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.     

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 p16INK4a expression in ThinPrep cervical specimens with the CINtec p16INK4a assay: correlation with biopsy follow-up results

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 (hc(2)) 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 (hc(2)) 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 hc(2) 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 hc(2). To increase p16(INK4a) immunostaining in abnormal cells, a modified kit version with improved staining performance has been developed and is currently being evaluated.     

p16INK4a immunohistochemistry is a promising biomarker to predict the outcome of low grade cervical intraepithelial neoplasia: comparison study with HPV genotyping

Objective

In cervical intraepithelial neoplasia (CIN), p16^INK4a immunohistochemistry has been reported to be a useful diagnostic biomarker. However, limited information is available about the association between the p16^INK4a immunohistochemistry and the outcomes of CIN. Here, we report p16^INK4a immunohistochemistry as an effective biomarker to predict the outcomes of CIN.

Methods

p16^INK4a immunohistochemistry was performed in patients with CIN from January 2000 to August 2009. Among these patients, we have performed a retrospective analysis of the medical records to evaluate the outcome of CIN 1-2 and performed statistical analysis to determine the correlation between p16^INK4a expression and the outcomes. We also performed HPV genotyping and analyzed the relation between the infecting human papillomavirus (HPV) genotype and the outcomes.

Results

A total of 244 patients, including 82 with CIN 1, 60 with CIN 2, and 102 with CIN 3, were examined. The rate of p16^INK4a overexpression increased with increasing CIN grade, 20.7% for CIN 1, 80.0% for CIN 2, and 89.2% for CIN 3, with significant differences between CIN 1 and CIN 2-3 group. In the 131 CIN 1-2 patients, the progression rate was significantly higher for the patients showing p16^INK4a overexpression than for those not showing p16^INK4a overexpression (p=0.005); the regression rate was also found to be significantly lower for the patients showing p16^INK4a overexpression (p=0.003). High-risk HPV genotypes were detected in 73 patients (73.7%). Both progression and regression rates were not significantly different between the high-risk HPV-positive and HPV-negative groups (p=0.401 and p=0.381, respectively).

Conclusion

p16^INK4a overexpression was correlated with the outcome of CIN 1-2, and p16^INK4a is considered to be a superior biomarker for predicting the outcome of CIN 1-2 compared with HPV genotyping.     

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.     

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 immunohistochemical staining and predictive value for progression of cervical intraepithelial neoplasia grade 1: A prospective study in China

p16^INK4A is strongly expressed in tissues diagnosed as cervical intraepithelial neoplasia (CIN) and cancer in women infected with human papillomavirus (HPV), but few prospective studies have evaluated p16^INK4A as a marker for the risk of low‐grade CIN (CIN1) progression. We investigated the prevalence of p16^INK4A immunostaining by CIN grade and whether overexpression of p16^INK4A in CIN1 predicts future risk for high‐grade CIN in Chinese women. 6,557 Chinese women aged 30–49 years were screened from 2003 to 2005 using cytology and carcinogenic HPV test. Colposcopy was performed on women with any abnormal result. p16^INK4A Immunostaining was performed on biopsies from all women with CIN1, as well as randomly selected women with normal or CIN grade 2 and worse (CIN2+) biopsies. Women with CIN1 were followed up without treatment. Colposcopy was performed on all untreated women at a 2‐year interval. The prevalence of p16^INK4A staining was 2.7%, 42.7%, 75.5%, 79.6% and 100% among women with normal, CIN1, 2, 3 and cancer biopsies, respectively (p < 0.001). HPV positivity was strongly associated with p16^INK4A staining [odds ratios (OR) = 12.8; 95% confidence intervals (CI): 5.2–31.6]. p16^INK4A staining of CIN1 biopsies at baseline was associated with an increased risk of finding high‐grade CIN over 2 years of follow‐up (OR = 1.43; 95% CI: 0.52–3.91). The two‐year cumulative incidence of CIN2+ for p16^INK4A positive women was higher at 10.71% than for p16^INK4A negative women at 1.30% (crude RR = 8.25, 95% CI: 1.02–66.62). p16^INK4A overexpression is strongly associated with grade of CIN and risk of progression to high‐grade CIN in women with low‐grade lesions.     

Diagnostic value of p16INK4A,Ki‐67, and human papillomavirus L1 capsid protein immunochemical staining on cell blocks from residual liquid‐based gynecologic cytology specimens

BACKGROUND:

This study was conducted to evaluate the reliability and role of cell block preparations in the diagnosis of neoplastic and preneoplastic lesions of the cervix and to improve the value of cell block preparations in diagnosing and predicting the prognosis of cervical lesions through immunostaining of p16INK4A (p16), Ki‐67, and human papillomavirus (HPV) L1 capsid protein (HPV L1).

METHODS:

In total, 138 specimens were diagnosed on liquid‐based cytology (LBC) and cell block preparations, and 63 specimens were subjected subsequently to tissue follow‐up and immunostaining for p16, Ki‐67, and HPV L1 on cell block sections.

RESULTS:

In 42 specimens that were diagnosed as low‐grade squamous intraepithelial lesion, high‐grade squamous intraepithelial lesion (HSIL), and squamous cell carcinoma (SCC) on cell blocks, 38 specimens (90.5%) were confirmed by histopathologic reports, and there was slightly better than 81.6% agreement between LBC and tissue follow‐up. Immunointensity and cells that were positive for p16 were enhanced according to increased pathologic grade and differed statistically between cervical intraepithelial neoplasia 1 (CIN‐1) and CIN‐2/CIN‐3 as well as SCC. The positive rates of HPV L1 decreased gradually according to the severity of cervical neoplasia, and HPV L1/p16 expression patterns were related to the severity of cervical lesions.

CONCLUSIONS:

The cell block preparation technique was complementary to LBC, and the authors concluded that the application of LBC combined with cell block preparations may improve the diagnostic accuracy of cytology. Immunostaining for p16 and Ki‐67 on cell block preparations can help to improve the diagnostic accuracy of HSIL and SCC. A combined expression pattern of p16 and HPV L1 may serve as a valuable index for predicting prognosis and follow‐up of cervical dysplastic lesions. Cancer (Cancer Cytopathol) 2010. © 2010 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.     

Performance of p16INK4a immunocytochemistry as a marker of anal squamous intraepithelial lesions

BACKGROUND:

Protein p16^INK4a immunocytochemistry (ICCp16) has the potential to reveal lesions at risk of progression to anal cancer. This study examined measures of diagnostic validity of ICCp16 in HIV‐positive patients treated at the Tropical Medicine Foundation of Amazonas in the coloproctology outpatient clinic.

METHODS:

One hundred ninety HIV‐positive patients were consecutively enrolled in 2007 and 2008. All patients underwent anal cytologic sampling to perform ICCp16 in conventional and GluCyte (Synermed International, Westfield, Indiana and S¸ao Paulo, Brazil) smears and also for genotyping of human papillomavirus (HPV). Patients were then subjected to anal biopsies monitored by high‐resolution anoscopy. Hematoxylin‐eosin and immunoperoxidase p16 (clone 6H12) stains were performed in slides with biopsied and cytological specimens, respectively. HPV genotyping on anal scrapings was performed by a polymerase‐chain reaction (PCR)‐based method. The immunochemical findings were compared with histopathological and PCR results in contingency tables and analyzed by nonparametric tests. Measures of diagnostic validity of ICCp16 were calculated. Statistical significance was set at P ≤ .5.

RESULTS:

There was no statistically significant association between the immunochemical results (conventional or GluCyte smears) and histopathological or HPV genotyping findings (P > .05). In the best scenario, ICCp16 presented 31% sensitivity and 81% specificity for the diagnosis of anal squamous intraepithelial lesion (ASIL) and 30% and 66%, respectively, for the diagnosis of infection with high‐risk HPV.

CONCLUSIONS:

There was no association between ICCp16 results and histopathological findings nor between ICCp16 and HPV genotyping. ICCp16 showed poor sensitivity and moderate specificity for the diagnosis of ASIL or high‐risk HPV. Cancer (Cancer Cytopathol) 2011. © 2011 American Cancer Society.     

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.     

p16INK4a overexpression predicts translational active human papillomavirus infection in tonsillar cancer

The causal role of human papillomaviruses (HPV) in squamous cell carcinogenesis of tonsillar cancers (TSCC) depends on the activity of the viral oncoproteins E6 and E7, leading to inactivation of the cellular tumor suppressor p53 and the retinoblastoma gene product pRb. Because of the negative feedback mechanisms, the pRb inactivation causes an increase of the inhibitor of the cyclin‐dependent kinases p16^INK4a. In 39 TSCC specimens, genotyping based on the amplification of HPV DNA was carried out using PCR by applying HPV type‐specific oligonucleotides. Subsequently, amplicons were hybridised with fluorescence‐labeled complementary probes using the Southern blot technology. For HPV E6/E7 mRNA expression, Northern hybridization and RT‐PCR were performed, and for p16^INK4a detection, immunohistochemistry was performed. With 21/39 (53%) HPV‐positives, the detection rate is within the range that can be expected in TSCC. The E6/E7 oncogene mRNA was detectable in 11 cases, 10 of which showed positive signals after p16^INK4a staining. Albeit the small study group was investigated, the correlation of the HPV DNA status with the p16^INK4a expression was of statistical significance (p = 0.02). Kaplan‐Meier estimations revealed better survival outcome for patients with HPV‐positive tumors with detectable E6/E7 mRNA and p16^INK4a overexpression (p = 0.02, median observation time 29 months). As mRNA expression tests are not routinely available in many clinical diagnostic laboratories, and based on the high correlation of p16^INK4a staining with HPV E6/E7 mRNA expression, in conclusion we suggest for a deeper exploration for the use of p16^INK4a as a surrogate marker with the potential to impact the standard of care of HPV DNA‐positive head and neck carcinomas.     

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.     

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.     

Diagnostic accuracy of p16INK4a immunohistochemistry in oropharyngeal squamous cell carcinomas: A systematic review and meta‐analysis

The accurate diagnosis of human papillomavirus (HPV) causality in oropharyngeal squamous cell carcinomas (OPSCC) is likely to influence therapeutic decisions in affected patients in the near future. We conducted a systematic review and meta‐analysis to determine the diagnostic accuracy of p16^INK4a immunohistochemistry (IHC) to identify HPV‐induced OPSCC. We identified all studies that performed p16^INK4a IHC (index test) and HPV E6/E7 mRNA detection using an amplification‐based method (gold standard to indicate a transforming relevance of HPV) in OPSCC. Testing with one or more comparator tests (HPV DNA PCR, HPV DNA in situ hybridization (ISH) and p16^INK4a IHC/HPV DNA PCR combined testing) was an optional criterion for inclusion. Among 1,636 retrieved studies 24 fulfilled the inclusion criteria. The pooled sensitivity of p16^INK4a IHC, HPV DNA PCR, HPV DNA ISH and p16^INK4a IHC/HPV DNA PCR combined testing was 94% (95%‐confidence interval (CI) 91–97%), 98% (CI 94–100%), 85% (CI 76–92%) and 93% (CI 87–97%), respectively. The pooled specificity was 83% (CI 78–88%), 84% (CI 74–92%), 88% (CI 78–96%) and 96% (CI 89–100%), respectively. p16^INK4a IHC/HPV DNA PCR combined testing was as sensitive as either p16^INK4a IHC or HPV DNA PCR alone but significantly more specific than either separate test. In conclusion, p16^INK4a IHC is highly sensitive but moderately specific to diagnose HPV‐transformed OPSCC when used as a single test. Combined p16^INK4a IHC and HPV DNA PCR testing significantly enhances specificity while maintaining high sensitivity. This diagnostic test combination thus represents an attractive testing strategy for the reliable diagnosis of HPV‐induced OPSCC in the clinical setting and may constitute an inclusion criterion for future therapeutic trials.     

Expression of the p16INK4a and Ki-67 in relation to the grade of cervical intraepithelial neoplasia and high-risk human papillomavirus infection

Objective

The purposes of this study were to evaluate the expression of p16^INK4a (referred as to p16) and Ki-67 in cervical intraepithelial neoplasia (CIN), and the correlation between high-risk human papillomavirus (HPV) infection and the above biomarkers.

Methods

We analyzed 31 patients who were diagnosed with CIN at Kwandong University Myongji Hospital from October 2006 to September 2007. CIN specimens (CIN1, 12; CIN2, 6; CIN3, 13) were obtained by colposcopy-directed biopsy (CDB) or loop electrical excision procedure (LEEP). The expressions of p16 and Ki-67 were evaluated by immunohistochemical methods with antibodies to p16 and Ki67. The immunohistochemical staining results were classified into four grades: 0, 1, 2 and 3. HPV genotyping or Hybrid Capture-II test was used to detect high-risk HPV.

Results

The expression of p16 (p<0.001) and Ki-67 (p=0.003) were positively associated with CIN grade. p16 expressions increased significantly with high-risk HPV infection (p=0.014), especially HPV type 16 and 58. Ki-67 expression was not related with high-risk HPV. There was positive correlation between the expression of the p16 and Ki-67 (p=0.007).

Conclusion

CIN grade were positively related to the expression of p16 and Ki-67. p16 expressions of high-risk HPV specimens significantly increased more than Ki-67. Therefore, in the diagnosis of CIN and high-risk HPV infection, p16 can be a useful biomarker.     

Human Papillomaviruses,p16INK4a and Akt expression in basal cell carcinoma

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.     

Inhibition of T-cell acute lymphoblastic leukemia proliferation in vivo by re-expression of the p16INK4a tumor suppressor gene

T-cell acute lymphoblastic leukemia (T-ALL) is characterized by the presence of differentiation-inhibited pro- and pre-T-cell blasts. The p16^INK4a tumor suppressor gene has been shown to be frequently deleted in human T-ALL cases. Deletion of p16^INK4a may be associated with poor prognosis and relapse of the disease. Radiation-induced murine T-ALL in C57B1/6 mice shares pathogenetic and molecular characteristics with the human disease. We used the murine disease as a model to study the status of the INK4/ARF gene locus and to examine the effect of p16^INK4a-re-expression in T-ALL cells on their leukemic potential in vivo. In 9 of 17 radiation-induced murine T-ALL cell lines, the p16^INK4a protein was not expressed as determined by immunoblotting. Southern blot analysis revealed homozygous deletions of the p16^INK4a gene locus in three of the nine lines, along with the genes encoding p15^INK4b and p19^ARF. Transduction of p16^INK4a-negative T-ALL lines with retrovirus encoding p16^INK4a significantly inhibited their in vitro proliferation by inducing G₁-arrest. Importantly, re-expression of p16^INK4a in p16^INK4a-negative T-ALL cells obliterated the induction of lethal disseminated leukemia in syngeneic mice. This is the first demonstration that re-establishment of p16^INK4a expression is critical for in vivo growth regulation of T-ALL cells.     

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.