Mutational landscape of gingivo-buccal oral squamous cell carcinoma reveals new recurrently-mutated genes and molecular subgroups

Abstract

Gingivo-buccal oral squamous cell carcinoma (OSCC-GB), an anatomical and clinical subtype of head and neck squamous cell carcinoma (HNSCC), is prevalent in regions where tobacco-chewing is common. Exome sequencing (n=50) and recurrence testing (n=60) reveals that some significantly and frequently altered genes are specific to OSCC-GB (USP9X, MLL4, ARID2, UNC13C and TRPM3), while some others are shared with HNSCC (for example, TP53, FAT1, CASP8, HRAS and NOTCH1). We also find new genes with recurrent amplifications (for example, DROSHA, YAP1) or homozygous deletions (for example, DDX3X) in OSCC-GB. We find a high proportion of C>G transversions among tobacco users with high numbers of mutations. Many pathways that are enriched for genomic alterations are specific to OSCC-GB. Our work reveals molecular subtypes with distinctive mutational profiles such as patients predominantly harbouring mutations in CASP8 with or without mutations in FAT1. Mean duration of disease-free survival is significantly elevated in some molecular subgroups. These findings open new avenues for biological characterization and exploration of therapies.

Figure 1. Data summary of 50 gingivo-buccal oral cancer patients.

Demographic characteristics, environmental exposures and landscape of genomic alterations are shown. These data have been organized in increasing order of the total number of mutations observed in each patient. Patient numbers that are underlined are female; the remaining are male. (a) Exposures to known risk factors, including HPV. Tobacco exposure includes all forms of tobacco use. Exposed patients are indicated as a filled square. (b) Ten genes found to be significantly altered, indicated in bold, are arranged in descending order of the percentage of patients who showed alterations (SNV and CNV, which are indicated with a ‘+’ separator). Types of alterations are colour-coded; the colour-coding scheme is indicated at the bottom of the figure. Six genes that were previously identified to be frequently altered in other cancers and were found to be present in at least 10% of the patients included in this study are also listed. Three genes that were found to be frequently mutated in an earlier study on head and neck squamous cell carcinoma are underlined. (c) Genes of relevance that are amplified (5 genes) or deleted (4 genes) in at least 10% of patients (note: CNVs detected in genes such as olfactory receptor genes are not listed because of lack of evidence of their involvement in cancers). All amplifications are full-gene amplifications; the deletion involving GSTT1 is a full-gene deletion, while the others are partial deletions. (d) Numbers of silent and non-silent mutations per Mb (note: for patient nos. 7 and 41, the total number of mutations per Mb, 12 and 29, respectively, exceeds the scale; hence, these numbers are displayed). (e) Spectrum of mutations for each patient: percent frequencies of various categories of SNVs and indels. The bars representing the frequency of C>G/T at non-CpG sites are highlighted for the three patients 2, 7 and 41 (see text for explanation), *Entire MMP gene-family on chromosome 11 was amplified.

Figure 2. Clustering of gingivo-buccal oral cancer patients based on mutational profiles.

Hierarchical clustering of 110 gingivo-buccal oral squamous cell carcinoma patients based on 10 significantly and frequently mutated genes form three broad clusters (C₁–C₃) with the following essential characteristics: (a) Patients with mutations in CASP8 with or without mutations in FAT1, (b) patients with mutations in TP53 and (c) patients with mutations in various other genes. Within each cluster, there are multiple subclusters. The duration (in months) of disease-free survival averaged over patients belonging to each subcluster is provided in the panel below. The mean duration of disease-free survival is long for three subclusters comprising patients with mutations in (i) CASP8, NOTCH1 and FAT1 (C_1.2), (ii) CASP8, NOTCH1 and ARID2 (C_1.4) and (iii) MLL4 with other genes (C_3.2). Filled boxes indicate DNA alterations; red and blue boxes indicate, respectively, nonsense/frame-shift/splice-site and missense/in-frame insertion-deletion.

Figure 3. Kaplan–Meier probability distributions of disease-free survival.

Results are shown for gingivo-buccal oral squamous cell carcinoma patients with (n=11; blue line) and without (n=99; green line) mutations in MLL4.