Biomarkers In Acute Lymphoblastic Leukemia Research Articles

A new insight updates in diagnosis and management of acute lymphoblastic leukemia, cytogenetics, immunophenotyping, and proteomic profile

Acute lymphoblastic leukemia (ALL) is a severely invasive hematological cancer that results from the rapid increase and accumulation of lymphoid blasts in the blood, bone marrow (BM), and other organs. The pervasiveness of ALL has made it the prevalent childhood acute leukemia making up approximately 80% of leukemia in children and about 20% in adults. Due to the prevalence of ALL, more accurate diagnosis and prognostic methods are required. Proteome analysis uses a variety of analytical techniques, including protein sequencing, structural or expression proteomics, protein modification, sub-cellular protein localization, protein-protein interactions, and biological functional proteomics. Analysis of cell-signaling pathways and activation/deactivation are crucial to follow up the development, remission, or relapse of ALL. As a result, this review emphasizes cytogenetics and immunophenotyping while also highlighting the proteomic profile, clinical symptoms, diagnosis, and management of ALL. Also, it evaluates the procedures and techniques for the testing of bodily fluids (peripheral blood, cerebrospinal fluid, and BM) from ALL patients at various stages of disease, as well as the use of proteomic platforms in discovering sensitive and specific biomarkers for ALL.

NLR, PLR, and HPR as novel diagnostic biomarkers for Acute Lymphoblastic Leukemia

NLR, PLR, and HPR as novel diagnostic biomarkers for Acute Lymphoblastic Leukemia

Nanopore sequencing of clonal IGH rearrangements in cell-free DNA as a biomarker for acute lymphoblastic leukemia.

Acute Lymphoblastic Leukemia (ALL) is the most common pediatric cancer, and patients with relapsed ALL have a poor prognosis. Detection of ALL blasts remaining at the end of treatment, or minimal residual disease (MRD), and spread of ALL into the central nervous system (CNS) have prognostic importance in ALL. Current methods to detect MRD and CNS disease in ALL rely on the presence of ALL blasts in patient samples. Cell-free DNA, or small fragments of DNA released by cancer cells into patient biofluids, has emerged as a robust and sensitive biomarker to assess cancer burden, although cfDNA analysis has not previously been applied to ALL. We present a simple and rapid workflow based on NanoporeMinION sequencing of PCR amplified B cell-specific rearrangement of the (IGH) locus in cfDNA from B-ALL patient samples. A cohort of 5 pediatric B-ALL patient samples was chosen for the study based on the MRD and CNS disease status. Quantitation of IGH-variable sequences in cfDNA allowed us to detect clonal heterogeneity and track the response of individual B-ALL clones throughout treatment. cfDNA was detected in patient biofluids with clinical diagnoses of MRD and CNS disease, and leukemic clones could be detected even when diagnostic cell-count thresholds for MRD were not met. These data suggest that cfDNA assays may be useful in detecting the presence of ALL in the patient, even when blasts are not physically present in the biofluid sample. The Nanopore IGH detection workflow to monitor cell-free DNA is a simple, rapid, and inexpensive assay that may ultimately serve as a valuable complement to traditional clinical diagnostic approaches for ALL.

Prospective quantitative gene expression analysis of kallikrein-related peptidase KLK10 as a diagnostic biomarker for childhood acute lymphoblastic leukemia.

BackgroundThe most common malignancy in children is acute lymphoblastic leukemia (ALL). This study aimed to explore KLK10 mRNA expression as a potential diagnostic biomarker for ALL in children and to examine the effect of chemotherapy on KLK10 mRNA expression following the induction and after three months of receiving chemotherapy.MethodsIn this prospective study, total RNA was extracted from blood samples of 23 pediatric ALL patients on diagnosis, after one month and three months of receiving chemotherapy. Healthy pediatric volunteers (n = 12) were selected as control individuals. After cDNA synthesis, KLK10 mRNA gene expression levels were quantified using quantitative real-time PCR (qRT-PCR).ResultsKLK10 mRNA expression levels were significantly decreased in leukemic cells compared to their levels in cells of normal blood samples (p = 0.0001). KLK10 expression levels in ALL patients after one month and three months of receiving chemotherapy decreased compared to normal blood samples (p < 0.0001 and p = 0.0175 respectively). The expression level of KLK10 mRNA in ALL patients after one month of chemotherapy was decreased compared to their level on diagnosis (p = 0.4413). KLK10 mRNA expression levels in ALL patients after three months of chemotherapy were increased compared to their level on diagnosis (p = 0.0602). The ROC curve illustrated that KLK10 mRNA expression could very efficiently discriminate ALL patients from normal counterparts (AUC=0.886, 95% CI [0.7720–1.000], SE = 0.0582, p = 0.0004).ConclusionKLK10 mRNA expression could serve as a potential diagnostic molecular biomarker for ALL in children.

Fabrication of Au/Fe3O4/RGO based aptasensor for measurement of miRNA-128, a biomarker for acute lymphoblastic leukemia (ALL).

Due to their high sensitivity, simplicity, portability, self‐contained, and low cost, the development of electrochemical biosensors is a beneficial way to diagnose and anticipate many types of cancers. An electrochemical nanocomposite‐based aptasensor is fabricated for the determination of miRNA‐128 concentration as the acute lymphoblastic leukemia (ALL) biomarker for the first time. The aptamer chains were immobilized on the surface of the glassy carbon electrode (GCE) through gold nanoparticles/magnetite/reduced graphene oxide (AuNPs/Fe3O4/RGO). Fast Fourier transform infrared (FTIR), X‐ray diffraction (XRD), vibrating sample magnetometer (VSM), and transmission electron microscopy (TEM) were used to characterize synthesized nanomaterials. Cyclic voltammetry (CV), square wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS) were used to characterize the modified GCE in both label‐free and labeled methods. The results indicate that the modified working electrode has high selectivity and for miRNA‐128 over other biomolecules. The hexacyanoferrate redox system typically operated at around 0.3 V (vs. Ag/AgCl), and the methylene blue redox system ran at about 0 V, were used as an electrochemical probe. The detection limit and linear detection range for hexacyanoferrate and methylene blue are 0.05346 fM, 0.1–0.9 fM, and 0.005483 fM, 0.01–0.09 fM, respectively. The stability and diffusion control analyses were performed as well. In both label‐free and labeled methods, the modified electron showed high selectivity for miRNA‐128. The use of methylene blue as a safer redox mediator caused miRNA‐128 to be detected with greater accuracy at low potentials in PBS media. The findings also show the substantial improvement in detection limit and linearity by using reduced graphene oxide‐magnetite‐gold nanoparticles that can be verified by comparing with previous studies on the detection of other miRNAs.

Serum MicroRNAs: -28-3p, -31-5p, -378a-3p, and -382-5p as novel potential biomarkers in acute lymphoblastic leukemia

IntroductionAcute lymphoblastic leukemia (ALL) is the most common malignancy in children and accounts for 20% of acute leukemia in adults. Recently, microRNAs (miRNAs) have been described as important molecules in hematologic malignancies. This study aimed to evaluate the expression levels of miR-28-3p, miR-31-5p, miR-378a-3p, miR-382-5p in patients with ALL. Materials and methodsIn this study, 21 patients with ALL (before and after treatment) and 21 healthy people were evaluated. After directly synthesizing cDNA from serum, miR-28-3p, miR-31-5p, miR-378a-3p, miR-382-5p expression were measured using the qRT-PCR method. Data were analyzed using SPSS 20, Genex 6.1, and GraphPad Prism8 software. ResultsThe expression levels of miR-378a-3p and miR-31-5p in patients with newly diagnosed ALL were significantly higher than in healthy individuals (P = 0.001, P = 0.004), and decreased significantly after treatment. (P = 0.04, P = 0.03). The expression of miR-28-3p and miR-382-5p in patients with newly diagnosed ALL were significantly lower than healthy individuals (P = 0.09, P = 0.01) and were increased significantly after treatment (P = 0.001, P = 0.006). ConclusionOur results indicated that miR-28-3p, miR-31-5p, miR-378a-3p, miR-382-5p may have a potential role in the pathogenesis of the disease; and also could be considered as a diagnostic marker and therapeutic target in ALL in future studies.

Circulating miR-146a expression as a non-invasive predictive biomarker for acute lymphoblastic leukemia

Dysregulation of non-coding microRNAs during the course of tumor development, invasion and/or progression to the distant organs, makes them a promising candidate marker for the diagnosis of cancer and associated malignancies. This exploratory study aims at evaluating the usefulness of plasma concentration of circulating mir-146a as a non-invasive biomarker for acute lymphoblastic leukemia (ALL). Total RNA including miRNA was isolated from 110 plasma samples of patients (n = 66), healthy controls (n = 24) and follow up (n = 20) cases and reverse transcribed. Relative concentrations were assessed using real-time quantitative PCR and fold-change was calculated by 2−ΔΔCt method. Finally, relative concentrations were correlated to clinicopathological factors. Patients (n = 66) were analyzed to determine fold expression of miR-146a in plasma samples of ALL. Before chemotherapy, pediatric (n = 42) and adult (n = 24) showed overexpression of miR-146a compared with healthy controls (P < 0.0001). There was no effect of age and gender on mir-146a expression in plasma. mirR-146a expression was independent of clinical and hematological features. Moreover, miR-146a levels in plasma of paired samples (n = 20) after treatment showed significant decrease in expression (P < 0.001). Expression of plasma miR-146a may be utilized as non-invasive marker to diagnose and predict prognosis in pediatric and adult patients with ALL. Moreover predicted targets may be utilized for ALL therapy in future.

MiR-181a and -b expression in acute lymphoblastic leukemia and its correlation with acute graft-versus-host disease after hematopoietic stem cell transplantation, COVID-19 and torque teno viruses.

Acute lymphoblastic leukemia (ALL), a malignant transformation and proliferation of the lymphoid line of blood cells, is characterized by chromosomal abnormalities and genetic changes. The purpose of this research was the evaluation of expression level of miR-181a and -b in patients with ALL compared to the control group. Furthermore, we examined their expression level in hematopoietic stem-cell transplantation (HSCT) patients who developed acute graft-versus-host disease (aGVHD) in comparison with those without aGVHD and explore the relationship between their expression level and cytogenetic abnormalities. In this cross-sectional study, 76 newly diagnosed adult De novo ALL patients were enrolled who were admitted to our referral hospital. All patients received standard chemotherapy, consisting of daunorubicin. A total of 37 patients underwent HSCT from the related human leukocyte antigen-matched donors. ALL patients have been diagnosed with the coronavirus disease 2019 (COVID-19) and Torque teno viruses (TTVs). We assessed the expression levels of miR-181a and -b in the peripheral blood sample of ALL patients at the time of diagnosis prior to chemotherapy, and healthy matched individuals by RT–PCR. TTVs and COVID-19 load were also determined via RT–PCR. In conclusion, the expression level of miR-181a and -b were significantly higher in ALL patients than healthy controls and also increased in patients who developed aGVHD in comparison with those without aGVHD. MiR-181a and -b can be a useful biomarker in ALL and a useful indicator of aGVHD. The expression level of miR-181a in ALL patients with COVID-19 is significantly up-regulated, while it is reduced in these patients with TTV.

Epigenetic Alterations of DLL4 and Hes5 in Acute Lymphoblastic Leukemia (ALL)

Acute lymphoblastic leukemia (ALL) is a hematologic condition with more than a quarter of pediatric cancers. Aberrant promoter methylation of Notch pathway genes causes the deactivation of TSGs. The pathway is also considered a crucial factor in the pathogenesis of ALL due to its active involvement in B and T cell development. Hypermethylation of Notch pathway genes has been reported previously. In this study, the promoter methylation frequency of genes DLL4 and Hes5 of the Notch pathway were studied using methylation-specific PCR in 30 pediatric ALL blood samples against 10 healthy controls. The objective of the study was to find the subtype-specific diagnostic biomarker for ALL. Hypermethylation frequency of DLL4 in pre-B ALL and T-ALL samples was found to be 84.21% and 100%, respectively. Whereas, Hes5 showed 100% mixed methylation in both diseased and control samples. The results predicted the possible epigenetic changes of Notch pathway and the possible role of DLL4 as a diagnostic biomarker of ALL.

Rs4846049 Polymorphism at the 3'-UTR of MTHFR Gene: Association with Susceptibility to Childhood Acute Lymphoblastic Leukemia.

Background Accumulating evidence has suggested the polymorphisms of methylenetetrahydrofolate reductase (MTHFR) were associated with susceptibility to childhood acute lymphoblastic leukemia (ALL). However, the known conclusions of currently known polymorphic loci (677 C > T and 1298 A > C) remain controversial. This study was to investigate new genetic biomarkers for ALL by analyzing the MTHFR polymorphisms at the 3′-untranslated region, which is a location bound by miRNAs. Methods Polymorphisms of rs4846049 (miR-555 binding) were assessed by PCR amplification and direct sequencing in 110 ALL patients and 105 healthy controls. The relative expression of MTHFR was detected by qRT-PCR. Results Overall, genotype distribution or allele carrier frequencies were not significantly different between patients with ALL and healthy controls (P > 0.05). Subgroup analysis results showed that T allele (OR = 0.134, 95% CI = 0.028–0.639; P=0.005) or genotypes with T allele (TT + GT) (OR = 0.133, 95% CI: 0.024–0.727; P=0.017) may be a protective factor for ALL susceptibility in patients with age >8 years. This conclusion was also true for the group only focusing on the precursor B-cell ALL patients. Furthermore, karyotype abnormality was more commonly observed in patients with the GG genotype (56.0%) compared to carriers of TT (0%) or GT (40.6%) genotypes, while c-myc break frequency was significantly higher in TT carriers (33%) than that of patients with GT (3.1%) or GG (0%) genotypes. PCR analysis showed patients carrying the GG genotype of rs4846049 exhibited the reduced mRNA expression of MTHFR. Conclusion MTHFR rs4846049 polymorphism may be associated with increased risk of childhood with ALL and MTHFR mRNA expression.

Critical Roles of Non-coding RNAs in Acute Lymphoblastic Leukemia

: MicroRNA (miRNA) is one of the non-coding RNA (ncRNA) molecules with 21 - 25-nucleotide length, playing an important role in gene control by transcriptional gene regulation, chromatin remodeling, genetic imprinting, and translation initiation. The deregulation of ncRNA can lead to several hematopoietic malignancies such as acute lymphoblastic leukemia (ALL). The study aimed to draw the crucial features of miRNA in the pathogenesis of ALL. The findings showed that miRNA expression changes in ALL are typical and involve several signaling pathways. The variations in miRNA gene expression can lead to the incomplete expression of oncoprotein or tumor suppressor gene (TSG). It seems that ncRNAs play pivotal roles in the ALL pathogenesis. However, ncRNAs might be interested as potential diagnostic and prognostic biomarkers in ALL.

Aberrant methylation of protocadherin 17 and its prognostic value in pediatric acute lymphoblastic leukemia.

The outcome of approximately 20% of patients with acute lymphoblastic leukemia (ALL) remains poor because of disease recurrence. We examined whether DNA methylation of cadherin superfamily genes is a useful biomarker for ALL relapse. We used Infinium Methylation 450K Arrays to assess genome-wide DNA methylation status. The methylation status of each individual gene was then determined by a combination of bisulfite restriction analysis and genome bisulfite sequencing. mRNA expression was evaluated by reverse-transcriptase PCR (RT-PCR) and quantitative real-time PCR. Cadherin superfamily genes including cadherin (CDH) 1, protocadherin (PCDH) 8, and PCDH17 were selected for analysis of methylation status. In 40 patient samples with B-cell precursor (BCP) ALL at diagnosis, the methylation frequencies of CDH1, PCDH8, and PCDH17 were 62.5, 55, and 30%, respectively. CDH1 and PCDH8 methylation was also detected in 80 and 20% of control bone marrow (BM) samples, respectively. On the contrary, PCDH17 was unmethylated in all control BM samples. There was a significant correlation between the methylation status of PCDH17 (but not CDH1 and PCDH8) and event-free survival or overall survival. Univariate and multivariate analyses showed that only PCDH17 methylation was associated with an increased risk for relapse and mortality in patients with BCP ALL. PCDH17 methylation at diagnosis was closely related to poor prognosis and thus could be used as a new biomarker to predict relapse in patients with BCP ALL.

Cyfip1 is downregulated in acute lymphoblastic leukemia and may be a potential biomarker in acute lymphoblastic leukemia.

The aim of the present study was to analyze the expression of Cyfip1 in acute lymphoblastic leukemia (ALL) and its correlations with clinical pathologic features. A total of 86 ALL samples and 32 normal peripheral blood lymphocyte (PBL) samples were enrolled in our study. mRNA expression of the Cyfip1 was assessed by real-time fluorescent relatively quantitative PCR, and Cyfip1 protein expression was evaluated by Western blot analysis. As a result, both mRNA and protein expression levels of Cyfip1 were significantly lower in ALL patients than those in the control samples (P = 0.025 and 0.000, respectively). Moreover, both mRNA and protein expression of both had an inverse relation with lymph node metastasis (P = 0.015 and 0.007, respectively), In conclusion, detecting mRNA and protein expression of Cyfip1 could provide clinically significant information relevant to diagnosis, progression, and treatment modalities for ALL, and Cyfip1 may serve as a potential biomarker for diagnosis and prognosis in ALL.

A Non-natural Nucleoside with Combined Therapeutic and Diagnostic Activities against Leukemia

Acute lymphoblastic leukemia (ALL) is the most common type of childhood cancer, presenting with approximately 5,000 new cases each year in the United States. An interesting enzyme implicated in this disease is terminal deoxynucleotidyl transferase (TdT), a specialized DNA polymerase involved in V(D)J recombination. TdT is an excellent biomarker for ALL as it is overexpressed in ~90% of ALL patients, and these higher levels correlate with a poor prognosis. These collective features make TdT an attractive target to design new selective anti-cancer agents against ALL. In this report, we evaluate the anti-leukemia activities of two non-natural nucleotides designated 5-nitroindolyl-2'-deoxynucleoside triphosphate (5-NITP) and 3-ethynyl-5-nitroindolyl-2'-deoxynucleoside triphosphate (3-Eth-5-NITP). Using purified TdT, we demonstrate that both non-natural nucleotides are efficiently utilized as TdT substrates. However, 3-Eth-5-NITP is poorly elongated, and this observation validates its activity as a chain-terminator for blunt-end DNA synthesis. Cell-based experiments validate that the corresponding non-natural nucleoside produces robust cytostatic and cytotoxic effects against leukemia cells that overexpress TdT. The strategic placement of the ethynyl moiety allows the incorporated nucleoside triphosphate to be selectively tagged with an azide-containing fluorophore via "click" chemistry. This reaction allows the extent of nucleotide incorporation to be quantified such that the anti-cancer effects of the corresponding non-natural nucleoside can be self-assessed. The applications of this novel nucleoside are discussed, focusing on its use as a "theranostic" agent that can improve the accuracy of dosing regimens and accelerate clinical decisions regarding therapeutic intervention.