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The etiological factors of squamous cell carcinomas of the head and neck have been well known for a long time. It is also well known that the incidence of oral cancer diagnosed in younger patients is on the rise. Due to the young age of these patients, the increase in the number of these cases and the fact that many of them neither smoke nor drink alcohol it has been suggested that other factors might be at play in the carcinogenesis of oral cancer. Thus, along the classic etiological factors of smoking and alcohol abuse certain molecular marker anomalies and the human papilloma virus (HPV) have emerged as potential factors. The aim of the present study is to verify the potential prognostic factors and to map the differences in biomarker expression between the young and the old patient groups. In the present study the immunohistochemical profile of samples obtained from oral squamous cell carcinomas was studied and compared with various clinico-pathological parameters. In 88 samples the expressions of p16, p53, Ki67, EGFR were studied with a tissue microarray technique under standard reaction conditions as well as the detection and typing of HPV infection with the Full Spectrum HPV DNA method. The biomarker expression profile of young patients with oral squamous cell carcinoma was compared to that of older patients (above 50). A significant difference was found between the immunohistochemical profile of the young and old patient groups in p16, Ki67 expression. The overall survival and progression free survival were influenced by p16 expression in young age., Competing Interests: MB was employed by Synlab Genoid Molecular Diagnostic Laboratory. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Csurgay, Zalatnai, Benczik, Csomó, Horváth, Lőrincz, Komlós and Németh.)

Gastric cancer (GC) is the most common gastrointestinal malignancy worldwide. However, the molecular mechanisms of the progression of GC are not fully understood. Ras-responsive element binding protein 1 (RREB1) is an oncogene in many types of cancer that is involved in various biological processes, such as DNA damage repair, cell growth and proliferation, cell differentiation, fat development, and fasting glucose balance. In this study, we demonstrate the role of RREB1 in gastric cancer. First, by immunohistochemistry staining (IHC) and bioinformatics analysis, we demonstrated the expression of RREB1 in gastric cancer and paired normal gastric tissues. Then, we established RREB1 overexpression and knockdown cell lines via lentiviral transfection and detected cell proliferation by using MTT, colony-forming, cell cycle and apoptosis assays in vitro. We demonstrated the effect of RREB1 on cell proliferation in vivo by using a subcutaneous xenograft tumor model in nude mice. Finally, by using Western blotting and IHC, we demonstrated the possible mechanism by which RREB1 affects cell proliferation. The IHC and bioinformatics analyses demonstrated that RREB1 was highly expressed in gastric cancer and showed that RREB1-expressing patients had a larger tumor size and more lymphovascular invasion than RREB1-negative patients. Knockdown of RREB1 inhibited cell proliferation in vivo and in vitro. Knockdown of RREB1 enhanced p16 expression in vivo and in vitro, and p16 expression was negatively related to RREB1 in gastric cancer tissue. RREB1 was highly expressed in gastric cancer, and knockdown of RREB1 inhibited cell proliferation via enhanced p16 expression.

Human papillomavirus (HPV)-induced anal intraepithelial neoplasia (AIN, graded 1-3) is highly prevalent in HIV-positive (HIV+) men who have sex with men (MSM), but only a minority of lesions progresses to cancer. Our study aimed to characterise comprehensively anal tissue samples from a cross-sectional series (n = 104) of HIV+ MSM and longitudinal series (n = 40) of AIN2/3 progressing to cancer using different biomarkers. The cross-sectional series consisted of 8 normal, 26 AIN1, 45 AIN2, 15 AIN3 and 10 anal squamous cell carcinoma. Tissue sections were immunohistochemically (IHC) stained for p16 (viral transformation marker), Ki-67 (cellular proliferation marker) and HPV-E4 (viral production marker). We evaluated the expression of IHC markers and compared it with DNA methylation, a marker for malignant transformation. E4 positivity decreased, whereas p16 and Ki-67 scores and methylation marker positivity increased (P values < .001) with increasing severity of anal lesions. Within AIN2, a heterogeneous biomarker pattern was observed concerning E4, p16 and methylation status, reflecting the biological heterogeneity of these lesions. In the longitudinal series, all AIN2/3 and carcinomas showed high p16 and Ki-67 expression, strong methylation positivity and occasional E4 positivity. We earlier showed that high methylation levels are associated with progression to cancer. The observed E4 expression in some AIN2/3 during the course of progression to cancer and absence of E4 in a considerable number of AIN1 lesions make the potential clinical significance of E4 expression difficult to interpret. Our data show that IHC biomarkers can help to characterise AIN; however, their prognostic value for cancer risk stratification, next to objective methylation analysis, appears to be limited., (© 2021 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

We defined the time course of ionizing radiation-induced senescence in lung compared to bone marrow of p16+/LUC mice in which the senescence-induced biomarker (p16) is linked to a luciferase reporter gene. Periodic in situ imaging revealed increased luciferase activity in the lungs of 20 Gy thoracic irradiated, but not 8 Gy total-body irradiated (TBI) mice beginning at day 75 and increasing to day 170. In serial sections of explanted lungs, senescent cells appeared in the same areas as did fibrosis in the 20 Gy thoracic irradiated, but not the 8 Gy TBI group. Lungs from 8 Gy TBI mice at one year did show increased RNA levels for p16, p21, p19 and TGF-β. Individual senescent cells in 20 Gy irradiated mouse lung included those with epithelial, endothelial, fibroblast and hematopoietic cell biomarkers. Rare senescent cells in the lungs of 8 Gy TBI mice at one year were of endothelial phenotype. Long-term bone marrow cultures (LTBMCs) were established at either day 60 or one year after 8 Gy TBI. In freshly removed marrow at both times after irradiation, there were increased senescent cells. In LTBMCs, there were increased senescent cells in both weekly harvested single cells and in colonies of multilineage hematopoietic progenitor cells producing CFU-GEMM (colony forming unit-granulocyte, erythrocyte, monocyte/macrophage, mega-karyocyte) that were formed in secondary cultures when these single cells were plated in semisolid media. LTBMCs from TBI mice produced fewer CFU-GEMM; however, the relative percentage of senescent cell-containing colonies was increased as measured by both p16-luciferase and β-galactosidase. Therefore, 20 Gy thoracic radiation, as well as 8 Gy TBI, induces senescent cells in the lungs. With bone marrow, 8 Gy TBI induced senescence in both hematopoietic cells and in colony-forming progenitors. The p16+/LUC mouse strain provides a valuable system in which to compare the kinetics of radiation-induced senescence between organs in vivo, and to evaluate the potential role of senescent cells in irradiation pulmonary fibrosis., (©2021 by Radiation Research Society. All rights of reproduction in any form reserved.)

Aims: Atypical polypoid adenomyoma (APAM) is an uncommon uterine lesion composed of complex endometrioid glands with frequent squamous morular metaplasia and fibromuscular stroma. On endometrial curettage, biopsy or polypectomy specimens, the admixture of endometrioid glands and smooth muscle raises the differential diagnosis of myoinvasive endometrioid carcinoma. Reproductive-age APAM patients may opt for fertility preservation, whereas myoinvasive carcinoma is treated surgically. One previous study reported an incidental finding that the stroma of APAM, in contrast to that of other polypoid lesions, was SATB2-positive. APAM has also been reported to show increased stromal p16 staining. We aimed to assess whether SATB2 and p16 are useful stains for the distinction of APAM from myoinvasive carcinoma and benign adenomyomatous polyps., Methods and Results: Cases of 'atypical polypoid adenomyoma' (n = 32), 'adenomyomatous polyp' (n = 39) and 'myoinvasive endometrioid carcinoma' (n = 30) were identified. Morphological features were assessed, along with the intensity and extent of SATB2 and p16 staining in the stromal component of each lesion. SATB2 expression was seen in the stromal components of 30 of 32 (94%) APAMs, versus none of 39 (0%) benign adenomyomatous polyps and five of 30 (17%) myoinvasive endometrioid carcinomas. Stromal p16 expression was seen in 31 of 31 (100%) APAMs, versus 20 of 39 (51%) benign adenomyomatous polyps and 12 of 30 (40%) myoinvasive endometrioid carcinomas., Conclusions: Patchy to diffuse SATB2 and block-type p16 staining of fibromuscular stroma separating atypical endometrioid glands is more consistent with APAM than with myoinvasive endometrioid carcinoma. These stains are potentially useful adjuncts to careful morphological evaluation of endometrial biopsies/curettings., (© 2021 John Wiley & Sons Ltd.)

Oncogene-induced senescence provides a barrier against malignant transformation. However, it can also promote cancer through the secretion of a plethora of factors released by senescent cells, called the senescence associated secretory phenotype (SASP). We have previously shown that in proliferating cells, nuclear lncRNA MIR31HG inhibits p16/CDKN2A expression through interaction with polycomb repressor complexes and that during BRAF-induced senescence, MIR31HG is overexpressed and translocates to the cytoplasm. Here, we show that MIR31HG regulates the expression and secretion of a subset of SASP components during BRAF-induced senescence. The SASP secreted from senescent cells depleted for MIR31HG fails to induce paracrine invasion without affecting the growth inhibitory effect. Mechanistically, MIR31HG interacts with YBX1 facilitating its phosphorylation at serine 102 (p-YBX1 S102 ) by the kinase RSK. p-YBX1 S102 induces IL1A translation which activates the transcription of the other SASP mRNAs. Our results suggest a dual role for MIR31HG in senescence depending on its localization and points to the lncRNA as a potential therapeutic target in the treatment of senescence-related pathologies.

The standard of age-related glomerulosclerosis is unclear. Both signal transducer and activator of transcription 3 (STAT3) and autophagy are involved in age-related kidney disease. Therefore, we aimed to explore the standard, as well as the potential mechanism(s). A total of 44 patients who underwent radical nephrectomy were enrolled. Pearson analysis was performed to investigate the parameters with ages. The patients were divided into the young- and aged-kidney groups. Kidney morphological changes were evaluated by histology staining, senescence was evaluated by senescence-associated-β-galactosidase (SA-β-gal) staining, and autophagosome was measured by transmission electron microscopy. Moreover, Western blot and/or immunohistochemistry were accomplished to assess the expression of p16, STAT3, and glycoprotein130 (GP130) and autophagy-related proteins. Furthermore, human glomerular mesangial cells were administrated with tocilizumab (TCZ) and/or IL-6, and then the above indexes were tested again. Sclerotic glomerular density and glomerular sclerosis rate were significantly higher in individuals more than 40 years old, and they were strongly correlated with ages. Moreover, the expression of p16, STAT3, GP130, and p62 was significantly increased, while LC3II and autophagosome were statistically decreased in the aged-kidney. Glomeruli were hardly to stain with SA-β-gal. For the in vitro experiments, we observed that IL-6 significantly increased p16, STAT3, GP130, and p62, induced higher SA-β-gal staining, while downregulated LC3II and autophagosome. Furthermore, TCZ statistically reversed the effects of IL-6 on the above expression of proteins. Glomerular sclerosis rate might be one standard for natural renal aging, and IL-6/STAT3-mediated autophagy may participate in the development of age-related glomerulosclerosis., (© 2020 Wiley Periodicals LLC.)

Follicular dendritic cell sarcoma (FDCS) is a rare entity which can share morphologic features with non-keratinizing squamous cell carcinoma. Recent reports suggest that up to half of FDCSs show immunohistochemical positivity for p16 (Zhang et al., in Hum Pathol 66:40-47, 2017), a stain that is conventionally used in the risk stratification of oropharyngeal squamous cell carcinoma (OPSCC). Herein, we report a case of p16-positive FDCS with clinical and histomorphologic overlap with human papilloma virus (HPV)-related OPSCC.

Background: Immortalization of primary prostate epithelial cells (PrEC) with just hTERT expression is particularly inefficient in the absence of DNA tumor viral proteins or p16 INK4A knockdown., Materials and Methods: Here, we describe the establishment of immortalized normal prostate epithelial cell line models using CRISPR technology to inactivate the CDKN2A locus concomitantly with ectopic expression of an hTERT transgene., Results: Using this approach, we have obtained immortal cell clones that exhibit fundamental characteristics of normal cells, including diploid genomes, near normal karyotypes, normal p53 and pRB cell responses, the ability to form non-invasive spheroids, and a non-transformed phenotype. Based on marker expression, these clones are of basal cell origin., Conclusions: Use of this approach resulted in the immortalization of independent clones of PrEC that retained normal characteristics, were stable, and non-transformed. Thus, this approach could be used for the immortalization of normal primary prostate cells. This technique could also be useful for establishing cell lines from prostate tumor tissues of different tumor grades and/or from patients of diverse ethnicities to generate cell line models that facilitate the study of the molecular basis of disease disparity.

Background: A possible oncogenic role of human papillomavirus (HPV) in head and neck cancers (mainly oropharynx tumors) has been suggested. This significant association has been considered true for oropharynx tumors; however, the association between HPV infection and laryngeal carcinomas is yet to be established. The aim of this study was to evaluate the relationship between p16 expression and long-term overall, disease-free, and disease-specific survival (OS, DF, and DSS, respectively) in patients surgically treated for laryngeal carcinoma., Materials and Methods: Seventy-four previously untreated laryngeal carcinoma patients who underwent surgical treatment were considered for this retrospective study. The tissue specimens were processed for immunohistochemical p16 protein (surrogate HPV marker) detection., Results: Survival analysis of the p16 expression of the primary tumor showed that the 5-year OS rates were 90% and 29.7% for the p16-positive and negative groups, respectively ( P = .003). The 5-year DFS and DSS also differed between both groups ( P < .001), whereas the 5-year DSS seemed to be related to tumor/lymph node classification and p16 expression. However, only p16 expression was identified as an independent prognostic factor associated with OS and DSS., Conclusions: Surgically treated p16-positive laryngeal cancer patients may represent a subset of patients with a better prognosis than their p16-negative counterparts.

Cell lines used in bioproduction are routinely engineered to improve their production efficiency. Numerous strategies, such as random mutagenesis, RNA interference screens, and transcriptome analyses have been employed to identify effective engineering targets. A genome-wide small interfering RNA screen previously identified the CASP8AP2 gene as a potential engineering target for improved expression of recombinant protein in the HEK293 cell line. Here, we validate the CASP8AP2 gene as an engineering target in HEK293 cells by knocking it out using CRISPR/Cas9 genome editing and assessing the effect of its knockout on recombinant protein expression, cell growth, cell viability, and overall gene expression. HEK293 cells lacking CASP8AP2 showed a seven-fold increase in specific expression of recombinant luciferase and a 2.5-fold increase in specific expression of recombinant SEAP, without significantly affecting cell growth and viability. Transcriptome analysis revealed that the deregulation of the cell cycle, specifically the upregulation of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene, contributed to the improvement in recombinant protein expression in CASP8AP2 deficient cells. The results validate the CASP8AP2 gene is a viable engineering target for improved recombinant protein expression in the HEK293 cell line., (© 2020 Wiley Periodicals LLC.)

Trop2 is an intracellular calcium signal transducer and a prognostic biomarker in many cancers. P16 is a cell cycle gene that negatively regulates cell proliferation and division in most human cancers. Oral squamous cell carcinoma (OSCC) is a common malignant tumor subgroup of head and neck squamous cell carcinoma worldwide. Both Ca 2+ -dependent and cell cycle signaling pathways play vital roles in OSCC, although the molecular mechanisms remain to be elucidated. We aimed to examine the function of Trop2 and P16 in regulating intracellular calcium ions and the cell cycle in OSCC cell lines. Furtherly, the mRNA and protein expression levels of Trop2 and P16 in OSCC tissue samples were assessed, and their function was evaluated as potential clinical prognostic biomarkers. Trop2 promoted intracellular calcium ion release in OSCC and induced S phase of the cell cycle. Moreover, Trop2-mediated Ca 2+ inhibited P16 expression through the AMP-activated protein kinase pathway in OSCC. Interestingly, P16 overexpression could not reverse these phenomena in vitro. We also demonstrated that human OSCC tissues showed high Trop2 mRNA and protein expression, and Trop2+/P16- expression is an independent prognostic marker for OSCC patients. Our data suggest that Trop2+/P16- may be a valuable prognostic marker for OSCC and that Trop2 inhibits P16 expression and induces S phase by promoting intracellular calcium release in OSCC., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Osteonecrosis is a harmful musculoskeletal disease. We aim to detect the effects of icariin (ICA) in MC3T3-E1 cell. MC3T3-E1 cell was pretreated with ICA and was subjected to hypoxia stimuli. The tumor-associated long noncoding RNA expressed on chromosome 2 (TALNEC2) overexpression or silencing vectors (pTALNEC2 or si-TALNEC2) was utilized for MC3T3-E1 cell transfection. Viability and apoptosis rate were individually tested by cell counting kit-8 and Annexin V-fluorescein isothiocyanate/propidium iodide kit untied with flow cytometry. The alkaline phosphatase activity (ALP) activity was tested through ALP assay. The quantitative reverse transcription PCR or Western blot was performed for elements detection at the RNA or protein level. Hypoxia treatment induced viability inhibition and CyclinD1 reduction, but elevation of p53 and p16. It also promoted apoptosis by increasing apoptotic cells, Bax, and cleaved-poly ADP-ribose polymerase but decreasing Bcl-2. Also, hypoxia stimuli restrained ALP activity, and osteopontin, osteocalcin, and Runt-related transcription factor 2 expression. Those effects caused by hypoxia stimuli were all reversed by ICA. TALNEC2 was downregulated by ICA, whose impacts were subsequently abolished by pTALNEC2. Silencing TALNEC2 displayed similar effects with ICA. But the apoptosis was not affected by si-TALNEC2. ICA blocked ste20-related proline/alanine-rich kinase/c-Jun N-terminal kinase (SPAK/JNK) but triggered phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway in MC3T3-E1 cell by suppressing TALNEC2. ICA relieved hypoxia-stimulated damage by restraining TALNEC2 through blocking SPAK/JNK and triggering PI3K/AKT/mTOR in the MC3T3-E1 cell., (© 2019 International Union of Biochemistry and Molecular Biology, Inc.)

Objectives: Tobacco and alcohol are the main etiological factors common to laryngeal cancers. However, the Human Papilloma Virus (HPV) constitutes an alternative risk factor according to several studies. In Tunisia, despite the annual increasing incidence of laryngeal squamous cell carcinoma (LSCC), the prevalence and prognostic significance of HPV have never been explored.In this study, we sought to highlight HPV DNA in 70 biopsies of laryngeal cancer, and to analyze the status of HPV infection in association with p53, p16, survivin, and IGF-1R expressions., Methods: HPV high risk (HPV HR) DNA was detected in tumors by in situ hybridization. However, the expression of p53, p16, survivin and IGF-1R were stained by immunohistochemistry test. The correlations of HPV status with clinicopathological parameters, overall survival, disease-free survival and proteins expressions were statistically evaluated., Results: HPV HR DNA was detected in 39 out of 70 (55.71%) laryngeal tumors. HPV+ patients have a better overall survival ( P  = .081) and long disease-free-survival ( P  = .016) with a low rate of recurrence ( P  = .006) than HPV - patients. No significant correlations were found between HPV HR status and clinicopathological parameters (all P  > .005). Moreover, HPV+ tumors were not associated with expression of p53, p16 and survivin. However, HPV HR status correlates with weak to moderate IGF-1R expression ( P =  .043)., Conclusion: The substantial detection of HPV HR in LSCC tumors suggest that this virus plays an important part in laryngeal cancer in Tunisia. It is a good prognostic factor. In addition, HPV infection could act to block the pathway of IGF-1R expression.

Triple-negative breast cancers (TNBCs) represent approximately 12-17% of all breast cancers and have distinctively aggressive clinical courses. Because routine biomarkers for breast cancer do not apply for TNBCs, it is essential to find novel prognostic markers and potential targets for therapeutic agents. p16 and SOX10 are emerging biomarkers with relatively unexplored expressions in TNBCs. We present an analysis of the expression of p16 and SOX10 in combination with that of androgen receptor (AR) and cytokeratin (CK) 5/6 in TNBCs. In addition, we used tissue microarrays (TMAs) to compare frequencies of p16 and SOX10 between TNBCs and non-TNBCs. Fifty-six TNBC samples with clinical data were stained immunohistochemically with p16, SOX10, AR, and CK5/6. Fifty-four cases (96.4%) were invasive ductal carcinoma, not otherwise specified, and 46 cases (82.1%) were Nottingham histologic grade 3. The majority of TNBC cases were positive for p16 (n = 44; 78.6%) and SOX10 (n = 48; 85.7%). AR was positive in 15 cases (26.8%). CK5/6 was positive in 24 cases (42.9%), which were classified as basal-like breast cancer (BLBC) subtype. The frequencies of p16 and SOX10 expression in BLBC and non-BLBC subtypes did not reveal significant statistical difference in a separate analysis. Using archived TNBC and non-TNBC TMAs, we observed that 56% of TNBC cases were positive for p16 compared with 16% of non-TNBC cases (p-value <0.0001). SOX10 was positive in 80% of TNBC cases compared with 35% of non-TNBC cases (p-value <0.0001). A significant correlation was observed between p16 and SOX10 coexpression in TNBC cases (n = 56/80, p = 0.02) but not in non-TNBC cases (n = 23/348; p = 0.626). In conclusion, p16 and SOX10 are frequently expressed in TNBC, regardless of CK5/6 expression. Furthermore, p16 and SOX10 are often coexpressed in TNBCs compared with non-TNBCs., (Copyright © 2020 Elsevier Inc. All rights reserved.)

White adipose tissue browning has emerged as a putative therapy of obesity, and studies in mice have shown that Cdkn2a is implicated in white-to-brown transition. However, the role of Cdkn2a product p16 has been never studied in human brown fat tissue. The aim of the study is to investigate the expression of p16 in normal brown fat and in hibernoma, a lipoma containing brown fat-like adipocytes. Ten normal brown fat tissues and 5 hibernomas were immunohistochemically studied for p16 expression. Nearby white adipose tissue was used for comparison. All brown fat and hibernomas specimens express p16 in a cytoplasmic manner. Neighboring white adipose tissue is negative for p16 expression. Thus, cytoplasmic p16 may be associated with fat tissue browning.

Objective: The incidence of oropharyngeal squamous cell carcinoma (OPSCC), especially human papillomavirus (HPV)-associated, is increasing worldwide. Immunotherapy become available for patients with carcinomas in the head and neck region, however without ideal biomarker. Markers like PD-L1 vary in the clone of the antibody used, and the method of evaluation. Adequate and reliable immune cells characterization and evaluation is still not found. Furthermore, studies analyzing representativeness of different tissue samples are scarce. We analyzed small biopsy, lymph node (LN) metastasis and resected OPSCC, in regards of tumor infiltrating lymphocyte (TIL) density, PD-L1 and p16 expression., Material and Methods: Patients with OPSCC diagnosed from 2000 to 2016, with small biopsy, resection specimen and LN metastasis samples were selected. We analyzed TILs on hematoxylin-eosin stain, and PD-L1 and p16 expression in tumor cells. Concordance between different tumor locations was evaluated., Results: 93 patients, with 65 small biopsies, 72 resection specimens, and 70 LN metastases were included. TILs, p16 and PD-L1 demonstrated very high concordance. Additionally, PD-L1 expression in the small biopsies was more representative of the PD-L1 expression in the resection specimens, than the LN samples., Conclusion: TILs density can be reliably assessed using hematoxylin-eosin stain with high concordance between the small biopsy, resection specimen and LN metastasis. Evaluation of concordance of p16 expression is very high, nevertheless some cases might be misdiagnosed on a small biopsy or lymph node metastasis. Evaluation of PD-L1 expression is very reliable on the biopsy specimen. Different PD-L1 clones and methods of evaluation still remain to be addressed., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Introduction: Human papillomavirus (HPV) infection is associated with the development of anogenital and head and neck cancers. In recent years a potential role of HPV in colorectal cancer (CRC) has been suggested., Objective: To investigate the presence of HPV in colorectal carcinomas and to study the role of p16INK4a as a marker of transcriptionally active HPV infection. In addition, to investigate the correlation between these findings and the CRC prognostic factors., Methods: Case control study with 92 cases of colorectal cancers, 75 controls of normal tissue adjacent to the tumor, and 30 controls of precursor lesions, including polyps and colorectal adenomas. Paraffinized samples were used, HPV detection and genotyping were performed by PCR and reverse hybridization by using the INNO LIPA kit, with SPF10 plus primers. The expression of the p16INK4a protein was investigated using immunohistochemistry. Data analysis was performed using descriptive, univariate statistics and survival curves were calculated by using the Kaplan Meier and log-rank method., Results: HPV was detected in 13% of the cases and the most prevalent genotype was HPV 16. HPV DNA was not detected in either control groups. The high expression of p16INK4a was observed in 30% of the cases, but it was not associated to the presence of HPV. The overall survival was 53.3% and was influenced by prognostic factors such as later stage, lymph node and distant metastasis., Conclusions: Based on these results, HPV is unlikely to be involved in colorectal carcinogenesis and p16INK4a expression is not a relevant marker of transcriptionally active HPV infection in CRC., Competing Interests: The authors have declared that no competing interests exist.

Liver progenitor cells (LPCs) contribute to liver regeneration during chronic damage and are implicated as cells of origin for liver cancers including hepatocellular carcinoma (HCC). The CDKN2A locus, which encodes the tumor suppressors alternate reading frame protein (ARF) and INK4A, was identified as one of the most frequently altered genes in HCC. This study demonstrates that inactivation of CDKN2A enhances tumorigenic transformation of LPCs. The level of ARF and INK4A expression was determined in a panel of transformed and nontransformed wild-type LPC lines. Moreover, the transforming potential of LPCs with inactivated CDKN2A was shown to be enhanced in LPCs derived from Arf -/- and CDKN2A fl/fl mice and in wild-type LPCs following CRISPR-Cas9 suppression of CDKN2A . ARF and INK4A abundance is consistently reduced or ablated following LPC transformation. Arf -/- and CDKN2A -/- LPCs displayed hallmarks of transformation such as anchorage-independent and more rapid growth than control LPC lines with unaltered CDKN2A . Transformation was not immediate, suggesting that the loss of CDKN2A alone is insufficient. Further analysis revealed decreased p21 expression as well as reduced epithelial markers and increased mesenchymal markers, indicative of epithelial-to-mesenchymal transition, following inactivation of the CDKN2A gene were required for tumorigenic transformation. Loss of ARF and INK4A enhances the propensity of LPCs to undergo a tumorigenic transformation. As LPCs represent a cancer stem cell candidate, identifying CDKN2A as a driver of LPC transformation highlights ARF and INK4A as viable prognostic markers and therapeutic targets for HCC.

Objectives: To review the significance of MDM2 and cyclin D1 expression and loss of p16 expression in malignant and borderline Brenner tumors (BTs) of the ovary., Methods: We describe 2 new cases of ovarian BT, 1 malignant and 1 borderline. We studied MDM2, p16, and cyclin D1 expression by immunohistochemistry in the benign, borderline, and malignant components of these 2 cases and in 5 additional cases of benign BT. We also reviewed and summarized the literature on the clinical, immunohistochemical and molecular characteristics of borderline and malignant BTs (BdBTs and MBTs)., Results: Nuclear expression of MDM2 was seen only in the MBT. Loss of p16 expression was seen in both BdBT and MBT. Cyclin D1 expression was in proportion to the degree of malignancy. Amplification of MDM2, loss of CDKN2A (p16-encoding gene), and amplification of CCND1 (cyclin D1-encoding gene) were confirmed by commercial next-generation sequencing in the case of MBT., Conclusions: We are the first to report immunohistochemical expression of MDM2 in an MBT. Amplification of MDM2 and loss of p16 expression may have a role in malignant transformation of BT., (© American Society for Clinical Pathology, 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Vulvar squamous cell carcinoma can be divided by human papillomaviruses (HPV) status into two distinct clinicopathological and molecular entities. New agents targeting the tumor surface expression of programmed cell death-1/programmed cell death-ligand-1 are becoming a therapeutic option in an increasing number of carcinomas. We evaluate CD274 (PD-L1), CDKN2A (p16), tumor protein p53 (TP53), and epidermal growth factor receptor (EGFR) immunoexpression in primary tumors, recurrences and lymph node metastases and its correlations with prognosis and HPV status. We report 93 cases of vulvar squamous cell carcinoma diagnosed between 2002 and 2016 with the description of their clinicopathological features and prognosis data. Immunohistochemistry for CD274, CDKN2A, TP53, and EGFR was performed on tissue microarrays collecting from primary tumor, recurrences and lymph node metastasis. Kaplan-Meier estimator and multivariable Cox regression analysis controlling for FIGO stage and age were used. Patients who underwent surgery had a superior overall survival (HR = 0.51, 95% CI = 0.26-0.99 p = 0.04). Lymph node metastasis size ≥5 mm was associated with an inferior overall survival (HR = 1.88, 95% CI = 1.22-2.92 p = 0.004). CDKN2A expression was correlated with an inferior rate of recurrent disease (p = 0.02). In high-risk HPV DNA+ vulvar squamous cell carcinomas patients with CDKN2A- carcinomas showed a significantly worse overall survival than women with CDKN2A+ tumors (56% vs.100%, p = 0.003). TP53 expression was associated with an increased rate of recurrent disease (p = 0.0005). CD274 expression was associated with lymph node metastasis (p = 0.04). In 16 patients the CD274, CDKN2A, TP53, and EGFR expression changed between primary tumors, recurrences and lymph node metastases during tumor progression. In conclusion, a significant percentage of vulvar squamous cell carcinoma has a heterogeneous biomarker expression during tumor progression. We highlight the importance of some of these markers to be used as prognostic biomarkers. This data brings new light to future treatment using targeted therapy to EGFR or CD274 to include retesting such biomarkers in recurrence and lymph nodes metastases.

The tumor-suppressor protein p16 is paradoxically overexpressed in cervical cancer (CC). Despite its potential as a biomarker, its clinical value and the reasons for its failure in tumor suppression remain unclear. Our purpose was to determine p16 clinical and biological significance in CC. p16 expression pattern was examined by immunohistochemistry in 78 CC cases (high-grade squamous intraepithelial lesions (HSILs) and squamous cell carcinomas of the cervix -SCCCs). CC cell proliferation and invasion were monitored by real-time cell analysis and Transwell ® invasion assay, respectively. Cytoplasmic p16 interactors were identified from immunoprecipitated extracts by liquid chromatography-tandem mass spectrometry, and colocalization was confirmed by double-immunofluorescence. We observed that SCCCs showed significantly more cytoplasmic than nuclear p16 expression than HSILs. Importantly, nuclear p16 absence significantly predicted poor outcome in SCCC patients irrespective of other clinical parameters. Moreover, we demonstrated that cytoplasmic p16 interacted with CDK4 and other unreported proteins, such as BANF1, AKAP8 and AGTRAP, which could sequester p16 to avoid nuclear translocation, and then, impair its anti-tumor function. Our results suggest that the absence of nuclear p16 could be a diagnostic biomarker between HSIL and SCCC, and an independent prognostic biomarker in SCCC; and explain why p16 overexpression fails to stop CC growth.

Senescent cells accumulate in tissues during aging or pathological settings. The semi-genetic or pharmacological targeting of senescent cells revealed that cellular senescence underlies many aspects of the aging-associated phenotype and diseases. We previously reported that cellular senescence contributes to aging- and disease-associated pulmonary dysfunction. We herein report that the elimination of Arf -expressing cells ameliorates cigarette smoke-induced lung pathologies in mice. Cigarette smoke induced the expression of Ink4a and Arf in lung tissue with concomitant increases in lung tissue compliance and alveolar airspace. The elimination of Arf -expressing cells prior to cigarette smoke exposure protected against these changes. Furthermore, the administration of cigarette smoke extract lead to pulmonary dysfunction, which was ameliorated by subsequent senescent cell elimination. Collectively, these results suggest that senescent cells are a potential therapeutic target for cigarette smoking-associated lung disease.

Tumor suppressor genes in the CDKN2A/B locus ( p15INK4b , p16INK4a , and p14ARF ) function as biological barriers to transformation and are the most frequently silenced or deleted genes in human cancers. This gene silencing frequently occurs due to DNA methylation of the promoter regions, although the underlying mechanism is currently unknown. We present evidence that methylation of p16INK4a promoter is associated with DNA damage caused by interference between transcription and replication processes. Inhibition of replication or transcription significantly reduces the DNA damage and CpGs methylation of the p16INK4a promoter. We conclude that de novo methylation of the promoter regions is dependent on local DNA damage. DNA methylation reduces the expression of p16INK4a and ultimately removes this barrier to oncogene-induced senescence.

Aim: Programmed death-ligand 1 (PD-L1) has become a widely used predictive biomarker for therapy with checkpoint inhibitors in a variety of cancers. Here, we studied the expression of PD-L1 in squamous cell carcinomas of the vulva (SCCV) with regard to HPV status via its surrogate marker p16. Additionally, the status of PD-L1 and p16 were analyzed for prognostic information and potential correlation to tumor-infiltrating lymphocytes (TILs)., Methods: PD-L1 was analyzed in 128 cases of SCCV using the tumor proportion score (TPS), the immune cell score (ICS) and the combined positive score (CPS). Cases were immunostained for p16 and analyzed for stromal TILs. PD-L1, p16, and TILs were compared to clinico-pathological parameters and patient's survival., Results: TPS ≥ 50% and CPS ≥ 50 were correlated to a worse grading (p = 0.028 and p = 0.031), but not to FIGO-stage. CPS ≥ 50 was associated to a worse prognosis with overall survival (p = 0.021) but was not correlated to the progression-free survival. P16-positivity was correlated to a longer progression-free survival (p = 0.006) and overall survival (p = 0.023). PD-L1 expression was independent from p16 status. TILs ≥ 50% were present in 24% of the cases and were strongly correlated to PD-L1 (TPS p = 0.02, ICS p < 0.001, CPS p = 0.001)., Conclusion: Our data demonstrate that PD-L1 expression is frequent in SCCV and independent from p16 status. High PD-L1 expression was associated with an unfavorable outcome whereas p16-positivity turned out to be an independent positive prognostic factor.

Aims: Previous studies have reported the presence of high-risk human papillomavirus (HR-HPV) in a subset of dysplastic oral epithelial lesions. Many cases show a histological spectrum of atypia similar to that seen in non-human papillomavirus (HPV) severe epithelial dysplasia, but some studies have suggested that HPV status can be inferred on the basis of histological features. We aimed to assess the utility of such histological features and p16 as surrogate markers of HPV infection in a retrospective cohort of 33 cases of severe epithelial dysplasia, with matched clinicopathological data and histological features., Methods and Results: Tissue sections were assessed for the expression of p16, minichromosome maintenance 2, HPV E4 and HPV L1 by the use of immunohistochemistry. HPV16/18 E6 and E7 expression was assessed by the use of RNA in-situ hybridisation (RNAScope). In the cohort, 18.2% of cases (6/33) were HR-HPV-positive, with no age/gender differences between the HPV-positive and HPV-negative groups. HPV E4 and HPV L1 were expressed in surface keratinocytes in four of six (66%) HPV-positive cases, indicative of productive HPV infection. Lack of p16 expression was predictive of HPV-negative status, but sensitivity and specificity varied according to the cut-off. Histologically, the presence of karyorrhectic nuclei and abnormal mitotic figures was higher in HPV-positive lesions (P < 0.05), but the predictive specificity and sensitivity were suboptimal (sensitivity, 0.75; specificity, 0.52)., Conclusions: This study demonstrates, for the first time, that a minority of severely dysplastic oral lesions harbour productive, biologically relevant HPV infection. Consideration should be given to the specific assessment of HPV status in severe epithelial dysplasia cases, as both p16 status and the presence of karyorrhectic cells are poor predictive markers of HPV status., (© 2019 John Wiley & Sons Ltd.)

P16 immunohistochemistry has been widely used in facilitating the diagnosis of human papillomavirus (HPV)-related usual type vulvar intraepithelial neoplasm. However, studies of p16 expression in primary vulvar extramammary Paget disease (EMPD) are limited. We assessed the p16 expression by immunohistochemistry in 40 cases of primary vulvar EMPD, including 34 cases of intraepithelial vulvar EMPD and 6 cases of invasive vulvar EMPD and correlated p16 expression patterns with disease progression. Overall, p16 expression was present in 36 cases (90%), including 20 cases (50%) with focal staining pattern and 16 cases (40%) with diffuse staining pattern. All 20 cases with focal p16 staining pattern were intraepithelial vulvar EMPD. Diffuse p16 staining pattern was present in 10/30 cases (33.3%) of intraepithelial EMPD and in 6/6 cases (100%) with invasive vulvar EMPD. Negative p16 staining was present in four intraepithelial EMPD cases. Using a highly sensitive RNA in situ hybridization method, we did not detect high-risk HPV in the selected 10 cases with diffuse p16 staining pattern, including 6 cases of intraepithelial EMPD and 4 cases of invasive EMPD. We also observed that intraepithelial EMPD had predominantly cytoplasmic p16 immunoreactivity, whereas nuclear p16 immunoreactivity was mainly seen in invasive EMPD components. Our study demonstrated that the p16 positive immunostaining was seen in the majority of primary vulvar EMPD which is not related to HPV infection. Therefore, knowing the overlapping p16 immunostaining patterns in vulvar EMPD and usual type vulvar intraepithelial neoplasm is important to render the correct diagnosis.

Purpose: To investigate the overexpression of genes in sebaceous gland carcinoma (SGC) of the eyelid compared to sebaceous adenoma of the eyelid in order to elucidate the molecular mechanism underlying pathogenesis., Methods: We performed histopathological examination of eyelid tissues surgically removed from four patients diagnosed with SGC (cases 1-3) and sebaceous adenoma (case 4) of the eyelid. Next, we performed global gene expression analysis of surgical tissue samples using a GeneChip ® system and the Ingenuity Pathways Knowledge Base. The results of the GeneChip ® analysis were explored with quantitative real-time polymerase chain reaction (qRT-PCR) analysis., Results: In the SGC samples, we found that 211, 199, and 199 genes, respectively, showed ≥ 2.0-fold higher expression than those in the sebaceous adenoma sample (case 4); 194 genes were common to all three SGC samples. For the 194 genes with upregulated expression, functional category analysis showed that SGC of the eyelid employed a unique gene network, including cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase 1 (CDK1), and cyclin E1 (CCNE1), which are related to cell cycle progression, incidence of tumor, and cell viability. Furthermore, qRT-PCR analysis showed that the expression levels of CDKN2A, CDK1, and CCNE1 were significantly upregulated in all SGC cases compared to those in the sebaceous adenoma case. These data were similar to the results of microarray analysis., Conclusion: Overexpression of cell cycle-related genes CDKN2A, CDK1, CCNE1, and their gene network may help elucidate the pathogenic pathway of SGC of the eyelid at the molecular level.

Cancer therapies that target a single protein or pathway may be limited by their specificity, thus missing key players that control cellular proliferation and contributing to the failure of the treatment. We propose that approaches to cancer therapy that hit multiple targets would limit the chances of escape. To this end, we have developed a bicistronic adenoviral vector encoding both the CDKN2A and p53 tumor suppressor genes. The bicistronic vector, AdCDKN2A-I-p53, supports the translation of both gene products from a single transcript, assuring that all transduced cells will express both proteins. We show that combined, but not single, gene transfer results in markedly reduced proliferation and increased cell death correlated with reduced levels of phosphorylated pRB, induction of CDKN1A and caspase 3 activity, yet avoiding the induction of senescence. Using isogenic cell lines, we show that these effects were not impeded by the presence of mutant p53. In a mouse model of in situ gene therapy, a single intratumoral treatment with the bicistronic vector conferred markedly inhibited tumor progression while the treatment with either CDKN2A or p53 alone only partially controlled tumor growth. Histologic analysis revealed widespread transduction, yet reduced proliferation and increased cell death was associated only with the simultaneous transfer of CDKN2A and p53. We propose that restoration of two of the most frequently altered genes in human cancer, mediated by AdCDKN2A-I-p53, is beneficial since multiple targets are reached, thus increasing the efficacy of the treatment.

As p53-binding protein 1 (53BP1) localizes to the sites of DNA double-strand breaks and rapidly forms nuclear foci (NF), and its presence may be an indicator of endogenous genomic instability (GIN). We previously showed that 53BP1 NF in cervical cells increase with neoplastic progression, indicating the significance of 53BP1 expression for the estimation of malignant potential during cervical carcinogenesis. This study aimed to further elucidate the impact of 53BP1 expression as a biomarker for cervical squamous intraepithelial lesion (SIL). A total of 81 tissue samples, including 17 of normal cervical epithelium, 22 of cervical intraepithelial neoplasia (CIN) 1, 21 of CIN2, and 21 of CIN3, from patients positive for high-risk human papillomavirus (HR-HPV) were used for double-label immunofluorescence of 53BP1 and Ki-67/p16 INK4a expression and HR-HPV in situ hybridization. We analyzed associations between 53BP1 expression type with parameters such as CIN grade, HR-HPV infection status, p16 INK4a expression, and CIN prognosis. Expression type of 53BP1 was significantly associated with histological grade of CIN and HR-HPV in situ hybridization signal pattern ( P < .0001). There was a significant correlation between 53BP1 and p16 INK4a expression levels ( r = .73, P < .0001). However, there was no association between 53BP1 expression type and CIN prognosis. We propose that 53BP1 expression type is a valuable biomarker for SIL, which can help estimate the grade and GIN of cervical lesions reflecting replication stress caused by the integration of HR-HPV to the host genome.

Stem cells derived from elderly donors or harvested by repeated subculture exhibit a marked decrease in proliferative capacity and multipotency, which not only compromises their therapeutic potential but also raises safety concerns for regenerative medicine. NANOG-a well-known core transcription factor-plays an important role in maintaining the self-renewal and pluripotency of stem cells. Unfortunately, the mechanism that NANOG delays mesenchymal stem cell (MSC) senescence is not well-known until now. In our study, we showed that both ectopic NANOG expression and PBX1 overexpression (i) significantly upregulated phosphorylated AKT (p-AKT) and PARP1; (ii) promoted cell proliferation, cell cycle progression, and osteogenesis; (iii) reduced the number of senescence-associated- β -galactosidase- (SA- β -gal-) positive cells; and (iv) downregulated the expression of p16, p53, and p21. Western blotting and dual-luciferase activity assays showed that ectopic NANOG expression significantly upregulated PBX1 expression and increased PBX1 promoter activity. In contrast, PBX1 knockdown by RNA interference in hair follicle- (HF-) derived MSCs that were ectopically expressing NANOG resulted in the significant downregulation of p-AKT and the upregulation of p16 and p21. Moreover, blocking AKT with the PI3K/AKT inhibitor LY294002 or knocking down AKT via RNA interference significantly decreased PBX1 expression, while increasing p16 and p21 expression and the number of SA- β -gal-positive cells. In conclusion, our findings show that NANOG delays HF-MSC senescence by upregulating PBX1 and activating AKT signaling and that a feedback loop likely exists between PBX1 and AKT signaling., Competing Interests: The authors declare no conflict of competing and financial interest., (Copyright © 2019 Feilin Liu et al.)

Bisphenol A (BPA), as synthetic monomer used in the production of polycarbonate plastic and epoxy resins, has endocrine disruptor properties and high risk on human health. Epigenetic alterations could act an important role in BPA-induced toxicity, but its mechanism has not been fully understood. We investigated the effects of BPA on gene expression of chromatin modifying enzymes, promoter methylation of tumor suppressor genes and histone modifications in human prostate carcinoma cells (PC-3). IC 50 value of BPA was determined as 217 and 190 μM in PC-3 cells by MTT and NRU tests, respectively. We revealed an increase in global levels of 5-methylcytocine and 5-hydroxymethylcytocine at 10 μM of BPA for 96 h. We observed a significant increase on promoter DNA methylation and decrease on gene expression of p16 gene while no change was observed for Cyclin D2 and Rassf1. Significant changes were observed in global histone modifications (H3K9ac, H3K9me3, H3K27me3, and H4K20me3) in PC-3 cells. According to these results, we investigated wide-range epigenetic modifications using PCR arrays. After 96 h BPA exposure, chromatin modifying enzymes including KDM5B and NSD1 were significantly downregulated. Also, promoter methylation of tumor suppressor genes including BCR, GSTP1, LOX, MGMT, NEUROG1, PDLIM4, PTGS2, PYCARD, TIMP3, TSC2 and ZMYDN10 altered significantly. ChIP results showed that H3K9ac, H3K9me3 and H3K27me3 modifications on p16 gene showed significant increases after 1 and 10 μM of BPA exposure. In conclusion, epigenetic signatures such as DNA methylation and histone modifications could be proposed as molecular biomarkers of BPA-induced prostate cancer progression., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Porocarcinomas are rare aggressive carcinomas that harbor tumor suppressor mutations and must be distinguished from benign entities such as poromas., Methods: To determine whether altered expression of these genes was diagnostically informative, we examined p53, Rb, and p16 staining patterns in 15 poromas and 16 porocarcinomas., Results: Poromas consistently displayed diffuse strong expression of Rb in all but one case that displayed focal loss (1/15, 7%), and no evidence of aberrancy in p53 or p16. Porocarcinomas displayed diffuse or focal loss of Rb expression in 9/16 (56%) cases, diffuse loss or overexpression of p53 in 8/15 (53%), and diffuse loss or overexpression of p16 in 6/14 (43%). Diffuse aberrancy in p53 and Rb expression correlated with tumor mutations in TP53 and RB1, respectively, whereas focal Rb loss was associated with wild type RB1. Diffuse p16 overexpression correlated with Rb loss rather than CDKN2A mutation. For porocarcinomas with all three stains evaluable, 10/13 (77%) displayed aberrancy in at least one marker., Conclusions: Our findings suggest that altered p53, p16, and/or Rb expression is relatively specific to porocarcinoma in comparison with poroma. Technical limitations of this panel, including possible focal Rb loss, must be kept in mind, especially in limited samples., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

We estimated the overall and type-specific prevalence of human papillomavirus (HPV) and p16 overexpression in vaginal cancer and vaginal intraepithelial neoplasia (VaIN). We conducted a systematic search of PubMed, Embase and Cochrane Library to identify studies published between 1986 and 2017 using PCR-based or Hybrid Capture 2 tests to evaluate the presence of HPV DNA and/or using any method to detect p16 overexpression in VaIN, vaginal squamous cell carcinoma (VaSCC), or other types of vaginal cancer. Applying a random effects model, we estimated the pooled prevalence of HPV and p16 overexpression along with 95% confidence intervals (CIs). The I 2 statistic was used to assess heterogeneity. We included 26 studies, reporting HPV prevalence and six studies evaluating p16 overexpression. The pooled HPV prevalences in VaSCC (n = 593) and VaIN (n = 1,374) were 66.7% (95% CI = 54.7-77.8) and 85.2% (95% CI = 78.2-91.0), respectively. Substantial inter-study heterogeneity was observed, and analyses stratified on geographic region, type of tissue, HPV detection method or PCR primer type did not fully explain the observed heterogeneity. The most predominant HPV type among the HPV positive VaSCC and VaIN cases was HPV16, followed by HPV33, and HPV45 (in VaIN) and HPV18, and HPV33 (in VaSCC). In pooled analyses, 89.9% (95% CI = 81.7-94.6) of HPV positive and 38.9% (95% CI = 0.9-90.0) of HPV negative vaginal cancers were positive for p16 overexpression. Our findings suggest that vaccination against HPV might prevent a substantial proportion of vaginal neoplasia and highlight the need for further studies of the possible clinical value of p16 testing in these patients., (© 2018 UICC.)

Background: At times, distinguishing Bowen disease (BD) and benign seborrheic keratosis (SK) is histologically challenging, especially when SK shows clonal features (clonal seborrheic keratosis [CSK]). While p16 is often reported as positive in BD and negative in SK, p16 expression in CSK is rarely studied. Here we investigate p16 immunohistochemistry in CSK, SK, and BD., Methods: p16 immunohistochemistry with pattern of expression was noted for 14 CSK, 12 SK, and 18 BD. The degree of inflammation among lesions with respect to p16 expression was also noted., Results: When examining p16 staining in clonal nests of CSK, 57% showed diffuse or patchy/diffuse positivity, 21% showed patchy positivity, and 21% showed clusters of single positive cells. 67% of BD showed diffuse positivity, 11% showed patchy/diffuse positivity, 17% showed patchy positivity, and 6% were negative. 25% of SK showed focal areas of patchy to full thickness positivity, 25% showed moderate number of positive single cells with or without patchy staining, and 50% showed negative/scattered single cell positivity., Conclusion: Our findings support that p16 positivity limited to clonal nests in CSK is normal. p16 positivity in clonal nests of CSK in isolation without concurrent atypical histologic features should not be used to support a diagnosis of BD., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

It has been previously demonstrated that hydrogen sulfide (H 2 S) prevents formaldehyde (FA)-induced neurotoxicity. However, the exact mechanisms underlying this protection remain to be fully elucidated. Neuronal senescence is involved in FA-induced neurotoxicity. Leptin signaling has anti-aging function. The present work was to investigate the protection of H 2 S against FA-induced neuronal senescence and the mediatory role of leptin signaling. FA-exposed HT-22 cells were used as the vitro model of FA-induced neuronal senescence. The senescence-associated β-galactosidase (SA-β-Gal) positive cell was detected by β-galactosidase staining. The expressions of P16 INK4a , P21 CIP1 , leptin, and lepRb (leptin receptor) were measured by western blot. The proliferation, viability, and apoptosis of cells were evaluated by Trypan blue exclusion assay, Cell Counting Kit-8 (CCK-8) assay, and Flow cytometry analysis, respectively. We found that H 2 S suppressed FA-induced senescence, as evidenced by the decrease in SA-β-Gal positive cells, the downregulations of P16 INK4a and P21 CIP1 , as well as decrease in cell growth arrest, in HT-22 cells. Also, H 2 S upregulated the expressions of leptin and lepRb in FA-exposed HT-22 cells. Furthermore, leptin tA (a specific inhibitor of the leptin) abolished the protective effects of H 2 S on FA-induced senescence and neurotoxicity (as evidenced by the increase in cell viability and the decrease in cell apoptosis) in HT-22 cells. These results indicated that H 2 S prevents FA-induced neuronal senescence via upregulation of leptin signaling. Our findings offer a novel insight into the mechanisms underlying the protection of H 2 S against FA-induced neurotoxicity. FA upregulates the expressions of P16 INK4a and P21 CIP1 via inhibiting leptin signaling, which in turn induces senescence in HT-22 cells; H 2 S downregulates the expressions of P16 INK4a and P21 CIP1 via reversing FA-downregulated leptin signaling, which in turn prevents FA-induced senescence in HT-22 cells.

The purpose of this study was to evaluate the prognostic impact of major histocompatibility complex (MHC) class I expression and programmed death-ligand 1 (PD-L1) expression in patients with head and neck squamous cell carcinoma (HNSCC). A total of 158 patients with HNSCC were evaluated retrospectively. The expression of MHC class I and PD-L1 was analyzed in tumor specimens using immunohistochemistry. The association between MHC class I/PD-L1 expression and clinical outcome was evaluated by Kaplan-Meier and Cox regression analyses. Among 158 patients, 103 (65.2%) showed positive PD-L1 expression, and 20 (12.7%) showed no detectable expression of MHC class I. The frequency of PD-L1 positive expression with concomitant MHC class I loss was 7.0%. In the PD-L1-positive group, MHC class I loss was associated with a significantly worse survival compared with MHC class I positivity (median overall survival 39.3 months vs. not reached; P = 0.005), whereas MHC class I status provided no prognostic impact in the PD-L1 negative group. Neither PD-L1 nor MHC class I alone showed a significant difference in overall survival. The loss of MHC class I expression in PD-L1-positive HNSCC was associated with a poor clinical outcome. This suggested that MHC class I expression status might be useful for the prognosis of tumor progression in HNSCC when combined with PD-L1 expression status. External validation with enough numbers of participants in such subgroup should be needed for validation.

Background: Women with atypical squamous cells of undetermined significance (ASC-US) can be triaged accurately with a high-risk human papillomavirus (hrHPV) test to identify those who need a referral. However, the triage of low-grade squamous intraepithelial lesion (LSIL) with hrHPV testing has very low specificity. Overexpression of p16, with or without Ki-67, indicates neoplastic transformation of human papillomavirus-infected cervical cells and may more accurately predict underlying cervical intraepithelial neoplasia of grade 3 or worse (CIN3+)., Methods: A literature search was conducted in 3 bibliographic databases. Studies were selected if they included women with ASC-US or LSIL who were triaged with dual staining (p16/Ki-67) and/or p16 staining and, if available, with a comparator hrHPV test to detect cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) or CIN3+., Results: Thirty-eight studies were eligible. The sensitivity of p16 staining for CIN3+ was significantly lower than that of hrHPV DNA testing (ratio for ASC-US, 0.87; 95% confidence interval [CI], 0.78-0.97; ratio for LSIL, 0.86; 95% CI, 0.80-0.93). In contrast, the specificity of p16 staining was substantially higher with relative specificities of 1.60 (95% CI, 1.35-1.88) and 2.29 (95% CI, 2.05-2.56) for ASC-US and LSIL respectively. Dual staining was as sensitive as hrHPV DNA testing but was more specific (ratio for ASC-US, 1.65; 95% CI, 1.42-1.92; ratio for LSIL, 2.45; 95% CI, 2.17-2.77)., Conclusions: This meta-analysis confirms that p16 staining and p16/Ki-67 staining are more specific for CIN2+/CIN3+ than hrHPV DNA testing. Although p16 staining is less sensitive for CIN3+ than hrHPV DNA testing, dual staining has similar sensitivity., (© 2019 American Cancer Society.)

p53-mediated cellular senescence has been intensively investigated, because it is important for tumor suppressive function. In addition, p16/INK4A is well known to be critical for cellular senescence. However, detailed molecular mechanism or relevance between p53 and p16-mediated senescence has not been demonstrated yet. Here we show that p53 induces p16 through Lamin A/C stabilization via direct interaction. Stabilized Lamin A/C promotes degradation of BMI-1 and MEL-18 (Polycomb repressor complex 1, PRC1), which sequesters p16 promotor. Increased p53 can reduce BMI-1/MEL-18 and induce p16 expression via Lamin A/C. Elimination of Lamin A/C can abolish p53-induced p16 expression and BMI-1/MEL-18 reduction. As Lamin A/C expression is increased during cell differentiation, this mechanism seems to be very useful for selective induction of senescence in non-stem cells. Our results suggest that Lamin A/C-p53 network is important for p16/INK4A-mediated cellular senescence.

Replicative capacity of normal human cells decreases as telomeric sequence is lost at each division. It is believed that when a subset of chromosomes reach a critically short length, an ATM-initiated and p53-mediated transcriptional response inhibits cell growth, promoting cell senescence. In addition to loss of telomeric sequence, senescence can be induced by other stresses including ionizing radiation, oxidative damage, chemical crosslinkers like the chemotherapeutic agent cisplatin, as well as overactivation of oncogenes and tumor suppressors. Our group found that the expression of an isoform of the INhibitor of Growth 1 gene called ING1a increases approximately 10-fold as fibroblasts approach senescence and that forced expression rapidly induces a senescent phenotype in primary diploid fibroblasts, epithelial and endothelial cells that resembles replicative senescence by most physical and biochemical measures. ING1a induces these changes through strongly inhibiting endocytosis to block mitogen signaling by inducing the expression of intersectin 2, a key scaffolding protein of the endosomal pathway. This, in turn increases the expression of Rb and of p57 Kip2 and p16 INK4a that serve to maintain Rb is an active, growth inhibitory state. The ING1a model is currently being used to better understand the mechanism(s) responsible for activating Rb to enforce the senescent state., (Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.)

Background: Hormonal changes in pregnancy are known to alter melanocytic lesions, with some nevi noted to have increased mitotic figures and increased Ki-67 proliferation index. Additionally, cytomorphologic changes have also been noted, referred to as superficial micronodules of pregnancy. These changes may alarm the pathologist for malignancy, particularly nevoid melanoma. Immunohistochemistry for p16 has been recently utilized to distinguish benign nevi from melanoma. We assessed the use of p16 immunohistochemistry for distinguishing melanocytic nevi of pregnant patients from nevoid melanomas., Methods: Fourteen nevomelanocytic lesions were obtained from pregnant or postpartum patients along with 20 nevoid melanomas for comparison. Immunohistochemistry with p16 was performed on each melanocytic lesion. The percentage of nuclear p16 staining of dermal melanocytes was grouped on a scale of <5%, 5% to 25%, >25% to 50%, and >50%., Results: The majority of nevi from pregnant patients (81%) showed staining of >5% for p16. In contrast, the majority of nevoid melanomas (65%) had staining of <5% for p16., Conclusion: The application of p16 as a potential immunohistochemistry diagnostic marker to distinguish nevi from pregnant patients vs nevoid melanomas may be useful., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HPV-related and HPV-unrelated oropharyngeal squamous cell carcinomas are two distinct entities according to the Union for International Cancer Control, with a better prognosis conferred to HPV-related oropharyngeal squamous cell carcinomas. However, variable clinical outcomes are observed among patients with p16 positive oropharyngeal squamous cell carcinoma, which is a surrogate marker of HPV infection. We aimed to investigate the prognostic value of RNA CISH against E6 and E7 transcripts (HPV RNA CISH) to predict such variability. We retrospectively included 50 histologically confirmed p16 positive oropharyngeal squamous cell carcinomas (p16 positive immunostaining was defined by a strong staining in 70% or more of tumor cells). HPV RNA CISH staining was assessed semi-quantitatively to define two scores: RNA CISH "low" and RNA CISH "high". Negative HPV RNA CISH cases were scored as RNA CISH "low". This series contained 29 RNA CISH low cases (58%) and 21 RNA CISH high cases (42%). Clinical and pathologic baseline characteristics were similar between the two groups. RNA CISH high staining was associated with a better overall survival in both univariate and multivariate analyses (p = 0.033 and p = 0.042, respectively). Other recorded parameters had no prognostic value. In conclusion, HPV RNA CISH might be an independent prognostic marker in p16 positive oropharyngeal squamous cell carcinomas and might help guide therapeutics.

Objectives: While cervical cytology is accepted triage for human papillomavirus (HPV)-positive women, the efficiency of cervical screening could be improved by exploiting disease markers with higher specificity., Methods: CINtec PLUS triage alone and combined with HPV 16/18 genotyping was performed on ThinPrep samples from HPV-positive women. Clinical performance and the potential to reduce or expedite colposcopy referrals were evaluated., Results: The 2-year sensitivity and specificity for cervical intraepithelial neoplasia grade 3 or worse (CIN3+) were 90% and 42%, respectively. Specificity was improved over HPV testing in equivocal cytology and could cut the referral rate by about 40%. When combined with HPV 16/18 genotyping, CINtec PLUS triage of the 12 other high-risk HPV genotypes generally demonstrated better sensitivity for CIN3+ than separate triage of non-type-specific HPV-positive women. This strategy could reduce colposcopy referrals by 31%., Conclusions: These findings highlight the potential of CINtec PLUS to improve management pathways in HPV-positive women. CINtec PLUS cytology represents a sensitive and efficient triage in HPV-positive women. The clinical performance of the dual-stain was most notable in women younger than 25 years and could potentially improve management pathways.

Introduction: Extramammary Paget disease (EMPD) of the vulva has been shown to express p16 by immunohistochemistry (IHC), however, p16 expression in the vulva and scrotum has not been extensively studied in relation to human papillomavirus (HPV) within EMPD of both the vulva and scrotum., Design: Twenty-two cases of EMPD (vulva, 16; scrotum, 6) were found in our laboratory information system. P16 and HPV IHC were performed. Any p16 reactivity less than 10% was considered negative. HPV in situ hybridization for both low- and high-risk HPV was also performed on all cases., Results: Of the 6 scrotal EMPD, 3 (50%) showed weak to moderate positive reactivity for p16 by IHC. Of the 16 vulvar EMPD, 13 (81%) were positive for p16, with at least moderate (2+) intensity with a mean expression of 33.3% (range = 10% to 80%) and 62% (range = 20% to 95%) in scrotal and vulvar EMPD, respectively. None of the scrotal or vulvar cases showed positive reactivity for HPV either by IHC or in situ hybridization., Conclusion: Both vulvar and scrotal EMPD can express p16 by IHC, more commonly vulvar than scrotal; however, no HPV was detected either by IHC or in situ hybridization. EMPD of vulva and scrotum does not appear to be related to HPV, and p16 expression may be regulated through a different mechanism.

Previous studies have suggested that cancer stem cells (CSCs) resisted radiotherapy and chemotherapy. P16 INK4A is a biomarker for cervical carcinogenesis and reduces proliferation of stem cells. We aimed to investigate the expression and clinical significance of cyclin-dependent kinase inhibitor 2A (P16 INK4A ), sex determining region Y-box 2 (SOX2), and Aldehyde dehydrogenase 1 family, member A1 (ALDH1A1) in cervical cancer treated with radiotherapy and cervical cell line models. The expressions of P16 INK4A , SOX2, and ALDH1A1 were performed by immunohistochemical staining of tumor samples from 139 cervical cancer patients with International Federation of Gynecology and Obstetrics stages Ib to IV. The staining showed high expression in 100, 107, and 13 patients with P16 INK4A (>80%), SOX2 (≥10%), and ALDH1A1 (50%), respectively. The high-P16 INK4A group had a higher five-year overall survival (OS) rate and disease-free survival (DFS) than the low-P16 INK4A group (OS: 62.0% and 35.2%, p = 0.016; DFS: 60.0% and 31.2%, p = 0.002). The low-P16 INK4A /high-SOX2 and low-P16 INK4A /high-ALDH1A1 groups had a worse five-year OS and DFS rate than the high-P16 INK4A /low-SOX2 and high-P16 INK4A /low-ALDH1A1 groups, respectively. Depletion of P16 INK4A promoted chemoresistance and radioresistance of cervical cancer cells increased the expression of SOX2 and ALDH1A1 and exhibited higher self-renewal ability. These results suggest that lower P16 INK4A expression associated with higher CSC markers predicts poor prognostic outcomes and is a promising target in patients with cervical cancer.

Background: Deregulation of cell cycle takes place during the development of many cancers as well as pancreatic ductal adenocarcinoma (PDA), which develops from precursor lesions, most frequently including pancreatic intraepithelial neoplasia (PanIN)., Aims: The aim of this study was to evaluate and compare the expression of p16, p21, and p53 proteins taking part in the regulation of the cell cycle in normal pancreatic ducts and pancreatic intraepithelial neoplasia at its various advancing stages., Methods: The expressions of p16, p21, and p53 were assessed immunohistochemically in 70 patients with different pancreatic diseases (pancreatic ductal adenocarcinoma, pancreatitis, and pancreatic cysts), showing also pancreatic intraepithelial neoplasia. The results correlated with chosen clinicopathological parameters., Results: Our study revealed a difference in p16, p21, and p53 expressions between normal pancreatic ducts and various stages of PanIN. p16 expression progressively decreased, whereas p21 and p53 increased from normal pancreas to PanIN 1, 2, and 3. The expression of p21 was associated with age, p53 with PanIN location in the pancreas and p16 with the type of primary diseases. Simultaneously, we observed a directly proportional relationship between the expression of p21 and p53 proteins and inversely proportional between the p16 and the p21 and p53 proteins., Conclusions: p16, p21, and p53 proteins play an important role in the deregulation of the cell cycle and participate in the development of pancreatic intraepithelial neoplasia. Immunohistochemical evaluation of their expressions may be helpful in the diagnosis of PanIN.

Long non-coding RNAs are known as key regulators in the progression and metastasis of breast cancer. MIAT originally has been considered as an lncRNA to be associated with a susceptibility to myocardial infarction. Here, we have detected the expression of MIAT in different cancer cells and a series of breast tumor tissue. MIAT expression was much higher in high-grade tumors compared to low-grade ones. Unlike P53 positive tumors, MIAT expression was upregulated in ER, PR, Her2 positive tumor tissues. Knockdown MIAT suppressed breast cancer cell proliferation and caused G1 arrest in cell cycle. Furthermore, downregulation of MIAT promoted apoptosis and significantly decreased migration of breast cancer cells. An increase in the expression of mir-302, mir-150, and a decrease in the expression of mir-29c were detected following MIAT silencing. More importantly, knockdown MIAT significantly elevated the expression of p16 Ink4A and Cox2, which commitment cellular senescence in breast cancer cells. Altogether, our results suggest that MIAT involved in breast cancer progression and could be candidate as a novel tumor marker for diagnosis and treatment of breast cancer., (© 2018 Wiley Periodicals, Inc.)

Quercetin is a flavonoid that has anti‑inflammatory, anti‑oxidant and lipid metabolic effects. It has also been reported to reduce the risk of cardiovascular disease. The present study measured the effects of quercetin on the expression of ATP‑binding cassette transporter 1 (ABCAl), ATP‑binding cassette sub‑family G member 1 (ABCG1), liver X receptor‑α (LXR‑α), proprotein convertase subtilisin/kexin type 9 (PCSK9), p53, p21 and p16 induced by oxidized low density lipoprotein (ox‑LDL). RAW264.7 macrophages were exposed to ox‑LDL with or without 20 µmol/l quercetin and cell proliferation and senescence were quantified using β‑gal staining. Superoxide dismutase (SOD), malondialdehyde (MDA) and lipid droplets were measured in the cytoplasm using oil red staining, while intracellular and total cholesterol (TC) were measured using filipin staining and a TC kit. Immunofluorescent studies and western blot analysis were performed to quantify the expression of ABCAl, ABCG1, LXR‑α, PCSK9, p53, p21 and p16. Quercetin increased RAW264.7 cell viability and reduced lipid accumulation, senescence, lipid droplets, intracellular cholesterol and TC. It was concluded that quercetin inhibits ox‑LDL‑induced lipid droplets in RAW264.7 cells by upregulation of ABCAl, ABCG1, LXR‑α and downregulation of PCSK9, p53, p21 and p16.

p16 is the most widely studied biomarker in lower anogenital tract squamous intraepithelial lesions and, currently the only recommended biomarker for histological grade assessment. The aim of this systematic review and meta-analysis was to evaluate p16-positive rates according to anal squamous intraepithelial lesions/anal intraepithelial neoplasia (AIN) grade. Two investigators independently searched four electronic databases: PubMed, Web of Sciences, Scopus, and Embase from inception until August 2017. Studies that evaluated p16 immunostaining in histological samples of anal and/or perianal squamous intraepithelial lesions and defined a p16-positive result as diffuse block staining with nuclear or nuclear plus cytoplasmic staining were included. A meta-analysis was performed using a random effects model. Fifteen studies consisting of 790 samples were included. The proportion of p16 expression increased with the severity of histological grade. p16 positivity was 2% (95% CI: 0.2-5%) in normal histology, 12% (95% CI: 2-27%) in low-grade squamous intraepithelial lesions (LSILs)/AIN1 (excluding condylomas), 7% (95% CI: 2-13%) in all LSIL (AIN1/LSIL/condyloma), 76% (95% CI: 61-88%) in AIN2, and 90% (95% CI: 82-95%) in AIN3. For anal high-grade squamous intraepithelial lesions (HSILs), in studies using a two-tiered nomenclature, p16 positivity was 84% (95% CI: 66-96%) and for all HSIL (AIN2, AIN3, HSIL combined) it was 82% (95% CI: 72-91%). In summary, p16 positivity in anal squamous intraepithelial lesions appears to be in a similar range to the commonly described cervical squamous intraepithelial lesions, however, for anal low-grade lesions positivity seems to be lower.

Background: Lichen Planus, LP, is an inflammatory disease of possible autoimmune origin affecting mainly oral and genital mucosa and skin. According to the WHO oral LP is considered a potentially malignant disorders. The p16 tumour suppressor protein can act as an inhibitor of cyclin dependent kinases 4 and 6 and thus down regulate cell cycle progression. Since the discovery of p16 several studies have evaluated its expression in various forms of human cancers. The aim of this study was to evaluate and compare the expression of p16 in oral and genital LP and corresponding healthy mucosa., Material and Methods: A total of 76 cases of oral LP (OLP), 34 cases of genital LP (GLP), 12 cases of healthy oral and 9 cases of healthy genital mucosa were analysed by the use of immunohistochemistry., Results: Data showed p16 to be highly expressed in both oral and genital LP, higher than in oral (p=0.000), and genital controls (p=0.002)., Conclusions: Results suggest that the over-expression of p16 seen in LP play a part in the histopathology of the disease.