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

2. 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

3. 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

4. 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

5. Rates cervical cancer
Rates Cervical Cancer
Goldie et al Vaccine 2006

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

7. 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

8. DR. PAPANICOLAOU INTRODUCED THE PAP TEST IN 1941

9. CONVENTIONAL PAP TEST

10. 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

11. PAP TEST-liquid
based
THINPREP

12. PAP TEST-liquid-based
THINPREP
PAP TEST-liquid-based

13. 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

14. CONVENTIONAL PAP SMEAR

15. Endocervical cells
THINPREP

16. 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.

17. Cervical Cancer Screening
Recent emphasis on molecular testing seeks to identify HPV strains considered high risk for carcinogenesis

18. 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

19. 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

20. 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

21. HPV Infection Currently HPV vaccination exist!
GARDASIL®Human Papillomavirus Quadrivalent (Types 6, 11, 16, and 18)
CERVARIX Human Papillomavirus Bivalent (Types 16 and 18)

22. 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

23. 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

24. 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

25. 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

26. 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)

27. 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

28. The Bethesda System for Reporting Cervical Cytology

29. 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

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

31. 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’.

32. TRICHOMONADS

33. CANDIDA SPP

34. HERPES SIMPLEX

35. 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

36. Cervical Intraepithelial Lesion
Is the precursor of invasive cancer

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

38. INTERRELATIONS OF NOMENCLATURE SYSTEMS
IN PREMALIGNANT CERVICAL DISEASE

39. SCJ
Squamous cells
Glandular cells

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

41. Low-Grade Squamous Intraepithelial Lesion (LSIL)

42. 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

43. Koilocyte

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

45. High-Grade Squamous Intraepithelial Lesion (HSIL)

46. High-Grade Squamous Intraepithelial Lesion (HSIL)

47. Colposcopic Guided Cervical Biopsy for HSIL
Mosaic &punctation in colposcopic view
CIN 3 in cervical biopsy

48. Invasive Squamous Cell Carcinoma
Is the most common type of cervical cancer

49. Features of Squamous Cell Carcinoma
Keratinized and dense cytoplasm
Cell pleomorphism: elongated, tadpole, oval
Irregular nuclear size and shape
India ink chromatin

50. Squamous Cell Carcinoma

51. Squamous Cell Carcinoma

52. Invasive Squamous Cell Carcinoma
Ulcerated cervical tumor
Cervical biopsy of tumor

53. 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

54. Endocervical Adenocarcinoma
Accounts for 20% of malignant cervical tumors

55. Atypical Endocervical Glands and Adenocarcinoma in Situ

56. Endometrial Carcinoma
Is the most common gynecologic cancer in the USA

57. ENDOMETRIAL ADENOCARCINOMA

58. 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

59. 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:

60. 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

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

62. 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

63. 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