Connecting the dots: (RANKL(+)) extracellular vesicle count in blood plasma in relation to bone metastases, skeletal related events and osimertinib treatment in patients with EGFR mutated non-small cell lung cancer.

BACKGROUND: The biological mechanisms responsible for the different incidences of bone metastases in molecular subgroups of non-small cell lung cancer (NSCLC) are not identified. Extracellular vesicles (EVs) may play a role, as they are involved in organotrophic metastasis. Phosphorylation of epidermal growth factor receptor (EGFR) in exosomes possibly leads to an increase in receptor activator of nuclear factor κB ligand (RANKL) triggering osteoclastogenesis. In search for new biomarkers with focus on EVs and RANKL, we studied in plasma of patients with EGFR (+) NSCLC the associations between the total concentration of EVs, RANKL(+) EVs, RANKL, and osteoprotegerin (OPG) protein levels, osimertinib treatment, presence of bone metastases and skeletal related events (SREs). METHODS: From the prospective biomarker cohort study START-TKI (NCT05221372), including patients with metastatic EGFR (+) NSCLC, we collected deep frozen plasma samples at initiation and during osimertinib treatment. Imaging flow cytometry (IFC) was used to determine the concentration of tetraspanin positive EVs and detection of RANKL on EVs. RANKL and OPG levels were measured by enzyme-linked immunosorbent assay (ELISA). Data on demographics, date of NSCLC diagnosis, date of initiation of osimertinib, presence of bone metastases and SREs were collected. Primary endpoint was the relation between (RANKL(+)) EV levels and bone metastases. RESULTS: Forty unique patients with in total 50 plasma samples (45% at initiation of osimertinib, 55% during osimertinib treatment) were included. Identification of EVs was possible in 38/40 patients, and determination of RANKL and OPG plasma levels in all samples. Of these 40 patients, 25 (63%) had bone metastases at sample collection. Both total EV and RANKL(+) EV concentrations were significantly higher in samples at initiation of osimertinib compared to samples during treatment [mean ± standard deviation (SD), 6.3×10(12)±2.1×10(12)/mL plasma vs. 3.2×10(12)±1.9×10(12)/mL plasma, P≤0.001 for total EV concentrations; and 2.2×10(10)±9.3×10(9)/mL plasma vs. 1.1×10(10)±8.0×10(9)/mL plasma, P=0.001 for RANKL(+) EVs]. Patients without a SRE had a significantly higher concentration of RANKL(+) EVs compared to patients with an SRE (mean ± SD, 1.8×10(10)±1.1×10(10)/mL plasma vs. 1.1×10(10)±7.4×10(9)/mL plasma, P=0.02). No association was found between the total EV concentration or RANKL(+) EVs, plasma levels of OPG and RANKL and bone metastases. CONCLUSIONS: No association was found between the presence of bone metastases and the total concentration of EVs, RANKL(+) EVs, or plasma values of RANKL and OPG. In patients without SREs the concentration of RANKL(+) EVs was significantly increased. Both the total EV and RANKL(+) EV concentrations significantly decreased during osimertinib treatment. This opens new perspectives for the role of (RANKL(+)) EVs as prognostic biomarkers for EGFR (+) NSCLC disease progression and response to therapy.