Stanford Medicine researchers have developed a new liquid biopsy that detects cancer, resistance to cancer therapies, and tissue damage from noncancerous conditions by analyzing messenger RNA (mRNA) circulating in the bloodstream. The research, published in Nature, details this new method called RARE-seq (random priming and affinity capture of cfRNA fragments for enrichment analysis by sequencing), which took more than six years of work to make circulating RNA a viable clinical biomarker.
“Just as archeologists can learn about ancient societies by studying the garbage they left behind, we can learn a lot about what is going on in the cells of a patient’s body based on the degraded RNA molecules that are cleared through the blood,” said co-lead author Maximilian Diehn, MD, PhD, a professor of radiation oncology at Stanford Medicine. “We have developed a sensitive, versatile new type of liquid biopsy that measures cell-free and circulating-tumor RNA and has the potential to enhance personalized medicine in cancer and noncancer diseases.”
Unlike many existing liquid biopsies, which focus on detecting tumor-derived DNA fragments and require the presence of mutations, the RARE-seq test analyzes cell-free messenger RNA (cfRNA). mRNA reflects real-time gene expression, providing insight into how cells behave, not just their genetic code. This allows detection of cancer and therapy resistance that arises from non-mutational changes, information DNA-based tests would detect.
Cell-free RNA in blood primarily consists of ribosomal RNA, with mRNA making up less than 5%. To improve the sensitivity of the test, the team focused on about 5,000 genes that are rarely expressed in healthy individuals’ blood. By targeting these “rare abundance genes,” the researchers increased cancer detection sensitivity over 50-fold.
“Analysis of the rare abundance genes lets us focus on the most relevant subset of RNA for detecting disease, just like archaeologists who want to learn about what people ate might focus on a subset of artifacts such as food containers or utensils,” said study co-leader Ash Alizadeh, MD, PhD, a professor of oncology and hematology and leader of the Cancer Genomics Program at Stanford Cancer Institute.
In the study, the researchers tested RARE-seq on 437 plasma samples from 369 individuals with either cancer or non-malignant conditions, as well as healthy controls. Among patients with non-small-cell lung cancer (NSCLC), the test detected RNA signatures of cancer in 30% of stage I cases, increasing to 83% sensitivity in stage IV, with 95% specificity. RARE-seq outperformed tumor-naive circulating tumor DNA (ctDNA) methods in sensitivity, particularly in earlier stages of disease.
The team was also able to use RARE-seq to detect mechanisms of resistance to EGFR tyrosine kinase inhibitors in lung cancer patients. These resistance mechanisms included both mutation-based changes and histological transformations to provide a more comprehensive view of disease progression and show the potential of this new testing method to monitor disease progression.
As Alizadeh explained: “Unfortunately, a significant fraction of our patients who are being treated for cancer go on to have their therapy stop working, and that resistance is often based on adaptations that do not involve genetic changes, but instead altering how the cells behave or even how the cells look under a microscope.”
Vital to the development of the test, the research team needed to find strategies that could ignore signals from the RNA from platelets. The platelets, which carry RNA but not DNA can interfere with the signals of circulating RNA that is derived from cancerous or diseased tissue.
“This approach means that the test can be used to examine blood samples currently in the freezer from a completed clinical trial, for example, and could help find a molecular signature that predicts who responded to a drug and who did not,” said Diehn.
The utility of the test extends beyond oncology. The test detected high levels of normal lung RNA in blood samples from patients who were intubated and on a ventilator for treatment of acute respiratory distress syndrome, a condition that severely damages the lungs and causes the cells to die.
The amount of normal lung RNA in blood samples from patients with COVID-19 reflected how sick they were. The researchers also found normal lung RNA in the blood of smokers who were healthy, possibly indicating microscopic injury to the lungs caused by smoking.
With their new test in hand, the researchers now plan to expand their clinical studies of the liquid biopsy to explore additional applications and develop standardized protocols to promote its integration into clinical care.
