Fusion InPipe, an integrative pipeline for gene fusion detection from RNA-seq data in acute pediatric leukemia.

RNA sequencing (RNA-seq) is a reliable tool for detecting gene fusions in acute leukemia. Multiple bioinformatics pipelines have been developed to analyze RNA-seq data, but an agreed gold standard has not been established. This study aimed to compare the applicability of 5 fusion calling pipelines (Arriba, deFuse, CICERO, FusionCatcher, and STAR-Fusion), as well as to define and develop an integrative bioinformatics pipeline (Fusion InPipe) to detect clinically relevant gene fusions in acute pediatric leukemia. We analyzed RNA-seq data by each pipeline individually and by Fusion InPipe. Each algorithm individually called most of the fusions with similar sensitivity and precision. However, not all rearrangements were called, suggesting that choosing a single pipeline might cause missing important fusions. To improve this, we integrated the results of the five algorithms in just one pipeline, Fusion InPipe, comparing the output from the agreement of 5/5, 4/5, and 3/5 algorithms. The maximum sensitivity was achieved with the agreement of 3/5 algorithms, with a global sensitivity of 95%, achieving a 100% in patients' data. Furthermore, we showed the necessity of filtering steps to reduce the false positive detection rate. Here, we demonstrate that Fusion InPipe is an excellent tool for fusion detection in pediatric acute leukemia with the best performance when selecting those fusions called by at least 3/5 pipelines.

A miRNA signature related to stemness identifies high-risk patients in paediatric acute myeloid leukaemia.

Clinical and biological variables like genetic aberrations at diagnosis and the levels of measurable residual disease (MRD) are the most powerful biomarkers to predict the outcome of paediatric leukaemia. Recently, a model integrating the genetic abnormalities, transcriptional identity, and leukaemia stemness measured as leukaemic stem cell score (pLSC6) has been proposed to identify high-risk paediatric acute myeloid leukaemia (AML) patients. However, the role of epigenetics in defining prognosis still needs to be established. We evaluated the role of 89 miRNAs regulating stemness and their contribution to predicting outcomes in 110 paediatric patients with acute leukaemia. We identified a 24-miRNA signature capable of distinguishing paediatric AML patients with excellent or poor outcomes. We validated these results in an independent cohort using public repository-based data. The 24-miRNA signature was significantly associated with the leukaemic stemness scores and the underlying genetics of patients. Notably, the combination of classical prognostic factors (MRD and genetics), the pLSC6 score and the 24-miRNA signature had a higher capacity to predict the overall and event-free survival than each variable individually. Our 24-miRNA signature provides epigenetic data to integrate into genetics, MRD and stemness-related leukaemic scores to refine risk stratification in paediatric AML patients.

Technical Validation and Clinical Utility of an NGS Targeted Panel to Improve Molecular Characterization of Pediatric Acute Leukemia.

Development of next-generation sequencing (NGS) has provided useful genetic information to redefine diagnostic, prognostic, and therapeutic strategies for the management of acute leukemia (AL). However, the application in the clinical setting is still challenging. Our aim was to validate the AmpliSeq™ for Illumina® Childhood Cancer Panel, a pediatric pan-cancer targeted NGS panel that includes the most common genes associated with childhood cancer, and assess its utility in the daily routine of AL diagnostics. In terms of sequencing metrics, the assay reached all the expected values. We obtained a mean read depth greater than 1000×. The panel demonstrated a high sensitivity for DNA (98.5% for variants with 5% variant allele frequency (VAF)) and RNA (94.4%), 100% of specificity and reproducibility for DNA and 89% of reproducibility for RNA. Regarding clinical utility, 49% of mutations and 97% of the fusions identified were demonstrated to have clinical impact. Forty-one percent of mutations refined diagnosis, while 49% of them were considered targetable. Regarding RNA, fusion genes were more clinically impactful in terms of refining diagnostic (97%). Overall, the panel found clinically relevant results in the 43% of patients tested in this cohort. To sum up, we validated a reliable and reproducible method to refine pediatric AL diagnosis, prognosis, and treatment, and demonstrated the feasibility of incorporating a targeted NGS panel into pediatric hematology practice.

Helpful Criteria When Implementing NGS Panels in Childhood Lymphoblastic Leukemia.

The development of Next-Generation Sequencing (NGS) has provided useful diagnostic, prognostic, and therapeutic strategies for individualized management of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients. Consequently, NGS is rapidly being established in clinical practice. However, the technology's complexity, bioinformatics analysis, and the different available options difficult a broad consensus between different laboratories in its daily routine introduction. This collaborative study among Spanish centers was aimed to assess the feasibility, pros, and cons of our customized panel and other commercial alternatives of NGS-targeted approaches. The custom panel was tested in three different sequencing centers. We used the same samples to assess other commercial panels (OncomineTM Childhood Cancer Research Assay; Archer®FusionPlex® ALL, and Human Comprehensive Cancer Panel GeneRead Panel v2®). Overall, the panels showed a good performance in different centers and platforms, but each NGS approach presented some issues, as well as pros and cons. Moreover, a previous consensus on the analysis and reporting following international guidelines would be preferable to improve the concordance in results among centers. Our study shows the challenges posed by NGS methodology and the need to consider several aspects of the chosen NGS-targeted approach and reach a consensus before implementing it in daily practice.

Measurable Residual Disease Assessed by Flow-Cytometry Is a Stable Prognostic Factor for Pediatric T-Cell Acute Lymphoblastic Leukemia in Consecutive SEHOP Protocols Whereas the Impact of Oncogenetics Depends on Treatment.

Robust and applicable risk-stratifying genetic factors at diagnosis in pediatric T-cell acute lymphoblastic leukemia (T-ALL) are still lacking, and most protocols rely on measurable residual disease (MRD) assessment. In our study, we aimed to analyze the impact of NOTCH1, FBXW7, PTEN, and RAS mutations, the measurable residual disease (MRD) levels assessed by flow cytometry (FCM-MRD) and other reported risk factors in a Spanish cohort of pediatric T-ALL patients. We included 199 patients treated with SEHOP and PETHEMA consecutive protocols from 1998 to 2019. We observed a better outcome of patients included in the newest SEHOP-PETHEMA-2013 protocol compared to the previous SHOP-2005 cohort. FCM-MRD significantly predicted outcome in both protocols, but the impact at early and late time points differed between protocols. The impact of FCM-MRD at late time points was more evident in SEHOP-PETHEMA 2013, whereas in SHOP-2005 FCM-MRD was predictive of outcome at early time points. Genetics impact was different in SHOP-2005 and SEHOP-PETHEMA-2013 cohorts: NOTCH1 mutations impacted on overall survival only in the SEHOP-PETHEMA-2013 cohort, whereas homozygous deletions of CDKN2A/B had a significantly higher CIR in SHOP-2005 patients. We applied the clinical classification combining oncogenetics, WBC count and MRD levels at the end of induction as previously reported by the FRALLE group. Using this score, we identified different subgroups of patients with statistically different outcome in both Spanish cohorts. In SHOP-2005, the FRALLE classifier identified a subgroup of high-risk patients with poorer survival. In the newest protocol SEHOP-PETHEMA-2013, a very low-risk group of patients with excellent outcome and no relapses was detected, with borderline significance. Overall, FCM-MRD, WBC count and oncogenetics may refine the risk-stratification, helping to design tailored approaches for pediatric T-ALL patients.

Paediatric patients with acute leukaemia and KMT2A (MLL) rearrangement show a distinctive expression pattern of histone deacetylases.

Histone deacetylase inhibitors (HDACi) had emerged as promising drugs in leukaemia, but their toxicity due to lack of specificity limited their use. Therefore, there is a need to elucidate the role of HDACs in specific settings. The study of HDAC expression in childhood leukaemia could help to choose more specific HDACi for selected candidates in a personalized approach. We analysed HDAC1-11, SIRT1, SIRT7, MEF2C and MEF2D mRNA expression in 211 paediatric patients diagnosed with acute leukaemia. There was a global overexpression of HDACs, while specific HDACs correlated with clinical and biological features, and some even predicted outcome. Thus, some HDAC and MEF2C profiles probably reflected the lineage and the maturation of the blasts and some profiles identified specific oncogenic pathways active in the leukaemic cells. Specifically, we identified a distinctive signature for patients with KMT2A (MLL) rearrangement, with high HDAC9 and MEF2D expression, regardless of age, KMT2A partner and lineage. Moreover, we observed an adverse prognostic value of HDAC9 overexpression, regardless of KMT2A rearrangement. Our results provide useful knowledge on the complex picture of HDAC expression in childhood leukaemia and support the directed use of specific HDACi to selected paediatric patients with acute leukaemia.