Expression & Epigenetic Silencing of MicroRNA for Predicting Therapeutic Response and Prognosis of HPV-negative HNSCC

Primary outcomes

1. Identify tumor specific microRNAs (miRs) whose expression increases or decreases by over 2 fold in fresh tumor vs. distant normal mucosa from patients with head and neck squamous cell carcinoma (HNSCC) negative for human papillomavirus (HPV)

   Time frame: As long as needed to collect 15 HPV-negative tumor-mucosal sample pairs and complete sequencing, up to 10 years

   SOLiD sequencing of fresh tumor-distant normal pairs will discover miRs whose expression is altered in HNSCC tumors. SOLiD sequencing will provide fully quantitative data for the expression of known and novel miRs. Because HPV-related tumors comprise approximately 20% of all HNSCC and are biologically distinct from HPV negative tumors classically associated with tobacco and alcohol, only HNSCC without p16 overexpression will be used in this discovery experiment. This restriction will increase homogeneity of the samples and the power to detect miRs associated with the etiology of HPV-negative HNSCC. The miRs identified will be validated using qPCR assay in the tumor-normal pairs and in p16-negative HNSCC cell lines (n>15). In addition, miRs previously reported to be dysregulated in HNSCC will be subjected to similar validation, even if not identified during the SOLiD sequencing discovery phase.

2. Identify miRs whose reduced expression in HPV-negative HNSCC is due to promoter methylation

   Time frame: As long as needed to complete sequencing and analyze results, up to 5 years

   miRs with reduced expression in tumor tissue identified under Outcome 1 will be scrutinized for CpG rich promoters by checking the UCSC database. The methylation status of CpG rich promoters of miRs will be examined by COBRA assay in tumor vs. normal pairs (n=15) and in p16-negative HNSCC cell lines (n>15). The heterogeneity of methylation status across the CpG rich promoter will be characterized by bisulfate sequencing using the COBRA PCR product. The silencing of miRs by promoter methylation will be examined by comparing mean miR expression levels between the methylation categories for the tumor-normal pairs and for the HNSCC cell lines. This is primarily a descriptive study.

3. Define the statistical association between the expression of tumor specific miRs, miR methylation, and the therapeutic response and prognosis of HPV-negative HNSCC using patient medical records

   Time frame: As long as needed to collect and analyze information from medical records, up to 5 years

   The therapeutic response and prognosis of HPV-negative HNSCC will be collected from two sources: the patient's case file from the New Mexico Tumor Registry (NMTR) and from the patient's UNMH medical record. qRT PCR will be designed to quantify the miR expression in RNA extracted from the formalin-fixed, paraffin embedded (FFPE) tissues. A methylation-specific PCR (MSP) will be designed to facilitate the large-scale methyl-typing in all retrospective clinical samples. The primary analysis will be performed in HPV-negative HNSCC. A secondary analysis will be conducted to include all HNSCC, with and without p16 overexpression. p16 overexpression status will be adjusted in the secondary analysis. The association between miR expression status (high or low) and miR methylation status (yes or no), and patient's PFS and OS will be examined using Kaplan-Meier survival plots and the log-rank test.