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Advanced Diagnostics

and Cytology
Joel L. Schwartz, D.M.D., D.M.Sc.
Director of Oral Maxillofacial Pathology
University of Illinois at Chicago
College of Dentistry

New Directions
The future of oral and pharyngeal cancers
is prevention
New screening techniques are progressing
that allow researchers to evaluate the risk
prior to developing lesions
Oral cytology testing using cells from the
tongue is both cost-effective and accurate
Researchers from UCLA report early
success using saliva to detect oral cancer

A Mechanism for Oral Cancer


Development
HPV
Environmental
Carcinogens
Tobacco
Carcinogens
Alcohol Abuse

Damage to DNA

DNA Repair

Cell Growth Regulation


DNA Content
Apoptosis
Nuclear Instability

Oral Cancer

Cell
Laboratory
Studies

Pre-Clinical
Oral Cancer
Model

Clinical
Translational
Early
Screening
Studies

Long Term Goal: To establish a set of markers


to screen at risk individuals for oral cancer
before a lesion is observed

Approach:
Test hypothesis for initial markers following
exposure to carcinogen in human oral
keratinocytes
Further evaluate markers during low dose oral
carcinogenesis and inhibition
Investigate expression of markers in at risk
populations for oral cancer (e.g., smokers)

Why Do We Want Markers?


Markers are required to:
reduce the mortality rate among oral
cancer patients (50% 5 year survival)
screen individuals before lesions
appear
help monitor therapy

Tools for Studying Oral Cancer


Prevention, Detection and Treatment
Cells- Growth of well differentiated oral
keratinocytes (normal, premalignant,
malignant)
-Transformation with HPV
-Transformation with PAH, tobacco
carcinogen, Betal Nut
Animal models
-Tobacco carcinogen induction of oral
cancer

Human Papillomavirus
Estimated: 35-55% of
oral cancers positive for
HPV
70 subtypes documented

High Risk
Types:
16,18
Lower Risk:
6,11,31

HPV 16 Role in Oral Cancer


HPV+

No Cancer

HPV+Tobacco or Environmental Carcinogen + Infection #2

Oral Cancer

Papilloma Lesions of the Oral Cavity


Squamous Papilloma:
Most common in 30 - 50 yr olds
Equally in males and females
HPV-6,11 in 50% of the lesions
Tongue and soft palate common
sites

Finger-like
projections with
fibrovascular core

Verruca Vulgaris(Common Wart)


Common Wart:
Found in children and middle
age
Found frequently on vermillion
border,labial mucosa, or
anterior tongue
HPV-2,4,40
Finger like projections with
chronic inflammatory cells
Cup-like appearance
Koilocytes
Eosinophilic intranuclear viral
inclusions

Condyloma Acuminatum (Venereal Wart)


STD associated lesion.
Mouth and genitalia.
HPV-6,11,16, 18

Koilocytes with keratohyalin


granules

Oral Keratinocyte Laboratory Response


to HPV Infection and/or PAH Exposure
Schwartz JL & Shklar. 1997. Eur J of Cancer 33: 431-438.
(Hamster oral keratinocytes)
Park NH, Gujuvula CN, Baek, JH. 1995. Intl J of Oncology 10: 2145-2153.
(Human oral keratinocytes)
HPV

No oral cancer
formation

HPV
HPV

ORAL CANCER FORMATION


PAH
PAH

PAH
PAH

PAH

PAH

Conclusions
The combination of HPV 16,18
infection and treatment with low doses
of environmental and/or tobacco
carcinogens is capable of changing a
non-cancer cell into a cancer cell

Common Interaction Sites of HPV


and Tobacco Carcinogens
A regulation of tumor suppression and cell
growth pathways (p53 pathway,
retinoblastoma,p300 complex proteins)
Influence upon cell protein chemistry (Ahr-Ahnt
complex formation)
Association with endocrine (hormonal effects :
estrogen, androgen and glucocorticoids )

Pre-Clinical Oral
Cancer Model
and Inhibition of Oral
Carcinogenesis

Tobacco Carcinogens

Mechanism For Induction and


Prevention of Oral Carcinogenesis
Early Events
Initiation

Later Events
Promotion

Cancer Formation

DNA Damage DNA Repair DNA


Apoptosis Nuclear
Content
Instability
Cell Growth
VEas Administration Inhibits Oral Carcinogenesis
Reduced DNA Damage Increased/Decreased Repair
Decreased
Cell Growth
Reduced DNA Content Increased Apoptosis Reduced
Nuclear

Clinical Translational
Early Screening Studies

We need to:
Screen before a lesion is observed
Change behavior
Provide prevention treatment

Variations of Oral Squamous


Carcinoma Presentations

Factors Influencing Mortality


and Survival
Time of diagnosis
Access to treatment
Success of treatment
State of health at initial detection

No improvement since 1973 in mortality or


morbidity for tongue and floor of mouth Sq. CA.

Early Screening and Detection of


Oral Mucosa Changes Before A
Lesion Appears

Screening and Detection of Oral Cancer


Oral Biopsies
-Pouch Biopsy
-Incisional Biospy
Oral Cytology of Lesions

State of the Art: Oral Cytology


Oral cytology = Exfoliative cytology, Pap Smear
Journal of the American Dental Association
Oral cytology should be a part of every oral
examination in which the dentist detects even the
least suspicious lesion-recommendations
published 30 years ago.

Some of the Problems: Oral Cytology


-10% of all dentists have ever done an oral

cytology smear
-42% were ever taught how to do a smear
-96.9 % of dental offices lack necessary
materials
Horowitz, et. al. JADA:131: 453-462, 2000

Determination of Malignancy
Evaluation of current lesion for malignancy
-analysis dependent on nuclear staining, pap
stain, toluidine blue, feulgen stain
-morphology-nuclear cytoplasmic ratio, bizarre
mitoses, micronuclei
Lack of specific genetic and molecular markers

Present Indications for Oral Cytology


A mucosal lesion is present but it
appears clinically innocuous and
otherwise would not be biopsied
Evaluation of an extensive mucosal
lesion when not possible to obtain
adequate sampling.

Additional Uses for Oral


Cytology
Patient too fragile for surgical biospy of lesion
or patient refuses surgery.
Follow-up for patients with a prior diagnosis of
premalignant or malignant lesion
Follow-up with patients, analyze single sites of
suspicion

NEED TO:
Combine current genetic and molecular
markers with the advantages of oral
cytology.
Screen for the risk for cancer before the
presence of a lesion.

Novel Extension of Current Method

Oral Cells
From Brush

Flow Cytometric
Analysis
1. DNA
Content-Ploidy
2. Cell Cycle,Apoptosis,
etc.

Phosphate Buffered Saline pH 7.4

Characteristics of Oral Cytology Samples


Viable cell number (Trypan blue dye exclusion (0.25%):
Smokers-2.6 X106 cells/ml. Among nucleated cells
16-25% non-viable,>80% viable.
Non-smokers-9.2X106 cell/ml. 5-8% non-viable,>90% viable.
Toluidine blue-Papanicolaou staining
Smokers-40-60% (red hue,upper layer),40-60% (blue hue,
lower layer, Nucleated cells about 90 -98%)
Non-smokers-80-90%(red hue, upper layer),10-20% (blue hue,
lower layer,Nucleated cells about 60 -85%)
Histomorphometric analysis: Kappa statistics analysis using
blinded determination for criteria: nuclear cytoplasmic reversal,
Hyperchromatism, pleomorphism, anaplasia, bizarre mitoses
And keratotic cells. 0,1 to 5 indicating relative scale % of cells

SIGNIFICANCE TO EXTENDED
ORAL CYTOLOGY METHODS
Non-invasive
Low cost
Sensitive
Reliable
Consistent
HIGH CORRELATION TO RISK (requires more
study)
Relevant to risk for other tobacco cancers (e.g.,
Lung, bladder, etc.)

Additional Validation Procedures


Clinical assessment among smokers of:
premalignant malignant lesion-laser
microdissection,
single cell suspensions,
DNA content staining, analysis using flow
and laser scanning cytometry
Exposure of keratinocytes in laboratory to
tobacco parent (B[a]P) and diol epoxide.
Cells analyzed using identical flow and laser
scanning procedures.

Non-smoker

8-OHdG Detection

(60-70%Nucleated)

Smoker
(90-95%Nucleated)

(3)Smoker

(3) Non-smoker

Mean %
44.26

3.14

Conclusion
Oral cytology which is relatively noninvasive, and low cost can provide a
genetic and molecular survey approach
of various markers linked to increased
risk for oral cancer
A base line of genetic and molecular
status can be obtained before a lesion is
observed. This information can be
associated with disease risk.
Prevention methods such as tobacco
control and chemoprevention can be
tested

Future Studies
Oral cytology validation requires further
study with a larger population of smokers,
former smokers, and non-smokers.
Development of novel approaches to regulate
tobacco carcinogen metabolism by controlling
oral bacteria
Synthesize novel chemoprevention agents
Molecular manipulation of proteins that block
carcinogen DNA damage

Future Studies
UCLA researchers report they can
measure elevated levels of four distinct
cancer-associated molecules in saliva and
distinguish with 91% accuracy between
healthy individuals and those diagnosed
with SCC using mRNA
Highlights the potential clinical value of
saliva as a diagnostic biofluid
http://www.nidcr.nih.gov/NewsAndReports/NewsRelease12202004.htm

Role for the Health Professional


Screen patients at risk
Provide dental care to improve response to
cancer treatment
Treat oral complications
Provide referral to other specialists

Prevention A Key Role for the


Health Professional
Health professionals will use oral cells to
Screen for an array of genetic and molecular
disorders
Assess prevention of tobacco related cancers
by various agents
Evaluate environmental carcinogens

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