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1、Advanced Diagnostics and CytologyJoel L. Schwartz, D.M.D., .Director of Oral Maxillofacial PathologyUniversity of Illinois at ChicagoCollege of DentistryNew DirectionsThe future of oral and pharyngeal cancers is prevention New screening techniques are progressing that allow researchers to evaluate t

2、he risk prior to developing lesionsOral cytology testing using cells from the tongue is both cost-effective and accurateResearchers from UCLA report early success using saliva to detect oral cancerA Mechanism for Oral Cancer Development Damage to DNAHPVEnvironmental CarcinogensTobacco CarcinogensAlc

3、ohol AbuseDNA Repair Cell Growth RegulationDNA Content Apoptosis Nuclear Instability Oral CancerLong 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 hu

4、man oral keratinocytesFurther evaluate markers during low dose oral carcinogenesis and inhibitionInvestigate 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 individual

5、s before lesions appearhelp monitor therapy Why Do We Want Markers?Tools for Studying Oral Cancer Prevention, Detection and TreatmentCells- Growth of well differentiated oral keratinocytes (normal, premalignant, malignant)-Transformation with HPV Transformation with PAH, tobacco carcinogen, Betal Nu

6、tAnimal modelsTobacco carcinogen induction of oral cancerHigh Risk Types:16,18Lower Risk:6,11,31Estimated: 35-55% of oral cancers positive for HPV70 subtypes documentedHuman PapillomavirusHPV+No CancerHPV 16 Role in Oral CancerHPV+Tobacco or Environmental Carcinogen + Infection #2 Oral CancerSquamou

7、s Papilloma:Most common in 30 - 50 yr oldsEqually in males and femalesHPV-6,11 in 50% of the lesionsTongue and soft palate common sites Papilloma Lesions of the Oral CavityFinger-like projections with fibrovascular coreVerruca Vulgaris(Common Wart)Common Wart:Found in children and middle ageFound fr

8、equently on vermillion border,labial mucosa, or anterior tongueHPV-2,4,40Finger like projections with chronic inflammatory cellsCup-like appearanceKoilocytesEosinophilic intranuclear viral inclusionsSTD associated lesion.Mouth and genitalia. HPV-6,11,16, 18Koilocytes with keratohyalin granulesCondyl

9、oma Acuminatum (Venereal Wart)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)HPVHPVHPVNo o

10、ral cancer formationPAHPAHPAHPAHPAHPAHORAL CANCER FORMATION ConclusionsThe 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 cellCommon Interaction Sites of HPV and Tobacco CarcinogensA r

11、egulation 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 Modeland Inhibi

12、tion of Oral Carcinogenesis Tobacco CarcinogensEarly Events Later EventsInitiationPromotion Cancer Formation Mechanism For Induction and Prevention of Oral CarcinogenesisDNA DamageDNA RepairCell GrowthDNA ContentApoptosisNuclear InstabilityVEas Administration Inhibits Oral Carcinogenesis Reduced DNA

13、 Damage Increased/Decreased Repair Decreased Cell GrowthReduced DNA Content Increased Apoptosis Reduced Nuclear InstabilityClinical Translational Early Screening Studies We need to:Screen before a lesion is observed Change behaviorProvide prevention treatmentVariations of Oral Squamous Carcinoma Pre

14、sentationsFactors Influencing Mortality and Survival Time of diagnosis Access to treatment Success of treatment State of health at initial detectionNo 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

15、Lesion AppearsScreening and Detection of Oral CancerOral Biopsies-Pouch Biopsy-Incisional Biospy Oral Cytology of Lesions State of the Art: Oral CytologyOral cytology = Exfoliative cytology, “Pap Smear“Journal of the American Dental Association“Oral cytology should be a part of every oral examinatio

16、n in which the dentist detects even the least suspicious lesion-recommendations published 30 years ago.Evaluation of current lesion for malignancy-analysis dependent on nuclear staining, pap stain, toluidine blue, feulgen stain-morphology-nuclear cytoplasmic ratio, bizarre mitoses, micronucleiLack o

17、f specific genetic and molecular markers Determination of MalignancyPresent Indications for Oral CytologyA mucosal lesion is present but it appears clinically innocuous and otherwise would not be biopsiedEvaluation of an extensive mucosal lesion when not possible to obtain adequate sampling. Additio

18、nal Uses for Oral CytologyPatient too fragile for surgical biospy of lesion or patient refuses surgery. Follow-up for patients with a prior diagnosis of premalignant or malignant lesionFollow-up with patients, analyze single sites of suspicionNEED TO:Combine current genetic and molecular markers wit

19、h the advantages of oral cytology.Screen for the risk for cancer before the presence of a lesion.Oral Cells From BrushPhosphate Buffered Saline pH 7.4Flow Cytometric AnalysisDNA Content-Ploidy2. Cell Cycle,Apoptosis, etc.Novel Extension of Current MethodCharacteristics of Oral Cytology SamplesViable

20、 cell number(Trypanblue dye exclusion (0.25%): Smokers-2.6 X106cells/ml. Among nucleated cells 16-25% non-viable,80% viable. Non-smokers-9.2X106cell/ml. 5-8% non-viable,90% viable.Toluidineblue-Papanicolaou stainingSmokers-40-60% (red hue,upper layer),40-60% (blue hue, lower layer, Nucleated cells a

21、bout 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 ke

22、ratotic cells. 0,1 to 5 indicating relative scale % of cells SIGNIFICANCE TO EXTENDED ORAL CYTOLOGY METHODSNon-invasiveLow costSensitiveReliableConsistentHIGH CORRELATION TO RISK (requires more study)Relevant to risk for other tobacco cancers (e.g., Lung, bladder, etc.)Additional Validation Procedur

23、esClinical assessment among smokers of:premalignant malignant lesion-laser microdissection, single cell suspensions, DNA content staining, analysis using flow and laser scanning cytometryExposure of keratinocytes in laboratory to tobacco parent (BaP) and diol epoxide. Cells analyzed using identical

24、flow and laser scanning procedures.Non-smoker (60-70%Nucleated)Smoker (90-95%Nucleated) (3)Smoker (3) Non-smoker Mean % 44.26 3.148-OHdG Detection ConclusionOral cytology which is relatively non-invasive, and low cost can provide a genetic and molecular survey approach of various markers linked to i

25、ncreased risk for oral cancerA 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 testedFuture 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 controlli