Rossitza Tacheva, MDAssistant Professor of Pathology, Anatomy

and Cell Biology

Gynecologic Cytopathology

OverviewCervical cancer screeningThe conventional Pap test Liquid based PapBethesda System for reporting Pap testExamples of normal and abnormal PapHPV and cervical cancerClinical utilization of HPV testingScreening guidelines in USACervical cancer prevention in the future

Screening Cervical CancerScreening Cervical Cancer

The Pap Test has been the most successful cancer screening program in history

Cervical cancer incidence and mortality have dropped in all populations with organized screening programs

In unscreened populations death from cervical cancer remains high, especially in developing countries

PAP (Papanicolaou) TestPAP (Papanicolaou) TestEarly detection of cervical cancer precursor lesions

(dysplastic changes) is the rationale for the Pap testNatural progress of invasive cervical cancer from pre-

invasive to invasive disease (average time 10 y) Cervical precancerous lesions are associated with

abnormalities in Pap smears that can be detected long before any abnormality is visible on gross inspection

Relatively easy to obtain the material and inexpensiveThe association with molecular test for HPV further

increases the sensitivity in the detection of lesionsSuccessfully treated

Rates Cervical Cancer Rates cervical cancer

Goldie et al Vaccine 2006

Cervical cancer USA 2014Cervical cancer USA 2014

13,000 cases per year4,000 deaths

Cervical Cancer ScreeningCervical Cancer Screening

Over time, cervical cancer screening has evolved from a single glass slide smear to a test involving liquid-based processing and molecular HPV testing

DR. PAPANICOLAOU INTRODUCED THE PAP TEST IN 1941

CONVENTIONAL PAP TEST

Pap TestPap Test

Most Pap tests in the United States are now processed from a liquid-based medium as opposed to having the cells smeared directly onto a glass slide

PAP TEST-liquidbased

THINPREP

PAP TEST-liquid-based

THINPREP

An Important ChangeAn Important Change

Cells are collected

in a vial of Preservcyt® solution

Fully automated

Minimizes blood, mucus, non-diagnostic debris

Dispersion Cellcollection

Cell transfer

The ThinPrep 2000 ProcessorThe ThinPrep 2000 Processor

The ThinPrep ProcessThe ThinPrep Process

CONVENTIONAL PAP SMEAR

THINPREP

Endocervical cells

HPV and Cervical CancerIn the last decade it has become clear that

infection with HIGH RISK HPV is required for the development of high grade precursor lesions and cervical cancer.

Dr. Harald zur Hausen in 2008 won Nobel Prize for demonstrating HPV as the etiological agent of cervical cancer.

Cervical Cancer Screening

Recent emphasis on molecular testing seeks to identify HPV strains considered high risk for carcinogenesis

HPV TypesHPV Types

Low- risk: 6,11,40,42,43,44,53,54,61,72,73,84

High-risk:16,18,31,33,35,39,45,51,52,56,58,59,68,82

Possible high-risk: 26,66,73

High-Risk HPV TypesHigh-Risk HPV TypesHPV 16 is found in half of all women with

cervical cancer; also the most commonly identified type in HGSIL and among women in the general population

The second most common HR virus is HPV 18

16 and 18 account for approximately 70% of cases of cervical intraepithelial neoplasia (CIN) and cervical carcinoma

HR HPV incorporate into the genome of the host cell

Low –Risk HPVLow –Risk HPV

HPV 6,11 are the most common typesAssociated with genital warts (condylomas)

of the lower genital tractDo not integrate into the host genome,

remaining instead as free episomal viral DNA

HPV InfectionHPV Infection

Currently HPV vaccination exist!

GARDASIL®Human Papillomavirus Quadrivalent (Types 6, 11, 16, and 18)

CERVARIX Human Papillomavirus Bivalent (Types 16 and 18)

Persistent HPV InfectionPersistent HPV Infection

Most HPV infections are transient and are eliminated within months by an acute and chronic inflammatory response

Half are persistent at 6 monthsOne third are persistent at 12 months9-20% are still persistent at 24 monthsSome of these progress to cervical

intraepithelial neoplasia (CIN), a precursor lesion from which most invasive cervical carcinomas develop

PathogenesisPathogenesis

HR HPVs express two potent oncoproteins encoded in the HPV genome called E6 and E7

The E6 and E7 oncoproteins bind and inactivate two critical tumor suppressors, p53 and Rb and promote growth and increased susceptibility to additional mutations that may eventually lead to carcinogenesis

Methods for HPV DNA Methods for HPV DNA TestingTesting

Residual material of liquid-based Pap test can be used for detection of high risk HPV DNA

Collection swab can also be used

Methods for HPV DNA TestingHybrid Capture 2

FDA approval 1999, used widely in clinical diagnostics laboratories

CervistaFDA approval 2009 for two tests:Detection of HR HPVGenotype 16 & 18Used in clinical diagnostics laboratories as well

PCR-COBAS 4800FDA approved 2011 for detection and genotyping

16 & 18Currently the only test approved for primary

screening

Key Objective HPV DNA TestingKey Objective HPV DNA Testing

Identify those women who have persisting HR HPV infections and are at risk for developing neoplasia

Women who become HPV DNA negative on follow-up are at low risk for having HSIL

Liquid based cytology or co-collection swab

required (cannot be done on conventional smears)

Clinical utilization of HR HPV DNA Clinical utilization of HR HPV DNA asas adjunct test to the Pap adjunct test to the Pap

Triage of women(>21) with ASCUS resultsPost-treatment surveillance of women with CIN2-3Adjunct to Pap test in primary cancer screening women 30 years or older

The Bethesda System for The Bethesda System for Reporting Cervical CytologyReporting Cervical Cytology

Bethesda System 2014Bethesda System 2014

Specimen AdequacySatisfactory -Sufficient number of well visualized epithelial cells

Unsatisfactory-Too few cells or more than 75% obscured by inflammation, blood

-Unlabeled

Interpretation / ResultInterpretation / Result

Negative for intraepithelial lesion or malignancy

Organisms

Epithelial abnormalities

OrganismsOrganisms

Trichomonas vaginalisFungal organisms morphologically

consistent with Candida sppShift in vaginal flora suggestive of bacterial

vaginosisBacteria morphologically consistent with

Actinomyces sppCellular changes consistent with Herpes

simplex virusCellular changes consistent with

Cytomegalovirus

TRICHOMONADS

CANDIDA SPP

HERPES SIMPLEX

• Squamous Cell• Atypical squamous cells of Undetermined Significance(ASCUS) cannot exclude HSIL (ASC-H)• Low Grade Squamous Intraepithelial lesion (LSIL)• High Grade Squamous Intraepithelial lesion (HSIL)• Squamous Cell Carcinoma

• Glandular Cell • Atypical Glandular cells (AGC) • Endocervical adenocarcinoma In Situ (AIS)• Adenocarcinoma

Bethesda System 2014 Bethesda System 2014 Epithelial Cell Epithelial Cell AbnormalitiesAbnormalities

Cervical Intraepithelial LesionCervical Intraepithelial Lesion

Is the precursor of invasive cancer

LSIL/HSIL/SCCLSIL/HSIL/SCCLow grade squamous intraepithelial

lesion (LSIL)

High grade squamous intraepithelial lesion (HSIL)

Squamous cell carcinoma

INTERRELATIONS OF NOMENCLATURE SYSTEMSINTERRELATIONS OF NOMENCLATURE SYSTEMS

IN PREMALIGNANT CERVICAL DISEASEIN PREMALIGNANT CERVICAL DISEASE

SCJ

Squamous cells

Glandular cells

Superficial cells

Intermediate cells

Endocervical cells

NORMAL PAP

Benign Epithelial Cells

Low-Grade Squamous Low-Grade Squamous Intraepithelial Lesion (LSIL)Intraepithelial Lesion (LSIL)

Features of LSIL/HPVFeatures of LSIL/HPVHPV viruses cause cytopathic changesMorphologic changes that can be

recognized under the microscopeKoilocyte- large cell with large dark

shrunken nucleus and cytoplasmic cavitation

KoilocyteKoilocyte

Features of HSILFeatures of HSIL

Immature cellsEnlarged nuclei, high nuclear to

cytoplasmic ratio (big nucleus/small cytoplasm)

Dark chromatinIrregular nuclear membrane

High-Grade Squamous High-Grade Squamous Intraepithelial Lesion (HSIL)Intraepithelial Lesion (HSIL)

High-Grade Squamous High-Grade Squamous Intraepithelial Lesion (HSIL)Intraepithelial Lesion (HSIL)

Colposcopic Guided Cervical Colposcopic Guided Cervical Biopsy Biopsy for HSIL for HSIL

Mosaic &punctation in colposcopic view CIN 3 in cervical biopsy

Invasive Squamous Cell Carcinoma

Is the most common type of cervical cancer

Features of Squamous Cell Features of Squamous Cell CarcinomaCarcinoma

Keratinized and dense cytoplasmCell pleomorphism: elongated, tadpole, ovalIrregular nuclear size and shapeIndia ink chromatin

Squamous Cell CarcinomaSquamous Cell Carcinoma

Squamous Cell CarcinomaSquamous Cell Carcinoma

Invasive Squamous Cell Invasive Squamous Cell CarcinomaCarcinoma

Ulcerated cervical tumor Cervical biopsy of tumor

Pap Test Glandular Cell Pap Test Glandular Cell AbnormalitiesAbnormalities

Detects glandular neoplams such as Endocervical carcinoma Endometrial carcinomaFeatures of Adenocarcinoma Abnormal glandular cells Variation in nuclear size Irregular chromatin distribution Prominent nucleoli Tumor necrosis

Endocervical Adenocarcinoma

Accounts for 20% of malignant cervical tumors

Atypical Endocervical Glands Atypical Endocervical Glands and Adenocarcinoma in Situand Adenocarcinoma in Situ

Endometrial Carcinoma

Is the most common gynecologic cancer in the USA

ENDOMETRIAL ADENOCARCINOMA

Use HPV co-testing in cancer screening

Approved as screening test in USA 2003In combination with Pap, not approved as

stand-aloneFor every 3-5 years useFor women 30 years old or olderEndorsed by ACS and ACOGA negative HPV test represents a low risk of

developing disease over 5 years and safely allows lengthening of testing intervals

Screening Guidelines ACS-ASCCP-ASCP<21 years: No screening21-29 years: Cytology alone every 3 years30-65 years: - HPV and Cytology every 5 years (preferred) - Cytology alone every 3 years (acceptable)>65 years: No screening following adequate negative

prior screeningAfter hysterectomy: No screening (if no history HSIL) HPV vaccinated: Follow age specific

recommendations (same as unvaccinated women) Am J Clin Pathol 2012:137:516-542

Molecular testing for primary cervical cancer screeningThe Roche Cobas 4800 became the first

HPV test approved by the FDA to replace the Pap test for primary screening of cervical cancer in 2014

The approval allows for HPV testing of women 25 and older and referral to colposcopy if the test is positive for HPV 16/18 and reflexed to a Pap test if positive for HPV but negative for HPV16/18

Guidelines document from ASCCP/SGO pending

Question

Is molecular testing more effective and efficient than co-testing for cervical cancer screening?

HPV TestingThe US Cancer Registry study has found

12.6 % of cervical cancers are either HPV negative or contain rare HPV subtypes

Quality control of HPV test : the internal control is not specific for cervical epithelial cells and could be wrongly deemed adequate

There is not sufficient information to say whether

primary HPV testing trumps co-testing

KEY Points The introduction of the Liquid based Pap improved

accuracy of the testThe Bethesda system simplified the diagnostic categories HPV is common yet it rarely can cause cancer Clinical use of HR HPV detection as an adjunct to the Pap HPV based screening program has been approved by the FDA for the Cobas HPV test in the USA No screening test is perfect The basis of screening requires doing it periodically and repeatedly whether it is a Pap test, co-testing or primary HPV screening