Gynecologic Cytopathology Rossitza Tacheva, MD Assistant Professor of Pathology, Anatomy and Cell Biology

Overview Cervical cancer screening The conventional Pap test Liquid based Pap Bethesda System for reporting Pap test Examples of normal and abnormal Pap HPV and cervical cancer Clinical utilization of HPV testing Screening guidelines in USA Cervical cancer prevention in the future

Screening 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) Test Early detection of cervical cancer precursor lesions (dysplastic changes) is the rationale for the Pap test Natural 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 inexpensive The association with molecular test for HPV further increases the sensitivity in the detection of lesions Successfully treated

Rates cervical cancer Rates Cervical Cancer Goldie et al Vaccine 2006

Cervical cancer USA 2014 13,000 cases per year 4,000 deaths

Cervical 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 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-liquid based THINPREP

PAP TEST-liquid-based THINPREP PAP TEST-liquid-based

The ThinPrep Process An Important Change The ThinPrep 2000 Processor Fully automated Minimizes blood, mucus, non- diagnostic debris Cells are collected in a vial of Preservcyt® solution This slide reviews the ThinPrep process. The cervical sample is collected in the conventional manner, but rather than smeared on a glass slide, the collection device is rinsed immediately into a vial of preservative fluid. In the laboratory, the ThinPrep 2000 Processor disperses the cells in the vial and then a gentle vacuum is applied across the filter membrane to collect the cells. The pores of the filter are sized to prevent the loss of any diagnostic cells. Once a specific number of cells have been collected on the filter membrane (~ 70,000), the filter is inverted and touched to the glass slide. The entire process is monitored by computers within the ThinPrep 2000 Processor to ensure a uniform preparation. Slides are fixed in alcohol and then stained and examined in the usual manner. The ThinPrep 2000 Processor can produce approximately 25 slides per hour, and a single operator can simultaneously run up to 3 processors. The ThinPrep 3000 is a fully automated batched processor capable of processing 4 batches of 80 slides in an 8 hour shift. Cell collection Cell transfer Dispersion 13

CONVENTIONAL PAP SMEAR

Endocervical cells THINPREP

HPV and Cervical Cancer In 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 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 Types HPV 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 HPV HPV 6,11 are the most common types Associated with genital warts (condylomas) of the lower genital tract Do not integrate into the host genome, remaining instead as free episomal viral DNA

HPV 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 Infection Most HPV infections are transient and are eliminated within months by an acute and chronic inflammatory response Half are persistent at 6 months One third are persistent at 12 months 9-20% are still persistent at 24 months Some of these progress to cervical intraepithelial neoplasia (CIN), a precursor lesion from which most invasive cervical carcinomas develop

Pathogenesis 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 Testing 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 Testing Hybrid Capture 2 FDA approval 1999, used widely in clinical diagnostics laboratories Cervista FDA approval 2009 for two tests: Detection of HR HPV Genotype 16 & 18 Used in clinical diagnostics laboratories as well PCR-COBAS 4800 FDA approved 2011 for detection and genotyping 16 & 18 Currently the only test approved for primary screening

Key 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 as adjunct test to the Pap Triage of women(>21) with ASCUS results Post-treatment surveillance of women with CIN2-3 Adjunct to Pap test in primary cancer screening women 30 years or older

The Bethesda System for Reporting Cervical Cytology

Bethesda System 2014 Specimen Adequacy Satisfactory -Sufficient number of well visualized epithelial cells Unsatisfactory -Too few cells or more than 75% obscured by inflammation, blood -Unlabeled

Interpretation / Result Negative for intraepithelial lesion or malignancy Organisms Epithelial abnormalities

Organisms Trichomonas vaginalis Fungal organisms morphologically consistent with Candida spp Shift in vaginal flora suggestive of bacterial vaginosis Bacteria morphologically consistent with Actinomyces spp Cellular changes consistent with Herpes simplex virus Cellular changes consistent with Cytomegalovirus Use Organisms rather than “Infection” because presence of organism does not always indicate ‘infection’.

TRICHOMONADS

CANDIDA SPP

HERPES SIMPLEX

Epithelial Cell Abnormalities Bethesda System 2014 Epithelial Cell Abnormalities 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 10-20% of ASCUS will have underlying CIN2/3. 1/1000 ASCUS will have invasive cancer. ASC-H is a mix of true HSIL and mimics and is approximately 5-10% of all ASCUS diagnoses. LSIL is generally a transient infection with HPV while HSIL is more often associated with viral persistence and progression. High glandular or squamous lesions are seen in 10-39% of AGC paps 35

Cervical Intraepithelial Lesion Is the precursor of invasive cancer

LSIL/HSIL/SCC Squamous cell carcinoma Low grade squamous intraepithelial lesion (LSIL) High grade squamous intraepithelial lesion (HSIL) Squamous cell carcinoma

INTERRELATIONS OF NOMENCLATURE SYSTEMS IN PREMALIGNANT CERVICAL DISEASE

SCJ Squamous cells Glandular cells

NORMAL PAP Superficial cells Endocervical cells Intermediate cells Benign Epithelial Cells Superficial cells Intermediate cells Endocervical cells

Low-Grade Squamous Intraepithelial Lesion (LSIL)

Features of LSIL/HPV HPV viruses cause cytopathic changes Morphologic changes that can be recognized under the microscope Koilocyte- large cell with large dark shrunken nucleus and cytoplasmic cavitation

Koilocyte

Features of HSIL Immature cells Enlarged nuclei, high nuclear to cytoplasmic ratio (big nucleus/small cytoplasm) Dark chromatin Irregular nuclear membrane

High-Grade Squamous Intraepithelial Lesion (HSIL)

High-Grade Squamous Intraepithelial Lesion (HSIL)

Colposcopic Guided Cervical Biopsy 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 Carcinoma Keratinized and dense cytoplasm Cell pleomorphism: elongated, tadpole, oval Irregular nuclear size and shape India ink chromatin

Squamous Cell Carcinoma

Squamous Cell Carcinoma

Invasive Squamous Cell Carcinoma Ulcerated cervical tumor Cervical biopsy of tumor

Pap Test Glandular Cell Abnormalities Detects glandular neoplams such as Endocervical carcinoma Endometrial carcinoma Features 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 and 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 2003 In combination with Pap, not approved as stand-alone For every 3-5 years use For women 30 years old or older Endorsed by ACS and ACOG A 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 screening 21-29 years: Cytology alone every 3 years 30-65 years: - HPV and Cytology every 5 years (preferred) - Cytology alone every 3 years (acceptable) >65 years: No screening following adequate negative prior screening After 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 screening The 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 Testing The 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 test The 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