Complement Genotype and Multiparametric Laboratory Analysis to Predict Response to Eculizumab and to Adjust Treatment in Patients with Paroxysmal Nocturnal Hemoglobinuria

Background: Paroxysmal Nocturnal Hemoglobinuria (PNH) is a disorder caused by the proliferation of hematopoietic stem cells carrying a somatic mutation in the gene PIG-A, which is necessary for the biosynthesis of the glycosyl phosphatidyl-inositol anchor. As a consequence, PNH erythrocytes lack, among other proteins, the complement regulators CD55 and CD59 and become susceptible to complement-mediated intravascular hemolysis. Before Eculizumab, a monoclonal antibody that prevents C5 cleavage, life expectancy of PNH patients was about 10-15 years from diagnosis, being thrombotic events the main cause of death. Despite the unquestionable success of Eculizumab therapy to treat PNH, the responses among PNH patients show heterogeneity, with some patients remaining transfusion-dependent for reasons not completely understood.

Methods: To get further insight into the different responses to Eculizumab treatment among patients, we have generated a PNH cohort including 12 PNH patients treated with Eculizumab. All patients included in the study had previously a minimum of ≥12 months on treatment with the drug (ten receiving 900 mg every 14 days, and two patients on 1200 mg every 14 days because breakthrough hemolysis). We have collected samples during two Eculizumab cycles (4 weeks) at different points during treatment: before and after Eculizumab injection and in the intermediate week. We have searched for correlations between clinical and hematological parameters, complement genetic findings, and deposition of C3 fragments on erythrocytes and plasma levels of complement activation components.

Results: We report the CR1 genotypes, complement and hematological determinations in 12 PNH patients during their treatment with Eculizumab. We have established that concentrations of free Eculizumab above 110 µg/mL completely inhibit serum hemolytic activity. Consistent with this, in our patient cohort, most patients with levels of free Eculizumab close or below 110 µg/mL exhibited traces of C5b9 and/or CH50. In general, lowest levels of free Eculizumab correlate with higher levels of C5b9. Our results indicate that proper monitoring of PNH patients would be useful to optimize Eculizumab dosage. We have 3 patients that showed symptoms of intravascular hemolysis (high LDH and hemosiderinuria) despite having a negative CH50 accompanied with sufficient free Eculizumab levels. Further studies are currently being done to elucidate the causes. We have found a correlation between CR1 haplotype, red cellsopsonization and C3 plasma levels: H/L individuals present higher % of C3 positive erythrocytes and low C3 plasma levels. Two of these patients also had low C4 plasma levels, one of them presenting also low levels of FH. These individuals are currently being studied to determine whether these low levels are due to genetic factors or consumption. We found a correlation between levels of bilirubin and % of reticulocytes; patients who had more than the 30% of their erythrocytes opsonized presented higher percentage of reticulocytes and bilirubin in plasma, which could be taken as indicatives of slight extravascular hemolysis. In this regard, individuals carrying the CR1 L allele might require special attention.