Expression of p16INK4A (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.

Purpose A major limitation of studies reporting a lower prevalence rate of human papilloma virus (HPV) in African American (AA) oropharyngeal cancer (OPSCC) patients than Caucasian Americans (CA), with corresponding worse outcomes, was adequate representation of HPV positive AA patients. This study examined survival outcomes in HPV positive and HPV negative AA with OPSCC Experimental Design The study cohort of 121 primary OPSCC had 42% AA. Variables of interest included age, race, gender, HPV status, stage, marital status, smoking, treatment, and date of diagnosis. Results CA 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 to 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 AA vs 1) HPV positive AA (HR=3.44, p=0.0012); 2) HPV positive CA (HR=3.11, p=<0.049); and 3) HPV negative CA (HR=2.21, p=0.049). Conclusions HPV has a substantial impact on overall survival in AA OPSCC. Among AA OPSCC, HPV positive patients had better survival than HPV negative. HPV negative AA also did worse than both, HPV positive CA and HPV negative CA. This study adds to the mounting evidence of HPV as a racially-linked sexual behavior life style risk factor impacting survival outcomes for both AA and CA OPSCC patients.

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 ligationdependent 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.

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 oropharyngeal SCC, have improved prognosis. For LSCC, 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 primary LSCC, HPV was determined using real-time quantitative PCR. Chi-square or Fisher’s exact test was used to test association of HPV+ve with 21 risk factors including race, stage, gender, age, smoking, alcohol, treatment, and health insurance. Kaplan-Meier and log rank test were used to study the association of HPV and LSCC survival outcome. Results HPV16 was detected in 27% LSCC. There was a trend towards higher HPV prevalence in Caucasian American (CA, 33%) vs African American (AA, 16%) (p=0.058). HPV was significantly associated with gender (p=0.016) and insurance type (p=0.001). HPV+ve LSCC had a slightly longer survival than HPV-negative (HPV−ve) patients, but the differences were not significant. There was no association with HPV and other risk factors including stage (early vs late). Conclusion We found high prevalence of HPV in males and lower prevalence of HPV infection in AA compared to CA. A slightly better survival for HPV+ve LSCC versus HPV−ve was noted but was not significant. Larger multi ethnic LSCC cohorts are needed to more clearly delineate HPV related survival across ethnicities.

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 methylationspecific 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 can

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 preinvasive 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.

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. Methylationspecific PCR was employed to confirm aberrant methylation detected by the methylation-specific multiplexligation-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.

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.