Platelet Defects in Acute Myeloid Leukemia—Potential for Hemorrhagic Events

Background and objectives: In acute myeloid leukemia (AML), extensive bleeding is one of the most frequent causes of death. Impaired activation and aggregation processes were identified in previous studies on platelet behaviour associated with this disease. This study’s aim was to examine platelet function in correlation with other haemorrhage risk factors (fever, sepsis, recent bleeding, uraemia, leucocytosis, haematocrit value, treatment). Design and methods: The analysis of platelet surface proteins (Glycoprotein Ib-IX (CD42b, CD42a), Glycoprotein IIb-IIIa (CD41, CD61), p-selectin (CD62P), granulophysin (CD63)) was conducted by flowcytometry from samples of whole blood in patients with acute myeloid leukaemia in different stages of diagnosis and therapy (n = 22) in comparison with healthy human controls (n = 10). Results and interpretations: Our results show a significant decrease in fluorescence level associated with platelet activation markers (CD63 (14.11% vs. 40.78 % p < 0.05); CD62P (15.26% vs. 28.23% p < 0.05)); adhesion markers (CD42b (69.08% vs. 84.41% p < 0.05)) and aggregation markers (CD61 (83.79% vs. 98.62% p < 0.001)) in patients compared to controls. The levels of CD41 (80.62% vs. 86.31%, p = 0.290) and CD42a (77.98% vs. 94.15%, p = 0.99) demonstrate no significant differences in the two groups. Conclusion: The AML patients present changes in adhesion receptors and activation markers, suggesting a functional defect or denatured intracellular signalling in platelets. The exposed data indicate that flow cytometry can effectively identify multiple functional platelet impairments in AML pathogenesis.

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Acute Myeloid Leukemia Cells Generate Leukemic Endothelial Cells with Leukemogenic Potential: Blood Vessels As Sanctuaries for Leukemia Relapse

Blood ◽

10.1182/blood.v118.21.241.241 ◽

2011 ◽

Vol 118 (21) ◽

pp. 241-241

Author(s):

Christopher R Cogle ◽

Gerard J Madlambayan ◽

Devorah C Goldman ◽

Azzah Al Masri ◽

Ronald P. Leon ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Endothelial Cell ◽

Blood Vessels ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Surface Proteins ◽

Nsg Mice ◽

Acute Myeloid ◽

Marrow Cells

Abstract Abstract 241 Human hematopoietic stem cells (HSCs) possess hemangioblast activity, which is defined as the ability to generate both blood and endothelium. Whether malignant HSC counterparts such as acute myeloid leukemia (AML) also display this bipotentiality remains to be defined. To test the hemangioblast potential of AML cells we first cultured primary human AML bone marrow in conditions established by Yoder and colleagues that support the growth of functional endothelial cell (EC) progenitors. AML cultured in endothelial colony forming cell (ECFC) media generated endothelial progenitor cell colonies that showed uptake of acetylated LDL and expressed several EC surface proteins, including CD105, CD146, UEA-1 and CD144. Importantly, ECFCs derived from AML bone marrow no longer expressed CD45 or myeloid surface proteins such as CD14. When placed in Matrigel, these AML derived ECFC generated capillary-like, tubular structures. Moreover, these ECFCs contained cytogenetic mutations associated with their parental leukemias. Thus, under the appropriate conditions, AML bone marrow cells can generate cells with an endothelial-like phenotype and harboring leukemia specific mutations that will be referred to as ‘L-ECFC.' To functionally define leukemia hemangioblast activity, a xenograft model of AML was employed. Sublethally irradiated NOD/scid/IL2Rγ−/− (NSG) mice were transplanted with primary human AML cells and then sacrificed at 8–36 weeks after transplant. Significant accumulations of human AML cells were found in perivascular regions of the liver. Both tight coupling and bona fide cell fusion between AML and ECs was observed. AML derived EC that were integrated into portal vein endothelium showed induction of CD105 expression Follow-up AML xenotransplant experiments with BrdU labeling revealed almost four-fold fewer (6%) of the AML cells incorporated within blood vessels were BrdU+, as compared to AML cells not integrated in blood vessels (22%) (P=0.01). These results suggest that AML cell incorporation within the endovascular lining induces cell quiescence. Thus, leukemia-integrated ECs may be less susceptible to cell cycle active agents like cytarabine. Results from these experiments also raised the possibility that AML cells adopting an endothelial-like phenotype may serve as a reservoir for leukemic relapse. To test this hypothesis, we injected CD105+CD45- L-ECFC derived from AML patients into NSG mice. These L-ECFC generated colonies of human CD105+CD45- within spleens and bone marrow of recipient mice. We also found a distinct population of human CD45+CD19- cells comprising 5–10% of bone marrow cells. Leukemia-derived cells were confirmed by detection of cytogenetically mutant cells consistent with the parent leukemia (e.g., MLL duplications). In conclusion, this study demonstrates that AML cells can functionally generate leukemic ECs that become quiescent after incorporation in blood vessel networks and can re-emerge with a leukemogenic phenotype. Together, our results raise the strong possibility that AML cells exhibit functional hemangioblast activity and that vascular endothelium may serve as a clinically important sanctuary for occult leukemia. Our data also support endothelial cell targeting strategies as a means to eradicate AML. Disclosures: No relevant conflicts of interest to declare.

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Circulating Endothelial Cells Are an Indicator of Response to Treatment in Acute Myeloid Leukemia Patients.

Blood ◽

10.1182/blood.v106.11.2776.2776 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2776-2776

Author(s):

Agnieszka Wierzbowska ◽

Tadeusz Robak ◽

Anna Krawczynska ◽

Agata Wrzesien-Kus ◽

Agnieszka Pluta ◽

...

Keyword(s):

Endothelial Cells ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Circulating Endothelial Cells ◽

Response To Treatment ◽

Control Group ◽

Healthy Controls ◽

Endothelial Progenitor ◽

Activation Markers ◽

Acute Myeloid

Abstract Introduction. The circulating endothelial cells (CEC) are proposed to be a noninvasive marker of angiogenesis. The number of CEC in peripheral blood of patients (pts) with acute myeloid leukemia (AML) has not been investigated so far. Patients and Methods. We evaluated the count of resting (rCEC) and activated (aCEC) CEC and circulating endothelial progenitor cells (CEPC) as well as apoptotic CEC (CECAnnV+) in 62 AML pts at the time of diagnosis and 30 healthy controls. Additionally in 26 pts measurements were performed at the time of response evaluation and in 15 pts also 24 h after the first and last dose of chemotherapy. The levels of CEC were correlated with known prognostic factors and response to treatment. CEC were evaluated by the four colour flow cytometry using a panel of previously described monoclonal antibodies and an appropriate analysis gate. CEPC were defined as negative for hematopoietic marker CD45 and positive for endothelial cells markers CD34, CD31 and the endothelial progenitor marker CD133. Resting CEC were defined as CD45−, CD133−, CD31+, CD34+, CD146+ and negative for activation markers (CD105, CD106). CD105 or CD106 positive mature endothelial cells were classified as activated CEC. Apoptotic CEC were CD146 and Annexin V positive. Results. In untreated AML pts we observed 10-fold higher CEC level (median 29,3/μL) than in the control group (2,95/μL) p<0,0001. The numbers of aCEC (12,7/μL), rCEC (12,3/μL) and CEPC (1,7/μL) were significantly higher in AML pts at diagnosis when compared to healthy controls (aCEC 0,9/μL, rCEC 1,6/μL and 0,1/μL; p<0,0001). CECAnnV+ count was also 10-fold higher in AML (1,5/μL) than in controls (0,15/μL; p<0,0001). Both CEC and CECAnnV+ counts did not correlate with WBC, hemoglobin and platelets count as well as percentage of blasts in bone marrow and absolute blast count. The positive correlations between CEC number and CEPC count (r=0,435; p<0,001), CECAnnV+ count (r=0,502; p<0,01) as well as LDH activity (r=0,328; p<0,02) were found. The significant decrease of aCEC and rCEC numbers 24 hours after the first dose of chemotherapy was noted in patients who achieved complete remission (CR)(p<0,04) but not in pts refractory to treatment. Moreover aCEC, rCEC, CEPC and CECAnnV+ counts determined at the time of response’s evaluation were significantly lower then at the time of diagnosis in pts who achieved CR (p<0,01) and did not differ in refractory AML. There was no difference between levels of both viable and apoptotic CEC in AML pts in CR and in the control group (p>0,05). Conclusions. The CEC and CECAnnV+ levels are significantly higher in AML patients than in healthy subjects and correlate with response to treatment. Further investigation should be undertaken to better determine their prognostic and therapeutic value.

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Conjugation of Lentivirus to Paramagnetic Particles via Nonviral Proteins Allows Efficient Concentration and Infection of Primary Acute Myeloid Leukemia Cells

Journal of Virology ◽

10.1128/jvi.79.20.13190-13194.2005 ◽

2005 ◽

Vol 79 (20) ◽

pp. 13190-13194 ◽

Cited By ~ 25

Author(s):

Lucas Chan ◽

Darren Nesbeth ◽

Taylor MacKey ◽

Joanna Galea-Lauri ◽

Joop Gäken ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Leukemia Virus ◽

Surface Proteins ◽

Virus Envelope ◽

Human Acute Myeloid Leukemia ◽

Acute Myeloid Leukemia Cells ◽

Acute Myeloid

ABSTRACT Nonviral producer cell proteins incorporated into retroviral vector surfaces profoundly influence infectivity and in vivo half-life. We report the purification and concentration of lentiviral vectors using these surface proteins as an efficient gene transduction strategy. Biotinylation of these proteins and streptavidin paramagnetic particle concentration enhances titer 400- to 2,500-fold (to 109 CFU/ml for vesicular stomatitis virus G protein and 5 × 108 for amphotropic murine leukemia virus envelope). This method also uses newly introduced membrane proteins (B7.1 and ΔLNGFR) directed to lentiviral surfaces, allowing up to 17,000-fold concentrations. Particle conjugation of lentivirus allows facile manipulation in vitro, resulting in the transduction of 48 to 94% of human acute myeloid leukemia blasts.

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Proteomic cell surface phenotyping of differentiating acute myeloid leukemia cells

Blood ◽

10.1182/blood-2010-02-271270 ◽

2010 ◽

Vol 116 (13) ◽

pp. e26-e34 ◽

Cited By ~ 66

Author(s):

Andreas Hofmann ◽

Bertran Gerrits ◽

Alexander Schmidt ◽

Thomas Bock ◽

Damaris Bausch-Fluck ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Surface ◽

Myeloid Leukemia ◽

Surface Protein ◽

Hematologic Malignancies ◽

Surface Proteins ◽

Model Systems ◽

Cell Surface Protein ◽

Cell Surface Proteins ◽

Acute Myeloid

Abstract Immunophenotyping by flow cytometry or immunohistochemistry is a clinical standard procedure for diagnosis, classification, and monitoring of hematologic malignancies. Antibody-based cell surface phenotyping is commonly limited to cell surface proteins for which specific antibodies are available and the number of parallel measurements is limited. The resulting limited knowledge about cell surface protein markers hampers early clinical diagnosis and subclassification of hematologic malignancies. Here, we describe the mass spectrometry based phenotyping of 2 all-trans retinoic acid treated acute myeloid leukemia model systems at an unprecedented level to a depth of more than 500 membrane proteins, including 137 bona fide cell surface exposed CD proteins. This extensive view of the leukemia surface proteome was achieved by developing and applying new implementations of the Cell Surface Capturing (CSC) technology. Bioinformatic and hierarchical cluster analysis showed that the applied strategy reliably revealed known differentiation-induced abundance changes of cell surface proteins in HL60 and NB4 cells and it also identified cell surface proteins with very little prior information. The extensive and quantitative analysis of the cell surface protein landscape from a systems biology perspective will be most useful in the clinic for the improved subclassification of hematologic malignancies and the identification of new drug targets.

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Phenotypic Aberrations of Myeloid CD34+ Cells in Patients with Myelodysplastic Syndrome (MDS): Clinical Significance and Correlation with Cytogenetics Abnormalities.

Blood ◽

10.1182/blood.v104.11.2374.2374 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2374-2374

Author(s):

Dolores Subira ◽

Patricia Font ◽

Eva Arranz ◽

Ramiro B Soraya ◽

Susana Castanon ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

High Risk ◽

Myeloid Leukemia ◽

Fish Analysis ◽

Aberrant Expression ◽

Number Of Patients ◽

Cd34 Cells ◽

Design And Methods ◽

Acute Myeloid

Abstract Expression of abnormal markers in myeloid CD34+ cells of patients with MDS is common, but few immunophenotypic data have been compiled so far. Based on the statement that CD7 and TdT are markers associated with a bad prognosis in acute myeloid leukemia, we intended to describe the incidence of their aberrant expression in CD34+ cells and its role helping to establish the diagnosis of MDS. OBJECTIVES: To explore the aberrant expression of TdT and CD7 in myeloid CD34+ cells of MDS patients and to describe their possible correlation with cytogenetic features. DESIGN AND METHODS: Bone marrow specimens from 45 patients with MDS were included in this study (17 RA, 12 RARS, 5 CMML, 9 RAEB, 2 RAEB-t). In addition, we analyzed 28 samples of bone marrow from patients with cytopenias, but no diagnosis of MDS, as a cohort control. Immunophenotyping was performed with the following combination of monoclonal antibodies: TdT FITC / CD7 PE/ CD34 PCy-5. A case was regarded as positive for any of these markers when their expression was described in at least, 25% of myeloid CD34+ cells. Besides, adequate cytogenetic data and FISH analysis of chromosomes 5, 7 and 8 were obtained from 42 out of the 45 MDS samples. RESULTS: The percentage of CD34+ myeloid cells in MDS samples was fewer than 2% in 25 cases, ranged from 2–5% in 6 cases and was equal or greater than 5% in 14 cases. Aberrant expression of CD7 and/or TdT was observed in 28 cases: 20 were positive for CD7, 5 cases were positive for TdT and co-expression of both antigens was described in 3 cases. Prevalence of these abnormal markers was much higher in patients with MDS (28/45; 62%) than in the cohort control (2/28; 7%) . Besides, we identified at least one abnormal marker in 14 of the 16 patients with high risk MDS (9 RAEB, 5 CMML, 2 RAEB.t). According to the IPSS, karyotypes were divided into subgroups of favorable, intermediate or unfavorable, being classified 29 patients with favorable karyotypes and 13 patients with no favorable cytogenetics. In the first group, the abnormal expression of CD7 and/or TdT was detected in 13/29 patients (44.8%) and in 10/13 patients in the non-favourable group (76.9%). CONCLUSIONS: CD7 and/or TdT expression in myeloid CD34 + cells may be helpful in establishing the diagnosis of MDS. Prevalence of these aberrancies seems to be higher in cases with no favorable karyotypes, but a larger number of patients will be required to state significant differences. Possible correlation between high risk MDS patients and immunophenotypic aberrancies suggests the convenience of following the outcome of those low risk MDS patients who have expression of any of these abnormal markers.

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