Comprehensive characterization of RNA cargo of extracellular vesicles in breast cancer patients undergoing neoadjuvant chemotherapy

Abstract

Extracellular vesicles (EVs) are g7aining increased attention as carriers of cancer-derived molecules for liquid biopsies. Here, we studied the dynamics of EV levels in the plasma of breast cancer (BC) patients undergoing neoadjuvant chemotherapy (NAC) and explored the relevance of their RNA cargo for the prediction of patients’ response to the therapy. EVs were isolated from serial blood samples collected at the time of diagnosis, at the end of NAC, and 7 days, 6, and 12 months after the surgery from 32 patients with locally advanced BC, and 30 cancer-free healthy controls (HCs) and quantified by nanoparticle tracking analysis. The pre-treatment levels of EVs in BC patients were higher than in HCs, significantly increased during the NAC and surgery, and decreased to the levels found in HCs 6 months after surgery, thus showing that a substantial fraction of plasma EVs in BC patients are produced due to the disease processes and treatment. RNA sequencing analysis revealed that the changes in the EV levels were associated with the alterations in the proportions of various RNA biotypes in EVs. To search for RNA biomarkers that predict response to the NAC, patients were dichotomized as responders and non-responders based on Miller-Payne grades and differential expression analyses were carried out between responders and non-responders, and HCs. This resulted in the identification of 6 miRNAs, 4 lncRNAs, and 1 snoRNA that had significantly higher levels in EVs from non-responders than responders at the time of diagnosis and throughout the NAC, and significantly lower levels in HCs, thus representing biomarkers for the prediction of response to NAC at the time of diagnosis. In addition, we found 14 RNAs representing piRNA, miRNA, lncRNA, snoRNA, and snRNA biotypes that were induced by NAC in non-responders and 2 snoRNAs and 1 piRNA that were induced by NAC in patients with early disease progression, thus warranting further functional studies on their role in chemoresistance and metastasis.

sRNAflow: A Tool for the Analysis of Small RNA-Seq Data

“…The identification of differentially expressed small RNAs holds considerable diagnostic potential, particularly in cancer and cardiovascular diseases [ 6 , 7 , 8 , 9 , 53 ]. A preliminary iteration of the presented program has been utilised in recent publications focusing on the analysis of extracellular vesicle contents, engaging in discussions about the biological interpretations of the results [ 51 , 53 , 54 ].…”

Unlocking the Potential of Circulating miRNAs in the Breast Cancer Neoadjuvant Setting: A Systematic Review and Meta-Analysis

“…Overall, nine studies (60%) [ 26 , 28 , 55 , 56 , 58 , 59 , 60 , 62 , 63 ] received a “low ROB”, four studies (27%) [ 57 , 61 , 65 , 66 ] were assigned an “unclear ROB” rating, and two studies (13%) [ 54 , 64 ] were assigned a “high ROB”. The omission of appropriate statistical analysis for predicting model validation and information in the first three domains (i.e., participants, predictors, and outcome) was a frequent source of bias in terms of high and unclear judgment.…”

Validation of potential RNA biomarkers for prostate cancer diagnosis and monitoring in plasma and urinary extracellular vesicles

“…Several studies, including our recent study of breast cancer, have shown that EV concentrations in body fluids are increased in cancer patients as compared to healthy controls and their levels reflect clinical events such as chemotherapy or surgery, thus suggesting that the excess EVs are produced in the body due to the disease process or treatment ( Lázaro-Ibáñez et al, 2014 ; Cappello et al, 2017 ; Krafft et al, 2017 ; Menck et al, 2017 ; König et al, 2018 ; Sadovska et al, 2022 ). Therefore, we expected that the EV numbers should decrease after the surgical removal of the tumor.…”