Bronchoscopic Cytology and Metagenomic Sequencing to Differentiate Cancer Treatment-related and Infectious Lung Injury

Summary

Here, we present a protocol to evaluate bronchoalveolar lavage fluid (BALF) through integrated bronchoscopy-guided collection, cytological analysis, and metagenomic next-generation sequencing (mNGS). This comprehensive workflow accurately distinguishes cancer treatment-related lung injury from pulmonary infections, facilitating precise and timely clinical decisions.

Abstract

Patients with cancer treatment-related lung injury (CTLI) frequently present with non-specific respiratory symptoms and radiological changes that closely mimic infectious pneumonia or tumor progression, presenting a significant challenge for a definitive diagnosis. Traditional diagnostic processes, mainly evaluated through blood biomarkers and standard microbial cultures, usually cannot make a clear diagnosis and take too much time. Here, we present a comprehensive protocol to diagnose CTLI by combining bronchoalveolar lavage fluid (BALF) cytological analysis with metagenomic next-generation sequencing (mNGS). The procedural workflow consists of three primary stages. First, standardized bronchoscopy is performed to obtain high-quality BALF samples. Second, conducting cytological analysis of the obtained BALF samples provides a snapshot of the lung microenvironment. This allows identification of inflammatory features and screening for malignant cells to exclude tumor progression. Finally, mNGS is utilized to identify or exclude active infectious etiologies. This advanced genomic technique achieves rapid, highly sensitive, and unbiased pathogen detection, successfully overcoming the limitations of traditional cultures. Representative results using this method demonstrate that this approach can effectively distinguish immune-related pneumonitis from active pulmonary infections or tumor progression. Compared with traditional diagnostic methods, this protocol has the advantage of quickly and accurately distinguishing CTLI from infectious etiologies and occult malignancies. Ultimately, this standardized workflow clarifies clinical diagnoses, guides critical treatment decisions, and improves patient outcomes.

Introduction

Advances in antitumor treatments, such as immune checkpoint inhibitors and targeted therapies, have significantly improved patient survival but also increased the incidence of non-infectious pulmonary toxicities, collectively known as cancer treatment-related lung injury (CTLI)^{1,2,3,4,5,6,7}. Notably, checkpoint inhibitor pneumonitis (CIP) has emerged as a fatal immune-related adverse event, with overall incidence rates of 2–19% and ....

Protocol

This protocol adheres to the principles of the Declaration of Helsinki and was approved by the institutional review board of our hospital. Informed consent was obtained from all participants prior to the procedure.

1. Preprocedure preparation

1. Patient evaluation (within 1 week of bronchoscopy)
   1. Obtain recent laboratory tests, including a complete blood count and coagulation profile, to ensure safety for an invasive procedure (Supplemental File 1).
   2. Perform a 12-lead electrocardiogram.
   3. Assess vital signs and ensure hemodynamic stability.
   4. Measure the patie....

Results

By following the above standardized protocol, clinicians can reproducibly obtain high-quality BALF samples and leverage both cellular and genomic analyses to distinguish CTLI from its mimics. Below, we present a representative result from our case to illustrate typical findings. A 68-year-old female with stage IV poorly differentiated lung cancer (right pleural region primary tumor) on pembrolizumab immunotherapy is presented to illustrate the BALF cytology + mNGS approach (Figure 1<.......

Discussion

In the current era of cancer therapy, treatment-related organ injuries, such as those induced by immune checkpoint inhibitors (ICIs), remain a major clinical concern that can limit treatment progression or even be life-threatening^3,4. Among ICI-induced immune-related adverse events (irAEs), pneumonitis has garnered significant attention as a primary cause of fatal outcomes¹⁰, accounting for 79% of irAE-related deaths in ICI-treated patient.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2% lidocaine	Hunan Kelun	N/A	Local anesthetic for airway mucosa.
0.5 mm glass beads	Sigma	N/A	Used for mechanical cell wall disruption of microbes.
BF-260 electronic bronchoscope	Olympus	N/A	The bronchoscope is used to obtain alveolar lavage fluid.
Burrows-Wheeler Alignment	Open Source	0.7.17(RRID:SCR_010910)	Algorithm for mapping sequences to the human genome.
High-flow humidified oxygen therapy instrument	BMC Medical	N/A	Provide a certain flow, heated and humidified breathing gas for patients with spontaneous breathing.
Vortex mixer	Scientific Industries	N/A	Scientific Industries
Midazolam	Jiangsu Enhua	N/A	Systemic sedative for patient comfort.
Nextseq 550 platform	Illumina	N/A	The gene sequence to sequence the genome in BALF
QIAseq Ultralow Input Library Kit	Tiangen	N/A	Kit for constructing metagenomic library
Qubit 4.0	Thermo Fisher Scientific	N/A	Instrument for detecting BALF nucleic acid content
TIANamp Micro DNA Kit DP316	Tiangen	N/A	The DNA extraction kit to extract total nucleic acids from BALF
Trypan blue	Biosharp	N/A	Dye used for cell viability assessment.
Water bath	Joan	N/A	Used for BALF sample inactivation at 65 °C.
Wright-Giemsa stain	yuanmu	N/A	Used for differential cell staining.
XN-9000 Hematology analyzer	Sysmex	SN-13699	Hematology analyzer to count total nucleated cells from BALF sample