Utility of P16 Expression and Ki-67 Proliferation Index in ASCUS and ASC-H Pap Tests Adam D. Toll, M.D., Deidra Kelly, C.T., and Zahra Maleki, M.D.*
Current cervical screening uses a combination of cytology and high-risk human papillomavirus (HR-HPV) analysis in cases of atypical squamous cells of undetermined significance (ASCUS) and atypical squamous cells cannot exclude high-grade intraepithelial lesion (ASC-H). These diagnoses are subject to interobserver variability and HR-HPV analysis can be limited by sampling inadequacy. This study correlates immunoexpression of P16 and Ki-67 in residual cervicovaginal material against cytology category and HR-HPV status. Eighteen pap tests were selected: 8 ASCUS, 4 ASC-H, and 6 controls (2 LSIL and 4 HSIL). Digene Hybrid Capture II test was used to detect HRHPV. The cytospins were stained for P16/Ki-67. Pap tests, P16, Ki-67, HR-HPV result and available biopsies were correlated. P16 expression correlated with HR-HPV status in 15/17 cases. Discordant cases (1 ASCUS and 1 ASC-H) were 1P16/–HRHPV. Ki-67 correlated with HR-HPV in 8/15 cases. Discordant cases were 1HR-HPV/– Ki-67 (HSIL, LSIL, and ASC-H one each), and –HR-HPV/1Ki-67 (3 ASCUS, 1 LSIL, 1 ASC-H). Two cases were 1 P16/1 Ki-67/– HR-HPV. None were - P16/– Ki-67/1 HR-HPV. Histologic follow-up in 13 cases varied from benign to CIN III. Two cases of 1P16/ – Ki-67/– HR-HPV had benign cervical biopcies. Although a small sample size, our findings show a utility for adjunct P16/ Ki-67 in addition to HRHPV testing in cases of squamous atypia when HR-HPVs are non-detected due to low DNA copies, or missed lesions in cervical biopsies. Diagn. Cytopathol. 2014;42:576–581. VC 2013 Wiley Periodicals, Inc.
Key Words:
ASCUS; ASC-H; Ki-67; P16; HR-HPV
Current management of cervical cancer screening uses cytology and molecular analysis of 13 strains of high-risk human papillomavirus (HR-HPV). Detection of HPV has
The study was presented as a poster at the ASC’s 60th Annual Scientific Meeting, Las Vegas, Las Vegas NV, November 2nd–6th, 2012. Division of Cytopathology, Department of Pathology, The Johns Hopkins Hospital, Baltimore, Maryland *Correspondence to: Zahra Maleki, M.D., Division of Cytology, Department of Pathology, 600 N. Wolfe Street/Carnegie 469C, Baltimore, MD 21287, USA. E-mail: [email protected] Received 28 February 2013; Accepted 29 October 2013 DOI: 10.1002/dc.23076 Published online 28 November 2013 in Wiley Online Library (wileyonlinelibrary.com).
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improved the false-negative rate of cytologic screening.1 Cases interpreted as cytologically benign with detected HPV have a 5–7% risk of a repeat screen being interpreted as abnormal vs. 0.5–1% risk if the HPV status is not detected.1 While the presence of HPV infection is found in virtually all cases of cervical dysplasia, the specificity for intraepithelial neoplasia is poor, as only 10% of infected women develop preneoplastic lesions.2,3 The presence of HPV infection in conjunction with a cytologic diagnosis of a squamous intraepithelial lesion (either low or high grade squamous intraepithelial lesions, LSIL and HSIL, respectively) strongly predicts dysplasia; however, this correlation is less predictive when the cytology shows either atypical squamous cells of undetermined significance (ASCUS), or atypical squamous cells where a highgrade intraepithelial lesion cannot be excluded (ASC-H).4 Complicating the issue is that colposcopic evaluation with biopsy has been found to underestimate high-grade cervical intraepithelial lesions in 26–42% of cases.5 Further, ASCUS diagnoses are subject to considerable interobserver variability and correlate to grade II or higher cervical intraepithelial neoplasia (CIN) in only 10% of cases.4,6–9 There may also be detrimental emotional and psychological impacts on the patient associated with these diagnoses. While detecting HPV is helpful, a lack of detection may be due to inadequate cellularity or a truly negative test. Additional markers of dysplasia could potentially increase the positive predictive value for these diagnoses. P16INK4A (P16) is a cyclin-dependent kinase inhibitor regulating the G1 checkpoint of cell division which becomes inactivated in several cancers either through genetic deletion or hypermethylation.10,11 Inactivation by the retinoblastoma gene from the HPV-related protein E7 leads to significant increases in P16, making it a potentially useful immunohistochemical screening marker for dysplasia arising from an HPV infection.10,12–15 In addition to the inactivation of tumor suppressor genes, HPV (notably genotypes 16 and 18) have been associated with C 2013 WILEY PERIODICALS, INC. V
Diagnostic Cytopathology DOI 10.1002/dc
P16 AND KI-67 IN ATYPICAL SQUAMOUS CELLS
increased proliferative activity of the squamous epithelium (as measured from Ki-67 immunohistochemical studies), and have also been shown to correlate with progression of dysplasia.11,16 P16 and Ki-67 are commonly used in cervical histology specimens as a means to distinguish reactive changes from dysplasia, and help separate low-grade from high-grade dysplasia. This study was designed to correlate the expression of P16 and Ki-67 proliferation index against the atypical cytologic diagnoses of ASCUS/ASC-H, as well as HPV status in cytology material. A secondary purpose was to confirm the feasibility of utilizing residual thin-layer cervicovaginal material for immunocytochemical staining.17
Methods and Material Institutional Review Board approval for this study was obtained. Eighteen random cervicovaginal cytology specimens with adequate cellularity and good preservation were selected based on diagnosis. The patients’ age, history of pregnancy, colposcopy findings, prior cervicovaginal pathology results, HPV testing results, and surgical biopsies were recorded for each patient if available. Eight cases of ASCUS and four cases of ASC-H were collected, along with six positive controls (two cases of LSIL and four HSIL). The original diagnoses were confirmed by a senior cytopathologist (ZM). Informed consents were not obtained since the Immunostains were performed on residual cervicovaginal material which was routinely discarded. Therefore the clinicians who collected the cervico-vagianl smears were not aware of the study and they obtained the specimens in a routine fashion. The Ki67 and P16 results were not reported to the patients or clinicians and did not have any effect on patient treatment or follow up. Pap smears were prepared in a liquid-based BD Surepath preparation. An in-house validated modified HC-2 HPV assay (Digene/Qiagen Corporation, Gaithersburg, Maryland, USA) was used to detect high risk HPV DNA. This test qualitatively screens for 13 high risk HPV types (including 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) by in vitro nucleic acid hybridization with signal amplification using chemiluminescence on a microplate. Sample scores were based on a ratio of the relative light unit (RLU) per positive control (CO). A RLU/CO score 2.5 was considered positive for the presence of highrisk HPV DNA. All RLU/CO scores 11
1 3 1 1
2 0 0 0
4 0 0 0
0 0 0 0
1 1 1 3
ASCUS ASC-H LSIL HSIL
the guidelines recommended. The cytology diagnosis of cervical pap smears, high risk HPV results, and P16 and Ki-67 immunostains were correlated together and with colposcopy and histologic findings.
Table II. Cytologic/histologic Correlation of Dysplastic Squamous Cells, HPV Status, Ki-67 Proliferation Index, and P16 Immunocytochemical Staining Cytology
Histology
HPV
P16
Ki-67
ASCUS ASCUS ASCUS ASCUS LSIL LSIL ASC-H ASC-H ASC-H HSIL HSIL HSIL HSIL
Benign CIN I Benign Benign CIN I Benign Benign CIN I-II Benign CIN III CIN III CIN III CIN I-II
Not Detected Detected Detected Detected Detected Not Detected Not Detected Detected Not Detected Detected Detected Detected NA
Positive Negative Positive Positive Positive Negative Positive Positive None Positive Positive Positive Positive
Low Low Rare High None High High None None High High None High
Results Cervical Pap Smears Eighteen liquid-based prepared Pap smears were reviewed. The average patient age was 35 years (range 22–60 years). Eight women were diagnosed with ASCUS (mean 36 years), four with ASC-H (mean 34 years), two with LSIL (mean 41 years), and four with HSIL (mean 30 years). The cervical cytology smears were reported according to the Bethesda system criteria for reporting cervical cytology.18 ASC-H cells were mostly single with the size of metaplastic cells. The nuclei were one and one half to two and one half times larger than normal intermediate cells. LSIL cells were single or in sheets. Cytologic changes were confined to cells with mature or superficialtype cytoplasm. The cells were large with enlarged nuclei more than three times the area of normal intermediate nuclei. Chromatin was coarsely granular with variable degrees of nuclear hyperchromasia. Cytoplasm showed distinctive perinuclear halos (koilocytosis). HSIL cells were single or in sheets recognized by marked high nuclear to cytoplasmic ratio, nuclear hyperchromasia, irregular nuclear contour, small cell size and immature, lacy or densely metaplastic cytoplasm.
HPV Testing Results HR HPV results obtained by hybrid capture II assay were available for 17 cases. One HSIL case did not have any HR HPV studies. HPV was reported as detected in nine cases, and not detected in the rest (Table II). In two cases (13%) HPV was not detected, while both P16 and Ki-67 were positive (one ASCUS and one ASC-H). There were no cases (0%) where HPV was detected, and both the P16 and Ki-67 were negative. Among the eight cases with no detectable HR HPV, four were called ASCUS (one of four cases biopsied; benign), three ASCH (two of three cases biopsied; benign squamous mucosa with chronic cervicitis), and one LSIL (biopsied: benign squamous mucosa). 578
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P16 Immunostaining Results P16 expression (Figures 1) correlated with HPV status in 15 of 17 cases (88% concordance; one case of HSIL did not have HPV testing). In one discordant case (ASCUS), HPV was not detected while P16 expression was identified. It was a 60-year old immunocompromised HIV positive patient that HR-HPVs were detected in her prior pap smears but not the concurrent HR-HPV studies which could be due to insufficient HPV DNA copies. P16 immunostain showed positive cells while the proliferation rate was low by Ki-67. The biopsy showed benign squamous mucosa. The second discordant case was an ASC-H case which appeared like CIN I-CIN II on colposcopy. P16 immunostain was weakly and focally positive and Ki-67 showed a high proliferation index. The biopsy showed benign squamous mucosa with marked acute and chronic cervicitis. There were three cases of 1P16 and –Ki-67 (1 LSIL and 1 ASC-H and 1 HSIL). Subsequent follow up biopsies of these cases showed CIN I, CIN I-II, and CIN III, respectively (Table III).
Ki-67 Immunostaining Results The Ki-67 proliferation index (Table I) scoring results were as follows: six negative (one ASCUS, three ASC-H, one LSIL, and one HSIL), two rare (both ASCUS), four mild (ASCUS), 0 moderate, and five high (1 LSIL, 1 ASC-H, and 3 HSIL). The Ki-67 index correlated with HPV in 8 of 15 cases (53% concordance). Discordant cases were as follows: three cases of 1 HPV / –Ki-67 (1 LSIL and 1 ASC-H and 1 HSIL), and five cases of –HPV/1Ki-67 (3 ASCUS, 1 LSIL, 1 ASC-H). Of 1HPV/ –Ki-67 cases, Subsequent biopsies of these cases showed CIN I, CIN I-II, and CIN III, respectively. Of –HPV/ 1Ki-67 cases, two ASCUS cases showed low proliferation index and no further biopsies. One ASCUS with low proliferation rate had subsequent benign biopsy and HR HPVs were detected several times in prior pap smears. The LSIL showed high proliferation index with
Diagnostic Cytopathology DOI 10.1002/dc
P16 AND KI-67 IN ATYPICAL SQUAMOUS CELLS
Fig. 1. Top row from left to right shows a concordant case of HSIL/ HR HPV 1/ Ki-67 1/ P16 1. HSIL pap cytology with detected HR HPV (a), Ki-67 highlights HSIL cells on immunostain (b); P16 stains a HSIL cell (c). Lower row from left to right shows a discordant case of ASC-H/ HR HPV –/ Ki-67 1/ P16 1. ASC-H pap cytology with non-detectable HR- HPV (d), Ki-67 highlights ASC-H cells on immunostain (e), P16 weakly stains an ASC-H cell (f). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Table III. Colposcopic Findings and Correlation with Cervical Cytology, HPV Studies, Surgical Biopsy, P16 Expression, and Ki-67 Proliferation Index Colposcopicfindings
Cervical cytology
Surgical biopsy
HR-HPV status
Normal Normal Normal Normal Normal CIN I CIN I – CIN II CIN II CIN II CIN III
ASCUS ASCUS ASCUS ASCUS HSIL ASCUS ASC-H ASC-H HSIL HSIL
None Benign Benign None CIN III CIN I Benign CIN I- CIN II CIN III CIN III
Detected Detected Not detected Not detected Detected Detected Not detected Detected Detected Detected
a subsequent benign biopsy. There were three cases of 1Ki-67 and –P16 (2 ASCUS and 1 LSIL). HR HPVs were not detected in any of three cases. Subsequent biopsy of LSIL case showed benign findings and inflammation.
Clinical Follow Up Colposcopic examination results were available for 10 patients (Table III). Colposcopic examination was unremarkable in five cases (4 ASCUS and 1 HGIL in cervical cytology material). One appeared as CIN II in colposcopy and showed HSIL in Pap cytology. One ASC-H was called CIN II on colposcopy and it was confirmed on subsequent biopsy. Colposcopy impression was CIN I-CIN II in an ASC-H case and the surgical biopsy showed benign findings with marked acute and chronic cervicitis. Colpo-
P16
Ki-67
Positive Positive Positive Negative Positive Positive Negative Positive Positive Positive
Rare High Low Low High Low High None High None
scopic examination was consistent with CIN I in an ASCUS case and surgical biopsy confirmed CIN I. Histologic follow-up (Table II) was available in 13 cases: four ASCUS (3 benign and 1 CIN I), two LSIL (1 benign and 1 CIN I), three ASC-H (2 benign and 1 CIN I-II), and four HSIL (3 CIN III and 1 CIN I-II). One HSIL P16 1/ Ki-67 1 with no HR HPV study on cytology material and a diagnosis of CIN I- CIN II on biopsy had immunohistochemical staining for P16 and Ki-67 were similar to of those in cytology material. The LSIL case with benign biopsy had prior LSIL on biopsy.
Discordant Cases One ASCUS, HR- HPV 1/ P16 1 (rare)/Ki-67 1 (rare) case with a benign cervical biopsy had multifocal VIN IIIII of right labia and VIN I of left labia which correlates Diagnostic Cytopathology, Vol. 42, No 7
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with our findings. There were two cases where HPV was not detected while P16 and Ki-67 (Figure 3) were positive; in both cases the corresponding cervical biopsy showed benign findings. One ASCUS, HR- HPV –/P16 1 (rare)/Ki-67 1 (low) case had a biopsy proven prior history of CIN I. One ASCUS, HR- HPV –/ P16 1/ Ki-67 1 (low) case with a subsequent benign biopsy, had a long history of HR HPV detected on her prior pap smears. Not detected HR -HPV was most likely due to sampling and low copies of HR -HPV DNA. One ASCH, HPV –/ P16 1 (rare) / Ki-67 1 (high) case with a CIN I-II on colposcopy had a subsequent biopsy showing marked acute and chronic cervicitis. Sampling problem was probably the only explanation for this case.
Discussion Our findings within this small sample population demonstrate a proof of principle for the adjunct immunocytochemical testing of P16 and Ki-67 in cases of ASCUS and ASC-H, and larger prospective studies with clinical follow-up are warranted to validate these findings. A secondary finding in this study was the further validation that residual thin-layer cervicovaginal material is potentially sufficient for immunocytochemical staining.17 The combination of P16 and / or Ki-67 was 100% sensitive for HPV infection. In two additional cases (1 ASCUS and 1 ASC-H) these surrogate markers were positive, while HPV was not identified. P16 has been shown to be a sensitive and specific marker for HPV infection in ASCUS lesions,19 potentially selecting patients at greater risk to progress from infection to dysplasia. Further, a retrospective examination of cytologically negative Pap smears preceding CIN II-III lesions found incipient HPV infection in only 80% of cases.20 The remaining cases were presumed to contain viral copy numbers below the limit of detection; however, other studies have emphasized the difficulty of detecting CIN III lesions both with cervicovaginal sampling and colposcopic examination.21 In our study, one HSIL case appeared normal on colposcopy and subsequent biopsy confirmed CIN III. While HPV detection in LSIL is approximately 80%, the sensitivity for ASCUS decreases significantly to 43%.22,23 These lesions in particular may benefit from adjunct P16 and Ki-67 testing which may help identify patients at risk to develop cervical neoplasia in spite of following screening guidelines. P16 and Ki-67 immunostaining on residual thinlayer cervicovaginal material make it possible to correlate the cytomorphology and immunoreactivity of the atypical cells at the same time. However, performing immunostains on residual thin-layer cervicovaginal material is another issue to be evaluated since the slides are not paraffin embedded and subsequently eliminate the 30 minute melting time in 80 C. The possible effect of heat elimina580
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tion on immunostaining outcome on cytospin slides should be investigated. Despite widespread screening programs and HPV testing, approximately 30% of newly diagnosed cervical cancers occur in women following the recommended guidelines.24 The HPV virus, notably HPV genotype 16, has been studied in an effort to predict which patients will develop persistent infection and ultimately disease progression. Viral integration into the host genome was cited as a significant factor in the development of invasive carcinoma.25 A related finding was that the expression of E6 and E7, critical in the progression to neoplasia, correlated much more with integration into the host genome rather than viral copy numbers.25 Of note, high viral loads were frequently observed in cases of benign and low-grade squamous lesions, attributed to high episomal levels unrelated to the oncogenic potential of HPV. This would support the utility of P16, rather than viral copy number, as a more appropriate adjunct test in the determination of which patients may show disease progression. Our results indicate that adjunct P16 and Ki-67 testing offers a potential benefit in addition to HPV testing, most notably in cases when the differential diagnosis includes reactive or atypical (either ASCUS or ASC-H) diagnoses. P16 and Ki-67 testing can potentially be improved by brushing the cervico-vaginal mucosa a few times more than standard or colleting cells separately for P16 and Ki16 staining. A consensus group recently recommended the use of P16 in histologic sections from anogenital lesions in the following cases: when the differential is between low grade and high grade (CIN21) dysplasia, when the differential is CIN21 vs. a benign mimic and in discordant cases when initial histologic sections are benign and the Pap was ASC-H or worse.5 In our study one case was called ASC-H and had a negative biopsy. P16 and Ki-67 performed on the biopsy were negative; however the specimen consisted almost entirely of endocervical tissue and cannot be considered adequate. The viral-related proteins E6 and E7, recognized as the oncogenic promoters of HPV, correlate well with P16 overexpression. Accompanied by a Ki-67 proliferation index, these markers may provide a benefit in cases with low viral copies, or in cases of less than optimal sampling. A meta-analysis of 17 studies examining p16 and HPV in cases of ASCUS and LSIL found p16 to show greater accuracy in cases of ASCUS for correlating with CIN2/ CIN3 on colposcopy (better specificity, similar sensitivity) than HPV-DNA testing. In cases of LSIL, p16 showed greater specificity but less sensitivity relative to HPV testing. Among the most significant issues facing p16 testing is the subjective, non-standardized manner p16 is evaluated. Nuclear and / or cytoplasmic expression may be interpreted as positive, and some advocate only evaluating
Diagnostic Cytopathology DOI 10.1002/dc
P16 AND KI-67 IN ATYPICAL SQUAMOUS CELLS
cells with abnormal nuclei to avoid counting benign metaplastic or endocervical cells. One potential solution is the use of a dual stain with Ki-67 proliferation. Most agree that a cell with positive nuclear Ki-67 and cytoplasmic p16 expression is pathologic. Studies examining this issue are underway, and may influence whether HPV testing remains the only ancillary study widely recommended for Pap smears.26
Acknowledgment The authors thank Dr. Dorothy Rosenthal for her critical review of this manuscript.
References 1. Brink AA, Zielinski GD, Steenbergen RD, Snijders PJ, Meijer CJ. Clinical relevance of human papillomavirus testing in cytopathology. Cytopathology 2005;16(1):7–12. 2. Ho GY, Bierman R, Beardsley L, Chang CJ, Burk RD. Natural history of cervicovaginal papillomavirus infection in young women. N Engl J Med 1998;338(7):423–428. 3. Schiffman MH, Bauer HM, Hoover RN, Glass AG, Cadell DM, Rush BB, et al. Epidemiologic evidence showing that human papillomavirus infection causes most cervical intraepithelial neoplasia. J Natl Cancer Inst 1993;85(12):958–964. 4. Berkhof J, de Bruijne MC, Zielinski GD, Bulkmans NW, Rozendaal L, Snijders PJ, et al. Evaluation of cervical screening strategies with adjunct high-risk human papillomavirus testing for women with borderline or mild dyskaryosis. Int J Cancer 2006; 118(7):1759–1768. 5. Darragh TM, Colgan TJ, Cox JT, Heller DS, Henry MR, Luff RD, et al. The Lower Anogenital Squamous Terminology 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;136(10):1266–1297. 6. Cuzick J, Szarewski A, Cubie H, Hulman G, Kitchener H, Luesley D, et al. Management of women who test positive for high-risk types of human papillomavirus: The HART study. Lancet 2003; 362(9399):1871–1876. 7. Denise Zielinski G, Snijders PJ, Rozendaal L, Voorhorst FJ, Runsink AP, de Schipper FA, et al. High-risk HPV testing in women with borderline and mild dyskaryosis: Long-term follow-up data and clinical relevance. J Pathol 2001;195(3):300–306. 8. Clavel C, Masure M, Bory JP, Putaud I, Mangeonjean C, Lorenzato M, et al. Human papillomavirus testing in primary screening for the detection of high-grade cervical lesions: A study of 7932 women. Br J Cancer 2001;84(12):1616–1623. 9. Group A-LTS. Results of a randomized trial on the management of cytology interpretations of atypical squamous cells of undetermined significance. Am J Obstet Gynecol 2003;188(6):1383–1392. 10. Yoshida T, Sano T, Kanuma T, Inoue H, Itoh T, Yazaki C, et al. Usefulness of CINtec(R) PLUS p16/Ki-67 double-staining in cytological screening of cervical cancer. Acta Cytol 2011;55(5):413– 420. 11. Longatto Filho A, Utagawa ML, Shirata NK, Pereira SM, Namiyama GM, Kanamura CT, et al. Immunocytochemical expression of p16INK4A and Ki-67 in cytologically negative and equivo-
cal pap smears positive for oncogenic human papillomavirus. Int J Gynecol Pathol 2005;24(2):118–124. 12. Meyer JL, Hanlon DW, Andersen BT, Rasmussen OF, Bisgaard K. Evaluation of p16INK4a expression in ThinPrep cervical specimens with the CINtec p16INK4a assay: Correlation with biopsy followup results. Cancer 2007;111(2):83–92. 13. Bibbo M, Klump WJ, DeCecco J, Kovatich AJ. Procedure for immunocytochemical detection of P16INK4A antigen in thin-layer, liquid-based specimens. Acta Cytol 2002;46(1):25–29. 14. Alameda F, Pijuan L, Lloveras B, Bellosillo B, Larrazabal F, Mancebo G, et al. The value of p16 in ASCUS cases: A retrospective study using frozen cytologic material. Diagn Cytopathol 2011; 39(2):110–114. 15. Klaes R, Friedrich T, Spitkovsky D, Ridder R, Rudy W, Petry U, et al. Overexpression of p16(INK4A) as a specific marker for dysplastic and neoplastic epithelial cells of the cervix uteri. Int J Cancer 2001;92(2):276–284. 16. Sahebali S, Depuydt CE, Segers K, Vereecken AJ, Van Marck E, Bogers JJ. Ki-67 immunocytochemistry in liquid based cervical cytology: Useful as an adjunctive tool? J Clin Pathol 2003;56(9): 681–686. 17. Akpolat I, Smith DA, Ramzy I, Chirala M, Mody DR. The utility of p16INK4a and Ki-67 staining on cell blocks prepared from residual thin-layer cervicovaginal material. Cancer Cytopathol 2004; 102(3):142–149. 18. Solomon D, Nayar R. The Bethesda System for Reporting Cervical Cytology, Definitions, Criteria, and Explanatory Notes. Springer; 2003. 19. Monsonego J, Pollini G, Evrard MJ, Sednaoui P, Monfort L, Quinzat D, et al. P16(INK4a) immunocytochemistry in liquid-based cytology samples in equivocal Pap smears: Added value in management of women with equivocal Pap smear. Acta Cytol 2007;51(5): 755–766. 20. Bulk S, Rozendaal L, Zielinski GD, Berkhof J, Daalmeijer NC, Snijders PJ, et al. High-risk human papillomavirus is present in cytologically false-negative smears: An analysis of "normal" smears preceding CIN2/3. J Clin Pathol 2008;61(3):385–389. 21. ACOG Practice Bulletin. Clinical Management Guidelines for Obstetrician-Gynecologists. Number 61, April 2005. Human papillomavirus. Obstet Gynecol 2005;105(4):905–918. 22. Maucort-Boulch D, Plummer M, Castle PE, Demuth F, Safaeian M, Wheeler CM, et al. Predictors of human papillomavirus persistence among women with equivocal or mildly abnormal cytology. Int J Cancer 2010;126(3):684–691. 23. Arbyn M, Sasieni P, Meijer CJ, Clavel C, Koliopoulos G, Dillner J. Chapter 9: Clinical applications of HPV testing: A summary of meta-analyses. Vaccine 2006;24 Suppl 3:S3/78–89. 24. Waxman AG, Zsemlye MM. Preventing cervical cancer: The Pap test and the HPV vaccine. Med Clin North Am 2008;92(5):1059– 1082. 25. Wanram S, Limpaiboon T, Leelayuwat C, Yuenyao P, Guiney DG, Lulitanond V, et al. The use of viral load as a surrogate marker in predicting disease progression for patients with early invasive cervical cancer with integrated human papillomavirus type 16. Am J Obstet Gynecol 2009;201(1):79 e1–e7. 26. Roelens J, Reuschenbach M, von Knebel Doeberitz M, Wentzensen N, Bergeron C, Arbyn M. p16INK4a immunocytochemistry versus human papillomavirus testing for triage of women with minor cytologic abnormalities: A systematic review and meta-analysis. Cancer Cytopathol;120(5):294–307.
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Current cervical screening uses a combination of cytology and high-risk human papillomavirus (HR-HPV) analysis in cases of atypical squamous cells of undetermined significance (ASCUS) and atypical squamous cells cannot exclude high-grade intraepithelial lesion (ASC-H). These diagnoses are subject to interobserver variability and HR-HPV analysis can be limited by sampling inadequacy. This study correlates immunoexpression of P16 and Ki-67 in residual cervicovaginal material against cytology category and HR-HPV status. Eighteen pap tests were selected: 8 ASCUS, 4 ASC-H, and 6 controls (2 LSIL and 4 HSIL). Digene Hybrid Capture II test was used to detect HRHPV. The cytospins were stained for P16/Ki-67. Pap tests, P16, Ki-67, HR-HPV result and available biopsies were correlated. P16 expression correlated with HR-HPV status in 15/17 cases. Discordant cases (1 ASCUS and 1 ASC-H) were 1P16/–HRHPV. Ki-67 correlated with HR-HPV in 8/15 cases. Discordant cases were 1HR-HPV/– Ki-67 (HSIL, LSIL, and ASC-H one each), and –HR-HPV/1Ki-67 (3 ASCUS, 1 LSIL, 1 ASC-H). Two cases were 1 P16/1 Ki-67/– HR-HPV. None were - P16/– Ki-67/1 HR-HPV. Histologic follow-up in 13 cases varied from benign to CIN III. Two cases of 1P16/ – Ki-67/– HR-HPV had benign cervical biopcies. Although a small sample size, our findings show a utility for adjunct P16/ Ki-67 in addition to HRHPV testing in cases of squamous atypia when HR-HPVs are non-detected due to low DNA copies, or missed lesions in cervical biopsies. Diagn. Cytopathol. 2014;42:576–581. VC 2013 Wiley Periodicals, Inc.
Key Words:
ASCUS; ASC-H; Ki-67; P16; HR-HPV
Current management of cervical cancer screening uses cytology and molecular analysis of 13 strains of high-risk human papillomavirus (HR-HPV). Detection of HPV has
The study was presented as a poster at the ASC’s 60th Annual Scientific Meeting, Las Vegas, Las Vegas NV, November 2nd–6th, 2012. Division of Cytopathology, Department of Pathology, The Johns Hopkins Hospital, Baltimore, Maryland *Correspondence to: Zahra Maleki, M.D., Division of Cytology, Department of Pathology, 600 N. Wolfe Street/Carnegie 469C, Baltimore, MD 21287, USA. E-mail: [email protected] Received 28 February 2013; Accepted 29 October 2013 DOI: 10.1002/dc.23076 Published online 28 November 2013 in Wiley Online Library (wileyonlinelibrary.com).
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improved the false-negative rate of cytologic screening.1 Cases interpreted as cytologically benign with detected HPV have a 5–7% risk of a repeat screen being interpreted as abnormal vs. 0.5–1% risk if the HPV status is not detected.1 While the presence of HPV infection is found in virtually all cases of cervical dysplasia, the specificity for intraepithelial neoplasia is poor, as only 10% of infected women develop preneoplastic lesions.2,3 The presence of HPV infection in conjunction with a cytologic diagnosis of a squamous intraepithelial lesion (either low or high grade squamous intraepithelial lesions, LSIL and HSIL, respectively) strongly predicts dysplasia; however, this correlation is less predictive when the cytology shows either atypical squamous cells of undetermined significance (ASCUS), or atypical squamous cells where a highgrade intraepithelial lesion cannot be excluded (ASC-H).4 Complicating the issue is that colposcopic evaluation with biopsy has been found to underestimate high-grade cervical intraepithelial lesions in 26–42% of cases.5 Further, ASCUS diagnoses are subject to considerable interobserver variability and correlate to grade II or higher cervical intraepithelial neoplasia (CIN) in only 10% of cases.4,6–9 There may also be detrimental emotional and psychological impacts on the patient associated with these diagnoses. While detecting HPV is helpful, a lack of detection may be due to inadequate cellularity or a truly negative test. Additional markers of dysplasia could potentially increase the positive predictive value for these diagnoses. P16INK4A (P16) is a cyclin-dependent kinase inhibitor regulating the G1 checkpoint of cell division which becomes inactivated in several cancers either through genetic deletion or hypermethylation.10,11 Inactivation by the retinoblastoma gene from the HPV-related protein E7 leads to significant increases in P16, making it a potentially useful immunohistochemical screening marker for dysplasia arising from an HPV infection.10,12–15 In addition to the inactivation of tumor suppressor genes, HPV (notably genotypes 16 and 18) have been associated with C 2013 WILEY PERIODICALS, INC. V
Diagnostic Cytopathology DOI 10.1002/dc
P16 AND KI-67 IN ATYPICAL SQUAMOUS CELLS
increased proliferative activity of the squamous epithelium (as measured from Ki-67 immunohistochemical studies), and have also been shown to correlate with progression of dysplasia.11,16 P16 and Ki-67 are commonly used in cervical histology specimens as a means to distinguish reactive changes from dysplasia, and help separate low-grade from high-grade dysplasia. This study was designed to correlate the expression of P16 and Ki-67 proliferation index against the atypical cytologic diagnoses of ASCUS/ASC-H, as well as HPV status in cytology material. A secondary purpose was to confirm the feasibility of utilizing residual thin-layer cervicovaginal material for immunocytochemical staining.17
Methods and Material Institutional Review Board approval for this study was obtained. Eighteen random cervicovaginal cytology specimens with adequate cellularity and good preservation were selected based on diagnosis. The patients’ age, history of pregnancy, colposcopy findings, prior cervicovaginal pathology results, HPV testing results, and surgical biopsies were recorded for each patient if available. Eight cases of ASCUS and four cases of ASC-H were collected, along with six positive controls (two cases of LSIL and four HSIL). The original diagnoses were confirmed by a senior cytopathologist (ZM). Informed consents were not obtained since the Immunostains were performed on residual cervicovaginal material which was routinely discarded. Therefore the clinicians who collected the cervico-vagianl smears were not aware of the study and they obtained the specimens in a routine fashion. The Ki67 and P16 results were not reported to the patients or clinicians and did not have any effect on patient treatment or follow up. Pap smears were prepared in a liquid-based BD Surepath preparation. An in-house validated modified HC-2 HPV assay (Digene/Qiagen Corporation, Gaithersburg, Maryland, USA) was used to detect high risk HPV DNA. This test qualitatively screens for 13 high risk HPV types (including 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) by in vitro nucleic acid hybridization with signal amplification using chemiluminescence on a microplate. Sample scores were based on a ratio of the relative light unit (RLU) per positive control (CO). A RLU/CO score 2.5 was considered positive for the presence of highrisk HPV DNA. All RLU/CO scores 11
1 3 1 1
2 0 0 0
4 0 0 0
0 0 0 0
1 1 1 3
ASCUS ASC-H LSIL HSIL
the guidelines recommended. The cytology diagnosis of cervical pap smears, high risk HPV results, and P16 and Ki-67 immunostains were correlated together and with colposcopy and histologic findings.
Table II. Cytologic/histologic Correlation of Dysplastic Squamous Cells, HPV Status, Ki-67 Proliferation Index, and P16 Immunocytochemical Staining Cytology
Histology
HPV
P16
Ki-67
ASCUS ASCUS ASCUS ASCUS LSIL LSIL ASC-H ASC-H ASC-H HSIL HSIL HSIL HSIL
Benign CIN I Benign Benign CIN I Benign Benign CIN I-II Benign CIN III CIN III CIN III CIN I-II
Not Detected Detected Detected Detected Detected Not Detected Not Detected Detected Not Detected Detected Detected Detected NA
Positive Negative Positive Positive Positive Negative Positive Positive None Positive Positive Positive Positive
Low Low Rare High None High High None None High High None High
Results Cervical Pap Smears Eighteen liquid-based prepared Pap smears were reviewed. The average patient age was 35 years (range 22–60 years). Eight women were diagnosed with ASCUS (mean 36 years), four with ASC-H (mean 34 years), two with LSIL (mean 41 years), and four with HSIL (mean 30 years). The cervical cytology smears were reported according to the Bethesda system criteria for reporting cervical cytology.18 ASC-H cells were mostly single with the size of metaplastic cells. The nuclei were one and one half to two and one half times larger than normal intermediate cells. LSIL cells were single or in sheets. Cytologic changes were confined to cells with mature or superficialtype cytoplasm. The cells were large with enlarged nuclei more than three times the area of normal intermediate nuclei. Chromatin was coarsely granular with variable degrees of nuclear hyperchromasia. Cytoplasm showed distinctive perinuclear halos (koilocytosis). HSIL cells were single or in sheets recognized by marked high nuclear to cytoplasmic ratio, nuclear hyperchromasia, irregular nuclear contour, small cell size and immature, lacy or densely metaplastic cytoplasm.
HPV Testing Results HR HPV results obtained by hybrid capture II assay were available for 17 cases. One HSIL case did not have any HR HPV studies. HPV was reported as detected in nine cases, and not detected in the rest (Table II). In two cases (13%) HPV was not detected, while both P16 and Ki-67 were positive (one ASCUS and one ASC-H). There were no cases (0%) where HPV was detected, and both the P16 and Ki-67 were negative. Among the eight cases with no detectable HR HPV, four were called ASCUS (one of four cases biopsied; benign), three ASCH (two of three cases biopsied; benign squamous mucosa with chronic cervicitis), and one LSIL (biopsied: benign squamous mucosa). 578
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P16 Immunostaining Results P16 expression (Figures 1) correlated with HPV status in 15 of 17 cases (88% concordance; one case of HSIL did not have HPV testing). In one discordant case (ASCUS), HPV was not detected while P16 expression was identified. It was a 60-year old immunocompromised HIV positive patient that HR-HPVs were detected in her prior pap smears but not the concurrent HR-HPV studies which could be due to insufficient HPV DNA copies. P16 immunostain showed positive cells while the proliferation rate was low by Ki-67. The biopsy showed benign squamous mucosa. The second discordant case was an ASC-H case which appeared like CIN I-CIN II on colposcopy. P16 immunostain was weakly and focally positive and Ki-67 showed a high proliferation index. The biopsy showed benign squamous mucosa with marked acute and chronic cervicitis. There were three cases of 1P16 and –Ki-67 (1 LSIL and 1 ASC-H and 1 HSIL). Subsequent follow up biopsies of these cases showed CIN I, CIN I-II, and CIN III, respectively (Table III).
Ki-67 Immunostaining Results The Ki-67 proliferation index (Table I) scoring results were as follows: six negative (one ASCUS, three ASC-H, one LSIL, and one HSIL), two rare (both ASCUS), four mild (ASCUS), 0 moderate, and five high (1 LSIL, 1 ASC-H, and 3 HSIL). The Ki-67 index correlated with HPV in 8 of 15 cases (53% concordance). Discordant cases were as follows: three cases of 1 HPV / –Ki-67 (1 LSIL and 1 ASC-H and 1 HSIL), and five cases of –HPV/1Ki-67 (3 ASCUS, 1 LSIL, 1 ASC-H). Of 1HPV/ –Ki-67 cases, Subsequent biopsies of these cases showed CIN I, CIN I-II, and CIN III, respectively. Of –HPV/ 1Ki-67 cases, two ASCUS cases showed low proliferation index and no further biopsies. One ASCUS with low proliferation rate had subsequent benign biopsy and HR HPVs were detected several times in prior pap smears. The LSIL showed high proliferation index with
Diagnostic Cytopathology DOI 10.1002/dc
P16 AND KI-67 IN ATYPICAL SQUAMOUS CELLS
Fig. 1. Top row from left to right shows a concordant case of HSIL/ HR HPV 1/ Ki-67 1/ P16 1. HSIL pap cytology with detected HR HPV (a), Ki-67 highlights HSIL cells on immunostain (b); P16 stains a HSIL cell (c). Lower row from left to right shows a discordant case of ASC-H/ HR HPV –/ Ki-67 1/ P16 1. ASC-H pap cytology with non-detectable HR- HPV (d), Ki-67 highlights ASC-H cells on immunostain (e), P16 weakly stains an ASC-H cell (f). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Table III. Colposcopic Findings and Correlation with Cervical Cytology, HPV Studies, Surgical Biopsy, P16 Expression, and Ki-67 Proliferation Index Colposcopicfindings
Cervical cytology
Surgical biopsy
HR-HPV status
Normal Normal Normal Normal Normal CIN I CIN I – CIN II CIN II CIN II CIN III
ASCUS ASCUS ASCUS ASCUS HSIL ASCUS ASC-H ASC-H HSIL HSIL
None Benign Benign None CIN III CIN I Benign CIN I- CIN II CIN III CIN III
Detected Detected Not detected Not detected Detected Detected Not detected Detected Detected Detected
a subsequent benign biopsy. There were three cases of 1Ki-67 and –P16 (2 ASCUS and 1 LSIL). HR HPVs were not detected in any of three cases. Subsequent biopsy of LSIL case showed benign findings and inflammation.
Clinical Follow Up Colposcopic examination results were available for 10 patients (Table III). Colposcopic examination was unremarkable in five cases (4 ASCUS and 1 HGIL in cervical cytology material). One appeared as CIN II in colposcopy and showed HSIL in Pap cytology. One ASC-H was called CIN II on colposcopy and it was confirmed on subsequent biopsy. Colposcopy impression was CIN I-CIN II in an ASC-H case and the surgical biopsy showed benign findings with marked acute and chronic cervicitis. Colpo-
P16
Ki-67
Positive Positive Positive Negative Positive Positive Negative Positive Positive Positive
Rare High Low Low High Low High None High None
scopic examination was consistent with CIN I in an ASCUS case and surgical biopsy confirmed CIN I. Histologic follow-up (Table II) was available in 13 cases: four ASCUS (3 benign and 1 CIN I), two LSIL (1 benign and 1 CIN I), three ASC-H (2 benign and 1 CIN I-II), and four HSIL (3 CIN III and 1 CIN I-II). One HSIL P16 1/ Ki-67 1 with no HR HPV study on cytology material and a diagnosis of CIN I- CIN II on biopsy had immunohistochemical staining for P16 and Ki-67 were similar to of those in cytology material. The LSIL case with benign biopsy had prior LSIL on biopsy.
Discordant Cases One ASCUS, HR- HPV 1/ P16 1 (rare)/Ki-67 1 (rare) case with a benign cervical biopsy had multifocal VIN IIIII of right labia and VIN I of left labia which correlates Diagnostic Cytopathology, Vol. 42, No 7
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TOLL ET AL.
with our findings. There were two cases where HPV was not detected while P16 and Ki-67 (Figure 3) were positive; in both cases the corresponding cervical biopsy showed benign findings. One ASCUS, HR- HPV –/P16 1 (rare)/Ki-67 1 (low) case had a biopsy proven prior history of CIN I. One ASCUS, HR- HPV –/ P16 1/ Ki-67 1 (low) case with a subsequent benign biopsy, had a long history of HR HPV detected on her prior pap smears. Not detected HR -HPV was most likely due to sampling and low copies of HR -HPV DNA. One ASCH, HPV –/ P16 1 (rare) / Ki-67 1 (high) case with a CIN I-II on colposcopy had a subsequent biopsy showing marked acute and chronic cervicitis. Sampling problem was probably the only explanation for this case.
Discussion Our findings within this small sample population demonstrate a proof of principle for the adjunct immunocytochemical testing of P16 and Ki-67 in cases of ASCUS and ASC-H, and larger prospective studies with clinical follow-up are warranted to validate these findings. A secondary finding in this study was the further validation that residual thin-layer cervicovaginal material is potentially sufficient for immunocytochemical staining.17 The combination of P16 and / or Ki-67 was 100% sensitive for HPV infection. In two additional cases (1 ASCUS and 1 ASC-H) these surrogate markers were positive, while HPV was not identified. P16 has been shown to be a sensitive and specific marker for HPV infection in ASCUS lesions,19 potentially selecting patients at greater risk to progress from infection to dysplasia. Further, a retrospective examination of cytologically negative Pap smears preceding CIN II-III lesions found incipient HPV infection in only 80% of cases.20 The remaining cases were presumed to contain viral copy numbers below the limit of detection; however, other studies have emphasized the difficulty of detecting CIN III lesions both with cervicovaginal sampling and colposcopic examination.21 In our study, one HSIL case appeared normal on colposcopy and subsequent biopsy confirmed CIN III. While HPV detection in LSIL is approximately 80%, the sensitivity for ASCUS decreases significantly to 43%.22,23 These lesions in particular may benefit from adjunct P16 and Ki-67 testing which may help identify patients at risk to develop cervical neoplasia in spite of following screening guidelines. P16 and Ki-67 immunostaining on residual thinlayer cervicovaginal material make it possible to correlate the cytomorphology and immunoreactivity of the atypical cells at the same time. However, performing immunostains on residual thin-layer cervicovaginal material is another issue to be evaluated since the slides are not paraffin embedded and subsequently eliminate the 30 minute melting time in 80 C. The possible effect of heat elimina580
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tion on immunostaining outcome on cytospin slides should be investigated. Despite widespread screening programs and HPV testing, approximately 30% of newly diagnosed cervical cancers occur in women following the recommended guidelines.24 The HPV virus, notably HPV genotype 16, has been studied in an effort to predict which patients will develop persistent infection and ultimately disease progression. Viral integration into the host genome was cited as a significant factor in the development of invasive carcinoma.25 A related finding was that the expression of E6 and E7, critical in the progression to neoplasia, correlated much more with integration into the host genome rather than viral copy numbers.25 Of note, high viral loads were frequently observed in cases of benign and low-grade squamous lesions, attributed to high episomal levels unrelated to the oncogenic potential of HPV. This would support the utility of P16, rather than viral copy number, as a more appropriate adjunct test in the determination of which patients may show disease progression. Our results indicate that adjunct P16 and Ki-67 testing offers a potential benefit in addition to HPV testing, most notably in cases when the differential diagnosis includes reactive or atypical (either ASCUS or ASC-H) diagnoses. P16 and Ki-67 testing can potentially be improved by brushing the cervico-vaginal mucosa a few times more than standard or colleting cells separately for P16 and Ki16 staining. A consensus group recently recommended the use of P16 in histologic sections from anogenital lesions in the following cases: when the differential is between low grade and high grade (CIN21) dysplasia, when the differential is CIN21 vs. a benign mimic and in discordant cases when initial histologic sections are benign and the Pap was ASC-H or worse.5 In our study one case was called ASC-H and had a negative biopsy. P16 and Ki-67 performed on the biopsy were negative; however the specimen consisted almost entirely of endocervical tissue and cannot be considered adequate. The viral-related proteins E6 and E7, recognized as the oncogenic promoters of HPV, correlate well with P16 overexpression. Accompanied by a Ki-67 proliferation index, these markers may provide a benefit in cases with low viral copies, or in cases of less than optimal sampling. A meta-analysis of 17 studies examining p16 and HPV in cases of ASCUS and LSIL found p16 to show greater accuracy in cases of ASCUS for correlating with CIN2/ CIN3 on colposcopy (better specificity, similar sensitivity) than HPV-DNA testing. In cases of LSIL, p16 showed greater specificity but less sensitivity relative to HPV testing. Among the most significant issues facing p16 testing is the subjective, non-standardized manner p16 is evaluated. Nuclear and / or cytoplasmic expression may be interpreted as positive, and some advocate only evaluating
Diagnostic Cytopathology DOI 10.1002/dc
P16 AND KI-67 IN ATYPICAL SQUAMOUS CELLS
cells with abnormal nuclei to avoid counting benign metaplastic or endocervical cells. One potential solution is the use of a dual stain with Ki-67 proliferation. Most agree that a cell with positive nuclear Ki-67 and cytoplasmic p16 expression is pathologic. Studies examining this issue are underway, and may influence whether HPV testing remains the only ancillary study widely recommended for Pap smears.26
Acknowledgment The authors thank Dr. Dorothy Rosenthal for her critical review of this manuscript.
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