Exploring the mutational spectrum of key kinase genes PIK3CA, BRAF, EGFR, ALK and ROS1 in oral squamous cell carcinoma

Oral Squamous Cell Carcinoma (OSCC) is the sixth most aggressive type of oral cancer. Mutations in cancer-driving genes such as protein kinase are well known in cancer progression. We selected candidate genes (PIK3CA, BRAF, EGFR, ALK, and ROS1) for mutations exploration in the OSCC patients belonging to Khyber Pakhtunkhwa (KP) through Next Generation-Whole Exome Sequencing (NG-WES) using Formalin Fixed Paraffin Embedded (FFPE) tissue blocks (27 tumor and 7 paired normal) for the 1st time followed by in-silico characterization. A total of 33 mutations were identified which constituted 28/33 (84.84%) SNVs, 4/33 (12.12%) frameshift deletions and 1/33 (3.03%) stop-gain mutation. While, of the 33 mutations, 12.6% (4/33) were novel and had not been previously reported in public mutation databases such as COSMIC or dbSNP. Among the total somatic mutations (24/33; 72.72%), 08/33 mutations were observed in multiple patients. Mutations of the ALK i.e. ALK^p.I1461V, ALK^p.K1491R and ALK^p.D1529E were found in 27/27, 21/27 and 20/27 tumor samples and hence can have potential biomarker applications. ISPRED-SEQ identified 07/33 interaction site mutations i.e. EGFR^p.R521K, EGFR^p.R831C, ROS1^p.S2229C, ROS1^p.E1902K, ROS1^p.K2228Q, ROS1^p.D2213N, and ROS1^p.P221S. SAAFEQ-SEQ predictions revealed that (28/29; 96.5%) SNVs destabilize protein except for ROS1^p.D2213N. ConSurf predictions indicated 17.3% (5/33) mutations (e.g., ROS1^p.N2240K (score 9) and ROS1^p.L567V (score 8), as highly conserved, likely disrupting kinase function and stability, unlike variable mutations with milder effects. MD simulations for interacting sites (IS) SNVs revealed structural deviations, with mutant proteins revealing larger gyration radius and fluctuations in root mean square deviation (RMSD) studies indicating a disrupted folding behavior. To conclude, we identified potential mutations on ROS1 that can have potential biomarker applications, however, we recommend studies in large Pakhtun cohorts in KP, Pakistan.