Non-transferrin-bound iron and oxidative stress during allogeneic hemopoietic stem cell transplantation in patients with or without iron overload
Authors: Lorena Duca, Maria Domenica Cappellini, Donatella Baronciani, Federica Pilo, Clara Targhetta, Giuseppe Visani, Isabella Nava, Emanuele Angelucci
Journal: American Journal of Hematology (2018)

In normal subjects serum transferrin saturation is 20%-35%, and “free-iron” better defined as non-transferrin-bound-iron (NTBI) is undetectable. The presence of NTBI has been reported in disorders associated with iron overload such as thalassemia and in serum of patients after intensive cytotoxic chemotherapy for the treatment of hematological malignancies1. Hematopoietic cell transplantation (HCT) is increasingly used as a curative therapy of a variety of malignant and nonmalignant diseases. Transplant conditioning with high doses of cytostatic drugs is associated with rapid transferrin saturation, changes in ferritin levels and accumulation of NTBI.2 The aim of our study was to evaluate the NTBI behavior in transfusion-dependent iron-overloaded thalassemia and non-iron-overloaded non-thalassemia (leukemia) patients during HCT and to verify its relationship to oxidative stress as expression of cellular damage.3 Eighteen patients (median age 8.8 years; range 4-16 years) affected by β-thalassemia major (8 female, 10 male) and 9 patients (median age 27.4 years; range 10-44 years) affected by different types of hematological malignancies (6 female, 3 male) were recruited for this study. There was only one splenectomized patient in each group. The control group consisted of 17 healthy subjects (9 female and 8 male aged 23-40 years). Institutional review board of Pesaro Hospital Hematology Unit approved this prospective observational non-interventional study as part of the ancillary research on HCT in iron loaded patients. All thalassemia patients were transfusion dependent and on deferoxamine subcutaneous infusion (20-40 mg/kg) according to the iron status. All patients stopped deferoxamine infusion at least 4 weeks before transplantation. Leukemia patients received a limited number of red blood cell units according to clinical management of their disease. No leukemia patient received chelation. All patients were conditioned with high dose oral Busulfan (14 mg/kg) from day −9 to −6, followed by cyclophosphamide (120-200 mg/kg) from day −5 to day −2 being day 0 the day of hemopoietic cell infusion. Starting from day −1 all patients were treated with intravenous Cyclosporine (3 mg/kg). NTBI and serum malondyaldehyde (MDA) as marker of lipid peroxidation that indirectly represent the tissue damage were analyzed prior to conditioning therapy (−9 “baseline values”), during therapy (day −5,−2), on the day of HCT (day 0) and up to 4 weeks later (day +7, +14 and + 28). Serum ferritin and hepatic iron concentration (HIC) were obtained from clinical laboratory procedures. Serum NTBI was assayed by high performance liquid chromatography (HPLC) after nitrilotriacetic acid (NTA) chelation. The solution was ultrafiltered and directly injected into the HPLC system with a titanium pump module (PerkinElmer S200, Boston, Massachusetts). NTBI in normal subjects is undetectable or even negative, because samples are measured in parallel with a corresponding blank formed by NTA and water which contains small amounts of iron which is not bound by transferrin; therefore the removal of blank makes normal samples negative. Free MDA was evaluated by means of a colorimetric assay for lipid peroxidation using a Bioxytech LPO-586 kit (Oxis International Inc, Portland, Oregon). Continuous variables were expressed as mean ± SD unless specifically indicated. Statistical differences among the groups were evaluated by Mann-Whitney test. Linear-regression analysis was performed for the correlation between 2 variables. At baseline the median ferritin in thalassemia patients was 1580 ng/mL (range 759-6360 ng/mL), while in non-thalassemia patients was 789 ng/mL (range 27-3916 ng/mL). The median HIC in thalassemia patients was 12.9 mg/g/dw (range 4.09-40.00 mg/g/dw, normal value <1.6 mg/g/dw), in non-thalassemia patients HIC was not measured. NTBI values were significantly higher in all patients compared with controls (−0.80 ± 0.70 μmol/L) pre and post HCT (thalassemia 1.76 ± 1.38 μmol/L P < .0001; leukemia 0.55 ± 1.45 μmol/L P = .013), although leukemia patients had overlapping NTBI values with respect to controls at baseline and at day 14. Significantly higher levels of NTBI were detected in thalassemia compared with leukemia patients at days −9 (baseline), 0 and 14 but not at the other time points during the study and particularly during and immediately after chemotherapy. NTBI levels were constantly higher in the thalassemia group but the relative increase compared to baseline was higher in leukemia patients (between 2.2- and 3-fold) than in thalassemia patients (between 1.6- and 2.5-fold). Early after transplant, consistently with erythropoietic recovery, NTBI returned to respective baseline values (Figure 1A). MDA values significantly correlated with NTBI values in leukemia patients independently from distance from transplant or chemotherapy (Figure 1B). No significant correlation was found in thalassemia group between MDA and NTBI values (data not shown). The markedly increase of NTBI in serum after myeloablative chemotherapy was originally attributed to suppression of erythropoietic activity. Other possible sources of extracellular iron after myeloablative conditioning include lysis of erythroid bone marrow cells by cytotoxic injury.4 Our study confirms reduction of the iron uptake by erythroid precursors during and immediately after chemotherapy. NTBI decrement in all the patients after HCT could be explained by high iron requirement by the reconstituting allogeneic erythroid marrow. The correlation between NTBI and MDA in leukemia and not in thalassemia patients may suggest that the previous iron overload in thalassemia and previous already elevated NTBI and MDA levels are able to attenuate changes in the oxidative state before and after transplantation. High levels of iron overload, in the absence of chelation, are responsible for the persistence of NTBI high levels even after marrow reconstitution in thalassemia patients. These data indicate that, although iron overload per se may lead to NTBI presence, the bone marrow ablation by cytotoxic drugs, in preparation of HCT is responsible for the appearance and increasing of NTBI in plasma, which may have a toxic effect causing organ damage even in non-thalassemia, non-iron loaded, patients. In conclusion these data support the concept of iron release from erythroid cells during bone marrow ablation by cytotoxic drugs even in the absence of systemic iron overload. The “free-iron” form may contribute to organ toxicity detectable during chemotherapy in preparation of HCT.5 In iron-overloaded patients with high NTBI levels a chelation period pre and immediately after bone marrow ablation and HCT could be beneficial in terms of organ toxicity and transplantation outcome.6 The possible clinical benefit must be demonstrated in a prospective controlled clinical trial. Dr Angelucci is Chair of the steering committee of the TELESTO trial by Novartis Oncology on iron chelation therapy in MDS.