Contributions
Abstract: S198
Type: Oral Presentation
Session title: New pathways and molecular mechanisms in MPN
Background
The classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are characterized by uncontrolled expansion of myeloid progenitor cells, elevated blood counts, phenotype-driver mutations in certain target-genes (JAK2, CALR, MPL) and an increased risk of leukemic transformation. Despite being considered stem-cell disorders, the phenotype of the disease-initiating neoplastic stem cells (NSC) in MPN remains unknown.
Aims
The aim of the study is to identify MPN-initiating NSC and characterize their phenotype in order to facilitate their enrichment and develop novel treatment strategies.
Methods
As assessed by multicolor flow cytometry and a panel of monoclonal antibodies (n=50), we were able to define target expression profiles of putative CD34+/CD38− NSC and CD34+/CD38+ progenitor cells in patients with chronic phase MPN (n=93) and post-MPN secondary acute myeloid leukemia (sAML, n=11). To confirm the disease-initiating ability of primary MPN NSC, xenotransplantation experiments with NSGS mice were performed.
Results
In almost all patients, the MPN NSC expressed several receptors involved in NSC-niche interaction, including CD97, CD99, CD117 and CD184. In contrast, NSC expressed CD26 only in a subset of patients with primary myelofibrosis (PMF). MPN NSC expressed significantly higher levels of CD184 compared to stem cells in healthy controls (p<0.0001) or sAML (p<0.01). Among the cytokine receptors tested, CD25 was identified on NSC in most patients with PMF and sAML, and in a few with essential thrombocythemia (ET), but not in patients with polycythemia vera (PV). PMF NSC also displayed higher levels of CD25 than the PMF progenitor cells (p<0.01). MPN NSC did not exhibit substantial amounts of IL-1RAP and erythropoietin receptor. We next examined the expression of various immunological targets and resistance-mediating immune checkpoints. In all patients tested, NSC were found to express CD33. We also found that CD34+/CD38+ progenitor cells in ET display higher levels of CD33 than NSC (p<0.01). However, neither NSC nor the progenitor cells in MPN expressed higher levels of CD33 compared to normal stem cells or normal progenitor cells. MPN NSC and sAML stem cells consistently expressed the “don’t eat me” checkpoint CD47 and the classical checkpoint CD274 (PD-L1). MPN NSC displayed significantly higher levels of CD274 compared to normal stem cells (p<0.001) or stem cells in sAML (p<0.05). No difference in the expression of surface antigens on MPN NSC were observed between patients harboring mutations in the JAK2 and CALR genes. Engraftment of human CD45+CD33+ cells in the bone marrow of NSGS mice was found in 15/15 mice injected with bulk mononuclear cells (MNC) containing CD34+ cells and in 0/15 NSGS mice injected with CD34-depleted MNC after 28 weeks. Whereas the CD38- sub-fraction of MPN MNC produced leukemic engraftment in 10/14 mice (71.4%) only 2/13 mice (15.4%) engrafted when injected with the CD38+ sub-fractions of MPN NSC. Engraftment of clonal MPN cells was confirmed by demonstrating the presence of JAK2-V617F mutation in engrafted human cells in mouse BM samples.
Conclusion
Together, we show that MPN NSC reside in a CD34+/CD38− fraction of the malignant clone and display a unique phenotype, including immune checkpoint molecules, cytokine receptors and other target antigens. Our results should support the isolation of MPN NSC and the development of NSC-eradicating therapies in MPN.
Keyword(s): Essential Thrombocytemia, Myelofibrosis, Polycythemia vera, Stem cell marker
Abstract: S198
Type: Oral Presentation
Session title: New pathways and molecular mechanisms in MPN
Background
The classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are characterized by uncontrolled expansion of myeloid progenitor cells, elevated blood counts, phenotype-driver mutations in certain target-genes (JAK2, CALR, MPL) and an increased risk of leukemic transformation. Despite being considered stem-cell disorders, the phenotype of the disease-initiating neoplastic stem cells (NSC) in MPN remains unknown.
Aims
The aim of the study is to identify MPN-initiating NSC and characterize their phenotype in order to facilitate their enrichment and develop novel treatment strategies.
Methods
As assessed by multicolor flow cytometry and a panel of monoclonal antibodies (n=50), we were able to define target expression profiles of putative CD34+/CD38− NSC and CD34+/CD38+ progenitor cells in patients with chronic phase MPN (n=93) and post-MPN secondary acute myeloid leukemia (sAML, n=11). To confirm the disease-initiating ability of primary MPN NSC, xenotransplantation experiments with NSGS mice were performed.
Results
In almost all patients, the MPN NSC expressed several receptors involved in NSC-niche interaction, including CD97, CD99, CD117 and CD184. In contrast, NSC expressed CD26 only in a subset of patients with primary myelofibrosis (PMF). MPN NSC expressed significantly higher levels of CD184 compared to stem cells in healthy controls (p<0.0001) or sAML (p<0.01). Among the cytokine receptors tested, CD25 was identified on NSC in most patients with PMF and sAML, and in a few with essential thrombocythemia (ET), but not in patients with polycythemia vera (PV). PMF NSC also displayed higher levels of CD25 than the PMF progenitor cells (p<0.01). MPN NSC did not exhibit substantial amounts of IL-1RAP and erythropoietin receptor. We next examined the expression of various immunological targets and resistance-mediating immune checkpoints. In all patients tested, NSC were found to express CD33. We also found that CD34+/CD38+ progenitor cells in ET display higher levels of CD33 than NSC (p<0.01). However, neither NSC nor the progenitor cells in MPN expressed higher levels of CD33 compared to normal stem cells or normal progenitor cells. MPN NSC and sAML stem cells consistently expressed the “don’t eat me” checkpoint CD47 and the classical checkpoint CD274 (PD-L1). MPN NSC displayed significantly higher levels of CD274 compared to normal stem cells (p<0.001) or stem cells in sAML (p<0.05). No difference in the expression of surface antigens on MPN NSC were observed between patients harboring mutations in the JAK2 and CALR genes. Engraftment of human CD45+CD33+ cells in the bone marrow of NSGS mice was found in 15/15 mice injected with bulk mononuclear cells (MNC) containing CD34+ cells and in 0/15 NSGS mice injected with CD34-depleted MNC after 28 weeks. Whereas the CD38- sub-fraction of MPN MNC produced leukemic engraftment in 10/14 mice (71.4%) only 2/13 mice (15.4%) engrafted when injected with the CD38+ sub-fractions of MPN NSC. Engraftment of clonal MPN cells was confirmed by demonstrating the presence of JAK2-V617F mutation in engrafted human cells in mouse BM samples.
Conclusion
Together, we show that MPN NSC reside in a CD34+/CD38− fraction of the malignant clone and display a unique phenotype, including immune checkpoint molecules, cytokine receptors and other target antigens. Our results should support the isolation of MPN NSC and the development of NSC-eradicating therapies in MPN.
Keyword(s): Essential Thrombocytemia, Myelofibrosis, Polycythemia vera, Stem cell marker