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© 2010 College of American Pathologists. All rights reserved. 1
This talk on “Bethesda System:
Integrating Cytology and HPV
Molecular Testing” is presented by
Mark H. Stoler, MD, FCAP
Your host is Jill Kaufman, PhD.
For comments about this webinar
or suggestions for upcoming
webinars, please contact
Jill Kaufman at [email protected]
THE WEBINAR WILL BEGIN MOMENTARILY. ENJOY!
Welcome to the PHC Webinar Series
© 2010 College of American Pathologists. All rights reserved. 2
Mark H. Stoler, MD, FCAP
• Professor of Pathology and Clinical Obstetrics and
Gynecology at the University of Virginia Health
System
• Associate Director of Surgical Pathology and
Cytopathology
• Board of Directors and is the Immediate Past-
President of the American Society for Clinical
Pathology
• Steering Committee and Pathology Quality Control
Committee for the NCI-sponsored ASCUS-LSIL Triage
Study
• author or co-author of over 200 peer-reviewed
publications and several book chapters
• editor-in-chief of Diagnostic Molecular Pathology
and the International Journal of Gynecological
Pathology; Associate Editor for the 5th Edition of
Sternberg’s Diagnostic Surgical Pathology
THE TRUE MEANING OF BETHESDA SYSTEM DIAGNOSES:
USING CYTOLOGY AND HPV TESTING TO PREDICT RISK OF CIN3
Mark H. Stoler, MD
• The College does not permit reproduction of any substantial
portion of the material in this Webinar without its written
authorization. The College hereby authorizes attendees of the
CAP Webinar to use the pdf presentation solely for educational
purposes within their own institutions. The College prohibits use
of the material in the Webinar – and any unauthorized use of the College’s name or logo – in connection with promotional
efforts by marketers of laboratory equipment, reagents, materials, or services.
• Opinions expressed by the speaker are the speaker’s own and do not necessarily reflect an endorsement by CAP of any
organizations, equipment, reagents, materials or services used by participating laboratories.
© 2010 College of American Pathologists. All rights reserved. 4
Declaration of COI
Consultant
Merck, Roche Molecular Systems, GenProbe, Hologic, Becton Dickinson,
Ventana Medical Systems and mtm Laboratories
The Bethesda System for Reporting
Cervical Cytology
Atypical squamous cells (ASC)of undetermined significance (ASC-US)cannot exclude HSIL (ASC-H)
Epithelial abnormalities: squamous (SIL)Low-grade squamous intraepithelial lesion (LSIL)High-grade squamous intraepithelial lesion (HSIL)Squamous cell carcinoma
Criteria Based Diagnosis
Wright and Schiffman (2003) NEJM
Natural History of HPV Infections
HPV Epithelial Biology
There are three basic biologic states:
Normal
plus infections, reactions, and neoplasia mimics
Low Grade = Viral infection/production
Characterized by viral and morph. transience
High Grade = HPV oncogene driven proliferation.
Characterized by viral and morph. persistence
Management Based on Risk of Precancer
There are only three management choices
INTERVAL FOLLOW UP AND REPEAT
COLPOSCOPY
DEFINITIVE THERAPY
Management Based on Risk of Precancer
There are only three management choices
<5%
5-50%
>50%
Squamocolumnar Junction
Normal = Orderly Squamous Maturation
NORMAL SQUAMOUS CELLS
Superficial and Intermediate Squamous Cells with Squamous Metaplasia
Parabasal Cells (Atrophy)
Normal
Normal
Normal
Normal
• Slow cell proliferation confined to parabasal zone
• Orderly coordinated nuclear maturation
• Orderly coordinated cytoplasmic maturation
• Absence of detectable HPV replication and
expression
Limitations of Cyto/Histomorphology
• How completely does the sample represent the biology?
• Lesion size and location
• Sampling
o Collector skill, sampling methods, # of samples, interpretive variability
• Pathology
o Cell locator function
o Cell and tissue interpretive variability
Probability of CIN2+
• Pre-colposcopic probability of biopsy proven
precancer (CIN2+) within 2 years of the index
Pap based on ALTS and other literature
• Am J Clin Pathol 2007;127:489-491
Probability of CIN2+: Normal
DX 18-25 26-35 36-45 >45Normal 0-5 0-5 0-5 0-5
Normal
HPV-
0-1 0-2 0-2 0-2
Normal
HPV+
5-10 5-10 5-10 5-10
The Bethesda System for Reporting
Cervical Cytology
Atypical squamous cells (ASC)of undetermined significance (ASC-US)cannot exclude HSIL (ASC-H)
Epithelial abnormalities: squamous (SIL)Low-grade squamous intraepithelial lesion (LSIL)High-grade squamous intraepithelial lesion (HSIL)Squamous cell carcinoma
LSIL
LOW-GRADE SQUAMOUS INTRAEPITHELIAL LESION
LOW-GRADE SQUAMOUS INTRAEPITHELIAL LESION
LOW GRADE SQUAMOUS
INTRAEPITHELIAL LESION
• Single cells and sheets• Abundant mature, well-defined cytoplasm• Nuclear enlargement• Variable nuclear hyperchromasia• Variations in nuclear size, number and shape• Evenly distributed chromatin• Nuclear membranes even or slightly irregular• Nucleoli inconspicuous or absent• Perinuclear cavitation (koilocytosis)
LSIL = CIN 1
HPV DNA in LSIL
LSIL p16
LSIL = Differentiation Dependent HPV Expression
Low Grade
• Differentiation dependent HPV gene expression
• E6/E7 expression under coordinate control
• Proliferation still confined to parabasal zone
• Abnormal nuclear & cytoplasmic maturation
• Viral productive phenotype
• Koilocytotic atypia is the histologic and cytologic hallmark
• Mild dysplasia = CIN 1 = LSIL
• No glandular correlate
Limitations of Cyto/Histomorphology
• How completely does the sample represent the biology?
• Lesion size and location
• Samplingo Collector skill, sampling methods, # of samples,
interpretive variability
• Pathologyo Cell locator function
o Cell and tissue interpretive variability
Probability of CIN2+: LSIL
DX 18-25 26-35 36-45 >45LSIL 25-30 20-25 20-25 15-20
The Bethesda System for Reporting
Cervical Cytology
Atypical squamous cells (ASC)of undetermined significance (ASC-US)cannot exclude HSIL (ASC-H)
Epithelial abnormalities: squamous (SIL)Low-grade squamous intraepithelial lesion (LSIL)High-grade squamous intraepithelial lesion (HSIL)Squamous cell carcinoma
HSIL
HIGH-GRADE SQUAMOUS INTRAEPITHELIAL LESION
HIGH-GRADE SQUAMOUS INTRAEPITHELIAL LESION
HIGH GRADE SQUAMOUS
INTRAEPITHELIAL LESION
• Cells smaller, less mature; cytoplasm variable• Single cells, sheets, syncytial-like aggregates• Hyperchromatic crowded groups• Hyperchromasia, variation in size and shape• Nuclear enlargement, increase in N/C ratios• Fine or coarsely granular, evenly distributed
chromatin; nucleoli absent• Irregular nuclear membranes
CIN3 = HSIL
HPV 16 mRNAs in HSIL
HSIL p16
HSIL = Proliferative Phenotype Driven by E6/E7 Expression
High Grade
• HPV oncogene driven cell proliferation
• Implies E6/E7 expressed in cells that can proliferate, coordinate control is broken
• Proliferating cells now clonally expand beyond normal compartment
• Lack of maturation, disordered growth, little differentiation, no viral production
• Proliferation dominates, mitotic activity
• Both squamous and glandular counterparts
• The immediate cancer precursor
• CIN 2/3 = moderate to severe dysplasia = carcinoma in situ = HSIL
CIN3 is In SITU CANCER
Invasive Keratinizing Carcinoma
Limitations of Cyto/Histomorphology
• How completely does the sample represent the biology?
• Lesion size and location
• Samplingo Collector skill, sampling methods, # of samples,
interpretive variability
• Pathologyo Cell locator function
o Cell and tissue interpretive variability
Probability of CIN2+: HSIL out of ASC
DX/ 18-25 26-35 36-45 >45HSIL(AS
C)
65-70 65-70 60-70 40-50
Probability of CIN2+: HSIL
DX 18-25 26-35 36-45 >45HSIL 80-85 85-90 85-90 80-85
HSIL(AS
C)
65-70 65-70 60-70 40-50
The Bethesda System for Reporting
Cervical Cytology
Atypical squamous cells (ASC)of undetermined significance (ASC-US)cannot exclude HSIL (ASC-H)
Epithelial abnormalities: squamous (SIL)Low-grade squamous intraepithelial lesion (LSIL)High-grade squamous intraepithelial lesion (HSIL)Squamous cell carcinoma
Equivocal Categories
• ASC-US, AGC-US, CIN2
• Atypia means morphologic changes less than
dysplasia/CIN/SIL
• Atypias - not diagnostic for true biology
• Inherent poor reproducibility
• Often a function of sample adequacy
• Important safety buffers
• Trade off of sensitivity vs. specificity
The Bethesda System for Reporting
Cervical Cytology
Atypical squamous cells (ASC)of undetermined significance (ASC-US)cannot exclude HSIL (ASC-H)
Epithelial abnormalities: squamous (SIL)Low-grade squamous intraepithelial lesion (LSIL)High-grade squamous intraepithelial lesion (HSIL)Squamous cell carcinoma
ATYPICAL SQUAMOUS CELLS OF
UNDETERMINED SIGNIFICANCE (ASC-US)
ATYPICAL SQUAMOUS CELLS OF
UNDETERMINED SIGNIFICANCE (ASC-US)
ATYPICAL SQUAMOUS CELLS OF
UNDETERMINED SIGNIFICANCE (ASC-US)
• Nuclear enlargement (2 ½ - 3X normal)• Slight increase in N/C ratios• Minimal nuclear hyperchromasia• Minimal irregularity in chromatin distribution or
nuclear shape• Minimal nuclear abnormalities associated with
dense orangeophilic cytoplasm
Probability of CIN2+: ASC
DX 18-25 26-35 36-45 >45ASC 15-20 10-15 5-10 5-10
ASC
HPV+
25-30 20-25 10-20 10-20
The Bethesda System for Reporting
Cervical Cytology
Atypical squamous cells (ASC)of undetermined significance (ASC-US)cannot exclude HSIL (ASC-H)
Epithelial abnormalities: squamous (SIL)Low-grade squamous intraepithelial lesion (LSIL)High-grade squamous intraepithelial lesion (HSIL)Squamous cell carcinoma
ATYPICAL SQUAMOUS CELLS, CANNOT
EXCLUDE HSIL (ASC-H)
ASC-H: Atypical Immature Squamous Metaplasia
ATYPICAL SQUAMOUS CELLS,
CANNOT EXCLUDE HSIL (ASC-H)
• Small cells singly or in small groups (atypical immature squamous metaplasia)
• Nuclei are 1 ½ to 2 ½ X normal• N/C ratios similar to that of HSIL• Nuclear abnormalities < HSIL• Hyperchromatic crowded groups
Probability of CIN2+: ASC-H
DX 18-25 26-35 36-45 >45ASC-H 45-50 60-65 30-40 30-40
LSIL 25-30 20-25 20-25 15-20
Hyperchromatic Crowded Groups = ???
ENDOCERVICAL ADENOCARCINOMA IN-SITU
HGIL
HPV oncogene driven AGC
HGIL
HPV oncogene driven AGC
HPV 18 expression in Adenocarcinoma
Hyperchromatic Crowded Groups
ASC-H HSIL
ENDOCERVICAL AIS AND HSIL
Limitations of Cyto/Histomorphology
• How completely does the sample represent the biology?
• Lesion size and location
• Samplingo Collector skill, sampling methods, # of samples,
interpretive variability
• Pathologyo Cell locator function
o Cell and tissue interpretive variability
Probability of CIN2+:AGC
DX 18-25 26-35 36-45 >45AGC 40-70 50-70 60-70 60-80
Probability of CIN2+
DX 18-25 26-35 36-45 >45Normal 0-5 0-5 0-5 0-5
Normal HPV- 0-1 0-2 0-2 0-2
Normal HPV+ 5-10 5-10 5-10 5-10
ASC 15-20 10-15 5-10 5-10
ASC HPV+ 25-30 20-25 10-20 10-20
LSIL 25-30 20-25 20-25 15-20
ASC-H 45-50 60-65 30-40 30-40
AGC 40-70 50-70 60-70 60-80
HSIL(ASC) 65-70 65-70 60-70 40-50
HSIL 80-85 85-90 85-90 80-85
DX 18-25 26-35 36-45 >45Normal 0-5 0-5 0-5 0-5
Normal HPV- 0-1 0-2 0-2 0-2
Normal HPV+ 5-10 5-10 5-10 5-10ASC 15-20 10-15 5-10 5-10ASC HPV+ 25-30 20-25 10-20 10-20
LSIL 25-30 20-25 20-25 15-20
ASC-H 45-50 60-65 30-40 30-40AGC 40-70 50-70 60-70 60-80HSIL(ASC) 65-70 65-70 60-70 40-50HSIL 80-85 85-90 85-90 80-85
Management Based on Risk of Precancer
There are only three management choices
<5%
5-50%
>50%
Management Based on Risk of Precancer
There are only three management choices
INTERVAL FOLLOW UP AND REPEAT
COLPOSCOPY
DEFINITIVE THERAPY
HPV Epithelial Biology
There are three basic biologic states:
Normal
plus infections, reactions, and neoplasia mimics
Low Grade = Viral infection/production
Characterized by viral and morph. transience
High Grade = HPV oncogene driven prolif.
Characterized by viral and morph. persistence
Wright and Schiffman (2003) NEJM
Natural History of HPV Infections
Two Tiered SIL: LSIL / HSIL
Scientific Rationale
• Data over last 10 years support modified paradigm for cervical cancer pathogenesis and confirms LSIL/HSIL dichotomy as scientifically valid
• Better diagnostic reproducibility (Kappa) for Bethesda cut points
The morphologic interpretation is only the beginning…
Next in the Series of Free PHC
Webinars
© 2010 College of American Pathologists. All rights reserved. 80
Source: Century Gothic, 9 pt, sentence case
• Molecular Markers in Breast Cancer—Thursday, January 20th, 11 am-
12 pm CT
o David Hicks, MD, FCAP
• Go to www.cap.org/institute For All Upcoming Webinars!
• Past Webinars Available Now Online at www.cap.org/instituteo Molecular Diagnosis for Lung Cancero Molecular Diagnosis for Colorectal Cancero Endoscopic Microscopy: Bridging the Radiology/Pathology Divide
Considerations in Setting up a Biorepositoryo Personalized Pathology: PHC in the General Pathology Practiceo Introduction to the Medical Homeo Personalized Medicine: Framing the Issues for Pathologyo Clinical Requests for Molecular Tests