AbstractBackgroundAcute Lymphoblastic Leukemia Research Articles

Clinical significance of serum CD155 levels in acute lymphoblastic leukemia patients

Abstract Background Acute lymphoblastic leukemia (ALL) is a hematologic malignancy characterized by poor outcomes in adults. Traditional diagnostic and prognostic markers take into account clinical data but also rely heavily on expensive and invasive tests such as bone marrow studies with cytogenetics. CD155 is an adhesion molecule that also modulates immune response in malignancy. Previous studies have shown that serum CD155 (sCD155) levels are higher in patients with malignancies, and have demonstrated a correlation between higher levels of sCD155 and poor outcomes. This study investigated the diagnostic and prognostic value of sCD155 in adult patients with acute lymphoblastic leukemia. Methodology 32 adult patients with acute lymphoblastic leukemia and 15 control patients were enrolled in this study. Clinical and outcome data were collected from patients and a blood sample from patients and controls was collected with ELISA testing for sCD155 levels. Results sCD155 was found to be significantly higher in patients than controls (P<0.05) and was significantly associated with bone marrow aspirate blast count. It was not associated with other clinical parameters including Sex, age, initial CBC parameter counts, presence of lymphadenopathy, presence of CNS infiltration, presence of Philadelphia chromosome, type of ALL, or risk stratification of ALL besides being associated with hepatosplenomegaly. ROC curve showed high sensitivity and specificity of sCD155 and Kaplan Meir curve showed a trend towards worse outcomes in patients with high sCD155 but it was not statistically significant. Conclusion sCD155 remains a promising marker for diagnosis of ALL but further studies are needed to confirm prognostic value.

Atopy manifestations in pediatric patients with acute lymphoblastic leukemia: correlation assessment with interleukin-4 (IL-4) and IgE level

BackgroundAcute lymphoblastic leukemia (ALL) is the most common type of cancer in the age range of under 15 years old and accounts for 25–30% of all childhood cancers. Although conventional chemotherapy regimens are used to improve the overall survival rate, it has been associated with some complications, amongst which allergic manifestations with unknown mechanisms are more common.MethodsOur study compared serum IgE and IL-4 concentration, as a hallmark of allergic responses in pediatric ALL patients before and after 6 months of intensive (high-dose) chemotherapy, to show whether changes in the level of these markers may be associated with atopy. Serum level of IL-4 and IgE was measured using enzyme-linked immunosorbent assay (ELISA) method.ResultsThe results showed that the level of IgE and IL-4 increased following chemotherapy in both ALL patients with and without atopy. In addition, post-chemotherapy treatment IgE and IL-4 levels were significantly elevated in patients with atopy compared to those without it. The difference between baseline and post-chemotherapy level of IgE and IL-4 was significantly higher in patients with atopy compared to those without it.ConclusionsTo the best of our knowledge, this is the first study that showed a connection between post-chemotherapy allergic manifestations in pediatric ALL patients and IL-4 and IgE level. Flow cytometry analysis of the T-helper 2 (Th2) lymphocytes and other allergy-related T cell subsets like Tc2 and Th9 as well as the study of the genetic variations in atopy-related genes like IL-4/IL-4R, IL-5, IL-9, IL-13, and high affinity FcεRI IgE receptor and also HLA genes is necessary to clearly define the underlying mechanism responsible for post-chemotherapy hypersensitivity reaction in pediatric ALL patients.

A case report of pediatric acute lymphoblastic leukemia with e8a2 BCR/ABL1 fusion transcript

BackgroundAcute lymphoblastic leukemia is the most common type of cancer in children. Most often it affects the age group between 2 and 5 years of age. Studies have shown an improvement in general survivability, more than 90% 5-year overall survival (OS). Current treatment protocols for acute lymphoblastic leukemia require verification of the presence of favorable and unfavorable genetic abnormalities, which help qualify patients to the appropriate risk group and select a more suitable treatment. The presence of the BCR/ABL1 fusion gene stratifies the patient into a high-risk group and requires special treatment with tyrosine kinase inhibitors (TKI). The three dominant mRNA transcripts are e1a2, e13a2, and e14a2. Nevertheless, cases of atypical BCR/ABL1 transcripts have also been reported.Case presentationThis paper presents the case of a pediatric patient with Ph + B-cell precursor acute lymphoblastic leukemia with rare atypical e8a2 BCR/ABL1 fusion transcript. Our patient achieved complete remission after 33 days of treatment. Molecular and cytogenetic studies in TP1 did not reveal the presence of the BCR/ABL1 transcript. The PCR-MRD test in TP1b was negative, the patient did not require hematopoietic stem cell transplantation.ConclusionGenetic evaluation of the bone marrow sample is crucial in the initial stage of the diagnosis. Fluorescent in situ hybridization and reverse transcriptase polymerase chain reaction with Sanger sequencing are the appropriate methods used in the detection of rare variants of BCR/ABL1 transcripts.

Serum Leptin Level in Children with Acute Lymphoblastic Leukemia

Abstract Background Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer .It is a disease of monoclonal proliferation of hematopoietic precursor cells of the lymphoid series within the bone marrow. Leptin is a peptide hormone which is predominantly produced by white adipose tissue. The main function of leptin in the human body is the regulation of energy expenditure and control of appetite .It is supposed to be involved in the pathogenesis of hematological malignancy through its proliferative, anti-apoptotic, and differentiating effects on hematopoietic neoplastic cells, in addition to its synergestic effect on vascular endothelial growth factor (VGEF) . Objectives To evaluate serum leptin level in children with acute lymphoblastic leukemia at diagnosis and at day 28 post induction chemotherapy,and to test the association between serum leptin levels and anthropometric measures of ALL patients and prognostic markers of ALL as age, gender, initial WBCS count, extramedullary infiltration to organs and CSF, cytogenetics, and response to treatment . Patients and Methods This is a case- control study performed at Clinical Pathology Department, Ain Shams University Hospitals. The study was conducted on 30 ALL pediatric patients admitted to Hematology & Oncology Unit, Pediatric Department, Ain Shams University Hospitals.The patients were evaluated at diagnosis before intake of chemotherapy and at day 28 post-induction chemotherapy, and 25 healthy controls matched with patients in age, sex, BMI. Results Serum leptin level is elevated in ALL patients at diagnosis as compared to controls .It has no significant relation with patients age, sex, BMI. It is elevated in patients who received cranial radiotherapy and those who relapsed. .However, it has no significant relation with clinical data (fever, lymphadenopathy and organomegaly) and laboratory characteristics (CBC, percentage of blasts infiltrating bone marrow and peripheral blood, immunophenotyping and cytogenetics) of ALL patients. Conclusion Data from our study showed that elevated leptin level in ALL patients can be considered a risk factor involved in ALL pathogenesis.It can be used as a biomarker for ALL diagnosis, a prognostic factor for ALL patients on chemotherapy, and a predicting factor for relapse.

The role of glutathione S-transferase omega gene polymorphisms in childhood acute lymphoblastic leukemia: a case-control study

BackgroundAcute lymphoblastic leukemia (ALL) is the most common childhood cancer. Glutathione-S-methyl transferase (GSTs) enzymes’ family is known to catalyze carcinogens detoxification. Overexpression of (GSTO) omega class was reported in cancer occurrence. The purpose of the study was to investigate the association of GSTO1*A140D (rs4925) and GSTO2*N142D (rs156697) polymorphisms with the susceptibility to childhood ALL and to evaluate their prognostic impact. Genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism technique in 96 Egyptian pediatric ALL patients and 99 controls.ResultsNo statistically significant different GSTO1*A140D genotype and allele distribution was observed among ALL cases and controls; however, a statistically significant different GSTO1*A140D genotype distribution was found between de novo ALL cases and controls [CC (37% vs. 56.6%), CA (47.8% vs. 40.4%), and AA (15.2% vs. 3.0%), respectively] (0.008). GSTO1*A140D variant genotypes’ frequency was significantly higher in de novo cases than in controls (63% vs. 43.4%) (0.028). The minor allele frequency (MAF) of GSTO1*A140D-A was significantly higher in de novo cases compared to controls (0.39 vs. 0.23) (0.005). Genotyping of GSTO2*N142D revealed a statistically significant difference of genotype distribution between ALL patients and controls [AA (26% vs. 36.3%), AG (62.5% vs. 61.6%), and GG (11.4% vs. 2.0%), respectively] (0.017) and between de novo ALL cases and controls [AA (37% vs. 36.3%), AG (45.7% vs. 61.6%), and GG (17.3% vs. 2.0%), respectively] (0.002). The MAF of GSTO2*N142D-G was significantly higher in ALL patients than in controls (0.42 vs. 0.32) (0.046). The high-risk ALL group had a higher frequency of GSTO1*A140D and GSTO2*N142D variant genotypes compared to corresponding wild genotypes and a higher frequency of combined polymorphisms compared to single polymorphisms and wild genotypes but with no statistically significant difference.ConclusionA statistically significant difference of GSTO1*A140D and GSTO2*N142D genotype distribution was detected between de novo ALL cases and controls. Compared to the control group, the MAF of GSTO1*A140D-A was overexpressed in de novo ALL cases and that of GSTO2*N142D-G was significantly higher in ALL patients. These findings suggest that the studied polymorphisms might play a significant role in the susceptibility to de novo childhood ALL in Egypt; however, GSTO1*A140D and/or GSTO2*N142D polymorphisms have no impact on ALL prognosis.

Expression of programmed death ligand-1 in B-cell acute lymphoblastic leukemia

Background Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy in the world. Patients with good prognostic factors have excellent survival rates with further improvement over the last decades. However, refractory ALL and relapsed ALL after hematopoietic stem cell transplantation are still associated with a poor prognosis. Immune checkpoints have gained attention in recent years in the field of oncology as a mechanism of cancer to evade immunity, but their status in B-ALL has yet to be investigated. Aim The aim was to assess programmed death ligand-1 (PDL-1) expression in newly diagnosed B-cell acute lymphoblastic leukemia (B-ALL). Materials and methods This study was directed on 45 patients with newly diagnosed B-ALL. Surface expression of PDL-1 on the blast cells was evaluated by multicolor flow cytometry. Results The study participants exhibited a mean PDL-1 expression of 16.28%, ranging from 8.5 to 70.45%. Conclusion This study has revealed that patients with newly diagnosed B-ALL could express PDL-1 to allow cancer cells to evade the immune system, demonstrating PD-1/PDL-1 as a universal target for therapy.

Differential network analysis and protein-protein interaction study reveals active protein modules in glucocorticoid resistance for infant acute lymphoblastic leukemia

BackgroundAcute lymphoblastic leukemia (ALL) is the most common type of cancer diagnosed in children and Glucocorticoids (GCs) form an essential component of the standard chemotherapy in most treatment regimens. The category of infant ALL patients carrying a translocation involving the mixed lineage leukemia (MLL) gene (gene KMT2A) is characterized by resistance to GCs and poor clinical outcome. Although some studies examined GC-resistance in infant ALL patients, the understanding of this phenomenon remains limited and impede the efforts to improve prognosis.MethodsThis study integrates differential co-expression (DC) and protein-protein interaction (PPI) networks to find active protein modules associated with GC-resistance in MLL-rearranged infant ALL patients. A network was constructed by linking differentially co-expressed gene pairs between GC-resistance and GC-sensitive samples and later integrated with PPI networks by keeping the links that are also present in the PPI network. The resulting network was decomposed into two sub-networks, specific to each phenotype. Finally, both sub-networks were clustered into modules using weighted gene co-expression network analysis (WGCNA) and further analyzed with functional enrichment analysis.ResultsThrough the integration of DC analysis and PPI network, four protein modules were found active under the GC-resistance phenotype but not under the GC-sensitive. Functional enrichment analysis revealed that these modules are related to proteasome, electron transport chain, tRNA-aminoacyl biosynthesis, and peroxisome signaling pathways. These findings are in accordance with previous findings related to GC-resistance in other hematological malignancies such as pediatric ALL.ConclusionsDifferential co-expression analysis is a promising approach to incorporate the dynamic context of gene expression profiles into the well-documented protein interaction networks. The approach allows the detection of relevant protein modules that are highly enriched with DC gene pairs. Functional enrichment analysis of detected protein modules generates new biological hypotheses and may help in explaining the GC-resistance in MLL-rearranged infant ALL patients.

Abstract LB-056: An integrated genomic, epigenetic, proteomic, and single cell analysis of pediatric B cell acute lymphoblastic leukemia to elucidate resistance mechanisms to CD19 CAR T cell therapy

Abstract Background Acute lymphoblastic leukemia (ALL) is the most common childhood cancer with a peak incidence at 3-5 years of age. Despite the improved survival rate of 90% for newly diagnosed children with ALL, the outcome for patients with relapsed disease is poor with a less than 30% overall survival. CD19 CAR T cell therapy has shown impressive response rates in relapsed/refractory disease. However, long-term survival analysis has shown that despite initial response rates exceeding 80%, durable response rates at one year are closer to 40%. Currently, little is known about molecular factors predicting durable response to CAR T therapy. We hypothesized that patients with CD19 CAR T therapy resistant ALL have a molecularly distinct disease compared to patients who respond to therapy, which can be identified in pre-treatment leukemia samples. Utilizing advanced genomic, epigenetic, proteomic, and single-cell techniques, we characterized the bone marrow of patients that were resistant or sensitive to therapy to identify mechanisms of resistance. Methods Patients enrolled in a phase I clinical trial at Seattle Children’s Hospital (PLAT-02) were categorized according to the durability of their response to CD19 CAR T therapy. Bone marrow aspirates from patients with leukemias resistant to therapy (4 pre-treatment with 2 paired post-treatment) were analyzed and compared to patients with therapy sensitive leukemias (5 pre-treatment). We performed bulk whole-exome sequencing and RNA-seq, single cell (sc) RNA-seq, scB cell receptor (BCR)-seq, methylation array, H3K27ac ChIP-seq, and ATAC-seq. Results Initial genomic analysis revealed a total of 5 previously reported recurrent hotspot mutations in ABL1, 2 x KRAS (Q61H), IKZF1, and EP300. RNA-seq analyses identified actionable fusions in 2 x ABL1, 2 x ETV6, 2 x ETV5, and 1x KMT2A with variable partners. Interestingly, a therapy-sensitive leukemia harbored a KMT2A-AFF1fusion that was previously shown to predispose patients treated with blinatumomab to leukemic plasticity and lineage switching. Additionally, we identified in-frame CREBBP-fusions in all leukemias that failed to achieve CD19 CAR T cell induced B cell aplasia. CREBBP perturbations have previously been associated with relapsed and refractory ALL. Integrated gene expression and epigenetic analyses identified several pathways associated with resistant disease. ATAC-seq and methylation data are being analyzed for lineage specification. Similarly, scRNA- and scBCR-seq data are being analyzed for the existence of mixed lineage and gene expression-based heterogeneity that may predict clonal selection under CAR T pressure. Conclusions This study establishes one of the most comprehensive approaches to genomic profiling for leukemia patient samples. Although our analysis is preliminary and sample number is small, in-depth analyses are highlighting crucial differences in leukemia that will allow improved prediction of responsiveness to CAR T therapy. Citation Format: Katherine E. Masih, Rebecca Gardner, Berkley E. Gryder, Justin Lack, Benjamin Z. Stanton, Ashley Wilson, Olivia Finney, Sivasish Sindiri, Young Song, Zachary Rae, Michael Kelly, Chaoyu Wang, Xinyu Wen, Adam Cheuk, Jun S. Wei, Michael Jensen, Rimas Orentas, Javed Khan. An integrated genomic, epigenetic, proteomic, and single cell analysis of pediatric B cell acute lymphoblastic leukemia to elucidate resistance mechanisms to CD19 CAR T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-056.

An improved advanced fragment analysis-based classification and risk stratification of pediatric acute lymphoblastic leukemia

BackgroundAcute lymphoblastic leukemia (ALL) contains cytogenetically distinct subtypes that respond differently to cytotoxic drugs. Therefore, subtype classification is important and indispensable in ALL diagnosis. In our previous study, we identified some marker genes in childhood ALL by means of microarray technology and, furthermore, detected the relative expression levels of 57 marker genes and built a comparatively convenient and cost-effective classifier with a prediction accuracy as high as 94% based on the advanced fragment analysis (AFA) technique.MethodsA more convenient improved AFA (iAFA) technique with one-step multiplex RT-PCR and an anti-contamination system was developed to detect 57 marker genes for ALL.ResultsThe iAFA assay is much easier and more convenient to perform than the previous AFA assay and has a prediction accuracy of 95.29% in ALL subtypes. The anti-contamination system could effectively prevent the occurrence of lab DNA contamination. We also showed that marker gene expression profiles in pediatric ALL revealed 2 subgroups with different outcomes. Most ALL patients (95.8%) had a good-risk genetic profile, and only 4.2% of ALL patients had a poor-risk genetic profile, which predicted an event-free survival (EFS) of 93.6 ± 1.3% vs 18.8 ± 9.8% at 5 years, respectively (P < 0.001).ConclusionsCompared to the previous AFA assay, the iAFA technique is more functional, time-saving and labor-saving. It could be a valuable clinical tool for the classification and risk stratification of pediatric ALL patients.

Genome-Wide Association Study of Susceptibility Loci for T-Cell Acute Lymphoblastic Leukemia in Children.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children and can arise in B or T lymphoid lineages. Although risk loci have been identified for B-ALL, the inherited basis of T-ALL is mostly unknown, with a particular paucity of genome-wide investigation of susceptibility variants in large patient cohorts. We performed a genome-wide association study (GWAS) in 1191 children with T-ALL and 12178 controls, with independent replication using 117 cases and 5518 controls. The associations were tested using an additive logistic regression model. Top risk variants were tested for effects on enhancer activity using luciferase assay. All statistical tests were two sided. A novel risk locus in the USP7 gene (rs74010351, odds ratio [OR] = 1.44, 95% confidence interval [CI] = 1.27 to 1.65, P = 4.51 × 10-8) reached genome-wide significance in the discovery cohort, with independent validation (OR = 1.51, 95% CI = 1.03 to 2.22, P = .04). The USP7 risk allele was overrepresented in individuals of African descent, thus contributing to the higher incidence of T-ALL in this race/ethnic group. Genetic changes in USP7 (germline variants or somatic mutations) were observed in 56.4% of T-ALL with TAL1 overexpression, statistically significantly higher than in any other subtypes. Functional analyses suggested this T-ALL risk allele is located in a putative cis-regulatory DNA element with negative effects on USP7 transcription. Finally, comprehensive comparison of 14 susceptibility loci in T- vs B-ALL pointed to distinctive etiology of these leukemias. These findings indicate strong associations between inherited genetic variation and T-ALL susceptibility in children and shed new light on the molecular etiology of ALL, particularly commonalities and differences in the biology of the two major subtypes (B- vs T-ALL).

Multidrug resistance gene 1 polymorphisms in pediatric patients with leukemia at a national referral hospital in Indonesia

Abstract Background Acute lymphoblastic leukemia (ALL) is the most prevalent cancer in the pediatric population. From 25% to 30% of patients with ALL will have a relapse that leads to death when they are teenagers. At Cipto Mangunkusumo Hospital, 40% of 126 pediatric patients with ALL relapsed from 2005 to 2011. A multiple variant of multidrug resistance gene 1 (MDR1) is C3435T, which can be used to understand the genetic basis of susceptibility to relapse. Objectives To identify the profile of MDR1 polymorphism in pediatric Indonesian patients with ALL. Methods We collected data from 44 patients with ALL who attended Cipto Mangunkusumo Hospital between January and June 2014. We investigated a silent C3435T polymorphism in MDR1 exon 26 with polymerase chain reaction- restriction fragment length polymorphism using MboI. Results There were 32 male and 12 female patient participants in this study. Eighteen patients were 1–3 years old and 26 were over 3 years. The mean age at 1–3 years was 2.4 ± 0.86, and over 3 years it was 6.3 ± 2.67 years. There were 27 patients with ALL in the standard risk group and 17 in the high risk group. We determined that the 25 samples from patients with ALL in the standard risk group were not digestible (allele T) and the 6 samples from patients with ALL in the high risk group were digestible (allele C). Conclusions The prevalence of the T allele was higher than that of the C allele in pediatric Indonesian patients with ALL.

Cluster of differentiation 97 as a biomarker for the detection of minimal residual disease in common acute lymphoblastic leukemia

Background Acute lymphoblastic leukemia (ALL) is a biologically heterogeneous disorder. Clinical parameters, immunophenotype, cytogenetic factors, and minimal residual disease (MRD) are among currently used factors in risk stratification and therapy determination of ALL patients. MRD is gaining importance nowadays for therapy efficacy, follow-up, and relapse risk estimation. Recent studies have highlighted potential markers that may improve the sensitivity of MRD detection by flow cytometry. Cluster of differentiation (CD) 97 is one of the markers that show overexpression in pediatric ALL. In this study, we aimed to assess the value of CD97 as a biomarker for MRD detection in pediatric ALL. Patients and methods This cohort study was conducted on 30 newly diagnosed patients with B-ALL. There were 16 male and 14 female patients with a mean age of 8.38±4.21. Twenty patients were in the low-risk group and 10 patients were in the high-risk group and were treated according to modified CCG 1991. A panel of monoclonal antibodies was used, with special emphasis on CD10, CD19, CD34, and CD97 at diagnosis and at day 14 postinduction of chemotherapy for MRD detection. Results The percentage of CD19/CD97, CD34/CD97, and CD10/CD97 at day 0 was 57.15±21.74, 57.73±21.20, and 57.87±20.77, whereas at day 14 it was 6.09±2.50, 10.67±8.89, and 5.97±2.44, respectively (P<0.001). CD97 was expressed in 81.5% of patients at diagnosis and was not detected at day 14 (P<0.001). One patient had blast counts more than 5% by light microscopy, whereas 29 patients had MRD more than 0.1 by flow cytometry at day 14 (P<0.001). Conclusion CD97 can be used for MRD tracing in pediatric ALL.

Increases in the numbers of cells with the phenotype of myeloid-derived suppressor and regulatory T cells in children with acute lymphoblastic leukemia

Abstract Background Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children. The precise mechanisms behind the relapse in this disease are not clearly known. One possible mechanism could be the accumulation of regulatory cells including myeloid-derived suppressor cells (MDSCs) and T regulatory cells (Tregs) which we and others have reported to mediate suppression of anti-tumor immune responses. Few previous studies investigated the roles of Treg cells in ALL, while no studies reported the emergence of MDSCs in ALL. Aim Therefore, we aimed to analyze the frequencies and phenotype of these cells in a group of Egyptian B-ALL pediatric patients (n=45). Materials and methods MDSCs were identified as Lin− HLA-DR− CD33+CD11b+; and Treg cells as CD4+CD25+CD127−/low using multiparametric flow cytometer. Results We found significant increases in the numbers of MDSCs and Treg cells in B-ALL patients as compared to healthy control volunteers. B-ALL patients showed significant increased numbers of MDSCs and Tregs before chemotherapy when compared to healthy volunteers. The numbers of MDSCs were more increased while the numbers of Treg cells were more decreased in patients during induction of chemotherapy. The highest increase in the numbers of MDSCs was observed in patients after induction of chemotherapy, while Treg cells showed the most significant decreased numbers after induction of chemotherapy. Conclusion Our results indicate that B-ALL patients harbor both MDSCs and Treg cells, opening a new avenue to investigate the mechanism mediating the emergence of these cells in larger numbers of patients at different treatment stages.

Neuron-specific enolase in cerebrospinal fluid as a neurochemical marker for brain damage in acute lymphoblastic leukemia

Background Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer with a cure rate approaching 80%. Neuron-specific enolase (NSE) is known to be a cell-specific isoenzyme of the glycolytic enzyme enolase. The expression of NSE is a late event in neural differentiation, thus making it a useful index of neural maturation. It is a highly specific marker for neurons and peripheral neuroendocrine cells. NSE in cerebrospinal fluid (CSF) is a reliable marker of neuronal damage. It is believed to signal for brain damage after hypoxic-ischemic and traumatic injury. Objective and methods This study aimed to measure CSF levels of NSE as a neurochemical marker for brain damage in response to chemotherapy at diagnosis and during induction using the enzyme-linked immunosorbent assay method. Thirty newly diagnosed ALL patients were enrolled in this study, comprising 19 male and 11 female patients. Results NSE level in the CSF samples increased from 9.3 ± 2.1 μg/l on day 0 (before the start of treatment) to 14.9 ± 1.85 μg/l on day 7 and then gradually decreased to 11.4 ± 1.46 μg/l on day 28. The increase in NSE levels in CSF on days 7 and 28 was considered highly significant when compared with those measured on day 0 (before induction therapy). Conclusion We concluded that the increase in NSE in CSF during induction therapy in children with ALL can be interpreted as an early sign of brain damage.

Safety of nalbuphine on neural tissues of rats and its efficacy in the treatment of acute herpetic pain in children with acute lymphoblastic leukemia

Background Acute lymphoblastic leukemia (ALL) has been previously shown to cause severe impairment in immunity, in turn making children more susceptible to viral infections, especially herpes zoster, which manifests with severe pain. Materials and methods The main purpose of this study was to experimentally evaluate the safety of nalbuphine on the neural tissues of rats treated with nalbuphine at doses of 0.5, 1, 2, 4, and 5 mg/kg injected intrathecally every alternate day for 14 days, and to determine the efficacy of caudal injection of nalbuphine with a minimal dose of oral paracetamol as an analgesic in pediatric patients with ALL suffering from acute herpetic pain. Results The results revealed that nalbuphine exerted no pathological changes in either the cerebellum or the spinal cord. However, the protective effect of nalbuphine with the minimal dose of paracetamol was associated with a significant analgesic effect in ALL children. Its analgesic effect was assessed by means of the Facial Pain Scale and the behavioral pain assessment scale, and motor block assessment was made on the basis of the Bromage score. Conclusion Caudal nalbuphine with a minimal dose of paracetamol has adequate analgesic effect on acute herpetic pain in pediatric patients with ALL, without any pathological changes in the cerebellum and spinal cord.

Expression of cassini, a murine gamma-satellite sequence conserved in evolution, is regulated in normal and malignant hematopoietic cells

BackgroundAcute lymphoblastic leukemia (ALL) cells treated with drugs can become drug-tolerant if co-cultured with protective stromal mouse embryonic fibroblasts (MEFs).ResultsWe performed transcriptional profiling on these stromal fibroblasts to investigate if they were affected by the presence of drug-treated ALL cells. These mitotically inactivated MEFs showed few changes in gene expression, but a family of sequences of which transcription is significantly increased was identified. A sequence related to this family, which we named cassini, was selected for further characterization. We found that cassini was highly upregulated in drug-treated ALL cells. Analysis of RNAs from different normal mouse tissues showed that cassini expression is highest in spleen and thymus, and can be further enhanced in these organs by exposure of mice to bacterial endotoxin. Heat shock, but not other types of stress, significantly induced the transcription of this locus in ALL cells. Transient overexpression of cassini in human 293 embryonic kidney cells did not increase the cytotoxic or cytostatic effects of chemotherapeutic drugs but provided some protection. Database searches revealed that sequences highly homologous to cassini are present in rodents, apicomplexans, flatworms and primates, indicating that they are conserved in evolution. Moreover, CASSINI RNA was induced in human ALL cells treated with vincristine. Surprisingly, cassini belongs to the previously reported murine family of γ-satellite/major satellite DNA sequences, which were not known to be present in other species.ConclusionsOur results show that the transcription of at least one member of these sequences is regulated, suggesting that this has a function in normal and transformed immune cells. Expression of these sequences may protect cells when they are exposed to specific stress stimuli.

Correlation of SRSF1 and PRMT1 expression with clinical status of pediatric acute lymphoblastic leukemia

BackgroundAcute lymphoblastic leukemia (ALL) is the most frequently-occurring malignant neoplasm in children, but the pathogenesis of the disease remains unclear. In a microarray assay using samples from 100 children with ALL, SFRS1 was found to be up-regulated. Serine/arginine-rich splicing factor 1 (SRSF1, also termed SF2/ASF), encoded by the SFRS1 gene, had been shown to be a pro-oncoprotein. Our previous study indicated that SRSF1 can be methylated by protein arginine methyltransferase 1 (PRMT1) in vitro; however, the biological function of SRSF1 and PRMT1 in pediatric ALL are presently unknown.MethodsMatched, newly diagnosed (ND), complete remission (CR) and relapse (RE) bone marrow samples from 57 patients were collected in order to evaluate the expression patterns of SRSF1 and PRMT1. The potential oncogenic mechanism of SRSF1 and PRMT1 in leukemogenesis was also investigated.ResultsWe identified significant up-regulation of SRSF1 and PRMT1 in the ND samples. Importantly, the expression of SRSF1 and PRMT1 returned to normal levels after CR, but rebounded in the RE samples. Our observation that SRSF1 could predict disease relapse was of particular interest, although the expression patterns of SRSF1 and PRMT1 were independent of the cytogenetic subtypes. In pre-B-cell lines, both SRSF1 and PRMT1 expression could be efficiently attenuated by the clinical chemotherapy agents arabinoside cytosine (Ara-c) or vincristine (VCR). Moreover, SRSF1 and PRMT1 were associated with each other in leukemia cells in vivo. Knock-down of SRSF1 resulted in an increase in early apoptosis, which could be further induced by chemotherapeutics.ConclusionsOur results indicate that SRSF1 serves as an anti-apoptotic factor and potentially contributes to leukemogenesis in pediatric ALL patients by cooperating with PRMT1.

Discovery and identification of potential biomarkers of pediatric Acute Lymphoblastic Leukemia

BackgroundAcute lymphoblastic leukemia (ALL) is a common form of cancer in children. Currently, bone marrow biopsy is used for diagnosis. Noninvasive biomarkers for the early diagnosis of pediatric ALL are urgently needed. The aim of this study was to discover potential protein biomarkers for pediatric ALL.MethodsNinety-four pediatric ALL patients and 84 controls were randomly divided into a "training" set (45 ALL patients, 34 healthy controls) and a test set (49 ALL patients, 30 healthy controls and 30 pediatric acute myeloid leukemia (AML) patients). Serum proteomic profiles were measured using surface-enhanced laser desorption/ionization-time-of-flight mass spectroscopy (SELDI-TOF-MS). A classification model was established by Biomarker Pattern Software (BPS). Candidate protein biomarkers were purified by HPLC, identified by LC-MS/MS and validated using ProteinChip immunoassays.ResultsA total of 7 protein peaks (9290 m/z, 7769 m/z, 15110 m/z, 7564 m/z, 4469 m/z, 8937 m/z, 8137 m/z) were found with differential expression levels in the sera of pediatric ALL patients and controls using SELDI-TOF-MS and then analyzed by BPS to construct a classification model in the "training" set. The sensitivity and specificity of the model were found to be 91.8%, and 90.0%, respectively, in the test set. Two candidate protein peaks (7769 and 9290 m/z) were found to be down-regulated in ALL patients, where these were identified as platelet factor 4 (PF4) and pro-platelet basic protein precursor (PBP). Two other candidate protein peaks (8137 and 8937 m/z) were found up-regulated in the sera of ALL patients, and these were identified as fragments of the complement component 3a (C3a).ConclusionPlatelet factor (PF4), connective tissue activating peptide III (CTAP-III) and two fragments of C3a may be potential protein biomarkers of pediatric ALL and used to distinguish pediatric ALL patients from healthy controls and pediatric AML patients. Further studies with additional populations or using pre-diagnostic sera are needed to confirm the importance of these findings as diagnostic markers of pediatric ALL.

Cytotoxic effect of 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) on childhood acute lymphoblastic leukemia (ALL) cells: implication for targeted therapy

BackgroundAcute lymphoblastic leukemia (ALL) is the most common hematological malignancy affecting children. Despite significant progress and success in the treatment of ALL, a significant number of children continue to relapse and for them, outcome remains poor. Therefore, the search for novel therapeutic approaches is warranted. The aim of this study was to investigate the AMP activated protein kinase (AMPK) as a potential target in childhood acute lymphoblastic leukemia (ALL) subtypes characterized by non-random translocation signature profiles. We evaluated the effects of the AMPK activator AICAR on cell growth, cell cycle regulators and apoptosis of various childhood ALL cells.ResultsWe found that treatment with AICAR inhibited cell proliferation, induced cell cycle arrest in G1-phase, and apoptosis in CCRF-CEM (T-ALL), NALM6 (Bp-ALL), REH (Bp-ALL, TEL/AML1) and SupB15 (Bp-ALL, BCR/ABL) cells. These effects were abolished by treatment with the adenosine kinase inhibitor 5'-iodotubericidin prior to addition of AICAR indicating that AICAR's cytotoxicity is mediated through AMPK activation. Moreover, we determined that growth inhibition exerted by AICAR was associated with activation of p38-MAPK and increased expression of the cell cycle regulators p27 and p53. We also demonstrated that AICAR mediated apoptosis through the mitochondrial pathway as revealed by the release of cytochrome C and cleavage of caspase 9. Additionally, AICAR treatment resulted in phosphorylation of Akt suggesting that activation of the PI3K/Akt pathway may represent a compensatory survival mechanism in response to apoptosis and/or cell cycle arrest. Combined treatment with AICAR and the mTOR inhibitor rapamycin resulted in additive anti-proliferative activity ALL cells.ConclusionAICAR-mediated AMPK activation was found to be a proficient cytotoxic agent in ALL cells and the mechanism of its anti-proliferative and apoptotic effect appear to be mediated via activation of p38-MAPK pathway, increased expression of cell cycle inhibitory proteins p27 and p53, and downstream effects on the mTOR pathway, hence exhibiting therapeutic potential as a molecular target for the treatment of childhood ALL. Therefore, activation of AMPK by AICAR represents a novel approach to targeted therapy, and suggests a role for AICAR in combination therapy with inhibitors of the PI3K/Akt/mTOR pathways for the treatment of childhood in ALL.