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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
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
HumanPapillomavirus 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
Mechanism For Induction and Prevention ofOral Carcinogenesis Early Events Later Events Initiation Promotion Cancer Formation DNA Damage DNA Repair DNA Content Nuclear Instability Apoptosis Cell Growth 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: • 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 OralCancer • 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.
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 lesionis 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.
AdditionalUses 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 ofCurrentMethod Oral Cells From Brush • Flow Cytometric Analysis • 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 cell/ml. 5 - 8% non - viable,>90% viable. 6 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.)
AdditionalValidation 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) 8-OHdG Detection Smoker (90-95%Nucleated) (3)Smoker (3) Non-smoker Mean % 44.26 3.14
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
FutureStudies • 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