Isolation of cfDNA and circulating extracellular vesicles allows for biomarker detection in a single aliquot of breast cancer patients' plasma

The last few years have witnessed exceptional advances in liquid biopsies based on the isolation and analysis of diverse tumor-derived material circulating in the blood, such as cell-free DNA (cfDNA) and extracellular vesicles (EVs). However, the isolation of different tumor-derived circulating components from a single aliquot of patients’ plasma and the consequent accurate detection of cancer biomarkers is still challenging. By taking advantage of a recently published nickel-based EVs isolation (NBI) protocol (1) that allows for recovery of cfDNA after EVs isolation, we generated a high-sensitivity molecular assay to accurately detect ERBB2 amplification and consequent HER2 overexpression on a limited volume of plasma collected from 20 breast cancer patients (stage I, II, and III) at diagnosis. The amplification of ERBB2 gene and the overexpression of the relevant encoded protein HER2 are currently used in the clinic for breast cancer classification at diagnosis and subsequent treatment decision with the anti-HER2 monoclonal antibody trastuzumab. By performing droplet digital PCR (ddPCR), we were able to detect ERBB2 amplifications in plasma cfDNA from 20 patients selected based on in situ evaluation of tissue biopsies (IHC and FISH). Moreover, we set up an antibody-based affinity reaction to detect HER2 protein on the surface of the isolated EVs that confirmed HER2 overexpression on a subset of patients. Most importantly, we also succeeded in absolute quantification of HER2 transcripts enclosed within EVs by performing ddPCR in samples of patients showing a range of circulating tumor-derived material. The yields and detection performance of this novel methodologic assay were tested on a cohort of healthy individuals (n=20) and on a cohort of triple-negative (and therefore HER2-negative) breast cancer patients (TNBC; n=20; stage: I, II, III). Overall, here we report a pilot study on a novel multimodal noninvasive method for breast cancer-specific biomarker detection from a minimal amount of plasma (1,5mL) integrating cfDNA-derived information with EVs-derived RNA and proteins analysis. This proof of concept may ultimately translate into relevant clinical applications for disease diagnosis as well as for therapy selection and monitoring of disease progression. Reference: 1. Notarangelo M et al. Ultrasensitive detection of cancer biomarkers by nickel-based isolation of polydisperse extracellular vesicles from blood. EBioMedicine 2019;43:114–26. Citation Format: Vera Mugoni, Caterina Nardella, Orsetta Quaini, Yari Ciani, Michela Notarangelo, Antonella Ferro, Orazio Caffo, Vito D9Agostino, Francesca Demichelis. Isolation of cfDNA and circulating extracellular vesicles allows for biomarker detection in a single aliquot of breast cancer patients’ plasma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B60.