Abstract 7506: Comprehensive mutational profile of true single circulating tumor cells compared with CtDNA in advanced pancreatic adenocarcinoma
Authors: Khandare Jayant , Bharde Atul , Khutale Ganesh , Andhari Saloni , Deshpande Richa , Hariramani Kanchan , Basavalingegowda Madhura , Jadhav Vikas , Uttarwar Mohan , Shafi Gowhar 
Published: March 2024

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is aggressive malignancy characterized by 5-year survival rate of &lt;10%. Comprehending PDAC's mutational landscape through NGS-based assays, acquiring representative tumor tissue biopsies remains arduous. Single circulating tumor cell (sCTC) genomics could provide clinically relevant personalized diagnostics. Here we demonstrate comparative analysis of sCTCs genomics and ctDNA outcomes in paired advanced PDAC patients.

Methods: Retrospectively, 25 live sCTCs and sCTC clusters were isolated from 10 advanced PDAC patients' blood using OncoRadar platform consisting glass beads conjugated with antiEPCam antibodies, tuned for releasing CTCs, without cell fixing, an assay in 96 well plate. Whole genomes from sCTCs were amplified using random degenerate primer-based PCR. Amplified genomes and paired ctDNA were subjected to CGP using clinical assay OncoIndx® to identify patient-specific genomic alterations in sCTC and paired ctDNA samples. The raw sequence alignment and variant calling was performed using iCare software®.

Results: Live 25 sCTCs and 8 CTC clusters were isolated (≥2 CTCs). Mutational profiling revealed multiple pathogenic (P) and likely pathogenic (LP) variants. sCTCs exhibited at least twice as many P variants compared to paired ctDNA samples, with CHD4, FGFR2, and HNF1α being the most common. The mutational profile of sCTCs showed concordance with at least one variant in paired ctDNA, and was enriched in dysregulated cytoskeleton and cell migration machinery. Specifically, COL7A, KIF5B, and RHOA mutations were prevalent in almost all sCTCs, with exclusive mutations (KIF5B and RHOA) in some cases. KRAS P variants were exclusive to 50% of ctDNA samples, paired sCTCs exhibited NRAS P variants. RAS isoforms were observed in 70% of sCTCs. Gene amplification occurred in CCND1/2, FGFR2, and ERBB2 in 90% of sCTCs, with CCND1 amplification more prevalent in sCTC clusters. Mutations in epigenetic repressors (HDAC1, KDM2B) were ubiquitous in sCTCs, co-occurring with DDR pathway mutations (F test, P 0.003, OR 11). Loss-of-function BRCA mutations were detected in 60% of sCTCs, and were absent in paired ctDNA. Additionally, 70% of sCTCs exhibited therapy-resistant variants (NF1, STK11, ARID1A, and PIK3CA), contrasting with a 10% frequency in paired ctDNA.

Conclusions: Genomic profile of sCTCs in advanced PDAC patients revealed a complex mutational landscape, providing additional clinically relevant insights that complemented ctDNA findings. sCTCs exhibited greater tumor mutational heterogeneity compared to paired ctDNA samples, and detected additional actionable pathogenic variants. Results suggest that sCTCs may offer therapeutically relevant molecular information for advanced PDAC patients for whom tissue is unavailable and ctDNA fails to provide actionable therapy options.

Citation Format: Jayant Khandare, Atul Bharde, Ganesh Khutale, Saloni Andhari, Richa Deshpande, Kanchan Hariramani, Madhura Basavalingegowda, Vikas Jadhav, Mohan Uttarwar, Gowhar Shafi. Comprehensive mutational profile of true single circulating tumor cells compared with CtDNA in advanced pancreatic adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7506.