Genotyping of Ebus-tbna Supernant Cell-free Dna in Nsclc

NCT ID: NCT04624373

Last Updated: 2025-04-03

Basic Information

• Recruitment Status: RECRUITING
• Total Enrollment: 50 participants
• Study Classification: OBSERVATIONAL
• Study Start Date: 2021-04-01
• Study Completion Date: 2025-12-31

Brief Summary

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.

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).

Conditions

Lung Cancer

Study Design

• Observational Model Type: COHORT
• Study Time Perspective: PROSPECTIVE

Study Groups

Identified mutation

The sensitivity of supernatant to identify the mutations detected on cell block (Gold standard).

Molecular analysis of surnatant

Intervention Type: OTHER

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).

Molecular analysis of surnatant

Intervention Type: OTHER

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).

Interventions

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).

Intervention Type: OTHER

Eligibility Criteria

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

• Minimum Eligible Age: 18 Years
• Maximum Eligible Age: 65 Years
• Eligible Sex: ALL
• Accepts Healthy Volunteers: No

Sponsors

University Hospital, Toulouse

OTHER

Sponsor Role: lead

Responsible Party

Responsibility Role: SPONSOR

Principal Investigators

Nicolas Guibert

Role: PRINCIPAL_INVESTIGATOR

University Hospital, Toulouse

Locations

Nicolas Guibert

Toulouse, , France

Site Status: RECRUITING

Countries

France

Central Contacts

Nicolas Guibert, MD

Role: CONTACT

guibert.n@chu-toulouse.fr

567778160 ext. +33

Facility Contacts

Nicolas Guibert

Role: primary

guibert.n@chu-toulouse.fr

5 67 77 81 60 ext. +33

Other Identifiers

RC31/19/0090

Identifier Type: -

Identifier Source: org_study_id