Molecular Analysis of Endoscopic Cytology Samples Supernatant in Pulmonary Nodules

Study Overview
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Brief Summary
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Lung cancer screening is based on low dose CT scan (LDCT), a highly sensitive but poorly specific tool. Complementary specific approaches are thus strongly needed, among which cell-free DNA (cfDNA) genotyping has been proven highly specific but of low sensitivity (25 to 50% for stage I diseases) due to inconstant tumor shed. Tumor biopsy is thus often required and radial endobronchial ultrasound (rEBUS) bronchoscopy is a minimally invasive approach (<3% complications) but of limited sensitivity in cases of nodules < 20 mm. The investigators hypothesized that methylation analysis on cfDNA floating in supernatant derived from rEBUS specimens could improve rEBUS sensitivity

Detailed Description
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Tumor biopsy is often required to characterize indeterminate nodules. Radial endobronchial ultrasound (rEBUS) bronchoscopy is often used due to a low rate of complications but its sensitivity is limited for small nodules. The investigators hypothesized that methylation analysis on cfDNA floating in supernatant derived from rEBUS specimens could improve rEBUS sensitivity. The primary outcome of this pilot, diagnostic validation, monocentric study is the sensitivity of targeted (9 genes panel) methylation analysis on supernatant cfDNA to detect a malignant nodule. Secondary outcomes include the comparison of supernatant to pathology and plasma cfDNA methylation analysis. Specificity, negative predictive value (NPV), positive predictive value (PPV) of targeted (9 genes panel) methylation analysis on supernatant cfDNA to detect a malignant nodule will be analyzed. 60 patients planned for a rEBUS bronchoscopy for one, two or three < 20 mm nodule, without mediastinal or extra thoracic lesions (cT1N0M0) will be included. The day of the rEBUS bronchoscopy, 2 7.5 mL blood samples are collected. rEBUS samples' supernatant, usually discarded, is saved. Cell free DNA is extracted from these biological specimens at the Laboratory of Oncological Medical Biology (LBMO) in Toulouse University Cancer Institute and tested for methylation (targeted analysis on 9 genes). The patients will be followed for one year to obtain a final diagnosis and correlate it with tissue, nodule supernatant and plasma methylation analyses.

Official Title
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Molecular Analysis of Endoscopic Cytology Samples Supernatant in Pulmonary Nodules (Cancer Or Benign Endoscopy) (KOBE)

Conditions
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Lung Cancer, Lung; Node

Intervention / Treatment
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* Biological: Blood samples


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: rEBUS bronchoscopy planned for one, two or three ≤ 20 mm nodule World Health Organization (WHO) Performance status 0-3 Informed signed consent Patient affiliated or beneficiary of a social security scheme (Social Security or Universal Medical Coverage). Exclusion Criteria: Lung cancer diagnosed before the date of the procedure Lung cancer strongly suspected due to mediastinal or extra thoracic lesions Patient under State Medical Assistance Patient deprived of liberty on administrative or judicial decision, or patient under guardianship, curators or safeguard of justice

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Diagnostic
Intervention Model: Single Group Assignment
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: blood sampling<br>blood sample (2 tubes of 7.5 mL of blood = 15 mL) during the preoperative check-up on the day of the ultrasound-bronchoscopy in order to compare the sensitivity of the analysis of free circulating DNA present in the supernatant of pulmonary nodules less than 20 mm samples taken under ultrasound-bronchoscopy to that present in the plasma | Biological: Blood samples<br>* Patients will be taken from an additional blood sample (2 tubes of 7.5 mL of blood = 15 mL) during the preoperative check-up on the day of the ultrasound-bronchoscopy in order to compare the sensitivity of the analysis of free circulating DNA present in the supernatant of pulmonary nodules less than 20 mm samples taken under ultrasound-bronchoscopy to that present in the plasma.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Sensitivity of targeted (9 genes panel) methylation analysis on supernatant cfDNA to detect a malignant nodule will be evaluated. | Promoter methylation rates of the 9 targeted genes in the free circulating DNA of the endoscopic samples supernatant is obtained by quantitative Polymerase Chain Reaction (PCR) of bisulfite-treated DNA fragments from the cytological samples supernatant after DNA concentration and separation by size at the Centre of Cancer Research of Toulouse (CRCT). These analyses will be performed blinded to the clinical data and the histopathology result of the lung nodule. | 1st day (D0) of patients' inclusion, on morning of their ultrasound-bronchoscopy | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Comparison of supernatant to pathology and plasma cfDNA methylation analysis. Specificity, negative predictive value (NPV), positive predictive value (PPV) of targeted (9 genes panel) methylation analysis on supernatant cfDNA to detect a malignant nodule | Methylation profiles of free circulating DNA from plasma obtained after centrifugation of blood samples will be obtained by the same method as described for analysis of cytopunction's supernatant methylation profile. These analyses will be performed blinded to the clinical data and the histopathology result of the lung nodule. | 1st day (D0) of patients' inclusion, on morning of their ultrasound-bronchoscopy | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Circulating free DNA, Methylation, Lung cancer, screening, lung nodule, radial EBUS, endobronchial ultrasound