Mutational profiling of true single circulating tumor cells (sCTC) in metastatic lung cancer to determine genomic heterogeneity and drug-resistance signatures.
Authors: Khandare Jayant , Bharde Atul , Tibdewal Anil , Bose Chirantan , Nadagouda Snigdha , Khutale Ganesh , Aland Gourishankar , Andhari Saloni , Agarwal Jaiprakash , Bhogadi Amani , Uttarwar Mohan , Vasudevan Aravindan , Chaturvedi Pankaj , Prabhash Kumar , Shafi Gowhar 
Published: June 2023

e21024

Background: Omics of single Circulating tumor cells (CTCs) is highly envisioned though challenging. . Genotyping of TRUE sCTC capture and omics data can determine the molecular heterogeneity of a single cell without the stringent need for deep sequencing compared to tissue or blood (1/100th fold compared to ctDNA). Here, we show the utility of sCTC genome profiling to determine inter and intra-patient mutational heterogeneity and compare the outcome with matched ensembled ctDNA mutational profiles. Methods: OncoRADAR an antibody-affinity-based platform was used to capture live sCTCs without cell fixing. 50 sCTCs (90 %) and clusters (10 %) were isolated from 20 metastatic lung cancer pts for mutational genotyping. Amplified whole genomes were subjected to targeted hybrid-capture comprehensive genome profiling. Libraries prepared using a custom-designed comprehensive gene panel OncoIndx, targeting 600 cancer-relevant exons including MSI and HRR pathway genes, were sequenced in paired-end mode on the Illumina platform. Variant calling was performed using an in-house developed bioinformatics pipeline. Pathway enrichment analysis was performed manually using data enriched from different gene pathway databases. Results: 81 mutations were detected in 56 genes across all sCTCs with pathogenic, likely pathogenic, and unknown significance. The mutational landscape included SNVs (38.6%), splice variants (10%), frameshift mutations (0.8%), and truncations (50.4%). From a subset of patients (10 %), multiple sCTCs showed PTEN variant cooccurring with ESR1 and MET. This suggest a clonal selection and emergence of subclones during progression. Pathways related to drug resistance, migration, deregulated cell cycle, and signaling were significantly enriched in multiple sCTCs when compared to plasma genotyping. Low average TMB (1.89), LOH (3.9) and HRD (14.67%), and high MSI (28.68%) scores were observed. In contrast, matching ctDNA had higher average LOH (18.67), HRD (45.08%), and lower MSI (0.99) scores. Multiple sCTCs from a small fraction of patients had comparable values for genome stability indicators, suggesting maintained genome stability. Genotyping of sCTC among patients showed a high variability compared to comprehensive analysis at the ctDNA level. Conclusions: Targeted gene profiling of true sCTCs revealed multiple variants that remained undetected in ctDNA, enabling a better understanding of tumor heterogeneity. Analysis of multiple sCTCs from the same patient underscores the importance of clonal evolution and plasticity during metastasis. Longitudinal monitoring of sCTCs may reveal personalized markers for disease prevalence, metastasis and relapse. sCTC genotyping may help design personalized therapy with better precision.