Developing and Validating a Multivariable Prognostic-Predictive Classifier for Treatment Escalation of Oropharyngeal Squamous Cell Carcinoma: The PREDICTR-OPC Study.

PURPOSE: While there are several prognostic classifiers, to date, there are no validated predictive models that inform treatment selection for oropharyngeal squamous cell carcinoma (OPSCC).Our aim was to develop clinical and/or biomarker predictive models for patient outcome and treatment escalation for OPSCC. EXPERIMENTAL DESIGN: We retrospectively collated clinical data and samples from a consecutive cohort of OPSCC cases treated with curative intent at ten secondary care centers in United Kingdom and Poland between 1999 and 2012. We constructed tissue microarrays, which were stained and scored for 10 biomarkers. We then undertook multivariable regression of eight clinical parameters and 10 biomarkers on a development cohort of 600 patients. Models were validated on an independent, retrospectively collected, 385-patient cohort. RESULTS: A total of 985 subjects (median follow-up 5.03 years, range: 4.73-5.21 years) were included. The final biomarker classifier, comprising p16 and survivin immunohistochemistry, high-risk human papillomavirus (HPV) DNA in situ hybridization, and tumor-infiltrating lymphocytes, predicted benefit from combined surgery + adjuvant chemo/radiotherapy over primary chemoradiotherapy in the high-risk group [3-year overall survival (OS) 63.1% vs. 41.1%, respectively, HR = 0.32; 95% confidence interval (CI), 0.16-0.65; P = 0.002], but not in the low-risk group (HR = 0.4; 95% CI, 0.14-1.24; P = 0.114). On further adjustment by propensity scores, the adjusted HR in the high-risk group was 0.34, 95% CI = 0.17-0.67, P = 0.002, and in the low-risk group HR was 0.5, 95% CI = 0.1-2.38, P = 0.384. The concordance index was 0.73. CONCLUSIONS: We have developed a prognostic classifier, which also appears to demonstrate moderate predictive ability. External validation in a prospective setting is now underway to confirm this and prepare for clinical adoption.

Clinico-pathologic conference: case 4. Langerhans cell histiocytosis (LCH).

A 13-month-old Japanese boy presented with painless swelling in a left mandible and cheek. Intraoral examination revealed swelling in the left mandible and hemorrhage of oral mucosa due to biting. CT images revealed a wide osteolytic lesion of the left mandible with floating teeth. Biopsy was carried out and histopathological diagnosis was discussed.

Extracapsular spread in oral squamous cell carcinoma.

BACKGROUND: Extracapsular spread (ECS) in the cervical lymph nodes represents the most significant adverse prognostic indicator in oral squamous cell carcinoma (OSCC). METHODS: In a consecutive cohort of OSCC treated by primary surgery, ECS was seen in 25% (101) of 400 patients. RESULTS: ECS doubled the incidence of local recurrence and distant metastases, but tripled regional failure. The recurrences occurred sooner in ECS than in non-ECS cases (206 vs 334 days, p = .04). Patients with macroscopic ECS had a 5-year overall survival (OS) of 19% compared with 31% in microscopic ECS. MRI neck staging offered poor sensitivity, especially in microscopic ECS. Age >75 years, smoking, and heavy use of alcohol were independent predictors of ECS, which may implicate a failure of immunosurveillance by the host as much as adverse biology of the tumor. CONCLUSIONS: Reporting of ECS is essential in accurate prognostication, and we advocate that all patients with OSCC and ECS should be grouped as pN3 on the basis of their prognosis. (c) 2009 Wiley Periodicals, Inc. Head Neck, 2010.

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.

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 > or =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 (Fisher's exact, P = 0.002) in a subset of patients.

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.

Tissue banking in head and neck cancer.

Progress in the molecular oncology in head and neck cancer (HNSCC) depends on high quality appropriate tissue samples for research. The expanding availability of new molecular platforms makes ever increasing demands on any available biospecimens. HNSCC offers several key advantages over other tumour sites to the cancer researcher such that, through effective tissue collection, clinicians will be of great help the basic scientist. Informed consent and ethical approval are pre-requisites for tissue banking and it is vital to develop protocols for collection and storage such that the best possible quality of tissue is utilised in future research.

Quantitative methylation analysis of resection margins and lymph nodes in oral squamous cell carcinoma.

Resection is the preferred treatment for oral squamous cell carcinoma, and pathological staging of the resected specimen is crucial. The role of molecular biology in the diagnosis of minimal residual disease has not been fully investigated and may improve staging. Multiple adjacent specimens were taken from the tumour, the invasive front, the surgical margin, and the lymph nodes of 20 specimens from patients with oral cancer. Bisulphite-treated DNA from these specimens was assayed quantitatively with pyrosequencing methylation assays (PMA) of CpG islands within the gene promoters of the p16 and CYGB genes. Results were recorded with histopathological results, and compared with clinical outcome. Biological and technical replicates confirmed the reliability of the techniques. PMA upgraded 13 of the 20 surgical margins, 6 of which subsequently had a recurrent tumour. Not all of these recurrences were predicted and the effects of adjuvant treatment make firm conclusions difficult.

CpG island methylation phenotype (CIMP) in oral cancer: associated with a marked inflammatory response and less aggressive tumour biology.

Studies in several tumour sites highlight the significance of the CpG island methylation phenotype (CIMP), with distinct features of histology, biological aggression and outcome. We utilise pyrosequencing techniques of quantitative methylation analysis to investigate the presence of CIMP in oral squamous cell carcinoma (OSCC) for the first time, and evaluate its correlation with allelic imbalance, pathology and clinical behaviour. Tumour tissue, control tissue and PBLs were obtained from 74 patients with oral squamous cell carcinoma. Pyrosequencing was used to analyse methylation patterns in 75-200 bp regions of the CpG rich gene promoters of 10 genes with a broad range of cellular functions. Allelic imbalance was investigated using a multiplexed panel of 11 microsatellite markers. Corresponding variables, histopathological staging and grading were correlated with these genetic and epigenetic aberrations. A cluster of tumours with a greater degree of promoter methylation than would be predicted by chance alone (P=0.001) were designated CIMP+ve. This group had less aggressive tumour biology in terms of tumour thickness (p=0.015) and nodal metastasis (P=0.012), this being apparently independent of tumour diameter. Further, it seems that these CIMP+ve tumours excited a greater host inflammatory response (P=0.019). The exact mechanisms underlying CIMP remain obscure but the association with a greater inflammatory host response supports existing theories relating these features in other tumour sites. As CIMP has significant associations with other well documented prognostic indicators, it may prove beneficial to include methylation analyses in molecular risk modelling of tumours.