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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 β‐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 β‐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 β‐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 β‐catenin in RNA and protein synthesis in T‐ALL and provide in vitro and in vivo experimental evidence that β‐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 β‐catenin inhibitors will enhance chemotherapy response and prevent disease relapse in T‐ALL patients. Synopsis A β‐catenin‐dependent RNA processing program is identified in human T‐ALL cells that is informative for the identification of refractory T‐ALL patients at diagnosis. Inhibition of β‐catenin activity prevents T‐ALL cell recovery and leukemia survival after chemotherapy. β‐catenin is a direct regulator of RNA processing in T‐ALL cells. The β‐catenin‐dependent RNA processing signature predicts response to therapy in T‐ALL patients at diagnosis. Inhibition of β‐catenin in vivo and in vitro improves response to chemotherapy in T‐ALL cells. Graphical Abstract A β‐catenin‐dependent RNA processing program is identified in human T‐ALL cells that is informative for the identification of refractory T‐ALL patients at diagnosis. Inhibition of β‐catenin activity prevents T‐ALL cell recovery and leukemia survival after chemotherapy.

B‐cell acute lymphoblastic leukemia (B‐ALL) is the commonest childhood cancer. High hyperdiploidy (HHD) identifies the most frequent cytogenetic subgroup in childhood B‐ALL. Although hyperdiploidy represents an important prognostic factor in childhood B‐ALL, the specific chromosome gains with prognostic value in HHD‐B‐ALL remain controversial, and the current knowledge about the hierarchy of chromosome gains, clonal heterogeneity and chromosomal instability in HHD‐B‐ALL remains very limited. We applied automated sequential‐iFISH coupled with single‐cell computational modeling to identify the specific chromosomal gains of the eight typically gained chromosomes in a large cohort of 72 primary diagnostic (DX, n = 62) and matched relapse (REL, n = 10) samples from HHD‐B‐ALL patients with either favorable or unfavorable clinical outcome in order to characterize the clonal heterogeneity, specific chromosome gains and clonal evolution. Our data show a high degree of clonal heterogeneity and a hierarchical order of chromosome gains in DX samples of HHD‐B‐ALL. The rates of specific chromosome gains and clonal heterogeneity found in DX samples differ between HHD‐B‐ALL patients with favorable or unfavorable clinical outcome. In fact, our comprehensive analyses at DX using a computationally defined risk predictor revealed low levels of trisomies +18+10 and low levels of clonal heterogeneity as robust relapse risk factors in minimal residual disease (MRD)‐negative childhood HHD‐B‐ALL patients: relapse‐free survival beyond 5 years: 22.1% versus 87.9%, P < 0.0001 and 33.3% versus 80%, P < 0.0001, respectively. Moreover, longitudinal analysis of matched DX‐REL HHD‐B‐ALL samples revealed distinct patterns of clonal evolution at relapse. Our study offers a reliable prognostic sub‐stratification of pediatric MRD‐negative HHD‐B‐ALL patients. Automated sequential‐iFISH coupled with single‐cell computational modeling in high‐hyperdiploid (HHD) B‐ALL samples revealed low levels of trisomies 18, 10 and clonal heterogeneity at diagnosis as robust predictive risk factors, properly classifying ~ 85% of patients according to relapse risk. We propose a simple and reliable protocol for improving patient stratification at diagnosis easily applicable into the clinical routine of hematoncology laboratories.

Pediatric acute B-cell lymphoblastic leukemia (B-ALL) constitutes a heterogeneous and aggressive neoplasia in which new targeted therapies are required. Long non-coding RNAs have recently emerged as promising disease-specific biomarkers for the clinic. Here, we identified pediatric B-ALL-specific lncRNAs and associated mRNAs by comparing the transcriptomic signatures of tumoral and non-tumoral samples. We identified 48 lncRNAs that were differentially expressed between pediatric B-ALL and healthy bone marrow samples. The most relevant lncRNA/mRNA pair was AL133346.1/CCN2 (previously known as RP11-69I8.3/CTGF), whose expression was positively correlated and increased in B-ALL samples. Their differential expression pattern and their strong correlation were validated in external B-ALL datasets (Therapeutically Applicable Research to Generate Effective Treatments, Cancer Cell Line Encyclopedia). Survival curve analysis demonstrated that patients with “high” expression levels of CCN2 had higher overall survival than those with “low” levels (p = 0.042), and this gene might be an independent prognostic biomarker in pediatric B-ALL. These findings provide one of the first detailed descriptions of lncRNA expression profiles in pediatric B-ALL and indicate that these potential biomarkers could help in the classification of leukemia subtypes and that CCN2 expression could predict the survival outcome of pediatric B-cell acute lymphoblastic leukemia patients.

B-cell precursor acute lymphoblastic leukaemia (B-ALL) is a malignancy of lymphoid progenitor cells with altered genes including the Janus kinase (JAK) gene family. Among them, tyrosine kinase 2 (TYK2) is involved in signal transduction of cytokines such as interferon (IFN) α/β through IFN−α/β receptor alpha chain (IFNAR1). To search for disease-associated TYK2 variants, bone marrow samples from 62 B-ALL patients at diagnosis were analysed by next-generation sequencing. TYK2 variants were found in 16 patients (25.8%): one patient had a novel mutation at the four-point-one, ezrin, radixin, moesin (FERM) domain (S431G) and two patients had the rare variants rs150601734 or rs55882956 (R425H or R832W). To functionally characterise them, they were generated by direct mutagenesis, cloned in expression vectors, and transfected in TYK2-deficient cells. Under high-IFNα doses, the three variants were competent to phosphorylate STAT1/2. While R425H and R832W induced STAT1/2-target genes measured by qPCR, S431G behaved as the kinase-dead form of the protein. None of these variants phosphorylated STAT3 in in vitro kinase assays. Molecular dynamics simulation showed that TYK2/IFNAR1 interaction is not affected by these variants. Finally, qPCR analysis revealed diminished expression of TYK2 in B-ALL patients at diagnosis compared to that in healthy donors, further stressing the tumour immune surveillance role of TYK2.

Acute myeloid leukemia (AML) with myelodysplasia-related changes is characterized by the presence of multilineage dysplasia (MLD), frequently related to high-risk cytogenetics and poor outcome. However, the presence of MLD does not modify the favorable prognostic impact of NPM1 mutation. The prognosis of patients with AML presenting marked dysplasia lacking high-risk cytogenetics and NPM1 mutation is uncertain. We evaluated the prognostic impact of MLD in 177 patients with intermediate-risk cytogenetics AML (IR-AML) and wild-type NPM1 . Patients were categorized as MLD-WHO (WHO myelodysplasia criteria; n  = 43, 24 %), MLD-NRW (significant MLD non-reaching WHO criteria; n  = 16, 9 %), absent MLD ( n  = 80, 45 %), or non-evaluable MLD ( n  = 38, 22 %). No differences concerning the main characteristics were observed between patients with or without MLD. Outcome of patients with MLD-WHO and MLD-NRW was similar, and significantly worse than patients lacking MLD. The presence of MLD (66 vs. 80 %, p  = 0.03; HR, 95 % CI = 2.3, 1.08–4.08) and higher leukocyte count at diagnosis was the only variable associated with lower probability of complete remission after frontline therapy. Concerning survival, age and leukocytes showed an independent prognostic value, whereas MLD showed a trend to a negative impact ( p  = 0.087, HR, 95 % CI = 1.426, 0.95–2.142). Moreover, after excluding patients receiving an allogeneic stem cell transplantation in first CR, MLD was associated with a shorter survival (HR, 95 % CI = 1.599, 1.026–2.492; p  = 0.038). In conclusion, MLD identifies a subgroup of patients with poorer outcome among patients with IR-AML and wild-type NPM1 .

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.

Background B-cell Acute Lymphoblastic Leukemia (B-ALL) remains an important cause of cancer-related death in children. Therefore, accurate identification at diagnosis of patients at high risk of relapse is crucial. In this context, long non-coding RNAs (lncRNAs) could be novel candidates with great potential. Hence, the aim of this study was to identify new prognostic biomarkers in pediatric B-ALL through an RNA sequencing (RNA-seq) approach that allows the detailed exploration of a wide range of lncRNAs. Methods Total RNA from two cohorts of B-ALL patients (C1 with 50 Spanish patients, and C2 with 72 Canadian patients) was sequenced with a depth of approximately 150 million paired-reads using Illumina technology. All protein coding and non-coding genes included in lncRNAKB annotation were studied to develop a gene expression-based 5-year Event Free Survival (EFS) prediction model. Results First, univariate Cox proportional hazards analyses identified 48 genes significantly associated with higher EFS risk in both cohorts. From these, ALASSO regression selected five genes, all of which are lncRNAs, as the most informative to develop the prediction model, which we have called surviBALL. Stratification of patients into three risk groups according to the surviBALL model revealed significantly poorer EFS in high-risk patients across C1, C2, and the integrated C1 + C2 cohort ( P  < 0.001). Validation in an independent cohort of 177 publicly available B-ALL samples confirmed surviBALL’s prediction capacity ( P  = 2.80 × 10 − 4 ) and its independence of both subtype and MRD. Conclusions These findings suggest that surviBALL has the potential to complement current risk stratification approaches, particularly by identifying patients at high risk of relapse at diagnosis. As a hypothesis-generating proof of concept, this study highlights the promise of more personalized treatment strategies and warrants further validation in independent cohorts.

BackgroundAlthough adoptive transfer of CD19-directed chimeric antigen receptor (CAR) T-cells (CD19-CAR T-cells) achieves high rates of complete response in patients with B-cell acute lymphoblastic leukemia (B-ALL), relapse is common. Bone marrow (BM) mesenchymal stem/stromal cells (BM-MSC) are key components of the hematopoietic niche and are implicated in B-ALL pathogenesis and therapy resistance. MSC exert an immunosuppressive effect on T-cells; however, their impact on CD19-CAR T-cell activity is understudied.MethodsWe performed a detailed characterization of BM-MSC from pediatric patients with B-ALL (B-ALL BM-MSC), evaluated their immunomodulatory properties and their impact on CD19-CAR T-cell activity in vitro using microscopy, qRT-PCR, ELISA, flow cytometry analysis and in vivo using a preclinical model of severe colitis and a B-ALL xenograft model.ResultsWhile B-ALL BM-MSC were less proliferative than those from age-matched healthy donors (HD), the morphology, immunophenotype, differentiation potential and chemoprotection was very similar. Likewise, both BM-MSC populations were equally immunosuppressive in vitro and anti-inflammatory in an in vivo model of severe colitis. Interestingly, BM-MSC failed to impair CD19-CAR T-cell cytotoxicity or cytokine production in vitro using B-ALL cell lines and primary B-ALL cells. Finally, the growth of NALM6 cells was controlled in vivo by CD19-CAR T-cells irrespective of the absence/presence of BM-MSC.ConclusionsCollectively, our data demonstrate that pediatric B-ALL and HD BM-MSC equally immunosuppress T-cell responses but do not compromise CD19-CAR T-cell activity.