ALBERT

Description: PNH is a rare acquired clonal disorder of the hematopoietic stem cell, characterized by a somatic mutation that inactivates the X-linked PIGA gene: this in turn results in deficiency on the cell surface of all proteins anchored by the glycosylphosphatidylinositol (GPI) molecule. Two of these proteins,CD55 andCD59, are complement regulators and their deficiency is responsible for the susceptibility of red cells (RBCs) from the mutant clone to lysis by activated complement. Since oxidative damage is another well-known mechanism of hemolysis (as in G6PD deficient red cells), we have investigated whether this plays a role also in PNH. To this end, we have carried out experiments on RBCs from healthy donors and on PNH-like RBCs (obtained in vitro from the same donors through the use of anti-CD55 and anti-CD59 blocking moAb). After exposure to AB0-compatible serum (in which thecomplement alternative pathway was activated by mild acidification) all PNH-like (but not normal) RBCs were lysed. In parallel experiments in which complement was blocked by eculizumab (ECU) - a moAb that binds to the complement component C5 and controls intravascular hemolysis in PNH patients - we measured the levels of reactive oxygen species (ROS) by the dichlorofluorescin diacetate assay. We found no significant difference of ROS levels between normal RBCs and PNH-like RBCs. We next tested in a similar way G6PD-deficient RBCs, because these are known to be exquisitely sensitive to oxidative damage. We found that ROS levels were significantly higher in the G6PD deficient RBCs that have been made PNH-like (Fig. 1). Thus, complement activation on the surface of PNH-like RBCs results in the production of ROS that can be demonstrated when C5-blockade prevents complement-mediated lysis of RBCs. The notion that G6PD deficiency can interact with PNH was strongly corroborated by the clinical observation of a 40yo woman from Sardinia (Italy) with a 2 years history of pancytopenia, who then developed florid hemolytic PNH: she had anemia with normal granulocyte and platelet counts, dark urine, high reticulocytosis, LDH up to 5x upper normal level, 95% GPI-negative granulocytes. When the patient was started on ECU. LDH levels promptly returned to normal, PNH RBCs rose from 20% (before ECU) to 42%, but reticulocyte count (~250x109/L) and blood transfusion requirement remained high (10 units in the last year). 39% of the GPI-negative RBCs had bound C3 fragments The peripheral blood smear revealed marked macro-anisocytosis, poikilocytosis, spherocytes, and hemighosts: a picture consistent with oxidative damage as seen in G6PD deficient patients during a hemolytic attack. The RBC G6PD activity was about one-half of normal (5 IU/g Hb), and DNA analysis revealed heterozygosity for the G6PD Mediterranean (Med) mutation. By mRNA sequence analysis we found that the GPI-negative clone expressed only the G6PD Med allele, suggesting that the PIG-A mutation took place in a stem cell in which the normal G6PD gene was on the inactive X-chromosome (G6PD, like PIG-A, is on the X chromosome); therefore, all the patients' PNH RBCs were also all G6PD deficient. We have previously shown that the clinical expression of PNH can be influenced by inherited factors: specifically, a polymorphism of the complement receptor 1 (CR1) gene correlates with the blood transfusion requirement of patients on ECU (Rondelli et al, Haematologica 2014). However, the patient here reported was homozygous for the more favorable allele ofCR1. Instead, in keeping with our experimental data, the poor response to ECU seen in this patient results probably from a unique interaction, within the same population of RBCs, between the acquired PNH abnormality and her inherited G6PD deficiency This type of interaction is novel and it seems to have pharmacogenetic implications. Indeed, on its own G6PD deficiency affects mildly the clinical expression of PNH, because complement activation causes RBC lysis regardless; however, paradoxically, when the lysis of PNH RBCs is prevented by C5 blockade, complement activation results in oxidative damage, with which PNH G6PD deficient RBCs are unable to cope. Except for one case previously reported by Oni et al (Blood 1970), this is the first detailed study of PNH associated with G6PD deficiency. Since in some parts of the world the frequency of G6PD deficiency can be as high as 30% or more, we expect that more cases of this association will be discovered in the future. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Many mutations of the housekeeping gene encoding glucose-6-phosphate dehydrogenase (G6PD) cause G6PD deficiency in humans. Some underlie severe forms of chronic nonspherocytic hemolytic anemia (CNSHA) for which there is no definitive treatment. By using retroviral vectors pseudotyped with the vesicular stomatitis virus G glycoprotein that harbor the human G6PD (hG6PD) complementary DNA, stable and lifelong expression of hG6PD was obtained in all the hematopoietic tissues of 16 primary bone marrow transplant (BMT) recipient mice and 14 secondary BMT recipients. These findings demonstrate the integration of a functional gene in totipotent stem cells. The average total G6PD in peripheral blood cells of these transplanted mice, measured as enzyme activity, was twice that of untransplanted control mice. This allowed the inference that the amount of G6PD produced by the transduced gene must be therapeutically effective. With the same vectors both the cloning efficiency and the ability to form embryoid bodies were restored in embryonic stem cells, in which the G6PD gene had been inactivated by targeted homologous recombination, thus effectively rescuing their defective phenotype. Finally, expression of normal human G6PD in hG6PD-deficient primary hematopoietic cells and in human hematopoietic cells engrafted in nonobese diabetic/severe combined immunodeficient mice was obtained. This approach could cure severe CNSHA caused by G6PD deficiency.

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: Many mutations of the housekeeping gene encoding glucose-6-phosphate dehydrogenase (G6PD) cause G6PD deficiency in humans. Some underlie severe forms of chronic nonspherocytic hemolytic anemia (CNSHA) for which there is no definitive treatment. By using retroviral vectors pseudotyped with the vesicular stomatitis virus G glycoprotein that harbor the human G6PD (hG6PD) complementary DNA, stable and lifelong expression of hG6PD was obtained in all the hematopoietic tissues of 16 primary bone marrow transplant (BMT) recipient mice and 14 secondary BMT recipients. These findings demonstrate the integration of a functional gene in totipotent stem cells. The average total G6PD in peripheral blood cells of these transplanted mice, measured as enzyme activity, was twice that of untransplanted control mice. This allowed the inference that the amount of G6PD produced by the transduced gene must be therapeutically effective. With the same vectors both the cloning efficiency and the ability to form embryoid bodies were restored in embryonic stem cells, in which the G6PD gene had been inactivated by targeted homologous recombination, thus effectively rescuing their defective phenotype. Finally, expression of normal human G6PD in hG6PD-deficient primary hematopoietic cells and in human hematopoietic cells engrafted in nonobese diabetic/severe combined immunodeficient mice was obtained. This approach could cure severe CNSHA caused by G6PD deficiency.

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.