Subject(s)
Gene Rearrangement , Histone-Lysine N-Methyltransferase , Myeloid-Lymphoid Leukemia Protein , Neoplasm, Residual , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Humans , Myeloid-Lymphoid Leukemia Protein/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Histone-Lysine N-Methyltransferase/genetics , Neoplasm, Residual/genetics , Neoplasm, Residual/diagnosis , Adult , Male , Female , Middle Aged , Young Adult , Aged , Adolescent , PrognosisABSTRACT
Pediatric acute myeloid leukemia (AML) is a highly heterogeneous disease making standardized measurable residual disease (MRD) assessment challenging. Currently, patient-specific DNA-based assays are only rarely applied for MRD assessment in pediatric AML. We tested whether quantification of genomic breakpoint-specific sequences via quantitative polymerase chain reaction (gDNA-PCR) provides a reliable means of MRD quantification in children with non-standardrisk AML and compared its results to those obtained with state-of-the-art ten-color flow cytometry (FCM). Breakpointspecific gDNA-PCR assays were established according to Euro-MRD consortium guidelines. FCM-MRD assessment was performed according to the European Leukemia Network guidelines with adaptations for pediatric AML. Of 77 consecutively recruited non-standard-risk pediatric AML cases, 49 (64%) carried a chromosomal translocation potentially suitable for MRD quantification. Genomic breakpoint analysis returned a specific DNA sequence in 100% (41/41) of the cases submitted for investigation. MRD levels were evaluated using gDNA-PCR in 243 follow-up samples from 36 patients, achieving a quantitative range of at least 10-4 in 231/243 (95%) of samples. Comparing gDNA-PCR with FCM-MRD data for 183 bone marrow follow-up samples at various therapy timepoints showed a high concordance of 90.2%, considering a cut-off of ≥0.1%. Both methodologies outperformed morphological assessment. We conclude that MRD monitoring by gDNA-PCR is feasible in pediatric AML with traceable genetic rearrangements and correlates well with FCM-MRD in the currently applied clinically relevant range, while being more sensitive below that. The methodology should be evaluated in larger cohorts to pave the way for clinical application.
Subject(s)
Genomics , Leukemia, Myeloid, Acute , Humans , Child , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/therapy , Flow Cytometry , Gene Rearrangement , Neoplasm, Residual/diagnosis , Neoplasm, Residual/geneticsABSTRACT
KMT2A-rearranged acute lymphoblastic infant leukemia (KMT2A-r iALL) is associated with outsize risk of relapse and relapse mortality. We previously reported strong upregulation of the immediate early gene EGR3 in KMT2A::AFF1 iALL at relapse; now we provide analyses of the EGR3 regulome, which we assessed through binding and expression target analysis of an EGR3-overexpressing t(4;11) cell culture model. Our data identify EGR3 as a regulator of early B-lineage commitment. Principal component analysis of 50 KMT2A-r iALL patients at diagnosis and 18 at relapse provided strictly dichotomous separation of patients based on the expression of four B-lineage genes. Absence of B-lineage gene expression translates to more than two-fold poorer long-term event-free survival. In conclusion, our study presents four B-lineage genes with prognostic significance, suitable for gene expression-based risk stratification of KMT2A-r iALL patients.
Subject(s)
Myeloid-Lymphoid Leukemia Protein , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Humans , Infant , Early Growth Response Protein 3/genetics , Early Growth Response Protein 3/metabolism , Myeloid-Lymphoid Leukemia Protein/genetics , Myeloid-Lymphoid Leukemia Protein/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Up-RegulationABSTRACT
The most frequent genetic aberration leading to infant ALL (iALL) is the chromosomal translocation t(4;11), generating the fusion oncogenes KMT2A:AFF1 and AFF1:KMT2A, respectively. KMT2A-r iALL displays a dismal prognosis through high relapse rates and relapse-associated mortality. Relapse occurs frequently despite ongoing chemotherapy and without the accumulation of secondary mutations. A rational explanation for the observed chemo-resistance and satisfactory treatment options remain to be elucidated. We found that elevated ICOSLG expression level at diagnosis was associated with inferior event free survival (EFS) in a cohort of 43 patients with t(4;-11) iALL and that a cohort of 18 patients with iALL at relapse displayed strongly increased ICOSLG expression. Furthermore, co-culturing t(4;11) ALL cells (ICOSLGhi) with primary T-cells resulted in the development of Tregs. This was impaired through treatment with a neutralizing ICOSLG antibody. These findings imply ICOSLG (1) as a relapse-predicting biomarker, and (2) as a therapeutic target involved in a potential immune evasion relapse-mechanism of infant t(4;11) ALL.
ABSTRACT
To fight the COVID-19 pandemic caused by the RNA virus SARS-CoV-2, a global vaccination campaign is in progress to achieve the immunization of billions of people mainly with adenoviral vector- or mRNA-based vaccines, all of which encode the SARS-CoV-2 Spike protein. In some rare cases, cerebral venous sinus thromboses (CVST) have been reported as a severe side effect occurring 4-14 days after the first vaccination and were often accompanied by thrombocytopenia. Besides CVST, splanchnic vein thromboses (SVT) and other thromboembolic events have been observed. These events only occurred following vaccination with adenoviral vector-based vaccines but not following vaccination with mRNA-based vaccines. Meanwhile, scientists have proposed an immune-based pathomechanism and the condition has been coined vaccine-induced immune thrombotic thrombocytopenia (VITT). Here, we describe an unexpected mechanism that could explain thromboembolic events occurring with DNA-based but not with RNA-based vaccines. We show that DNA-encoded mRNA coding for Spike protein can be spliced in a way that the transmembrane anchor of Spike is lost, so that nearly full-length Spike is secreted from cells. Secreted Spike variants could potentially initiate severe side effects when binding to cells via the ACE2 receptor. Avoiding such splicing events should become part of a rational vaccine design to increase safety of prospective vaccines.
Subject(s)
COVID-19 Vaccines/adverse effects , COVID-19/prevention & control , Sinus Thrombosis, Intracranial/etiology , Thrombocytopenia/etiology , Vaccines, DNA/adverse effects , ChAdOx1 nCoV-19/adverse effects , Drug-Related Side Effects and Adverse Reactions/etiology , Humans , Pandemics , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Syndrome , Vaccination/adverse effects , Venous Thrombosis/etiologyABSTRACT
About 25% of the patients with the translocation t(11;19)(q23;p13.3)/KMT2A-MLLT1 present three-way or more complex fusions, associated with a worse prognosis, suggesting that a particular mechanism creates functional KMT2A fusions for this condition. In this work, we show a cryptic three-way translocation t(9;11;19). Interestingly, long-distance inverse polymerase chain reaction sequencing revealed a KMT2A-MLLT1 and the yet unreported out-of-frame SEC16A-KMT2A fusion, associated with low SEC16A expression and KMT2A overexpression, in an infant with B-acute lymphoblastic leukemia presenting a poor prognosis. Our case illustrates the importance of molecular cytogenetic tests in selecting cases for further investigations, which could open perspectives regarding novel therapeutic approaches for poor prognosis childhood leukemias.
Subject(s)
Endoplasmic Reticulum , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Child , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Humans , Infant , Myeloid-Lymphoid Leukemia Protein/genetics , Myeloid-Lymphoid Leukemia Protein/metabolism , Neoplasm Proteins/genetics , Nuclear Proteins/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Transcription Factors/genetics , Translocation, Genetic , Vesicular Transport ProteinsABSTRACT
BACKGROUND: The detection of KMT2A gene rearrangements have an important impact on the prognosis and management of acute leukemias. These alterations most commonly involve reciprocal translocations at specific breakpoint regions within KMT2A. To date, more than 100 translocation partner genes of KMT2A have been identified, with different effects on risk stratification. METHODS AND RESULTS: We report the case of a mature plasmacytoid dendritic cells proliferation associated with B lymphoblasts harboring a KMT2A-ARHGEF12 fusion. This rare rearrangement, resulting from a cryptic deletion on the long arm of chromosome 11, is located outside the known major and minor breakpoint regions of KMT2A, not reported to date. The review of the few cases of KMT2A-ARHGEF12 reveals the tendency of this deletion to occur in therapy related hematologic neoplasm and confer unfavorable prognosis. CONCLUSION: This review sheds light into the rare KMT2A-ARHGEF12 fusion in leukemia. Reporting rare chimeras is essential to improve knowledge about the biological mechanism and associated clinical consequences.
Subject(s)
Leukemia, Myeloid, Acute , Oncogene Proteins, Fusion/genetics , Bone Marrow/pathology , Fatal Outcome , Follow-Up Studies , Gene Rearrangement , Humans , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Male , Middle Aged , Neoplasm, Residual/diagnosis , PrognosisABSTRACT
Therapy-related acute myeloid leukemia (t-AML) following treatment with topoisomerase-II inhibitors has been increasingly reported. These compounds (e.g. etoposide) promote DNA damage and are associated with KMT2A rearrangements. They are widely used as first-line treatment in hemophagocytic lymphohistiocytosis (HLH). Here we describe a newborn who developed t-AML after HLH treatment. We provide detailed clinical, cytogenetic, and molecular characteristics of this patient, including the identification of a novel gene fusion - KMT2A-SNX9 - in t-AML. Considering the dismal outcome of this case, we discuss the side-effects of etoposide administration during HLH treatment in infants.
Subject(s)
Diploidy , Karyotype , Leukemia, Myeloid, Acute/chemically induced , Leukemia, Myeloid, Acute/genetics , Lymphohistiocytosis, Hemophagocytic/drug therapy , Oncogene Proteins, Fusion/genetics , Base Sequence , Child , Fatal Outcome , Humans , Infant , Infant, Newborn , MaleABSTRACT
Several studies demonstrated a relevant role of polymorphisms located within the HLA-B and -C loci and the Killer Immunoglobulin Receptors (KIRs) 3DL1 and 3DS1 in controlling HIV-1 replication. KIRs are regulatory receptors expressed at the surface of NK and CD8+ T-cells that specifically bind HLA-A and -B alleles belonging to the Bw4 supratype and all the -C alleles expressing the C1 or C2 supratype. We here disclose a novel signature associated with the Elite Controller but not with the long-term nonprogressor status concerning 2DS activating KIRs and HLA-C2 alleles insensitive to miRNA148a regulation. Overall, our findings support a crucial role of NK cells in the control of HIV-1 viremia.
Subject(s)
HIV Infections/immunology , HIV Infections/metabolism , HIV Infections/virology , HIV-1/immunology , HLA-C Antigens/immunology , Host-Pathogen Interactions/immunology , Receptors, KIR/agonists , Alleles , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/virology , Chromosomes, Human, Pair 19 , Chromosomes, Human, Pair 6 , Disease Progression , Genetic Predisposition to Disease , Genotype , HIV Infections/genetics , HLA-C Antigens/genetics , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/immunology , Host-Pathogen Interactions/genetics , Humans , Odds Ratio , Polymorphism, Genetic , Receptors, KIR/genetics , Receptors, KIR/metabolismABSTRACT
Usually, HLA typing has been performed either by serology-based typing incubating a panel of known anti-HLA antibodies with viable lymphocytes of unknown HLA type or by molecular typing including medium-resolution HLA typing by Sequence Specific Oligonucleotide Probes (SSOP) or high-resolution HLA typing by Sequence Based Typing (SBT). Traditionally, HLA antigens have been defined using serological techniques, but these methods have several disadvantages, such as low resolution, the requirement for viable cells, and cell surface expression of HLA molecules. HLA type screening methods are categorized as low, medium, and high resolution, and only sequencing-based typing methods provide the highest resolution and are considered the gold standard for HLA typing.Among the HLA SBT based-methods, the Pyrosequencing(®) technique is an extremely versatile and accurate real-time sequencing technique with some advantages compared to classic Sanger method.Here, we describe a quick and inexpensive method that allows through the use of Pyrosequencing subtyping of HLA class I molecules, into HLA-Bw6, -Bw4 I80, or -Bw4 T80 and HLA-C1, or -C2 groups. In particular, this analysis is focused on the amino acids around residue 80. This method demonstrated good sensitivity, specificity, and reproducibility. Using a quantitative allele acquisition mode, the method provides accurate sequence information required for the definition of heterozygous and/or homozygous samples.
Subject(s)
HLA Antigens/genetics , HLA Antigens/immunology , HLA-C Antigens/genetics , HLA-C Antigens/immunology , Histocompatibility Testing/methods , Sequence Analysis, DNA/methods , Alleles , Genetic Loci/genetics , Genomics , Humans , Polymerase Chain Reaction , Polymorphism, Single NucleotideABSTRACT
OBJECTIVE: To test the ability of dorzolamide hydrochloride and timolol maleate to display antioxidant effects. METHODS: Antioxidant activity was tested in whole trabecular meshwork (TM) tissue as collected from corneal donors' biopsy specimens, young (third passage) and old (10th passage) human TM cells, and acellular systems composed of pure DNA and subcellular fractions containing or devoid of mitochondria. Oxidative stress was induced by hydrogen peroxide. Monitored end points included DNA fragmentation as evaluated by the halo test, oxidative DNA damage in terms of 8-hydroxy-2'-deoxyguanosine, and mitochondrial function as evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide test. RESULTS: The antioxidant effect of dorzolamide and timolol were observed on TM biopsy specimens and human TM cells exposed to hydrogen peroxide. As evaluated in cell subfractions, timolol displays antioxidant activity regardless of mitochondria presence. Conversely, the antioxidant activity of dorzolamide was maximized in the presence of mitochondria-containing subcellular fractions and in young human TM cells with functional mitochondria. CONCLUSIONS: The antioxidant effect of timolol was direct. The antioxidant effect of dorzolamide involves mitochondria and is likely to be exerted mainly during the early glaucoma phases when the mitochondrial damage in the TM tissue still occurs at low levels. Clinical Relevance Timolol has an antioxidant effect on the entire cell, whereas dorzolamide exerts protective activity toward oxidative stress only in the presence of intact mitochondria (ie, in endothelial cells that are younger when the cellular damage is still limited). The important role of mitochondrial damage in primary open- angle glaucoma is supported by the finding that mutant myocilin impairs mitochondrial functions in human TM meshwork cells.
Subject(s)
Antioxidants/pharmacology , Glaucoma, Open-Angle/drug therapy , Mitochondria/drug effects , Sulfonamides/pharmacology , Thiophenes/pharmacology , Timolol/pharmacology , Trabecular Meshwork/drug effects , 8-Hydroxy-2'-Deoxyguanosine , Autoradiography , Cell Line , Chromatography, Thin Layer , DNA Fragmentation , Deoxyguanosine/analogs & derivatives , Deoxyguanosine/metabolism , Humans , Hydrogen Peroxide/toxicity , Oxidative Stress/drug effects , Tissue DonorsABSTRACT
We have investigated alterations of microRNA expression profiles in the apparently healthy lung of mice and rats as an early response to exposure to cigarette smoke, either mainstream (MCS) or environmental, and/or to treatment with chemopreventive agents. Further on, we evaluated microRNA alterations at a later stage, when lung tumors were detectable in MCS-exposed mice. Lung samples were available from previous studies, in which strain H mice had been exposed to MCS for 4 months, starting immediately after birth, and then kept in filtered air for an additional 3 months. Some samples were from MCS-exposed mice treated either with N-acetyl-l-cysteine during pregnancy or with phenethyl isothiocyanate after weaning. The analysis of 576 mouse microRNAs showed that MCS strongly dysregulated microRNA expression and that both chemopreventive agents efficiently attenuated this trend, especially in noncancer tissue. MicroRNA expression was affected by histopathology, with specific signatures related to occurrence of pneumonia, adenoma, or bronchoalveolar carcinoma. Within pairs of samples from individual mice, microRNA analysis discriminated adenomatous tissue and especially carcinomatous tissue from the surrounding normal appearing tissue. A series of microRNA alterations characterized the sequential stages of pulmonary carcinogenesis. The involved functions included oncogene activation, inhibition of oncosuppressor genes, recruitment of undifferentiated stem cells, inflammation, inhibition of gap-junctional intercellular communications, angiogenesis, invasiveness, and metastatization. Thus, microRNA expression profiles in lung are dysregulated by MCS along all steps of the carcinogenesis process and depend on the interplay among exposure to noxious agents, treatment with dietary and pharmacological agents, and occurrence of pulmonary diseases.
Subject(s)
Anticarcinogenic Agents/metabolism , Lung Neoplasms , Lung/pathology , Lung/physiology , Nicotiana/toxicity , Pneumonia , Tobacco Smoke Pollution/adverse effects , Animals , Anticarcinogenic Agents/pharmacology , Female , Gene Expression Profiling , Humans , Lung/drug effects , Lung Neoplasms/chemically induced , Lung Neoplasms/pathology , Lung Neoplasms/physiopathology , Mice , Pneumonia/chemically induced , Pneumonia/pathology , Pneumonia/physiopathology , Pregnancy , Principal Component Analysis , Rats , Smoke/adverse effects , Smoking/adverse effectsABSTRACT
Our previous studies demonstrated that exposure to cigarette smoke (CS), either mainstream or environmental, results in a remarkable downregulation of microRNA expression in the lung of both mice and rats. The goals of the present study were to evaluate the dose responsiveness to CS and the persistence of microRNA alterations after smoking cessation. ICR (CD-1) neonatal mice were exposed whole-body to mainstream CS, at the doses of 119, 292, 438, and 631mg/m(3) of total particulate matter. Exposure started within 12h after birth and continued daily for 4 weeks. The levels of bulky DNA adducts and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) were measured by (32)P postlabeling procedures, and the expression of 697 mouse microRNAs was analyzed by microarray. The highest CS dose was lethal. Exposure to CS caused a dose-dependent increase of DNA alterations. DNA adducts and, even more sharply, 8-oxodGuo were reverted 1 and 4 weeks after smoking cessation. Exposure to CS resulted in an evident dysregulation of microRNA expression profiles, mainly in the sense of downregulation. The two lowest doses were not particularly effective, while the highest nonlethal dose produced extensive microRNA alterations. The expression of most downregulated microRNAs, including among others 7 members of the let-7 family, was restored one week after smoking cessation. However, the recovery was incomplete for a limited array of microRNAs, including mir-34b, mir-345, mir-421, mir-450b, mir-466, and mir-469. Thus, it appears that microRNAs mainly behave as biomarkers of effect and that exposure to high-dose, lasting for an adequate period of time, is needed to trigger the CS-related carcinogenesis process in the experimental animal model used.
Subject(s)
Gene Expression Regulation/drug effects , Lung/drug effects , Lung/physiology , MicroRNAs/metabolism , Nicotiana/toxicity , Smoke/adverse effects , Tobacco Smoke Pollution/adverse effects , 8-Hydroxy-2'-Deoxyguanosine , Animals , Animals, Newborn , Biomarkers/metabolism , Cluster Analysis , DNA Adducts/chemistry , Deoxyguanosine/analogs & derivatives , Deoxyguanosine/chemistry , Dose-Response Relationship, Drug , Female , Mice , MicroRNAs/genetics , Microarray Analysis , Pregnancy , Principal Component Analysis , Rats , Smoking/adverse effects , Smoking CessationABSTRACT
Although microRNAs (miRNA) have extensively been investigated in cancer research, less attention has been paid to their regulation by carcinogens and/or protective factors in early stages of the carcinogenesis process. The present study was designed to evaluate the modulation of mRNA expression as related to exposure of neonatal mice to environmental cigarette smoke (ECS) and to treatment with chemopreventive agents. Exposure to ECS started immediately after birth and for 2 weeks after weaning. Thereafter, groups of mice received daily either budesonide (BUD) or phenethyl isothiocyanate (PEITC) with the diet. The expression of 576 miRNAs was evaluated by miRNA microarray in liver and lung. In sham-exposed mice, the expression of miRNAs tended to be higher in liver than in lung. ECS downregulated the expression of a number of miRNAs in lung, whereas mixed alterations were observed in liver. PEITC and BUD did not substantially affect the physiological situation in lung, whereas both agents caused intense variations in liver, reflecting the occurrence of damage mechanisms, such as inflammation, DNA and protein damage, cellular stress, proliferation and apoptosis. PEITC and BUD protected the lung from ECS-induced alterations of miRNA expression but exhibited some adverse effects in liver.
Subject(s)
Anticarcinogenic Agents/pharmacology , Budesonide/pharmacology , Isothiocyanates/pharmacology , Liver/drug effects , Lung/drug effects , MicroRNAs/analysis , Nicotiana/adverse effects , Smoke/adverse effects , Animals , Female , Gene Expression Regulation/drug effects , Liver/metabolism , Lung/metabolism , MiceABSTRACT
Mice are particularly susceptible to carcinogens when exposure starts early in life. We evaluated the expression of stem cell antigen-1 (Sca-1) gene in the lung of variously aged CD-1 mice, either untreated or exposed to environmental cigarette smoke (ECS) and/or to a light source. Sca-1 expression progressively decreased with age. The expression of Sca-1 gene and the amount of Sca-1 protein, which was exclusively localized in endothelial cells of the pulmonary vasculature, were significantly upregulated in mice exposed either to ECS or ECS plus light throughout the weaning period, starting at birth. These findings may contribute to explain the high vulnerability of mouse lung early in life.
Subject(s)
Antigens, Ly/genetics , Lung/metabolism , Membrane Proteins/genetics , Tobacco Smoke Pollution , Age Factors , Animals , Animals, Newborn , Antigens, Ly/analysis , Immunohistochemistry , Lung/chemistry , Membrane Proteins/analysis , Mice , Up-RegulationABSTRACT
Interferon (IFN)-alpha is a cytokine with marked therapeutic activity in transplantable tumor models, that is in part due to angiogenesis inhibition. Aim of this study was to investigate the effects of IFN-alpha during the early phases of tumor development in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. To provide sustained IFN-alpha production, TRAMP mice were injected intraperitoneally with lentiviral vectors. IFN-alpha administration resulted in rapid and protracted upregulation of IFN-alpha-regulated genes associated with antiangiogenic and antiproliferative functions in the prostate of TRAMP mice, including guanylate-binding protein 1 (GBP-1), IFI204 and CXCL10-11. These transcriptional changes were accompanied by effects on the tumor vasculature, including significant reduction of intraductal microvessel density and increased pericyte coverage, and marked reduction of tumor cell proliferation, without induction of tumor necrosis. Intriguingly, GBP-1 and myxovirus resistance A, two IFN-regulated proteins, were found expressed in approximately 40% of human prostate cancer samples analyzed, suggesting expression of endogenous IFN-alpha. Overall, these findings demonstrate that IFN-alpha is able to counteract the angiogenic switch and impairs tumor cell proliferation in preinvasive lesions. Since the angiogenic switch also marks progression of human prostatic cancer, these results highlight the potential of angiogenesis inhibitors for the development of chemoprevention strategies in high-risk individuals.