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

• 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

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

To establish a set of markers to screen at risk individuals for oral cancer before a lesion is observed
•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)

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

Estimated: 35-55% of oral cancers positive for
HPV
70 subtypes documented
High Risk
Types:
16,18
Lower Risk:
6,11,31

HPV+
HPV 16 Role in Oral Cancer
No Cancer
HPV+Tobacco or Environmental Carcinogen + Infection #2
Oral Cancer

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

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
HPV
HPV
No oral cancer formation
ORAL CANCER FORMATION
PAH
PAH
PAH
PAH
PAH
PAH

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

• 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 Later Events
Initiation Promotion
DNA Damage DNA Repair
Cancer Formation
DNA
Content
Apoptosis
Nuclear
Instability
Cell Growth
VE as
Administration Inhibits Oral Carcinogenesis
Reduced DNA Damage Increased/Decreased Repair
Decreased
Cell Growth
Reduced DNA Content Increased Apoptosis Reduced
Nuclear
Instability

Clinical Translational
Early Screening Studies

• Screen before a lesion is observed
• Change behavior
• Provide prevention treatment

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.

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

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

• 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

• 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.

•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

• 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 X10 6 cells/ml. Among nucleated cells
16-25% non-viable,>80% viable.
Non-smokers-9.2X10
6 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

• 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.)

• 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
(60-70%Nucleated)
8-OHdG Detection
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

• 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

• 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

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

• 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