Clinical significance of mucin-like high molecular weight glycoprotein originated from lung cancer as tumor marker

Clinica Chimica Acta, 180 (1989) 45-58 Elsevier

45

CCA 04372

Clinical significance of mucin-like high molecular weight glycoprotein originated from lung cancer as tumor marker Minoru

Imokawa

‘, Hidetoshi

,Okabe I, Yukio Ochi ’ and Yoshihiro

Kajita 2

I Central Clinical Laboratory, Shiga University of Medical Science, Otsu, Shiga, and ’ Department of Internal Medicine, Nantan General Hospital, Yagi, Kyoto (Japan) (Received 20 May 1988; revision received 18 October 1988; accepted 24 October 1988) Key words: Mucin; Glycoprotein; Tumor-associated antigen

A high molecular weight, mucous glycoprotein (MG) from the pleural fluid of lung adenocarcinoma was purified by the DEAE-cellulose, gel-filtration and wheat germ agglutinin affinity chromatography. Protein portion of the molecule was composed of amino acids rich in serine, threonine and proline, but methionine and tyrosine concentrations were relatively low. About 65% of the weight, was composed of galactose, galactosamine, glucosamine, fucose and sialic acid. The gel-filtration pattern on Sepharose 4B revealed M, > lo6 Da. The SDS-PAGE pattern revealed a main band at the position of the it4, about 350 kDa under the reducing condition. Rabbit antibody against this molecule recognized mainly the peptide portion, and the radioimmunoassay (RIA) using the double antibody method was developed by this antibody. Serum MG level was low in healthy subjects and in benign diseases (0.8 + 0.7 U/ml; mean + SD and 1.1 f 2.3 U/ml, respectively). Thus, 3 U/ml was used as the cut-off value. The mean of serum MG levels and positive rates in malignant diseases were significantly high; 4.4 U/ml and 32.3% in lung cancer, 20.1 U/ml and 77.5% in pancreas cancer 11.6 U/ml and 64.3% in gastric cancer, 12.9 U/ml and 57.1% in hepatoma, 12.3 U/ml and 77.8 in colon cancer. Other malignancies such as ovarial and uterus cancer showed also high levels. Elevated values in these malignancies were observed frequently in patients with metastasis. On the other hand, the false Correspondence to: Dr. Y. O&i, Central Clinical Laboratory, Shiga University of Medical Science, Otsu, Shiga, 520-21, Japan.

0009-8981/89/$03.50

0 1989 Elsevier Science Publishers B.V. (Biomedical Division)

positive cases were found in 10% of benign diseases. Determination of MG seems to be useful for the detection of several kinds of malignancies, but it is not adequately sensitive as a screening method for early cancer detection.

Introduction Mucins which are major components of mucus, are produced and secreted from epithelial cells throughout the bronchial and gastrointestinal tract, including salivary glands, esophagus, stomach and intestine. Changes of characteristics in mucin originated from tumor have been suggested from the changed density of carbohydrate moeities on the peptide core and increased lectin binding in the colon cancer-associated mucin [1,2]. The usefulness of colon-specific glycoprotein (M, > 4 x 106) as a tumor marker for colorectal cancer had been suggested [3]. There are many reports discussing the relation between mucus glycoproteins and malignancies [l-6]. Recently, tumor-associated carbohydrate antigens detected by monoclonal antibodies (MoAbs) has been reported in succession [7-121. CA 19-9, CA 50, CA 12-5, CA 15-3, DU-PAN-2 and ST-439 have been introduced [7-121. It has been reported that several antigens detected by MoAbs recognize the carbohydrate epitopes of mucin-like high molecular weight (M,) glycoprotein. In the present experiment, we purified the mucin-like high M, glycoprotein (MG) from the pleural fluid of a patient with adenocarcinoma of the lung. The clinical usefulness as tumor marker, and the relation with tumor-associated carbohydrate antigens defined by MoAbs were investigated. Materials and methods Sample The pleural fluid (total protein about 0.5 g/dl) of a patient with adenocarcinoma of the lung was collected and kept frozen until use. The sample had blood group 0 antigen and Lewis [a( -)b( +)] antigen. Tumor markers in the sample were as follows; AFP (alpha-fetoprotein), < 2 ng/ml, CEA, 5 300 ng/ml, CA 19-9, 6 300 U/ml, SCC (squama cell carcinoma), 990 ng/ml. Purification of the high molecular weight mucous glycoprotein The sample (500 ml) was dialysed against 0.05 mol/l phosphate buffer, pH 8.0, for 2 days at 5 “C and the supernatant was obtained after centrifugation at 20000 x g. for 30 min. The supematant was applied to a 4 X 40 cm column of DEAE-cellulose (James River Co. Berlin) which had been equilibrated with 0.05 mol/l phosphate buffer, pH 8.0. After the elution of the unabsorbed fraction with 0.05 mol/l phosphate buffer, the eluted fraction with 0.2 mol/l phosphate buffer was collected. Then, this fraction was concentrated to 100 ml by lyophilization, and the contents of several tumor markers were measured by the radioimmunoassay (RIA).

Aliquots of 5 ml of the supematant were applied on Sepharose 4B (Pharmacia Co., Uppsala, Sweden) column (2.6 x 105 cm) equilibrated with the elution buffer (0.5 mol/l phosphate buffer, pH 7.8). The eluted fraction in void volume was rechromatographied on Sepharose 4B column. The void volume fraction containing about 50% of carbohydrate contents in the applied sample contained a large amount of CA 19-9 antigen but no CEA. The void volume fraction was applied to wheat germ agglutinin (WGA)-Sepharose 4B (Honen Co. Tokyo) column (2.6 X 20 cm; binding capacity is about 130 nmol N-acetyl-D-galactosamin/ml) equilibrated with 0.05 mol/l phosphate buffer pH 7.8. The unbound fraction was washed out by the same buffer, and then the bound fraction was eluted by the same buffer containing 20% (w/v) N-acetylglucosamin (Nakarai Co., Kyoto, Japan). The small amount of MG (about 4 mg) was found in the eluted fraction. The eluted material was dialysed against the distilled water at 4” C and then lyophilized. This preparation was used as the purified MG. Antisera to MG Rabbits were immunized once a week for 5 wk with 0.5 mg of the purified MG emulsified with Freund’s complete adjuvant (Nakarai). Antisera were treated at 56” C for 30 min, and absorbed with normal erythrocytes having 0 antigen and Lewis b antigen. Absorbed antisera were used for the RIA. Analytical methods Protein content of purified MG preparation was determined by the method of Lowry [13]. Total neutral hexose was assayed with the anthrone reagent [14] using galactose as the standard. Fucose was determined by the method of Gibbons [15]. Total hexosamine released by hydrolysis with 3 mol/l hydrochloric acid for 16 h at 100 ’ C was estimated by the procedure of Gardell [16] using D( + )-glucosamine. Galactosamine was determined according to Ludowieg and Benmaman [17]. Sialic acid content was assayed by the method of Jourdian [18] with neuraminic acid as the standard. Tests for uranic acid and sulfate content were carried out according to the method of Bitter and Muir [19] and Terho [20], respectively. With JEOL JLC-200A amino acid analyzer (Nihondenshi Co., Japan), amino acid analyses were performed on samples which had been hydrolysed with 6 mol/l hydrochloric acid for 72 h, at 110 o C. Electrophoretic studies Polyacrylamide gel electrophoresis (PAGE) was performed on polyacrylamide gradient gels (5-128) in the sodium dodecyl sulfate as described by Laemmli [21]. Samples labeled by the Bolton-Hunter method [22] (specific activity 20 pCi/pg protein) were diluted with an equal volume of 4% SDS with or without reducing agent (2% mercaptoethanol) and heated (90 o C, 5 min) before application to the gel. After electrophoresis, the gels were dried and autoradiography were carried out at -70°C using Kodak X-OMAT RP film and Comex lighting-plus enhancing screens (DuPont).

48

Con A binding 100 ~1 (2536

U) of MG fraction purified by WGA affinity column was applied on 1 ml of Con A affinity column (Pharmacia) equilibrated with 100 ml of 20 mmol/l Tris buffer, pH 7.8, containing 0.5 mol/l NaCl. The unbound fraction was collected in 5 ml and then washed out completely. Subsequently the bound fraction was dissociated by the same buffer containing 0.5 mol/l cY-methyl-D-mannoside and collected in 5 ml. After dialysing with 50 mmol/l phosphate buffer, pH 7.8, both samples were examined for MG immunoreactivity. Enzyme susceptibility

MG preparations (0.1 mg) were incubated at 37” C for 16 h with or without trypsin (0.5 mg/5 BAEEU; Sigma Chemical Co., St. Louis, MO, USA) in phosphate buffer, pH 7.8, pepsin (0.5 mg/1.3 U; Sigma) in acetate buffer pH 2.5, protease (0.5 mg/350 mu; Sigma) in phosphate buffer pH 7.8, and neuraminidase (25 mu; Nakarai) in acetate buffer, pH 5.0. After enzyme treatment, each sample was determined by RIA for MG after boiling for 5 min. Radioimmunoassay

(RIA) for MG

RIA was performed by mean of the double antibody method using ‘251-labeled antigen. Assay medium contained 50 ~1 of test serum or the standard solution, 100 ~1 of the diluted antibody ( x 2000) and 300 ~1 of 0.05 mol/l phosphate buffer, pH 7.8, containing 0.1% bovine serum albumin, and 50 ~1 of ‘251-labeled MG. Incubation was performed at room temperature for 12 h, and 500 ~1 of anti-rabbit gamma globulin solution containing 4% polyethylene glycol (PEG; No. 6000) was added. After incubation at 37 o C for 2 h, the precipitated radioactivity was examined after centrifugation at 3000 rpm for 30 min. The MG concentration of samples was expressed as U/ml. Test sample

The study population consisted of 321 patients with malignant diseases (lung cancer; 96 cases, pancreatic cancer; 40 cases, gastric cancer; 42 cases, colon cancer; 27 cases), 128 patients with benign diseases (chronic pancreatitis; 6 cases, gall stone; 4 cases, uterus myoma; 34 cases, endometriosis; 9 cases, others; 75 cases) and normal human sera 50 cases as control (male 25 and female 25). Determination

of tumor marker

alpha-Fetoprotein (AFP), CEA and SCC (squamous cell carcinoma) were determined by RIA kit of Eiken Co. (Tokyo, Japan), Roche Lab. (NJ, USA) and Dainabot Co. (Tokyo, Japan), respectively. CA 19-9, CA 12-5 and CA 15-3 were determined by RIA kit of CIS (France). Experiment

using anti-MC

coated bead

The bead was coated by incubation with 0.14 mg/ml of anti-MG (rabbit IgG purified by Protein A-Sepharose 4B) for 2 days, and 0.1% bovine serum albumin (BSA) was added to block the unsaturated part for 5 days, and then washed out by

49

phosphate buffer. This coated bead was incubated with MG standard or sera of malignant patients for 16 h at room temperature. After 3 times washing with phosphate buffer, pH 7.8 containing 0.1% BSA, the bead was incubated with ‘251-anti-CA 19-9, ‘251-anti-CA 12-5, ‘251-anti-CA 15-3 and ‘251-anti-sialylyl SSEA-1 (Otsuka Assay Lab., Tokyo, Japan), at 37 o C for 3 h. After 3 times washing with the buffer, the radioactivity of the beads was counted. Results Purification and characterization of MG

At the typical purification step each content of protein, and carbohydrate was shown in Table I. The typical elution pattern on Sepharose 4B column was shown in Fig. 1A. More than 50% of the neutral sugar and 66% of the immunoreactivity of MG in the applied sample were recovered in the void volume fraction. But the recovery of protein was < 10%. Further purification was performed by Sepharose 4B rechromatography (Fig. 1B). More than 80% and about 10% of CA 19-9 antigen in the applied sample were found in the void volume fraction and the fraction of mol wt of approximately 500 000, respectively. Changes of chemical composition of MG after WGA-Sepharose 4B affinity chromatography were shown in Table II. After this purification the increase of carbohydrate content, fucose and sialic acid content, and the decrease of protein content were observed. The purified component showed the positive reactivity with anti-CA 19-9, anti-blood group 0 antigen and anti-Lewis b antigen, but not with anti-human serum, anti-CEA, anti-AFP. Analysis of amino acid in the purified MG was shown in Table III. The predominant amino acids comprising the peptide core were serine, threonine, proline, alanine, glycine and valine. The serine and threonine residues was composed of nearly one-third of the total amino acids.

TABLE I Purification of mucin-like glycoprotein Purification procedure

Total protein ’ (mg)

Total neutral hexose a (mg)

Total sialic acid ’ (mg)

Total antigen b (U)

Specific activity D/mS protein

DEAE-cellulose Sepharose 4B (1st) Sepharose 4B (2nd) WGA

235.9 69.1 6.8 0.38

187.2 29.1 14.7 0.98

10.7 4.9 2.1 0.17

52 x104 41 x104 27 x104 1.65 x lo4

2.2 x 6.0 x 39.6 x 43.9 x

10s lo3 10’ 10s

’ Total protein, neutral hexose and sialic acid were determined by the method of Lowry [13], Seifer [14] and Jourdian (281, respectively. b MG was determined by IUA.

50

100

50 fraction

No.

Fig. 1. Gel-filtration of mucus glycoprotein (MG) using Sepharose 4B. A. The purified fraction by DEAE-cellulose was applied. B. Rechromatography of the void volume fraction eluted from Sepharose 4B. The elution position of the markers are indicated by arrows. a,-macroglobulin (820,000); Cl% (200,000).

SDS-PAGE of ‘251-labeled MG was performed under the reducing or the non-reducing conditions. As shown in Fig. 2, under the non-reducing condition most of radioactivity were in the stacking gel and a part of activity was found in the TABLE Chemical

II composition

Substance

Total hexose Hexosamine Glucosamine Galactosamine Fucose Siahc acid Sulfate Uranic acid Protein Lipid Others

of MG Chemical composition (gW) WGA affinity chromatography Before

After

19.7 16.7 11.3 5.4 1.3 0.6

24.5 33.9 27.5 6.4 8.3 4.3 0.9 nd. 9.4 n.d.

2.8 30.9 n.d.

51

TABLE

III

Amino

acid analysis

Amino

acid

of MG Amount

g/l~ Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine

in MG

g

nmol/mg 10.9 10.7 18.8 16.3 159.6 78.7 27.5 78.4 38.5 59.1 44.3 1.8 13.7 27.8 4.0 8.9

0.16 0.17 0.33 0.22 1.90 0.83 0.40 0.90 0.29 0.53 0.52 0.03 0.18 0.36 0.07 0.17

(Electrophoresis reducing

in the

presence

and

the

I 350K

-

150K

--_)

50K

Fig. 2. SDS-PAGE

abcence

of

agent) Stacking get

Separating

-

and autoradiography

of 12$I-MG. The dried gel was subjected after SDS-PAGE (5-12X).

to autoradiography

52

position of M, of about 350 kDa without other radioactive band. On the other hand, under reducing conditions larger amounts of radioactivity at the position of M, of about 350 kDa and a faint radioactive band of i’t4,of 50-100 kDa by dissociation of disulfide bond were observed. In immunoelectrophoresis (IEP) using the purified MG as antigen and antiserum a single immunoprecipitation arc with p mobility was observed (data not shown). In the Con A-binding experiment, 2.6% (67.3 U) of the total immunoreactivity (2 536 U) applied to Con A-Sepharose 4B column was retained. The immunologic stability of MG was examined by treatment with perchloric acid (PCA) and various enzymes. Treatment with protease, trypsin and pepsin resulted in remarkable loss (80-90%) of MG immunoreactivity. However, PCA extract, neuraminidase treatment and boiling for 5-30 min exhibited slight effect on the immunologic activity of MG. Clinical evaluation A typical standard curve of MG-RIA was shown in Fig. 3. This RIA had the sensitivity enough to detect 1 U/ml (about 250 ng/ml) of MG. When 3 different MG concentrations were assayed in 5 consecutive MG RIAs (over a period of 3 mth) the intraassay and interassay coefficient of variation (CV) were 7.1% and 9.2%, respectively. Clinical significance of MG determination was shown in Fig. 4. Serum MG levels of normal subjects ranged from 0 to 2.9 U/ml, with 0.8 f 0.7 U/ml (mean f SD). Thus, 3 U/ml corresponding to mean f 3 SD for healthy subjects were chosen as the cut-off value. The mean values of a serum MG and positive rates in 321 malignant diseases were found as follows; 4.4 U/ml and 32.3% in lung cancer, 20.1 U/ml and 77.5% in pancreatic cancer, 11.6 U/ml and 64.3% in gastric cancer, 12.9

1

2

345

10

20

304050

100

MG (U/ml) Fig. 3. A standard curve of MG using RIA. Each point represents mean f 2 SD.

53

1

_.5

D&eases

100

10

1 g 1 Heoatoma

1

/ Pancreatitis

1

i_---Other

Fig.

4. Serum

chr.

K>

benig2r

MG levels in various d&eases. The scxeen area denotes nomxaI range. (I, patients without clinically detectable metastasis; X, patients with metastasis.

Anti-MG solid sandwich method

CPM 2,ooo

15.6

M

i%Anti-sialyl

O.-O

‘“l-Anti-CA

SEA-1 19-9

-

‘“l-Anti-CA

12-5

M

‘“l-Anti-CA

i

31.3 62.5

125

250

Fig. 5. The biding of ‘251-siaIyl SEA-l, ‘*‘I-anti-CA 19.9, ‘ZSI-anti-CA 12-5 and ‘25f-anti-CA 15-3 with MG. The bead coated with anti-MG was incubated with MG standard, and then the bead was incubated with1231-anti-sialyl SSEA-1, anti-CEA 19-9, anti-CA 12-J and a&CA 15-3.

54

Anti-MG

I

CPM 2,000 i

solid sandwich

-

serum

of gastric

-

serum

of pancreatic

-

serum

of colon

method

cancer cancer

cancer

J

lJooo: II

X16X8

1

I

I

I

x4

x1-

x2

dilution

of test

serum

Fig. 6. The binding of iz51-anti-CA 19-9 with MG in the serum of mahgnant patients. Experimental procedure was the similar as Fig. 5. Sera of malignant patients were used instead of MG standard.

U/ml and 57.1% in hepatoma (primary hepatocellular cancer), and 12.3 U/ml and 77.8% in colon cancer. Increased MG levels (> 3 U/ml) were observed frequently in patients with metastasis. However, the normal MG levels in patients with metastasis were fairly observed. On the other hand, serum MG level of 90% (115/128) in benign diseases was under the cut-off value. Thirteen false positive cases were uterus myoma (2 cases), endometriosis (2 cases) and others (9 cases included liver cirrhosis and diabetes mellitus). Existence of the epitope recognized by MoAbs for various tumor associated carbohydrate antigens into the carbohydrate chain of MG was examined by TABLE IV Serum MG and CA 19-9 levels in Lewis negative patients with malignant tumor No.

Diseases

CA 19-9 (U/ml)

MG (U/ml)

1 2 3 4 5 6 I 8 9 z

Gastric ca Hepatoma Lung ca Gastric ca Colon ca Gastric ca Gastric ca Gastric ca Tongue ca

< 5.0

13.9 8.7 16.5 19.0 2.1 10.6 5.2 0.1 15.6 10.3

< < < < < < < < <

5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0

55

anti-MG solid sandwich method. ‘251-anti-CA 19-9 and ‘251-anti-sialyl SSEA-1 showed positive binding with MG molecule, but ‘251-anti-CA 12-5 or ‘2SI-anti-CA 15-3 showed negative binding (Fig. 5). ‘251-anti-CA 19-9 showed also positive binding with MG molecules in sera from gastric cancer, pancreatic cancer and colon cancer (Fig. 6). In Lewis negative malignant patients comparison of serum CA 19-9 with serum MG levels was shown in Table IV. Serum CA 19-9 levels of 5 gastric cancers and other malignants were c 5 U/ml. On the other hand, serum, MG levels were significantly elevated in many cases of these malignant patients. Discussion

Several studies have indicated that antigenic determinant site of a certain group of the tumor-associated carbohydrate antigen detected by MoAbs may be the carbohydrate portions of mucin-like glycoprotein < lo6 Da [23-271. The increased production of mucin-like glycoprotein from carcinoma cells was suggested from markedly increases of these carbohydrate antigens in sera of various cancers [23-271. In the present study we purified mucin-like high molecular weight glycoprotein from the pleural fluid in a patient with lung adenocarcinoma, and investigated clinical significance of the glycoprotein as tumor marker by the RIA using the polyclonal antibody. Final purification method using WGA affinity chromatography which possesses specificity for N-acetylglucosamine residue and a terminal sialic acid residue was effective. The chemical composition analysis of the isolated protein was composed of about 65% of carbohydrate (24.5% neutral hexose, 33.9% hexosamine and 4.3% sialic acid) and small amounts of peptide including serine, threonine, proline, alanine, glycine and valine as the predominant amino acids. This glycoprotein did not bind with Con A. These facts indicate that the purified glycoprotein is a mucin-like glycoprotein in which the linkage of the oligosaccharides to the peptide core is 0-glycosidic as shown in trachea-bronchial origin [28,29]. The M, of MG was thought to be over lo6 Da as shown by gel-filtration. The analysis of SDS-PAGE revealed that a major band of M, about 350 kD and a faint band of M, of 50-100 kD were detected under the reducing condition. Pearson et al. [30] reported that the protein of M, 70 kD was the constituent part of pig gastric mucous glycoprotein through disulfide bridges. The appearance of a low molecular weight protein after reducing a mucous glycoprotein is found often. Tumor associated mucous antigens such as POA by Gelder et al [31] and PCAA by Shimano et al. [32] are thought to possess characteristic similar as MG. However, MG was different from these glycoproteins in the point of the carbohydrate content, heat-stability, PCA extraction and Con A binding ability. Interestingly, existence of both CA 19-9 and sialyl SSEA-1 antigen was found on the carbohydrate chains of MG by the experiment of 12%labeled anti-tumor associated carbohydrate antigen binding with MG in sera of malignant patients. Kannagi et al. [35] have reported that circulating mucous glycoprotein could be

56

classified into several groups by PCA treatment method of serum. MG is thought to be one of mucous glycoproteins bearing CA 19-9 and sialyl SSEA-1 antigen. Clinical significance of MG as tumor marker was examined by the RIA using the double antibody method. False positive cases in benign diseases were observed in 108, especially gynecological diseases and liver cirrhosis. The significant increase and high positivity were observed in many malignancies, especially the digestive organ cancer. Although the increased MG levels were observed highly in patients with metastasis, the normal MG levels were observed fairly in patients with metastasis. Thus, the detection of cancer in early stage seems to be difficult. The tendency of high levels in the digestive organ cancer was similar to that of tumor associated carbohydrate antigens such as CA 19-9, CA-50, DUPAN-2 and etc [7-11,33,34], except the incidence of abnormal high levels in lung carcinoma of sialyl SSEA-1 and also in lung carcinoma, breast cancer of Le’, LeY antigens [35]. Koprowski et al. [36] reported that Lewis negative cases can not synthesize Lewis antigens for lack of the fucosyltransferase. Thus, no increase of serum CA 19-9 levels in Lewis negative malignant cases was suggested. In the present experiment serum CA 19-9 levels of Lewis negative malignant patients were completely negative. This fact and enzyme susceptible experiments of MG indicate that the polyclonal antibody for MG may be mainly produced for the peptide core of MG. Thus, measurement of MG in cancerous states by the RIA using the polyclonal antibody is thought to show mainly the quantitative change in circulating blood. On the other hand, determination of tumor associated carbohydrate antigen recognized by MoAbs is thought to show mainly the qualitative changes of mucin-like glycoprotein. Further study of purification of the antigen and MoAb for MG may need to improve the sensitivity and specificity as the tumor marker. Acknowledgments

We thank Mr. Hamazu, M (Depart. of Radiology) for sample separation Miss Murai, K. and Mrs. Ishizumi, T. for assistance with the manuscript.

and

References 1 Gold DV, Miller F. Comparison of human colonic mucoprotein antigen from normal and neoplastic mucosa. Cancer Res 1978;38:3204-3211. 2 Boland CR, Montgomery CK, Kim YS. A cancer-associated mucin alteration in benign colonic polps. Gastroenterology 1982;82:664-672. 3 Pant KD, Shochat D, Nelson MO, Goldenberg DM. Colon-specific antigen-p (CSAp). Cancer 1982;50:919-926. 4 Kawaswki H, Kimoto E. Mucosal glycoproteins in carcinoma cells of gastrointestinal tract, as detected by immtmofluorescence. technique. Acta Path01 Jpn 1974;24:481-494. 5 Goldenberg DM, Pant KD, Dahlman HL. Antigens associated with normal and malignant gastrointestinal tissues. Cancer Res 1976;36:3455-3463. 6 Boland CR Ahnen DJ. Binding of lectins to goblet cell mucin in malignant and premalignant colonic epithelium in the CF-1 mouse. gastroenterology 1985;89:127-137. 7 Koprowski H, Steplewski Z, Mitchell K, Herlyn M, Herlyn D, Fuhrer P. Colorectal carcinoma antigens detected by hybridoma antibodies. Somatic Cell Genet 1979;5:957-972.

8 Lidholm L, Hohngren J, Sevennerholm L, et al. Monoclonal antibodies against gastrointestinal tumor-associates antigens isolated as monosialogliosides. Int Arch Allergy Appl Immunol 1983;71:178-181. 9 Bast Jr RC, Freeney M, Lazarus H, Nadler LM, Colvin RB, Kanapp RC. Antibody with human ovarian carcinoma. J Clin Invest 1981;68:1331-1337. 10 Hilkens J, Buijs F, Hilgers J, et al. Monoclonal antibodies against human milk-fat globule menbranes detecting differentiation antigens of the mammary gland and its tumors. Int J Cancer 1984;34:197-206. 11 Metzgar RS, Gaillard MT, Levine SJ, Tuck FL, Bossen EH, Borowitz MJ. Antigens of human pancreatic adenocarcinoma cell defined by murine monoclonal antibodies. Cancer Res 1982;42:601-608. 12 Watanabe W, Hirohashi S, Shimosato Y, et al. Carbohydrate antigen defined by a monoclonal antibody raised against a gastric cancer xenograft. Jpn J Cancer Res (Gann) 1985;76:43-52. 13 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-275. 14 Seifer S, Dayton S, Novic B, Muntwyler E. The estimation of glycogen with the anthrone reagent. Arch Biochem Biophys 1950;25:191-200. 15 Gibbons MN. The determination of metylpentoses. Analyst 1955;80:268-276. 16 Garde11 S. Separation on dowex 50 ion exchange resin of glucosamine and glactosamine and their quantitative determination. Acta Chem Stand 1953;7:207-215. 17 Ludowieg J, Benmaman JD. Calorimetric differentitation of hexosamines. Anal Biol Chem 1967;19:80-88. 18 Jourdian GW, Dean L, Roseman S. The sialic acids. J Biol Chem 1971;246:430-435. 19 Bitter T, Muir HM. A modified uranic acid carbazole reaction. Anal Biochem 1962;4:330-334. 20 Terho TT, Hartiala K. Method for determination of the sulfate content of glycosaminoglycana Anal Biohem 1971;41:471-476. 21 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-685. 22 Bolton AE, Hanter WM. The labelling of proteins to high specific radioactivities by conjugation to a ‘251-containing acetylating agent. Biochem J 1973;133:529-539. 23 Magnani JL, Steplewski 2, Koprowski H, Ginsburg V. Identification of the gastrointestinal and pancreatic cancer-associated antigen detected by monoclonal antibody 19-9 in the sera of patients as a mucin. Cancer Res 1983;43:5489-5492. 24 Sekine H, Ohno T, Kufe DW. Purification and characterization of a high molecular weight glycoprotein detectable in human milk and breast carcinomas. J Immunol 1985;135:3610-3615. 25 Lan MS, Finn OJ, Fernsten PD, Metzgar RS. Isolating and properties of a human pancreatic adenocarcinoma-associated antigen, DU-PAN-2. Cancer Res 1985;45:305-310. 26 Hanish FG, Uhlenbruck G, Dienst C, Stottrop M, Hippauf E. CA 125 and CA 19-9: two cancer-associated sialylsaccharide antigen on a mucus glycoprotein from human milk. Eur J Biochem 1985;249:323-330. 27 Lan Jr MS, Bast RC, Colnaghi MI, et al. Co-expression of human cancer-associated epitopes on mucin molecules. Int J Cancer 1987;39:68-72. 28 Roberts GP. Isolation and characterisation of glycoprotein from sputum. Eur J Biochem 1974;50:265-280. 29 Feldhoff PA, Bhavanandan VP, Davidson EA. Purification, properties, and analysis of human asthmatic bronchial mucin. Biochemistry 1979;18:2430-2436. 30 Pearson JP, Allen A, Parry S. A 70000-molecularweight protein isolated from purified pig gastric mucus glycoprotein by reduction of disulphide bridges and its implication in the polymeric structure. Biochem J 1981;197:155-162. 31 Gelder FB, Reese CJ, Moossa AR Hall T, Hunter R. Purification, partial characterization, and clinical evaluation of pancreatic oncofetal antigen. Cancer Res 1978;38:313-324. 32 Shimano T, Loor RM, Papsidero LD, et al. Isolation, characterization and clinical evaluation of a pancreas cancer-associated antigen. Cancer 1981;47:1602-1613. 33 Metzgar RS, Rodriguez N, Finn OJ, et al. Detection of a pancreatic cancer-associated antigen (DU-PAN-2 antigen) in serum and ascites of patients with adenocarcinoma. Proc Nat1 Acad Sci USA 1984;81:5242-5246.

58 34 Koprowski H, Herlyn M, Steplewski Z. Specific antigen in serum of patients with colon carcinoma. Science 1981;212:53,54. 35 Karmagi R, Fukushi Y, Tachikawa T, et al. Quantitative and qualitative characterization of human cancer-associated serum glycoprotein antigens expressing fucosyl or sialyl-fucosyl type 2 chain polylactosamine. Cancer Res 1986;46:2619-2626. 36 Koprowski H, Brockhaus M, Blaszczyk M, Magnani J, Steplewski Z, Ginsburg V. Lewis blood-type may affect the incidence of gastrointestinal cancer. Lancet 1982;1:1332-1333.