Using live true single-circulating tumor cell comprehensive genomics to show clonal evolution and tumor heterogeneity in pancreatic cancer management.

Abstract

4164 Background: Traditional tissue and CtDNA biopsies have limitations in pancreatic ductal adenocarcinoma (PDAC) due to sampling bias and inability to capture tumor evolution effectively. ctDNA offers Tx decisions, longitudinal monitoring for recurrence, real-time tumor evolution, and responses. However, ctDNA success is limited by its low sensitivity often with NMDs. While single circulating tumor cell (sCTC) genomics may offer higher sensitivity, but challenged with selective capture of live sCTCs without leukocyte contamination. We address limitations by identifying both clonal and rare sub-clonal mutations in PDAC. We report CellBiopsy assay to capture and release of sCTCs, facilitated by ctDNA-integrated comprehensive genomic profiling (CGP) at a true single-cell. Methods: In an observational study, ten advanced PDAC patients receiving SOC were accrued with MCW IRB approved protocol (PREDICT-MCW NCT ID:NCT05802069). All patients gave consent to investigational interventions. Live sCTCs were isolated at baseline (BL) and follow-up (FL) using OncoIndx Ikon sCTC assay in 10 mL of blood. Live CTCs were captured using glass beads with anti EpCam antibody and released in 96 well plate assay. DNA from individually captured sCTCs was linearly amplified, followed by target enrichment using OncoIndx CGP assay. Sequencing libraries were prepared and sequenced on Illumina NextSeq2000 (500× depth). ctDNA underwent deeper sequencing at 10000x coverage. Data was processed using iCare software for sequence alignment and variant calling. Results: Prospectively, 74 live sCTCs were isolated at baseline and follow up (mean sCTC distribution 7). Post SOC treatment, 50% of the patients (5/10) exhibited a 30% reduction in sCTC count at FL. NRAS mutations were the most frequent alteration observed in sCTCs (40.5%), followed by HRAS (27%) and TP53 (23%). Paired ctDNA predominantly revealed KRAS G12 variants (40%). Additional divergent molecular alterations in sCTCs were accounted for in NRAS, TP53, SMAD4, and PIK3CA-MTOR-AKT pathways, providing insights into mechanisms of treatment resistance and disease aggressiveness. Samples with co-occurring NRAS and TP53 , or SMAD4 , mutations along with ERBB2 amplification, were associated with aggressive disease. In FL sCTCs genomics revealed evolving molecular profiles enriched for activating variants in the PIK3CA-MTOR-AKT pathway compared to bulk tissue and ctDNA genomics at BL. Conclusions: Compared to ctDNA, sCTC CGP revealed heterogeneous molecular profile in PDAC, offering precise insights into tumor heterogeneity, clonal evolution, disease progression, and treatment outcome. Integrating paired DNA profiling of ctDNA and sCTC DNA may provide a more CGP landscape of PDAC. Ongoing analyses aim to evaluate temporal dynamics of CGP using ctDNA and sCTC DNA assay for advancing personalized management of PDAC. Clinical trial information: NCT05802069 .