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Dina R. Mody, MDDirector of Cytology Labs

Houston Methodist Hospital and Bioreference LabsThe Ibrahim Ramzy Chair in Pathology

Department of Pathology and Genomic MedicineProfessor of Pathology and Laboratory Medicine

Weill Medical College of Cornell University

Spectrum and Pitfalls in Cervicovaginal Cytology: Squamous Lesions

For This lecture…

Adequacy

LSIL: Patterns and Mimicks

HSIL: Patterns and Mimicks

Squamous Carcinomas: Types and Mimicks

ASC-US and ASC-H

Reporting rates

Cytology-Histology correlations

Bethesda Updates

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PAP Classes(1954)

Class I, absence of atypical or abnormal cells

Class II, atypical cytology but no evidence of malignancy

Class III, suggestive of but not conclusive for malignancy

Class IV, strongly suggestive of malignancy

Class V, Conclusive for malignancy

Normal

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Endometrials

AtrophyPost Partum/depo Provera

Endometrials Exodus, day 7

From M J Thrall in Diagnostic Pathology: Cytopathology Mody: Amirsys 2014

Bethesda 3 Atlas (2015)

66% greater page count Addressed issues raised post TBS 2001

on adequacy and terminology No major terminology changes Updated and refined criteria Added to pitfalls and images Management and references updated Risk stratification

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2015

2004

1993

E- version already available in April

For this talk I will discuss….

Normal, Adequacy Issues

Repair and atypical repair, radiation

Mimics of SIL and Squamous Ca

Recognizing Diathesis in various preparations

Pitfalls in Squamous cell carcinoma diagnosis (over and Under calls)

Bethesda Updates with each topic

Published simultaneously in May issue of Acta Cytologica (IAC), Cancer Cytopathology,Journal of the American Society of Cytopathology (ASC)and Journal of Lower Genital Tract Disorders (ASCCP)

JASCyto May 2015

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Chapter 1: AdequacyGeorge G. Birdsong and Diane D. Davey

Important quality assurance of TBS

TBS 2014 adds guidance Clarified cellularity criteria for vaginal/post

radiation samples

Added data on lubricant/blood interference

HPV testing in unsatisfactory specimens

TZ component/2012 ASCCP

From ASC webinar by Nayar and Wilbur, April 28, 2015

Adequacy

Conventional Smears: 8000-12000 well preserved well visualized squamous or squamous metaplastic cells

5000 squamous cells minimum for liquid based for cervicovaginal specimens

This works out to 8-9cells/HPF on SP and 3-4on TP, 10 fields across

Mention endocervical/TZ component 2000 squamous cells is the absolute minimum for

vaginal or s/p XRT/Rx smears*, anything less is Unsat

*See Bethesda 2014 for details

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Adequacy Issues with Lubricants

Carbomers and Carbopol polymers impact adequacy onThinpreps

No effect on Surepaths

Not much on conventionals either

Water soluble lubricants recommended

Adequacy Issues with Blood

Especially problematic with Thinprepspecimens

Filter clogged

Glacial acetic acid wash and reprocessing

Wash may affect HPV testing using some HPV platforms

HPV testing on Unsatisfactory Paps

Negative HPV test on Unsat Pap is unreliable Some HPV testing platforms have no internal

control(HCII)

Internal control may not be epithelial cell specific

A positive HPV test still requires additional follow up

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Management Guidelines related to Adequacy

Unsatisfactory cytology…repeat in 2-4 months(may treat atrophy or inflammation prior to repeat) If Unsat due to low cellularity but recent

negative screening history, can adjust repeat test at a longer interval than 2-4 months Two consecutive Unsats then colposcopy Women>30 with Unsat pap and +HPV then

colpo If 16, 18 types+ then colposcopy

Management Guidelines related to TZ / Endocervical component

Women 21-29 with no TZ/EC…routine screening Women 30 and over with no TZ/EC, HPV

testing is prefered. If negative HPV or no HPV, then routine screening in 3 yrs Women 30 and over with HPV +, repeat in

1yr Women 30 and over, HPV+ and genotyping

16 or 18+, then colposcopy. Other types, Pap in a year

Repair Criteria

Repair Flat sheets with distinct

cellular outlines, non overlapping nuclei

Streaming pattern, PMNs Smooth, round nuclear

outlines, slight nuclear enlargement

Normo or hypochromic, rarely mild hyperchromasia

Regular nucleoli Rounding on LBPs Bi and multinucleation

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Radiation

Increased cell size without change in N:C ratio

Bizzare shapes

Degenerative changes, vacuoles in nu/cytopl

Mild hyperchromasia, variable nucleoli

Polychromatic staining

Atypical Repair

Many features of repair

Large nucleoli

Nuclear features and overlap brings carcinoma in differential

Often interpreted as atypical glandulars

How to Report Equivocal SIL in TBS 2014

ASC-H + LSIL when definite LSIL in background (preferred)

Or SIL of uncertain grade with comment as to why

Should occur only in a small % of cases

Chapter on squamous lesions also expanded to show more problematic patterns and pitfalls and diathesis

From ASC webinar by Wilbur and Nayar. April 28 2015. www.cytopathology.org

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Examples of LSIL

LSIL Criteria

Changes limited to “Mature cells” Nuclear enlargement >3X normal

intermediate cell nucleus Variable hyperchromasia, (exception in

liquid based) nu size, number, shape Slight nuclear membrane irregularity Koilocytosis Must have nuclear abnormalities to qualify Note differences in liquid based

Mimics of LSIL

Pseudokoilocytosis

Radiation

Herpes

Hyperkeratosis

Tight halos

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Mimics of LSIL

Navicular cells/Pseudokoilocytosis

Nuclear features of lSILare not present

Glycogenation/yellow tinge

No distinct condensation

Tight halos may also be seen

Mimics of LSIL

Tight Halos of Reactive changes

Small tight halo usually due to organisms

No peripheral condensation of cytoplasm

Equal distance between edge of nucleus and halo rim(unlike lSIL)

Lack of nuclear features of LSIL

Mimics of LSIL

Radiation

Increased cell size without change in N:C ratio

Bizzare shapes

Degenerative changes, vacuoles in nu/cytopl

Mild hyperchromasia, variable nucleoli

Polychromatic staining

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Mimics of LSIL

Herpes Multinucleation, Molding

and margination of the chromatin

These changes in mature cells, if not well developed may be mistaken for LSIL

Pay attention to other cells for classic features of herpes

Both can co-exist

Mimics of LSIL

Hyperkeratosis

Anucleate squames

Tight halos

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HSIL Criteria

Small less mature cells affected

Single, sheets or syncytial-like aggregates

Nuclear hyperchromasia, irregularity, variation in size and shape, occasional prominent folds

Nucleoli generally absent except gland extension

Cytoplasm may be immature/lacy, dense or rarely densely keratinized

Patterns of HSIL

Dispersed

Syncytial

Endocervical Gland Involvement

Hypo chromatic

Stripped nuclei

Keratinizing

Repair – like/ stromal cells like

Conventional

Liquid Based (Thin prep

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TP TP

TP

SP

Hypo chromatic HSIL on TP

LSIL+HSIL/CIN II

TP

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The HSIL Hurricane Pattern!

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Mimics of HSIL

Isolated epithelial cells Reserve cells, Parabasal cells, immature metaplasia

IUD cells Isolated cells with herpes Exfoliated endometrial cells Endometrial stromal cells Histiocytes Isolated bizarre cells with atrophy Hyper chromatic crowded groups of benign cells Uncommon malignancies

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Mimics of HSIL

Transitional Metaplasia

Postmenopausal women

Atrophic background

Few groups

Fine even chromatin

Linear/longitudinal grooves

P16 and HPV negative

Mimics of HSIL

Benign HyperchromaticCrowded Groups (HCGs)

Follicular cervicitis

Atrophy

Histiocytes

Epithelial Cell Abnormalities

Squamous CellAtypical Squamous cells of undetermined significance(ASC-US) Cannot exclude High grade SIL(ASC-H)Low Grade Squamous Intraepithelial Lesion HPV and CIN IHigh Grade Squamous Intraepithelial Lesion CINII, CIN III, CIS, Susp for invasionSquamous Cell carcinoma

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Squamous Cell Carcinoma

Non Keratinizing and Keratinizing types

Features and diathesis vary by preparation type

Cellularity also variable

Diathesis usually subtle in liquid based

Diathesis Liquid Based

TP TP

SP

Subtle Diathesis Liquid BasedSP

TPTP

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Keratinizing Squamous Cell Carcinoma

Isolated cells or in aggregates

Variable size, shape, tadpoles, spindles

Variation in nuclear size, shape, hyperchromasia, granularity

Macronucleoliuncommon

Diathesis less than in non keratinizing types, clinging diathesis in liquid based

Non Keratinizing Squamous Cell Carcinoma

Syncytia with ill defined cell borders

Features of HSIL but cells usually smaller

Variation in nuclear size, shape, hyperchromasia, granularity

Macronucleoli and basophilic cytoplasm in large cell variant

Diathesis more obvious, clinging diathesis in liquid based

Overcalling Squamous cell carcinomas

Pseudo diathesis of atrophic vaginitis

Irritated and ulcerated endocervical Polyps

Lubricant simulating diathesis

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Atrophic Vaginitis with Pseudodiathesis and Random atypia

Under calling Squamous cell carcinomas

Low cellularity

Obscuring inflammation or blood

Repair like features

Beware of the Bloody Unsat!Dilute/Lyse and reprep the case!

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Atypical Squamous Cells

ASC refers to cytologic changes suggestive of SIL, which are qualitatively and quantitatively insufficient for a definitive interpretation. 3 essential features

Squamous differentiation

Increased N:C ratio

Minimal hyperchromasia, ch clumping, irregularity, smudging or multinucleation

Note: Applies to entire specimen not individual cells

Atypical Squamous Cells- of Undetermined Significance (ASC-US)

Mature Cell type (superficial or intermediate)

Nuclei 2.5-3X the area of normal intermediate cell nucleus

Slightly increased N:C ratio

Minimal nuclear hyperchromasia, irregularity in chromatin distribution or shape

Nuclear abnormality with dense orangeophilic cytoplasm (atypical parakeratosis)

Note: Applies to entire specimen not individual cells

Common Patterns Classified as ASC-US

Atypical parakeratosis

Atypical repair

Atypia in Postmenopausal women with atrophy

Decidua

Trophoblastic cells

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Atypical Squamous Cells, Cannot exclude HSIL(ASC-H)

Immature Cell types Single cells or small fragments of <10 cellsSmall cells with high N:C ratios(Atypical

immature metaplasia)Metaplastic cells with nu 1.5-2.5 X normalN:C ratio closer to HSIL but other nuclear

abnormalities fall shortIn liquid based, cells small and 2-3X

neutrophil nuclei

Mis classified ASC-H

Isolated endocervical cells

Endometrial cells

Histiocytes

IUD cells

Decidual cells

Artefacts

PAP interpretations preceding HSIL Biopsies (conventionals, 1990s or earlier data) ASCUS 39%

HSIL 31%

LSIL 20%

AGUS 10%

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PAP interpretations preceding HSIL Biopsies (Liquid Based with Imaging)

Khan K, Smith D, Thrall M. Archives of Pathology and Lab medicine; 2012

HPV Reporting Rates

Category 5th 25th 50th 75th 90th 95th

ASC-US 4 26 37 48 53 62.2

ASC-H 0 1.8 50 71 89 98.7

>30scr 0 1.9 4 11 25 26

Moriarty A, Schwartz M et al. Arch pathol lab med-vol 132, pp 1290-94 august 2008

CAP HPV Reporting rates 2014

Table 5. High-risk HPV Positive Rate Percentage

Categories N Mean 10th 25th 50th 75th 90th Total HPV tested volume reported positive

463 22.60 10.0 13.8 20.0 28.4 38.5

ASC-US in women 30 years of age or older (CHPV)

43 31.91 19.8 25.0 30.7 38.3 53.3

ASC-US in women younger than 30 years of age (CHPV)

32 47.73 25.9 42.1 51.3 56.5 63.2

ASC-US (PAP) 110 37.05 11.8 26.4 38.3 47.8 54.7 ASC-H 103 39.87 0.0 1.0 53.8 68.1 79.0 NILM Pap test co-test in women over 30 years of age

81 10.91 2.1 4.4 6.5 11.0 22.5

AGC 90 16.47 0.0 0.0 13.2 27.0 39.3 LSIL in postmenopausal women

41 31.15 0.0 0.8 20.0 64.1 76.8

ASC-US indicates atypical squamous cells of undetermined significance; ASC-H, atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion; LSIL, low-grade intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; AGC, atypical glandular cells.

From CAP Cytopathology Resource Committee, Archives of Pathology and Lab medicine

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ASC-H with HR HPV and Histologic Follow-up (%)

Age HR-HPV+ HR-HPV-

CIN2+

CINI CIN2+

CINI

20-29 33.9 31.4 0* 24.1

30-39 46.3 26.9 4.1* 9.6

40-49 24.2 30.3 0* 3.8

50-59 25 33.3 0 19

Total 32.7 29.2 1.2* 12.9*

*Statisticallysignificant

Arch Pathol Lab Med132:1874-1881 2008

Cytology Histology Correlations

In the US, mandated for HSIL and carcinomas (CLIA88)

Good QA practice

Good patient care

Different ways and timelines for doing the correlations

Varies by institutions and practice settings

Cytology Histology Correlations

Probes study by CAP of 22,439 correlations in 348 labs

94.3% of US labs

2.3% Canada, 3.4% Australia, Belgium, UK and others

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Colposcopic Sampling, 52

Cytologic Screening, 3

Interpretive, 25

Histotechnical, 4

Cytologic Sampling, 9

Combination, 7

Etiologies for Non-Correlating Cervical Cytologies and Biopsies

Tritz D et al. Am J of Clin Path. Vol 103; 1995 594-597

What is considered a positive correlation? Cytology and tissue diagnosis match or

are within one grade of each other Reasons for non correlation Tissue(colpo sampling) Tissue Interpretive Cytology Sampling Cytology Screening Cytology Interpretive Other/technical

LSIL HSIL

HSIL/CIN II-III

1 2

3

4

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Cervical Biopsy–Cytology Correlation, a CAP Q probes study of 22439 correlations in 348 labs

Cytology Diagnosis(%)Biopsy Dx Neg ASC/G I II+

NILM 67 37 21 5

LSIL 26 44 60 7

HSIL+ 7 14 18 88

Jones B, Novis D Arch pathol lab med vol 120 1996, 523-531

Cytology Histology Correlation at Methodist Hospital

Reasons for Discrepancy

Tissue/Colposcopic sampling

Cytology Interpretation or screening

Tissue Interpretation

Technical Issues

Cytology sampling

Difference of two categories considered significant

Cytology Histology Correlation (HSIL+) at Methodist Hospital (2000-2007)

Correlating

NonCorrelating

N= 1477

76%

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Reasons Cytology Histology Non Correlation (HSIL+) at Methodist Hospital (2000-2007)

ColpoSampling

Cytosampling

Cyto diagnosis

TissueDiagnosis

Technical

N=344, Colpo sampling 87%, cytodiagnosis 9.3%

Factors Influencing Accuracy of Colposcopy Guided Biopsy Severity of Referal Pap

Patient age and menopausal status

Visibility of Squamo columnar junction

Lesion Size

Endocervical extension

Training and experience of colposcopist

Type of clinicianGage J et all. Obstetrics & Gynecology Vol 108, No2, 2006 pp264-272Costa et all. Gynecologic oncology Vol 90, 2003. pp 57-63

Number of colposcopically Directed Biopsies and Outcomes (ALTS)Bx result one Bx 2Bxs 3 or >CIN3+ 52% 63% 57%

CIN2+ 68%* 82%* 83%

Aty+CINI 13% 10% 10%

Cum 81.3%* 91.7%* 93%*

* P<0.01 between one Bx Vs >1 Bx

Gage J et all. Obstetrics & Gynecology Vol 108, No2, 2006 pp264-272

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Interobserver Diagnostic Agreement for Colpo QC using Digitized Colposcopic Images (ALTS)

QC reviewers Colpo agreement kappa1&2(NL) 63% 0.211&3 57% 0.23*2&3 63% 0.35*

1&2(CIN2>) 51% 0.321&3 55% 0.392&3 55% 0.38*

*statistically significant, (NL) histologic diagnosis normal, (CIN2>) histologic diagnosis of CIN 2 or worse

Ferris D, Litaker M for ALTS group J of lower gen tract dis Vol9, no1, 2005, 29-35

Intraobserver Variability Colposcopy

First Session

Nl(%) CINI II III

Second session

NL 56(81) 12 3 3

CINI 10 23(49) 5 6

CIN2 3 9 31(60) 12

CIN3 0 3 13 48(70)

Hopman E et al. Gynecologic oncology. Vol 58, 1995, pp206-209

LAST reference

Darragh TM, Colgan TJ, Cox JT, Heller DS, Henry MR, Luff RD, McCalmont T,Nayar R, Palefsky JM, Stoler MH, Wilkinson EJ, Zaino RJ, Wilbur DC; Members of LAST Project Work Groups. The Lower Anogenital SquamousTerminology Standardization Project for HPV-Associated Lesions: background and consensus recommendations from the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology. Arch Pathol Lab Med. 2012 Oct;136(10):1266-97.

Epub 2012 Jun 28. Erratum in: Arch Pathol Lab Med. 2013 Jun;137(6):738. PubMed PMID: 22742517.

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Bethesda Interobserver Reproducibility Study-TBS 2014 (BIRST-2)

85 unknown or new images from the new atlas were included

1290 individuals attempted, 833 completed(41% outside USA)

Histograms will be placed on TBS2014 website and results published in JASC

BIRST-2 website will golive in summer/fall of 2015

TBS Contributions

Initiation of research and clinical trials

Alignment of management with terminology

Prototype for standardized reporting terminology in pathology Thyroid Bethesda, Urinary Paris,

Pancreaticobiliary

Histopathology LAST/WHO