BACKGROUND: Immune-mediated mechanisms substantially contribute to the Rasmussen encephalitis (RE) pathology, but for unknown reasons, immunotherapy is generally ineffective in patients who have already developed intractable epilepsy; overall laboratory data regarding the effect of immunotherapy on patients with RE are limited. We analyzed multiple samples from seven differently treated children with RE and evaluated the effects of immunotherapies on neuroinflammation. Immunotherapy was introduced to all patients at the time of intractable epilepsy and they all had to undergo hemispherothomy. METHODS: Immunohistochemistry, flow cytometry, Luminex multiplex bead and enzyme-linked immunosorbent assay techniques were combined to determine: 1) inflammatory changes and lymphocyte subpopulations in 45 brain tissues; 2) lymphocyte subpopulations and the levels of 12 chemokines/cytokines in 24 cerebrospinal fluid (CSF) samples and 30 blood samples; and 3) the dynamics of these parameters in four RE patients from whom multiple samples were collected. RESULTS: Sustained T cell-targeted therapy with cyclophosphamide, natalizumab, alemtuzumab, and intrathecal methotrexate (ITMTX), but not with azathioprine, substantially reduced inflammation in brain tissues. Despite the therapy, the distributions of CD8+ T cells and the levels of C-X-C motif ligand (CXCL) 10, CXCL13, and B cell activating factor (BAFF) in patients' CSF remained increased compared to controls. A therapeutic approach combining alemtuzumab and ITMTX was the most effective in producing simultaneous reductions in histopathological inflammatory findings and in the numbers of activated CD8+ T cells in the brain tissue, as well as in the overall CD8+ T cell population and chemokine/cytokine production in the CSF. CONCLUSIONS: We provide evidence that various T cell-targeted immunotherapies reduced inflammation in the brains of RE patients. The observation that intractable epilepsy persisted in all of the patients suggests a relative independence of seizure activity on the presence of T cells in the brain later in the disease course. Thus, new therapeutic targets must be identified. CXCL10, CXCL13 and BAFF levels were substantially increased in CSF from all patients and their significance in RE pathology remains to be addressed.
- MeSH
- CD8-pozitivní T-lymfocyty imunologie MeSH
- cytokiny imunologie MeSH
- dítě MeSH
- ELISA MeSH
- encefalitida patologie terapie MeSH
- imunoterapie metody MeSH
- lidé MeSH
- mozek patologie MeSH
- předškolní dítě MeSH
- zánět terapie MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
PURPOSE: The aim of this study was to test the possibility of using specimens obtained by a cavitron ultrasonic surgical aspirator (CUSA) in flow and mass cytometry investigations of pediatric brain tumors. METHODS: CUSA specimens obtained from 19 pediatric patients with brain tumors were investigated. Flow and mass cytometry methods were applied to analyze the composition of material collected using the CUSA. Cell suspensions were prepared from CUSA aspirates. Then sample viability was assessed by conventional flow cytometry and subsequently stained with a panel of 31 metal-labeled antibodies. RESULTS: Viability assessment was performed using conventional flow cytometry. Viability of cells in the acquired samples was below 50% in 16 of 19 cases. A mass cytometry investigation and subsequent analysis enabled us to discriminate brain tumor cells from contaminating leukocytes, whose proportions varied across the specimens. The addition of the viability marker cisplatin directly into the mass cytometry panel gave the means to selecting viable cells only for subsequent analyses. The proportion of non-viable cells was higher among tumor cells compared leukocytes. CONCLUSIONS: When the analysis of the tumor cell immunophenotype is performed with markers for determining viability, the expression of the investigated markers can be evaluated. Suitable markers can be selected by high-throughput methods, such as mass cytometry, and those that are diagnostically relevant can be investigated using flow cytometry, which is more flexible in terms of time.
- MeSH
- analýza jednotlivých buněk MeSH
- cisplatina metabolismus MeSH
- leukocyty metabolismus patologie MeSH
- lidé MeSH
- nádorové biomarkery metabolismus MeSH
- nádory mozku diagnóza metabolismus patologie chirurgie MeSH
- neurochirurgické výkony přístrojové vybavení MeSH
- průtoková cytometrie MeSH
- ultrazvuková terapie přístrojové vybavení MeSH
- viabilita buněk MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Novel biological subtypes and clinically important genetic aberrations (druggable lesions, prognostic factors) have been described in B-other acute lymphoblastic leukemia (ALL) during the last decade; however, due to a lack of studies on unselected cohorts, their population frequency and mutual associations still have to be established. We studied 110 consecutively diagnosed and uniformly treated childhood B-other patients using single nucleotide polymorphism arrays and whole exome/transcriptome sequencing. The frequency of DUX4-rearranged, BCR-ABL1-like, ZNF384-rearranged, ETV6-RUNX1-like, iAMP21 and MEF2D-rearranged subtypes was 27%, 15%, 5%, 5%, 4%, and 2%, respectively; 43% of cases were not classified into any of these subtypes (B-rest). We found worse early response to treatment in DUX4-rearranged leukemia and a strong association of ZNF384-rearranged leukemia with B-myeloid immunophenotype. Of the druggable lesions, JAK/STAT-class and RAS/RAF/MAPK-class aberrations were found in 21% and 43% of patients, respectively; an ABL-class aberration was found in one patient. A recently described negative prognostic factor, IKZF1plus , was found in 14% of patients and was enriched in (but not exclusive for) BCR-ABL1-like subtype. PAX5 fusions (including 4 novel), intragenic amplifications and P80R mutations were mutually exclusive and only occurred in the B-rest subset, altogether accounting for 20% of the B-other group. PAX5 P80R was associated with a specific gene expression signature, potentially defining a novel leukemia subtype. Our study shows unbiased European population-based frequencies of novel ALL subtypes, recurrent (cyto)genetic aberrations and their mutual associations. This study also strengthens and widens the current knowledge of B-other ALL and provides an objective basis for optimization of current genetic diagnostics.
- MeSH
- chromozomální aberace * MeSH
- dítě MeSH
- fúzní onkogenní proteiny genetika MeSH
- genomika metody MeSH
- kohortové studie MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- mutace * MeSH
- nádorové biomarkery genetika MeSH
- následné studie MeSH
- pre-B-buněčná leukemie epidemiologie genetika patologie MeSH
- předškolní dítě MeSH
- prognóza MeSH
- regulace genové exprese u nádorů MeSH
- transkriptom * MeSH
- Check Tag
- dítě MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
Implementation of cytarabine into induction therapy became standard of care for younger patients with mantle cell lymphoma (MCL). On the basis of its beneficial impact, many centers incorporated cytarabine at lower doses also into first-line treatments of elderly patients. We conducted a multicenter observational study that prospectively analyzed safety and efficacy of alternating 3 + 3 cycles of R-CHOP and R-cytarabine for newly diagnosed transplant-ineligible MCL patients. A total of 73 patients were enrolled with median age 70 years. Most patients had intermediate (39.7%) and high-risk (50.7%) disease according to MCL international prognostic index. Rituximab maintenance was initiated in 58 patients. Overall response rate reached 89% by positron emission tomography-computed tomography, including 75.3% complete remissions. Two patients (2.7%) did not complete the induction therapy because of toxicity. Three patients (4.1%) were considered nonresponders, which led to therapy change before completion of induction. Estimated progression-free survival and overall survival were 51.3% and 68.6% at 4 years, respectively. Mantle cell lymphoma international prognostic index, bulky disease (≥ 5 cm), and achievement of positron emission tomography-negativity independently correlated with progression-free survival. Grade 3 to 4 hematologic and nonhematologic toxicity was documented in 48% and 20.5% patients, respectively. Alternation of R-CHOP and R-cytarabine represents feasible and very effective regimen for elderly/comorbid MCL patients. This study was registered at GovTrial (clinicaltrials.gov) NCT03054883.
- MeSH
- cyklofosfamid farmakologie terapeutické užití MeSH
- cytarabin farmakologie terapeutické užití MeSH
- doxorubicin farmakologie terapeutické užití MeSH
- lidé středního věku MeSH
- lidé MeSH
- lymfom z plášťových buněk farmakoterapie patologie MeSH
- myší monoklonální protilátky farmakologie terapeutické užití MeSH
- prednison farmakologie terapeutické užití MeSH
- protokoly antitumorózní kombinované chemoterapie farmakologie terapeutické užití MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- vinkristin farmakologie terapeutické užití 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
Rituximab maintenance (RM) prolongs survival of elderly patients with mantle cell lymphoma (MCL). Persistent minimal residual disease (MRD) after induction repeatedly correlated with shorter progression-free survival (PFS). However, none of the published studies analyzed patients treated with RM. The main purpose was to analyze prognostic significance of MRD in the elderly patients with newly diagnosed MCL treated according to the recently published observational trial protocol (alternation of R-CHOP and R-cytarabine, 3 + 3 cycles, GovTrial number NCT03054883) at the centers that implemented RM. Minimal residual disease was evaluated by a EuroMRD standardized real-time PCR approach after 3 and 6 cycles of the induction therapy. Prognostic significance of MRD was analyzed in a subcohort of patients treated at the centers that implemented RM as a standard approach. Bone marrow proved to be a significantly more sensitive source for MRD detection than peripheral blood. In either compartment MRD (positive versus negative) after 3 or 6 cycles of the induction therapy did not correlate with PFS. The observed loss of prognostic significance of MRD after the R-CHOP-based induction appears to be a consequence of RM immune control over the residual lymphoma.
- MeSH
- cyklofosfamid aplikace a dávkování MeSH
- doxorubicin aplikace a dávkování MeSH
- lidé středního věku MeSH
- lidé MeSH
- lymfom z plášťových buněk * krev farmakoterapie mortalita MeSH
- míra přežití MeSH
- myší monoklonální protilátky aplikace a dávkování MeSH
- následné studie MeSH
- prednison aplikace a dávkování MeSH
- přežití po terapii bez příznaků nemoci MeSH
- protokoly antitumorózní kombinované chemoterapie aplikace a dávkování MeSH
- reziduální nádor MeSH
- rituximab aplikace a dávkování MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- udržovací chemoterapie * MeSH
- vinkristin aplikace a dávkování 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
- klinické zkoušky MeSH
- multicentrická studie MeSH
- pozorovací studie MeSH
- MeSH
- cytodiagnostika metody přístrojové vybavení MeSH
- hmotnostní spektrometrie metody přístrojové vybavení MeSH
- klinické laboratorní techniky MeSH
- monoklonální protilátky izolace a purifikace MeSH
- nádory mozku * diagnóza imunologie patologie MeSH
- regulace genové exprese u nádorů MeSH
- Publikační typ
- práce podpořená grantem MeSH
In mass cytometry, the isolation of pure lymphocytes is very important to obtain reproducible results and to shorten the time spent on data acquisition. To prepare highly purified cell suspensions of peripheral blood lymphocytes for further analysis on mass cytometer, we used the new CD81+ immune affinity chromatography cell isolation approach. Using 21 metal conjugated antibodies in a single tube we were able to identify all basic cell subsets and compare their relative abundance in final products obtained by density gradient (Ficoll-Paque) and immune affinity chromatography (CD81+ T-catch™) isolation approach. We show that T-catch isolation approach results in purer final product than Ficoll-Paque (P values 0.0156), with fewer platelets bound to target cells. As a result acquisition time of 105 nucleated cells was 3.5 shorter. We then applied unsupervised high dimensional analysis viSNE algorithm to compare the two isolation protocols, which allowed us to evaluate the contribution of unsupervised analysis over supervised manual gating. ViSNE algorithm effectively characterized almost all supervised cell subsets. Moreover, viSNE uncovered previously overseen cell subsets and showed inaccuracies in Maxpar™ Human peripheral blood phenotyping panel kit recommended gating strategy. These findings emphasize the use of unsupervised analysis tools in parallel with conventional gating strategy to mine the complete information from a set of samples. They also stress the importance of the impurity removal to sensitively detect rare cell populations in unsupervised analysis. © 2016 International Society for Advancement of Cytometry.
- MeSH
- antigeny CD81 chemie metabolismus MeSH
- Ficoll chemie MeSH
- leukocyty mononukleární cytologie MeSH
- lidé MeSH
- lymfocyty cytologie imunologie MeSH
- obrazová cytometrie metody MeSH
- protilátky chemie imunologie MeSH
- separace buněk metody MeSH
- viabilita buněk imunologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
