RNA Sequencing Demonstrates Accurate Diagnosis of B-ALL Subtypes in Pediatric Patients


According to the authors, the test has promise in guiding treatment but needs further refining to obtain information beyond molecular subtyping.

Study results published in Elsevier confirm that RNA sequencing analysis (RNA-Seq) workflow for clinical diagnosis of molecular subtypes in pediatric B-acute lymphoblastic leukemia (B-ALL) is a feasible method. ALL is the most common childhood cancer, making up more than 30% of all pediatric cancer cases.

Stone blocks with letters ALL held by doctor's hands -- Image credit: Sviatlana | stock.adobe.com

Image credit: Sviatlana | stock.adobe.com

Because ALL contains a plethora of diverse molecular subtypes that have their own drug sensitivity pattern, treatment response, and prognosis, it is important to identify the specific subtype of the disease. This is particularly important in the current state of personalized medicine; however, identifying the subtype requires a multitude of profiling tools, which can be an expensive and lengthy process.

“The goal of the study was to address limitations of current standard-of-care (SOC) testing for pediatric B-ALL, which cannot identify multiple recently discovered subtypes of the disease,” said co-lead lead investigator Gordana Raca, MD, PhD, department of pathology and laboratory medicine, Children’s Hospital Los Angeles, in a press release. “We also wanted to take advantage of expression data generated by the enrichment-based RNA-Seq assay clinically validated in our laboratory for detection of oncogenic fusions in cancer to evaluate if additional data analyses could help determine subtype classification in addition to fusion information for our B-ALL cases.”

Study results were analyzed from 61 newly diagnosed patients and 25 patients with relapsed or refractory B-ALL who underwent cytogenetic and molecular characterization as part of their standard clinical. The investigators hypothesized that RNA-Seq—which is used in the discovery of novel molecular subtypes of B-ALL—would be a clinically beneficial tool for diagnosing the classification of B-ALL cases. To test this, following clinical karyotype analysis, fluorescence in situ hybridization, chromosomal microarray, and next-generation sequencing DNA and RNA fusion panel testing, the investigators analyzed RNA-Seq data in 28 cases of B-ALL with known subtype and 48 with undetermined subtypes.

“The study showed that B-ALL cases with known subtypes by SOC testing were fully concordant with the RNA-Seq–based classification. In addition, RNA-Seq analysis allowed us to successfully classify a large proportion of cases that remained unknown upon comprehensive SOC testing,” said co-lead investigator Zhaohui Gu, PhD, department of computational and quantitative medicine & systems biology, Beckman Research Institute of City of Hope, Duarte, in the press release.

RNA-Seq analysis accurately identified the subtypes in all 28 known cases as well as determining approximately 78% of the subtypes (n = 38) in the previously unknown cases. Further, RNA-Seq analysis was also able to detect oncogenic fusions, large copy number abnormalities, oncogenic hot-spot sequence variants, and deletions of intragenic IKZF1.

“RNA-Seq analysis enables accurate determination of the genetic subtype for an increased proportion of pediatric B-ALL cases, which will allow more accurate risk stratification and optimization of patient management. Accurate subtype determination at diagnosis will also grow our knowledge about morphologic, immunophenotypic, and clinical characteristics of novel B-ALL subtypes,” said Raca in the press release. “We were surprised by a relatively high frequency of some novel B-ALL subtypes (like PAX5Alt and DUX4) in our patient cohort, which remain undiagnosed by SOC testing. The study also resulted in discovery of several previously unreported fusions.”

The study authors also note that the classification has the potential to be a powerful clinical assessment method of pediatric B-ALL. As a single test, it is able to determine subtypes in a higher proportion of cases compared to previous SOC testing methods and has the potential to increase the diagnostic efficiency of testing within B-ALL.

Further, a similar study notes that RNA-Seq is 1 of the most powerful single tools for somatic profiling in pediatric B-ALL; however, it remains contentious whether RNA-Seq can be the lone means of classification. The authors note that there may be some limitations, particularly in locations that may not have as strong analytical methods.

“Overall, RNA-Seq as a single somatic genomic ALL diagnostic platform holds immense promise, although still with associated gaps. In some cases, validation of positive cases by an alternative method—such as oncogene fusion test and DNA sequencing—may still be arguably a sounder approach. The test needs further refining to comprehensively obtain mutation information beyond that of molecular subtyping to make it globally accessible,” said Shawn H.R. Lee, MD, an author of the similar study, Khoo Teck Puat–National University Children’s Medical Institute, National University Health System, Singapore, and department of pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, in the press release. “As we move further into the era of precision medicine, the information provided by this test holds excellent promise in guiding relevant and risk-stratified treatment in this childhood cancer.”


Elsevier. RNA sequencing analysis may hold the key to more accurate diagnosis and targeted treatment of pediatric B-acute lymphoblastic leukemia. News release. January 29, 2024. Accessed February 1, 2024. https://www.eurekalert.org/news-releases/1032702

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