SAENTA-fluoresceins: New Flow Cytometry Reagents for Assessing Transport of Nucleoside Drugs in Acute Leukemia

1992 ◽  
pp. 65-71
Author(s):  
J. S. Wiley ◽  
G. P. Jamieson ◽  
A. M. Brocklebank ◽  
M. B. Snook ◽  
W. H. Sawyer ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Leukemia ◽  
Nucleoside Drugs

P-53 PROTEIN EXPRESSION BY FLOW CYTOMETRY AND RELATION TO K1-67 AND CD34 EXPRESSIONS IN 45 ACUTE LEUKEMIA

1993 ◽  
Vol 79 (3) ◽  
pp. 285-285
Author(s):  
Marc Maynadie ◽  
Philippe Moskovtchenko ◽  
Rémi Gandois ◽  
Paule-Marie Carli
Keyword(s):  
Flow Cytometry ◽  
Acute Leukemia ◽  
Protein Expression ◽  
P 53

Flow Cytometry in Acute Leukemia: Practical Aspects

2008 ◽  
pp. 258-258
Author(s):  
Kumar A ◽  
Gujral S ◽  
Badrinath Y
Keyword(s):  
Flow Cytometry ◽  
Acute Leukemia

Neurotrophin Receptors and Ligands Are Highly Expressed in Acute Leukemia and Promote Leukemogenesis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 696-696
Author(s):  
Zhixiong Li ◽  
Gernot Beutel ◽  
Johann Meyer ◽  
Christian Koenecke ◽  
Thomas Neumann ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Leukemia ◽  
Protein Tyrosine Kinase ◽  
De Novo ◽  
Statistical Significance ◽  
Point Mutations ◽  
Receptor Protein ◽  
Clear Correlation ◽  
Receptor System ◽  
Induced Apoptosis

Abstract The neurotrophins (NTs), which include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT-3, NT-4, and NT-6, play a major role in neuronal survival. NTs are unique in that they utilize two different classes of receptors: the TRK (tropomyosin-related kinase) receptor protein tyrosine kinase (TRKA, TRKB, TRKC) and the low affinity NGF receptor (LNGFR=p75NTR). Recently, we and others have obtained evidence for potential involvement of this receptor system in leukemia. In the present study, we demonstrate for the first time expression of the three TRKs on the protein level in blasts from patients with newly diagnosed de novo or secondary acute leukemia. TRK expression was detected by flow cytometry using monoclonal antibodies previously validated on cell lines expressing retrovirally encoded TRK and considered positive if >20% of leukemic blasts expressed the respective receptor. 93 adult patients (41 female, 52 male) with a mean age of 53.8 years and diagnosis of AML (87%), ALL (12%), or AUL (1%) were enrolled after informed consent. Positive expression for at least one TRK was found in 55% of the analyzed cases without statistically meaningful differences in expression rate between AML (42/81) and ALL (8/11). Interestingly, while TRKB was expressed alone in blasts, TRKA or TRKC expression always occurred concomitantly with TRKB. In contrast to a previous study, we established a clear correlation of TRK expression pattern and FAB classification. In particular, TRKA expression occurred in 19 of 32 myelo-monocytic/monocytic leukemias (59%) whereas only 5 of 39 non-myelo-monocytic/monocytic leukemias (13%) were positive (p<0.005). The same observation was made for TRKC expression (44% vs. 10%, p<0.02). However, TRKB expression was more widespread, but tended to correlate with secondary AML (p=0.05). While only very few point mutations were found in TRK sequences by RT-PCR, we observed co-expression of BDNF (ligand for TRKB) in over half of TRKBpos cases (53%, 16/30) by flow cytometry. Expression of NGF or NT3 was also observed. Interestingly, high expression of TRKB or BDNF was associated with higher leukemic blast percentages (p< 0.0001). Remarkably, we found a trend for better mean overall survival in the BDNFneg subgroup (908 days vs. 527 days for BDNFpos). However, due to heterogeneity of clinical data and currently short follow-up, our results have not reached statistical significance. Activation of TRKA or TRKB by NGF and BDNF, respectively, efficiently rescued murine myeloid 32D cells from irradiation-induced apoptosis. Moreover, co-expression of TRKB/BDNF or TRKA/NGF in murine hematopoietic cells induced leukemia (n=6) in mouse models. Since BDNF is expressed by stromal cells in bone marrow, our findings suggest that TRKs might play an important role in leukemogenesis, with autocrine or paracrine stimulation as potential oncogenic mechanisms. Thus, TRKs and their downstream effectors might serve as novel therapeutic targets in acute leukemia.

CHILDHOOD Biphenotypic ACUTE LEUKEMIA with High Incidence of Basophilic Differentiation: The CONTRIBUTION of Ultrastructural Methods to Diagnosis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4698-4698
Author(s):  
Peretz Resnitzky ◽  
Drorit Luria ◽  
Dina Shaft ◽  
Gali Avrahami ◽  
Marta Jeison ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Leukemia ◽  
Nuclear Membrane ◽  
Conflicts Of Interest ◽  
Medical Center ◽  
Leukemic Cells ◽  
Ultrastructural Studies ◽  
High Incidence ◽  
B Lineage ◽  
Biphenotypic Acute Leukemia

Abstract Abstract 4698 The diagnosis of Biphenotypic Acute Leukemia (BAL) is still a challenge in clinical hematology. In spite of the use of the score formulation of the European Group for the Immunological Classification of Leukemias (EGIL) there are still cases difficult to define. We assessed the contribution of ultrastructural morphology and cytochemistry to the diagnosis of BAL in pediatric leukemic patients. Twenty six patients diagnosed at Schneider Children's Medical Center of Israel between the years 1989 to 2004 have been classified as BAL in a retrospective analysis evaluated by various combinations of light-microscopy (LM) morphology, immunophenotype flow-cytometry, cytogenetics and electron-microscopy (EM) including myeloperoxidase (MPO) and platelet peroxidase (PPO) reaction. The identification of myeloid features by EM was based on the presence of 1) MPO positive granules and MPO reactivity in the nuclear membrane, profiles of endoplasmic reticulum (ER) and in the membranes of the Golgi system; 2) PPO reaction in the nuclear membrane and profiles of ER, and 3) the presence of primary basophilic granules in the cytoplasm of the blasts. The EGIL scoring system has been used with the addition of the ultrastructural findings for the definition of BAL. In 24 cases the morphologic appearance and cytochemistry by LM of the leukemic cells were that of undifferentiated blasts. By cytogenetic evaluation abnormal karyotypes were detected in 16 patients, normal in 4 and unknown in 6 patients. Out of 26 patients 15 had the combination of T-ALL with myeloid phenotype and 11 had the B-lineage with myeloid features. Unexpectedly the blasts of 9 of the 26 BAL patients had basophilic differentiation as indicated by the presence of typical primary basophilic granules with MPO reactivity in a scattered pattern as previously reported in basophylic leukemia. Interestingly 7 of these 9 cases had a T lymphoid component. By contrast among 15 patients who had FAB M0, 9 had the B lineage markers. The remaining 2 patients had M1 and M7 features with T lymphoid phenotype. The contribution of EM studies enabled to establish the diagnosis of BAL in 11 out of 26 patients, in other 6 patients it allowed for the refinement of the AML subgroup such as basophilic leukemia and in the remaining 9 patients EM confirmed the diagnosis of BAL as defined by flow cytometry. Conclusions Our study identified a subgroup of children with acute leukemia in whom BAL could be suggested only by the addition of ultrastructural studies. EM could further refine and confirm the diagnosis of BAL in all the other cases. Of interest is the high incidence of basophylic differentiation among BAL pediatric patients, and its association with T lymphoid phenotype. Disclosures: No relevant conflicts of interest to declare.

Flow Cytometry Thresholds of Myeloperoxidase Detection to Discriminate Between Acute Lymphoblastic or Myeloblastic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1450-1450 ◽  
Author(s):  
Julien Guy ◽  
Iléana Antony-Debré ◽  
Isabelle Arnoux ◽  
Chantal Fossat ◽  
Emmanuel Benayoun ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Leukemia ◽  
Fluorescence Intensity ◽  
Conflicts Of Interest ◽  
Specific Binding ◽  
Negative Control ◽  
Analysis Method ◽  
Isotype Control ◽  
Blast Cells ◽  
Negative Controls

Abstract Abstract 1450 Background: The WHO 2008 classification emphasizes the role of myeloperoxidase (MPO) detection as the only requirement for assigning myeloid lineage to a blast population, notably for the diagnosis of acute leukemia of ambiguous lineage. WHO highlights flow cytometry (FCM) as the preferred method for MPO detection and EGIL proposed a 10% cut-off for FCM but 3% for cytochemistry. Here we performed a reevaluation of the MPO positivity threshold by FCM comparing as background reference isotype controls or the autofluorescence of residual normal lymphocytes. Methods: A multicenter retrospective trial was set-up which compared retrospectively 128 acute lymphoblastic leukemias (ALL) and 75 acute myeloid leukemias without maturation (AML1), defined as MPO negative or positive by benzidine-based cytochemistry. Blasts were considered MPO positive by flow cytometry when their mean fluorescence intensity exceeded that of blast cells incubated with an isotype control or that of residual lymphocytes in the MPO-stained sample. In order to homogenize the interpretation of MPO staining, five various cut-offs were assessed for both negative controls, respectively 2%, 1%, 0.75%, 0.5% and 0.25%. Besides, as Ratio Fluorescence Intensity (RFI) may be a useful parameter to analyze staining for markers with unimodal distribution, we evaluated the RFI of blast cells MPO fluorescence relative to both controls, comparing mean and median intensities. Results: The harmonized method that was developed to interpret MPO staining by FCM between 4 French centers can be applied regardless of antibodies, permeabilization reagents or instruments used. For both negative controls, the 1% cut-off provided the best discrimination by ROC curve, and was used to assess the percentage of stained blasts in the 203 cases. The EGIL 10% threshold of stained blasts to discriminate between ALL and AML, using the isotype control to assess positivity, provided 100% sensitivity and 85.4% specificity, the optimal threshold being 13% (sensitivity 95.1%, specificity 91.7%). Residual normal lymphocytes proved to be an advantageous alternative reference, a threshold of 28% yielding improved 97.4% sensitivity and 96.1% specificity (Figure). The correlation between both methods was excellent, yet the percentage of positive blasts was higher using lymphocytes as control compared to isotype control. Using the RFI method, the isotype control appeared as the more relevant negative control to discriminate ALL against AML1 with a threshold of 3.42. However, with 90% sensivity and 95,83% specificity, the RFI method was less performing than percentages in this study. Finally, we assessed the relevance of this analysis method and positivity thresholds on 18 AMLs with minimal differentiation (AML0), MPO-negative by definition in cytochemistry, yet liable to be positive in FCM. Interestingly, with these new appropriate thresholds, MPO staining was positive for 10 of 18 AML0 when using lymphocytes as negative controls and only 3 of 17 cases when using an isotype control. Conclusion: i) MPO detection by flow cytometry can be interpreted indifferently of the negative control used if an appropriate threshold is applied; ii) Our analysis method of MPO expression is relevant for ALL and AML discrimination and can be useful for AML0 or mixed phenotype acute leukemia assessment, especially using residual lymphocytes as reference, since the latter bypass the higher non-specific binding of isotype controls on blast cells. Disclosures: No relevant conflicts of interest to declare.

Peptide-Receptor Radiotherapy with CXCR4-Targeting Pentixather Reduces Leukemia Burden in Acute Leukemia PDX and Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4055-4055 ◽  
Author(s):  
Stefan Habringer ◽  
Peter Herhaus ◽  
Margret Schottelius ◽  
Constantin Lapa ◽  
Rouzanna Istvanffy ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Leukemia ◽  
Cell Line ◽  
Pet Imaging ◽  
Treatment Options ◽  
Individual Treatment ◽  
Peptide Receptor ◽  
Colony Forming Unit ◽  
Established Treatment ◽  
Pdx Models

Abstract Introduction: The G-protein coupled CXC-motif Chemokine Receptor 4 (CXCR4) and its ligand CXCL12 are master regulators of cell migration, organogenesis and maintenance of the hematopoietic stem/progenitor cell (HSPC) niche. However, CXCR4 also drives survival, proliferation and metastasis of cancer cells and its expression is associated with adverse prognosis in a broad range of malignancies, including acute myeloid and lymphoblastic leukemia (AML, ALL). Despite of high rates of complete remissions after induction chemotherapy, AML, and to a lesser extend ALL, frequently relapse with a more aggressive phenotype and require highly active therapies to reduce leukemic burden before allogeneic stem cell transplantation (alloSCT). We recently showed that CXCR4-directed PET imaging with 68Ga-Pentixafor is feasible in AML patients, providing first evidence for the potential of CXCR4-directed theranostics (Herhaus, Habringer et al., Haematologica 2016). Methods: We used patient-derived xenograft (PDX) and cell line xenograft mouse models of AML and ALL to evaluate the efficacy and toxicity of a CXCR4-targeted peptide receptor radiotherapy (PRRT) theranostic approach with the CXCR4-binding PET tracer 68Ga-Pentixafor and its b-emitting therapeutic counterpart 177Lu-Pentixather. We analyzed bone marrow (BM), spleen, blood (PB) and liver of treated PDX mice by flow cytometry, immunohistochemistry and radioactive biodistribution assays. The toxicity to the murine BM HSPC and the hematopoietic niche was assessed via flow cytometry, colony forming unit assays, isolation and differentiation of BM mesenchymal stem cells (MSCs) and co-culture experiments. We provide first evidence for this highly innovative CXCR4-directed theranostic approach in patients with AML who relapsed after alloSCT and had no other established treatment options. Results: We generated PDX models of acute leukemia patients in NSG mice that required intact CXCR4 signaling for disease initiation and progressively infiltrated spleen, BM and PB. 68Ga-Pentixafor PET imaging enabled visualization of CXCR4-positive leukemic burden in spleen and BM of acute leukemia PDX and cell line xenografts. In ALL PDX, CXCR4-directed PRRT with 177Lu-Pentixather rapidly distributed to leukemia-harboring organs, which lead to significant accumulation of radioactivity in spleen and BM. 177Lu-Pentixather therapy resulted in efficient eradication or significant reduction of leukemic infiltration in PB, spleen and BM. Spleen size was reduced dramatically as early as 24hr after initiation of PRRT. Treated mice suffered severe suppression of BM function as evidenced by therapy-induced severe cytopenia affecting mature CD45+ blood cells and colony-forming unit potential of progenitors in the BM. To assess the damage to the BM niche, we isolated MSCs of 177Lu-Pentixather treated mice compared to control. We found that MSCs from 177Lu-Pentixather-treated mice were still viable and proliferated in vitro. Importantly, treated MSCs were still capable of supporting normal lineage-marker negative murine HSPC and induced their differentiation into mature leukocytes in co-culture. Finally, we treated patients with refractory AML after alloSCT and multiple failed treatment regimens, who had no further established treatment options with CXCR4 PRRT followed by a conventional conditioning regimen and second alloSCT. Our data indicate that CXCR4 targeting and the inevitable and desired cross-fire effect of 177Lu-Pentixather PRRT could be a highly efficient means to eradicate leukemia and to induce myeloablation. This approach could serve as a valuable addition to conditioning protocols in alloSCT. Conclusion: In conclusion, our work provides first evidence for the efficacy of the novel CXCR4-directed agent 177Lu-Pentixather in acute leukemia PDX models and in a proof-of-concept individual treatment approach in one patient relapsed after standard alloSCT. Importantly, these findings can directly be translated into clinical studies in patients and provide crucial information regarding efficacy and toxicity. A phase I/II study to integrate CXCR-directed PRRT into conditioning regimens is planned. Disclosures Peschel: MophoSys: Honoraria. Wester:Scintomics GmbH: Employment, Other: CEO.

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