Implementation of RNA sequencing and array CGH in the diagnostic workflow of the AIEOP-BFM ALL 2017 trial on acute lymphoblastic leukemia

Abstract

Risk-adapted therapy has significantly contributed to improved survival rates in pediatric acute lymphoblastic leukemia (ALL) and reliable detection of chromosomal aberrations is mandatory for risk group stratification. This study evaluated the applicability of panel-based RNA sequencing and array CGH within the diagnostic workflow of the German study group of the international AIEOP-BFM ALL 2017 trial. In a consecutive cohort of 117 children with B cell precursor (BCP) ALL, array analysis identified twelve cases with an IKZF1 plus profile of gene deletions and one case of masked hypodiploidy. Genetic markers BCR-ABL1 (n = 1), ETV6-RUNX1 (n = 25), and rearrangements involving KMT2A (n = 3) or TCF3 (n = 3) were assessed by established conventional techniques such as karyotyping, FISH, and RT-PCR. Comparison of these results with RNA sequencing analysis revealed overall consistency in n=115/117 cases, albeit with one undetected AFF1-KMT2A fusion in RNA sequencing and one undetected ETV6-RUNX1 fusion in conventional analyses. The combined application of RNA sequencing, FISH, and CGH+SNP array reliably detected all genetic markers necessary for risk stratification and will be used as the diagnostic standard workflow for BCP-ALL patients enrolled in the AIEOP-BFM ALL 2017 study. Prospectively, consistent collection of genome-wide CGH+SNP array as well as RNA sequencing data will be a valuable source to elucidate new prognostic lesions beyond established markers of pediatric ALL. In this respect, RNA sequencing identified various gene fusions in up to half of the IKZF1 plus (n = 6/12) and Bother (n = 19/36) cases but not in cases with hyperdiploid karyotypes (n = 35). Among these fusions, this study reports several previously undescribed in frame PAX5 fusions, including PAX5-MYO1G and PAX5-NCOA6.

Clinical application of whole transcriptome sequencing for the classification of patients with acute lymphoblastic leukemia

“…Multiple entity-defining fusion transcripts are known in BCP-ALLs ( BCR-ABL1 , KMT2A-AFF1 , TCF3-PBX1 , ETV6-RUNX1 ), and a reliable detection is mandatory for every diagnostic workflow. The applied fusion calling procedure identified 97% of the fusions, as detected by gold standard techniques, which is in line with previous RNA-Seq studies in pediatric ALL cohorts that reported detection rates between 91 and 97% [ 3 , 39 – 41 ]. The number of true positive calls can be increased by considering the overlap of different callers [ 42 ], while simultaneously reducing the number of false negative ones.…”

The long-term outcome and risk factors for precursor B cell acute lymphoblastic leukemia without specific fusion genes in Chinese children: experiences from multiple centers

“…Specific fusion genes have yet to be clarified in at least one-third of B-ALL patients [5,7,8], and our study and the literature verified that early treatment response was the main prognostic factor in these patients [4,5,6,17,21,22,23,24]. In recent studies, NGS has been used to discover more genetic abnormalities in B-ALL, and advanced studies are exploring the relationships among these genetic abnormalities, pathogenesis and outcomes [25,27,28,29]. In this study, NGS detected abnormalities in 62.5% of BM relapsed patients, rare or unreported fusion genes and/or gene mutations were detectable, and adverse molecular genetic abnormalities such as TP53, CREBBP and IKZF1 [30,31] were detected in BM relapsed patients.…”

Detection of EP300-ZNF384 fusion in patients with acute lymphoblastic leukemia using RNA fusion gene panel sequencing

“…Targeted RNA fusion-related gene panel sequencing could be more efficient in clinical applications. Reliable gene fusions could be detected with higher performance price ratio [17,18]. The RNA fusion gene panel used in this study, covered 225 genes, could detect over 900 fusion isoforms, and reached a high validation rate.…”