Ludovica Marando

Complement fraction 3 binding on erythrocytes as additional mechanism of disease in paroxysmal nocturnal hemoglobinuria patients treated by eculizumab

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 (51)Cr labeling of RBCs was carried out while on eculizumab, we have demonstrated reduced RBC half-life in vivo, with excess (51)Cr 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.

Hemoglobin normalization after splenectomy in a paroxysmal nocturnal hemoglobinuria patient treated by eculizumab.

To the editor: In paroxysmal nocturnal hemoglobinuria (PNH), hemolysis is due to the absence on red blood cell (RBC) surface of the 2 complement regulators CD55 and CD59,[1][1],[2][2] which causes uncontrolled complement activation and consequent chronic intravascular hemolysis via the membrane

From perpetual haemosiderinuria to possible iron overload: iron redistribution in paroxysmal nocturnal haemoglobinuria patients on eculizumab by magnetic resonance imaging

Paroxysmal nocturnal haemoglobinuria (PNH) was initially named ‘chronic haemolytic anaemia with perpetual haemosiderinuria’, and often results in iron deficiency secondary to urinary iron loss (Parker et al, 2005). Eculizumab is an inhibitor of complement component 5, which has been shown effective in the control of complement-mediated intravascular haemolysis of PNH (Hillmen et al, 2006; Kelly et al, 2011; Risitano et al, 2011). We investigated iron compartmentalization in 20 haemolytic PNH patients: two untreated, 14 on eculizumab, and four analysed before and during eculizumab treatment (Table I). Standard biochemical testing, including iron parameters and flow cytometry for complement component 3 (C3) on erythrocytes, was combined with magnetic resonance imaging (MRI) to assess calculated iron content (CIC) of kidneys, liver and spleen (Gandon et al, 1994; Deugnier & Turlin, 2007). The six patients not (yet) treated with eculizumab had overt intravascular haemolysis (high lactate dehydrogenase (LDH) levels and haemosiderinuria) and normal/low serum ferritin levels (Table I). Regardless of transfusion requirement, all of them showed a homogeneous pattern of iron compartmentalization (Mathieu et al, 1995), characterized by renal cortex siderosis (renal CIC > 200) and absence of hepato-splenic iron deposition (liver and spleen CIC were normal) (Fig. 1A). In contrast, three distinct patterns of iron tissue deposition were identified in the PNH patients receiving eculizumab (Table I; Fig. 1E): (i) Low iron levels in liver, spleen and kidneys, (n = 5; Fig. 1B); (ii) high iron levels in liver and/or spleen and low in kidneys (n = 9; Fig. 1C); (iii) high iron levels in liver, spleen and kidney (n = 3). In one of the treated patients, the iron deposition pattern was similar to that of untreated patients. All the four patients studied before and during eculizumab showed resolution or reduction of renal siderosis, with two of them developing a progressive liver iron overload. Looking for possible explanations for distinct patterns of iron tissue deposition we correlated tissue specific CICs with measures of iron status (ferritin, transferrin saturation and haemosiderinuria) and markers of intravascular haemolysis. Kidney iron content, expressed as CIC, did not correlate with any parameter but with haemosiderinuria (P < 0·001). Very high renal CIC levels (>100 units), similar to untreated patients, were present in only 22% (4 out 18) of patients on eculizumab: three patients had signs of chronic residual intravascular haemolysis, whereas one had experienced a recent breakthrough episode. Liver and spleen CIC directly correlated each other (P < 0,001, r = 0·62) but not with renal CIC. Both liver and spleen CIC showed a direct correlation with serum ferritin (P < 0·001, r = 0·79; P < 0·001, r = 0·81) but not with transferrin saturation, LDH and haemosiderinuria. Fifteen patients showed some degree of liver iron overload that, remarkably, was not restricted to patients still requiring blood transfusions: it was mild in 8, moderate in six and severe only in 1 (Table I). In contrast with the low/normal serum ferritin levels present in untreated patients (regardless of transfusion requirement), 10 patients on eculizumab showed high serum ferritin levels ( 300 lg/l), which was associated with liver iron overload (mild, moderate and severe in 4, 5 and 1 cases, respectively). Iron tissue content was studied by liver biopsy in two patients: severe hepatocellular iron overload was found in the transfusiondependent patient RM003 (high ferritinaemia and transferrin saturation) but not in the transfusion independent patient NA003 (moderate increase of ferritinaemia and normal transferrin saturation). To unravel the mechanisms underlying iron overload in PNH patients on eculizumab, we correlated liver CIC with haematological parameters during anti-complement therapy and response to treatment. Liver CIC did not correlate with the duration of eculizumab treatment or with others biomarkers of response (including LDH) with the exception of the inverse correlation with haemoglobin levels (P = 0·02, r = 0·28). Accordingly, patients with suboptimal response (Hb < 110 g/l; Risitano et al, 2009a) had higher liver CIC than patients with optimal response (P = 0·02). Suboptimal response has been associated with the development of variable degrees of extravascular haemolysis, which is probably secondary to C3 opsonization of PNH erythrocytes (Risitano et al, 2009a, 2011). Indeed, we found that liver CIC correlated directly with the absolute reticulocyte count (ARC; P = 0·02, r = 0·26), which is an index of residual haemolysis, and with the percentage of C3-opsonized PNH erythrocytes (P = 0·01, r = 0·31), which has been previously identified as a surrogate marker of extravascular haemolysis in PNH patients on eculizumab (Risitano et al, 2009a). Increased ferritin has been described recently as a possible finding in PNH patients on eculizumab (Risitano et al, 2009b; Röth et al, 2011); here we demonstrate that such

Glutaminolysis is a metabolic dependency in FLT3ITDacute myeloid leukemia unmasked by FLT3 tyrosine kinase inhibition

FLT3 internal tandem duplication (FLT3ITD) mutations are common in acute myeloid leukemia (AML) associated with poor patient prognosis. Although new-generation FLT3 tyrosine kinase inhibitors (TKI) have shown promising results, the outcome of FLT3ITD AML patients remains poor and demands the identification of novel, specific, and validated therapeutic targets for this highly aggressive AML subtype. Utilizing an unbiased genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screen, we identify GLS, the first enzyme in glutamine metabolism, as synthetically lethal with FLT3-TKI treatment. Using complementary metabolomic and gene-expression analysis, we demonstrate that glutamine metabolism, through its ability to support both mitochondrial function and cellular redox metabolism, becomes a metabolic dependency of FLT3ITD AML, specifically unmasked by FLT3-TKI treatment. We extend these findings to AML subtypes driven by other tyrosine kinase (TK) activating mutations and validate the role of GLS as a clinically actionable therapeutic target in both primary AML and in vivo models. Our work highlights the role of metabolic adaptations as a resistance mechanism to several TKI and suggests glutaminolysis as a therapeutically targetable vulnerability when combined with specific TKI in FLT3ITD and other TK activating mutation-driven leukemias.

Paroxysmal Nocturnal Hemoglobinuria after Autologous Stem Cell Transplantation: Extinction of the Clone during Treatment with Eculizumab – Pathophysiological Implications of a Unique Clinical Case

The clinical and biological spectrum of paroxysmal nocturnal hemoglobinuria (PNH) is variable, ranging from classical hemolytic forms to PNH associated with aplastic anemia or other bone marrow (BM) failure syndromes. We report a previously undescribed case of PNH occurring after autologous stem cell transplantation (ASCT) in a patient affected by relapsing non-Hodgkin’s lymphoma. The intensive chemotherapy and the ASCT resulted in a contraction of the effective hematopoietic stem cell (HSC) pool and a derangement of the immune system. The delayed engraftment and the BM hypoplasia represented a favorable environment for the expansion of the pathological clone. This case is paradigmatic even for the unexpected trend of the PNH clone during treatment with the terminal complement inhibitor eculizumab; in fact, the clone reduced until undergoing unexpected extinction, i.e. the recovery of normal hematopoiesis. Eculizumab seems not to play a direct role in HSC kinetics; the clinical remission probably occurred because the environmental conditions that led to the expansion of the PNH clone were transient and disappeared.