Identification of Diagnostic and Prognostic Biomarkers in the Pathological Continuum of Bronco Chronic Obstructive Pulmonary Disease, Idiopathic Pulmonary Fibrosis and Pulmonary Neoplasia

Identification of Diagnostic and Prognostic Biomarkers in the Pathological Continuum of Bronco Chronic Obstructive Pulmonary Disease, Idiopathic Pulmonary Fibrosis and Pulmonary Neoplasia

Primary Objective:

To evaluate the association between inflammatory, immunological, genetic, and epigenetic biomarkers measured at enrollment and the clinical, functional, and phenotypic characteristics of patients with chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and lung cancer.

Secondary Objective:

To assess the prognostic value of the identified biomarkers by evaluating their ability to predict clinical outcomes at 12 months.

Primary Outcome Measure:

Association between baseline inflammatory, immunological, genetic, and epigenetic biomarkers and disease-specific clinical, functional, and phenotypic characteristics assessed at enrollment, including:

COPD: current or former smokers, stratified according to the predominant phenotype (emphysema or bronchiolitis); IPF: rapid progressors, slow progressors, and patients with combined pulmonary fibrosis and emphysema (CPFE); Lung cancer: current smokers, former smokers who quit less than 15 years before enrollment, former smokers who quit 15 years or more before enrollment, and never-smokers.

Secondary Outcome Measure:

Predictive performance of baseline inflammatory, immunological, genetic, and epigenetic biomarkers for 12-month clinical outcomes.

This is a non-profit, interventional pilot study without an investigational medicinal product. The study consists of two phases: a prospective phase (Phase I) and a retrospective phase (Phase II).

Study Timeline

The overall study duration will be 30 months, as follows:

Patient enrollment and biological sample collection: 18 months. Follow-up visit: 12 months after enrollment for each participant. Laboratory analyses: 20 months. Statistical analyses: at Month 24 (primary endpoint) and at Month 30 (12-month follow-up analyses).

Phase I - Prospective

Participants enrolled in the prospective phase will undergo study assessments according to the following schedule:

T0 (Baseline): Enrollment. T1: 12-month follow-up after enrollment. Clinical and laboratory data will be collected at baseline (T0) and at the 12-month follow-up visit (T1), as specified in the study schedule of assessments.

Phase II - Retrospective Archived tissue samples will be used.

Study Setting Phase I

The prospective phase will enroll:

40 patients with chronic obstructive pulmonary disease (COPD), current or former smokers; 40 patients with idiopathic pulmonary fibrosis (IPF), current or former smokers; 30 never-smoking patients with resectable Stage I or II lung adenocarcinoma undergoing surgical resection; 30 current or former smoking patients with resectable Stage I, II, or III lung adenocarcinoma undergoing surgical resection.

Patients with COPD and IPF will be recruited during outpatient visits. Patients with lung cancer will be recruited either during the preoperative outpatient evaluation or upon hospital admission for surgical treatment.

Phase II Archived tissue biopsy specimens collected during routine clinical practice, either fresh-frozen or formalin-fixed paraffin-embedded (FFPE).

The retrospective cohort will include samples from:

40 patients with COPD; 40 patients with IPF; 30 never-smoking patients with Stage I or II lung adenocarcinoma; 30 smoking patients with Stage I or II lung adenocarcinoma. Study Procedures Phase I - Prospective

Following written informed consent, all participants will undergo collection of approximately 40 mL of peripheral blood:

Three EDTA tubes of whole blood; Two serum tubes.

Peripheral blood mononuclear cells (PBMCs) will be isolated by Ficoll-Paque Plus density-gradient centrifugation, washed with phosphate-buffered saline (PBS), counted, and processed as follows:

Immunophenotypic characterization of innate and adaptive immune cell markers using monoclonal antibody staining and flow cytometry; In vitro stimulation with culture medium alone and with lipopolysaccharide (LPS) plus nigericin to evaluate NLRP3 inflammasome activation; Cryopreservation of a PBMC aliquot in dimethyl sulfoxide (DMSO) at -140°C until analysis.

Serum samples will undergo:

Automated extraction of microRNAs using commercially available column-based extraction kits; Reverse transcription into complementary DNA (cDNA), with storage at -20°C until analysis.

PBMCs will also be used for:

Genomic DNA extraction using the phenol-chloroform method, followed by storage at -20°C until analysis.

Phase II - Retrospective

Archived fresh-frozen and FFPE tissue samples collected during routine clinical care will be retrieved from the participating biobank and pathology department.

Patients whose biological samples are eligible for inclusion will be contacted to obtain specific informed consent for the use of their archived specimens for the present research project. Only samples from participants providing written informed consent will be included.

From FFPE tissue samples:

Genomic DNA will be extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen) and an automated extraction platform, then stored at -20°C until analysis.

MicroRNAs will be extracted using the RNeasy FFPE Kit (Qiagen) and an automated extraction platform, reverse-transcribed into cDNA, and stored at -20°C until analysis.

In addition to the translational research analyses specified in the protocol, results from routine molecular diagnostic testing previously performed on retrospective lung tumor specimens will also be collected. These include PD-L1 expression analysis in squamous cell carcinomas and Myriapod next-generation sequencing (NGS) mutational profiling (DNA and RNA) in lung adenocarcinomas. These molecular variables will be included as covariates in the statistical analyses.

Inclusion Criteria:

• Age ≥ 18 years.
• Clinical diagnosis of: Chronic Obstructive Pulmonary Disease (COPD), current or former smokers; and/or Idiopathic Pulmonary Fibrosis (IPF), current or former smokers; and/or Resectable lung adenocarcinoma (Stage I-III, according to clinical indication for surgical resection).
• Ability to comply with study procedures and follow-up visits.

Exclusion Criteria:

• Inability or unwillingness to provide informed consent.
• Active respiratory infection or acute exacerbation of COPD or IPF at the time of enrollment.
• Previous or concomitant malignant disease (except non-melanoma skin cancer) that could interfere with study objectives.
• Prior systemic immunosuppressive or anti-inflammatory therapy that may significantly alter immune profiling within a defined washout period (if applicable per protocol).
• Severe comorbid conditions limiting life expectancy or ability to complete follow-up (e.g., advanced heart failure, severe renal or hepatic disease).

Inadequate biological sample quality or impossibility to obtain required blood samples.