Clinical Significance of Serum Levels of a Carbohydrate Antigen, Sialyl SSEA-1, in Patients with Fibrosing Lung Disease 1 , 2
HIROAKI SATOH, HIROSHI KAMMA, TAKESABURO OGATA, HEIICHI VANO, MORIO OHTSUKA, and SHIZUO HASEGAWA Introduction
In 1978, Solter and Knowles (1)first established a monoclonal antibody against one of the polylactosamine antigens, stage-specific embryonic antigen (SSEA-l) or LewisX, which is specific to the preimplantation stage of murine embryos. Kannagi and colleagues (2) determined the structure of SSEA-l to be a carbohydrate carrying both Le X-hapten and i-antigenic structure, and they induced various relatives from this antigen. SSEA-l and its relatives were frequently detected in human cancers of various organs. Among these relatives, sialyl SSEA-l was most frequently elevated in sera of patients with lung cancers, especially adenocarcinoma (3- 5). Imm unohistochemical studies confirmed the expression of the antigen in cancer cells in patients with adenocarcinoma of the lung (6, 7). Recently, the measurement of serum sialyl SSEA-l has become common in Japan as one of the tumor-markers for lung cancer. With regard to pulmonary nonmalignant diseases, the serum levels of sialyl SSEA-l are believed to be less than 38.0 units/ml, which has been estimated as the cutoff value according to the results of a large survey of 1,105 healthy Japanese subjects (8). Two surveys of nonmalignant lung diseases reported that serum sialyl SSEA-l was elevated in about 10070 of those patients (9, 10). The serum sialyl SSEA-l in nonmalignant lung diseases has not been investigated in detail (11), and its clinical significance is not apparent. Although the physiologic significance of SSEA-l antigen and its relatives has not yet been determined, it has been suggested that the antigens could be related to the facilitation of cell-to-cell interaction in proliferating cells (1). In the developing lungs of human embryos and fetuses, the antigens are specifically expressed in the most immature cells, which undergo the process of vigorous proliferation (7). If the antigens are correlated
SUMMARY Serum levels of sialyl SSEA-1 antigen, a carbohydrate antigen, were measured by radioimmunoassay In 142patients with nonmalignant lung diseases. In 20 of 41patients with fibrosing lung disease, either Idiopathic or associated with collagen disease, the serum sialyl SSEA-1 levels were abnormally elevated. In patients with other lung diseases, the serum levels were almost within normal limits, less than 38.0 units/mi. In fibrosing lung disease the serum levels ranged from 13.8 to 147.0 unlts/ml and were largely concurrent with the degree of disease activity. The therapeutic effects of corticosteroid, which were evaluated with clinical-radiographic-physiologic scores and survivals in the patients with elevated serum levels, were significantly lower than those of the patients with the normal range of antigen levels. An immunohistochemical study performed on autopsied lungs from five patients with flbrosing lung disease Indicated that the antigen was selectively expressed In the pulmonary epithelial cells that covered the remodeling alveolar septi In the lungs. No antigen was detectable by immunostaining in normal pulmonary epithelium among five normal lungs. From these findings, It Is thought that the elevated levels of serum sialyl SSEA-1 may be derived from proliferating epithelial cells that were dominant In the late stage of fibrosing lung disorders. The measurements of serum slalyl SSEA·1 In patients with fibrosing lung disease may be clinically useful In establishing the degree of disease activity that has an influence on patient prognosis and therapeutic response. AM REV RESPIR DIS 1991; 144:1177-1181
with the process of epithelial cell growth in lungs, it is expected that the antigens might be expressed not only in cancer cells but also in the epithelial cells frequently proliferating in fibrosing lungs as a process of epithelial repair. We examined the serum levels of sialyl SSEA-l antigen in patients with nonmalignant lung diseases with special reference to those with fibrosing lung disease. In order to determine the source of increased serum levels of sialyl SSEA-l, an immunohistochemical study was performed using the monoclonal antibody against the antigen. Finally, the clinical significance of serum levels of the antigen is discussed especially with regard to the prognosis of patients and the response to corticosteroid therapy. Methods Patients The study population consisted only of consecutivenew patients with nonmalignant lung diseases who were seen at our institution between June 1987 and August 1990. All the patients were clinically negative for various cancer tests, including bronchoscopy or enteroscopy, computed tomography, and echography. Patients with cancer in any organs and those with its suspicion were excluded from this study.
The diagnosis of idiopathic pulmonary fibrosis (IPF) was established by a combination of medical history, physicalexamination, laboratory tests, chest radiograph, pulmonary function tests, arterial blood analyses, and the results of lung biopsies according to previously described criteria (12, 13). Patients with IPF had no known causes of pulmonary fibrosis such as organic or inorganic dust inhalation, drug ingestion, or primary granulomatous lung disorders. Histologic confirmation of the diagnosis was obtained by transbronchial biopsy (n = 16)or by autopsy (n = 6). The diagnoses of collagen diseases were made according to the criteria of those diseases (14-18). The 142 patients studied here were composed of 41 with fibrosing lung disease, 40 with alveolar pneumonia, 43 with bronchial asthma, and 18 with emphysema. The patients with fibrosing lung disease were composed of 22 with IPF and 19 with various collagen
(Received in original form September 4, 1990and in revised form June 11, 1991) I From the Departments of Internal Medicine and Pathology, University Hospital, University of Tsukuba, Tsukuba-city, Ibaraki, Japan. 2 Correspondence and requests for reprints should be addressed to Dr. Takesaburo Ogata, Department of Pathology, Institute of Basic Medical Sciences,University of Tsukuba, Tsukuba-city, Ibaraki 305, Japan.
SATOH, KAMMA, OGATA, VANO, OHTSUKA, AND HASEGAWA
diseases involving lungs. The latter group of patients included eight with rheumatoid arthritis, five with polymyositis, three with Sjogren disease,two with progressivesystemic sclerosis, and one with mixed connective tissue disease. All the patients with fibrosing lung disease had bilateral diffuse interstitial patterns on chest radiographs. Nine patients with fibrosing lung disease died of progressive respiratory failure during the study period, and autopsies were carried out on six of them. Seventeen patients with fibrosing lung disease weretreated with prednisone. The initial dose was 1 mg/kg/day or its equivalent until their pulmonary function stabilized, and then the dose was tapered gradually based on clinical response. The activities of the fibrosing lung disease were evaluated according to the composite clinical-radiographic-physiologic score (CRP) proposed by Watters and colleagues (19), using seven variables: dyspnea, chest radiograph, spirometry, lung volume, diffusing capacity, resting alveolar-arterial P0 2 and exercise O 2 saturation. The improvement or aggravation of the disease was assessed using CRP score at 4 to 12 wk after the corticosteroid therapy began. The assessment was made according to whether the following changes had occurred; a change in dyspnea score of more than 6, or a change in CRP score of more than 10 (20). Measurement of Sialyl SSEA-I Antigen in Serum Serum samples were obtained from the patients at admission. Seventeen patients with fibrosing lung disease were examined more than two times. The control samples wereobtained from 14 healthy adult volunteers. An immunoradiometric assay of serum sialyl SSEA-l antigen was performed using anti-sialyl SSEA-l monoclonal antibody FH-6 (Otsuka Assay Laboratory, Tokushima, Japan). The levels of the antigen in the sera of patients weredetermined by a binding of 1251_ labeled monoclonal antibody FH-6 on polystyrene plastic beads that had been coated with the monoclonal antibody and incubated with sera. Serum samples (20 ul) were mixed with 200 ul of 50 mM citrate buffer at pH 4.5 and incubated for 18 h with beads coated with the monoclonal antibody at room temperature. The beads were washed three times and incubated with 200 J,ll of the 125 1_ labeled monoclonal antibodies for 1h at room temperature. The beads that had absorbed 125 1 were measured after three washes with the same buffer. Duplicate assays wereperformed for each sample. The cutoff levels in serum were determined to be 38.0 units/ml, corresponding to the mean ± 2 SD (standard deviation) calculated in a survey of a Japanese group of 1,105 healthy subjects (8). Immunohistochemical Study in Lung Tissues In order to determine the expression of sialyl SSEA-1 antigen in the lungs, an immuno-
38.0units/ml of serum sialylSSEA-l, was 48.8% (20 of 41) in fibrosing lung disease, 7.50/0 (three of 40) in alveolar pneumonia, 7.00/0 (three of 43) in bronchial asthma, and 0% (zero of 18) in emphysema (figure 1). The antigen levels in patients with fibrosing lung disease ranged widely from 13.8 to 147.0 units/ml, with a mean of 49.4 ± 34.6 units/ml, and these were significantly higher than those in the healthy control subjects. The mean levels of the antigen in patients with alveolar pneumonia, bronchial asthma, and emphysema were 28.5 ± 6.5,26.2 ± 9.7, and 24.0 ± 6.5 units/ml, respectively. There was no statistical difference in the levels of sialyl SSEA-l between IPF (48.0 ± 29.6 units/ml) and pulmonary involvement with various collagen diseases (50.8 ± 32.9 units/mI).
histochemical study was performed using monoclonal antibody FH -6 on five autopsy lung specimens from fivepatients with fibrosing lung disease (three with IPF and two with collagen diseases involving lung) and on five normal lungs as control. Paraffin sections were used for this study because there are no significant differences in immunostaining between frozen sections and paraffin sections (21).After the deparaffinization and blocking with normal horse serum, the specimens were incubated overnight with FH-6 (Otsuka Assay Lab) in a moist chamber at 4 0 C and washed three times with phosphate-buffered saline (PBS). The sections were incubated with biotinylated horse antimouse antibody (Vector Laboratories, Burlingame, CAl for 30 min, and then they were incubated with avidin-biotinylated peroxidase complex (Vector Lab) for 45 min. After washing three times in PBS, the specimens were colored by addition of a solution of 3,3'-diaminobenzidine tetrahydrochloride (Wako Pure Chemicals Ind., Osaka, Japan) and 0.005070 hydrogen peroxide in 0.05 mol/L TRIS buffer at pH 7.6.
Sialyl SSEA-I and Clinical Features in Patients with Fibrosing Lung Disease The correlations between serum levels of sialyl SSEA-l and various clinical features including age, sex, smoking history, duration of symptoms, physiologic data, and number of patients who died during the study period are shown in table 1. There were no significant differences concerning those factors between the patients with higher sialyl SSEA-l (more than 38.0 units/ml) and those with lower sialyl SSEA-l (less than 38.0 units/ rnl), except for the number of patients who died during the study period. The patients with fibrosing lung disease were followed throughout the study period. Survival curves of 41 patients with the disease are shown in figure 2. There was a significant difference in survival rate between the two groups of pa-
Statistical Analysis Comparison between two groups was made using the Mann-Whitney U test and the chisquare test. Survival curves were plotted according to the method of Kaplan and Meier, and the prognostic significance was evaluated by means of the generalized Wilcoxon's test. Data were expressed as the mean ± SD.
Serum Levels of Sialyl SSEA-I in Patients with Nonmalignant Lung Diseases The serum levels of sialyl SSEA-l in the healthy control group ranged from 10.9 to 30.5 units/ml, with a mean of 20.4 ± 6.2 units/ml. There were no healthy persons showing elevated serum levels of sialyl SSEA-l higher than 38.0 units/ml. The ratio of patients with more than
sialyl SSEA-l (unit/mt)
o Fibrosing lung disease (n=4l) Alveolar pneumonia (n =40) Bronchial asthma (n=43) Emphysema (n=18)
• ::.1:•••, ::- -.: -.: -.
.:.-::,... •••':.';-:0 --+--
;. "oi o\) --+-
Healthy control (n=14)
0 Fig. 1. Serum levels of sialyl SSEA-1 in patients with nonmalignant lung diseases and in healthy control subjects. Bars represent mean ± SO in each group of the diseases.
SIALYL CARBOHYDRATE ANTIGEN IN FIBROSING WNG DISEASE
tients based on serum sialyl SSEA-llevels (p < 0.05).
TABLE 1 DEMOGRAPHIC AND PHYSIOLOGIC CHARACTERISTICS OF THE PATIENTS WITH FIBROSING LUNG DISEASE· Patients with Lower Sialyl SSEA-1 « 38.0 units/ml) (n = 21)
Patients with Higher Sialyl SSEA-1 (> 38.0 units/ml) (n = 20)
58.5 ± 10.1 8/13 9/12
61.8 ± 10.4 10/10 9/11
11.4 ± 23.4
17.0 ± 26.1
± 15.5 ± 40.4 ± 14.3
NS NS NS NS
Age, yr Sex, male/female Smokers/nonsmokers Duration of symptoms, months Physiology Pao2, mm Hg AaPo 2, mm Hg VC, % pred Dleo, % pred Patients who died during the study period, n (%)
73.3 36.0 73.4 54.1
± ± ± ±
12.2 21.0 20.5 26.7
70.2 44.3 68.1 48.0
P < 0.05
• Data are expressed as the mean ± SO.
sialyl SSEA-l .:;:
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sialyl SSEA-l ~38 (unit/m£ )
~ 40 ::J