Abstract 2311: Extensive molecular heterogeneity in circulating tumor cells: A key player in oral squamous cell carcinoma prognosis
Authors: Chauhan Anshika , Boora Geeta S. , Ghosh Sahana , Parsana Monil , Patra Subrata K. , Verma Roshan K. , Bakshi Jaimanti , Chatterjee Debajyoti , Srinivasan Radhika , Ghoshal Sushmita , Maitra Arindam , Pal Arnab 
Published: June 2023

Abstract
Background: Oral Squamous cell carcinoma (OSCC) often results in unfavorable outcomes due to locoregional relapse. While circulating tumor cells (CTCs) may be attributed to these locoregional metastases, detecting CTCs and their molecular profiling remains challenging. Here we report a novel strategy for isolating CTCs in OSCC. Secondly, we characterized the CTCs to prognosticate the disease outcome using their transcriptomic signature.
Methods: Keeping in view the inherent disadvantages of currently available CTC-isolation techniques, like the use of single marker or size-based isolation, we developed a novel FACS–based strategy, combining pre-enrichment by immunomagnetic depletion of CD45+ cells, followed by positive selection of CTCs using multiple FITC-conjugated epithelial markers (EpCAM, EGFR, CK) and EMT marker (Vimentin), with a novel gating strategy for isolating pure CTCs. Isolated CTCs were reconfirmed using various methods. CTCs and Primary tumors were isolated in 63 OSCC patients, and their transcriptomic signature was correlated with disease outcome.
Results: Seventy-one percent (45/63) of patients were positive for CTCs, and associated with poor OS and DFS, while there is no difference between the proportion of CTC-positivity between early (23/33;69%) and late (22/30;73%) TNM stage. CTC-specific markers (EpCAM, EGFR, CK, and Vim) are expressed in various combinations, indicating extensive phenotypic heterogeneity. Importantly, EpCAM-positive CTCs were detected only in 46% (29/63) patients, and patients with EpCAM-negative CTCs had worse OS (p=0.044*), indicating the importance of a multi-marker approach. On unsupervised hierarchical clustering of RNAseq data of isolated CTCs and primary tumors, we found that CTCs and primary tumors clustered separately, indicating that the CTCs are transcriptionally different from the primary tumors. Moreover, CTCs were clustered into two distinct sub-clusters with significantly different OS (p=0.0004***) and DFS (p=0.0097***), suggesting that the inter-patient transcriptomic heterogeneity of CTCs is an important prognostic factor. We found coagulation, IL-6 JAK STAT3 signaling, allograft rejection, complement, EMT, inflammatory response, heme metabolism, UV response, and ECM receptor interaction pathways significantly enriched in CTCs compared to the primary tumor. TNF-alpha signaling via NF-kB and KRAS signaling pathways were also positively enriched in CTCs with poor prognostic traits. Interestingly, for the first time, we found various Cancer/Testis (CT) antigens upregulated in CTCs, while not detected in primary tumors.
Conclusion: Here we report a novel, technologically less difficult, cost-effective CTC isolation technique suitable for developing countries. We explored the prognostic implications of the extensive heterogeneity in CTCs isolated from OSCC patients for the first time.
Citation Format: Anshika Chauhan, Geeta S. Boora, Sahana Ghosh, Monil Parsana, Subrata K. Patra, Roshan K. Verma, Jaimanti Bakshi, Debajyoti Chatterjee, Radhika Srinivasan, Sushmita Ghoshal, Arindam Maitra, Arnab Pal. Extensive molecular heterogeneity in circulating tumor cells: A key player in oral squamous cell carcinoma prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2311.