GA trial is registered under EudraCT#: 2013-001639-38.

• MeSH
• azacytidin * terapeutické užití   MeSH
• faktor stimulující kolonie granulocytů MeSH
• lidé MeSH
• myelodysplastické syndromy * farmakoterapie   MeSH
• protinádorové antimetabolity terapeutické užití   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• dopisy MeSH
• práce podpořená grantem MeSH
• randomizované kontrolované studie MeSH
• Názvy látek
• azacytidin * MeSH
• faktor stimulující kolonie granulocytů MeSH
• protinádorové antimetabolity MeSH

The mechanisms by which myelodysplastic syndrome (MDS) cells resist the effects of hypomethylating agents (HMA) are currently the subject of intensive research. A better understanding of mechanisms by which the MDS cell becomes to tolerate HMA and progresses to acute myeloid leukemia (AML) requires the development of new cellular models. From MDS/AML cell lines we developed a model of 5-azacytidine (AZA) resistance whose stability was validated by a transplantation approach into immunocompromised mice. When investigating mRNA expression and DNA variants of the AZA resistant phenotype we observed deregulation of several cancer-related pathways including the phosphatidylinosito-3 kinase signaling. We have further shown that these pathways can be modulated by specific inhibitors that, while blocking the proliferation of AZA resistant cells, are unable to increase their sensitivity to AZA. Our data reveal a set of molecular mechanisms that can be targeted to expand therapeutic options during progression on AZA therapy.

• Klíčová slova
• Azacytidine, CDX mice, PI3K/AKT signaling, myelodysplastic syndrome, resistance,
• MeSH
• anotace sekvence MeSH
• azacytidin farmakologie   MeSH
• biologické modely * MeSH
• chemorezistence * účinky léků   genetika   MeSH
• DNA nádorová genetika   MeSH
• fosfatidylinositol-3-kinasy metabolismus   MeSH
• myši SCID MeSH
• myši MeSH
• protoonkogenní proteiny c-akt metabolismus   MeSH
• reprodukovatelnost výsledků MeSH
• signální transdukce účinky léků   MeSH
• transkriptom genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• azacytidin MeSH
• DNA nádorová MeSH
• protoonkogenní proteiny c-akt MeSH

Azacitidine (AZA) for higher risk MDS patients is a standard therapy with limited durability. To monitor mutation dynamics during AZA therapy we utilized massive parallel sequencing of 54 genes previously associated with MDS/AML pathogenesis. Serial sampling before and during AZA therapy of 38 patients (reaching median overall survival 24 months (Mo) with 60% clinical responses) identified 116 somatic pathogenic variants with allele frequency (VAF) exceeding 5%. High accuracy of data was achieved via duplicate libraries from myeloid cells and T-cell controls. We observed that nearly half of the variants were stable while other variants were highly dynamic. Patients with marked decrease of allelic burden upon AZA therapy achieved clinical responses. In contrast, early-progressing patients on AZA displayed minimal changes of the mutation pattern. We modeled the VAF dynamics on AZA and utilized a joint model for the overall survival and response duration. While the presence of certain variants associated with clinical outcomes, such as the mutations of CDKN2A were adverse predictors while KDM6A mutations yield lower risk of dying, the data also indicate that allelic burden volatility represents additional important prognostic variable. In addition, preceding 5q- syndrome represents strong positive predictor of longer overall survival and response duration in high risk MDS patients treated with AZA. In conclusion, variants dynamics detected via serial sampling represents another parameter to consider when evaluating AZA efficacy and predicting outcome.

• Klíčová slova
• AML, MDS, NGS, azacitidine, somatic mutation,
• Publikační typ
• časopisecké články MeSH

Primary cutaneous T-cell lymphomas (CTCL) affect the skin and tend to transform and spread. CTCL involves primarily the Mycosis fungoides (MF) and more aggressive Sezary syndrome (SS). Oncogenic microRNAs (miRs) are stable epigenetic inhibitors often deregulated in the tumour and detectable as biomarkers in non-cellular fractions of peripheral blood. The tumour-specific expression of miR-155, miR-203, and miR-205 was shown to correctly diagnose CTCL. We herein asked whether these microRNAs can be used as plasma biomarkers for clinical CTCL monitoring. Patients with CTCL (n = 10) and controls with non-malignant conditions (n = 11) repeatedly donated plasma samples every ca. five months. MicroRNAs were detected in the plasma samples by specifically-primed RT-PCR followed by multivariate analyses of the miR expression dynamics. We herein established the plasma miR-classifier for detecting CTCL based on the miR-155 upregulation and miR-203/miR-205 downregulation with 100% specificity and 94% sensitivity. The 3-miR-score in the consecutive samples coincided with the clinical outcome of MF and SS patients such as the therapy response or changes in the clinical stage or tumor size. Quantitation of the selected microRNAs in plasma is a specific and straightforward approach for evaluating CTCL outcome representing, thus, a valuable tool for CTCL diagnostics and therapy response monitoring.

• Klíčová slova
• Psoriasis vulgaris, Sezary syndrome, atopic dermatitis, cutaneous T-cell lymphomas (CTCL), microRNA, mycosis fungoides,
• MeSH
• cirkulující mikroRNA * MeSH
• kožní T-buněčný lymfom krev   diagnóza   genetika   terapie   MeSH
• kůže patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mikro RNA genetika   MeSH
• nádorové biomarkery * MeSH
• prognóza MeSH
• protokoly protinádorové kombinované chemoterapie terapeutické užití   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• staging nádorů MeSH
• stanovení celkové genové exprese MeSH
• studie případů a kontrol MeSH
• tekutá biopsie MeSH
• výsledek terapie MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cirkulující mikroRNA * MeSH
• mikro RNA MeSH
• MIRN155 microRNA, human MeSH Prohlížeč
• MIRN203 microRNA, human MeSH Prohlížeč
• MIRN205 microRNA, human MeSH Prohlížeč
• nádorové biomarkery * MeSH

The imitation switch nuclear ATPase Smarca5 (Snf2h) is one of the most conserved chromatin remodeling factors. It exists in a variety of oligosubunit complexes that move DNA with respect to the histone octamer to generate regularly spaced nucleosomal arrays. Smarca5 interacts with different accessory proteins and represents a molecular motor for DNA replication, repair, and transcription. We deleted Smarca5 at the onset of definitive hematopoiesis (Vav1-iCre) and observed that animals die during late fetal development due to anemia. Hematopoietic stem and progenitor cells accumulated but their maturation toward erythroid and myeloid lineages was inhibited. Proerythroblasts were dysplastic while basophilic erythroblasts were blocked in G2/M and depleted. Smarca5 deficiency led to increased p53 levels, its activation at two residues, one associated with DNA damage (S15Ph °s ) second with CBP/p300 (K376Ac ), and finally activation of the p53 targets. We also deleted Smarca5 in committed erythroid cells (Epor-iCre) and observed that animals were anemic postnatally. Furthermore, 4-hydroxytamoxifen-mediated deletion of Smarca5 in the ex vivo cultures confirmed its requirement for erythroid cell proliferation. Thus, Smarca5 plays indispensable roles during early hematopoiesis and erythropoiesis. Stem Cells 2017;35:1614-1623.

• Klíčová slova
• Cell cycle progression, Erythroid differentiation, Fetal liver erythropoiesis, Hematopoietic stem and progenitor cells, Hypoxia, Imitation switch, Smarca5, p53 pathway,
• MeSH
• adenosintrifosfatasy nedostatek   metabolismus   MeSH
• anemie patologie   MeSH
• buněčná diferenciace * MeSH
• buněčný cyklus MeSH
• chromozomální proteiny, nehistonové nedostatek   metabolismus   MeSH
• delece genu MeSH
• erytroidní buňky cytologie   MeSH
• erytropoéza MeSH
• genotyp MeSH
• hematopoetické kmenové buňky cytologie   metabolismus   MeSH
• hematopoéza MeSH
• messenger RNA genetika   metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• nádorový supresorový protein p53 metabolismus   MeSH
• poškození DNA genetika   MeSH
• proliferace buněk MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• adenosintrifosfatasy MeSH
• chromozomální proteiny, nehistonové MeSH
• messenger RNA MeSH
• nádorový supresorový protein p53 MeSH
• Smarca5 protein, mouse MeSH Prohlížeč

Richter syndrome represents the transformation of the chronic lymphocytic leukemia (CLL) into an aggressive lymphoma, most frequently the diffuse large B-cell lymphoma (DLBCL). In this report we describe a patient with CLL, who developed a clonally-related pleomorphic highly-aggressive mantle cell lymphoma (MCL) after five cycles of a fludarabine-based second-line therapy for the first relapse of CLL. Molecular cytogenetic methods together with whole-exome sequencing revealed numerous gene alterations restricted to the MCL clone (apart from the canonical t(11;14)(q13;q32) translocation) including gain of one copy of ATM gene or emergence of TP53, CREBBP, NUP214, FUBP1 and SF3B1 gene mutations. Similarly, gene expression analysis revealed vast differences between the MCL and CLL transcriptome, including overexpression of cyclin D1, downregulation of cyclins D2 and D3, or downregulation of IL4R in the MCL clone. Backtracking analysis using quantitative PCR specifically detecting an MCL-restricted focal deletion of TP53 revealed that the pre-MCL clone appeared in the bone marrow and peripheral blood of the patient approximately 4 years before the clinical manifestation of MCL. Both molecular cytogenetic and sequencing data support the hypothesis of a slow development of the pre-MCL clone in parallel to CLL over several years, and thereby exclude the possibility that the transformation event occurred at the stage of the CLL relapse clone by mere t(11;14)(q13;q32) acquisition.

• Klíčová slova
• Richteŕs transformation, chronic lymphocytic leukemia, clonal evolution, mantle cell lymphoma, transforming genes,
• MeSH
• chronická lymfatická leukemie genetika   metabolismus   patologie   MeSH
• difúzní velkobuněčný B-lymfom genetika   metabolismus   patologie   MeSH
• hybridizace in situ fluorescenční MeSH
• imunohistochemie MeSH
• lidé středního věku MeSH
• lidé MeSH
• lidské chromozomy, pár 11 MeSH
• lidské chromozomy, pár 14 MeSH
• lymfom z plášťových buněk genetika   metabolismus   patologie   MeSH
• nádorové biomarkery genetika   metabolismus   MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• translokace genetická MeSH
• ztráta heterozygozity MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• Názvy látek
• nádorové biomarkery MeSH
• nádorový supresorový protein p53 MeSH
• TP53 protein, human MeSH Prohlížeč

PURPOSE: Here, we studied whether amplicon next-generation deep sequencing (NGS) could improve the detection of emerging BCR-ABL1 kinase domain mutations in chronic phase chronic myeloid leukemia (CML) patients under tyrosine kinase inhibitor (TKI) treatment and discussed the clinical relevance of such sensitive mutational detection. METHODS: For NGS data evaluation including extraction of biologically relevant low-level variants from background error noise, we established and applied a robust and versatile bioinformatics approach. RESULTS: Results from a retrospective longitudinal analysis of 135 samples of 15 CML patients showed that NGS could have revealed emerging resistant mutants 2-11 months earlier than conventional sequencing. Interestingly, in cases who later failed first-line imatinib treatment, NGS revealed that TKI-resistant mutations were already detectable at the time of major or deeper molecular response. Identification of emerging mutations by NGS was mirrored by BCR-ABL1 transcript level expressed either fluctuations around 0.1 %(IS) or by slight transcript level increase. NGS also allowed tracing mutations that emerged during second-line TKI therapy back to the time of switchover. Compound mutants could be detected in three cases, but were not found to outcompete single mutants. CONCLUSIONS: This work points out, that next-generation deep sequencing, coupled with a robust bioinformatics approach for mutation calling, may be just in place to ensure reliable detection of emerging BCR-ABL1 mutations, allowing early therapy switch and selection of the most appropriate therapy. Further, prospective assessment of how to best integrate NGS in the molecular monitoring and clinical decision algorithms is warranted.

• MeSH
• bcr-abl fúzní proteiny genetika   MeSH
• benzamidy terapeutické užití   MeSH
• chronická myeloidní leukemie farmakoterapie   genetika   MeSH
• dospělí MeSH
• imatinib mesylát MeSH
• inhibitory proteinkinas terapeutické užití   MeSH
• lidé středního věku MeSH
• lidé MeSH
• longitudinální studie MeSH
• mutace účinky léků   MeSH
• piperaziny terapeutické užití   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• protinádorové látky terapeutické užití   MeSH
• pyrimidiny terapeutické užití   MeSH
• retrospektivní studie MeSH
• senioři MeSH
• tyrosinkinasy antagonisté a inhibitory   MeSH
• výpočetní biologie MeSH
• vysoce účinné nukleotidové sekvenování * MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bcr-abl fúzní proteiny MeSH
• benzamidy MeSH
• imatinib mesylát MeSH
• inhibitory proteinkinas MeSH
• piperaziny MeSH
• protinádorové látky MeSH
• pyrimidiny MeSH
• tyrosinkinasy MeSH

Mantle cell lymphoma (MCL) is an aggressive type of B-cell non-Hodgkin lymphoma (NHL) associated with poor prognosis. Animal models of MCL are scarce. We established and characterized various in vivo models of metastatic human MCL by tail vein injection of either primary cells isolated from patients with MCL or established MCL cell lines (Jeko-1, Mino, Rec-1, Hbl-2, and Granta-519) into immunodeficient NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ mice. MCL infiltration was assessed with immunohistochemistry (tissues) and flow cytometry (peripheral blood). Engraftment of primary MCL cells was observed in 7 out of 12 patient samples. The pattern of engraftment of primary MCL cells varied from isolated involvement of the spleen to multiorgan infiltration. On the other hand, tumor engraftment was achieved in all five MCL cell lines used and lymphoma involvement of murine bone marrow, spleen, liver, and brain was observed. Overall survival of xenografted mice ranged from 22 ± 1 to 54 ± 3 days depending on the cell line used. Subsequently, we compared the gene expression profile (GEP) and phenotype of the engrafted MCL cells compared with the original in vitro growing cell lines (controls). We demonstrated that engrafted MCL cells displayed complex changes of GEP, protein expression, and sensitivity to cytotoxic agents when compared with controls. We further demonstrated that our MCL mouse models could be used to test the therapeutic activity of systemic chemotherapy, monoclonal antibodies, or angiogenesis inhibitors. The characterization of MCL murine models is likely to aid in improving our knowledge in the disease biology and to assist scientists in the preclinical and clinical development of novel agents in relapsed/refractory MCL patients.

• MeSH
• imunofenotypizace MeSH
• imunohistochemie MeSH
• játra metabolismus   MeSH
• Kaplanův-Meierův odhad MeSH
• kostní dřeň metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• lymfom z plášťových buněk farmakoterapie   genetika   metabolismus   MeSH
• modely nemocí na zvířatech * MeSH
• mozek metabolismus   MeSH
• myši inbrední NOD MeSH
• myši knockoutované MeSH
• myši SCID MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádorové buňky kultivované MeSH
• receptory interleukinů - společná gama-podjednotka nedostatek   genetika   MeSH
• regulace genové exprese u nádorů * MeSH
• senioři MeSH
• slezina metabolismus   MeSH
• transkriptom genetika   MeSH
• transplantace heterologní MeSH
• zvířata MeSH
• Check Tag
• 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
• Názvy látek
• Il2rg protein, mouse MeSH Prohlížeč
• receptory interleukinů - společná gama-podjednotka MeSH

BACKGROUND: Mantle cell lymphoma (MCL) is an aggressive type of B-cell non-Hodgkin lymphoma associated with poor prognosis. Implementation of high-dose cytarabine (araC) into induction therapy became standard-of-care for all newly diagnosed younger MCL patients. However, many patients relapse even after araC-based regimen. Molecular mechanisms responsible for araC resistance in MCL are unknown and optimal treatment strategy for relapsed/refractory MCL patients remains elusive. METHODS: Five araC-resistant (R) clones were derived by long-term culture of five MCL cell lines (CTRL) with increasing doses of araC up to 50 microM. Illumina BeadChip and 2-DE proteomic analysis were used to identify gene and protein expression changes associated with araC resistance in MCL. In vitro cytotoxicity assays and experimental therapy of MCL xenografts in immunodeficient mice were used to analyze their relative responsiveness to a set of clinically used anti-MCL drugs. Primary MCL samples were obtained from patients at diagnosis and after failure of araC-based therapies. RESULTS: Marked downregulation of deoxycytidine-kinase (DCK) mRNA and protein expression was identified as the single most important molecular event associated with araC-resistance in all tested MCL cell lines and in 50% primary MCL samples. All R clones were highly (20-1000x) cross-resistant to all tested nucleoside analogs including gemcitabine, fludarabine and cladribine. In vitro sensitivity of R clones to other classes of clinically used anti-MCL agents including genotoxic drugs (cisplatin, doxorubicin, bendamustine) and targeted agents (bortezomib, temsirolimus, rituximab) remained unaffected, or was even increased (ibrutinib). Experimental therapy of immunodeficient mice confirmed the anticipated loss of anti-tumor activity (as determined by overall survival) of the nucleoside analogs gemcitabine and fludarabine in mice transplanted with R clone compared to mice transplanted with CTRL cells, while the anti-tumor activity of cisplatin, temsirolimus, bortezomib, bendamustine, cyclophosphamide and rituximab remained comparable between the two cohorts. CONCLUSIONS: Acquired resistance of MCL cells to araC is associated with downregulation of DCK, enzyme of the nucleotide salvage pathway responsible for the first phosphorylation (=activation) of most nucleoside analogs used in anti-cancer therapy. The data suggest that nucleoside analogs should not be used in the therapy of MCL patients, who relapse after failure of araC-based therapies.

• MeSH
• 2D gelová elektroforéza MeSH
• buněčné klony MeSH
• chemorezistence účinky léků   MeSH
• cytarabin farmakologie   MeSH
• deoxycytidin analogy a deriváty   farmakologie   MeSH
• deoxycytidinkinasa metabolismus   MeSH
• down regulace účinky léků   MeSH
• gemcitabin MeSH
• hmotnostní spektrometrie MeSH
• kladribin farmakologie   MeSH
• lidé MeSH
• lymfom z plášťových buněk farmakoterapie   enzymologie   genetika   MeSH
• myší monoklonální protilátky farmakologie   terapeutické užití   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• proteomika MeSH
• protinádorové látky farmakologie   terapeutické užití   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• rituximab MeSH
• stanovení celkové genové exprese MeSH
• vidarabin analogy a deriváty   farmakologie   MeSH
• western blotting MeSH
• xenogenní modely - testy protinádorové aktivity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytarabin MeSH
• deoxycytidin MeSH
• deoxycytidinkinasa MeSH
• fludarabine MeSH Prohlížeč
• gemcitabin MeSH
• kladribin MeSH
• myší monoklonální protilátky MeSH
• protinádorové látky MeSH
• rituximab MeSH
• vidarabin MeSH

Elevated levels of microRNA miR-155 represent a candidate pathogenic factor in chronic B-lymphocytic leukemia (B-CLL). In this study, we present evidence that MYB (v-myb myeloblastosis viral oncogene homolog) is overexpressed in a subset of B-CLL patients. MYB physically associates with the promoter of miR-155 host gene (MIR155HG, also known as BIC, B-cell integration cluster) and stimulates its transcription. This coincides with the hypermethylated histone H3K4 residue and spread hyperacetylation of H3K9 at MIR155HG promoter. Our data provide evidence of oncogenic activities of MYB in B-CLL that include its stimulatory role in MIR155HG transcription.

• MeSH
• chromatin chemie   genetika   metabolismus   MeSH
• chronická lymfatická leukemie genetika   metabolismus   MeSH
• genetická transkripce fyziologie   MeSH
• HeLa buňky MeSH
• lidé MeSH
• mikro RNA genetika   MeSH
• mikročipová analýza MeSH
• nádorové buňky kultivované MeSH
• onkogenní proteiny v-myb metabolismus   fyziologie   MeSH
• promotorové oblasti (genetika) MeSH
• regulace genové exprese u leukemie MeSH
• shluková analýza MeSH
• stanovení celkové genové exprese MeSH
• transfekce MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromatin MeSH
• mikro RNA MeSH
• MIRN155 microRNA, human MeSH Prohlížeč
• onkogenní proteiny v-myb MeSH