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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