Identification of Potential Salivary Biomarkers and Enriched Protein Domain/Motif Families for Early Detection and Lymph Node Invasion in OSCC Patients of the Indian Population

Oral squamous cell carcinoma (OSCC) is a major public health concern in India, ranking first in cancer-related mortality among men and fourth among women, causing about nine deaths per hour. Despite advances in clinical management, prognosis remains poor due to late-stage detection and a lack of reliable biomarkers for early diagnosis and disease monitoring. This study aimed to identify salivary biomarkers and enriched protein domain/motif families for early OSCC detection and progression assessment, including lymph node invasion, in the Indian population. Using diaPASEF-based comparative salivary proteomics, 45 saliva samples from healthy individuals, premalignant lesions (PM), and OSCC patients (with/without nodal invasion) were analyzed and validated through public data sets and targeted proteomics. A total of 1068 proteins were identified, with significant differential expression across disease stages. Enriched domain/motif families included ITIs, lipocalins, calcium-binding EF-hand motifs, SERine Proteinase Inhibitors (SERPINs), trypsin-like serine proteases, and annexin repeats. Functional enrichment revealed pathways related to endopeptidase regulation, wound healing, and calcium ion binding. Key multiclass biomarkers, NUCB2, ITIH4, ITIH2, RBP4, CALU, and SDF4, achieved area under the curve (AUC) > 0.7 in receiver operating characteristic (ROC) models across our data, The Cancer Genome Atlas (TCGA), and Clinical Proteomic Tumor Analysis Consortium (CPTAC), and were validated using parallel reaction monitoring (PRM). These findings highlight novel non-invasive salivary biomarkers with potential for early OSCC detection and lymph node invasion prediction.