Abstract IA18: Tumor suppressor gene silencing by somatic mutations and promoter methylation leads to genomic instability in head and neck squamous cell carcinoma
Authors: Guerrero-Preston Rafael 
Published: October 2015

For years studies have attempted to come up with measures and markers that predict and are linked to cancer survival. Alterations in a subgroup of genes, tumor suppressor genes (TSG), have recently emerged as important in linkage to cancer survival, particularly the silencing that can occur by somatic mutations and promoter DNA methylation. However, the link between TSG silencing and cancer survival is not well understood. If we can understand how TSG silencing is molecularly linked to cancer survival we can improve clinical management and pave the wave for personalized treatment solutions. Head and neck squamous cell carcinoma (HNSCC) is one of the first tumors in which independent somatic mutations and promoter DNA methylation events were shown to inactivate the same gene, CDKN2a, a well-known TSG. HNSCC is also a tumor in which the linkage between causative agents, molecular alterations, and signaling pathways has been studied. A better understanding of the link between survival and TSG silencing will lead to improvements in clinical management and outcomes of HNSCC patients, across ethnic and racial groups.

Black American men and men in Puerto Rico have a higher HNSCC burden than Non-Latino White American (NLW) men, but the biological basis for these differences are poorly understood. Persons of African ancestry have a higher incidence of oral cavity and pharynx tumors compared to persons of European ancestry (NCI SEER: age-adjusted incidence rates 1975-2009: 13.4 vs. 11.9 per 100,000, respectively). Similarly for larynx cancers, the incidence is higher among Blacks compared to NLW (NCI SEER: age-adjusted incidence rates 1975-2009: 6.8 vs. 4.4 per 100,000 respectively). Moreover, the gap in 5-year relative survival rates in oral cavity and pharynx cancer between NLW and Blacks has increased from 18% to 22% from 1975 to 2008. Notably, Black patients consistently have lower five-year survival rates as compared to NLW patients (all HNSCC sites: 41% vs 60.8%, oral cavity and pharynx: 45% vs. 67%, larynx: 51% vs. 65%). Moreover, the gap in 5-year relative survival rates in oral cavity and pharynx cancer of NLWs and Blacks has increased from 18% to 22% from 1975 to 2008. In addition, the Annual Percentage Change in oral and pharyngeal cancer mortality rates between 1998-2002 for men in Puerto Rico was higher (1.2 x 100,000) than for NLW (-2.1 x 100,000), Latinos in US (-6.3 x 100,000) and Blacks (-6.5 x 100,000). The incidence of soft palate/oropharyngeal cancer among men in Puerto Rico was 2.8 times higher than in Latino men in US (p&lt;0.05) and about 1.4 times higher than in NLW men but 21% lower than in Black men (p&gt;0.05) during the same period.

Most HNSCC cases are attributable to smoking and alcohol consumption. The incidence of HNSCC overall has been decreasing over the past 30 years due to a reduction in the use of tobacco. However, there has been a dramatic increase in the incidence rates of oropharyngeal (OPSCC) cancers because of infection with high-risk human papillomavirus (HPV). The main molecular determinants in HNSCC are the abrogation of p53 and retinoblastoma (pRb) pathways that lead to uncontrolled cell replication. The current molecular classification distinguishes only HPV-positive and HPV-negative HNSCC tumors. HPV-positive HNSCC have different patterns of genomic and epigenomic alterations, when compared to HPV negative HNSCC. Both tumor types exhibit alterations in TSG pathways, caused either by viral oncogenes or somatic mutations, which lead to genomic instability, uncontrolled cell replication and aberrant survival signaling. Mutations in TP53, EGFR-MEK, NOTCH, PI3K, PTEN and AKT pathways are frequently observed in HPV-negative HNSCC. These mutations cooperate to create aberrant mitogenic/survival signaling. No TP53 mutations were seen in HPV-positive HNSCC on exome sequencing, and the overall mutation rate was approximately half of that seen in HPV-negative samples. In addition, in contrast to HPV-negative tumors, the expression of CDKN2A, encoding p16INK4A, is highly upregulated and amplification of cyclin D is infrequent in HPV-positive HNSCC.

Survival rates for HPV-negative and HPV-positive HNSCC patients are strikingly different. Molecular alterations linked with HNSCC survival, such as TP53 mutations, loss of heterozygosity, microsatellite instability, and differential DNA methylation are associated with exposure to cigarette smoke and alcohol consumption in HPV-negative HNSCC. Most HPV-positive HNSCC patients show superior responses to chemotherapy and radiotherapy, and are less likely to be diagnosed in Black patients. Together this evidence suggests that diverse genomic and epigenomic landscapes arise in response to distinct exposure gradients in HNSCC leading to distinct entities with poorer survival. HNSCC has long been known to be a disease of smokers and older men. Recently, the number of younger never-smokers developing HNSCC has increased and is now considered a distinct clinical and molecular entity. HPV infection does not explain the rising incidence trend of never-smokers with oral tongue squamous cell carcinoma, whom are typically HPV-negative. The majority of HPV-negative HNSSC patients with smoking history usually have a higher genome-wide burden of mutations in TSG and poorer survival rates than never smokers. However, survival is poorer in never-smokers rather than in smokers with oral tongue HPV-negative tumors, a distinct new oral cavity HNSCC entity that is not clinically or molecularly understood.

We recently uncovered 186 downregulated genes harboring cancer specific promoter methylation including PAX1 and PAX5 and we identified 10 key tumor suppressor genes (GABRB3, HOXC12, PARP15, SLCO4C1, CDKN2A, PAX1, PIK3AP1, HOXC6, PLCB1 and ZIC4) inactivated by both promoter methylation and/or somatic mutation in HNSCC. We also found evidence of interplay between mutations in the TP53 and NOTCH1 pathways with gene downregulation associated to PAX5 and PAX1 promoter methylation in HNSCC. Survival analyses revealed that patients with PAX5 (p= 0.001) or NID2 (p=0.05) promoter methylation had a worse outcome than those without it. In subsequent analyses of TCGA data we found that combined somatic TP53 mutations and PAX5 promoter methylation are linked to worse outcomes when compared to patients with either alteration alone (p&lt;0.0001). The next step is to examine the association between survival and combined somatic TP53 mutations and PAX5 promoter methylation in the major HNSCC anatomic subsites: oral cavity, oropharynx and pharynx. This research will provide an understanding of sub-site specific molecular and pathways alterations linked to HNSCC survival, and a blueprint for personalized therapy solutions.

Genomic and post-genomic research has shifted to focus on biomarker discovery for diagnosis, prognosis and prediction of treatment response, alongside the development of targeted therapies, with the ultimate goal of creating early detection and personalized therapy for HNSCC. Genetic, epigenetic and molecular advances are revealing new links between TSG silencing and survival in pathways involved in the development and progression of HNSCC. These advances are creating new opportunities to enable increased treatment choices and reduce survival disparities in HNSCC. Similar approaches are being implemented in other tumors with genome instability that affect underserved populations.

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Citation Format: Rafael Guerrero-Preston. Tumor suppressor gene silencing by somatic mutations and promoter methylation leads to genomic instability in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr IA18.