
–
EGFR/TGF-α
TGFβ/SMAD
c-MET
PI3K/Akt/mTOR
Stat3
Molecular Targets
in Oral Cancer
Howard et al. Oral Oncol. 2012;48:10-7
Stat3 signaling in oral neoplasia
TGF-α
gp80
IL6
Src
JAK
JAK
EGFR
Stat3
JAK
gp130
MEKKs
Mek1/2
MEK4/7
Mek3/6
Stat3
ERK
JNK
p38
SOCS
PIAS3
Stat3
Bcl-XL
Cyclin D1
Survivin
IL-6
1a
TGF-α
1a
2a
IL-6R/
1b
gp130
JAKJAK
P
P
P
P
P
P
P
P
STAT
P
SRC
Cytoplasmic
negative regulators
(e.g. SHP, SOCS)
P
1b
P
EGFR
P
STAT
2b
STAT
dimer
P
Additional regulation
(e.g crosstalk with MAPKs,
serine phosphorylation)
2a
Nuclear
negative regulators
(e.g. PIAS,
phosphatase)
P
Transcription of specific
target genes
(e.g. Bcl-XL, Cyclin D1,
VEGF)
STAT
dimer
P
Transcriptional
Coactivators (e.g. p300/CBP)
Nikitakis et al, Current Cancer Drug Targets, 2004, 4, 637-651
Overactivity of
upstream
kinases and
insufficient
negative
regulation
Availability of
STAT molecules
Recruitment and
phosphorylation
1
P
STAT
dimer
2
Dimerization
3
P
Nuclear translocation
4
P
5
DNA binding
STAT
dimer
P
6
Interaction with
transcriptional
coactivators
Transcriptional
activation
7
Nikitakis et al, Current Cancer Drug Targets, 2004, 4, 637-651
Molecular targets in Oral
Cancer
Yedida et al. OOOO. 2013;33:482-9
Limitations and challenges


Current inconsistency and lack
of validation of obtained results
hampers the use of biomarkers
in a clinical setting
Reasons for inconsistency:
– Molecular heterogeneity

Patient-specific and lesion-specific
– Differences in methodology

Use of different protocols and
methods for analysis and
interpretation of data
Limitations and challenges

Differences in experimental design
– Patient and case selection

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Epidemiological factors, site, clinical
characteristics, histological features
Effects of diagnostic and therapeutic
interventions
Follow-up
Study of a limited number of genes
and their proteins underestimates the
complexity of carcinogenesis:
– Limited capacity of any single molecular
biomarker to reliably predict outcome in
individual patients
– No “silver bullet” for the treatment of all
oral cancers – Need for an individualized
approach
Conclusions


Significant correlations
between specific
molecular markers and
oral cancer prognosis and
susceptibility to treatment
have been established
Molecular targeted
therapies for oral cancer
have been introduced and
rapidly evolving
Conclusions


Improved understanding of
the molecular basis of oral
carcinogenesis, refinement of
methodology and study
design and further technologic
advances will facilitate the
discovery of reliable molecular
predictors and targets
Clinical application awaits
further validation through
large scale, multicenter, well
controlled studies