THE PROLIFERATIVE ACTIVITY OF THE BONE MARROW CELLS INVESTIGATED IN VITRO CELL CULTURE OF PATIENTS WITH CHRONIC MYELOID LEUKEMIA TREATED WITH TYROSINE KINASE INHIBITORS
(Abstract release date: 05/19/16)
EHA Library. Perekhrestenko T. 06/09/16; 135365; LB2254
Dr. Tetiana Perekhrestenko
Contributions
Contributions
Abstract
Abstract: LB2254
Type: Eposter Presentation
Background
The mechanisms of CML progression were postulated based on in vitro modeling even before they were detected in the clinical practice. CML progression can be driven by different mechanisms on tissue, cell, chromosomal and molecular level. Recent in vitro studies of CML bone marrow cells also suggest that disease progression can be caused by functional changes in pool of more matured hematopoietic progenitor cells rather than in population of HSCs. Such evolution of HPCs during CML progression can be identified by changes in their clonogenic potential during cultivation in semi-solid agar.
Aims
To analyze the changes in functional properties of HSCs and HPCs, such as ability to form cell aggregates during cultivation in CFU assay with further evaluation of cell composition of individually picked-up cell aggregates, with respect of correlation with CML progression in each individual patient.
Methods
Totally 51 samples of bone marrow were analyzed from patients with CML in chronic phase, who were treated with TKI, Imatinib. CFU-assay: Mononuclear fraction was separated from bone marrow aspirates by centrifugation over Ficoll-Hypaque gradient. All results were expressed as the mean number obtained from quadruplicate cultures. Clones of > 40 cells were counted as colony forming units granulocyte monocyte (CFU-GM) and those with 5 - 40 cells as cluster forming units (ClFU).
Results
CFU assay indicated that there was a significant (p < 0.05) difference in clonogenic potential (number of CFU-GM and ClFU) between groups of patients with different response to the therapy. CFU numbers were lower in group of patients with optimal response to the TKI therapy (mean value of 25.54±4.6) and for patients with TKI treatment failure (26.00±9.70) when compared to CFU numbers for groups of patients before TKI treatment (70.40±22.51) and patients with suboptimal response to TKI treatment (72.37±15.32). Significant decline (p < 0.05) was also indicated for mean numbers of ClFU for group of patients with optimal response for TKI (24.05±6.75) when compared to groups of patients with suboptimal response to TKI (49.94±7.89), but also for group of patients with TKI treatment failure (51.17±9.22). The mean number of ClFU for patients before TKI treatment was 41.80±9.78 and was not significantly different from other groups (p > 0.05). As to colony to cluster ratio our results showed, that there is a correlation (ρ = 0.67) between CCR index and number of bone marrow cells with Ph-chromosome. To have a possibility of identification of cells, composing aggregates obtained in CFU assay, a positive correlation was also found between numbers of colonies, formed during cultivation in CFU assay and level of CD34+ cells in bone marrow (ρ = 0.46) and between number of clusters and CD33+ cells of bone marrow (ρ = 0.76). To evaluate changes in the differentiation potential of HSCs and HPCs that can be related to pathologic process of CML, we picked-up individual cells aggregates, formed during in vitro CFU assay of bone marrow mononuclear cells of CML patients. Cells, composing those aggregates were analyzed with calculation of maturation index (MI) for each individual sample. It was indicated, that MI correlates with level of bone marrow cells, containing Ph-chromosome. CFU assay is one of the standard methods used to identify functional properties of HSCs and HPCs of bone marrow. Our data indicated alteration of clonogenic activity of bone marrow hemopoietic cells for patients with different response to TKI treatment. Thus, CFU and ClFU numbers were increased in case of examination of bone marrow of patients before TKI treatment, so as for patients with suboptimal response and TKI treatment failure.
Conclusion
In summary, obtained results suggest that different mechanisms (BCR-ABL dependent and independent) may be involved in CML progression process in the same time. Disease prognosis should be preferably carried out on an individual basis.
Session topic: E-poster
Type: Eposter Presentation
Background
The mechanisms of CML progression were postulated based on in vitro modeling even before they were detected in the clinical practice. CML progression can be driven by different mechanisms on tissue, cell, chromosomal and molecular level. Recent in vitro studies of CML bone marrow cells also suggest that disease progression can be caused by functional changes in pool of more matured hematopoietic progenitor cells rather than in population of HSCs. Such evolution of HPCs during CML progression can be identified by changes in their clonogenic potential during cultivation in semi-solid agar.
Aims
To analyze the changes in functional properties of HSCs and HPCs, such as ability to form cell aggregates during cultivation in CFU assay with further evaluation of cell composition of individually picked-up cell aggregates, with respect of correlation with CML progression in each individual patient.
Methods
Totally 51 samples of bone marrow were analyzed from patients with CML in chronic phase, who were treated with TKI, Imatinib. CFU-assay: Mononuclear fraction was separated from bone marrow aspirates by centrifugation over Ficoll-Hypaque gradient. All results were expressed as the mean number obtained from quadruplicate cultures. Clones of > 40 cells were counted as colony forming units granulocyte monocyte (CFU-GM) and those with 5 - 40 cells as cluster forming units (ClFU).
Results
CFU assay indicated that there was a significant (p < 0.05) difference in clonogenic potential (number of CFU-GM and ClFU) between groups of patients with different response to the therapy. CFU numbers were lower in group of patients with optimal response to the TKI therapy (mean value of 25.54±4.6) and for patients with TKI treatment failure (26.00±9.70) when compared to CFU numbers for groups of patients before TKI treatment (70.40±22.51) and patients with suboptimal response to TKI treatment (72.37±15.32). Significant decline (p < 0.05) was also indicated for mean numbers of ClFU for group of patients with optimal response for TKI (24.05±6.75) when compared to groups of patients with suboptimal response to TKI (49.94±7.89), but also for group of patients with TKI treatment failure (51.17±9.22). The mean number of ClFU for patients before TKI treatment was 41.80±9.78 and was not significantly different from other groups (p > 0.05). As to colony to cluster ratio our results showed, that there is a correlation (ρ = 0.67) between CCR index and number of bone marrow cells with Ph-chromosome. To have a possibility of identification of cells, composing aggregates obtained in CFU assay, a positive correlation was also found between numbers of colonies, formed during cultivation in CFU assay and level of CD34+ cells in bone marrow (ρ = 0.46) and between number of clusters and CD33+ cells of bone marrow (ρ = 0.76). To evaluate changes in the differentiation potential of HSCs and HPCs that can be related to pathologic process of CML, we picked-up individual cells aggregates, formed during in vitro CFU assay of bone marrow mononuclear cells of CML patients. Cells, composing those aggregates were analyzed with calculation of maturation index (MI) for each individual sample. It was indicated, that MI correlates with level of bone marrow cells, containing Ph-chromosome. CFU assay is one of the standard methods used to identify functional properties of HSCs and HPCs of bone marrow. Our data indicated alteration of clonogenic activity of bone marrow hemopoietic cells for patients with different response to TKI treatment. Thus, CFU and ClFU numbers were increased in case of examination of bone marrow of patients before TKI treatment, so as for patients with suboptimal response and TKI treatment failure.
Conclusion
In summary, obtained results suggest that different mechanisms (BCR-ABL dependent and independent) may be involved in CML progression process in the same time. Disease prognosis should be preferably carried out on an individual basis.
Session topic: E-poster
Abstract: LB2254
Type: Eposter Presentation
Background
The mechanisms of CML progression were postulated based on in vitro modeling even before they were detected in the clinical practice. CML progression can be driven by different mechanisms on tissue, cell, chromosomal and molecular level. Recent in vitro studies of CML bone marrow cells also suggest that disease progression can be caused by functional changes in pool of more matured hematopoietic progenitor cells rather than in population of HSCs. Such evolution of HPCs during CML progression can be identified by changes in their clonogenic potential during cultivation in semi-solid agar.
Aims
To analyze the changes in functional properties of HSCs and HPCs, such as ability to form cell aggregates during cultivation in CFU assay with further evaluation of cell composition of individually picked-up cell aggregates, with respect of correlation with CML progression in each individual patient.
Methods
Totally 51 samples of bone marrow were analyzed from patients with CML in chronic phase, who were treated with TKI, Imatinib. CFU-assay: Mononuclear fraction was separated from bone marrow aspirates by centrifugation over Ficoll-Hypaque gradient. All results were expressed as the mean number obtained from quadruplicate cultures. Clones of > 40 cells were counted as colony forming units granulocyte monocyte (CFU-GM) and those with 5 - 40 cells as cluster forming units (ClFU).
Results
CFU assay indicated that there was a significant (p < 0.05) difference in clonogenic potential (number of CFU-GM and ClFU) between groups of patients with different response to the therapy. CFU numbers were lower in group of patients with optimal response to the TKI therapy (mean value of 25.54±4.6) and for patients with TKI treatment failure (26.00±9.70) when compared to CFU numbers for groups of patients before TKI treatment (70.40±22.51) and patients with suboptimal response to TKI treatment (72.37±15.32). Significant decline (p < 0.05) was also indicated for mean numbers of ClFU for group of patients with optimal response for TKI (24.05±6.75) when compared to groups of patients with suboptimal response to TKI (49.94±7.89), but also for group of patients with TKI treatment failure (51.17±9.22). The mean number of ClFU for patients before TKI treatment was 41.80±9.78 and was not significantly different from other groups (p > 0.05). As to colony to cluster ratio our results showed, that there is a correlation (ρ = 0.67) between CCR index and number of bone marrow cells with Ph-chromosome. To have a possibility of identification of cells, composing aggregates obtained in CFU assay, a positive correlation was also found between numbers of colonies, formed during cultivation in CFU assay and level of CD34+ cells in bone marrow (ρ = 0.46) and between number of clusters and CD33+ cells of bone marrow (ρ = 0.76). To evaluate changes in the differentiation potential of HSCs and HPCs that can be related to pathologic process of CML, we picked-up individual cells aggregates, formed during in vitro CFU assay of bone marrow mononuclear cells of CML patients. Cells, composing those aggregates were analyzed with calculation of maturation index (MI) for each individual sample. It was indicated, that MI correlates with level of bone marrow cells, containing Ph-chromosome. CFU assay is one of the standard methods used to identify functional properties of HSCs and HPCs of bone marrow. Our data indicated alteration of clonogenic activity of bone marrow hemopoietic cells for patients with different response to TKI treatment. Thus, CFU and ClFU numbers were increased in case of examination of bone marrow of patients before TKI treatment, so as for patients with suboptimal response and TKI treatment failure.
Conclusion
In summary, obtained results suggest that different mechanisms (BCR-ABL dependent and independent) may be involved in CML progression process in the same time. Disease prognosis should be preferably carried out on an individual basis.
Session topic: E-poster
Type: Eposter Presentation
Background
The mechanisms of CML progression were postulated based on in vitro modeling even before they were detected in the clinical practice. CML progression can be driven by different mechanisms on tissue, cell, chromosomal and molecular level. Recent in vitro studies of CML bone marrow cells also suggest that disease progression can be caused by functional changes in pool of more matured hematopoietic progenitor cells rather than in population of HSCs. Such evolution of HPCs during CML progression can be identified by changes in their clonogenic potential during cultivation in semi-solid agar.
Aims
To analyze the changes in functional properties of HSCs and HPCs, such as ability to form cell aggregates during cultivation in CFU assay with further evaluation of cell composition of individually picked-up cell aggregates, with respect of correlation with CML progression in each individual patient.
Methods
Totally 51 samples of bone marrow were analyzed from patients with CML in chronic phase, who were treated with TKI, Imatinib. CFU-assay: Mononuclear fraction was separated from bone marrow aspirates by centrifugation over Ficoll-Hypaque gradient. All results were expressed as the mean number obtained from quadruplicate cultures. Clones of > 40 cells were counted as colony forming units granulocyte monocyte (CFU-GM) and those with 5 - 40 cells as cluster forming units (ClFU).
Results
CFU assay indicated that there was a significant (p < 0.05) difference in clonogenic potential (number of CFU-GM and ClFU) between groups of patients with different response to the therapy. CFU numbers were lower in group of patients with optimal response to the TKI therapy (mean value of 25.54±4.6) and for patients with TKI treatment failure (26.00±9.70) when compared to CFU numbers for groups of patients before TKI treatment (70.40±22.51) and patients with suboptimal response to TKI treatment (72.37±15.32). Significant decline (p < 0.05) was also indicated for mean numbers of ClFU for group of patients with optimal response for TKI (24.05±6.75) when compared to groups of patients with suboptimal response to TKI (49.94±7.89), but also for group of patients with TKI treatment failure (51.17±9.22). The mean number of ClFU for patients before TKI treatment was 41.80±9.78 and was not significantly different from other groups (p > 0.05). As to colony to cluster ratio our results showed, that there is a correlation (ρ = 0.67) between CCR index and number of bone marrow cells with Ph-chromosome. To have a possibility of identification of cells, composing aggregates obtained in CFU assay, a positive correlation was also found between numbers of colonies, formed during cultivation in CFU assay and level of CD34+ cells in bone marrow (ρ = 0.46) and between number of clusters and CD33+ cells of bone marrow (ρ = 0.76). To evaluate changes in the differentiation potential of HSCs and HPCs that can be related to pathologic process of CML, we picked-up individual cells aggregates, formed during in vitro CFU assay of bone marrow mononuclear cells of CML patients. Cells, composing those aggregates were analyzed with calculation of maturation index (MI) for each individual sample. It was indicated, that MI correlates with level of bone marrow cells, containing Ph-chromosome. CFU assay is one of the standard methods used to identify functional properties of HSCs and HPCs of bone marrow. Our data indicated alteration of clonogenic activity of bone marrow hemopoietic cells for patients with different response to TKI treatment. Thus, CFU and ClFU numbers were increased in case of examination of bone marrow of patients before TKI treatment, so as for patients with suboptimal response and TKI treatment failure.
Conclusion
In summary, obtained results suggest that different mechanisms (BCR-ABL dependent and independent) may be involved in CML progression process in the same time. Disease prognosis should be preferably carried out on an individual basis.
Session topic: E-poster
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