# Highly Sensitive Detection of miR-200c in Metastatic Lymph Nodes Using Scanning Single-Molecule Counting

## TL;DR

A new method called SSMC can detect miR-200c in lymph nodes with high sensitivity, potentially improving lung cancer staging.

## Contribution

SSMC offers a faster and more sensitive alternative to RT-qPCR for miR-200c detection in metastatic lymph nodes.

## Key findings

- SSMC detected miR-200c with 85.7% sensitivity and 83.3% specificity for lymph node metastasis.
- SSMC results correlated strongly with RT-qPCR (R2 = 0.81, p < 0.0001).
- The rapid SSMC method provided results in 30 minutes, outperforming conventional RT-qPCR.

## Abstract

The novel 9th TNM classification of lung cancer distinguishes between single-station N2 (N2a) and multi-station N2 (N2b) disease, refining nodal staging. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a highly diagnostic, minimally invasive tool for this purpose, with rapid on-site evaluation recommended for improved efficiency. To reduce false-negative diagnoses, we explored microRNA biomarkers as a complementary approach to EBUS-TBNA. This study assessed the feasibility and clinical utility of scanning single-molecule counting (SSMC) for diagnostic samples, offering faster and more convenient miRNA detection than conventional RT-qPCR. We evaluated SSMC’s ability to detect miR-200c in metastatic lymph nodes, demonstrating its potential for highly sensitive molecular nodal staging.

Background/Objectives: Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a well-established, minimally invasive method for sampling mediastinal and hilar lymph nodes. It has high sensitivity and specificity for diagnosing lymph node involvement. However, achieving a definitive cytopathological diagnosis can be challenging owing to the limited presence of atypical tumor cells. The scanning single-molecule counting (SSMC) method enables rapid measurement of target molecule expression. This study assessed the correlation between miR-200c expression measured by SSMC and lymph node metastasis confirmed by cytopathology. Methods: Following EBUS-TBNA in patients with lung cancer or suspected lung cancer, we flushed 22-gauge biopsy needles with 1 mL saline and extracted microRNA (miRNA) from the lavage fluid. The quality of SSMC results for miR-200c was evaluated and compared with that of quantitative real-time polymerase chain reaction (RT-qPCR). Results: Linear regression analysis of miR-200c between SSMC and RT-qPCR showed a statistically significant positive correlation (R2 = 0.81, p < 0.0001), demonstrating the feasibility of SSMC for evaluating miRNA in lymph nodes. Based on these findings, we enrolled and analyzed 100 lymph nodes from 86 patients. High miR-200c expression detected lymph node metastasis with high sensitivity (85.7%) and specificity (83.3%) and an AUC of 0.88. No discordance was observed between the standard SSMC and rapid methods. Conclusions: The expression of miR-200c, as measured by SSMC, may serve as a biomarker for molecular nodal staging. The rapid SSMC method provides results within 30 min, significantly faster than conventional RT-qPCR, and may complement rapid on-site cytological evaluation.