Evaluation of enzyme-linked immunosorbent assay plasma p16INK4a protein in squamous cell carcinoma in uterine cervix: A case-control study.

Background: Cervical cancer is one of the common cancers in females. The common method of screening is Pap test which has low sensitivity. Hence, better methods are explored with different biomarkers, of which estimation of P16 protein can be opted in early detection of cervical cancer. Materials and Methods: Seventy cases and seventy controls were considered for the study. Cases were invasive squamous cell carcinoma (SCC) of cervix confirmed by histopathology. Controls were healthy age-matched females. The blood sample of cases and controls was collected in K2 Ethylenediaminetetraacetic acid vacutainer, and the separated plasma was subjected to estimation of P16 protein by quantitative sandwich Enzyme-Linked ImmunoSorbent Assay method. The data were analyzed for the association between p16 protein in plasma in cases and controls. Results: The age among cases and controls ranged from 30 to 80 years. The P16 levels among cases ranged from 3.4 to 19.6 ng/ml with a mean of 7.24 ± 2.35 ng/ml. The plasma P16 level in controls ranged between 0.9 and 9.7 ng/ml with mean of 4.1 ± 2.22 ng/ml. At cutoff more than 4.8 ng/ml in cases, the sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy were 92.86%, 72.86%, 77.4%, 91.1%, and 82.86%, respectively. The specificity increased with increase in plasma p16 levels. The P16 levels were maximum in stage IV disease. Conclusion: This was a pilot study to detect the plasma p16INK4a levels in SCC of cervix. The levels of plasma p16 protein between 3.9 and 5 ng/ml can be considered as the range for the test to be positive. In clinically suspected cases of cervical cancer, levels more than 4.8 ng/ml can be considered for the diagnosis as point of care test.

Probing the epigenetic signatures in subjects with coronary artery disease.

Depletion of S-adenosyl methionine and 5-methyltetrahydrofolate; and elevation of total plasma homocysteine were documented in CAD patients, which might modulate the gene-specific methylation status and alter their expression. In this study, we have aimed to delineate CAD-specific epigenetic signatures by investigating the methylation and expression of 11 candidate genes i.e. ABCG1, LIPC, PLTP, IL-6, TNF-α, CDKN2A, CDKN2B, F2RL3, FGF2, P66 and TGFBR3. The methylation-specific PCR and qRT-PCR were used to assess the methylation status and the expression of candidate genes, respectively. CAD patients showed the upregulation of IL-6, TNF-α, CDKN2A, CDKN2B, F2RL3, FGF2, P66, and TGFBR3. Hypomethylation of CDKN2A loci was shown to increase risk for CAD by 1.79-folds (95% CI 1.22-2.63). Classification and regression tree (CART) model of gene expression showed increased risk for CAD with F2RL3 > 3.4-fold, while demonstrating risk reduction with F2RL3 < 3.4-fold and IL-6 < 7.7-folds. This CAD prediction model showed the excellent sensitivity (0.98, 95% CI 0.88-1.00), specificity (0.91, 95% CI 0.86-0.92), positive predictive value (0.82, 95% CI 0.75-0.84), and negative predictive value (0.99, 95% CI 0.94-1.00) with an overall accuracy of 92.8% (95% CI 87.0-94.1%). Folate and B12 deficiencies were observed in CAD cases, which were shown to contribute to hypomethylation and upregulation of the prime candidate genes i.e. CDKN2A and F2RL3. Early onset diabetes was associated with IL-6 and TNF-α hypomethylation and upregulation of CDKN2A. The expression of F2RL3 and IL-6 (or) hypomethylation status at CDKN2A locus are potential biomarkers in CAD risk prediction. Early epigenetic imprints of CAD were observed in early onset diabetes. Folate and B12 deficiencies are the contributing factors to these changes in CAD-specific epigenetic signatures.

Relationship closeness buffers the effects of perceived stress on transcriptomic indicators of cellular stress and biological aging marker p16INK4a.

Chronic stress can accelerate biological aging, offering one mechanism through which stress may increase age-related disease risk. Chronic activation of the sympathoadrenal system increases cellular energy production, resulting in cell stress that can initiate cellular senescence, a permanent state of cell growth arrest. Our previous research linked psychosocial stress with increased expression of senescence marker p16INK4a; however, less is known about the role of protective psychosocial factors in biological aging. We examined relationship closeness (perceived interconnectedness with one's spouse) as a protective buffer of the effects of stress on expression of the p16INK4a-encoding gene (CDKN2A) and transcription control pathways activated under cell stress. Seventy parents (Mage=43.2) completed interview-based and questionnaire measures of psychosocial stress and relationship closeness. Blood samples assessed CDKN2A expression and inferred activity of a priori-selected transcription factors Nrf2 and heat shock factors (HSFs) via genome-wide transcriptome profiling. Random intercept models adjusting for age, sex, and ethnicity/race revealed that perceived stress was associated with elevated CDKN2A expression for parents with low but not high closeness. Secondary bioinformatics analyses linked the interaction of perceived stress and relationship closeness to Nrf2 and HSF-1 activity. Findings identify relationship closeness as a protective factor that may buffer the impact of stress on cellular stress and senescence pathways.

Association between Nrf2 and CDKN2A expression in patients with end-stage renal disease: a pilot study.

Patients with end-stage renal disease (ESRD) display phenotypic features of premature biological aging, characterized by disproportionately high morbidity and mortality at a younger age. Nuclear factor erythroid 2-related factor 2 (Nrf2) activity, a master regulator of antioxidative responses, declines with age and is implicated in the pathogenesis of age-related disorders; however, little is known about the association between Nrf2 and premature biological aging in ESRD patients. In a cross-sectional pilot cohort of 34 ESRD patients receiving maintenance hemodialysis, we measured the expression of Nrf2 and cyclin-dependent kinase inhibitor 2A (CDKN2A, or p16INK4a, a biomarker of biological aging) genes in whole blood and examined the association of Nrf2 with CDKN2A expression, using Spearman's rank correlation and multivariable linear regression models with adjustment for potential confounders. There was a significant negative correlation between Nrf2 and CDKN2A expression (rho=-0.51, P=0.002); while no significant correlation was found between Nrf2 expression and chronological age (rho=-0.02, P=0.91). After multivariable adjustment, Nrf2 expression remained significantly and negatively associated with CDKN2A expression (ß coefficient=-1.51, P=0.01), independent of chronological age, gender, race, and diabetes status. These findings suggest a potential contribution of Nrf2 dysfunction to the development of premature biological aging and its related morbidities in ESRD patients.

Comparing serum protein levels can aid in differentiating HPV-negative and -positive oropharyngeal squamous cell carcinoma patients.

BACKGROUND: The surrogate immunohistochemical marker, p16INK4a, is used in clinical practice to determine the high-risk human papillomavirus (HPV) status of oropharyngeal squamous cell carcinomas (OPSCC). With a specificity of 83%, this will misclassify some patients compared with direct HPV testing. Patients who are p16INK4a-positive but HPV DNA-negative, or RNA-negative, may be unsuitable for treatment de-escalation aimed at reducing treatment-related side effects. We aimed to identify cost-effective serum markers to improve decision making for patients at risk of misclassification by p16INK4a alone. METHODS: Serum proteins from pre-treatment samples of 36 patients with OPSCC were identified and quantified using label-free mass spectrometry-based proteomics. HPV-status was determined using p16INK4a/HPV DNA and E6/E7 mRNA. Serum protein expressions were compared between groups of patients according to HPV status, using the unpaired t-test with a Benjamini-Hochberg correction. ROC curves (AUC) were calculated with SPSS (v25). RESULTS: Of 174 serum proteins identified, complement component C7 (C7), apolipoprotein F (ApoF) and galectin-3-Binding Protein (LGALS3BP) significantly differed between HPV-positive and -negative tumors (AUC ranging from 0.84-0.87). ApoF levels were more than twice as high in the E6/E7 mRNA HPV-positive group than HPV-negative. CONCLUSIONS: Serum C7, ApoF and LGALS3BP levels discriminate between HPV-positive and HPV-negative OPSCC. Further studies are needed to validate these host immunity-related proteins as markers for HPV-associated OPSCC.

Assessment of HPV 16, HPV 18, p16 expression in advanced stage laryngeal cancer patients and prognostic significance / Avaliação da expressão do HPV 16, HPV 18, e p16 em pacientes com câncer de laringe em estágio avançado e significado prognóstico

Abstract Introduction: Human papilloma virus is an etiological risk factor for a subset of head and neck squamous cell carcinomas. HPV has been proven to be a powerful prognostic biomarker for oropharyngeal cancer, but its role in the larynx has not been explored in depth. The developmental mechanisms of laryngeal carcinomas are quite complex and controlled by various factors. Smoking and alcohol are most important risk factors. Recent studies indicate that HPV infection also plays an important role in larynx carcinomas. HPV related laryngeal carcinomas especially occur at the supraglottic region of larynx. Objective: We aimed to determine the frequency of HPV/protein16 positivity in patients with laryngeal carcinoma and association of HPV and/or p16 positivity with variables such as age, sex, smoking habits, tumor localization, lymph node metastasis, recurrence and survival in advanced stage laryngeal carcinoma in our study. Methods: This retrospective study included 90 patients with advanced laryngeal carcinoma. The Control group was 10 normal larynx mucosa specimens. The presence of HPV was investigated polyclonally by polymerase chain reaction, and protein16 with immunohistochemical method. In HPV positive cases, the presence of HPV types 16, 18 were evaluated by polymerase chain reaction. Demographic features of patients were noted. Patient survival and association with HPV/protein16 was determined. Results: Polyclonal HPV positivity was detected in 11 (12.2%) of 90 cases. Out of these 11 cases, HPV 16 was positive in 6, HPV 18 in 4, and both HPV 16 and 18 were positive in 1. In 18 (20%) of the cases, p16 was positive. Six of the cases (6.6%) had both HPV and protein16 positivity. In cases where protein16 alone or HPV and protein16 were co-positive, alcohol use was less and the tumor was found more likely to be localized in the supraglottic area. These ratios were statistically significant. Supraglottic localization of tumor was determined to be increased in protein16 positive cases. The correlation between protein16 positivity and supraglottic area location was determined to be statistically significant (p= 0.011). 55.6% of protein16 positive cases was located in the supraglottic region, 33.3% was glottic and 11.1% was transglottic. Although life expectancy over 5 years were numerically higher in HPV and protein16 positive cases, this was not found to be statistically significant. There was no statistically significant relationship between HPV positivity and mean age, differentiation, smoking and alcohol use, tumor progression, lymph node metastasis, localization, recurrence, cause of mortality and treatment methods in our study. The mean follow-up period of our patients was 6.7 years. Conclusion: The close relationship between HPV and oropharyngeal squamous cell carcinoma could not be shown in larynx malignancy in many studies, including our study. Our findings support a limited role of HPV in laryngeal carcinogenesis. Protein16 is not a reliable surrogate for HPV status in laryngeal cancers and is not a predictor of laryngeal cancer survival. Supraglottic localization of tumor was determined to be increased in protein16 positive cases. The correlation between protein16 positivity and supraglottic area location was determined to be statistically significant. There is a need for more populated clinical trials, where neoplastic proliferation is better demonstrated and the accuracy of the results obtained is supported by different techniques.

Resumo Introdução: O papilomavírus humano é um fator de risco etiológico para um subconjunto de carcinoma espinocelular de cabeça e pescoço. Tem sido demonstrado que o HPV é um poderoso biomarcador prognóstico para o câncer de orofaringe, mas seu papel na laringe ainda não foi explorado em profundidade. Os mecanismos de desenvolvimento dos carcinomas de laringe são bastante complexos e controlados por vários fatores. Tabagismo e álcool são os fatores de risco mais importantes. Estudos recentes indicam que a infecção pelo HPV também desempenha um papel importante nos carcinomas da laringe. Os carcinomas laríngeos relacionados ao HPV ocorrem especialmente na região supraglótica. Objetivo: Nosso objetivo foi determinar a frequência da positividade para o HPV / proteína 16 em pacientes com carcinoma da laringe e a associação da positividade para o HPV e /ou proteína 16 com variáveis como idade, sexo, tabagismo, localização do tumor, metástase linfonodal, recidiva e sobrevivência de carcinoma da laringe em estágio avançado em nosso estudo. Método: Este estudo retrospectivo incluiu 90 pacientes com carcinoma laríngeo avançado. O grupo controle incluiu 10 amostras de mucosa laríngea normal. A presença de HPV foi inves-tigada por anticorpo policlonal através de reação de polimerase em cadeia e a proteína 16 por método imunohistoquímico. Nos casos positivos para o HPV, a presença dos tipos 16 e 18 do foi avaliada por reação de polimerase em cadeia. As características demográficas dos pacientes foram observadas. A sobrevida dos pacientes e a associação com HPV / proteína 16 foram determinadas. Resultados: A positividade com anticorpo policlonal do HPV foi detectada em 11 (12,2%) dos 90 casos. Desses 11 casos, o HPV 16 foi positivo em 6, o HPV 18 em 4 e o HPV 16 e 18 foram positivos em 1. Em 18 (20%) dos casos, a proteína 16 foi positiva. Seis dos casos (6,6%) apresentaram positividade para HPV e proteína16. Nos casos positivos apenas para a proteína 16 ou quando HPV e a proteína 16 foram co-positivos, a ingestão de álcool foi menor e o tumor apresentou maior probabilidade de estar localizado na área supraglótica. Essas proporções foram estatisticamente significantes. A localização supraglótica do tumor foi maior em casos positivos para proteína 16. A correlação entre positividade para proteína 16 e localização da área supraglótica foi estatisticamente significante (p = 0,011). Dos casos positivos para proteína 16, 55,6% foram supraglóticos, 33,3% glóticos e 11,1% transglóticos. Embora a expectativa de vida acima de 5 anos tenha sido numericamente maior nos casos positivos para HPV e proteína 16, isso não foi estatisticamente significante. Não houve relação estatisticamente significante entre positividade do HPV e média de idade, diferenciação, tabagismo e uso de álcool, progressão tumoral, metástase linfonodal, localização, recidiva, causa de mortalidade e métodos de tratamento em nosso estudo. O período médio de seguimento de nossos pacientes foi de 6,7 anos. Conclusão: A estreita relação entre HPV e carcinoma espinocelular orofaríngeo não pôde ser demonstrada na laringe em muitos estudos, inclusive no nosso estudo. Nossos achados confirmam um papel limitado do HPV na carcinogênese da laringe. A proteína 16 não é um substituto confiável para o status do HPV nos cânceres de laringe e não é preditor da sobrevida do câncer de laringe. A localização supraglótica do tumor foi maior em casos positivos para proteína16. A correlação entre positividade para proteína 16 e localização na área supraglótica foi determinada como estatisticamente significante. Há necessidade de ensaios clínicos com amostras maiores, nos quais a proliferação neoplásica seja melhor demonstrada e a precisão dos resultados obtidos seja apoiada por diferentes técnicas.

Middle-Aged Indians with Type 2 Diabetes Are at Higher Risk of Biological Ageing with Special Reference to Serum CDKN2A.

Sedentary lifestyle and high visceral adiposity have elevated the risk of type 2 diabetes (T2DM) among Indians at younger age. In this study, we aimed to investigate the association of oxidative stress and chronic inflammatory mediators with ageing with special reference to the biological ageing marker cyclin-dependent kinase inhibitor 2A (CDKN2A) among middle-aged (31-50 years) Indian healthy and T2DM subjects. Malondialdehyde (MDA), oxidized LDL (oxLDL), interleukin-6 (IL-6), interleukin 1ß (IL-1ß), tumor necrosis factor α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and CDKN2A were measured in T2DM patients (n = 80) and controls (n = 80) aged 31-50 years, further grouped into G1: 31-40 years and G2: 41-50 years. IL-6, TNF-α, MCP-1, and CDKN2A showed a significant association with ageing among both T2DM patients and controls. But the strength of the association of MCP-1 and CKDN2A with ageing was significantly stronger in T2DM patients than the controls. All the oxidative stress and proinflammatory mediators showed nonsignificant associations with CDKN2A in the controls. However, IL-6, TNF-α, and MCP-1 showed a strong association with CDKN2A in T2DM patients. An increased risk of high levels of CDKN2A was found in G1 T2DM patients (OR: 3.484 (95% CI: 1.246-9.747) p = 0.017) and G2 T2DM patients (OR: 5.000 (95% CI: 1.914-13.061), p = 0.001) with reference to the respective control groups. Our study reveals that the middle-aged Indians with T2DM are at higher risk of biological ageing. The development of T2DM is more common among middle-aged Indians. T2DM may exacerbate the ageing process and may subsequently predispose Indians to various age-related complications at a much early age.

Assessment of HPV 16, HPV 18, p16 expression in advanced stage laryngeal cancer patients and prognostic significance.

INTRODUCTION: Human papilloma virus is an etiological risk factor for a subset of head and neck squamous cell carcinomas. HPV has been proven to be a powerful prognostic biomarker for oropharyngeal cancer, but its role in the larynx has not been explored in depth. The developmental mechanisms of laryngeal carcinomas are quite complex and controlled by various factors. Smoking and alcohol are most important risk factors. Recent studies indicate that HPV infection also plays an important role in larynx carcinomas. HPV related laryngeal carcinomas especially occur at the supraglottic region of larynx. OBJECTIVE: We aimed to determine the frequency of HPV/protein16 positivity in patients with laryngeal carcinoma and association of HPV and/or p16 positivity with variables such as age, sex, smoking habits, tumor localization, lymph node metastasis, recurrence and survival in advanced stage laryngeal carcinoma in our study. METHODS: This retrospective study included 90 patients with advanced laryngeal carcinoma. The Control group was 10 normal larynx mucosa specimens. The presence of HPV was investigated polyclonally by polymerase chain reaction, and protein16 with immunohistochemical method. In HPV positive cases, the presence of HPV types 16, 18 were evaluated by polymerase chain reaction. Demographic features of patients were noted. Patient survival and association with HPV/protein16 was determined. RESULTS: Polyclonal HPV positivity was detected in 11 (12.2%) of 90 cases. Out of these 11 cases, HPV 16 was positive in 6, HPV 18 in 4, and both HPV 16 and 18 were positive in 1. In 18 (20%) of the cases, p16 was positive. Six of the cases (6.6%) had both HPV and protein16 positivity. In cases where protein16 alone or HPV and protein16 were co-positive, alcohol use was less and the tumor was found more likely to be localized in the supraglottic area. These ratios were statistically significant. Supraglottic localization of tumor was determined to be increased in protein16 positive cases. The correlation between protein16 positivity and supraglottic area location was determined to be statistically significant (p =  0.011). 55.6% of protein16 positive cases was located in the supraglottic region, 33.3% was glottic and 11.1% was transglottic. Although life expectancy over 5 years were numerically higher in HPV and protein16 positive cases, this was not found to be statistically significant. There was no statistically significant relationship between HPV positivity and mean age, differentiation, smoking and alcohol use, tumor progression, lymph node metastasis, localization, recurrence, cause of mortality and treatment methods in our study. The mean follow-up period of our patients was 6.7 years. CONCLUSION: The close relationship between HPV and oropharyngeal squamous cell carcinoma could not be shown in larynx malignancy in many studies, including our study. Our findings support a limited role of HPV in laryngeal carcinogenesis. Protein16 is not a reliable surrogate for HPV status in laryngeal cancers and is not a predictor of laryngeal cancer survival. Supraglottic localization of tumor was determined to be increased in protein16 positive cases. The correlation between protein16 positivity and supraglottic area location was determined to be statistically significant. There is a need for more populated clinical trials, where neoplastic proliferation is better demonstrated and the accuracy of the results obtained is supported by different techniques.

Senescence and Inflammatory Markers for Predicting Clinical Progression in Parkinson's Disease: The ICICLE-PD Study.

BACKGROUND: Cognitive decline is a frequent complication of Parkinson's disease (PD) and the identification of predictive biomarkers for it would help in its management. OBJECTIVE: Our aim was to analyse whether senescence markers (telomere length, p16 and p21) or their change over time could help to better predict cognitive and motor progression of newly diagnosed PD patients. We also compared these senescence markers to previously analysed markers of inflammation for the same purpose. METHODS: This study examined the association of blood-derived markers of cell senescence and inflammation with motor and cognitive function over time in an incident PD cohort (the ICICLE-PD study). Participants (154 newly diagnosed PD patients and 99 controls) underwent physical and cognitive assessments over 36 months of follow up. Mean leukocyte telomere length and the expression of senescence markers p21 and p16 were measured at two time points (baseline and 18 months). Additionally, we selected five inflammatory markers from existing baseline data. RESULTS: We found that PD patients had shorter telomeres at baseline and 18 months compared to age-matched healthy controls which also correlated to dementia at 36 months. Baseline p16 levels were associated with faster rates of motor and cognitive decline over 36 months in PD cases, while a simple inflammatory summary score at baseline best predicted cognitive score over this same time period in PD patients. CONCLUSION: Our study suggests that both inflammatory and senescence markers (p16) are valuable predictors of clinical progression in PD patients.

Identification of CDKN2A variants in breast cancer patients in Pakistan.

The role of cyclin-dependent kinase inhibitor 2A gene (CDKN2A) variants in breast cancer is not well understood, here we investigated their possible effects on breast cancer in Pakistani women attending the NORI Hospital, Islamabad. Direct DNA sequencing of CDKN2A identified an already known polymorphism in the 3' UTR, c.*29G>C (rs11515), in 5.88% patients and two novel variants. One, a deep intronic substitution (c.458-554T>G) in 1.96% patients, is also detected as a compound heterozygous form along with c.*29G>C in 1.96% patients (c.[458-554T>G; *29G>C]). The other is a novel deletion (c.458-82delG) occurring as a compound variant with two other identified variants c.[458-554T>G; 458-82delG; *29G>C] in 1.96% patients. In silico pathogenicity prediction analyses did not predict pathogenic effects on breast cancer for these individual variants. We conclude that variations in CDKN2A are not the major genetic cause of breast cancer in the enrolled Pakistani patients.

Roles of Ki-67 and p16 as biomarkers for unknown primary head and neck squamous cell carcinoma.

PURPOSE: Treatment guidelines have not been established for unknown primary head and neck squamous cell carcinoma (SCC). For these patients, chemoradiotherapy (CRT) can provide a better prognosis than that for patients with other head and neck cancers. The presence of HPV in the tumor is associated with a better outcome. However, not all patients with HPV-positive unknown primary head and neck SCC experience good treatment outcomes in actual clinical settings. METHODS: We thus retrospectively determined the Ki-67 proliferation index and p16 expression status to assess the associations of these parameters with treatment outcomes of patients with unknown primary head and neck SCC. RESULTS: The subjects were 13 patients who underwent CRT after surgery or excision biopsy between 1999 and 2016. The 2- and 5-year overall survival (OS) rate was 76.9% and 68.4%, respectively. The prognostic factor was age. There was no significant difference in survival between patients with a high Ki-67 vs. low Ki-67 or between patients with p16-positive vs. p16-negative metastases OS. However, all p16-positive patients with low Ki-67 showed good locoregional control. CONCLUSIONS: The combination of ki67 expression and p16 expression status may allow prediction of local control more accurately than p16 expression status alone.

Evaluation of Relationship between Occurrence of Liver Cancer and Methylation of Fragile Histidine Triad (FHIT) and P16 Genes.

BACKGROUND We examined the level of fragile histidine triad (FHIT) and p16 gene methylation in patients with hepatocellular carcinoma and explored the relationship to liver cancer. MATERIAL AND METHODS There were 56 patients with primary liver cancer who were admitted to the hospital from July 2015 to October 2017 included in the liver cancer group, and 24 non-hepatoma patients (hepatitis A/hepatitis B/hepatitis C, liver cirrhosis, liver fibrosis, and fatty liver, alcoholic liver identified as a control group. Fasting venous blood samples were collected from the 2 groups. Methylation-specific PCR (MSP) was used to detect the methylation of FHIT and p16 genes in the 2 groups. The risk factors for lung cancer were analyzed by logistic regression. In addition, the effects of FHIT and p16 gene methylation on the diagnostic accuracy of liver cancer were calculated. RESULTS The incidence of FHIT and p16 methylation in serum from the liver cancer group was 51.8% and 67.9%, respectively. The incidence of FHIT and p16 methylation in the non-hepatoma group was 16.7% and 25.0%. There was a statistical statistically correlated with gender, and the methylation of FHIT and p16 genes (P<0.05). From logistic regression analysis results, methylation of p16 and FHIT genes was an independent risk factor for hepatocellular carcinoma with odds ratio (OR) values of 10.550 (2.313~48.116) and 8.239 (2.386~28.455), respectively. CONCLUSIONS The methylation of p16 and FHIT genes was an independent risk factor for hepatocellular carcinoma.

p16 Influence on Laryngeal Squamous Cell Carcinoma Relapse and Survival.

OBJECTIVE: (1) To identify p16 protein in laryngeal squamous cell carcinoma (LSCC) specimens and to correlate it with the presence of human papillomavirus (HPV) found in these specimens from a previous study. (2) To analyze p16 impact on 10-year overall and disease-free survival. STUDY DESIGN: Retrospective case series with oncologic database chart review. SETTING: Academic tertiary care hospital. SUBJECTS: A total of 123 samples of LSCC (taken from the glottis only) from patients treated with primary surgical resection between 1977 and 2005. METHODS: p16 protein expression was analyzed through immunohistochemistry and compared with the presence of HPV established in our previous studies. Results were compared with histologic, clinicopathologic, and survival parameters, with a 10-year follow-up. RESULTS: Of the samples, 39.02% were positive for p16, but only 11.38% were positive for both p16 and HPV. The p16+ cohort showed a significant improvement in disease-free survival ( P = .0022); statistical significance was not achieved for overall survival. p16+ cases had fewer relapses over time, with no relapses after a 2-year follow-up. Age at the time of diagnosis and tobacco consumption were the only epidemiologic factors that influenced overall survival. CONCLUSION: The expression of p16 protein was a beneficial prognostic factor for disease-free survival among patients with LSCC of the glottis, with no relapses after a 2-year follow-up.

Is human papillomavirus and p16 expression associated with survival outcomes in nasopharyngeal cancer?: A systematic review and meta-analysis.

INTRODUCTION: Human papillomavirus (HPV) is a known prognostic indicator in oropharyngeal cancer. Not much is known about the prognostic role of HPV in Nasopharyngeal cancer (NPC). Here, we performed a systematic review and meta-analysis of the literature to investigate if HPV status was a prognostic factor for NPC. METHODS: PubMed (via the web), Embase, Scopus, and the Cochrane Library were searched. A systematic review and meta-analysis was done to generate the pooled Hazard Ratios (HR) for Overall Survival (OS). RESULTS: A total of 7 studies from 2014 to 2018, reporting data on 2646 patients (range 43-1328) were included in this meta-analysis. The pooled data showed that HPV/p16 status was not associated with OS in NPC with HR of 0.77 (95% CI: 0.55-1.09, p = 0.14). The test for heterogeneity showed little to no heterogeneity of results (I2 = 4%, p = 0.38). Subgroup analysis showed that in large sample sizes, HPV was significantly associated with survival. CONCLUSION: Despite the finding in the pooled HR, we could not draw a definitive conclusion as to the prognostic significance of HPV in NPC. Recommendations for future research are given.

P16INK4a gene promoter methylation as a biomarker for the diagnosis of non-small cell lung cancer: An updated meta-analysis.

BACKGROUND: This meta-analysis was conducted to investigate the diagnostic performance of P16INK4a gene promoter methylation as a biomarker of non-small cell lung cancer (NSCLC). METHODS: Two reviewers independently searched the Web of Science, PubMed, Cochrane, Embase, China National Knowledge Infrastructure, and Chinese Biomedical Literature databases. Publications relevant to P16INK4a gene promoter methylation in serum or bronchoalveolar fluid/sputum were screened and included in this meta-analysis. Pooled diagnostic sensitivity, specificity, and symmetric receiver operating characteristic curve were calculated. RESULTS: Twenty-six publications with 1768 lung cancer cases and 1323 controls were included. The pooled sensitivity, specificity, positive and negative likelihood ratios, and diagnostic odds ratio were 0.46 (95% confidence interval [CI] 0.43-0.48), 0.90 (95% CI 0.88-0.91), 6.33 (95% CI 3.89-10.30), 0.57 (95% CI 0.50-0.65) and 10.72 (95% CI 6.94-16.56), respectively, for P16INK4a gene promoter methylation as a biomarker for the diagnosis of NSCLC. The area under the symmetric receiver operating characteristic curve was 0.75 with a standard error of 0.004. No publication bias was detected via line regression test (t = 0.95; P = 0.35) and Begg's funnel plot. CONCLUSION: P16INK4a gene promoter methylation detection in serum or bronchoalveolar fluid/sputum may be a potential biomarker for NSCLC diagnosis; however, the sensitivity was relatively low, which is not suitable for NSCLC screening.

Cancer stage and pack-years, but not p16 or HPV, are relevant for survival in hypopharyngeal and laryngeal squamous cell carcinomas.

PURPOSE: Recently, p16 has been included in the TNM guideline for oropharyngeal carcinomas. The role of HPV and p16 in hypopharyngeal and laryngeal carcinomas has not yet been established sufficiently. METHODS: Hundred and thirty-four patients with hypopharyngeal and laryngeal carcinomas were included in this retrospective analysis. Only patients with known HPV status were eligible for the investigation. Survival probabilities were estimated for different risk factors. RESULTS: Eighty-five patients presented with laryngeal carcinoma and 49 patients with hypopharyngeal carcinoma. 8% were HPV positive (10.6% laryngeal, 4.1% hypopharyngeal carcinoma). Median follow-up time was 58 months. We observed a significantly better overall survival for patients with an early tumor stage compared to advanced carcinoma. One of the hypopharyngeal HPV positive carcinomas was also p16 positive and one was p16 negative. Of the nine HPV positive laryngeal carcinomas, four were p16 positive and five p16 negative. Neither patients who were HPV positive nor patients positive for p16 showed a significantly better outcome than HPV or p16 negative patients. In contrast, nicotine pack-years showed a highly significant correlation with survival in our patient collective. CONCLUSIONS: The data suggest that tumor stage and nicotine exposure seem to have the highest impact on survival in hypopharyngeal and laryngeal squamous cell carcinoma patients. There is no evidence for a better survival for p16 positive or HPV positive patients with hypopharyngeal or laryngeal squamous cell carcinoma. HPV seems to play a minor role in these entities of head and neck carcinoma.

Methylation analysis of p16, SLIT2, SCARA5, and Runx3 genes in hepatocellular carcinoma.

This study is to investigate the methylation status of multiple tumor suppressor 1 (p16), secreted glycoprotein 2 (SLIT2), scavenger receptor class A, member 5 putative (SCARA5), and human runt-related transcription factor 3 (Runx3) genes in the peripheral blood of hepatocellular carcinoma (HCC).This is a case-control study. The peripheral blood samples were collected from 25 HCC patients, 25 patients with high risk of HCC (defined as "internal control group"), and 25 healthy individuals (defined as "external control group"), respectively. Then the methylation status of p16, SLIT2, SCARA5, and Runx3 genes in the blood samples were analyzed by pyrosequencing. The relationship between the methylation and the clinical features of HCC patients were evaluated.The methylation levels in the 7 CpG loci of p16 gene in HCC patients were low and without statistically significant difference (P > .05) compared to the control groups. Although the methylation levels of CpG3 and CpG4 in SLIT2 gene loci were higher than those of the control groups, there was no statistically significant difference (P > .05). However, the methylation rate of CpG2 locus in SCARA5 gene in HCC patients was significantly higher (P < .05). And the methylation rates of CpG1, CpG2, CpG3, CpG4, CpG5, and CpG8 in Runx3 gene in HCC patients were significantly different to that of control groups (P < .05). We also have analyzed the correlations between the CpG islands methylation of Runx3 or SCARA5 genes and the age, gender, hepatitis B, liver cirrhosis, alpha fetal protein, or hepatitis B surface antigen (HBsAg) of the HCC patients, which all showed no significant correlations (P > .05).The methylation status of SCARA5 and Runx3 genes are abnormal in HCC patients, which may further be used as molecular markers for early auxiliary diagnosis of liver cancer.

Role of senescence marker p16 INK4a measured in peripheral blood T-lymphocytes in predicting length of hospital stay after coronary artery bypass surgery in older adults.

Adults older than 65 years undergo more than 120,000 coronary artery bypass (CAB) procedures each year in the United States. Chronological age alone, though commonly used in prediction models of outcomes after CAB, does not alone reflect variability in aging process; thus, the risk of complications in older adults. We performed a prospective study to evaluate a relationship between senescence marker p16(INK4a) expression in peripheral blood T-lymphocytes (p16 levels in PBTLs) with aging and with perioperative outcomes in older CAB patients. We included 55 patients age 55 and older, who underwent CAB in Johns Hopkins Hospital between September 1st, 2010 and March 25th, 2013. Demographic, clinical and laboratory data following outline of the Society of Thoracic Surgeons data collection form was collected, and p16 mRNA levels in PBTLs were measured using TaqMan® qRT-PCR. Associations between p16 mRNA levels in PBTLs with length of hospital stay, frailty status, p16 protein levels in the aortic and left internal mammary artery tissue, cerebral oxygen saturation, and augmentation index as a measure of vascular stiffness were measured using regression analyses. Length of hospital stay was the primary outcome of interest, and major organ morbidity, mortality, and discharge to a skilled nursing facility were secondary outcomes. In secondary analysis, we evaluated associations between p16 mRNA levels in PBTLs and interleukin-6 levels using regression analyses. Median age of enrolled patients was 63.5 years (range 56-81 years), they were predominantly male (74.55%), of Caucasian descent (85.45%). Median log2(p16 levels in PBTLs) were 4.71 (range 1.10-6.82). P16 levels in PBTLs were significantly associated with chronological age (mean difference 0.06 for each year increase in age, 95% CI 0.01-0.11) and interleukin 6 levels (mean difference 0.09 for each pg/ml increase in IL-6 levels, 95% CI 0.01-0.18). There were no significant associations with frailty status, augmentation index, cerebral oxygenation and p16 protein levels in blood vessels. Increasing p16 levels in PBTLs did not predict length of stay in the hospital (HR 1.10, 95% CI 0.87-1.40) or intensive care unit (HR 1.02, 95% CI 0.79-1.32). Additional evaluation of p16 levels in PBTLs as predictor of perioperative outcomes is required and should include additional markers of immune system aging as well as different outcomes after CAB in addition to length of hospital stay.

Serum biomarkers for detection of head and neck squamous cell carcinoma.

BACKGROUND: Detection of hypermethylated circulating tumor DNA has the potential to be a minimally invasive, low cost, and reproducible method for cancer detection. METHODS: We evaluated serum from 100 patients with known head and neck squamous cell carcinoma (HNSCC) and 50 healthy control patients for 3 previously described methylation targets, endothelin receptor type B (EDNRB), cyclin-dependent kinase inhibitor 2A (CDKN2A or p16), and deleted in colorectal carcinoma (DCC), using quantitative methylation specific polymerase chain reaction (qMSPCR). RESULTS: EDNRB hypermethylation was identified in the serum of 10% of the patients with HNSCC but in none of the control patients. DCC hypermethylation was detected in 2 serum samples from patients with cancer that also amplified EDNRB and one of these samples also had p16 hypermethylation. EDNRB hypermethylation was statistically significant by Fisher's exact test (p = .03) when comparing HNSCC to controls. CONCLUSIONS: Serum EDNRB hypermethylation is a highly specific but not sensitive serum biomarker for HNSCC.