Frequency and Early Prognostic Significance of Gene-Class Fusions Identified By RNA-Seq in Children and Adolescent with T-Cell Acute Lymphoblastic Leukemia Enrolled on AIEOP-BFM ALL 2017 Study
Authors: Gotti Giacomo , Saitta Claudia , Silvestri Daniela , Colombo Arianna , Colnaghi Federica , Rebellato Stefano , Marzorati Matilde , Peccatori Nicolò , Bertorello Nicoletta , Lo Nigro Luca , Locatelli Franco , Parasole Rosanna , Putti Maria Caterina , Conter Valentino , Colombini Antonella , Buldini Barbara , Biondi Andrea , Valsecchi Maria Grazia , Cazzaniga Giovanni , Fazio Grazia , Rizzari Carmelo 
Published: December 2024

Introduction
T-cell acute lymphoblastic leukemia (T-ALL) has been historically associated with inferior outcome compared to B-cell precursor ALL with slower treatment response and lack of recurrent prognostic cytogenetic alterations. With the introduction of next-generation sequencing analyses, a deeper characterization of childhood ALL has been achieved. However, if for B-ALL the identification of fusion-class genes has clinical implication, the frequency and relevance of gene fusions in T-ALL still remain poor. Here we describe the incidence of gene-class fusions in the pediatric cohort of T-ALL patients treated according to the AIEOP-BFM ALL 2017 protocol and explore the association with presenting patient characteristics and early treatment response.
Methods
From Dec 1st 2021 to Mar 31st 2024, in AIEOP Italian centers 132 consecutive patients with T-ALL have been enrolled in the trial AIEOP-BFM ALL 2017 Study. According to material availability, Whole transcriptome RNA-seq was performed at disease onset in 128 (97%) patients (Universal RNA-seq kit, Tecan with paired-end sequencing on Nextseq2000). The analysis was performed with 4 independent pipelines with the purpose to detect fusion genes (Dragen RNA, Star, Fusion Catcher, Arriba) and results were evaluable in 123 (96%) patients (5 cases were excluded due to poor library quality).
Results
Among 123 evaluable patients, at least one fusion gene was identified in 51 (41.5%) patients and validated through real-time PCR. Most common gene-class fusions were STIL::TAL1 (n=10, 8.1%), KMT2A-rearranged (KMT2Ar, n=9, 7.3%), ABL-class (n=8, 6.5%) and MLLT10-class (n=8, 6.5%). Other fusions were identified in 16 (13.0%) patients, with ETV6-class in 4 patients (3.3%) and CDK6-class in 2 patients (1.6%), in addition to single non recurrent fusion/cases. All gene-class fusions were mutually exclusive.
Patient characteristics at diagnosis were evaluated in association with the presence of gene-class fusions. ABL-class and MLLT10-class fusions were more common in females and patients with MLLT10-class fusion tended to be older (age ≥ 10 years). Patients with STIL::TAL1 fusion were more likely to have hyperleukocytosis (white blood cell counts ≥ 100 × 109/L) and central nervous system disease (CNS3).
We investigated whether the presence of gene-class fusion was associated with treatment response in term of prednisone poor response (PPR), high level of bone marrow disease (≥10% of blasts) measured by flow-cytometry (FCM-MRD) at induction day 15, end of induction (EOI) PCR-MRD, end of consolidation (EOC) PCR-MRD and final risk group classification. Patients carrying a KMT2Ar were characterized by higher proportion of PPR, FCM-MRD with ≥10% of blasts and higher EOI MRD levels: of note, approximately half (44.4%) of them had EOI MRD ≥5x10-3. The presence of MLLT10-class fusion was associated with high EOI MRD, with 50% of patients having levels ≥5x10-3, as well as for STIL::TAL1 patients (40% of patients with ≥5x10-3). No association was found at EOC between PCR-MRD levels and gene-class fusions. Interestingly, all patients carrying KMT2Ar except one were classified as HR while a lower proportion of HR patients was found among patients with ABL-class fusion. Indeed, the presence of ABL-class fusion was not associated with any unfavorable early treatment responses.
Conclusion
Our study showed a high incidence of gene-class fusions in children and adolescents with newly diagnosed T-ALL. We found that KMT2Ar, STIL::TAL1 and MLLT10-class fusions are associated with high risk features in a prospective cohort of uniformly treated patients with pediatric T-ALL. On the contrary, patients carrying ABL-class fusions did not show adverse characteristics of early treatment response. Despite a longer follow-up is required, prognostic relevant gene-class fusions might integrate current risk stratification and identify patients who might qualify for early interventional studies or targeted therapies.