103
Cancer Letters, 67 (1992) 103 - 112 Elsevier Scientific Publishers Ireland Ltd.
Cytosol and serum ferritin in breast carcinoma G. Giiner, Department
G. Kirkali (Bilgin), C. Yenisey, of Biochemistry,
(Received 28 January 1992) (Revision received 24 August (Accepted 25 August 1992)
Faculty
of Medicine,
Dokuz Eyliil University,
35340 Inciralti, Izmir (Turkey)
1992)
Keywords: ferritin; cytosol; tumour marker
Summary
This study was undertaken to determine tissue and serum ferritin levels in different stages of breast carcinoma. Eighty-nine cases have been evaluated, the groups inuestigated being breast carcinoma, benign breast disease and healthy controls. Ferritin leuels in both the sera and the tissue cytosols were measured by an enzyme immunoassay method, while totat proteins were assayed by Lowry’s procedure and the ferritin concentrations given in ng ferritin/mg cytosol protein. No significant difference has been determined for serum ferritin between any of the groups studied, while the tissue cytosol ferritins were found to be 91.6 f 50.9, 565.0 * 48.3, 142.7 f 93.3, 683.3 f 212.9 and 655.5 f 100.4 ng/mg cytosol protein for the benign, malign (globaD, malign (stage 11, malign (stage ZlJ and malign (stage 111)groups, respectively. The differences between the malign groups and the benign group were found to be highly significant (P < 0.001) except for the stage 1 subgroup, which was fairly significant (P < 0.05). A sensitivity of 90% was eualuated for tissue cytosol ferritin
in breast carcinoma, the ‘in&a-patient’ sensitiuity being 100%. In conclusion, we state that tissue ferritin is more ualuable than serum ferritin as a tumour marker of diagnosis for breast carcinoma. Correspondence to: Giil Giiner, Department of Biochemistry, Faculty of Medicine, Dokuz Eyliil University, 353Kl lnciralti Izmir, Turkey.
0304-3835/92/$05.00 Printed and Published
i.R. T&e
0 1992 Elsevier Scientific Publishers in Ireland
breast
carcinoma;
Introduction
Several biological and clinical parameters have been employed in the investigation of breast cancer biology, evaluation of prognosis and in the choice of therapeutic strategy [2,7,9,10,12,14,18,21,26,31]. The use of serum ferritin as a biochemical tumour marker in carcinoma of the breast has its limitations because of: (1) low incidence of serum elevations in early stages of disease [16]; (2) high rate of overlapping values in patients with carcinoma of the breast, benign cases and normal controls [6,16,29]; (3) inutility as a prognostic indicator in low stage of malignancy [ 161. Tissue tumour markers are among the least studied biological parameters, in other types of cancer as well as in breast carcinoma. Recently, there has been a growing interest in the determination of tumour markers in tissue in different carcinoma types [10,12,13,19,22, 31,321 and most studies on tumor markers in breast cancer tissue have employed immunohistochemical methods [3,4,24]. This work was undertaken to determine tissue ferritin levels, using the quantitative enzymo-immunoassay procedure, in patients with various stages of breast carcinoma and to compare with serum values, as well as with Ireland Ltd
104
analogue values in a benign tumour group and in healthy controls. The rationale behind this study was that ferritin concentrations of tissues of different stages of tumour growth would contribute to the elucidation of the biological features of the tumour, as well as to tumour diagnosis and prognosis. In addition, the alterations in ferritin observed in breast carcinoma might be reflected earlier and more directly by tissue ferritin than by serum ferritin. Materials
and Methods
Patients and turnout-s Inclusion in this investigation required the following conditions: (1) untreated primary breast carcinoma, or, benign breast disease; (2) no anemia (Hb L 12 g/d]); (3) no evidence of liver disease; (4) no iron overload or hemotransfusion in the last 6 months, which, in some way, might interfere with ferritin levels. The breast carcinoma group consisted of 30 women, with lesions histopathologically diagnosed as ‘carcinoma of the breast’, of whom 6 had stage I, 13, stage II and 11, stage III pathology, according to the TNM classification system [34]. The benign tumour group consisted of 27 patients with lesions diagnosed as ‘non-malign’ [ 111. Eleven fibroadenoma, 13 fibrocystic disease, 1 intraductal papilloma, 1 non-specific hyperplasia and 1 lipoma case were included in the benign group. Thirty-two-control subjects, all of whom were women, were also included in the study in order to obtain a normal range of values for serum ferritin under laboratory conditions. This control group consisted of the nursing, medical and laboratory staff, diagnosed to be ‘healthy’ from routine laboratory check-up. They ranged in age from 30 to 65 years (mean 48 years) and also conformed to the above mentioned ‘conditions of inclusion’ to our study. Tissue samples were divided into three groups: (1) carcinoma tissue and (2) normal
breast tissue not adjacent to the carcinoma, both obtained from the carcinoma patients as well as (3) benign tissue samples. All samples of breast tissue (0.3 - 1.0 g) were obtained from mastectomy specimens, immediately chilled on ice, dissected away from fat and necrotic tissue and stored at - 70°C. A portion of each sample was kept for histopathological investigation.
Cy tosol preparation Cytosols were prepared using the procedure described by GION et al. [13]. To begin with, defrozen tissue samples were resuspended with 2 volumes of phosphate buffer solution (0.1 M; pH 8.4) and then homogenized at 4OC for 10 min. Cytosols were prepared by centrifugation of the homogenates at 50 000 x g for 1 h at 4OC and subsequent collection of the supernatants.
Protein assay The total protein in the cytosol was measured by Lowry’s method [23] using bovine albumin as standard.
Collection of sera Blood samples for ferritin assays were taken preoperatively between 08:OO and 09:OO h on the morning of the mastectomy, before any anaesthetic drugs had been given. Blood samples were allowed to clot at 4OC and then sera were obtained by centrifugation at 2000 x g for 10 min and kept at - 70°C until analysis.
Ferritin assay Ferritin levels in both sera and the tissue cytosols were measured by an enzyme immunoassay method (BioMerieux) and presented as ng/ml for sera and ng/mg cytosol protein for the tissue cytosols. Standards were prepared to cover the range 0 - 800 ng/ml. Samples with concentrations exceeding 800 ng/ml were diluted and reassayed. The lower limit of detection at the 99% confidence level was 2 ng/ml. The enzyme immunoassay is based on a sandwich
105
technique using two monoclonal antibodies. Briefly, it is performed in two steps: (1) In the immunological step, samples are incubated 30 min at 18- 25*C with the horseradish peroxidase-labelled monoclonal anti-ferritin, in monoclonal anti-ferritin coated tubes. Ferritin present in samples binds, on one hand, to the monoclonal anti-ferritin on the tubes and, on the other hand, to the labelled monoclonal anti-ferritin. (2) In the enzymatic step, the complex obtained is incubated 30 min at 18- 25*C with the chromogen substrate (orthophenylenediamine - O.P.D./H,O,) .The color obtained is measured at 492 nm and the concentrations determined from the calibration curve obtained under identical conditions. Samples were analyzed in batches. As a means of quality control, some serum and cytosol samples were analyzed repetitively in a single batch as well as being reassayed in every batch. The intra- and interassay coefficients of variation of the quality control sera were 2.5 - 4.5% and 3.2 - 4.8%) respectively, while those of cytosols were 2.9 -4.8% and 4.2 - 5.1% ,respectively. To ensure that there were no matrix effects in the case of analysis of tissue cytosols the following experiments were carried out: to a given cytosol, with previously determined ferritin concentration, divided into six portions, the same volume of increasing concentrations of ferritin standard (50, 100, 150, 200, 250, 300 ng/ml) were added and the ferritin assayed. The obtained results represented the summation of the ferritin levels for easy assay, which would not be expected in case of the presence of matrix effects, due to an expected variable interference of the matrix. The cytosol ferritin levels were expressed as ng ferritin/mg cytosol protein using the total protein data of the cytosols, obtained by the Lowry procedure. Statistical analysis Ferritin levels between groups were analyzed using Student’s t-test [20]. As is indicated in the Results section, the t-test for paired variables was employed whenever necessary.
Histological eualuation The pathological classification of tumours was done according to the criteria of the World Health Organization [34]. Furthermore, a portion of the sample used for biochemical analysis was stored in liquid nitrogen for reevaluation. Results Eighty nine cases have been evaluated in this study, of which 30 were breast carcinoma, 27 showed benign breast disease and 32 were normal controls. The distribution of serum ferritin in the benign and the malign tumour group (stage I, II and III), as well as in the healthy controls, is skewed and is shown in Fig. 1. It is clear from the figure that the values are overlapping and that cut-off values are not employable. Table I shows the tissue cytosol and the serum ferritin levels (means f S.D.) for all the groups. For serum ferritin, no significant difference has been determined between any of the groups studied. On the other hand, tissue ferritins displayed significant differences, at the level of P < 0.0001 between the malign group evaluated globally and the benign group, as well as between the malign subgroups stage II and stage III and the benign group. As the normal breast tissue samples have been obtained in parallel with the malign samples, that is, for each malign case studied, one normal and one malign tissue sample have been obtained, the normal tissue ferritins underwent separate statistical evaluation for stage II and stage III, using the t-test for paired variables, the results of which are represented in Table II. Significant differences, at the level of P < 0.001 have been determined between the malign tumour ferritin and its normal counterpart, for both stage II and stage III (Table II). The distribution of tissue cytosol ferritins for single cases are schematized in Fig. 2. As is seen on the Figure, there is an excellent separation between the normal and the malign tissue ferritin, with no overlap. There was no
106
S;erum Ferritin
(ng/ml)
19cI-
. rea I_
17cI_
.
.
160 I_
. 8
1X I_
. . 146
I_
. 130I_
. 120
110I_
100
90
60
A i
. .
.
v : .:
. .
0 .. 8
.
oh
.
. .
70
.:.
t .
.
..
60
.
.
.
50
.
8. .
... .
40
8
30
.
. 20
t
10
0 NWlll6l
I
Benign
Fis. 1. Serum ferritin concentrations 1, Stage II and Stage III).
I
stage
I
I
stage
II
I
stage
Ill
in controls and patients with benign breast disease and breast carcinoma
(Stage
107
Tabla 1. Serum and tissue cytosol ferritin concentrations in breast carcinoma, benign breast disease, and normal controls (data are given as means l SD.). Group
N
Serum ferritin (ng/mII
Normal Benign Malign Stage I Stage II Stage 111
32 27 30 6 13 11
89.5 f 19.1 86.6 * 38.6” 86.6 zt 48.Sb
Tissue ferritin (ng/mg cytosol protein) 91.6 565.0 142.7 683.3 655.5
A 50.9 zt 48.3d ziz 93.3’ zt 212.gd z+z100.4’
“P > 0.05 compared with the normal group. bP > 0.05 compared with the normal and the benign group. “P < 0.05 compared with the benign group. dP < 0.001 compared with the normal group, and stage I subgroup. ‘P > 0.05 compared with the stage II subgroup.
tumour case which had lower ferritIn level in tumour than in normal tissue, which corresponds to a sensitivity of 100% whether we use, as a cut-off value: (1) the highest value found in normal tissue samples (120.0 ng/mg cytosol protein), or, (2) mean plus two standard deviations (84.3 + 51.0 = 135.1). This sensitivity level is regarded as ‘intra-patient’ sensitivity. We also evaluated the ‘inner-patients’ sensitivity of ferritin as a tumour marker. To this end, the distribution of tissue cytosol ferritin levels in all the groups and subgroups, including the benign group and stage I subgroup, is presented in Fig. 3. Using the cut-off value of 120.00 ng/mg cytosol protein, 7 of the total 27 benign cases fall above the line, while,
Table Il. * SD.).
using the cut-off value of 135.1 ng/mg cytosol protein, only 3 are observed above the line. For the Stage I subgroup, using any of the two cut-off points, 3 of the cases fall below the line (Fig. 3). The interpatient sensitivity calculated for tissue cytosol ferritin is 90%. Dimcussion Ferritin(s) are a ‘family’ of isomeric proteins - isoferritins - that have an important function in iron storage, metabolism and detoxification [33]. Ferritin is found almost anywhere in the body; its concentration is high in liver, spleen and hematopoetic tissue [30]. The protein moiety of ferritin has a molecular weight of approximately 450 000 Da [4] and the entire
Malign tissue cytosol ferritin levels compared with the normal counterparts
(data are given as means
Group
N
Malign tissue
Normal tissue
Malign (Globally) Stage II Stage III
24
691.5 + 158.3’
84.3 i 20.5
13 11
712.8 zt 196.6’ 666.0 l 99.3’
77.3 f 22.1 92.6 zt 15.6
‘Very significant increase (P
c
0.0001) compared with the normal tissue ferritin.
108
N
C -
1260
-
1200
-
1140
-
1080
-
1020
-
960
-
900
-
640
-
780
-
720
-
660
-
600
-
540
-
460
135 -
-
420
120 -
-
360
105-
-
300
90 _
-
240
60 -
-
180
45 -
-
120
-
60
m
cytosol
protein
p < 0.0001
Fig. 2.
Tissue ferritin: distribution
of single cases.
Dots, normal tissue cytosol (N); stars, breast cancer cytosol (C)
109
ng Ferritin / mg cytosol protein
1300-
l
125012001150-, llOO1050-, lOOO9.50-, 900-, 850-
l
800-,
l .
750-m
a ..
700650600-
.
550soo-,
.
8
450-
.
400-, 350-, 300-,
..
250-, 200-
.
150-, loo-.
0
4
T
50-,
’
________ ___---__ -________
=a=
________-
?f’ .
:r .
Normal
I I
Benign
. I I
I
stage
I
,
I
stage
II
’
stage
III
Control
Fis. 3.
Tissue cytosol ferritin concentrations (Stage I, Stage II and Stage III).
in controls and patients with benign breast disease and breast carcinoma
110
molecule is composed of the protein shell, consisting of multiple subunits, which enclosed a variable amount of iron. Ferritin was originally detected in sera of patients with severe liver disease in 1956 [24] and then in Hodgkin’s disease in 1968 [3]. In 1974, Marcus and Zinberg [25] observed a marked increase in ferritin content of several mammary carcinomas and suggested that measurement of serum ferritin levels might be useful in the clinical evaluation and management of patients with these tumours. In the years to follow, up to the present, serum ferritin levels have been intensely investigated in mammary carcinomas [ 1,2,9, 16,25,26,29,31] with the idea that the estimation may be of empirical value in cancer immunodiagnosis. Serum ferritin has been claimed to be a non-specific marker for breast cancer, due to the fact that it may also be found in high concentrations in the cases of phlogistic and hepatic diseases, or when necrotic foci are present [13,28]. The present study was designed to measure both tissue and serum ferritin levels in breast carcinoma, with the understanding that in primary breast cancer, the source of the ferritin level in blood is the tumour mass and that the tissue level might reflect more directly the ferritin status of the tumour, due to the fact that serum ferritin level is also influenced by clearance by the metabolism of the patient. Cytosol was used mainly because it is a well standardized, reproductible method yielding a preparation without any cellular debris which could interfere with the immunochemical technique and also because ferritin has been demonstrated in cytosplasm of breast cancer by immunohistochemical methods [25]. In our work, we did not observe significant differences in the serum ferritin values between the cases of breast carcinoma and those with benign tumours, as well as the normal controls. This finding is not in keeping with the data of JACOBS et al. [16] who have reported higher ferritin concentrations in early breast cancer than in normal women. This may be due to the difference in the methods used. However, the fact that the actual number of
patients in each group in our study is relatively small should also be noted. The ferritin molecule is composed of a protein shall, consisting of multiple subunits, which encloses a variable amount of iron. Differences in the subunit composition of isoferritins from various tissues are responsible for the molecule’s electrophoretic mobility [33]. It is also known that considerable variation existed between isoferritins of tumors of different histological origin, thus causing immunological dissimilarities. This may, in part, help explain the above-mentioned discrepancies in serum ferritin in these two studies. It is interesting to note that Marcus and Zinberg [25] have reported higher serum ferritin levels in preoperative sera of only 41% of women with mammary carcinoma. On the other hand, Bezwoda et al. [2] have found serum concentrations of ferritin > 400 ng/ml in breast cancer patients invariably with hepatic metastases. This is why we chose to investigate directly the breast cancer tissue; to eliminate the interferences from other sources than breast tissue. If fact, serum ferritin levels may be influenced by a number of factors, including total body iron stores, release of ferritin from inflamed tissues and production of ferritin by proliferating tumour cells. The use of breast cancer tissue for ferritin assays discards two of the above mentioned sources. Ferritin has been demonstrated on the surface of T-cells in Hodgkin’s disease and breast cancer and has been found to suppress in vitro lymphocyte function. The presence of high concentrations of ferritin in breast carcinoma tissue has been suggested to raise the possibility of local and/or systemic immunoregulation [321. Our data on tissue cytosol ferritin emphasize the fact that breast cancer tissue shows very significantly high ferritin levels compared with benign or normal tissue. The sensitivity of tissue ferritin, calculated to be 90%, was higher than the reported by Gion et al [12], their values being between 51.0% and 82.3%) depending on the choice of the cut-off value. In addition, we evaluated the intra-patient
111
sensitivity for tissue ferritin, that is, the ability of ferritin to distinguish the tumour from the normal tissue of the same breast. Our intra-patient sensitivity value determined to be 100% for tissue ferritin is a little higher than that of Gion et al. [X2].
8 9
10
Conclusions
Serum ferritin values can not be evaluated as a reliable indicator in the breast carcinoma cases. A remarkable (about 7-fold) increase is observed in the ferritin concentration of malignant breast tissue, compared to benign lesions of the breast. Tissue ferritin, even in the stage I malignancy was determined to be significantly higher than that of benign lesions. Follow-up of patients, using serum ferritin levels, seemed to be necessary in order to elucidate the role of this tumour marker as a prognostic indicator. This study has brought up clear evidence that tissue cytosol ferritin is definitely more useful than serum ferritin as a tumour marker of diagnosis.
11
12
13
14
References 15 Aungst, C.W. (1968) Fenitin in body fluids, J. Lab. Cjin. Med., 71, 517-522. Bezwoda, W., Derman, D., Bothwell, T., Macphil, P., Levin, J. and De Moor, N. (1981) Significance of serum concentrations of carcinoembryonic antigen, ferritin and calcitonin in breast cancer. Cancer, 48, 1623 - 1628. Bjorklund, V. and Bjorklund, B. (1979) Localization of synthesis of TPA in normal and malignant human tissue by immuno-histological techniques. In: Protides the Biological Fluids, pp. 223-32. Editor: H. Peters. Pergamon Press, Oxford. Bonetti, F., Maconi, A., Pisa, R., Menestrina, F., Novelli, P., Iannucci, A., Chilisi, M. and Rasniewskij, K. (1981) L’identificazione istologica dell’ antigene carcinoembrionario nei tumori mammari. Pathologica, 23, 639 - 47. Cazzola, M., Arosio, P., Belloti, V., Bergamaschi, G., Dezza, L. and lacobeilo, C. (1985) Immunological reactivity of serum ferritin in patients with malignancy. Tumori, 71,547-554. Coombes, R.C., Powles, T., Gazet, J.C., Ford, H.T., Nash, A.G. and Sloane, J.P. (1977) A biochemical approach to the staging of breast cancer. Cancer, 40, 937 - 944. Cooper, E.H., Forbes, M.A., Hancock, A.K., Price, J.J. and Parker, D. (1989) An evaluation of mucin-like car-
16
17
18
19
20 21
22
cinema associated antigen (MCA) in breast cancer. Br. J. Cancer, 59, 797 - 800. Crichton, R.R. (1971) Structure and function of ferritin. Angew Chem. (Engl.), 12, 57-65. Van Dalen, A. (1990) Pre-operative tumour marker levels in patients with breast cancer and their prognosis. Tumour Biol., 11 (4), 189-95. Duffy, M.J., O’Connell, M., O’Sullivan, F., McKenna, B., Allen, M.A. and McDonnell, L. (1983) CEA - like material in cytosol from human breast carcinomas. Cancer, 51, 121- 123. Ellerhorst-Ryan, J.M., Turba, E.P., Stahl, D.L. (1988) Evaluating benign breast disease. Nurse Practitioner, 13(g), 13-20. Gion, M., Mione, R., Dittadi, R., Griggio, L., Munegato, G., Valsecchi, M., Del Maschio, 0. and Bruscagnin, G. (1986) Carcinoembryonic antigen, ferritin, tissue polypeptide antigen, and CA 15-3 in breast cancer: relationship between carcinoma and normal breast tissue. Int. J. Biol. Markers, 1, 33 - 38. Gion, M., Mione, R., Dittadi, R., Griggio, L., Munegato, G., Valsecchi, M., Del Maschio, O., Fasan, S. and Bruscagnin, G. (1985) Estrogen and progesterone receptors in breast carcinoma and in nonmalignant breast tissue. Tumori, 71, 477 - 81. Gozdz, S.S., Kowalska, M.M., Slusniak, J.T., Slusniak, A., Korejba, W., Bukowski, J., Bansinska, E., al Jazzaf, H. and Szymendera, J.J. (1989) Pretreatment concentrations of breast carcinoma antigen (CA 15.3) and mucin-like carcinoma-associated antigens in patients with carcinoma of the breast. Tumour Bioj, 10(2), 103- 108. Hazard, J.T. and Drysdale, J.W. (1977) Ferritinaemia in cancer. Nature, 265, 755. Jacobs, A., Jones, B., Ricketts, C., Bulbrook, R.D. and Wang, D.Y. (1976) Serum ferritin concentration in earIy breast cancer. Br. J. Cancer, 34, 286-290. Jacobs, A. and Worwood, M. (1975) Ferritin in serum: Clinical and Biochemical Implications. N. Engl. J. Med., 292, 951- 956. Jensen, J.L., MacLean, G.D., Suresh, M.R., Almeida, A., Jette, D., Lloyd, S., Bodnar, D., Krantz, M. and Longenecker, B.M. Possible utility of serum determinations of CA 125 and CA 27.29 in breast cancer management. (1991) Mt. J. Biol. Markers, 6 (l), l-6. Kew, M.C., Torrance, J.D. Derman, D., Simon, M., Macnab, G.M. and Charlton, R.W. (1978) Serum and tumor ferritins in primary liver cancer. Gut, 19, 294 - 299. Koopmans, L.H. (1987) Introduction to Contemporary Statistical Methods. Duxbury Press, Boston. Kuhajda, F.P., Offutt, L.E. and Mendelshon, G. (1983) The distribution of carcinoembryonic antigen in breast carcinoma. Cancer, 52, 1257 - 64. Loganath, A., Peh, K.L., Gunasegaram R., Thiagaraj D., Cheah E., Kottegoda S.R. and Ratnam S.S. (1988) Comparison of AFP and beta-h CG levels in infiltrating duct mammary carcinoma at different stages of malignancy. Pathology, 20, 275- 278.
112
23
24
25
26
27
28
Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem., 193, 265- 275. Mansour, E.G., Heartert, M., Park, C.H., Koeheler, K.A. and Petrelli, M. (1983) Tissue and plasma carcinoembryonit antigen in early breast cancer. Cancer, 51, 1243 - 1248. Marcus, D.M. and Zindberg, N. (1974) Isolation of ferritin from human mammary and pancreatic carcinomas by means of antibody immunoadsorbents. Arch. Biochem. Biophys., 162, 493- 501. Miserez, A.R., Gunes, I., Muller-Brand, J., Walther, E., Frtdrich, R. and Macke, H. (1991) Clinical value of a mucin-like carcinoma-associated antigen in monitoring breast cancer patients in comparison with CA 15 - 13. Eur. J. Cancer, 27(2), 126- 131. Nicolini, A., Carpi, A., Menconi, F., Ianni, A. and Bianchi, R. (1983) Clinical value of seum ferritin, calcitonin and CEA measurements in patients with lung cancer. J. Nucl. Med. All. Sci., 27, 165. Reissman, K.R. and Dietrich, M.R. (1956) On the presence of ferritin in the peripheral blood of patients with hepatocellular disease. J. Clin Invest., 35, 588- 595.
29
30
31
32
33
34
Tappin, J.A., George, W.B. and Bellingham, A.J. (1979) Effect of surgery on serum ferritin concentration in patients with breast cancer. Br. J. Cancer, 40, 658- 660. Volpino, P., Cangemi, V., Caputo, V., Mazzarino, E. and Galati, G. (1984) Clinical usefulness of serum ferritin measurements in lung cancer patients. J. Nucl. Med. All. Sci., 28(l), 28-30. Waharen, B., Lidbrink, E., Wallgren, A., Eneroth, P. and Zajicek, J. (1978) Carcino-embryonic antigen and other tumor markers in tissue and serum or plasma of patients with primary breast carcinoma. Cancer, 42, 1870- 1878. Weinstein, R.E., Bond, B.H. Silberberg, B.K., Vaughn, C.B., Subbaiah, P. and Pieper, D.R. (1989) Tissue ferritin concentration and prognosis in carcinoma of the breast. Breast Cancer Res. Treat., 14, 349-53. Williams, M.R., Turkes, A., Pearson, D., Griffiths, K. and Blarney, R.W. (1990) Serum ferritin as a marker of therapeutic response in stage III and IV breast cancer. Eur. J. Surg. Oncol., 16, 22-27. The World Health Organization. The WHO histological typing of breast tumours, Edn. 2. (1982) Am. J. Clin. Pathol., 78, 806 - 816.
Cancer Letters, 67 (1992) 103 - 112 Elsevier Scientific Publishers Ireland Ltd.
Cytosol and serum ferritin in breast carcinoma G. Giiner, Department
G. Kirkali (Bilgin), C. Yenisey, of Biochemistry,
(Received 28 January 1992) (Revision received 24 August (Accepted 25 August 1992)
Faculty
of Medicine,
Dokuz Eyliil University,
35340 Inciralti, Izmir (Turkey)
1992)
Keywords: ferritin; cytosol; tumour marker
Summary
This study was undertaken to determine tissue and serum ferritin levels in different stages of breast carcinoma. Eighty-nine cases have been evaluated, the groups inuestigated being breast carcinoma, benign breast disease and healthy controls. Ferritin leuels in both the sera and the tissue cytosols were measured by an enzyme immunoassay method, while totat proteins were assayed by Lowry’s procedure and the ferritin concentrations given in ng ferritin/mg cytosol protein. No significant difference has been determined for serum ferritin between any of the groups studied, while the tissue cytosol ferritins were found to be 91.6 f 50.9, 565.0 * 48.3, 142.7 f 93.3, 683.3 f 212.9 and 655.5 f 100.4 ng/mg cytosol protein for the benign, malign (globaD, malign (stage 11, malign (stage ZlJ and malign (stage 111)groups, respectively. The differences between the malign groups and the benign group were found to be highly significant (P < 0.001) except for the stage 1 subgroup, which was fairly significant (P < 0.05). A sensitivity of 90% was eualuated for tissue cytosol ferritin
in breast carcinoma, the ‘in&a-patient’ sensitiuity being 100%. In conclusion, we state that tissue ferritin is more ualuable than serum ferritin as a tumour marker of diagnosis for breast carcinoma. Correspondence to: Giil Giiner, Department of Biochemistry, Faculty of Medicine, Dokuz Eyliil University, 353Kl lnciralti Izmir, Turkey.
0304-3835/92/$05.00 Printed and Published
i.R. T&e
0 1992 Elsevier Scientific Publishers in Ireland
breast
carcinoma;
Introduction
Several biological and clinical parameters have been employed in the investigation of breast cancer biology, evaluation of prognosis and in the choice of therapeutic strategy [2,7,9,10,12,14,18,21,26,31]. The use of serum ferritin as a biochemical tumour marker in carcinoma of the breast has its limitations because of: (1) low incidence of serum elevations in early stages of disease [16]; (2) high rate of overlapping values in patients with carcinoma of the breast, benign cases and normal controls [6,16,29]; (3) inutility as a prognostic indicator in low stage of malignancy [ 161. Tissue tumour markers are among the least studied biological parameters, in other types of cancer as well as in breast carcinoma. Recently, there has been a growing interest in the determination of tumour markers in tissue in different carcinoma types [10,12,13,19,22, 31,321 and most studies on tumor markers in breast cancer tissue have employed immunohistochemical methods [3,4,24]. This work was undertaken to determine tissue ferritin levels, using the quantitative enzymo-immunoassay procedure, in patients with various stages of breast carcinoma and to compare with serum values, as well as with Ireland Ltd
104
analogue values in a benign tumour group and in healthy controls. The rationale behind this study was that ferritin concentrations of tissues of different stages of tumour growth would contribute to the elucidation of the biological features of the tumour, as well as to tumour diagnosis and prognosis. In addition, the alterations in ferritin observed in breast carcinoma might be reflected earlier and more directly by tissue ferritin than by serum ferritin. Materials
and Methods
Patients and turnout-s Inclusion in this investigation required the following conditions: (1) untreated primary breast carcinoma, or, benign breast disease; (2) no anemia (Hb L 12 g/d]); (3) no evidence of liver disease; (4) no iron overload or hemotransfusion in the last 6 months, which, in some way, might interfere with ferritin levels. The breast carcinoma group consisted of 30 women, with lesions histopathologically diagnosed as ‘carcinoma of the breast’, of whom 6 had stage I, 13, stage II and 11, stage III pathology, according to the TNM classification system [34]. The benign tumour group consisted of 27 patients with lesions diagnosed as ‘non-malign’ [ 111. Eleven fibroadenoma, 13 fibrocystic disease, 1 intraductal papilloma, 1 non-specific hyperplasia and 1 lipoma case were included in the benign group. Thirty-two-control subjects, all of whom were women, were also included in the study in order to obtain a normal range of values for serum ferritin under laboratory conditions. This control group consisted of the nursing, medical and laboratory staff, diagnosed to be ‘healthy’ from routine laboratory check-up. They ranged in age from 30 to 65 years (mean 48 years) and also conformed to the above mentioned ‘conditions of inclusion’ to our study. Tissue samples were divided into three groups: (1) carcinoma tissue and (2) normal
breast tissue not adjacent to the carcinoma, both obtained from the carcinoma patients as well as (3) benign tissue samples. All samples of breast tissue (0.3 - 1.0 g) were obtained from mastectomy specimens, immediately chilled on ice, dissected away from fat and necrotic tissue and stored at - 70°C. A portion of each sample was kept for histopathological investigation.
Cy tosol preparation Cytosols were prepared using the procedure described by GION et al. [13]. To begin with, defrozen tissue samples were resuspended with 2 volumes of phosphate buffer solution (0.1 M; pH 8.4) and then homogenized at 4OC for 10 min. Cytosols were prepared by centrifugation of the homogenates at 50 000 x g for 1 h at 4OC and subsequent collection of the supernatants.
Protein assay The total protein in the cytosol was measured by Lowry’s method [23] using bovine albumin as standard.
Collection of sera Blood samples for ferritin assays were taken preoperatively between 08:OO and 09:OO h on the morning of the mastectomy, before any anaesthetic drugs had been given. Blood samples were allowed to clot at 4OC and then sera were obtained by centrifugation at 2000 x g for 10 min and kept at - 70°C until analysis.
Ferritin assay Ferritin levels in both sera and the tissue cytosols were measured by an enzyme immunoassay method (BioMerieux) and presented as ng/ml for sera and ng/mg cytosol protein for the tissue cytosols. Standards were prepared to cover the range 0 - 800 ng/ml. Samples with concentrations exceeding 800 ng/ml were diluted and reassayed. The lower limit of detection at the 99% confidence level was 2 ng/ml. The enzyme immunoassay is based on a sandwich
105
technique using two monoclonal antibodies. Briefly, it is performed in two steps: (1) In the immunological step, samples are incubated 30 min at 18- 25*C with the horseradish peroxidase-labelled monoclonal anti-ferritin, in monoclonal anti-ferritin coated tubes. Ferritin present in samples binds, on one hand, to the monoclonal anti-ferritin on the tubes and, on the other hand, to the labelled monoclonal anti-ferritin. (2) In the enzymatic step, the complex obtained is incubated 30 min at 18- 25*C with the chromogen substrate (orthophenylenediamine - O.P.D./H,O,) .The color obtained is measured at 492 nm and the concentrations determined from the calibration curve obtained under identical conditions. Samples were analyzed in batches. As a means of quality control, some serum and cytosol samples were analyzed repetitively in a single batch as well as being reassayed in every batch. The intra- and interassay coefficients of variation of the quality control sera were 2.5 - 4.5% and 3.2 - 4.8%) respectively, while those of cytosols were 2.9 -4.8% and 4.2 - 5.1% ,respectively. To ensure that there were no matrix effects in the case of analysis of tissue cytosols the following experiments were carried out: to a given cytosol, with previously determined ferritin concentration, divided into six portions, the same volume of increasing concentrations of ferritin standard (50, 100, 150, 200, 250, 300 ng/ml) were added and the ferritin assayed. The obtained results represented the summation of the ferritin levels for easy assay, which would not be expected in case of the presence of matrix effects, due to an expected variable interference of the matrix. The cytosol ferritin levels were expressed as ng ferritin/mg cytosol protein using the total protein data of the cytosols, obtained by the Lowry procedure. Statistical analysis Ferritin levels between groups were analyzed using Student’s t-test [20]. As is indicated in the Results section, the t-test for paired variables was employed whenever necessary.
Histological eualuation The pathological classification of tumours was done according to the criteria of the World Health Organization [34]. Furthermore, a portion of the sample used for biochemical analysis was stored in liquid nitrogen for reevaluation. Results Eighty nine cases have been evaluated in this study, of which 30 were breast carcinoma, 27 showed benign breast disease and 32 were normal controls. The distribution of serum ferritin in the benign and the malign tumour group (stage I, II and III), as well as in the healthy controls, is skewed and is shown in Fig. 1. It is clear from the figure that the values are overlapping and that cut-off values are not employable. Table I shows the tissue cytosol and the serum ferritin levels (means f S.D.) for all the groups. For serum ferritin, no significant difference has been determined between any of the groups studied. On the other hand, tissue ferritins displayed significant differences, at the level of P < 0.0001 between the malign group evaluated globally and the benign group, as well as between the malign subgroups stage II and stage III and the benign group. As the normal breast tissue samples have been obtained in parallel with the malign samples, that is, for each malign case studied, one normal and one malign tissue sample have been obtained, the normal tissue ferritins underwent separate statistical evaluation for stage II and stage III, using the t-test for paired variables, the results of which are represented in Table II. Significant differences, at the level of P < 0.001 have been determined between the malign tumour ferritin and its normal counterpart, for both stage II and stage III (Table II). The distribution of tissue cytosol ferritins for single cases are schematized in Fig. 2. As is seen on the Figure, there is an excellent separation between the normal and the malign tissue ferritin, with no overlap. There was no
106
S;erum Ferritin
(ng/ml)
19cI-
. rea I_
17cI_
.
.
160 I_
. 8
1X I_
. . 146
I_
. 130I_
. 120
110I_
100
90
60
A i
. .
.
v : .:
. .
0 .. 8
.
oh
.
. .
70
.:.
t .
.
..
60
.
.
.
50
.
8. .
... .
40
8
30
.
. 20
t
10
0 NWlll6l
I
Benign
Fis. 1. Serum ferritin concentrations 1, Stage II and Stage III).
I
stage
I
I
stage
II
I
stage
Ill
in controls and patients with benign breast disease and breast carcinoma
(Stage
107
Tabla 1. Serum and tissue cytosol ferritin concentrations in breast carcinoma, benign breast disease, and normal controls (data are given as means l SD.). Group
N
Serum ferritin (ng/mII
Normal Benign Malign Stage I Stage II Stage 111
32 27 30 6 13 11
89.5 f 19.1 86.6 * 38.6” 86.6 zt 48.Sb
Tissue ferritin (ng/mg cytosol protein) 91.6 565.0 142.7 683.3 655.5
A 50.9 zt 48.3d ziz 93.3’ zt 212.gd z+z100.4’
“P > 0.05 compared with the normal group. bP > 0.05 compared with the normal and the benign group. “P < 0.05 compared with the benign group. dP < 0.001 compared with the normal group, and stage I subgroup. ‘P > 0.05 compared with the stage II subgroup.
tumour case which had lower ferritIn level in tumour than in normal tissue, which corresponds to a sensitivity of 100% whether we use, as a cut-off value: (1) the highest value found in normal tissue samples (120.0 ng/mg cytosol protein), or, (2) mean plus two standard deviations (84.3 + 51.0 = 135.1). This sensitivity level is regarded as ‘intra-patient’ sensitivity. We also evaluated the ‘inner-patients’ sensitivity of ferritin as a tumour marker. To this end, the distribution of tissue cytosol ferritin levels in all the groups and subgroups, including the benign group and stage I subgroup, is presented in Fig. 3. Using the cut-off value of 120.00 ng/mg cytosol protein, 7 of the total 27 benign cases fall above the line, while,
Table Il. * SD.).
using the cut-off value of 135.1 ng/mg cytosol protein, only 3 are observed above the line. For the Stage I subgroup, using any of the two cut-off points, 3 of the cases fall below the line (Fig. 3). The interpatient sensitivity calculated for tissue cytosol ferritin is 90%. Dimcussion Ferritin(s) are a ‘family’ of isomeric proteins - isoferritins - that have an important function in iron storage, metabolism and detoxification [33]. Ferritin is found almost anywhere in the body; its concentration is high in liver, spleen and hematopoetic tissue [30]. The protein moiety of ferritin has a molecular weight of approximately 450 000 Da [4] and the entire
Malign tissue cytosol ferritin levels compared with the normal counterparts
(data are given as means
Group
N
Malign tissue
Normal tissue
Malign (Globally) Stage II Stage III
24
691.5 + 158.3’
84.3 i 20.5
13 11
712.8 zt 196.6’ 666.0 l 99.3’
77.3 f 22.1 92.6 zt 15.6
‘Very significant increase (P
c
0.0001) compared with the normal tissue ferritin.
108
N
C -
1260
-
1200
-
1140
-
1080
-
1020
-
960
-
900
-
640
-
780
-
720
-
660
-
600
-
540
-
460
135 -
-
420
120 -
-
360
105-
-
300
90 _
-
240
60 -
-
180
45 -
-
120
-
60
m
cytosol
protein
p < 0.0001
Fig. 2.
Tissue ferritin: distribution
of single cases.
Dots, normal tissue cytosol (N); stars, breast cancer cytosol (C)
109
ng Ferritin / mg cytosol protein
1300-
l
125012001150-, llOO1050-, lOOO9.50-, 900-, 850-
l
800-,
l .
750-m
a ..
700650600-
.
550soo-,
.
8
450-
.
400-, 350-, 300-,
..
250-, 200-
.
150-, loo-.
0
4
T
50-,
’
________ ___---__ -________
=a=
________-
?f’ .
:r .
Normal
I I
Benign
. I I
I
stage
I
,
I
stage
II
’
stage
III
Control
Fis. 3.
Tissue cytosol ferritin concentrations (Stage I, Stage II and Stage III).
in controls and patients with benign breast disease and breast carcinoma
110
molecule is composed of the protein shell, consisting of multiple subunits, which enclosed a variable amount of iron. Ferritin was originally detected in sera of patients with severe liver disease in 1956 [24] and then in Hodgkin’s disease in 1968 [3]. In 1974, Marcus and Zinberg [25] observed a marked increase in ferritin content of several mammary carcinomas and suggested that measurement of serum ferritin levels might be useful in the clinical evaluation and management of patients with these tumours. In the years to follow, up to the present, serum ferritin levels have been intensely investigated in mammary carcinomas [ 1,2,9, 16,25,26,29,31] with the idea that the estimation may be of empirical value in cancer immunodiagnosis. Serum ferritin has been claimed to be a non-specific marker for breast cancer, due to the fact that it may also be found in high concentrations in the cases of phlogistic and hepatic diseases, or when necrotic foci are present [13,28]. The present study was designed to measure both tissue and serum ferritin levels in breast carcinoma, with the understanding that in primary breast cancer, the source of the ferritin level in blood is the tumour mass and that the tissue level might reflect more directly the ferritin status of the tumour, due to the fact that serum ferritin level is also influenced by clearance by the metabolism of the patient. Cytosol was used mainly because it is a well standardized, reproductible method yielding a preparation without any cellular debris which could interfere with the immunochemical technique and also because ferritin has been demonstrated in cytosplasm of breast cancer by immunohistochemical methods [25]. In our work, we did not observe significant differences in the serum ferritin values between the cases of breast carcinoma and those with benign tumours, as well as the normal controls. This finding is not in keeping with the data of JACOBS et al. [16] who have reported higher ferritin concentrations in early breast cancer than in normal women. This may be due to the difference in the methods used. However, the fact that the actual number of
patients in each group in our study is relatively small should also be noted. The ferritin molecule is composed of a protein shall, consisting of multiple subunits, which encloses a variable amount of iron. Differences in the subunit composition of isoferritins from various tissues are responsible for the molecule’s electrophoretic mobility [33]. It is also known that considerable variation existed between isoferritins of tumors of different histological origin, thus causing immunological dissimilarities. This may, in part, help explain the above-mentioned discrepancies in serum ferritin in these two studies. It is interesting to note that Marcus and Zinberg [25] have reported higher serum ferritin levels in preoperative sera of only 41% of women with mammary carcinoma. On the other hand, Bezwoda et al. [2] have found serum concentrations of ferritin > 400 ng/ml in breast cancer patients invariably with hepatic metastases. This is why we chose to investigate directly the breast cancer tissue; to eliminate the interferences from other sources than breast tissue. If fact, serum ferritin levels may be influenced by a number of factors, including total body iron stores, release of ferritin from inflamed tissues and production of ferritin by proliferating tumour cells. The use of breast cancer tissue for ferritin assays discards two of the above mentioned sources. Ferritin has been demonstrated on the surface of T-cells in Hodgkin’s disease and breast cancer and has been found to suppress in vitro lymphocyte function. The presence of high concentrations of ferritin in breast carcinoma tissue has been suggested to raise the possibility of local and/or systemic immunoregulation [321. Our data on tissue cytosol ferritin emphasize the fact that breast cancer tissue shows very significantly high ferritin levels compared with benign or normal tissue. The sensitivity of tissue ferritin, calculated to be 90%, was higher than the reported by Gion et al [12], their values being between 51.0% and 82.3%) depending on the choice of the cut-off value. In addition, we evaluated the intra-patient
111
sensitivity for tissue ferritin, that is, the ability of ferritin to distinguish the tumour from the normal tissue of the same breast. Our intra-patient sensitivity value determined to be 100% for tissue ferritin is a little higher than that of Gion et al. [X2].
8 9
10
Conclusions
Serum ferritin values can not be evaluated as a reliable indicator in the breast carcinoma cases. A remarkable (about 7-fold) increase is observed in the ferritin concentration of malignant breast tissue, compared to benign lesions of the breast. Tissue ferritin, even in the stage I malignancy was determined to be significantly higher than that of benign lesions. Follow-up of patients, using serum ferritin levels, seemed to be necessary in order to elucidate the role of this tumour marker as a prognostic indicator. This study has brought up clear evidence that tissue cytosol ferritin is definitely more useful than serum ferritin as a tumour marker of diagnosis.
11
12
13
14
References 15 Aungst, C.W. (1968) Fenitin in body fluids, J. Lab. Cjin. Med., 71, 517-522. Bezwoda, W., Derman, D., Bothwell, T., Macphil, P., Levin, J. and De Moor, N. (1981) Significance of serum concentrations of carcinoembryonic antigen, ferritin and calcitonin in breast cancer. Cancer, 48, 1623 - 1628. Bjorklund, V. and Bjorklund, B. (1979) Localization of synthesis of TPA in normal and malignant human tissue by immuno-histological techniques. In: Protides the Biological Fluids, pp. 223-32. Editor: H. Peters. Pergamon Press, Oxford. Bonetti, F., Maconi, A., Pisa, R., Menestrina, F., Novelli, P., Iannucci, A., Chilisi, M. and Rasniewskij, K. (1981) L’identificazione istologica dell’ antigene carcinoembrionario nei tumori mammari. Pathologica, 23, 639 - 47. Cazzola, M., Arosio, P., Belloti, V., Bergamaschi, G., Dezza, L. and lacobeilo, C. (1985) Immunological reactivity of serum ferritin in patients with malignancy. Tumori, 71,547-554. Coombes, R.C., Powles, T., Gazet, J.C., Ford, H.T., Nash, A.G. and Sloane, J.P. (1977) A biochemical approach to the staging of breast cancer. Cancer, 40, 937 - 944. Cooper, E.H., Forbes, M.A., Hancock, A.K., Price, J.J. and Parker, D. (1989) An evaluation of mucin-like car-
16
17
18
19
20 21
22
cinema associated antigen (MCA) in breast cancer. Br. J. Cancer, 59, 797 - 800. Crichton, R.R. (1971) Structure and function of ferritin. Angew Chem. (Engl.), 12, 57-65. Van Dalen, A. (1990) Pre-operative tumour marker levels in patients with breast cancer and their prognosis. Tumour Biol., 11 (4), 189-95. Duffy, M.J., O’Connell, M., O’Sullivan, F., McKenna, B., Allen, M.A. and McDonnell, L. (1983) CEA - like material in cytosol from human breast carcinomas. Cancer, 51, 121- 123. Ellerhorst-Ryan, J.M., Turba, E.P., Stahl, D.L. (1988) Evaluating benign breast disease. Nurse Practitioner, 13(g), 13-20. Gion, M., Mione, R., Dittadi, R., Griggio, L., Munegato, G., Valsecchi, M., Del Maschio, 0. and Bruscagnin, G. (1986) Carcinoembryonic antigen, ferritin, tissue polypeptide antigen, and CA 15-3 in breast cancer: relationship between carcinoma and normal breast tissue. Int. J. Biol. Markers, 1, 33 - 38. Gion, M., Mione, R., Dittadi, R., Griggio, L., Munegato, G., Valsecchi, M., Del Maschio, O., Fasan, S. and Bruscagnin, G. (1985) Estrogen and progesterone receptors in breast carcinoma and in nonmalignant breast tissue. Tumori, 71, 477 - 81. Gozdz, S.S., Kowalska, M.M., Slusniak, J.T., Slusniak, A., Korejba, W., Bukowski, J., Bansinska, E., al Jazzaf, H. and Szymendera, J.J. (1989) Pretreatment concentrations of breast carcinoma antigen (CA 15.3) and mucin-like carcinoma-associated antigens in patients with carcinoma of the breast. Tumour Bioj, 10(2), 103- 108. Hazard, J.T. and Drysdale, J.W. (1977) Ferritinaemia in cancer. Nature, 265, 755. Jacobs, A., Jones, B., Ricketts, C., Bulbrook, R.D. and Wang, D.Y. (1976) Serum ferritin concentration in earIy breast cancer. Br. J. Cancer, 34, 286-290. Jacobs, A. and Worwood, M. (1975) Ferritin in serum: Clinical and Biochemical Implications. N. Engl. J. Med., 292, 951- 956. Jensen, J.L., MacLean, G.D., Suresh, M.R., Almeida, A., Jette, D., Lloyd, S., Bodnar, D., Krantz, M. and Longenecker, B.M. Possible utility of serum determinations of CA 125 and CA 27.29 in breast cancer management. (1991) Mt. J. Biol. Markers, 6 (l), l-6. Kew, M.C., Torrance, J.D. Derman, D., Simon, M., Macnab, G.M. and Charlton, R.W. (1978) Serum and tumor ferritins in primary liver cancer. Gut, 19, 294 - 299. Koopmans, L.H. (1987) Introduction to Contemporary Statistical Methods. Duxbury Press, Boston. Kuhajda, F.P., Offutt, L.E. and Mendelshon, G. (1983) The distribution of carcinoembryonic antigen in breast carcinoma. Cancer, 52, 1257 - 64. Loganath, A., Peh, K.L., Gunasegaram R., Thiagaraj D., Cheah E., Kottegoda S.R. and Ratnam S.S. (1988) Comparison of AFP and beta-h CG levels in infiltrating duct mammary carcinoma at different stages of malignancy. Pathology, 20, 275- 278.
112
23
24
25
26
27
28
Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem., 193, 265- 275. Mansour, E.G., Heartert, M., Park, C.H., Koeheler, K.A. and Petrelli, M. (1983) Tissue and plasma carcinoembryonit antigen in early breast cancer. Cancer, 51, 1243 - 1248. Marcus, D.M. and Zindberg, N. (1974) Isolation of ferritin from human mammary and pancreatic carcinomas by means of antibody immunoadsorbents. Arch. Biochem. Biophys., 162, 493- 501. Miserez, A.R., Gunes, I., Muller-Brand, J., Walther, E., Frtdrich, R. and Macke, H. (1991) Clinical value of a mucin-like carcinoma-associated antigen in monitoring breast cancer patients in comparison with CA 15 - 13. Eur. J. Cancer, 27(2), 126- 131. Nicolini, A., Carpi, A., Menconi, F., Ianni, A. and Bianchi, R. (1983) Clinical value of seum ferritin, calcitonin and CEA measurements in patients with lung cancer. J. Nucl. Med. All. Sci., 27, 165. Reissman, K.R. and Dietrich, M.R. (1956) On the presence of ferritin in the peripheral blood of patients with hepatocellular disease. J. Clin Invest., 35, 588- 595.
29
30
31
32
33
34
Tappin, J.A., George, W.B. and Bellingham, A.J. (1979) Effect of surgery on serum ferritin concentration in patients with breast cancer. Br. J. Cancer, 40, 658- 660. Volpino, P., Cangemi, V., Caputo, V., Mazzarino, E. and Galati, G. (1984) Clinical usefulness of serum ferritin measurements in lung cancer patients. J. Nucl. Med. All. Sci., 28(l), 28-30. Waharen, B., Lidbrink, E., Wallgren, A., Eneroth, P. and Zajicek, J. (1978) Carcino-embryonic antigen and other tumor markers in tissue and serum or plasma of patients with primary breast carcinoma. Cancer, 42, 1870- 1878. Weinstein, R.E., Bond, B.H. Silberberg, B.K., Vaughn, C.B., Subbaiah, P. and Pieper, D.R. (1989) Tissue ferritin concentration and prognosis in carcinoma of the breast. Breast Cancer Res. Treat., 14, 349-53. Williams, M.R., Turkes, A., Pearson, D., Griffiths, K. and Blarney, R.W. (1990) Serum ferritin as a marker of therapeutic response in stage III and IV breast cancer. Eur. J. Surg. Oncol., 16, 22-27. The World Health Organization. The WHO histological typing of breast tumours, Edn. 2. (1982) Am. J. Clin. Pathol., 78, 806 - 816.
