p16INK4a Overexpression Research Articles

Human Papillomaviruses 16 and 18 E6 Oncoprotein Detection Test in Primary Oropharyngeal Carcinomas and Metastatic Lymph Nodes: A Cross-Sectional Study.

Accuracy in the diagnosis of HPV-associated oropharyngeal carcinoma (OPSCC) of a rapid, low-cost lateral flow immunochromatographic assay for detecting E6 oncoprotein of HPV-16 and HPV-18 was previously evaluated in a small pilot study. This cross-sectional study aimed to assess on a large case series the sensitivity and specificity of E6 oncoprotein as a diagnostic marker for HPV-associated carcinogenesis in OPSCC. 137 consecutive patients with histologically confirmed OPSCC were enrolled in two hospitals in Northeast Italy. HPV status was determined by PCR for HPV DNA and p16INK4a immunohistochemistry on primary tumor biopsies. An OPSCC was defined as HPV-associated when double positive for high-risk HPV-DNA and p16INK4a overexpression in primary lesion. Cytological samples from primary tumors and metastatic lymph nodes were obtained and tested for HPV16/18 E6 oncoproteins using the lateral flow immunochromatographic assay, which requires between 90 and 120 min to provide a result. Diagnostic performances were calculated as percentage with confidence intervals (CI). Of the 137 OPSCC cases, 68 (49.6%) were HPV-associated, testing positive for both high-risk HPV-DNA and p16INK4a, with HPV16 predominating (82.4%). An average waiting time of 22 days was observed to obtain the results of p16INK4a and HPV-DNA after primary lesions biopsy. In patients with HPV16/18-associated OPSCC, the HPV16/18 E6 oncoprotein was detected in 59 out of 60 cytological samples from the primary lesion (sensitivity: 98.3%; 95% CI: 91.1-100%) and in 45 out of 51 cytological samples from lymph node metastases (sensitivity: 88.2%; 95% CI: 76.1-95.6%). The E6 oncoprotein assay showed a specificity of 100% in both primary tumors and lymph node metastases. The low-cost lateral flow immunochromatographic assay for detecting HPV16/18 E6 oncoproteins confirmed high accuracy for identifying HPV-associated OPSCC, particularly in primary tumors, suggesting its potential as a valuable diagnostic tool in clinical practice. Its rapid diagnostic capability could significantly accelerate the process of treatment decision-making, enhancing the timely management of patients.

Human papillomavirus (HPV) and Epstein-Barr virus (EBV) in Penile Cancer in Thailand.

Human papillomavirus (HPV) and Epstein-Barr virus (EBV) are important etiological factors for several cancers. This study aimed to determine the prevalence of HPV and EBV infection in penile cancer. Forty-three formalin-fixed paraffin-embedded penile cancer tissue samples were analyzed for the HPV-induced p16INK4A protein by immunohistochemistry and Epstein-Barr encoding region in situ hybridization. Demographic data and overall survival were analyzed. The median age of patients was 59 years, ranging from 23 to 91 years old. Most of the tumors (86%) were located at the tip of the penis. HPV infection was positive in 12/43 (27.9%) patients. EBV infection was observed in 2/43 (4.6%) of cases and there was no co-infection detected in this cohort. Patients who had p16INK4A overexpression had a trend toward longer survival compared to those without; the median survival time of 104.4 vs 89 months, the hazard ratio of 0.48 (95% CI: 0.16-1.42, p = 0.173). One-third of penile cancer patients were positive for HPV-induced p16INK4A expression and there was a trend toward better survival in HPV-positive patients. EBV infection was infrequent in penile cancer in Thailand.

Copy Number Profiling Implicates Thin High-Grade Squamous Intraepithelial Lesions as a True Precursor of Cervical Human Papillomavirus-Induced Squamous Cell Cancer

Full-thickness high-grade squamous intraepithelial lesions (HSIL) are precursors of invasive cervical squamous cell carcinoma (SCC). The World Health Organization and Lower Anogenital Squamous Terminology Standardization Project for human papilloma virus (HPV)–associated lesions divide full-thickness HSIL of the cervix into thin HSIL with thickness of 1 to 9 cell layers and the typical full-thickness HSIL of >10 cell layers. Although HPV oncogene transcripts and p16ink4a overexpression, as markers of transforming HPV infection, are detectable in thin HSIL, the biological significance of thin HSIL in cervical carcinogenesis remains poorly understood. To further characterize thin HSIL, we performed a comparative study of chromosomal copy number variations (CNV), an analysis of dysregulated genes present in the segments with CNV, and a generalized genetic complexity calculation for 31 thin HSIL, 31 thick HSIL, 24 microinvasive SCC (pT1a SCC), and 22 highly invasive SCC samples. Thin HSIL share various CNV and specific dysregulated gene pathways with thick HSIL and invasive SCC. Thin HSIL exhibited an average CNV of 11.6% compared with 14.1% for thick HSIL, 15.5% for pT1a SCC, and 26.6% for highly invasive SCC. The CNV included gains at 1q and 3q (40% and 43%, respectively), partial loss of 3p, and loss of chromosomes 11 (18%), 16 (50%), 20 (35%), and 22 (40%). Pathways affected solely in thin HSIL were those enhancing immune evasion and primarily involved the (interleukin) IL6, IL21, and IL23 genes. ILs are transiently upregulated in response to infection and play a crucial role in mounting antitumor T-cell activity. Deregulation reflects an attempt by the HPV to evade the initial immune response of the host. The primary pathways shared by thick HSIL and invasive SCC were interactions between lymphoid and nonlymphoid cells, NOTCH2 signaling, tight junction interactions (primarily of the claudin family), and FGR2 alternative splicing. Our results show that thin HSIL carry similar genetic changes as thick HSIL and SCC, indicating that thin HSIL are true precursor lesions that can progress to thick HSIL and SCC.

Relationship between P16INK4a Overexpression in Human Papillomavirus Infection in High Grade CIN and Cervical Carcinoma

Background: High-risk HPV strains are a well-known contributor to the carcinogenesis of cervical squamous cell carcinoma (SCC). Indeed, there has been a high detection of HPV DNA in SCC and precancerous lesions. Objective: The aim of the study was to assess the Relationship Between Human Papillomavirus Infection and P16INK4a Overexpression in Cervical Squamous Cell Carcinoma. Methodology: This descriptive cross-sectional study was carried out in the Pathology Department of DMC with collaboration of Bangabandhu Sreikh Mujib Medical University (BSMMU), Microbiology Department and Bangladesh Agricultural University (BAU), Mymansingh, during July 2017 to June 2018. A total 50 VIA positive and clinically suspected premalignant and malignant patient’s attendant at DMC included as study population. Colposcopic biopsy specimens were sent to the Department of Pathology, DMCH, for histopathological examination. Some portion of biopsy samples were preserved at -4ºC for HR-HPV DNA detection. Statistical analyses were done by using the SPSS 26.0. Result: Highest number of cases 20 (40%) were in fourth decade and their mean age was found to be 44.20±11.20, ranging from 28 to 67 years. Among 50 positive cases, maximum number of 32 (62%) cases were married between 16 to 20 years of age, 12 (22%) cases were married in less than 15 years of age and 6 (8%) cases were married at more than 20 years of age. The findings of colposcopic examinations of total 50 cases. Common diagnoses were CIN II in 24 (48%) cases, 8(16%) case represented CIN III, Squamous cell carcinoma in 16 (32%) cases and Adeno Carcinoma 2(4%) cases. In comparison of p16 score with cervical lesions in study subjects there were 26 true positive cases and 13 true negative cases. According to p16 score a total 37 (26 true positive+ 11 false positive) cases were p16 positive. Out of which 16(32.0%) and 21(42.0%) were HR-HPV positive and negative respectively. Similarly according to p16 score 13 nevative cases ........

SCREENING FOR HUMAN PAPILLOMAVIRUS USING SELF-COLLECTED CERVICAL SAMPLES FROM WOMEN DEPRIVED OF LIBERTY IN THE STATE OF AMAZONAS, BRAZIL

Objective: The aim of the study was to identify the prevalence of human papillomavirus (HPV) infections using a self-collection device and also describe the frequency of P16INK4a overexpression in women deprived of liberty (WDL). Methods: This observational, cross-sectional, descriptive, analytical, and epidemiological study of the prevalence of HPV infection included 268 women deprived of liberty (WDL) in the state of Amazonas who performed self-collection using the Roche® HPV CTNG test. Patients with positive HPV results were submitted to a Pap smear in liquid medium, colposcopy, an immunocytochemistry evaluation using P16INK4a, and a biopsy, when necessary. Results: The age of the 268 women ranged from 19 to 64 years (mean±standard deviation: 33.5±9.1 years). HPV was detected in 66 (24.6%) women: 12 (4.5%) had HPV 16, 3 (1.1%) had HPV 18, 51 (19%) had other high-risk HPV genotypes, 21 (7.8%) had inconclusive results and 181 (67.5%) had negative results. Two women had a high-grade lesion and 13 had a low-grade lesion. Eight women showed alterations in their Pap smear and 32 (64%) presented overexpression of P16INK4a. Self-collection was well-accepted by 95.9% of the WDL. Conclusion: This study shows that the prevalence of HPV among the sample of WDL was high, that self-collection as a screening method was well-accepted and that the prison population has peculiar characteristics since there is a higher prevalence of other high-risk HPV genotypes.

Box A of HMGB1 Maintains the DNA Gap and Prevents DDR-induced Kidney Injury in D-galactose Induction Rats.

Disability and mortality rates for renal failure are still increasing. DNA damage and oxidative stress intoxication from body metabolism, high blood glucose, or the environment cause significant kidney damage. Recently, we reported that Box A of HMGB1 (Box A) acts as molecular scissors, producing DNA gaps that prevent DNA damage in kidney cell lines and ultimately reverse aging phenotypes in aging rat models. The present study aimed to demonstrate the potency of Box A in preventing D-galactose (D-gal)-induced kidney injury. A Box A expression plasmid was constructed and administered to a rat model. D-gal was injected subcutaneously for eight weeks. Serum was collected to study renal function, and white blood cells were collected for DNA gap measurement. Kidney tissue was also collected for γ-H2AX and NF-κB immunostaining; Senescence-associated (SA)-beta-gal staining; and analysis of the mRNA expression of p16INK4A, TNF-α, and IL-6. Moreover, histopathology analysis was performed using hematoxylin & eosin and Masson trichome staining. Pretreatment with Box A administration prevented the reduction of DNA gaps and the consequences of the DNA damage response, which include elevated serum creatinine; high serum BUN; an increased positive SA-beta-gal staining area; overexpression of p16INK4A, NF-κB and senescence-associated secretory phenotype molecules, including IL-6, TNF-α; and histological alterations, including tubular dilation and collagen accumulation. Box A effectively prevents DNA gap reduction and all D-gal-induced kidney pathological changes at the molecular, histological, and physiological levels. Therefore, Box A administration is a promising novel therapeutic strategy to prevent DNA-damaging agent-induced kidney failure.

Diagnostic Performance of p16ink4a in the Presence of E6/E7 mRNA in Formalin-fixed Paraffin Embedded Specimens: A Cross-sectional Study

Introduction: Clinically significant High-risk Human Papillomavirus (HR-HPV) infection in Oral Cavity Squamous Cell Carcinoma (OCSCC) is not clearly defined. There is uncertainty regarding the true impact of HPV-related variability (ranging from 0% to 100%) when using the GP5+/GP6+/MY09/11 (L1) primer. The identification of HPV DNA in tumours is insufficient to establish a causal viral association; therefore, a gold standard test is required to determine HPV’s role in malignant cells by identifying the transcripts of the E6/E7 mRNA viral oncogene and the p16ink4a biomarker. Aim: To assess the diagnostic performance of p16ink4a in the presence of E6/E7 mRNA in Formalin-fixed, Paraffin-embedded (FFPE) specimens. Materials and Methods: This cross-sectional study was conducted in the Department of Pathology at JIPMER in Puducherry, India, from February 2019 to April 2022. Combined molecular markers p16ink4a and E6/E7 mRNA (considered the gold standard) from FFPE specimens of newly diagnosed Squamous Cell Carcinoma (SCC) were analysed to identify biologically and clinically relevant Infections using Immunohistochemistry (IHC) and real-time Polymerase Chain Reaction (PCR). Results: This study indicates that tissue samples with p16 positive expression (score of 2 or higher) showed the presence of HPV E6/E7 mRNA, with a sensitivity of 100% and a specificity of 91.67%. The relationship between p16ink4a and E6/E7 mRNA expression in all OCSCC cases was found to be significant (p-value=0.003). Conclusion: The study found a significant relationship between mRNA and p16ink4a overexpression in tongue SCC. This finding suggests that alterations in mRNA expression levels are associated with changes in p16ink4a expression in tongue cancer. Such understanding could potentially aid in the development of diagnostic or therapeutic strategies targeting these molecular pathways in tongue SCC.

The Spectrum of HPV-independent Penile Intraepithelial Neoplasia: A Proposal for Subclassification.

Compared with vulva, precursor lesions of human papillomavirus (HPV)-independent invasive squamous cell carcinoma (SCC) of the penis are insufficiently characterized. We analyzed the histologic and immunohistochemical characteristics of 70 peritumoral precursor lesions and correlated them with the histology and mutational profile of the adjacent HPV-negative invasive penile SCC. Atypical basal keratinocyte proliferation with variously elongated epithelial rete with premature squamatiziation, but regular superficial cornification, termed differentiated penile intraepithelial neoplasia (d-PeIN), were identified adjacent to 42/70 (60%) SCC (36/42 keratinizing (P<0.001); 3 papillary, and 1 each verrucous, clear cell, sarcomatoid SCC). d-PeIN were associated with chronic inflammatory dermatoses (32/42; P<0.001), p53 overexpression (26/42; P<0.001), and hotspot mutations in TP53 (32/42; P<0.001), CDKN2A (26/42; P<0.001) or both (21/42; P=0.003) in the adjacent SCC. Cytoplasmic p16ink4a overexpression in 5/42 d-PeIN correlated with CDKN2A missense mutations in the adjacent SCC. In all, 21/70 (30%) cornified verrucous or glycogenated verruciform precursors with minimal atypia and wild-type p53 (18/21; P<0.001) occurred adjacent to verrucous or papillary SCC (17/21; P<0.001) and keratinizing (4/21) SCC, which harbored mutations in HRAS and/or PIK3CA (12/21; P<0.004). Undifferentiated p16ink4a-negative full-thickness precursors were identified in 7/70 (10%) SCC. Four histologically different HPV-independent penile precursor lesions can be assigned to 2 major genetic/biological pathways with characteristic highly differentiated precursors requiring different clinical management decisions. These include d-PeIN in chronic inflammatory dermatoses, with p53 overexpression and TP53/CDKN2A mutations, and the p53 wild-type verrucous and verruciform precursors unassociated with dermatoses, but with mutations in oncogenes PIK3CA and HRAS.

NKG2D-CAR T cells eliminate senescent cells in aged mice and nonhuman primates.

Cellular senescence, characterized by stable cell cycle arrest, plays an important role in aging and age-associated pathologies. Eliminating senescent cells rejuvenates aged tissues and ameliorates age-associated diseases. Here, we identified that natural killer group 2 member D ligands (NKG2DLs) are up-regulated in senescent cells in vitro, regardless of stimuli that induced cellular senescence, and in various tissues of aged mice and nonhuman primates in vivo. Accordingly, we developed and demonstrated that chimeric antigen receptor (CAR) T cells targeting human NKG2DLs selectively and effectively diminish human cells undergoing senescence induced by oncogenic stress, replicative stress, DNA damage, or P16INK4a overexpression in vitro. Targeting senescent cells with mouse NKG2D-CAR T cells alleviated multiple aging-associated pathologies and improved physical performance in both irradiated and aged mice. Autologous T cells armed with the human NKG2D CAR effectively delete naturally occurring senescent cells in aged nonhuman primates without any observed adverse effects. Our findings establish that NKG2D-CAR T cells could serve as potent and selective senolytic agents for aging and age-associated diseases driven by senescence.

Rising Trend in the Prevalence of HPV-Driven Oropharyngeal Squamous Cell Carcinoma during 2000-2022 in Northeastern Italy: Implication for Using p16INK4a as a Surrogate Marker for HPV-Driven Carcinogenesis.

The prevalence and incidence of oropharyngeal squamous cell carcinomas (OPSCCs) driven by human papillomavirus (HPV) infection are increasing worldwide, being higher in high-income countries. However, data from Italy are scanty. p16INK4a overexpression is the standard in determining HPV-driven carcinogenesis, but disease prevalence impacts on its positive predictive value. This is a multicenter retrospective study enrolling 390 consecutive patients aged ≥18 years, diagnosed with pathologically confirmed OPSCC in Northeastern Italy between 2000 and 2022. High-risk HPV-DNA and p16INK4a status were retrieved from medical records or evaluated in formalin-fixed paraffin-embedded specimens. A tumor was defined as HPV-driven when double positive for high-risk HPV-DNA and p16INK4a overexpression. Overall, 125 cases (32%) were HPV-driven, with a significant upward temporal trend from 12% in 2000-2006 to 50% in 2019-2022. The prevalence of HPV-driven cancer of the tonsil and base of the tongue increased up to 59%, whereas it remained below 10% in other subsites. Consequently, the p16INK4a positive predictive value was 89% for the former and 29% for the latter. The prevalence of HPV-driven OPSCC continued to increase, even in the most recent period. When using p16INK4a overexpression as a surrogate marker of transforming HPV infection, each institution should consider the subsite-specific prevalence rates of HPV-driven OPSCC as these significantly impact on its positive predictive value.

Prognostic Impact of Human Papilloma Virus Infection on Cervical Cancer Patients Reflected by p16ink4a Expression: Single Institution Experience

Background: Human Papilloma virus (HPV) is the main cause of cervical cancer. Infection with HPV can be reflected by overexpression of p16ink4a. Aim of the study: we aimed at evaluating the expression of p16ink4a in cervical cancer patients and its prognostic significance. Patients and methods: This retrospective registry and follow up study was conducted on 95 women diagnosed with cervical cancer. After screening of patients presented to our hospitals; 50 patients were eligible for inclusion in the study. P16ink4a was assessed by immunohistochemistry on archived tumor’s samples and correlation with different epidemiological, clinical and pathological data was performed. Prognostic impact of P16ink4a on overall survival (OS) and event free survival (EFS) was evaluated.Results: Median age of patients was 55 years. The main presenting symptom was bleeding. Most of the patients presented with late FIGO staging 64% with stage IIIb and 10% with stage IIb. P16ink4a was positive in 80% of patients. Correlation between P16ink4a and different clinic-epidemiological data revealed positive significant correlation with tumor grade and tumor size (P values of 0.03 and 0.05 respectively). Considering the effect of p16ink4a expression and EFS and OS, our study failed to show any significant correlation.Conclusion: The late stage at presentation of our population encourages the need for national screening program. The main cause of cervical cancer is HPV reflected by positive P16 similar to international literature. In our study the failure to reach a significant correlation with survival may be due the small sample size.

P16INK4a Regulates ROS-Related Autophagy and CDK4/6-Mediated Proliferation: A New Target of Myocardial Regeneration Therapy.

Neonatal mice achieve complete cardiac repair through endogenous myocardial regeneration after apical resection (AR), but this capacity is rapidly lost 7 days after birth. As an upstream inhibitor of cyclin-dependent kinase 4/6- (CDK4/6-) mediated cell cycle activity, p16INK4a is widely involved in regulating tumor and senescence. Given that p16INK4a had a significant negative regulation on cell proliferation, targeting cardiomyocytes (CMs) to inhibit p16INK4a seems to be a promising attempt at myocardial regeneration therapy. The p16INK4a expression was upregulated during perimyocardial regeneration time. Knockdown of p16INK4a stimulated CM proliferation, while p16INK4a overexpression had the opposite effect. In addition, p16INK4a knockdown prolonged the proliferation time window of newborn myocardium. And p16INK4a overexpression inhibited cell cycle activity and deteriorated myocardial regeneration after AR. The quantitative proteomic analysis showed that p16INK4a knockdown mediated the cell cycle progression and intervened in energy metabolism homeostasis. Mechanistically, overexpression of p16INK4a causes abnormal accumulation of reactive oxygen species (ROS) to induce autophagy, while scavenging ROS with N-acetylcysteine can alleviate autophagy and regulate p16INK4a, CDK4/6, and CyclinD1 in a covering manner. And the effect of inhibiting the proliferation of p16INK4a-activated CMs was significantly blocked by the CDK4/6 inhibitor Palbociclib. In summary, p16INK4a regulated CM proliferation progression through CDK4/6 and ROS-related autophagy to jointly affect myocardial regeneration repair. Our study revealed that p16INK4a might be a potential therapeutic target for myocardial regeneration after injury.

P16 and EGFR analyses in cervical carcinoma on paraffin material in Egyptian females: the NCI experience

Abstract Introduction Cervical cancer is considered the fourth most prevalent malignant tumor in women worldwide. In Egypt, it ranks as the 14th most frequent cancer among women. Epidermal growth factor receptor has been an attractive target for anticancer therapy. Although EGFR mutations are less commonly seen in cervical cancer, there is a potential correlation between EGFR expression level and clinical response. Overexpression of p16INK4a is associated with increased overall and disease-free survival in cervical cancer, thus denoting a better prognosis. Aim of the work To investigate the expression of EGFR and p16 by immunohistochemistry as well as amplification of EGFR by FISH technique in cervical carcinoma among Egyptian females. Material and methods Seventy-seven cases of invasive cervical carcinomas were retrieved from paraffin blocks. EGFR and p16 expression were assessed by immunohistochemistry. EGFR gene amplification was assessed by FISH analysis. Results p 16 Positivity was detected in 62.3% of cases. Positive reaction to EGFR (score 3+) was detected in 20.8% of cases. The EGFR gene was not amplified in 77.9% of cases. Statistical significance was found between histological type, p16, and EGFR expression. A fair agreement was detected between EGFR expression and EGFR amplification. Poor agreement was detected between p16, EGFR expression, and EGFR amplification. Conclusion EGFR and p16 have an independent role in the development of cervical cancer in Egyptian females. EGFR overexpression and EGFR amplification represent two different genetic events; in other words, an increase in gene copy number does not mean that there will be an increase in the function of the gene as expressed by an increase in protein expression.

Dissecting the influence of cellular senescence on cell mechanics and extracellular matrix formation in vitro.

Tissue formation and healing both require cell proliferation and migration, but also extracellular matrix production and tensioning. In addition to restricting proliferation of damaged cells, increasing evidence suggests that cellular senescence also has distinct modulatory effects during wound healing and fibrosis. Yet, a direct role of senescent cells during tissue formation beyond paracrine signaling remains unknown. We here report how individual modules of the senescence program differentially influence cell mechanics and ECM expression with relevance for tissue formation. We compared DNA damage-mediated and DNA damage-independent senescence which was achieved through over-expression of either p16Ink4a or p21Cip1 cyclin-dependent kinase inhibitors in primary human skin fibroblasts. Cellular senescence modulated focal adhesion size and composition. All senescent cells exhibited increased single cell forces which led to an increase in tissue stiffness and contraction in an in vitro 3D tissue formation model selectively for p16 and p21-overexpressing cells. The mechanical component was complemented by an altered expression profile of ECM-related genes including collagens, lysyl oxidases, and MMPs. We found that particularly the lack of collagen and lysyl oxidase expression in the case of DNA damage-mediated senescence foiled their intrinsic mechanical potential. These observations highlight the active mechanical role of cellular senescence during tissue formation as well as the need to synthesize a functional ECM network capable of transferring and storing cellular forces.

A comprehensive analysis of penile cancer in the region with the highest worldwide incidence reveals new insights into the disease

BackgroundAlthough penile cancer (PC) is uncommon in developed countries, it is widespread in developing countries. The state of Maranhão (Northeast, Brazil) has the highest global incidence recorded for PC, and, despite its socioeconomic vulnerability, it has been attributed to human papillomavirus (HPV) infection. This study aimed to determine the histopathological features, the prevalence of HPV infection, and the immunohistochemical profile of PC in Maranhão.MethodsA retrospective cohort of 200 PC cases were evaluated. HPV detection was performed using nested-PCR followed by direct sequencing for genotyping. Immunohistochemistry (IHC) was performed using monoclonal antibodies anti-p16INK4a, p53, and ki-67.ResultsOur data revealed a delay of 17 months in diagnosis, a high rate of penile amputation (96.5%), and HPV infection (80.5%) in patients from Maranhão (Molecular detection). We demonstrated the high rate of HPV in PC also by histopathological and IHC analysis. Most patients presented koilocytosis (75.5%), which was associated with those reporting more than 10 different sexual partners during their lifetime (p = 0.001). IHC revealed frequent p16INK4a overexpression (26.0%) associated with basaloid (p < 0.001) and high-grade tumors (p = 0.008). Interestingly, p16 appears not to be a better prognostic factor in our disease-free survival analysis, as previously reported. We also demonstrated high ki-67 and p53 expression in a subset of cases, which was related to worse prognostic factors such as high-grade tumors, angiolymphatic and perineural invasion, and lymph node metastasis. We found a significant impact of high ki-67 (p = 0.002, log-rank) and p53 (p = 0.032, log-rank) expression on decreasing patients’ survival, as well as grade, pT, stage, pattern, and depth of invasion (p < 0.05, log-rank).ConclusionsOur data reaffirmed the high incidence of HPV infection in PC cases from Maranhão and offer new insights into potential factors that may contribute to the high PC incidence in the region. We highlighted the possible association of HPV with worse clinical prognosis factors, differently from what was observed in other regions. Furthermore, our IHC analysis reinforces p16, ki-67, and p53 expression as important diagnosis and/or prognosis biomarkers, potentially used in the clinical setting in emerging countries such as Brazil.

Prevalence of HPV Infection and p16INK4a Overexpression in Surgically Treated Laryngeal Squamous Cell Carcinoma.

Background: The mucosal high-risk (HR) human papillomavirus (HPV) is associated with oropharyngeal carcinogenesis. Aims of this study were to evaluate the prevalence of HR-HPV infection in laryngeal squamous cell carcinoma (LSCC) from different subsites, and the clinico-biological meaning of p16 overexpression. Methods: Ninety-seven LSCCs submitted to primary surgery (n = 75) or to post-irradiation salvage laryngectomy (n = 22) were evaluated for HR-HPV DNA and RNA using Luminex-based assays. p16 immunohistochemistry was performed. Results: HR-HPV DNA from HPV16 was detected in seven cases (8.75%), without significant differences between supraglottic and glottic lesions. HPV RNA was never detected. p16 overexpression correlated with HR-HPV DNA, but the kappa agreement score was poor. HPV DNA showed no impact on prognosis. p16 overexpression was associated with a better survival (OS, RFS) in primarily operated cases, while an inverse association with OS was observed in the salvage surgery group. Conclusions: HR-HPV infection appears to have a marginal role in LSCC independent of the anatomical subsite. p16 expression is deregulated in LSCC independent of HPV but displays a prognostic role in patients submitted to primary surgery. The negative predictive role of p16 overexpression in patients undergoing salvage surgery deserves more investigations for validation and elucidation of its clinical relevance.

P16ink4a overexpression ameliorates cardiac remodeling of mouse following myocardial infarction via CDK4/pRb pathway

BackgroundP16ink4a can accumulate in senescent cells and can be induced by different oncogenic stimulations. These functions make p16ink4a a biomarker of senescence and cancer. However, the exact role of p16ink4a remains unclear in cardiovascular disease. This study was aimed to investigate the role of p16ink4a in cardiac remodeling after myocardial infarction (MI). MethodsIn vivo, gain and loss of function experiments using p16ink4a overexpression and knockdown adenovirus were induced to determine the effect of p16ink4a on cardiac structure and function after MI. The in vitro effects of p16ink4a were evaluated by overexpression and knockdown adenovirus of p16ink4a on isolated neonatal mouse cardiac myocytes (NMCMs) and neonatal mouse cardiac fibroblasts (NMCFs). ResultsExpression level of p16ink4a was increased after MI and enriched in the infarction area. In vivo, overexpression of p16ink4a protected, while knockdown of p16ink4a worsened cardiac function. In vitro, p16ink4a did not influence the hypertrophy of NMCMs. Overexpression of p16ink4a inhibited the proliferation and migration of NMCFs and reduced the level of collagen I and α-SMA. Consistently, knockdown of p16ink4a in vitro displayed the opposite effects. Further mechanism studies revealed that p16ink4a affected the expression level of cyclin-dependent kinase 4 (CDK4) and phosphorylation of retinoblastoma (pRb), which could be a potential pathway in regulating cardiac remodeling after MI. ConclusionOverexpression of 16ink4a in cardiac fibroblasts can ameliorate cardiac dysfunction and attenuate pathological cardiac remodeling in mice after MI by regulating the p16ink4a/CDK4/pRb pathway.

Understanding the Pattern of Oropharyngeal Cancers from North-East Romanian Patients

Background: Human papilloma virus (HPV) is acknowledged as a risk factor for oropharyngeal squamous cellular cancers (OPSCC), of which the dominant types are tonsillar (TSCC) and base of tongue cancer (BOTSCC). Objective: To assess the role of HPV in selected OPSCC cases, from Romanian patients by sensitive and complementary molecular assays. Material and Methods: Fifty-four formalin fixed paraffin embedded (FFPE) OPSCC samples were analyzed for HPV DNA by a PCR-based bead-based multiplex-assay. Thirty-four samples were tested for HPV RNA and for overexpression of p16INK4a by immunohistochemistry. Twenty samples were evaluated by Competitive Allele-Specific Taqman PCR (CAST-PCR) for fibroblast growth factor receptor 3 protein (FGFR3) status. Results: A total of 33.3% (18/54) OPSCC samples were positive for HPV DNA. HPV16 was the most frequent type (30%, 16/54); followed by HPV18 (3.7%, 2/54); and 1 sample (1.8%) was positive for both HPV16 and 18. HPV18 E6*I was detected in a HPV18 DNA-positive oropharynx tumor. Four samples positive for HPV16 were also positive for p16INK4a. All the tested samples were negative for FGFR3. Conclusions: The increased HPV16 prevalence is in line with similar studies and is a new confirmation that HPV16 is the most prevalent type in our country; supporting the potential benefit of prophylactic vaccines. Overall, there is no concordance between DNA and any of the two other analytes that are considered being markers of HPV-driven cancers. There is a need to explore novel screening strategies that could be broadly used in the clinical routine to initiate preventive measures.

Genotype Distribution and Prevalence of Human Papillomavirus in Head and Neck Cancer Samples from Istanbul, Turkey.

Human papillomavirus (HPV)-associated tumors account for a significant proportion of head and neck squamous cell carcinomas (HNSCC) in developed countries. In recent years, there has been a rise of HPV infections associated with HNSCC, especially HPV16, which is the most commonly detected type in oral and oropharyngeal cancers. To investigate the frequency of HPV-driven HNSCC among patients living in Turkey, HPV DNA positivity and p16INK4A expression were assessed in primary tumor biopsies (n = 106). Eighteen out of one hundred and six (19%) HNSCC tumors showed p16INK4A overexpression, and 26/106 cases (24.5%) were positive for HPV DNA. Sixteen out of twenty-six samples were positive for both HPV DNA and p16INK4A staining. HPV16 could be isolated from 22/26 samples (84.6%) and was found to be the most frequently detected HPV type. This study represents the largest cohort of Turkish patients with HNSCC characterized according to HPV status and p16INK4A expression. Our data suggest that HPV16 infection, along with smoking, contribute to the development of HNSCC.

The Isothermal Amplification AmpFire Assay for Human Papillomavirus (HPV) Detection and Genotyping in Formalin-Fixed, Paraffin-Embedded Oropharyngeal Cancer Samples

Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinomas (OPSCCs) represent a distinct clinical entity compared with HPV-negative tumors with particular regard to treatment response and survival outcome. The aim of this study was to assess the AmpFire Multiplex HR-HPV tests, for the detection and genotyping of 15 high-risk (HR) HPV types in formalin-fixed, paraffin-embedded (FFPE) samples and identify HPV-driven OPSCC. DNA from 160 OPSCC FFPE specimens plus 23 samples from other head and neck primary sites was tested. Results were compared with those obtained using Linear Array HPV-DNA Genotyping Test. Linear Array and AmpFire Multiplex HR-HPV tests showed, for all samples and specifically for OPSCCs, an overall concordance agreement of 98.9% and 99.4% and a Cohen κ coefficient of 0.972 and 0.984, respectively. A concordance of 100% for HPV16 and HPV18 was observed. The overall agreement between p16INK4a overexpression and HPV detection by the AmpFire Multiplex HR-HPV assay in 145 OPSCC samples was 93.8%, with a Cohen κ coefficient of 0.848. The AmpFire HPV Tests are simple assays for detection and genotyping of HPV-DNA in OPSCC FFPE samples and can be easily implemented in the clinical practice setting for HPV-DNA detection.