Abstract 1941: Development of a multiomic approach for the detection of actionable biomarkers in metastatic breast cancer
Authors: Martin Blankfard, David Bourdon, Raj Srikrishnan, Alessandra Cunsolo, Kenneth Valkenburg, Cynthia Maddox, Renee Sears, Taylor Jensen
Journal: Cancer Research (2025)

Abstract Background: Thirty percent of all primary breast cancer patients develop metastatic disease with a 5-year survival rate of 22%. At the forefront of treatment are molecular diagnostic tests that guide treatment options for patients. The multiomic approaches of utilizing protein biomarker detection and next generation genomic analysis has improved blood-based diagnostics. We have coupled the characterization of circulating tumor cells (CTC) by immunofluorescence (IF) assays and single cell measurements of chromosomal instability with next-generation sequencing (NGS) of ctDNA in this multiomic approach. The combined assays identify actionable genomic variants, CTC chromosomal instability and protein biomarkers to characterize MBC. Methods: Contrived samples were created with cell line cells (CLCs) spiked into noncancerous blood from healthy donors for analytical validation. 46 patients with complete pathology and tissue biopsies were included in the clinical validation. Whole blood was collected in Streck® tubes and centrifuged for the isolation of plasma. cfDNA was extracted from plasma, and actionable variants were identified with PGDx elio plasma complete™ (EPC) NGS assay. EPC is a 521-gene NGS test, validated in a CAP/CLIA laboratory, that detects SNVs, Indels, fusions, CNVs, MSI, and bTMB. Red blood cells were lysed, and all nucleated cells were deposited on glass slides at a density of 3M cells per slide. CTC protein biomarkers were analyzed on the glass slides with IF assays. CTCs were identified as cytokeratin-positive and CD45/CD31-negative cells. CTCs were probed with HER2 and ER IF assays to determine biomarker status. CTCs were isolated and DNA was extracted for single cell sequencing and analysis for chromosomal instability and ERBB2 amplification. Results: EPC exhibited greater than 93% analytical sensitivity, and specificity. Overall precision was greater than 85%. An LOD of 1 CTC in 6 million cells and range of 1 - 300 cells/slide was reported. Sensitivity, specificity, accuracy and precision of the CTC assays was greater than 90%. 100% of the healthy donors exhibited no presence of CTCs and exhibited no actionable variants by the NGS ctDNA assays. Conversely, 19 (59%) cancer cases were found to have actionable alterations. CTCs were identified in 28 (88%) of the cases. Chromosomally unstable CTCs were found in the 24 (75%) of the patients. HER2-overexpressing CTC were found in 14 (43%) of cases, and concordant with 59% of matched tissue cases by HER2 (IHC). ER-overexpressing CTCs were found in 23 (71%) of the cases and concordant with 68% of matched tissue cases by IHC. Conclusion: This multiomic approach allowed for the identification of biomarker status in patients with MBC and for identification of actionable variants by NGS ctDNA. These data show the value of using a multiomic approach in liquid biopsy to provide an informed decision for further treatment in the metastatic setting. Citation Format: Martin Blankfard, David Bourdon, Raj Srikrishnan, Alessandra Cunsolo, Kenneth Valkenburg, Cynthia Maddox, Renee Sears, Taylor Jensen. Development of a multiomic approach for the detection of actionable biomarkers in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1941.