Josena K. Stephen

Epigenetic Modulation of Signal Transduction Pathways in HPV-Associated HNSCC

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.

Human papilloma virus prevalence in a multiethnic screening population.

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 χ2 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.

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.

Molecular characterization of late stomal recurrence following total laryngectomy

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 1s and 2s 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.

Methylation Markers for Early Detection and Differentiation of Follicular Thyroid Cancer Subtypes

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.

Human Papilloma Virus (HPV) Modulation of the HNSCC Epigenome

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).

The biological significance of methylome differences in human papilloma virus associated head and neck cancer

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. IPAs 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.

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.