When Less Is More: Specific Capture and Analysis of Tumor Exosomes in Plasma Increases the Sensitivity of Liquid Biopsy for Comprehensive Detection of Multiple Androgen Receptor Phenotypes in Advanced Prostate Cancer Patients

Abstract

We evaluated the advantages and the reliability of novel protocols for the enrichment of tumor extracellular vesicles (EVs), enabling a blood-based test for the noninvasive parallel profiling of multiple androgen receptor (AR) gene alterations. Three clinically relevant AR variants related to response/resistance to standard-of-care treatments (AR-V7 transcript, AR T878A point mutation and AR gene amplification) were evaluated by digital PCR in 15 samples from patients affected by Castration-Resistant Prostate Cancer (CRPC). Plasma was processed to obtain circulating RNA and DNA using protocols based on tumor EVs enrichment through immuno-affinity and peptide-affinity compared to generic extraction kits. Our results showed that immuno-affinity enrichment prior to RNA extraction clearly outperforms the generic isolation method in the detection of AR-V7, also allowing for a distinction between responder (R) and non-responder (NR) patients. The T878A mutation was detected, overall, in nine out of 15 samples and no approach alone was able to reveal mutations in all harboring samples, showing that the employed methods complement each other. AR amplification was detected in the majority of CRPC samples analysed using either cell-free DNA (cfDNA) or exosome isolation kits (80%). We demonstrated that selective isolation of a subset of circulating exosomes enriched for tumor origin, rather than analysis of total plasma exosomes, or total plasma nucleic acids, increases sensitivity and specificity for the detection of specific alterations.

Isolation of extracellular vesicles improves the detection of mutant DNA from plasma of metastatic melanoma patients

“…Furthermore, studies to identify mutation-rich EV fractions in plasma or other body fluids 36 and to improve the efficiency and stringency of the affinity isolation with novel tumor-specific binding agents (eg. antibodies or aptamers) are warranted [37][38][39][40] .…”

Isolation of Extracellular Vesicles from Biological Fluids via the Aggregation–Precipitation Approach for Downstream miRNAs Detection

“…Thus, a comparison between the published reports and the obtained data led to the conclusion that precipitation-based protocols for EV isolation are effective and useful for subsequent miRNA analysis in different biological fluids. However, it should be noted that the developed method, as well as other protocols for polymer based EVs precipitation, UC, ultrafiltration and the range of other methods allow nonspecific isolation of EVs from biofluids, whereas the EVs enrichment through immune-affinity and peptide-affinity may isolate tumor exosomes specifically [60] and potentially detect tumor-specific molecules more sensitively.…”

Exosomes and Extracellular Vesicles in Myeloid Neoplasia: The Multiple and Complex Roles Played by These “Magic Bullets”

“…The first evidence of this arose from the study of circulating vesicles in solid tumors. Two of the principal mechanisms described are the delivery of chemo-resistant molecules [13,132,133] and the modulation of immunotherapy [134,135]. Successively, recent investigations have confirmed the presence of both mechanisms in myeloid malignancies as well, with special regard to AMLs.…”

“…Their involvement in intercellular communication is due to their capability of carrying and delivering different markers from the ancestral cell, in particular, proteins, DNA, and RNA, Intercellular communication through exosome release seems to be involved in several physiological and pathological mechanisms [6,9], as mentioned above. In the last decade, exosomes have been investigated and associated with various disorders [10][11][12], including cancer [13,14], neurodegeneration [15], and inflammatory diseases [16,17]. The continuous evolvement of new technologies, such as next-generation sequencing (NGS) [18], digital PCR (dPCR) [19], and proteomic strategies [20], have enabled deep investigations of exosomes from different points of view.…”