Optimization of RNA Sequencing Workflow in Brush Swabs of Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) is associated with poor survival due to challenges with timely diagnosis and surveillance. Noninvasive biopsy methods, such as brush swab biopsy, have emerged as a promising approach with several clinical advantages over surgical biopsies, which may help overcome challenges in diagnosis, prognostication, and surveillance. We aimed to evaluate a noninvasive brush swab method for nucleic acid collection suitable for RNA sequencing in patients with OSCC. Brush swabs were collected from tumor sites before surgery, followed by corresponding fresh-frozen tissue samples from the same sites during surgical resection. Thirty-nine patients were enrolled (median age 65 years (IQR, 55-73)). As expected, tissue samples yielded higher RNA concentration and integrity than swabs (456.3 ng/ul (IQR, 291.3 - 651.3) vs 73.2 ng/ul (IQR, 34.9 - 106.3); DV200 (91.6% (IQR, 85.9 - 93.0) vs 82.0% (IQR, 77.3 - 87.8)); both p < 0.0001). Despite lower RNA quality, swab samples showed a high sequencing mapping rate after bacterial rRNA depletion (84.3% (IQR, 67.3 - 91.1)). Gene expression profiles were strongly correlated between matched tissues and swabs (r = 0.969; 95% CI, 0.967-0.969; p < 2.2e-16). We developed and validated a protocol for total transcriptomic profiling of brush swab samples and demonstrated excellent concordance with paired tissue samples in OSCC patients. These findings support the use of brush swabs as a noninvasive alternative for biomarker discovery, with potential applications in OSCC diagnosis and prognostication.