We are currently assisting at the explosion of the epitranscriptomics, which studies the functional role of chemical modifications into RNA molecules. Among more than 100 RNA modifications, the N6-methyladenosine (m6A), in particular, has attracted the interest of researchers all around the world. m6A is the most abundant internal chemical modification in mRNA and it can control any aspect of mRNA post-transcriptional regulation. m6A is installed by “writers”, removed by “erasers”, and recognized by “readers”, thus, it can be compared to the reversible and dynamic epigenetic modifications in histones and DNA. Given its fundamental role in determining the way mRNAs are expressed, it comes as no surprise that alterations to m6A modifications have a deep impact in cell differentiation, normal development and human diseases. Here, we review the proteins involved in m6A modification in mammals, m6A role in gene expression and its contribution to cancer development. In particular, we will focus on AML that, among first, has indicated how alteration in m6A modification can disrupt normal cellular differentiation and lead to cancer.

Acute lymphoblastic leukaemia is currently treated with bacterial L-Asparaginase; however, its side effects bring up the special need for improved and efficient novel enzymes development. Previously, we obtained low anti-asparaginase antibody production and high serum enzyme half-life in mice with the P40S/S206C mutant; however, its specific activity was significantly reduced. Thus, our aim was to test single mutants, S206C and P40S, for in vitro and in vivo assays. Our results showed that the drop in specific activity was caused by P40S substitution. Besides, our single mutants were highly stable in biological environment simulation, unlike double mutant P40S/S206C. The in vitro cell viability assay demonstrated that mutant enzymes have higher cytotoxic effect than WT on T-cells derived ALL and on some solid cancer cell lines. The in vivo assays were performed in mice to identify toxicological effects, to evoke immunological response and to study the enzymes pharmacokinetics. From these tests, none of the enzymes was toxic; however, S206C evoked lower physiological changes and immune responses. In relation to pharmacokinetic profile, S206C has two-fold higher activity than WT and P40S two hours after injection. In conclusion, we present bioengineered E. coli asparaginases with high specific enzyme activity and lesser side effects.

Background: Breast cancer and other neoplasms, such as acute lymphocytic leukaemia, are a severe hazard to public health globally. L-glutamine-degrading enzymes are required for the treatment of malignancies such as acute lymphocytic leukemia and breast cancer. L-glutaminase, an amidohydrolase, has been shown to be an effective chemotherapeutic tool for treating a variety of malignancies. The aim of the study: Molecular characterization is used in the study and production of bacterial L- glutaminase as an anticancer agent from a variety of Egyptian soil locales. The type of the study: Screening experimental study. Methodology: In the current study, many bacterial isolates were tested on mineral L-glutamine agar selective medium (MGA) to evaluate if they generated L-glutaminase. The most effective positive bacterial isolates that produce L-glutaminase, however, were discovered by morphological and biochemical analysis. The primary positive isolate producing L-glutaminase was also identified utilizing molecular detection by 16S rRNA sequencing homology technique. The properties of bacteria-produced L- glutaminase were investigated. The anticancer activity was assessed with an MTT assay. Results: Sole bacterial isolates that only needed L-glutamine for metabolic nitrogen grew well on MGA. The optimal conditions for producing positive bacterial isolates were pH 7.4 and 37 ̊C. The morphological and biochemical investigations of soil samples from diverse soil conditions in Egypt revealed that Bacillus subtilis subsp. niger (ATCC 9372) was the primary positive bacterial isolate producing L-glutaminase. At a pH of 7.4 and a temperature of 37 °C, the activators KCL, ZnSO4, FeSO4, K2HPO4, and MgSO4 offered ideal conditions for defining L-glutaminase production. L-glutaminase from Bacillus subtilis was detected to be a highly bioavailable and efficient anticancer treatment. The original yield [productivity] from MGA was 6.3 U/ml, but invitro coupled transcription and translation production raised it to 89± 1 U/ml. L-glutaminase had a molecular mass of approximately 36 kDa. The purification of L-glutaminase resulted total activity of 21,883 ± 15.67 (U), specific activity of 379.14 ± 7.35 (U/mg of protein), and purification fold of 1.82 ± 3.06. The enzymatic activators Ni2+ and Mg 2+ increased L-glutaminase activity by 20%, 27%, respectively. The compound showed significant anticancer activity (IC50 = 38.72, 8.6, 6.41, 19.02, 49.83, and 9.45 μg/ml) against cancer cell lines from the liver (HepG-2), colon (HCT- 116), breast (MCF-7), lung (A-549), lymphocytic (CCL-120), and cervical (Hela). The kinetic parameters Km and Vmax were 15.2^ 10 -3 M and 119.86 μmol/ml/min, indicating a higher affinity for the substrate. The enzyme demonstrated high efficacy at pH 6.5 and temperature 35. The enzyme was thermostable at 58 ̊C for 1 hour. Conclusion: Acute lymphocytic leukemia and breast cancer are two instances of auxotrophic malignancies for which L-glutamine is the sole metabolic source, and Bacillus subtilis- derived L-glutaminase was a suitable enzyme in their treatment.

Background: Breast cancer and other neoplasms like acute lymphocytic leukemia pose a serious threat to public health worldwide. Treatment for malignancies including acute lymphocytic leukemia and breast cancer requires enzymes that break down L-glutamine. An amidohydrolase called L-glutaminase can be an effective chemotherapeutic tool for treating various cancers. Methods: On mineral L-glutamine agar selective medium (MGA), a few bacterial isolates were examined in the current investigation to see if they produced L-glutaminase. However, the most effective positive bacterial isolates generating L-glutaminase were identified using morphological and biochemical testing. Molecular detection using the direct Southern blotting approach was also used to determine the main positive isolate that produces L-glutaminase. L-glutaminase synthesis by bacteria was evaluated for its characteristics. Using an MTT test, the anticancer activity was evaluated. Results: exclusive bacterial isolates that used L-glutamine as their sole source of metabolic nitrogen exhibited favorable growth on MGA. PH 7.4 and a temperature of 37 0C were the ideal environmental and physiological conditions for developing positive bacterial isolates. From the soil samples taken from various soil conditions in Egypt, the morphological and biochemical analyses showed that Bacillus cereus 14579 was the main positive bacterial generating L-glutaminase isolate. At a pH of 7.4 and a temperature of 37 °C, the following activators—KCL, ZnSO4, FeSO4, K2HPO4, and MgSO4—provided the high-grade circumstances for characterizing L-glutaminase synthesis. Bacillus cereus-produced L-glutaminase has shown excellent bioavailability and effectiveness as an anticancer therapy. The yield [productivity] was 5.2 U/ml during the original manufacture from MGA, but it increased to 42.96 U/ ml by bacterial recombinant DNA manufacturing. The molecular mass of L-glutaminase was roughly 37 KDa. L-glutaminase was purified, yielding final enzyme recovery of 55 1.23%, total activity 12,990± 19.76 ( U), specific activity 384.66± 8.92( U/ mg of protein), and purification fold 2± 2.99. Furthermore, the L-glutaminase activity was raised by 19%, 23%, 15%, and 9%, respectively, by the enzymatic activators Mn2+, K+, Na+, and Fe3+. It demonstrated strong DPPH scavenging activity (IC50 = 61 μg/ mL) and anticancer activity( IC50 = 40.72, 9.7, 7.39, 20.61, 51.28, and 11.55 μg/ mL, respectively) against human liver (HepG-2), colon ( HCT-116), breast ( MCF-7), lung( A-549), lymphocytic( CCL-120), and cervical( Hela) cancer cell lines. A stronger affinity for its substrate was shown by the kinetic parameters of Km and Vmax, which were 13.2 10-5 M and 119.86 μmol/ ml/ min, respectively. Conclusion: Acute lymphocytic leukemia and breast cancer are examples of auxotrophic malignancies for which L-glutamine serves as the only metabolic source, and L-glutaminase generated by Bacillus cereus was an appropriate enzyme in the therapy of these diseases.

Acute myeloid leukaemia (AML) is characterized by the accumulation of undifferentiated blast cells in the bone marrow and blood. In most AMLs, relapse frequently occurs due to resistance to chemotherapy. Compelling research results indicate that drug resistance in cancer cells is highly dependent on the intracellular levels of reactive oxygen species (ROS). Modulating ROS levels is therefore a valuable strategy to overcome the chemotherapy resistance of leukemic cells. In this study, we evaluated the efficiency of diphenyleneiodonium (DPI), a well-known inhibitor of ROS production, in targeting AML cells. Results showed that although inhibiting cytoplasmic ROS production, DPI triggered an increase in the mitochondrial ROS levels caused by the disruption of the mitochondrial respiratory chain. We also demonstrated that DPI blocks the mitochondrial oxidative respiration (OxPhos) in a dose-dependent manner and that AML cells with high OxPhos status were highly sensitive to treatment with DPI, which synergizes with the chemotherapeutic agent cytarabine (Ara-C). Thus, our results suggest that targeting mitochondrial function by DPI might be exploited to target AML cells with high OxPhos status.

Parents’ attitudes and practices may support the children’s reactions to treatments for leukemia and their general adjustment. This study has two aims: to explore parenting depending on child's age and to develop and test a model on how family processes influence the psycho-social development of children with leukemia. Patients were 118 leukemic children and their parents recruited at the Haematology-Oncologic Clinic of the Department of Pediatrics, University of Padua. All parents were Caucasian with a mean age of 37.39 years (SD = 6.03). Children’s mean age was 5.89 years (SD = 4.21). After the signature of the informed consent, the parents were interviewed using the EFI-C from which we derived Parenting dimension and 3 parental perceptions on child’s factors. One year later, the clinical psychologist interviewed again parents using the VABS scales. The analyses revealed the presence of a significant difference in parenting by child’s age: Infants required a higher and more intensive parenting. Child’s coping to medical procedures at the second week after the diagnosis, controlled for parenting effect, impacted upon child’s adaptation one-year post diagnosis. Specific intervention programmes are proposed in order to help children more at risk just after the diagnosis for developmental delays.

Early childhood is considered to be a period of rapid development, with the acquisition of abilities predicting future positive school competences. Motor, cognitive and social difficulties related to cancer therapies heavily impact the development of children with cancer. This study focused on two main aims: to assess the developmental pathways of preschool children with acute lymphoblastic leukaemia one year post-treatment and to compare these abilities both with those of a control group of healthy peers and with Italian norms. Forty-four children and their families, recruited through the Haematology-Oncologic Clinic of the Department of Child and Woman Health (University of Padua), agreed to participate to this study. The children’s mean age was 4.52 years (SD = 0.94, range = 2.5-6 years), equally distributed by gender, all diagnosed with Acute Lymphoblastic Leukaemia. Matched healthy peers were recruited through paediatricians’ ambulatories. Each family was interviewed adopting the Vineland Adaptive Behaviour Scales. Paired sample Wilcoxon tests revealed that children were reported to have significantly more developmental difficulties than their healthy peers. When compared with Italian norms they scored particularly low in verbal competence, social and coping skills. No significant association were found between treatment variables and developmental abilities. These findings suggest that the creation of specialized interventions both for parents and children may fill the possible delays in children’s development probably due to stress, lack of adequate stimulation or difficult adaptation.

Early childhood is considered to be a period of rapid development, with the acquisition of abilities predicting future positive school competences. Motor, cognitive and social difficulties related to cancer therapies heavily impact the development of children with cancer. This study focused on two main aims: to assess the developmental pathways in preschool children with leukaemia one year post-treatment; and to compare these abilities with those of a control group of healthy peers. Forty-eight children and their families, recruited through the Haematology-Oncologic Clinic of the Department of Child and Woman Health (University of Padua), agreed to participate in this study. The children’s mean age was 4.36 years (SD = 1.07, range = 1.91–6 years), equally distributed by gender, most of whom were diagnosed with Acute Lymphoblastic Leukaemia (N = 44). Matched healthy peers were recruited through paediatricians’ ambulatories. Each family was interviewed adopting the Vineland Adaptive Behaviour Scales. Paired sample t-tests revealed that children, especially aged 42–72 months, were reported to have significantly more developmental difficulties than their healthy peers, particularly in verbal competence, social and coping skills and gross motor abilities. These findings suggest that the creation of specialized interventions for both parents and children may fill the possible delays in children’s development due to toxic therapies and their associated hospitalisation.

Mesenchymal stem cells (MSCs) are one of the main residents in the bone marrow (BM), having an essential role in the regulation of hematopoietic stem cell (HSCs) differentiation and proliferation. Myelodysplastic syndromes (MDS) are a group of myeloid disorders impacting hematopoietic stem and progenitor cells (HSCPs) and characterised by BM failure, ineffective haematopoiesis, cytopenia, a high risk of transformation through the expansion of MDS clones together with additional genetic defects. It has been indicated that MSCs play anti-tumorigenic roles such as cell cycle arrest and pro-tumorigenic roles including induction of metastasis in MDS and leukaemia. Growing evidence has shown that MSCs have impaired functions in MDS, such as decreased proliferation capacity, differentiation ability, haematopoiesis support, immunomodulation function, and increased inflammatory alterations within the BM through some intracellular pathways such as Notch and Wnt and extracellular modulators secreted by MSCs abnormally, including increased expressions of inflammatory factors and decreased expressions of hematopoietic factors, contributing to the development and progression of MDS. Therefore, MSCs can be targeted for the treatment of MDS and leukaemia. However, it remains unclear what drives MSCs to behave abnormally. In this review, dysregulations in MSCs and their contributions to myeloid haematological malignancies will be discussed.

Chronic myeloid leukaemia (CML) is a haematological neoplasm driven by the BCR/ABL fusion oncogene. The monogenic aspect of the disease and the feasibility of ex vivo therapies in haematological disorders make CML an excellent candidate for gene therapy strategies. The ability to abolish any coding sequence by CRISPR-Cas9 nucleases offers a powerful therapeutic opportunity to CML patients. However, a definitive cure can only be achieved when only CRISPR-edited cells are selected. A gene-trapping approach combined with CRISPR technology would be an ideal approach to ensure this. Here, we have developed a CRISPR-Trap strategy that efficiently inserts a donor gene trap (SA-CMV-Venus) cassette into the BCR-ABL1-specific fusion point in the CML K562 human cell line. The trapping cassette interrupts the on-cogene coding sequence and expresses a reporter gene that enables the selection of edited cells. Quantitative expression analyses showed significantly higher level of expression of the BCR-Venus allele coupled with a drastically lower level of BCR/ABL expression in Venus+ cell fractions. Functional in vitro experiments showed cell proliferation arrest and apoptosis in selected Venus+ cells. Finally, xenograft experiments with the selected Venus+ cells showed a large reduction in tumour growth, thereby demonstrating a therapeutic benefit in vivo. This study is a proof of concept for the therapeutic potential of a CRISPR-Trap system as a novel strategy for gene elimination in haematological neoplasms.

Balínt Syndrome is an acquired disorder manifesting in the inability to recognize several objects at once (simultagnosia), inaccurate visually guided limb movements despite intact motor function (optic ataxia) and the inability to make accurate voluntary saccades to visual targets despite demonstrating unrestricted range of eye movements (ocular motor apraxia). Here we report the first case of a patient presenting with Balínt Syndrome caused by a platelet-derived growth factor receptor A mutation (PDGFRA)-induced Hypereosinophilic Syndrome (HES).

Background: The implementation of tyrosine kinase inhibitors (TKIs) in the treatment of chronic myeloid leukemia (CML) has brought a significant improvement in the prognosis for CML patients and a decrease in the number of patients requiring allogeneic hematopoietic stem cell transplantation (allo-HCT). Nevertheless, the impact of TKIs on allo-HCT outcomes have not been thoroughly explored. Objectives: The main endpoint of our research was to assess the impact of prior TKI treatment on acute graft-versus-host disease (aGvHD) and chronic graft-versus-host disease (cGvHD). Methods: In our retrospective analysis we included 240 patients treated between 1993 and 2013, and divided them into 3 groups according to the therapy administered prior to matched related or matched unrelated donor allo-HCT (imatinib group n=41, dasatinib/nilotinib group n=28, TKI naïve group n=171). Results: Both the cumulative incidence of aGvHD (p=0.044) and cGvHD (p<0.001) in individuals receiving second generation TKIs (2G-TKIs) prior to allo-HCT compared to patients receiving no TKIs or imatinib (IMA) (40.7% vs 61.4% vs 70.7%, p=0.044; 25.0% vs 76.4% vs 51.2%, p<0.001, respectively). In case of the 2G-TKI cohort, the number of low-grade aGvHD and cGvHD was significantly lower compared to the IMA and TKI-naïve groups (p=0.018, p=0.004; p<0.001 versus TKI-naïve, respectively). In terms of 3-year overall survival (OS) there were no important variations between TKI-naïve, IMA and 2G-TKI (55% vs 49.9% vs 69.6%, p=0.740). Conclusions: The results of our study suggest that TKI treatment prior to allo-HCT may have protective impact on immune-mediated outcomes.

There is still little research on psychological wellbeing, life satisfaction and reported problems in preadolescents and adolescents under therapy for leukaemia, and also little research comparing them with their healthy peers. The present study aims to analyse the life satisfaction, hope, psychological wellbeing and reported problems’ intensity in patients aged 8-18 during the first year of therapy, to identify those more at risk and to compare their reports with matched healthy peers. After the parental written consent signature, a battery of self-reported questionnaires was administered during hospitalisation or day hospital admissions post 6 months and post 12 months from the diagnosis. Younger patients (aged 8–13 years) were more at risk than older ones in their problems’ intensity and psychological symptoms; females and Acute Myeloid Leukaemia patients reported lower current life satisfaction perceptions; hope was associated with lower depression symptoms and mood problems. Healthy peers have a better perception of current life, but reported a lower hope score, more anxiety symptoms and more cognitive problems than patients. The first 6 months were more critical for patients’ psychological health. The clinical aim was to identify the patients more at risk in order to prepare ad hoc psychological interventions.

Artificial intelligence methods may help in unveliling information hidden in high-dimensional oncological data. Flow cytometry studies of haematological malignancies provide quantitative data with the potential to be used for the construction of response biomarkers. Many computational methods from the bioinformatics toolbox can be applied to these data but have not been exploited in their full potential in leukaemias, specifically for the case of childhood B-cell acute lymphoblastic leukemia. In this paper we analysed flow cytometry data obtained on diagnosis from 54 paediatric B-cell acute lymphoblastic leukemia patients from two local institutions. We constructed classifiers based on the Fisher’s Ratio to quantify differences in expression levels of immunophenotypical markers between patients with relapsing and non-relapsing disease. The distribution of the marker CD38 was found and validated to have a strong discriminating power between both patient cohorts, thus providing a classifier.

Following the development of tyrosine kinase inhibitors (TKI), the survival of patients with chronic myeloid leukaemia (CML) drastically improved. With the introduction of these agents, CML is now considered a chronic disease, for some patients. Taking into consideration the side effects, toxicity, and high cost, discontinuing TKIs became a goal for patients with chronic phase CML. Patients who achieved deep molecular response (DMR) and discontinued TKI, remained in treatment-free remission (TFR). Currently, the data from the published literature demonstrate that 40-60% of patients achieve TFR, with relapses occurring within the first six months. In addition, almost all patients who relapsed regained a molecular response upon re-treatment, indicating TKI discontinuation is safe. However, there is still a gap in the understanding the mechanisms behind TFR, and whether there are prognostic factors that can predict the best candidates who qualify for TKI discontinuation with a view to keeping them in TFR. Furthermore, the information about a second TFR attempt and the role of gradual de-escalation of TKI before complete cessation is limited. This review highlights the factors predicting success or failure of TFR. In addition, it ex-amines the feasibility of a second TFR attempt after the failure of the first one, and the current guidelines concerning TFR in clinical practice.

Chronic lymphocytic leukemia (CLL) is characterized by dysfunctional B cells. Immune checkpoint molecules such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and programmed death-1 (PD-1) are upregulated in patients with CLL and may correlate with prognostic markers such as beta-2 microglobulin (B2M). The aim of this study was to evaluate the levels of immune checkpoints on B-cell subsets and to further correlate them with B2M levels in patients with CLL. We recruited 21 patients with CLL and 12 controls. B cell subsets and the levels of immune checkpoint expression were determined using conventional multi-color flowcytometry. Basal levels of B2M in patients with CLL were measured using an Enzyme-linked immunosorbent assay. Patients with CLL had increased levels of activated B cells when compared to the control group, p< 0.001. The expression of PD-1 and CTLA-4 were increased on activated B cells and memory B cells, p< 0.05. There were no associations between B2M levels and the measured immune checkpoints on B cell subsets, after adjusting for sex and age. African patients with CLL express elevated levels of PD-1 and CTLA-4 immune checkpoints on activated and memory B cell subsets. However, there is no correlation between these immune checkpoint expressions and B2M levels.

Bioactive lipids are involved in cellular signalling events with links to human disease. Many of these are involved in inflammation under normal and pathological conditions. Despite being attractive molecules from a pharmacological point of view, detection and quantification of lipids has been a major challenge. Here, we have optimised a liquid chromatography dynamic multiple reaction monitoring targeted mass spectrometry (LC-dMRM-MS) approach to profile eicosanoids and fatty acids in biological samples. In particular, by applying this analytic workflow to study a cellular model of Chronic Myeloid Leukaemia (CML), we found that intra- and extra-cellular 2-Arachidonoylglycerol (2-AG), intracellular Arachidonic Acid (AA), and extracellular Prostaglandin F_2α (PGF_2α), 5-Hydroxyeicosatetraenoic acid (5-HETE), Palmitic acid (PA, C16:0) and Stearic acid (SA, C18:0) were altered in response to immunomodulation by type I Interferon (IFN-I), a currently approved treatment for CML. Our observations indicate changes in eicosanoid and fatty acid metabolism with potential relevance in the context of cancer inflammation and CML.

The use of chimeric antigen receptors (CAR-T cells) for the treatment of patients with malignant haematological diseases has become a well-established application, for conditions such as refractory or relapsed B cell acute lymphoblastic leukaemia (B-ALL), B cell lymphomas (BCL), and multiple myeloma (MM). Nearly 35,000 patients have received autologous CAR-T cells for the treatment of these conditions only in the USA. Since their approval by the Food and Drug Administration (FDA) in 2017, over 1200 clinical trials have been initiated globally and there are at least 10 different CAR-T cells with approval by different regulatory agencies around the globe. In the USA the Food and Drug Administration (FDA) has approved 6 commercial CAR-T cells that are widely distributed worldwide. At the time of this writing, several clinical trials have been performed in patients with solid tumours such as glioblastoma, renal and pancreatic cancer, as well as in patients with autoimmune conditions such as systemic lupus erythematosus (SLE), idiopathic inflammatory myositis (IIM), and systemic sclerosis (SS). There are also several studies showing the potential benefit of CAR-T cells for other non-malignant diseases such as asthma and even fungal infections. In this review, without pretending to cover all the current areas of treatments with CAR-T cells, we offer a brief summary of some of the most relevant aspects of the use of CAR-T cells for some of these conditions.