Despite recent advances and development of new therapeutic targets and personalized medicine, acute myeloid leukemia (AML) treatment remains defiant. Identification of residual leukemic cells ...(measurable residual disease -MRD) by Multiparametric Flow Cytometry has been a fast and effective tool, but disease heterogeneity makes MRD assessment a clinical challenge, especially in cases with uncommon features, needing clinical and technical expertise to be accurately performed and interpreted.
To emphasize how absent CD33 expression impacts on interpreting MDR in AML.
We report three clinical cases of AML with absent CD33 assessed for FLT3 and NPM1 mutation, RUNX1/RUNX1T1 and CBFB-MYH11 rearrangements, karyotype and an 8-color panel immunophenotyping (Table 1). Empty gating strategy was used to identify LAIPs (Leukemia Associated-Immunophenotype), different from normal and leukemic stem cells, compared to healthy and regenerating non-AML bone marrow (BM) from diagnosis and follow up (Figures 1 and 2). Assays were performed at the Hematology Laboratory of Ribeirão Preto the Medical School, University of São Paulo, in accordance to Local Ethical Boards.
Patient's demographic and clinical data are summarized on Table 2. MFC revealed completely negative CD33 from diagnosis to follow-ups. Treatment consisted of 2 induction cycles (cycle 1: 3 days of Daunorubicin 60 mg/m2 and 7 days of cytarabine 200 mg/m2; cycle 2: 6 days of cytarabine 1 g/m2 twice a day). Consolidation consisted of 1 or 2 cycles of 6 days of cytarabine 1 g/m2 twice a day. BMs were obtained at diagnosis, after 1st and 2nd induction, after 1st and 2nd Consolidation, and 3, 9, 12 and 15 months after consolidation of chemotherapy. Even with different number of follow-ups for each patient, time analysis was preserved. Complete remission (complete or incomplete) was achieved by day 30 after 1st induction and no change in the outcome was reported, even though an altered maturational phenotype persisted with complete absent CD33 expression, during and after treatment, suggesting a very rare polymorphism of CD33 receptor that mimicked MRD positivity. Since CD33 is a common myeloid antigen expressed on malignant blasts in AML, it is prominent evaluate and carefully analyze this marker, once is known that polymorphisms, for example, rs12459419 can affect CD33-antibody conjugated based therapy. CD33low blasts are associated to a more mature AML and its high expression is related to adverse landscapes in pediatric AML, highlighting the importance of CD33 evaluation. Regarding vulnerable steps of MRD assessment, Post –analytical phase must embrace a set of standards in order to prevent report errors. Here we present three cases of AML with absent CD33 from diagnosis till follow-ups visits. CD33 absence mimics positivity for MRD studies. This data emphasizes the importance of caution in MFC analysis and correlation of diagnostic and follow-ups results and interpretation, as constant training of the team.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Lipid rafts are highly ordered membrane domains rich in cholesterol and sphingolipids that provide a scaffold for signal transduction proteins; altered raft structure has also been implicated in ...cancer progression. We have shown that 25 μm 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets high cholesterol domains in model membranes and induces apoptosis in leukemia cells but spares normal hematopoietic and epithelial cells under the same conditions. We performed a quantitative (SILAC) proteomic screening of ODPC targets in a lipid-raft-enriched fraction of leukemic cells to identify early events prior to the initiation of apoptosis. Six proteins, three with demonstrated palmitoylation sites, were reduced in abundance. One, the linker for activation of T-cell family member 2 (LAT2), is an adaptor protein associated with lipid rafts in its palmitoylated form and is specifically expressed in B lymphocytes and myeloid cells. Interestingly, LAT2 is not expressed in K562, a cell line more resistant to ODPC-induced apoptosis. There was an early loss of LAT2 in the lipid-raft-enriched fraction of NB4 cells within 3 h following treatment with 25 μm ODPC. Subsequent degradation of LAT2 by proteasomes was observed. Twenty-five μm ODPC inhibited AKT activation via myeloid growth factors, and LAT2 knockdown in NB4 cells by shRNA reproduced this effect. LAT2 knockdown in NB4 cells also decreased cell proliferation and increased cell sensitivity to ODPC (7.5×), perifosine (3×), and arsenic trioxide (8.5×). Taken together, these data indicate that LAT2 is an early mediator of the anti-leukemic activity of alkylphospholipids and arsenic trioxide. Thus, LAT2 may be used as a target for the design of drugs for cancer therapy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Na Leucemia Mieloide Crônica (LMC), pacientes que alcançam resposta molecular profunda sustentada (RMP) com inibidor de tirosinoquinase (ITQ) podem ter o tratamento descontinuado. A remissão livre de ...tratamento (RLT) ocorre na minoria dos pacientes e não há biomarcadores preditores de manutenção ou perda de resposta. A imunidade antitumoral mediada por Linfócitos T (LT) e Natural Killer (NK) pode contribuir para manutenção e resultar em respostas distintas durante e após a descontinuação. Correlacionamos o fenótipo T e NK com a cinética de doença residual mensurável (uso de ITQ) e com a perda da RLT (descontinuação-DES). Foram incluídos 10 controles saudáveis (CT) e 23 pacientes com LMC: 8 ao diagnóstico (DX), 4 tratados por mais de 3 anos com RM menor que log 3 (RM<3), 13 com RMP sustentada (RM>4,5) em DES, divididos e acompanhados em meses (m), no qual, 3m (n = 13), 6m (n = 8) e 9m (n = 7), e 8 que recaíram (R) no DES. Até o ponto 6m, os pacientes estavam com 50% da dose do ITQ e, após este ponto, tiveram o ITQ suspenso. Foram avaliados frequência, subtipos e maturação de NK e LT por citometria de fluxo do sangue periférico. Os subtipos NK (CD45hiCD19−CD3−) foram definidos como secretório (CD56brightCD16−) ou citotóxico (CD56dimCD16+), e a maturação funcional das células CD19−CD3−CD56+ foi classificada: CD11b−CD27− (tolerante); CD27+CD11b− (secretória imatura); CD27+CD11b+(secretória madura) e CD11b+CD27− (citotóxica), além dos marcadores de ativação CD57 e NKp80. A frequência destas subpopulações foi avaliada em subgrupos de pacientes de acordo com o nível de RM por RT-qPCR para o gene BCR::ABL1. Em comparação aos CT, amostras de LMC em atividade (DX, RM<3 e R) exibiram queda de LT e NK totais, enquanto pacientes em uso de ITQ com boa resposta (DES) têm maior frequência de LT (p = 0,0002) e NK (p = 0,0061). DX, RM<3 e R tiveram redução de CD3+ e CD4+ e tendência de aumento de CD8+, e DES teve maior frequência de CD3+ (p =0,0004) e CD4+ (p = 0,0418) e tendência de redução de CD8+(p >0,05). Em relação aos subtipos de NK, DX, RM<3 e R, mostraram diminuição de CD56dimCD16+, entretanto DES apresentaram maior frequência (p = 0,0017). O subtipo CD56brightCD16− não diferiu entre os grupos (p > 0,05). Na maturação funcional de NK, DX, RM<3 e R, houve aumento de células secretórias imaturas e tolerantes, enquanto em DES diminuição de secretórias imaturas (p = 0,0387) e tolerantes (p = 0,0023) e tendência de aumento de secretórias madura (p > 0,05). Na ativação de NK, DX, RM<3 e R, exibiram queda de CD57+ (p = 0,0358) e NKp80 (p = 0,0036), e em DES evidenciaram valores elevados. Pacientes saudáveis, RM>4,5 e DES, possuem perfil mais maduro de NK, no qual a atividade citotóxica é semelhante aos CT. Entretanto, pacientes maus respondedores (RM<3 e R) exibem um perfil mais imaturo, maturação semelhante ao DX com aumento de secretórias imaturas e tolerantes. A frequência de NK citotóxicas CD56dim (antitumorais) está reduzida em pacientes com LMC em atividade e que não atingiram RM maior. O uso de ITQ leva à redução de BCR::ABL1 concomitante ao estímulo imune, com recuperação das células citotóxicas. A ativação de NK por meio de CD57 e NKp80 está prejudicada na presença de doença molecular e durante o tratamento, sugerindo que a monitorização de CD4 e NK citotóxicas durante a descontinuação poderá ser útil para predizer a recaída da RLT.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
CD56 expression has been associated with a poor prognosis in lymphoid neoplasms, including T-cell acute lymphoblastic leukemia (T-ALL). MicroRNAs (miRNAs) play an important role in lymphoid ...differentiation, and aberrant miRNA expression has been associated with treatment outcome in lymphoid malignancies. Here, we evaluated miRNA expression profiles in normal thymocytes, mature T-cells, and T-ALL samples with and without CD56 expression and correlated microRNA expression with treatment outcome.
The gene expression profile of 164 miRNAs were compared for T-ALL/CD56+ (n=12) and T-ALL/CD56- (n=36) patients by Real-Time Quantitative PCR. Based on this analysis, we decided to evaluate miR-221 and miR-374 expression in individual leukemic and normal samples.
miR-221 and miR-374 were expressed at significantly higher levels in T-ALL/CD56+ than in T-ALL/CD56- cells and in leukemic blasts compared with normal thymocytes and peripheral blood (PB) T-cells. Age at diagnosis (15 or less vs grater than 15 years; HR: 2.19, 95% CI: 0.98-4.85; P=0.05), miR-221 expression level (median value as cut off in leukemic samples; HR: 3.17, 95% CI: 1.45-6.92; P=0.004), and the expression of CD56 (CD56-vs CD56+; HR: 2.99, 95% CI: 1.37-6.51; P=0.006) were predictive factors for shorter overall survival; whereas, only CD56 expression (HR: 2.73, 95% CI: 1.03-7.18; P=0.041) was associated with a shorter disease-free survival rate.
miR-221 is highly expressed in T-ALL and its expression level may be associated with a poorer prognosis.
Two separate petroleum systems have been identified in the Austrian sector of the North Alpine Foreland Basin: a lower Oligocene – Cenomanian/Eocene oil and thermogenic gas system; and an ...Oligocene‐Miocene microbial gas system. Recent studies by both academic and industry‐based research groups have resulted in an improved understanding of these petroleum systems, which are reviewed in this paper.
Lower Oligocene organic‐rich intervals (up to 12 %TOC; HI: 400–600 mgHC/gTOC), capable of generating slightly more than 1 t of hydrocarbons/m2, are the source rocks for the thermogenic petroleum system in the Austrian sector of the North Alpine Foreland Basin. The present‐day distribution of this source rock is controlled by submarine mass movements which removed a large part of the organic‐rich interval from its depositional location during the late early Oligocene. The transported material was redeposited in locations to the south which are at the present day buried beneath Alpine thrust sheets. In addition, source rock units were incorporated into Molasse imbricates during Alpine deformation. Hydrocarbon generation began during the Miocene, and the oil kitchen was located to the south of the Alpine thrust front. Hence, lateral migration over distances of up to 50 km was required to charge the mainly Eocene and Cenomanian non‐ and shallow‐marine sandstone reservoir units. Hydrocarbons are in general trapped in structures related to east‐west trending normal faults, and differences in source rock facies resulted in the development of separate western and eastern oil families. Surprisingly, with the exception of some fields in the eastern part of the study area, associated gas contains varying (and sometimes very high) percentages of primary and secondary microbial methane. The composition of oil in some fields is influenced by both biodegradation and water washing. Post‐Miocene uplift in the Austrian sector of the basin had further effects on biodegradation and the consequent formation of secondary microbial gas, and also resulted in re‐migration.
The upper Oligocene to lower Miocene succession (Puchkirchen Group, Hall Formation) provides both source and reservoir rocks for the microbial petroleum system in the Austrian sector of the North Alpine Foreland Basin. TOC contents (<1.0 %) and HI values (<140 mgHC/gTOC) of pelitic source rocks are typically low. Microbial gas was generated shortly after deposition during early diagenesis and was subsequently fixed in gas hydrates. Basin subsidence and high sedimentation rates resulted in decomposition of the hydrates below their stability zone, and reservoirs were filled during the early Miocene. Subsequent mixing of microbial gas with thermogenic gas and condensates is widespread. However, biodegradation has prevented precise determination of the fraction of thermogenic hydrocarbons present in gas samples. Reservoir sandstones were deposited within a deep‐marine channel belt along the axis of the North Alpine Foreland Basin, and reservoir quality depends on the precise position within this belt. In the study area, gas is trapped in compaction anticlines or at channel margin pinch‐outs and additional traps are formed by imbrication structures.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Background: The Jak2V617F mutation represents the most common molecular abnormality present in Philadelphia chromosome-negative Classical Myeloproliferative Neoplasms (Ph-neg MPNs). The differential ...expression of genes/proteins between the hematopoietic stem cells (HSC) and the more committed bone marrow progenitors may reveal, at least in part, the contribution of each cell type to the pathogenesis of the disease. Recently, it has been shown that the deregulation of molecules of the BCL2 family in MPN increases the survival of the transformed hematopoietic stem and progenitor cells (HSPCs), which may contribute the progression of the disease (Figueiredo-Pontes et al, Blood #122:2852 2013). In this context, it is possible that the inhibition of Bcl-2 proteins may increase the sensibility of transformed HSPC to apoptosis leading to the control of myeloid proliferation and potential reduction of the Jak2 mutation allele burden. To date, the treatment of Ph-neg MPNs remains non-curative despite the development of Jak2 inhibitors, which improve the clinical features but fail to eliminate leukemic initiating cells. Aim: Therefore, we aimed to study if the modulation of Bcl-2 proteins could have an impact in the control of MPN. Methods: Wild type CD45.1 C57BL/6 mice (age: 10-12 weeks) were transplanted with 5x106 total bone marrow from CD45.2 C57BL/6 mice (age: 10-12 weeks old) harboring the Jak2V617F mutation after lethal irradiation. All transplanted mice presented at least 80% of chimerism and developed MPN features (increased red blood cells (RBC), white blood cells (WBC) and platelets counts, elevated hematocrit and hemoglobin) 6 weeks post-transplantation. Treatment with vehicle (5% dimethylacetamide 0.5% methylcellulose, n=5) or Bcl-2 inhibitor (GX15-070 - the BH3 mimetic obatoclax 3 mg/Kg/day, n=6) once a day by oral gavage was started 7 weeks after transplant for a time period of 4 weeks. Spleen and bone marrow were collected from euthanized mice to evaluate both chimerism and frequency of HSC by using fluorescence-conjugated antibodies against CD45.1, CD45.2, Ter119, CD19, CD4, CD8 CD3, CD71, cKit, Sca-1, CD48, CD150, CD16/32 and CD34. Data are represented as means and standard error of mean (mean ± SEM). The statistical significance was defined as p≤0.05 and analyzed by the Mann Whitney test. Results: Mice treated with obatoclax exhibited a reduction of 32% in spleen weight compared to the vehicle group, but did not reach statistical significance (Control=0.19 ±0.01 g vs Obatoclax=0.13 ±0.03 g; p=0.3). No differences were observed in blood counts (RBC, WBC or platelets) between vehicle and obatoclax-treated mice. In addition, treatment with the pan-Bcl-2 inhibitor did not affect the number of Lineage negative, Sca-1+, c-Kit+ (LSK) cells (Control=5.8x104±2.8x104vs Obatoclax=4.7x104±1.2x104, p=0.9), MPP (Control=1.9x104±2.8x104vs Obatoclax=1.9x104±8.5x103, p=0.9), ST-HSC (Control=2.8x104±1.9x104vs Obatoclax=4.4x104±4x103, p=0.2) and LT-HSC (Control=9.8x103±4.7x103vs Obatocalx=1x104±2.4x103, p=0.9). Similarly, no differences were observed in the number of myeloid progenitors between the groups: (Control=6x105±1.7x105vs Obatoclax=1x106±1.9x105, p=0.1), MEP (Control=2.7x105±7.7x104vs Obatoclax=2.9x105±6.3x104,p=0.9), CMP (Control=9.8x104±2.1x104vs Obatoclax=2.3x105±4.4x104, p=0.06) and GMP (Control=1.5x105±6.5x104vs Obatoclax=3.6x105±8.8x104, p=0.08). The frequency of erythroid progenitors in the BM (Early progenitors, Control=18.0% ±3% vs Obatoclax=26.0%±1.4%, p=0.052, and late erythroid cells, Control=38.4%±9.5% vs Obatoclax=49.0%±2.7%, p=0.4) was also not altered by Bcl-2 inhibition. Conclusion: Our findings are in accordance with a phase II clinical trial demonstrating that obatoclax has no clinical activity in patients with Myelofibrosis. (Parikh et al, CLML 2010). Thus, we concluded that obatoclax did not reduce MPN burden in our model, when used as a single agent administrated orally. Yet, we cannot exclude the possibility that that the modulation of Bcl-2 overexpression at MPN initiating cells level could render these cells sensitive to the JAK2 inhibition. Hence, further studies will be necessary to assess whether the combination of obatoclax with other drugs such as JAK2 inhibitors could result in improved therapeutic response.
Mullally:Janssen: Research Funding. Kobayashi:Taiho Pharmaceutical: Research Funding; MiNA therapeutics: Research Funding; Pfizer: Consultancy; Ono Pharmaceutica: Consultancy; Chugai Pharmaceutical: Honoraria; Boehringer Ingelheim: Honoraria; Roche Diagnostic: Honoraria. Figueiredo-Pontes:Novartis: Honoraria.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Magnetic optical sensor particles with multifunctional cores and shells are synthesized via a facile nanoprecipitation method and the subsequent modification of the particle shell. The hydrophobic ...particle core includes optical oxygen indicators, a light harvesting system, photosensitizers, and magnetic nanoparticles. Further functionalities are introduced by modifying the shell with enzymes, antibodies, multiple layers of polyelectrolytes, stimuli‐responsive polymers, and luminescent indicator dyes. The hydrodynamic diameter is tunable by varying different precipitation parameters.
Multifunctional magnetic optical sensor particles with sizes between 50 and 180 nm are synthesized by an emulsifier‐free nanoprecipitation method. Through surface and core modifications these particles are suitable for optical oxygen‐ and pH‐sensing and imaging, for singlet oxygen production, as magnetic carriers for biosensors, and many other applications.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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