Background Breast cancer (BC) represents the most prevalent cancer and is a leading cause of cancer related mortality among women worldwide. Despite notable therapeutic advancements, concern for metastatic recurrence remains a significant challenge. Liquid biopsy has emerged as a promising method for monitoring cancer treatment efficacy and disease progression. However, existing technologies predominantly focus on ctDNA, and their clinical utility is hindered by limited detection sensitivity and accuracy. To address these challenges, NVIGEN has developed NVIGEN X® comprehensive liquid biopsy, a novel minimally invasive approach that integrates ctDNA, CTC, and protein biomarkers. This comprehensive method could enable longitudinal monitoring of tumor dynamics, offering valuable insights into cancer biology, treatment responses, and emerging therapeutic targets. Here, we showcase compelling patient case studies, and the potential clinical implications of our technology for an exciting and evolving field in cancer diagnostics. Method Patients with advanced breast cancer were prospectively identified and consented. 10mL blood per timepoint was collected in Streck tubes. ctDNA, CTC and proteins were captured from a single tube of blood using NVIGEN magnetic nanoparticles according to established protocols. Following DNA extraction, ctDNA and CTC samples underwent NVIGEN’s highly sensitive sample workflow, optimized for NGS data accuracy and efficiency, employing hybridization capture with a 180 gene customizable cancer panel. Sequenced data were processed with NVIGEN’s proprietary high-sensitivity data analysis pipeline. Gene alterations identified from both ctDNA and CTC samples were analyzed and integrated to provide a comprehensive view of the tumor profile. Protein biomarkers of interest were detected using NVIGEN’s on-beads ELISA assay. Correlation analysis was performed to examine the relationship between the detected gene alterations, protein biomarkers, ctDNA/CTC data, and clinicopathologic features. Treatment response and identification of potential new treatment targets were evaluated based on the integrated analysis of ctDNA, CTC, protein biomarkers, and patient information. Result We collected 94 blood samples from 85 patients. Here we will present data from initial 36 patient samples. Of these 36 patients, 18 were tested with ctDNA/protein biomarkers (duo assays), and 18 were tested with ctDNA/CTC/protein biomarkers (trio assays). Comparison between duo and trio samples revealed that trio samples provided 2.5 times the number of detected variants compared to duo samples, with the median of variant numbers in duo assays being 52 (range 22-442), and in trio assays being 130 (range 73-703). Over 50% of the detected variants fell within the 0.1-0.5% variant allele frequency (VAF) range (number 2599) with total variant number being 5123. Several patient case studies were conducted using serial blood samples. These case studies revealed notable patterns of important gene alterations, such as ESR1, ERBB2 and PIK3CA, and demonstrated trends in the presence of these genes in ctDNA, CTC, or their coexistence. Protein biomarker information was correlated with ctDNA, CTC NGS data, and patient clinical status. Conclusions The analysis of the presented data from 36 patient samples with advanced breast cancer highlight the clinical relevance and potential significance of utilizing ctDNA, CTC and protein biomarkers in monitoring disease dynamics, treatment response, and guiding personalized treatment approaches. Our results suggest that individual protein biomarker analysis may be crucial in supporting personalized diagnostics and enabling more effective personalized medicine strategies.

Citation Format: Aihua Fu, Minh Ton, Kevan Wang, Weiwei Gu, Henry Jin, Nuzhat Shaikh, Sasha Madan, Shreya Perepa, Tianhong Li, Minetta Liu, Heather Parsons, George Sledge, Fauzia Riaz. NVIGEN X® Comprehensive Liquid Biopsy for Sensitive ctDNA, Circulating Tumor Cells (CTC), and Protein Detection in Breast Cancer: Next-Generation Sequencing, Patient Case Studies, and Clinical Implications [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-15-06.