Characterisation of FLT3 alterations in childhood acute lymphoblastic leukaemia.

BACKGROUND: Alterations of FLT3 are among the most common driver events in acute leukaemia with important clinical implications, since it allows patient classification into prognostic groups and the possibility of personalising therapy thanks to the availability of FLT3 inhibitors. Most of the knowledge on FLT3 implications comes from the study of acute myeloid leukaemia and so far, few studies have been performed in other leukaemias. METHODS: A comprehensive genomic (DNA-seq in 267 patients) and transcriptomic (RNA-seq in 160 patients) analysis of FLT3 in 342 childhood acute lymphoblastic leukaemia (ALL) patients was performed. Mutations were functionally characterised by in vitro experiments. RESULTS: Point mutations (PM) and internal tandem duplications (ITD) were detected in 4.3% and 2.7% of the patients, respectively. A new activating mutation of the TKD, G846D, conferred oncogenic properties and sorafenib resistance. Moreover, a novel alteration involving the circularisation of read-through transcripts (rt-circRNAs) was observed in 10% of the cases. Patients presenting FLT3 alterations exhibited higher levels of the receptor. In addition, patients with ZNF384- and MLL/KMT2A-rearranged ALL, as well as hyperdiploid subtype, overexpressed FLT3. DISCUSSION: Our results suggest that specific ALL subgroups may also benefit from a deeper understanding of the biology of FLT3 alterations and their clinical implications.

CircRNAome of Childhood Acute Lymphoblastic Leukemia: Deciphering Subtype-Specific Expression Profiles and Involvement in TCF3::PBX1 ALL.

Childhood B-cell acute lymphoblastic leukemia (B-ALL) is a heterogeneous disease comprising multiple molecular subgroups with subtype-specific expression profiles. Recently, a new type of ncRNA, termed circular RNA (circRNA), has emerged as a promising biomarker in cancer, but little is known about their role in childhood B-ALL. Here, through RNA-seq analysis in 105 childhood B-ALL patients comprising six genetic subtypes and seven B-cell controls from two independent cohorts we demonstrated that circRNAs properly stratified B-ALL subtypes. By differential expression analysis of each subtype vs. controls, 156 overexpressed and 134 underexpressed circRNAs were identified consistently in at least one subtype, most of them with subtype-specific expression. TCF3::PBX1 subtype was the one with the highest number of unique and overexpressed circRNAs, and the circRNA signature could effectively discriminate new patients with TCF3::PBX1 subtype from others. Our results indicated that NUDT21, an RNA-binding protein (RBP) involved in circRNA biogenesis, may contribute to this circRNA enrichment in TCF3::PBX1 ALL. Further functional characterization using the CRISPR-Cas13d system demonstrated that circBARD1, overexpressed in TCF3::PBX1 patients and regulated by NUDT21, might be involved in leukemogenesis through the activation of p38 via hsa-miR-153-5p. Our results suggest that circRNAs could play a role in the pathogenesis of childhood B-ALL.

Diverse mutations and structural variations contribute to Notch signaling deregulation in paediatric T-cell lymphoblastic lymphoma.

BACKGROUND: T-cell lymphoblastic lymphoma (T-LBL) is an aggressive neoplasm closely related to T-cell acute lymphoblastic leukaemia (T-ALL). Despite their similarities, and contrary to T-ALL, studies on paediatric T-LBL are scarce and, therefore, its molecular landscape has not yet been fully elucidated. Thus, the aims of this study were to characterize the genetic and molecular heterogeneity of paediatric T-LBL and to evaluate novel molecular markers differentiating this entity from T-ALL. PROCEDURE: Thirty-three paediatric T-LBL patients were analyzed using an integrated approach, including targeted next-generation sequencing, RNA-sequencing transcriptome analysis and copy-number arrays. RESULTS: Copy number and mutational analyses allowed the detection of recurrent homozygous deletions of 9p/CDKN2A (78%), trisomy 20 (19%) and gains of 17q24-q25 (16%), as well as frequent mutations of NOTCH1 (62%), followed by the BCL11B (23%), WT1 (19%) and FBXW7, PHF6 and RPL10 genes (15%, respectively). This genetic profile did not differ from that described in T-ALL in terms of mutation incidence and global genomic complexity level, but unveiled virtually exclusive 17q25 gains and trisomy 20 in T-LBL. Additionally, we identified novel gene fusions in paediatric T-LBL, including NOTCH1-IKZF2, RNGTT-SNAP91 and DDX3X-MLLT10, the last being the only one previously described in T-ALL. Moreover, clinical correlations highlighted the presence of Notch pathway alterations as a factor related to favourable outcome. CONCLUSIONS: In summary, the genomic landscape of paediatric T-LBL is similar to that observed in T-ALL, and Notch signaling pathway deregulation remains the cornerstone in its pathogenesis, including not only mutations but fusion genes targeting NOTCH1.

Variants in vincristine pharmacodynamic genes involved in neurotoxicity at induction phase in the therapy of pediatric acute lymphoblastic leukemia.

Vincristine is an important drug of acute lymphoblastic leukemia (ALL) treatment protocols that can cause neurotoxicity. Patients treated with LAL/SHOP protocols often suffer from vincristine-related neurotoxicity in early phases of treatment. A genetic variant in CEP72, a gene involved in vincristine pharmacodynamics, was recently associated with neurotoxicity after prolonged vincristine treatment. This association was not replicated in our Spanish population during induction phase. To test the possibility that other variants in genes involved in vincristine pharmacodynamics were associated with vincristine neuropathy in early phases of the treatment, we evaluated the correlation with toxicity of 24 polymorphisms in 9 key genes in a large cohort of 152 Spanish children with B-ALL homogeneously treated. Results showed no association between any genetic variant in the TUBB1, TUBB2A, TUBB2B, TUBB3, TUBB4, MAPT, MIR146a, MIR202, and MIR411 genes and vincristine-related neurotoxicity. These results are in line with the hypothesis that there are different mechanisms causing pheripheral neurotoxicity after prolonged and short-term vincristine treatments.

Mir-pharmacogenetics of Vincristine and peripheral neurotoxicity in childhood B-cell acute lymphoblastic leukemia.

Vincristine (VCR), an important component of childhood acute lymphoblastic leukemia (ALL) therapy, can cause sensory and motor neurotoxicity. This neurotoxicity could lead to dose reduction or treatment discontinuation, which could in turn reduce survival. In this line, several studies associated peripheral neurotoxicity and polymorphisms in genes involved in pharmacokinetics (PK) and pharmacodynamics (PD) of VCR. Nowadays, it is well known that these genes are regulated by microRNAs (miRNAs) and SNPs in miRNAs could modify their levels or function. Therefore, the aim of this study was to determine whether SNPs in miRNAs could be associated with VCR-induced neurotoxicity. To achieve this aim, we analyzed all the SNPs in miRNAs (minor allele frequency (MAF) ≥ 0.01) which could regulate VCR-related genes in a large cohort of Spanish children with B-cell precursor ALL (B-ALL) homogeneously treated with LAL/SHOP protocols. We identified the A allele of rs12402181 in the seed region of miR-3117-3p, that could affect the binding with ABCC1 and RALBP1 gene, and C allele of rs7896283 in pre-mature sequence of miR-4481, which could be involved in peripheral nerve regeneration, significantly associated with VCR-induced neurotoxicity. These findings point out the possible involvement of two SNPs in miRNA associated with VCR-related neurotoxicity.

The miR-1206 microRNA variant is associated with methotrexate-induced oral mucositis in pediatric acute lymphoblastic leukemia.

Five-year survival rates of pediatric acute lymphoblastic leukemia (ALL) have reached 90% in the developed countries. However, toxicity because of methotrexate (MTX) occurs frequently. Variety in the occurrence of toxicity is partly determined by single nucleotide polymorphisms (SNPs) in coding regions. Recently, five SNPs in non-coding pre-microRNAs and microRNA processing (miRNA) genes were identified in association with MTX-induced oral mucositis. This study aimed to replicate the association of these miRNA variants in relation to MTX-induced oral mucositis in a prospective childhood ALL cohort. Three out of five SNPs with a minor allele frequency more than 0.15 [CCR4-NOT transcription complex (CNOT4) rs3812265, miR-1206 rs2114358, miR-2053 rs10505168] were analyzed in 117 pediatric ALL patients treated with 5 g/m MTX (DCOG ALL-10). Oral mucositis was defined as grade more than or equal to 3 according to the National Cancer Institute criteria. rs2114358 in miR-1206 was associated with oral mucositis [odds ratio (OR): 3.6; 95% confidence interval (CI): 1.1-11.5], whereas we did not confirm the association of CNOT4 rs3812265 (OR: 0.69; 95% CI: 0.27-1.80) and miR-2053 rs10505168 (OR: 2.50; 95% CI: 0.76-8.24). Our results replicate the association between rs2114358 in miR-1206 and MTX-induced oral mucositis in childhood ALL. Genetic variation in miR-1206 has potential as a novel biomarker to predict MTX-induced toxicity.

MiR-pharmacogenetics of methotrexate in childhood B-cell acute lymphoblastic leukemia.

OBJECTIVES: Methotrexate (MTX), the key drug in childhood B-cell acute lymphoblastic leukemia (B-ALL) therapy, often causes toxicity. An association between genetic variants in MTX transport genes and toxicity has been found. It is known that these transporters are regulated by microRNAs (miRNAs), and miRNA single nucleotide polymorphisms (SNPs) interfere with miRNA levels or function. With regard to B-cell ALL, we have previously found rs56103835 in miR-323b that targets ABCC4 associated with MTX plasma levels. Despite these evidences and that nowadays a large amount of new miRNAs have been annotated, studies of miRNA polymorphisms and MTX toxicity are almost absent. Therefore, the aim of this study was to determine whether there are other variants in miRNAs associated with MTX levels. PATIENTS AND METHODS: Blood samples of 167 Spanish patients with pediatric B-cell ALL treated with the LAL-SHOP protocol were analyzed. We selected all the SNPs described in pre-miRNAs with a minor allele frequency more than 1% (213 SNPs in 206 miRNAs) that could regulate MTX transporters because the miRNAs that target MTX transporter genes are not completely defined. Genotyping was performed with VeraCode GoldenGate platform. RESULTS: Among the most significant results, we found rs56292801 in miR-5189, rs4909237 in miR-595, and rs78790512 in miR-6083 to be associated with MTX plasma levels. These miRNAs were predicted, in silico, to regulate genes involved in MTX uptake: SLC46A1, SLC19A1, and SLCO1A2. CONCLUSION: In this study, we detected three SNPs in miR-5189, miR-595, and miR-6083 that might affect SLC46A1, SLC19A1, and SLCO1A2 MTX transport gene regulation and could affect MTX levels in patients with pediatric B-cell ALL.

Lack of association of the CEP72 rs924607 TT genotype with vincristine-related peripheral neuropathy during the early phase of pediatric acute lymphoblastic leukemia treatment in a Spanish population.

Vincristine is a component of acute lymphoblastic leukemia (ALL) treatment with the potential to induce peripheral neuropathy. Recently, the CEP72 rs924607 TT genotype was found to be associated with vincristine-induced toxicity during the continuation phase in pediatric ALL patients treated on the Total XIIIB and COG AALL0433 protocols at St Jude Children's Research Hospital and Children's Oncology Group. This finding could provide a base for safer dosing of vincristine. Nevertheless, there are variations in vincristine regimens among ALL treatment protocols and phases in different populations. Therefore, the aim of this study was to determine whether the CEP72 rs924607 TT genotype is a useful marker of vincristine neuropathy during induction therapy among Spanish children with B-ALL treated on the LAL-SHOP protocols. No association was found between neurotoxicity during the induction phase and the rs924607 TT genotype. This lack of association could be because of population differences and/or differences in neurotoxicity etiology between induction and continuation phases of treatment.

Polymorphisms in miRNA processing genes and their role in osteosarcoma risk.

BACKGROUND: The possible associations between genetic variants and osteosarcoma risk have been analyzed without conclusive results. Those studies were focused mainly on genes of biologically plausible pathways. However, recently, another pathway has acquired relevance in cellular transformation and tumorigenesis, the microRNA (miRNA) processing pathway. Dysregulation of the expression levels of genes in this pathway has been described in cancer. Consequently, single nucleotide polymorphisms (SNPs) in genes that codify for proteins involved in the miRNA processing pathway may affect miRNAs, and therefore their target genes, which might be associated with cancer development and progression. The aim of this study was to evaluate whether SNPs in miRNA processing genes confer predisposition to osteosarcoma. PROCEDURE: We analyzed 72 SNPs in 21 miRNA processing genes in a total of 99 osteosarcoma patients and 387 controls. RESULTS: A total of three SNPs were associated with osteosarcoma susceptibility. Interestingly, these SNPs were located in miRNA processing genes (CNOT1, CNOT4 and SND1) which are part of the RISC complex. Among them, the association of rs11866002 in CNOT1 was nearly significant after Bonferroni correction. CONCLUSIONS: This study suggests that SNPs in RISC complex genes may be involved in osteosarcoma susceptibility, especially rs11866002 in CNOT1.

Noncoding RNA-related polymorphisms in pediatric acute lymphoblastic leukemia susceptibility.

BACKGROUND: Evidence for an inherited genetic risk for pediatric acute lymphoblastic leukemia has been provided in several studies. Most of them focused on coding regions. However, those regions represent only 1.5% of the entire genome. In acute lymphoblastic leukemia (ALL), it has been suggested that the expression of microRNAs (miRNAs) is dysregulated, which suggests that they may have a role in ALL risk. Changes in miRNA function may occur through single-nucleotide polymorphisms (SNPs). Therefore, the aim of this study was to evaluate whether polymorphisms in pre-miRNAs, and/or miRNA-processing genes, contribute to a predisposition for childhood ALL. METHODS: In this study, we analyzed 118 SNPs in pre-miRNAs and miRNA-processing genes in 213 B-cell ALL patients and 387 controls. RESULTS: We found 11 SNPs significantly associated with ALL susceptibility. These included three SNPs present in miRNA genes (miR-612, miR-499, and miR-449b) and eight SNPs present in six miRNA biogenesis pathway genes (TNRC6B, DROSHA, DGCR8, EIF2C1, CNOT1, and CNOT6). Among the 118 SNPs analyzed, rs12803915 in mir-612 and rs3746444 in mir-499 exhibited a more significant association, with a P value <0.01. CONCLUSION: The results of this study indicate that SNP rs12803915 located in pre-mir-612, and SNP rs3746444 located in pre-mir-499, may represent novel markers of B-cell ALL susceptibility.

The Role of the Dysregulation of Long Non-Coding and Circular RNA Expression in Medulloblastoma: A Systematic Review.

Medulloblastoma (MB) is the most common malignant brain tumor in childhood. Although recent multi-omic studies have led to advances in MB classification, there is still room for improvement with regard to treatment response and survival. Therefore, identification of new and less invasive biomarkers is needed to refine the diagnostic process and to develop more personalized treatment strategies. In this context, non-coding RNAs (ncRNAs) could be useful biomarkers for MB. In this article, we reviewed the role of two types of ncRNAs, long non-coding (lncRNAs) and circular RNAs (circRNAs), as biomarkers for the diagnosis, subgroup classification, and prognosis of MB. We also reviewed potential candidates with specific functions and mechanisms of action in the disease. We performed a search in PubMed and Scopus using the terms ("long non coding RNAs" OR "lncRNAs") and ("circular RNAs" OR "circRNAs") AND "medulloblastoma" to identify biomarker discovery or functional studies evaluating the effects of these ncRNAs in MB. A total of 26 articles met the inclusion criteria. Among the lncRNAs, the tumorigenic effects of the upregulated lnc-IRX3-80 and lnc-LRRC47-78 were the most studied in MB. Among the circRNAs, the upregulation of circSKA3 and its functional impact in MB cell lines were the most consistent results, so this circRNA could be considered a potential biomarker in MB. Additional validation is required for many deregulated lncRNAs and circRNAs; therefore, further studies are warranted.

microRNA sequencing for biomarker detection in the diagnosis, classification and prognosis of Diffuse Large B Cell Lymphoma.

Despite being considered a single disease, Diffuse Large B Cell Lymphoma (DLBCL) presents with variable backgrounds, which results in heterogeneous outcomes among patients, with 40% of them still having primary refractory disease or relapse. Thus, novel biomarkers are needed. In addition, multiple factors regarding its pathogenesis remain unclear. In this context, recent investigations point to the relevance of microRNAs (miRNAs) in cancer. However, regarding DLBCL, there is inconsistency in the data reported. Therefore, in this work, the main goals were to determine a miRNA set with utility as biomarkers for DLBCL diagnosis, classification, prognosis and treatment response, as well as to decipher the mechanism of action of deregulated miRNAs in the origin of the disease. We analyzed miRNA expression in a cohort of 78 DLBCL patients and 17 controls using small RNA sequencing and performed a miRNA-mRNA interaction network analysis. This way, we were able to define new miRNA expression signatures for diagnosis, classification, treatment response and prognosis, and we identified plausible mechanisms of action by which deregulated miRNAs could be involved in DLBCL pathogenesis. In summary, our study remarks that miRNAs could play an important role in DLBCL.

lncRNA deregulation in childhood acute lymphoblastic leukemia: A systematic review.

Childhood acute lymphoblastic leukemia (ALL), the most common pediatric cancer, is a heterogeneous disease comprised of multiple molecular subtypes with distinct somatic genetic alterations, which results in different outcomes for the patients. Accurate patient risk stratification through genetic markers could increase survival rates, but the identification of reliable biomarkers is needed, as 20­30% of B­ALL patients cannot be classified in the clinic with routine techniques and some patients classified as low­risk and good­responders to treatment will eventually relapse. Long non­coding RNAs (lncRNAs) can represent novel candidates with diagnostic, classification, prognosis, and treatment response potential. However, regarding childhood ALL, there is inconsistency in the data reported due to the lack of a consensus nomenclature for lncRNA naming and the methodology and designing applied for their study. Therefore, the aim of the article is to clarify the potential of lncRNAs as biomarkers in childhood ALL through a systematic review. From a revision of 23 manuscripts, it was found that AWPPH overexpression could represent a novel marker for ALL diagnosis, including both B and T immunophenotypes, and 18 lncRNAs were specifically associated with B­cell ALL (B­ALL) patients. We identified subtype­specific signatures for ETV6­RUNX1, hyperdiploidy and KMT2A subtypes. These signatures hold promise as novel diagnostic markers and could refine the classification of patients.

Prognostic and therapeutic value of somatic mutations in diffuse large B-cell lymphoma: A systematic review.

Diffuse large B-cell lymphoma (DLBCL), the most common type of Non-Hodgkin lymphoma (NHL), is a highly heterogeneous and aggressive disease. Regardless of this heterogeneity, all patients receive the same first-line therapy, which fails in 30-40 % of patients, who are either refractory or relapse after remission. With the aim of stratifying patients to improve treatment outcome, different clinical and genetic biomarkers have been studied. The present systematic review aimed to identify somatic mutations that could serve as prognosis biomarkers or as therapeutic target mutations in DLBCL. Regarding their role as prognostic markers, mutations in CD58 and TP53 seem the most promising predictors of poor outcome although the combination of different alterations and other prognostic factors could be a more powerful strategy. On the other hand, different approaches regarding targeted therapy have been proposed. Therefore, mutational analysis could help guide treatment choice in DLBCL yet further studies and clinical trials are needed.

miRNA deregulation in childhood acute lymphoblastic leukemia: a systematic review.

Despite remarkable improvements in survival of childhood acute lymphoblastic leukemia (ALL), nonresponding or relapsing patients still represent one of the most frequent causes of death by disease in children. Accurate patient risk stratification based on genetic markers could increases survival rates. miRNAs can represent novel candidates with diagnostic, predictive and prognostic potential; however, many groups investigated their involvement with contradictory results. Aim: To clarify the role of miRNAs as biomarkers through a systematic review. Results: From a revision of 45 manuscripts, we found that miR-128 and miR-181 overexpression could represent markers for ALL diagnosis and underexpression of miR-708 and miR-99a could be markers for bad prognosis. Conclusion: These signatures could refine classification and risk stratification of patients and improve ALL outcome.

Role of rs10406069 in miR-5196 in hyperdiploid childhood acute lymphoblastic leukemia.

Aim: To determine the role of single nucleotide polymorphisms (SNPs) in noncoding RNAs in childhood acute lymphoblastic leukemia (ALL) subtypes. Materials & methods: We screened all SNPs in 130 pre-miRNA genes to assess their role in the susceptibility of the most common subtypes of ALL: hyperdiploid and ETV6-RUNX1. Results: In two independent cohorts, we found a significant association between rs10406069 in miR-5196 and the risk of developing hyperdiploid ALL. This observation could be explained by the impact of the SNP on miR-5196 expression and in turn, in its target genes. Indeed, rs10406069 was associated with expression changes in SMC1A, a gene involved in sister chromatin cohesion. Conclusion: rs10406069 in miR-5196 may have a relevant role in hyperdiploid ALL risk.

Role of miRNAs in treatment response and toxicity of childhood acute lymphoblastic leukemia.

Childhood acute lymphoblastic leukemia survival rates have increased remarkably during last decades due, in part, to intensive treatment protocols. However, therapy resistance and toxicity are still two important barriers to survival. In this context, pharmacoepigenetics arises as a tool to identify new predictive markers, required to guide clinicians on risk stratification and dose individualization. The present study reviews current evidence about miRNA implication on childhood acute lymphoblastic leukemia therapy resistance and toxicity. A total of 12 studies analyzing differential miRNA expression in relation to drug resistance and six studies exploring the association between miRNAs-related SNPs and drug-induced toxicities were identified. We pointed out to miR-125b together with miR-99a and/or miR-100 overexpression as markers of vincristine resistance and rs2114358 in mir-1206 as mucositis marker as the most promising results.

Involvement of miRNA polymorphism in mucositis development in childhood acute lymphoblastic leukemia treatment.

AIM: Mucositis, linked to methotrexate, daunorubicin or cyclophosphamide, is a frequent childhood acute lymphoblastic leukemia (ALL) therapy side effect. miRNAs regulate the expression of pharmacokinetic/pharmacodynamic pathway genes. SNPs in miRNAs could affect their levels or function, and affect their pharmacokinetic/pharmacodynamic pathway target genes. Our aim was to determine the association between miRNA genetic variants targeting mucositis-related genes and mucositis-developing risk. PATIENTS & METHODS: We analyzed 160 SNPs in 179 Spanish children with B-cell precursor ALL homogeneously treated with LAL/SHOP protocols. RESULTS: We identified three SNPs in miR-4268, miR-4751 and miR-3117 associated with mucositis, diarrhea and vomiting, respectively. CONCLUSION: The effect of these SNPs on genes related to drug pharmacokinetics/pharmacodynamics could explain mucositis, diarrhea and vomiting development during ALL therapy.

Pharmacoepigenetics in childhood acute lymphoblastic leukemia: involvement of miRNA polymorphisms in hepatotoxicity.

AIM: Hepatotoxicity is one of the most common drug-related toxicities during the treatment of childhood acute lymphoblastic leukemia (ALL). Many genes involved in liver-specific signaling pathways are tightly controlled by miRNAs, and miRNA function could be modulated by SNPs. As a consequence, we hypothesized that variants in miRNAs could be associated with drug-induced hepatotoxicity. METHODS: We analyzed 213 SNPs in 206 miRNAs in a cohort of 179 children with ALL homogeneously treated. RESULTS: rs2648841 in miR-1208 was the most significant SNP during consolidation phase after false discovery rate correction, probably through an effect on its target genes DHFR, MTR and MTHFR. CONCLUSION: These results point out the possible involvement of SNPs in miRNAs in toxicity to chemotherapy in children with ALL.

Involvement of SNPs in miR-3117 and miR-3689d2 in childhood acute lymphoblastic leukemia risk.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Numerous studies have shown that microRNAs (miRNAs) could play a role in this disease. Nowadays, more than 2500 miRNAs have been described, that regulate more than 50% of genes, including those involved in B-cell maturation, differentiation and proliferation. Genetic variants in miRNAs can alter their own levels or function, affecting their target gene expression, and then, may affect ALL risk. Therefore, the aim of this study was to determine the role of miRNA genetic variants in B-ALL susceptibility. We analyzed all variants in pre-miRNAs (MAF > 1%) in two independent cohorts from Spain and Slovenia and inferred their functional effect by in silico analysis. SNPs rs12402181 in miR-3117 and rs62571442 in miR-3689d2 were associated with ALL risk in both cohorts, possibly through their effect on MAPK signalling pathway. These SNPs could be novel markers for ALL susceptibility.