Investigating mechanisms of lymph node metastasis in oral cancer


Integrative analysis of whole-genome, transcriptome and Assay for Transposase-Accessible Chromatin (ATAQ) sequencing data to assess genome-wide chromatin accessibility, genomic lesions in chromatin regulators and inform chromatin states that drive critical gene expression patterns.



BACKGROUND: Oral squamous cell carcinoma (OSCC) is the common head and neck cancer with a five-year survival rate of only 50% in North America. About 40% of OSCC patients are diagnosed with existing cervical lymph-node metastasis (LNM), further exacerbating outcomes. Many of these patients present with small but aggressive tumors (T1/T2) defying conventional wisdom that LNM is usually associated with larger tumors (T3/T4). Surgical resection is an integral component of curative therapy and involves large resections that frequently require reconstruction of portions of the oral cavity and other facial features. When LNM is present, radical neck dissection is performed in addition to oral surgery. The morbidity associated with these massive resections is severe and life-long. Targeted therapies have reduced treatment-associated morbidity and revolutionized the management of many cancer types, but few are currently used in OSCC management; this is predominantly attributed to a limited understanding of mechanisms underlying LNM and that a majority of identified genome alterations in OSCC affect tumour suppressors that are refractory to conventional targeting approaches.

Cancer cells are incredibly diverse not only due to genomic and transcriptomic heterogeneity, but also epigenomic versatility. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) can identify regions of open chromatin, transcription factor binding motifs and gene regulatory elements including enhancers and promoters. Integration of ATAC-seq, whole genome sequencing (WGS) and RNA sequencing (RNA-seq) data can help assess genome-wide chromatin accessibility, genomic lesions in chromatin regulators and inform chromatin states that drive critical gene expression patterns.

HYPOTHESIS: Investigating the genomic landscape, chromatin states and associated gene expression patterns in fresh-frozen OSCC samples will identify novel oncogenic mechanisms that regulate progression and LNM.


To perform ATAC-seq, WGS and RNA-seq on banked OSCC samples: The Ohlson Research Initiative (ORI) has generated a biobank of fresh-frozen OSCC samples with prospectively collected and regularly updated clinical data. We have identified primary tumor samples and matched normal tissue from 30 OSCC patients with small (T1/2) tumors; 15 presenting with and 15 without LNM. Intact nuclei, DNA and RNA will be extracted from separate tissue scrolls cut from each of the embedded tissue blocks to perform ATAC-seq, WGS and RNA-seq assays, respectively. Sequencing will be performed on the available Illumina NovaSeq platform at the Centre for Health Genomics and Informatics (CHGI). We will perform analysis of the ATAC-seq data to identify differential peaks, motifs, nucleosome accessibility, and transcription factor footprints in both tumor vs. normal tissues, and samples with or without LNM. WGS will provide a comprehensive view of the unique mutations and genomic alterations in OSCC tissue and those driving LNM. WGS data will be integrated with ATAC-seq to infer lesions in chromatin regulatory regions. Finally, RNA-seq analyses will inform specific gene expression changes associated with tumorigenesis and LNM driven by epigenomic changes identified by ATAC-seq.

SIGNIFICANCE: These comprehensive and integrative analyses of the OSCC genome, transcriptome and epigenome will provide important insights into mechanisms driving OSCC progression and LNM. The proposed study may also identify signaling axes that may be targeted to transform OSCC management.