Combination Of P16 Research Articles

Immunohistochemistry as an adjunct for challenging histological patterns of borderline Brenner tumors: An illustrative study of 4 cases

Borderline Brenner tumors (BBT) have a range of morphology that shows considerable overlap with that of malignant Brenner tumors (MBT). In particular, two histological patterns of BBT can be particularly challenging: 1) BBT with intraepithelial carcinoma (BBT-IEC) and 2) BBT with a small nested pattern (BBT-SNP). BBT-IEC is characterized by a tumor with the low-power non-infiltrative silhouette of a conventional BBT, but with increased cytological atypia and mitotic activity similar to that of MBT. Conversely, BBT-SNP is characterized by a complex proliferation of small tumor nests that closely resemble the infiltrative growth pattern of MBT, but without the obligate cytologic atypia and mitotic activity of MBT.We suggest that the combination of p16, p53 and Ki-67 may be helpful in distinguishing these 2 patterns of BBT from both conventional BBT and from MBT. While both conventional BBT and BBT-IEC show a null pattern of p16 expression, our case of BBT-IEC showed aberrant p53 overexpression, albeit with a maturation pattern similar to that described for TP53 mutant mucinous ovarian carcinoma and differentiated vulvar intraepithelial neoplasia (dVIN). Similarly, while BBT-SNP shows an infiltrative-like growth pattern similar to that of MBT, our case also showed a wild-type pattern of p53 expression and a Ki-67 proliferative index similar to areas with conventional BBT histology. In conclusion, in our small case series, we show that the use of immunohistochemistry for p53 and Ki-67 may help to distinguish challenging patterns of BBT from MBT. Further studies are needed to validate this finding in a larger case cohort.

An Evaluation of High-Risk HPV in Squamous Cell Carcinomas of the Lip in a South African Cohort

BackgroundTo determine the prevalence of HR-HPV in a series of lip SCC from South African patients, using currently accepted HPV-testing methodologies and to define the clinical and histomorphologic features of HPV-associated lip SCC.MethodsFifty SCC of lip and 50 control cases were tested for HR-HPV using p16 and HR-HPV DNA PCR. p16-equivocal/positive and HPV DNA PCR-positive SCC were further evaluated for the expression of HPV-16 and HPV-18 mRNA transcripts using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to confirm transcriptionally active HPV.Resultsp16 was positive in 22% (n = 11) and equivocal in 4% (n = 2) of the SCC. One p16-positive case showed positivity for both HPV-16 DNA and HPV-16 E6/E7 mRNA transcripts (HPV prevalence rate of 2%). The HPV-positive case was non-keratinizing and occurred in an 80-year-old female. The two p16-equivocal cases were HR-HPV DNA positive and mRNA PCR negative. p16 was found to have a positive predictive value of 9%.ConclusionFindings from our cohort of lip SCC suggest that HR-HPV may have an insignificant role in the pathogenesis of SCC at this site. Due to its low ppv, p16 is insufficient to establish HR-HPV infection in SCC of the lip. The combination of p16 and DNA PCR appears to correlate with the presence of transcriptionally active virus. HPV E6/E7 mRNA detection is the gold standard for identifying HR-HPV. mRNA testing is not widely available in sub-Saharan Africa due to technical and financial constraints; however, the test appears to be of great value in p16-equivocal lip SCC.

Immunohistochemical Expression of p16 and CDK4 in Soft Tissue Tumors.

The aim of this study was to determine the immunohistochemical expression of p16 (p16INK4a) and cyclin-dependent kinase 4 (CDK4) and CDK4 markers in various lineages of soft tissue tumors and to evaluate their role in differentiating atypical lipomatous tumors/well-differentiated liposarcomas from benign lipomas. A total of 70 cases of both excisional and incisional biopsies of soft tissue tumors were included in this study. Histopathological examination was done by using formalin-fixed, paraffin-embedded tissue samples. After that, we performed IHC expressions of p16 and CDK4 markers on the unstained slides of these soft tissue tumors. Results: Immunohistochemical study showed that positive expressions of p16, CDK4, and combined (p16+CDK4) markers were 51.4%, 10.0%, and 12.9%, respectively in soft tissue tumors. Positive p16 expression was observed among a higher proportion of malignant cases (66.7%) as compared to benign (20.0%) and intermediate (50.0%) cases. This difference was found to be statistically significant (p=0.009). Negative expression of only CDK4 and combined (p16 and CDK4) were observed among a higher proportion of benign as compared to malignant and intermediate cases (90.0% vs. 78.6% & 75.0%, p=0.393 and 65.0% vs. 26.2% & 37.5%, p=0.028, respectively). This difference was not found to be statistically significant. For adipocytic tumors, the majority of malignant and intermediate tumors had positive p16 (7/7; 100%) and CDK4 (6/7; 85.7%) immunohistochemical expression. These differences were found to be statistically significant. Immunohistochemical marker p16 can be used to differentiate between malignant and benign soft tissue tumors. Amongst adipocytic tumors, combination of p16 and CDK4 immunohistochemical expression can be used to differentiate liposarcomasfrom benign ones.

Evaluation of variable p16 immunostaining patterns, Ki-67 indices and HPV status in cervical SILs and squamous cell carcinomas: An institutional experience.

The present study evaluates the immunoexpression of p16 and Ki-67 in cervical squamous intraepithelial lesion (SIL) and carcinomas and correlates their expression with clinicopathological features and HPV-DNA status. A total 36 included cases of SIL and squamous cell carcinoma (SCC) were subjected to p16 and Ki-67 immunostaining. p16 staining was evaluated depending on grading, distribution, localization pattern, intensity and IHC score. Ki-67 expression was graded based on percentage of positive cells. Incidence of HSIL and SCC cases was found to be significantly increased with parity > 5. p16 grade III diffuse nucleocytoplasmic immunostaining was observed in 62.5% LSIL, 80% HSIL and 87% SCC cases. Significant association of p16 staining intensity, IHC score and Ki-67 indices was noted with increasing grades of SILs and carcinomas. Our experience indicates that a combination of p16 and Ki-67 immunostaining may be useful to determine the severity of dysplastic change.

P16 Regulates Transcription Factor Sp1 Function to Affect Outcome in Oropharyngeal Cancer

<h3>Purpose/Objective(s)</h3> Our group has previously demonstrated that p16 activates a ubiquitin mediated pathway to repress homologous recombination via activation of HUWE1, potentially via the transcription factor Sp1. However, the specific mechanism of this interaction and its clinical significance remain to be determined. We performed <i>in vitro</i> analysis of p16 and Sp1 interaction in multiple contexts and performed correlative analysis using the head and neck TCGA clinical data, as well as analysis of HPV+ and HPV- oropharyngeal cancer (OPC) treated with chemoradiation (CRT) to evaluate the combined importance of p16 and Sp1 on outcome. We hypothesized that p16 increases the Sp1-HUWE1 interaction. <h3>Materials/Methods</h3> For clinical outcome, 100 patients with oropharyngeal cancer evenly divided between p16+ and p16- and treated with chemoradiation were evaluated for gene expression using targeted multiplex analysis platform. Additional clinical correlative analyses between HPV, p16, HUWE1 and Sp1 were performed using the TCGA Head and Neck cohort (n=530). UM-SCC-47 p16 CRISPR KO and HN5 cells harboring doxycycline-inducible p16 were used to perform <i>in vitro</i> studies. Immunoprecipitation (IP) was used to confirm the protein-protein interaction between protein p16 and transcription factor Sp1. Chromatin immunoprecipitation (ChIP) assay was used to determine the existence of the Sp1 protein-HUWE1 DNA promoter interaction. <h3>Results</h3> Sp1 (p=7.9e⁻¹⁶) and HUWE1 (p=7.9e⁻⁴) were expressed at significantly higher levels in HPV+ tumors and Sp1 and HUWE1 gene expression were highly correlated (p=5.3e10⁻⁴⁵) in the TCGA Head and Neck cohort. In addition, Sp1 expression was significantly higher in p16 positive OPC tumors in a separate patient cohort (p=3e⁻³). In OPC patents treated uniformly with CRT, the combination of p16 and Sp1 expression were associated with reduced locoregional recurrence following CRT (p=7e⁻⁶), effectively delineating good prognosis p16- patients and poor prognosis p16+ patients. IP of p16 in HN5 cells showed increased interaction with Sp1 after doxycycline-induced p16INK4a expression, and in combination with IR. ChIP assay demonstrated SP1 dependent enrichment in 2 of 5 GC box regions of the human HUWE1 promoter. More importantly, these SP1 protein-HUWE1 promoter interactions were significantly enhanced after p16 overexpression. <h3>Conclusion</h3> We show Sp1 directly interacts with HUWE1 promoter resulting in an increase of HUWE1 expression, and this interaction is mediated by p16. This increase of HUWE1 expression then leads to down-regulation of homologous recombination, sensitizing cells to DNA damage. Clinical data shows the combined expression of p16, Sp1 and HUWE1 are highly correlated and that the combination of p16 and Sp1 may be an effective biomarker in OPC.

Oropharyngeal cancer outcomes correlate with p16 status, multinucleation and immune infiltration

AbstractOropharyngeal squamous cell carcinoma (OPSCC), largely fueled by the human papillomavirus (HPV), has a complex biological and immunologic phenotype. Although HPV/p16 status can be used to stratify OPSCC patients as a function of survival, it remains unclear what drives an improved treatment response in HPV-associated OPSCC and whether targetable biomarkers exist that can inform a precision oncology approach. We analyzed OPSCC patients treated between 2000 and 2016 and correlated locoregional control (LRC), disease-free survival (DFS) and overall survival (OS) with conventional clinical parameters, risk parameters generated using deep-learning algorithms trained to quantify tumor-infiltrating lymphocytes (TILs) (OP-TIL) and multinucleated tumor cells (MuNI) and targeted transcriptomics. P16 was a dominant determinant of LRC, DFS and OS, but tobacco exposure, OP-TIL and MuNI risk features correlated with clinical outcomes independent of p16 status and the combination of p16, OP-TIL and MuNI generated a better stratification of OPSCC risk compared to individual parameters. Differential gene expression (DEG) analysis demonstrated overlap between MuNI and OP-TIL and identified genes involved in DNA repair, oxidative stress response and tumor immunity as the most prominent correlates with survival. Alteration of inflammatory/immune pathways correlated strongly with all risk features and oncologic outcomes. This suggests that development of OPSCC consists of an intersection between multiple required and permissive oncogenic and immunologic events which may be mechanistically linked. The strong relationship between tumor immunity and oncologic outcomes in OPSCC regardless of HPV status may provide opportunities for further biomarker development and precision oncology approaches incorporating immune checkpoint inhibitors for maximal anti-tumor efficacy.

High-risk HPV-related Squamous Cell Carcinoma in the Conjunctiva and Lacrimal sac: Clinicopathologic Characteristics and Diagnostic Utility of p16 and Rb Immunohistochemistry.

High-risk human papillomavirus (HPV) infection in conjunctival and lacrimal sac squamous cell carcinomas (SCCs) has been sporadically reported; however, its prevalence, clinicopathologic significance and surrogate markers have not been fully elucidated. Here, we attempted to clarify these questions in Japanese patients with conjunctiva and lacrimal sac SCCs. We retrospectively collected 51 conjunctival SCC and 7 lacrimal sac SCC samples and analyzed them for (1) transcriptionally active high-risk HPV infection using messenger RNA in situ hybridization and (2) protein expressions of p16 and Rb using immunohistochemistry (IHC). Among a total of 58 cases, 25 (43.1%) and 16 (27.6%) tumors were positive for p16-IHC and HPV in situ hybridization, respectively. Ten (19.6%) of the 51 conjunctival SCCs, especially in the palpebral conjunctiva, and 6 (85.7%) of the 7 lacrimal sac SCCs were positive for high-risk HPV. High-risk HPV infection was significantly associated with younger patients, nonkeratinizing SCC histology, p16-positivity and partial loss of Rb expression, but not with recurrence risk. Notably, p16-IHC was not a perfect surrogate marker for high-risk HPV infection; only 64% (16/25) of p16-positive tumors were positive for high-risk HPV. In contrast, the p16+/Rb partial loss pattern was exclusively correlated with high-risk HPV-positivity. The results suggest that the combination of p16 and Rb expression patterns by IHC could be a useful method to predict high-risk HPV infection in conjunctival and lacrimal sac SCCs. HPV infection may be of less prognostic value in this field of cancers.

Evaluation of Cervical High-Grade Squamous Intraepithelial Lesions-Correlated Markers as Triage Strategy for Colposcopy After Co-Testing.

BackgroundColposcopy was referred in cases with severe abnormalities in co-testing. Although p16/Ki67 dual staining reduced the referral rate, its sensitivity and specificity need to be enhanced.MethodsThe expressions of p16, Ki-67, SMAD3, YAP1, RELA were evaluated in the colposcopy referral population. The inclusion criteria included 30–60 years and diagnosed with HPV16/18-positive, other HR-HPV-positive with ASCUS, LSIL, AGC (atypical glandular cell) in co-testing. Colposcopies, endocervical curettages of cervical biopsies were also collected. Cases were excluded if there were no biopsies, if the interval between a cervical screening test and biopsies was more than 6 months, or if insufficient tissue was available as a formalin-fixed paraffin-embedded block. The pathology was independently reviewed by two pathologists. Discrepant interpretations were adjudicated by a third pathologist.ResultsIn total, 1194 of 1273 cases who were referred to colposcopy were evaluated in the present study. The sensitivity and specificity of p16+ combined with Ki-67+ for predicting CIN2+ were 62.1% and 89.5%, respectively. p16+ combined with YAP1+ and/or RELA+ provided a sensitivity and specificity of 70.9% and 89.5%, respectively, while 72.8% and 86.4% were achieved by p16+ combined with YAP1+ and/or SMAD3+ and/or RELA+. In HPV16/18+ and LSIL subgroups, the sensitivity and specificity of p16+ combined with Ki-67+ for predicting CIN2+ were 67.7% and 87.6%, respectively, for the former group and 58.6%, 88.8%, respectively, for the latter group. p16+, YAP1+/RELA+ showed a better performance for predicting CIN2+ with a better sensitivity and considerable specificity in the other HPV+ combined with ASCUS group than were achieved by p16+ combined with Ki-67+. RELA+ and the combination of p16 and RELA/YAP1 also provided the Max AUC area.ConclusionOur study shows that RELA and the combination of p16 and RELA/YAP1 achieved better sensitivity and specificity for detecting morphologically CIN2+ lesions.

Clinical significance of P16-positive status and high index of proliferative activity in patients with oropharyngeal squamous cell carcinoma

Introduction. In accordance with uICC and AJCC 8th edition TNM classifications, there is a strong evidence for division of oropharyngeal squamous cell carcinoma (OPSCC) into 2 molecular subtypes by HPV-status with distinct prognosis depending on biological differences. Such a division leads to differences in staging OPSCC and in future it will lead to implementation of preventive measures and new therapeutic strategies against HPV-positive cancer. Aim of the study : to assess the clinical and prognostic significance of the combination of P16, a surrogate marker for HPV-positivity, and high proliferative activity in patients with oropharyngeal carcinoma. Material and Methods . Immunohistochemical (ICH) analysis with monoclonal antibodies specific for P16 and Ki67 proteins was used to detect expression patterns in the formalin-fixed, paraffin-embedded tumor samples obtained from 104 patients with squamous cell carcinoma of the tongue and oropharynx, treated at Oncological Dispencery № 1 in Krasnodar from 2011 to 2016. HPV-positive status was determined if more than 70 % of tumor cells had moderate or strong nuclear and cytoplasmic P16-staining. High index of proliferative activity (PA) was detected if more than 50 % tumor cells expressed Ki67 nuclear antigen. Results. P16-positivie status was associated with tonsillar cancer ( р =0.002), female gender ( р =0.015), age &lt;60 years ( р &lt;0.001), non - keratinizing morphology ( р =0.022), and high index of PA (p=0.01).The combination of P16&gt;70 % with high PA demonstrated correlation with tonsillar cancer ( р &lt;0.001), female gander ( р =0.015), age under 60 years ( р &lt;0.001) and non - keratinizing morphology ( р =0.012). HPV-positive patients and patients with a combination of P16&gt;70 % and high index of PA at N1-2 had an overall survival benefit (p=0.021). Conclusion. The correlation between IHC-complex for P16&gt;70 %/Ki67&gt;50 % and clinicopathologicl parameters and overall survival confirms the biological features of HPV-associated cancer. The evaluation of this IHC-complex can increase the diagnostic accuracy of IHC-analysis of HPV-status and predict the prognosis of patients with OPSCC.

A Combination of p16 and MIB-1 Immunostainings is Possibly a Useful Method for Diagnosing Verrucous Carcinoma of the Foot

A Combination of p16 and MIB-1 Immunostainings is Possibly a Useful Method for Diagnosing Verrucous Carcinoma of the Foot

Highlights of This Issue

Current biomarkers for predicting the response to anti-PD-1 therapy require tissue biopsy and have not shown sufficient predictive values. Kim and colleagues discovered, in anti-PD-1-treated patients with metastatic or refractory thymic epithelial tumors and in patients with non-small cell lung cancer, a higher fold-change in the percentage of Ki-67+ cells among PD-1+CD8+ T cells 7 days after the first dose predicts superior durable clinical benefit and survival rates. This result was validated in an independent cohort of anti-PD-1-treated patients with non-small cell lung cancer. In summary, the first-week proliferative response of peripheral blood PD-1+CD8+ T cells predicts the response to anti-PD-1 therapy in solid tumors.CDK4/6 inhibitors (palbociclib, ribociclib, and abemaciclib) are a new class of drugs that are approved alone or in combination with endocrine therapy for advanced estrogen receptor positive breast cancer. Clark and colleagues sought to combine palbociclib with paclitaxel using a novel dosing schedule taking advantage of the mechanism of action of both drugs to potentially increase the efficacy of single agent paclitaxel. Administering the drugs on an alternating schedule potentially enables G1-synchronization by palbociclib, a few days rest without drug allows cells to re-enter the cell cycle, increasing the percentage of cells in M phase when exposed to paclitaxel. Alternating sequential palbociclib/paclitaxel in patients with Rb+ ABC is feasible and safe, without evidence of additive toxicity.Nowicki and colleagues describe a clinical trial using dual cell immunotherapy, consisting of genetically modified lymphocytes with NY-ESO-1-specific T-cell receptor adoptively transferred together with NY-ESO-1 peptide-pulsed dendritic cell vaccination with or without CTLA-4 immune checkpoint blockade with ipilimumab. The authors report that these protocols are generally safe, feasible, and result in high antitumor activity in patients with advanced sarcoma and melanoma. However, further improvements in durability of these cellular therapies are needed because patients frequently relapse after an initially vigorous antitumor response.Cancer of the esophagus claims more than 400,000 lives annually year. Without efficient, reliable screening, diagnosis often occurs too late to save the patient. To this end, Wang and colleagues developed a minimally invasive, rapid test using a swallowable, retrievable sponge (EsophaCap) in conjunction with epigenetic biomarkers to detect precancerous changes (Barrett's esophagus) in cells lining the esophagus. The EsophaCap was administered to 94 people over the course of the study. The combination of p16, NELL1, AKAP12, and TAC1 methylation yielded high sensitivity and specificity in diagnosing Barrett's esophagus. These findings indicate the need for large prospective studies testing this early diagnostic modality.

Strong SOX10 expression in human papillomavirus–related multiphenotypic sinonasal carcinoma: report of 6 new cases validated by high-risk human papillomavirus mRNA in situ hybridization test

Human papillomavirus (HPV)-related multiphenotypic sinonasal carcinoma (HMSC) is associated with high-risk HPV (HR-HPV) infection. Using HR-HPV messenger RNA (mRNA) in situ hybridization (ISH), we reported 6 new HMSC cases and compared their histopathology with that of sinonasal adenoid cystic carcinoma. Using p16 immunohistochemistry (IHC) and HR-HPV ISH, we retrospectively identified 6 HMSC cases. All HMSC cases were positive for HR-HPV mRNA ISH and p16 IHC. Two HMSC cases had overlying atypical squamous epithelium, and 1 had invasive squamous cell carcinoma (SCC). All HMSC cases were SOX10 positive, whereas the overlying atypical squamous epithelium and the SCC were SOX10 negative. One atypical HMSC-like case was also identified, which was positive for HR-HPV mRNA ISH, HR-HPV DNA ISH, and SOX10 IHC, but negative for p16 IHC. This study showed that HR-HPV mRNA ISH was a useful tool to diagnose HMSC and had stronger signals compared with HR-HPV DNA ISH. HR-HPV E6/E7 mRNA could be identified in the overlying atypical squamous epithelium and the invasive SCC. A combination of p16 and SOX10 IHC will be a useful screening panel for HMSC followed by confirmatory HR-HPV mRNA ISH test.

Prevalence of Human Papillomavirus (HPV) in Women with Normal Colposcopy or Cytology Status Northeast Brazil

s S49 Results: All eighteen cases were found to have adequate cells with good preservation. P16 expression correlated with HR-HPV status in 13 of 15 cases (87% concordance; 3 cases of HSIL did not have HR-HPV testing). In each discordant case (1 ASCUS and 1 ASC-H), the HR-HPV was not detected, while P16 expression was identified. The Ki-67 index scoring was as follows: 6 negative (ASCUS, 3 ASC-H, 1 LSIL, and 1 HSIL), 2 rare (both ASCUS), 4 mild (ASCUS), 0 moderate, and 5 high (1 LSIL, 1 ASCH, and 3 HSIL). The Ki-67 index correlated with HR-HPV in 8 of 15 cases (53% concordance). Discordant cases were as follows: 2 cases of + HRHPV / Ki-67 (one LSIL and one ASC-H), and 5 cases of HR-HPV / + Ki-67 (3 ASCUS, 1 LSIL, 1 ASC-H). In 2 cases (13%), the HR-HPV was not detected, while both P16 and Ki-67 were positive. There were no cases (0%) where HR-HPV was detected and both the P16 and Ki-67 were negative. Histologic follow-up was available in 7 cases: 2 ASCUS (benign and CIN I), 1 LSIL (CIN I), 2 ASC-H (benign and CIN I-II) and 2 HSIL (CIN III). There were 2 cases where HR-HPV was not detected while P16 and Ki-67 were positive, in both cases the corresponding cervical biopsy showed benign findings. Conclusions: The combination of P16 and Ki-67 expression was 100% sensitive for HR-HPV status. In 2 cases (13%) with P16 expression and Ki67 immunoreactivity, HR-HPV was not detected, and the follow-up histology was benign. Although limited by a small sample size, our preliminary findings show a utility for adjunct P16 and Ki-67 in cases of cytologic atypia in addition to HR-HPV testing. Our results indicate that adjunct P16 and Ki-67 testing offers additional benefit in cases that HRHPVs are not detected due to low numbers of DNA copies or missed lesions in small cervical biopsies or endocervical curretings.

Inhibition of Breast Tumor Cell Growth by Ectopic Expression of p16/INK4A Via Combined Effects of Cell Cycle Arrest, Senescence and Apoptotic Induction, and Angiogenesis Inhibition

p16-mediated inhibition of cancer cell proliferation and tumor suppression have been studied before,; the common consensus is that p16's cell-cycle arrest function plays a primary role in these actions, with some additional apoptotic induction by p16. However, other effects of p16 that may potentially contribute to p16-mediated anti-tumor ability have not been well studied. The emerging data including ours indicated that p16 contributes its anti-cancer ability by inducing tumor cells to senescence. Moreover, we showed that p16 inhibits breast cancer cell growth by inhibiting the VEGF signaling pathway and angiogenesis. In this study, we used adenoviral-mediated p16 expression (AdRSVp16) and breast cancer cell line MDA-MB-231 as the model to simultaneously analyze all these p16's anti-tumor functions. We demonstrated that adenoviral-mediated p16 expression exhibited multiple anti-tumor functions by simultaneously suppressing in vitro growth and in vivo angiogenesis of breast cancer cells, blocking cell division, as well as inducing senescence and apoptosis. The in vivo study implies that p16's effect on anti-angiogenesis may play a more significant role than its anti-cell proliferation in the overall suppression of tumor growth. These results suggest, for the first time, that AdRSVp16-mediated tumor suppression results from a combination of p16's multiple anti-tumor functions including p16's well-known anti-proliferation/cell division function, apoptotic and senescence induction function, and its lesser-known/under-investigated anti-angiogenesis function. These combined results strongly indicate that p16 gene therapy has a multi-module platform with different anti-tumor functions; therefore, this study justifies and promotes the viral-mediated p16 gene therapy as a promising and powerful treatment approach for cancer patients due to p16's multiple anti-tumor functions.

Evaluation of Human Papilloma Virus Diagnostic Testing in Oropharyngeal Squamous Cell Carcinoma: Sensitivity, Specificity, and Prognostic Discrimination

Human papillomavirus-16 (HPV16) is the causative agent in a biologically distinct subset of oropharyngeal squamous cell carcinoma (OPSCC) with highly favorable prognosis. In clinical trials, HPV16 status is an essential inclusion or stratification parameter, highlighting the importance of accurate testing. Fixed and fresh-frozen tissue from 108 OPSCC cases were subject to eight possible assay/assay combinations: p16 immunohistochemistry (p16 IHC); in situ hybridization for high-risk HPV (HR HPV ISH); quantitative PCR (qPCR) for both viral E6 RNA (RNA qPCR) and DNA (DNA qPCR); and combinations of the above. HPV16-positive OPSCC presented in younger patients (mean 7.5 years younger, P = 0.003) who smoked less than HPV-negative patients (P = 0.007). The proportion of HPV16-positive cases increased from 15% to 57% (P = 0.001) between 1988 and 2009. A combination of p16 IHC/DNA qPCR showed acceptable sensitivity (97%) and specificity (94%) compared with the RNA qPCR "gold standard", as well as being the best discriminator of favorable outcome (overall survival P = 0.002). p16 IHC/HR HPV ISH also had acceptable specificity (90%) but the substantial reduction in its sensitivity (88%) impacted upon its prognostic value (P = 0.02). p16 IHC, HR HPV ISH, or DNA qPCR was not sufficiently specific to recommend in clinical trials when used in isolation. Caution must be exercised in applying HPV16 diagnostic tests because of significant disparities in accuracy and prognostic value in previously published techniques.

Usefulness of p16ink4a, ProEX C, and Ki-67 for the Diagnosis of Glandular Dysplasia and Adenocarcinoma of the Cervix Uteri

Although the diagnostic criteria of in-situ and invasive adenocarcinomas of the cervix uteri are well established, the differentiation from benign mimics may be difficult and the morphologic features of the precursors of endocervical adenocarcinoma are still debated. In this study, we evaluated the usefulness of p16ink4a (p16), ProEX C, and Ki-67 for the diagnosis of endocervical adenocarcinoma and its precursors. Immunohistochemistry with p16, ProEX C, and Ki-67 was performed in 82 glandular lesions including 15 invasive adenocarcinomas, 29 adenocarcinomas in situ (AIS), 22 non-neoplastic samples, and 16 cases of glandular dysplasia (GD), which showed significant nuclear abnormalities but did not meet the diagnostic criteria for AIS. The immunohistochemical expression pattern was scored according to the percentage of the stained cells (0, 1+, 2+, and 3+ when 0% to 5%, 6% to 25%, 26% to 50%, and more than 50% of the cells were stained, respectively) and was evaluated for each antibody. p16 was at least focally expressed (1+ or more) in 14 of 15 invasive adenocarcinomas, in all AIS and in 7 negative samples. ProEX C and Ki-67 both scored 1+ or more in all adenocarcinomas and AIS and in 8 and 6 negative samples, respectively. Of the GD 15, 14, and 15 expressed p16, ProEX C, and Ki-67, respectively. The score differences between neoplastic and non-neoplastic samples were highly significant for each marker (P<0.001); however, the score distribution by marker differed significantly only in GD (P=0.006) in which, compared with the other markers, p16 showed more often a 3+ pattern. Our study shows that p16, Ki-67, and ProEX C may be helpful for the diagnosis of glandular lesions of the cervix uteri and may also improve the diagnostic accuracy of endocervical GD. In particularly problematic cases, the combination of p16 and a proliferation marker can provide additional help for the interpretation of these lesions.

High-Grade Endometrial Carcinoma: Serous and Grade 3 Endometrioid Carcinomas Have Different Immunophenotypes and Outcomes

High-grade endometrial carcinomas are a heterogeneous group of tumors and include grade 3 endometrioid (EC-3), serous (SC), and clear cell carcinomas (CCC). There are conflicting data about the prognosis of these subtypes of high-grade endometrial carcinoma; this may be a result of lack of reproducibility in classifying tumor cell type. The purpose of this study was to examine differences in immunophenotype and outcome in a series of high-grade endometrial carcinomas, focusing on the comparison of EC-3 versus SC. We selected 180 endometrial carcinomas of SC, EC, or CCC type. No mixed carcinomas were included in the study. We chose the following immunohistochemical markers, estrogen receptor (ER), insulin-like growth factor 2 mRNA-binding protein 3 (IMP3), p16, p53, progesterone receptor (PR), and phosphatase and tensin homolog (PTEN) as being significantly differentially expressed in endometrial carcinoma subtypes. The tumors were stratified into 4 groups on the basis of their cell type and grade: EC grade 1 or 2, EC-3, SC, and CCC. Univariate survival analysis revealed significant differences in outcome between the 4 groups (P<0.0001), with significantly longer disease-specific survival for grade 1 or 2 EC versus EC-3 (P=0.0001), and EC-3 versus SC (P=0.0003). p16, PTEN, and IMP3 expression was observed more frequently in SC compared with EC-3 (P<0.0001, P=0.021, and P=0.031, respectively). These 3 markers showed the highest sensitivity and specificity in distinguishing between EC-3 and SC, with receiver operating characteristics area under the curve of 0.85, 0.69, and 0.71, respectively. ER and p53 approached but did not reach significance for differential expression in EC-3 versus SC (P=0.055 and P=0.068, respectively). A combination of p16 and PTEN predicts EC-3 versus SC with a sensitivity of 90.0% and specificity of 96.8%. p16 and PTEN can aid in distinguishing between EC-3 and SC of the endometrium, and are superior to ER, PR, and p53 for this purpose. EC-3 carcinomas have a significantly better prognosis than SC carcinomas of the endometrium.

Potent synergy of dual antitumor peptides for growth suppression of human glioblastoma cell lines

Molecular targeting agents have become formidable anticancer weapons, which show much promise against the refractory tumors. Functional peptides are among the more desirable of these nanobio-tools. Intracellular delivery of multiple functional peptides forms a basis for potent, non-invasive mode of delivery, providing distinctive therapeutic advantages. Here, we examine growth suppression efficiency of human glioblastomas by dual-peptide targeting. We did simultaneous introduction of two tumor suppressor peptides (p14(ARF) and p16(INK4a) or p16(INK4a) and p21(CIP1) functional peptides) compared with single-peptide introduction using Wr-T-mediated peptide delivery. Wr-T-mediated transport of both p14(ARF) and p16(INK4a) functional peptides (p14-1C and p16-MIS, respectively) into human glioblastoma cell line, U87DeltaEGFR, reversed specific loss of p14 and p16 function, thereby drastically inhibiting tumor growth by >95% within the first 72 h, whereas the growth inhibition was approximately 40% by p14 or p16 single-peptide introduction. Additionally, the combination of p16 and p21(CIP1) (p21-S154A) peptides dramatically suppressed the growth of glioblastoma line Gli36DeltaEGFR, which carries a missense mutation in p53, by >97% after 120 h. Significantly, our murine brain tumor model for dual-peptide delivery showed a substantial average survival enhancement (P < 0.0001) for peptide-treated mice. Wr-T-mediated dual molecular targeting using antitumor peptides is highly effective against growth of aggressive glioblastoma cells in comparison with single molecule targeting. Thus, jointly restoring multiple tumor suppressor functions by Wr-T-peptide delivery represents a powerful approach, with mechanistic implications for development of efficacious molecular targeting therapeutics against intractable human malignancies.

Combined analysis of HPV‐DNA, p16 and EGFR expression to predict prognosis in oropharyngeal cancer

Molecular prognostic indicators for oropharyngeal squamous cell carcinoma (OSCC), including HPV-DNA detection, epidermal growth factor receptor (EGFR) and p16 expression, have been suggested in the literature, but none of these are currently used in clinical practice. To compare these predictors, 106 newly diagnosed OSCC for the presence of HPV-DNA and expression of p16 and EGFR were analyzed. The 5-year disease-free survival (DFS) and overall survival (OS) were calculated in relation to these markers and a multivariate Cox analysis was performed. Twenty-eight percent of the cases contained oncogenic HPV-DNA and 30% were positive for p16. The p16 expression was highly correlated with the presence of HPV-DNA (p < 0.001). Univariate analysis of the 5-year DFS revealed a significantly better outcome for patients with p16-positive tumors (84% vs. 49%, p = 0.009). EGFR-negative tumors showed a tendency toward a better prognosis in DFS (74% vs. 47%, p = 0.084) and OS (70% vs. 45%, p = 0.100). Remarkable and highly significant was the combination of p16 and EGFR expression status, leading to 5-year DFS of 93% for p16+/EGFR- tumors vs. 39% for p16-/EGFR+ tumors (p = 0.003) and to a 5-year OS of 79% vs. 38%, respectively (p = 0.010). In multivariate analysis p16 remained a highly significant prognostic marker for DFS (p = 0.030) showing a 7.5-fold increased risk for relapse in patients with p16-negative tumors. Our data indicate that p16 expression is the most reliable prognostic marker for OSCC and further might be a surrogate marker for HPV-positive OSCC. HPV+/p16+ tumors tended to have decreased EGFR expression, but using both immunohistological markers has significant prognostic implications.

Frequent Deletions of Tumor Suppressor Genes in Pure Pancreatic Juice from Patients with Tumoral or Nontumoral Pancreatic Diseases

Background/Aims: K-ras codon 12 mutation is the most frequent genetic alteration in pancreatic cancer. Sensitivity and specificity of K-ras are not high enough to detect all pancreatic cancers, especially at early stage. This study investigated whether detection of p16 and/or DPC4 deletions along with K-ras mutation in DNA samples could improve the definition of patients at risk of pancreatic cancer. Methods: K-ras mutations were investigated by sequencing. p16 and DPC4 homozygous deletions were studied using comparative multiplex polymerase chain reaction of DNA in pancreatic juice sampled during endoscopic retrograde pancreatography in 57 patients with either pancreatic cancer (group I, 18 patients), chronic pancreatitis (group II, 20 patients), or nontumoral pancreatobiliary disease (group III, 19 patients). Results: The frequencies of Ki-ras mutations were 61% in group I, 10% in group II, and 10.5% in group III. The frequencies of p16 exon 2 and DPC4 deletions were, respectively, 28 and 36% in group I, 50 and 58% in group II, and 24 and 36% in group III. Conclusions: The combination of p16 and DPC4 deletions with K-ras mutation does not improve the diagnosis of pancreatic cancer based on K-ras mutation alone. These data suggest that tumor suppressor gene inactivation can occur with a high frequency during nonmalignant pancreatic diseases.