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Purpose: The incidence of thyroid cancer (TC) is increasing. Cytology by itself cannot distinguish TC from some benign nodules especially in certain subtypes of TC. Our immediate goal is to identify DNA methylation markers for early detection of TC and to molecularly differentiate TC subtypes from benign nodules., Methods: Promoter methylation status of 21 candidate genes was examined on formalin-fixed paraffin-embedded tissue (FFPE) utilizing quantitative methylation-specific polymerase chain reaction (QMSP) in a retrospective cohort of 329 patients (56% white, 29% African American, 61% female) comprising 71 normal thyroid, 83 benign nodules [follicular adenomas (FA)], 90 follicular TC (FTC) and 85 papillary TC (PTC). All genes were analyzed individually (Kruskal-Wallis and Wilcoxon rank sum tests) and in combination (logistic regression models) to identify genes whose methylation levels might best separate groups., Results: Combination gene panels TPO and UCHL1 (ROC = 0.607, sensitivity 78%) discriminated FTC from FA, and RASSF1 and TPO (ROC = 0.881, sensitivity 78%) discriminated FTC from normal. Methylation of TSHR distinguished PTC from FTC (ROC = 0.701, sensitivity 84%) and PTC from FA (ROC = 0.685, sensitivity 70%). The six gene panel of TIMP3, RARB2, SERPINB5, RASSF1, TPO and TSHR, which differentiates PTC from normal thyroid, had the best combination sensitivity (91%) and specificity (81%) of the panels addressing discrimination of cancer tissue., Conclusions: Aberrant gene methylation used in combination panels may be useful clinically in differentiating FTC and PTC from benign nodules. If confirmed in additional studies, these findings could help reduce the over diagnosis of thyroid cancer and surgeries related to over diagnosis.

In recent years, studies have suggested that promoter methylation in human papilloma virus (HPV) positive head and neck squamous cell carcinoma (HNSCC) has a mechanistic role and has the potential to improve patient survival. The present study aimed to replicate key molecular findings from previous analyses of the methylomes of HPV positive and HPV negative HNSCC in an independent cohort, to assess the reliability of differentially methylated markers in HPV-associated tumors. HPV was measured using real-time quantitative PCR and the biological significance of methylation differences was assessed by Ingenuity Pathway Analysis (IPA). Using an identical experimental design of a 450K methylation platform, 7 of the 11 genes were detected to be significantly differentially methylated and all 11 genes were either hypo- or hypermethylated, which was in agreement with the results of a previous study. IPA's enriched networks analysis identified one network with msh homeobox 2 (MSX2) as a central node. Locally dense interactions between genes in networks tend to reflect significant biology; therefore MSX2 was selected as an important gene. Sequestration in the top four canonical pathways was noted for 5-hydroxytryptamine receptor 1E (serotonin signaling), collapsin response mediator protein 1 (semaphorin signaling) and paired like homeodomain 2 (bone morphogenic protein and transforming growth factor-β signaling). Placement of 9 of the 11 genes in highly ranked pathways and bionetworks identified key biological processes to further emphasize differences between HNSCC HPV positive and negative pathogenesis.

Thyroid cancer has the fastest rising incidence rates and is the fifth most common cancer in women. There are four main types of which the papillary and follicular types together account for >90%, followed by medullary cancers (3%-5%) and anaplastic carcinomas (<3%). For individuals who present with early stage disease of papillary and follicular cancers, there are no accurate markers to predict whether they will develop metastatic or recurrent disease. Our immediate goal is to molecularly differentiate follicular cancer subtypes for enhanced classification. Promoter methylation status of genes with reported associations in thyroid cancer ( CASP8, CDKN2A, DAPK1, ESR1, NIS, RASSF1 and TIMP3 ) were examined in a cohort of follicular thyroid cancers comprising of 26 Hurthle and 27 Classic subtypes utilizing quantitative methylation-specific PCR. RASSF1 was differentially methylated in Classic tumor tissue compared to Hurthle (p<0.001). Methylation of RASSF1 pointed to racial group differences between African Americans and Caucasian Americans (p=0.05). Extra thyroidal extension was found to be associated with DAPK1 (p=0.014) and ESR1 (p=0.036) methylation. Late stage disease was associated with older age (p<0.001) and methylation of DAPK1 (p=0.034) and ESR1 (p=0.035). The methylation status of RASSF1, DAPK1 and ESR1 suggests the utility of methylation markers to molecularly differentiate thyroid cancer subtypes for enhanced classification and early detection of thyroid cancer.

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.

Currently, the human papilloma virus (HPV), in addition to tobacco and alcohol, is considered another independent risk factor for oropharyngeal squamous head and neck cancer (OPSCC), where the prevalence of HPV-16 increases to 50-90 % for the oropharynx. Also, incidence and mortality in head and neck SCC (HNSCC) continue to be higher in African Americans (AA) than in Caucasian Americans (CA). A recent study found that poorer survival outcomes for AA versus CA with oropharyngeal tumors were attributable to racial differences in the prevalence of HPV positive (+) tumors; HPV negative (-) AA and CA patients had similar outcomes (Settle et al., Cancer Prev Res (Phila) 2:776-781, 2009). Evidence indicates that a HPV+ diagnosis has significant prognostic implications; these patients have at least half the risk of death when compared with the HPV- patient, due in part to a better response to chemoradiotherapy (Fakhry et al., J Natl Cancer Inst 100:261-269, 2008).Epigenetic events of promoter hypermethylation are emerging as promising molecular strategies for cancer detection, representing tumor-specific markers occurring early in tumor progression. HPV infection is now recognized to play a role in the pathogenesis of OPSCC, where HPV+ and HPV- patients appear to be clinically and biologically distinct with reported genome-wide hypomethylation and promoter hypermethylation in HPV+ HNSCC tumors. A recent study from our group applying pathway analysis to investigate the biological role of the differentially methylated genes in HPV+ and HPV- HNSCC reported 8 signal transduction pathways germane to HNSCC (Worsham et al., Otolaryngol Head Neck Surg 149:409-416, 2013).

A tissue field of somatic genetic alterations precedes the histopathological phenotypic changes of carcinoma. Genomic changes could be of potential use in the diagnosis and prognosis of pre-invasive squamous head and neck carcinoma (HNSCC) lesions and as markers for cancer risk assessment. Studies of sequential molecular alterations and genetic progression of pre-invasive HNSCC have not been clearly defined. Studies have shown recurring alterations at chromosome 9p21 (location of the CDKN2A) and TP53 mutations in the early stages of HNSCC. However, gene silencing via hypermethylation is still a relatively new idea in the development of HNSCC and little is known about the contribution of epigenetics to the development of neoplasia, its transformation, progression, and recurrence in HNSCC. This review examines the role of promoter hypermethylation of tumor suppressor genes in the progression continuum from benign papillomas to malignancy in HNSCC., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Objective: Human papilloma virus (HPV) positive and HPV negative head and neck squamous cell cancer (HNSCC) are biologically distinct with a prognostic advantage for HPV positive patients compared to HPV negative cases. DNA promoter methylation is central to human diseases such as cancer, including HNSCC, with reported genome-wide hypomethylaton and promoter hypermethylation in HPV positive HNSCC tumors. The goal of this study was to identify differentially methylated genes in HPV positive versus HPV negative primary HNSCC genomes with clues to signaling networks., Study Design: Laboratory-based study., Setting: Primary care academic health care system., Subjects and Methods: DNA from 4 HPV positive and 4 HPV negative freshly frozen primary HNSCC were subject to comprehensive genome-wide methylation profiling. Differentially methylated gene lists were examined using the Signal Transduction Pathways (canonical) filter in the Genomatix Pathway System (GePS)., Results: Twofold methylation differences were observed between HPV positive and HPV negative cases for 1168 genes. Pathway analysis applied to investigate the biological role of the 1168 differentially methylated genes revealed 8 signal transduction pathways forming a network of 66 genes, of which 62% are hypermethylated., Conclusion: Our study reveals a predominant hypermethylation profile for genes in signal transduction pathways of HPV positive HNSCC tumor genomes. Because signaling events in the cell play a critical role in the execution of key biological functions, insights into how complex cellular signaling cascades and networks may be programmed in HNSCC are likely to be critical in the development of new biological agents designed to hit multiple targets.

Purpose: A major limitation of studies reporting a lower prevalence rate of human papilloma virus (HPV) in African American patients with oropharyngeal squamous cell cancer (OPSCC) than Caucasian Americans, with corresponding worse outcomes, was adequate representation of HPV-positive African American patients. This study examined survival outcomes in HPV-positive and HPV-negative African Americans with OPSCC., Experimental Design: The study cohort of 121 patients with primary OPSCC had 42% African Americans. Variables of interest included age, race, gender, HPV status, stage, marital status, smoking, treatment, and date of diagnosis., Results: Caucasian Americans are more likely to be HPV positive (OR = 3.28; P = 0.035), as are younger age (age < 50 OR = 7.14; P = 0.023 compared with age > 65) or being married (OR = 3.44; P = 0.016). HPV positivity and being unmarried were associated with being late stage (OR = 3.10; P = 0.047 and OR = 3.23; P = 0.038, respectively). HPV-negative patients had 2.7 times the risk of death as HPV-positive patients (P = 0.004). Overall, the HPV-race groups differed (log-rank P < 0.001), with significantly worse survival for HPV-negative African Americans versus (i) HPV-positive African Americans (HR = 3.44; P = 0.0012); (ii) HPV-positive Caucasian Americans (HR = 3.11; P = < 0.049); and (iii) HPV-negative Caucasian Americans (HR = 2.21; P = 0.049)., Conclusions: HPV has a substantial impact on overall survival in African American patients with OPSCC. Among African American patients with OPSCC, HPV-positive patients had better survival than HPV negative. HPV-negative African Americans also did worse than both HPV-positive Caucasian Americans and HPV-negative Caucasian Americans. This study adds to the mounting evidence of HPV as a racially linked sexual behavior life style risk factor impacting survival outcomes for both African American and Caucasian American patients with OPSCC., (©2013 AACR.)

Objective: The goal was to determine the prevalence of high-risk HPV16 using saliva in a screening population in Detroit, Michigan., Materials and Methods: Real-time quantitative polymerase chain reaction was applied to detect HPV16 in saliva DNA from 349 screening subjects without head and neck cancer (HNC), 156 with HNC, and 19 controls. Cut points for human papilloma virus (HPV) positivity were >0 and >0.001 copy/cell. Proportions were compared between groups using exact χ(2) or Fisher exact tests (P < .05)., Results: At a cut point >0, each group had an overall HPV prevalence of more than 5%, with a higher prevalence of 30.8% in the HNC patient group. At a cut point >0.001, the prevalence was lower: 0% in the control, 1.2% in the screening, and 16.7% in the HNC group. In the latter, for both cut points, HPV prevalence was different across sites (<0.001) and significantly higher in the oropharynx than larynx or site as other after Hochberg's adjustment. At >0, women in the screening group had a higher prevalence of HPV than did men (P = .010), and at >0.001, the prevalence was higher for men in the HNC group than for women (P = .035). In the screening group, at >0, only African Americans had a higher prevalence than Caucasian Americans (P = .025)., Conclusions: In the screening group, a 6.9% and 1.2% screening rate was noted at cut points >0 and >0.001, respectively. The results provide data to inform public health considerations of the feasibility of saliva as a screening tool in at-risk populations with the long-term goal of prophylactic vaccination against oral HPV.

Expression of p16 INK4A (p16 positive) is highly correlated with human papilloma virus (HPV) infection in head and neck squamous cell carcinoma (HNSCC), however, p16-positivity is not limited to HPV positive tumors and therefore, not a perfect surrogate for HPV. p16 survival outcomes are best documented for the oropharyngeal site (OP); non-OP sites such as the oral cavity (OC), larynx, and hypopharynx (HP) are understudied. The goal of this study was to evaluate p16 in the context of HPV16 and examine p16 survival outcomes in HPV16 positive and HPV16 negative site-specific HNSCC. p16 and HPV16 status were determined by immunohistochemistry and qPCR respectively, on 80 primary HNSCC from four sites: OC, OP, larynx and HP. p16 expression was different across sites (p<0.001), was more frequent in OP than non-OP sites (p<0.0001), and was different between Caucasian Americans (CA) and African Americans (AA) (p=0.031), similar to HPV (p=0.013). p16 was associated with marital status (p=0.008) and smoking (p=0.014). p16 positive patients had improved survival (similar to HPV16 positive cases). Patients with p16 negative/HPV16 negative status had the worst survival for all sites combined as well as for OP. p16 status is an important prognostic indicator in both OPSCC and non-OPSCC and the p16 positive/HPV16 negative group is likely a distinct subgroup lacking any HPV genotype. Cohorts with larger representations of non-OP sites examining multiple molecular markers will be key to deciphering and dissecting out p16's role as a useful prognostic indicator when assessed in combination with HPV status.

Objective: There is a lack of consensus regarding the causes of the differences in the higher incidence of and the mortality from head and neck squamous cell carcinoma (HNSCC) in African Americans (AA) versus Caucasian Americans (CA). We examined a comprehensive array of risk factors influencing health and disease in an access-to-care, racially diverse, primary HNSCC cohort., Study Design: Cross-sectional study., Setting: Primary care academic health care system., Subjects and Methods: The cohort of 673 patients comprised 391 CA and 282 AA (42%). Risk variables included demographic, histopathology, and clinical/epidemiologic factors. Tumor DNA was interrogated for loss and gain of 113 genes with known involvement in HNSCC/cancer. Logistic regression for univariate analysis was followed by multivariate modeling with determination of model predictability (c-index)., Results: Of the 39 univariate differences between AA and CA, multivariate modeling (c-index = 0.81) retained 7 differences (P < .05). AA were less likely to be married and more likely to have tumor lymphocytic response, undergo radiation treatment, and smoke. Insurance type was a significant predictor of race. AA were more likely to have Medicaid, Medicare, and other HMO types. AA tumors were more likely to have loss of CDKN2A and gain of SCYA3 versus CA., Conclusions: Multivariate modeling indicated significant differences between AA and CA HNSCC for histopathology, treatment, smoking, marital status, type of insurance, 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.

Objective: Human papillomavirus (HPV), particularly HPV16, is a causative agent for 25% of head and neck squamous cell cancer, including laryngeal squamous cell cancer (LSCC). HPV-positive (HPV+ve) patients, particularly those with oropharyngeal SCC, have improved prognosis. For LSCC patients, this remains to be established. The goal was to determine stage and survival outcomes in LSCC in the context of HPV infection., Study Design: Historical cohort study., Setting: Primary care academic health system., Subjects and Methods: In 79 patients with primary LSCC, HPV was determined using real-time quantitative polymerase chain reaction. χ(2) or Fisher exact test was used to test the association of HPV+ve with 21 risk factors including race, stage, gender, age, smoking, alcohol, treatment, and health insurance. Kaplan-Meier and log-rank tests were used to study the association of HPV and LSCC survival outcome., Results: HPV16 was detected in 27% of LSCC patients. Caucasian American (CA) patients had higher HPV prevalence (33%) than did African American (AA) LSCC patients (16%; P = .058). HPV was significantly associated with gender (P = .016) and insurance type (P = .001). There were no differences in survival between HPV+ve and HPV-negative (HPV-ve) patients. There was no association with HPV and other risk factors including stage (early vs late)., Conclusion: We found a high prevalence of HPV in men and a lower prevalence of HPV infection in AA compared with CA. Despite the strikingly better survival of patients with HPV+ve oropharyngeal tumors, even when adjusted for smoking, this correlation does not seem to hold true in the larynx. Larger multiethnic LSCC cohorts are needed to more clearly delineate HPV-related survival across ethnicities.

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.

Genetic events specific to the pathogenesis of malignancy can offer clues to the tumorigenesis process. The objective of this study was to identify gene alterations that differentiate tumor and nontumor lesions in squamous head and neck cancer (HNSCC). DNA from 220 primary HNSCC with concurrently present tumor and nontumor lesions from the same patient was interrogated for genomic alterations of loss or gain of copy. Conditional logistic regression dealt with tumor and non-tumor records within a patient. Of 113 genes, 53 had univariate effects (P < 0.01), of which 16 genes remained in the multivariable model with P < 0.01. The model had a C-index (ROC) of 0.93. Loss of CDKN2B and gain of BCL6, FGF3, and PTP4A3 predicted tumor. Loss of BAK1 and CCND1 and gain of STCH predicted nontumor. This highly powered model assigned alterations in 16 genes specific for malignant versus nonmalignant lesions, supporting their contribution to the pathogenesis of HNSCC as well as their potential utility as relevant targets for further evaluation as markers of early detection and progression.

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 model's 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.

Thyroid cancer is the most common endocrine cancer with 1,690 deaths each year. There are four main types of which the papillary and follicular types together account for >90% followed by medullary cancers with 3% to 5% and anaplastic carcinomas making up <3%. Epigenetic events of DNA hypermethylation are emerging as promising molecular targets for cancer detection. Our immediate and long term goal is to identify DNA methylation markers for early detection of thyroid cancer. This pilot study comprised of 21 patients to include 11 papillary thyroid cancers (PTC), 2 follicular thyroid cancers (FTC), 5 normal thyroid cases, and 3 hyperthyroid cases. Aberrant promoter methylation was examined in 24 tumor suppressor genes using the methylation specific multiplex ligation-dependent probe amplification (MS-MLPA) assay and in the NIS gene using methylation-specific PCR (MSP). The frequently methylated genes were CASP8 (17/21), RASSF1 (16/21) and NIS (9/21). In the normal samples, CASP8, RASSF1 and NIS were methylated in 5/5, 4/5 and 1/5 respectively. In the hyperthyroid samples, CASP8, RASSF1 and NIS were methylated in 3/3, 2/3 and 1/3 respectively. In the thyroid cancers, CASP8, RASSF1, and NIS were methylated in 9/13, 10/13, and 7/13 respectively. CASP8, RASSF1 and NIS were also methylated in concurrently present normal thyroid tissue in 3/11, 4/11 and 3/11 matched thyroid cancer cases (matched for presence of both normal thyroid tissue and thyroid cancer), respectively. Our data suggests that aberrant methylation of CASP8, RASSF1, and NIS maybe an early change in thyroid tumorigenesis regardless of cell type.

Aberrant methylation of promoter CpG islands is a hallmark of human cancers and is an early event in carcinogenesis. We examined whether promoter hypermethylation contributes to the pathogenesis of benign breast lesions along a progression continuum to invasive breast cancer. The exploratory study cohort comprised 17 breast cancer patients with multiple benign and/or in situ lesions concurrently present with invasive carcinoma within a tumor biopsy. DNA from tumor tissue, normal breast epithelium when present, benign lesions (fibroadenoma, hyperplasia, papilloma, sclerosing adenosis, apocrine metaplasia, atypical lobular hyperplasia or atypical ductal hyperplasia), and in situ lesions of lobular carcinoma and ductal carcinoma were interrogated for promoter methylation status in 22 tumor suppressor genes using the multiplex ligation-dependent probe amplification assay (MS-MLPA). Methylation specific PCR was performed to confirm hypermethylation detected by MS-MLPA. Promoter methylation was detected in 11/22 tumor suppressor genes in 16/17 cases. Hypermethylation of RASSF1 was most frequent, present in 14/17 cases, followed by APC in 12/17, and GSTP1 in 9/17 cases with establishment of an epigenetic monocloncal progression continuum to invasive breast cancer. Hypermethylated promoter regions in normal breast epithelium, benign, and premalignant lesions within the same tumor biopsy implicate RASSF1, APC, GSTP1, TIMP3, CDKN2B, CDKN2A, ESR1, CDH13, RARB, CASP8, and TP73 as early events. DNA hypermethylation underlies thepathogenesis of step-wise transformation along a monoclonal continuum from normal to preneoplasia to invasive breast cancer.

The goal was to determine recurrent or second primary status for late stomal malignancies, 16 and 17 years post-total laryngectomy in two laryngeal squamous cell carcinoma (LSCC) patients, based on DNA methylation signatures and HPV typing. Adopting a literature review based definition of late stomal recurrences as new primaries at the site of the stoma or neopharynx occurring >5 years after total laryngectomy, we employed a multi-gene candidate approach to examine promoter methylation in 24 tumor suppressor genes and PCR-based assays for HPV status offered additional insights into whether the late stomal tumors post-total laryngectomy were related or not. The primary tumor for Patient 1 was negative for HPV but had aberrant hypermethylation of APC, MLH1 and BRCA1. The stomal biopsy 17-years later showed presence of HPV-16 without any methylated genes. In Patient 2, HPV-11 and promoter methylation of APC identified in the primary tumor was also observed in the stomal malignancy 16 years post-total laryngectomy. Additional information provided by molecular typing for HPV and methylation markers underscored Patient 1's and 2's late stomal presentation as most likely a second primary and recurrence, respectively. DNA methylation markers are particularly advantageous because DNA methylation is an early event in tumorigenesis, and the epigenetic modification, 5-methylcytosine, is a stable marker. Molecular marks to discern genetic heterogeneity or relatedness of stomal malignancies several years post-total laryngectomy can provide clues to their status as either second primaries or likely recurrences. Our results support the hypothesis that a subset of stomal recurrences after total laryngectomy represents second primary tumors.

This study examined molecular (DNA hypermethylation), clinical, histopathological, demographical, smoking, and alcohol variables to assess diagnosis (early versus late stage) and prognosis (survival) outcomes in a retrospective primary laryngeal squamous cell carcinoma (LSCC) cohort. The study cohort of 79 primary LSCC was drawn from a multi-ethnic (37% African American), primary care patient population, diagnosed by surgical biopsies in the Henry Ford Health System from 1991-2004, and followed from 5-18 years (through 2009). Of the 41 variables, univariate risk factors of p<0.10 were tested in multivariate models (logistic regression {diagnosis} and Cox {survival} models {p<0.05}). Aberrant methylation of ESR1 (p=0.01), race as African American (p=0.04), and tumor necrosis (extensive) (p=0.02) were independent predictors of late stage LSCC. Independent predictors of poor survival included presence of vascular invasion (p=0.0009), late stage disease (p=0.03) and methylation of the HIC1 gene (p=0.0002). Aberrant methylation of ESR1 and HIC1 signified independent markers of poorer outcome. In this multi-ethnic, primary LSCC cohort, race remained a predictor of late stage disease supporting disparate diagnosis outcomes for African American patients with LSCC.

Objective: Promoter hypermethylation is emerging as a promising molecular strategy for early detection of cancer. We examined promoter methylation status of 1143 cancer-associated genes to perform a global but unbiased inspection of methylated regions in head and neck squamous cell carcinoma (HNSCC)., Study Design: Laboratory-based study., Setting: Integrated health care system., Subjects and Methods: Five samples, two frozen primary HNSCC biopsies and three HNSCC cell lines, were examined. Whole genomic DNA was interrogated using a combination of DNA immunoprecipitation (IP) and Affymetrix whole-genome tiling arrays., Results: Of the 1143 unique cancer genes on the array, 265 were recorded across five samples. Of the 265 genes, 55 were present in all five samples, and 36 were common to four of five samples, 46 to three of five, 56 to two of five, and 72 to one of five samples. Hypermethylated genes in the five samples were cross-examined against those in PubMeth, a cancer methylation database combining text mining and expert annotation (http://www.pubmeth.org). Of the 441 genes in PubMeth, only 33 are referenced to HNSCC. We matched 34 genes in our samples to the 441 genes in the PubMeth database. Of the 34 genes, eight are reported in PubMeth as HNSCC associated., Conclusion: This pilot study examined the contribution of global DNA hypermethylation to the pathogenesis of HNSCC. The whole-genome methylation approach indicated 231 new genes with methylated promoter regions not yet reported in HNSCC. Examination of this comprehensive gene panel in a larger HNSCC cohort should advance selection of HNSCC-specific candidate genes for further validation as biomarkers in HNSCC., (2010 American Academy of Otolaryngology-Head and Neck Surgery Foundation. Published by Mosby, Inc. All rights reserved.)

INTRODUCTION: This study examined the contribution of promoter hypermethylation to the pathogenesis of respiratory papillomatosis (RP), including recurrences (RRP) and progression to squamous cell carcinoma (SSC). MATERIALS AND METHODS: A retrospective cohort of 25 laryngeal papilloma cases included 21 RRP, two of which progressed to SCC. Aberrant methylation status was determined using the multi-gene (22 tumor suppressor genes) methylation-specific multiplex ligation-dependent probe amplification assay and confirmed using methylation specific PCR. RESULTS: Twenty genes had altered DNA methylation in 22 of 25 cases. Aberrant methylation of CDKN2B and TIMP3 was most frequent. Promoter hypermethylation of BRCA2, APC, CDKN2A and CDKN2B was detected in 2 RRP cases with subsequent progression to SCC. Of the 25 cases, 22 were positive for HPV-6, 2 for HPV-11 and 1 for HPV-16 and 33. CONCLUSIONS: Consistent aberrant methylation of multiple tumor suppressor genes contributes to the pathogenesis of laryngeal papillomas. Persistent aberrant DNA methylation events in 2 RRP cases that progressed to cancer indicate an epigenetic monoclonal progression continuum to SCC.

Gene silencing through promoter hypermethylation is a growing concept in the development of human cancers. In this study, we examined the contribution of aberrant methylation of promoter regions in methylation-prone tumor suppressors to the pathogenesis of vulvar cancer. Thirteen cell lines from 12 patients with squamous cell carcinoma of the vulva were evaluated for aberrant methylation status and gene copy number alterations, concomitantly, using the methylation-specific multiplex ligation-dependent probe amplification assay. Of the 22 tumor suppressor genes examined, aberrant methylation was observed for 9 genes: tumor protein p73 (TP73), fragile histidine triad (FHIT), von Hippel-Lindau (VHL), adenomatosis polyposis coli (APC), estrogen receptor 1 (ESR1), cyclin-dependent kinase inhibitor 2B (CDKN2B), death-associated protein kinase 1 (DAPK1), glutathione S-transferase pi (GSTP1), and immunoglobin superfamily, member 4 (IGSF4). The most frequently methylated genes included TP73 in 9 of 13 cell lines, and IGSF4, DAPK1, and FHIT in 3 of 13 cell lines. Methylation-specific polymerase chain reaction was performed for TP73 and FHIT to confirm aberrant methylation by methylation-specific multiplex ligation-dependent probe amplification. In the context of gene copy number and methylation status, both copies of the TP73 gene were hypermethylated. Loss or decreased mRNA expression of TP73 and IGSF4 by reverse transcription polymerase chain reaction confirmed aberrant methylation. Frequent genetic alterations of loss and gain of gene copy number included gain of GSTP1 and multiple endocrine neoplasia type 1 (MEN1), and loss of malignant fibrous histiocytoma amplified sequence 1 (MFHAS1) and IGSF4 in over 50% of the squamous cell carcinoma of the vulva cell lines. These findings underscore the contribution of both genetic and epigenetic events to the underlying pathogenesis of squamous cell carcinoma of the vulva.

Objective: To examine epigenetic events of aberrant promoter methylation as diagnostic markers in primary head and neck squamous cell carcinoma using a novel multigene approach. Promoter methylation-mediated silencing is a hallmark of several established tumor suppressor genes. Changes in DNA methylation have been reported to occur early in carcinogenesis and therefore are potentially important early indicators of existing disease., Design: A multicandidate gene probe panel interrogated DNA for aberrant methylation status in 22 cancer genes using the methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay. Aberrant promoter hypermethylation was confirmed using methylation-specific polymerase chain reaction after bisulfite treatment., Setting: Primary care medical center., Subjects: We examined fresh-frozen primary head and neck tumor specimens from 28 patients, including 21 late-stage (19 stage IV and 2 stage III) and 7 early-stage (6 stage II and 1 stage I) tumors., Results: Promoter hypermethylation was observed in 14 of the 28 patients (50%). Genes for RARB, APC, and CHFR were most frequently hypermethylated, occurring in 11 (39%) for RARB, 7 (25%) for CHFR, and 6 (21%) for APC. Aberrant methylation of CHFR was solely a stage IV event. Methylation-specific polymerase chain reaction after bisulfite treatment with conventional and real-time polymerase chain reaction confirmed aberrant methylation for RARB and CHFR., Conclusions: Promoter methylation profiling of primary head and neck squamous cell carcinoma using multiple target genes identified RARB, APC, and CHFR as frequent epigenetic events. The clinical implications of these genes as diagnostic and treatment biomarkers are highly relevant as attractive targets for cancer therapy, given the reversible nature of epigenetic gene silencing.

Benign inverted papillomas have been reported as monoclonal but lacking common genetic alterations identified in squamous cell carcinoma of the head and neck. Epigenetic changes alter the heritable state of gene expression and chromatin organization without change in DNA sequence. We investigated whether epigenetic events of aberrant promoter hypermethylation in genes known to be involved in squamous head and neck cancer underlie the pathogenesis of sinonasal papillomas. Ten formalin-fixed paraffin DNA samples from three inverted papilloma cases, two exophytic (everted) papilloma cases, and two cases with inverted and exophytic components were studied. DNA was obtained from microdissected areas of normal and papilloma areas and examined using a panel of 41 gene probes, designed to interrogate 35 unique genes for aberrant methylation status (22 genes) using the methylation-specific multiplex-ligation-specific polymerase assay. Methylation-specific PCR was employed to confirm aberrant methylation detected by the methylation-specific multiplex-ligation-specific polymerase assay. All seven cases indicated at least one epigenetic event of aberrant promoter hypermethylation. The CDKN2B gene was a consistent target of aberrant methylation in six of seven cases. Methylation-specific PCR confirmed hypermethylation of CDKN2B. Recurrent biopsies from two inverted papilloma cases had common epigenetic events. Promoter hypermethylation of CDKN2B was a consistent epigenetic event. Common epigenetic alterations in recurrent biopsies underscore a monoclonal origin for these lesions. Epigenetic events contribute to the underlying pathogenesis of benign inverted and exophytic papillomas. As a consistent target of aberrant promoter hypermethylation, CDKN2B may serve as an important epigenetic biomarker for gene reactivation studies.

Objective: To investigate the contribution of promoter methylation-mediated epigenetic events in recurrent respiratory papillomatosis tumorigenesis., Design: Archival tissue DNA, extracted from microdissected papilloma lesions, was interrogated for methylation status by means of the novel, multigene methylation-specific multiplex ligation-dependent probe amplification assay., Subjects: Fifteen subjects with recurrent respiratory papillomatosis, 3 females and 12 males, all with adult onset of illness (age range, 23-73 years) except for 1 female patient with juvenile onset (1 year old)., Results: Promoter hypermethylation was recorded in 14 of 15 cases, and 19 of 22 unique methylation-prone cancer genes in the multigene panel had altered DNA methylation in at least 1 laryngeal papilloma biopsy specimen. Identical abnormally methylated genes were found in 5 of 15 recurrent cases, of which the CDKN2B gene was hypermethylated in all 5 cases. Dissimilar epigenetic events were noted in the remaining cases., Conclusions: A clonal origin was derived for 5 of 15 recurrent respiratory papillomatosis biopsy specimens based on identical epigenetic events. The high frequency of epigenetic events, characterized by consistent promoter hypermethylation of multiple tumor suppressor genes, points to the use of gene silencing mechanisms in the pathogenesis of recurrent respiratory papillomatosis.