Med. OncoL & TumorPharmacother. Vol. 1, No. 3, pp. 149-156, 1984 0736-0118/84 $3.00 + .00 Printed in Great Britain Pergamon Press Ltd.

SERUM F E R R I T I N A N D M A L I G N A N T T U M O U R S

ALLAN JACOBS Department of Haematology, Welsh National School of Medicine, Cardiff, U.K.

(A ccepted 29 A ugust 1983)

Increased concentrations of serum ferritin are common in patients with malignant disease and appear to be a non-specific response. The present evidence does not suggest that serum ferritin assay is useful either for specific diagnosis or monitoring of malignant disease. A special role for acidic isoferritins in malignancy remains to be substantiated.

Key words: Ferritin, Isoferritin, Cancer, Leukaemia.

INTRODUCTION

In recent years there has been an increasing interest in serum ferritin. An awareness that its concentration may be increased in patients with malignant disease has led to considerable discussion regarding its possible significance and its value in the diagnosis of leukaemia and solid tumours. Ferritin is an iron storage protein synthesised in all animal cells 1 and it is not restricted to cells with a specialised iron storage role, though there is variation between different cell types in the amount of protein produced. The molecule consists of a roughly spherical protein shell about 12 nm in diameter with a central cavity which can contain over 4000 atoms of iron as a ferric hydroxide phosphate complex. The iron-free protein, apoferritin, has a mol. wt of about 450,000. Iron enters and leaves the molecule through six channels which penetrate the shell along the molecular four-fold axes. Tissue concentrations of ferritin increase in response to iron loading and are decreased following iron loss. Details of the structure of the molecule and its role in iron metabolism have been given in recent reviewsJ -3

There is much heterogeneity amongst ferritin molecules. Their iron content may vary, polymerisation may occur and tissue ferritins show heterogeneity on isoelectric focusing, the pattern of which varies from tissue to tissue. Human liver and spleen ferritins show a range of isoelectric points between 5.3 and 5.8 while heart ferritin shows more acidic proteins with isoelectric points between 4.8 and 5.2. These isoferritins are composed of varying proportions of two subunit types which have different sizes and charges and have been called H (heart) subunits, with a tool. wt of 21,000 and

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L (liver) subunits, with a mol. wt of 19,0007 There is a correlation between the proportion of H and L subunits in a particular tissue ferritin and its isoelectric po in t :

The first specific identification of circulating ferritin was made by Reissmann and Dietrich in 1956 by precipitating the protein with a rabbit antibody against human liver ferritin, s They did not find ferritin to be present in normal subjects but found detectable amounts in patients with acute liver disease and concluded that this was a result of hepato-cellular damage. These findings were later confirmed by Wohlers and Schonlau 6 who also found ferritinaemia in patients with haemochromatosis, infectious mononucleosis, pernicious anaemia and acute myeloblastic leukaemia. Aungst v measured the concentra- tion of ferritin in serum immunologically by a double gel diffusion technique and found it to be present in a variety of benign and malignant diseases, the highest concentrations being found in patients with hepatic necrosis and severe haemolytic anaemias, though high concentrations were also found in patients with lymphatic malignancy.

The development of a sensitive immunoradiometric assay for ferritin by Addison et al. 8 made it possible to measure circulating ferritin concentration in normal subjects and experience soon indicated that serum ferritin concentration was normally related to the iron stores of the body. 9:~ Normal subjects had a concentration of about 15-300 /lg 1 -l (males) and 15-200 #g 1-1 (females). Patients with iron deficiency had lower levels than normal and patients with iron overload had greatly elevated serum ferritin concentrations. There are a number of recent reviews of serum ferritin which may be consulted. 3'n'12

150 A . J a c o b s

It has been suggested that malignant tissue itself could be a source of the increased amounts of ferritin found in the plasma in many patients with tumours and that ferritin arising in malignant cells may be abnormal in type. Evidence on this point remains conflicting and there is certainly no clear indication at the present time that ferritin can be considered as a specific tumour marker. More probably there has been insufficient appreciation of the variation in isoferritin patterns between tissues and within the same tissue under different conditions.

The detection of ferritin in the spleens of patients with Hodgkin's disease by Bieber and Bieber 13 led to their assumption that this protein was a tumour associated antigen. Furthermore, increased splenic ferriti'n synthesis in Hodgkin's disease led to the suggestion that their high serum ferritin concentrations were derived from the tumour tissue. 14 Sarcione e t al. is

showed that peripheral blood mononuclear cells from patients with Hodgkin's disease synthesised ferritin at a higher rate than normal mononuclear cells. None of these studies suggested that the ferritin protein produced by the tumour cells was in any way abnormal and the authors' surprising conclusions linking the presence of ferritin with the malignant process appeared to arise more from their failure to realise the ubiquity of ferritin in all tissues than from any evidence of an abnormality associated with Hodgkin's disease. Leukaemic cells in patients with acute myeloblastic leukaemia contain higher levels of ferritin than normal leucocytes 16 and both ferritin and total protein synthesis is increased in these cells when compared to normal leucocytes) 7 In this case it was considered possible that the very high levels of circulating ferritin were related both to the increased synthesis, the considerable increase in leucocyte mass, and a release of ferritin from these cells on their death. Biochemical and immunological characterisation of ferritin from leukaemic cells showed that these had similar properties to normal tissue ferritin) 8'19

A number of workers have shown changes in the isoelectric focusing pattern of ferritin isolated from hepatoma tissue compared to normal liver ferritin. Alpert e t al. :~ showed that the more acidic hepatoma ferritin isolated in two cases resembled some of the isoferritin bands found in foetal liver on isoelectric focusing and suggested that a unique hepatoma isoferritin existed in which the turnout not only synthesises some of the components of normal ferritin but also a specific 'carcinofoetal' ferritin subunit. Massover 21 characterised ferritin derived from mouse hepatoma tissue and found that although it differed in charge from the homologous liver protein all the ferritins were composed of the same two types of subunit and differences seemed to relate to different proportions of the two subunits. Similar acidic isoferritin bands have been found in human breast

cancer, 22 colon cancer 23 and neuroblastoma 24 as well as renal and pancreatic carcinomas. 2s

FERRITIN ASSAYS

The first sensitive immunoradiometric assays for serum ferritin were based on antibodies against purified human liver or spleen ferritins. 8'26 These contain mainly more basic isoferritins with a high proportion of L subunits. The earlier work on serum ferritin concentration in patients with malignant disease was carried out using such assays. More recently it has been possible to obtain specific antibodies to acidic isoferritins with a high proportion of H subunits by using an antigen derived either from heart tissuel HeLa cells or placenta. 27-29 Hazard and Drysdale 27 pointed out that assays for liver or spleen type ferritins would have only a low reactivity for the more acidic 'carcinofoetal' isoferritins which they suggested were more typical of cancer tissues. They therefore tested sera from a variety of cancer patients using antibodies derived from liver ferritin and HeLa type ferritin and found that in breast cancer, pancreatic cancer, stomach cancer and a few other isolated cases a greater concentration of ferritin was detectable with the HeLa assay than with the liver assay. They suggested that this high HeLa/liver ratio found in cancer patients was quite different from the situation found in non- malignant conditions, though it was interesting that this pattern was not found in a small number of cases of acute leukaemia and multiple myeloma. As some cancer cells appeared to produce specific isoferritins. Hazard and Drysdale 27 suggested the use of serum ferritin measurements in cancer diagnosis. Selective assay of these isoferritins would clearly be feasible, as the different ferritin populations could be distinguished immunologically, apparently on the basis of their subunit composition. Subsequent studies 2a'29 using antibodies to HeLa cell ferritin and to heart ferritin found no evidence that patients with cancer had a high concentration of circulating acidic isoferritins. Niitsu e t al. 3~ also found high levels of acidic ferritin inpatients with iron deficiency but not in all patients with cancer, and Mori e t al. al

using antibodies against heart, HeLa cell and placental ferritin found it difficult to derive a parameter for the biochemical or immunological diagnosis of cancer.

PHYSIOLOGY OF SERUM FERRITIN

The abundant data on serum ferritin concentrations in a variety of malignant states need to be considered in the light of our present knowledge of the physiology as well as the heterogeneity of the circulating protein. The normal presence of ferritin in the plasma suggests that it may be a secreted protein. Indirect evidence suggests that it originates from the cells of the reticulo- endothelial system and the relationship between body

iron stores and serum ferritin supports this. Mack e t al. 3z

have shown that the perfused rat liver will release ferritin and maintain the concentration of ferritin in the perfusate at a level appropriate to liver iron stores. In patients with liver damage direct release of cellular ferritin into the circulation may occu r , 33 resulting in grossly increased concentrations. Increased concentrations are also found in patients with inflammatory disease, either due to increased synthesis or secondary to an increase in iron stores resulting from the associated anaemia. Experi- mental work has shown how both acute inflammation 34 and microinfarcts 3s can increase ferritin production.

The major part of normal circulating ferritin binds concanavalin A and it is this glycosylation of the protein which accounts for the acidic bands seen on isoelectric focusing. Where ferritin is released into the circulation as a result of tissue damage this produces an increase in the fraction which does not bind to concanavalin A. 36 Serum ferritin normally reacts with an antibody to liver or spleen ferritin and is rich in L subunits. Under normal circumstances none of the circulating ferritin reacts with an antibody to the acidic isoferritin of heart or HeLa cells, suggesting that very little H subunit type ferritin is found in the plasma, z8'29 Ferritin turnover in plasma has recently been demonstrated in two experiments. In the first, purified human serum ferritin from patients with idiopathic haemochromatosis was labelled with 131 I and injected into two normal volunteers. Clearance was found to be relatively slow, with 50% of the 131I ferritin remaining in the plasma at 27-30 h. When Con A binding ferritin was measured separately this was found to clear only slowly, with a T% of approximately 50 h while the non-Con A binding component had a T~/;_ of approximately 5 h. a7 In two additional experiments when purified spleen ferritin was labelled with ~31I this was cleared from the plasma rapidly with a 7hA of approximately 9 min and surface counting showed that this accumulated rapidly in the liver. 38 Rats' hepatocytes appear to have ferritin receptors on their surface membranes a9 and this may provide a specific mechanism for the clearance of plasma ferritin. Covell 4~ has shown that in the rabbit ferritin is cleared to RE cells in the liver.

SERUM FERRITIN IN MALIGNANT DISEASE

Many patients with solid turnouts have an elevated serum ferritin concentration when this is measured by the normal assay techniques using an anti-spleen ferritin antibody. However, in the majority of patients serum ferritin concentration is in the normal range. High levels are found particularly in pancreatic cancer, lung cancer and hepatoma. 3~

Patients with untreated acute leukaemia generally have elevated serum ferritin concentrations. 16'41-4a There

F e r r i t i n a n d m a l i g n a n c y 1 51

are particularly high concentrations in myelomonocytic leukaemia. High values have also been reported in children with acute lymphoblastic leukaemia (ALL). 42'4r Some- what lower concentrations are found in patients with chronic leukaemias, a6,41,4a Parry e t al. 42 examined the effects of therapy on the serum ferritin concentration in leukaemic patients. In children with ALL serum ferritin concentrations were raised at presentation and usually rose even further during chemotherapy but in children in complete remission and no longer receiving chemotherapy, serum ferritin concentrations fell to within the normal range. Slimes e t al . , a4 however, found that serum ferritin concentrations in childhood ALL fell, even while the patients were receiving chemotherapy. Attempts to use serum ferritin concentration in ALL patients as a means of predicting relapse have not been successful. 4s,~s In acute myeloblastic leukaemia serum ferritin concentrations remained high even when patients were in complete remission. 42

Lang e t a t 4~ measured serum ferritin concentration during the course of chronic myeloid leukaemia and concluded that it was a helpful parameter in the follow- up of such patients. It is difficult, however, to find the basis for this conclusion as the data simply indicates that, in those cases where plasma radio iron is largely deviated to non-erythroid tissues, there is an associated high concentration of serum ferritin. This phenomenon would be expected in any case where there is decreased erythropoiesis and it is unlikely that serum ferritin concentration would give a more useful index of the progress of the disease than direct haematological investigation. In patients with siderobtastic anaemia there is no difference in the serum ferritin concentration between those with no associated haematological abnormality and those with a pre-leukaemic state. 4s

Raised levels of circulating ferritin have been known in Hodgkin's disease for many years, s-7 Ferritin concentrations measured by immunoradiometric assay were found to be related to the stage of the disease, increasing from stage 1 to stage 4, but were not related to histological type. 4a'49 Patients in complete remission have normal serum ferritin concentrations 43 (also Jacobs, unpublished data). In non-Hodgkin's lymphoma, Matzner e t al. 4a found a correlation between ferritin and turnout histology with the highest values in active histiocytic lymphoma and the lowest in lymphocytic lymphoma. Cases with mixed histology had intermediate values.

Marcus and Zinberg 22 showed that ferritin isolated from breast and pancreatic tumour tissue contained acidic isoferritins not found in adult liver and later reported increased serum ferritin concentrations in patients with this disease, s~ Jacobs e t aL sl measured serum ferritin concentration in 250 normal adult women and compared these with 229 serum samples from

152 A . J a c o b s

women presenting with early breast cancer. The results showed that concentrations above 200 /.tg 1-1 were found in 10% of breast cancer subjects and that these patients had a significantly higher tumour recurrence rate during the subsequent 4 years. However, while a few patients with early breast cancer undoubtedly have serum ferritin concentrations somewhat higher than normal this could result from the non-specific effect of malignancy on reticuloendothelial iron metabolism and the degree of elevation of serum ferritin is certainly not comparable to the very high levels found in patients with acute leukaemia. Coombes e t aL 52 examined sera from 42 patients with active breast cancer using ten biochemical parameters, all of which had been individually advocated as turnout markers. All patients with overt metastatic breast cancer had abnormalities of at least three of these markers including ferritin in 88%, carcino-embryonic antigen (CEA) in 81% and C reactive protein in 81%. However, the ferritin assay proved to have little value in the prediction of the development of metastatic disease, s3 Bezwoda e t al. s4 estimated serum concentrfftions of CEA, ferritin and calcitonin in 107 patients with breast cancer and found CEA and ferritin values to be statistically higher in those with metastases. Ferritin was the leaast sensitive of these two parameters though metastases were present in all 9 patients having a concentration higher than 400 pg I -~ . Such metastases were invariably in the liver and the increased circulating concentration of ferritin may well have been due to liver damage.

Niitsu e t aL 3 found increased concentrations of serum ferritin in patients with pancreatic cancer a~ but Nitti e t al. ss do not consider that this can be used as a marker for pancreatic malignancy. In cancer of the uterine cervix, Ito et aL s6 have shown that serum ferritin concentration is related to the stage of the disease and the turnout volume; they suggest that if the ferritinaemia is caused by ferritin synthesis in the tumour cells the relationship between tumour and serum concentration must be similar to that between choriocarcinoma and human chorionic gonadotrophin (HCG) and they suggest that periodic measurement of the serum ferritin levels may be useful in monitoring the clinical response to therapy. Grail e t al. s7 have in fact compared serum ferritin concentrations with alphafetoprotein (AFP) and the/3 subunit of HCG as markers of the disease process in 12 patients with germ cell tumours. High concentrations of serum ferritin were found in l 0 patients, high AFP in 10 and high HCG in 6. A poor prognosis was associated with a persistently raised serum ferritin and either, or both, an elevated AFP or/3 HCG. Decreasing concentra- tions of serum ferritin indicated a favourable response to treatment, while rising values were associated with a recurrence or spread of the tumour. These authors felt that while serum ferritin could not be considered a

specific tumour marker it might still be helpful in following clinical progress of disease.

Hann e t al. 24 found that in children with neuro- blastoma increased serum concentrations of ferritin were related to the presence of active disease. Longitudinal studies showed that serum ferritin concentrations varied with the activity of the disease, with a return to normal levels during clinical remission. In a more detailed study of biological differences between neuroblastoma with visceral metastases, which normally has a high probability of spontaneous regression, and tumour with bone metastases, which is usually fatal, serum ferritin concentrations were found to be high in 15 out of 17 children with the potentially fatal form of the disease but not in children with the more favourable variant. High serum ferritin was associated with inhibition of lymphocyte E rosette formation, s8

In primary liver cell cancer Kew e t aL s9 found increased serum ferritin concentrations in 58 out of 76 patients. There was a negative correlation between serum ferritin and AFP in these patients. Chapman e t aL, 6~ however, who also found high serum ferritin concentrations in 19 out of 30 patients with primary liver cell cancer, also found AFP levels to be raised in 21 of these patients. There was no relationship between AFP concentration and serum ferritin concentration and it was felt that in view of the very high specificity of AFP for primary liver cell cancer and the non- specificity of an increased serum ferritin concentration AFP was the superior screening test for this tumour. However, in those patients who were negative for AFP, serum ferritin might have a role in monitoring therapy. Patients with cirrhosis who had normal AFP and ferritin concentrations, were unlikely to have primary liver cell cancer. Melia e t aL 61 a!so conclude that serum ferritin has no role in the differential diagnosis of liver cell cancer.

Raised serum ferritin concentrations in patients with malignant disease appear to be due to a number of causes. The anaemia found in such patients is poorly understood and the reduction in red cell mass is accompanied by a transfer of iron to the storage compartment, with a consequent increase in ferritin synthesis in the reticuloendothelial system. Tumour cells, like all other cells, contain ferritin and necrosis occurring either spontaneously or as a result of therapy gives rise to a direct release of ferritin from damaged cells and possibly very high plasma levels. Similarly, hepatic deposits of tumours result in liver damage and the release of parenchymal ferritin into the circulation. Haemorrhage into tumours will result in excessive haem breakdown by macrophages with a resultant increase in ferritin synthesis and release into the circulation.

ACIDIC ISOFERRITINS

Following the suggestion of Hazard and Drysdale 27 that since many tumours produced acidic isoferritins rich in H subunits and their demonstration that many sera from patients with malignant disease had high circulating concentrations of HeLa type ferritin, a number of further reports have emerged. Jones and Worwood 28 devised an immunoradiometric assay for acidic ferritins based on an antibody against the acidic isoferritin of human heart. Ferritin was undetectable in normal serum using this assay, though it was found in samples from patients with breast cancer and acute leukaemia. It was also present in the sera from patients with idiopathic haemochromatosis, transfusional iron overload and acute myocardial infarction. In all cases where ferritin was detected using an anti-heart inamuno- assay there were very high circulating levels of ferritin using the conventional spleen type assay, usually a 10- to 100-fold excess. It seems likely that most of the ferritin detected with the heart assay in these cases was due to cross-reactivity with the large amounts of circulating spleen type ferritin. Only in patients who had recently had a myocardial infarction was there a somewhat higher proportion of heart reacting ferritin in the circulation, though even in this case it was usually less than one third the concentration measured with the conventional assay. These results directly contradicted those of Hazard and Drysdale and, in a subsequent study using an assay based on antibodies to HeLa cell ferritin, similar results were found in normal subjects and patients with carcinoma of the bronchus, hepatoma, carcinoma of the oesophagus, and carcinoma of the stomach. In this case no HeLa reacting ferritin was found in two patients with idiopathic haemochromatosis and very high spleen type concentrations, though some HeLa type ferritin was detected in patients with myocardial infarction. A study of 443 patients with breast tumours and 100 normal control subjects, showed no evidence of an increase in acidic ferritins disporportionate to the total ferritin concentration in either patients with benign breast tumours, early cancer or advanced cancer. 29 Similarly, in a study of 100 patients with acute lympho- blastic leukaemia there was no significant rise in HeLa type ferritin in the serum and no difference between those patients remaining in remission and those whose disease relapsed. 29 Similar results were found in 112 patients with acute myeloblastic leukaemia at varying stages of the disease. It was concluded that although the serum of many patients may contain isoferritins which are acidic on isoelectric focusing because of their glycosylation, none of these react to antisera raised against ferritins with a high proportion of H subunits. It was felt that there was little application for antibodies to acidic isoferritins from HeLa cells or heart in the diagnosis or monitoring of cancer. Niitsu e t al., 3~ using

Ferritin and malignancy 153

an assay against heart isoferritin confirmed a high concentration in the serum of patients following myocardial infarction but also found increased levels of heart reacting ferritin in serum compared to spleen type ferritin in patients with hepatoma and pancreatic carcinoma. Slightly less clear cut results were obtained by Arosio et al. 62 in patients with colon cancer and lung cancer and they suggested that the simultaneous measurement of both types of ferritin in serum might increase the usefulness of this marker for monitoring lung cancer. More recently Cazzola et a[ 63 have estimated both basic and acidic isoferritins in the sera of patients with Hodgkin's disease using antibodies against liver and HeLa cell ferritin respectively. They found raised levels of serum ferritin with the conventional assay only in patients with systemic disease and these were always associated with low serum iron concentrations. Ferritin concentrations returned to normal when the patients achieved remission. The findings suggested that this phenomenon was associated with the reticuloendothelial abnormality found in patients with many malignant or inflammatory disease processes and was not specific for Hodgkin's disease. Raised concentrations of serum ferritin with the HeLa assay were found in 94% of all untreated patients and this was not related to the presence of systemic symptoms or alterations in iron metabolism. Ferritin measured by this assay did not return to normal until 1 -2 years after complete remission and it was suggested that this ferritin may be derived from abnormal leucocytes or monocytes, possibly including the malignant cells.

CONCLUSIONS

Serum ferritin concentration measured by con- ventional assays is increased in a wide variety of malignant states. In some conditions, such as breast cancer, only a few patients may show minimal increases while in the acute leukaemias very high concentrations may be found. There appeared to be many contributing causes for this including tumour necrosis and haemor- rhage, liver damage and the generalised disorder that occurs not only with cancer but also with chronic inflammatory states. There is no direct evidence for ferritin secretion by tumours. Tumour tissue invariably contains ferritin and although in a few cases the isoferritin profile has been shown to be different from that in the tissue from which the tumour arises, no specific tumour ferritin has been demonstrated. Although some of the tumour ferritins are somewhat acidic and this has encouraged a search for acidic isoferritins in the serum from such cases, no consistent picture has emerged. One of the problems here is the specificity of the assays used. Some cross-reactivity between liver and heart type ferritins may occur and many of the reports give no

154 A. Jacobs

indication as to how much of the ferritin detected with the heart assay may be a result of such cross reactivity. In addition to this, while there is now a considerable degree of standardisation of the conventional ferritin assay 64 no such standardisation has been attempted for assays of acidic isoferritins. There is considerable variation in the assay techniques reported, variable purity of the antigens used and variable specificity and affinity of the antibodies produced. The antibodies have been raised in a number of different species and m onoclonal antibodies have been reported.

Experience in this laboratory 2s'29 has suggested that acidic isoferritins are not detectable in normal serum and when found in patients with cancer this is a result of cross reactivity with the far larger amounts of basic isoferritins which are present. This suggests to us either that malignant tissues in general do not produce acidic isoferritins or that if they do they are not secreted into the plasma in amounts large enough to be detected. Oertel 6s has examined serum ferritin from patients with malignant diseases by anion exchange chromatography and has found no indication that it is in any way different from normal serum ferritin. However, Worwood et al. 6e

have shown that when human ferritins with a high proportion of H subunits are injected into rabbits, these are cleared extremely rapidly compared to more basic ferritins and failure to detect them in serum may simply be a reflection of their extremely fast turnover. There is, of course, the theoretical possibility that tumours may produce a ferritin variant so different in properties from the normal protein that it has so far escaped detection.

At the present time there does not appear to be a good basis for suggesting that the serum ferritin assay, whether for basic or acidic ferritins can be used for the specific diagnosis or monitoring of malignant disease. While serum ferritin concentrations may reflect disease activity, this is no more specific than other parameters such as the erythrocyte sedimentation rate.

REFERENCES

I. Harrison P M, Clegg A A, May K: Ferritin structure and function, in Jacobs A, Worwood M (eds): Iron in Biochemistry and Medicine, vol. II, pp. 131-171. London, Academic Press (1980).

2. Bomford A B, Munro H N: Biosynthesis of ferritin and isoferritins, in Jacobs A, Worwood M (eds): Iron in Biochemistry and Medicine, vol. II, pp. 173-202. London, Academic Press (1980).

3. Worwood M: Ferritin in human tissues and serum. Clin Haemat 11,275 (1982).

4. Arosio P, Adelman T G, Drysdale J W: On ferritin heterogeneity. Further evidence for heteropolymers. J biol Chem 253, 4471 (1978).

5. Reissmann K R, Dietrich M R: On the presence of ferritin in the peripheral blood of patients with hepatocellular disease. J Clin lnvest 35,588 (1956).

6. Wohlers S, Schonlau F: Uber das Vorkommen von Ferritin in Serum. Klin Woch 37,445 (1959).

7. Aungst C W: Ferritin in body fluids. J Lab Clin Med 71,517 (1968).

8. Addison G M, Beamish M R, Hales C N, Hodgkins M, Jacobs A, Llewellin P: An immunoradiometric assay for ferritin in the serum of normal subjects and patients with iron deficiency and iron overload. J Clin Path 25,326 (1972).

9. Waiters G O, Miller S M, Worwood M: Serum ferritin in concentration and iron stores in normal subjects. J Clin Path 26,770 (1973).

t0. Jacobs A, Worwood M: Ferritin in serum. Clinical and biochemical applications. New Engl J Med 292, 951 (1975).

11. Worwood M: Serum ferritin, in CR C Critical R eviews in Clinical Laboratory Sciences, pp. 171-204.

12. Worwood M: Serum ferritin, in Cook J D (ed): Methods in Haematology, vol. 1, pp. 59-89. London, Churchill-Livingstone.

13. Bieber C P, Bieber M M: Detection of ferritin as a circulating tumour-associated antigen in Hodgkin's disease. Nat Cancer Inst Monogr 36, 147 (1973).

14. Sarcione D J, Stutzman L, Mittelman A: Ferritin synthesis by splenic tumour tissue of Hodgkin's disease. Experien tin 31, "1334 ( 1975).

15. Sarcione E J, Smalley J R, Lema M J, Stutzman L: Increased ferritin synthesis and release by Hodgkin's disease peripheral blood lymphocytes. Int J Cancer 20,339 (1977).

16. Worwood M, Summers M, Miller S, Jacobs A, Whittaker J A: Ferritin in blood ceils from normal subjects and patients with leukaemia. Brit J Haemat 28, 27 (1974).

17. White G P, Worwood M, Parry'D H, Jacobs A: Ferritin synthesis in normal and leukaemic lympho- cytes. Nature 240, 584 (1974).

18. Cragg S J, Jacobs A, Parry D H, Wagstaff M, Worwood M: Isoferritins in acute leukaemia. Brit J Cancer 35,635 (1977).

19. Wagstaff M, Worwood M, Jacobs A: Biochemical and immunological characterisation of ferritin from leukaemic ~ells. Brit J Haemat 45,263 (1980).

20. Alpert E, Coston, R L, Drysdale J W: Carcinofoetal human liver ferritins. Nature 242, 194 (1980).

21. Massover W H: Mouse hepatoma and liver ferritins. Comparative structural studies. Biochim biophys Acta 579,169 (1979).

22. Marcus D M, Zinberg N: Isolation of ferritin from human mammary and pancreatic carcinomas by means of antibody immunoabsorbence. Arch Biochem Bioph),s 162,493 (1974).

23. Alpert E, Quaroni A, Goldenberg D M Alterations in triptic peptide atoms of ferritins purified from human colon carcinoma. Biochim biophys A eta 581, 193 (1979).

24. Hann H-W L, Levey H M, Evans A E: Serum ferritin as a guide to therapy in neuroblastoma. Cancer Res 40, 1411 (1980).

25. Halliday J W, McKeering L V, Powell L W: Isoferritin composition of tissues and serum in human cancers. CancerRes 36, 4486 (1976).

26. Miles L E M, Lipschitz D A, Bieber C P, Cook J D: Measurement of serum ferritin by a 2-site immuno- radiometric assay. A naly t Biochem 61, 209 (1974).

27. Hazard J T, Drysdale J W: Ferri t inaemia in cancer. Nature 265,755 (1977).

28. Jones B M, Worwood M: An immunoradiometr ic assay for the acidic ferritin of human heart: applica- tion to human tissues~ cells and serum. Clin Chim Acta 85, 81 (1978).

29. Jones B M, Worwood M, Jacobs A: Serum ferritin in patients with cancer: determination with antibodies to HeLa ceils and spleen ferritin. Clin Chim A cta 166,203 (1980).

30. Niitsu Y, Goto Y, Kohgo Y, Adachi C, Onodera Y, Urushizaki I: Evaluation of heart isoferritin assay for diagnosis of cancer, in Albertini A (ed): Radio- immunoassay of'Hormones, Proteins and Enzymes, pp. 256 -266 . Amsterdam, Excerpta Medica.

31. Mori W, Asakawa H, Taguchi T, Sakai R: Human isoferritin and malignant neoplasms. A cta Haemat Jap 41, 1309 (1978).

32. Mack U, Cooksley W G E, Ferris R A, Powell L W, Halliday J W: Regulation of plasma ferritin in isolated perfused rat liver. Brit J Haemat 47, 403 (1981).

33. Prieto J, Barry M, Sherlock S: Serum ferritin in patients with iron overload and acute and chronic liver diseases. Gastroenterology 68,525 (1975).

34. Konijn A M, Hershko C: Ferrit in synthesis in inflammation. 1. Pathogenesis of impaired iron release. Brit J Haemat 37, 7 (1977).

35. Gregg P J, Eastham E J, Bell J I, Walder D N: Serum ferritin and dysbaric osteonecrosis. Undersea Biomed Res 4, 75 (1977).

36. Worwood M, Cragg S J, Wagstaff M, Jacobs A: Binding of human serum ferritin to concanavalin A. Clin Sei 56, 83 (1979).

37. Worwood M, Cragg S J, Williams A M, Wagstaff M, Jacobs A: The clearance of '3t l -human plasma ferritin in man. Blood 60, 8.27 (1982).

38. Cragg S J, Covell A M, Burch A, Owen G M, Jacobs A, Worwood M: Turnover of 13'I-human spleen ferritin in plasma. Brit J Haemat (in press).

39. Mack U, Powell L W, Halliday J W: A receptor for ferritin on hepatocytes. J biol Chem 258, 4672 (1983).

40. Covell A M: Turnover of ferritin in plasma. PhD thesis. University of Wales (1983).

41. Jones P A E, Miller S M, Worwood M, Jacobs A: Ferri t inaemia in leukaemia and Hodgkin's disease. Bri tJCancer 27 ,212 (1973).

42. Parry D H, Worwood M, Jacobs A: Serum ferritin in acute leukaemia at presentation and during remissions. Brit Med J 1 ,245 (1975).

43. Matzner Y, Konijn A M, Hershko C: Serum ferritin in haematological malignancies. A m J Hemat 9, 13 (1980).

44. Slimes M A, Wang W C, Dalman P R: Elevated serum ferritin in children with malignancies. Scand JHaemat 19, 153 (1977).

45. Parry D H, Ricketts, Jacobs A: Serum ferritin

Ferritin and malignancy 155

during unmaintained remission in acute lympho- blastic leukaemia. Brit Med J 2, 1341 (1978).

46. Koller M-E, Romslo P H, Finne P H, Hanneberg B: Serial determinations of serum ferritin in children with acute lymphoblast ic leukaemia. A cta Paediat Scand 68, 93 (1979).

47. Lang J M, Abet M, Methlin G, Oberling F: Serum ferritin during the course of chronic myeloid leukaemia. Increase of serum ferritin as a marker of dyserythropoiesis. A cta Haemat 67, 145 (1982).

48. May A, De Souza P, Barnes K, Kaaba S, Jacobs A: Erythroblast iron metabolism in sideroblastic marrows. Brit JHaem 52, 611 (1982).

49. Jacobs A, Slater A, Whit taker J A, Cannelos G, Wiernik P H: Serum ferritin concentrat ion in untreated Hodgkin's disease. Brit J Cancer 34, 162 (1976).

50. Marcus D M, Zinberg N: Measurement of serum ferritin by radioimmunoassay: results in normal individuals and patients with breast cancer. J Nat CaneerInst 55,791 (1975).

51. Jacobs A, Jones B, Ricketts C, Bulbrook R D, Wang D Y: Serum ferritin concentrat ion in early breast cancer. Brit J Cancer 34, 286 (1976).

52. Coombes R C, Powles T J, Gazer J C, Ford H G, Slone J P, Laurence D J R, Neville A M: Biochemical markers in human breast cancer. Lancet 1, 132 (1977).

53. Coombes R C, Powles T J, Gazer J C, Ford H T, McKinna A, Gehrke C W, Keyser J W, Mitchell P E G, Patel S, Stimson W H, Worwood M, Jones M, Neville A M: Screening for metastases in breast cancer: an assessment of biological and physical methods. Cancer 48 ,310 (1981).

54. Bezwoda W, Derman D, Bothwell T, MacPhail P, Levin J, DeMoor N: Significance of serum con- centrations of carcino embryonic antigen, ferritin and calcitonen in breast cancer. Cancer 48, 1623 (1981).

55. Nitti D, Fabris C, Del Favero G, Farini R, Crassi F, Farini A, Baccaglini U, Pedrazzoli S, Piccoli A, Lise M, Naccarato R: Serum ferritin in pancreatic disease. An accurate test of malignancy? Digestion 25, 258 (1982).

56. l to H, Takagi Y, Ando Y, Kubo A, Hashimoto S, Tsutsui F, Kurihara S: Serum ferritin levels in patients with cervical cancer. Obst Gyn 55, 358 (1980).

57. Grail A, Bates G, Milford Ward A, Jones W G, Hancock B W: Serum ferritin as a third marker in germ cell tumours. Eur J Cancer Clin Oncol 18, 261 (1982).

58. Harm H-W L, Evans A E, Cowin I J, Leitmeyer J E: Biological differences between neuroblastoma stages IV-S and IV: measurement of serum ferritin and E rosette inhibition in 30 children. N e w Engl J Med 305,425 (1981).

59. Kew M C, Torrance J D, Derman D, Simon M, Macnab G M, Charlton R W, Bothwell T H: Serum and tumour ferritins in pr imary liver cancer. Gut 19, 294 (1978).

60. Chapman R W, Bassendine M S, Laulicht M, Gorman

156 A. Jacobs

A, Thomas H C, Sherlock S, Hoffbrand A V: Serum ferritin and binding of serum ferritin to concanavalin A as a tumour marker in patients with primary liver cell cancer and chronic liver disease. Digest Dis Sci 27, 111 (1982).

61. Melia, W M, Bullock S, Johnson P J, Williams R: Serum ferritin in hepatocellular carcinoma: a comparison with alpha foetoprotein. Cancer 51, 2112 (1982).

62. Arosio P, Iacobello C, Montesoro E, Albert ini A: Serum ferritin evaluation with radioimmunoassays specific for HeLa and liver ferritin types, lmmun Lett 3 ,309 (1981).

63. Cazzola M, Arosio P, Gobbi P G, Barosi G, Ber- gamaschi G, Dezza L, IacobeUo C, Ascari E: Basic

and acidic isoferritins in the serum of patients with Hodgkin's disease. Eur J Cancer Clin Oncol 19, 339 (1983).

64. International Commit tee for Standardisation in Haematology. Preparation, characterisation and storage of human ferritin for use as a standard for the assay of serum ferritin (in preparation).

65. Oertel J: An ion-exchange chromatography of serum ferritin in patients with malignant diseases. TumourDiagnost 2, 99 (1981).

66. Worwood M, CoveU A M, Cragg S J, Jacobs A: Turnover of ferritin in plasma. Proc Sixth lnt Con.[" Proteins o f Iron Storage and Transport. Sapporo (1983).