Neuroblastomas contain iron-rich ferritin

zy zyxwvuts zyx Neuroblastomas Contain Iron-Rich Ferritin zyxwvutsrq zyxwvu zyx THEODORE C. IANCU, MD, HANNA SHILOH, MSC, AND AMOS KEDAR, MD The ultrastructure of neuroblastoma was examined using unstained sections so that ferritin particles could be identified by the electron density of their iron cores. Ferritin and hemosiderin were found in ten of 11 neuroblastomas that were examined when the patients first presented. The study was therefore expanded to an additional group of children, including some diagnosed by noninvasive procedures and given chemotherapy before the excision of their tumors. In this second group 12 of 20 specimens contained ferritin and hemosiderin in variable amounts. In both groups there was a tendency for patients with advanced disease to have increased amounts of iron compounds in the tumor tissue (Stage I11 and particularly Stage IV). Most Stage IV patients also had elevated serum ferritin levels. However, based on the available heterogenous material, no absolute relationship could be established between age, disease stage, tumoral storage iron, and the level of serum ferritin. The presence of ferritin in neuroblastoma may be linked to the elevated serum ferritin levels and may be implicated in tumorigenesis. Cancer 61:2497-2502,1988. E have been reported in iron storage disorders as well as in a number of infections and inflammatory and malignant conditions.’.2The presence of excess iron is known to induce the synthesis of (apo)ferritin in various cells3followed by segregation of the inorganic iron within the protein shell.4 In contrast, the origin of the iron-poor serum ferritin is not clear. Putative mechanisms include “secretion” of serum femtin by reticuloendothelial cells as well as the release of ferritin into the circulation from disintegrating cells. Recent reports have provided evidence that primary neuroblastomas and cultured neuroblastoma cells produce ferritin.’-* In addition, correlations were found between serum femtin levels and the stage of neuroblastoma. In Stages IV and IVS, despite similar extensive LEVATED LEVELS OF SERUM FERRITIN ’ disease burdens, the prognosis is quite different. Stage IV neuroblastoma, the prognosis of which is unfavorable, is associated with markedly elevated serum ferritin levels. In contrast, Stage IVS (without bone involvement) has a good prognosis, and normal levels of serum ferritin are usually found. Patients with early-stage neuroblastoma (Stages I and 11) have low levels of serum ferritin and favorable outcome^.^ Previous studies concerning ferritin in neuroblastoma were directed at identifying the protein (apoferritin) regardless of its iron content. Because of the suspected role of iron in tumorigenesis and the reported relationship between serum and tumor ferritin,’-1° we investigated its presence in neuroblastoma. An electron microscope examination of unstained biopsy sections has helped to identify electron-dense ferritin cores containing variable amounts of iron oxyhydroxide.’ I Patients and Methods From the Department of Pediatrics and Pediatric Research Unit, Carmel Hospital and Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel. Supported in part by the Milman Fund for Pediatric Research. The authors thank Jonathan Pritchard, MB, FRCP and R.A. Risdon, MD, FRCPath, from the Department of Hematology and Oncology and the Department of Histopathology, the Hospital for Sick Children, Great Ormond Street, London, UK, for providing the clinical and pathologic material concerning the second group of patients. We also thank A. Luder, MD, for help with the manuscript and Jeffrey Davis and Judith Regev for technical assistance. Address for reprints: Theodore C. Iancu, MD, Department of Pediatrics and Pediatric Research Unit, Carmel Hospital, 34362 Haifa, Israel. Accepted for publication November 20, 1987. Patients zyx zy We examined the ultrastructure of neuroblastoma first in a group of 11 patients aged 5 days to 6 years referred to the Carmel Hospital in Haifa, Israel. The diagnosis was established by a light microscope examination of the primary tumor, and the stage was established according to the classification of Evans et al.l 2 Since most biopsies were obtained before awareness of the value of serum ferritin in the assessment of neuroblastoma, levels were available in only two patients with 2497 zyxwvu zyxwv zy zyxwvuts zy CANCERJune 15 1988 2498 Vol. 61 TABLE1. Patients in the First Group Patient no. 1 2 3 4 5 6 7 8 9 10 11 zyxwvutsrqpo Age Sex 5d 2 mo 2 mo 6 mo 6 mo 1 Yr 1yr3mo 1yr7mo 3 yr 4 Yr 6 vr M F M M M M M F F F M Diagnosis Stage NB NB NB NB NB NB NB GNB NB GNB NB IVS I1 NB: neuroblastoma; G N B ganglioneuroblastoma; Neg: negative. * Storage iron in tumor: Negative: no femtin or hemosiderin visible or the amount so small as to be considered of no significance; 1+: few femtin particles in some cells in either cytosol or siderosomes; 2+: Stage IV and one patient with Stage I1 neuroblastoma. A two-site immunoradiometric assay (RAMCO, Houston, TX) was used for the serum ferritin determinations (normal range, 14 to 125 ng/ml). In the second group of patients 19 children aged 2 days to 6 years referred to the Hospital for Sick Children, Great Ormond Street, London, were included. Most of these patients had been diagnosed by noninvasive procedures at various times before a morphologic diagnosis was made. In this group serum ferritin levels were obtained from patients, but not necessarily at time of diagnosis or surgery. 111 11 111 111 111 111 IV I1 IV Serum fenitin ng/ml Storage iron in tumor* I+ I+ Neg 2+ 2+ I72 I+ 2+ I+ 3+ 2+ 3+ 576 650 more fenitin particles dispersed in the cytosol and in more cells, also occasional siderosomes; 3+: numerous fenitin particles in many cells, some with features of macrophages, but mainly in tumoral cells and typical cytoplasmic neuritic processes; also frequent siderosomes. Morphologic Studies Tumor specimens were routinely processed and stained with Perls' stain for iron. Semi-thin sections ( 1 pm) were stained with 1% toluidine blue and examined for orientation, degree of tumor maturation, and hemorrhage. For electron microscopy the specimens were immediately fixed in 2.5% glutaraldehyde buffered with 0.1 mol/l sodium cacodylate (pH 7.4) and then were postfixed in 2% osmium tetroxide buffered with phosphate zyx TABLE2. Patients in the Second Group Patient no. 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28t 29t 30 31 Age at diagnosis Sex Diagnosis Stage 2d 2 mo 3 mo 4 mo 7 mo 9 mo 1yr3mo 1 y r 8 mo 1 yr 1 1 mo 2 y r 1 mo 2yr2mo 2yr8mo 3yr4mo 3yr4mo 4yr3mo 4yr5mo 4yrSmo 4yr5mo 5 Yr 6yr3mo F M F F M M F M F M F F F M M M F F M M NB NB NB NB NB NB NB NB NB NB GNB NB NB NB NB GNB NB NB NB NB IVS IV I1 IV I1 I1 IV IV IV IV I1 1v IV NB: neuroblastoma; GNB: ganglioneuroblastoma; Neg: negative. * Storage iron in tumor: Negative: no fenitin or hemosiderin visible or the amount so small as to be considered of no significance; I +: few femtin particles in some cells in either cytosol or siderosomes; 2+: more femtin particles dispersed in the cytosol and in more cells, also 111 IV 111 IV IV IV IV Age at biopsy 17 d 2 mo 2yr2mo 10 mo 7 mo 10 mo I yr4mo 2yr3mo 2yr4mo 2yr6mo 2yr5mo 3 yr 1 mo 3 yr 10 mo 3 yr 10 mo 4yr9mo 4 yr 10 mo 4yr5mo 5 Yr 5yr5mo 6yr9mo Serum femtin ng/ml Storage iron in tumor* 5 10 330 478 148 23 1 255 42 84 occasional siderosomes; 3+: numerous femtin particles in many cells, some with features of macrophages, but mainly in tumoral cells and typical cytoplasmic neuritic processes; also frequent siderosomes. t Patients 28 and 29: two specimens from the same patient. No. 12 - NEUROBLASTOMAS CONTAIN FERRITIN zyxwvut zyxw zyxw sr Zuncu et al. 2499 FIG. 1. A femtin cluster in a neuroblastoma cell (arrow), which also shows numerous, randomly dispersed cytosolic particles. These are smaller and less electron-dense than the clustered particles (Patient 1 1 ) . Inset: within immature neuroblastoma cells clusters of typical iron-rich ferritin may be seen even in the absence of cytosolic ferritin (unstained, X 140,000). to pH 7.2. They were dehydrated and embedded in Polarbed 8 12 (Polaron, Watford, UK). Five blocks were prepared from each specimen, and six grids from each block were cut at 60 nm with a diamond knive and mounted on 300-mesh copper grids. Two grids were conventionally stained with uranyl acetate and lead citrate and examined for ultrastructure features of neuroblastoma, ganglioneuroblastoma, or ganglioneuroma. Four grids from each specimen were left unstained and were examined for the presence of electron-dense particles or aggregates consistent with ferritin molecules or hemosiderin. Specimens were viewed and photographed with Jeol JEM 100 S and Philips 300 electron microscopes. Results The relationship between age, stage of neuroblastoma, and the presence of ferritin, as documented by electron microscopy in the initial group of patients is shown in Table I. The data on the second group of patients are given in Table 2. An electron microscope examination showed that ferritin iron cores were in most biopsies but not in all cells, and that there were less in the very young patients with limited disease. The particles were in clusters or randomly dispersed in the cytosol of tumor cells (Fig. I ) and cytoplasmic neuritic processes (Fig. 2). The particles were more conspicuous in tumors from patients older than 6 months of age in Stages I11 and IV. Most cytosolic femtin cores had a diameter of about 6 nm and a medium electron density (Type 11). Thus, these particles had an intermediate iron content between the smaller (5.5 nm, “iron-poor,” Type I) and the larger (6.5 nm, “iron rich,” Type 111) particle^.'^^'^ There were fewer iron-containing membrane-bound bodies (lysosomes-siderosomes) than in iron-loading conditions (e.g., thalassemia major) or experimental iron overload. Nevertheless, the siderosomes displayed the typical features of ferritin segregation. In some the iron-rich ferritin was densely packed without any symmetrical arrangement (Fig. 3). These ferritin cores were quite different from the smaller particles seen in the cytosol or within lipofuscin-like bodies. Hexagonal paracrystalline arrangements, frequently reported in macrophages in various iron-loading conditions, were found in siderosomes with an irregular shape (Fig. 4).In such organelles particles other than those forming paracrystalline arrangements were smaller and could not be resolved because they were too densely packed. Other siderosomes contained multi-layered femtin arrays (Fig. 5). The center-to-center distance of the array-forming individual cores, as well as the interval between layers were equal to those of the arrays found in iron-loaded hepatocytes. zyxwv zy 2500 zyxwvu zyxwvutsrq CANCERJune15 1988 Vol. 61 Discussion The main contribution of our present study is the finding of iron-rich fenitin and hemosiderin aggregates in neuroblastoma cells and cytoplasmic neuritic processes. The tumor cells had features of iron overload similar to other cells exposed to iron either locally or systemically. With continuing exposure to iron, cytosolic ferritin is transported into lysosomes (siderosomes). Large amounts of iron are sequestered in these organelles as ferritin and its degradation product, hemosiderin.1’-’6 The origin of iron in the ferritin molecules of neuroblastoma cells is not clear. The simplest explanation relates to the extravasated erythrocytes frequently present in these tumors either spontaneously or after therapy. Heme-iron could induce fenitin synthesis in both tumor cells and macrophages. In many tumors hemorrhage was indeed noted by macroscopic, light, and electron microscope examinations, but no storage iron was found. Alternatively, in a number of neuroblastomas ferritin particles were seen in the absence of hemorrhage. Some cells with features of macrophages contained erythrocytes, but the transfer of ferritin to tumor zyxw FIG. 3. Segregation of ferritin particles in siderosomes of various types. Lipid droplets, apparently lipofuscin (arrows), are seen in bodies that also contain small and medium-sized electron-dense particles. Larger femtin cores are seen in a different type of siderosome (arrowhead) (Patient 6 ) (lead citrate, X75,OOO). cells by rhopheocytosis, as documented in bone marrow normoblasts,” was not noted. Even if extravasated erythrocytes contribute to an iron-rich milieu in some neuroblastomas, this mechanism cannot explain the increased synthesis of fenitin in nonhemorrhagic tumors such as breast carcinoma.18For tumors like the neuroblastoma described in this article in which femtin contains significant amounts of iron, the numerous transferrin receptors identified on the surface of tumor cells could provide malignant cells with the iron needed for growth and development. This investigation also shows a trend toward the presence of more storage iron in immature and expansive tumors, i.e., in patients with Stage 111 and particularly Stage IV disease. The fact that some specimens from patients in the latter stages were either negative for iron or had only a minor iron content may be related to age or the time between a noninvasive diagnosis and the actual removal of tumor tissue for diagnosis. This possibility is exemplified by a Stage IV patient from whom two specimens (No. 28 and 29) were available. The first specimen showed a medium degree of iron content (2+), while the second specimen taken after 6 months of chemotherapy was negative. zyxwvutsr FIG.2 . Part of a cytoplasmic neuritic process with neurosecretory granules (arrow) and cytosolic femtin. Part of an adjacent cell (arrowhead) contains neurosecretory granules but no femtin (Patient 5 ) (unstained, X62,OOO). zyxwvutsr zyxw zyxw zyxwvutsrqpo zyxwvutsrqponml NEUROBLASTOMAS CONTAIN FERRITIN No. 12 - Zancu et al. 2501 zyxwvutsrq FIG. 4. In this cell the siderosomes are similar to those of macrophages, i.e., either round (broad arrow) or irregular and contain paracrystalline hexagonal ferritin arrangements (arrow) (patient 7). Unstained, x 100,000. Our observations on morphology are complementary to previous reports documenting the secretion of femtin by neuroblastoma cells and stage-related elevation of serum f e ~ ~ i t iAll n .patients ~ ~ ~ ~with ~ marked tumor iron FIG.5. A siderosome with multi-layered ferritin arrays (arrow) identical to those found in iron-ladenhepatocytes (Patient 7). Smaller, less electron-dense particles are seen in the cytosol (C) or as individual or coalesced particles within the siderosome (S) (unstained, X 100,000). overload (3+) had Stage IV disease, and all patients with elevated serum ferritin levels also had Stage IV disease. However, some Stage IV patients had no elevated ferritin levels and/or increased tumor iron content. In addition to the already mentioned timing of specimen examination, chemotherapy given to some of the children before tissue removal could also influence the results. Therefore, although a relationship between disease stage, serum fenitin, and the presence of storage iron in the tumors is apparent in some, no absolute relationship could be established. A major question is the relevance of the presence of iron in these tumors with regard to the malignant growth process. Rapidly growing cells require iron for growth and metaboli~m,~,’~ and evidence accumulated in recent years suggests that iron has a more direct role of the iron within t u m o r i g e n e s i ~ .A~ ~“breakdown ~~ holding mechanism”20has been described as a potential factor in malignancy. Surplus iron inhibits the tumoricidal activity of mouse macrophages2’ and can modulate critical functions of the immune system such as natural killer and antibody-dependent cellular cytotoxicity.22In Hodgkin’s disease it has been suggested that the malignant cell is a phagocyte whose excessive avidity for iron or inability to utilize the metal in a normal manner leads to its malignant tran~formation.~~ A recent study on the influence of blood transfusions on neuroblastoma’ confirms the relationship between high zyxwvu zyxwvu CANCERJune15 1988 2502 serum femtin levels and poor clinical outcome. The authors suggest that the tumor benefits from the transfusions through their documented immunosupressive effect, or that the tumor utilizes transfusional iron overload as a stimulus for its own production and secretion of femtin and expands proportionally to the amount of blood transfused. Studies on the role of iron in tumorigenesis, including those on the “decompartmentalized” iron,’’ have not hitherto utilized morphologic evidence for the presence of iron in tumors. The ferritin iron cores as well as the hemosiderin aggregates seen in neuroblastoma cells and cytoplasmic neuritic processes document the failure of tumor cells to keep iron outside the cell. The ultrastructural documentation of iron-rich fenitin in neuroblastoma may also be relevant to the recent attempts to use antiferritin antibodies as a therapeutic agent.24The fact that not all cancers show increased iron or ferritin concentrations, that in some malignant tumors iron apparently accumulates around the as well as our finding that not all neuroblastoma cells contain ironrich ferritin point towards the difficulties of establishing a unifying concept for the role of iron in tumorigenesis. REFERENCES Vol. 61 7. Potaznik D, de Sousa M, Helson L, Bagin R, Groshen S, Bhalla RB. 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