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

Cancer. 2021 May 15;127(10):1576-1589. doi: 10.1002/cncr.33393. Epub 2020 Dec 21.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biomarkers, Tumor
  • Carcinoma, Squamous Cell* / diagnosis
  • Carcinoma, Squamous Cell* / genetics
  • DNA, Neoplasm* / genetics
  • DNA, Neoplasm* / isolation & purification
  • Humans
  • Mouth Neoplasms* / diagnosis
  • Mouth Neoplasms* / genetics
  • Mutation
  • Saliva*


  • Biomarkers, Tumor
  • DNA, Neoplasm