International Journal of Gynecological Pathology 00:1–6, Lippincott Williams & Wilkins, Baltimore r 2015 International Society of Gynecological Pathologists
Original Article
p16INK4a Immunohistochemical and Histopathologic Study of Pap Test Cases Interpreted as HSIL Without CIN2-3 Identification in Subsequent Cervical Specimens Felipe J. Solano,
M.D.
and Edward J. Wilkinson,
M.D.
Summary: Management guidelines 2012 recognized high-grade cervical lesion (HSIL) Pap tests without subsequent high-grade cervical intraepithelial lesion (CIN2-3) in cervical specimens. It is known that the risk of CIN2-3 is higher with CIN1 after HSIL compared with low-grade cervical lesion, and that p16INK4a is a surrogate high-risk human papillomavirus marker proved to improve CIN2-3 interpretation. Two pathologists rereviewed Pap and hematoxylin and eosin preparations from the preceding 3 yr and selected cases with a HSIL Pap test with subsequent cervical specimens that did not demonstrate a HSIL/CIN2-3. Immunohistochemical study for p16INK4a was performed and graded on representative sections. The results were tabulated and analyzed. Of the identified 596 HSIL Pap cases, 82% had HSIL on initial cervical specimens. Table 1 shows the 56 cases included in the study with graded and stratified p16INK4a results. On review of the p16INK4a slides, only 2 cases could be upgraded to HSIL/CIN2-3 from the original diagnosis. p16INK4a 2-3+ was expressed more frequently in cases initially interpreted on Pap as low-grade cervical lesion as compared with benign (24 of 35 cases). In the younger than 24-yr-old group p16 2-3+ reactivity was more frequent in benign and low-grade cervical lesion/CIN1 groups (benign: 3 of 5 cases, and CIN1: 6 of 8), and p16 negative reactivity was not seen. p16INK4a was graded 0-1+ more frequently in specimens interpreted as benign in the older than 25 yr olds (10 of 16 cases). The study suggests some diagnostic benefit from the use of p16INK4a immunohistochemical study on cervical specimens from women with a HSIL Pap test without HSIL/CIN2-3 on original hematoxylin and eosin review. Key Words: p16INK4a—HSIL—CIN1—CIN2-3—Cervical specimen.
The Pap test, combined with appropriate clinical management, has proven to decrease the prevalence of intraepithelial and invasive neoplastic epithelial
cervical diseases. Cervical squamous intraepithelial lesions [low-grade and high-grade SIL/cervical intraepithelial lesion (CIN) lesions] and invasive tumors are known to be related to ‘‘high risk’’ oncogenic types of human papillomavirus (HR-HPV). Studies of the natural history of HPV indicate that the great majority of related cervical squamous intraepithelial neoplastic lesions (SIL/CIN), including low-grade (CIN1) and high-grade (CIN2-3) lesions, may regress (1–3). Regression is more common in low-grade cervical lesion (LSIL)/CIN1 lesions, especially in women less than 24 yr of age. This young population has a higher rate of LSIL lesions, higher rate of HRHPV, and higher reported rate of clearing lesions as
From the Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, Florida. Supported by the Department of Pathology and Laboratory Medicine at the University of Florida, College of Medicine, Gainesville, Florida. The authors declare no conflict of interest. Address correspondence and reprint requests to Edward J. Wilkinson, MD, Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, P.O. Box 100275, Gainesville, FL 32610-0275. E-mail: wilkinso@ pathology.ufl.edu.
1
DOI: 10.1097/PGP.0000000000000159
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
2
F.J. SOLANO AND E.J. WILKINSON
compared with older ages. High-grade cervical lesion (HSIL)/CIN2-3 cases, however, have a recognized risk of progressing to invasive squamous cell carcinoma (1–5). In the United States, CIN2 is the accepted cut off to manage a lesion as a high-grade lesion (1–5). In the 2012 ‘‘consensus guidelines for the management of abnormal cervical cancer screening test and cancer precursors,’’ (1) HSIL/CIN2-3 is identified in cervical specimens in approximately 60% of women with a HSIL Pap test. The risk of HSIL/CIN2-3 is substantially higher in women with LSIL/CIN1 after an HSIL Pap test (5 yr risk of CIN3+ of 15%) compared with women with ASC-US/HR-HPV positive or LSIL Pap test result (5-yr risk for CIN3+ of 3.8%) (1). It is recommended that women with LSIL/CIN1, or no lesion, preceded by an HSIL Pap test be managed with either a diagnostic excisional procedure or, if the colposcopic examination is adequate and the endocervical sampling is negative, follow-up with observation and contesting at 12 and 24 mo is acceptable; independent of age or parity status (1). The consensus recognized there is room for improvement of management guidelines including increased scientific knowledge on the use of immunohistochemical studies, such as the use of p16INK4a, in the study of women with HSIL Pap without HSIL/CIN2-3 (1). p16INK4a is a surrogate immunohistochemical marker for HR-HPV and is proposed to be of use to improve the subjective pathology interpretative diagnosis of cervical high-grade squamous intraepithelial lesion (CIN2-3) (2–23). A specific grading pattern of p16INK4a immunostain has been associated with the presence of a CIN2-3 lesion in a cervical specimen (2–23). It has been reported that for HSIL/CIN3 the sensitivity and specificity of diffuse p16INK4a immunostaining was 100% and 95%, respectively; for HSIL/CIN2, it was 81.1% and 95.4%, respectively. When the values where generalized to a large cohort of woman, the positive predictive and negative predictive values were 13.9% and 100% for CIN3, and 20.4% and 99.7% for CIN2 or CIN3, respectively (7). Recommendations for the use of this biomarker were addressed in the Lower Anogenital Squamous Lesion Terminology (LAST) study (2). In summary, p16INK4a is recommended when the hematoxylin and eosin (H&E) morphologic differential diagnosis is between precancer (HSIL/CIN2-3) and a mimic of precancer-like squamous metaplasia (2). Strong and diffuse ‘‘block positive’’ p16 immunostaining findings support a categorization of precancerous lesion (2).
In the same study the use of p16, in cases where a diagnosis of CIN2 is contemplated, is also recommended. In such cases, negative or nonblock staining favors an interpretation of CIN1 or no-CIN lesion (2). The test of p16 immunostaining is recommended as an adjunctive assessment tool for morphologic assessment of biopsies with LSIL/CIN1 or negative findings when preceded by a HSIL, ASC-H, ASCUS/HPV16+, or AGC-NOS Pap test with the caveat that any p16-positive area must meet the morphologic criteria for HSIL/CIN2-3 on H&E (2). Our objectives are to describe immunohistochemical staining patterns of p16INK4a in cervical specimens in a set of women with a prior Pap test interpreted as HSIL with subsequent cervical specimens interpreted as negative or LSIL/CIN1. To identify previously unidentified HSIL/CIN2-3 by review and by resectioning and staining cervical tissue initially interpreted as negative for a highgrade lesion. The final purpose is to help improve guidelines by improving pathology diagnostic methods and interpretations used to direct, stratify, and support the management of women with a Pap test of HSIL and a subsequent negative, or LSIL/CIN1 lesion, on cervical specimens. METHODS With institutional review board approval a retrospective and descriptive study was conducted starting with computer-based data search of cytology Pap test cases with an interpretation of HSIL (ICD9 code 795.04) performed over the prior 3 yr (from May 15, 2009 to May 15, 2012). Patients with a HSIL Pap test and subsequent cervical specimens without interpretation of HSIL/CIN2-3 were identified. A computer search of all additional cervical pathology and cytology history on the study cases was also performed. Immunohistochemical study for p16INK4a was performed on cervical specimens from study cases and graded in squamous or metaplastic epithelium from 0+ to 3+. The scoring of p16 included both nuclear and cytoplasmic staining, and was taken from Hampl et al. (8). Graded as 0 (no staining), 1 (rare singly dispersed cells staining), 2 (patchy but strong staining, often not continuous from basement membrane), and 3 (strong and diffuse staining, usually continuous staining from basement membrane and extending upward in proportion to lesion grade). Four-micron paraffin sections were cut and placed on plus slides and dried for 2 hr in a 601C oven. The
Int J Gynecol Pathol Vol. 00, No. 00, ’’ 2015
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
P16INK4A IMMUNOHISTOCHEMICAL AND HISTOPATHOLOGIC STUDY slides were then placed on the Ventana Benchmark automated immunostainer where they were dewaxed. Heat-induced epitope retrieval was performed with Ventana’s CC1 retrieval solution for 30 min at 95 to 1001C. Primary mouse monoclonal antibody antip16INK4a, clone JC8 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), at 1:25 dilution was applied to sections at 371C for 32 min. Presence of the antigen was visualized by using the Ultra View DAB detection kit (Ventana Medical Systems Inc., Tucson, AZ). Slides were counterstained with Ventana hematoxylin, taken off the stainer, dehydrated, cleared, mounted with permanent mounting media, and labeled. The available Pap-stained Pap test slides previously interpreted as HSIL, and the original H&E-stained slides from cervical specimens, were reviewed by the principal investigator and the coinvestigator. Cervical specimens (biopsies and excisions) with only negative, or LSIL/CIN1, interpretations and with representative transformation zone, adequate amounts of tissue for p16INK4a immunostaining, and preceding Pap test interpreted as HSIL were included. On review of H&E-stained slides from cervical specimens, mitoses above mid squamous epithelium, nuclear hyperchromatism, prominent nucleoli, radial dispersion of nuclear chromatin, abundance of cytoplasm, cellular and nuclear pleomorphism, disorganization, overlapping of cells, inflammation, and immature versus mature metaplasia were evaluated. Analysis of H&E morphologic features, p16INK4a immunostaining graded cases and history data were recorded and tabulated in an Excel table. The information, graphics, and sum tables were created using Microsoft Office software programs and analyzed by simple comparison. RESULTS A total of 596 HSIL Pap test cases were identified from 57,285 total reported Pap tests from May 15, 2009 to May 15, 2012, corresponding to a 1.0% of Pap test interpreted as HSIL. ASC/SIL ratios and ASC percentages for 2009 to 2012 were 1.0% and 5%, on average, respectively, per year. No discrepancies on the amount of tissue material available on H&E-stained slides from cervical specimens versus p16INK4a immunostained slides were observed by naked eye and microscopic examination. Of the HSIL cases, 488 (82%) were excluded in this study due to a subsequent diagnosis of a high-grade squamous intraepithelial lesion (CIN2-3) on cervical specimen
3
(biopsy or excision). Of the total 108 (18%) remaining patient/cases with Pap tests with HSIL all had diagnoses of benign cervical findings or LSIL/CIN1 on cervical specimens; 14 were excluded from the study due to absence of a Pap slide available for evaluation of the HSIL, 36 (6.4%) were excluded due to underrepresentation of the T zone on the cervical specimen, and 1 case was excluded for marked ulceration. Two (0.33%) cases with a HSIL interpretation on Pap test were excluded on review due to HSIL not confirmed in the interpreted slide. Of those 2, 1 case was reinterpreted as LSIL and the second case as ASC-H. A total of 56 HSIL cases were identified for the study. The Pap tests were from women with ages ranging from 18 to 62 yr of age. The p16INK4a study cases were graded and stratified for results by age and H&E interpretations (Table 1). Twenty-one (21) HSIL Pap cases had an interpretation of negative on subsequent cervical specimen and 35 had a diagnosis of LSIL/CIN1. Photographs demonstrating p16INK4a grading on study representative cases are presented (Fig. 1). Independent of the age, p16INK4a Grade 2-3+ was present more frequently in cervical specimens originally interpreted as LSIL/CIN1 (24 of 35 cases) as compared with negative (9 of 21 cases) (Table 1). In contrast, p16INK4a Grade 0-1+ was more frequently found in cervical specimens with negative interpretations (12 of 21 cases) compared with LSIL/ CIN1 interpretations (11 of 35 cases). In the older than 25-yr-old group, this was observed in 10 of 16 negative cases. In the younger than 24-yr-old group, negative and LSIL/CIN1 interpretation groups, p16INK4a reactivity was always seen (Grade 0 not seen). In this younger than 24-yr-old group p16INK4a 2-3+ reactivity was more frequently observed in the negative and LSIL/CIN1 groups (3 of 5 cases in negative group, and 6 of 8 cases in CIN1) (Table 1). In total, there were 11 cases with p16 Grade 3+ (Table 1). Nine p16 Grade 3+ cases had a LSIL/ CIN1 interpretation and only 2 p16 Grade 3+ cases were in women aged less than 24 yr. Of the 22 cases with p16 Grade 2+, 15 were interpreted as LSIL/ CIN1. Of these, 7 were from women less than 24-yrold with 5 interpreted as LSIL/CIN1 and 2 interpreted as negative (Table 1). On review of the original H&E cervical specimens from the 56 cases, 2 cases were upgraded to HSIL/CIN2-3 from the original diagnoses of benign and LSIL/CIN1, respectively. Both cases had a Int J Gynecol Pathol Vol. 00, No. 00, ’’ 2015
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
4
F.J. SOLANO AND E.J. WILKINSON TABLE 1. p16INK4a staining grade results stratified by original interpretation and patient age
Interpretation on studied cervical specimens
Negative
CIN1
o24 yr old 425 yr old Total cases o24 yr old 425 yr old Total cases Total no. cases
Age groups p16INK4a staining result and grade Negative staining 0+ 1+ Positive staining 2+ 3+ Total no. cases
0 2
2 8
2 10
0 2
3 6
3 8
5 18
2 1 5
5 1 16
7 2 21
5 1 8
10 8 27
15 9 35
22 11 56
p16INK4a Grade of 3+. One of the cases was from a older than 25-yr-old woman who subsequently had a HSIL Pap test 240 d after the biopsy specimen in this study. The second case was from a woman younger than 24-yr-old woman who had no further follow-up. A total of 11 of 56 study cases had additional subsequent cervical specimens reported (Table 2). Six cases with p16 Grade 0-1+ had a subsequent cervical specimen interpreted as negative, or LSIL/CIN1, 34 to 600 d after the original cervical specimen interpretation. Four of the 11 patients had p16 Grade 2+ and subsequently had HSIL/CIN3 on cervical specimens obtained from 90 to 360 d after the original cervical specimen interpretation; all these cases were in women aged older than 25 yr of age (Table 2). Four cases with p16 Grade 1+ had a prior cervical specimen interpreted as HSIL/CIN2-3 240 to 365 d before the study p16 stained cervical specimen case. In 33 cases with p16INK4a Grade 2-3+, nuclear hyperchromasia was found in 24 cases (11 of 11 cases had 3+ on p16). In contrast, 6 of 21 cases with p16
grade of 0-1+ had some nuclear hyperchromasia on H&E sections. Focal mitoses in the mid portion of the epithelium or above was seen more frequently in cases with p16 Grade 2-3+. Of 33 cases, 14 had p16 2-3+, 9 of which had 3+ on p16. In contrast, in 21 cases with no mitoses in the mid epithelium or above, all had p16 graded 0-1+. All the other H&E variables evaluated and collected including prominent nucleoli, radial dispersion of nuclear chromatin, abundance of cytoplasm, cellular and nuclear pleomorphism, disorganization and overlapping of cells, inflammation, and immature versus mature metaplasia were equally represented in all the groups without a recognized trend by simple data comparison. CONCLUSIONS H&E interpretations of cervical specimens are the gold standard for the interpretation of abnormalities of the uterine cervix including epithelial preneoplastic
Case 2
Case 3
Case 4
CIN1 interpretation
Negative interpretation
Negative interpretation
0+
1+
2+
3+
p16INK4a stain scale 20X power view
H&E stain 20X power view
Case 1 Negative interpretation
FIG. 1. Magnification of 20 of microscopic photograph examples of cases showing the grading scale for p16INK4a in squamous epithelium 0 (no staining), 1 (rare singly dispersed cells staining), 2 (patchy but strong staining, often not continuous from basement membrane), and 3 (strong and diffuse staining, usually continuous staining from basement membrane and extending upward in proportion to lesion grade).
Int J Gynecol Pathol Vol. 00, No. 00, ’’ 2015
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
P16INK4A IMMUNOHISTOCHEMICAL AND HISTOPATHOLOGIC STUDY
5
TABLE 2. Summary of available additional past and new cervical specimen history collected from patients enrolled in the study stratified by original interpretation and patient age Age groups Negative cases o24 425 CIN1 cases o24 425
p16INK4a grade
Past cervical specimen
New cervical specimen
Time between interpretation and new specimen (d)
1+ 0+ 1+ 2+
NA NA NA NA
CIN1 Negative CIN1 CIN3
188 360 600 90
1+ 1+ 1+ 0+ 2+ 2+ 2+
CIN3 NA NA Negative NA CIN1 NA
CIN2 Negative CIN1 CIN1 CIN3 CIN3 CIN3
60 34 210 180 360 200 300
NA indicates not available.
conditions. Described mimickers of HSIL on Pap test include basal cell hyperplasia, immature and mature squamous metaplasia, reactive/inflamed cervical epithelium, and atrophy. The use the use of p16INK4a has proven to be of significant value in distinguishing these changes from a HSIL/CIN2-3 lesions (2–23). This study addresses, defines, and confirms the value of the current CAP-ASCCP LAST Committee recommendations to perform p16INK4a in cases with a HSIL Pap test where no HSIL/CIN2-3 is identified on a subsequent cervical specimen (2). This study confirms the value in review of the HSIL Pap test initiating the cervical sampling, but also suggests that review of the specimen interpreted as negative may identify a high-grade lesion on review. In cases with a history of HSIL on Pap test with subsequent cervical specimens with pathology interpretations of negative or CIN1, review of the initial slides, resectioning of the index block, and immunohistochemical study for p16INK4a may be of value to properly classify a specific specimen as having, or not having, a high-grade lesion. The negative predictive value for diffuse p16INK4a immunostaining to support the diagnosis of HSIL/CIN2-3 has been reported as >97% (7). The finding of p16 Grade 3+ will strongly suggest HSIL/CIN2-3 with supportive morphologic pathologic findings. In such cases, management per ASCCP guidelines of CIN2-3 is proposed. Also, our findings in cases from women aged older than 25 yr of age with 2+ p16INK4a and additional cervical specimen history, suggest classifying a lesion originally perceived on H&E as LSIL/ CIN1 as HSIL/CIN2-3. Precaution should be taken not to reinterpret a biopsy from a younger than 24yr-old woman with histopathologic features of LSIL that is p16 Grade 2+ as HSIL (CIN2-3). Such cases
we think should be classified as ‘‘p16-positive LSIL (p16+LSIL),’’ although some argument could be made to classify such a biopsy as ‘‘ungraded CIN lesion.’’ The absence of a transformation zone in cervical specimens may be of value as a quality indicator for further close follow-up in patients with a preceding high-grade Pap test result. In our study group, approximately one third (36 of 108 cases) identified women with an HSIL Pap with subsequent LSIL/ CIN1 or negative cervical specimen did not have representative transformation zone in the cervical sample. Two HSIL Pap test cases from the study group were considered as potential overinterpreted for HSIL, corresponding to 0.33% of all the HSIL. In our institution the concordance percentage rate is >60% (488 of 596 cases for 82%) for HSIL Pap cases with diagnosis of HSIL/CIN2-3 on subsequent cervical specimens. p16 antibody is a commercially available marker that is derived from different clones. One study has demonstrated a potential difference in staining patterns depending of the clone and tissue that is studied (12). More studies in this matter are needed and validation studies are suggested if p16INK4a is in use. In summary, this study suggests some diagnostic benefit from the use of p16INK4a immunohistochemical study on cervical specimens from women with HSIL Pap test where HSIL/CIN2-3 is not identified on original H&E review. Acknowledgments: The authors thank the technical and clerical staff of the University of Florida UF-Path labs and UF-Health Shands at Rocky Point cytology and histology laboratories. Int J Gynecol Pathol Vol. 00, No. 00, ’’ 2015
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
6
F.J. SOLANO AND E.J. WILKINSON REFERENCES
1. Massad LS, Einstein MH, Huh WK, et al, 2012 ASCCP Consensus Guidelines Conference. 2012 updated consensus guidelines for the management of abnormal cervical cancer screening tests and cancer precursors. J Low Genit Tract Dis 2013;17(suppl 1): S1–S27. 2. Darragh TM, Colgan TJ, Cox JT, et al, Members of LAST Project Work Groups. 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. J Low Genit Tract Dis 2012;16:205–42. 3. Waxman AG, Chelmow D, Darragh TM, et al. Revised terminology for cervical histopathology and its implications for management of high-grade squamous intraepithelial lesions of the cervix. Obstet Gynecol 2012;120:1465–71. 4. Saslow D, Solomon D, Lawson HW, et al, ACS-ASCCPASCP Cervical Cancer Guideline Committee. American Cancer Society, American Society for Colposcopy and Cervical Pathology, and American Society for Clinical Pathology screening guidelines for the prevention and early detection of cervical cancer. CA Cancer J Clin 2012;62:147–72. 5. Walker JL, Wang SS, Schiffman M, et al, ASCUS LSIL Triage Study Group. Predicting absolute risk of CIN3 during postcolposcopic follow-up: results from the ASCUS-LSIL Triage Study (ALTS). Am J Obstet Gynecol 2006;195:341–8. 6. Bergeron C, Ordi J, Schmidt D, et al, European CINtec Histology Study Group. Conjunctive p16INK4a testing significantly increases accuracy in diagnosing high-grade cervical intraepithelial neoplasia. Am J Clin Pathol 2010;133:395–406. 7. Wang SS, Trunk M, Schiffman M, et al. Validation of p16INK4a as a marker of oncogenic human papillomavirus infection in cervical biopsies from a population-based cohort in Costa Rica. Cancer Epidemiol Biomarkers Prev 2004;13:1355–60. 8. Hampl M, Wentzensen N, Vinokurova S, et al. Comprehensive analysis of 130 multicentric intraepithelial female lower genital tract lesions by HPV typing and p16 expression profile. J Cancer Res Clin Oncol 2007;133:235–45. 9. Guo M, Hu L, Baliga M, et al. The predictive value of p16(INK4a) and hybrid capture 2 human papillomavirus testing for high-grade cervical intraepithelial neoplasia. Am J Clin Pathol 2004;122:894–901. 10. Galgano MT, Castle PE, Atkins KA, et al. Using biomarkers as objective standards in the diagnosis of cervical biopsies. Am J Surg Pathol 2010;34:1077–87. 11. Samarawardana P, Dehn DL, Singh M, et al. p16(INK4a) is superior to high-risk human papillomavirus testing in cervical
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
cytology for the prediction of underlying high-grade dysplasia. Cancer Cytopathol 2010;118:146–56. Sawicka M, Pawlikowski J, Wilson S, et al. The specificity and patterns of staining in human cells and tissues of p16INK4a antibodies demonstrate variant antigen binding. PLoS One 2013;8:e53313. Brown CA, Bogers J, Sahebali S, et al. Role of protein biomarkers in the detection of high-grade disease in cervical cancer screening programs. J Oncol 2012;2012:289315. Negri G, Bellisano G, Zannoni GF, et al. p16 ink4a and HPV L1 immunohistochemistry is helpful for estimating the behavior of low-grade dysplastic lesions of the cervix uteri. Am J Surg Pathol 2008;32:1715–20. Tsoumpou I, Arbyn M, Kyrgiou M, et al. p16(INK4a) immunostaining in cytological and histological specimens from the uterine cervix: a systematic review and meta-analysis. Cancer Treat Rev 2009;35:210–20. Qiao X, Bhuiya TA, Spitzer M. Differentiating high-grade cervical intraepithelial lesion from atrophy in postmenopausal women using Ki-67, cyclin E, and p16 immunohistochemical analysis. J Low Genit Tract Dis 2005;9:100–7. Iaconis L, Hyjek E, Ellenson LH, et al. p16 and Ki-67 immunostaining in atypical immature squamous metaplasia of the uterine cervix: correlation with human papillomavirus detection. Arch Pathol Lab Med 2007;131:1343–1349Erratum in: Arch Pathol Lab Med. 2008;132(1):13. Negri G, Vittadello F, Romano F, et al. p16INK4a expression and progression risk of low-grade intraepithelial neoplasia of the cervix uteri. Virchows Arch 2004;445:616–20. Yildiz IZ, Usubu¨tu¨n A, Firat P, et al. Efficiency of immunohistochemical p16 expression and HPV typing in cervical squamous intraepithelial lesion grading and review of the p16 literature. Pathol Res Pract 2007;203:445–9. Kostopoulou E, Samara M, Kollia P, et al. Different patterns of p16 immunoreactivity in cervical biopsies: correlation to lesion grade and HPV detection, with a review of the literature. Eur J Gynaecol Oncol 2011;32:54–61. Lambert AP, Anschau F, Schmitt VM. p16INK4A expression in cervical premalignant and malignant lesions. Exp Mol Pathol 2006;80:192–6. Kalof AN, Cooper K. Our approach to squamous intraepithelial lesions of the uterine cervix. J Clin Pathol 2007;60:449–55. Wentzensen N, Schwartz L, Zuna RE, et al. Performance of p16/Ki-67 immunostaining to detect cervical cancer precursors in a colposcopy referral population. Clin Cancer Res 2012;18:4154–62.
Int J Gynecol Pathol Vol. 00, No. 00, ’’ 2015
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
Original Article
p16INK4a Immunohistochemical and Histopathologic Study of Pap Test Cases Interpreted as HSIL Without CIN2-3 Identification in Subsequent Cervical Specimens Felipe J. Solano,
M.D.
and Edward J. Wilkinson,
M.D.
Summary: Management guidelines 2012 recognized high-grade cervical lesion (HSIL) Pap tests without subsequent high-grade cervical intraepithelial lesion (CIN2-3) in cervical specimens. It is known that the risk of CIN2-3 is higher with CIN1 after HSIL compared with low-grade cervical lesion, and that p16INK4a is a surrogate high-risk human papillomavirus marker proved to improve CIN2-3 interpretation. Two pathologists rereviewed Pap and hematoxylin and eosin preparations from the preceding 3 yr and selected cases with a HSIL Pap test with subsequent cervical specimens that did not demonstrate a HSIL/CIN2-3. Immunohistochemical study for p16INK4a was performed and graded on representative sections. The results were tabulated and analyzed. Of the identified 596 HSIL Pap cases, 82% had HSIL on initial cervical specimens. Table 1 shows the 56 cases included in the study with graded and stratified p16INK4a results. On review of the p16INK4a slides, only 2 cases could be upgraded to HSIL/CIN2-3 from the original diagnosis. p16INK4a 2-3+ was expressed more frequently in cases initially interpreted on Pap as low-grade cervical lesion as compared with benign (24 of 35 cases). In the younger than 24-yr-old group p16 2-3+ reactivity was more frequent in benign and low-grade cervical lesion/CIN1 groups (benign: 3 of 5 cases, and CIN1: 6 of 8), and p16 negative reactivity was not seen. p16INK4a was graded 0-1+ more frequently in specimens interpreted as benign in the older than 25 yr olds (10 of 16 cases). The study suggests some diagnostic benefit from the use of p16INK4a immunohistochemical study on cervical specimens from women with a HSIL Pap test without HSIL/CIN2-3 on original hematoxylin and eosin review. Key Words: p16INK4a—HSIL—CIN1—CIN2-3—Cervical specimen.
The Pap test, combined with appropriate clinical management, has proven to decrease the prevalence of intraepithelial and invasive neoplastic epithelial
cervical diseases. Cervical squamous intraepithelial lesions [low-grade and high-grade SIL/cervical intraepithelial lesion (CIN) lesions] and invasive tumors are known to be related to ‘‘high risk’’ oncogenic types of human papillomavirus (HR-HPV). Studies of the natural history of HPV indicate that the great majority of related cervical squamous intraepithelial neoplastic lesions (SIL/CIN), including low-grade (CIN1) and high-grade (CIN2-3) lesions, may regress (1–3). Regression is more common in low-grade cervical lesion (LSIL)/CIN1 lesions, especially in women less than 24 yr of age. This young population has a higher rate of LSIL lesions, higher rate of HRHPV, and higher reported rate of clearing lesions as
From the Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, Florida. Supported by the Department of Pathology and Laboratory Medicine at the University of Florida, College of Medicine, Gainesville, Florida. The authors declare no conflict of interest. Address correspondence and reprint requests to Edward J. Wilkinson, MD, Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, P.O. Box 100275, Gainesville, FL 32610-0275. E-mail: wilkinso@ pathology.ufl.edu.
1
DOI: 10.1097/PGP.0000000000000159
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
2
F.J. SOLANO AND E.J. WILKINSON
compared with older ages. High-grade cervical lesion (HSIL)/CIN2-3 cases, however, have a recognized risk of progressing to invasive squamous cell carcinoma (1–5). In the United States, CIN2 is the accepted cut off to manage a lesion as a high-grade lesion (1–5). In the 2012 ‘‘consensus guidelines for the management of abnormal cervical cancer screening test and cancer precursors,’’ (1) HSIL/CIN2-3 is identified in cervical specimens in approximately 60% of women with a HSIL Pap test. The risk of HSIL/CIN2-3 is substantially higher in women with LSIL/CIN1 after an HSIL Pap test (5 yr risk of CIN3+ of 15%) compared with women with ASC-US/HR-HPV positive or LSIL Pap test result (5-yr risk for CIN3+ of 3.8%) (1). It is recommended that women with LSIL/CIN1, or no lesion, preceded by an HSIL Pap test be managed with either a diagnostic excisional procedure or, if the colposcopic examination is adequate and the endocervical sampling is negative, follow-up with observation and contesting at 12 and 24 mo is acceptable; independent of age or parity status (1). The consensus recognized there is room for improvement of management guidelines including increased scientific knowledge on the use of immunohistochemical studies, such as the use of p16INK4a, in the study of women with HSIL Pap without HSIL/CIN2-3 (1). p16INK4a is a surrogate immunohistochemical marker for HR-HPV and is proposed to be of use to improve the subjective pathology interpretative diagnosis of cervical high-grade squamous intraepithelial lesion (CIN2-3) (2–23). A specific grading pattern of p16INK4a immunostain has been associated with the presence of a CIN2-3 lesion in a cervical specimen (2–23). It has been reported that for HSIL/CIN3 the sensitivity and specificity of diffuse p16INK4a immunostaining was 100% and 95%, respectively; for HSIL/CIN2, it was 81.1% and 95.4%, respectively. When the values where generalized to a large cohort of woman, the positive predictive and negative predictive values were 13.9% and 100% for CIN3, and 20.4% and 99.7% for CIN2 or CIN3, respectively (7). Recommendations for the use of this biomarker were addressed in the Lower Anogenital Squamous Lesion Terminology (LAST) study (2). In summary, p16INK4a is recommended when the hematoxylin and eosin (H&E) morphologic differential diagnosis is between precancer (HSIL/CIN2-3) and a mimic of precancer-like squamous metaplasia (2). Strong and diffuse ‘‘block positive’’ p16 immunostaining findings support a categorization of precancerous lesion (2).
In the same study the use of p16, in cases where a diagnosis of CIN2 is contemplated, is also recommended. In such cases, negative or nonblock staining favors an interpretation of CIN1 or no-CIN lesion (2). The test of p16 immunostaining is recommended as an adjunctive assessment tool for morphologic assessment of biopsies with LSIL/CIN1 or negative findings when preceded by a HSIL, ASC-H, ASCUS/HPV16+, or AGC-NOS Pap test with the caveat that any p16-positive area must meet the morphologic criteria for HSIL/CIN2-3 on H&E (2). Our objectives are to describe immunohistochemical staining patterns of p16INK4a in cervical specimens in a set of women with a prior Pap test interpreted as HSIL with subsequent cervical specimens interpreted as negative or LSIL/CIN1. To identify previously unidentified HSIL/CIN2-3 by review and by resectioning and staining cervical tissue initially interpreted as negative for a highgrade lesion. The final purpose is to help improve guidelines by improving pathology diagnostic methods and interpretations used to direct, stratify, and support the management of women with a Pap test of HSIL and a subsequent negative, or LSIL/CIN1 lesion, on cervical specimens. METHODS With institutional review board approval a retrospective and descriptive study was conducted starting with computer-based data search of cytology Pap test cases with an interpretation of HSIL (ICD9 code 795.04) performed over the prior 3 yr (from May 15, 2009 to May 15, 2012). Patients with a HSIL Pap test and subsequent cervical specimens without interpretation of HSIL/CIN2-3 were identified. A computer search of all additional cervical pathology and cytology history on the study cases was also performed. Immunohistochemical study for p16INK4a was performed on cervical specimens from study cases and graded in squamous or metaplastic epithelium from 0+ to 3+. The scoring of p16 included both nuclear and cytoplasmic staining, and was taken from Hampl et al. (8). Graded as 0 (no staining), 1 (rare singly dispersed cells staining), 2 (patchy but strong staining, often not continuous from basement membrane), and 3 (strong and diffuse staining, usually continuous staining from basement membrane and extending upward in proportion to lesion grade). Four-micron paraffin sections were cut and placed on plus slides and dried for 2 hr in a 601C oven. The
Int J Gynecol Pathol Vol. 00, No. 00, ’’ 2015
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
P16INK4A IMMUNOHISTOCHEMICAL AND HISTOPATHOLOGIC STUDY slides were then placed on the Ventana Benchmark automated immunostainer where they were dewaxed. Heat-induced epitope retrieval was performed with Ventana’s CC1 retrieval solution for 30 min at 95 to 1001C. Primary mouse monoclonal antibody antip16INK4a, clone JC8 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), at 1:25 dilution was applied to sections at 371C for 32 min. Presence of the antigen was visualized by using the Ultra View DAB detection kit (Ventana Medical Systems Inc., Tucson, AZ). Slides were counterstained with Ventana hematoxylin, taken off the stainer, dehydrated, cleared, mounted with permanent mounting media, and labeled. The available Pap-stained Pap test slides previously interpreted as HSIL, and the original H&E-stained slides from cervical specimens, were reviewed by the principal investigator and the coinvestigator. Cervical specimens (biopsies and excisions) with only negative, or LSIL/CIN1, interpretations and with representative transformation zone, adequate amounts of tissue for p16INK4a immunostaining, and preceding Pap test interpreted as HSIL were included. On review of H&E-stained slides from cervical specimens, mitoses above mid squamous epithelium, nuclear hyperchromatism, prominent nucleoli, radial dispersion of nuclear chromatin, abundance of cytoplasm, cellular and nuclear pleomorphism, disorganization, overlapping of cells, inflammation, and immature versus mature metaplasia were evaluated. Analysis of H&E morphologic features, p16INK4a immunostaining graded cases and history data were recorded and tabulated in an Excel table. The information, graphics, and sum tables were created using Microsoft Office software programs and analyzed by simple comparison. RESULTS A total of 596 HSIL Pap test cases were identified from 57,285 total reported Pap tests from May 15, 2009 to May 15, 2012, corresponding to a 1.0% of Pap test interpreted as HSIL. ASC/SIL ratios and ASC percentages for 2009 to 2012 were 1.0% and 5%, on average, respectively, per year. No discrepancies on the amount of tissue material available on H&E-stained slides from cervical specimens versus p16INK4a immunostained slides were observed by naked eye and microscopic examination. Of the HSIL cases, 488 (82%) were excluded in this study due to a subsequent diagnosis of a high-grade squamous intraepithelial lesion (CIN2-3) on cervical specimen
3
(biopsy or excision). Of the total 108 (18%) remaining patient/cases with Pap tests with HSIL all had diagnoses of benign cervical findings or LSIL/CIN1 on cervical specimens; 14 were excluded from the study due to absence of a Pap slide available for evaluation of the HSIL, 36 (6.4%) were excluded due to underrepresentation of the T zone on the cervical specimen, and 1 case was excluded for marked ulceration. Two (0.33%) cases with a HSIL interpretation on Pap test were excluded on review due to HSIL not confirmed in the interpreted slide. Of those 2, 1 case was reinterpreted as LSIL and the second case as ASC-H. A total of 56 HSIL cases were identified for the study. The Pap tests were from women with ages ranging from 18 to 62 yr of age. The p16INK4a study cases were graded and stratified for results by age and H&E interpretations (Table 1). Twenty-one (21) HSIL Pap cases had an interpretation of negative on subsequent cervical specimen and 35 had a diagnosis of LSIL/CIN1. Photographs demonstrating p16INK4a grading on study representative cases are presented (Fig. 1). Independent of the age, p16INK4a Grade 2-3+ was present more frequently in cervical specimens originally interpreted as LSIL/CIN1 (24 of 35 cases) as compared with negative (9 of 21 cases) (Table 1). In contrast, p16INK4a Grade 0-1+ was more frequently found in cervical specimens with negative interpretations (12 of 21 cases) compared with LSIL/ CIN1 interpretations (11 of 35 cases). In the older than 25-yr-old group, this was observed in 10 of 16 negative cases. In the younger than 24-yr-old group, negative and LSIL/CIN1 interpretation groups, p16INK4a reactivity was always seen (Grade 0 not seen). In this younger than 24-yr-old group p16INK4a 2-3+ reactivity was more frequently observed in the negative and LSIL/CIN1 groups (3 of 5 cases in negative group, and 6 of 8 cases in CIN1) (Table 1). In total, there were 11 cases with p16 Grade 3+ (Table 1). Nine p16 Grade 3+ cases had a LSIL/ CIN1 interpretation and only 2 p16 Grade 3+ cases were in women aged less than 24 yr. Of the 22 cases with p16 Grade 2+, 15 were interpreted as LSIL/ CIN1. Of these, 7 were from women less than 24-yrold with 5 interpreted as LSIL/CIN1 and 2 interpreted as negative (Table 1). On review of the original H&E cervical specimens from the 56 cases, 2 cases were upgraded to HSIL/CIN2-3 from the original diagnoses of benign and LSIL/CIN1, respectively. Both cases had a Int J Gynecol Pathol Vol. 00, No. 00, ’’ 2015
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
4
F.J. SOLANO AND E.J. WILKINSON TABLE 1. p16INK4a staining grade results stratified by original interpretation and patient age
Interpretation on studied cervical specimens
Negative
CIN1
o24 yr old 425 yr old Total cases o24 yr old 425 yr old Total cases Total no. cases
Age groups p16INK4a staining result and grade Negative staining 0+ 1+ Positive staining 2+ 3+ Total no. cases
0 2
2 8
2 10
0 2
3 6
3 8
5 18
2 1 5
5 1 16
7 2 21
5 1 8
10 8 27
15 9 35
22 11 56
p16INK4a Grade of 3+. One of the cases was from a older than 25-yr-old woman who subsequently had a HSIL Pap test 240 d after the biopsy specimen in this study. The second case was from a woman younger than 24-yr-old woman who had no further follow-up. A total of 11 of 56 study cases had additional subsequent cervical specimens reported (Table 2). Six cases with p16 Grade 0-1+ had a subsequent cervical specimen interpreted as negative, or LSIL/CIN1, 34 to 600 d after the original cervical specimen interpretation. Four of the 11 patients had p16 Grade 2+ and subsequently had HSIL/CIN3 on cervical specimens obtained from 90 to 360 d after the original cervical specimen interpretation; all these cases were in women aged older than 25 yr of age (Table 2). Four cases with p16 Grade 1+ had a prior cervical specimen interpreted as HSIL/CIN2-3 240 to 365 d before the study p16 stained cervical specimen case. In 33 cases with p16INK4a Grade 2-3+, nuclear hyperchromasia was found in 24 cases (11 of 11 cases had 3+ on p16). In contrast, 6 of 21 cases with p16
grade of 0-1+ had some nuclear hyperchromasia on H&E sections. Focal mitoses in the mid portion of the epithelium or above was seen more frequently in cases with p16 Grade 2-3+. Of 33 cases, 14 had p16 2-3+, 9 of which had 3+ on p16. In contrast, in 21 cases with no mitoses in the mid epithelium or above, all had p16 graded 0-1+. All the other H&E variables evaluated and collected including prominent nucleoli, radial dispersion of nuclear chromatin, abundance of cytoplasm, cellular and nuclear pleomorphism, disorganization and overlapping of cells, inflammation, and immature versus mature metaplasia were equally represented in all the groups without a recognized trend by simple data comparison. CONCLUSIONS H&E interpretations of cervical specimens are the gold standard for the interpretation of abnormalities of the uterine cervix including epithelial preneoplastic
Case 2
Case 3
Case 4
CIN1 interpretation
Negative interpretation
Negative interpretation
0+
1+
2+
3+
p16INK4a stain scale 20X power view
H&E stain 20X power view
Case 1 Negative interpretation
FIG. 1. Magnification of 20 of microscopic photograph examples of cases showing the grading scale for p16INK4a in squamous epithelium 0 (no staining), 1 (rare singly dispersed cells staining), 2 (patchy but strong staining, often not continuous from basement membrane), and 3 (strong and diffuse staining, usually continuous staining from basement membrane and extending upward in proportion to lesion grade).
Int J Gynecol Pathol Vol. 00, No. 00, ’’ 2015
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
P16INK4A IMMUNOHISTOCHEMICAL AND HISTOPATHOLOGIC STUDY
5
TABLE 2. Summary of available additional past and new cervical specimen history collected from patients enrolled in the study stratified by original interpretation and patient age Age groups Negative cases o24 425 CIN1 cases o24 425
p16INK4a grade
Past cervical specimen
New cervical specimen
Time between interpretation and new specimen (d)
1+ 0+ 1+ 2+
NA NA NA NA
CIN1 Negative CIN1 CIN3
188 360 600 90
1+ 1+ 1+ 0+ 2+ 2+ 2+
CIN3 NA NA Negative NA CIN1 NA
CIN2 Negative CIN1 CIN1 CIN3 CIN3 CIN3
60 34 210 180 360 200 300
NA indicates not available.
conditions. Described mimickers of HSIL on Pap test include basal cell hyperplasia, immature and mature squamous metaplasia, reactive/inflamed cervical epithelium, and atrophy. The use the use of p16INK4a has proven to be of significant value in distinguishing these changes from a HSIL/CIN2-3 lesions (2–23). This study addresses, defines, and confirms the value of the current CAP-ASCCP LAST Committee recommendations to perform p16INK4a in cases with a HSIL Pap test where no HSIL/CIN2-3 is identified on a subsequent cervical specimen (2). This study confirms the value in review of the HSIL Pap test initiating the cervical sampling, but also suggests that review of the specimen interpreted as negative may identify a high-grade lesion on review. In cases with a history of HSIL on Pap test with subsequent cervical specimens with pathology interpretations of negative or CIN1, review of the initial slides, resectioning of the index block, and immunohistochemical study for p16INK4a may be of value to properly classify a specific specimen as having, or not having, a high-grade lesion. The negative predictive value for diffuse p16INK4a immunostaining to support the diagnosis of HSIL/CIN2-3 has been reported as >97% (7). The finding of p16 Grade 3+ will strongly suggest HSIL/CIN2-3 with supportive morphologic pathologic findings. In such cases, management per ASCCP guidelines of CIN2-3 is proposed. Also, our findings in cases from women aged older than 25 yr of age with 2+ p16INK4a and additional cervical specimen history, suggest classifying a lesion originally perceived on H&E as LSIL/ CIN1 as HSIL/CIN2-3. Precaution should be taken not to reinterpret a biopsy from a younger than 24yr-old woman with histopathologic features of LSIL that is p16 Grade 2+ as HSIL (CIN2-3). Such cases
we think should be classified as ‘‘p16-positive LSIL (p16+LSIL),’’ although some argument could be made to classify such a biopsy as ‘‘ungraded CIN lesion.’’ The absence of a transformation zone in cervical specimens may be of value as a quality indicator for further close follow-up in patients with a preceding high-grade Pap test result. In our study group, approximately one third (36 of 108 cases) identified women with an HSIL Pap with subsequent LSIL/ CIN1 or negative cervical specimen did not have representative transformation zone in the cervical sample. Two HSIL Pap test cases from the study group were considered as potential overinterpreted for HSIL, corresponding to 0.33% of all the HSIL. In our institution the concordance percentage rate is >60% (488 of 596 cases for 82%) for HSIL Pap cases with diagnosis of HSIL/CIN2-3 on subsequent cervical specimens. p16 antibody is a commercially available marker that is derived from different clones. One study has demonstrated a potential difference in staining patterns depending of the clone and tissue that is studied (12). More studies in this matter are needed and validation studies are suggested if p16INK4a is in use. In summary, this study suggests some diagnostic benefit from the use of p16INK4a immunohistochemical study on cervical specimens from women with HSIL Pap test where HSIL/CIN2-3 is not identified on original H&E review. Acknowledgments: The authors thank the technical and clerical staff of the University of Florida UF-Path labs and UF-Health Shands at Rocky Point cytology and histology laboratories. Int J Gynecol Pathol Vol. 00, No. 00, ’’ 2015
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
6
F.J. SOLANO AND E.J. WILKINSON REFERENCES
1. Massad LS, Einstein MH, Huh WK, et al, 2012 ASCCP Consensus Guidelines Conference. 2012 updated consensus guidelines for the management of abnormal cervical cancer screening tests and cancer precursors. J Low Genit Tract Dis 2013;17(suppl 1): S1–S27. 2. Darragh TM, Colgan TJ, Cox JT, et al, Members of LAST Project Work Groups. 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. J Low Genit Tract Dis 2012;16:205–42. 3. Waxman AG, Chelmow D, Darragh TM, et al. Revised terminology for cervical histopathology and its implications for management of high-grade squamous intraepithelial lesions of the cervix. Obstet Gynecol 2012;120:1465–71. 4. Saslow D, Solomon D, Lawson HW, et al, ACS-ASCCPASCP Cervical Cancer Guideline Committee. American Cancer Society, American Society for Colposcopy and Cervical Pathology, and American Society for Clinical Pathology screening guidelines for the prevention and early detection of cervical cancer. CA Cancer J Clin 2012;62:147–72. 5. Walker JL, Wang SS, Schiffman M, et al, ASCUS LSIL Triage Study Group. Predicting absolute risk of CIN3 during postcolposcopic follow-up: results from the ASCUS-LSIL Triage Study (ALTS). Am J Obstet Gynecol 2006;195:341–8. 6. Bergeron C, Ordi J, Schmidt D, et al, European CINtec Histology Study Group. Conjunctive p16INK4a testing significantly increases accuracy in diagnosing high-grade cervical intraepithelial neoplasia. Am J Clin Pathol 2010;133:395–406. 7. Wang SS, Trunk M, Schiffman M, et al. Validation of p16INK4a as a marker of oncogenic human papillomavirus infection in cervical biopsies from a population-based cohort in Costa Rica. Cancer Epidemiol Biomarkers Prev 2004;13:1355–60. 8. Hampl M, Wentzensen N, Vinokurova S, et al. Comprehensive analysis of 130 multicentric intraepithelial female lower genital tract lesions by HPV typing and p16 expression profile. J Cancer Res Clin Oncol 2007;133:235–45. 9. Guo M, Hu L, Baliga M, et al. The predictive value of p16(INK4a) and hybrid capture 2 human papillomavirus testing for high-grade cervical intraepithelial neoplasia. Am J Clin Pathol 2004;122:894–901. 10. Galgano MT, Castle PE, Atkins KA, et al. Using biomarkers as objective standards in the diagnosis of cervical biopsies. Am J Surg Pathol 2010;34:1077–87. 11. Samarawardana P, Dehn DL, Singh M, et al. p16(INK4a) is superior to high-risk human papillomavirus testing in cervical
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
cytology for the prediction of underlying high-grade dysplasia. Cancer Cytopathol 2010;118:146–56. Sawicka M, Pawlikowski J, Wilson S, et al. The specificity and patterns of staining in human cells and tissues of p16INK4a antibodies demonstrate variant antigen binding. PLoS One 2013;8:e53313. Brown CA, Bogers J, Sahebali S, et al. Role of protein biomarkers in the detection of high-grade disease in cervical cancer screening programs. J Oncol 2012;2012:289315. Negri G, Bellisano G, Zannoni GF, et al. p16 ink4a and HPV L1 immunohistochemistry is helpful for estimating the behavior of low-grade dysplastic lesions of the cervix uteri. Am J Surg Pathol 2008;32:1715–20. Tsoumpou I, Arbyn M, Kyrgiou M, et al. p16(INK4a) immunostaining in cytological and histological specimens from the uterine cervix: a systematic review and meta-analysis. Cancer Treat Rev 2009;35:210–20. Qiao X, Bhuiya TA, Spitzer M. Differentiating high-grade cervical intraepithelial lesion from atrophy in postmenopausal women using Ki-67, cyclin E, and p16 immunohistochemical analysis. J Low Genit Tract Dis 2005;9:100–7. Iaconis L, Hyjek E, Ellenson LH, et al. p16 and Ki-67 immunostaining in atypical immature squamous metaplasia of the uterine cervix: correlation with human papillomavirus detection. Arch Pathol Lab Med 2007;131:1343–1349Erratum in: Arch Pathol Lab Med. 2008;132(1):13. Negri G, Vittadello F, Romano F, et al. p16INK4a expression and progression risk of low-grade intraepithelial neoplasia of the cervix uteri. Virchows Arch 2004;445:616–20. Yildiz IZ, Usubu¨tu¨n A, Firat P, et al. Efficiency of immunohistochemical p16 expression and HPV typing in cervical squamous intraepithelial lesion grading and review of the p16 literature. Pathol Res Pract 2007;203:445–9. Kostopoulou E, Samara M, Kollia P, et al. Different patterns of p16 immunoreactivity in cervical biopsies: correlation to lesion grade and HPV detection, with a review of the literature. Eur J Gynaecol Oncol 2011;32:54–61. Lambert AP, Anschau F, Schmitt VM. p16INK4A expression in cervical premalignant and malignant lesions. Exp Mol Pathol 2006;80:192–6. Kalof AN, Cooper K. Our approach to squamous intraepithelial lesions of the uterine cervix. J Clin Pathol 2007;60:449–55. Wentzensen N, Schwartz L, Zuna RE, et al. Performance of p16/Ki-67 immunostaining to detect cervical cancer precursors in a colposcopy referral population. Clin Cancer Res 2012;18:4154–62.
Int J Gynecol Pathol Vol. 00, No. 00, ’’ 2015
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
