The relapsed acute lymphoblastic leukemia network (ReALLNet): a multidisciplinary project from the spanish society of pediatric hematology and oncology (SEHOP).

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, with survival rates exceeding 85%. However, 15% of patients will relapse; consequently, their survival rates decrease to below 50%. Therefore, several research and innovation studies are focusing on pediatric relapsed or refractory ALL (R/R ALL). Driven by this context and following the European strategic plan to implement precision medicine equitably, the Relapsed ALL Network (ReALLNet) was launched under the umbrella of SEHOP in 2021, aiming to connect bedside patient care with expert groups in R/R ALL in an interdisciplinary and multicentric network. To achieve this objective, a board consisting of experts in diagnosis, management, preclinical research, and clinical trials has been established. The requirements of treatment centers have been evaluated, and the available oncogenomic and functional study resources have been assessed and organized. A shipping platform has been developed to process samples requiring study derivation, and an integrated diagnostic committee has been established to report results. These biological data, as well as patient outcomes, are collected in a national registry. Additionally, samples from all patients are stored in a biobank. This comprehensive repository of data and samples is expected to foster an environment where preclinical researchers and data scientists can seek to meet the complex needs of this challenging population. This proof of concept aims to demonstrate that a network-based organization, such as that embodied by ReALLNet, provides the ideal niche for the equitable and efficient implementation of "what's next" in the management of children with R/R ALL.

A human genome editing-based MLL::AF4 ALL model recapitulates key cellular and molecular leukemogenic features.

Cellular ontogeny and MLL breakpoint site influence the capacity of MLL-edited CD34+ hematopoietic cells to initiate and recapitulate infant patients' features in pro-B-cell acute lymphoblastic leukemia (B-ALL). We provide key insights into the leukemogenic determinants of MLL-AF4+ infant B-ALL.

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.

ß-Catenin activity induces an RNA biosynthesis program promoting therapy resistance in T-cell acute lymphoblastic leukemia.

Understanding the molecular mechanisms that contribute to the appearance of chemotherapy resistant cell populations is necessary to improve cancer treatment. We have now investigated the role of ß-catenin/CTNNB1 in the evolution of T-cell Acute Lymphoblastic Leukemia (T-ALL) patients and its involvement in therapy resistance. We have identified a specific gene signature that is directly regulated by ß-catenin, TCF/LEF factors and ZBTB33/Kaiso in T-ALL cell lines, which is highly and significantly represented in five out of six refractory patients from a cohort of 40 children with T-ALL. By subsequent refinement of this gene signature, we found that a subset of ß-catenin target genes involved with RNA-processing function are sufficient to segregate T-ALL refractory patients in three independent cohorts. We demonstrate the implication of ß-catenin in RNA and protein synthesis in T-ALL and provide in vitro and in vivo experimental evidence that ß-catenin is crucial for the cellular response to chemotherapy, mainly in the cellular recovery phase after treatment. We propose that combination treatments involving chemotherapy plus ß-catenin inhibitors will enhance chemotherapy response and prevent disease relapse in T-ALL 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.

CD34+CD19-CD22+ B-cell progenitors may underlie phenotypic escape in patients treated with CD19-directed therapies.

CD19-directed immunotherapies have revolutionized the treatment of advanced B-cell acute lymphoblastic leukemia (B-ALL). Despite initial impressive rates of complete remission (CR) many patients ultimately relapse. Patients with B-ALL successfully treated with CD19-directed T cells eventually relapse, which, coupled with the early onset of CD22 expression during B-cell development, suggests that preexisting CD34+CD22+CD19- (pre)-leukemic cells represent an "early progenitor origin-related" mechanism underlying phenotypic escape to CD19-directed immunotherapies. We demonstrate that CD22 expression precedes CD19 expression during B-cell development. CD34+CD19-CD22+ cells are found in diagnostic and relapsed bone marrow samples of ∼70% of patients with B-ALL, and their frequency increases twofold in patients with B-ALL in CR after CD19 CAR T-cell therapy. The median of CD34+CD19-CD22+ cells before treatment was threefold higher in patients in whom B-ALL relapsed after CD19-directed immunotherapy (median follow-up, 24 months). Fluorescence in situ hybridization analysis in flow-sorted cell populations and xenograft modeling revealed that CD34+CD19-CD22+ cells harbor the genetic abnormalities present at diagnosis and initiate leukemogenesis in vivo. Our data suggest that preleukemic CD34+CD19-CD22+ progenitors underlie phenotypic escape after CD19-directed immunotherapies and reinforce ongoing clinical studies aimed at CD19/CD22 dual targeting as a strategy for reducing CD19- relapses. The implementation of CD34/CD19/CD22 immunophenotyping in clinical laboratories for initial diagnosis and subsequent monitoring of patients with B-ALL during CD19-targeted therapy is encouraged.

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.

Comprehensive Custom NGS Panel Validation for the Improvement of the Stratification of B-Acute Lymphoblastic Leukemia Patients.

BACKGROUND: B-acute lymphoblastic leukemia (B-ALL) is a hematological neoplasm of the stem lymphoid cell of the B lineage, characterized by the presence of genetic alterations closely related to the course of the disease. The number of alterations identified in these patients grows as studies of the disease progress, but in clinical practice, the conventional techniques frequently used are only capable of detecting the most common alterations. However, techniques, such as next-generation sequencing (NGS), are being implemented to detect a wide spectrum of new alterations that also include point mutations. METHODS: In this study, we designed and validated a comprehensive custom NGS panel to detect the main genetic alterations present in the disease in a single step. For this purpose, 75 B-ALL diagnosis samples from patients previously characterized by standard-of-care diagnostic techniques were sequenced. RESULTS: The use of the custom NGS panel allowed the correct detection of the main genetic alterations present in B-ALL patients, including the presence of an aneuploid clone in 14 of the samples and some of the recurrent fusion genes in 35 of the samples. The panel was also able to successfully detect a number of secondary alterations, such as single nucleotide variants (SNVs) and copy number variations (CNVs) in 66 and 46 of the samples analyzed, respectively, allowing for further refinement of the stratification of patients. The custom NGS panel could also detect alterations with a high level of sensitivity and reproducibility when the findings obtained by NGS were compared with those obtained from other conventional techniques. CONCLUSIONS: The use of this custom NGS panel allows us to quickly and efficiently detect the main genetic alterations present in B-ALL patients in a single assay (SNVs and insertions/deletions (INDELs), recurrent fusion genes, CNVs, aneuploidies, and single nucleotide polymorphisms (SNPs) associated with pharmacogenetics). The application of this panel would thus allow us to speed up and simplify the molecular diagnosis of patients, helping patient stratification and management.

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.

FMS-like tyrosine kinase 3 (FLT3) modulates key enzymes of nucleotide metabolism implicated in cytarabine responsiveness in pediatric acute leukemia.

Treatment of pediatric acute leukemia might involve combined therapies targeting the FMS-like tyrosine kinase 3 (FLT3) receptor (i.e. quizartinib - AC220) and nucleotide metabolism (cytarabine - AraC). This study addressed the possibility of FLT3 modulating nucleoside salvage processes and, eventually, cytarabine action. Bone marrow samples from 108 pediatric leukemia patients (B-cell precursor acute lymphoblastic leukemia, BCP-ALL: 83; T-ALL: 9; acute myeloid leukemia, AML: 16) were used to determine the mRNA expression levels of FLT3, the cytarabine activating kinase dCK, and the nucleotidases cN-II and SAMHD1. FLT3 mRNA levels positively correlated with dCK, cN-II and SAMHD1 in the studied cohort. FLT3 inhibition using AC220 promoted the expression of cN-II in MV4-11 cells. Indeed, inhibition of cN-II with anthraquinone-2,6-disulfonic acid (AdiS) further potentiated the synergistic action of AC220 and cytarabine, at low concentrations of this nucleoside analog. FLT3 inhibition also down-regulated phosphorylated forms of SAMHD1 in MV4-11 and SEM cells. Thus, inhibition of FLT3 may also target the biochemical machinery associated with nucleoside salvage, which may modulate the ability of nucleoside-derived drugs. In summary, this contribution highlights the need to expand current knowledge on the mechanistic events linking tyrosine-kinase receptors, likely to be druggable in cancer treatment, and nucleotide metabolism, particularly considering tumor cells undergo profound metabolic reprogramming.

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.

FLT3 is implicated in cytarabine transport by human equilibrative nucleoside transporter 1 in pediatric acute leukemia.

FLT3 abnormalities are negative prognostic markers in acute leukemia. Infant leukemias are a subgroup with frequent MLL (KMT2A) rearrangements, FLT3 overexpression and high sensitivity to cytarabine, but dismal prognosis. Cytarabine is transported into cells by Human Equilibrative Nucleoside Transporter-1 (hENT1, SLC29A1), but the mechanisms that regulate hENT1 in acute leukemia have been scarcely studied.We explored the expression and functional link between FLT3 and main cytarabine transporters in 50 pediatric patients diagnosed with acute lymphoblastic leukemia and MLL rearrangement (ALL-MLL+) and other subtypes of leukemia, and in leukemia cell lines.A significant positive correlation was found between FLT3 and hENT1 expression in patients. Cytarabine uptake into cells was mediated mainly by hENT1, hENT2 and hCNT1. hENT1-mediated uptake of cytarabine was transiently abolished by the FLT3 inhibitor PKC412, and this effect was associated with decreased hENT1 mRNA and protein levels. Noticeably, the cytotoxicity of cytarabine was lower when cells were first exposed to FLT3 inhibitors (PKC412 or AC220), probably due to decreased hENT1 activity, but we observed a higher cytotoxic effect if FLT3 inhibitors were administered after cytarabine.FLT3 regulates hENT1 activity and thereby affects cytarabine cytotoxicity. The sequence of administration of cytarabine and FLT3 inhibitors is important to maintain their efficacy.