High-precision, low-cost pediatric leukemia diagnostics

Researchers have introduced a novel diagnostics method that can more sensitively detect gene fusions in B-cell acute lymphoblastic leukemia (B-ALL), the most common type of pediatric cancer, compared with other publicly available fusion detection algorithms. The tool, detailed in an article appearing in The Journal of Molecular Diagnostics, AMP's official journal, 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), was developed to detect these fusion genes. It uses Oxford Nanopore Technologies’ long-read sequencing, which looks at larger DNA and RNA fragments and is easier to implement across various resource contexts compared with short-read sequencing technology.

The researchers working on this new tool 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.

Jeremy R. Wang, Ph.D, at the Lineberger Comprehensive Cancer Center at the University of North Carolina at Chapel Hill was the senior investigator for the project.

“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,“ Wang said.

He continued: “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 researchers 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,” Wang explained.

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

Additionally, the 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,” Wang said.

Wang concluded: “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.”

About The Journal of Molecular Diagnostics

The Journal of Molecular Diagnostics, the official publication of the Association for Molecular Pathology, co-owned by the American Society for Investigative Pathology, and published by Elsevier, seeks to publish high-quality original papers on scientific advances in the translation and validation of molecular discoveries in medicine into the clinical diagnostic setting, and the description and application of technological advances in the field of molecular diagnostic medicine.

The editors welcome review articles that contain: novel discoveries or clinicopathologic correlations, including studies in oncology, infectious diseases, inherited diseases, predisposition to disease, or the description of polymorphisms linked to disease states or normal variations; the application of diagnostic methodologies in clinical trials; or the development of new or improved molecular methods for diagnosis or monitoring of disease or disease predisposition.