Abstract
Aims: Extracellular vesicles (EVs) are potential biomarkers for immunotherapy response. Building on our preliminary study linking a gene signature to outcomes, this study validates the signature in melanoma and non-small lung cancer (NSCLC) and explores links to tertiary lymphoid structures (TLS) and B cells.
Methods: Baseline plasma samples from 114 patients (65 melanoma, 49 NSCLC) treated with pembrolizumab or ipilimumab/nivolumab were collected. EVs were isolated using a modified Qiagen exoEasy protocol. EV-RNA was extracted (miRNeasy kit), converted to cDNA, and analysed for qRT-PCR on the ViiA 7 system targeting a 20-gene signature. Gene expression differences between responders/stable patients (≥6 months) and progressors were assessed using the Mann-Whitney U-test. Logistic regression, AUROC, and cox models evaluated prediction and survival. TLS in tumour samples are being analysed by Akoya Phenocycler Fusion; and B cell subsets in PBMCs via Cytoflex SRT.
Results: In melanoma patients, a predictive model combining all 20 genes achieved an AUC of 0.94, sensitivity of 92%, and specificity of 73%. Gene signature expression correlated with improved PFS (HR: 0.09, 95% CI: 0.02–0.42, p<0.0001) and OS (HR: 0.12, 95% CI: 0.03–0.54, p<0.0001). In NSCLC patients, the model had an AUC of 0.943, 100% sensitivity, and 71% specificity. Gene signature expression was also linked to better PFS (HR: 0.2, 95% CI: 0.07–0.51, p<0.0001) and OS (HR: 0.38, 95% CI: 0.17–0.90, p=0.0041). Ongoing analyses aim to elucidate whether the gene signature is associated with TLS in the tumour and/or B-cell subset in peripheral blood.
Conclusion: Our validation study underscores the potential of the EV-gene signature as a predictive biomarker for immunotherapy responses.