Download Advanced Diagnostics and Cytology

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
Cell
Laboratory
Studies
Pre-Clinical
Oral Cancer
Model
Clinical
Translational
Early
Screening
Studies
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
Human Papillomavirus
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
Tobacco Carcinogens
Mechanism For Induction and
Prevention of Oral Carcinogenesis
Early Events
Initiation
Later Events
Promotion
Cancer Formation
DNA Damage DNA Repair DNA
Apoptosis Nuclear
Instability
Content
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 Oral Cancer
•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.
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
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 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.
Additional Uses 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 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 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 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.)
Additional Validation 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
8-OHdG Detection
(60-70%Nucleated)
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
Future 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 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