Advanced Diagnostics Advanced Diagnostics and Cytologyand Cytology

Joel L. Schwartz, D.M.D., D.M.Sc.Director of Oral Maxillofacial Pathology

University of Illinois at ChicagoCollege of Dentistry

New DirectionsNew 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 A Mechanism for Oral Cancer DevelopmentDevelopment

Damage to DNAHPV

Environmental Carcinogens

Tobacco Carcinogens

Alcohol Abuse

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:Long Term Goal: To establish a set of markers to screen at risk individuals for oral

cancer before a lesion is observed

Approach: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)

Markers are required to:

•reduce the mortality rate among oral cancer patients (50% 5 year survival)

•screen individuals before lesions appear

•help monitor therapy

Why Do We Want Markers?Why Do We Want Markers?

Tools for Studying Oral Cancer Tools for Studying Oral Cancer Prevention, Detection and TreatmentPrevention, 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

High Risk Types:

16,18

Lower Risk:

6,11,31

Estimated: 35-55% of oral cancers positive for HPV

70 subtypes documented

HumanHuman PapillomavirusPapillomavirus

HPV+ No Cancer

HPV 16 Role in Oral CancerHPV 16 Role in Oral 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

Papilloma Lesions of the Oral CavityPapilloma Lesions of the Oral Cavity

Finger-like projections with fibrovascular core

Verruca Vulgaris(Common Wart)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

STD associated lesion.

Mouth and genitalia.

HPV-6,11,16, 18

Koilocytes with keratohyalin granules

Condyloma Acuminatum (Venereal Wart)Condyloma Acuminatum (Venereal Wart)

Oral Keratinocyte Laboratory Response Oral Keratinocyte Laboratory Response to HPV Infection and/or PAH Exposureto 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

HPV

HPV

No oral cancer formation

PAH

PAH

PAH

PAHPAH

PAH

ORAL CANCER FORMATION

ConclusionsConclusions

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 Common Interaction Sites of HPV and Tobacco Carcinogensand 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

Early Events Later Events

Initiation Promotion Cancer Formation

Mechanism For Induction and Prevention of Oral Carcinogenesis

DNA Damage DNA Repair

Cell Growth

DNA Content

Apoptosis Nuclear Instability

VEas 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

We need to:need to:

•Screen before a lesion is observed

•Change behavior

•Provide prevention treatment

Variations of Oral Squamous Variations of Oral Squamous

Carcinoma PresentationsCarcinoma Presentations

Factors Influencing Mortality Factors Influencing Mortality

and Survivaland 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 Early Screening and Detection of Oral Mucosa Changes Before A Oral Mucosa Changes Before A

Lesion AppearsLesion Appears

Screening and Detection of OralScreening and Detection of Oral CancerCancer

•Oral Biopsies

-Pouch Biopsy

-Incisional Biospy

•Oral Cytology of Lesions

State of the Art: Oral CytologyState 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.

-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

Some of the Problems: Oral Cytology

•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

Determination of MalignancyDetermination of Malignancy

Present Indications for Oral CytologyPresent 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.

AdditionalAdditional Uses for Oral Uses for Oral CytologyCytology

•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: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.

Oral Cells

From Brush

Phosphate Buffered Saline pH 7.4

Flow Cytometric Analysis

1. DNA Content-”Ploidy”

2. Cell Cycle,Apoptosis, etc.

Novel Extension ofNovel Extension of CurrentCurrent MethodMethod

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 stainingSmokers-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 mitosesAnd keratotic cells. 0,1 to 5 indicating relative scale % of cells

SIGNIFICANCE TO EXTENDED SIGNIFICANCE TO EXTENDED ORAL CYTOLOGY METHODSORAL 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.)

AdditionalAdditional Validation ProceduresValidation 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

(60-70%Nucleated)

Smoker

(90-95%Nucleated)

(3)Smoker (3) Non-smoker

Mean %

44.26 3.14

8-OHdG Detection

Conclusion

•Oral cytology which is relatively non-invasive, 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

FutureFuture StudiesStudies

•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 StudiesFuture 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 ProfessionalRole 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 thePrevention A Key Role for the

Health ProfessionalHealth 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