Tracking the Response to Immunotherapy: Blood microRNA Dynamics in Patients With Advanced Non-Small Cell Lung Cancer

Abstract

Purpose: Despite the significant improvement in outcomes for patients with advanced non-small cell lung cancer (NSCLC) treated with immune checkpoint inhibitors (ICIs), resistance, whether primary or secondary, remains a substantial challenge. Currently, reliable biomarkers to monitor ICI response are lacking, highlighting the need for minimally invasive tools like liquid biopsy to track treatment efficacy. This study aimed to identify circulating microRNAs (miRNAs) as potential biomarkers to track ICI response in patients with NSCLC.

Materials and methods: The Apollo longitudinal study enrolled patients with advanced NSCLC receiving ICI in first or subsequent lines. Plasma samples were collected at baseline and follow-up to prospectively assess miRNA profiles until progressive disease (PD). Using a custom reverse transcription-quantitative polymerase chain reaction platform, 276 ratios among 24 lung cancer-related miRNAs were analyzed. The generalized estimating equation and joint models were applied to select the miRNA ratios most associated with PD over time. To control for multiple testing, the Benjamini-Yekutieli method was applied setting a 10% false discovery rate threshold.

Results: From the 211 patients, a total of 454 plasma samples were analyzed. Clinical and biochemical variables had little effect on miRNAs' profile. The analysis identified nine miRNA ratios, all involving miR-145-5p, as significant biomarkers for monitoring treatment response, even after adjustment for the line of therapy. These ratios exhibited a longitudinal modulation pattern consistent with radiologic response, particularly in patients who initially benefited from ICI treatment. In addition, in an independent set of 32 plasma samples from 10 patients receiving ICI as maintenance therapy, the same trends were observed.

Conclusion: A focused panel of miRNA ratios, driven by miR-145-5p, effectively reflects response to ICI therapy in patients with advanced NSCLC, highlighting their potential as biomarkers for treatment monitoring.

FIG 1.

Kaplan-Meier plot for (A) progression-free survival and (B) overall survival. Shadows represent the 95% CIs.

FIG 2.

Over-time plasma sample collection and circulating miRNA profiling. (A) Map of samples collected at BL, first radiologic examination (R1), and subsequent years of follow-up (Y1, Y2, Y3) from the 211 patients with NSCLC overall and the 121 considered for longitudinal analysis. Arrows' size reflects the number of available plasma samples. Green background is for samples collected while patients benefit from ICI treatment (stable or responsive disease). Red background is for samples collected at the time of PD. Gray background is for censored patients. Box plot reporting the distribution of values (B) before and (C) after miRNA ratio transformation in each plasma sample. (D) Hierarchical unsupervised clustering analysis considering 450 samples with available molecular profiling and the 276 unique miRNA ratios generated. Samples were classified in strata of characteristics such as batch number, year of sample collection, and time of treatment: BL, R1, Y1, Y2, Y3, PD, and last radiologic examination before PD. (E) Radar plots reporting the association, as by the Spearman correlation test, of BL clinical variables with the first three PCs derived from PC analysis of plasma miRNA profiling. BL, baseline; ECOG, Eastern Cooperative Oncology Group; ICI, immune checkpoint inhibitor; LDH, lactate dehydrogenase; miRNA, microRNA; NLR, neutrophil-lymphocyte ratio; NSCLC, non–small cell lung cancer; PC, principal component; PD, progressive disease; PS, performance status.

FIG 3.

Modulation over time of (A) the PFS hazard versus (B-J) selected miRNA ratio patterns, and their distributions by longitudinal plasma sample occurrences considering (K) the 121 patients with NSCLC with longitudinal data receiving ICI in first or subsequent lines, and (L) 10 on ICI maintenance. Box plot colors represent the time of plasma sample collection: the BL; the first radiologic examination in the presence of response or stable disease (R1); annual follow-up (Y1, Y2, Y3); and the last radiologic examination before PD, PD, and on maintenance (M1, M2). BL, baseline; ICI, immune checkpoint inhibitor; miRNA, microRNA; NSCLC, non–small cell lung cancer; PD, progressive disease; PFS, progression-free survival.

FIG 4.

Representative images describing the longitudinal modulation of nine selected microRNA ratios, as well as their average value (ratio's mean), in four patients with lung ADC or SCC who received a benefit from treatment with immune checkpoint inhibitors. (A and B) Two patients with low (<50%) and (C and D) two patients with high (≥50%) PD-L1 expression were selected. The diameter of the main lung lesion (orange arrow) was considered as the parameter of the radiologic response. For each ratio, the –ΔΔCt values were calculated using the lowest expressor as a calibrator and multiplied by the corresponding β1 coefficient identified by the generalized estimating equation model. ADC, adenocarcinoma; SCC, squamous cell carcinoma.

FIG A1.

CONSORT diagram describing the Apollo study population considered for the present study. ICI, immune checkpoint inhibitor; NSCLC, non–small cell lung cancer.