Welcome to the PHC Webinar Series

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!
2010 College of American Pathologists. All rights reserved. 1

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 PastPresident 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 Sternbergs Diagnostic Surgical Pathology
2

2010 College of American Pathologists. All rights reserved.

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 Colleges name or logo in connection with promotional efforts by marketers of laboratory equipment, reagents, materials, or services.

Opinions expressed by the speaker are the speakers 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.

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

Natural History of HPV Infections

Wright and Schiffman (2003) NEJM

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 Normal Normal HPVNormal HPV+ 18-25 0-5 0-1 26-35 0-5 0-2 36-45 0-5 0-2 >45 0-5 0-2

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

Sampling

o Collector skill, sampling methods, # of samples, interpretive variability o Cell locator function o Cell and tissue interpretive variability

Pathology

Probability of CIN2+: LSIL

DX LSIL 18-25 25-30 26-35 20-25 36-45 20-25 >45 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

Sampling

o Collector skill, sampling methods, # of samples, interpretive variability o Cell locator function o Cell and tissue interpretive variability

Pathology

Probability of CIN2+: HSIL out of ASC

DX/ 18-25 HSIL(AS 65-70 C) 26-35 65-70 36-45 60-70 >45 40-50

Probability of CIN2+: HSIL

DX 18-25 HSIL 80-85 HSIL(AS 65-70 C) 26-35 85-90 65-70 36-45 85-90 60-70 >45 80-85 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 - 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 ASC ASC HPV+ 18-25 15-20 25-30 26-35 10-15 20-25 36-45 5-10 10-20 >45 5-10 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 to 2 X normal

N/C ratios similar to that of HSIL Nuclear abnormalities < HSIL Hyperchromatic crowded groups

Probability of CIN2+: ASC-H

DX ASC-H LSIL 18-25 45-50 25-30 26-35 60-65 20-25 36-45 30-40 20-25 >45 30-40 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

Sampling

o Collector skill, sampling methods, # of samples, interpretive variability o Cell locator function o Cell and tissue interpretive variability

Pathology

Probability of CIN2+:AGC
DX AGC 18-25 40-70 26-35 50-70 36-45 60-70 >45 60-80

Probability of CIN2+
DX
DX Normal Normal

18-25
18-25 0-5

26-35
26-35 0-5

36-45
36-45 0-5 0-5 0-2

>45
>450-5 0-5 0-2

Normal HPVNormal HPV-

0-1

0-5 0-1

0-2 5-10 5-10 10-15 10-15

20-25 20-25 0-2

0-5

0-2

0-2

Normal HPV+ +5-10 5-10 Normal HPV ASC ASC LSIL

LSIL

5-10 5-10 5-10 5-10

10-20 10-20 20-25

5-10 5-10 5-10 5-10

10-20 10-20 15-20

15-20 15-20 25-30

25-30

25-30 ASC ASC HPV+ 25-30 HPV+

ASC-H

ASC-H AGC
HSIL HSIL(ASC)

45-50 40-70
80-85 65-70

45-50

20-25 60-65 50-70

65-70 50-70 85-90 65-70 60-65

20-25

30-40

20-25

30-40 60-70
60-70 60-70 85-90 60-70

30-40

15-20

30-40 60-80
40-50 60-80 80-85 40-50

AGCHSIL(ASC) 40-70 65-70 HSIL

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

Natural History of HPV Infections

Wright and Schiffman (2003) NEJM

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

Molecular Markers in Breast CancerThursday, January 20th, 11 am12 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/institute o Molecular Diagnosis for Lung Cancer o Molecular Diagnosis for Colorectal Cancer
o Endoscopic Microscopy: Bridging the Radiology/Pathology Divide Considerations in Setting up a Biorepository o Personalized Pathology: PHC in the General Pathology Practice o Introduction to the Medical Home o Personalized Medicine: Framing the Issues for Pathology o Clinical Requests for Molecular Tests
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2010 College of American Pathologists. All rights reserved.