Gillian L. Hall

Extracapsular spread in oral squamous cell carcinoma.

Extracapsular spread (ECS) in the cervical lymph nodes represents the most significant adverse prognostic indicator in oral squamous cell carcinoma (OSCC).

P16 Promoter Methylation Is a Potential Predictor of Malignant Transformation in Oral Epithelial Dysplasia

Management of the patient with oral epithelial dysplasia depends on the ability to predict malignant transformation. Histologic grading of this condition fails in this regard and is also subject to interpathologist and intrapathologist variability. This study uses longitudinal clinical samples to explore the prognostic value of a previously validated panel of methylation biomarkers in a cohort of patients with histologically proven oral dysplasia. Methylation enrichment pyrosequencing assays were used to provide the sensitivity of traditional methylation-specific PCR with the additional specificity advantages of a subsequent confirmatory sequencing reaction. In 57% (8 of 14) patients with a lesion that transformed to oral squamous cell carcinoma, 26% (26 of 100) of longitudinal samples collected over ≥3 years showed p16 methylation. Only 1% (2 of 184) of samples from 8% of patients (2 of 24) not undergoing malignant transformation within 3 years had p16 methylation. Both of these samples with p16 promoter methylation were the most recently collected and the patients remain under continuing clinical review. Promoter methylation of MGMT, CYGB, and CCNA1 did not correlate with malignant progression. We thus conclude that methylation of the p16 gene promoter shows promise as a predictor for malignant transformation (Fishers exact, P = 0.002) in a subset of patients. (Cancer Epidemiol Biomarkers Prev 2008;17(8):2174–9)

Cytoglobin is upregulated by tumour hypoxia and silenced by promoter hypermethylation in head and neck cancer

Background:Cytoglobin (Cygb) was first described in 2002 as an intracellular globin of unknown function. We have previously shown the downregulation of cytoglobin as a key event in a familial cancer syndrome of the upper aerodigestive tract.Methods:Cytoglobin expression and promoter methylation were investigated in sporadic head and neck squamous cell carcinoma (HNSCC) using a cross-section of clinical samples. Additionally, the putative mechanisms of Cygb expression in cancer were explored by subjecting HNSCC cell lines to hypoxic culture conditions and 5-aza-2-deoxycitidine treatment.Results:In clinically derived HNSCC samples, CYGB mRNA expression showed a striking correlation with tumour hypoxia (measured by HIF1A mRNA expression P=0.013) and consistent associations with histopathological measures of tumour aggression. CYGB expression also showed a marked negative correlation with promoter methylation (P=0.018). In the HNSCC cell lines cultured under hypoxic conditions, a trend of increasing expression of both CYGB and HIF1A with progressive hypoxia was observed. Treatment with 5-aza-2-deoxycitidine dramatically increased CYGB expression in those cell lines with greater baseline promoter methylation.ConclusionWe conclude that the CYGB gene is regulated by both promoter methylation and tumour hypoxia in HNSCC and that increased expression of this gene correlates with clincopathological measures of a tumours biological aggression.

The Role of Pyrosequencing in Head and Neck Cancer Epigenetics : Correlation of Quantitative Methylation Data With Gene Expression

OBJECTIVE To evaluate promoter methylation quantitation using recently described pyrosequencing techniques by correlation with messenger RNA (mRNA) expression. DESIGN DNA was extracted from tissue samples and was subjected to bisulphite conversion. Quantitative methylation data for multiple CpG sites in each of 9 gene promoters were obtained for tumors using pyrosequencing. RNA was extracted and converted to complementary DNA, and this formed the template for relative quantitation assays of the expression of each gene by real-time reverse transcription-polymerase chain reaction. SETTING Academic research. PATIENTS Thirty-seven patients with head and neck squamous cell carcinoma. MAIN OUTCOME MEASURES The genes studied were P16 (OMIM 600160), cyclin A1 (OMIM 604036), RARB (OMIM 180220), E-cadherin (OMIM 192090), MGMT (OMIM 156569), STAT1 (OMIM 600555), ATM (OMIM 607585), hMLH1 (OMIM 120436), and TIMP3 (OMIM 188826). Immunohistochemistry was also performed for p16. RESULTS STAT1, TIMP3, ATM, and hMLH1 promoters were essentially unmethylated in all cases. The data for cyclin A1 (Spearman rank correlation, rho = -0.53; P < .001), MGMT (rho = -0.53, P < .001), and RARB (rho = -0.34, P =.02) showed the expected negative correlation between levels of methylation and mRNA expression. The data relating to E-cadherin were inconclusive. Surprisingly, P16 expression was statistically significantly greater in those cases with higher levels of methylation (rho = 0.57, P < .001), a finding at odds with assumptions usually made in the literature relating gene promoter methylation to reduced gene expression. The results from p16 immunohistochemistry were in keeping with the mRNA data, but the number of positive staining samples proved too few for statistical analysis. CONCLUSIONS These data present a novel perspective on head and neck cancer epigenetics and reveal new and some unexpected associations and findings. The advantages of pyrosequencing over nonquantitative techniques are discussed in analyses of this nature.

Quantitative promoter methylation differentiates carcinoma ex pleomorphic adenoma from pleomorphic salivary adenoma

Background:Potential epigenetic biomarkers for malignant transformation to carcinoma ex pleomorphic adenoma (Ca ex PSA) have been sought previously with and without specific comparison with the benign variant, pleomorphic salivary adenoma (PSA). Previous analysis has been limited by a non-quantitative approach. We sought to demonstrate quantitative promoter methylation across a panel of tumour suppressor genes (TSGs) in both Ca ex PSA and PSA.Methods:Quantitative methylation-specific real-time polymerase chain reaction (qMSP) analysis of p16INK4A, CYGB, RASSF1, RARβ, human telomerase reverse transcriptase (hTERT), Wilms’ tumour 1 (WT1) and TMEFF2 gene promoters was undertaken on bisulphite-converted DNA, previously extracted from archival fixed tissue specimens of 31 Ca ex PSA and an unrelated cohort of 28 PSA. All target regions examined had formerly been shown to be hypermethylated in salivary and/or mucosal head and neck malignancies.Results:The qMSP demonstrated abnormal methylation of at least one target in 20 out of 31 (64.5%) Ca ex PSA and 2 out of 28 (7.1%) PSA samples (P<0.001). RASSF1 was the single gene promoter for which methylation is shown to be a statistically significant predictor of malignant disease (P<0.001) with a sensitivity of 51.6% and a specificity of 92.9%. RARβ, TMEFF2 and CYGB displayed no apparent methylation, while a combinatory epigenotype based on p16, hTERT, RASSF1 and WT1 was associated with a significantly higher chance of detecting malignancy in any positive sample (odds ratio: 24, 95% CI: 4.7–125, P<0.001).Conclusions:We demonstrate the successful application of qMSP to a large series of historical Ca ex PSA samples and report on a panel of TSGs with significant differences in their methylation profiles between benign and malignant variants of pleomorphic salivary adenoma. qMSP analysis could be developed as a useful clinical tool to differentiate between Ca ex PSA and its benign precursor.

Determinants of outcome following surgery for oral squamous cell carcinoma

The recent changes in incidence and prevalence of oral squamous cell carcinoma in relation to gender and age mirror the changing patterns of exposure to tobacco and alcohol, the main etiological agents. Most cases of oral cancer are managed by surgery, often combined with radiotherapy. Histopathological assessment of the resection specimen provides information vital for postoperative management and prognosis. This review considers the full range of histological determinants of outcome in relation to the primary oral tumor and any metastatic involvement of the cervical lymphatic system, together with an outline of more general patient factors that may also impact on morbidity and mortality rates.

1 UICC/AJCC pTNM staging in oral squamous cell carcinoma: ECS should now be grouped as pN3

Background: The role of TNM staging is to aid the clinician in planning treatment and to indicate prognosis. It serves to evaluate and compare results of treatments as well as stratification of patients into clinical trials. Existing treatments for OSCC favour surgery with widespread use of neck dissections. Currently pTNM takes no account of extracapsular spread (ECS). Methods: From a consecutive cohort of 489 OSCC patients treated by primary surgery, pN was recorded and the presence, pattern and severity (micro/macro) of ECS was noted in those 400 receiving neck dissections. Results: Using the current staging, 221 (55%) were pN0, 72 (18%) pN1, 105 (26%) pN2 but only 2 (0.005%) were pN3. 101 (25%) of the patients had ECS. Extracapsular spread (ECS) in the cervical lymph nodes was the single most significant adverse prognostic indicator of all those recorded. ECS doubled the incidence of local recurrence and distant metastases, but tripled regional failure. Patients with macroscopic ECS had a 5-year OS of 19% compared with 31% in microscopic ECS. The single change of reclassifying ECS to pN3 resolved the obvious imbalance in nodal stage cased by “underuse” of pN3 in the existing system. With this change 48 (12%) were pN1, 30 (8%) were pN2 and 101 (25%) were pN3 and these stagings were reflected by progressive and uniform worsening of OS and DSS. This reclassification is currently under consideration by AJCC/UICC. Conclusions: Reporting of ECS is essential in accurate prognostication and we advocate that all patients with OSCC and ECS should be grouped as pN3.