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. 2025 Mar 31;14(3):761-774.
doi: 10.21037/tlcr-24-1007. Epub 2025 Mar 19.

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

Anita J W M Brouns  1   2   3 , Iris J Robbesom-van den Berge  4 , Sophie M Ernst  5 , Christi M J Steendam  5 , Wouter W Woud  6 , Liang Wu  7 , Anne-Marie C Dingemans  5 , Lizza E L Hendriks  2   3 , Marjolein van Driel  4
Affiliations

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

Anita J W M Brouns et al. Transl Lung Cancer Res. .

Abstract

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), 6.3×1012±2.1×1012/mL plasma vs. 3.2×1012±1.9×1012/mL plasma, P≤0.001 for total EV concentrations; and 2.2×1010±9.3×109/mL plasma vs. 1.1×1010±8.0×109/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, 1.8×1010±1.1×1010/mL plasma vs. 1.1×1010±7.4×109/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.

Keywords: Bone metastases; epidermal growth factor receptor+ non-small cell lung cancer (EGFR+ NSCLC); extracellular vesicles (EVs); osteoprotegerin (OPG); receptor activator of nuclear factor κB ligand (RANKL).

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Conflict of interest statement

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-24-1007/coif). A.J.W.M.B. has received a travel grant from the ELCC, and she have participated in an advisory board for Jansen (payment was paid to A.J.W.M.B.). C.M.J.S. has received research funding from AstraZeneca, stichting NVALT studies, ICON Clinical Research/Arcus biosciences, VitroScan Leiden (payment was paid to institution), consulting fees from BMS (payment was paid to institution), honoraria from iDoctor and Benecke (payment was paid to institution) and C.M.J.S. received uncompensated relationships from Eli Lily (payment was paid to C.M.J.S.). A.M.C.D. has received grants or contracts from Amgen, Dutch Cancer Society and Hanart, consulting fees from Amgen, Bayer, Boehringer Ingelheim, Sanofy, Roche, Johnson&Johnson, Astra Zeneca, Pfizer and Mirati, honor from Janssen, Pfizer, Astra Zeneca, Lilly and Amgen. All payments were paid to the institution. A.M.C.D. takes part in a data safety monitoring board or advisory board from Takeda, Roche and Lilly (all payments were paid to the institution). A.M.C.D. is chair from EORTC LCG, unpaid. L.E.L.H. has received research fundings from Roche Genentech, AstraZeneca, Boehringer Ingelheim, Takeda, Merck, Pfizer, Novartis, Gilead (all payments were paid to institution), honor for speaker educationals/webinars from AstraZeneca, Bayer, Lilly, MSD, high5oncology, Takeda, Janssen, GSK, Sanofi, Pfizer (all payments were paid to institution) and from Medtalks, Benecke, VJOncology, Medimix (payments were paid to LEL Hendriks). She took part in advisory boards from Abbvie, Amgen, Anhearth, AstraZeneca, Bayer, BMS, Boehringer Ingelheim, Daiichi, GSK, Janssen, Lilly, Merck, MSD, Novartis, Pfizer, Pierre Fabre, Roche, Sanofi, Summit Therapeutics, Takeda (all payments were paid to institution). L.E.L.H. is the member of guideline committees of Dutch guidelines on NSCLC, brain metastases and leptomeningeal metastases (payment to L.E.L.H.), ESMO guidelines on metastatic NSCLC, non-metastatic NSCLC and SCLC (non-financial), other (non-financial): secretary NVALT studies foundation, subchair EORTC metastatic NSCLC systemic therapy, vice-chair scientific committee Dutch Thoracic Group. L.E.L.H. is local PI of clinical trials from AstraZeneca, GSK, Novartis, Merck, Roche, Takeda, Blueprint, Mirati, Abbvie, Gilead, MSD, Amgen, Boehringer Ingelheim and Pfizer (all payments were paid to institution). The other authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
Distribution of EVs in plasma samples of patients at initiation of osimertinib therapy and during osimertinib therapy. (A) Total concentration of EVs. (B) Concentration of RANKL+ EVs. (C) Intensity of RANKL expression on RANKL+ EVs. (D) Percentage of RANKL+ EVs of total EVs per patient. (E) Representative dot plot images of IFC data analysis of plasma EVs from a patient at initiation and a patient during treatment with osimertinib. Intensity_MC_Ch05 (Y-axis) shows the APC-conjugated tetraspanin signal and Intensity_MC_Ch02 (X-axis) shows the AF488 conjugated RANKL signal. Red boxes indicate the tetraspanin positive particles, blue boxes indicate particles that are tetraspanin positive and also RANKL positive (double positive). Green boxes show the RANKL positive particles that are not tetraspanin positive, in orange the negative particles are shown for both fluorochromes. All settings are based upon a series of controls shown in the Appendix 1. EVs are characterized as tetraspanin positive particles, RANKL+ EVs as RANKL positive and tetraspanin positive particles (double positive). An asterisk (*) indicates a significant difference (P<0.05) between two groups, a horizontal line represents the mean in (A,B,D) or the median in (C). APC, allophycocyanin; a.u., arbitrary units; EVs, extracellular vesicles; MFI, median fluorescence intensity; OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor κB ligand.
Figure 2
Figure 2
Distribution of EVs in plasma samples of patients with and without bone metastases at the time of sample collection. (A) Total concentration of EVs. (B) Concentration of RANKL+ EVs. (C) Intensity of RANKL expression on RANKL+ EVs. (D) Percentage of RANKL+ EVs of total EVs per patient. A horizontal line represents the mean in (A,B,D) or the median in (C). A.u., arbitrary units; EVs, extracellular vesicles; MFI, median fluorescence intensity; OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor κB ligand.
Figure 3
Figure 3
Distribution of EVs in plasma of patients with and without SREs at time of sample collection. (A) Total concentration of EVs. (B) Concentration of RANKL+ EVs. (C) Intensity of RANKL expression on RANKL+ EVs. (D) Percentage of RANKL+ EVs of total EVs per patient. An asterisk (*) indicates a significant difference (P<0.05), a horizontal line represents the mean in (A,B,D) or the median in (C). A.u., arbitrary units; EVs, extracellular vesicles; MFI, median fluorescence intensity; OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor κB ligand; SRE, skeletal related event.
Figure 4
Figure 4
Distribution of OPG and RANKL protein levels, and RANKL:OPG ratio in plasma of patients at initiation of osimertinib therapy and during osimertinib therapy. (A) Plasma level of OPG (pg/mL). (B) Plasma level of RANKL (pg/mL). (C) RANKL:OPG ratio in plasma. A horizontal line represents the median in (A,B), or the mean in (C). RANKL and OPG protein levels were measured via ELISA. ELISA, enzyme-linked immunosorbent assay; OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor κB ligand.
Figure 5
Figure 5
Distribution of OPG and RANKL protein levels, and RANKL:OPG ratio in plasma of patients with and without bone metastases at time of sample collection. (A) Plasma level of OPG (pg/mL). (B) Plasma level of RANKL (pg/mL). (C) RANKL:OPG ratio in plasma. An asterisk (*) indicates a significant difference between the two groups (P<0.05). A horizontal line represents the median in (A,B), or the mean in (C). OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor κB ligand.
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