Abstract 2504: Functional characterization of chromosomal deletions in head and neck squamous cell carcinoma
Authors: Farhad Ramin , Palop Vicente Planells , Tward Aaron 
Published: July 2021

Abstract
Introduction: Large-scale chromosomal deletions frequently occur in the progression of head and neck squamous cell carcinoma (HNSCC). Many studies have linked these deletions with particular patient outcomes. However, their precise contributions to tumorigenesis have yet to be elucidated. Here, we introduce an in vitro model for studying large-scale chromosomal deletions to establish their role as driver or passenger events in HNSCC progression.
Methods: CRISPR/Cas9 vectors coding for guide RNAs (gRNAs) that flank two of the commonly deleted regions in HNSCC tumors were transfected into primary human keratinocytes. Eighteen different combinations of gRNAs were utilized to create deletions of various sizes at two different genomic loci. Genomic DNA (gDNA) was harvested 24 hours post-transfection and analyzed via endpoint PCR and Sanger sequencing to assess for the presence of the deletions. Edited cells were further sorted via fluorescence-activated cell sorting (FACS) and the efficiency of the deletions were analyzed via performing TaqMan copy number assay qPCR on extracted gDNA. Subsequently, the invasive properties of the genome-edited cells were then investigated using organotypic cultures, a near-physiological three-dimensional model system for studying squamous epithelial biology.
Results: The feasibility of performing large-scale deletions was confirmed via endpoint PCR and Sanger sequencing, in which all 18 gRNA combinations created successful deletions. We further sought to create a pure population of the cells harboring the deletions by isolating the edited cells via FACS. TaqMan copy number assay qPCR on gDNA harvested from these cells indicated a 10-45% reduction in the copy number of loci of interest. Genome-edited keratinocytes containing deletions at two of the most commonly deleted sites in HNSCC tumors were used for organotypic assays, in which the preliminary results suggested that deletion of these sites can lead to a gain of invasive behavior.
Conclusion: Chromosomal deletions occur at a high frequency in HNSCC tumors. However, there is no established model to determine whether these deletions exist as driver or passenger events. Here, we demonstrate the introduction of successful large-scale deletions in a primary human cell culture system that can be further utilized to study the role of these events in HNSCC tumorigenesis. Furthermore, we show that two of the most common deletions in HNSCC may act as driver events in tumor progression.
Support: This study was supported by Program in Breakthrough Biological Research, UCSF School of Dentistry OHNRG pilot grant, and an R01 grant (DE029890-01 - Defining the Mutational pathogenesis of Oral Preneoplasia).
Citation Format: Ramin Farhad, Vicente Planells Palop, Aaron Tward. Functional characterization of chromosomal deletions in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2504.