ALBERT

Description: In paroxysmal nocturnal hemoglobinuria (PNH) hemolytic anemia is due mainly to deficiency of the complement regulator CD59 on the surface of red blood cells (RBCs). Eculizumab, an antibody that targets complement fraction 5 (C5), has proven highly effective in abolishing complement-mediated intravascular hemolysis in PNH; however, the hematologic benefit varies considerably among patients. In the aim to understand the basis for this variable response, we have investigated by flow cytometry the binding of complement fraction 3 (C3) on RBCs from PNH patients before and during eculizumab treatment. There was no evidence of C3 on RBCs of untreated PNH patients; by contrast, in all patients on eculizumab (n = 41) a substantial fraction of RBCs had C3 bound on their surface, and this was entirely restricted to RBCs with the PNH phenotype (CD59−). The proportion of C3+ RBCs correlated significantly with the reticulocyte count and with the hematologic response to eculizumab. In 3 patients in whom 51Cr labeling of RBCs was carried out while on eculizumab, we have demonstrated reduced RBC half-life in vivo, with excess 51Cr uptake in spleen and in liver. Binding of C3 by PNH RBCs may constitute an additional disease mechanism in PNH, strongly enhanced by eculizumab treatment and producing a variable degree of extravascular hemolysis.

Description: PNH is a hematological disorder characterized by complement-mediated intravascular hemolysis due to lack on RBCs of the complement regulators CD55 and CD59 and subsequent activation of the membrane attack complex (MAC). Eculizumab (EC) is an anti-complement fraction 5 antibody which abolishes intravascular hemolysis in PNH patients, leading to reduction of transfusion requirement and of anemia. Although the effector complement pathway is completely blocked by eculizumab in all patients, response to this agent in terms of transfusion requirement and hemoglobin level varies considerably. We have investigated the notion that patients with suboptimal hematological response may suffer from residual hemolysis mediated by mechanisms other than intravascular hemolysis via MAC. After the initial observation of a positive C3d Coombs test in some PNH patients on EC, we extensively studies C3 coating by flow cytometry in 56 PNH patients (41 of them while receiving EC). We found that in all cases on EC treatment a significant proportion of RBCs were coated with complement fraction 3 (C3); by converse, in 28 untreated PNH patients we found no evidence of C3 on red cells. C3 coating was strictly limited to CD59-neg RBCs, as CD59+/C3+ RBCs (as those seen in cold agglutinine disease patients, positive control) were never found. C3 coating was quite different among EC-treated patients, and correlated with the PNH RBC population. The percentage of C3+ cells within the PNH RBC population (the only subjected to C3 coating) was quite different in individual patients (0.5–61.3%, median 22.6%) and substantially preserved over time. We compared the level of C3 coating with the hematological response: all the 41 EC-treated patients showed marked LDH reduction with a substantial improvement of anemia, leading to transfusion independence in 34/41 patients (83%) and stable resolution of anemia in 14 (34%, defined optimal responders). The optimal responders showed a lower percentage of C3+ cells in comparison to suboptimal responders (20.9±19.0 vs 32.2±17.8; p=0.04). Indeed, patients with lower C3 coating (below the median value of 22.6%) showed a significantly higher rate of optimal response (51% in comparison to 15% of those with coating above the median, p=0.01). C3 coating also correlated with the absolute reticulocyte count (p=0.03), clearly suggesting that C3 coating was associated with ongoing residual extravascular hemolysis in vivo, and with pre-treatment LDH level (p=0.001). To confirm the presence of extravascular hemolysis, in 3 index patients with suboptimal response reduced RBC half-life was demonstrated in vivo by 51Cr RBC survival study, which showed reduced RBC half-life and excess uptake in liver and spleen. One of these patients underwent video-laparoscopic splenectomy, which led to transfusion independence and significant increase in Hb level. These data demonstrate that C3 coating of PNH RBCs is a common phenomenon in PNH patients on EC; in addition, we provide evidence that this leads to C3-mediated extravascular hemolysis through the reticulo-endothelial system. This novel mechanism of disease may account for residual hemolysis and suboptimal clinical benefit in some EC treated PNH, paving the way for additional therapeutic strategies to optimize the hematological response to this agent.