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.

To examine the promoter methylation status of the 22 cancer genes and their contribution to disease progression in 6 head and neck squamous cell carcinoma (HNSCC) cell lines. Design: A panel of 41 gene probes, designed to interrogate 35 unique genes with known associations to cancer including HNSCC, was interrogated for alterations in gene copy number and aberrant methylation status (22 genes) using the methylation-specific multiplex ligationdependent probe amplification assay.

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.

Objective: To identify the extent and the smallest region of loss for CDKN2B INK4b , CDKN2A ARF,INK4a , and MTAP. Homozygous deletions of human chromosome 9p21 occur frequently in malignant cell lines and are common in squamous cell carcinoma of the head and neck (HNSCC). This complex region encodes the tumor suppressor genes cyclin-dependent kinase 2B (CDKN2B) (p15 INK4b ) and CDKN2A (p14 ARF , p16 INK4a ) and the housekeeping gene methylthioadenosine phosphorylase (MTAP).Design: A targeted probe panel designed to finely map the region of 9p21 loss comprised 3 probes for CDKN2B INK4b , 7 for CDKN2A ARF, INK4a , and 3 for MTAP and was interrogated using the multiplex ligationdependent probe amplification assay (MLPA). The MLPA genomic copy number alterations for CDKN2A were validated using real-time polymerase chain reaction. Subjects: Six HNSCC primary (A) and recurrent or metastatic (B) cell lines were examined: UMSCC-11A/11B, UMSCC-17A/17B, and UMSCC-81A/81B. Results: Cell line UMSCC-11B retained all 9p loci tested in the region. Cell lines UMSCC-17A/B indicated homozygous deletion of CDKN2A ARF, INK4a starting at p16 INK4 exon 1␣ to include exons 2 and 3. Homozygous loss was indicated for CDKN2B INK4b and CDKN2A ARF,INK4a in UMSCC-11A, and UMSCC-81A. Cell line UMSCC-81B indicated retention of all 9p loci except for exon 1␣ (p16 INK4a ). Selective loss of the 3Ј end of MTAP was observed in UMSCC-11A. Genomic alterations by fine-mapping MLPA were validated at the DNA level for CDKN2A.Conclusions: We identified exon 1␣ (p16 INK4a ) as the smallest region of loss in the CDKN2A ARF, INK4a gene. The frequency and precise loss of CDKN2B INK4b , CDKN2A ARF, INK4a , and MTAP in the prognosis of 9p21-deleted HNSCC may provide impetus for use of these targets as therapeutic biomarkers in head and neck cancer.

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.