Scientists develop a superior diagnostic tool to spot hidden gene fusions in pediatric cancer

Researchers have introduced a novel diagnostic method that can more sensitively detect gene fusions in B-cell acute lymphoblastic leukemia (B-ALL), the most common type of pediatric cancer, compared to other publicly available fusion detection algorithms.

The tool, detailed in an article appearing in The Journal of Molecular Diagnostics, published by Elsevier, enables a higher diagnostic yield from low-coverage, low-cost sequencing.

Current treatment of B-ALL is classified according to risk levels depending on age, white blood cell count, response to therapy, central nervous system status, and genomic subtype. Pediatric B-ALL is primarily driven by chromosomal abnormalities or structural variants, which typically result in fusion oncogenes that cause cancer cells to grow and multiply. It is critical to diagnose B-ALL genomic subtypes for appropriate risk-stratified treatment.

The novel algorithm, called FUSILLI (FUSions In Leukemia for Long-read sequencing Investigator), has been developed to detect these fusion genes. It uses Oxford Nanopore Technologies' (ONT) long-read sequencing, which looks at larger DNA and RNA fragments and is easier to implement across various resource contexts compared to short-read sequencing technology.

The researchers working on this new tool have previously demonstrated the use of nanopore RNA sequencing to classify B-ALL. Now, the sensitive and accurate method for detecting gene fusion subtypes presented in FUSILLI closes a critical gap in using these data for clinical fusion detection.

Long-read sequencing, and nanopore sequencing specifically, represent a new era of sequencing compared to more conventional short-read sequencing approaches. It has been around for about a decade but is now becoming mature enough for clinical applications."

Jeremy R. Wang, PhD, Study Senior Investigator and Associate Professor, Department of Genetics, Department of Pathology and Laboratory Medicine, and Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina

"Compared to traditional short-read next-generation sequencing, nanopore sequencing has dramatically lower capital and consumables costs and much faster turnaround time, making it particularly advantageous in resource-limited diagnostic settings. Our research builds on this technology to show the potential of diagnosing genomic subtypes of pediatric cancers, which are traditionally resolved through several different expertise- and resource-intensive assays."

The investigators took a supervised approach at what filtering parameters were needed to detect true B-ALL gene fusions as compared to results obtained from clinical testing. For gene fusion detection, there can be false positives that result from both technical and computational artifacts.

"From our experience, we've seen sequencing chimeras (artificial DNA sequences created during the sequencing process), producing long reads that resemble true gene fusions. These are rare events. With careful filtering and sufficient sequencing depth, we discriminate these from true B-ALL gene fusions supported by a minimum of two reads," explains Dr. Wang.

The study further established a limit of detection, finding that approximately 10 million reads per sample are required to reliably detect B-ALL fusions using this approach.

Additionally, researchers compared results against other publicly available fusions callers (with default parameters) and demonstrated superior sensitivity without significant loss of specificity for clinically relevant fusion events. Also, because they limited the data to clinically relevant B-ALL gene fusions, the researchers achieved a much smaller search space and faster computation times.

While the primary leukemic-driving fusions are the dominant fusion detected in most cases, the team observed an unexpected number of suggestive secondary alterations in the cohorts' data. "For instance, we see PAX5::ZCCHC7 in several cases, which is a known secondary alteration, but less is known about its clinical relevance. A better understanding of these lesser-known genomic events that are not well captured by existing diagnostic tools has the potential to further improve risk stratification and personalized medicine."

Dr. Wang concludes, "With the development of FUSILLI, we show the potential of using a single low-cost sequencing assay for diagnosing gene fusion subtypes of B-ALL, with faster turnaround time. Modern genomic subtyping in pediatric B-ALL informs risk-stratification and targeted therapy, improving treatment response rate and reducing unnecessary treatment-related toxicity."