• Klíčová slova
• mutace NPM1,
• MeSH
• akutní myeloidní leukemie * diagnóza   genetika   MeSH
• imunohistochemie MeSH
• lidé MeSH
• mutace * MeSH
• myelodysplastické syndromy diagnóza   genetika   MeSH
• nukleofosmin genetika   škodlivé účinky   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• dopisy MeSH

• Klíčová slova
• mutace NPM1, inhibitory meninu,
• MeSH
• akutní myeloidní leukemie * diagnóza   farmakoterapie   genetika   patofyziologie   MeSH
• antitumorózní látky terapeutické užití   MeSH
• farmakoterapie klasifikace   MeSH
• imunohistochemie MeSH
• imunoterapie adoptivní metody   MeSH
• lidé MeSH
• mutace * MeSH
• nukleofosmin genetika   škodlivé účinky   MeSH
• prognóza MeSH
• protein exportin 1 antagonisté a inhibitory   MeSH
• reziduální nádor MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• přehledy MeSH

The nucleophosmin 1 (NPM1) gene is one of the most commonly mutated genes in acute myeloid leukemia (AML), occurring in approximately 60% of adult cytogenetically normal AML (CN-AML). To date, these mutations have only been detected in cells of the myeloid lineage, whereas the potential clonal involvement of the lymphoid lineage is controversial. In our study, NPM1 mutations were analyzed using the highly sensitive real-time quantitative polymerase chain reaction (RQ-PCR) method on fluorescence-activated cell-sorted (FACS) purified different circulating mature cell populations in patients with NPM1-mutated CN-AML. As expected, NPM1 mutations were found in myeloid blood cells, including CD14(+) monocytes and CD66b(+) granulocytes. However, we were also able to detect NPM1 mutations in CD19(+) B cells and CD3(-)14(-)16(+)56(+) natural killer (NK) cells, albeit at lower levels. Surprisingly, mutations were also detected in CD3(+) T cells from all analyzed patients. Our data demonstrate that NPM1-mutated CN-AML originates in an early stem cell with both lymphoid and myeloid differentiation potential.

• MeSH
• akutní myeloidní leukemie diagnóza   farmakoterapie   genetika   MeSH
• fenotyp MeSH
• genová dávka MeSH
• imunofenotypizace MeSH
• jaderné proteiny genetika   MeSH
• karyotypizace MeSH
• leukocyty metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mutace * MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Anaplastic large cell lymphoma (ALCL) is a T-cell malignancy predominantly driven by a hyperactive anaplastic lymphoma kinase (ALK) fusion protein. ALK inhibitors, such as crizotinib, provide alternatives to standard chemotherapy with reduced toxicity and side effects. Children with lymphomas driven by nucleophosmin 1 (NPM1)-ALK fusion proteins achieved an objective response rate to ALK inhibition therapy of 54% to 90% in clinical trials; however, a subset of patients progressed within the first 3 months of treatment. The mechanism for the development of ALK inhibitor resistance is unknown. Through genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) activation and knockout screens in ALCL cell lines, combined with RNA sequencing data derived from ALK inhibitor-relapsed patient tumors, we show that resistance to ALK inhibition by crizotinib in ALCL can be driven by aberrant upregulation of interleukin 10 receptor subunit alpha (IL10RA). Elevated IL10RA expression rewires the STAT3 signaling pathway, bypassing otherwise critical phosphorylation by NPM1-ALK. IL-10RA expression does not correlate with response to standard chemotherapy in pediatric patients, suggesting that a combination of crizotinib and chemotherapy could prevent ALK inhibitor resistance-specific relapse.

• MeSH
• anaplastický velkobuněčný lymfom farmakoterapie   genetika   metabolismus   patologie   MeSH
• antitumorózní látky farmakologie   terapeutické užití   MeSH
• biologické modely MeSH
• buněčné linie MeSH
• chemorezistence genetika   MeSH
• CRISPR-Cas systémy MeSH
• editace genu MeSH
• exprese genu MeSH
• imunohistochemie MeSH
• inhibitory proteinkinas farmakologie   terapeutické užití   MeSH
• krizotinib farmakologie   terapeutické užití   MeSH
• lidé MeSH
• receptor interleukinu-10 - alfa-podjednotka genetika   metabolismus   MeSH
• signální transdukce účinky léků   MeSH
• transkripční faktor STAT3 metabolismus   MeSH
• tyrosinkinasy genetika   metabolismus   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The immune system is important for elimination of residual leukemic cells during acute myeloid leukemia (AML) therapy. Anti-leukemia immune response can be inhibited by various mechanisms leading to immune evasion and disease relapse. Selected markers of immune escape were analyzed on AML cells from leukapheresis at diagnosis (N = 53). Hierarchical clustering of AML immunophenotypes yielded distinct genetic clusters. In the absence of DNMT3A mutation, NPM1 mutation was associated with decreased HLA expression and low levels of other markers (CLIP, PD-L1, TIM-3). Analysis of an independent cohort confirmed decreased levels of HLA transcripts in patients with NPM1 mutation. Samples with combined NPM1 and DNMT3A mutations had high CLIP surface amount suggesting reduced antigen presentation. TIM-3 transcript correlated not only with TIM-3 surface protein but also with CLIP and PD-L1. In our cohort, high levels of TIM-3/PD-L1/CLIP were associated with lower survival. Our results suggest that AML genotype is related to blast immunophenotype, and that high TIM-3 transcript levels in AML blasts could be a marker of immune escape. Cellular pathways regulating resistance to the immune system might contribute to the predicted response to standard therapy of patients in specific AML subgroups and should be targeted to improve AML treatment.

• MeSH
• akutní myeloidní leukemie * diagnóza   genetika   MeSH
• antigeny CD274 genetika   MeSH
• biologické markery MeSH
• buněčný receptor 2 viru hepatitidy A genetika   MeSH
• DNA methyltransferasa 3A * genetika   MeSH
• lidé MeSH
• mutace MeSH
• nukleofosmin * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: NPM1 plasmid standards are required for absolute quantification of minimal residual disease in acute myeloid leukemia patients. The standards are usually obtained, next to commercially constructed gene fragments, from transgenic bacteria colonies. However, this procedure is laborious and very time consuming. METHODS AND RESULTS: We have developed a PCR method that speeds up, simplifies, and streamlines the process of preparing NPM1 plasmid standards. The method is based on a combination of three primers, two surrounding the usual NPM1 mutation position and one over the mutation site. With this method, we were able to clearly distinguish plasmids with at least 15 different NPM1 mutations from the wild-type NPM1 plasmid. CONCLUSIONS: With the new approach, preparing NPM1 plasmid standards is easier, identifying NPM1-positive colonies is possible in less than a day and moreover, for a lower price than commercially constructed gene fragments.

• MeSH
• akutní myeloidní leukemie * genetika   MeSH
• jaderné proteiny * genetika   MeSH
• lidé MeSH
• mutace genetika   MeSH
• nukleofosmin MeSH
• plazmidy genetika   MeSH
• reziduální nádor genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Nucleophosmin (NPM1) mutations in exon 12 are the most common genetic alternation in cytogenetically normal AML (CN-AML). Although mutation types A, B, and D represent the majority of cases, rare mutation variants of the NPM1 gene in individual patients do occur. In this study, we have evaluated a novel, DNA-based real-time quantitative polymerase chain reaction (RQ-PCR) for the detection of three of the most commonly occurring mutations and for six rare patient-specific mutation types, which represent 28% of all of the NPM1 mutations in our group of 25 CN-AML patients. Furthermore, the prognostic relevance of NPM1-based monitoring of minimal residual disease (MRD) in peripheral blood (PB), bone marrow (BM), and in specific cell subsets (CD34(+), CD34(-), CD34(dim)) of BM were evaluated. In 80% of the evaluable patients, a molecular relapse preceded a hematological relapse. Moreover, in this subset of patients, the molecular relapse occurred at a median of 97 days before the hematological relapse. Our compartment analysis showed a strong correlation between BM and PB (r = 0.907, P < 0.001) as well as a high copy number of mutated NPM1 in CD34(+) BM cells. In conclusion, we have demonstrated applicability of our presented RQ-PCR method for a large percentage of mutated NPM1 patients with CN-AML as well as the usefulness for long-term follow-up monitoring of MRD and the prediction of hematological relapse.

• MeSH
• akutní myeloidní leukemie diagnóza   genetika   MeSH
• antigeny CD34 MeSH
• buňky kostní dřeně MeSH
• dospělí MeSH
• genová dávka MeSH
• jaderné proteiny genetika   MeSH
• krevní buňky MeSH
• lidé středního věku MeSH
• lidé MeSH
• mutace MeSH
• mutační analýza DNA metody   MeSH
• prediktivní hodnota testů MeSH
• prognóza MeSH
• recidiva MeSH
• reziduální nádor diagnóza   genetika   MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

OBJECTIVES: Interaction of leukemia cells with the bone marrow extracellular matrix promotes cell survival and resistance to chemotherapy. In this work, we analyzed integrin expression and adhesivity to fibronectin in primary cells from patients with acute myeloid leukemia. METHODS: Surface expression of integrins β1 and αVβ3 on primary leukemia cells (N = 46) was correlated with the stem cell marker CD34, as well as with cell adhesivity to fibronectin. The results were analyzed with regard to the mutational status of NPM1 and FLT3 genes. RESULTS: The integrin β1 was omnipresent, whereas αVβ3 was often more expressed on CD34-positive cells. In particular, higher αVβ3 expression on CD34+ cells was associated with NPM1 mutation (P = .0018). Monocytic leukemias had significantly higher αVβ3 expression compared to less maturated cases (P = .0008). Cells from patients with internal tandem duplications in FLT3 (FLT3-ITD) had lower adhesivity to fibronectin compared to cells with wild-type FLT3 (P = .031), specifically in less differentiated myeloblasts. Inhibition of a putative FLT3-ITD target, EZH2, increased cell adhesivity in MV4-11 cell line (P = .024). CONCLUSIONS: The integrin αVβ3 is expressed in particular on CD34+ cells with NPM1 mutation and might have a prognostic value in patients with mutated NPM1. FLT3-ITD is associated with lower cell adhesivity, especially in patients with less differentiated leukemias.

• MeSH
• akutní myeloidní leukemie genetika   metabolismus   MeSH
• buněčná adheze MeSH
• buněčná membrána metabolismus   MeSH
• duplikace genu MeSH
• exprese genu MeSH
• fibronektiny metabolismus   MeSH
• integriny genetika   metabolismus   MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• lidé MeSH
• mutace MeSH
• nádorové buněčné linie MeSH
• tyrosinkinasa 3 podobná fms genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• akutní myeloidní leukemie diagnóza   genetika   terapie   MeSH
• alely MeSH
• exony MeSH
• genotyp MeSH
• jaderné proteiny genetika   MeSH
• kombinovaná terapie metody   MeSH
• lidé MeSH
• mladý dospělý MeSH
• mutace * MeSH
• protokoly antitumorózní kombinované chemoterapie terapeutické užití   MeSH
• transplantace hematopoetických kmenových buněk MeSH
• Check Tag
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• Publikační typ
• dopisy MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH

PURPOSE: Nucleophosmin 1 (NPM1) mutations are associated with a favorable prognosis in acute myeloid leukemia (AML) when an internal tandem duplication (ITD) in the fms-related tyrosine kinase 3 gene (FLT3) is absent (FLT3-ITDneg) or present with a low allelic ratio (FLT3-ITDlow). The 2017 European LeukemiaNet guidelines assume this is true regardless of accompanying cytogenetic abnormalities. We investigated the validity of this assumption. METHODS: We analyzed associations between karyotype and outcome in intensively treated patients with NPM1mut/FLT3-ITDneg/low AML who were prospectively enrolled in registry databases from nine international study groups or treatment centers. RESULTS: Among 2,426 patients with NPM1mut/FLT3-ITDneg/low AML, 2,000 (82.4%) had a normal and 426 (17.6%) had an abnormal karyotype, including 329 patients (13.6%) with intermediate and 83 patients (3.4%) with adverse-risk chromosomal abnormalities. In patients with NPM1mut/FLT3-ITDneg/low AML, adverse cytogenetics were associated with lower complete remission rates (87.7%, 86.0%, and 66.3% for normal, aberrant intermediate, and adverse karyotype, respectively; P < .001), inferior 5-year overall (52.4%, 44.8%, 19.5%, respectively; P < .001) and event-free survival (40.6%, 36.0%, 18.1%, respectively; P < .001), and a higher 5-year cumulative incidence of relapse (43.6%, 44.2%, 51.9%, respectively; P = .0012). These associations remained in multivariable mixed-effects regression analyses adjusted for known clinicopathologic risk factors (P < .001 for all end points). In patients with adverse-risk chromosomal aberrations, we found no significant influence of the NPM1 mutational status on outcome. CONCLUSION: Karyotype abnormalities are significantly associated with outcome in NPM1mut/FLT3-ITDneg/low AML. When adverse-risk cytogenetics are present, patients with NPM1mut share the same unfavorable prognosis as patients with NPM1 wild type and should be classified and treated accordingly. Thus, cytogenetic risk predominates over molecular risk in NPM1mut/FLT3-ITDneg/low AML.

• MeSH
• akutní myeloidní leukemie genetika   MeSH
• chromozomální aberace MeSH
• dospělí MeSH
• jaderné proteiny genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mutace MeSH
• prognóza MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• tyrosinkinasa 3 podobná fms genetika   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH