Shaleta Havard

IGSF4 Methylation as an Independent Marker of Human Papillomavirus–Positive Oropharyngeal Squamous Cell Carcinoma

IMPORTANCE Human papillomavirus (HPV) is a known causative agent for oropharyngeal squamous cell carcinoma (OPSCC). Whereas it is becoming more firmly established that HPV-positive head and neck squamous cell carcinoma is associated with better survival outcomes, believed to be because of better response to chemoradiation therapy, the specific mechanisms for these improved survival outcomes remain underexplored. OBJECTIVE To examine the relationship between HPV status and promoter methylation in an OPSCC cohort. DESIGN, SETTING, AND PARTICIPANTS Real-time quantitative polymerase chain reaction was used to examine oncogenic HPV type 16 in a retrospective cohort of 121 patients with primary OPSCC. Aberrant promoter methylation of IGSF4, DAPK1, and ESR1 genes, known to be methylated in head and neck squamous cell carcinoma, including OPSCC, was examined by means of quantitative methylation-specific polymerase chain reaction. INTERVENTIONS Patients received standard therapy. MAIN OUTCOMES AND MEASURES Univariate associations between HPV and methylation were analyzed using Fisher exact tests followed by multivariable logistic regression. Cox proportional-hazards regression was used to model the risk of death given age, race, sex, HPV status, methylation, stage, smoking, and treatment. RESULTS In univariate logistic regression analyses, HPV-positive status was significantly associated with Caucasian race (P = .02), treatment (radiotherapy only, P = .01; chemoradiotherapy, P = .007), and IGSF4 methylation (P = .005). The final multivariate logistic model, after controlling for patient characteristics (sex, age, smoking, race, and treatment) with backward variable selection among genes, retained IGSF4 methylation (OR, 4.5 [95% CI, 1.6-12.8]; P = .005), Caucasian race (OR, 2.9 [95% CI, 1.0-8.3]; P = .053), treatment (radiotherapy only vs neither: OR, 11.62 [95% CI, 2.02-66.82]; P = .02; chemoradiotherapy vs neither: OR, 11.15 [95% CI, 1.92-64.65]; P = .01), male sex (OR, 4.7 [95% CI, 1.3-17.0]; P = .02), and younger age (OR, 0.9 [95% CI, 0.90-1.0]; P = .008) as independent predictors of HPV-positive status. Cox regression modeling indicated HPV-negative status, age, male sex, smoking, and radiation treatment as independent predictors of mortality. CONCLUSIONS AND RELEVANCE Methylation of IGSF4 is an independent predictor of HPV-positive status. DNA methylation in conjunction with HPV infection appears to play a role in OPSCC.

Promoter Methylation in Head and Neck Tumorigenesis

In addition to genetic alterations of gains and losses, epigenetic events of promoter methylation appear to further undermine a destabilized genomic repertoire in squamous head and neck carcinoma (HNSCC). This chapter provides an overview of frequently methylated tumor suppressor genes in benign head and neck papillomas, primary HNSCC tumors, and HNSCC cell lines and their relevance as epigenetic markers in head and neck tumorigenesis.

Disparate Molecular, Histopathology, and Clinical Factors in HNSCC Racial Groups

Objective: Causes of differences in the higher incidence of and mortality from squamous head and neck cancer (HNSCC) in African American (AA) vs Caucasian Americans (CA) lack a consensus. We examined a comprehensive array of risk factors influencing health and disease in an access to care, racially diverse, primary HNSCC cohort. Method: The study cohort of 673 primary HNSCC comprised 391 CA and 282 AA. Risk variables included demographic, histopathology, and clinical/epidemiologic risk factors. Tumor DNA was interrogated for loss and gain of 113 genes with known involvement in HNSCC/cancer. Univariate analysis was followed by multivariate modeling with determination of c-index. Results: Of the 39 univariate differences in race as AA and CA, multivariate modeling (c-index = 0.81) retained 6 variables (pCDKN2A) and aneupoidy of SCYA3 than CA tumors. AA were more likely to be unmarried, to have radiation treatment, and be more likely to be current and past smokers. Insurance type as HAP (Health Alliance Plan), Blue Cross, Medicaid, Medicare, and other was a significant determinant of race. AA were more likely to have Medicaid and Medicare. Insurance status by stage and radiation by stage were not significant. Conclusion: Molecular modeling indicated significant differences between AA and CA for histopathology, patient factors, and tumor gene copy number alterations. Our data reiterate that for HNSCC, as in the case of other complex diseases, tumor genetics or biology is only one of many potential contributors to differences among racial groups.

Distinct Gene Profiles for Tumor and Non-Tumor Tissue in the Head and Neck: An Analytical Approach

In a study of genetic alterations, the Multiplex Ligation-dependent Probe Amplification (MLPA) assay was used to measure gain or loss of 113 gene-probes in tumor and non-tumor tissue samples collected from each of the 220 patients with squamous head and neck cancer (HNSCC). Conditional and marginal models were available; both models account for correlated data but have different aspects. The conditional logistic regression model was proposed to estimate the subject-specific risk of tumor based on the paired tumor and non-tumor data collection, which was in contrast with the marginal model to estimate population-average risk.The modeling process included rigorous variable selection, an initial multivariable model, a final model selection, and model validation. Genes with individual effect (p<0.01) were considered as candidates for the initial multivariable model for tumor. The final model included gene-probes with p<0.01 and estimations of odds ratios (OR) 95% Confidence Intervals (CIs) and the models predictive ability, measured by the receiver operating characteristic curve (ROC). A 10-fold cross-validation was performed to validate the model. Of 113 gene-probes, using the conditional approach, 16 genes in 7 chromosomes, remained in the final multivariable model with p<0.01 and an ROC score of 0.94. The cross-validation showed ROC mean (SD) score of 0.96(0.04). The marginal model, in contrast, ended with 8 gene-probes and had an observed ROC of 0.81. CONCLUSION: The conditional approach appears to be the model of choice when assessing gene-probe risks of subjects with paired data collection and fewer missing covariates, compared to the marginal approach. This multiple gene model demonstrated excellent ability to discriminate tumor from non-tumor, and supports its contribution to the pathogenesis of HNSCC as well as their potential utility for further markers of early tumor detection.

Abstract 150: HPV and aberrant DNA methylation status in paired saliva and tumor samples in HNSCC

To examine the utility of non-invasive detection of high risk HPV and aberrant DNA methylation in matched saliva and tumor DNA in squamous head and neck cancer (HNSCC). In a cohort of 35 primary HNSCC with matched tumor (cases) and saliva specimens, HPV status was initially determined using the Linear Array HPV Genotyping kit and confirmed by PCR for HPV subtypes 16, 31, 33 and 45. Aberrant methylation of 24 tumor suppressor genes was interrogated using the methylation specific multiplex ligation probe amplification (MS-MLPA) assay. Of the 35 cases, 20 were negative for HPV in the saliva and tumor, 9 were positive for HPV in both and in 6 cases HPV was positive in the tumor specimen but not detected in the corresponding saliva. In the 9 cases that were positive for HPV in the tumor and matched saliva, 6 were HPV 16 positive and 1 case each was positive for HPV 31, 33 and 45. The sensitivity and specificity for HPV in this cohort were 100% and 60%, respectively. Promoter hypermethylation frequency in tumor samples was 60% (21/35) as compared to 14% (5/35) in saliva. The most frequently methylated genes in tumors included RARB-10/21, APC- 9/21, TIMP3 and CDKN2B- 7/21, CDKN2A and IGSF4- 5/21 tumor samples. The most frequently methylated genes in saliva samples included TIMP3, ESR1 and IGSF4 (3/5 saliva samples), followed by APC, CDKN2B and DAPK1 (2/3). Two of the 35 tumor and saliva pairs had commonly methylated genes, APC in one pair, and TIMP3, CDKN2B, ESR1 and IGSF4 in the other. There was no association between HPV and methylation. PCR analysis of HPV DNA in salivary rinses allows for detection of HPV and promoter methylation status in HNSCC and is a feasible noninvasive alternative to more invasive procedures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 150.

A Multiparametric Gene-Based Diagnosis Model for HNSCC

Problem A current shortcoming in the diagnosis, prognosis and treatment of HNSCC is a lack of methods that adequately addresses the complexity and diversity of the disease. Diagnostic and prognostic marker systems based on single parameters have generally proven inadequate. Multiparametric methods, which rely on many pieces of information, are ideally suited to the grouping of tumor subtypes, identification of specific patterns of disease progression, and in predicting clinical outcomes. Methods In a retrospective multi-ethnic primary HNSCC cohort drawn from a primary healthcare setting, and constructed through re-review of the primary biopsy, gene alterations (104 genes) and clinical variables (9 histopathology and 3 demographic variables) were evaluated as predictors of stage (TNM, early versus late). Statistical analysis compared logistic regression and Classification and Regression Tree (CART®) analyses to derive the most predictive model, assessed using receiver operating characteristic (ROC) curve analysis. Results Considering all clinical and gene variables for the 360 primary HNSCC study cohort, the multivariate logistic regression model retained only tumor grade, sample type (radical dissection) and their interaction with ROC as 64%. CART® generated a multivariable model with 12 variables: clinical variables of age, pattern of invasion, and tumor grade and gene variables of TP53, F3, TFF1, CDKN2A, KIAA0170, HS222808, TANK, MYC, and UTY1, with an ROC of 0.82%. Conclusion A group of clinical variables in multiparametric combinations with molecular alterations discriminated early and late stage HNSCC. CART® improved the models performance. Significance Validation of this initial multiparametric strategy for predicting late stage HNSCC comprising several genes and clinical factors, currently underway, should yield a multiparametric, comprehensive genome-wide molecular algorithm integrated with clinical risk factors in order to refine HNSCC diagnosis and prognosis associated with clinical and pathological staging to aid in the clinical management of patients at the earliest stages. Support NIH R01 DE15990.

11:00: Genetic Differentiation of Tumor and Nontumor in HNSCC

neck. Hypermethylation of RARB gene was seen in both saliva and corresponding tumor in one HNSCC patient. CONCLUSION: Noninvasive detection of genomic and epigenetic alterations in saliva by MLPA were highly specific to HNSCC patients and represented the tumor in vivo. SIGNIFICANCE: Genomic and epigenetic alterations detected by MLPA in saliva offer promising biomarkers to serve as molecular signatures for noninvasive HNSCC screening and for tumor surveillance. Additionally these contribute to our understanding of the underlying pathogenesis of HNSCC. SUPPORT: HFHS Dev Grant 06 (SS) R01 NIH DE 15990 (MJW).