Oncology 34 : 267-272 ( 1977)
Carcinoembryonic Antigen, ai-Fetoprotein, Ferritin and 012-Pregnancy Associated Glycoprotein in the Serum of Lung Cancer Patients and its Demonstration in Lung Tumor Tissues C. G ropp, F. G. Lehmann, H. W. Bauer, and K. Havemann Medizinische UniversitatskUnik Marburg
Key Words. CEA - Ferritin - rxi-F'etoprotein - SP3 - Lung cancer
Introduction In recent years several proteins, often associated with fetal growth, have been recognised in the serum and other body fluids of patients with a variety of different tumors. G old and Freedman [1] described the carcinoembryonic antigen (CEA) which was first thought to be specific for colonic cancer and A belew [2] and Tatarinow [3] classified the alpha 1fetoproteins as a carcinofetal-antigen associated with hepa toma. There are other proteins which levels were sometimes found to be elevated in cancer patients, for example the fer ritin [4-6] and the pregnancy-associated 012-glycoprotein (alpha-2-PAG) [7-9], The immunological detection of these tumor-associated antigens had drawn particular attention to their possible role in diagnosis and treatment of neoplasms and it is hoped that their presence might serve as an indicator for tumor formation and might contribute to earlier and more precise diagnosis. In patients with lung cancer, the tumor is often so far extended at the time of diagnosis that the efficiency of surgery or radio therapy is limited. Furthermore the result of treatment is dif ficult to evaluate in this malignancy. We therefore intended to define the usefulness of the determination of tumor-associated antigens like CEA, ou-fetoprotein, ferritin and cd-PAG for the detection of lung cancer and especially of metastases. It was also the purpose of this report to study the role of serial anti gen determinations for the therapeutic monitoring of lung cancer patients. We furthermore tried to determine these antigens, detectable in the sera of lung cancer patients, in the tissue extracts of a variety of lung tumors.
Materials and Methods CEA and AFP CEA and AFP were measured by radioimmunoassay. CEA was tested by the commercial radioimmunoassay of Ire Sorin. For AFP the radioimmunoassay of the Abbot Company was used. The CEA assay was considered positive if the CEA con centration was 10 ng/ml or more. All lower values were con sidered negative. AFP levels of more than 1000 ng are con sidered to be highly suspicious for cancer. Ferritin Serum ferritin levels were determined by the one-dimensional Laurell electrophoresis, using a rabbit antiserum (Behringwerke Marburg) to human placenta ferritin. A placenta fer ritin extract containing 2 mg/ml ferritin served as a standard. Ferritin levels are expressed in ug/ml. Alpha-2-PAC Alpha-2-PAG levels were measured by a modified Laurell technique. The experimental details are described elsewhere [9], The values of Alpha-2-PAG are expressed in mg/100 ml. Preparation o f tumor tissue For the detection of the described tumor associated antigens and proteins in the tumor tissue, tumor extracts were pre pared according to Yachi et al. [10]. The tumor tissue was minced and suspended in buffer. A single cell suspension was prepared using a Potter-Elvehjem. The preparation was fil tered through gauze and the cell-suspension centrifuged for 10 minutes at 300 x g. Cells were then resuspended in 10 mM Tris-buffer and homogenised using a Potter-Elvehjem again. After centrifugation for 10 minutes at 400 x g an equal volume of 3 M KC1 was given to the supernatant. After ultracentri fugation for 1 hour at 100000 x g the supernatant was made 50% saturated in (NEL) 2 SO4 by adding an equal volume of the saturated solution. The resulting precipitation was re moved by centrifugation for 1 hour at 30000 x g. The super natant was dialysed against running water for 24 hours. For the detection of the specific antigens and proteins, extracts from these preparations were then tested with antisera against CEA, AFP, ferritin and Alpha-2-PAG in the Ouchterlony and Laurell technique. Tumor tissue preparations were also tested for their content of CEA by radioimmunoassay.
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Summary. Levels of carcinoembryonic antigen (CEA), alpha-feto protein (AFP), ferritin and co-pregnancy associated glycoprotein (Alpha-2-PAG) were determined in patients with confirmed lung cancer at the time of diagnosis and in serial determinations during and after radio- or chemotherapy. Whereas AFP levels were not elevated in patients with lung cancer, increased levels of CEA, fer ritin and Alpha-2-PAG were found in more than 50% of the pa tients. The results suggest that determination of CEA, ferritin and Alpha-2-PAG in the serum of patients with lung cancer may be useful to detect metastases or recurrences and to monitor the results of treatment. Furthermore, in this study CEA and ferritin could be demonstrated in extracts of lung tumor tissues by specific antisera.
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Figure 1. Carcinoembryonic antigen (CEA) levels in lung cancer patients with and without métastasés. The middle column shows CEA levels of patients in which métastasés were confirmed shortly after the elevated antigen levels were determined (n = 90).
Figure 3. Serial carcinoembryonic antigen (CEA) levels in lung cancer patients (n = 18) who received complete or partial remission during radiotherapy. The dotted lines means the time of radiotherapy ( | | = time of recurrence).
Results
has to be assumed that in these patients metastases were al ready present at the time of diagnosis but were too small for clinical demonstration. CEA levels of lung cancer patients in relationship to the histological type of carcinoma and to the extent of disease are shown in figure 2. Nearly all patients with metastatic large cell carcinoma had high CEA levels. The highest CEA levels were found in patients with bone and/ or liver metastases. Serial CEA determinations during and after therapy are shown in figures 3-5.
CEA
CEA was found increased, i.e. values of 10 ng/ml or higher in 47 out of 90 (52%) patients with confirmed lung cancer (figure I). In patients who had no clinically detectable metastases at the time of diagnosis only two had elevated CEA levels of more than 10 ng/ml but below 20 ng/ml. On the contrary, elevated CEA values were found in 37 out of 42 patients with metastases. Only two of these patients had CEA values below 10 ng/ml. In three patients with metastases no CEA could be detected. Seven patients with CEA concentrations of 10 ng/ml or higher had no clinically detectable metastases at the time of diagnosis but metastases could be demonstrated shortly thereafter in all cases. CEA values increased in further determinations. So it
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squamaus
oat cell
lorge cell
Patients with metastoses
Figure 2. Carcinoembryonic antigen (CEA) levels in lung cancer patients in relationship to the histologic type of carcinoma and to the extent of disease (n = 90).
Figure 4. Serial carcinoembryonic antigen (CEA) levels in lung cancer patients (n = 10) with progressive tumor growth during radio therapy. All patients died shortly after the last CEA determination.
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Figure 3 shows serial CEA determinations in all responders to radiotherapy. CEA levels decreased after therapy or re mained undetectable but increased at the time of recurrence and tumor progression. In figure 4 serial CEA determinations of patients who did not respond to radiotherapy are shown. They all had tumor pro gression during therapy and had no further treatment. Figure 5 shows some examples of serial CEA determinations in patients with métastasés treated with chemotherapy. Pa tients who responded to therapy had temporary decreased CEA levels but at the time of recurrence CEA levels in creased. Ferritin Ferritin was detectable by the Laurell technique in 33 out of 50 patients (66 %) with confirmed lung cancer at the time of diagnosis. In 11 out of 28 patients without clinically proven métastasés, ferritin was present in the serum but levels were not higher than 5 ug/ml. The ferritin values were significantly higher in the group with clinically demonstrated métastasés (figure 6). In comparison, out of 30 hospitalised patients without carcinoma 5 had ferritin in the serum. One of these five patients suffered from liver cirrhosis, the others had severe pneumonia. Ferritin, however, was not elevated in normal controls. Serial ferritin determinations (figure 7) showed decreased or unchanged ferritin values when patients respond ed to chemo- or radiotherapy. Non-responders showed a rise of ferritin values or sometimes values remained unchanged (figure 8). Alpha-2-PAG Alpha-2-PAG was positive in 15 out of 25 male patients (60%) with confirmed lung cancer at the time of diagnosis. Levels of Alpha-2-PAG were between 0,5 and 3,7 mg%. Patients with métastasés had higher levels of Alpha-2-PAG
G .K , 58 years, large cell corcinoma
S W , 63years, oat cellcorcinoma
Figure 6. Ferritin levels in lung cancer patients in relationship to the extent of disease (n = 50).
than patients without métastasés but the difference was not significant. In serial determinations before, during and after therapy there seems to exist some correlation between Alpha2-PAG values and the clinical course of patients (figures 9 and 10). In responders to therapy Alpha-2-PAG levels decreased and in patients with tumor progression an increase of Alpha-2PAG levels in the serum could be observed. AFP AFP levels were not elevated in patients with lung cancer but were as high as in normal controls. Also patients with con firmed liver métastasés had no elevated serum AFP values. Tumor tissue preparations Tumor extracts of squamous, oat-cell and large cell car cinomas were tested with antisera against CEA, ferritin, AFP and Alpha-2-PAG in the Ouchterlony and Laurell technique. There were positive results with anti-CEA and anti-ferritin in all tumor extracts investigated. No reaction with an AFP
Months W K , 61 years, squamous carcinoma
R .K., 61 years, lorge cell carcinoma >320 -
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50-
60-
60-
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10-
30-
R
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Months
6
201 ■ 6
8
10
12
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Figure 5. Serial cardnoembryonic antigen (CEA) levels in four pa tients with metastatic lung cancer during chemotherapy. PR = partial remission. R = recurrence.
Figure 7. Serial ferritin levels in lung cancer patients (n = 16) who received complete or partial remission during radiotherapy. The dotted lines mean the time of radiotherapy (]'(= time of recurrence).
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30-
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antiserum and Alpha-2-PAG antiserum could be detected. Antisera against the tumor extracts prepared in rabbits showed a precipitation line with placenta ferritin but there was no reaction to AFP. We had no opportunity to test this tumor antisera against purified CEA as well as to Alpha-2PAG.
Figure 8. Serial ferritin levels in lung cancer patients (n = 15) with progressive tumor growth during radiotherapy. All patients died shortly after the last ferritin determination.
Figure 9. Serial oo-pregnancy-associated glycoprotein (ou-PAG) levels in lung cancer patients during radio- and chemotherapy (n = 11). All patients received partial or complete remission. = pa tients with chemotherapy. Î I = time of recurrence).
Months
Figure 10. Serial ot2-pregnancy-associated glycoprotein (« 2-PAG) levels in lung cancer patients (n = 9) with progressive tumor growth during radio- and chemotherapy. = patients with chemo therapy).
The CEA was first described as an antigen specific for color ectal carcinoma [1, 11, 12]. In recent years many data have been reported indicating elevated CEA levels in patients with non-digestive cancer or in benign digestive disease [13-20], However, it has been confirmed that the determina tion of CEA may be useful in the management of patients with colorectal cancer by aiding the detection of cancer and especially of metastases and in monitoring patients during the post-therapeutic follow-up period [21], In lung cancer Lo G erfo et al. [22] demonstrated CEA in the sera of patients with this malignancy and Pusztaszeri and Mach [18] could isolate CEA from lung cancer tissues. It was Concannon [23] who reported high CEA levels in lung cancer patients with metastases and he concluded that high CEA levels may ex clude patients from surgical therapy. However, the results of CEA determination in lung cancer patients seemed to be dis couraging because of 50% false negative results. Recently the Roche collaborative study [24] did show positive results in 75 % and false negative in 25 % of patients with confirmed lung cancer. In our study 54% of lung cancer patients had elevated CEA levels while in 46% CEA could not be de monstrated. In accordance with Concannon [10] most of the patients with metastases had elevated CEA titers. The highest CEA levels were found in patients with bone and liver me tastases. There were also elevated CEA levels in some patients without detectable metastases at the time of diagnosis. Shortly after, in all these patients, metastases could be de monstrated by other clinical methods. Therefore CEA deter minations may be useful by aiding detection of metastases in lung cancer patients. Because of the high proportion of false negative results it is quite clear that the CEA assay is not useful as a cancer screening test for patients with lung cancer. This is a result also well confirmed in other types of cancer [24], Increasing interest is put into the use of serial CEA deter minations in cancer patients to monitor the effectiveness of various modes of treatment (i.e. surgical, chemical, ir radiation). Especially in gastrointestinal, but also in breast cancer serial CEA determination has become a good adjunct to management [21], Our results may indicate that serial CEA determinations are useful also in patients with lung cancer. Patients who responded to chcmo- or radiotherapy showed a fall in CEA when values were raised before therapy or CEA remained undetectable. An increase of CEA levels in further determinations indicated a progressive tumor spread. On the other hand, patients who did not respond to therapy showed a rise in CEA levels during therapy because of tumor pro gression. There were some cases in which tumors did not pro duce CEA at any time. Therefore CEA determinations should be used only in context with other clinical and laboratory findings.
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Discussion
Gropp et al.: Carcinoembryonic Antigen, ai-Fetoprotein, Ferritin and co-Pregnancy Associated Glycoprotein
References 1
G old . P. and F reedman, S. O.: Demonstration of tumor-speci
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fic antigens in human colonic carcinomata by immunological tolerance and absorption techniques. J. exp. Med. 121: 439462(1965). A belev, G. 1.: Alpha-fetoprotein as a marker of embryospecific differentiation in normal and tumor tissues. Transplantn Rev. 20:3(1974). T atarinov. Y. S.: Content of embryo specific alphaglobulin in the blood of human fetus, new-born and adult man in primary cancer of the liver. Vop. med. Khim. 11:20 (1965). B ieber , C. P. and Bieber , M. M.: Detection of ferritin as a circulating tumor-associated antigen in Hodgkin's disease. Natn. Cancer Inst. Monogr. 36: 147-157 (1973). Jones, P. A.; M iller. F. M„ Worwood , et al.: Ferritinaemia in leukaemia and Hodgkin’s disease. Br. J. Cancer 27:212-217(1973). Marcus, D. M. and Z inberg, N.: Measurement of scrum fer ritin by radioimmunoassay: results in normal individuals and patients with breast cancer. J. natn. Cancer Inst. 55: 791— 795(1975). Bauer , H. W.; G ropp, C.; Sieber , A., und Bohn , H.: Alpha-2schwangerschaftsassoziiertes Glykoprotein: Bestimmungen zur Verlaufskontrolle des Bronchialkarzinoms. Thoraxchirurgie (in press). Bauer , H. W. und H useeld, K. J.: Alpha-2 schwangerschaftsassoziiertes Glykoprotein, ein biochemischer Parameter im Therapievcrlauf des Mammacarcinoms. VIII. Tag. Ges. Immun. Abstract in Z. Immun. Forsch. 152:74-75 (1976). Bohn , H.: Immunochemical determination of human pre gnancy proteins. Arch. Gynaek. 217:219-231 (1974). Yachi, A.; M atsuura, Y.; Carpenter , C. M., and H yde, L.: Immunochemical studies on human lung cancer antigens soluble in 50% saturated ammonium sulfate. J. natn. Cancer Inst. 40:663-682 (1968). K rupey, J.; G old , P., and F reedman , S. O.: Purification and characterization of carcincembryonic antigens of the human digestive system. Nature. Lond. 2/5:67-68 (1967). Krupey, J.; W ilson, T.; F reedman, S. O., and G old , P.: The preparation of purified carcinoembryonic antigen of the human digestive system from large quantities of tumor tissue. Immunochemistry 9 :6 17-622 (1972). Chu, T. M. and NEMOTOT, T.: Evaluation of carcinoembryonic antigen in human mammary carcinoma. J. natn. Cancer Inst. 5 1 :1119-1122(1973). K110 0 , S. K. and Mackay, C. V.: Carcinoembryonic antigenic activity of tissue extracts: A quantitative study of malignant and benign neoplasms, cirrhotic liver, normal adult and fetal organs. Int. J. Cancer II: 681-687 (1973). Lo G er io , P.; Krupey, J„ and H ansen, H. J.: Demonstration of an antigen common to several varieties of neoplasia. New Engl. J. Med. 285:138-141(1971). Lo G erfo , P.; Krupey , J.. and H ansen, H. J.: Demonstration of a common neoplastic antigen (abstract). Clin. Res. 19: 143 (1971). L urie , B. B.; Lowenstein, M. S., and Z amcheck, N.: Elevated carcinoembryonic antigen in benign biliary obstruction. Clin. Res. 22:363 A (1974). P usztaszeri, G. and M ach, J. P.: Carcinoembryonic antigen
19
in non digestive cancerous and normal tissues. Immunochemistry 10:197-204(1973). Steward , A. M.; Nixon, D. W.; Z amcheck, N. et al.: Car
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cinoembryonic antigen in breast cancer patients: Serum levels and disease progress. Cancer 3 3 :1246-1252 (1974). Steward , A. M.; N ixon, D. W.; Z amcheck, N„ and A isen-
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Ferritin is found in different molecular forms (isoferritin) in many tissues [25], In serum, ferritin was detected in patients with liver [26] and Hodgkin’s [4] disease. Elevated serum ferritin levels were also reported in patients with acute leu kemia [5, 27] and in breast cancer [6], Measurement of ferritin levels was described as being useful in monitoring the response to therapy [6], So far there are no reports in patients with lung cancer. Serial measurements of serum ferritin showed a fall or persistence in patients who responded to therapy. In patients with tumor progression during therapy, ferritin values in creased. Our results indicate that ferritin determinations may be useful as an additional method for the detection of metastases and in monitoring therapy of lung cancer patients. Similar results have been described in breast cancer and in Hodgkin’s disease. Pregnancy-associated aa-glycoprotein (Alpha-2-PAG) is an a2-glycoprotein of a molecular weight of 360000 which is present in trace amounts in normal sera and is found strongly elevated during the first three months of pregnancy [28, 29], Elevated levels Alpha-2-PAG also occur in patients with various non-malignant diseases and in patients with malignant disease [9]. The highest levels were found in patients with prostate carcinoma and in cancer patients with bone me tastases. In lung cancer patients there was no statistically significant difference in 012 -PAG levels of patients with and without metastases although levels were higher in extended disease. Serial determinations of serum Alpha-2-PAG showed a cor relation between the Alpha-2-PAG concentration and the clinical course of lung cancer patients. Similar results had been reported for breast cancer patients [7], Although up to now the number of patients investigated is small the results are encouraging to use Alpha-2-PAG determination in further investigations. CEA and ferritin could be demonstrated in extracts of lung tumor tissues of various histology. No AFP and Alpha-2-PAG could be detected in those preparations. CEA was first isolated from lung tumors by PUSZTASZERI and M a c h [18] who could show an immunological identity with the CEA isolated from colonic cancer tissues. Later on CEA was described in other tumors but also in normal tissues. So it was supposed that CEA has to be considered as a glycoprotein common to various epithelial tissues whose concentration could be increased in carcinomas arising from these tissues. In our investigation CEA was only identified in preparations of tumor tissues but not in preparations of normal lung tissue by radioimmunoas say. The origin of the serum ferritin is uncertain. The only direct evidence for increased synthesis of ferritin by a malignant cell was given by W h it e et al. [27]. Recently ferritin has been de tected in mammary carcinomas by immunofluorescence. In our study ferritin was demonstrated in lung cancer but not in normal lung tissue preparations. This may indicate that fer ritin is synthetised by the malignant cells. Alpha-2-PAG could not be detected in our tumor extracts. The origin and the biological function of this protein is still uncertain.
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Gropp et al.: Carcinoembryonic Antigen, ai-Fetoprotein, Ferritin and cu-Pregnancy Associated Glycoprotein A.: Serum carcinoembryonic antigen (CEA) levels in breast cancer patients - Correlation with disease progress and response to chemotherapy. Clin. Res. 21:983 (1973). Z amcheck, N.: The present status of CEA in diagnosis, pro gnosis and evaluation of therapy. Cancer 36: 2460-2468 (1975). Lo G erfo , P.; H erter , F. P.; B raun , J., and H ansen, H. J.: Tumor associated antigen with pulmonary neoplasms. Ann. Surg. / 75:495-500 (1972). K. E.; W eil , C. S., and Copper , J. W.: The carcinoembryonic antigen assay in bronchogenic carcinoma. Cancer 34: 184192(1974). Roche Collaborative study. Data on file. Hoffmann-La Roche & Co. AG, Basel. A ungst, C. W.: Ferritin in body fluids. J. Lab. clin. Med. 71: 517-522(1968). R eissmann, K. R. and D ietrich , M. R.: On the presence of ferritin in the peripheral blood of patients with hepatocellular disease. J. clin. Invest. 35:588-595 (1956). berg ,
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22
24 25 26
27
28
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W hite , G. P.; Worwood, M.; P arry , D. H. et al.: Ferritin synthesis in normal and leukaemic leukocytes. Nature 250: 584-586(1974). Bohn , H.: Quantitative immunologische Bestimmung von Alpha-2 AP-Glykoprotein (SP.i) und vergleichende Unter suchungen über das Stcroid-bindende-Globulin (SP:). Beh ringinst. Mitt. 54:56(1974). Bohn , H.: Charakterisierung der schwangerschaftsassozi ierten Glykoproteine als akute Phase-Proteine. Arch. Gynaek. 213:54 (1972).
Request reprints from: C. G ropp, Medizinische Universitätsklinik, Emil Mannkopffstraße 1, 3550 Marburg/Lahn (FRG)
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Carcinoembryonic Antigen, ai-Fetoprotein, Ferritin and 012-Pregnancy Associated Glycoprotein in the Serum of Lung Cancer Patients and its Demonstration in Lung Tumor Tissues C. G ropp, F. G. Lehmann, H. W. Bauer, and K. Havemann Medizinische UniversitatskUnik Marburg
Key Words. CEA - Ferritin - rxi-F'etoprotein - SP3 - Lung cancer
Introduction In recent years several proteins, often associated with fetal growth, have been recognised in the serum and other body fluids of patients with a variety of different tumors. G old and Freedman [1] described the carcinoembryonic antigen (CEA) which was first thought to be specific for colonic cancer and A belew [2] and Tatarinow [3] classified the alpha 1fetoproteins as a carcinofetal-antigen associated with hepa toma. There are other proteins which levels were sometimes found to be elevated in cancer patients, for example the fer ritin [4-6] and the pregnancy-associated 012-glycoprotein (alpha-2-PAG) [7-9], The immunological detection of these tumor-associated antigens had drawn particular attention to their possible role in diagnosis and treatment of neoplasms and it is hoped that their presence might serve as an indicator for tumor formation and might contribute to earlier and more precise diagnosis. In patients with lung cancer, the tumor is often so far extended at the time of diagnosis that the efficiency of surgery or radio therapy is limited. Furthermore the result of treatment is dif ficult to evaluate in this malignancy. We therefore intended to define the usefulness of the determination of tumor-associated antigens like CEA, ou-fetoprotein, ferritin and cd-PAG for the detection of lung cancer and especially of metastases. It was also the purpose of this report to study the role of serial anti gen determinations for the therapeutic monitoring of lung cancer patients. We furthermore tried to determine these antigens, detectable in the sera of lung cancer patients, in the tissue extracts of a variety of lung tumors.
Materials and Methods CEA and AFP CEA and AFP were measured by radioimmunoassay. CEA was tested by the commercial radioimmunoassay of Ire Sorin. For AFP the radioimmunoassay of the Abbot Company was used. The CEA assay was considered positive if the CEA con centration was 10 ng/ml or more. All lower values were con sidered negative. AFP levels of more than 1000 ng are con sidered to be highly suspicious for cancer. Ferritin Serum ferritin levels were determined by the one-dimensional Laurell electrophoresis, using a rabbit antiserum (Behringwerke Marburg) to human placenta ferritin. A placenta fer ritin extract containing 2 mg/ml ferritin served as a standard. Ferritin levels are expressed in ug/ml. Alpha-2-PAC Alpha-2-PAG levels were measured by a modified Laurell technique. The experimental details are described elsewhere [9], The values of Alpha-2-PAG are expressed in mg/100 ml. Preparation o f tumor tissue For the detection of the described tumor associated antigens and proteins in the tumor tissue, tumor extracts were pre pared according to Yachi et al. [10]. The tumor tissue was minced and suspended in buffer. A single cell suspension was prepared using a Potter-Elvehjem. The preparation was fil tered through gauze and the cell-suspension centrifuged for 10 minutes at 300 x g. Cells were then resuspended in 10 mM Tris-buffer and homogenised using a Potter-Elvehjem again. After centrifugation for 10 minutes at 400 x g an equal volume of 3 M KC1 was given to the supernatant. After ultracentri fugation for 1 hour at 100000 x g the supernatant was made 50% saturated in (NEL) 2 SO4 by adding an equal volume of the saturated solution. The resulting precipitation was re moved by centrifugation for 1 hour at 30000 x g. The super natant was dialysed against running water for 24 hours. For the detection of the specific antigens and proteins, extracts from these preparations were then tested with antisera against CEA, AFP, ferritin and Alpha-2-PAG in the Ouchterlony and Laurell technique. Tumor tissue preparations were also tested for their content of CEA by radioimmunoassay.
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Summary. Levels of carcinoembryonic antigen (CEA), alpha-feto protein (AFP), ferritin and co-pregnancy associated glycoprotein (Alpha-2-PAG) were determined in patients with confirmed lung cancer at the time of diagnosis and in serial determinations during and after radio- or chemotherapy. Whereas AFP levels were not elevated in patients with lung cancer, increased levels of CEA, fer ritin and Alpha-2-PAG were found in more than 50% of the pa tients. The results suggest that determination of CEA, ferritin and Alpha-2-PAG in the serum of patients with lung cancer may be useful to detect metastases or recurrences and to monitor the results of treatment. Furthermore, in this study CEA and ferritin could be demonstrated in extracts of lung tumor tissues by specific antisera.
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Figure 1. Carcinoembryonic antigen (CEA) levels in lung cancer patients with and without métastasés. The middle column shows CEA levels of patients in which métastasés were confirmed shortly after the elevated antigen levels were determined (n = 90).
Figure 3. Serial carcinoembryonic antigen (CEA) levels in lung cancer patients (n = 18) who received complete or partial remission during radiotherapy. The dotted lines means the time of radiotherapy ( | | = time of recurrence).
Results
has to be assumed that in these patients metastases were al ready present at the time of diagnosis but were too small for clinical demonstration. CEA levels of lung cancer patients in relationship to the histological type of carcinoma and to the extent of disease are shown in figure 2. Nearly all patients with metastatic large cell carcinoma had high CEA levels. The highest CEA levels were found in patients with bone and/ or liver metastases. Serial CEA determinations during and after therapy are shown in figures 3-5.
CEA
CEA was found increased, i.e. values of 10 ng/ml or higher in 47 out of 90 (52%) patients with confirmed lung cancer (figure I). In patients who had no clinically detectable metastases at the time of diagnosis only two had elevated CEA levels of more than 10 ng/ml but below 20 ng/ml. On the contrary, elevated CEA values were found in 37 out of 42 patients with metastases. Only two of these patients had CEA values below 10 ng/ml. In three patients with metastases no CEA could be detected. Seven patients with CEA concentrations of 10 ng/ml or higher had no clinically detectable metastases at the time of diagnosis but metastases could be demonstrated shortly thereafter in all cases. CEA values increased in further determinations. So it
>320»
>320.
>320.
2 9 o :306
150-
100-
50to -
_ 30E
5 20 -
- .
t
O-1- -----1--------- i - ---------1----squomous
oat celt
targe cell
Potients without metastases
squamaus
oat cell
lorge cell
Patients with metastoses
Figure 2. Carcinoembryonic antigen (CEA) levels in lung cancer patients in relationship to the histologic type of carcinoma and to the extent of disease (n = 90).
Figure 4. Serial carcinoembryonic antigen (CEA) levels in lung cancer patients (n = 10) with progressive tumor growth during radio therapy. All patients died shortly after the last CEA determination.
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Figure 3 shows serial CEA determinations in all responders to radiotherapy. CEA levels decreased after therapy or re mained undetectable but increased at the time of recurrence and tumor progression. In figure 4 serial CEA determinations of patients who did not respond to radiotherapy are shown. They all had tumor pro gression during therapy and had no further treatment. Figure 5 shows some examples of serial CEA determinations in patients with métastasés treated with chemotherapy. Pa tients who responded to therapy had temporary decreased CEA levels but at the time of recurrence CEA levels in creased. Ferritin Ferritin was detectable by the Laurell technique in 33 out of 50 patients (66 %) with confirmed lung cancer at the time of diagnosis. In 11 out of 28 patients without clinically proven métastasés, ferritin was present in the serum but levels were not higher than 5 ug/ml. The ferritin values were significantly higher in the group with clinically demonstrated métastasés (figure 6). In comparison, out of 30 hospitalised patients without carcinoma 5 had ferritin in the serum. One of these five patients suffered from liver cirrhosis, the others had severe pneumonia. Ferritin, however, was not elevated in normal controls. Serial ferritin determinations (figure 7) showed decreased or unchanged ferritin values when patients respond ed to chemo- or radiotherapy. Non-responders showed a rise of ferritin values or sometimes values remained unchanged (figure 8). Alpha-2-PAG Alpha-2-PAG was positive in 15 out of 25 male patients (60%) with confirmed lung cancer at the time of diagnosis. Levels of Alpha-2-PAG were between 0,5 and 3,7 mg%. Patients with métastasés had higher levels of Alpha-2-PAG
G .K , 58 years, large cell corcinoma
S W , 63years, oat cellcorcinoma
Figure 6. Ferritin levels in lung cancer patients in relationship to the extent of disease (n = 50).
than patients without métastasés but the difference was not significant. In serial determinations before, during and after therapy there seems to exist some correlation between Alpha2-PAG values and the clinical course of patients (figures 9 and 10). In responders to therapy Alpha-2-PAG levels decreased and in patients with tumor progression an increase of Alpha-2PAG levels in the serum could be observed. AFP AFP levels were not elevated in patients with lung cancer but were as high as in normal controls. Also patients with con firmed liver métastasés had no elevated serum AFP values. Tumor tissue preparations Tumor extracts of squamous, oat-cell and large cell car cinomas were tested with antisera against CEA, ferritin, AFP and Alpha-2-PAG in the Ouchterlony and Laurell technique. There were positive results with anti-CEA and anti-ferritin in all tumor extracts investigated. No reaction with an AFP
Months W K , 61 years, squamous carcinoma
R .K., 61 years, lorge cell carcinoma >320 -
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50-
60-
60-
tumor progression
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10-
30-
R
\7 Vv * 2
Months
6
201 ■ 6
8
10
12
10Q- “ Ó
6 6
8 10 12
Months
Figure 5. Serial cardnoembryonic antigen (CEA) levels in four pa tients with metastatic lung cancer during chemotherapy. PR = partial remission. R = recurrence.
Figure 7. Serial ferritin levels in lung cancer patients (n = 16) who received complete or partial remission during radiotherapy. The dotted lines mean the time of radiotherapy (]'(= time of recurrence).
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antiserum and Alpha-2-PAG antiserum could be detected. Antisera against the tumor extracts prepared in rabbits showed a precipitation line with placenta ferritin but there was no reaction to AFP. We had no opportunity to test this tumor antisera against purified CEA as well as to Alpha-2PAG.
Figure 8. Serial ferritin levels in lung cancer patients (n = 15) with progressive tumor growth during radiotherapy. All patients died shortly after the last ferritin determination.
Figure 9. Serial oo-pregnancy-associated glycoprotein (ou-PAG) levels in lung cancer patients during radio- and chemotherapy (n = 11). All patients received partial or complete remission. = pa tients with chemotherapy. Î I = time of recurrence).
Months
Figure 10. Serial ot2-pregnancy-associated glycoprotein (« 2-PAG) levels in lung cancer patients (n = 9) with progressive tumor growth during radio- and chemotherapy. = patients with chemo therapy).
The CEA was first described as an antigen specific for color ectal carcinoma [1, 11, 12]. In recent years many data have been reported indicating elevated CEA levels in patients with non-digestive cancer or in benign digestive disease [13-20], However, it has been confirmed that the determina tion of CEA may be useful in the management of patients with colorectal cancer by aiding the detection of cancer and especially of metastases and in monitoring patients during the post-therapeutic follow-up period [21], In lung cancer Lo G erfo et al. [22] demonstrated CEA in the sera of patients with this malignancy and Pusztaszeri and Mach [18] could isolate CEA from lung cancer tissues. It was Concannon [23] who reported high CEA levels in lung cancer patients with metastases and he concluded that high CEA levels may ex clude patients from surgical therapy. However, the results of CEA determination in lung cancer patients seemed to be dis couraging because of 50% false negative results. Recently the Roche collaborative study [24] did show positive results in 75 % and false negative in 25 % of patients with confirmed lung cancer. In our study 54% of lung cancer patients had elevated CEA levels while in 46% CEA could not be de monstrated. In accordance with Concannon [10] most of the patients with metastases had elevated CEA titers. The highest CEA levels were found in patients with bone and liver me tastases. There were also elevated CEA levels in some patients without detectable metastases at the time of diagnosis. Shortly after, in all these patients, metastases could be de monstrated by other clinical methods. Therefore CEA deter minations may be useful by aiding detection of metastases in lung cancer patients. Because of the high proportion of false negative results it is quite clear that the CEA assay is not useful as a cancer screening test for patients with lung cancer. This is a result also well confirmed in other types of cancer [24], Increasing interest is put into the use of serial CEA deter minations in cancer patients to monitor the effectiveness of various modes of treatment (i.e. surgical, chemical, ir radiation). Especially in gastrointestinal, but also in breast cancer serial CEA determination has become a good adjunct to management [21], Our results may indicate that serial CEA determinations are useful also in patients with lung cancer. Patients who responded to chcmo- or radiotherapy showed a fall in CEA when values were raised before therapy or CEA remained undetectable. An increase of CEA levels in further determinations indicated a progressive tumor spread. On the other hand, patients who did not respond to therapy showed a rise in CEA levels during therapy because of tumor pro gression. There were some cases in which tumors did not pro duce CEA at any time. Therefore CEA determinations should be used only in context with other clinical and laboratory findings.
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Discussion
Gropp et al.: Carcinoembryonic Antigen, ai-Fetoprotein, Ferritin and co-Pregnancy Associated Glycoprotein
References 1
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Ferritin is found in different molecular forms (isoferritin) in many tissues [25], In serum, ferritin was detected in patients with liver [26] and Hodgkin’s [4] disease. Elevated serum ferritin levels were also reported in patients with acute leu kemia [5, 27] and in breast cancer [6], Measurement of ferritin levels was described as being useful in monitoring the response to therapy [6], So far there are no reports in patients with lung cancer. Serial measurements of serum ferritin showed a fall or persistence in patients who responded to therapy. In patients with tumor progression during therapy, ferritin values in creased. Our results indicate that ferritin determinations may be useful as an additional method for the detection of metastases and in monitoring therapy of lung cancer patients. Similar results have been described in breast cancer and in Hodgkin’s disease. Pregnancy-associated aa-glycoprotein (Alpha-2-PAG) is an a2-glycoprotein of a molecular weight of 360000 which is present in trace amounts in normal sera and is found strongly elevated during the first three months of pregnancy [28, 29], Elevated levels Alpha-2-PAG also occur in patients with various non-malignant diseases and in patients with malignant disease [9]. The highest levels were found in patients with prostate carcinoma and in cancer patients with bone me tastases. In lung cancer patients there was no statistically significant difference in 012 -PAG levels of patients with and without metastases although levels were higher in extended disease. Serial determinations of serum Alpha-2-PAG showed a cor relation between the Alpha-2-PAG concentration and the clinical course of lung cancer patients. Similar results had been reported for breast cancer patients [7], Although up to now the number of patients investigated is small the results are encouraging to use Alpha-2-PAG determination in further investigations. CEA and ferritin could be demonstrated in extracts of lung tumor tissues of various histology. No AFP and Alpha-2-PAG could be detected in those preparations. CEA was first isolated from lung tumors by PUSZTASZERI and M a c h [18] who could show an immunological identity with the CEA isolated from colonic cancer tissues. Later on CEA was described in other tumors but also in normal tissues. So it was supposed that CEA has to be considered as a glycoprotein common to various epithelial tissues whose concentration could be increased in carcinomas arising from these tissues. In our investigation CEA was only identified in preparations of tumor tissues but not in preparations of normal lung tissue by radioimmunoas say. The origin of the serum ferritin is uncertain. The only direct evidence for increased synthesis of ferritin by a malignant cell was given by W h it e et al. [27]. Recently ferritin has been de tected in mammary carcinomas by immunofluorescence. In our study ferritin was demonstrated in lung cancer but not in normal lung tissue preparations. This may indicate that fer ritin is synthetised by the malignant cells. Alpha-2-PAG could not be detected in our tumor extracts. The origin and the biological function of this protein is still uncertain.
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Gropp et al.: Carcinoembryonic Antigen, ai-Fetoprotein, Ferritin and cu-Pregnancy Associated Glycoprotein A.: Serum carcinoembryonic antigen (CEA) levels in breast cancer patients - Correlation with disease progress and response to chemotherapy. Clin. Res. 21:983 (1973). Z amcheck, N.: The present status of CEA in diagnosis, pro gnosis and evaluation of therapy. Cancer 36: 2460-2468 (1975). Lo G erfo , P.; H erter , F. P.; B raun , J., and H ansen, H. J.: Tumor associated antigen with pulmonary neoplasms. Ann. Surg. / 75:495-500 (1972). K. E.; W eil , C. S., and Copper , J. W.: The carcinoembryonic antigen assay in bronchogenic carcinoma. Cancer 34: 184192(1974). Roche Collaborative study. Data on file. Hoffmann-La Roche & Co. AG, Basel. A ungst, C. W.: Ferritin in body fluids. J. Lab. clin. Med. 71: 517-522(1968). R eissmann, K. R. and D ietrich , M. R.: On the presence of ferritin in the peripheral blood of patients with hepatocellular disease. J. clin. Invest. 35:588-595 (1956). berg ,
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Request reprints from: C. G ropp, Medizinische Universitätsklinik, Emil Mannkopffstraße 1, 3550 Marburg/Lahn (FRG)
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