Flow cytometry in detection of Nucleophosmin 1 mutation in acute myeloid leukemia patients: A reproducible tertiary hospital experience

2020 ◽  
Author(s):  
Rasha Abd El‐Rahman El‐Gamal ◽  
Azza El‐Sayed Hashem ◽  
Deena Mohamed Habashy ◽  
Menna Allah Zakareya Abou Elwafa ◽  
Noha Hussein Boshnak
Keyword(s):  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Tertiary Hospital ◽  
Hospital Experience ◽  
Acute Myeloid

scholarly journals Development of siRNA-Loaded Lipid Nanoparticles Targeting Long Non-Coding RNA LINC01257 as a Novel and Safe Therapeutic Approach for t(8;21) Pediatric Acute Myeloid Leukemia

Pharmaceutics ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1681
Author(s):  
Patrick Connerty ◽  
Ernest Moles ◽  
Charles E. de Bock ◽  
Nisitha Jayatilleke ◽  
Jenny L. Smith ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Mononuclear Cells ◽  
Sirna Delivery ◽  
Standard Of Care ◽  
Lipid Nanoparticles ◽  
Protein Product ◽  
Peripheral Blood Mononuclear ◽  
Acute Myeloid

Standard of care therapies for children with acute myeloid leukemia (AML) cause potent off-target toxicity to healthy cells, highlighting the need to develop new therapeutic approaches that are safe and specific for leukemia cells. Long non-coding RNAs (lncRNAs) are an emerging and highly attractive therapeutic target in the treatment of cancer due to their oncogenic functions and selective expression in cancer cells. However, lncRNAs have historically been considered ‘undruggable’ targets because they do not encode for a protein product. Here, we describe the development of a new siRNA-loaded lipid nanoparticle for the therapeutic silencing of the novel oncogenic lncRNA LINC01257. Transcriptomic analysis of children with AML identified LINC01257 as specifically expressed in t(8;21) AML and absent in healthy patients. Using NxGen microfluidic technology, we efficiently and reproducibly packaged anti-LINC01257 siRNA (LNP-si-LINC01257) into lipid nanoparticles based on the FDA-approved Patisiran (Onpattro®) formulation. LNP-si-LINC01257 size and ζ-potential were determined by dynamic light scattering using a Malvern Zetasizer Ultra. LNP-si-LINC01257 internalization and siRNA delivery were verified by fluorescence microscopy and flow cytometry analysis. lncRNA knockdown was determined by RT-qPCR and cell viability was characterized by flow cytometry-based apoptosis assay. LNP-siRNA production yielded a mean LNP size of ~65 nm with PDI ≤0.22 along with a >85% siRNA encapsulation rate. LNP-siRNAs were efficiently taken up by Kasumi-1 cells (>95% of cells) and LNP-si-LINC01257 treatment was able to successfully ablate LINC01257 expression which was accompanied by a significant 55% reduction in total cell count following 48 h of treatment. In contrast, healthy peripheral blood mononuclear cells (PBMCs), which do not express LINC01257, were unaffected by LNP-si-LINC01257 treatment despite comparable levels of LNP-siRNA uptake. This is the first report demonstrating the use of LNP-assisted RNA interference modalities for the silencing of cancer-driving lncRNAs as a therapeutically viable and non-toxic approach in the management of AML.

Clinical relevance of P-glycoprotein expression in de novo acute myeloid leukemia

Blood ◽  
1996 ◽  
Vol 87 (5) ◽  
pp. 1997-2004 ◽  
Author(s):  
G Del Poeta ◽  
R Stasi ◽  
G Aronica ◽  
A Venditti ◽  
MC Cox ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Survival Rates ◽  
Disease Free Survival ◽  
Normal Karyotype ◽  
Free Survival ◽  
Negative Phenotype ◽  
Acute Myeloid

Abstract Cytofluorimetric detection of the multidrug resistance (MDR)-associated membrane protein (P-170) was performed at the time of diagnosis in 158 patients with acute myeloid leukemia using the C219 monoclonal antibody (MoAb). In 108 of these cases the JSB1 MoAb was also tested. An improved histogram subtraction analysis, based on curve fitting and statistical test was applied to distinguish antigen-positive from antigen-negative cells. A marker was considered positive when more than 20% of the cells were stained. At onset, P-170 was detected in 43% of cases with C219 and in 73% of cases with JSB1. There was a strict correlation between C219 and JSB1 positivity, as all C219+ cases were also positive for JSB1 MoAb (P < .001). No relationship was found between sex, age, organomegaly, and MDR phenotype. Significant correlation was found between CD7 and both C219 and JSB1 expression (P < .001 and .001, respectively). C219-negative phenotype was more often associated with a normal karyotype (24 of 55 with P = .030). Rhodamine 123 (Rh123) staining and flow cytometry analysis showed a significantly decreased mean fluorescence in 51 C219+ and 38 JSB1+ patients compared to 42 MDR negative ones (P < .001). The rate of first complete remission (CR) differed both between C219+ and C219- cases and between JSB+ and JSB- ones (30.9% v 71.1% and 35.4% v 93.1%, respectively, P < .001). Of the 21 C219+ patients who had yielded a first CR, 19 (90.4%) relapsed, compared with 28 of 64 (43.7%) C219- patients (P < .001). Of the 28 JSB1+ patients in first CR, 17 (60.7%) relapsed relative to 8 (29.6%) of 27 JSBI- ones (P = .021). A higher rate of relapses among MDR+ compared with MDR- patients was observed both for C219 and JSB1 MoAbs taken separately (C219 80% v 44%; JSB1 52% v 27%), with no relationship to age. The survival rates (Kaplan-Meyer method) were significantly shorter both in C219+ patients and in JSB1+ cases (P < .001). Disease-free survival curves followed this same trend. The combination (C219- JSB1+) identified a subset of patients with an intermediate outcome compared to C219 positive cases. The prognostic value of both markers (C219 and JSB1) was confirmed in multivariate analysis. These results suggest that the assessment of MDR phenotype by flow cytometry may be an important predictor of treatment outcome.

CD8 T-cell responses to Wilms tumor gene product WT1 and proteinase 3 in patients with acute myeloid leukemia

Blood ◽  
2002 ◽  
Vol 100 (6) ◽  
pp. 2132-2137 ◽  
Author(s):  
Carmen Scheibenbogen ◽  
Anne Letsch ◽  
Eckhard Thiel ◽  
Alexander Schmittel ◽  
Volker Mailaender ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Myeloid Leukemia ◽  
Wilms Tumor ◽  
Proteinase 3 ◽  
Ifn Γ ◽  
Tumor Gene ◽  
Acute Myeloid

Abstract Wilms tumor gene product WT1 and proteinase 3 are overexpressed antigens in acute myeloid leukemia (AML), against which cytotoxic T lymphocytes can be elicited in vitro and in murine models. We performed this study to investigate whether WT1- and proteinase 3-specific CD8 T cells spontaneously occur in AML patients. T cells recognizing HLA-A2.1-binding epitopes from WT1 or proteinase 3 could be detected ex vivo in 5 of 15 HLA-A2–positive AML patients by interferon-γ (IFN-γ) ELISPOT assay and flow cytometry for intracellular IFN-γ and in 3 additional patients by flow cytometry only. T cells producing IFN-γ in response to proteinase 3 were further characterized in one patient by 4-color flow cytometry, identifying them as CD3+CD8+CD45RA+ CCR7−T cells, resembling cytotoxic effector T cells. In line with this phenotype, most of the WT1- and proteinase-reactive T cells were granzyme B+. These results provide for the first time evidence for spontaneous T-cell reactivity against defined antigens in AML patients. These data therefore support the immunogenicity of WT1 and proteinase 3 in acute leukemia patients and the potential usefulness of these antigens for leukemia vaccines.

The Growth Inhibition and Apoptosis Induction of Acute Myeloid Leukemia Blast Population by the Blocking IGF-IR Pathway.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4515-4515
Author(s):  
Si Sun ◽  
Yanli He ◽  
Xingbing Wang ◽  
Wei Liu ◽  
Jun Liu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Pi3k Pathway ◽  
Leukemia Cell Line ◽  
Free System ◽  
Acute Myeloid ◽  
Marrow Cells

Abstract The insulin-like growth factor-1receptor (IGF-1R) is overexpressed in a variety of tumors and has been associated with cancer development. Here, we analysis the IGF-IR expression on the bone marrow cells from 45 newly diagnosed patients with acute myeloid leukemia (AML) by flow cytometry. IGF-1R universally expressed on AML blasts and the leukemia cell line HL-60, did not show significant correlation with FAB subtypes. However, the bone marrow cells from AML patients with high myeloblast counts (&gt;80%) generally showed brighter IGF-IR expressions, which indicated the IGF-IR pathway might play an important role for AML blast proliferation and survival. Indeed, blocking the IGF-1R pathway by neutralizing monoclonal antibodies could reduce the proliferation of HL-60 cells by 38.28% at 48 hr. This inhibitory effect on blast growth was observed in 4 of 5 AML samples. In the same IGF-1R blocking treatment, the apoptosis of HL-60 cells was significantly induced, resulting in apoptosis of 57% of the cell population with the measurement of Annexin V vs PI staining by flow cytometry. The control contained only 20% apoptotic cells. We also demonstrated that the blockade of the IGF-1R pathway inhibited the phophorylation of the PI3K pathway component Akt in HL-60 cells when cultured in a serum free system with a supplement of 50ng/ml exogenous IGF. Since PI3K pathway activation greatly contributes to the proliferation, survival and drug resistance of AML, it is of interest to study whether blockading IGF-IR could also inhibit the PI3K pathway in primary AML blasts and synergize other anti-leukemia agents to improve the therapeutic effectiveness. Conclusions: IGF-IR may play an important role in the proliferation and survival of the AML blast population; Blocking the IGF-IR pathway could significantly inhibit the growth of AML blasts and considerably induce the apoptosis of AML blasts; IGF-IR could become a critical molecular target in anti-leukemia drug discovery.

Clinical Characterization of Acute Myeloid Leukemia with Myelodysplasia-Related Changes as Defined by the 2008 WHO Classification System.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 922-922
Author(s):  
Olga K Weinberg ◽  
Mahesh Seetharam ◽  
Li Ren ◽  
Lisa Ma ◽  
Katie Seo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Overall Survival ◽  
Acute Myeloid Leukemia ◽  
Clinical Outcome ◽  
Myeloid Leukemia ◽  
Who Classification ◽  
Risk Group ◽  
Who Criteria ◽  
Multilineage Dysplasia ◽  
Acute Myeloid

Abstract Background: Although some studies have validated the 2001 WHO classification of acute myeloid leukemia (AML), including the importance of multilineage dysplasia, others have suggested that multilineage dysplasia correlates with unfavorable cytogenetics but has no independent impact on prognosis. In 2008, the revised WHO classification system has expanded this category into “AML with myelodysplasia-related changes” (AML-MRC) that now includes 1) AML arising from myelodysplastic syndrome (MDS), 2) AML with MDS-related cytogenetic abnormalities, and 3) AML with multilineage dysplasia. An individual case may fall into this category by meeting any of the criteria. The goal of the current study is to clinically characterize this newly defined AML-MRC subgroup. Methods: One-hundred consecutive AML patients diagnosed at Stanford University Hospital between 2005 and 2007 with adequate material for mutation analysis were studied. Cases were classified using the 2008 WHO criteria. Diagnostic cytogenetic findings were reviewed and patients were stratified into risk groups using Southwest Oncology Group criteria. Available flow cytometry immunophenotyping results were reviewed and all samples were tested for NPM, FLT3 (ITD and D835) and CEBPA mutations. Clinical parameters including hemogram data at time of diagnosis were reviewed. Clinical follow-up including overall survival (OS), progression free survival (PFS) and complete remission (CR) rates were retrospectively determined. Kaplan-Meier methods and univariate and multivariate Cox proportional hazards regression analysis were used to compare the clinical data. Results: The cases included 57 males and 43 females with a median age of 56 (range 17–81). Cytogenetic risk-group stratification resulted in 9 patients with favorable, 65 with intermediate and 19 with unfavorable risk status. Using the 2008 WHO criteria, there were 48 AML-MRC, 40 AML not otherwise specified (AML-NOS), 9 AML with either t(8;21), inv(16) or t(15;17), and 3 therapy related AMLs. Overall, 26 patients had a NPM1 mutation (16 of which were FLT3 mutated), 25 had FLT3-ITD, 8 had FLT3-D835 and 9 had a CEBPA mutation (3 of which were FLT3 mutated). Compared to AML-NOS, patients with AML-MRC were significantly older (59 vs 51 years, p=0.014) and presented with lower hemoglobin (9 vs 11.2 g/dL, p=0.044), lower platelets (47 vs 54 K/uL, p=0.059), unfavorable cytogenetics (14/46 vs 3/36, p=0.014) and exhibited a decreased frequency of CEBPA mutation (0/46 vs 7/40, p=0.001) as compared to AML-NOS. Based on the flow cytometry immunophenotyping, the blasts from patients with AML-MRC more frequently expressed CD14 compared to AML-NOS (10/46 vs 4/36, p=0.048). Clinical outcome data showed that patients with AML-MRC had a significantly worse OS, PFS and CR compared to AML-NOS (Figure, all p<0.0001). Even after excluding the 14 patients with unfavorable cytogenetics from the AML-MRC group, the remaining patients with AML-MRC (defined solely by the presence of multilineage dysplasia) had worse outcomes compared to all AML-NOS patients (OS, p=0.013; PFS, p=0.012; CR, p=0.0076). Among 65 patients with intermediate risk cytogenetics, the outcome difference between the AML-MRC and AML-NOS groups remained significant (OS, p=0.0292; PFS, p=0.0232), also indicating prognostic significance of multilineage dysplasia. Within the AML-MRC group, univariate analysis showed that low platelets (<20,000/mm3), FLT3-D835 mutation and MDS-related cytogenetics correlated with OS (p=0.0456, p=0.0265, p=0.002 respectively) and PFS (p=0.0478, p=0.0626, p=0.001). A multivariate Cox proportional hazard analysis, performed on the entire group, identified unfavorable cytogenetic risk group, advanced age (> 60), FLT3-ITD and AML-MRC status as significant predictors of worse OS with the following respective hazard ratios: 2.82 (95% CI, 1.52–5.26), 2.11 (1.01–4.42), 1.98 (1.01–3.90), 1.92 (1.01–3.65). Conclusion: The newly defined WHO category of AML-MRC exhibits a significantly worse clinical outcome compared to AML-NOS and is predictive of worse overall survival in the multivariate analysis of AML patients, independent of age or cytogenetic risk group. These findings support the clinical, morphologic and cytogenetic criteria for this 2008 WHO AML category. Figure Figure

Flow Cytometry Quantification of Leukemic Stem Cells Is Associated with Risk Stratification and May Be Useful for Minimal Residual Disease in Acute Myeloid Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1470-1470
Author(s):  
Lorena Lobo Figueiredo Pontes ◽  
Leandro F. Dalmazzo ◽  
Luciana Correa Oliveira de Oliveira ◽  
Bárbara A Santana-Lemos ◽  
Felipe M Furtado ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Risk Group ◽  
Leukemic Stem Cells ◽  
The Poor ◽  
Hla Dr ◽  
Minimal Residual ◽  
Acute Myeloid

Abstract Abstract 1470 Investigation of minimal residual disease (MRD) after remission induction (RI) therapy provides important information for risk assessment in patients with acute myeloid leukemia (AML). The presence of immature and chemotherapy-resistant leukemic stem cells (LSC) within the bulk of AML blasts at diagnosis and in post-induction bone marrow (BM) may lead to relapses. Nevertheless, whether the frequency of cells with LSC characteristics and their clearance after induction is correlated with prognosis has not been established. Using four-color multiparametric flow cytometry (MFC), BM quantification of leukemic associated phenotypes (LAPs) and LSCs was performed at diagnosis and after the first RI from 37 AML patients, excluding acute promyelocytic leukemia, with a median age of 48 years and a male/female ratio of 0.95. All patients received the conventional daunorrubicin and cytarabin (3 + 7) RI chemotherapy. Complete remission (CR) was defined as BM blast count inferior to 5%. Thirty-three patients were classified according to the European Leukemia Net recommendation and 9/32 (28.1%) patients were allocated in favorable, 14/32 (43.8%) in intermediate and 9/32 (28.1%) in poor cytogenetic/molecular risk group. FLT3-ITD mutation was detected in 9/33 (27.3%) and 5/28 (17.9%) carried NPM1 mutation. MRD identification was performed on 20 erythrocyte-lysed whole BM samples after staining with a panel of directly conjugated monoclonal antibodies. Five patients were excluded from this analysis because a LAP could not be identified. Blasts gating was performed considering the low expression of CD45 and sideward scatter (SSC) and CD34 expression (these latter, when more than 20% of blasts were detected at diagnosis). Within this population, the following LAPs were investigated: CD15/CD117, HLA-DR/CD13, HLA-DR/CD33, CD2/CD56, CD19/CD11b, CD42a/CD33, CD64/CD11c or CD14/CD11c. LSCs were selected by the same CD45dim × SSC gating strategy and defined as CD34+/CD38−/CD123+. After staining procedures, at least one hundred thousand events were acquired in a FACScalibur flow cytometer and analysis was performed using the Cell Quest software. LAP and LSC quantification, at diagnosis and at days 21 to 30 of RI, was analyzed as a categorical variable defined as lower or higher than the median and was compared to the following variables: age (< or > 60 years old); WBC count (< or > 30 × 103); cytogenetic/molecular risk; and morphological CR. The comparison between LAP and LSC quantification at both time points was also assessed. Comparison of categorical variables was performed using Fisher's exact test or Yates' corrected chi-square for two or more variables, respectively. Statistical analyses were performed using SPSS 13.0 software and P < 0.05 was considered to be significant. LSC quantification at diagnosis was found at varying frequencies across different cytogenetic/molecular risk groups, being higher at the poor risk group (P = 0.041). Of note, 100% of the poor risk patients had high levels of LSC at diagnosis. In addition, the presence of FLT3-ITD mutation was associated with higher amounts of the LSC population at diagnosis (P = 0.043). The most frequent detected LAP was CD45dim/CD34+/HLA-DR+/CD13+. Low or high expression of the LAP was not correlated with the prognostic variables at diagnosis. CR rate was 83.33% and was not different in the groups with high or low levels of LSC at diagnosis. However, LAP and LSC quantification after RI were found to be correlated (P = 0.018), suggesting that the LSC subpopulation can be useful for MRD monitoring at this early treatment time-point. Therefore, LSC quantification by MFC at diagnosis can identify patients at high risk of relapse and offers the opportunity to study the stem cell compartment after chemotherapy. These findings are particularly important for the intermediate normal karyotype risk group patients, who frequently do not have specific molecular targets for MRD monitoring. Disclosures: No relevant conflicts of interest to declare.

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