ALBERT

Description: Background and aim: Treatment with erythropoietin stimulating agents (ESA) is first-line treatment for anemic low-risk MDS patients, but although clinical variables such as endogenous serum erythropoietin levels have been associated with response, the bone marrow (BM) cell populations targeted during ESA-induced erythroid improvement have not been identified. Initiating SF3B1 mutations in MDS-RS arise in the multipotent hematopoietic stem cell (HSC), and we previously showed that most SF3B1-mutated MDS-RS patients retain a small residual wildtype HSC population, something that we propose may have clinical relevance (Mortera-Blanco, et al, Blood 2017). In this study, we aimed to map clonal dynamics of mutant and wildtype hematopoietic stem and progenitor cells (HSPCs) during steady-state and ESA treatment. Methodology: We used advanced flow cytometry to sort and characterize HSPC populations in BM samples of SF3B1-mutated MDS-RS patients with stable anemia, before and during ESA response, and during chronic transfusion dependency. In addition to conventional HSPC markers, we added CD105 to the antibody panel as it has been reported to distinguish committed erythroid progenitors (Mori, et al, PNAS 2015). Variant allele frequency (VAF) of the known SF3B1 mutations was determined using droplet digital PCR. Results: Expression patterns of mature CD38+ progenitors; common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs) and megakaryocytic-erythroid progenitors (MEPs), mostly followed published patterns with CD105 expression as expected in a subset of MEPs. The immature CD38- HSCs, multipotent progenitors (MPPs), and lymphoid-primed MPPs (LMPPs) should be negative for the CD105 surface marker (Mori, et al, PNAS 2015) but we confirmed a very low expression (

Description: Abstract 276 Cord blood mononuclear cells (CBMNCs) are an ideal cell source for therapeutic applications although maintenance of fetal globin in expanded erythroid cells is problematic. We have previously developed a 3D bone marrow (BM) biomimicry through the use of a synthetic scaffold made of polyurethane (PU) coated with collagen type I which expanded CBMNCs in a cytokine-free environment for at least 28 days, with or without addition of serum. Addition of near physiological concentrations (0.2U/mL) recombinant human erythropoietin (rhuEPO) to the 3D CBMNCs serum- and cytokine-free cultures at day 7 enhanced normal erythropoiesis to enucleation (from CD45+/CD71+/CD235− to CD45−/CD71+/CD235+ stages) and promoted erythroid clonogenic capacity (CFU-E and BFU-E). However, mechanisms of erythroid expansion and globin-maturity of the cells are ill-defined. We have now extended our investigation to evaluate in situ hemoglobin and cytokine gene expression and detect cellular hemoglobin protein and cytokine production in culture supernatants. CBMNCs were separated by Ficoll-Paque density gradient centrifugation and seeded onto collagen-coated PU 3D scaffolds at 2.5×106cells/scaffold (5×5×5mm3). Cultures were established with full-medium exchange every other day in 4 conditions: 2 controls (serum-free and serum-containing cultures without rhuEPO) and serum-free cultures with addition of rhuEPO at 1.845U/mL or 0.2U/mL from day 7 onwards. Culture output was evaluated by in situ analysis and by physically extracting cells from the scaffolds. β-globin (HBB) and γ-globin (HBG) genes were detected by in situ PCR from day 0 and were consistently expressed throughout the culture period in all conditions (HBB expression was consistently higher). A decline in expression of both globins was noted at days 21 and 28 only in serum-free cultures without addition of rhuEPO. Western blot analysis of extracted cells confirmed both HBB and HBG expression in the serum-containing rhuEPO-free culture. In serum-free conditions, both HBB and HBG proteins were noted only at day 7; once rhuEPO was added to these cultures, a hemoglobin switch occurred with persistence of only HBB at days 21 and 28. Even in serum-free and rhuEPO-free cultures, HBB was present, although in lower amounts than in the rhuEPO-exposed cells, which suggested that fetal globin switching and maturation towards adult erythropoiesis occurred primarily in the serum-free cultures exposed to rhuEPO. Increased expression of GM-CSF, IL-1β and TNF was observed to day 7 and declined thereafter while IL-6 expression was observed only at day 7. There was constant high expression of TGF-β throughout the culture in all conditions, whereas TPO was not detected in any condition. The erythropoietin-receptor (EPO-R) gene was detected in all conditions, yet EPO-R was shed into the supernatant mostly in the first 7 days (maximum 42pg/ml) of serum-free cultures, declining by day 14, which corresponded to the timing of rhuEPO addition. Flt-3 was consistently detected in supernatant of the serum-containing cultures, yet declined after day 14 in serum-free cultures with and without rhuEPO. Stem cell factor (SCF), critical in early stages of erythropoiesis, was not present in culture supernatants at any time-point. Interestingly, endogenous production of EPO (maximum 0.8 mU/ml) was detected by ELISA in the first 2 weeks of both serum-containing and serum-free cultures, prior to addition of rhuEPO. High EPO concentration (maximum 2600mU/ml) was still observed in the serum-free, rhuEPO-containing cultures over the entire 28 days, suggesting that EPO was not completely utilized by the maturing cells during culture and that even lower concentrations of EPO could be beneficial. EPO detection was maximal at day 21 for serum-free cultures exposed to rhuEPO; this day 21 peak, in conjunction with the known erythroid maturation kinetics within the 3D culture, the detection of endogenous EPO production at day 14, the shedding profile of EPO-R and the hemoglobin switch, suggested that the 14–21 day time-points of culture will be important for the future study of erythroid physiology within this system. In conclusion, the 3D BM biomimicry is a good model to study erythropoiesis ex vivo, using physiological concentrations of rhuEPO and serum-free conditions rendering it suitable for future clinical applications. Disclosures: No relevant conflicts of interest to declare.

Description: Background The popular concept that human cancers might be driven by rare self-renewing cancer stem cells (CSCs) has extensive implications for cancer biology and modelling, as well for development of more efficient and targeted therapies. However, experimental support for the existence of distinct and rare CSCs in human malignancies remain contentious, particularly in light of compelling evidence that cancer-propagating cells frequently fail to read out in existing human stem cell assays. Therefore, to unequivocally establish the existence and identity of human CSCs, the challenge is first to identify candidate CSCs, and to establish their unique ability to self-renew and replenish molecularly and functionally distinct non-tumorigenic progeny followed by functional in situ validation within the patients themselves. Methods We have in the hematological malignancy myelodysplastic syndromes (MDS) characterize candidate hematopoietic stem and progenitor stages in the bone marrow of low-intermediate risk MDS patients by flow cytometry. Distinct cell populations were functionally characterised for lineage commitment in standard colony forming cell (CFC) assays, and for self-renewal potential in long-term culture initiating cell (LTC-IC) assays and in immune-deficient (NSG) mice. Moreover, we tracked the cellular origin of all identified somatic genetic lesions identified in each patient by targeted next-generation sequencing of genomic DNA isolated from each purified MDS stem and progenitor cell population. Results In low-intermediate risk MDS patients, regardless whether they were del(5q) (n=19) or non-del(5q) (n=11), we could identify rare but distinct Lin-CD34+CD38-CD90+CD45RA- candidate stem cells, granuclocyte-monocyte progenitors (GMPs) and megakaryocyte-erythroid progenitors (MEPs) with frequencies within total BM similar to that of normal age-matched controls. Global gene expression analysis by RNA sequencing of MDS stem cells, GMPs and MEPs suggested that these are molecularly distinct populations. Myeloid and erythroid gene expression signatures were restricted to the GMPs and MEPs, respectively, whereas a transcriptional stem cell signature was restricted to the MDS stem cells. GMPs and MEPs isolated from del(5q) (n=12) and non-del(5q) (n=8) MDS patients displayed lineage-restricted myeloid and erythroid differentiation potentials, respectively. Self-renewal in LTC-IC assay was restricted exclusively to MDS Lin-CD34+CD38-CD90+CD45RA- stem cells in del(5q) (n=11) and non-del(5q) (n=8) MDS patients. Xenotransplantation into NSG mice also confirmed that only Lin-CD34+CD38-CD90+CD45RA- MDS stem cells have in vivo self-renewal potential, and these experiments also demonstrated their ability to replenish downstream CMPs, GMPs and MEPs, establishing the hierarchical relationship of MDS stem and progenitor cells. Targeted DNA sequencing of 88 genes recurrently mutated in MDS and other myeloid malignancies was pursued to identify somatic genetic lesions within the bulk bone marrow of MDS patients (n=13). In total we identified 30 presumed genetic driver lesions, including del(5q) and mutations in key transcription factors (RUNX1), signalling pathways (JAK2, CSF3R), epigenetic regulators (TET2, ASXL1), apoptosis regulators (TP53), and spliceosome components (SF3B1, SRSF2, U2AF2, SRSF6). Importantly, in support of their unique ability to self-renew and replenish lineage-restricted MDS progenitors, all stable somatic genetic lesions identified could in each MDS patient be backtracked to the rare stem cell population as defined phenotypically by flow cytometry and functionally by LTC-IC or xenograft potential, unequivocally establishing their unique stem cell identity within the malignant clone. Conclusions These findings provide definitive evidence for the existence of rare and distinct stem cells in MDS, a finding with extensive implications for therapeutic strategies in MDS and other cancers whose existence might also strictly depend on the persistence of rare CSCs. MDS stem cells typically acquire multiple driver mutations, together conferring a competitive advantage over normal stem cells, while even in combination failing to inflict self-renewal ability on MDS myelo-erythroid progenitor cells. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points SF3B1 mutations in MDS-RS have a multipotent lymphomyeloid origin. Transplantation of SF3B1 mutated MDS-RS HSCs into immune-deficient mice results in generation of characteristic ring sideroblasts.

Description: Deletions involving chromosome 7 constitute the second most common cytogenetic abnormality in myelodysplastic syndromes (MDS), associated with an overall poor outcome and high risk of transformation to acute myeloid leukemia (AML). Previous studies focusing on low to intermediate risk MDS, including cases with del(5q) as the only cytogenetic abnormality, have revealed a remarkably preserved stem and myeloid progenitor cell hierarchy, more characteristic of normal hematopoiesis than that of AML. Herein we explored this in a group of seven higher-risk MDS cases, all with an isolated deletion of the entire chromosome 7 (-7; monosomy 7), as the only cytogenetic abnormality. In all investigated isolated monosomy 7 cases, the expression of cell surface markers used to define the normal hematopoietic stem and myeloid progenitor cell hierarchy in bone marrow resembled more the expression pattern observed in AML than in low-risk MDS, irrespectively of the -7 MDS patients blast counts. Specifically, when compared to healthy age-matched controls, we observed a 15-fold reduction of LIN-CD34+CD38-CD45RA-CD90+ cells (hematopoietic stem cells; HSCs, p=0.010), a 15-fold increase in LIN-CD34+CD38+CD123+CD45RA+ cells (Granulocyte-macrophage progenitors; GMPs, p=0.038) and a 300-fold increase of LIN-CD34+CD38-CD90-CD45RA+ cells (lymphoid-primed multipotent progenitors; LMPPs, p=0.050) within total bone marrow mononuclear cells of MDS patients with isolated monosomy 7. Fluorescence in situ hybridization (FISH) analysis revealed high -7 clonal involvement (〉80%) for all investigated progenitor populations, whereas it was much lower (

Description: The MDS subgroup refractory anemia with ring sideroblasts (RARS) is characterised by aberrant mitochondrial ferritin accumulation in erythroblasts that fail to mature into erythrocytes. Recently, dominant mutations in SF3B1, a core component of the spliceosome were demonstrated in 〉75% of RARS, but only in a minority of other MDS subtypes. Many RARS patients also carry other driver mutations, such as epigenetic mutations in DNMT3A and TET2, but the order of occurrence and cooperation between these mutations have not been established. We recently showed that SF3B1 suppresses the expression of the mitochondrial transporter protein ABCB7, which in turn mediates erythroid failure in RARS, but the link to clonal advantage of RARS hemopoietic stem cells (HSC) remains unclear. To explore this link, as well as the impact of additional mutations, we studied RARS with normal karyotype. Screening for 111 recurrently mutated genes in myeloid malignancies revealed SF3B1 in 12 out of 13 patients, TET2 mutations in 3 of these patients (Q916*, H1881Y, Q690*, and R1404*), and DNMT3A mutations in 3 patients(E240fs*8, F414L, W305*, E285*). Other mutations occurred only once. The frequencies of phenotypically defined RARS stem and myeloid-erythroid progenitor cells in the bone marrow (BM) did not differ from that of normal BM controls, whereas pro-B cells were significantly reduced in the RARS samples (p

Description: Refractory anemia with ring sideroblasts (RARS) is characterized by severe anemia, erythroid apoptosis and aberrant mitochondrial ferritin accumulation in erythroblasts. Dominant mutations in SF3B1, a core component of the splicing machinery are detected in more than 75% of patients. Of the recurrent mutations, SF3B1 alone is associated with the RARS phenotype. We recently showed that SF3B1 suppresses the expression of the mitochondrial transporter protein ABCB7, which in turn mediates erythroid failure in RARS. The exact mechanisms remain unclear, hence potential targets for treatment have not been identified. Fifty-six patients with RARS or RCMD-RS were subjected to targeted sequencing of which 52 (93%) had SF3B1 mutations and 43 also other recurrent mutations mostly involving epigenetic regulators. To explore the mechanisms behind the disrupted RARS erythropoiesis molecular and biological features of erythroid progenitors from SF3B1 mutated RARS and normal bone marrow (NBM) were monitored in vitro throughout a well-established 14-days liquid culture system. Normal and RARS transcriptomes (RNAseq) were analyzed at two time points (CD34+ cells at day 0 and early erythroblasts at day 4) during differentiation, followed by validation in an extended cohort of 11 RARS and 4 NBM using Taqman Low Density Array (TLDA). CD34+ RARS progenitors were characterized by activation of genes involved in the oxidative stress pathway and in particular genes involved in defense against oxidative stress, such as SEPP-1. This activation dropped dramatically at day 4. By contrast, early differentiation in RARS displayed a marked failure to up-regulate genes in the autophagy pathway, which is essential for removal of organelles and terminal maturation to erythrocytes. We then followed the clone size of SF3B1 in 8 erythroid cultures, two of which also underwent myeloid differentiation. 4 cultures used RARS patients with stable untreated anemia (Hb 107 g/L ± 15), a high percentage of ring sideroblasts and a relatively normal reticulocyte count. SF3B1 allelic burden was assessed by pyrosequencing at day 0, 4, 7 and 14. To exclude the possibility that the culture system itself promoted survival of mutated progenitors we also analyzed isolated GPA+ progenitors from uncultured mononuclear BM, and finally we analyzed blood reticulocytes from the 4 untreated patients. We observed a normal expansion of both erythroid and myeloid progenitors during culture, with a stable allele burden (± 10%) in all patients. In fact, cultured and non-cultured erythroid progenitors showed a higher allelic burden than the corresponding myeloid cells. However, the allele burden in reticulocytes was only 60% of that found in the freshly isolated GPA+ fraction, indicating that reticulocytes to a greater extent derived from unmutated cells (Figure 1). In summary, we show that genes involved in autophagy are markedly down-regulated during early erythroid differentiation in RARS. While SF3B1 mutations do not confer a growth disadvantage to differentiating erythroid bone marrow progenitors, the final step of erythroid maturation to reticulocytes is clearly affected. Hence, we demonstrate a novel interference of the SF3B1 mutation with terminal erythroid maturation. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 503 The ability to expand cord blood (CB) cells ex vivo overcomes an important limitation to its wider clinical application in cellular therapies. The current practice of hematopoietic cell culture is based on two-dimensional (2D) tissue culture flasks or well plates which require either co-culture with allogeneic or xenogeneic stromal cells and the exogenous provision of several cytokines. This 2D culture environment is artificial and lacks the 3D cellular niches that characterise the in vivo hematopoietic inductive microenvironment. Specifically, the cultured cells are exposed to abnormally high cytokine concentrations, which may result in differentiation and loss of pluripotency. We have previously developed a 3D bone marrow biomimicry through the use of synthetic scaffolds made of poly (D,L-lactide-co-glycolide) (PLGA) and polyurethane (PU) coated with collagen type I. Our previous work has shown that these scaffolds, which were seeded with cord blood (CB) mononuclear cells (MNCs) at a cell density of 3-6×106cells per scaffold (5×5×5mm3), could successfully support long-term culture in the absence of exogenous growth factors for over 4 weeks. Specifically, the 3D biomimicry facilitated a 53-fold total MNC expansion, with an increase in the BFU-E and CFU-GM progenitor cell population. However, these cultures, although cytokine-free, contained 20-30% (v/v) fetal calf serum which can have both conducive and inhibitory effects on hematopoietic cell cultures due to the unknown composition and concentration of humoral factors contained within. Inclusion of serum in expansion-type cultures can limit the clinical application of the derived product. The serum-free and cytokine-free culture and expansion of hematopoietic cells has not been achieved until now. Herein, we report that for at least 4 weeks the polyurethane (PU) scaffolds coated with collagen type I were able to maintain and expand human CB MNCs. Furthermore the progenitor population, as determined by the colony forming unit assay, was also maintained and preferentially directed towards the granulocytic lineage, even though the CFU-GEMMs declined. Immunophenotypic analysis of the extracted cells confirmed the presence of erythroid precursors (CD71+CD45-) as well as early maturing myeloid cells. In contrast, the 2D cytokine- and serum-free cultures collapsed within 3-4 days. We hypothesized that the 3D biomimicry was able to facilitate serum- and cytokine-free conditions because it can recapitulate the three-dimensional architecture of the human bone marrow. This hypothesis was supported by scanning electron microscopy of the central sections of the scaffolds that showed the migration of cells within the pores and establishment of “niche-like” structures. In conclusion, this novel 3D culture system is capable of long-term, cytokine- and serum-free expansion of haematopoietic cells from cord blood, enabling the study of haematopoiesis as well as facilitating the expansion of cells for future clinical applications. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4896 Current in vitro models for the study of AML rely on cell lines or, if primary cells are used, require abnormally high concentrations of exogenous cytokines and/or stromal cells in a co-culture system. These conditions introduce bias and artifact by selecting for culture-responsive cells and do not account for the 3D leukemic growth observed in the bone marrow. We have previously shown that human AML cell lines can be cultivated in 3D synthetic polymeric scaffolds coated with collagen and fibronectin for 8 weeks. We have also shown that human cord blood mononuclear cells can be cultured in the same 3D in vitro system with good viability (〉90%), proliferation and clonogenic capacity in the absence of exogenous cytokines over a period of 4–6 weeks. Herein, we evaluate the potential for human primary AML cells to survive in a cytokine-free environment on 3D polyurethane (PU) scaffolds rendered bioactive by collagen type I over 35 days. After informed consent, peripheral blood (PB) and/or bone marrow (BM) aspirate samples were obtained from patients with AML of different subtypes, some also with abnormal karyotype. Mononuclear cells were separated by standard Ficoll-Paque density gradient centrifugation, seeded directly at a density of 2×106 cells per scaffold (5×5×5 mm3) and then cultivated without exogenous cytokines for 35 days with full medium exchange performed every other day. Standard two-dimensional (2D) Dexter cultures were used as controls. Cultures were assessed at 2 hours for seeding efficiency, and then weekly for viability, proliferative capacity, and cellular phenotype. Of 19 patient samples studied, 11 exhibited good viability prior to seeding and were successfully cultured in the scaffolds for 35 days. Seeding efficiency was high (82 ± 0.22%). Although cells were extracted manually from the scaffolds for assessment, necessitating some cell death due to the extraction technique, average viability was higher than 75 ± 0.15% over the first 21 days, with 54 ± 0.11% still viable at 35 days. In contrast, AML controls in standard 2D-flask cultures did not survive beyond 14 days. In situ, the proliferation rates were different for each AML sample, yet growth kinetics showed a similar trend, peaking at day 14–21 in all AML subtypes. Interestingly, there were no differences in the growth kinetics of mononuclear cells seeded into the 3D scaffolds from PB and BM of the same patient. The similarity of the kinetic profile to that observed previously in the 3D normal cord blood cultures suggested that the 2–3 week time period was critical for the establishment and stabilization of both types of cultures and that in future it may be an informative period to investigate further. Scanning electron microscopy (SEM) showed that in situ cellular density increased with culture time and cells had migrated within the inner scaffold pores and appeared organized in colonies and clusters within distinct niches. Retention of the initial AML phenotype and morphology was observed by immunohistochemistry of sectioned scaffolds. Wright-Giemsa staining of the extracted cells every 7 days confirmed fidelity of the AML population in culture over time (in the absence of exogenous cytokines), with leukemic blasts and dysplastic maturation similar in appearance to those observed in the original patient bone marrow sample, even after 35 days. Maintenance of the original AML karyotype in 2 patients with chromosome 7 abnormalities was confirmed by interphase FISH with 55.2% and 76.4% of the output leukemic cells exhibiting a signal pattern consistent with del(7q) after 21 days (10% and 73% blasts in the original input cell population, respectively), indicating culture fidelity. This in vitro 3D platform can be used to study primary human AML in the absence of exogenous cytokines and xenogeneic or allogeneic antigens, thereby abrogating the introduction of bias in the study of leukemogenesis, AML clonal hierarchy and the microenvironment. The model may be widely applicable to a broad range of leukemia types for the study of disease, and as a biomimetic tool for drug discovery and chemotherapeutic or small molecule inhibitor drug targeting within the 3D microenvironment. Disclosures: No relevant conflicts of interest to declare.