8 Advanced Diagnostics and Cytology.ppt
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Transcript of 8 Advanced Diagnostics and Cytology.ppt
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