Ultrasensitive detection of tumor-specific mutations in saliva of patients with oral cavity squamous cell carcinoma

Abstract

Background: Oral cavity squamous cell carcinoma (OCSCC) is the most common head and neck malignancy. Although the survival rate of patients with advanced-stage disease remains approximately 20% to 60%, when detected at an early stage, the survival rate approaches 80%, posing a pressing need for a well validated profiling method to assess patients who have a high risk of developing OCSCC. Tumor DNA detection in saliva may provide a robust biomarker platform that overcomes the limitations of current diagnostic tests. However, there is no routine saliva-based screening method for patients with OCSCC.

Methods: The authors designed a custom next-generation sequencing panel with unique molecular identifiers that covers coding regions of 7 frequently mutated genes in OCSCC and applied it on DNA extracted from 121 treatment-naive OCSCC tumors and matched preoperative saliva specimens.

Results: By using stringent variant-calling criteria, mutations were detected in 106 tumors, consistent with a predicted detection rate ≥88%. Moreover, mutations identified in primary malignancies were also detected in 93% of saliva samples. To ensure that variants are not errors resulting in false-positive calls, a multistep analytical validation of this approach was performed: 1) re-sequencing of 46 saliva samples confirmed 88% of somatic variants; 2) no functionally relevant mutations were detected in saliva samples from 11 healthy individuals without a history of tobacco or alcohol; and 3) using a panel of 7 synthetic loci across 8 sequencing runs, it was confirmed that the platform developed is reproducible and provides sensitivity on par with droplet digital polymerase chain reaction.

Conclusions: The current data highlight the feasibility of somatic mutation identification in driver genes in saliva collected at the time of OCSCC diagnosis.

Keywords: early detection; liquid biopsy. mutation; next-generation sequencing (NGS); oral cavity squamous cell carcinoma (OCSCC); oral rinse; saliva.

Figure 1:. Selection of genes for the targeted OCSCC panel.

A. A minimal set of genes where mutations would represent >85% distinct samples in the dataset were identified from three different WES studies on OCSCC patients, namely the TCGA-HNSC dataset restricted to OCSCC data (n=329), the ICGC (n=50) and the MD Anderson dataset (n=40). The intersection on the Venn diagram represents the core set of genes (TP53, FAT1 and CASP8) that captures the highest number of OCSCC samples across the three datasets. PIK3CA, HRAS, NOTCH1, and CDKN2A were manually curated for their clinical and biological significance in OCSCC, after evaluating the top 20 genes in each dataset. B. Bar chart shows the proportion of distinct samples (in each dataset and all three datasets combined) carrying at least one mutation in the 7 genes panel.

Figure 2:. Targeted sequencing of primary OCSCC malignancies.

A. Heatmap shows a sample-wise mutation distribution across the sequenced primary tumor specimens (106 subjects where at least one mutation was detected by the targeted 7 gene panel). Top panel: number of mutations per patient; Right panel: mutational frequency for each gene included in the targeted sequencing panel. The gender and histopathological stage classification are indicated in the strip chart below the heat map. B. Histogram comparing frequency distribution of mutations per gene in our study cohort and combined (TCGA, MD Anderson and ICGC) public datasets (n=419).

Figure 3:. Analytical validation of the assay performance for low frequency variant detection.

A. A positive control containing synthetic loci with 7 known mutations in TP53 and PIK3CA genes (at 0.25% VAF) was sequenced across 8 independent sequencing runs (solid colored lines). The same input material was also assessed by ddPCR assay (dashed black line). All mutations were detected in each one of the sequencing runs with expected VAF and remarkable concordance between ddPCR and NGS generated VAF values. B. Mutation free genomic loci containing a region of 665 bases in HRAS gene from a well-characterized contemporary normal NA12878 cell line was sequenced across 9 independent sequencing runs. Table summarizes the number of false positive calls detected in each sequencing run at >0.1% VAF.

Figure 4:. Concordance between primary tumor and oral rinse specimens.

Percentage of OCSCC samples with functional mutations identified in primary tumor biopsies that were also detected in paired pre-treatment oral rinse specimens. Bar chart indicates concordance seen in patients with early stage (I and II) disease, late stage (III and IV) disease, and combined concordance across the entire cohort. Samples with more than one functional mutation in the primary tumor were considered to be concordant if any one of the mutations was detected in the paired oral rinse specimen.

Figure 5.. Mutations distribution in primary tumor biopsies and matched oral rinse specimens.

A-G. Lollipop plots show the landscape of genetic aberrations detected in primary tumors (top) and matched oral rinses (bottom) in each gene included in the sequencing panel. The variants are color coded by the mutation type (red – nonsense, green – missense, blue – deletion, violet – insertion). Gene domains are indicated in the bottom of each panel. H. Table depicts a dynamic increase in cumulative detection in the oral rinse with addition of each gene to the sequencing panel.