ALBERT

Description: Introduction: Myelodysplastic Syndromes (MDS) are clonal neoplasms. Whether the transforming event occurs in a myeloid committed cell or in earlier hematopoietic progenitor/stem cell it is still not ascertained. We evaluated the repopulating ability and stem cell potential of hypoxia maintained primary bone marrow (BM) progenitors derived from Myelodysplastic Syndromes (MDS) patients, and their capacity of engraftment in sublethally irradiated NOD-SCID mice. Methods: Thirty eight BM samples were obtained at diagnosis from MDS patients (WHO: 9 RA, 12 RCMD, 7 RAEB, 8 AML/post MDS, 2 Del(5)q). Mononuclear BM cells were isolated and cultured with TPO, FLT3-L, SCF, IL-3 in severe hypoxic conditions (0,3%O2, 5%CO2, 95%N2), for 10-13 days (LC1). The stem cell potential of these cultures was explored by transferring cells to growth-permissive secondary cultures in normoxia (LC2), in the presence of SCF, G-CSF, IL-6, IL-3, and according to the Culture-Repopulating Ability (CRA) assay methodology, cell proliferation was evaluated daily. Expression of CD34, CD38, CD117, CD133 was determined before and after hypoxia cultures, and when present, the persistence of chromosomal aberrations was analyzed by FISH at various stages of cell culture. In parallel, MDS mononuclear BM cells were intravenously injected in sublethally irradiated NOD-SCID mice before hypoxic culture and after LC1. Mice were sacrificed at day 56 and 90 after graft. The presence of CD45+ human cells in the peripheral blood of injected mice was evaluated by flow cytometry analysis every two weeks, and quantitated to evaluate the repopulating ability and the different engraftment capacity of cells before hypoxic culture and after hypoxic incubation. Mice marrow trephines as well as spleens were also evaluated morphologically to test the engraftment of human MDS cells. Results: In all 38 MDS cases studied, cultured cell number decreased of one log or more after 10-13 days of culture in hypoxic conditions. In 12/38 MDS cases we observed a significant repopulating ability at day 17 of LC2, all classified as Low/INT-1 IPSS risk category. In IPSS high and INT-2 risk cases, repopulating ability according to CRA method was absent; CD34+ cells were always decreased after hypoxia and did not coexpress CD38, confirming the selection of earlier hematopoietic progenitor cells. We demonstrated the persistence of chromosomal aberrations in CD34pos/CD38neg cells after hypoxic culture in 5 MDS cases (del5q, +8, del20q, -Y, complex karyotype). Sublethally irradiated NOD-SCID mice transplanted with hypoxia cultured cells showed in peripheral blood an higher percentage of CD45+ human cell than mice transplanted with non hypoxia selected cells. In particular, mice transplanted with hypoxia cultured BM mononuclear cells derived from a 5q- MDS patient showed an higher percentage of CD45+ human cells than mice transplanted with non-hypoxia selected cells at day 28 after transplant (5,5% vs 0,56 %); mice transplanted with hypoxia cultured BM mononuclear cells derived from a CRDM MDS patient showed an higher percentage of CD45+ human cells than mice transplanted with cells before hypoxia at day 56 (13,3% vs 1,4%), and at day 71 (43,05% vs 12,7%). Mice bone marrow cytometry analysis confirmed higher percentage of CD45+ cells in bone marrow derived from mice transplanted with hypoxia cultured cells than non-hypoxia cultured (1,9 % vs 0,1%). Conclusions: Severe hypoxic culture conditions are maintaining a sub-population of MDS cells endowed with increased repopulating capacity in vitro and in vivo. Most importantly, in our cohort of cases studied, repopulating ability was observed exclusively in IPSS lower risk MDS cases. Disclosures Santini: celgene, Janssen, Novartis, Onconova: Honoraria, Research Funding.

Description: C5-blockade with eculizumab prevents complement-mediated intravascular hemolysis in PNH patients and its clinical consequences. However, a distinct population of PNH red blood cells bound with C3 fragments appears in almost all treated patients. This C3 binding results in extravascular hemolysis that in some patients reduces the clinical benefit from eculizumab. In each PNH patients on eculizumab there are always two distinct populations of PNH red blood cells, one with (C3+) and one without (C3-) C3 binding. This phenomenon is somehow paradoxical since the glycosylphosphatidylinositol (GPI)-linked complement regulators, CD55 and CD59, are uniformly deficient on the surface of PNH red cells. To investigate this phenomenon, we have modeled in vitro the C3 binding in the context of C5 blockade by incubating red blood cells from PNH patients with AB0-matched sera from patients on eculizumab. Complement alternative pathway has been activated by mild acidification (in presence of Mg/EGTA to prevent the activation of complement classical pathway) and C3 binding has been assessed by flow cytometry at serial time points. In these experimental conditions a fraction of PNH red blood cells, similar to what happens in vivo, become promptly C3+ and its size increases with the time: from 9.4±2.7% after 5 minutes to 21.2±9.5% after 24 hours. The membrane defects of PNH cells suggested that the deficiency of CD55, which regulates the formation and accelerates the dissociation of C3 convertases, should be responsible for C3 binding to PNH red blood cells in presence of eculizumab (Parker CJ. Hematology Am Soc Hematol Educ Program. 2011;2011:21-29). In order to verify experimentally this hypothesis we have inactivated CD55 or CD59 on normal red blood cells by using blocking monoclonal antibodies (moAb - listed in the figure legend), and we have tested them in vitro upon activation of complement alternative pathway by mild acidification in presence or absence of C5 blockade. We found that CD55 inactivation on normal red blood cells results neither in hemolysis (without C5 blockade) nor in any C3 binding (with C5 blockade). As expected without C5 blockade CD59-inactivated normal red blood cells undergo hemolysis but, surprisingly, we found that in presence of C5 blockade they become bound with C3 fragments (Figure 1), just as it occurs in vivo in PNH patients on eculizumab. The simultaneous inactivation of both CD55 and CD59 further increased the level of C3 binding. Thus, at variance with the starting hypothesis, the deficiency of CD59, not that of CD55, plays the major role in C3 binding to PNH red cells of patients on eculizumab. Therapeutic C5 blockade in PNH patients has unmasked a novel function of CD59: in addition to prevent MAC formation, it plays a central role also in the regulation of C3 activation on cell surface through molecular mechanisms not elucidated yet. It remains to be established the physiological role, if any, of this novel function of CD59 and whether it play a role in determining the pleomorphic clinical features of the congenital CD59 deficiency. Finally, these findings may lead to investigate innovative approaches to reduce C3 binding and extravascular hemolysis in PNH patients on eculizumab and, in a broader context, to modulate complement activity. Figure 1 Figure 1. Disclosures Risitano: Novartis: Research Funding; Alexion Pharmaceuticals: Other: lecture fees, Research Funding; Rapharma: Research Funding; Alnylam: Research Funding.

Description: Abstract 983 Eculizumab, a monoclonal antibody that binds to the complement component C5, controls intravascular hemolysis in PNH patients and causes significant clinical improvement. However, the extent to which patients respond is variable in terms of hemoglobin levels, and some patients remain red blood cell (RBC) transfusion-dependent in spite of regular eculizumab treatment. We have hypothesized that genetic polymorphisms of complement-related genes might contribute to determining this variability of response to eculizumab. In order to test this hypothesis, we have analyzed polymorphic alleles of the Complement Receptor 1 (CR1) gene and of the Complement Component 3 (C3) gene. We have genotyped by standard methods (1) the HindIII restriction length polymorphism (RFLP) in the CR1 intron 27 (rs11118133 A〉T) whose co-dominant alleles, H (common) and L (rare), are associated with high and low levels of CR1 expression on RBC, respectively; (2) the single nucleotide polymorphism rs2230199 C〉G of the C3 gene, responsible for the allelic electrophoretic variants, S (slow, common allele) and F (fast, rare allele), that influence the complement alternative pathway activity. We have studied 72 patients with hemolytic PNH treated with eculizumab for at least 6 months. Patients with clear evidences of bone marrow failure (platelets ≤ 50,000/μL, neutrophils ≤1000/μL) have not been included in this study because this condition may affect the clinical response independently of the control of hemolysis. In order for our criterion for response to be stringent and objective we used blood transfusion requirement: patients who received no RBC transfusion have been defined as Responders; patients who received any RBC transfusion have been defined as Poor Responders. In this series of 72 hemolytic PNH patients on eculizumab the allelic frequency of the CR1 rare allele L was 0.31, and that of the C3 rare allele F was 0.19. We found no correlation between response and the C3 genotype (Chi square for trend: p=0.939). On the other hand the proportion of Poor Responders is significantly higher in patients who were heterozygotes (H/L) for the CR1 polymorphism, and even higher in those who were homozygous for the rare allele (L/L) of the CR1 HindIII RFLP polymorphism (Chi square for trend: p=0.016): see figure 1. We do not know yet the precise mechanism whereby the CR1 genotype affects the response to eculizumab. CR1 enhances the decay of C3 and C5 convertase by binding to C3b and C4b and it plays a role in the clearance of immune complex and in phagocytosis. Thus, the variable levels of CR1 expression on RBC membrane associated with the HindIII RFLP polymorphism may affect the regulation of complement alternative pathway. Moreover, it is well established that in almost all PNH patients on eculizumab a significant fraction of GPI-negative RBCs are opsonized by C3 and therefore may undergo extravascular hemolysis. Thus, a possible explanation of our findings is that the lower levels of CR1 expression on RBCs associated with H/L and L/L genotypes are responsible for a more intense extravascular hemolysis in PNH patients on eculizumab. In conclusion, our data show that the CR1 HindIII polymorphism may predict the poor response to eculizumab in PNH patients and provide additional insight in the mechanisms responsible for the variable clinical effectiveness of complement blockade. Disclosures: Risitano: Alexion: Membership on an entity's Board of Directors or advisory committees, Research Funding. Peffault de Latour:Alexion: Consultancy. Iori:Alexion: invited speaker Other. Socie:Alexion: Consultancy. Luzzatto:Alexion: Consultancy. Notaro:Alexion: Consultancy.

Description: Abstract 5055 Classical chronic myeloproliferative neoplasms (MPNs) are amongst the best-characterized neoplasms associated with one of a set of specific somatic mutations, the most common of which is in the JAK2 gene. MPN is a relatively rare condition, and it could be surmised that it develops preferentially in people who have an increased tendency to somatic mutations, i. e. an increased somatic mutation rate (μ). Since measuring μ is rather labor-intensive, in this study we have measured instead the frequency of peripheral blood granulocytes that have inactivating mutations in a reporter gene, namely the gene PIG-A, a gene whose protein product is required for numerous glycosyl-phosphatidylinositol-anchored proteins to become surface bound. Since several such proteins are displayed by normal granulocytes, mutant cells can be numbered accurately by flow cytometry; and the frequency (ƒ) of such mutant cells may be a good surrogate of μ (Peruzzi et al. Mutation Research. 2010, 705:3). We have determined ƒ in 74 patients with MPN (including 29 with polycythemia vera (PV), 17 with essential thrombocythemia (ET), 16 with primary myelofibrosis (PMF) 12 with post-PV/post-ET (PPV/PET) MF. Overall the proportion of those with a JAK2(V617F) mutation was 90%. In a subset of 59 of these patients, who had “uncomplicated” MPNs, the median of ƒ was no different compared to 142 healthy controls (4. 74×10−6vs. 4. 87×10−6: Figure 1). However, in a subset of 15, consisting of patients who in addition to MPN had at least one primary malignancy (2 had lymphomas and 13 had one of several types of solid tumors), the median value of ƒ was 15. 22×10−6, i. e. significantly elevated (Mann Whitney test: MPNs vs. MPNs with a second neoplasm, p