Immune checkpoint inhibitors (ICIs) are largely used in the treatment of patients with advanced non-small-cell lung cancer (NSCLC). Novel biomarkers that provide biological information that could be useful for clinical management are needed. In this respect, extracellular vesicles (EV)-associated microRNAs (miRNAs) that are the principal vehicle of intercellular communication may be important sources of biomarkers. We analyzed the levels of 799 EV-miRNAs in the pretreatment plasma of 88 advanced NSCLC patients who received anti-PD-1 therapy as single agent. After data normalization, we used a two-step approach to identify candidate biomarkers associated to both objective response (OR) by RECIST and longer overall survival (OS). Univariate and multivariate analyses including known clinicopathologic variables and new findings were performed. In our cohort, 24/88 (27.3%) patients showed OR by RECIST. Median OS in the whole cohort was 11.5 months. In total, 196 EV-miRNAs out 799 were selected as expressed above background. After multiplicity adjustment, abundance of EV-miR-625-5p was found to be correlated with PD-L1 expression and significantly associated to OR by RECIST (p = 0.0366) and OS (p = 0.0031). In multivariate analysis, PD-L1 staining and EV-miR-625-5p levels were constantly associated to OR and OS. Finally, we showed that EV-miR-625-5p levels could discriminate patients with longer survival, in particular in the class expressing PD-L1 ≥50%. EV-miRNAs represent a source of relevant biomarkers. EV-miR-625-5p is an independent biomarker of response and survival in ICI-treated NSCLC patients, in particular in patients with PD-L1 expression ≥50%.

Immunotherapy has revolutionized the treatment paradigm of non-small cell lung cancer and improved patients’ prognosis. Immune checkpoint inhibitors have quickly become standard frontline treatment for metastatic non-oncogene addicted disease, either as a single agent or in combination strategies. However, only a few patients have long-term benefits, and most of them do not respond or develop progressive disease during treatment. Thus, the identification of reliable predictive and prognostic biomarkers remains crucial for patient selection and guiding therapeutic choices. In this review, we provide an overview of the current strategies, highlighting the main clinical challenges and novel potential biomarkers.

Background/Objectives: Despite advances in immunotherapy, reliable biomarkers beyond PD-L1 expression are urgently needed to optimize treatment decisions in advanced non-squamous NSCLC. Thyroid Transcription Factor-1 (TTF-1), a biomarker associated with favorable prognosis in chemotherapy-treated patients, has unclear prognostic implications in the immunotherapy era. Methods: We conducted a multicenter retrospective study involving 163 advanced non-squamous NSCLC patients treated with first-line immunotherapy or chemo-immunotherapy and an additional historical chemotherapy-only cohort (n = 37). We evaluated the prognostic significance of TTF-1 expression for progression-free survival (PFS) and overall survival (OS). A systematic review and meta-analysis, performed following PRISMA guidelines, integrated our findings with existing evidence. Hazard ratios (HRs) were calculated using Cox proportional hazards models. Results: TTF-1 negativity was associated with significantly worse median PFS (6.7 vs. 16 months; HR 2.22, 95% CI 1.59–3.13; p < 0.001) and OS (11.5 vs. 26.4 months; HR 2.33, 95% CI 1.64–3.45; p < 0.001) compared to TTF-1 positivity. The prognostic value of TTF-1 was independent of PD-L1 status, with limited predictive relevance of PD-L1 expression observed within TTF-1-negative tumors. Meta-analysis (9 studies, 14 cohorts, n = 2019 patients) confirmed significantly inferior outcomes for TTF-1-negative patients across multiple immunotherapy-based regimens (pooled HR for PFS: 1.75, 95% CI 1.50–2.04; OS: 1.76, 95% CI 1.45–2.14). Conclusions: TTF-1 negativity independently predicts poor prognosis in advanced non-squamous NSCLC treated with immunotherapy-based regimens, identifying patients with limited benefit despite high PD-L1 expression. Integrating TTF-1 status into clinical practice may guide personalized treatment strategies, highlighting the need for prospective validation.

Background Receptor activator of nuclear factor kappa-B ligand (RANKL) can directly promote tumor growth and indirectly support tumor immune evasion by altering the tumor microenvironment and immune cell responses. This study aimed to assess the prognostic significance of soluble RANKL in patients with advanced non-small cell lung cancer (NSCLC) receiving programmed cell death 1 (PD1)/programmed death-ligand 1 (PDL1) checkpoint inhibitor therapy. Methods Plasma RANKL levels were measured in 100 patients with advanced NSCLC without bone metastases undergoing monotherapy with PD1/PDL1 checkpoint inhibitors. To establish the optimal cut-off value, we used the Cutoff Finder package in R. Survival curves for four distinct patient groups, according to their RANKL and PDL1 levels (high or low), were generated using the Kaplan-Meier method and compared with the log-rank test. The Cox regression model calculated HRs and 95% CIs for overall survival (OS) and progression-free survival (PFS). Results The optimal RANKL cut-off was established at 280.4 pg/mL, categorizing patients into groups with high or low RANKL levels. A significant association was observed between increased RANKL concentrations and decreased survival rates at 24 months, only within the subgroup expressing high levels of PDL1 (p=0.002). Additionally, low RANKL levels in conjunction with elevated PDL1 expression correlated with improved PFS (median 22 months, 95% CI 6.70 to 50 vs median 4 months, 95% CI 3.0 to 7.30, p=0.009) and OS (median 26 months, 95% CI 20 to not reached vs median 7 months, 95% CI 6 to 13, p=0.003), indicating RANKL’s potential as an indicator of adverse prognosis in these patients. Multivariate analysis identified RANKL as an independent negative prognostic factor for both PFS and OS, regardless of other clinicopathological features. Conclusion These results highlight the prognostic and predictive value of RANKL specifically in patients with high PDL1 expression.

Background: Chemoimmunotherapy combinations represent the standard first-line treatment for non-oncogene addicted metastatic NSCLC (mNSCLC). However, evidence in elderly patients remains limited and conflicting. We conducted an analysis of the efficacy and safety of chemoimmunotherapy in patients aged ≥75 years enrolled in the Real-Combo Lung study, an observational study including patients with non-oncogene-addicted mNSCLC and PD-L1 expression < 50%. Patients and Methods: The primary objective of the study was to compare progression-free survival (PFS) and overall survival (OS) between patients aged ≥75 (elderly cohort) and those aged <75 years (non-elderly cohort). Safety outcomes were evaluated as a secondary objective. Results: A total of 495 patients were enrolled, with 89 (18%) aged ≥75 and 406 (82%) aged <75 years. No significant differences in PFS and OS were observed between the two cohorts. The median PFS was 13.3 months (95% CI: 9.3–NR) in the elderly cohort and 10.5 months (95% CI: 9.5–12.9) in the non-elderly cohort (unadjusted HR 0.84, 95% CI: 0.61–1.16, p = 0.29). The median OS was 17.5 months (95% CI: 14.7–NR) versus 21.4 months (95% CI: 17–NR), respectively (unadjusted HR 1.09, 95% CI: 0.76–1.56, p = 0.63). In multivariable analysis, ECOG PS ≥ 2 and baseline use of steroids were significantly associated with a worse outcome in the elderly cohort for both PFS and OS. Safety data did not differ significantly between cohorts. Conclusions: In this real-world study, elderly patients with mNSCLC derived outcomes comparable to those of younger patients, with similar efficacy and a manageable safety profile when treated with chemoimmunotherapy combinations.