Abstract 1934: Gene expression profiling of circulating tumor cells provides prognostic insights in oral squamous cell carcinoma

Abstract Background: Oral Squamous Cell Carcinoma (OSCC) often presents with bad prognosis due to high frequency of loco-regional metastasis and relapse. Circulating Tumour Cells (CTCs) are precursors for metastasis, can be identified through liquid biopsy, but their molecular analysis has been challenging due to limitations in current isolation and downstream analysis techniques. To address this, we developed an innovative FACS-based method for CTC isolation and explored the prognostic potential of CTC gene expression in OSCC. Methods: We developed an innovative technique that isolates CTCs by depleting CD45+ hematogenous cells and positively selecting CTCs using four markers— EpCAM, EGFR, CK, and Vimentin (Chauhan et al., 2024) using FACS. Next, we performed whole transcriptome analysis of isolated CTCs and paired primary tumours from 24 OSCC patients. Gene expression signatures in CTCs were validated in separate cohort of 60 patients. Results: Data analysis revealed distinct gene expression profiles between CTCs and primary tumours, with 521 differentially expressed genes. Interestingly, PADI4 gene, an enzyme that is involved in the citrullination of various proteins was significantly upregulated in CTCs (p<0.0001). Validation of this finding in the separate patient cohort showed that patients with detectable PADI4 levels in CTCs had significantly poorer disease-free survival (p=0.038, HR=2.571). Interestingly, PADI4 expression was more in CTCs which having EMT signatures. Upon unsupervised hierarchical clustering of CTC transcriptome data, two subclusters of CTCs (namely C1A and C1B) with a total of 372 differentially- expressed between genes between them were found. Patients in CTC cluster C1B had significantly poorer overall survival (p=0.0028) and disease-free survival (p=0.0185), with a higher incidence of loco-regional metastasis (p=0.0082). We also validated topmost upregulated genes in separate cohort of patients and identified that TNFAIP2, NFKBIZ, CTSS and PTGS2 are significantly associated with increased probability of cancer relapse. Multiple logistic regression modelling using these genes identified that cumulative expression of baseline TNFAIP2, NFKBIZ, CTSS and PTGS2 expression can successfully predict recurrence in our cases. The developed model was validated using calibration plot analysis. Conclusion: Our study demonstrates that the gene expression profiles of CTCs can serve as prognostic indicators in OSCC and we successfully developed a four-gene panel model for predicting relapse in OSCC patients. Citation Format: Anshika Chauhan, Geeta S. Boora, Sushmita Ghoshal, Arindam Maitra, Arnab Pal. Gene expression profiling of circulating tumor cells provides prognostic insights in oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1934.