Genotyping of Ebus-tbna Supernant Cell-free Dna in Nsclc (CELTICS)

Molecular Analysis of the Surnantant of Echoguidated Bronchoscopic Cytopunctions in Lung Cancer

The wide uptake of "liquid biopsy" diagnostics in the care of advanced cancer patients highlights the desire for improved access to tumor allowing accurate tumor genotyping (1). Genotyping of plasma cfDNA is now routine for detection of EGFR driver mutations at diagnosis of NSCLC, or for detection of the EGFR T790M mutation after TKI resistance, and is an emerging approach for the detection of other drivers (HER2 or BRAF mutations, ALK or ROS1 fusions…) (2) or the estimation of tumor mutation burden (TMB) (3). However, the most sensitive plasma genotyping platforms still have a sensitivity of only 70%-80%, such that a negative result requires tissue biopsy confirmation.

Study Overview

• Status: Recruiting
• Conditions: Lung Cancer
• Intervention / Treatment: Other: Molecular analysis of surnatant

Detailed Description

The wide uptake of "liquid biopsy" diagnostics in the care of advanced cancer patients highlights the desire for improved access to tumor allowing accurate tumor genotyping (1). Genotyping of plasma cfDNA is now routine for detection of EGFR driver mutations at diagnosis of NSCLC, or for detection of the EGFR T790M mutation after TKI resistance, and is an emerging approach for the detection of other drivers (HER2 or BRAF mutations, ALK or ROS1 fusions…) (2) or the estimation of tumor mutation burden (TMB) (3). However, the most sensitive plasma genotyping platforms still have a sensitivity of only 70%-80%, such that a negative result requires tissue biopsy confirmation. This poses a clinical challenge because negative plasma genotyping is correlated with more limited metastatic spread and lower tumor burden, such that biopsy of these patients may be even more challenging. Because invasive biopsy remains an integral part of the diagnostic strategy, methods are needed for maximizing the yield from these biopsy procedures.

There is a current paradox between the need for large amounts of tissue for multiplex analysis of an increasing number of targetable drivers and markers of response to immune therapy (PD-L1, TMB) and the development of minimally invasive biopsy procedures that results in limited specimens. Up to 25% of patients are thus treated without knowledge of the molecular profile of their tumor (4). In particular, 20% of endobronchial ultrasonography transbronchial needle aspiration (EBUS-TBNA) are rejected from genotyping due to lack of tissue (5) after time and tissue consuming diagnostics steps that are sometimes not required (resistance setting). Circulating tumor DNA is an emerging approach for cancer genotyping but sensitivity is limited to 70-80% (6) by inconsistent tumor shed and low DNA concentrations, so that tissue biopsy is still routine. Also, feasibility of TMB assessment on tissue is only 60% (likely much less on EBUS-TBNA specimens) (7) and approximately 80% in plasma (blood TMB, bTMB) (3).

The presence of cfDNA in several biological fluids and the feasibility of detecting mutations of interest (usually targeting only EGFR) in these fluids (urine, pleural fluid, CSF) have been clearly demonstrated (8-12), while blood is the most widely studied liquid biopsy substrate in advanced NSCLC.

Furthermore, we showed in a proof of concept study, investigating various FNA specimens in a limited numbers of patients that cytology samples' supernatant (usually discarded) is a rich source of DNA. Our results suggest that supernatant free DNA (sfDNA) can be used for baseline and resistance genotyping (13).

• Study Type: Observational
• Enrollment (Estimated): 50

Contacts and Locations

• Study Contact: Name: Nicolas Guibert, MD; Phone Number: +33 567778160; Email: guibert.n@chu-toulouse.fr

Study Locations

• France
  • Toulouse, France
    • Recruiting
    • Nicolas Guibert
    • Contact: Nicolas Guibert; Phone Number: +33 5 67 77 81 60; Email: guibert.n@chu-toulouse.fr

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 18 years to 65 years (Adult, Older Adult)
• Accepts Healthy Volunteers: No

• Sampling Method: Probability Sample

Study Population

Patients will be recruited in our Department in Toulouse University Hospital, during appointment needed for information and planning of the EBUS-TBNA procedure.

Description

Inclusion Criteria:

• Age > 18 years-old

• Patients planned for an EBUS-TBNA for

  1. Suspicion of stage IV lung cancer (PET+ mediastinal node(s)) (Cohort 1)
  2. Stage IV NSCLC with an EGFR, BRAF, HER2, MET mutation or ALK, RET or ROS1 rearranged NSCLC and acquired resistance to targeted therapy (Cohort 2)
• Performance status 0-3
• Informed consent

Exclusion Criteria:

• Refusal to participate
• Patient under legal tutelage

Study Plan

How is the study designed?

Design Details

• Observational Models: Cohort
• Time Perspectives: Prospective

Number of groups / cohorts

2

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
Identified mutation; The sensitivity of supernatant to identify the mutations detected on cell block (Gold standard).	Other: Molecular analysis of surnatant; The interventional pulmonologist selects the most suspect node. The corresponding TBNA is placed in Cytolyt and tagged using a sticker to indicate the specimen from which supernatant must be saved after the initial spin. The supernatant is transferred to the "Laboratoire de Biologie Médicale Oncologique" where it undergoes a further hard spin. The remaining supernatant is stored at -80°C before to send it to Foundation One for DNA extraction from 3 ml of supernatant and genotyping. Two 7,5 mL blood tubes are transferred to the laboratory to extract plasma. Plasma was stored at -80°C and then sent to Foundation One for DNA extraction from 2 mL of plasma and genotyping. 10 slides from the cell block are shipped to Foundation One. These specimens are tested by FoundationOne®CDX (tissue), and FoundationOne®Liquid (supernatant and plasma) for genomic and TMB analyses (hybrid-capture based next generation sequencing).
Non identified mutation; The sensitivity of supernatant to identify the mutations detected on cell block (Gold standard).	Other: Molecular analysis of surnatant; The interventional pulmonologist selects the most suspect node. The corresponding TBNA is placed in Cytolyt and tagged using a sticker to indicate the specimen from which supernatant must be saved after the initial spin. The supernatant is transferred to the "Laboratoire de Biologie Médicale Oncologique" where it undergoes a further hard spin. The remaining supernatant is stored at -80°C before to send it to Foundation One for DNA extraction from 3 ml of supernatant and genotyping. Two 7,5 mL blood tubes are transferred to the laboratory to extract plasma. Plasma was stored at -80°C and then sent to Foundation One for DNA extraction from 2 mL of plasma and genotyping. 10 slides from the cell block are shipped to Foundation One. These specimens are tested by FoundationOne®CDX (tissue), and FoundationOne®Liquid (supernatant and plasma) for genomic and TMB analyses (hybrid-capture based next generation sequencing).

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
main aim of the study	to investigate the sensitivity of sfDNA genotyping in various clinical settings and to compare it to cell block	18 months

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
TMB estimation	To assess the feasibility of TMB estimation on this specimen	18 months
sensitivity of plasma	To compare the sensitivity of plasma genotyping to cell block	18 months
concordance between plasma and supernatant	To investigate the concordance between plasma and supernatant for mutation detection and TMB estimation	18 months
mutation rate	To calculate the rate of patients with at least one additional mutation detected on supernatant compared to cell block	18 months
Sensitivity of supernatant and plasma	To compare the sensitivity of supernatant and plasma to cell block for the detection of specific alterations (EGFR, HER2, BRAF, PIK3CA, KRAS, MET mutations, ALK, ROS1, NTRK, RET fusions)	18 months
Turnaround time of supernatant	To compare the turnaround time of supernatant, plasma and cell block for genotyping	18 months

Collaborators and Investigators

• Sponsor: University Hospital, Toulouse
• Investigators: Principal Investigator: Nicolas GUIBERT, University Hospital, Toulouse

Study record dates

Study Major Dates

• Study Start (Actual): April 1, 2021
• Primary Completion (Estimated): December 31, 2025
• Study Completion (Estimated): December 31, 2025

Study Registration Dates

• First Submitted: November 9, 2020
• First Submitted That Met QC Criteria: November 9, 2020
• First Posted (Actual): November 10, 2020

Study Record Updates

• Last Update Posted (Actual): April 3, 2025
• Last Update Submitted That Met QC Criteria: March 31, 2025
• Last Verified: March 1, 2025

More Information

Terms related to this study

• Keywords: NSCLC; liquid biopsy; genotyping; cytology supernatant
• Additional Relevant MeSH Terms: Neoplasms by Site; Neoplasms; Respiratory Tract Diseases; Lung Diseases; Respiratory Tract Neoplasms; Thoracic Neoplasms; Lung Neoplasms
• Other Study ID Numbers: RC31/19/0090

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No
• product manufactured in and exported from the U.S.: No