Triage of women with ASCUS and LSIL cytology: use of qualitative assessment of p16INK4a positive cells to identify patients with high-grade cervical intraepithelial neoplasia

BACKGROUND: The identification of a small percentage of high-grade cervical intraepithelial neoplasias (HGCIN) among patients with minor cytological abnormalities (atypical squamous cells of undetermined significance [ASCUS] and/or low-grade squamous intraepithelial lesions [LSIL] group) is a major problem in cytology-based cervical cancer screening. The authors investigated the efficacy of p16INK4a as a biomarker to identify samples of patients with HGCIN among those with an ASCUS or LSIL result in Papanicolaou cytology. METHODS: Consecutive liquid-based cytology specimens of 137 ASCUS and 88 LSIL results were selected from gynecologists who adopted a triage regimen with biopsy under colposcopy 2 months later, independent of the p16INK4a result. p16INK4a stained slides were prepared and independently read by 2 observers, who used a recently described score to categorize p16INK4a stained squamous cells. The endpoint of the study was detection of a biopsy-confirmed HGCIN. RESULTS: The overall sensitivity and specificity of p16INK4a positive cells with a nuclear score >2 for diagnosis of HGCIN in ASCUS and LSIL cases combined was 96% and 83%, respectively. The sensitivity and specificity in the ASCUS group was 95% and 84%, and 100% and 81% in the LSIL group, respectively. Two observers had a high concordance in assessing p16INK4a stained cells (kappa value of 0.841). CONCLUSIONS: These data suggested that the use of p16INK4a as a biomarker combined with nuclear scoring of p16INK4a positive cells in cervical cytology to triage ASCUS and/or LSIL cases allows identification of HGCIN with good sensitivity and specificity.     

p16/ki-67 dual-stain cytology in the triage of ASCUS and LSIL papanicolaou cytology: results from the European equivocal or mildly abnormal Papanicolaou cytology study

BACKGROUND:

The objective of this study was to analyze the diagnostic performance of a newly established immunocytochemical dual‐stain protocol, which simultaneously detects p16^INK4a and Ki‐67 expression in cervical cytology samples, for identifying high‐grade cervical intraepithelial neoplasia (CIN2+) in women with Papanicolaou (Pap) cytology results categorized as atypical squamous cells of undetermined significance (ASCUS) or low‐grade squamous intraepithelial lesions (LSIL).

METHODS:

Residual liquid‐based cytology material from 776 retrospectively collected ASCUS/LSIL cases that were available from a recent study evaluating p16 cytology and HPV testing were subjected to p16/Ki‐67 dual staining. The presence of 1 or more double‐immunoreactive cell(s) was regarded as a positive test outcome, irrespective of morphology. Test results were correlated to histology follow‐up.

RESULTS:

Sensitivity of p16/Ki‐67 dual‐stain cytology for biopsy‐confirmed CIN2+ was 92.2% (ASCUS) and 94.2% (LSIL), while specificity rates were 80.6% (ASCUS) and 68.0% (LSIL), respectively. Similar sensitivity/specificity profiles were found for both age groups of women aged <30 years versus women aged ≥30 years. Dual‐stain cytology showed comparable sensitivity, but significantly higher specificity, when compared with human papillomavirus (HPV) testing.

CONCLUSIONS:

The results of this study show that p16/Ki‐67 dual‐stain cytology provided a high sensitivity for the detection of underlying CIN2+ in women with ASCUS or LSIL Pap cytology results, comparable to the rates previously reported for HPV testing and p16 single‐stain cytology. However, the specificity of this morphology‐independent interpretation of p16/Ki‐67 dual‐stain cytology testing was further improved compared with the earlier p16 single‐stain cytology approach, which required morphology interpretation, and it is significantly higher when compared with HPV testing. Cancer (Cancer Cytopathol) 2011;. © 2011 American Cancer Society.     

Immunocytochemical colocalization of P16(INK4a) and Ki-67 predicts CIN2/3 and AIS/adenocarcinoma

BACKGROUND:

Although previous studies have shown that p16^INK4a and Ki‐67 are sensitive and specific markers for high‐grade lesions (≥CIN2) on cervical biopsies, limited information is available regarding the performance of a dual‐staining approach as a diagnostic adjunct in cervical cytology. We evaluated a dual p16^INK4a/Ki‐67 immunocytochemistry (ICC) assay to determine its sensitivity and specificity versus that of high‐risk HPV (HR‐HPV) in a US‐based pilot cytology study.

METHODS:

ThinPrep specimens from 122 cervical cytology specimens encompassing 23 negative (NILM), 20 ASC‐US, 22 LSIL, 17 ASCH, 22 HSIL, and 18 AGC cases were processed for multiplexed ICC staining using a CINtec Plus Kit. Dual‐positive assay results were defined based on the detection of 1 or more epithelial cells that were stained for both p16^INK4a and Ki‐67 without regard to cellular morphology. HR‐HPV testing was performed by multiplex PCR with capillary electrophoresis genotyping.

RESULTS:

Dual staining for p16^INK4a and Ki‐67 was frequently detected in HSIL and AGC but was rarely detected in NILM cases. The HR‐HPV assay showed a sensitivity of 76.2% and a specificity of 55.8% for the detection of clinically significant cervical squamous or endometrial lesions. In contrast, the colocalization of p16^INK4a plus Ki‐67 maintained a high sensitivity of 81.8% and improved specificity to 81.8% for biopsy‐confirmed CIN2/3, endocervical adenocarcinoma, or endometrial adenocarcinoma.

CONCLUSIONS:

Dual staining for p16^INK4a/Ki‐67 immunocytochemistry dramatically increased specificity and maintained high‐level sensitivity for the diagnosis of CIN2/3 or glandular lesions compared with PCR‐based testing for HR‐HPV. Cancer (Cancer Cytopathol) 2012. © 2011 American Cancer Society.     

p16INK4a immunocytochemistry versus human papillomavirus testing for triage of women with minor cytologic abnormalities

The best method for identifying women who have minor cervical lesions that require diagnostic workup remains unclear. The authors of this report performed a meta‐analysis to assess the accuracy of cyclin‐dependent kinase inhibitor 2A (p16^INK4a) immunocytochemistry compared with high‐risk human papillomavirus DNA testing with Hybrid Capture 2 (HC2) to detect grade 2 or greater cervical intraepithelial neoplasia (CIN2+) and CIN3+ among women who had cervical cytology indicating atypical squamous cells of undetermined significance (ASC‐US) or low‐grade cervical lesions (LSIL). A literature search was performed in 3 electronic databases to identify studies that were eligible for this meta‐analysis. Seventeen studies were included in the meta‐analysis. The pooled sensitivity of p16^INK4a to detect CIN2+ was 83.2% (95% confidence interval [CI], 76.8%‐88.2%) and 83.8% (95% CI, 73.5%‐90.6%) in ASC‐US and LSIL cervical cytology, respectively, and the pooled specificities were 71% (95% CI, 65%‐76.4%) and 65.7% (95% CI, 54.2%‐75.6%), respectively. Eight studies provided both HC2 and p16^INK4a triage data. p16^INK4a and HC2 had similar sensitivity, and p16^INK4a has significantly higher specificity in the triage of women with ASC‐US (relative sensitivity, 0.95 [95% CI, 0.89‐1.01]; relative specificity, 1.82 [95% CI, 1.57‐2.12]). In the triage of LSIL, p16^INK4a had significantly lower sensitivity but higher specificity compared with HC2 (relative sensitivity, 0.87 [95% CI, 0.81‐0.94]; relative specificity, 2.74 [95% CI, 1.99‐3.76]). The published literature indicated the improved accuracy of p16^INK4a compared with HC2 testing in the triage of women with ASC‐US. In LSIL triage, p16^INK4a was more specific but less sensitive. Cancer (Cancer Cytopathol) 2012. © 2012 American Cancer Society.     

p16INK4a is superior to high‐risk human papillomavirus testing in cervical cytology for the prediction of underlying high‐grade dysplasia

BACKGROUND

The primary goal of this study was to compare the clinical performance of an optimized and rigorously controlled immunocytochemical (ICC) assay for p16^INK4a to high‐risk (HR) human papillomavirus (HPV) detection by polymerase chain reaction (PCR) as diagnostic adjuncts for cytology specimens from colposcopy patients.

METHODS:

The study included 403 cervical cytology specimens collected within 3 months of colposcopy. The colposcopic impression and cervical biopsy diagnosis served as the standards for correlation with cytological, p16^INK4a, and HPV data. p16^INK4a was evaluated using an immunoperoxidase‐based assay that was linear over 4 logs for the detection of HeLa‐spiked positive control cytology specimens, using a threshold for positive test results that was based on receiver operating characteristic curve analysis. HR‐HPV was detected by multiplex PCR using genotype‐specific primers.

RESULTS:

In all combined diagnostic categories (negative for intraepithelial lesion and malignancy, atypical glandular cells, atypical squamous cells of undetermined significance, atypical squamous cells cannot exclude high‐grade squamous intraepithelial lesion, low‐grade squamous intraepithelial lesion, and high‐grade squamous intraepithelial lesion), the p16^INK4a ICC and HR‐HPV assays, respectively, had sensitivity of 81.7% and 83.3% (P = .81) and specificity of 78.1% and 50.9% (P < .001) for the detection of underlying ≥grade 2 cervical intraepithelial neoplasia (CIN) lesions on biopsy. Furthermore, the positive predictive value of p16^INK4a ICC was greater than that of HR‐HPV for patients with biopsies ≥CIN‐2 (41.2% and 24.2%, respectively, P = .001).

CONCLUSIONS:

This p16^INK4a immunocytochemical assay has superior specificity but similar sensitivity to HR‐HPV testing to predict underlying high‐grade dysplastic lesions in patients who are referred for colposcopy. The determination of the overall performance characteristics of p16^INK4a immunocytochemistry, as an independent test or in combination with HPV testing in low‐risk screening populations, however, will require subsequent large‐scale prospective clinical trials. Cancer (Cancer Cytopathol) 2010. © 2010 American Cancer Society.     

Value of p16(INK4a) in the diagnosis of low-grade urothelial carcinoma of the urinary bladder in urinary cytology

BACKGROUND:

The sensitivity of urinary cytology for the diagnosis of urothelial carcinomas is low, particularly in low‐grade carcinomas. The UroVysion test is a fluorescent in situ hybridization multiprobe assay that increases the sensitivity of urinary cytology. However, this test is not widely available. P16^INK4a, a protein involved in cell cycle progression, is overexpressed in urothelial carcinoma. Immunocytochemical expression of p16^INK4a has been examined in biopsy samples from urothelial carcinomas, but few studies have addressed this protein in urine cytology.

METHODS:

The authors compared the results of p16^INK4a immunoreactivity in cytology and biopsy samples from 83 cases, including low‐grade urothelial carcinomas, reactive epithelial lesions, and negative cases.

RESULTS:

p16^INK4a assessment of in urine cytology samples showed a sensitivity of 66.7% and a specificity of 82.8% in the diagnosis of low‐grade urothelial carcinomas.

CONCLUSIONS:

On the basis of these results, the authors propose that immunocytochemical detection of p16^INK4a is a reliable tool in urine cytology, both for the diagnosis of low‐grade urothelial carcinomas and for follow‐up purposes. More retrospective and prospective studies are required to verify these results. Cancer (Cancer Cytopathol) 2012. © 2012 American Cancer Society.     

Evaluation of p16INK4a expression in ThinPrep cervical specimens with the CINtec p16INK4a assay

BACKGROUND.

The aim of this study was to examine p16^INK4a protein expression in ThinPrep (Cytyc Corporation, Marlborough, Mass) cervical specimens by using the CINtec p16^INK4a Cytology Kit (Dako, Glostrup, Denmark). The ability of this assay to accurately identify underlying high‐grade lesions was assessed by using follow‐up biopsies and comparing these results with Hybrid Capture 2 (Digene, Gaithersburg, Md) high‐risk HPV (hc₂) results.

METHODS.

Three hundred ninety‐eight residual ThinPrep samples were collected, and histological follow‐up data were retrieved for abnormal cytology specimens. After preparation of a Papanicolaou‐stained slide, a second slide was processed in preparation for p16^INK4a immunostaining. High‐risk human papillomavirus testing (hc₂) was also performed.

RESULTS.

Of the 163 cytologically abnormal samples, 6‐month biopsy follow‐up data were available for 45% of the specimens. At initial blinded evaluation, 21 of the 26 cases with cervical intraepithelial neoplasia (CIN) II/III follow‐up were positive for p16^INK4a, yielding an overall diagnostic sensitivity of 81%; 29 of the 47 cases diagnosed as CIN I or less were p16^INK4a negative, yielding a diagnostic specificity of 62%. In comparison, the hc₂ test results indicated a diagnostic sensitivity of 100% with a diagnostic specificity of 15%. After review of selected cases with CIN II/III follow‐up, 25 of 26 slides were deemed to be positive for p16^INK4a, increasing the diagnostic sensitivity to 96%.

CONCLUSIONS.

The CINtec p16^INK4a Cytology Kit, in combination with ThinPrep cervical samples, allowed clear evaluation of p16^INK4a protein overexpression. Diagnostic specificity of the CINtec p16^INK4a assay was significantly improved relative to hc₂. To increase p16^INK4a immunostaining in abnormal cells, a modified kit version with improved staining performance has been developed and is currently being evaluated. Cancer (Cancer Cytopathol) 2007. © 2007 American Cancer Society.     

Evaluation of p16INK4a/Ki‐67 dual stain in comparison with an mRNA human papillomavirus test on liquid‐based cytology samples with low‐grade squamous intraepithelial lesion

BACKGROUND:

The objective of the current study was to investigate the clinical performance of detecting high‐grade lesions with the CINtec PLUS p16^INK4a/Ki‐67 dual stain and the APTIMA human papillomavirus (HPV) Assay in a cohort of women with low‐grade squamous intraepithelial lesion (LSIL) cytology. The authors also assessed the reproducibility of the evaluation of immunocytochemical staining.

METHODS:

The 2 tests were performed on liquid‐based residual material from 469 women with LSILs. The samples had at least 5 years of follow‐up and the gold standard used was high‐grade cervical intraepithelial neoplasia (CIN2+/CIN3+) proven on histology.

RESULTS:

Approximately 69% of all the women included in the study had a positive test for HPV mRNA and 56% was positive for the dual stain. The 2 tests demonstrated high sensitivities. When examining the specificities, the APTIMA HPV Assay performed with significantly lower values than the CINtec PLUS test. For patients with CIN2+, the APTIMA HPV Assay had a specificity of 36.1% versus 51.3% for the CINtec PLUS test, and for women with CIN3+, the specificity was 33.8% versus 48.2%, respectively. The difference was even more pronounced when analyzing women aged < 30 years separately. The kappa values between the 3 observers in scoring the dual stain ranged from 0.43 to 0.49 and improved in a second evaluation round to values ranging from 0.50 to 0.66.

CONCLUSIONS:

The CINtec PLUS p16^INK4a/Ki‐67 dual‐staining test in LSIL cytology samples demonstrated high sensitivity that was similar to that of the APTIMA HPV Assay in the detection of underlying high‐grade disease but with enhanced specificity, especially among women aged < 30 years. The kappa value for the evaluation of the CINtec PLUS dual‐staining test was moderate but could be improved through training. Cancer (Cancer Cytopathol) 2013. © 2012 American Cancer Society.     

Evaluation of quantity and staining pattern of human papillomavirus (HPV)‐infected epithelial cells in thin‐layer cervical specimens using optimized HPV‐CARD assay

BACKGROUND.

Testing for human papillomavirus (HPV) is used in the triage of women with a cervical cytology of atypical squamous cells of undetermined significance (ASCUS). A fluorescent in situ hybridization assay was developed for the detection of HPV using the catalyzed receptor deposition technique (HPV‐CARD). In this study, the utility of this assay was tested for the detection of HPV in liquid‐based cervical cytology specimens.

METHODS.

A total of 195 liquid‐based cytology specimens were analyzed using the HPV‐CARD assay. The results from the assay were compared with HPV polymerase chain reaction (PCR) and typing results. The number of HPV‐infected cells and the staining pattern was correlated with the cytology classification.

RESULTS.

A 91% concordance between HPV‐CARD and PCR was observed for the detection of high‐risk HPV viruses. A 78% concordance was observed for specimens that were negative for HPV. In ASCUS, low‐grade squamous intraepithelial lesion (LSIL), and high‐grade squamous intraepithelial lesion (HSIL) categories, the average number of HPV‐positive cells per slide was 19 cells, 127 cells, and 450 cells, respectively. The number of cells with a punctate staining, suggestive of HPV integration, was 21% in ASCUS, 34% in LSIL, and 46% in HSIL specimens.

CONCLUSIONS.

The results of the current study indicate positive correlations between the severity of the disease and the increased overall quantity of HPV‐positive epithelial cells in cervical cytology specimens and accumulation of cells with punctate staining suggestive of integrated HPV. In summary, the developed HPV‐CARD assay was found to provide novel information regarding the proportion and staining pattern of HPV‐infected epithelial cells in different cytologic categories of cervical specimens. Cancer (Cancer Cytopathol) 2007. © 2007 American Cancer Society.     

The clinical impact of using p16INK4a immunochemistry in cervical histopathology and cytology: An update of recent developments

Cervical cancer screening test performance has been hampered by either lack of sensitivity of Pap cytology or lack of specificity of Human Papillomavirus (HPV) testing. This uncertainty can lead to unnecessary referral and treatment, which is disturbing for patients and increases costs for health care providers. The identification of p16^INK4a as a marker for neoplastic transformation of cervical squamous epithelial cells by HPVs allows the identification of HPV‐transformed cells in histopathology or cytopathology specimens. Diagnostic studies have demonstrated that the use of p16^INK4a immunohistochemistry substantially improves the reproducibility and diagnostic accuracy of histopathologic diagnoses. p16^INK4a cytology has substantially higher sensitivity for detection of cervical precancer in comparison to conventional Pap tests. Compared to HPV DNA tests, immunochemical detection of p16^INK4a‐stained cells demonstrates a significantly improved specificity with remarkably good sensitivity. About 15 years after the initial observation that p16^INK4a is overexpressed in HPV‐transformed cells we review the accumulated clinical evidence suggesting that p16^INK4a can serve as a useful biomarker in the routine diagnostic work up of patients with HPV infections and associated lesions of the female anogenital tract.     

The role of human papillomavirus type 16/18 genotyping in predicting high‐grade cervical/vaginal intraepithelial neoplasm in women with mildly abnormal Papanicolaou results

BACKGROUND:

The authors compared the predictive value of type 16 and/or 18 human papillomavirus (HPV) versus non‐16/18 HPV types for high‐grade (grade ≥2) cervical neoplasm/vaginal intraepithelial neoplasm and carcinoma (CIN/VAIN2+) in women with mildly abnormal Papanicolaou (Pap) results (ie, atypical squamous cells of undetermined significance [ASCUS] or low‐grade squamous epithelial lesion [LSIL]).

METHODS:

The authors retrospectively selected Pap specimens with HPV testing results obtained from 243 women (155 with ASCUS and 88 with LSIL Pap results) in their Department of Pathology. HPV genotyping was performed using the EasyChip HPV blot assay. The Pap specimens with HPV16/18 and non‐16/18 HPV types were compared with follow‐up biopsy results. Follow‐up duration ranged from 1 month to 58 months (mean, 26 months).

RESULTS:

In total, 58 of 155 specimens (37%) that had ASCUS and 29 of 88 specimens (33%) that had LSIL were positive for HPV16/18. CIN/VAIN2+ biopsies were identified in 43 of 155 women (28%) with ASCUS and in 28 of 88 women (32%) with LSIL. Women with ASCUS and HPV16/18 had a significantly higher rate (43%) of CIN/VAIN2+ than women with ASCUS and non‐16/18 HPV types (19%; P = .003; odds ratio, 3.10; 95% confidence interval, 1.48‐6.53). There was no statistically significant difference in the rate of CIN/VAIN2+ between women who had LSIL and HPV16/18 (45%) and those who had LSIL and non‐16/18 HPV types (29%; P = .16; odds ratio, 1.96; 95% confidence interval, 0.77‐4.97).

CONCLUSIONS:

HPV genotyping for HPV16/18 improved risk assessment for women with ASCUS Pap results and may be used to predict the risk of CIN/VAIN2+ to better guide follow‐up management. Cancer (Cancer Cytopathol) 2013. © 2012 American Cancer Society.     

Comparison of ProEx C with p16INK4a and Ki‐67 immunohistochemical staining of cell blocks prepared from residual liquid‐based cervicovaginal material

BACKGROUND.

Although liquid‐based cervicovaginal cytology has high sensitivity for detecting dysplastic/malignant lesions, many pitfalls exist. Cell blocks can be prepared from residual liquid‐based cervicovaginal material and used for immunohistochemistry. The aim of this study was to evaluate a new marker, ProEx C, on cell blocks and its ability to distinguish dysplastic/malignant lesions from morphologically abnormal but benign cells. The results of this study were compared with previously reported results for p16 and Ki‐67 on the same material.

METHODS.

ProEx C is a cocktail of monoclonal antibodies against proteins associated with aberrant S phase cell cycle induction (topoisomerase IIA, minichromosome maintenance protein 2). ThinPrep (CytycCorp., Boxborough, Mass) cervicovaginal specimens from 79 patients were selected. Four cases had no residual abnormal cells in the cell block. On the basis of the cell block diagnosis, 29 cases were negative for intraepithelial lesion or malignancy (NILM), 27 had low‐grade squamous intraepithelial lesions (LSIL), 16 had high‐grade squamous intraepithelial lesions (HSIL), and 3 had squamous cell carcinomas (SCC). Cell block sections were immunostained with ProEx C.

RESULTS.

Thirteen of 16 (81%) cases of HSIL stained positively with ProEx C. Two of 27 (7%) LSIL stained positively, and 2 (7%) cases of NILM stained positively. All 3 cases of SCC were strongly positive (100%). Staining for ProEx C showed a higher positive predictive value compared with p16.

CONCLUSIONS.

ProEx C can be used on cell blocks prepared from residual liquid‐based cervicovaginal cytologic specimens. Being a nuclear only stain, it is cleaner and easier to interpret even in scant specimens. Cancer (Cancer Cytopathol) 2008. © 2008 American Cancer Society.     

Combined p16INK4a and human papillomavirus testing improves the prediction of cervical intraepithelial neoplasia (CIN II-III) in Thai patients with low-grade cytological abnormalities

Thailand is in the process of developing a national cervical screening program. This study examined p16INK4a staining and HPV prevalence in abnormal cervical samples with atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL), to evaluate the efficacy of combined HPV and p16INK4a detection to predict CIN II-III. Totals of 125 ASCUS and 87 LSIL cases were re-evaluated by Pap test and cervical cells of ASCUS and LSIL cases were prepared on slides for p16INK4a detection by immunocytochemistry. HPV genotyping of DNA extracts was performed by GP5+/6+ PCR and reverse line blot hybridization. Histopathologic tests were performed to identify cervical lesion. Total of 212 cases were diagnosed to normal (20), ASCUS (112), LSIL (78) and HSIL (2). HPV was detected in ASCUS (49/112, 43.8%), LSIL (60/78, 76.9%) and HSIL (2/2, 100%) cases. The majority of HPV positive samples typed for high-risk HPV. 55.7% (107/192) of abnormal cases (ASCUS, LSIL and HSIL) were positive p16INK4a. For the 111 HPV DNA positive cases, 34 of 49 (69.4%) ASCUS cases and 49 of 60 (81.7%) LSIL cases were p16INK4a positive. 140 biopsies were taken and histological classified: CIN negative (65 cases), CIN I (56 cases) and CIN II-III (19 cases). HPV DNA detection predicted CIN II-III with sensitivity and specificity of 84% and 49%, whereas p16INK4a staining showed higher sensitivity (89.5%) and specificity (56.2%). The prediction of CIN II-III was significantly better by combination of positive HPV DNA and p16INK4a with 93.8% sensitivity and 59.2% specificity. Detection of HPV DNA combined with p16INK4a in cervical cells can predict CIN II-III and may improve the screening diagnosis of Thai women at risk for CIN II-III or cancer.     

Accuracy of liquid‐based cytology

BACKGROUND:

In the New Technologies for Cervical Cancer Screening (NTCC) randomized controlled trial, no significant increase in the sensitivity of liquid‐based cytology (LBC) was observed compared with conventional cytology. Both were interpreted by cytologists who had limited previous LBC experience. The objective of the current study was to assess whether different results could be expected with experienced LBC interpreters.

METHODS:

A stratified, random sample of 818 LBC slides from the NTCC study was obtained. These slides were reviewed blindly and independently by 3 international experts who did not participate in the NTCC. The sensitivity and specificity of external experts were estimated for cervical intraepithelial neoplasia grade 2 or greater (CIN2+) and for CIN3+ histology, and the differences were compared with the sensitivity and specificity of the original cytologic interpretation using cutoffs of atypical squamous cells of undetermined significance (ASCUS) and low‐grade squamous intraepithelial lesion (LSIL).

RESULTS:

With the endpoint of CIN2+ histology, the difference in sensitivity between external experts and the original interpretation was ?5.3 (95% confidence interval [CI], ?16.0 to 5.4) with ASCUS as the cutoff and 3.8 (95% CI, ?8.2 to 15.8) with LSIL as the cutoff. External experts had slightly lower specificity using ASCUS as the cutoff (?3.4; 95% CI, ?3.9 to ?2.9) and LSIL as the cutoff (?0.7; 95% CI, ?1.0 to ?0.4).

CONCLUSIONS:

The accuracy of the external experts' interpretation was similar to that of the original interpretation. Therefore, the current results indicated that LBC is not expected to increase sensitivity even if it is used by interpreters who have extensive experience with this technique. Cancer (Cancer Cytopathol) 2010. © 2010 American Cancer Society.     

The role of deeper levels and ancillary studies (p16Ink4a and ProExC) in reducing the discordance rate of Papanicolaou findings of high‐grade squamous intraepithelial lesion and follow‐up cervical biopsies

BACKGROUND:

Discordant results of cervical biopsy histology after a cytologic diagnosis of high‐grade squamous intraepithelial lesion (HSIL) are often attributed to sampling variation. The purpose of the current study was to determine whether deeper levels and ancillary staining (p16^Ink4a and ProExC) reduce the discordant rate.

METHODS:

A total of 246 cases of HSIL were retrieved from the computerized database from 2005 and 2006. Of these cases, 151 were followed by cervical biopsy. There was cytologic‐histologic correlation in 87 cases, as defined by the presence of high‐grade (2 or 3) cervical intraepithelial neoplasia (HGCIN). For each discordant biopsy (n = 64), 2 deeper levels for hematoxylin and eosin (H&E) were taken at 30‐μ and 90‐μ depths, and 4 sections for p16^Ink4a and ProExC staining were taken at a 60‐μ depth. All cytologic and histologic material from these 64 cases was reviewed by 3 cytopathologists. In 2 cases, the original HSIL diagnoses were downgraded and the cases censored from the study.

RESULTS:

Fifty‐seven of the 62 discordant cases had sufficient tissue for deeper levels and ancillary staining. Two of 57 cases were reclassified to HGCIN. In both of these cases, reclassification was suggested by results of immunostains; however, the H&E sections were necessary for definitive interpretation of the immunostain results.

CONCLUSIONS:

In the current study, deeper levels and ancillary staining with p16^Ink4a and ProExC did not significantly reduce the discordance rate. Although there are many known causes of sampling variation, including factors related to colposcopic technique, regression of infection, and insufficient histologic sectioning, sampling variation remains a valid justification of noncorrelation in women with HSIL followed up by cervical biopsy alone. Cancer (Cancer Cytopathol) 2009. © 2009 American Cancer Society.     

p16INK4A immunohistochemical staining may be helpful in distinguishing branchial cleft cysts from cystic squamous cell carcinomas originating in the oropharynx

BACKGROUND:

We investigated p16^INK4A expression in branchial cleft cysts and its utility in distinguishing branchial cleft cysts from metastatic head and neck squamous cell carcinomas (SCCs) in fine‐needle aspiration biopsies (FNABs).

METHODS:

A study set comprising 41 resections (15 SCC and 26 branchial cleft cysts) and a test set of 15 FNABs (11 SCC and 4 branchial cleft cysts) were analyzed with p16^INK4A immunohistochemistry and human papillomavirus (HPV) polymerase chain reaction (PCR)/pyrosequencing. Cases with discrepant p16^INK4A and PCR/pyrosequencing results were further evaluated with HPV in situ hybridization (ISH). SCCs were divided into keratinizing SCC and nonkeratinizing SCC groups and site of origin.

RESULTS:

Metastatic oropharyngeal nonkeratinizing SCC in the study set exhibited diffuse, strong p16^INK4A (7 of 7) and HPV16 DNA positivity (6 of 6), while keratinizing SCC from the larynx and oral cavity was negative for p16^INK4A. p16^INK4A reactivity in the branchial cleft cyst study set was characterized by focal, strong staining (6 of 21) involving the superficial squamous epithelium. HPV DNA was identified in 7 of 19 branchial cleft cyst study set cases by PCR/pyrosequencing, but these cases were negative by HPV ISH. In the test set, oropharyngeal nonkeratinizing SCC exhibited diffuse, strong p16^INK4A (3 of 3) and HPV16 DNA (2 of 2), while metastatic keratinizing SCC was negative for p16^INK4A and HPV DNA. All 4 FNABs of branchial cleft cysts were negative for p16^INK4A. Diffuse, strong p16^INK4A correlated with oropharyngeal origin (P = .001) and nonkeratinizing morphology (P = .0001).

CONCLUSIONS:

Branchial cleft cysts can exhibit focal strong reactivity limited to the superficial squamous epithelium and glandular epithelium. Although p16^INK4A immunohistochemistry may be helpful in distinguishing oropharyngeal nonkeratinizing SCC from branchial cleft cysts in FNAB specimens, it is not helpful in cases of keratinizing SCC because these cases are typically negative for p16^INK4A. Cancer (Cancer Cytopathol) 2009. © 2009 American Cancer Society.     

The mechanisms by which polyamines accelerate tumor spread

Background

The pathogenic role of beta-HPVs in non melanoma skin cancer (NMSC), is not still completely understood, and literature data indicate that they might be at least cofactors in the development of certain cutaneous squamous cell carcinomas. However, only few reports contain data on basal cell carcinoma (BCC). The HPVs interact with many cellular proteins altering their function or the expression levels, like the p16^INK4a and Akt. Our study aimed to determine the presence of different beta -HPV types and the expression of p16^INK4a and Akt in BCC, the commonest NMSC, in the normal appearing perilesional skin and in forehead swab of 37 immunocompetent patients.

Methods

The expression of p16^INK4a and Akt, by immunohistochemistry, and the HPV DNA, by nested PCR, were investigated in each sample.

Results

No correspondence of HPV types between BCC and swab samples was found, whereas a correspondence between perilesional skin and BCC was ascertained in the 16,7% of the patients. In BCC, 16 different types of beta HPV were found and the most frequent types were HPV107 (15,4%), HPV100 (11,5%) and HPV15 (11,5%) all belonging to the beta HPV species 2. Immunohistochemistry detected significant p16^INK4a expression in almost all tumor samples (94,3%) with the highest percentages (> 30%) of positive cells detected in 8 cases. A statistically significant (p = 0,012) increase of beta HPV presence was detected in p16^INK4a strongly positive samples, in particular of species 2. pAkt expression was detected in all tumor samples with only 2 cases showing rare positive cells, whereas Akt2 expression was found in 14 out of 35 BCC (40%); in particular in HPV positive samples over-expressing p16^INK4a.

Conclusions

Our data show that p16^INK4a and pAkt are over-expressed in BCC and that the high expression of p16^INK4a and of Akt2 isoform is often associated with the presence of beta-HPV species 2 (i.e. HPV 15). The association of these viruses with the up-regulation of p16^INK4a and Akt/PI3K pathway suggests that in a subtype of BCC these viruses may exert a role in the carcinogenesis or in other, still undefined, biological property of these tumors. If this particular type of BCC reflects a different biology it will remain undisclosed until further studies on a larger number of samples will be performed.     

p16INK4a/Ki‐67 co‐expression specifically identifies transformed cells in the head and neck region

p16^INK4a immunohistochemical overexpression is an overall reliable surrogate marker of human papillomavirus (HPV)‐associated head and neck squamous cell carcinomas (HNSCC). However, cases of ambiguous p16^INK4a overexpression are regularly detected in the head and neck: p16^INK4a expression can be observed in non‐malignant tissue, such as tonsillar crypt epithelium and a proportion of branchial cleft cysts. Additionally, diverse patterns of p16^INK4 expression can complicate interpretation of “p16^INK4a‐positivity”. These aspects impede the unrestricted application of p16^INK4a as a diagnostic marker in the head and neck. We hypothesized that combined detection of p16^INK4a and the proliferation marker Ki‐67 could support clarification of ambiguous p16^INK4a expression in the head and neck by specifically indicating p16^INK4a‐expressing cells with proliferative activity. p16^INK4a/Ki‐67 co‐expression in a combined staining procedure was correlated to distinct p16^INK4a expression patterns and HPV status (HPV DNA followed by E6*I oncogene mRNA detection) in 147 HNSCC and 50 non‐malignant head and neck samples. p16^INK4a/Ki‐67 co‐expression only occurred in transformed cells of the head and neck. Co‐expression was never detected in non‐transformed cells. Combined p16^INK4a/Ki‐67 expression was stringently associated with a diffuse p16^INK4a expression pattern. All HPV oncogene‐expressing HNSCC showed p16^INK4a/Ki‐67 co‐expression. We demonstrate that p16^INK4a/Ki‐67 co‐expression occurs exclusively in transformed cells of the head and neck. Our findings indicate a substantial impact of combined p16^INK4a/Ki‐67 expression in the assessment of ambiguous p16^INK4a expression in the head and neck by specifically identifying p16^INK4a‐expressing cells with proliferative activity. This property will be of considerable significance for head and neck histo‐ and cytopathology.     

Introduction of p16<Superscript>INK4a</Superscript> as a surrogate biomarker for HPV in women with invasive cervical cancer in Sudan

Background

Cervical cancer is the fourth most common cancer in women worldwide with highest incidence reported in Eastern Africa in 2012. The primary goal of this study was to study the expression of p16^INK4a in squamous cell carcinoma (SCC) of the cervix by immunohistochemistry (IHC) and determine relation with clinico-pathological parameters. This study further explored the correlation of p16^INK4a immunostaining with another proliferation marker, Ki-67 and to study if human papillomavirus (HPV) IHC can be used as a marker for detection of virus in high-grade dysplasia.

Methods

A total of 90 samples, diagnosed for cervical cancer, were included in the study. Fixed Paraffin Embedded (FFPE) tissue sections were stained with anti-p16^INK4a, anti-Ki-67 and anti-HPV antibodies using automated immunohistochemistry platform (ASLink 48-DAKO).

Results

Immunohistochemical protein expression of p16^INK4a positivity was found to be highest in SCC (92.2%, n = 71) than other HPV tumors (76.9%, n = 10). The majority of cases (97.4%) were p16^INK4a positive in the age group 41–60 years. In addition, a statistically significant difference between p16^INK4a and HPV was observed among total cervical tumor cases and SCC cases.

Conclusions

As expected staining of invasive cervical cancer with anti-HPV showed rare positivity because HPV heralds active infection in dysplastic lesions and not of frank cervical carcinoma. In contrast, anti-p16^INK4a IHC results showed positive correlation in SCC and other cervical tumors.

     

Should LSIL‐H be a distinct cytology category?

BACKGROUND:

The 2001 Bethesda System for gynecologic cervical cytology reporting classifies squamous intraepithelial lesions into low‐grade (LSIL) and high‐grade (HSIL) lesions. An intermediate term, “low‐grade squamous intraepithelial lesion, cannot exclude high‐grade squamous intraepithelial lesion (LSIL‐H),” has been used in a small percentage of LSIL cases. To the authors' knowledge, little is known regarding the human papillomavirus (HPV) status in patients with LSIL‐H.

METHODS:

A total of 808 SurePath specimens obtained between December 2009 and April 2011 were tested for 40 HPV genotypes using DNA microarray, followed by a confirmatory DNA sequencing assay.

RESULTS:

The infection rate for high‐risk HPV in women with LSIL‐H (92%) was strikingly close to that for women with HSIL (91%), which was higher than that for those with LSIL (74%); atypical squamous cells, cannot rule out high‐grade lesion (ASC‐H) (78%); or LSIL and ASC‐H combined (74%). HPV type 16, the most common carcinogenic HPV genotype, was detected in 36% of women with LSIL‐H, which was significantly higher than that in women with LSIL and ASC‐H combined (13.8%), but less than that in women with HSIL (44.6%). Patients with LSIL‐H and HSIL had similar infection rates for low‐risk/intermediate‐risk HPV genotypes, which were lower than those in LSIL or LSIL and ASC‐H combined.

CONCLUSIONS:

Women found to have LSIL‐H on a Papanicolaou test appear to have a unique HPV distribution pattern that clearly differs from LSIL and is comparable to that for HSIL, suggesting an increased risk of high‐grade lesions over that of women with LSIL. Recognizing LSIL‐H as an independent diagnostic category may help in the early identification of the high‐risk subgroup that may require a management algorithm comparable to that for patients with HSIL. Cancer (Cancer Cytopathol) 2012. © 2012 American Cancer Society.