Method for ultrasensitive detection of cell-free RNA

Commentary on "An ultrasensitive method for detection of cell-free RNA"

The research article "An ultrasensitive method for detection of cell-free RNA," by Nesselbush et al. published in Nature (2025), describes RARE-seq, a novel method optimized for cell-free RNA (cfRNA) analysis. This method offers significantly enhanced sensitivity for detecting tumor-derived cfRNA compared to traditional whole-transcriptome RNA sequencing. The study highlights the potential of cfRNA analysis for non-invasive gene expression profiling and disease monitoring.  

The development of RARE-seq has several key implications

• Enhanced sensitivity for cancer detection: RARE-seq demonstrates a limit of detection of 0.05% for tumor-derived cfRNA, which is approximately 50-fold more sensitive than whole-transcriptome RNA sequencing. This increased sensitivity allows for the detection of non-small-cell lung cancer (NSCLC) expression signatures across different stages, with higher sensitivity observed in later stages. The method also shows promise for detecting other cancer types, such as pancreatic adenocarcinoma, prostate adenocarcinoma, and liver hepatocellular carcinoma.  
• Improved liquid biopsy: The ultrasensitive nature of RARE-seq enhances the capabilities of blood-based liquid biopsies for non-invasive characterization of cancers, complementing existing methods that primarily focus on circulating tumor DNA (ctDNA). The study indicates that tumor-naive ctRNA analysis using RARE-seq can be more sensitive than tumor-naive ctDNA analysis for NSCLC detection.  
• Detection of resistance mechanisms: RARE-seq can identify both genetic and non-genetic mechanisms of resistance to therapies like EGFR TKIs in NSCLC, including histological transformation and the emergence of specific mutations or pathway activations. This is particularly significant as histological transformation is not detectable by mutation-based ctDNA approaches.  
• Potential for tissue-of-origin determination: The study demonstrates the potential utility of RARE-seq for identifying the tissue of origin of a malignancy, which could be valuable for patients with cancers of unknown primary origin.  
• Applications beyond oncology: RARE-seq shows promise for monitoring non-malignant conditions involving tissue injury, such as benign pulmonary conditions like COPD, COVID-19 infections, and ARDS. It can also be used to track RNA-based therapeutics, such as mRNA vaccines, and assess host responses.  

Future Directions

The findings suggest several avenues for future research and application:

• Validation in larger cohorts: Further studies evaluating large clinical cohorts of patients with early-stage cancers and risk-matched controls are needed to rigorously test the utility of cfRNA for cancer screening applications.  
• Combination with other liquid biopsy approaches: It would be interesting to compare and potentially combine RARE-seq with other liquid biopsy methods, such as methylation-based and fragmentomics-based approaches.  
• Exploring diverse clinical applications: Further research is required to fully establish the utility of cfRNA analysis using RARE-seq in each of the potential clinical applications explored in the study.  
• Improving pre-analytical factors: While RARE-seq includes a computational approach to address platelet contamination, further optimization of blood collection and processing protocols could potentially further minimize this confounding factor.  

Impact on Current Methods for RNA Analysis

RARE-seq significantly impacts current methods for cfRNA analysis by addressing key challenges:

• Overcoming platelet contamination: The study identifies platelet contamination as a major confounder in cfRNA analysis and proposes both pre-analytical and computational approaches to mitigate its effects. The computational method to remove platelet contributions broadly applies to future cfRNA studies.  
• Improving sensitivity for rare transcripts: By selectively capturing "rare abundance genes" (RAGs) that are lowly expressed or absent in healthy cfRNA, RARE-seq significantly enhances the detection sensitivity for transcripts originating from non-hematopoietic tissues and diseases. This is a notable improvement over whole-transcriptome RNA sequencing, which is less sensitive for detecting these rare transcripts due to the high background of transcripts from hematopoietic cells.  
• Enabling tumor-naive detection: The RAG-focused approach allows for tumor-naive detection of ctRNA, meaning it does not require prior knowledge of tumor-specific alterations. This is advantageous for applications like cancer screening and identification of tissue of origin.  
• Facilitating comprehensive resistance monitoring: Unlike methods solely focused on mutations, RARE-seq's ability to simultaneously detect somatic mutations and transcriptional changes provides a more comprehensive approach to monitoring therapeutic resistance.  

In summary, RARE-seq represents a significant advancement in cfRNA analysis by providing an ultrasensitive and versatile method with broad potential for clinical applications in oncology and beyond. The insights gained regarding platelet contamination and the targeting of rare transcripts are likely to influence future developments in the field of liquid biopsy and RNA analysis.

For further information visit here: An ultrasensitive method for detection of cell-free RNA (https://doi.org/10.1038/s41586-025-08834-1)