BIOLOGICAL AND CLINICAL FEATURES OF IMMUNOSUPPRESSIVE THERAPY IN PATIENTS AFFECTED BY LARGE GRANULAR LYMPHOCYTE LEUKEMIA
(Abstract release date: 05/19/16)
EHA Library. Teramo A. 06/09/16; 132564; E1015
Dr. Antonella Teramo
Contributions
Contributions
Abstract
Abstract: E1015
Type: Eposter Presentation
Background
Large Granular Lymphocyte Leukemia (LGLL) is a rare and heterogeneous lymphoproliferative disorder characterized by the chronic proliferation of clonal Large Granular Lymphocytes (LGLs) with cytotoxic activity. Recently, activating STAT3 mutations have been discovered in 30-40% of T-LGLL patients. The treatment of LGLL is based on immunosuppressive drugs, such as Methotrexate (MTX), Cyclophosphamide (CTX) and Cyclosporin A (CyA), used at low doses. These therapies have limited efficacy and the Overall Response Rate (ORR) is near 50%. Furthermore, in vitro studies on the molecular effects of these drugs on leukemic lymphocytes are not available in the literature and the biological rational of LGLL treatment has yet not been investigated.
Aims
The aim of this study was to analyze the in vivo efficacy of treatment on a cohort of patients referred to the Hematology Unit at Padua University. In addition, we performed in vitro study of the molecular effect of these drugs on pathological LGLs in order to better understand their efficacy/specificity, in particular considering their effect on STAT3 pathway.
Methods
Clinical characteristics and treatment response were collected from 17 LGLL patients. For in vitro analysis a cohort accounting 35 patients was studied. Cell cultures of patients’ peripheral blood mononuclear cells (PBMCs) were set up with CTX (5µM), CyA (10µM) or MTX (100µM). On collected culture cells apoptosis by Annexin V assay and phosphorylation of STAT3 by Western Blot were investigated.
Results
Clinical results demonstrated that ORR for each drug ranged from 35.7% to 57.1%, CTX treatment showing the higher ORR independently from the therapy line. Fifteen out of the 17 treated patients were STAT3 mutated, indicating a strong correlation between STAT3 mutation and symptomatic disease. Interestingly, consistent with literature data, patients carrying Y640F STAT3 mutation showed a better ORR on MTX treatment as compared with patients with different STAT3 mutation (66% versus 14.2%).In vitro, CTX and CyA showed a strong increase of LGL apoptosis, leading to the complete disappearance of LGL clone within 3-6 days of culture. Anyway, concerning the specificity of the killing leukemic LGL cells, while sparing non leukemic PBMCs, our data showed that both CTX and CyA were also partially toxic for non-LGLs population. As regards MTX treatment, no increase of apoptosis was revealed, but, interestingly, we observed that MTX induced apoptosis when LGL were stimulated to proliferate by IL-15, suggesting a predominant effect on activated cells.Considering the key role of STAT3 activation in leukemic LGL survival, we observed a decrease of STAT3 phosphorylation levels only after CyA treatment.Finally, adding autologous plasma (10%) to cell culture the cytotoxic effect of drugs was partially reduced (~50%), suggesting that plasma might contain cytokines preserving LGL survival.
Conclusion
In summary, CTX showed a good efficacy in vivo and in vitro, even if in this last setting the compound did not exhibit a good LGL specificity. MTX might be more efficient when LGLs are proliferating and when LGLs carry activating mutations, even if it does not affect STAT3 activation. Finally, we demonstrated that CyA expresses its cytotoxic effect through a mechanism that down-modulates STAT3 activation.Our results contribute to get insights into the molecular mechanisms of immunosuppressive drugs used in LGLL treatment and suggest that STAT3 mutation/activation might represent a suitable target for therapy.
Session topic: E-poster
Keyword(s): Cyclophosphamide, Cyclosporin A, Large granular lymphocytic leukaemia, Methotrexate
Type: Eposter Presentation
Background
Large Granular Lymphocyte Leukemia (LGLL) is a rare and heterogeneous lymphoproliferative disorder characterized by the chronic proliferation of clonal Large Granular Lymphocytes (LGLs) with cytotoxic activity. Recently, activating STAT3 mutations have been discovered in 30-40% of T-LGLL patients. The treatment of LGLL is based on immunosuppressive drugs, such as Methotrexate (MTX), Cyclophosphamide (CTX) and Cyclosporin A (CyA), used at low doses. These therapies have limited efficacy and the Overall Response Rate (ORR) is near 50%. Furthermore, in vitro studies on the molecular effects of these drugs on leukemic lymphocytes are not available in the literature and the biological rational of LGLL treatment has yet not been investigated.
Aims
The aim of this study was to analyze the in vivo efficacy of treatment on a cohort of patients referred to the Hematology Unit at Padua University. In addition, we performed in vitro study of the molecular effect of these drugs on pathological LGLs in order to better understand their efficacy/specificity, in particular considering their effect on STAT3 pathway.
Methods
Clinical characteristics and treatment response were collected from 17 LGLL patients. For in vitro analysis a cohort accounting 35 patients was studied. Cell cultures of patients’ peripheral blood mononuclear cells (PBMCs) were set up with CTX (5µM), CyA (10µM) or MTX (100µM). On collected culture cells apoptosis by Annexin V assay and phosphorylation of STAT3 by Western Blot were investigated.
Results
Clinical results demonstrated that ORR for each drug ranged from 35.7% to 57.1%, CTX treatment showing the higher ORR independently from the therapy line. Fifteen out of the 17 treated patients were STAT3 mutated, indicating a strong correlation between STAT3 mutation and symptomatic disease. Interestingly, consistent with literature data, patients carrying Y640F STAT3 mutation showed a better ORR on MTX treatment as compared with patients with different STAT3 mutation (66% versus 14.2%).In vitro, CTX and CyA showed a strong increase of LGL apoptosis, leading to the complete disappearance of LGL clone within 3-6 days of culture. Anyway, concerning the specificity of the killing leukemic LGL cells, while sparing non leukemic PBMCs, our data showed that both CTX and CyA were also partially toxic for non-LGLs population. As regards MTX treatment, no increase of apoptosis was revealed, but, interestingly, we observed that MTX induced apoptosis when LGL were stimulated to proliferate by IL-15, suggesting a predominant effect on activated cells.Considering the key role of STAT3 activation in leukemic LGL survival, we observed a decrease of STAT3 phosphorylation levels only after CyA treatment.Finally, adding autologous plasma (10%) to cell culture the cytotoxic effect of drugs was partially reduced (~50%), suggesting that plasma might contain cytokines preserving LGL survival.
Conclusion
In summary, CTX showed a good efficacy in vivo and in vitro, even if in this last setting the compound did not exhibit a good LGL specificity. MTX might be more efficient when LGLs are proliferating and when LGLs carry activating mutations, even if it does not affect STAT3 activation. Finally, we demonstrated that CyA expresses its cytotoxic effect through a mechanism that down-modulates STAT3 activation.Our results contribute to get insights into the molecular mechanisms of immunosuppressive drugs used in LGLL treatment and suggest that STAT3 mutation/activation might represent a suitable target for therapy.
Session topic: E-poster
Keyword(s): Cyclophosphamide, Cyclosporin A, Large granular lymphocytic leukaemia, Methotrexate
Abstract: E1015
Type: Eposter Presentation
Background
Large Granular Lymphocyte Leukemia (LGLL) is a rare and heterogeneous lymphoproliferative disorder characterized by the chronic proliferation of clonal Large Granular Lymphocytes (LGLs) with cytotoxic activity. Recently, activating STAT3 mutations have been discovered in 30-40% of T-LGLL patients. The treatment of LGLL is based on immunosuppressive drugs, such as Methotrexate (MTX), Cyclophosphamide (CTX) and Cyclosporin A (CyA), used at low doses. These therapies have limited efficacy and the Overall Response Rate (ORR) is near 50%. Furthermore, in vitro studies on the molecular effects of these drugs on leukemic lymphocytes are not available in the literature and the biological rational of LGLL treatment has yet not been investigated.
Aims
The aim of this study was to analyze the in vivo efficacy of treatment on a cohort of patients referred to the Hematology Unit at Padua University. In addition, we performed in vitro study of the molecular effect of these drugs on pathological LGLs in order to better understand their efficacy/specificity, in particular considering their effect on STAT3 pathway.
Methods
Clinical characteristics and treatment response were collected from 17 LGLL patients. For in vitro analysis a cohort accounting 35 patients was studied. Cell cultures of patients’ peripheral blood mononuclear cells (PBMCs) were set up with CTX (5µM), CyA (10µM) or MTX (100µM). On collected culture cells apoptosis by Annexin V assay and phosphorylation of STAT3 by Western Blot were investigated.
Results
Clinical results demonstrated that ORR for each drug ranged from 35.7% to 57.1%, CTX treatment showing the higher ORR independently from the therapy line. Fifteen out of the 17 treated patients were STAT3 mutated, indicating a strong correlation between STAT3 mutation and symptomatic disease. Interestingly, consistent with literature data, patients carrying Y640F STAT3 mutation showed a better ORR on MTX treatment as compared with patients with different STAT3 mutation (66% versus 14.2%).In vitro, CTX and CyA showed a strong increase of LGL apoptosis, leading to the complete disappearance of LGL clone within 3-6 days of culture. Anyway, concerning the specificity of the killing leukemic LGL cells, while sparing non leukemic PBMCs, our data showed that both CTX and CyA were also partially toxic for non-LGLs population. As regards MTX treatment, no increase of apoptosis was revealed, but, interestingly, we observed that MTX induced apoptosis when LGL were stimulated to proliferate by IL-15, suggesting a predominant effect on activated cells.Considering the key role of STAT3 activation in leukemic LGL survival, we observed a decrease of STAT3 phosphorylation levels only after CyA treatment.Finally, adding autologous plasma (10%) to cell culture the cytotoxic effect of drugs was partially reduced (~50%), suggesting that plasma might contain cytokines preserving LGL survival.
Conclusion
In summary, CTX showed a good efficacy in vivo and in vitro, even if in this last setting the compound did not exhibit a good LGL specificity. MTX might be more efficient when LGLs are proliferating and when LGLs carry activating mutations, even if it does not affect STAT3 activation. Finally, we demonstrated that CyA expresses its cytotoxic effect through a mechanism that down-modulates STAT3 activation.Our results contribute to get insights into the molecular mechanisms of immunosuppressive drugs used in LGLL treatment and suggest that STAT3 mutation/activation might represent a suitable target for therapy.
Session topic: E-poster
Keyword(s): Cyclophosphamide, Cyclosporin A, Large granular lymphocytic leukaemia, Methotrexate
Type: Eposter Presentation
Background
Large Granular Lymphocyte Leukemia (LGLL) is a rare and heterogeneous lymphoproliferative disorder characterized by the chronic proliferation of clonal Large Granular Lymphocytes (LGLs) with cytotoxic activity. Recently, activating STAT3 mutations have been discovered in 30-40% of T-LGLL patients. The treatment of LGLL is based on immunosuppressive drugs, such as Methotrexate (MTX), Cyclophosphamide (CTX) and Cyclosporin A (CyA), used at low doses. These therapies have limited efficacy and the Overall Response Rate (ORR) is near 50%. Furthermore, in vitro studies on the molecular effects of these drugs on leukemic lymphocytes are not available in the literature and the biological rational of LGLL treatment has yet not been investigated.
Aims
The aim of this study was to analyze the in vivo efficacy of treatment on a cohort of patients referred to the Hematology Unit at Padua University. In addition, we performed in vitro study of the molecular effect of these drugs on pathological LGLs in order to better understand their efficacy/specificity, in particular considering their effect on STAT3 pathway.
Methods
Clinical characteristics and treatment response were collected from 17 LGLL patients. For in vitro analysis a cohort accounting 35 patients was studied. Cell cultures of patients’ peripheral blood mononuclear cells (PBMCs) were set up with CTX (5µM), CyA (10µM) or MTX (100µM). On collected culture cells apoptosis by Annexin V assay and phosphorylation of STAT3 by Western Blot were investigated.
Results
Clinical results demonstrated that ORR for each drug ranged from 35.7% to 57.1%, CTX treatment showing the higher ORR independently from the therapy line. Fifteen out of the 17 treated patients were STAT3 mutated, indicating a strong correlation between STAT3 mutation and symptomatic disease. Interestingly, consistent with literature data, patients carrying Y640F STAT3 mutation showed a better ORR on MTX treatment as compared with patients with different STAT3 mutation (66% versus 14.2%).In vitro, CTX and CyA showed a strong increase of LGL apoptosis, leading to the complete disappearance of LGL clone within 3-6 days of culture. Anyway, concerning the specificity of the killing leukemic LGL cells, while sparing non leukemic PBMCs, our data showed that both CTX and CyA were also partially toxic for non-LGLs population. As regards MTX treatment, no increase of apoptosis was revealed, but, interestingly, we observed that MTX induced apoptosis when LGL were stimulated to proliferate by IL-15, suggesting a predominant effect on activated cells.Considering the key role of STAT3 activation in leukemic LGL survival, we observed a decrease of STAT3 phosphorylation levels only after CyA treatment.Finally, adding autologous plasma (10%) to cell culture the cytotoxic effect of drugs was partially reduced (~50%), suggesting that plasma might contain cytokines preserving LGL survival.
Conclusion
In summary, CTX showed a good efficacy in vivo and in vitro, even if in this last setting the compound did not exhibit a good LGL specificity. MTX might be more efficient when LGLs are proliferating and when LGLs carry activating mutations, even if it does not affect STAT3 activation. Finally, we demonstrated that CyA expresses its cytotoxic effect through a mechanism that down-modulates STAT3 activation.Our results contribute to get insights into the molecular mechanisms of immunosuppressive drugs used in LGLL treatment and suggest that STAT3 mutation/activation might represent a suitable target for therapy.
Session topic: E-poster
Keyword(s): Cyclophosphamide, Cyclosporin A, Large granular lymphocytic leukaemia, Methotrexate
{{ help_message }}
{{filter}}