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P-007 Novel epigenetic and genetic alterations in cytogenetically normal refractory cytopenia with multilineage dysplasia and chronic myelomonocytic leukemia
Poster Presentations – 12th International Symposium on Myelodysplastic Syndromes / Leukemia Research 37 S1 (2013) S1–S117
Division of Pathology, Karolinska University Hospital Huddinge, Stockholm, Sweden Background: Refractory anemia with ring sideroblasts (RARS) is a myelodysplastic syndrome subgroup distinguished by anemia, erythroid apoptosis and mitochondrial ferritin accumulation in erythroblasts, which fail to mature into erythrocytes. Introduction: It is poorly understood at which stage and by which mechanism erythroid failure occurs; hence possible targets for therapy are yet to be discovered. Recently, heterozygous mutations in SF3B1, a core component of the spliceosome, were identified in >70% of RARS. This discovery may provide clues to how the failure of terminal differentiation is executed, and how the hematopoietic stem cells (HSC) gain their clonal advantage. Purpose: To explore the molecular and cellular mechanisms behind clonal expansion of immature progenitors and defective erythroid maturation, we investigated transcriptome profiling, splice patterns and SF3B1 allelic burden during erythroid differentiation in 11 RARS patients SF3B1 mutated, of which the majority also carried other driver mutations. Materials and Methods: Initially, normal and RARS transcriptomes from two time points during early erythroid differentiation were analyzed and compared, and in a second phase, transcriptome findings were validated using Taqman Low Density Array (TLDA) analysis in an extended cohort. Results: We observed the activation of genes involved in defence against oxidative stress in CD34+ progenitors. By contrast, erythroblasts were characterized by a failure to up-regulate genes in the autophagy pathway, essential for terminal maturation to erythrocytes, and by a maintained expression over time of regulators of transcription, apoptosis and adhesion, indicating that transcription in general is not hampered by SF3B1 mutation. We also report marked alterations in gene expression during RARS differentiation (FDR <5%) and altered splicing of genes involved in hematopoiesis. The exon usage pattern was very heterogeneous within the RARS population, while showing limited variation in the NBM cohort. Finally, we demonstrated that SF3B1 mutation does not confer a growth disadvantage to the erythroid cells until final maturation to reticulocytes, the stage at which autophagy becomes essential for erythroid differentiation. Conclusions: Our study suggests oxidative stress defence, potentially caused by mitochondrial iron accumulation, as an underlying mechanism for the clonal advantage of RARS stem and early progenitor cells and provides novel insights into the erythroid differentiation process. By showing that anemia develops during terminal maturation into erythrocytes, we provide important insights into how the severe anemia in RARS patients may be addressed.
P-006 Prognostic value of TP53 gene mutations in higher-risk MDS treated with azacitidine (AZA) C. Bally 1 , L. Ades 1 , A. Renneville 2 , M.J. Mozziconacci 3 , C. Preudhomme 2 , H. DeThé 4 , J. Lehmann-Che 4 , P. Fenaux 1 . 1 Service d’Hématologie clinique, Hopital Avicenne, Bobigny, France; 2 Service d’Hématologie biologique, CHU de Lille, Lille, France; 3 Service d’Hématologie biologique, Institut Paoli Calmette, Marseille, France; 4 Biologie moléculaire, Hopital Saint Louis, Paris, France Background: TP53 gene mutations, in MDS and AML, are associated with resistance to chemotherapy (Wattel, Blood, 1994). Introduction: Their correlation with results of treatment with hypomethylating agents has not been studied. Purpose: We assessed the prognostic value of TP53 mutations for response to AZA in higher risk MDS, AML (20-30% blasts) or advanced CMML (ie EU label for AZA).
S25
Materials and Methods: Patients received AZA (75 mg/m2 /d ×7 days/4 w) in 2 GFM centers. TP53 mutations were detected by the functional FASAY technique, whose sensitivity is 10-15% (Flaman, PNAS 1995) and confirmed by direct sequencing (1% sensitivity NGS technique using pyrosequencing, GS Junior System). Results: 72 pts treated with AZA (median n¡ of cycles 4, range 17) were analyzed, including 34 MDS, 33 AML (20-30% blasts) and 5 CMML. Karyotype was fav in 27%. int in 13% and unfav in 60% pts, including 47% complex karyotypes. 51% of pts responded (IWG 2006 criteria), including 34% CR, 6% PR, 4% marrow CR, 7% Stable disease with HI. Median OS from onset of AZA was 15.6 months 29 (40%) pts had TP53 mutation by FASAY, always confirmed by NGS, including 22/34 (67%) complex karyotypes. ORR to AZA (41% vs 57%, p=0.227) and CR rate (21% vs 42%, p=0.073) did not significantly differ between mutated and unmutated pts, but OS was shorter in pts with TP53 mutation (median 13 vs 21 months, p=0.0022, Figure 1), complex cytogenetics (median 14 vs 18 months, p=0.0120), WHO AML (median 13 vs 21 months for -MDS or CMML, p=0.0014). By multivariate analysis, marrow blast % (HR 1.03 (p<10-4)) and TP53 mutational status (HR 2.29 p=0.035) were the only 2 factors retaining significance for OS.
Figure 1
Conclusions: In higher risk MDS, AML 20-30% blasts and CMML treated with AZA, TP53 mutation is an independent prognostic parameter of poorer survival.
P-007 Novel epigenetic and genetic alterations in cytogenetically normal refractory cytopenia with multilineage dysplasia and chronic myelomonocytic leukemia J.X. Cheng 1 , J. Anastasi 2 , J.Q. Shen 3 , A. Rodrigues 4 , D. Roulston 1 , J.W. Vardiman 2 . 1 Pathology, University of Michigan, Ann Arbor, USA; 2 Pathology, University of Chicago, Chicago, USA; 3 Bioinformatics, University of Nevada, Las Vegas, USA; 4 Bioinformatics, University of Michigan, Ann Arbor, USA Background: Refractory cytopenia with multilineage dysplasia (RCMD), a subtype of myelodysplastic syndrome (MDS), is characterized by multilineage dysplasia, pancytopenia and no increase in blasts. Chronic myelomonocytic leukemia (CMML), a subtype of myelodysplastic/myeloproliferative neoplasm (MDS/MPN), usually has a proliferative component manifested by absolute monocytosis, in addition to some dysplastic features. The different characteristics of these two diseases may suggest that they are molecularly distinct entities caused by different molecular mechanisms and/or evolution of varying molecular abnormalities.
S26
Poster Presentations – 12th International Symposium on Myelodysplastic Syndromes / Leukemia Research 37 S1 (2013) S1–S117
Introduction: PU.1, an ETS transcription factor, is critical for myelopoiesis, especially for determining erythroid/megakaryocytic vs. lymphoid/myeloid differentiation. In a recent study, we uncovered a novel epigenetic mechanism that involves an increase in the repressing epigenetic modification of Histone 3 lysine 27 trimethylation (H3K27me3) at PU.1 and its downstream genes, resulting in inactivation of the PU.1 and lymphoid/myeloid potent progenitor (PU.1/LMPP) pathway in some cytogenetically normal RCMDs (CN-RCMDs) (J.X. Cheng et al. Leukemia in press). Here we extend our prior study to cytogenetically normal chronic myelomonocytic leukemia (CN-CMML). Purpose: To compare the epigenetic and genetic mechanisms underlying the pathogenesis of CN-RCMD and CN-CMML Materials and Methods: Both primary bone marrow cells from normal, CN-RCMD and CN-CMML patients and cell lines derived from MDS and monocytic leukemia were used. Genome-wide chromatin immunoprecipitation (ChIP-on-chip), sequential ChIP, methylated DNA immunoprecipitation (MeDIP-onchip), chromosome conformation capture (3-C), real-time RT-PCR, flow cytometry, immunohistochemistry, si-RNA technology, histone methytransferase inhibitors, genome-wide exome-sequencing and Sanger sequencing were employed. Results: Our study revealed distinct chromatin patterns of the PU.1 and its down-stream genes in CN-CMML vs. CN-RCMD. In contrast to an increase in the repressing epigenetic modification of H3K27me3 and inactivation of the PU.1/LMPP pathway in CN-RCMD, an increase in both the repressing modification of H3K27me3 and the activating modification of H3K4me3, i.e. a “bivalent” chromatin and a hyperactivation of the PU.1/LMPP pathway was observed in CN-CMML. The methyltransferase inhibitors for H3K27 or H3K4 methylation had an inhibitory effect on monocytic leukemia cells. Reduction of PU.1 expression by si-RNA effectively inhibited growth of monocytic leukemia cells, but not MDS-derived erythroid/myeloid leukemia cells. Exome-sequencing revealed a large number of novel recurrent gene mutations in CN-RCMD (n=10) and CN-CMML (n=8); specifically, 325 mutations in CN-RCMD only, 449 in CN-CMML only, and 393 mutations shared by both. Sanger sequencing was performed to confirm the exome-sequencing results and identified rare subtype-specific mutations in these diseases. Conclusions: We propose two hypothetical models of dysregulation of the PU.1/LMPP pathway by epigenetic/genetic alterations in CNRCMD and CN-CMML. See Figure 1 below.
P-008 Hedgehog signaling pathway and stem cell markers are linked with dysplastic changes of myelodysplastic syndrome B. Alinger, R. Kemmerling, E. Klieser, R. Illig, T. Kiesslich, D. Neureiter. Institute of Pathology, Salzburger Landeskliniken/Paracelsus Medical University, Salzburg, Austria Background: Assumed role of the Hedgehog (HH) signaling pathway within the regulation of hematopoietic stem cells (HSCs) maintenance as well as in the clonal disease of myelodysplastic syndrome (MDS) still remains unclear. Introduction: Recent studies revealed an aberrant activation of the hedgehog signaling pathway and cancer stem cells in a wide variety of human cancers including malignant hematological neoplasms. Purpose: Therefore, we investigated the expression pattern of subset HH signaling members and different hematopoietic stem cell markers at different stages of MDS compared to control samples from healthy donors. Materials and Methods: Bone marrow samples of 18 normal versus 80 patients with MDS (subgroups according to WHO: n=40 refractory cytopenia (RA) with multilineage dysplasia, n=13/22 refractory anemia with excess of blasts I/II, and n=5 secondary acute myeloid leukemia) were investigated. All MDS samples were fully characterized for dysplastic change of bone marrow as well as for risk factors (IPSS), laboratory parameters and follow-up. mRNA and protein expression pattern of HSC makers (CD34, CD133, Bmi1, Nanog, and Oct4) as well as of HH cascade members such as ligands (Desert, Sonic, and Indian hedgehog), receptors (Smoothened, Patched, and HIP) and transcription factors (Gli 1-3) were analyzed by PCR and immunohistochemistry, respectively. Results: Heterogeneous hematopoietic-lineage and MDS-stage dependent expression pattern of HH members as well as of hematopoietic stem cell markers were observed: mRNA and protein levels of HH signaling members were low and increased from ligands to transcription factors in MDS. Most significant differences were observed in granulo- followed by megakaryo- and erythropoesis, respectively (ANOVA P<0.05) comparing controls versus MDS-sampels as well as different IPSS scoring levels. Analysis revealed significant independent predictors of the HH signaling family for survival probability. Finally, the expression pattern of the HH signaling members were mostly positive correlated to CD34 expression and heterogeneously to other stem cell markers applied. Conclusions: Our investigation revealed that (i) HH signaling pathway was linked to hematopoietic-lineage specific expression pattern in normal and dysplastic hematopoiesis; and that (ii) the expression of some HH signaling members is partially associated with stem cell differentiation in MDS.
P-009 Distinct epigenetic abnormalities distinguish therapy-related myeloid neoplasms (t-MN) following Hodgkin lymphoma (HL) vs. breast cancer (BC) M. Figueroa 1 , J. Sotzen 1 , E. Fabiani 2 , L. Flanchi 2 , S. Hohaus 2 , G. Leone 2 , M.T. Voso 2 . 1 Pathology, University of Michigan, Ann Arbor, USA; 2 Hematology, Universita Cattolica del Sacro Cuore, Rome, Italy
Figure 1
Background: t-MN occur as a late complication after treatment of several primary hematological and non-hematological malignancies, including HL and BC. Introduction: t-MN are classically divided into two categories, those secondary to ionizing radiation/alkylating agents and those secondary to topoisomerase II inhibitors. However, in practice most patients receive polychemotherapy regimens. Aside from the therapy received, the type of primary neoplasm may also help determine the phenotype of the t-MN. Recently, we have shown that t-MN in pa-
Division of Pathology, Karolinska University Hospital Huddinge, Stockholm, Sweden Background: Refractory anemia with ring sideroblasts (RARS) is a myelodysplastic syndrome subgroup distinguished by anemia, erythroid apoptosis and mitochondrial ferritin accumulation in erythroblasts, which fail to mature into erythrocytes. Introduction: It is poorly understood at which stage and by which mechanism erythroid failure occurs; hence possible targets for therapy are yet to be discovered. Recently, heterozygous mutations in SF3B1, a core component of the spliceosome, were identified in >70% of RARS. This discovery may provide clues to how the failure of terminal differentiation is executed, and how the hematopoietic stem cells (HSC) gain their clonal advantage. Purpose: To explore the molecular and cellular mechanisms behind clonal expansion of immature progenitors and defective erythroid maturation, we investigated transcriptome profiling, splice patterns and SF3B1 allelic burden during erythroid differentiation in 11 RARS patients SF3B1 mutated, of which the majority also carried other driver mutations. Materials and Methods: Initially, normal and RARS transcriptomes from two time points during early erythroid differentiation were analyzed and compared, and in a second phase, transcriptome findings were validated using Taqman Low Density Array (TLDA) analysis in an extended cohort. Results: We observed the activation of genes involved in defence against oxidative stress in CD34+ progenitors. By contrast, erythroblasts were characterized by a failure to up-regulate genes in the autophagy pathway, essential for terminal maturation to erythrocytes, and by a maintained expression over time of regulators of transcription, apoptosis and adhesion, indicating that transcription in general is not hampered by SF3B1 mutation. We also report marked alterations in gene expression during RARS differentiation (FDR <5%) and altered splicing of genes involved in hematopoiesis. The exon usage pattern was very heterogeneous within the RARS population, while showing limited variation in the NBM cohort. Finally, we demonstrated that SF3B1 mutation does not confer a growth disadvantage to the erythroid cells until final maturation to reticulocytes, the stage at which autophagy becomes essential for erythroid differentiation. Conclusions: Our study suggests oxidative stress defence, potentially caused by mitochondrial iron accumulation, as an underlying mechanism for the clonal advantage of RARS stem and early progenitor cells and provides novel insights into the erythroid differentiation process. By showing that anemia develops during terminal maturation into erythrocytes, we provide important insights into how the severe anemia in RARS patients may be addressed.
P-006 Prognostic value of TP53 gene mutations in higher-risk MDS treated with azacitidine (AZA) C. Bally 1 , L. Ades 1 , A. Renneville 2 , M.J. Mozziconacci 3 , C. Preudhomme 2 , H. DeThé 4 , J. Lehmann-Che 4 , P. Fenaux 1 . 1 Service d’Hématologie clinique, Hopital Avicenne, Bobigny, France; 2 Service d’Hématologie biologique, CHU de Lille, Lille, France; 3 Service d’Hématologie biologique, Institut Paoli Calmette, Marseille, France; 4 Biologie moléculaire, Hopital Saint Louis, Paris, France Background: TP53 gene mutations, in MDS and AML, are associated with resistance to chemotherapy (Wattel, Blood, 1994). Introduction: Their correlation with results of treatment with hypomethylating agents has not been studied. Purpose: We assessed the prognostic value of TP53 mutations for response to AZA in higher risk MDS, AML (20-30% blasts) or advanced CMML (ie EU label for AZA).
S25
Materials and Methods: Patients received AZA (75 mg/m2 /d ×7 days/4 w) in 2 GFM centers. TP53 mutations were detected by the functional FASAY technique, whose sensitivity is 10-15% (Flaman, PNAS 1995) and confirmed by direct sequencing (1% sensitivity NGS technique using pyrosequencing, GS Junior System). Results: 72 pts treated with AZA (median n¡ of cycles 4, range 17) were analyzed, including 34 MDS, 33 AML (20-30% blasts) and 5 CMML. Karyotype was fav in 27%. int in 13% and unfav in 60% pts, including 47% complex karyotypes. 51% of pts responded (IWG 2006 criteria), including 34% CR, 6% PR, 4% marrow CR, 7% Stable disease with HI. Median OS from onset of AZA was 15.6 months 29 (40%) pts had TP53 mutation by FASAY, always confirmed by NGS, including 22/34 (67%) complex karyotypes. ORR to AZA (41% vs 57%, p=0.227) and CR rate (21% vs 42%, p=0.073) did not significantly differ between mutated and unmutated pts, but OS was shorter in pts with TP53 mutation (median 13 vs 21 months, p=0.0022, Figure 1), complex cytogenetics (median 14 vs 18 months, p=0.0120), WHO AML (median 13 vs 21 months for -MDS or CMML, p=0.0014). By multivariate analysis, marrow blast % (HR 1.03 (p<10-4)) and TP53 mutational status (HR 2.29 p=0.035) were the only 2 factors retaining significance for OS.
Figure 1
Conclusions: In higher risk MDS, AML 20-30% blasts and CMML treated with AZA, TP53 mutation is an independent prognostic parameter of poorer survival.
P-007 Novel epigenetic and genetic alterations in cytogenetically normal refractory cytopenia with multilineage dysplasia and chronic myelomonocytic leukemia J.X. Cheng 1 , J. Anastasi 2 , J.Q. Shen 3 , A. Rodrigues 4 , D. Roulston 1 , J.W. Vardiman 2 . 1 Pathology, University of Michigan, Ann Arbor, USA; 2 Pathology, University of Chicago, Chicago, USA; 3 Bioinformatics, University of Nevada, Las Vegas, USA; 4 Bioinformatics, University of Michigan, Ann Arbor, USA Background: Refractory cytopenia with multilineage dysplasia (RCMD), a subtype of myelodysplastic syndrome (MDS), is characterized by multilineage dysplasia, pancytopenia and no increase in blasts. Chronic myelomonocytic leukemia (CMML), a subtype of myelodysplastic/myeloproliferative neoplasm (MDS/MPN), usually has a proliferative component manifested by absolute monocytosis, in addition to some dysplastic features. The different characteristics of these two diseases may suggest that they are molecularly distinct entities caused by different molecular mechanisms and/or evolution of varying molecular abnormalities.
S26
Poster Presentations – 12th International Symposium on Myelodysplastic Syndromes / Leukemia Research 37 S1 (2013) S1–S117
Introduction: PU.1, an ETS transcription factor, is critical for myelopoiesis, especially for determining erythroid/megakaryocytic vs. lymphoid/myeloid differentiation. In a recent study, we uncovered a novel epigenetic mechanism that involves an increase in the repressing epigenetic modification of Histone 3 lysine 27 trimethylation (H3K27me3) at PU.1 and its downstream genes, resulting in inactivation of the PU.1 and lymphoid/myeloid potent progenitor (PU.1/LMPP) pathway in some cytogenetically normal RCMDs (CN-RCMDs) (J.X. Cheng et al. Leukemia in press). Here we extend our prior study to cytogenetically normal chronic myelomonocytic leukemia (CN-CMML). Purpose: To compare the epigenetic and genetic mechanisms underlying the pathogenesis of CN-RCMD and CN-CMML Materials and Methods: Both primary bone marrow cells from normal, CN-RCMD and CN-CMML patients and cell lines derived from MDS and monocytic leukemia were used. Genome-wide chromatin immunoprecipitation (ChIP-on-chip), sequential ChIP, methylated DNA immunoprecipitation (MeDIP-onchip), chromosome conformation capture (3-C), real-time RT-PCR, flow cytometry, immunohistochemistry, si-RNA technology, histone methytransferase inhibitors, genome-wide exome-sequencing and Sanger sequencing were employed. Results: Our study revealed distinct chromatin patterns of the PU.1 and its down-stream genes in CN-CMML vs. CN-RCMD. In contrast to an increase in the repressing epigenetic modification of H3K27me3 and inactivation of the PU.1/LMPP pathway in CN-RCMD, an increase in both the repressing modification of H3K27me3 and the activating modification of H3K4me3, i.e. a “bivalent” chromatin and a hyperactivation of the PU.1/LMPP pathway was observed in CN-CMML. The methyltransferase inhibitors for H3K27 or H3K4 methylation had an inhibitory effect on monocytic leukemia cells. Reduction of PU.1 expression by si-RNA effectively inhibited growth of monocytic leukemia cells, but not MDS-derived erythroid/myeloid leukemia cells. Exome-sequencing revealed a large number of novel recurrent gene mutations in CN-RCMD (n=10) and CN-CMML (n=8); specifically, 325 mutations in CN-RCMD only, 449 in CN-CMML only, and 393 mutations shared by both. Sanger sequencing was performed to confirm the exome-sequencing results and identified rare subtype-specific mutations in these diseases. Conclusions: We propose two hypothetical models of dysregulation of the PU.1/LMPP pathway by epigenetic/genetic alterations in CNRCMD and CN-CMML. See Figure 1 below.
P-008 Hedgehog signaling pathway and stem cell markers are linked with dysplastic changes of myelodysplastic syndrome B. Alinger, R. Kemmerling, E. Klieser, R. Illig, T. Kiesslich, D. Neureiter. Institute of Pathology, Salzburger Landeskliniken/Paracelsus Medical University, Salzburg, Austria Background: Assumed role of the Hedgehog (HH) signaling pathway within the regulation of hematopoietic stem cells (HSCs) maintenance as well as in the clonal disease of myelodysplastic syndrome (MDS) still remains unclear. Introduction: Recent studies revealed an aberrant activation of the hedgehog signaling pathway and cancer stem cells in a wide variety of human cancers including malignant hematological neoplasms. Purpose: Therefore, we investigated the expression pattern of subset HH signaling members and different hematopoietic stem cell markers at different stages of MDS compared to control samples from healthy donors. Materials and Methods: Bone marrow samples of 18 normal versus 80 patients with MDS (subgroups according to WHO: n=40 refractory cytopenia (RA) with multilineage dysplasia, n=13/22 refractory anemia with excess of blasts I/II, and n=5 secondary acute myeloid leukemia) were investigated. All MDS samples were fully characterized for dysplastic change of bone marrow as well as for risk factors (IPSS), laboratory parameters and follow-up. mRNA and protein expression pattern of HSC makers (CD34, CD133, Bmi1, Nanog, and Oct4) as well as of HH cascade members such as ligands (Desert, Sonic, and Indian hedgehog), receptors (Smoothened, Patched, and HIP) and transcription factors (Gli 1-3) were analyzed by PCR and immunohistochemistry, respectively. Results: Heterogeneous hematopoietic-lineage and MDS-stage dependent expression pattern of HH members as well as of hematopoietic stem cell markers were observed: mRNA and protein levels of HH signaling members were low and increased from ligands to transcription factors in MDS. Most significant differences were observed in granulo- followed by megakaryo- and erythropoesis, respectively (ANOVA P<0.05) comparing controls versus MDS-sampels as well as different IPSS scoring levels. Analysis revealed significant independent predictors of the HH signaling family for survival probability. Finally, the expression pattern of the HH signaling members were mostly positive correlated to CD34 expression and heterogeneously to other stem cell markers applied. Conclusions: Our investigation revealed that (i) HH signaling pathway was linked to hematopoietic-lineage specific expression pattern in normal and dysplastic hematopoiesis; and that (ii) the expression of some HH signaling members is partially associated with stem cell differentiation in MDS.
P-009 Distinct epigenetic abnormalities distinguish therapy-related myeloid neoplasms (t-MN) following Hodgkin lymphoma (HL) vs. breast cancer (BC) M. Figueroa 1 , J. Sotzen 1 , E. Fabiani 2 , L. Flanchi 2 , S. Hohaus 2 , G. Leone 2 , M.T. Voso 2 . 1 Pathology, University of Michigan, Ann Arbor, USA; 2 Hematology, Universita Cattolica del Sacro Cuore, Rome, Italy
Figure 1
Background: t-MN occur as a late complication after treatment of several primary hematological and non-hematological malignancies, including HL and BC. Introduction: t-MN are classically divided into two categories, those secondary to ionizing radiation/alkylating agents and those secondary to topoisomerase II inhibitors. However, in practice most patients receive polychemotherapy regimens. Aside from the therapy received, the type of primary neoplasm may also help determine the phenotype of the t-MN. Recently, we have shown that t-MN in pa-