Characterization of functionally active gene fusions in human papillomavirus related oropharyngeal squamous cell carcinoma

Abstract

The Cancer Genome Atlas (TCGA) sequencing analysis of head and neck squamous cell carcinoma (HNSCC) recently reported on gene fusions, however, few human papillomavirus (HPV) positive samples were included, and the functional relevance of identified fusions was not explored. We therefore performed an independent analysis of gene fusions in HPV-positive oropharyngeal SCC (OPSCC). RNA sequencing was performed on 47 HPV-positive OPSCC primary tumors and 25 normal mucosal samples from cancer unaffected controls on an Illumina TruSeq platform. MapSplice2 was used for alignment and identification of fusion candidates. Putative fusions with less than five spanning reads, detected in normal tissues, or that mapped to the same gene were filtered out. Selected fusions were validated by RT-PCR and Sanger sequencing. Within 47 HPV-positive OPSCC tumors, 282 gene fusions were identified. Most fusions (85.1%) occurred in a single tumor, and the remaining fusions recurred in 2–16 tumors. Gene fusions were associated with significant up regulation of 16 genes (including EGFR and ERBB4) and down regulation of four genes (PTPRT, ZNF750, DLG2, SLCO5A1). Expression of these genes followed similar patterns of up regulation and down regulation in tumors without these fusions compared to normal tissue. Five of six gene fusions selected for validation were confirmed through RT-PCR and sequencing. This integrative analysis provides a method of prioritizing functionally relevant gene fusions that may be expanded to other tumor types. These results demonstrate that gene fusions may be one mechanism by which functionally relevant genes are altered in HPV-positive OPSCC.

A Novel Functional Splice Variant of AKT3 Defined by Analysis of Alternative Splice Expression in HPV-Positive Oropharyngeal Cancers

“…(13) For comparison, normal oropharynx mucosal tissue from uvulopalatopharyngoplasty (UPPP) surgical specimens were obtained from 25 cancer unaffected controls. All tissue samples were collected from the Johns Hopkins Tissue Core under an approved IRB protocol (#NA_00036235) and written informed consent was obtained from each patient prior to collection of samples in accordance to ethical guidelines according to the Belmont Report.…”

“…(13) Samples were required to achieve an RNA Integrity Number (RIN) of at least 7.0. Barcoded, stranded RNA-seq libraries from ribosomal RNA depleted total RNA (400 ng) was prepared using the Illumina TruSeq stranded total RNA Seq Gold kit (San Diego, CA).…”

“…Gene expression values were quantified from RNA sequencing data using RSEM version 1.2.9 and upper quartile normalization according to the TCGA RSEM v2 normalization pipeline. (1,13)…”

Integrated Analysis of Whole-Genome ChIP-Seq and RNA-Seq Data of Primary Head and Neck Tumor Samples Associates HPV Integration Sites with Open Chromatin Marks

“…Primary tumor tissue samples were obtained from a cohort of 47 patients with HPV-related oropharyngeal squamous cell carcinoma, as previously described (13). For comparison, healthy oropharynx mucosal tissue from uvulopalatopharyngoplasty (UPPP) surgical specimens were obtained from 25 cancer-unaffected controls (13).…”

“…We have adjusted various processing parameters and extensively validated this optimized protocol in tumor cell lines, primary patients’ samples, and patient-derived xenograft (PDX) models. The chromatin data generated in this study was coupled with matched RNA-Seq data that include samples profiled as part of a larger cohort of 72 HPV+ tumors (13). We performed integrated bioinformatics analysis of the ChIP-Seq and RNA-Seq data to determine the potential functional role of chromatin alterations in HPV+ HNSCC.…”