Study of single live circulating tumor cells capture and their genomic profile and enriched mutations of PIK3CA and HRR pathway in patients with breast cancer.
Authors: Khandare Jayant , Bharde Atul , Kishore Sirish , Andhari Saloni , Khutale Ganesh , Jadhav Bhagwat , Prajapati Sangeeta , Zhou Wenhui , Mobeen Fauzul , Hariramani Kanchan , Basavalingegowda Madhura , Jadhav Vikas Leelavati Balasaheb , uttarwar mohan , Joshi Ashish , Ghadyalpatil Nikhil , Velkuru Sai vivek , Thirumalairaj Raja , Shafi Gowhar 
Published: June 2024

e13161

Background: Capturing live single circulating tumor cells (sCTC) for intra-cellular compressive genomic profiling and concordance with ctDNA is highly challenging. Utilization of single sCTC genomics holds promise for detecting clinically relevant, actionable insights to tailor personalized therapy, especially when No Mutations Detected (NMDs) using CfDNA. Despite numerous studies providing valuable insights on mutational landscapes from breast tumors, predicting cancer progression, metastasis, and relapse continue to pose major challenges. ctDNA is suboptimal for 30-40% population with disease relapse/progression after a primary therapy. We show comprehensive genetic profile (CGP) of live sCTCs and paired ctDNA from a diverse breast cancer population. Methods: 26 live sCTCs (2 CTC clusters) were isolated from 12 patients. Live sCTCs were isolated from 12 Breast Cancer patients using OncoRadar live sCTC capture method. Whole genomes were amplified using multiple displacement amplification, target enriched with hybridization capture using OncoIndx (CGP 1080 gene panel) to obtain sequencing libraries, and sequenced on Illumina platform in paired end mode (depth 500x). The raw sequence alignment and variant calling was performed using iCare software. Paired ctDNA samples were processed the same way, except for sequencing depth (5500x). Results: Compared to paired ctNDA, genomic profiling of sCTCs revealed complex mutational patterns. Whereas sCTCs showed least two pathogenic (P) or likely pathogenic (LP) variants, paired ctDNA was negative for 25% cases (3/12). 92% sCTCs showed mutations in the PIK3CA-MTOR pathway with PIK3CA being the most prevalent (73 %). A significant association was observed between co-occurrence of HER2 amplification and PIK3CA mutations (P =0.04, OR=10). Similarly, sCTCs showed a strong correlation between HRR pathway and PIK3CA pathway mutations (r=0.76, P =0.003, OR=3.43). Genomic profile of sCTCs and paired ctDNA showed concordance with at least one gene mutation when compared at individual patient level. Additionally, at individual mutation level, PIK3CA exon 10 mutations were most frequent (61.5%) along with BRCA2 pathway variants, followed by IDH1/2(54%), ARID1A (50%), NRAS (46%), FGFR2 (42%) and TP53 (42%). Interestingly, 70% sCTCs showed the presence of therapy resistance conferring variants (NF1, ARID1A, ESR1, RB1 and PTEN), compared to only 25 % observed in paired ctDNA. Conclusions: The genomic profile of sCTCs represents true tumor heterogeneity and can be combined with ctDNA for better clinical outcome. sCTCs exhibited high mutational heterogeneity compared to paired ctDNA. Therapy resistance signatures were more prevalent in sCTCs and may offer valuable clinical insights for patients unable to provide tissue biopsy or show negative ctDNA.