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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 jkaufma@cap.org

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