Diagnosis and treatment of acute myeloid leukemia (AML) remains difficult to this day, cure rates remain low and measurable residual disease (MRD) assessment is still challenging. Early detection of ...residual leukemic cells is essential to predict therapeutic success and Multiparametric Flow Cytometry (MFC) has been an effective tool for it. Here, we focus on the importance in caution when interpreting MDR in AML.
Bone marrow (BM) sample was assessed to FLT3 and NPM1 mutation, RUNX1/RUNX1T1 and CBFB-MYH11 rearrangements, karyotype (KT) and immunophenotyping. Using an 8-colour antibody panel (CD45, CD34, CD117, CD 33, HLA-DR, CD38, CD123, CD13, CD19, CD7, CD11b, CD4, CD56, CD64, CD11c, CD15, CD133, CD41a and NG2), we applied a gating strategy with fixed gates comparing normal to AML BM, settled to catch LAIPs (Leukemia Associated-Immunophenotypes), abnormal cell maturation pattern and Leukemic Stem Cell, that were detected on diagnosis and evaluated for MRD. Assays were performed at the Hematology Laboratory of the Medical School of Ribeirão Preto, University of São Paulo, in accordance to Local Ethical Boards.
A 21-year-old male patient with AML without maturation, diagnosed after 60 days of symptoms, presented anemia (7.9 g/dL), thrombocytopenia (26 000/μL) and white blood cell count of 5710/μL with 73% blasts in peripheral blood and 84% in BM. MFC revealed CD45dim and low side scatter; positive staining for CD117, CD34, HLA-DR, CD38, CD123, CD11c, CD64, CD15 and CD133; negative staining for CD33, CD14, CD7, CD11b, CD4, CD56, CD19, CD41a and NG2. We found no metaphases (KT), no molecular abnormality and he failed risk stratification. 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) and 1 or 2 consolidation cycles (6 days of cytarabine 1 g/m2 twice a day). BM was obtained at diagnosis, 30 and 33 days after 1st and 2nd induction, and 3, 9, 12 and 15 months after consolidation of chemotherapy. The positivity of a specific phenotype (CD117+/CD34+/CD33wk/CD13+ ≥ 0.1%) was preserved at diagnosis and follow-ups: 0,506%, 0,681%, 0,402%, 0,101%, 0,158% and 0,122%. Complete remission was achieved by day 30 after 1st induction and no change in the outcome was reported. Absent CD33 was observed in diagnosis and follow-ups, during and after treatment.
Post-analytical phase of MFC is the most vulnerable to wrong interpretations. CD33 is a common myeloid antigen expressed on malignant blasts in AML and had been reported as a potential target therapy. CD33low blasts are associated to a more mature AML and its high expression is related to adverse landscapes, highlighting the importance of CD33 evaluation. Standard care for AML enrolls MRD monitoring and failure to achieve an MRD-negative, CR or detected MRD during or after therapy is associated with relapse or poor outcomes. Still, MRD analysis must embrace a set of standards in order to prevent post-analytic errors. Here we present a case report of AML with absent CD33 maintained from diagnosis to MRD follow-ups, mimicking MRD positivity. This data emphasizes the importance of caution in MFC analysis and correlation of diagnostic and follow-ups results interpretation, as well 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
A collection of new reversible glycosidase inhibitors of the iminoalditol type featuring N-substituents containing perfluorinated regions has been prepared for evaluation of physicochemical, ...biochemical and diagnostic properties. The vast variety of feasible oligofluoro moieties allows for modular approaches to customised structures according to the intended applications, which are influenced by the fluorine content as well as the distance of the fluorous moiety from the ring nitrogen. The first examples, in particular in the D-galacto series, exhibited excellent inhibitory activities. A preliminary screen with two human cell lines showed that, at subinhibitory concentrations, they are powerful pharmacological chaperones enhancing the activities of the catalytically handicapped lysosomal D-galactosidase mutants associated with GM1 gangliosidosis and Morquio B disease.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
10-(Octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC) is an alkylphospholipid that can interact with cell membranes because of its amphiphilic character. We describe here the interaction ...of ODPC with liposomes and its toxicity to leukemic cells with an ED-50 of 5.4, 5.6 and 2.9
μM for 72
h of treatment for inhibition of proliferation of NB4, U937 and K562 cell lines, respectively, and lack of toxicity to normal hematopoietic progenitor cells at concentrations up to 25
μM. The ED-50 for the non-malignant HEK-293 and primary human umbilical vein endothelial cells (HUVEC) was 63.4 and 60.7
μM, respectively. The critical micellar concentration (CMC) of ODPC was 200
μM. Dynamic light scattering indicated that dipalmitoylphosphatidylcholine (DPPC) liposome size was affected only above the CMC of ODPC. Differential calorimetric scanning (DCS) of liposomes indicated a critical transition temperature (
T
c) of 41.5
°C and an enthalpy (∆H) variation of 7.3
kcal mol
−
1. The presence of 25
μM ODPC decreased
T
c and ∆H to 39.3
°C and 4.7
kcal mol
−
1, respectively. ODPC at 250
μM destabilized the liposomes (36.3
°C, 0.46
kcal mol
−
1). Kinetics of 5(6)-carboxyfluorescein (CF) leakage from different liposome systems indicated that the rate and extent of CF release depended on liposome composition and ODPC concentration and that above the CMC it was instantaneous. Overall, the data indicate that ODPC acts on in vitro membrane systems and leukemia cell lines at concentrations below its CMC, suggesting that it does not act as a detergent and that this effect is dependent on membrane composition.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Acute myeloid leukemia with monocytic blast differentiation (AML-MC) is a rare malignancy (5-10% incidence) with a challenging diagnosis, classified as “non otherwise specified” AML (AML NOS), unless ...recurrent genetic abnormalities, therapy-related or associated myelodysplasia-related changes are reported (WHO 2017). We observed a high incidence of AML-MC and aimed to assess its molecular and immunophenotypic features in a multicenter study in Brazil.
387 AML bone marrow samples (18-84 years old) from 10 Brazilian centers enrolled in International Consortium of Acute Leukemias (ICAL) Study – IC-AML2015, is in accordance Local Ethical Boards committee. From which, 149 were morphologically diagnosed with AML-MC. FLT3 and NPM1 mutations, RUNX1/RUNX1T1 and CBFB-MYH11 rearrangements, karyotype and immunophenotype were assessed. Mononuclear cells were stained with CD45, CD34, CD117, CD11c, CD64 and CD15. Cell acquisition/analysis was performed in FACS Canto II /FACS Diva software (BD Bioscience). Positivity was considered when ≥20% of the leukemic cells. Treatment consisted on 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) and 1 or 2 consolidation cycles (6 days of cytarabine 1 g/m2 twice a day) and/or BM transplantation.
AML-MC corresponded to 38.50% of cases enrolled in the study. Stratifying according to ELN2017 (without TP53 and ASXL1), 23% were Favorable, 22% Intermediate and 19% Adverse risk. 35% of patients failed Stratification or had Not Assessed Information. Molecular Assessment showed positive detections for FLT3 (17.48%), RUNX1-RUNX1T1 (2.80%), CBFB-MYH11 (8.51%) and NPM1 (23.77%). According to treatment response evalutation, complete remission CR was achieved by 49,14%, 55,8%, 53,70% and 59,46% of patients on 1st,2ndInductionof remission cycle of chemotherapy, 1st and 2sn cycle of Consolidaton of chemotherapy, and 26.37% of patients relapsed. Multiparametric Flow Cytometry (all risks) showed 39% CD34−, 61% CD34+,11% CD34−/CD117−, 13% CD117, 87% CD117+, 95% CD33+, 89% CD38+, 47% CD123+, 8% CD14+, 21% CD7+, 44% CD11b+, 19% CD56+, 29% CD13−, 2% CD19+, 27% CD64+, 74% CD11c+ 27%, CD64+CD11c+, 19% CD15+/CD133+ and 60% CD15+ cells. Among AML-NOS patients, 26% presented CD15+ and none were stratified as Favorable risk (6% Intermediate, 17% Adverse and 4% Failed Stratification).
Since AML-MC is known to be rare, accounting for 5-10% of AML cases worldwide, its high incidence in our study seemed to be odd and raised curiosity in the molecular and phenotypic context. The fact that a great number of these cases are intermediate ELN2017 risk brings up the necessity to look at them closer, since this is a challenging group when it comes to therapy decisions. Although good response to treatment was noticed, immunophenotypically CD15 expression without molecular abnormalities was more frequent in intermediate and adverse risk. These findings may direct that high rates of good treatment response and favorable outcome in these patients are more likely to be due to favorable molecular abnormalities, while expression of CD15 alone may be associated with a more aggressive disease. Knowing that, it should be of value to investigate whether CD15 could be a good prognostic marker.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Here, we evaluated whether the overexpression of transcriptionally inactive ΔNp73 cooperates with PML/RARA fusion protein in the induction of an APL-leukemic phenotype, as well as its role in vitro ...in proliferation, myeloid differentiation, and drug-induced apoptosis. Using lentiviral gene transfer, we showed in vitro that ΔNp73 overexpression resulted in increased proliferation in murine bone marrow (BM) cells from hCG-PML/RARA transgenic mice and their wild-type (WT) counterpart, with no accumulation of cells at G2/M or S phases; instead, ΔNp73-expressing cells had a lower rate of induced apoptosis. Next, we evaluated the effect of ΔNp73 on stem-cell self-renewal and myeloid differentiation. Primary BM cells lentivirally infected with human ΔNp73 were not immortalized in culture and did not present significant changes in the percentage of CD11b. Finally, we assessed the impact of ΔNp73 on leukemogenesis or its possible cooperation with PML/RARA fusion protein in the induction of an APL-leukemic phenotype. After 120 days of follow-up, all transplanted mice were clinically healthy and, no evidence of leukemia/myelodysplasia was apparent. Taken together, our data suggest that ΔNp73 had no leukemic transformation capacity by itself and apparently did not cooperate with the PML/RARA fusion protein to induce a leukemic phenotype in a murine BM transplantation model. In addition, the forced expression of ΔNp73 in murine BM progenitors did not alter the ATRA-induced differentiation rate in vitro or induce aberrant cell proliferation, but exerted an important role in cell survival, providing resistance to drug-induced apoptosis.
Diagenesis is an essential tool to reconstruct the development of reservoir rocks. Diagenetic processes - precipitation and dissolution - have an influence on pore space. The present paper aims to ...study the diagenetic history of deep-marine sandstones of the Austrian Alpine Foreland Basin. To reach that goal, sediment petrology and diagenetic features of more than 110 sandstone samples from water- and gas-bearing sections from gas fields within the Oligocene-Miocene Puchkirchen Group and Hall Formation has been investigated. Special emphasis was put on samples in the vicinity of the gas-water contact (GWC). The sediment petrography of sandstones of Puchkirchen Group and Hall Formation is similar; hence their diagenesis proceeded the same way. In fact, primary mineralogy was controlled by paleo-geography with increasing transport distance and diverse detrital input.
Sediment petrographically, investigated sandstones from the water-bearing horizon seemed quite comparable to the gas-bearing sediments. In general, they can be classified as feldspatic litharenites to litharenites and display porosities of up to 30% and permeabilities of up to 1300 mD. The carbon and oxygen isotopic composition of bulk carbonate cements from these sandstones range from−3.8 to +2.2 and from −7.5 to +0.2‰ VPDB. However, near the Gas-Water Contact (GWC) a horizon with low porosities (<3%) and permeabilities (<0.1 mD) is present. This zone is completely cemented with calcite, which has a blocky/homogenous morphology. A slight, but significant negative shift in δ18O isotopy (−2.5‰) is evident.
During early diagenesis the first carbonate generations formed. First a fibrous calcite and afterwards a micritic calcite precipitated. Further siliciclastic minerals, such as quartz and feldspar (K-feldspar and minor plagioclase), exhibit corroded grains. Occasionally, clay minerals (illite; smectite, chlorite) formed as rims around detrital grains. Late diagenesis is indicated by the formation of a low permeable zone at the GWC.
•Diagenesis of Puchkirchen Group and Hall Formation proceeded the same way.•Water-bearing sandstones are diagenetically comparable to gas-bearing zones.•Early diagenetic carbonate cementation impeded further compaction.•Corrosion of early carbonate account for higher porosity and permeability.•A low permeable zone between the water- and gas-bearing horizons developed.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
A Leucemia Mieloide Aguda (LMA) se origina de anormalidades genéticas ao nível das células-tronco hematopoéticas (CTH), que adquirem propriedades de células-tronco leucêmicas (CTL) com capacidade ...aumentada de sobrevivência e bloqueio de maturação. Hipotetizamos que a parada de maturação leucêmica por meio de definição fenotípica das células de origem possa acrescentar informações importantes sobre a biologia da LMA e contribuir para a predição de desfechos. Assim, analisamos retrospectivamente em coorte brasileira de pacientes com LMA o perfil fenotípico de maturação de blastos leucêmicos e sua associação com achados clássicos de morfologia, risco genético e informações clínicas. Foram incluídos dados de imunofenotipagem de 359 indivíduos com LMA encaminhados para o estudo ICAML2015, entre 18 e 70 anos, acompanhados durante oito anos, exceto os casos de Leucemia Promielocítica Aguda (LPA) que só foram incluídos após 2020. A expressão de CD34, CD117, CD13, CD33, CD38, (c)MPO e HLA-DR foi avaliada para a categorização nos subtipos HSC: Hematopoietic stem cell; MPP: Multipotent progenitors; CMP: Common myeloid progenitors; GMP: Granulocyte-monocyte progenitors; MP: Monocyte progenitors; GP: Granulocyte progenitors. LMAs tipo HSC foram definidas por CD34+, CD117−/+, HLA-DR+, CD13/33−, MPO−. A expressão de CD13 e 33 definiu o subtipo MPP. A expressão de cMPO entre 10-70% definiu o subtipo CMP e > 70% GMP. Os subtipos MP/GP foram diferenciados pela expressão de HLA-DR no subtipo GP. A reclassificação dos casos demonstrou a seguinte distribuição: HSC (n = 31, 8.64%); MPP (n = 77, 21.45%); CMP (n = 37, 10.31%); GMP (n = 75, 20.89%); MP (n = 50, 13.93%); GP (n = 89, 24.79%). De acordo com a estratificação de risco da European-Leukemia Net 2017, dentre os subtipos HSC, MPP, CMP e GMP predominaram o risco favorável, seguidos de intermediário e adverso. Nos subtipos MP e GP, o risco intermediário foi mais prevalente em relação ao favorável e adverso. A correlação com a classificação morfológica demonstrou que LMA sem maturação, com maturação, mielomonocítica e monocítica se distribuíram de forma equivalente nos subtipos MPP, CMP, GMP, MP e GP; no subtipo HSC não se encontrou leucemia monocítica ou promielocítica, sendo esta caracteristicamente do tipo GP e raramente GMP. Para 325 pacientes, os dados dos genes NPM1, FLT3 -ITD, BAALC, EVI1 e CEBPA estavam disponíveis, sendo encontrada com maior frequência a mutação FLT3 -ITD no subtipo MPP (26/325) e NPM1 em GP (44/325). Em algum ponto de seu seguimento 57/359 pacientes recaíram, representando nos subtipos: HSC (5/31, 16%), MPP (15/77, 19%), CMP (6/37, 16%), GMP (16/75, 21%), MP (6/50, 12%) e GP (9/89, 10%). A sobrevida global foi avaliada em 309/359 pacientes, tendo-se observado maior taxa para o subtipo GP (66%) e menor para CMP (11%) aos 24 meses de seguimento (p = 0.03). Os achados apontam que as correlações entre o estado de parada de maturação da LMA e dados clínicos de estratificação de risco e sobrevida merecem atenção e melhor exploração. É possível que a combinação dos dados genéticos com a classificação imunofenotípica de origem leucêmica seja útil para aferir prognóstico na LMA, pois é plausível que as leucemias mais maduras tenham um microambiente inflamatorio exuberante, capaz de modificar a imunidade antitumoral e contribuir para os desfechos diversos.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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