Julie Ahn

NF-κB and STAT3 Transcription Factor Signatures Differentiate HPV-positive and HPV-negative Head and Neck Squamous Cell Carcinoma

Using high‐throughput analyses and the TRANSFAC database, we characterized TF signatures of head and neck squamous cell carcinoma (HNSCC) subgroups by inferential analysis of target gene expression, correcting for the effects of DNA methylation and copy number. Using this discovery pipeline, we determined that human papillomavirus‐related (HPV+) and HPV− HNSCC differed significantly based on the activity levels of key TFs including AP1, STATs, NF‐κB and p53. Immunohistochemical analysis confirmed that HPV− HNSCC is characterized by co‐activated STAT3 and NF‐κB pathways and functional studies demonstrate that this phenotype can be effectively targeted with combined anti‐NF‐κB and anti‐STAT therapies. These discoveries correlate strongly with previous findings connecting STATs, NF‐κB and AP1 in HNSCC. We identified five top‐scoring pair biomarkers from STATs, NF‐κB and AP1 pathways that distinguish HPV+ from HPV− HNSCC based on TF activity and validated these biomarkers on TCGA and on independent validation cohorts. We conclude that a novel approach to TF pathway analysis can provide insight into therapeutic targeting of patient subgroup for heterogeneous disease such as HNSCC.

Outlier analysis defines zinc finger gene family DNA methylation in tumors and saliva of head and neck cancer patients

Head and Neck Squamous Cell Carcinoma (HNSCC) is the fifth most common cancer, annually affecting over half a million people worldwide. Presently, there are no accepted biomarkers for clinical detection and surveillance of HNSCC. In this work, a comprehensive genome-wide analysis of epigenetic alterations in primary HNSCC tumors was employed in conjunction with cancer-specific outlier statistics to define novel biomarker genes which are differentially methylated in HNSCC. The 37 identified biomarker candidates were top-scoring outlier genes with prominent differential methylation in tumors, but with no signal in normal tissues. These putative candidates were validated in independent HNSCC cohorts from our institution and TCGA (The Cancer Genome Atlas). Using the top candidates, ZNF14, ZNF160, and ZNF420, an assay was developed for detection of HNSCC cancer in primary tissue and saliva samples with 100% specificity when compared to normal control samples. Given the high detection specificity, the analysis of ZNF DNA methylation in combination with other DNA methylation biomarkers may be useful in the clinical setting for HNSCC detection and surveillance, particularly in high-risk patients. Several additional candidates identified through this work can be further investigated toward future development of a multi-gene panel of biomarkers for the surveillance and detection of HNSCC.

Integrative computational analysis of transcriptional and epigenetic alterations implicates DTX1 as a putative tumor suppressor gene in HNSCC

Over a half million new cases of Head and Neck Squamous Cell Carcinoma (HNSCC) are diagnosed annually worldwide, however, 5 year overall survival is only 50% for HNSCC patients. Recently, high throughput technologies have accelerated the genome-wide characterization of HNSCC. However, comprehensive pipelines with statistical algorithms that account for HNSCC biology and perform independent confirmatory and functional validation of candidates are needed to identify the most biologically relevant genes. We applied outlier statistics to high throughput gene expression data, and identified 76 top-scoring candidates with significant differential expression in tumors compared to normal tissues. We identified 15 epigenetically regulated candidates by focusing on a subset of the genes with a negative correlation between gene expression and promoter methylation. Differential expression and methylation of 3 selected candidates (BANK1, BIN2, and DTX1) were confirmed in an independent HNSCC cohorts from Johns Hopkins and TCGA (The Cancer Genome Atlas). We further performed functional evaluation of NOTCH regulator, DTX1, which was downregulated by promoter hypermethylation in tumors, and demonstrated that decreased expression of DTX1 in HNSCC tumors maybe associated with NOTCH pathway activation and increased migration potential.

Xenograft Model for Therapeutic Drug Testing in Recurrent Respiratory Papillomatosis

Objective: Identifying effective treatment for papillomatosis is limited by a lack of animal models, and there is currently no preclinical model for testing potential therapeutic agents. We hypothesized that xenografting of papilloma may facilitate in vivo drug testing to identify novel treatment options. Methods: A biopsy of fresh tracheal papilloma was xenografted into a NOD-scid-IL2Rgammanull (NSG) mouse. Results: The xenograft began growing after 5 weeks and was serially passaged over multiple generations. Each generation showed a consistent log-growth pattern, and in all xenografts, the presence of the human papillomavirus (HPV) genome was confirmed by polymerase chain reaction (PCR). Histopathologic analysis demonstrated that the squamous architecture of the original papilloma was maintained in each generation. In vivo drug testing with bevacizumab (5 mg/kg i.p. twice weekly for 3 weeks) showed a dramatic therapeutic response compared to saline control. Conclusion: We report here the first successful case of serial xenografting of a tracheal papilloma in vivo with a therapeutic response observed with drug testing. In severely immunocompromised mice, the HPV genome and squamous differentiation of the papilloma can be maintained for multiple generations. This is a feasible approach to identify therapeutic agents in the treatment of recurrent respiratory papillomatosis.

The PD-1 and PD-L1 pathway in recurrent respiratory papillomatosis

Generation of an immunosuppressive microenvironment may enable a persistent human papillomavirus infection in the setting of an otherwise normal immune system. We hypothesized that expression of the T‐lymphocyte co‐inhibitory receptor programmed death 1 (PD‐1) and its ligand PD‐L1 would be increased in the recurrent respiratory papillomatosis (RRP) microenvironment compared to normal controls.

Immunologic responses to a novel DNA vaccine targeting human papillomavirus‐11 E6E7

Recurrent respiratory papillomatosis (RRP) is a benign disease caused by human papillomavirus (HPV) types 6 and 11. Although a prophylactic vaccine against RRP is available, a therapeutic vaccine is needed to treat those already infected. The objective of our study was to design and test a DNA vaccine targeting HPV11 proteins.

Voice and Swallowing Dysfunction in Neurofibromatosis 2

Objective Neurofibromatosis 2 (NF2) is a neuro-oncologic condition that presents with bilateral vestibular schwannomas of the cerebellopontine angle (CPA). Voice and swallowing impairment can occur from direct involvement or compression of the vagus nerve or as the result of surgical excision of CPA tumors. The objectives in this study are to (1) assess the prevalence of voice and swallowing impairments and (2) analyze the effects of vagal dysfunction in patients with NF2. Study Design Cross-sectional. Setting Academic tertiary care center. Subjects and Methods Patients at a neurofibromatosis center were mailed Voice Handicap Index and Sydney Swallow Questionnaire surveys. Stroboscopic, voice, and swallowing evaluations were performed for patients who elected to participate in screening exams. Results There were high rates of self-assessed and objective voice and swallowing handicaps in this population. Fourteen of 40 (35%) patients had a self-assessed voice handicap, and 20 of 40 (50%) patients had a self-assessed swallow handicap. Vocal fold motion impairment (VFMI) was observed in 22 of 31 (71%) patients examined, with 27 of 62 (44%) possible vocal cords affected. Velopharyngeal insufficiency (45%) and piriform sinus pooling or residue (39%) were seen in a significant percentage of patients. There was a significant relationship between vocal cord motion impairment and CPA surgical intervention ipsilateral to the impairment (P = .002). The presence of VFMI was strongly associated with voice (P = .002) and swallowing (P = .01) impact on quality of life. Conclusion Speech and swallowing impairments are highly prevalent in patients with NF2, cause significant impact on quality of life, and are most commonly related to surgical interventions in the CPA region.

Prophylactic immunization with human papillomavirus vaccines induces oral immunity in mice

Although it has been shown that prophylactic vaccination can induce genital immunity, there is inadequate information on human papillomavirus (HPV) vaccine‐induced oral immunity, which is of particular interest due to HPV‐associated oropharyngeal malignancies and recurrent respiratory papillomatosis. Therefore, we assessed the efficacy of various HPV vaccines against oral HPV pseudovirus (PsV) infection in mice.

Adjuvant and Novel Treatment of Recurrent Respiratory Papillomatosis

Recurrent respiratory papillomatosis (RRP) is a rare benign neoplasm of the respiratory tract caused by human papillomavirus types 6 and 11. Surgical removal of the papilloma by microdebridement or laser is the current standard of care, but in most cases the disease recurs and patients require repeated surgical intervention. Over time, repeated procedures can lead to complications such as significant vocal cord scarring and laryngeal stenosis. There is therefore an urgent need for adjuvant treatments or alternative therapies that can control the disease or prevent recurrence, but unfortunately there is currently no known universally effective adjuvant therapy. Challenges for evaluating the effectiveness of adjuvant treatment in RRP include variable success rates in patients, the highly unpredictable underlying disease process, and potential for side effects. The current adjuvant treatment and novel approaches for RRP are presented.

Abstract 2880: The discovery of novel GSN alternative splicing in head and neck squamous cell carcinoma

Alternative splice events (ASES) are significant components of potential oncogenic pathways alterations and play a critical role in malignant cell transformation in a variety of solid and liquid tumors, including head and neck squamous cell carcinoma (HNSCC). However, high throughput analyses performed to date have not considered ASEs. Therefore, they have detected a limited number of genetic alterations for HNSCC, which incompletely describe the HNSCC specific pathway alterations. The heterogeneous nature of these alterations has made the discovery of reliable HNSCC biomarkers and therapeutic targets for this disease challenging. We performed alternative splice events (ASEs) analysis to enhance our understanding of HNSCC biology. To define ASEs specific to HNSCC we designed a novel bioinformatics pipeline from RNA-sequencing data of HNSCC tumors and independent normal samples. Evaluating the top scoring candidates, we have found several highly promising ASE candidates, including GSN, Gelsolin, an actin-binding protein, a key regulator of actin filament assembly and disassembly. Previously published literature proposes that GSN demonstrates tumor-suppressor properties by reducing cell proliferation in vivo and in vitro via suppression of protein kinase C (PKC, part of the PI3K pathway which is altered in HNSCC). The alternative splicing event involves an insertion of 110 bp from the 14th intron. This insertion contains a stop codon in frame, and the splice variant gives rise to a truncated (562 amino acids(aa)) protein with only 4 Gelsolin domains (instead of the full-length 731 aa protein with 6 Gelsolin domains). Using RNA-Seq data we demonstrated that 40% of tumor samples harbor the GSN-ASE. QRT-PCR confirmed that while total expression of GSN is decreased in HNSCC samples, GSN is expressed in the alternative truncated form only in HNSCC tumors and not normal tissues. Accordingly, total GSN expression is also seen to be downregulated in breast, lung and colon cancers. Evaluation of TCGA data confirmed the pre-dominant expression of the truncated GSN isoform over the wt GSN in HNSCC, bladder urothelial carcinoma, colon adenocarcinoma, lung SCC, breast invasive carcinoma, cervical SCC and endocervical adenocarcinoma. Moreover, we confirmed that that the expression of the truncated GSN is important for the migration and invasion of the cancer cells in vitro. These data suggest that alternative splicing plays an important role in the GSN gene for multiple tumor types. Citation Format: Daria A. Gaykalova, Dylan Kelley, Theresa Guo, Craig Bohrson, Ilse Tiscareno, Veronika Zizkova, Michael Considine, Ludmila Danilova, Emily Flam, Justin Bishop, Julie Ahn, Samantha Merritt, Marla Goldsmith, Chi Zhang, Wayne Koch, William Westra, Zubair Khan, Michael Ochs, Sarah Wheelan, Elana Fertig, Joseph Califano. The discovery of novel GSN alternative splicing in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2880.