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1.
Int J Mol Sci ; 22(18)2021 Sep 09.
Article in English | MEDLINE | ID: mdl-34575904

ABSTRACT

The KMT2A/AFF1 rearrangement is associated with an unfavorable prognosis in infant acute lymphocytic leukemia (ALL). Discordant ALL in monozygotic twins is uncommon and represents an attractive resource to evaluate intrauterine environment-genetic interplay in ALL. Mutational and epigenetic profiles were characterized for a discordant KMT2A/AFF1-rearranged infant monozygotic twin pair and their parents, and they were compared to three independent KMT2A/AFF1-positive ALL infants, in which the DNA methylation and gene expression profiles were investigated. A de novo Q61H NRAS mutation was detected in the affected twin at diagnosis and backtracked in both twins at birth. The KMT2A/AFF1 rearrangement was absent at birth in both twins. Genetic analyses conducted at birth gave more insights into the timing of the mutation hit. We identified correlations between DNA methylation and gene expression changes for 32 genes in the three independent affected versus remitted patients. The strongest correlations were observed for the RAB32, PDK4, CXCL3, RANBP17, and MACROD2 genes. This epigenetic signature could be a putative target for the development of novel epigenetic-based therapies and could help in explaining the molecular mechanisms characterizing ALL infants with KMT2A/AFF1 fusions.


Subject(s)
DNA-Binding Proteins/genetics , Epigenesis, Genetic , Gene Expression Regulation , Gene Rearrangement , Histone-Lysine N-Methyltransferase/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , Transcriptional Elongation Factors/genetics , Twins, Monozygotic/genetics , Alleles , Computational Biology/methods , CpG Islands , DNA Methylation , Epigenomics/methods , Female , Genotype , Humans , Infant, Newborn , Male , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Exome Sequencing
2.
Blood ; 130(5): 643-654, 2017 08 03.
Article in English | MEDLINE | ID: mdl-28550041

ABSTRACT

In acute lymphoblastic leukemia (ALL), central nervous system (CNS) involvement is a major clinical concern. Despite nondetectable CNS leukemia in many cases, prophylactic CNS-directed conventional intrathecal chemotherapy is required for relapse-free survival, indicating subclinical CNS manifestation in most patients. However, CNS-directed therapy is associated with long-term sequelae, including neurocognitive deficits and secondary neoplasms. Therefore, molecular mechanisms and pathways mediating leukemia-cell entry into the CNS need to be understood to identify targets for prophylactic and therapeutic interventions and develop alternative CNS-directed treatment strategies. In this study, we analyzed leukemia-cell entry into the CNS using a primograft ALL mouse model. We found that primary ALL cells transplanted onto nonobese diabetic/severe combined immunodeficiency mice faithfully recapitulated clinical and pathological features of meningeal infiltration seen in patients with ALL. ALL cells that had entered the CNS and were infiltrating the meninges were characterized by high expression of vascular endothelial growth factor A (VEGF). Although cellular viability, growth, proliferation, and survival of ALL cells were found to be independent of VEGF, transendothelial migration through CNS microvascular endothelial cells was regulated by VEGF. The importance of VEGF produced by ALL cells in mediating leukemia-cell entry into the CNS and leptomeningeal infiltration was further demonstrated by specific reduction of CNS leukemia on in vivo VEGF capture by the anti-VEGF antibody bevacizumab. Thus, we identified a mechanism of ALL-cell entry into the CNS, which by targeting VEGF signaling may serve as a novel strategy to control CNS leukemia in patients, replacing conventional CNS-toxic treatment.


Subject(s)
Central Nervous System Neoplasms/metabolism , Leukemic Infiltration/metabolism , Neoplasm Proteins/metabolism , Neoplasms, Experimental/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Vascular Endothelial Growth Factor A/metabolism , Animals , Bevacizumab/pharmacology , Cell Survival/drug effects , Central Nervous System Neoplasms/drug therapy , Central Nervous System Neoplasms/pathology , Endothelial Cells/metabolism , Endothelial Cells/pathology , Heterografts , Humans , Leukemic Infiltration/drug therapy , Leukemic Infiltration/pathology , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Transplantation , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/pathology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Transendothelial and Transepithelial Migration/drug effects , Vascular Endothelial Growth Factor A/antagonists & inhibitors
3.
Haematologica ; 103(6): 1008-1017, 2018 06.
Article in English | MEDLINE | ID: mdl-29519870

ABSTRACT

In contrast to well-established hierarchical concepts of tumor stem cells, leukemia-initiating cells in B-cell precursor acute lymphoblastic leukemia have not yet been phenotypically identified. Different subpopulations, as defined by surface markers, have shown equal abilities to reconstitute leukemia upon transplantation into immunodeficient mice. Using a non-obese diabetes/severe combined immunodeficiency human acute lymphoblastic leukemia mouse model and cell cycle analysis annotating cells to distinct cycle phases, we functionally characterized leukemia-initiating cells and found that cells in all stages of the cell cycle are able to reconstitute leukemia in vivo, with early cycling cells (G1blow population) exhibiting the highest leukemia-initiating potential. Interestingly, cells of the G2/M compartment, i.e. dividing cells, were less effective in leukemia reconstitution. Moreover, G1blow cells were more resistant to spontaneous or drug-induced cell death in vitro, were enriched for stem cell signatures and were less metabolically active, as determined by lower levels of reactive oxygen species, compared to G2/M stage cells. Our data provide new information on the biological properties of leukemia-initiating cells in B-cell precursor acute lymphoblastic leukemia and underline the concept of a stochastic model of leukemogenesis.


Subject(s)
Cell Cycle , Energy Metabolism , Leukemia/etiology , Leukemia/metabolism , Animals , Biomarkers , Cell Cycle/genetics , Disease Models, Animal , Disease Susceptibility , Gene Expression Profiling , Humans , Immunophenotyping , Leukemia/mortality , Leukemia/pathology , Mice , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Oxidative Stress , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/etiology , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Prognosis
4.
Autism Res ; 17(2): 311-323, 2024 02.
Article in English | MEDLINE | ID: mdl-38108559

ABSTRACT

The term "toe walking" describes walking on the toes with a lack of heel strike upon initiation of the stance phase of gait. In individuals with autism spectrum disorder (ASD), this phenomenon, or "tip-toe behavior" (TTB), can be present in a substantial proportion of subjects even during standing. In this study, we investigated TTB in 50 persons with ASD (age range 4-26 years). We evaluated TTB through an observational/report-based assessment protocol. Subsequently, we employed a new structured video-based coding protocol based on standardized video recordings, focusing on static and dynamic conditions. Finally, the findings of the two protocols were compared. Twenty-four subjects with TTB were identified and classified according to three functional groups: TTB1, present only during running (6 subjects); TTB2, present during walking and running (11 subjects); and TTB3, present during standing, walking, and running (7 subjects). Moreover, we found that TTB3 subjects exhibited a significantly higher quantity of TTB compared with subjects in the TTB1 and TTB2 groups during both standing and walking tests. Additionally, a high quantity of TTB in the static test was found to be related to a high quantity of TTB in the dynamic test. Variables such as age, autism severity, intellectual disability, and gender were not significantly associated with the mean percent of TTB both in static and dynamic tests in multivariate analysis. This structured video-based coding approach appears feasible and useful for assessing TTB in individuals with ASD and it has the potential to provide insights into TTB trajectories and aid in designing possible interventions.


Subject(s)
Autism Spectrum Disorder , Intellectual Disability , Humans , Child, Preschool , Child , Adolescent , Young Adult , Adult , Autism Spectrum Disorder/complications , Cross-Sectional Studies , Intellectual Disability/complications , Toes , Gait
5.
Tumori ; 110(5): 375-385, 2024 Oct.
Article in English | MEDLINE | ID: mdl-39101541

ABSTRACT

BACKGROUND: Colorectal cancer is a worldwide leading cause of death accounting for high-rate mortality. Mutations in the epidermal growth factor receptor and RAS/MAPK pathways, as well as altered methylation genes profiles, have been described as molecular mechanisms promoting and sustaining tumour development and progression. Aberrant methylation is a well-known epigenetic mechanism involved in gene regulation; particularly several genes were reported as hypermethylated in CRC. Recently, it was shown that epigenetic alterations in genes such as neuropeptide y, proenkephalin and Wnt inhibitory factor 1 can be used as promising disease biomarkers. Almost all methods developed for the DNA methylation analysis combined next generation sequencing, conventional qRT-PCR or ddPCR with the prior DNA modification with sodium bisulfite. METHODS AND RESULTS: We implemented a ddPCR method to assess the methylation status of Wnt inhibitory factor 1 and neuropeptide y using the methylation sensitive restriction enzyme approach that does not impact on DNA quality and guarantees the discrimination of DNA methylation independent of bisulfite conversion. CONCLUSIONS: We showed that this method is robust and sensitive also allowing the monitoring of CRC disease progression when applied to circulating free DNA samples from liquid biopsies, proving to be a fast and easy to implement assay to be used for the monitoring of the methylation pattern of clinically relevant target genes.


Subject(s)
Adaptor Proteins, Signal Transducing , Biomarkers, Tumor , Colorectal Neoplasms , DNA Methylation , Neuropeptide Y , Repressor Proteins , Humans , Colorectal Neoplasms/genetics , Neuropeptide Y/genetics , Adaptor Proteins, Signal Transducing/genetics , Biomarkers, Tumor/genetics , Repressor Proteins/genetics , Polymerase Chain Reaction/methods , Epigenesis, Genetic , Female
6.
Recenti Prog Med ; 112(4): 243-249, 2021 04.
Article in Italian | MEDLINE | ID: mdl-33877085

ABSTRACT

Therapeutic plans (TPs) were introduced in Italy in 2004 in order to ensure the continuity in the prescription of new drugs between specialist physicians and general practitioners (GPs). Over the years this prescription tool was updated several times: starting from a paper form without any template ("paper TP") to a template defined by AIFA to collect specific clinical information, up to a web-based form to collect all information into a database. Over time-critical issues concerning its usefulness have been raised, especially when AIFA established several extensions for TP validity to ensure the social distancing required by the covid-19 pandemic. Therefore, after several years from their establishment, pending adoption of necessary implementing of Ministerial Decree of 25th March 2020, a check of the actual impact of TPs is required, in order to plan their review. This study provide a detailed overview of all TPs active in Italy at the 11th May 2020. From Farmadati database, all drugs reimbursed by the National Health Service (class A drugs) and requiring TP were selected. The consumption of these drugs has been derived from OsMed Reports that make available data of medicines consumption and expenditure in the general population in Italy. The analysis showed that TP is required for the prescription of 935 medicinal products (9.6% of class-A drugs) and 147 different active substances (belonging to 34 different Therapeutic Groups and 66 subgroups). Out of these, 67 (46%) required a paper TP without any template, 72 (49%) a paper TP on AIFA template, and 8 (5%) a web-based TP. The Therapeutic Group with the largest number of active ingredients with TP were antidiabetics (19.7%), followed by immunomodulating and immunosuppressants (9.5%) and medications for asthma and COPD (6.8%). Consumption analysis of drugs with TP showed that this prescription tool covers 943,899,598 DDD per year, equal to 2,586,026 DDD/day. This means that TPs have a very high impact in terms of the prevalence of patients treated on the entire care process. Of all annual DDDs prescribed on TP, 46.8% concerned drugs with TP on template AIFA, 34.5% drugs with web-based TP, while the remaining 18.7% drugs with paper TP without a template. This analysis may provide the basis for an analytical case-by-case review of TP maintenance needs, trying to maximize the benefits of this tool and to reduce its possible adverse effects. This review could be helpful to ensure the appropriateness in the drug uses, to enhance the role of general medicine, and to simplify the pathways of millions of patients ensuring the continuity of their care.


Subject(s)
Clinical Protocols , Drug Prescriptions/standards , Humans , Italy
7.
J Orthop ; 21: 444-448, 2020.
Article in English | MEDLINE | ID: mdl-32982098

ABSTRACT

BACKGROUND: About 20% of individuals with autism spectrum disorders (ASD) showed tip-toe behavior (TTB). This behavior may be related to a decreased ankle joint range of motion (ROM) in dorsiflexion. Physiologically, gastrocnemius (GM) and soleus (SM) muscles influence ankle ROM independently. However, no studies investigated the relationship between the amount of time individuals with ASD spend in TTB and GM and SM muscle lengths. OBJECTIVE: To evaluate the relationship between three mutually exclusive clinical patterns of TTB i.e., during standing, walking and running (TTB Class 1), or during walking and running (TTB Class 2), or only when running (TTB Class 3), and GM and SM muscle lengths. METHODS: Sixty-nine individuals with ASD (average age: 14.1 ± 3.6 years, 56 males) were enrolled. In a clinical setting, SM and GM muscle lengths of both legs were assessed through a manual goniometer. Measurements were performed by two trained assessors blinded to TTB classifications. RESULTS: Individuals with ASD classified as TTB Class 1 demonstrated a shortening of both GM and SM compared with NO-TTB and TTB Class 3 individuals. CONCLUSIONS: Our results support the relationship between TTB severity and GM and SM shortening assessed by a decreased ankle joint ROM in dorsiflexion. Further studies are needed to determine the factors associated with TTB and decreased ankle ROM.

8.
Eur J Haematol ; 83(5): 406-19, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19558506

ABSTRACT

OBJECTIVE: Gene expression profiles become increasingly more important for diagnostic procedures, allowing clinical predictions including treatment response and outcome. However, the establishment of specific and robust gene signatures from microarray data sets requires the analysis of large numbers of patients and the application of complex biostatistical algorithms. Especially in case of rare diseases and due to these constrains, diagnostic centers with limited access to patients or bioinformatic resources are excluded from implementing these new technologies. METHOD: In our study we sought to overcome these limitations and for proof of principle, we analyzed the rare t(4;11) leukemia disease entity. First, gene expression data of each t(4;11) leukemia patient were normalized by pairwise subtraction against normal bone marrow (n = 3) to identify significantly deregulated gene sets for each patient. RESULT: A 'core signature' of 186 commonly deregulated genes present in each investigated t(4;11) leukemia patient was defined. Linking the obtained gene sets to four biological discriminators (HOXA gene expression, age at diagnosis, fusion gene transcripts and chromosomal breakpoints) divided patients into two distinct subgroups: the first one comprised infant patients with low HOXA genes expression and the MLL breakpoints within introns 11/12. The second one comprised non-infant patients with high HOXA expression and MLL breakpoints within introns 9/10. CONCLUSION: A yet homogeneous leukemia entity was further subdivided, based on distinct genetic properties. This approach provided a simplified way to obtain robust and disease-specific gene signatures even in smaller cohorts.


Subject(s)
Chromosomes, Human, Pair 11/metabolism , Chromosomes, Human, Pair 4/metabolism , Gene Expression Regulation, Leukemic , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Translocation, Genetic , Chromosomes, Human, Pair 11/genetics , Chromosomes, Human, Pair 4/genetics , Female , Gene Expression Profiling/methods , Histone-Lysine N-Methyltransferase , Homeodomain Proteins/biosynthesis , Homeodomain Proteins/genetics , Humans , Male , Myeloid-Lymphoid Leukemia Protein/biosynthesis , Myeloid-Lymphoid Leukemia Protein/genetics , Oligonucleotide Array Sequence Analysis/methods , Oncogene Proteins, Fusion/biosynthesis , Oncogene Proteins, Fusion/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/classification , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics
9.
Cancers (Basel) ; 11(11)2019 Nov 11.
Article in English | MEDLINE | ID: mdl-31717959

ABSTRACT

Direct antiviral agents (DAAs) have excellent efficacy against chronic hepatitis C virus (HCV) infection. Despite this strength, recent studies raised concerns about an unexpected hepatocellular carcinoma (HCC) occurrence rate after DAA therapy. In this exploratory case-control study, we evaluated the potential use of miRNAs as serum biomarkers for the detection of early HCC in DAA-treated patients. In the discovery phase, the circulating miRNome was assessed in 10 matched patients with (HCC+) or without HCC (HCC-) occurrence. Microarray analysis was performed before (T0) and after one month of the DAA therapy (T1). MiRNAs discriminating HCC+ and HCC- patients were validated in 60 samples by means of RT-qPCR. We estimated the time-averaged difference of a given miRNA between HCC+ and HCC- patients using a bootstrapped random-effect generalized least square regression model (RE-GLS). At T0, miR-1207-5p, miR-1275, miR-3197, miR-4443, miR-3178, miR-483-5p, miR-4706, miR-4793-3p and miR-1246 discriminated HCC+ from HCC- patients (p < 0.05). At T1, only miR-1180-3p, miR-1228-3p, miR-4329 and miR-4484 (p < 0.05) discriminated HCC+ from HCC- patients. The subsequent validation phase identified miR-3197 as changing with both disease and time. Our results suggest that patients might be already committed to HCC occurrence before DAA therapy. MiR-3197 shows some potential for the identification of patients at risk of HCC during DAA treatments.

10.
Sci Rep ; 9(1): 14670, 2019 10 11.
Article in English | MEDLINE | ID: mdl-31605010

ABSTRACT

Circular RNAs (circRNAs) are abundantly expressed in the haematopoietic compartment, but knowledge on their diversity among blood cell types is still limited. Nevertheless, emerging data indicate an array of circRNA functions exerted through interactions with other RNAs and proteins, by translation into peptides, and circRNA involvement as regulatory molecules in many biological processes and cancer mechanisms. Interestingly, the role of specific circRNAs in leukemogenesis has been disclosed by a few studies, mostly in acute myeloid leukemia. In this study, circRNA expression in B-cells, T-cells and monocytes of healthy subjects is described, including putative new circRNA genes. Expression comparison considered 6,228 circRNAs and highlighted cell population-specific expression and exon usage patterns. Differential expression has been confirmed by qRT-PCR for circRNAs specific of B-cells (circPAX5, circAFF3, circIL4R, and circSETBP1) or T-cells (circIKZF1, circTNIK, circTXK, and circFBXW7), and for circRNAs from intronic (circBCL2) and intergenic regions that were overexpressed in lymphocytes. Starting from this resource of circRNA expression in mature blood cell populations, targeted examination identified striking and generalized upregulated expression of circPAX5, circPVT1 and circHIPK3 in pediatric B-precursor acute lymphoblastic leukemia, and disclosed circRNAs with variable expression across cytogenetic subtypes.


Subject(s)
Cell Lineage/genetics , Gene Expression Profiling/methods , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , RNA, Circular/genetics , Blood Cells/pathology , Cell Line, Tumor , Child , Female , Gene Expression Regulation, Neoplastic/genetics , Humans , Male , Pediatrics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/classification , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , RNA, Circular/classification
11.
Theranostics ; 9(17): 4860-4877, 2019.
Article in English | MEDLINE | ID: mdl-31410187

ABSTRACT

HIF-1α has been suggested to interplay with Wnt signaling components in order to activate a neuronal differentiation process in both normal brain and glioblastoma (GBM). Based on these data, we explored the molecular mechanisms underlying the observed capability of GBM cells to acquire a neuronal phenotype upon Wnt signaling stimulation and how the microenvironment, particularly hypoxia, modulates this process. Methods: here, the employment of ChIP-seq techniques together with co-immunoprecipitation approaches allowed to reconstruct the molecular interactions responsible for activating specific pro-differentiating transcriptional programs in GBM cells. Moreover, gene silencing/over-expression approaches coupled with the functional analysis of cell phenotype were applied to confirm ChIP-driven hypotheses. Finally, we combined the use of publicly available gene expression datasets with protein expression data by immunohistochemistry to test the clinical relevance of obtained results. Results: our data clearly suggest that HIF-1α is recruited by the ß-catenin/TCF1 complex to foster neuronal differentiation gene transcription in hypoxic GBM cells. Conversely, at higher oxygen levels, the increased expression of TCF4 exerts a transcriptional inhibitory function on the same genomic regions, thus counteracting differentiation. Moreover, we demonstrate the existence of a positive correlation between the expression levels of HIF-1α, TCF1 and neuronal phenotype in GBM tumors, accompanied by the over-expression of several Wnt signaling components, finally affecting patient prognosis. Conclusion: we unveiled a peculiar mechanism by which TCF1 and HIF-1α can induce a reminiscent neuronal differentiation of hypoxic GBM cells, which is hampered, in normoxia, by high levels of TCF4, thus not only de facto controlling the balance between differentiation and stemness, but also impacting on intra-tumoral heterogeneity and eventually patient outcome.


Subject(s)
Brain Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Glioblastoma/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Neoplastic Stem Cells/metabolism , Neurogenesis , Wnt Signaling Pathway , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Hypoxia , Glioblastoma/metabolism , Glioblastoma/pathology , Hepatocyte Nuclear Factor 1-alpha/genetics , Hepatocyte Nuclear Factor 1-alpha/metabolism , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Neoplastic Stem Cells/cytology , Neurons/cytology , Neurons/metabolism , Oxygen/metabolism , Tumor Cells, Cultured
12.
Autism Res ; 11(10): 1404-1415, 2018 10.
Article in English | MEDLINE | ID: mdl-30199607

ABSTRACT

There is increasing evidence that autism spectrum disorder (ASD) subjects have also motor impairments. Toe walking (TW) is a phenomenon that can be found in ASD subjects during gait, even if this condition was found not to be necessarily related only to walking, since these children often also stand and run on their tiptoes. Since persistent TW in ASD subjects may contribute to secondary shortening of the Achilles's tendon, it becomes important to have an assessment tool and/or outcome measure for both the clinical and rehabilitative settings. The aim of this systematic review is to critically evaluate and describe the methods employed to assess toe walking in ASD subjects. The systematic review protocol was previously registered on PROSPERO. We conducted an extensive literature search in PubMed, CINAHL, PsycINFO, The Cochrane Library, and Scopus databases. There were no restrictions on the types of study design eligible for inclusion. Ten studies were included in the systematic review. Risk of bias of the included studies was conducted using the following instruments depending on the study types: STROBE Statement, Cochrane risk of bias tool, and CARE checklist. Almost all the included studies (8/10) proposed a tip-toe behavior (TTB) assessment only during walking. Nine out of ten of the included studies assessed TTB using a qualitative methodology. The results evidenced the heterogeneity of qualitative methods and a lack of a structured quantitative test to assess toe walking in ASD subjects. Autism Res 2018, 11: 1404-1415. © 2018 International Society for Autism Research, Wiley Periodicals, Inc. LAY ABSTRACT: Toe walking (TW) is a phenomenon that can be found during ASD subject's gait. The persistence of this behavior may contribute to secondary Achilles's tendon shortening. In this perspective it becomes important to have an assessment tool and/or outcome measure for both the clinical and rehabilitative settings. The current systematic review aimed to describe the methods employed to assess TW. The results evidenced the lack and the need of a structured quantitative test to assess TW in ASD subjects.


Subject(s)
Autism Spectrum Disorder/complications , Gait Analysis/methods , Movement Disorders/complications , Movement Disorders/diagnosis , Child , Female , Gait/physiology , Humans , Male , Toes , Walking/physiology
13.
Biochem Pharmacol ; 153: 230-241, 2018 07.
Article in English | MEDLINE | ID: mdl-29408328

ABSTRACT

Dysregulation of the cyclin D1-CDK4/CDK6 complex is frequently observed in almost all human cancer and contributes to aberrant cell proliferation and consequent tumorigenesis. Although many reports described the importance of CDK4/CDK6 in different set of human tumors, only few studies have been performed on leukemia. By gene expression analysis performed in a cohort of childhood patients affected by B-acute lymphoblastic leukemia (B-ALL) we found that both CDK4 and CDK6 are highly expressed. Moreover, reverse phase protein array (RPPA) analysis showed that cyclin D1 levels are higher in patients undergoing relapse. Starting from these considerations, we evaluated the effect of dual inhibition of CDK4/CDK6 in B-ALL and if this inhibition could enhance cytotoxic killing of leukemia cells after combination treatment with dexamethasone. We treated B-ALL cell lines with ribociclib, a highly specific CDK4/6 inhibitor. As expected, treatment with ribociclib induced growth inhibition of B-ALL cell lines, accompanied by strong cell cycle arrest in G1 phase, along with a dose-dependent decrease in phosphorylated retinoblastoma protein. Ribociclib exposure strongly synergizes with dexamethasone in SEM and RCH-ACV, two dexamethasone-resistant cell lines, along with a strong decrease in proliferation and a significant increase in apoptotic cell death. These results were also confirmed on primary cultures derived from bone marrow of pediatric patients affected by B-ALL. Immunoblot analysis showed a significant increase in glucocorticoid receptor (GR) along with some of its target genes, after combined treatment with ribociclib and dexamethasone. Altogether our findings support the concept that pharmacologic inhibition of CDK4/CDK6 may represent a useful therapeutic strategy to control cell proliferation in B-ALL and provide new insight in understanding potential mechanism of glucocorticoid resistance.


Subject(s)
Aminopyridines/administration & dosage , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Glucocorticoids/administration & dosage , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/enzymology , Protein Kinase Inhibitors/administration & dosage , Purines/administration & dosage , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/physiology , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 6/metabolism , Dexamethasone/administration & dosage , Dose-Response Relationship, Drug , Humans , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy
14.
Mol Cancer Ther ; 17(8): 1705-1716, 2018 08.
Article in English | MEDLINE | ID: mdl-29748211

ABSTRACT

MLL-rearranged acute lymphoblastic leukemia (ALL) occurring in infants is a rare but very aggressive leukemia, typically associated with a dismal prognosis. Despite the development of specific therapeutic protocols, infant patients with MLL-rearranged ALL still suffer from a low cure rate. At present, novel therapeutic approaches are urgently needed. Recently, the use of small molecule inhibitors targeting the epigenetic regulators of the MLL complex emerged as a promising strategy for the development of a targeted therapy. Herein, we have investigated the effects of bromodomain and extra-terminal (BET) function abrogation in a preclinical mouse model of MLL-AF4+ infant ALL using the BET inhibitor I-BET151. We reported that I-BET151 is able to arrest the growth of MLL-AF4+ leukemic cells in vitro, by blocking cell division and rapidly inducing apoptosis. Treatment with I-BET151 in vivo impairs the leukemic engraftment of patient-derived primary samples and lower the disease burden in mice. I-BET151 affects the transcriptional profile of MLL-rearranged ALL through the deregulation of BRD4, HOXA7/HOXA9, and RUNX1 gene networks. Moreover, I-BET151 treatment sensitizes glucocorticoid-resistant MLL-rearranged cells to prednisolone in vitro and is more efficient when used in combination with HDAC inhibitors, both in vitro and in vivo Given the aggressiveness of the disease, the failure of the current therapies and the lack of an ultimate cure, this study paves the way for the use of BET inhibitors to treat MLL-rearranged infant ALL for future clinical applications. Mol Cancer Ther; 17(8); 1705-16. ©2018 AACR.


Subject(s)
Myeloid-Lymphoid Leukemia Protein/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Animals , Humans , Mice , Mice, Inbred NOD , Mice, Mutant Strains , Mice, SCID , Transcriptome
15.
BMC Genomics ; 8: 188, 2007 Jun 22.
Article in English | MEDLINE | ID: mdl-17587440

ABSTRACT

BACKGROUND: Microarray gene expression (MAGE) signatures allow insights into the transcriptional processes of leukemias and may evolve as a molecular diagnostic test. Introduction of MAGE into clinical practice of leukemia diagnosis will require comprehensive assessment of variation due to the methodologies. Here we systematically assessed the impact of three different total RNA isolation procedures on variation in expression data: method A: lysis of mononuclear cells, followed by lysate homogenization and RNA extraction; method B: organic solvent based RNA isolation, and method C: organic solvent based RNA isolation followed by purification. RESULTS: We analyzed 27 pediatric acute leukemias representing nine distinct subtypes and show that method A yields better RNA quality, was associated with more differentially expressed genes between leukemia subtypes, demonstrated the lowest degree of variation between experiments, was more reproducible, and was characterized with a higher precision in technical replicates. Unsupervised and supervised analyses grouped leukemias according to lineage and clinical features in all three methods, thus underlining the robustness of MAGE to identify leukemia specific signatures. CONCLUSION: The signatures in the different subtypes of leukemias, regardless of the different extraction methods used, account for the biggest source of variation in the data. Lysis of mononuclear cells, followed by lysate homogenization and RNA extraction represents the optimum method for robust gene expression data and is thus recommended for obtaining robust classification results in microarray studies in acute leukemias.


Subject(s)
Gene Expression Profiling , Gene Expression Regulation, Leukemic , Genetic Techniques , Leukemia/genetics , Leukemia/metabolism , RNA, Neoplasm , Analysis of Variance , Biomarkers, Tumor/genetics , Humans , Image Processing, Computer-Assisted , Leukocytes, Mononuclear/metabolism , Models, Biological , Models, Genetic , Oligonucleotide Array Sequence Analysis , RNA/metabolism , Solvents/chemistry
16.
Oncol Rep ; 18(2): 417-23, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17611665

ABSTRACT

Hypermethylation of CpG islands is the most well defined epigenetic change in neoplasia and plays an important role in the inactivation or silencing of cancer related genes. DLX genes (1-7), with large CpG islands in their 5' region, are implicated in a number of processes among which haematopoiesis. They are characterized by highly dynamic spatio-temporal expression and supposed to be involved in resistance to apoptosis of several tumor cell lines. In acute lymphoblastic leukemia (ALL) hypermethylation is a common phenomenon frequently associated with poor prognosis in specific genetic childhood leukemia subgroups. These data together with the presence of large CpG islands in the up-stream regions of the DLX genes make them attractive candidates for methylation regulated gene expression and leukemia related aberrancies. To validate the role of DLX genes in paediatric B-ALL cells, we studied two cell lines and two groups of patients with paediatric chromosomal rearrangements: MLL-AF4 and TEL-AML1, respectively. Analysis of methylation and gene expression patterns of DLX3 in 64 specimens of B-lineage ALL revealed that DLX3 presents aberrant methylation in paediatric B-ALL patients. In vitro experiments with 5-Aza-2'dC on leukemia cell lines, confirmed by Western blot analysis, indicated that the methylation of DLX3 CpG islands has a functional role and interferes with the DLX3 gene and DLX3 protein expression in B-ALL cells. Importantly, hypermethylation of DLX3 significantly reduces its expression in MLL-AF4 rearranged leukemias while methylation is almost absent in TEL-AML1 positive ALL specimens. These results show that differential DLX3 methylation could be a new epigenetic marker for genotypic B-cell leukemia subgroup with high-risk features.


Subject(s)
Burkitt Lymphoma/pathology , DNA Methylation , Homeodomain Proteins/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , Oncogene Proteins, Fusion/genetics , Promoter Regions, Genetic/genetics , Transcription Factors/genetics , Blotting, Western , Burkitt Lymphoma/genetics , Burkitt Lymphoma/metabolism , Cell Line, Tumor , Child , Down-Regulation , Gene Expression Regulation, Neoplastic , HL-60 Cells , Homeodomain Proteins/metabolism , Humans , Reverse Transcriptase Polymerase Chain Reaction/methods , Transcription Factors/metabolism , U937 Cells
17.
Autism Res ; 10(9): 1547-1557, 2017 Sep.
Article in English | MEDLINE | ID: mdl-28383150

ABSTRACT

We assessed presentation patterns and characteristics of tip-toe behavior (TTB), more commonly known as toe walking, in a cohort of severe autism spectrum disorder (ASD) subjects with intellectual disability in two studies. The first study included 69 consecutive ASD subjects (57 males, mean age = 14 years-3.7 SD) under observation at our institute. A therapist assessed the presence of TTB during standing, walking, and running through direct observation and an interview with the subjects main caregiver. The prevalence of TTB was 32%. We found three clinical presentation patterns of TTB: (1) present when standing, walking and running (45.5%), (2) present when walking and running (18.4%), or (3) present only when running (36.4%). TTB subjects were more frequently nonverbal than those without TTB (72.7% vs. 44.6%-P = 0.03). On the other hand, no significant difference in ASD severity according to the ADOS scale was found between TTB and non-TTB subjects. In the second study, carried out in a subgroup of 14 ASD subjects (7 TTB and 7 non-TTB), we evidenced that a soft floor surface (foam mats) made a substantial difference in reducing the TTB phenomenon. TTB is frequently present in ASD individuals and may occur in three mutually exclusive modalities, which ultimately defines what is commonly known as toe walking. The presence of TTB seems correlated to the severity of language delay. Foot contact on soft surfaces reduces TTB both during static and/or dynamic tasks. Further evaluation is needed to clarify the potential pathophysiological implications of this phenomenon. Autism Res 2017, 10: 1547-1557. © 2017 International Society for Autism Research, Wiley Periodicals, Inc.


Subject(s)
Autism Spectrum Disorder/complications , Autism Spectrum Disorder/physiopathology , Intellectual Disability/complications , Intellectual Disability/physiopathology , Walking/physiology , Adolescent , Adult , Child , Cohort Studies , Cross-Sectional Studies , Female , Humans , Male , Posture/physiology , Running/physiology , Severity of Illness Index , Toes , Young Adult
18.
Sci Rep ; 6: 34449, 2016 10 04.
Article in English | MEDLINE | ID: mdl-27698462

ABSTRACT

To induce and sustain the leukaemogenic process, MLL-AF4+ leukaemia seems to require very few genetic alterations in addition to the fusion gene itself. Studies of infant and paediatric patients with MLL-AF4+ B cell precursor acute lymphoblastic leukaemia (BCP-ALL) have reported mutations in KRAS and NRAS with incidences ranging from 25 to 50%. Whereas previous studies employed Sanger sequencing, here we used next generation amplicon deep sequencing for in depth evaluation of RAS mutations in 36 paediatric patients at diagnosis of MLL-AF4+ leukaemia. RAS mutations including those in small sub-clones were detected in 63.9% of patients. Furthermore, the mutational analysis of 17 paired samples at diagnosis and relapse revealed complex RAS clone dynamics and showed that the mutated clones present at relapse were almost all originated from clones that were already detectable at diagnosis and survived to the initial therapy. Finally, we showed that mutated patients were indeed characterized by a RAS related signature at both transcriptional and protein levels and that the targeting of the RAS pathway could be of beneficial for treatment of MLL-AF4+ BCP-ALL clones carrying somatic RAS mutations.


Subject(s)
GTP Phosphohydrolases/genetics , High-Throughput Nucleotide Sequencing , Leukemia/genetics , Membrane Proteins/genetics , Mutation , Myeloid-Lymphoid Leukemia Protein , Oncogene Proteins, Fusion , Proto-Oncogene Proteins p21(ras)/genetics , Female , GTP Phosphohydrolases/metabolism , Humans , Leukemia/metabolism , Leukemia/pathology , Male , Membrane Proteins/metabolism , Proto-Oncogene Proteins p21(ras)/metabolism
19.
Oncotarget ; 6(3): 1382-95, 2015 Jan 30.
Article in English | MEDLINE | ID: mdl-25682198

ABSTRACT

Despite increasingly successful treatment of pediatric ALL, up to 20% of patients encounter relapse. By current biomarkers, the majority of relapse patients is initially not identified indicating the need for prognostic and therapeutic targets reflecting leukemia biology. We previously described that rapid engraftment of patient ALL cells transplanted onto NOD/SCID mice (short time to leukemia, TTLshort) is indicative of early patient relapse. Gene expression profiling identified genes coding for molecules involved in mTOR signaling to be associated with TTLshort/early relapse leukemia. Here, we now functionally address mTOR signaling activity in primograft ALL samples and evaluate mTOR pathway inhibition as novel treatment strategy for high-risk ALL ex vivo and in vivo. By analysis of S6-phosphorylation downstream of mTOR, increased mTOR activation was found in TTLshort/high-risk ALL, which was effectively abrogated by mTOR inhibitors resulting in decreased leukemia proliferation and growth. In a preclinical setting treating individual patient-derived ALL in vivo, mTOR inhibition alone, and even more pronounced together with conventional remission induction therapy, significantly delayed post-treatment leukemia reoccurrence in TTLshort/high-risk ALL. Thus, the TTLshort phenotype is functionally characterized by hyperactivated mTOR signaling and can effectively be targeted ex vivo and in vivo providing a novel therapeutic strategy for high-risk ALL.


Subject(s)
Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/metabolism , Animals , Child , Child, Preschool , Female , Humans , Infant , Infant, Newborn , Male , Mice , Mice, Inbred NOD , Mice, SCID , Molecular Targeted Therapy , Phosphatidylinositol 3-Kinases/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Random Allocation , Risk Factors , TOR Serine-Threonine Kinases/genetics , Transcriptome , Xenograft Model Antitumor Assays
20.
J Clin Oncol ; 28(11): 1919-27, 2010 Apr 10.
Article in English | MEDLINE | ID: mdl-20231685

ABSTRACT

PURPOSE Juvenile myelomonocytic leukemia (JMML) is a rare early childhood myelodysplastic/myeloproliferative disorder characterized by an aggressive clinical course. Age and hemoglobin F percentage at diagnosis have been reported to predict both survival and outcome after hematopoietic stem cell transplantation (HSCT). However, no genetic markers with prognostic relevance have been identified so far. We applied gene expression-based classification to JMML samples in order to identify prognostic categories related to clinical outcome. PATIENTS AND METHODS Samples of 44 patients with JMML were available for microarray gene expression analysis. A diagnostic classification (DC) model developed for leukemia and myelodysplastic syndrome classification was used to classify the specimens and identify prognostically relevant categories. Statistical analysis was performed to determine the prognostic value of the classification and the genes identifying prognostic categories were further analyzed through R software. RESULTS The samples could be divided into two major groups: 20 specimens were classified as acute myeloid leukemia (AML) -like and 20 samples as nonAML-like. Four patients could not be assigned to a unique class. The 10-year probability of survival after diagnosis of AML-like and nonAML-like patients was significantly different (7% v 74%; P = .0005). Similarly, the 10-year event-free survival after HSCT was 6% for AML-like and 63% for nonAML-like patients (P = .0010). CONCLUSION Gene expression-based classification identifies two groups of patients with JMML with distinct prognosis outperforming all known clinical parameters in terms of prognostic relevance. Gene expression-based classification could thus be prospectively used to guide clinical/therapeutic decisions.


Subject(s)
Biomarkers, Tumor/metabolism , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Leukemia, Myelomonocytic, Juvenile/classification , Leukemia, Myelomonocytic, Juvenile/diagnosis , Neoplasm Recurrence, Local/diagnosis , Adolescent , Biomarkers, Tumor/genetics , Child , Child, Preschool , Female , Humans , Infant , Leukemia, Myelomonocytic, Juvenile/therapy , Male , Neoplasm Recurrence, Local/therapy , Neoplasm Staging , Oligonucleotide Array Sequence Analysis , Survival Rate , Treatment Outcome
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