Insights on Dissemination of Lung Cancer

In patients with resectable NSCLC treated with surgery alone or with surgery as part of a multimodal treatment, CTC, ctDNA, STAS, and lymph node micrometastasis levels will be evaluated to see if they are associated with recurrence patterns and long-term outcomes.

To achieve this objective, the presence and count of CTC and ctDNA will be analyzed from peripheral venous blood samples collected at three time points (before and after surgery and during follow-up). In addition, the following aspects will be evaluated as secondary objectives:

1. Evaluate the consistency of spatial transcriptomics between tumor cells located in the primary lung tumor and those present in STAS and/or lymph node metastases, as well as differences in the molecular profile between primary lung tumor cells and circulating tumor cells. 2. Quantify the impact of intraoperative variables on the release of CTCs and ctDNA into the bloodstream.

The main intraoperative variables are:

* The sequence of resection of hilar structures (e.g., vein first vs. artery first). * Lung manipulation techniques (open surgery, VATS RATS). * The extent of resection (segmentectomy, lobectomy, pneumonectomy). CTCs will be analyzed using the current gold standard CTCs will be analyzed using the current gold standard platform CTC (CellSearch system, Menarini Silicon Biosystems, Italy), while ctDNA will be evaluated using Next Generation Sequencing (NGS) technology (ILLUMINA platform).

For spatial transcriptomics assessment, eosin and hematoxylin-stained slides of the primary tumor and STAS and/or lymph node metastases will be analyzed using the Xenium platform (10x Genomics, USA) to spatially quantify mRNA species with the Xenium Human Immuno-Oncology profiling panel.

For project endpoints, continuous variables will be described as median (IQR) and compared using t-tests or Mann-Whitney tests; categorical variables will be described as n (%) and compared using χ²/Fisher tests. OS and DFS will be estimated using Kaplan-Meier, compared using log-rank tests, and analyzed using multivariate Cox models (verification of proportionality assumption; results as HR and 95% CI). For the incidence of recurrence considering death as a competing event (DFI), we will use cumulative incidence functions and Gray tests; for multivariate analysis, the Fine-Gray model (sub-hazard ratio, 95% CI). Associations between biomarkers (CTC/ctDNA as continuous and/or dichotomized, e.g., CTC ≥1 cell; VAF%/ng•mL) and time-dependent outcomes will be evaluated with Cox/Fine-Gray; the probability of STAS and lymph node micrometastases with logistic regression. Pre-/post-/follow-up CTC/ctDNA dynamics will be analyzed using paired tests (Wilcoxon) and linear/mixed models with repeated measures or ANCOVA adjusting for baseline values. The effect of intraoperative variables on ΔCTC/ΔctDNA will be estimated using linear/mixed models; on the risk of recurrence/death with Cox/Fine-Gray, adjusting for age, sex, stage, histotype, grade (G3), visceral pleural invasion, PD-L1, approach (Open/VATS/RATS), extent of resection, and adjuvant therapies (with possible stratification by stage). Missing data will be treated with multiple imputation (MAR assumption) and sensitivity analyses will be performed (e.g., alternative biomarker thresholds). Significance threshold α = 0.05 two-tailed; p-values and 95% CIs will be reported; when appropriate, correction for multiple comparisons (Benjamini-Hochberg). Analysis with R/Stata/SPSS.