Iron overload: causes, assessment methods, significance in transplantation setting and therapeutically approaches

Introduction

Iron is an essential microelement in human body [17]. It is quite important co-factor of enzymes active in mitochondrial respiratory chain, citrate cycle, DNA synthesis, and plays a central role in oxygen binding to hemoglobin and myoglobin; iron-containing proteins are indispensable for collagen, tyrosine and catecholamine metabolism. But free, non-chelated iron, due to its catalytic action in a redox-reaction may form dangerous hydroxyl radicals that may cause cell death resulting from peroxidative damage of cell membranes. In the course of evolutionary development, this was resolved by formation of specialized molecules adapted to its intestinal absorption from food, its transport and deposition in soluble, non-toxic form.

Three crucial mechanisms are important for iron homeostasis, as follows:

•    Iron absorption from food products in small intestine which, generally, compensates for physiological losses;

•    IRE/IRP (Iron Responsive Element/Iron Regulatory Protein) system means intracellular iron uptake or its deposition in complex with ferritin. These processes provide cellular demands of iron and, at the same time, preventing toxic effect of iron overload;

•    Erythrophagocytosis and recirculation of erythrocyte-derived iron, thus ensuring main requirements for iron during erythropoiesis.

These three mechanisms provide stabilization of iron stores in the organism, as well as its intracellular metabolism and bioavailability [17, 34].

The key point of intestinal iron absorption is hepcidin-mediated regulation system.  Hepcidin, a peptide hormone made in the liver, is the principal regulator of systemic iron homeostasis. Hepcidin controls plasma iron concentration and tissue distribution of iron by inhibiting intestinal iron absorption, iron recycling by macrophages, and iron mobilization from hepatic stores. Hepcidin acts by inhibiting cellular iron efflux through binding to and inducing the degradation of ferroportin, the sole known cellular iron exporter. Synthesis of hepcidin is homeostatically increased by iron loading and decreased by anemia and hypoxia. Hepcidin is also elevated during infections and inflammation, causing a decrease in serum iron levels and contributing to the development of anemia of inflammation, probably as a host defense mechanism to limit the availability of iron to invading microorganisms [58].

Disorders associated with altered iron metabolism may be arbitrarily classified in two large groups: iron-deficient and iron overload states.

Iron deficiencies may be caused by alimentary factors (low amount of iron in food), increased iron losses (e.g., due to bleedings) and disturbed iron absorption (Crohn’s or celiac disease, surgical removal of part of intestine, the use of some medications etc).

The most common reasons for conditions, associated with iron overload, are as follows [60, 74]:

1. Genetic alterations of iron metabolism

•    Hereditary haemochromatosis types 1-4

•    Acoeruloplasminaemia

•    Atransferrinaemia  

2. Genetic disturbances of erythropoiesis, and/or life cycle of red blood cells (hereditary dyserythropoietic, haemolytic anaemias, including thalassemias, sickle cell anaemia (SCA),  Blakmond-Diamond anaemia)

3. Acquired conditions requiring long-term, repeated haemotransfusions (myelodysplastic syndrome (MDS), acute leukaemias, myelofibrosis, multiple myeloma etc)

4. Chronic liver diseases (hepatitis, alcohol abuse, nonalcoholic steatohepatitis etc)

Transfusion-associated iron overload

Iron overload due to RBC transfusions is a relatively common complication related to long-term treatment of various haematological diseases. Each unit of blood contains 200 mg of haeme iron, more than 100-fold than a daily amount of iron normally absorbed from the diet. Transfused red blood cells, upon their senescence, are degraded and their iron is re-utilized by resident macrophages. These fractions of bioavailable iron, after binding to transferring, are released into the circulation, and dissipate to body tissues. Iron overload is an inevitable consequence of regular transfusion therapy [37]. After receiving 20 lifetime units of packed red blood cells, patients can become iron overloaded. No matter how many years pass between transfusions, the dangers are the same, because the body has no way actively excrete excess iron. Iron overload is proportional to transfusion burden [67, 57].

When iron overload is evident, transferrin becomes saturated, thus leading to increased non-transferrin bound iron (NTBI) in blood plasma. Whereas tissue uptake of transferrin-bound iron is regulated by expression of membrane-bound transferrin receptor (responsible for transport of transferrin into cells [4]), excess of NTBI is often absorbed by susceptible tissues. This leads to pools of unbound iron within cells, which mediate toxicity by the formation of reactive oxygen species (ROS) [30]; Figure 1.

Figure 1.

ROS react with cellular components such as the plasma membrane, lysosomes, and organelle membranes, leading to cellular leakage, dysfunction, and, ultimately, cell death [33].

The organ damage that occurs with transfusional iron overload is similar to that seen in hereditary haemochromatosis, although iron accumulation occurs more rapidly, and deposition of iron in resident tissue macrophages is proportionally greater [43, 74, 77]. Hereditary haemochromatosis is characterized by iron accumulation primarily in the liver, heart, and pancreas. However, whereas chronic anaemias are related to errors in erythropoiesis, the main cause of haemochromatosis is a reduction or absence in expression of hepcidin (in particular, due to the haemochromatosis gene (HFE) mutations) [43, 74, 77]. 

Under steady-state conditions, hepcidin prevents excessive intestinal absorption of iron and its release from macrophages. Therefore it minimizes the amount of iron that enters into plasma. However, in conditions of chronic iron overload, blood transfusion leads to an increase in erythrocytes, which are broken down by macrophages. This initially results in an increase in iron levels in resident tissue macrophages. Hepcidin expression also downregulates iron release from this system, leading to a “build-up” in the reticuloendothelial system and its possible saturation (a stage that is reduced or absent in patients with haemochromatosis). Subsequently, iron is deposited in various tissues, thus leading to tissue damage [43, 46, 63].

It is well established that, prior to advent of iron-chelating therapy, cardiomyopathy and liver fibrosis associated with iron overload were among the leading causes of death among children with thalassemia [15, 61]. The role of iron overload in MDS patients is also well established. There are different data concerning overall survival and progression free survival in MDS patients according to transfusion dependency and serum ferritin level [49, 50, 51, 75, 78]. Studies have indicated that iron overload following RBC transfusions was an independent, adverse prognostic factor for overall survival (OS) and leukemia-free survival (LFS): OS and LFS were significantly shorter in transfusion-dependent patients with MDS than in those who were not transfusion dependent.

Accurate assessment of body iron burden is necessary to diagnose iron overload and also to effectively manage therapy.

Assessment of body iron stores

There are numerous methods available for the detection and assessment of iron levels, both as total body and specific tissue levels. Because transfusions may lead to rapid iron accumulation, monitoring a patient's number of transfused blood units, serum ferritin levels, and/or liver iron concentrations can play an essential role in the management of iron overload. Each method is associated with advantages and disadvantages [19, 52].

Diagnosis of postransfusional iron overload:

•    Established
      –    Ferritin
      –    Liver iron concentration (biopsy)

•     Investigational
      –    Biomagnetic liver susceptometry (SQUID)
      –    Magnetic resonance imaging (MRI)

Ferritin is inexpensive, noninvasive, and widely available method, which provides reliable estimates of iron burden when performed on a serial basis. Serum ferritin levels consistently >1000 mcg/L are suggestive of iron overload [62, 70], and in the absence of appropriate therapy are associated with adverse clinical outcomes in iron overload [50, 62, 75]. Ferritin is the simplest method for assessment of iron overload, but it has limitations of the value [13]. The advantages and disadvantages of the ferritin measure are summarized
in Table 1.

Table 1. Ferritin as a marker of iron overload

Liver biopsy provides direct information about the structure, function, and extent of iron deposition within the liver, and may also have prognostic value.

Liver iron concentration (LIC) predicts total body storage iron [5, 13], but measuring LIC by Liver Biopsy has its limitations, too
(Table 2).

Table 2. LIC as a marker of iron overload

Magnetic resonance imaging (MRI) provides a noninvasive, quantitative method of estimating parenchymal iron levels. In principle, MRI can be used to quantify iron stores wherever they exist in the body. In practice, MRI has been investigated in the assessment of hepatic, cardiac, and anterior pituitary iron stores. MRI measures tissue iron concentration indirectly via the detection of the paramagnetic influences of storage iron (ferritin and haemosiderin) on the proton resonance behavior of tissue water [39]. The longitudinal (R1) and transverse (R2) nuclear magnetic relaxation rates of nearby solvent water protons can then be calculated. Both R1 and R2 rates are increased when interacting with paramagnetic particles such as iron. R2 (or spin-echo imaging) is preferable to R1 for determining LIC, since ferritin enhances the relaxation of both R1 and R2, while haemosiderin only has a strong R2 relaxation accelerating effect. Gradient echo imaging produces images for calculating T2* and R2*, where R2* = 1000/T2*. A T2* of 20 ms is equivalent to an R2* of 50 Hz. MRI provides a non-invasive alternative to liver biopsy, and may actually be more accurate in patients with heterogeneous liver iron deposition (such as those with cirrhosis) since it measures iron in the whole organ. In addition, the pathologic status of the liver can also be assessed using MRI [3, 25, 27]. MRI remains the only noninvasive modality in clinical use with the ability to detect cardiac iron deposition. T2* MRI is rapidly becoming the new standard for measuring cardiac iron levels. One study found that below a myocardial T2* of 20 ms, there was a progressive and significant decline in left ventricular ejection fraction (LVEF). In general, the lower the T2*, the higher the risk of cardiac dysfunction, with a T2* <8 ms suggestive of severe iron overload [3].

SQUID stands for Superconducting Quantum Interference Device. This imaging modality uses a very low-power magnetic field with sensitive detectors that measure the interference of iron within the field. The sensor requires a cryogenic environment, since it must be superconducting to operate. Although SQUID is still considered investigational, linear correlations have been demonstrated between SQUID measurements and liver biopsy LIC levels [14, 16, 59, 66].

Although SQUID directly measures the magnetic susceptibility of ferritin and haemosiderin, at present it does not have sufficient spatial or temporal resolution to evaluate myocardial iron. In a large clinical trial, LIC data obtained by SQUID were shown to underestimate LIC values obtained from biopsy by a factor of 0.46 [64].

Both SQUID and MRI have linear correlations with LIC assessed by biopsy, but they provide indirect measurement of LIC, have high cost and as highly specialized equipment requires dedicated technician [3, 7]. Limitations of these methods are summarized in Table 3.

Table 3. SQUID and MRI as a markers of iron overload

Various additional laboratory tests have been developed to assess iron overload. While not widely available, they may hold promise of providing additional clinical information.

•    Serum ferritin iron may be less susceptible than serum ferritin to confounding factors such as inflammation [36].

•    Serum transferrin receptor concentration has been used to detect both iron deficiency and excess iron. In the presence of iron overload, cells downregulate transferrin receptor expression, and therefore serum transferrin receptor concentration would be expected to be reduced [41].

•    Non-transferrin-bound iron (NTBI) - which is normally not found in plasma, increased rapidly during conditioning therapy and contribute to .oxidative stress after high-dose radiochemotherapy [24].

•    Labile plasma iron (LPI) quantifies the oxidative activity of the patient's plasma-borne NTBI. In theory, this test can be used as a direct measure of iron overload. One approach to measuring LPI is to measure the reactive radicals generated in the subject's blood by exposure to ascorbate, compared to those generated after the addition of a chelating agent (which blocks the oxidative activity of the NTBI) [26].

•    Directly chelatable iron (DCI) is not a test for iron overload per se, but rather an experimental assay for assaying the plasma pool of non-transferrin-bound iron (NTBI).

If to speak about recommendations three guidelines concerning MDS, SCD and thalassemia should be mentioned.

Consensus recommendations developed by leading MDS researchers and clinicians recommend baseline assessment of body iron stores at diagnosis of MDS and at regular intervals – at least every 3 months – thereafter [28]. They recommend that monitoring be performed with a combination of serum ferritin, serum transferrin, and liver MRI [28].

U.S. National Heart, Lung, and Blood Institute (NHLBI) guidelines for the management of sickle cell disease state that, even in patients who receive intermittent transfusions, "a comprehensive program to monitor and treat iron overload is necessary" [56]. NHLBI guidelines state that [56]:

•    Screening for iron overload with serum ferritin testing is recommended at the onset of transfusions.

•    Iron overload is likely to be detected after 20 transfusions.

•    Liver biopsy is the most accurate test for confirming iron overload in SCD.

According to the Thalassaemia International Federation (TIF) Guidelines for Clinical Management, iron overload constitutes "the most important complication in β-thalassemia and the major focus of clinical management") [21].

TIF guidelines recommend screening for iron overload at the onset of transfusions. Iron overload is likely to be detected after the first 10-20 transfusions (near age 3 years) [22]:

•    Monitor serum ferritin at least every 3 months

•    Do not rely on serum ferritin alone

•    Liver iron concentration – determined by biopsy or noninvasively by MRI or SQUID – is the reference standard for estimating iron loading

Thus, summarized these guidelines,  it can be recommended for all haematological conditions to accurately evaluate and record the iron input, start screening for iron overload at the time of beginning the initial treatment then  after each 10-20 transfusions and every three months.  Serum ferritin can be used as the basic parameter, but though it is not highly specific marker, it shouldn’t be used alone. LIC measurement by biopsy (if indicated) or by MRI or SQUID (if available) is desirable. Assessment of the heart iron by MRI T2* (cardiac risk), at least once is also recommended, If positive, it should be used as the main result to set treatment [16].

Iron overload in transplantation setting

Iron overload commonly accompanies bone marrow transplantation [73]. Haematopoetic cell transplant recipients - both allogenic and autologous – often present with iron overload because of exposure to RBC transfusions, both during the initial treatment of their disease and in the post transplant period [48]. Despite these there are some conditions which contribute to increased risk for iron overload: the genetic background (heterozygous C282Y mutations can be identified in ~30% of patients with MDS); chemo/radiotherapy (total body irradiation (TBI)/alkylator-induced alterations which increase NTBI and decrease total radical-trapping antioxidant activity in plasma); chronic infections and inflammation; haemolytic disorders induced by immunosuppressive agents (cyclosporine and tacrolimus) [60]. Even in patients without a history of transfusions, a predictable increase in serum iron, transferring saturation, ferritin and non-transferrin bound iron has been observed in the early post transplantat period [11, 31, 71].

There are several studies concerning importance of iron overload in transplantation setting [9, 20, 35, 47, 53, 60, 69, 73, 76]. The majority of data are devoted to thalassemia and MDS patients. Concerning thalassemia patients hepatomegaly, hepatic fibrosis due to iron overload and poor compliance with iron chelation were identified as being independent prognostic factors, and a classification system according to these factors was developed and adopted by most centers [71]. Age is also an important factor when predicting outcome, with adult patients usually having a poorer outcome when compared to children [44]. In MDS the recently defined WHO classification–based Prognostic Scoring System (WPSS) (which include the transfusion dependency as a prognostic factor) was able to identify five risk groups of untreated MDS patients with different survival and risk of  leukaemic progression, compared with the four groups defined by IPSS [32]. It was observed in addition that WPSS has an independent prognostic significance on both overall survival (OS) and probability of relapse in MDS patients undergoing allo-HSCT. This score appeared to improve post transplantation prognostic stratification with respect to the International prognosis scoring system (IPSS). Considering MDS patients without excess of  blasts, the WPSS identified two groups of patients (low vs. intermediate risk) with a significant difference in OS and treatment related mortality (TRM), whereas in the same group of patients IPSS failed to stratify the prognosis [49]. Interestingly, both WHO classification and WPSS maintained their prognostic effect on post transplantation outcome also in specific subsets of patients, such as patients older than 50 years as well as patients receiving reduce intensity chemotherapy (RIC). This observation might be relevant in the light of the increased number of RIC performed in MDS in most recent years, after the demonstration of their efficacy in allowing engraftment and in decreasing TRM in patients ineligible for standard conditioning allo-HSCT [49]. In particular, WHO classification and WPSS show a relevant prognostic value in post transplantation outcome of MDS patients and might help decision making in transplantation.

Armand et al. estimated that iron overload could be a significant contributor to TRM for patients with haematologic malignancies undergoing HSCT. They studied 590 patients who underwent myeloablative allogeneic HSCT at their institution, and on whom pre-transplantation serum ferritin was available. An elevated pretransplantation serum ferritin level was strongly associated with lower OS and DFS. Subgroup multivariable analyses demonstrated that this association was restricted to patients with acute leukemia or MDS [9].

Altes et al. concluded that very high level (VHL) of ferritin and transferring saturation (TS) >/=100% at the time of conditioning are associated with an increase in toxic deaths after transplant [2].

Several other studies also confirm that iron overload can increase morbidity and mortality in transplanted patients [9, 20, 35, 47, 53, 60, 71, 73, 76].

Early and late complications of HCT that have been associated with iron overload are summarized in Table 4.