New Fusili tool improves gene fusion detection in pediatric leukemia

Researchers have introduced a new diagnostic method that can more sensitively detect gene fusions in B-cell acute lymphoblastic leukemia (B-ALL), the most common type of childhood cancer, than other publicly available fusion detection algorithms. Tool, details of which are given in an article Journal of Molecular DiagnosticsPublished by Elsevier, enables high diagnostic yield from low-coverage, low-cost sequencing.

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

A novel algorithm called FUSILI (Fusions in Leukemia for Long-Read Sequencing Investigators) 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 in different resource contexts than short-read sequencing technology.

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 of detecting gene fusion subtypes presented in FUSILLI closes an important gap in using these data for clinical fusion identification.

Senior investigator Jeremy R. “Long-read sequencing, and especially nanopore sequencing, represents a new era of sequencing compared to more traditional short-read sequencing approaches. It has been around for a decade but is now becoming mature enough for clinical applications,” says Wang, PhD, Department of Genetics, Pathology and Laboratory Medicine, and Linebarger Comprehensive Cancer Center, School of Medicine, University of North Carolina, Chapel Hill.

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 clinical settings. “Our research builds on this technology to show the potential for diagnosing genomic subtypes of pediatric cancer that are traditionally resolved through multiple, separate expertise and resource-intensive tests.”

Jeremy R. Wang, University of North Carolina

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

Dr. Wang explains, “From our experience, we have seen sequencing chimeras (artificial DNA sequences created during the sequencing process) that produce long reads that resemble genuine gene fusions. These are rare occurrences. With careful filtering and sufficient sequencing depth, we distinguish these from genuine B-ALL gene fusions, which are supported by a minimum of two reads.”

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

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

While primary leukemic-driving fusions are the predominant fusions found in most cases, the team observed an unexpected number of suggestive secondary alterations in the cohort’s data. “For example, we see PAX5::ZCCHC7 In many cases, there is a known secondary change, but less is known about its clinical relevance. “A better understanding of these lesser-known genomic events, which are not well captured by existing clinical tools, has the potential to further improve risk stratification and personalized medicine.”

Dr. Wang concluded, “With the development of Fusili, we show the potential to use a single low-cost sequencing assay to diagnose gene fusion subtypes of B-ALL with rapid turnaround times. Modernized genomic subtyping in pediatric B-ALL informs risk-stratification and targeted therapy, improving treatment response rates and reducing unnecessary treatment-related toxicities.”