Abstract A01: Characterization of NOTCH1 mutations in oral cavity squamous cell carcinomas
Authors: Koo Kendrick , Angel Christopher , Kershaw Nadia , Mouradov Dmitri , Chueh Anderly , Wiesenfeld David , Iseli Tim A. , McCullough Michael , Sieber Oliver , Burgess Antony W. 
Published: May 2017

Abstract
Introduction: Oral cavity squamous cell carcinomas (OSCCs) are the most prevalent head and neck malignancy and are a significant cause of mortality and morbidity, especially in the developing world. A better understanding of the molecular pathogenesis of OSCCs is essential for improved management of this disease. Mutations in the NOTCH1 gene have been identified in 9-18% patients with head and neck malignancies. A large proportion of these mutations lie in its extracellular domain and are thought to be inactivating, in contrast to the activating NOTCH1 mutations found in hematological malignancies such as T-ALL. This dichotomy raises interesting questions about the role of the Notch pathway in malignancies. In this study, we aim to characterize NOTCH1 mutations in a cohort of OSCC patients and use an in vitro model to investigate the role of NOTCH1 inactivation in OSCC tumorigenesis.
Methods: Targeted next-generation sequencing of a custom 69 gene panel was carried out on fresh frozen and formalin fixed paraffin embedded (FFPE) tissue from prospectively and retrospectively recruited OSCC patients presenting to a tertiary cancer center (n = 94). Detailed clinical annotation was collected for all patients. The BWA/GATK pipeline was used for sequence alignment and mutation calling. Downstream sequencing pipelines were designed in the R statistical environment to optimize identification of somatic mutations in the FFPE samples. Notch pathway mutations were interrogated, and comparisons made against NOTCH1 mutations across all cancer subtypes in The Cancer Genome Atlas (TCGA) database.
Five OSCC cell lines (H314, H357, H400, BICR16, BICR56) were sequenced and Notch1 expression characterized by Western blotting. A lentiviral CRISPR/Cas9 system using custom designed guides was used to knock out NOTCH1 in the cell lines expressing wild-type Notch1. Wild-typeNOTCH1-mCherry was expressed in the cell lines with mutated NOTCH1. Non-adherent clonal growth assays and RNAseq were used to assess the impact of these modifications.
Results: In our patient cohort, a high frequency of mutations were identified in known tumor suppressor genes TP53 (57%) and CDKN2A (22%). Sixteen patients (17%) were NOTCH1 mutant. The majority of these mutations fell within the extracellular domain and 10 were predicted to be nonsense mutations. Ten patients (11%) had a NOTCH3 mutation.
Two of the five OSCC cell lines were NOTCH1 mutant and expressed no detectable Notch1 protein. A wild-type Notch1-mCherry construct was introduced into the mutant cell lines and Notch1 was knocked out in the three cell lines in which functional Notch1 protein was detected. The results of the growth assays and RNA sequencing for all of the pre- and post- modification cell lines will be presented.
Conclusions: We have built a bioinformatics pipeline to successfully analyze FFPE tissue and have identified potentially inactivating NOTCH1 mutations in our patients at similar frequencies to international cohorts. We have identified NOTCH3 mutations, which are of uncertain significance. Successful knockout of NOTCH1 in OSCC cell lines was achieved.
Citation Format: Kendrick Koo, Christopher Angel, Nadia Kershaw, Dmitri Mouradov, Anderly Chueh, David Wiesenfeld, Tim A. Iseli, Michael McCullough, Oliver Sieber, Antony W. Burgess. Characterization of NOTCH1 mutations in oral cavity squamous cell carcinomas. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A01.