Targeted Next-Generation Sequencing of Cancer-Related Genes in a Norwegian Patient Cohort With Head and Neck Squamous Cell Carcinoma Reveals Novel Actionable Mutations and Correlations With Pathological Parameters

Abstract

BackgroundTargeted next-generation sequencing (NGS) is increasingly applied in clinical oncology to advance personalized treatment. Despite success in many other tumour types, use of targeted NGS panels for assisting diagnosis and treatment of head and neck squamous cell carcinomas (HNSCC) is still limited.AimThe focus of this study was to establish a robust NGS panel targeting most frequent cancer mutations in long-term preserved formalin-fixed paraffin-embedded (FFPE) tissue samples of HNSCC from routine diagnostics.Materials and MethodsTumour DNA obtained from archival FFPE tissue blocks of HNSCC patients treated at Haukeland University Hospital between 2003-2016 (n=111) was subjected to mutational analysis using a custom made AmpliSeq Library PLUS panel targeting 31 genes (Illumina). Associations between mutational burden and clinical and pathological parameters were investigated. Mutation and corresponding clinicopathological data from HNSCC were extracted for selected genes from the Cancer Genome Atlas (TCGA) and used for Chi-square and Kaplan-Meier analysis.ResultsThe threshold for sufficient number of reads was attained in 104 (93.7%) cases. Although the specific number of PCR amplified reads detected decreased, the number of NGS-annotated mutations did not significantly change with increased tissue preservation time. In HPV-negative carcinomas, mutations were detected mainly in TP53 (73.3%), FAT1 (26.7%) and FLG (16.7%) whereas in HPV-positive, the common mutations were in FLG (24.3%) FAT1 (17%) and FGFR3 (14.6%) genes. Other less common pathogenic mutations, including well reported SNPs were reproducibly identified. Presence of at least one cancer-specific mutations was found to be positively associated with an extensive desmoplastic stroma (p=0.019), and an aggressive type of invasive front (p=0.035), and negatively associated with the degree of differentiation (p=0.041). Analysis of TCGA data corroborated the association between cancer-specific mutations and tumour differentiation and survival analysis showed that tumours with at least one mutation had shorter disease-free and overall survival (p=0.005).ConclusionsA custom made targeted NGS panel could reliably detect several specific mutations in archival samples of HNSCCs preserved up to 17 years. Using this method novel associations between mutational burden and clinical and pathological parameters were detected and actionable mutations in HPV-positive HNSCC were discovered.

Understanding the Pattern of Oropharyngeal Cancers from North-East Romanian Patients

“…By NGS (next generation sequencing), FGFR3 was found mutated in 15% of Norwegian HNSCC samples analyzed. This finding confirms the possibility of advanced personalized treatment, using targeted therapy against FFR3 [7]. Besides FGFR3, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) was found to be most commonly mutated in HNSCC.…”

The TP53 Codon 72 Arginine Polymorphism Is Found with Increased TP53 Somatic Mutations in HPV(−) and in an Increased Percentage among HPV(+) Norwegian HNSCC Patients

“…In two of these patients, pathogenic mutations were observed in the TRAF3 gene and in the FGFR3 gene, respectively (Table 6). In the other three patients, no further mutations were detected with the employed NGS panel [17] (Table 6).…”

“…DNA isolation, HPV detection, and our custom-made next-generation sequencing (NGS) panel were previously described [17]. Briefly, an experienced pathologist selected representative tumor samples.…”

“…Carcinogenesis is considered a multistep process from dysplasia to cancer [16]. Reports published on the genomic analysis of HPV(−) HNSCC demonstrated findings of mutations such as TP53, CDKN2A, MLL2/3, NOTCH1, PIK3CA, NSD1, FBXW7, DDR2, and CUL3 [13,15,17,18]. The reported mutational profile varies to some extent, but TP53 is the most common mutated gene in HNSCC [18,19].…”

RNA-Based Liquid Biopsy in Head and Neck Cancer

“…The basic diagnostic path for head and neck cancers includes primarily imaging diagnostics (endoscopy, head MRI, CT of the sinuses and head, panoramic dental x-ray, dental cone beam CT, PET/CT) supported by histopathological tests [19]. At the same time, the pursuit of faster, more effective and less invasive diagnostic methods leads to the introduction of innovative tests such as immunochemistry [20,21], molecular analyses [22][23][24], ultrasound elastography [25,26] or Raman spectroscopy [27]. There are even reports of the use of Artificial Intelligence-based methods in head and neck cancer diagnosis [28].…”