MAB4133 Sigma-AldrichAnti-p16 Antibody, clone D25

A significant proportion of squamous cell carcinomas of the oropharynx (OP-SCC) are related to human papillomavirus (HPV) infection and p16 overexpression. This subgroup proves better prognosis and survival but no evidence exists on the correlation between HPV and p16 overexpression based on diagnostic measures and definition of p16 overexpression. We evaluated means of p16 and HPV diagnostics, and quantified overexpression of p16 in HPV-positive and -negative OP-SCCs by mode of immunohistochemical staining of carcinoma cells.PubMed, Embase, and the Cochrane Library were searched from 1980 until October 2012. We applied the following inclusion criteria: a minimum of 20 cases of site-specific OP-SCCs, and HPV and p16 results present. Studies were categorised into three groups based on their definition of p16 overexpression: verbal definition, nuclear and cytoplasmatic staining between 5 and 69%, and ≥70% staining.We identified 39 studies with available outcome data (n=3926): 22 studies (n=1980) used PCR, 6 studies (n=688) used ISH, and 11 studies (n=1258) used both PCR and ISH for HPV diagnostics. The methods showed similar HPV-positive results. Overall, 52.5% of the cases (n=2062) were HPV positive. As to p16 overexpression, 17 studies (n=1684) used a minimum of 5-69% staining, and 7 studies (n=764) used ≥70% staining. Fifteen studies (n=1478) referred to a verbal definition. Studies showed high heterogeneity in diagnostics of HPV and definition of p16. The correlation between HPV positivity and p16 overexpression proved best numerically in the group applying ≥70% staining for p16 overexpression. The group with verbal definitions had a significantly lower false-positive rate, but along with the group applying 5-69% staining showed a worse sensitivity compared with ≥70% staining.There are substantial differences in how studies diagnose HPV and define p16 overexpression. Numerically, p16 staining is better to predict the presence of HPV (i.e. larger sensitivity), when the cutoff is set at ≥70% of cytoplasmatic and nuclear staining.

Latent infection by Epstein-Barr virus (EBV) is highly associated with the endemic form of Burkitt lymphoma (eBL), which typically limits expression of EBV proteins to EBNA-1 (Latency I). Interestingly, a subset of eBLs maintain a variant program of EBV latency - Wp-restricted latency (Wp-R) - that includes expression of the EBNA-3 proteins (3A, 3B and 3C), in addition to EBNA-1. In xenograft assays, Wp-R BL cell lines were notably more tumorigenic than their counterparts that maintain Latency I, suggesting that the additional latency-associated proteins expressed in Wp-R influence cell proliferation and/or survival. Here, we evaluated the contribution of EBNA-3A. Consistent with the enhanced tumorigenic potential of Wp-R BLs, knockdown of EBNA-3A expression resulted in abrupt cell-cycle arrest in G0/G1 that was concomitant with conversion of retinoblastoma protein (Rb) to its hypophosphorylated state, followed by a loss of Rb protein. Comparable results were seen in EBV-immortalized B lymphoblastoid cell lines (LCLs), consistent with the previous observation that EBNA-3A is essential for sustained growth of these cells. In agreement with the known ability of EBNA-3A and EBNA-3C to cooperatively repress p14(ARF) and p16(INK4a) expression, knockdown of EBNA-3A in LCLs resulted in rapid elevation of p14(ARF) and p16I(NK4a). By contrast, p16(INK4a) was not detectably expressed in Wp-R BL and the low-level expression of p14(ARF) was unchanged by EBNA-3A knockdown. Amongst other G1/S regulatory proteins, only p21(WAF1/CIP1), a potent inducer of G1 arrest, was upregulated following knockdown of EBNA-3A in Wp-R BL Sal cells and LCLs, coincident with hypophosphorylation and destabilization of Rb and growth arrest. Furthermore, knockdown of p21(WAF1/CIP1) expression in Wp-R BL correlated with an increase in cellular proliferation. This novel function of EBNA-3A is distinct from the functions previously described that are shared with EBNA-3C, and likely contributes to the proliferation of Wp-R BL cells and LCLs.

Recently, the management of head and neck squamous cell carcinoma (HNSCC) has focused considerable attention on biomarkers, which may influence outcomes. Tests for human papilloma infection, including direct assessment of the virus as well as an associated tumour suppressor gene p16, are considered reproducible. Tumours from familial melanoma syndromes have suggested that nuclear localisation of p16 might have a further role in risk stratification. We hypothesised p16 staining that considered nuclear localisation might be informative for predicting outcomes in a broader set of HNSCC tumours not limited to the oropharynx, human papilloma virus (HPV) status or by smoking status.Patients treated for HNSCC from 2002 to 2006 at UNC (University of North Carolina at Chapel Hill) hospitals that had banked tissue available were eligible for this study. Tissue microarrays (TMA) were generated in triplicate. Immunohistochemical (IHC) staining for p16 was performed and scored separately for nuclear and cytoplasmic staining. Human papilloma virus staining was also carried out using monoclonal antibody E6H4. p16 expression, HPV status and other clinical features were correlated with progression-free (PFS) and overall survival (OS).A total of 135 patients had sufficient sample for this analysis. Median age at diagnosis was 57 years (range 20-82), with 68.9% males, 8.9% never smokers and 32.6% never drinkers. Three-year OS rate and PFS rate was 63.0% and 54.1%, respectively. Based on the p16 staining score, patients were divided into three groups: high nuclear, high cytoplasmic staining group (HN), low nuclear, low cytoplasmic staining group (LS) and high cytoplasmic, low nuclear staining group (HC). The HN and the LS groups had significantly better OS than the HC group with hazard ratios of 0.10 and 0.37, respectively, after controlling for other factors, including HPV status. These two groups also had significantly better PFS than the HC staining group. This finding was consistent for sites outside the oropharynx and did not require adjustment for smoking status.Different p16 protein localisation suggested different survival outcomes in a manner that does not require limiting the biomarker to the oropharynx and does not require assessment of smoking status.

We evaluated X-ray repair complementing defective repair in Chinese hamster cells 1 (XRCC1) protein in head and neck squamous cell carcinoma (HNSCC) patients in association with outcome.XRCC1 protein expression was assessed by immunohistochemical (IHC) staining of pretreatment tissue samples in 138 consecutive HNSCC patients treated with surgery (n = 31), radiation (15), surgery and radiation (23), surgery and adjuvant chemoradiation (17), primary chemoradiation (51), and palliative measures (1).Patients with high XRCC1 expression by IHC (n = 77) compared with patients with low XRCC1 expression (n = 60) had poorer median overall survival (OS; 41.0 months vs. OS not reached, P = 0.009) and poorer progression-free survival (28.0 months vs. 73.0 months, P = 0.031). This association was primarily due to patients who received chemoradiation (median OS of high- and low-XRCC1 expression patients, 35.5 months and not reached respectively, HR 3.48; 95% CI: 1.44-8.38; P = 0.006). In patients treated with nonchemoradiation modalities, there was no survival difference by XRCC1 expression. In multivariable analysis, high XRCC1 expression and p16(INK4a)-positive status were independently associated with survival in the overall study population (HR = 2.62; 95% CI: 1.52-4.52; P less than 0.001 and HR = 0.21; 95% CI: 0.06-0.71; P = 0.012, respectively) and among chemoradiation patients (HR = 6.02; 95% CI: 2.36-15.37; P less than 0.001 and HR = 0.26; 95% CI: 0.08-0.92, respectively; P = 0.037).In HNSCC, high XRCC1 protein expression is associated with poorer survival, particularly in patients receiving chemoradiation. Future validation of these findings may enable identification of HNSCC expressing patients who benefit from chemoradiation treatment.

We describe 2 nonsmoking, nondrinking couples who developed human papillomavirus (HPV)-associated tonsillar cancer within 12 months of each other. After histopathologic evaluation, HPV L1, E2, E6, and LCR regions were amplified, and phylogenetic analysis of amplimers was determined. Quantitative polymerase chain reaction targeting HPV-16/18 L1 and E7 regions and P16 immunohistochemistry were performed. Tissues were HPV-16 positive, with distinct intercouple nucleotide differences and multiple unique intracouple similarities identified. Diffuse nuclear P16 expression was detected in tumor cells. This report demonstrates matching HPV-16 strains in 2 couples with concurrent development of tonsillar carcinoma who did not have other risk factors, revealing the potential infectious nature of oropharyngeal cancer.