Differential mechanisms of <i>CDKN2A</i> (p16) alteration in oral tongue squamous cell carcinomas and correlation with patient outcome

Lim, Annette; Do, Hongdo; Young, Richard J.; Wong, Stephen Q.; Angel, Christopher; Collins, Marnie; Takano, Elena A.; Corry, June; Wiesenfeld, D.; Kleid, Stephen; Sigston, Elizabeth; Lyons, Bernard; Fox, Stephen B.; Rischin, Danny; Dobrovic, Alexander; Solomon, Benjamin

doi:10.1002/ijc.28727

Cited by 54 publications

(25 citation statements)

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“…Details of the full cohort of 131 patients have previously been described [ 96 ]. The clinical and tumor characteristics of the 115 patients with bisulfite modified DNA available for methylation analyses are presented in Table 2 .…”

Section: Resultsmentioning

confidence: 99%

“…A summary of the methylation sensitive high resolution melting (MS-HRM) results for the panel of interrogated genes is presented in Table 3 , including results from our previous study on CDKN2A [ 96 ].…”

Section: Resultsmentioning

confidence: 99%

“…The patient cohort of 131 OTSCC patients has been described previously [ 96 ]. Briefly, patients included consisted of those who we could confirm had OTSCC, had comprehensive clinicopathological and outcome data available, and had pre-treatment FFPE blocks containing invasive squamous cell carcinoma.…”

Section: Methodsmentioning

confidence: 99%

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Quantitative methodology is critical for assessing DNA methylation and impacts on correlation with patient outcome

et al. 2014

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BackgroundDNA hypermethylation is reported as a frequent event and prognostic marker in head and neck squamous cell carcinomas (HNSCC). Methylation has been commonly assessed with non-quantitative methodologies, such as methylation-specific PCR (MSP). We investigated previously reported hypermethylated genes with quantitative methodology in oral tongue squamous cell carcinomas (OTSCC).ResultsThe methylation status of 12 genes in 115 OTSCC samples was assessed by one or more of three quantitative analyses: methylation sensitive high resolution melting (MS-HRM), sensitive-melting analysis after real time-methylation specific PCR (SMART-MSP), and bisulfite pyrosequencing.In contrast to much of the literature, either no or infrequent locus-specific methylation was identified by MS-HRM for DAPK1, RASSF1A, MGMT, MLH1, APC, CDH1, CDH13, BRCA1, ERCC1, and ATM. The most frequently methylated loci were RUNX3 (18/108 methylated) and ABO (22/107 methylated). Interrogation of the Cancer Genome Atlas (TCGA) HNSCC cohort confirmed the frequency of significant methylation for the loci investigated.Heterogeneous methylation of RUNX3 (18/108) and ABO (22/107) detected by MS-HRM, conferred significantly worse survival (P = 0.01, and P = 0.03). However, following quantification of methylation levels using pyrosequencing, only four tumors had significant quantities (>15%) of RUNX3 methylation which correlated with a worse patient outcome (P <0.001), while the prognostic significance of ABO hypermethylation was lost. RUNX3 methylation was not prognostic for the TCGA cohort (P = 0.76).ConclusionsWe demonstrated the critical need for quantification of methylation levels and its impact on correlative analyses. In OTSCC, we found little evidence of significant or frequent hypermethylation of many loci reported to be commonly methylated. It is likely that previous reports have overestimated the frequency of significant methylation events as a consequence of the use of non-quantitative methodology.Electronic supplementary materialThe online version of this article (doi:10.1186/1868-7083-6-22) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Quantitative methodology is critical for assessing DNA methylation and impacts on correlation with patient outcome

et al. 2014

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“…Demokan’s study noted that promoter hypermethylation levels were associated with decreased p16 expression in the tumor samples [ 22 ]. Interestingly, Annette’s study found that 95% (110/116) of OSCC did not overexpress p16, while p16 INK4a promoter methylation was 18% (20/113) [ 50 ]. Human papillomaviruses (HPV) is also an important factor in carcinogenesis of HNSCC.…”

Section: Discussionmentioning

confidence: 99%

Association between P16INK4a Promoter Methylation and HNSCC: A Meta-Analysis of 21 Published Studies

et al. 2015

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BackgroundThe p16INK4a is an important tumor suppressor gene (TSG) and aberrant methylation of promoter is known to be a major inactivation mechanism of the tumor suppressor and tumor-related genes. Aberrant TSG methylation was considered an important epigenetic silencing mechanism in the progression of head and neck squamous cell carcinoma (HNSCC). However, some studies have reported differences in the methylation frequencies of P16INK4a promoter between cancer and the corresponding control group. Therefore, we conducted a meta-analysis to better identify the association.MethodsPubMed, Ovid, ISI Web of Science, and EMBASE were searched to identify eligible studies to evaluate the association of p16INK4a promoter methylation and HNSCC. Odds ratio (ORs) and 95% confidence intervals (95%CI) were calculated to evaluate the strength of association between p16INK4a promoter methylation and HNSCC.ResultsA total of twenty-one studies with 1155 cases and 1017 controls were included in the meta-analysis. The frequencies of p16INK4a promoter methylation in the cancer group were significantly higher than those in the control group (cancer group: median: 46.67%, range = 7.84%-95.12%; control group: median: 18.37%, range = 0–83.33%; respectively). The pooled odds ratio was 3.37 (95%CI = 2.32–4.90) in the cancer group versus the corresponding control group under the random-effects model.ConclusionThis meta-analysis of 21 published studies identified that aberrant methylation of p16INK4a promoter was found to be significantly associated with HNSCC.

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“…Four micron FFPE sections from the TMAs or whole slides were stained for p16 using Ventana Ultraview Detection reagents on a Benchmark Ultra instrument (Ventana Medical Systems, Tucson, AZ, USA) as previously described ( Lim et al , 2014 ). In brief, antigen retrieval was performed with CC1 high-pH retrieval solution for 52 min followed by incubation at 36 °C for 32 min in primary antibody (mouse monoclonal p16, Clone E6H4; Ventana Medical Systems) and then detection with UltraView Universal DAB detection kit (Ventana Medical Systems).…”

Section: Methodsmentioning

confidence: 99%

Frequency and prognostic significance of p16INK4A protein overexpression and transcriptionally active human papillomavirus infection in laryngeal squamous cell carcinoma

et al. 2015

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Background:Human papillomavirus (HPV) infection is a powerful prognostic biomarker in a subset of head and neck squamous cell carcinomas, specifically oropharyngeal cancers. However, the role of HPV in non-oropharyngeal sites, such as the larynx, remains unconfirmed.Methods:We evaluated a cohort of 324 laryngeal squamous cell carcinoma (LSCC) patients for the expression of p16INK4A (p16) protein by immunohistochemistry (IHC) and for high-risk HPV E6 and E7 mRNA transcripts by RNA in situ hybridisation (ISH). p16 expression and HPV status were correlated with clinicopathological features and outcomes.Results:Of 307 patients assessable for p16 IHC, 20 (6.5%) were p16 positive. Females and node-positive patients were more likely to be p16 positive (P<0.05). There were no other significant clinical or demographic differences between p16-positive and -negative cases. There was no difference in overall survival (OS) between p16-positive and -negative patients with 2-year survival of 79% in each group (HR=0.83, 95% CI 0.36–1.89, P=0.65). There was no statistically significant difference in failure-free survival (FFS) with 2-year FFS of 79% and 66% for p16-positive and -negative patients, respectively (HR=0.60, 95% CI 0.26–1.36, P=0.22). Only seven cases were found to be HPV RNA ISH positive, all of which were p16 IHC positive. There was no statistically significant difference in OS between patients with HPV RNA ISH-positive tumours compared with -negative tumours with 2-year survival of 86% and 71%, respectively (HR=0.76, 95% CI 0.23–2.5, P=0.65). The 2-year FFS was 86% and 59%, respectively (HR=0.62, 95% CI 0.19–2.03, P=0.43).Conclusions:p16 overexpression is infrequent in LSCC and the proportion of cases with high-risk HPV transcripts is even lower. There are no statistically significant correlations between p16 IHC or HPV RNA ISH status and OS or disease outcomes.

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Differential mechanisms of CDKN2A (p16) alteration in oral tongue squamous cell carcinomas and correlation with patient outcome

Cited by 54 publications

References 30 publications

Quantitative methodology is critical for assessing DNA methylation and impacts on correlation with patient outcome

Quantitative methodology is critical for assessing DNA methylation and impacts on correlation with patient outcome

Association between P16INK4a Promoter Methylation and HNSCC: A Meta-Analysis of 21 Published Studies

Frequency and prognostic significance of p16INK4A protein overexpression and transcriptionally active human papillomavirus infection in laryngeal squamous cell carcinoma

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