76 Arch Pathol Lab Med Vol 138, January 2014 p16 Utilization in Cervical Biopsies Singh et al

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1 Variability of Pathologists Utilization of p16 and Ki-67 Immunostaining in the Diagnosis of Cervical Biopsies in Routine Pathology Practice and Its Impact on the Frequencies of Cervical Intraepithelial Neoplasia Diagnoses and Cytohistologic Correlations Charanjeet Singh, MD; J. Carlos Manivel, MD; Alexander M. Truskinovsky, MD; Kay Savik, MS; Samy Amirouche, CT(ASCP); Jana Holler, CT(ASCP); Bharat Thyagarajan, MD; H. Evin Gulbahce, MD; Stefan E. Pambuccian, MD Context. The use of p16 in cervical biopsies improves the accuracy of cervical intraepithelial neoplasia (CIN) diagnosis and grading and decreases its interpathologist variability. Objective. To determine the impact of the frequency of use of p16 immunostains in cervical biopsies on pathologists diagnoses of CIN grade 1 and grade 2 or above (CIN1 and CIN2þ) and on cytohistologic correlations. Design. We identified all cervical biopsy specimens with cytologic correlations subjected or not to p16 staining from January 1, 2005, to September 30, 2010; calculated each pathologist s percentage of p16 use; and correlated it with their major cytohistologic discrepancy rates, CIN2þ diagnoses, and CIN1/CIN2þ ratios. Results. During the study period, each of the 23 pathologists interpreted 59 to 1811 (mean, 518) of cervical biopsy specimens, used p16 for 0% to 21.31% (mean, 10.14%) of these, had CIN2þdetection rates of 9.5% to 24.1% (mean, 18.9%), and CIN1/CIN2þ ratios of 0.7 to 4.5 (mean, 1.5). Compared to the 12 low users of p16, who used p16 fewer times than the institution s mean for p16 use, the 11 high users of p16 diagnosed more biopsies (8391 versus 3459), had a lower rate of major cytohistologic discrepancies (12.62% versus 14.92%, P,.001), a higher rate of CIN2þ diagnoses (19.9% versus 16.4%, P,.001), a lower range of CIN2þ rates (15.0% 23.1% versus 9.5% 24.1%), and lower CIN1/CIN2þ ratios (1.2 versus 2.3). Conclusions. We found a high intrainstitutional variability of p16 use in cervical biopsies, CIN2þ rates, and CIN1/CIN2þ ratios. Use of p16 for greater than 10% of cervical biopsies was associated with improved cytohistologic correlation rates and with lower variability in the frequencies of histologic diagnoses. (Arch Pathol Lab Med. 2014;138:76 87; doi: / arpa oa) A successful cervical cancer screening program relies on the consistent detection and treatment of significant cervical squamous lesions, defined as cervical intraepithelial neoplasia grade 2 (CIN2) and above (CIN2þ), in order to Accepted for publication April 25, From the Department of Laboratory Medicine and Pathology, University of Minnesota Medical School (Drs Singh, Truskinovsky, Thyagarajan, Gulbahce, Manivel, and Pambuccian); the School of Nursing, University of Minnesota (Ms Savik); and the Cytology Laboratory, University of Minnesota Medical Center-Fairview (Mr Amirouche and Ms Holler), Minneapolis. Dr Pambuccian is now at the Department of Pathology, Loyola University Medical Center, Maywood, Illinois. The authors have no relevant financial interest in the products or companies described in this article. Presented in part at the 59th Annual Scientific Meeting of the American Society of Cytopathology; November 4 8, 2011; Baltimore, Maryland. Reprints: Charanjeet Singh, MD, Laboratory Medicine and Pathology, University of Minnesota, 420 Delaware St SE, C-445 Mayo, MMC76, Minneapolis, MN ( singh. charanjeet21@gmail.com). prevent their potential progression to invasive squamous cell carcinoma. Cervical intraepithelial neoplasia grade 2, the threshold to institute ablative treatment, 1 is one of the least reproducible cervical biopsy diagnostic categories, 2,3 but its recognition is aided by the use of immunohistochemical surrogate markers such as p16 ink4a and Ki-67 (MIB1), which detect the impact of high-risk human papillomavirus (HPV) infection and integration on the maturation and proliferation of the squamous epithelium. 4,5 The use of these biomarkers has been shown to reduce the interobserver variability 6 and improve the reliability of the histologic diagnosis of cervical squamous dysplasia, and to increase the sensitivity of cytologic screening 7 and the accuracy of the histologic diagnosis of CIN2þ We hypothesized that the use of p16 and Ki-67 in routine biopsy diagnosis may also decrease the variability in the frequency of CIN2þ diagnoses by individual pathologists and improve their cytohistologic correlation frequencies by increasing the reliability of gold standard biopsy diagnosis against which Papanicolaou (Pap) test diagnoses are judged. The aim of this study was to determine the variability in the use of p16 ink4a (with or without the additional use of Ki-67, 76 Arch Pathol Lab Med Vol 138, January 2014 p16 Utilization in Cervical Biopsies Singh et al

2 henceforth referred to as p16) immunostains in routine cervical biopsy practice by pathologists working at a multihospital health care system, and to determine its causes and its impact on the interpathologist variability in the frequencies of cervical biopsy diagnoses and cytohistologic correlation frequencies. MATERIALS AND METHODS After obtaining the approval of the University of Minnesota s Institutional Review Board (Minneapolis) (study No. 1101E95102), the electronic records of our institution were searched for all cervical biopsies with prospectively performed cytohistologic correlations, with specimens subjected or not subjected to p16 immunostaining during the period from January 1, 2005 to September 30, All biopsy specimens from a patient obtained on the same date were considered a unique case; if other cervical biopsy specimens from the same patient were obtained within the 6-months interval of the index Pap test, they were also included in the study, as they may have resulted in a different diagnosis, may have been diagnosed by a different pathologist, and may have been subjected, or not, to p16 staining. Since p16 staining may also be seen in tubal metaplasia and other glandular lesions, all the glandular lesions of cervix were excluded from our study. The following data were recorded for each patient: (1) age, (2) results of the preceding Pap test, (3) when available, results of HPV genotyping studies performed on the preceding Pap test, (4) number and type of cervical histopathologic specimens (ie, single or multiple cervical biopsies, endocervical curettage, loop electrosurgical excision procedure [LEEP]/cone biopsy, hysterectomy), (5) date of the biopsy and preceding Pap test to calculate the interval between them, (6) the most severe grade of dysplasia or malignancy in any of the biopsy specimens obtained, (7) whether or not p16, with or without Ki-67 immunostaining, was performed, and (8) the number of biopsy specimens for which immunostaining was performed. The data were entered into a spreadsheet, together with a numeric code assigned to each pathologist and additional information about the pathologist (subspecialty training in cytopathology or gynecologic pathology, years of posttraining pathology experience, and pathologist s community or academic hospital practice group). All pathologists who had diagnosed more than 50 cervical biopsies during the study period were included for further analysis. The percentage of cases in which p16 staining was used was calculated for each pathologist and was then correlated with the age of the patients (dichotomized as younger than 30 years and 30 years or older), prior Pap test diagnosis (dichotomized as,lsil [low-grade squamous intraepithelial lesion] and LSIL), prior HPV test results (classified as negative, low-risk HPV, and high-risk HPV [hr-hpv]), the number of cervical biopsies interpreted by each pathologist, their frequency of biopsy diagnoses (no CIN, CIN1 and CIN2, CIN3 or squamous cell carcinoma, henceforth referred to as CIN2þ), and the ratios of CIN1/CIN2þ and of CIN2þ/ (CIN1 þ CIN2þ). Correlations were performed for each pathologist and for groups of pathologists classified as high users and low users of p16 from their percentage of p16 use above or below the institution s mean p16 use. According to the standard histopathology protocol used during the study period, 3 slides each containing three 4- to 5-lm-thick serial sections with step sectioning at 40 to 60 lm between slides (levels) were cut from the formalin-fixed, paraffin-embedded blocks in all cervical specimens, LEEP/cone biopsy specimens, and endocervical curettage specimens and stained with hematoxylineosin (H&E). Additional deeper levels and intradepartmental consultations were requested at the discretion of the sign-out pathologist. Immunostaining was performed at the centralized immunohistochemistry laboratory, which serves pathologists working at 5 hospitals belonging to Fairview Health Services in Minnesota (an academic health center and 4 community hospitals). Staining for Ki-67 (clone 30-9, Ventana Medical Systems, Inc, Tucson, Arizona) and p16 (clone 16P04, Cell Marque, Hot Springs, Arizona, from January 1, 2005, to June 30, 2008; and clone E6H4 [CINtec], mtm laboratories, Westborough, Massachusetts, from July 1, 2008, to September 30, 2010) were performed on 4-lm-thick sections on a Ventana NexES immunostainer according to the manufacturers instructions. Positive and negative controls were run with each sample. The centralized Fairview Health Services Cytopathology Laboratory serves a relatively low-risk, predominantly suburban population and processes about SurePath (BD, Franklin Lakes, New Jersey) liquid-based Pap tests annually, resulting in the following diagnoses: approximately 5% for atypical squamous cells of undetermined significance (ASC-US), 2% for low-grade squamous intraepithelial lesion (LSIL), and 0.4% for high-grade squamous intraepithelial lesion (HSIL). During the study period, all Pap tests were screened by 1 of 10 cytotechnologists, and abnormal Pap test results were diagnosed according to the 2001 Bethesda System 11 by pathologists from 1 of 5 hospitalbased pathology groups. Diagnosis of biopsy specimens was usually performed by the same pathology group, but not necessarily by the same pathologist. All but 3 pathologists included in this study (all academic pathologists) signed out Pap tests, in addition to biopsies. No subspecialty sign-out was practiced in any of the pathology groups. Cytohistologic correlations (CHCs) were performed prospectively at the time of biopsy diagnosis by the biopsy pathologist, were reviewed after 6 months of the index Pap test to allow for accrual of follow-up biopsies, and entered into the cytology laboratory s prospectively maintained CHC database. Biopsies were considered to correlate with prior Pap test results when the highest grade diagnosis for any of the biopsy specimens was the same (ie, no evidence of squamous intraepithelial lesion or malignancy [NILM] versus negative for dysplasia, LSIL versus CIN1, HSIL, and CIN2þ). Discrepancies were classified as minor when there was 1-step discrepancy between the Pap test result and the biopsy result (ie, NILM versus CIN1, LSIL versus CIN2þ, or in ASC-US cases in which the biopsy diagnosis was negative for dysplasia or CIN1) and major when a 2-step discrepancy between Pap test result and biopsy result existed (ie, NILM versus CIN2þ). Since the focus of this study was to assess the value of p16 in the diagnosis of cervical squamous lesions and its impact on CHC, all cases showing glandular neoplasms (in situ or invasive) were excluded from further analysis. Human papillomavirus DNA testing was performed by a homebrew polymerase chain reaction based test by using the MY09/11 primer set and restriction fragment length polymorphism genotyping, as a reflex test on residual SurePath samples in cases diagnosed as ASC-US, as part of routine cotesting with Pap and HPV for women aged 30 years and older, and in selected cases when specifically requested by the clinicians. Internal controls (bglobin) were run on each sample, and positive and negative controls, including low-quantity controls, were included with each run. Human papillomavirus results were reported as positive for hr- HPV if HPV type(s) 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, or 68 were identified, and negative for hr-hpv when no HPV or any other HPV type(s) were identified. The presence of low-risk HPV types was included in a comment. Human papillomavirus results were available for review by the pathologists at the time the biopsy results were interpreted. No significant changes occurred in the patient population served by our laboratory, or in the biopsy, immunohistochemistry, Pap test, or HPV DNA methodology. Only minor changes occurred during the study period in the cytotechnologist staffing (replacement of 3 of 10 cytotechnologists) and pathologist staffing (replacement of 3 of 26 pathologists). To assess the reasons for ordering and the criteria used for interpretation of p16 staining, we reviewed the surgical pathology reports of every 10th case subjected to p16 staining and every 100th case not subjected to p16 staining, arranged in chronologic order, for information regarding the use of additional levels, intradepartmental consultation, the reasons for ordering p16 (6Ki-67) stains, and the criteria used for interpreting these staining results. The slides from 30 consecutive cases in which p16 was used were Arch Pathol Lab Med Vol 138, January 2014 p16 Utilization in Cervical Biopsies Singh et al 77

3 Figure 1. Exclusion criteria (n ¼ number of cases). Abbreviations: AdenoCA, adenocarcinoma; AIS, adenocarcinoma in situ; CHC, cytohistologic correlation; Pap, Papanicolaou. retrieved and reviewed to assess the pattern of staining and interpretation of the p16 (6Ki-67) staining results. All statistical calculations were performed by using PASW Statistics 18 (SPSS Inc, Chicago, Illinois). All tests were 2-sided. A P value of,.05 was considered significant. RESULTS After excluding 646 cases (Figure 1), cervical surgical pathology specimens from women aged 14 to 83 years (mean, 33 years; SD, years) (5750 women younger than 30 years and 6100 women aged 30 years or older), interpreted by 23 pathologists, were further analyzed (Table 1). At the end of the study period, the 23 pathologists, all board-certified in anatomic pathology, had 4 to 35 years (mean, years) of posttraining experience. Eight pathologists practiced at an academic health center, while 15 pathologists practiced at 1 of 4 community hospitals. Seven pathologists had completed a fellowship in either cytopathology (n ¼ 6) or gynecologic pathology (n ¼ 1). The number of biopsies, frequencies of diagnosis of CIN1 and CIN2þ, use of p16, and major cytohistologic discrepancy frequencies for individual pathologists are shown in Table 2. During the entire study period, p16 staining was used in 1201 of cases (10.14%): in 813 cases (67.7%) on a single biopsy and in 388 (33.3%) on 2 or more biopsies per case. In 1009 cases (84.01%), p16 was used in conjunction with Ki-67. The use of p16 stains increased over time, peaking in frequency after 2 years, and was associated with a slight, statistically not significant increase in the rate of CIN2þ diagnoses (Table 3). As a percentage of all Pap diagnoses preceding the index biopsy, p16 stains were used most often in atypical squamous cells, cannot exclude high grade (ASC-H), followed by HSIL or above (Table 4). The frequency of ordering p16 was higher when the prior Pap diagnosis was LSIL or above than when it was for a grade below LSIL (11.4% versus 8.7%, P,.001). p16 staining was performed slightly less frequently (but not statistically significant) for women aged 30 years or older (10.8% in women 30 years or older versus 12.9% in younger than 30 years; P ¼.10). p16 stains were used more frequently when the biopsy specimen and the Pap test were interpreted by the same pathologist (12% versus 9.6%; P,.001), when the prior HPV test was hr-hpv positive (13.9% versus 10.3%; P ¼.003), and when the final diagnosis was CIN 2þ (12.9% versus 9.5%; P,.001). Conversely, the percentage of cases diagnosed as CIN2þ was higher in cases for which p16 immunostaining was performed (24.1% versus 18.3%; P,.001), in cases preceded by a Pap test diagnosis of LSIL or above (26.9% versus 9.6%; P,.001), in cases that tested positive for hr- HPV on the preceding Pap test (27.6% versus 8.1%; P,.001), for women younger than 30 years (22.5% versus 12.3%; P,.001), and when the same pathologist interpreted both the Pap test and the biopsy specimen (22.6% versus 17.9%; P,.001). Individual pathologists used p16 in 0% to 21.31% (mean, 10.14%) of cervical biopsy cases. There was significant variability in the use of p16 according to the hospital in which the pathologist practiced and it was associated with a similar variability in the frequencies of CIN2þ diagnoses (Table 5). There was a significant inverse correlation 78 Arch Pathol Lab Med Vol 138, January 2014 p16 Utilization in Cervical Biopsies Singh et al

4 Table 1. Specimen Types, Histologic Diagnoses, Prior Papanicolaou (Pap) Test Results, and Human Papillomavirus (HPV) DNA Test Results No. Percentage Cervical surgical specimens Single biopsies Multiple biopsies Biopsies þ ECC ECC only LEEP/cone Hysterectomy Total Histologic diagnoses No dysplasia or malignancy CIN CIN2 or CIN SCC Total Prior Pap test results a NILM ASC-US LSIL ASC-H LSIL-H AGCs HSIL SCC AdenoCA Total HPV DNA test results b Negative for HPV Positive for low-risk HPV Positive for high-risk HPV Total Abbreviations: AdenoCA, adenocarcinoma; AGCs, atypical glandular cells; ASC-H, atypical squamous cells, cannot rule out a high-grade squamous intraepithelial lesion; ASC-US, atypical squamous cells of undetermined significance; CIN 1 3, cervical intraepithelial neoplasia 1 to 3; ECC, endocervical curettage; HSIL, high-grade squamous intraepithelial lesion; LEEP, loop electrosurgical excision procedure; LSIL, low-grade squamous intraepithelial lesion; LSIL-H, low-grade squamous intraepithelial lesion, cannot rule out a high-grade squamous intraepithelial lesion; NILM, no evidence of intraepithelial lesion or malignancy; SCC, squamous cell carcinoma. a A total of 5463 cases had Pap test diagnoses graded below LSIL (NILM, ASC-US) and 6387 had diagnoses of LSIL or above (LSIL, ASC- H, LSIL-H, AGC, HSIL, and SCC). b HPV DNA tests were performed as a reflex test for ASC-US in 2231 cases (76.2%). between the experience of the pathologists in terms of years of pathology practice and the use of p16 in the diagnosis of the cervical biopsies (r ¼ 0.436, P ¼.04). Pathologists with specialized training in cytopathology or gynecologic pathology used p16 stains more frequently (204 of 1480 [13.78%] versus 997 of [9.61%], P,.001). There was no statistically significant difference in the frequency of p16 staining ordered by academic and community pathologists (200 of 1983 [10.09%] versus 1001 of 9867 [10.14%], P ¼.90). Although the mean frequency of use of p16 by all pathologists was 10.14%, we classified the pathologists as high users and low users, using a slightly lower value (9.76%) to divide the pathologists into similar-sized groups, composed of 12 and 11 pathologists, respectively (Table 6). There were no significant differences in the age or prior Pap test results of women whose biopsies were diagnosed by the 2 groups of pathologists. However, compared to low users, high users of p16 diagnosed a population with a significantly higher frequency overall of HPVþ women and hr-hpvþ women. High users interpreted a significantly higher volume of cervical biopsies than low users, diagnosed CIN2þ more frequently but diagnosed CIN (all grades) less frequently, owing to their markedly lower frequency of CIN1 diagnoses (Table 6 and Figure 2), thereby resulting in a higher proportion of CIN cases diagnosed as CIN2þ and a lower CIN1/CIN2þ ratio. High users of p16 had a lower rate of major discrepancies on CHC, diagnosed CIN2þ more frequently in biopsy specimens from women with a prior diagnosis of hr-hpvþ ASC-US, and had a higher percentage of CIN2þ diagnoses for biopsies obtained in the follow-up of women with Pap diagnosis of LSIL or above. Of all cases in which p16 stains were used, 23.98% (288 of 1201) were diagnosed as CIN2þ. A similar percentage of cases in which p16 was used were diagnosed as CIN2þ by low users and high users (11 of 63 [17.46%] versus 277 of 1138 [24.34%], P ¼.30). The percentage of CIN2þ diagnoses made by individual pathologists in cases subjected to p16 staining showed a large variation (4.76% 44.44%) even among high users. Controlling for the preceding Pap test diagnosis (,LSIL and LSIL), age of the patient (younger than 30 years and 30 years or older) and HPV status (high-risk, low-risk, or none), the high users of p16 were 1.3 times more likely to diagnose CIN2þ lesions (odds ratio, 1.3; 95% confidence interval, ; P ¼.03). The pathologists volume of cervical biopsies diagnosed during the study period could not be used in this multivariate analysis because it was highly associated with their p16 use. There was a lower range of variability in the frequencies of CIN2þ diagnoses made by individual pathologists classified as high users (Table 5). Comparing the standard deviations of CIN2þ diagnosis frequencies of individual pathologists to that of their group ( low users or high users ), the low users of p16 had a higher variability in CIN2þ diagnoses (0.35 [range, ] versus 0.40 [range, ], t test, equal variances not assumed, P ¼.003) (Figure 3). High users of p16 also had a lower CIN1/ CIN2þ ratio than low users and a lower range of variability of the CIN1/CIN2þ ratios between individual pathologists (Table 6). Using a cutoff volume of 300 biopsies, we classified 13 pathologists who diagnosed a total of 2549 cases as having a low volume, and 10 pathologists who diagnosed a total of 9301 cases as having a high volume (Table 7). Compared to low volume pathologists, high volume pathologists used p16 more frequently and diagnosed CIN1 less frequently and CIN2þ more frequently, resulting in lower CIN1/CIN2þ ratios. However, there was no difference between the major discrepancy frequencies of high volume and low volume pathologists. The review of a total of 239 surgical pathology reports, 119 with p16 staining and 120 without p16 staining, showed that among cases in which p16 immunostaining was performed, there were significantly more single cervical biopsies (26.67% versus 12.61%, P ¼.01) and fewer LEEP/cone biopsies or hysterectomies (5.83% versus 17.65%, P ¼.004). A total of 36 CIN2þ diagnoses (21 CIN2, 12 CIN3, 2 nongradable high-grade squamous intraepithelial lesion, and 1 squamous cell carcinoma) were made for the cases that were subjected to p16 staining, while only 22 CIN2þ diagnoses (11 CIN2, 10 CIN3, and 1 squamous cell carcinoma) were made for the cases that were not subjected Arch Pathol Lab Med Vol 138, January 2014 p16 Utilization in Cervical Biopsies Singh et al 79

5 Table 2. Pathologist Number of Biopsies, Frequency of p16 Use, Frequency of Cervical Intraepithelial Neoplasia (CIN), CIN1, and CIN2þ Diagnoses, and Frequency of Major Cytohistologic Discrepancies by Pathologist High User or Low User of p16/ki-67 Hospital All Biopsies p16 Staining Performed, No. (%) Biopsy Diagnosis CIN1, No. (%) CIN1, No. (%) CIN2þ, No. (%) 1 Low user UH (0.00) 161 (49.08) 119 (36.28) 42 (12.80) 2 Low user CH (0.63) 167 (52.51) 126 (39.62) 41 (12.89) 3 Low user UH (0.78) 163 (63.17) 120 (46.51) 43 (16.67) 4 Low user CH (0.98) 101 (49.50) 66 (32.35) 35 (17.16) 5 Low user CH (1.69) 152 (51.52) 107 (36.27) 45 (15.25) 6 Low user CH (1.69) 318 (59.77) 210 (39.47) 108 (20.30) 7 Low user CH (1.76) 381 (60.86) 230 (36.74) 151 (24.12) 8 Low user CH (2.37) 106 (35.93) 78 (26.44) 28 (9.49) 9 Low user CH (3.19) 148 (40.78) 115 (40.78) 33 (11.70) 10 Low user CH (3.91) 106 (59.21) 78 (43.58) 28 (15.64) 11 a Low user UH 59 3 (5.08) 33 (55.93) 27 (45.76) 6 (10.17) 12 a Low user CH (7.23) 39 (46.98) 31 (37.35) 8 (9.64) 13 High user CH (9.76) 769 (42.18) 424 (23.26) 345 (18.92) 14 a High user UH (9.89) 192 (70.32) 138 (50.55) 54 (19.78) 15 a High user UH (10.29) 111 (54.41) 71 (34.80) 40 (19.61) 16 High user CH (10.82) 611 (35.33) 287 (16.60) 324 (18.74) 17 High user CH (11.19) 318 (56.48) 215 (38.19) 103 (18.29) 18 High user CH (11.5) 65 (57.52) 48 (42.48) 17 (15.04) 19 a High user UH (12.64) 105 (57.69) 65 (35.71) 40 (21.98) 20 High user CH (17.48) 708 (39.66) 295 (16.53) 413 (23.14) 21 a High user UH (17.59) 323 (57.98) 202 (36.27) 121 (21.72) 22 High user CH (18.27) 420 (40.38) 230 (22.12) 190 (18.27) 23 a High user UH (21.31) 78 (63.93) 53 (21.31) 25 (20.49) Total (10.14) 5575 (47.04) 3335 (28.14) 2240 (18.90) Abbreviations: CH 1 4, community hospital 1 to 4; CIN1, cervical intraepithelial neoplasia 1; CIN2þ, cervical intraepithelial neoplasia 2 or above; N/A, not applicable; UH, university hospital. a Specialized training in cytopathology or gynecologic pathology. to p16 staining. Additional levels were examined more frequently (51 of 120 [42.5%] versus 19 of 119 [16%], P,.001) and intradepartmental consultations were reported more often (42 of 120 [35%] versus 7 of 119 [5.9%], P,.001) in cases in which p16 staining was performed. The reasons for ordering the p16 staining were stated in 75 of the 120 reports reviewed; it was indicated as atrophy (n ¼ 3), metaplasia (n ¼ 8), atypical metaplasia (n ¼ 21), inflammation with reactive/reparative changes (n ¼ 9), small focus of abnormality and/or poor orientation (n ¼ 13), distinction between CIN1 and CIN2 (n ¼ 7), or discrepancy between the histologic preliminary diagnosis and a prior Pap test diagnosis of ASC-H, low-grade squamous intraepithelial lesion, cannot exclude high grade (LSIL-H), or HSIL (n ¼ 15). The staining pattern of p16 (and of Ki-67, if performed) was described in 60 of the 120 reports reviewed; 3 cases had no tissue of interest present on the immunostained slide(s), while the remaining reports described the pattern of staining and reason for the interpretation of the staining as positive (strong, diffuse, nuclear and cytoplasmic blocklike p16 staining, and full-thickness Ki-67 staining of nuclei or the presence of clusters of suprabasal nuclei) or as negative (no staining or weak, focal, or patchy staining for p16 or only focal basal layer staining for Ki-67). The review of the slides of 30 consecutive random cases subjected to p16 (6Ki-67) staining confirmed the patterns of staining described (Figure 4, A through D). Table 3. Annual Numbers and Percentages COMMENT The main findings of our study were that pathologists who used p16 more frequently than the institutional mean frequency had a higher rate of CIN2þ diagnoses, less variability in the frequencies of CIN2þ diagnoses, and a lower rate of major cytohistologic discrepancies, owing at least in part to their higher rate of CIN2þ diagnoses in cases in which the preceding Pap test diagnosis was hr-hpvþ of p16/ki-67 Stains and of Diagnoses for Cervical ASC-US or was LSIL or above. No previous study Intraepithelial Neoplasia Grade 2 or Above (CIN2þ) specifically addressed the impact of biomarkers applied to p16 CIN2þ cervical biopsy specimens on CHC frequencies. However, a Year No. of Cases % No. of Cases % recent study that examined the value of deeper levels and ancillary staining with p16 and ProExC (BD) found a modest / / increase in CHC frequencies by identifying CIN2þ in 2 of / / / / discordant biopsy specimens obtained for a prior cytologic / / / / diagnosis of HSIL. 12 A higher frequency of p16 immunostain use correlated / / positively with the volume of cervical biopsies examined by P value versus versus the pathologist, and with specialized training in cytopathology or gynecologic pathology, and correlated inversely with ; P, ; P ¼ Arch Pathol Lab Med Vol 138, January 2014 p16 Utilization in Cervical Biopsies Singh et al

6 CIN2þ in p16 Stained, No. (%) Table 2. CIN1/ CIN2þ Ratio Extended CIN2þ/ CIN1þ Fraction, % Major Discrepancies, No. (%) N/A (18.60) 0 (0.00) (21.38) 0 (0.00) (17.44) 2 (100) (12.75) 0 (0.00) (16.95) 3 (33.33) (10.90) 0 (0.00) (14.38) 5 (71.43) (14.92) 0 (0.00) (13.48) 1 (14.29) (10.61) 0 (0.00) (18.64) 0 (0.00) (7.23) 63 (35.39) (8.12) 12 (44.44) (15.75) 6 (28.57) (12.25) 48 (25.67) (17.06) 3 (4.76) (9.77) 1 (7.69) (2.65) 10 (43.48) (10.44) 69 (22.12) (14.17) 40 (40.82) (13.64) 21 (11.05) (12.12) 4 (15.38) (13.11) 288 (23.98) (13.29) the experience of the pathologist in terms of years of practice of surgical pathology. The use of p16 did not correlate with the type of pathology practice (academic versus community) or with the characteristics of the patient population in terms of age and prior Pap test diagnoses. We believe that our results suggest that the use of p16 stains may have helped pathologists detect additional cases of CIN2þ that may not have otherwise been confidently diagnosed on routine H&E examination Other factors may have contributed to the higher CIN2þ frequencies for pathologists who used p16 staining more frequently. Despite the fact that our institution routinely uses 9 serial sections/levels per biopsy specimen beyond which finding additional CIN2þ lesions would be unusual 20 and that examination of additional levels frequently preceded the immunostaining step, p16 stains may have highlighted CIN2þ lesions that would have otherwise been detected on deeper H&E-stained levels. 21,22 The use of p16 stains may have also led to a systematic overdiagnosis of CIN2þ. However, if such overdiagnosis occurred, its magnitude was likely small, since p16 was frequently used in combination with Ki-67, to increase its specificity; moreover, the criteria used for p16 and Ki-67 positivity were consistent with those recommended by previous publications and with the recently published consensus recommendations from the College of American Pathologists (CAP) and the American Society for Colposcopy and Cervical Pathology (ASCCP). 4,6,10,17,23 26 Although p16 staining can be seen in CIN1 caused by hr- HPV, in such cases the morphology is typical for LSIL/CIN1, and no Ki-67 staining beyond the lower third of the squamous epithelium is seen. In our study, pathologists who used p16 stains more frequently also diagnosed a lower percentage of their cases as CIN1, which may be related to their concomitant use of Ki-67, which has been shown to increase the accuracy of CIN1 diagnoses. 24 Although CIN1 is currently not considered a premalignant condition, the documented tendency for pathologists to overdiagnose CIN1 24,27 may lead to overtreatment and, because it implies the presence of a sexually transmitted, potentially oncogenic virus, it can have profound psychologic, sexual, reproductive, and social ramifications. 24 In general, pathologists tended to order more p16 stains in cases in which the prior Pap test diagnosis was LSIL or above, for women who were hr-hpvþ, and in cases in which the final biopsy diagnosis did not correlate with the prior Pap test result. Patient age did not appear to play a significant role in the decision to order stains. The tendency to order p16 stains more frequently in cases preceded by a cytologic diagnosis of atypical squamous cells may be due to the fact that these biopsy specimens are more difficult to grade. 28 Our study also found that even among high users of p16 stains, there was an almost 10-fold (4.76% 44.44%) variation in CIN2þ diagnoses among individual pathologists for biopsy specimens stained with p16. This suggests that some pathologists may have used these stains primarily to confirm the suspected diagnosis of CIN2þ or to differentiate between CIN1 and CIN2þ, whereas others may have used them primarily to rule out CIN2þ. Prior Pap Test Diagnosis Table 4. Biopsies, No. Use of p16 and Cervical Intraepithelial Neoplasia Grade 2 or Above (CIN2þ) Diagnoses According to Prior Papanicolaou (Pap) Test Results Total Biopsies, % p16/ki-67 Staining Performed, No. p16 Staining Performed According to Prior Pap Test Diagnosis, % Prior Pap Test Diagnosis of All p16 Staining Procedures Performed, % CIN2þ Diagnoses, No. CIN2þ Diagnoses, % NILM ASC-US LSIL ASC-H LSIL-H AGCs HSILþ a Total Abbreviations: AGCs, atypical glandular cells; ASC-H, atypical squamous cells, cannot rule out a high-grade lesion; ASC-US, atypical squamous cells of undetermined significance; HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion; LSIL-H, lowgrade squamous intraepithelial lesion, cannot rule out a high-grade lesion; NILM, negative for intraepithelial lesion or malignancy. a HSILþ¼HSIL þ carcinoma (includes 2 cases with Pap test diagnoses of adenocarcinoma and 19 with Pap test diagnoses of squamous cell carcinoma). Arch Pathol Lab Med Vol 138, January 2014 p16 Utilization in Cervical Biopsies Singh et al 81

7 Hospital Table 5. No. (%) of Pathologists Use of p16/ki-67 and Cervical Intraepithelial Neoplasia Grade 2 or Above (CIN2þ) Diagnoses in Different Hospital-Based Pathologist Groups a No. (%) of Biopsies No. (%) of All p16/ki-67 Staining Procedures Performed p16/ki-67 Staining Performed as % of Biopsies Range of p16/ki-67 Staining Performed by Individual Pathologists, % (x-fold Variation) CIN2þ, No. CIN2þ as % of Biopsies Range of CIN2þ Diagnoses Made by Individual Pathologists, % (x-fold Variation) UH 8 (34.78) 1983 (16.73) 200 (16.65) b (2.2) CH1 6 (26.09) 1452 (12.25) 36 (3.00) (11.5) (1.6) CH2 5 (21.74) 6581 (55.54) 869 (72.36) (17.8) (1.2) CH3 3 (13.04) 1721 (14.52) 83 (6.91) (6.6) (1.3) CH4 1 (4.35) 113 (0.95) 13 (1.08) Total (100) 1201 (100) b (2.4) Abbreviations: CH 1 4, community hospital 1 to 4; UH, university hospital. a Differences for all comparisons for p16/ki-67 use and for CIN2þ rates were statistically significant (P,.01). b x-fold variation cannot be calculated. One of the most worrisome findings of our study was that, despite the similar patient population served, with similar pretest risk factors, such as age and prior Pap test diagnoses (but slightly different hr-hpv positivity status), the proportion of total cervical biopsy specimens diagnosed as CIN2þ by different pathologists showed significant (2.5-fold) variability and was accompanied by a similarly marked variability in CIN1 diagnoses, resulting in CIN1/CIN2þ ratios showing a 6-fold variation. These findings are similar to those of a recent study, 29 currently available only in abstract form, which found that, even after adjusting for population age and referral cytology, there was a significant variability in the proportion of cases diagnosed as CIN2 (6.7% 16.5%; median, 10.3%) and CIN3 ( %; median, 4.2%) and in the CIN1/CIN2þ ratio ( ) among biopsy specimens diagnosed in 11 pathology laboratories in New Mexico. An even greater variation between laboratories in their frequencies of CIN2þ diagnoses and CIN1/ CIN2þ ratios was found in a large study from Finland. 30 Despite the apparent simplicity of the criteria used for the diagnosis and grading of CIN, even experts may disagree on their practical application. 31,32 The large interobserver variability found by multiple studies makes it clear that pathologists encounter significant challenges in the diagnosis and grading of CIN. This may be due to technical factors (small biopsy sample; poor processing, staining, or sectioning; poor orientation; incomplete representation; significant epithelial denudation; thermal, crush, and other artifacts), patient-related factors (pregnancy, postpartum status, menopause, exogenous hormone administration, coexistent infections, prior radiation), or to pathologistrelated factors. The latter appear to be the major source of the observed variability in cervical dysplasia grading. 33 Since studies have shown that the subjective judgment of proportions may be inherently difficult, it is not surprising that pathologists may have difficulty in determining the exact third of the squamous epithelium to which the atypical epithelium lacking differentiation extends, Table 6. Differences Between Pathologists Who Were High Users (9.76%) and Low Users (,9.76%) of p16 Biomarker All Pathologists All Pathologists, Percentage Range and Variability (x-fold) High Users of p16/ki-67 Pathologists, No. (%) 23 (100) 11 (47.8) Average pathologist experience, y 6 SD (8.8) Biopsies diagnosed, No. (%) (100) (30.9) 8391 (70.8) Preceding Pap tests LSIL, No. (%) 6387/ (53.90) (1.31) 4486/8391 (53.46) Women 30 y, No. (%) 6100/ (51.48) (2.25) 4301/8391 (51.26) Preceding Pap tests with hr-hpv, a No. (%) 1369/2904 (47.1) (3.24) 1113/2290 (48.6) p16 stains ordered, No. (%) 1201/ (10.13) b 1138/8391 (13.56) All CIN diagnoses, No. (%) 5575/ (47.04) (2) 3700/8391 (44.09) CIN1 diagnoses, No. (%) 3335/ (28.14) (3.1) 2028/8391 (24.17) CIN2þ diagnoses, No. (%) 2240/ (18.9) (2.5) 1672/8391 (19.9) CIN1/CIN2þ ratio (6.3) 1.21 CIN2þ diagnoses with preceding hr-hpvþ ASC-US, No. (%) 277/1161 (23.9) b 233/921 (25.3) Cases with major cytohistologic discrepancies, No. (%) 1575/ (13.29) (8) 1059/8391 (12.62) CIN2þ diagnoses with preceding Pap diagnosis of.lsil (ASC-H, LSIL-H, HSIL, AGCs, SCC), No. (%) 1208/2895 (41.72) (3.62) 890/2037 (43.69) Abbreviations: AGCs, atypical glandular cells; ASC-H, atypical squamous cells, cannot rule out a high-grade lesion; ASC-US, atypical squamous cells of undetermined significance; CIN, cervical intraepithelial neoplasia; CIN1, cervical intraepithelial neoplasia 1; CIN2þ, cervical intraepithelial neoplasia 2 or above; hr-hpv, high-risk human papillomavirus; HSIL, high-grade squamous intraepithelial neoplasia; LSIL, low-grade squamous intraepithelial lesion; LSIL-H, low-grade squamous intraepithelial lesion, cannot rule out a high-grade lesion; NS, not statistically significant; Pap, Papanicolaou; SCC, squamous cell carcinoma; SD, standard deviation. a Of women who were tested for HPV. b x-fold variation could not be calculated. 82 Arch Pathol Lab Med Vol 138, January 2014 p16 Utilization in Cervical Biopsies Singh et al

8 Figure 2. Comparison of percentage of cervical biopsy diagnoses (y-axis) of no cervical intraepithelial neoplasia (CIN), CIN1, and CIN2þ for low users and high users of p16. especially in thin, atrophic, metaplastic, denuded, or keratinized epithelia. In addition, pathologists may also use other poorly reproducible criteria to differentiate between CIN1 and CIN2, such as the presence and degree of nuclear atypia or pleomorphism, the level of mitotic figures within the squamous epithelium, and the presence of abnormal mitotic figures, and may give different weights to these criteria. 37 Different pathologists may use different thresholds to diagnose CIN2, and such differences in diagnostic thresholds can lead to systematic diagnostic bias (underdiagnosis or overdiagnosis of CIN2þ), 40 which may be corrected by the use of objective biomarkers such as p Although the pathologists diagnostic decision-making process is largely unknown, 41 it is believed that most of the pathologists day-to-day diagnostic decisions are made implicitly, through Gestalt cognition 42 (type 1 decisionmaking process), rather than explicitly, through the analytic use of diagnostic criteria (type 2 decision-making process). The Gestalt or holistic approach to the diagnosis and classification of lesions is developed through training and experience, is based on pattern analysis of the lesion as a Table 6. Extended High Users of p16/ki-67, Percentage Range and Variability (x-fold) Low Users of p16/ki-67 Low Users of p16/ki-67, Percentage Range and Variability (x-fold) Difference Between High Users and Low Users of p16/ki-67, P Value 12 (52.2) 4 20 (5) (8.8) (15.8) 3459 (39.2) (10.6), (1.22) 1901/3459 (54.96) (1.31) NS (P ¼.14) (2.25) 1799/3459 (52.02) (1.84) NS (P ¼.50) (1.75) 256/614 (41.7) (3.24) (2.2) 63/3459 (0.02) b, (1.8) 1875/3459 (54.20) (1.8), (2.6) 1307/3459 (37.79) (1.8), (1.5) 568/3459 (16.4) (2.5), (4) (3), /240 (18.3) b (6.4) 516/3459 (14.92) (3), (1.62) 318/858 (37.06) (3.62),.001 Arch Pathol Lab Med Vol 138, January 2014 p16 Utilization in Cervical Biopsies Singh et al 83

9 Figure 3. Comparison of cervical intraepithelial neoplasia grade 2 (CIN2þ) rates and standard deviations of low users and high users of p16 (mean and range of variance are shown on y-axis). whole, is intuitive, fast, and automatic, and usually serves the pathologist well. However, in the absence of feedback, it is vulnerable to biases, 43 since it relies on implicit weights and thresholds. We believe that the more frequent use of an objective marker such as p16, in routine and histologically difficult cases where CIN2 is a differential diagnostic consideration, offers an opportunity for immediate feedback and allows pathologists a better opportunity to modify their diagnostic thresholds than do intradepartmental consultations, because pathologists from the same group frequently have developed similar (but not necessarily accurate) thresholds through training at the same institutions and working together for an extended period of time. Repeated objective feedback can also result in the convergence of the diagnostic thresholds of individual pathologists and can explain the tendency for pathologists who used p16 more frequently to have a narrower range of variation in their CIN2þ frequencies and CIN1/CIN2þ ratios. In the absence of such feedback, experienced pathologists may develop overconfidence and may be more susceptible to premature closure, 44 which could help explain their tendency to rely less often on p16 stains. Our study also found that the rate of p16 use was highly correlated with the volume of cervical biopsies signed out by individual pathologists, which could have several explanations. Since grading of cervical dysplasia based on p16 staining is reportedly easier than that based on H&E staining, 9 pathologists who signed out a higher volume of cervical biopsies may have performed more p16 immunostaining procedures to avoid making time-consuming diagnostic decisions in difficult cases because they were overworked. However, this is unlikely, since all pathologists included in this study had similar workloads, and p16 staining was frequently preceded by the examined additional levels and intradepartmental consultation. We believe that pathologists exposed to higher volumes of cervical biopsies were more aware of the diagnostic difficulties posed by grading cervical dysplasia and used p16 more liberally. Our study showed that higher pathologist volumes of cervical biopsies, in combination with more liberal use of Table 7. Differences Between Pathologists Who Interpreted a High Volume (300) and Low Volume (,300) of Cervical Biopsies All Pathologists All Pathologists, Percentage Range and Variability (x-fold) High Volume Pathologists Percentage Range High Volume Pathologists Percentage Range and Variability (x-fold) Pathologists, No. (%) 23 (100) 10 (43.4) Biopsies diagnosed, No. (%) (100) (30.9) 9301 (78.49) (5.7) p16 stains ordered, No. (%) 1201/ (10.14) a 1050/9301 (11.29) a All CIN diagnoses, No. (%) 5575/ (47.04) (2) 4176/9301 (44.9) (1.72) CIN1 diagnoses, No. (%) 3335/ (28.14) (3.1) 2338/9301 (25.14) (2.4) CIN2þ diagnoses, No. (%) 2240/ (18.9) (2.5) 1838/9301 (19.76) (1.9) CIN1/CIN2þ ratio (6.3) (4.3) Cases with major cytohistologic discrepancies, No. (%) 1575/ (13.29) (8) 1230/9301 (13.22) (2.6) Abbreviations: CIN, cervical intraepithelial neoplasia; CIN1, cervical intraepithelial neoplasia 1; CIN2þ, cervical intraepithelial neoplasia 2 or above; NS, not statistically significant. a x-fold variation could not be calculated. 84 Arch Pathol Lab Med Vol 138, January 2014 p16 Utilization in Cervical Biopsies Singh et al

10 biomarkers, were associated with a decrease in the cytohistologic major discrepancy frequencies and the interpathologist range of CIN2þ and CIN1/CIN2þ ratios. This suggests that higher volumes of cervical biopsies per pathologist, which could be achieved by subspecialty signout, in institutions where the conditions (number of pathologists and case volume) allow it, could be a valuable solution to decrease diagnostic variability. This is the first study to address the variability of pathologists immunostain ordering behavior in the diagnosis and grading of cervical dysplasia. Similar to prior studies addressing the immunostain ordering behavior of pathologists in surgical pathology 45 and autopsy pathology, 46 we found a significant variability of pathologists p16 use in the interpretation of cervical biopsies. Our study found that one of the factors that appeared to play a role in the pathologists decision to use p16 stains was the desire to confirm their own prior Pap test diagnoses ( anchoring and confirmation bias 47 ). The use of p16 and Ki-67 may also be influenced by the degree of pathologist s familiarity with the value or staining patterns of these stains through exposure during training, practice of colleagues, articles in medical journals, and presentations at scientific meetings, by their personal positive or negative experience with the marker, and by other factors, including the desire of objectivity, compulsiveness to achieve an accurate diagnosis, insecurity, degree of confidence in interpretation of the stains, and tolerance for risk taking. We found that pathologists who used p16 in more than approximately 10% of cervical biopsy cases diagnosed CIN2þ in a higher percentage of their cases and had better CHC frequencies. This percentage is lower than the 19% estimated by the recent consensus recommendations from the CAP and ASCCP 26 and it is unclear whether this value is an optimal cutoff for this population or for other pathology practices serving populations with similar or different risk profiles. Whether the optimal percentage of cases subjected to p16 staining is dependent on the baseline HPV positivity, age, and risk profile of the population deserves further study. The findings of this study reflect the real-world routine use of p16 by pathologists involved in cervical biopsy diagnosis. The pathologists included in this study worked in both community and academic settings, had various levels of training and expertise in cervical biopsy diagnosis, had a Low Volume Pathologists Table 7. Extended Difference Between Low Volume High Volume Pathologists and and Low Volume Variability (x-fold) Pathologists, P Value 13 (56.5) 2549 (21.51) (5.00), /2549 (5.92) (27.5), /2549 (53.59) (2), /2549 (39.11) (1.9), /2549 (15.77) (2.3), (2.8), /2549 (13.53) (7) NS (P ¼.70) broad range of years of practice of surgical pathology, and were involved in the diagnosis of Pap tests and follow-up cervical biopsies for a population with a frequency of abnormal Pap test diagnoses similar to the national 50th percentile, 48 and a CIN2þ rate comparable to that reported from large national studies. 49 We therefore believe that the results of this study are generalizable to similar pathology practices involved in screening relatively low-risk populations, which are not using subspecialty sign-out. We believe that the observed interpathologist variability does not reflect negatively on the pathologists included in the study but rather on the existing published grading systems, and on the unrealistic expectations that they raise, 50 despite the lack of consensus on the nomenclature, diagnostic criteria, and the use of biomarkers in the histologic diagnosis and grading of anogenital squamous dysplasia. It is likely that the recently published explicit consensus recommendations from the CAP and the ASCCP 26 will result in a reduction of this unacceptably high variability in the frequencies of biopsy diagnoses rendered by different pathologists and groups of pathologists. Monitoring the frequency of p16 use and the adoption of quality assurance measures similar to those used in gynecologic cytology 51 for intralaboratory and interlaboratory comparisons could help determine the presence and causes of variability and help find solutions to address this problem. We believe that, similar to the widely used monitoring of the frequencies of abnormal Pap test diagnoses 49 and the atypical squamous cells to squamous intraepithelial lesions ratios, 48 monitoring the frequencies of CIN1 and CIN2þ diagnoses rendered by different pathologists and different groups of pathologists serving populations with similar risk profiles (and their resulting CIN1/CIN2þ ratios) could serve this purpose. The main limitations of our study were due to its retrospective nature and the inclusion of a very large number of cases that made the review of all slides or reports impractical. We therefore could not evaluate the exact reasons for ordering the stains, the diagnostic criteria for a positive p16 and Ki-67 staining result used by each pathologist, or the relative contribution of H&E histology, levels, intradepartmental consultation, and of p16 and Ki67 results in establishing the final diagnosis. Another limitation of the study was that we considered biopsies that were performed at different dates for the same women, within 6 months of a Pap test, as separate cases, which most likely has led to an overestimation of the incidence of CIN2þ, since such additional biopsies were frequently LEEP/cone biopsies following cervical biopsies diagnosed as CIN2þ. Another limitation of the study is the large variation in the number of cases diagnosed by individual pathologists during the study period, which is explained by the variation in the hospitals cervical biopsy volumes and pathologists full-time or part-time involvement in clinical service activities or participation in only a part of the study period. In conclusion, our study shows that the use of p16 staining in the diagnosis and grading of dysplasia is helpful in more than approximately 10% of cervical biopsies, since it results in improved cytohistologic correlation frequencies, by lowering the frequencies of major cytohistologic discrepancies, and in lower frequencies of CIN1 and higher frequencies of CIN2þ diagnoses, with a lower range of interpathologist CIN2þ frequencies. Arch Pathol Lab Med Vol 138, January 2014 p16 Utilization in Cervical Biopsies Singh et al 85

11 Figure 4. Papanicolaou (Pap) test and biopsy findings of a case interpreted as cervical intraepithelial neoplasia grade 2 after p16 staining. A and B, SurePath (BD, Franklin Lakes, New Jersey) liquid-based Pap test interpreted as atypical squamous cells, cannot exclude high grade. C and D, Followup biopsy specimen shows an area of atypical metaplasia. E and F, Uniform, strong, blocklike staining for p16 (original magnifications [A and B], 340 [C and E], and 3200 [D and F]). The authors wish to thank Her Sceng and Jonathan Henriksen, MS, for their help with data management and graphics, and Gabriela Oprea-Ilies, MD, Department of Pathology, Emory University, Atlanta, Georgia, for her insightful review of the manuscript and suggestions. References 1. Wright TC Jr, Massad LS, Dunton CJ, Spitzer M, Wilkinson EJ, Solomon D consensus guidelines for the management of women with cervical intraepithelial neoplasia or adenocarcinoma in situ. J Low Genit Tract Dis. 2007;11(4): Castle PE, Stoler MH, Solomon D, Schiffman M. The relationship of community biopsy-diagnosed cervical intraepithelial neoplasia grade 2 to the quality control pathology-reviewed diagnoses: an ALTS report. Am J Clin Pathol. 2007;127(5): Galgano MT, Castle PE, Stoler MH, Solomon D, Schiffman M. Can HPV-16 genotyping provide a benchmark for cervical biopsy specimen interpretation? Am J Clin Pathol. 2008;130(1): Keating JT, Cviko A, Riethdorf S, et al. Ki-67, cyclin E, and p16ink4 are complimentary surrogate biomarkers for human papilloma virus-related cervical neoplasia. Am J Surg Pathol. 2001;25(7): Klaes R, Friedrich T, Spitkovsky D, 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): Arch Pathol Lab Med Vol 138, January 2014 p16 Utilization in Cervical Biopsies Singh et al