Scand. J. Immunol, Vol. 5, 1976.
Carcinoembryonic Antigen and Cystic Fibrosis Protein in Blood from Cystic Fibrosis Homozygotes and Heterozygote Carriers G. B. WILSON, N. M. BURDASH, P. ARNAUD, M. T. MONSHER & H. H. FUDENBERG Department of Basic and Clinical Immimology and Microbiology and Department of Laboratory Medicine, Medical University of South Carolina, Charleston, South Carolina, USA Wilson, G.B., Burdash, N.M., Arnaud, P., Monsher, M.T. & Fudenberg, H.H. Carcinoembryonic Antigen and Cystic Fibrosis Protein in Blood from Cystic Fibrosis Homozygotes and Heterozygote Carriers. Scand. J. Immunol. 5, 829-836, 1976. Carcinoembryonic antigen (CEA) activity was measured by radioimmunoassay in blood from c> stic fibrosis (CF) homozygotes, heterozygote carriers of CF, normal healthy controls, and other patient controls with carcinomas involving gastrointestinal organs. All samples were also screened by electrofocusing for cystic fibrosis protein (CFP), a metabolic marker previously shown to be associated with the CF gene. Significantly increased levels of CEA activity were found in all CFP-positive groups; however, viith one exception all patient controls with marked increases in CEA activity were CFP-negative. Immunodiffusion of perchloric acid extracts of CEA-like material from heterozygote carrier blood indicated that the CEA-like material, which was elevated in homozygotes and heterozygotes for CF, showed only partial identity with two separate CEA preparations obtained from colon carcinomas and was not identical to either A, B, or O (H) blood group substances. Tlus glycoprotein material did, however, react with three different anti-CEA antisera. Our finding of an abnormally increased glycoprotoin in cystic fibrosis, taken together with previous reports demonstrating abnormalities in the carbohydrate portion of glycoproteins found in various exocrine secretions in CF, further suggests that the primary defect in this disease is manifested partly as a defect in glycoprotein metabolism. This defect may result from an abnormality in one or more of the glycosyltransferases, possibly caused by a more primary defect in polyamine metabolism. G.B. Wilson, Deparimenl of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, Charleston, SC 29401, USA Cystic fibrosis (CF) is a genetically inherited lethal disorder resulting from an unknown inborn error of metabolism (26). Generalized dysfunction of exocrine and apocrine glands is a common feature of the disease (9, 12). Fibrosis, degeneration, and failure of the exocrine glands to develop appear to result from the accumulation of 'abnormal' mucous secretions, leading to dilatation and ultimate distortion of anatomy and function (9). The more common pathologic findings in CF, in-
eluding pancreatic insufficiency, bronchial obstruction, hepatic cirrhosis, dysgenesis of the vas deferens, meconium ileus, and rectal prolapse, may result from this sequence of events (9, 29, 33). The observation (17) of goblet cell hyperplasia and of occlusion of goblet cells in exocrine tissues by inspissated material (1) adds support to the possibility that abnormal exocrine glycoprotein metabolism may be a major factor in the clinical progession of the disease.
830
G. B. Wilson, N. M. Burdash, P. Arnaud, M. T. Monsher & H. H. Fudenberg
The metabolic defect responsible for the abnormal exocrine secretions has not been delineated (9, 11, 29); however, several abnormal proteins and glycoproteins have been shown to be associated with cystic fibrosis. Among these are the CF ciliary dyskinesia factor (6) and CF protein factor (35, 36) found in scrum, a related substance (26) found in sweat (23) and saliva (24), and a calcium-binding protein found in subniaxillary saliva (4). In addition, many of the glycoproteins found in bronchial mucus (28), salivary glands (21), sputum (16, 32), duodenal fluid, and rectal mucus (10, 30) are abnormal, primarily in their carbohydrate compositions, which may render them more viscous and less soluble (10) and could result in obstruction of exocrine gland ducts. In the present paper we report results showing that a carcinoembryonic antigen (CEA)like substance is found in increased concentrations in blood from both cystic fibrosis patients (CF homozygotes) and heterozygote carriers of CF as determined by radioimmunoassay. This CEA-reactive material was shown to be associated with the CE gene by concurrently screening for cystic fibrosis protein (CFP) in a blind fashion all individuals tested for CEA activity (36). CFP has previously been shown to be a metabolic marker apparently found specifically in the blood of individuals homozygous or heterozygous for CF (35, 36). Two lines of evidence suggested to us that the CFA-reactive material we found elevated in the blood from homozygotes and heterozygotes for CF is antigenically nonidentical to the glycoprotein originally described as carcinoembryonic antigen (CEA) by Gold et al. (13, 14) in fetal colon extracts and adult human carcinomas and later shown to be elevated in various neoplastic diseases and other inflammatory and regenerative disorders of nonmalignant origin (15, 27). [1] Perchloric acid (PCA)-extracted CEA-reactive material from obligate heterozygote carrier blood showed a reaction of partial identity when compared with two different preparations of purified CEA by double immunodiffusion, yet gave a single precipitin line when tested against three different anti-CEA antisera. [2] Several 'nor-
mal patient controls' with diagnosed carcinomas of gastrointestinal organs or with increased levels of CEA activity, or with both, were not CFP-positive by electrofocusing. The significance of our findings is discussed. MATERIALS AND METHODS Collection and processing of samples. Vetious
blood samples were collected from each individual and processed as described previously (36); after analysis for the presence of cystic fibrosis protein by isoelectric focusing they were stored at -2O''C until assayed for CEA levels by radioimmunoassay. Twenty-four patients with cystic fibrosis (homozygotes for CF) and 22 of their parents (obligate heterozygote carriers of cystic fibrosis) were chosen at random for this study from individual families who attended a Cystic Fibrosis Clinic located at either the Department of Pediatrics, University of California, San Francisco; Children's Hospital at Stanford; or Children's Hospital at Oakland. The diagnosis was confirmed in all CF patients by the clinical history and the presence of an elevated concentration of chloride in the sweat (33). The ages of the CE patients ranged from 3 to 33 years (tnean, 11 years). Twenty-one healthy individuals (normal control subjects) with no known family history or clinical history indicative of cystic fibrosis were also studied. These samples were selected from among 110 normal controls who had previously been screened (35) for the presence of CEP by the isoeiectric focusing assay (36). Five normal control samples positive for the presence of CEP and 16 negative for CEP were included in this study. The five I'ormal controls positive for CFP may in fact be heterozygote carriers of the CF gene and are referred to as 'presumed heterozygote carriers' in this report. In addition, 11 samples from individuals with a disease other than cystic fibrosis were studied. None of the patients had a known family history or clinical history indicative of cystic fibrosis. The 11 normal patients analyzed as controls for CEA and CEP were obtained
CEA in Cystic Fibrosis 831 Table I. Carcinoembryonic antigen (CEA) activity and cystic fibrosis (CF) protein (CFP) in human blood Sample from Normal individuals Presumed heterozygote carriers for CF Obligate heterozygotes for CF Cystic fibrosis Patients
Number tested
CEA activity (ng/ml)*
16
2.46 ± 0.07 4.76 ± 0.37* 7.59 ± 0.28* 6.35 ± 0.16* 1.2, 2.4
5 22 22 2
Cancer Cancer of colon after resection Dukes's class-D cancer of the colon Recurrent cancer of the rectum Cancer of the rectum after resection Carcinoid tumor of the rectum Cancer of liver Cancer of pancreas
No. positive for CFP** 0/16 5/5
22/22 22/22 0/2
2 1
2.1,2.1, 1.3,2.0 6400*, 5400* 18.8*
0/4 0/2 l/l
1 1
2.0 1.7
0/1
I 1
52* 1.8
4
0/1 0/1
o/i
* Mean level of CEA is significantly elevated {P
Carcinoembryonic Antigen and Cystic Fibrosis Protein in Blood from Cystic Fibrosis Homozygotes and Heterozygote Carriers G. B. WILSON, N. M. BURDASH, P. ARNAUD, M. T. MONSHER & H. H. FUDENBERG Department of Basic and Clinical Immimology and Microbiology and Department of Laboratory Medicine, Medical University of South Carolina, Charleston, South Carolina, USA Wilson, G.B., Burdash, N.M., Arnaud, P., Monsher, M.T. & Fudenberg, H.H. Carcinoembryonic Antigen and Cystic Fibrosis Protein in Blood from Cystic Fibrosis Homozygotes and Heterozygote Carriers. Scand. J. Immunol. 5, 829-836, 1976. Carcinoembryonic antigen (CEA) activity was measured by radioimmunoassay in blood from c> stic fibrosis (CF) homozygotes, heterozygote carriers of CF, normal healthy controls, and other patient controls with carcinomas involving gastrointestinal organs. All samples were also screened by electrofocusing for cystic fibrosis protein (CFP), a metabolic marker previously shown to be associated with the CF gene. Significantly increased levels of CEA activity were found in all CFP-positive groups; however, viith one exception all patient controls with marked increases in CEA activity were CFP-negative. Immunodiffusion of perchloric acid extracts of CEA-like material from heterozygote carrier blood indicated that the CEA-like material, which was elevated in homozygotes and heterozygotes for CF, showed only partial identity with two separate CEA preparations obtained from colon carcinomas and was not identical to either A, B, or O (H) blood group substances. Tlus glycoprotein material did, however, react with three different anti-CEA antisera. Our finding of an abnormally increased glycoprotoin in cystic fibrosis, taken together with previous reports demonstrating abnormalities in the carbohydrate portion of glycoproteins found in various exocrine secretions in CF, further suggests that the primary defect in this disease is manifested partly as a defect in glycoprotein metabolism. This defect may result from an abnormality in one or more of the glycosyltransferases, possibly caused by a more primary defect in polyamine metabolism. G.B. Wilson, Deparimenl of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, Charleston, SC 29401, USA Cystic fibrosis (CF) is a genetically inherited lethal disorder resulting from an unknown inborn error of metabolism (26). Generalized dysfunction of exocrine and apocrine glands is a common feature of the disease (9, 12). Fibrosis, degeneration, and failure of the exocrine glands to develop appear to result from the accumulation of 'abnormal' mucous secretions, leading to dilatation and ultimate distortion of anatomy and function (9). The more common pathologic findings in CF, in-
eluding pancreatic insufficiency, bronchial obstruction, hepatic cirrhosis, dysgenesis of the vas deferens, meconium ileus, and rectal prolapse, may result from this sequence of events (9, 29, 33). The observation (17) of goblet cell hyperplasia and of occlusion of goblet cells in exocrine tissues by inspissated material (1) adds support to the possibility that abnormal exocrine glycoprotein metabolism may be a major factor in the clinical progession of the disease.
830
G. B. Wilson, N. M. Burdash, P. Arnaud, M. T. Monsher & H. H. Fudenberg
The metabolic defect responsible for the abnormal exocrine secretions has not been delineated (9, 11, 29); however, several abnormal proteins and glycoproteins have been shown to be associated with cystic fibrosis. Among these are the CF ciliary dyskinesia factor (6) and CF protein factor (35, 36) found in scrum, a related substance (26) found in sweat (23) and saliva (24), and a calcium-binding protein found in subniaxillary saliva (4). In addition, many of the glycoproteins found in bronchial mucus (28), salivary glands (21), sputum (16, 32), duodenal fluid, and rectal mucus (10, 30) are abnormal, primarily in their carbohydrate compositions, which may render them more viscous and less soluble (10) and could result in obstruction of exocrine gland ducts. In the present paper we report results showing that a carcinoembryonic antigen (CEA)like substance is found in increased concentrations in blood from both cystic fibrosis patients (CF homozygotes) and heterozygote carriers of CF as determined by radioimmunoassay. This CEA-reactive material was shown to be associated with the CE gene by concurrently screening for cystic fibrosis protein (CFP) in a blind fashion all individuals tested for CEA activity (36). CFP has previously been shown to be a metabolic marker apparently found specifically in the blood of individuals homozygous or heterozygous for CF (35, 36). Two lines of evidence suggested to us that the CFA-reactive material we found elevated in the blood from homozygotes and heterozygotes for CF is antigenically nonidentical to the glycoprotein originally described as carcinoembryonic antigen (CEA) by Gold et al. (13, 14) in fetal colon extracts and adult human carcinomas and later shown to be elevated in various neoplastic diseases and other inflammatory and regenerative disorders of nonmalignant origin (15, 27). [1] Perchloric acid (PCA)-extracted CEA-reactive material from obligate heterozygote carrier blood showed a reaction of partial identity when compared with two different preparations of purified CEA by double immunodiffusion, yet gave a single precipitin line when tested against three different anti-CEA antisera. [2] Several 'nor-
mal patient controls' with diagnosed carcinomas of gastrointestinal organs or with increased levels of CEA activity, or with both, were not CFP-positive by electrofocusing. The significance of our findings is discussed. MATERIALS AND METHODS Collection and processing of samples. Vetious
blood samples were collected from each individual and processed as described previously (36); after analysis for the presence of cystic fibrosis protein by isoelectric focusing they were stored at -2O''C until assayed for CEA levels by radioimmunoassay. Twenty-four patients with cystic fibrosis (homozygotes for CF) and 22 of their parents (obligate heterozygote carriers of cystic fibrosis) were chosen at random for this study from individual families who attended a Cystic Fibrosis Clinic located at either the Department of Pediatrics, University of California, San Francisco; Children's Hospital at Stanford; or Children's Hospital at Oakland. The diagnosis was confirmed in all CF patients by the clinical history and the presence of an elevated concentration of chloride in the sweat (33). The ages of the CE patients ranged from 3 to 33 years (tnean, 11 years). Twenty-one healthy individuals (normal control subjects) with no known family history or clinical history indicative of cystic fibrosis were also studied. These samples were selected from among 110 normal controls who had previously been screened (35) for the presence of CEP by the isoeiectric focusing assay (36). Five normal control samples positive for the presence of CEP and 16 negative for CEP were included in this study. The five I'ormal controls positive for CFP may in fact be heterozygote carriers of the CF gene and are referred to as 'presumed heterozygote carriers' in this report. In addition, 11 samples from individuals with a disease other than cystic fibrosis were studied. None of the patients had a known family history or clinical history indicative of cystic fibrosis. The 11 normal patients analyzed as controls for CEA and CEP were obtained
CEA in Cystic Fibrosis 831 Table I. Carcinoembryonic antigen (CEA) activity and cystic fibrosis (CF) protein (CFP) in human blood Sample from Normal individuals Presumed heterozygote carriers for CF Obligate heterozygotes for CF Cystic fibrosis Patients
Number tested
CEA activity (ng/ml)*
16
2.46 ± 0.07 4.76 ± 0.37* 7.59 ± 0.28* 6.35 ± 0.16* 1.2, 2.4
5 22 22 2
Cancer Cancer of colon after resection Dukes's class-D cancer of the colon Recurrent cancer of the rectum Cancer of the rectum after resection Carcinoid tumor of the rectum Cancer of liver Cancer of pancreas
No. positive for CFP** 0/16 5/5
22/22 22/22 0/2
2 1
2.1,2.1, 1.3,2.0 6400*, 5400* 18.8*
0/4 0/2 l/l
1 1
2.0 1.7
0/1
I 1
52* 1.8
4
0/1 0/1
o/i
* Mean level of CEA is significantly elevated {P
