- MeSH
- bcr-abl fúzní proteiny genetika MeSH
- chronická myeloidní leukemie * diagnóza genetika MeSH
- lidé MeSH
- myeloidní leukemie * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- dopisy MeSH
- práce podpořená grantem MeSH
Lipocalin 2 (LCN2), a proinflammatory mediator, is involved in the pathogenesis of myeloproliferative neoplasms (MPN). Here, we investigated the molecular mechanisms of LCN2 overexpression in MPN. LCN2 mRNA expression was 20-fold upregulated in peripheral blood (PB) mononuclear cells of chronic myeloid leukemia (CML) and myelofibrosis (MF) patients vs. healthy controls. In addition, LCN2 serum levels were significantly increased in polycythemia vera (PV) and MF and positively correlated with JAK2V617F and mutated CALR allele burden and neutrophil counts. Mechanistically, we identified endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) as a main driver of LCN2 expression in BCR-ABL- and JAK2V617F-positive 32D cells. The UPR inducer thapsigargin increased LCN2 expression >100-fold, and this was not affected by kinase inhibition of BCR-ABL or JAK2V617F. Interestingly, inhibition of the UPR regulators inositol-requiring enzyme 1 (IRE1) and c-Jun N-terminal kinase (JNK) significantly reduced thapsigargin-induced LCN2 RNA and protein expression, and luciferase promoter assays identified nuclear factor kappa B (NF-κB) and CCAAT binding protein (C/EBP) as critical regulators of mLCN2 transcription. In conclusion, the IRE1-JNK-NF-κB-C/EBP axis is a major driver of LCN2 expression in MPN, and targeting UPR and LCN2 may represent a promising novel therapeutic approach in MPN.
- Publikační typ
- časopisecké články MeSH
Myeloproliferative neoplasms (MPN), comprising essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF), are hematological disorders of the myeloid lineage characterized by hyperproliferation of mature blood cells. The prediction of the clinical course and progression remains difficult and new therapeutic modalities are required. We conducted a CD34+ gene expression study to identify signatures and potential biomarkers in the different MPN subtypes with the aim to improve treatment and prevent the transformation from the rather benign chronic state to a more malignant aggressive state. We report here on a systematic gene expression analysis (GEA) of CD34+ peripheral blood or bone marrow cells derived from 30 patients with MPN including all subtypes (ET (n = 6), PV (n = 11), PMF (n = 9), secondary MF (SMF; post-ET-/post-PV-MF; n = 4)) and six healthy donors. GEA revealed a variety of differentially regulated genes in the different MPN subtypes vs. controls, with a higher number in PMF/SMF (200/272 genes) than in ET/PV (132/121). PROGENγ analysis revealed significant induction of TNFα/NF-κB signaling (particularly in SMF) and reduction of estrogen signaling (PMF and SMF). Consistently, inflammatory GO terms were enriched in PMF/SMF, whereas RNA splicing-associated biological processes were downregulated in PMF. Differentially regulated genes that might be utilized as diagnostic/prognostic markers were identified, such as AREG, CYBB, DNTT, TIMD4, VCAM1, and S100 family members (S100A4/8/9/10/12). Additionally, 98 genes (including CLEC1B, CMTM5, CXCL8, DACH1, and RADX) were deregulated solely in SMF and may be used to predict progression from early to late stage MPN.
- MeSH
- antigeny CD34 genetika MeSH
- esenciální trombocytemie genetika MeSH
- lidé MeSH
- myeloproliferativní poruchy genetika MeSH
- polycythaemia vera genetika MeSH
- primární myelofibróza genetika MeSH
- regulace genové exprese u nádorů MeSH
- transkriptom * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Pegylated interferon-α (peg-IFNa) treatment induces molecular responses (MR) in patients with myeloproliferative neoplasms (MPNs), including partial MR (PMR) in 30-40% of patients. Here, we compared the efficacy of IFNa treatment in JAK2V617F- vs. calreticulin (CALR)-mutated cells and investigated the mechanisms of differential response. Retrospective analysis of MPN patients treated with peg-IFNa demonstrated that patients harboring the JAK2V617F mutation were more likely to achieve PMR than those with mutated CALR (p = 0.004), while there was no significant difference in hematological response. In vitro experiments confirmed an upregulation of IFN-stimulated genes in JAK2V617F-positive 32D cells as well as patient samples (peripheral blood mononuclear cells and CD34+ hematopoietic stem cells) compared to their CALR-mutated counterparts, and higher IFNa doses were needed to achieve the same IFNa response in CALR- as in JAK2V617F-mutant 32D cells. Additionally, Janus-activated kinase-1 (JAK1) and signal transducers and activators of transcription 1 (STAT1) showed constitutive phosphorylation in JAK2V617F-mutated but not CALR-mutated cells, indicating priming towards an IFNa response. Moreover, IFN-induced growth arrest was counteracted by selective JAK1 inhibition but enhanced by JAK2 inhibition. In conclusion, our data suggest that, clinically, higher doses of IFNa are needed in CALR-mutated vs. JAK2V617F-positive patients and we suggest a model of JAK2V617F-JAK1/STAT1 crosstalk leading to a priming of JAK2V617F-positive cells to IFNa resulting in differential sensitivity.
- MeSH
- antivirové látky farmakologie MeSH
- apoptóza MeSH
- dospělí MeSH
- interferon alfa farmakologie MeSH
- Janus kinasa 1 genetika metabolismus MeSH
- Janus kinasa 2 genetika MeSH
- kalretikulin genetika MeSH
- lidé středního věku MeSH
- lidé MeSH
- mutace * MeSH
- myeloproliferativní poruchy farmakoterapie genetika metabolismus patologie MeSH
- myši MeSH
- nádorové biomarkery genetika metabolismus MeSH
- nádorové buňky kultivované MeSH
- následné studie MeSH
- prognóza MeSH
- proliferace buněk MeSH
- retrospektivní studie MeSH
- senioři MeSH
- transkripční faktor STAT1 genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- senioři MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: High-throughput bioinformatics analyses of next generation sequencing (NGS) data often require challenging pipeline optimization. The key problem is choosing appropriate tools and selecting the best parameters for optimal precision and recall. RESULTS: Here we introduce ToTem, a tool for automated pipeline optimization. ToTem is a stand-alone web application with a comprehensive graphical user interface (GUI). ToTem is written in Java and PHP with an underlying connection to a MySQL database. Its primary role is to automatically generate, execute and benchmark different variant calling pipeline settings. Our tool allows an analysis to be started from any level of the process and with the possibility of plugging almost any tool or code. To prevent an over-fitting of pipeline parameters, ToTem ensures the reproducibility of these by using cross validation techniques that penalize the final precision, recall and F-measure. The results are interpreted as interactive graphs and tables allowing an optimal pipeline to be selected, based on the user's priorities. Using ToTem, we were able to optimize somatic variant calling from ultra-deep targeted gene sequencing (TGS) data and germline variant detection in whole genome sequencing (WGS) data. CONCLUSIONS: ToTem is a tool for automated pipeline optimization which is freely available as a web application at https://totem.software .
Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic diseases characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. The disease is driven by somatic mutations in exon 9 of CALR or exon 10 of MPL or JAK2-V617F in >90% of the cases, whereas the remaining cases are termed "triple negative." We aimed to identify the disease-causing mutations in the triple-negative cases of ET and PMF by applying whole-exome sequencing (WES) on paired tumor and control samples from 8 patients. We found evidence of clonal hematopoiesis in 5 of 8 studied cases based on clonality analysis and presence of somatic genetic aberrations. WES identified somatic mutations in 3 of 8 cases. We did not detect any novel recurrent somatic mutations. In 3 patients with clonal hematopoiesis analyzed by WES, we identified a somatic MPL-S204P, a germline MPL-V285E mutation, and a germline JAK2-G571S variant. We performed Sanger sequencing of the entire coding region of MPL in 62, and of JAK2 in 49 additional triple-negative cases of ET or PMF. New somatic (T119I, S204F, E230G, Y591D) and 1 germline (R321W) MPL mutation were detected. All of the identified MPL mutations were gain-of-function when analyzed in functional assays. JAK2 variants were identified in 5 of 57 triple-negative cases analyzed by WES and Sanger sequencing combined. We could demonstrate that JAK2-V625F and JAK2-F556V are gain-of-function mutations. Our results suggest that triple-negative cases of ET and PMF do not represent a homogenous disease entity. Cases with polyclonal hematopoiesis might represent hereditary disorders.
- MeSH
- esenciální trombocytemie genetika metabolismus MeSH
- exom * MeSH
- genomika metody MeSH
- inaktivace chromozomu X genetika MeSH
- Janus kinasa 2 genetika metabolismus MeSH
- lidé MeSH
- mutace * MeSH
- mutační analýza DNA MeSH
- myeloproliferativní poruchy genetika metabolismus MeSH
- primární myelofibróza genetika metabolismus MeSH
- receptory thrombopoetinu genetika metabolismus MeSH
- variabilita počtu kopií segmentů DNA MeSH
- vysoce účinné nukleotidové sekvenování * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
ATM abnormalities are frequent in chronic lymphocytic leukemia and represent an important prognostic factor. Sole 11q deletion does not result in ATM inactivation by contrast to biallelic defects involving mutations. Therefore, the analysis of ATM mutations and their functional impact is crucial. In this study, we analyzed ATM mutations in predominantly high-risk patients using: i) resequencing microarray and direct sequencing; ii) Western blot for total ATM level; iii) functional test based on p21 gene induction after parallel treatment of leukemic cells with fludarabine and doxorubicin. ATM dysfunction leads to impaired p21 induction after doxorubicin exposure. We detected ATM mutation in 16% (22 of 140) of patients, and all mutated samples manifested demonstrable ATM defect (impaired p21 upregulation after doxorubicin and/or null protein level). Loss of ATM function in mutated samples was also evidenced through defective p53 pathway activation after ionizing radiation exposure. ATM mutation frequency was 34% in patients with 11q deletion, 4% in the TP53-defected group, and 8% in wild-type patients. Our functional test, convenient for routine use, showed high sensitivity (80%) and specificity (97%) for ATM mutations prediction. Only cells with ATM mutation, but not those with sole 11q deletion, were resistant to doxorubicin. As far as fludarabine is concerned, this difference was not observed. Interestingly, patients from both these groups experienced nearly identical time to first treatment. In conclusion, ATM mutations either alone or in combination with 11q deletion uniformly led to demonstrable ATM dysfunction in patients with chronic lymphocytic leukemia and mutation presence can be predicted by the functional test using doxorubicin.
- MeSH
- ATM protein antagonisté a inhibitory genetika fyziologie MeSH
- chromozomální delece MeSH
- chronická lymfatická leukemie diagnóza genetika patologie MeSH
- dospělí MeSH
- doxorubicin farmakologie MeSH
- kohortové studie MeSH
- leukocyty mononukleární účinky léků patologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- lidské chromozomy, pár 11 genetika MeSH
- mutace genetika MeSH
- retrospektivní studie MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- viabilita buněk účinky léků fyziologie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Vyd. 1. 219 s. ; 20 cm
V autentickém životním příběhu sledujeme desetiletý zápas těžce zkoušené ženy o život a elementární lidskou důstojnost, a to v situaci, kdy ji lékaři, společnost i manžel už odepsali. Příznaky závažné nemoci se u Ivety Kollertové začaly projevovat v šestatřiceti letech. V té době byli ona i její manžel bez zaměstnání a téměr v bezdomoví se starali o dvě děti, přičemž svým zdravotním problémům zprvu nevěnovala pozornost. Trvalo delší dobu, než lékaři vyřkli ortel: rakovina vnitřních orgánů ve velmi pokročilém stadiu, vyhlídky na další život mizivé. Iveta leží několik let takřka sama v bytě, nepohyblivá, s rozpadávající se tělesnou schránkou, až si jednoho dne řekne, že to nevzdá a rozhodne se něco změnit, rozhodne se pro život... Autorčino vyprávění, proložené jejími básněmi a dopisy s přáteli, vzniklo ve spolupráci se spisovatelkou Blankou Kubešovou, která patří k několika málo lidem, kteří Ivetě dokázali nabídnout pomocnou ruku a zasadit se za její práva u příslušných úřadů a institucí. Kniha je také svědectvím o selhávání sociálního systému v naší zemi i poděkováním těm, kteří se nezištně zasloužili o to, aby se Ivetin život stal snesitelným.
- MeSH
- nádory psychologie terapie MeSH
- Publikační typ
- autobiografie MeSH
- Konspekt
- Patologie. Klinická medicína
- Biografie
- NLK Obory
- humanitní vědy a umění
- lékařství
- O autorovi
- Kollertová, Iveta, 1963- Autorita
