ALBERT

Description: Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) that leads to progressive bone marrow (BM) fibrosis. Although the cellular mutations involved in the pathogenesis of PMF have been extensively investigated, the sequential events that drive stromal activation and fibrosis by hematopoietic–stromal cross-talk remain elusive. Using an unbiased approach and validation in patients with MPN, we determined that the differential spatial expression of the chemokine CXCL4/platelet factor-4 marks the progression of fibrosis. We show that the absence of hematopoietic CXCL4 ameliorates the MPN phenotype, reduces stromal cell activation and BM fibrosis, and decreases the activation of profibrotic pathways in megakaryocytes, inflammation in fibrosis-driving cells, and JAK/STAT activation in both megakaryocytes and stromal cells in 3 murine PMF models. Our data indicate that higher CXCL4 expression in MPN has profibrotic effects and is a mediator of the characteristic inflammation. Therefore, targeting CXCL4 might be a promising strategy to reduce inflammation in PMF.

Description: Using high-throughput sequencing, an increased number of gene mutations has been identified in cancer. Among the up to hundreds of acquired mutations in cancer clones, only a few cooperating mutations are believed to be needed for initiation of the malignant disease. Recently, we reported a single amino acid substitution at position 676 (N676K) within the FLT3 kinase domain as the sole cause of resistance to PKC412 in one patient with FLT3-ITD associated acute myeloid leukemia (AML). The FLT3-N676K mutation was more recently identified independently in up to 6% of de novo AML patients with inv(16) by other groups. As FLT3-TKD mutations are strongly associated with inv(16) in AML and particularly FLT3-N676K was found almost exclusively in AML patients with inv(16), this prompted us to investigate the transforming activity of FLT3-N676K and to test whether FLT3-N676K would cooperate with inv(16) to promote AML. First, we analyzed in vivo leukemogenesis mediated by FLT3-N676K. Retroviral expression of FLT3-N676K in myeloid 32D cells induced AML in syngeneic C3H/HeJ mice (n=11/13, latency ~8 weeks), with a transforming activity similar to FLT3-ITD (n=8/8), FLT3-TKD D835Y (n=8/9), and FLT3-ITD-N676K (n=9/9) mutations. Three out of 14 C57BL/6J mice transplanted with FLT3-N676K-transduced primary lineage negative (Lin-) bone marrow cells died of acute leukemia (latency of 68, 77, and 273 days), while none of 16 animals in the control groups including FLT3-ITD and CBFß-SMMHC developed any hematological malignancy. Secondly, co-expression of FLT3-N676K and CBFß-SMMHC did not promote acute leukemia in 3 independent experiments using C3H/HeJ and C57BL/6J mice (n=16). So far only 1 out of 11 C57BL/6J mice co-expressing FLT3-N676K and CBFß-SMMHC developed acute leukemia (AML with latency of 166 days). In comparison with FLT3-ITD, FLT3-N676K tended to result in stronger phosphorylation of FLT3, MAPK and AKT, and diseased animals carrying FLT3-N676K demonstrated much lower frequency of leukemic stem cells in the majority of analyzed cases. Importantly, leukemic cells co-expressing FLT3-N676K and CBFß-SMMHC were still highly sensitive to the FLT3 inhibitor AC220. Taken together, we show that FLT3-N676K mutant is potent to transform murine hematopoietic stem/progenitor cells in vivo independently of the inv(16) chimeric gene CBFB-MYH11. This is the first report of acute leukemia induced by an activating FLT3 mutation in C57BL/6J mice. Moreover, our data suggest that targeting FLT3-N676K mutation may be an attractive treatment option for FLT3-N676K-positive patients without concurrent ITD. Our data emphasize more careful analysis of the cooperating network of mutations identified in AML by high-throughput sequencing. This work was supported by DJCLS (grant: 13/22) and the Deutsche Forschungsgemeinschaft (grant: Li 1608/2-1). KH and ZP were supported by the China Scholarship Council (2011638024 and 201406100008). Disclosures Heidel: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Imatinib may reverse bone marrow fibrosis (MF) in chronic myeloid leukemia (CML). The risk of emergence or relapse of MF and its prognostic relevance are not known as yet. 110 patients with Ph+ CML recruited in two trials and treated with 400 mg imatinib / day were examined for the risk of MF and its prognostic significance. Imatinib effectively reversed pretreatment MF (P = 0.0002), and pretreatment fiber increase did not significantly reduce the probability of achieving a hematologic, cytogenetic and major molecular response. However, during a follow-up period of 4 years, small foci with fiber increase (FFI) and MF relapsed in 13 % (5 / 38) of patients, and among patients with an initial normal fiber content (n = 72), FFI emerged in 26 % with transformation to full-blown MF in 38 % of them. FFI occurred in 3 / 27 cases with a major molecular response. The risk of emergence or relapse of FFI amounted to 13.7 % and of full-blown MF to 5.9 % per year, and the risk of acceleration and death increased to 90 % two years after occurrence of MF. FFI could be predicted neither by the risk score (Sokal, Hasford) nor the hematologic, cytogenetic and molecular response, nor by clonal evolution during imatinib treatment. In multivariate analysis, emergence or relapse of FFI and MF turned out to be a significant independent predictor of imatinib failure (P = 0.0003) as well as of acceleration and death of patients (P 〈 0.0009). CONCLUSION: Although imatinib reverses initial MF in CML, it does not guarantee against relapse or emergence of MF which affects ~ 6 % of patients per year and therefore, may become a major problem during long-term therapy of CML. Focal evolution of MF should be included in the definitions of imatinib failure and accelerated phase, and continuing imatinib treatment at the current dose is no longer appropriate for patients with emergence of FFI or MF during therapy - regardless of the degree of hematologic, cytogenetic and molecular response.

Description: Abstract 2826 Background of anemia in myelodysplastic syndromes (MDS) with 5q deletion (del(5q)) is a haplo-insufficiency of the RPS14 gene. However, degree of anemia and quantity of erythropoiesis vary markedly between MDS patients with del(5q), even with a similar degree of RPS14 insufficiency, so that the question arises whether other influences may play a further relevant role in erythroid failure in this disease. From 35 patients with low or intermediate-1 risk MDS with del(5q) who developed a transfusion-dependent anemia and were recruited and treated with lenalidomide (10 mg / d) within the MDS-003 study 38 +/− 33 months after diagnosis of MDS, bone marrow biopsies taken at 6 to 12-month time intervals after diagnosis of disease were evaluated for changes of erythropoiesis having occurred prior to lenalidomide therapy by morphometric, immunohistochemical, and molecular methods. A second group of patients with MDS treated with best supportive care (n = 307) served as a control. From each bone marrow biopsy, 20 000 nucleated cells were evaluated marking (1) nucleated erythroid cells (NEC) immunohistochemically using anti-HbA, glycophorin-C (GPC), CD71, and CD34 antibodies and (2) marrow macrophages by anti-CD68 antibody. By analogy to burst and colony forming units from in-vitro models, the numbers of erythroid precursor cells (EPC) were estimated by the numbers of erythroblastic islands (IslE) within marrow. Morphometrically assessed erythropoiesis was related to the mRNA expression of RPS14, SPARC, and TNF-alpha genes, nuclear p53 protein expression of NEC and their relevance for transfusion dependence and AML-free survival time of patients. 5q deletion predicted erythroid hypoplasia independently of the type of MDS, evidence of marrow fibrosis and AML transformation (P 〈 0.0005). During early-phase MDS with del(5q), anemia correlated with p53 overexpression of NEC and decreased numbers of HbA+ NEC (P 〈 0.00005). After the first year of disease, two alterations of erythropoiesis became more relevant which did not correlate to p53 overexpression of NEC: (1) a decline in the numbers of GPC+HbA- NEC indicating impaired differentiation of EPC to proerythroblasts which correlated with RPS14 insufficiency, and (2) a progressive decrease in the number of IslE indicating a block of differentiation at an earlier stage, the IslE forming EPC, which did not correlate to RPS14 gene expression, but to two other independent factors, a reduced SPARC and an increased TNF-alpha gene expression (P 〈 0.00005). The degree of TNF-alpha expression correlated to the number of a central component of the IslE, the (iron-presenting) macrophages, the potential source of TNF-alpha overexpression. Both alterations contributed significantly to erythroid hypoplasia and progressive transfusion dependence (P 〈 0.00005). Response to lenalidomide therapy continued for 〉 4 years (100% AML-free survival) when started during a phase with high numbers of p53 overexpressing NEC, but was short or poor with 〈 20 % AML-free survival when started during a phase when differentiation of erythropoiesis was impaired at a less mature stage (P = 0.0001). On the basis of these observations, a score predicting the efficacy of lenalidomide in individual patients could be constructed. Conclusions: In MDS with del(5q), erythropoiesis significantly changes over time. In the early stage of disease, maturation defect of erythropoiesis mainly affects HbA+ NEC and correlates to p53 overexpression of NEC. In the course of disease, erythropoiesis becomes hypoplastic due to differentiation defects at the level of more immature erythroid (precursor) cells relating to a p53-independent mechanism of RPS14 insufficiency and altered SPARC as well as TNF-alpha gene expression. Efficacy of lenalidomide therapy is significantly impaired in the case of vanishing IslE, reduced SPARC and increased TNF-alpha gene expression. Disclosures: Giagounidis: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Knight:Celgene Corp.: Employment.

Description: Heterozygous deletion of RPS14 occurs in del(5q) MDS and has been linked to impaired erythropoiesis, characteristic of this disease subtype. We previously generated a mouse model with conditional inactivation of Rps14 and demonstrated a p53-dependent erythroid differentiation defect with apoptosis at the transition from polychromatic to orthochromatic erythroblasts resulting in age-dependent progressive anemia, megakaryocyte dysplasia, and loss of hematopoietic stem cell (HSC) quiescence. We now sought to determine the mechanistic basis for the anemia using unbiased, quantitative mass spectrometry in erythroid progenitor cells. We found powerful induction of proteins involved in innate immune signaling, particularly the danger associated molecular pattern (DAMP) heterodimeric S100A8/S100A9 proteins. We found significantly increased S100a8 in the erythroid progenitor populations affected by the differentiation block (RIII-RIV population) and in monocytes and macrophages of Rps14 haploinsufficient bone marrows, all representing cells of the erythroblastic niche. Recombinant S100A8 was sufficient to impair erythropoiesis in wild-type cells. We rescued the erythroid differentiation defect in Rps14 haploinsufficient HSCs by genetic inactivation of S100a8 expression using CRISPR/Cas-mediated gene inactivation in primary mouse Rps14 haploinsufficient HSPC. We validated the association between induction of S100A8 and a severe erythroid phenotype in bone marrow samples of patients with del(5q) MDS. To examine whether ribosomal haploinsufficiency also leads to activation of S100A8 in patients with del(5q) MDS, we measured S100A8 expression using immunofluorescence in bone marrow biopsies from MDS patients with and without del(5q). In del(5q) MDS, the frequency of S100A8-positive cells was associated with disease severity, as reflected by transfusion burden. RPS14, CSNK1A1 and miR-145 are universally co-deleted in the 5q- syndrome and each represent different clinical features of del(5q) MDS in murine models. Haploinsufficiency of miR-145 or -146a also induces inappropriate activation of innate immune signaling. To analyze the combinatorial effect of haploinsufficiency Rps14, Csnk1a1 and miRNA-145, we transduced hematopoietic stem and progenitor cells (HSPC) from compound haploinsufficient Rps14 and Csnk1a1 mice and stably knocked down both miR-145/miR-146a by retrovirus-mediated overexpression of respective target sequences. Compound haploinsufficiency of Rps14, Csnk1a1 and miR-145/146a led to a progressive anemia comparable to Rps14 haploinsufficiency with splenomegaly and an erythroid differentiation defect at the RIII/RIV population, indicating that the anemia is mainly driven by Rps14 haploinsufficiency. Bone marrow histology demonstrated the typical 5q-phenoytpe of megakaryocytes, in line with significant thrombocytosis. At 10 months of age, hematopoietic stem and progenitor cells were significantly increased (lineagelow ckit+ Sca1+; LSK), in particular multipotent progenitor cells (MPPs; lineagelow ckit+ Sca1+ CD48- CD150+) to significantly higher extents than in solely Rps14 or Csnk1a1 haploinsufficient cells. We next asked if compound haploinsufficiency of the three 5q-genes has combinatorial or synergistic effects on S100a8 expression. Compound haploinsufficiency of Csnk1a1, Rps14 and miR-145/146a induced the highest expression of S100a8 in monocytes, while haploinsufficiency of Rps14 alone induced the highest expression of S100a8 in the RIII erythroid population, suggesting that cell-type specific induction mediates the phenotype. Our data indicate an unexpected link between haploinsufficiency for a ribosomal gene, Rps14, activation of S100A8, and inhibition of erythropoiesis. We demonstrate that compound haploinsufficiency for Csnk1a1 and miR145/146a with Rps14 haploinsuffciency increases the expression of S100a8, mainly in monocytes, and recapitulates the phenotype of del(5q) MDS by cooperating, cell-type specific effects. Disclosures Platzbecker: Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Boehringer: Research Funding.

Description: Introduction Myelofibrosis is a hematopoetic stem cell neoplasm characterized by bone marrow inflammation, reactive marrow fibrosis and extramedullary hematopoiesis. Myelofibrosis is associated with a chronic inflammatory state, including, but not limited to the bone marrow space. Chronic inflammation is triggering the initiation of fibrogenesis, and bone marrow fibrosis is a hallmark of terminal phase myelofibrosis. Positron emission tomography/computed tomography (PET/CT) using the glucose analogue 18F-fluorodeoxyglucose (18F-FDG) is widely used for imaging of both inflammatory and malignant processes due to increased glucose consumption in inflammatory and neoplastic cells. A noninvasive method to visualize and quantify the extent of active myelofibrosis would be highly desirable, e.g. for therapy monitoring studies. Therefore, the aim of this study was to assess if 18F-FDG PET/CT provides noninvasive insights into the metabolic implications of the disease. Methods In 30 patients, the biodistribution of the glucose analogue 18F-FDG was analyzed 60 min after intravenous injection of 350 MBq of 18F-FDG. The extent of bone marrow involvement was graded using a four-point scale. Bone marrow metabolism was quantified by measuring the mean and maximum standardized uptake value (SUV) in the bone marrow space. Imaging findings were compared with laboratory, cytogenetic and histopathological data. Results Retention of 18F-FDG was observed in bone marrow and spleen. Bone marrow involvement varied, and 4 different patterns could be found. Ten (33.3%) of the 30 patients showed only mildly increased 18F-FDG uptake in the central skeleton and the proximal extremities (PET grade 1). Three (10%) patients showed markedly increased tracer uptake in the central skeleton and the proximal extremities extending into the distal half of the femoral bone (PET grade 2). In 8 (26.7%) patients, increased tracer uptake in the central skeleton and the extremities extending into the tibial bone was found (PET grade 3). Nine (30.0%) patients demonstrated increased 18F-FDG uptake in the central skeleton and the extremities extending into the small bones of the feet (PET grade 4). Extent of bone marrow involvement (PET grade) decreased over time from initial diagnosis (rs = -0.43, p = 0.019). Metabolic activity of the bone marrow decreased as the histopathological grade of fibrosis increased (rs = -0.37, p = 0.04) and as splenic volume increased (rs = -0.40, p = 0.03). There was a significant positive correlation between the metabolic activity of the bone marrow and splenic metabolic activity (p = 0.04), indicating that splenic uptake is parainflammatory. Conclusions 18F-FDG PET/CT emerges as a promising technique for visualization and quantitation of bone marrow metabolism in myelofibrosis. We conclude that the increased bone marrow metabolism mainly reflects inflammatory activity within the bone marrow space. Further evaluation in prospective studies is required to determine the potential clinical impact and prognostic significance of PET. Disclosures No relevant conflicts of interest to declare.

Description: Marrow fibrosis (MF) is rarely considered in myelodysplastic syndromes (MDS) although the frequency of this complication ranges from 10 to 50 % in the few reports on this issue, and there are no data on occurrence and significance of this complication in the context of the International Prognostic Scoring System (IPSS) and the World Health Organization (WHO) classification of disease. In a retrospective study, diagnostic bone marrow biopsies from a total of 936 patients with MDS were examined for MF and its relevance to the course of disease. Frequency of MF varied markedly between different types of MDS ranging from 3 % (RARS) to 37 % (MDS, therapy-related; WHO classification, P 〈 0.000005). Risk of MF furthermore correlated with multilineage dysplasia (P 〈 0.000005). However, there was no obvious correlation to the IPSS or to karyotype abnormalities. The survival time of patients was significantly reduced by about 50 % from 11 (RAEB-1/-2) - 55 (RARS, RCMD-RS) down to 6 (RAEB-1/-2) - 33 months (RARS, RCMD-RS) in median when MF was detected independently of the IPSS and the classification of disease (FAB, WHO; P = 0.0001). We conclude that MF is an unfavorable complication of MDS significantly shortening the survival time of patients independently of the IPSS and the classification of disease.