Cases Of Childhood Acute Lymphoblastic Leukemia Research Articles

Application of prime editing system to introduce TP53 R248Q hotspot mutation in acute lymphoblastic leukemia cell line.

In childhood acute lymphoblastic leukemia (ALL), TP53 gene mutation is associated with chemoresistance in a certain population of relapsed cases. To directly verify the association of TP53 gene mutation with chemoresistance of relapsed childhood ALL cases and improve their prognosis, the development of appropriate human leukemia models having TP53 mutation in the intrinsic gene is required. Here, we sought to introduce R248Q hotspot mutation into the intrinsic TP53 gene in an ALL cell line, 697, by applying a prime editing (PE) system, which is a versatile genome editing technology. The PE2 system uses an artificial fusion of nickase Cas9 and reverse-transcriptase to directly place new genetic information into a target site through a reverse transcriptase template in the prime editing guide RNA (pegRNA). Moreover, in the advanced PE3b system, single guide RNA (sgRNA) matching the edited sequence is also introduced to improve editing efficiency. The initially obtained MDM2 inhibitor-resistant PE3b-transfected subline revealed disrupted p53 transactivation activity, reduced p53 target gene expression, and acquired resistance to chemotherapeutic agents and irradiation. Although the majority of the subline acquired the designed R248Q and adjacent silent mutations, the insertion of the palindromic sequence in the scaffold hairpin structure of pegRNA and the overlap of the original genomic DNA sequence were frequently observed. Targeted next-generation sequencing reconfirmed frequent edit errors in both PE2 and PE3b-transfected 697 cells, and it revealed frequent successful edits in HEK293T cells. These observations suggest a requirement for further modification of the PE2 and PE3b systems for accurate editing in leukemic cells.

Evidence of spatial clustering of childhood acute lymphoblastic leukemia cases in Greater Mexico City: report from the Mexican Inter-Institutional Group for the identification of the causes of childhood leukemia.

A heterogeneous geographic distribution of childhood acute lymphoblastic leukemia (ALL) cases has been described, possibly, related to the presence of different environmental factors. The aim of the present study was to explore the geographical distribution of childhood ALL cases in Greater Mexico City (GMC). A population-based case-control study was conducted. Children <18 years old, newly diagnosed with ALL and residents of GMC were included. Controls were patients without leukemia recruited from second-level public hospitals, frequency-matched by sex, age, and health institution with the cases. The residence address where the patients lived during the last year before diagnosis (cases) or the interview (controls) was used for geolocation. Kulldorff's spatial scan statistic was used to detect spatial clusters (SCs). Relative risks (RR), associated p-value and number of cases included for each cluster were obtained. A total of 1054 cases with ALL were analyzed. Of these, 408 (38.7%) were distributed across eight SCs detected. A relative risk of 1.61 (p<0.0001) was observed for the main cluster. Similar results were noted for the remaining seven ones. Additionally, a proximity between SCs, electrical installations and petrochemical facilities was observed. The identification of SCs in certain regions of GMC suggest the possible role of environmental factors in the etiology of childhood ALL.

Optimized treatment of childhood B-lineage acute lymphoblastic leukemia

Childhood acute lymphoblastic leukemia (ALL) accounts for about 75% of childhood leukemia cases, and B-lineage acute lymphoblastic leukemia (B-ALL) accounts for more than 80% of childhood ALL cases. Over the past half century, new molecular biological targets discovered by new techniques have been used in precise stratification of disease prognosis, and there has been a gradual increase in the 5-year overall survival rate of childhood ALL. With the increasing attention to long-term quality of life, the treatment of childhood B-ALL has been constantly optimized from induction therapy to the intensity of maintenance therapy, including the treatment of extramedullary leukemia without radiotherapy, which has been tried with successful results. The realization of optimized treatment also benefits from the development of new techniques associated with immunology and molecular biology and the establishment of standardized clinical cohorts and corresponding biobanks. This article summarizes the relevant research on the implementation of precise stratification and the intensity reduction and optimization treatment of B-ALL in recent years, providing reference for clinicians.

Investigating DNA methylation as a mediator of genetic risk in childhood acute lymphoblastic leukemia.

Genome-wide association studies have identified a growing number of single nucleotide polymorphisms (SNPs) associated with childhood acute lymphoblastic leukemia (ALL), yet the functional roles of most SNPs are unclear. Multiple lines of evidence suggest that epigenetic mechanisms may mediate the impact of heritable genetic variation on phenotypes. Here, we investigated whether DNA methylation mediates the effect of genetic risk loci for childhood ALL. We performed an epigenome-wide association study (EWAS) including 808 childhood ALL cases and 919 controls from California-based studies using neonatal blood DNA. For differentially methylated CpG positions (DMPs), we next conducted association analysis with 23 known ALL risk SNPs followed by causal mediation analyses addressing the significant SNP-DMP pairs. DNA methylation at CpG cg01139861, in the promoter region of IKZF1, mediated the effects of the intronic IKZF1 risk SNP rs78396808, with the average causal mediation effect (ACME) explaining ~30% of the total effect (ACME P = 0.0031). In analyses stratified by self-reported race/ethnicity, the mediation effect was only significant in Latinos, explaining ~41% of the total effect of rs78396808 on ALL risk (ACME P = 0.0037). Conditional analyses confirmed the presence of at least three independent genetic risk loci for childhood ALL at IKZF1, with rs78396808 unique to non-European populations. We also demonstrated that the most significant DMP in the EWAS, CpG cg13344587 at gene ARID5B (P = 8.61×10-10), was entirely confounded by the ARID5B ALL risk SNP rs7090445. Our findings provide new insights into the functional pathways of ALL risk SNPs and the DNA methylation differences associated with risk of childhood ALL.

Variation in the rate of diagnosis of childhood acute lymphoblastic leukemia in about to the COVID-19 Pandemic: a single-center study

Background. Greaves delayed infection hypothesis states that acute lymphoblastic leukemia (ALL) in children develops in two critical steps, with the first step (first hit) occurring in utero and the second step (second hit) occurring in the postnatal period and involving leukemia-associated genetic changes, depending upon the timing of exposure to common childhood infections. In this study, we investigated whether isolation of children at home as part of the lockdown during the Coronavirus Disease 2019 (COVID-19) pandemic resulted in a reduction in the number of new childhood ALL cases by avoiding the second hit. Materials and metods. This retrospective study included all newly diagnosed pediatric ALL patients aged 1–18 years. The study periods were from 15 March 2020 to 31 December 2020 (COVID-19 lockdown period) and from 15 March 2019 to 31 December 2019 (control period). Results. Acute leukemia was diagnosed in 73 children between January 2019 and December 2020. Of these, there were 58 ALL cases and 15 acute myeloid leukemia cases. During the COVID-19 lockdown period, 14 of the children were diagnosed with ALL. In the control period, 21 children were diagnosed with ALL. No pediatric patients diagnosed with ALL in August, September, and October 2020. Conclusions. We observed a reduced incidence within the lockdown period, possibly related to the potential role of SARS-CoV-2 infection as a second hit in childhood ALL.

Contribution of Cyclin-dependent Kinase Inhibitor 1B Genotypes to Childhood Leukemia Risk.

Although genetic differences in cell-cycle control genes have been associated with cancer risk, to our knowledge, no report has specifically examined the role of gene variants in childhood acute lymphoblastic leukemia (ALL). Cyclin-dependent kinase inhibitor 1B (CDKN1B; also known as p27/KIP1) is a cell-cycle regulating gene. This study aimed at investigating the association between CDKN1B genotypes and childhood ALL risk. In 266 childhood ALL cases and 266 healthy controls, the CDKN1B rs34330 and 2066827 polymorphisms were genotyped, and the association of CDKN1B genotypes with childhood ALL risk were analyzed. The genotypes of CDKN1B rs34330 and 2066827 were similarly distributed between the control and case groups (p for trend=0.8718 and 0.4030, respectively). The allelic frequency also exhibited no statistical difference (p=1.0000 and 0.6666, respectively). There was no significant interaction between CDKN1B genotypes and age or sex. CDKN1B genotypes were not found to be minor contributors to childhood ALL susceptibility in Taiwan.

The Molecular Landscape of KMT2A-Rearranged Leukemia from Infancy to Adulthood Reveals Age and Leukemia-Specific Mutational Patterns

Genetic rearrangements involving the KMT2A gene (KMT2A-R) are seen in around 10% of acute leukemia overall. KMT2A-R occurs in all ages and usually correlates with high-risk clinical features, in particular in infants aged 0-12 months of age with acute lymphoblastic leukemia (ALL). To uncover age- and leukemia-subtype specific molecular patterns in KMT2A-R ALL and acute myeloid leukemia (AML), we performed whole genome (WGS), whole exome (WES), and RNA-sequencing on a well-annotated Nordic KMT2A-R cohort of 104 patients, including infant ALL (n=33), childhood ALL (n=18), adult ALL (n=15), and pediatric AML (n=38) patients. For 77 patients, we performed WGS (40x) at diagnosis and remission as well as WES (140x) on the diagnostic sample, and remaining patients underwent WES only (n=27). RNA-sequencing was performed on 58 cases with available RNA.Twenty-two genes were recurrently altered and remarkably, NRAS, KRAS, FLT3, PAX5, TP53, CDKN2A/B and IKZF1 accounted for 70% of mutations. The landscape of mutations suggested the presence of leukemia and age-specific associations with MYST4, PTPN11, and SETD2 uniquely altered in AML and PIK3CD, DNAH11, NOTCH1, CSMD3 and CDKN2A/B in ALL. Some genes were mutated in both KMT2A-R ALL and AML, but were more common in one disease, such as FLT3 and KRAS in AML and PAX5, TP53 and IKZF1 in ALL. Moreover, age-associated patterns were seen in ALL with NRAS more frequently mutated than KRAS in infant ALL (26% vs 15%), and KRAS more frequently mutated than NRAS in childhood ALL (24% vs 18%), with adult ALL having fewer such mutations (NRAS 13%; KRAS 7%). Alterations of CDKN2A/B and TP53 were absent in infant ALL, detected in childhood and adult ALL only. PAX5 alterations were primarily detected in childhood ALL (22%, 9% infant ALL, 7% adult ALL), with all three PAX5-altered infant cases having the KMT2A-MLLT3 fusion gene. Finally, KMT2A-R pediatric AML had the highest fraction of FLT3 mutations (24%, 9% infant ALL, 11% childhood ALL, 0% adult ALL) and all but one mutation occurred in KMT2A-MLLT3 rearranged cases and most were kinase domain point mutations.We next expanded our analysis to include non-recurrent alterations. PI3K/RAS pathway alterations were detected across ages and subtypes with the highest fraction in pediatric AML (63%) and the lowest in adult ALL (27%, 43% infant ALL, 41% childhood ALL). Further, cell cycle related genes were primarily mutated in childhood (39%) and adult ALL cases (33%) and rarer in infant ALL (12%) and pediatric AML (16%) and genes within the B-cell pathway were more commonly altered in childhood ALL (29%) than in infant ALL (9%). Finally, in line with our previous study (Andersson et al, Nat Genet 2015) epigenetic mutations were absent in infant ALL, but present in 20-35% of the other patients.RNA-sequencing identified the KMT2A-fusion in 56/58 cases, with low exonic coverage preventing detection of the fusion in two cases. The reciprocal KMT2A fusion was only expressed in 13/39 cases where it was predicted to be expressed based on karyotype or whole genome sequencing data with 11/13 cases having the KMT2A-AFF1 fusion gene. In addition, RNA-sequencing identified 6 in-frame and 12 out-of-frame fusion genes that had formed either as part of the KMT2A-R itself or that were independent genetic events. Further, a novel in-frame KMT2A-ACIN1 fusion was identified in a child aged 1 year with B-precursor ALL. ACIN1 encodes Apoptotic Chromatin Condensation Inducer 1 and the fusion was formed through an insertion of 14q11 into 11q23. ACIN1 is also rearranged as part of the ACIN1-NUTM1 that we identified in an infant with ins(15;14)(q22;q11.2q32.1) (Andersson et al Nat Genet 2015). To study the ability of KMT2A-ACIN1 to induce leukemia in mice, we injected retrovirally transduced mouse bone marrow cells containing the fusion into syngeneic mice and KMT2A-MLLT3 was used as control. All mice succumbed to disease at an average of 112 days for KMT2A-ACIN1 (n=12) and 63 days for KMT2A-MLLT3 (n=5) and mice displayed splenomegaly and leukocytosis with an immunophenotype indicative of AML. Primary leukemia cells isolated from moribund mice gave rise to leukemia in sublethally irradiated recipients with reduced disease latency. In conclusion, these results highlight the differential molecular patterns in KMT2A-R leukemia across infancy to adulthood thereby providing novel pathogenetic insight. DisclosuresNo relevant conflicts of interest to declare.

Significant Association of CCND1 Genotypes With Susceptibility to Childhood Acute Lymphoblastic Leukemia.

This study investigated whether genetic variations in cyclin D1 (CCND1) are associated with susceptibility to childhood acute lymphoblastic leukemia (ALL). A total of 266 childhood ALL cases and 266 healthy controls were genotyped for CCND1 rs9344 and rs678653. There was a significant difference in the genotypic distribution of rs9344 between childhood ALL patients and healthy controls (p=0.0077). Compared to the AA genotype, AG and GG genotypes were associated with significantly decreased risks of childhood ALL with odds ratio (OR) of 0.65 [95% confidence interval (CI)=0.44-0.94, p=0.0234] and 0.45 (95%CI=0.26-0.78, p=0.0040), respectively. Supporting this, allelic frequency distributions between childhood ALL patients and controls was significantly different (OR=0.68, 95%CI=0.53-0.88, p=0.0025). There was no significant difference in the genotypic and allelic distributions of rs678653 between cases and controls. CCND1 rs9344, but not rs678653, may serve as a predictive marker of susceptibility for childhood ALL.

The Association of PRKRAP1 Pseudogene with Acute Lymphoblastic Leukemia Risk

Acute Lymphoblastic Leukemia (ALL) consists of excessive proliferation of lymphoblasts. The frequency of Human Leukocyte Antigen (HLA)-DR53 (DRB4) homozygosity and allele was found to be higher in male childhood ALL cases. The aim of this study was to identify the HLA-DR53 allele frequency; interferon-inducing double chain ribonucleic acid binding protein kinase A pseudogene 1 (PRKRAP1) positivity; rs2395185 allele and genotype frequencies in ALL patients.

Dasatinib in the Management of Pediatric Patients With Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia.

Acute leukemia is the most common cancer in childhood; in particular, acute lymphoblastic leukemia (ALL) represents roughly up to 80% of all cases of acute leukemias in children. Survival of children with ALL has dramatically improved over the last few decades, and is now over 90% (versus 40% of adult patients) in developed countries, except for in infants (i.e., children < 1 year), where no significant improvement was registered. Philadelphia positive ALL (Ph+ALL) accounts for around 3% of cases of childhood ALL, its incidence increasing with patient’s age. Before the era of tyrosine-kinase inhibitors (TKIs), pediatric Ph+ALL showed a worse prognosis in comparison to other forms of ALL, and was managed with intensive chemotherapy, followed, whenever possible, by allogenic hematopoietic stem cell transplantation (HSCT) in first morphological complete remission. TKIs have revolutionized the current clinical approach, which involves combinations of imatinib plus standard chemotherapy that can abrogate the negative prognostic impact conferred by the presence of BCR/ABL1 rearrangement, resulting in the probability of event-free survival (EFS) being significantly better than that recorded in the pre-TKI era. Long-term follow-up confirms these data, questioning the role of a real advantage offered by HSCT over intensive chemotherapy plus TKI in all Ph+ALL pediatric patients. Imatinib was the first generation TKI and the prototype of targeted therapy, but over the years second- (dasatinib, nilotinib, bosutinib) and third-generation (ponatinib) TKIs showed a capacity to overcome resistance to imatinib in Ph+ hematological neoplasms. Given the effectiveness of the first-in-class TKI, imatinib, also the second-generation TKI dasatinib was incorporated in the treatment regimens of Ph+ALL. In this manuscript, we will discuss the role of this drug in pediatric Ph+ALL, analyzing the available data published to date.

Reduced intensity of early intensification does not increase the risk of relapse in children with standard risk acute lymphoblastic leukemia - a multi-centric clinical study of GD-2008-ALL protocol

BackgroundThe prognosis of childhood acute lymphoblastic leukemia (ALL) is optimistic with a 5-year event-free survival (EFS) rate of 70–85%. However, the major causes of mortality are chemotherapy toxicity, infection and relapse. The Guangdong (GD)-2008-ALL collaborative protocol was carried out to study the effect of reduced intensity on treatment related mortality (TRM) based on Berlin-Frankfurt-Münster (BFM) 2002 backbone treatment. The study was designed to elucidate whether the reduced intensity is effective and safe for children with ALL.MethodsThe clinical data were obtained from February 28, 2008 to June 30, 2016. A total of 1765 childhood ALL cases from 9 medical centers were collected and data were retrospectively analyzed. Patients were stratified into 3 groups according to bone marrow morphology, prednisone response, age, genotype, and karyotype information: standard risk (SR), intermediate risk (IR) and high risk (HR). For SR group, daunorubicin was decreased in induction IA while duration was reduced in Induction Ib (2 weeks in place of 4 weeks). Doses for CAM were same in all risk groups - SR patients received one CAM, others got two CAMs.ResultsThe 5-year and 8-year overall survival (OS), event-free survival (EFS) and cumulative incidence of relapse (CIR) were 83.5±0.9% and 83.1±1.0%, 71.9±1.1% and 70.9±1.2%, and 19.5±1.0% and 20.5±1.1%, respectively. The 2-year treatment-related mortality (TRM) was 5.2±0.5%. The 5-year and 8-year OS were 90.7±1.4% and 89.6±1.6% in the SR group, while the 5-year and 8-year EFS were 81.5±1.8% and 80.0±2.0%. In the SR group, 74 (15.2%) patients measured minimal residual disease (MRD) on Day 15 and Day 33 of induction therapy. Among them, 7 patients (9.46%) were MRD positive (≥ 0.01%) on Day 33. The incidence of relapse in the MRD Day 33 positive group (n=7) was 28.6%, while in the MRD Day 33 negative group (n=67) was 7.5% (p=0.129).ConclusionsThe results of GD-2008-ALL protocol are outstanding for reducing TRM in childhood ALL in China with excellent long term EFS. This protocol provided the evidence for further reducing intensity of induction therapy in the SR group according to the risk stratification. MRD levels on Day 15 and Day 33 are appropriate indexes for stratification.

Philadelphia Chromosome-Positive (Ph+) Childhood Acute Lymphoblastic Leukemia: Good Initial Steroid Response Allows Early Prediction of a Favorable Treatment Outcome

Among 4,760 acute lymphoblastic leukemia (ALL) patients enrolled from 1986 to 1995 in two subsequent trials of the BFM and AIEOP study group, 61 patients were found to have Philadelphia chromosome-positive (Ph+) ALL. These patients were analyzed for presenting features and treatment outcome to identify specific prognostic factors. Treatment stratification was based on initial cell mass and early response as determined by blast count in peripheral blood after a 7-day induction prephase with prednisone and one dose of intrathecal methotrexate on day 1. All patients were treated by similar intensive Berlin-Frankfurt-Münster (BFM) protocols. The median age of Ph+ patients was 7.5 years, the median white blood cell count (WBC) was 75 × 109/L, 77% of patients had common ALL, and 29% coexpressed myeloid markers. After a median observation time of 4.2 years, 29 of 61 patients are alive (survival probability [pSUR] at 4 years, 0.49; standard error [SE], 0.06), and 24 of 61 are in first complete remission (CR1; probability of event-free survival [pEFS] at 4 years, 0.38; SE, 0.06). Twenty (35%) of 57 evaluable patients had ≥1,000 leukemic blasts per microliter of blood on day 8 of induction (defined as prednisone-poor-response [PPR]). These patients were older (10.0v 6.88 years; P = .02) and had a higher WBC (144v 29 × 109/L; P = .0016) as compared with patients with prednisone good response (PGR; <1,000 blasts/μL at day 8). Only 2 of 20 patients (10%) with PPR remained in CR1 and alive: 6 patients with PPR did not survive after allogeneic bone marrow transplantation (BMT) due to recurring disease (n = 3) and toxicity (n = 3), and 12 nontransplanted patients died due to progression (n = 5) or relapse (n = 7). In contrast, 26 (70%) of the 37 patients with PGR are alive. Of 18 patients transplanted by allo-BMT, 1 relapsed (now in CR2) and 4 died after BMT. Among the 19 patients with PGR treated by chemotherapy alone, 8 remained in CR1 and 11 relapsed, of which 4 are in CR2 or CR3. The prednisone response emerged as the only independent prognostic factor for survival in Cox regression analysis. Thus, two thirds of Ph+ childhood ALL cases can be identified early by PGR, which, when treated with intensive BFM chemotherapy, with or without BMT, have a significantly lower risk of treatment failure. With a median continuous complete remission (CCR) time of 4.1 years, pEFS for PGR is 0.55 (SE, 0.08) compared with 0.10 (SE, 0.07) in patients with PPR (P = .0001). PGR is also an indicator for treatment responsiveness and durable second remission after relapse, which in turn may provide a second chance for BMT.© 1998 by The American Society of Hematology.

Mutational landscape and patterns of clonal evolution in relapsed pediatric acute lymphoblastic leukemia.

Relapse of acute lymphoblastic leukemia (ALL) remains a leading cause of childhood death. Prior studies have shown clonal mutations at relapse often arise from relapse-fated subclones that exist at diagnosis. However, the genomic landscape, evolutionary trajectories and mutational mechanisms driving relapse are incompletely understood. In an analysis of 92 cases of relapsed childhood ALL, incorporating multimodal DNA and RNA sequencing, deep digital mutational tracking and xenografting to formally define clonal structure, we identify 50 significant targets of mutation with distinct patterns of mutational acquisition or enrichment. CREBBP, NOTCH1, and Ras signaling mutations rose from diagnosis subclones, whereas variants in NCOR2, USH2A and NT5C2 were exclusively observed at relapse. Evolutionary modeling and xenografting demonstrated that relapse-fated clones were minor (50%), major (27%) or multiclonal (18%) at diagnosis. Putative second leukemias, including those with lineage shift, were shown to most commonly represent relapse from an ancestral clone rather than a truly independent second primary leukemia. A subset of leukemias prone to repeated relapse exhibited hypermutation driven by at least three distinct mutational processes, resulting in heightened neoepitope burden and potential vulnerability to immunotherapy. Finally, relapse-driving sequence mutations were detected prior to relapse using deep digital PCR at levels comparable to orthogonal approaches to monitor levels of measurable residual disease. These results provide a genomic framework to anticipate and circumvent relapse by earlier detection and targeting of relapse-fated clones.

Clinico-epidemiological features and response in childhood acute lymphoblastic leukemia at regional cancer center of Northeast India.

Introduction:Acute lymphoblastic leukemia (ALL) comprises 19.3% of all childhood cancers in Northeast India.Methods:We analyzed clinicoepidemiological features and early response to the treatment of all the cases of childhood ALL (age <15 years) diagnosed and treated at Dr. B Borooah Cancer Institute over 1 year.Results:Of 52 eligible cases, 69% were male (male:female ratio of 2.2:1) and the mean age was 7.1 years. Thirty-three children (63%) had baseline white blood cell count ≥20 × 109/L. Precursor B-cell was most the common subtype seen in 61% of children. Seven cases (14%) had high-risk (HR) cytogenetics, with t (9,22) being the most common one. Male sex and HR cytogenetics were significantly associated with poor early responses.Conclusion:ALL is a common childhood malignancy with high cure rates. However, poor socioeconomic status and the presence of higher proportions of disease-related factors lead to poor outcome in this part of the country.

Curcumin Induces Apoptotic Cell Death via Inhibition of PI3-Kinase/AKT Pathway in B-Precursor Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is a significant cancer of children resulting from the clonal proliferation of lymphoid precursors with arrested maturation. Although chemotherapeutic approaches have been achieving successful remission for the majority of cases of childhood ALL, development of resistance to chemotherapy has been observed. Thus, new therapeutic approaches are required to improve patient's prognosis. Therefore, we investigated the anticancer potential of curcumin in ALL. We tested a panel of B-precursor ALL (B-Pre-ALL) cell lines with various translocations after treatment with different doses of curcumin. Curcumin suppresses the viability in a concentration-dependent manner in 697, REH, SupB15, and RS4;11 cells (doses from 0 to 80 μM). Curcumin induces apoptosis in B-Pre-ALL cell lines via activation of caspase-8 and truncation of BID. Curcumin treatment increased the ratio of Bax/Bcl-2 and resulted in a leaky mitochondrial membrane that led to the discharge of cytochrome c from the mitochondria to the cytoplasm, the activation of caspase 3 and the cleavage of PARP. Curcumin treatment of B-Pre-ALL cell lines induced a dephosphorylation of the constitutive phosphorylated AKT/PKB and a down-regulation of the expression of cIAP1, and XIAP. Moreover, curcumin mediates its anticancer activity by the generation of reactive oxygen species. Finally, the suboptimal doses of curcumin potentiated the anticancer activity of cisplatin. Altogether, these results suggest an important therapeutic role of curcumin, acting as a growth suppressor of B-Pre-ALL by apoptosis via inactivation of AKT/PKB and down-regulation of IAPs and activation of intrinsic apoptotic pathway via generation of Reactive Oxygen Species (ROS). Our interesting findings raise the possibility of considering curcumin as a potential therapeutic agent for the treatment of B-Pre-ALL.

Proteogenomics and Hi-C reveal transcriptional dysregulation in high hyperdiploid childhood acute lymphoblastic leukemia

Hyperdiploidy, i.e. gain of whole chromosomes, is one of the most common genetic features of childhood acute lymphoblastic leukemia (ALL), but its pathogenetic impact is poorly understood. Here, we report a proteogenomic analysis on matched datasets from genomic profiling, RNA-sequencing, and mass spectrometry-based analysis of >8,000 genes and proteins as well as Hi-C of primary patient samples from hyperdiploid and ETV6/RUNX1-positive pediatric ALL. We show that CTCF and cohesin, which are master regulators of chromatin architecture, display low expression in hyperdiploid ALL. In line with this, a general genome-wide dysregulation of gene expression in relation to topologically associating domain (TAD) borders were seen in the hyperdiploid group. Furthermore, Hi-C of a limited number of hyperdiploid childhood ALL cases revealed that 2/4 cases displayed a clear loss of TAD boundary strength and 3/4 showed reduced insulation at TAD borders, with putative leukemogenic effects.

A genetic variant in miR-100 is a protective factor of childhood acute lymphoblastic leukemia.

BackgroundIn the past decade, miR‐100, miR‐146a, and miR‐210 were reported to be dysregulated in childhood acute lymphoblastic leukemia (ALL). However, effects of genetic variants in these three microRNAs have not been investigated in Chinese population.MethodsIn this study, we conducted a case‐control study to evaluate the relationship between genetic variants in miR‐100, miR‐146a, and miR‐210 and the risk of childhood ALL in Chinese population. Subsequently, plasma expression level of miR‐100 was also detected.ResultWe found that subjects carrying mutant homozygous TT genotype of miR‐100 rs543412 had a statistically significantly decreased risk of childhood ALL (adjusted odds ratio [OR] = 0.73, 95% confidence interval [CI] = 0.55‐0.97, P = 0.029). This protective effect was also observed among subjects whose parents were ever drinkers (adjusted OR = 0.53, 95% CI = 0.29‐0.94), or whose living house were ever painted (adjusted OR = 0.57, 95% CI = 0.34‐0.94). Besides, rs543412 variant homozygous TT had a significantly protective role in patients with childhood B‐ALL. Finally, we found that expression level of miR‐100 in plasma of childhood ALL cases was significantly higher than that of noncancer controls.ConclusionOur study suggested that there was significant association between the polymorphisms in miR‐100 (rs543412) and decreased susceptibility to childhood ALL.

Leukocyte-associated immunoglobulin-like receptor-1, T-cell leukemia/lymphoma protein-1, and nuclear factor κB expression in childhood acute lymphoblastic leukemia

Background Immune regulation is crucial for the pathogenesis of childhood acute lymphoblastic leukemia (ALL). Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is an immune regulator, expressed by T and B immune cells. T-cell leukemia/lymphoma protein-1 (TCL1) is a coactivator of α-serine/threonine-protein kinase, and when dysregulated, it causes lymphomagenesis and cancer progression. Nuclear factor-κB (NF-κB) is a transcription factor that regulates genes involved in immune responses. This study aimed to study the expression of LAIR-1, TCL1, and NF-κB on blasts in childhood ALL with evaluation of their prognostic value.Patients and methods This descriptive cross-sectional study was conducted on 60 newly diagnosed childhood ALL cases. They were divided into group 1 (n=47) of B-cell ALL and group 2 (n=13) of T-cell ALL. The expression pattern of LAIR-1, TCL1, and NF-κB on blasts was assessed using flow cytometry.Results Correlation studies in total patients with ALL (n=60) revealed significant positive correlation between percentage of blasts expressing LAIR-1% and total leukocytic count (r=0.262, P=0.043) and percentage of blasts infiltrating the peripheral blood (r=0.292, P=0.025). A significant positive correlation of LAIR-1 mean fluorescence intensity with percentage of blasts expressing CD13 (r=0.293, P=0.026) and CD33 (r=0.373, P=0.004) was found. In group 2, a significant positive correlation was seen between percentage of blasts expressing TCL1 and LAIR-1 mean fluorescence intensity (r=0.566, P=0.044). According to Cox regression analysis, the percentage of blasts expressing NF-κB was seen to significantly increase the risk of death, in childhood ALL (hazard ratio=1.085, 95% confidence interval=1.02–1.16, P=0.01).Conclusion LAIR-1 expression carries the potentiality of being a bad prognostic marker in childhood ALL. A relation is suggested between the expression of both LAIR-1 and TCL1 on blasts. NF-κB expression might negatively influence the survival rate in childhood ALL.

A Comprehensive FISH- and Array-Based Diagnostic Screeningstrategy for the Assessment of Clinically Relevant Genetic Parameters: A Prospective Analysis of 205 Childhood ALL Cases

Background: The detailed definition of causative genomic alterations is not only an indispensable prerequisite for the predictive and prognostic subdivision of childhood acute lymphoblastic leukemia (ALL) but increasingly also one, on which individualized treatment approaches will be based on. Apart from the already well-established genetic categories, the recent identification of several new classes of potentially relevant alterations together with the increasing availability of novel therapeutic options therefore necessitates a diagnostic workflow that is able to satisfy the ensuing clinical needs in a comprehensive manner. The two most interesting changes in this context are the therapeutically targetable recurrent but rare and heterogeneous tyrosine kinase and JAK2 pathway-activating (TKA) gene fusions and the more elusive cohort of apparently relapse risk-prone cases with hitherto only vaguely defined combinations of gene region-specific copy number alterations (CNA). Despite the availability of a multitude of applicable techniques, the fast and cost-efficient identification of the entire expectable abnormality patterns still remains a challenge, especially if one needs to perform the diagnostic work-up on a single case basis. We previously proposed that these diagnostic requirements could be covered best with a systematic hierarchical FISH screening approach for the identification of gene fusions together with array (combined SNP and non-polymorphic probes) analyses of genome-wide quantitative and qualitative large- and small-scale copy number aberrations (CNAs).Material and Methods: Since June 2015 we have therefore evaluated the feasibility of such a workflow in a prospective manner and screened so far 205 patients (i.e. 184 with B- and 21 with T-ALL, including 18 relapses with 14 diagnostic/relapse pairs) that were consecutively enrolled in the Austrian AIEOP-BFM 2009 treatment study. Cytogenetic preparations served as backup, since metaphase spreads were used for further FISH clarification of otherwise unresolvable complex rearrangements or ploidy patterns if deemed necessary. All identified gene fusions were subsequently validated with single or multiplex RT-PCR analyses.Results: FISH screening was positive in 90% (184/205) of cases and provided already the most essential diagnostic clues. CNAs were present in all T-ALL and 97% (179/184) of B-ALL cases, including 13 with an IKZF1pluspattern and three with ERG deletions, which both will be used das stratifying markers in the upcoming treatment trial. Taken together, our screening strategy allowed the unambiguous classification of the vast majority of B-ALLs: 70 hyperdiploid, 3 hypodiploid, 35 ETV6-RUNX1, 6 KMT2A-rearranged, 8 TCF3-PBX1, 2 BCR-ABL1, 4 dic(9;20), 5 iAMP21, 8 IGH-rearranged, 6 P2RY8-CRLF2, 3 PAX5-rearranged, 3 ZNF384-rearranged, 2 ETV6-rearranged, 3 TKA fusion-positive and 10 so called “B-other” cases without any apparent specific abnormality. RNA-sequencing analyses of these ten cases revealed that seven of them harbored a DUX4 gene rearrangement.Conclusions: Apart from its proven practical diagnostic value, our combined FISH/array approach has also several additional advantages, especially if one considers the amount of achievable information. Both procedures require only little amount of material and are highly standardized, reproducible and robust technologies. Moreover, arrays deliver DNA-sequence-based data, the coordinates of which can be efficiently stored, processed and analyzed. As such, they not only serve as a pure diagnostic tool but also as a valuable discovery platform. DisclosuresNo relevant conflicts of interest to declare.

PDGFRB mutation and tyrosine kinase inhibitor resistance in Ph-like acute lymphoblastic leukemia

AbstractPhiladelphia chromosome (Ph)-like acute lymphoblastic leukemia (ALL) comprises ∼10% to 15% of childhood ALL cases, many of which respond exquisitely to tyrosine kinase inhibitors (TKIs), for example, imatinib in PDGFRB-rearranged ALL. However, some cases developed drug resistance to TKIs and the mechanisms are poorly understood. In this study, we identified a novel PDGFRB fusion gene, namely AGGF1-PDGFRB, and functionally characterized its oncogenic potential in vitro. Further genomic profiling of longitudinally collected samples during treatment revealed the emergence of a mutation, PDGFRBC843G, which directly conferred resistance to all generations of ABL TKIs, including imatinib, dasatinib, nilotinib, and ponatinib. PDGFRB-mutant leukemia cells are highly sensitive to multitarget kinase inhibitor CHZ868, suggesting potential therapeutic options for some patients resistant to ABL TKIs. In summary, we describe a complex clonal evolution pattern in Ph-like ALL and identified a novel PDGFRB point mutation that drives leukemia relapse after ABL TKI treatment.