Contributions
Abstract: PB2288
Type: Publication Only
Background
Myeloproliferative neoplasms (MPN) are chronic myeloid cancers that are characterized by the overproduction of mature blood cells, and that may evolve into acute myeloid leukaemia. In solid tumours, calreticulin (CALR) overexpression produces a pro-phagocytic signal and is counteracted by concomitant expression of anti- phagocytic CD47, reflecting an apoptosis vs survival mechanism
Aims
To investigate the expression and cellular location of CALR and CD47 in patients with MPN in comparison with healthy controls.
Methods
Mononuclear cells were obtained from peripheral blood of 6 MPN patient samples (2 Polycythaemia Vera, 1 Myelofibrosis, 3 Essential Thrombocythemia), along with 3 healthy controls by FICOLL separation.Cells were fractionised into 4 compartments: Membrane, cytoplasm, cytosol and nucleus. Proteins were extracted using TRIzol extraction andCALR and CD47 protein expression was analysed by western blotting
Results
Overall CALR expression was unchanged in MPN samples comparing with controls (3.23 vs 2.95 fold, respectively) (Fold changes are seen through normalization against the housekeeping protein). In contrast, CD47 significantly increased in MPN samples vs. controls (3.66 vs 0.06 fold, respectively). CALR and CD47 showed similar patterns of cellular localization in controls: membrane (51% and 58.7%, respectively), cytosol (38.4% and 36.2%, respectively), cytoplasm (9.6% and 4.5%, respectively), and nucleus (1% and 0.7%, respectively). In MPN samples CALR and CD47 moved into the membrane (CALR=77.2%; CD47=66.6%) and cytoplasm (CALR=12.5%; CD47=14.8%), reducing in cytosol (CALR=5.4%; CD47=16.4%) and no changes in nuclear expression (CALR=1.3%; CD47=2.2%) were observed.
Conclusion
CD47, but not CALR, is overexpressed in patients with MPN comparing with controls. This opposes previous studies in solid tumours, which show significant increases of both CALR and CD47, suggesting a role for CD47 as a strong anti-phagocytic signal responsible for immune survival in MPN. We have also shown that in contrast to cells from healthy controls, MPN cells mainly express CALR and CD47 on the cell surface with possibly a slightly higher relative CALR expression. No differences in CALR and CD47 expression was noted in different MPN subtypes however a larger cohort of patients, treated and untreated, is required to confirm our findings and identify CALR/CD47 pattern changes in response to therapies.
Session topic: 16. Myeloproliferative neoplasms - Clinical
Keyword(s): Myeloproliferative disorder
Abstract: PB2288
Type: Publication Only
Background
Myeloproliferative neoplasms (MPN) are chronic myeloid cancers that are characterized by the overproduction of mature blood cells, and that may evolve into acute myeloid leukaemia. In solid tumours, calreticulin (CALR) overexpression produces a pro-phagocytic signal and is counteracted by concomitant expression of anti- phagocytic CD47, reflecting an apoptosis vs survival mechanism
Aims
To investigate the expression and cellular location of CALR and CD47 in patients with MPN in comparison with healthy controls.
Methods
Mononuclear cells were obtained from peripheral blood of 6 MPN patient samples (2 Polycythaemia Vera, 1 Myelofibrosis, 3 Essential Thrombocythemia), along with 3 healthy controls by FICOLL separation.Cells were fractionised into 4 compartments: Membrane, cytoplasm, cytosol and nucleus. Proteins were extracted using TRIzol extraction andCALR and CD47 protein expression was analysed by western blotting
Results
Overall CALR expression was unchanged in MPN samples comparing with controls (3.23 vs 2.95 fold, respectively) (Fold changes are seen through normalization against the housekeeping protein). In contrast, CD47 significantly increased in MPN samples vs. controls (3.66 vs 0.06 fold, respectively). CALR and CD47 showed similar patterns of cellular localization in controls: membrane (51% and 58.7%, respectively), cytosol (38.4% and 36.2%, respectively), cytoplasm (9.6% and 4.5%, respectively), and nucleus (1% and 0.7%, respectively). In MPN samples CALR and CD47 moved into the membrane (CALR=77.2%; CD47=66.6%) and cytoplasm (CALR=12.5%; CD47=14.8%), reducing in cytosol (CALR=5.4%; CD47=16.4%) and no changes in nuclear expression (CALR=1.3%; CD47=2.2%) were observed.
Conclusion
CD47, but not CALR, is overexpressed in patients with MPN comparing with controls. This opposes previous studies in solid tumours, which show significant increases of both CALR and CD47, suggesting a role for CD47 as a strong anti-phagocytic signal responsible for immune survival in MPN. We have also shown that in contrast to cells from healthy controls, MPN cells mainly express CALR and CD47 on the cell surface with possibly a slightly higher relative CALR expression. No differences in CALR and CD47 expression was noted in different MPN subtypes however a larger cohort of patients, treated and untreated, is required to confirm our findings and identify CALR/CD47 pattern changes in response to therapies.
Session topic: 16. Myeloproliferative neoplasms - Clinical
Keyword(s): Myeloproliferative disorder