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Evaluation of tumor-associated antigen (2H6 antigen) in detecting early stages of gastric cancer
Clilh
Clliea
Acts 228 (1994) 101-112
XF
Evahation of tumor-associated antigen (2H6 antigen) in detecting early stages of gastric cancer
AsaodwicbcoqmaI-liokcdionnuoosor beat assay(ELBA) wasdevelopedby usingmonoclonal antibody2H6 (2H6 MoAb). MoAb 2H6 could be usedto detect the 2H6 antigenin the em of severalauxcra, showingpositiveratesof 65.4%,66.7%.47.4%.SOB%,45.2%and 16.7%in gastriccancer.hepatoahlar carcinoma,cancersof thecolon,esophays, breastand m rerpsctively.On the other hand, the positive rates in benigndii or hepIthy donon were 3.4%,3.4%,4.2%,8.3%.6.9%,and 1.2%in myasthoia gmvis, polymyositis, gastritis,gastricuker, oth benigndireraa and normalhealthydonors,ra~pcctively.Fiftytwo patientawith gastriccancerw investigatedin detail.The positiveratesof serum2H6 antigen,CE& AFP, CAW9 and CA125 in patientawith gastriccancerwere 65.4%,9.W’ 2.3%,25.0%and 8.1% rapechly. Among thesetumormarkers,serum2H6 antigenkvels aloDewcrt aignifiitIy ekvatcd in patientswith earlystage(I andII) gastriccancer.Furthermore, combiningthe mewnxunt for merum 2H6 with CA19-9 increasedthe percentageof gastriccancerslightly.No correlationbctwcco2H6 antigenand theseother tumor markers was obecrved.The acrnmkv& of 2H6 antigenwere monitoredpost-sqically in 1I patients for12mekrondtbey~foundtodiminilhgradually.Theftndin~~suggertthatthemeasuremntof~2H6~ti~lrraybeau#fulmarlrerforan~y~~oflFastriccawer. Akinwiolkml: CEA, cudaoanbryonic antipn; CHS. choksterol hemisuccinatc; ELBA, mzymcdlinked immunoaodmnt anay; MoAb. monoclond antibody. lConerpading ruthof. OM9.8981/94&37.00 8 1994 Ekvk SSDI 0009-898 1(94)05865-P
Science B.V. All righta rwavcd
102
H. Harada et al. / Clin. Chim. Acta 228 (1994) 101-112
Keyword: Moneclonalantibody 2Hb; 2Hb antigen; Early gastriccancer;Tumotu mark !krological diagnosis
1. Intrluh&ion Gastric cancer is one of the most common cancers worldwide and in Japan it is still the leading type of cancer despite a declining trend [ 1,2J.The early stage of gastric cancer is often complicated and extremely difficult to diagnose. The develop ment of additional methods for diagnosis, is being influenced by the desire for efficient, non-invasive diagnostic procedures such as the identification of serum markers specifically associated with patients with gastric cancer. Carcinoembryonic antigen (CEA) and CA19-9 are widely used as serum markers for the diagnosis of gastric cancer. Analysis has shown that serum levels of CEA and CA19-9 are elevated in patients with gastric cancer [3-61. In a recent study, TAG-72 was found to be elevated in the serum of patients with primary gastric cancer [7,8].These serum markers are limited to use in the early diagnosis of gastric carcinoma [7,9- 111.Stein et al. [12] and Oldham and Herberman [13] reported that the immunogenicity of tumor cells is typically weak and has no clinically useful potential. Shinitxky and colleagues reported that augmentation of the immunogenicity of the tumor cells could be achieved by increasing their membrane microviscosity by the incorporation of cholesterol or its hydrophilic esters and that cholesterol bemisuccinate (CHS)treated tumor cells promoted the expression of latent tumor associated antigen [ 14,151. We hence attempted to produce a new monoclonal antibody which recognizes the early stage of cancer by immunixation with CHS-treated MKN-45 cells. Monoclonal antibody (MoAb) 2H6 was established by immunization with CHS-treated MKN-45 cells. The 2H6 antigen recognized by MoAb 2H6 was a glycoprotein consisting of several molecular species: 250,000, 75,000,68,009 and 50,000 V61. We investigated by testing with MoAb 2H6 whether various cancers in the early stage can be detected from serum, especiahy in serum from patients with gastric cancer. The present study evaluated the presperative serum levels of 2H6 antigen, CEA, CA19-9 and AFP, alone or in combination, in patients with cancer or benign disease. The findings suggest the potential utility of the 2H6 assay in detecting serum 2H6 antigen for the diagnosis of early stage gastric cancer. 2.MateriaIsatldmethods 2.1. Preparationof MoAb CHStreated MKN-45 cells were prepared by a modifmtion of the methods of Skomick et al. [17]. Establishment and purification of MoAb 2H6 were performed as described by Harada et al. [ 161. 2.2. 2H6 antigen preparation To prepare the standard antigen for sandwich enxymed-linked immunosorbent
assay (ELISA) a detergent extract of 1 x 10’ MKN-45 cells was collected. For the
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et al. /Ch.
Chk. Acta 228 (1994) 101-112
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extraction of cell membrane proteins, ext~tion buffer intoning 50 mM Tris-HCl (pH 7.5), 0.15 M NaCl and 0.5% Nonidet P-40 (Nakarai Co, Lad., Tokyo) was used. The crude extracts were adjusted to a concentration of 1 mghnl in 50 mlb4Tris-NC1 containing 0.15 M NaCl and stored at -8OOCuntil assayed. 2H6 antigen-containing crude extracts were stable for at least 6 months. Crude extracts of 0.1 m&l represent 1,000units/ml. Crude extracts of 0.1 mg/ml corresponded to a ~n~ntration of a~ro~~tely 0.5 &ml of the 2H6 antigen purified from serum. 2.3. Sandwich ELISA Serum levels of 2H6 antigen were determined by a sandwich ELISA. 96Well plates (Nunc, 442402) coated with 10 &ml of 2H6 MoAb were incubated at 4OC overnight. The wells were then blocked with 50 n&i Tris-HCl (PH 7.5) containing 1% BSA, 0.15 M NaCl and 0,050/oTween 20, designated as the assay buffer, for 2 h at room temperature. One hundred microliters of standard antigen diluted 2-fold with assay buffer or patient’s serum diluted 5-fold with assay buffer were added to the wells and the plates were incubated for 3 h at room temperature. One hundred microliters of 2,000-fold diluted biotin-conjugated anti-2H6 MoAb were added and then the wells were incubated for 2 h at room tem~~t~. Biotinylation of 2H6 MoAb was performed by a modification of the method of Guesdon et al. [18]. One hundred microliters of 2,000-fold diluted peroxidase conjugated with streptavidin (Tago, 4025)were added and then the wells were incubated for 1.5 h at room temperature. Two hundred microliters of o-phenylenediamine solution were added and the wells were incubated for 20 min at room temperature. The enzyme reaction was stop ped with 50 &well of 5 N H#O., and absorbance at 492 nm was measured on a microplate reader (Dynatech, MR 5000). Serum 2H6 antigen levels (units/ml) in 5fold diluted samples were determined from a standard calibration curve. 2.4. CEA, CA19-9, CA125 and AFP radioimmunoassay Serum levels of CEA were determined with the CEA RIA BeadsfM Kit (Dainabot Co,, Tokyo, Japan). CA19-9 and CA125 serum levels were measured, respectively, with a CA19-9 and CA125 RIA Kit (Centocor, Malvern, PA). AFP serum levels were determined with alpha-Feto RIA BeadsTM(Dainabot Co., Tokyo, Japan). 2.5. Human ser4 Serum from human patients was kindly provided by Kanaxawa U~ve~ty (Cancer Res. Inst.). Serum samples from 83 healthy donors (50 males, 33 females; aged 21-56 years) and 353 patients (166 males; 187 females; aged 18-78 years) were examined. The panels of patients’ sera included 58 from patients with autoimmune diseases (myasthenia gravis, 29; polymyositis, 29), 95 from patients with benign gastric disease (gastritis 71; ulcer, 24) and 58 from patients with other benign diseases (duodenal ulcers, 9; chol~~holi~i~s, 8; chol~o~t~~s, 8; polyps of colon, 8; chronic hepatitis, 8; liver cirrhosis, 8; diabetes mellitus, 9). Sera obtained from malignancies included 71 from patients with gastric cancer, 9 from patients with hepatocellular carcinoma, 19 from patients with colon cancer, 5 from patients with w of the esophagus, 31 from patients with breast cancer and 7 from patients with cancer of the pancreas. Fifty-two of 72 with malignant gastric d&eases were
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101-112
pathologically staged: etage I (n = 28), stage II (n = 5), stage III (n = 6), stage IV (n = 13).Serum samples were obtained within 12 weeksprior to surgery.All sgdcs were stored at -40°C until assay. Assay performed was in a blind fashion. 3. Raladta 3.1. Standard curve of sandwich ELBA using 2H6 MoAb The extracts obtained from MKN-45 cells were serially diluted, and the levels of 2H6 antigen were determined by a sandwich ELISA using 2H6 MoAb. As shown in Fig. 1, the value of Am corresponded to the dilution rate of the extracts. The assay was detectable within the range of 8-500 units/ml. In&a-assay coefIicients of variation for the standard ranged from 3.5 to 8.4O/o, indicating s&kient reproducibility (data not shown). These results showed that 2H6 antigen levels in sera could be quantitatively measured by this sandwich ELISA. 3.2. Assay of sewn levels of 2H6 antigen in patients and normal controls The 2H6 levels in sera of the healthy donors, patients with carcinoma and those with various benign diseases were determined by sandwich ELISA. As shown in Fig. 2, only one of the test samples from the 83 healthy donors showed a serum level of
2
10’
1d
2Ii6 lntlgon (U/ml) Fig. 1. Standard owva of undwbb
ELISA udqj 2H6 MoAb. The 2H6 antiSen lmd,
of toriallydihttad
2Ii6 antigen in excess.of 300 ~~~. The mean and standa& d~iation from normal heaIthy donors was 140.6 f 163.7 unit&l. From this result wt classified cases with 2H6 levels above 300 units/ml as 2H6 antigen positive. 2H6 antigen levels were elevated in 60.6% in the sera from patients with various cancers. Among patients diagnosed with carcinomas, 65.4%, 66.7?&,47,40/o,80.~~, 45.2% and 16.7% of the sera from patients with gastric cancer, hepatocellular carcinoma, cancers of the colon, esophagus, breast and pancreas had elevated serum 2H6 antigen levels (> 300 units/ml), respectively. In contrast, 5.2% of the sera from patients with various
l/b3 (1.2%)
34152 i (65.4%) I ,
Fig. 2. Senun Icvclsof 2H6 antigen in patientswith variouscancerdiseasesand healthy donors.The 2H6 antigenassay was perfomwdas describedin Materials and methods.The straightline denotes the cut-off k&s ofZH6 antigen(300 units/ml).Ca., c8aar, Upperordiite, number~~n~~ tested(positive ratio).
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bedpdi~hadeiev~2H62n~lewls.Amoagpotiantawith~ cIimams, 3.4%,3.4%,4.2%,8.3%and6.9?Aof the
sera from patients with myasthenia gravis, polymyositis, gastritis, gastric ulcer and gastrointestinal disease (pancreas, liver, colon) showed slightly positive results (Fig. 3). 3.3. Serum 2H6 levelsinpatientswithvariousstagesof gastriccancer The relationship between serum 2H6 levels and the clinical staging of 52 patients with gastric cancer were evaluated (Fig. 4). The positive rates summa&d according to stage were as follows: stage I, 57.1%; stage II, 60.0%; stage III, 83.3%; stage IV, 76.9%. In particular, relatively high serum 2H6 levels were found in patients diagnosed with early stage (I) gastric cancer. The positive rates observed in the advanced stages (III and IV) were higher than those observed in the relatively early stages (I and II).
1000
1000
5 f
200
900
. I i: t. .. . .
B
1
100
100
! . ..
10
: Yyatonlr
Polymyoath’
10
I
Oartritlr
Qartrlc Ulcer
Othorr
Qrrvlr Fi~3.~bwlrof2H6ra~inprtiontrwithvrriour~dicarec,Ths2H6oati~nuuywu performed u dadbed in Fig. 1.
H. Haraab et al. /Clin. Chim. Acta 228 (1994) 101-112
107
3.4. Comparison of various tumor markers in patients with gastric cancer The relationships between various tumor markers (2H6 antigen, CEA, CAl9-9,
AFP and CA123 and the clinical stage of the patients with gastric cancer were evaluated (Table 1). Positive serum 2H6 levels were found in a higher percentage of patients with various stages of gastric cancer than were those of CEA, CAl9-9, AFP, or CA125 In particular, serum 2H6 was elevated in the highest percentage of patients with early stap (I) gastric cancer. On the other hand, elevated serum CAl9-9, CEA and CA125 were observed predominantly in serum samples from patients with advanced stage (III and IV) gastric cancer. When the levels of CAl9-9 and 2H6 were analyzed in relation to gastric cancer,
3/s BO.O%
516
IO/13
B3.W
r6.BV.l
- 100
10 1
10
I
II
m
IV
Qartric Cmcor 6trgo Fig. 4. Serum Ievclc of 2H6 antigen in patient8 with various stages of gastric cancer. The assay was perfornwd 91 de&bed in Fig. 1. The dashed line denotes the cut-off levels of the 2H6 antigen. Upper ordinate, number positivdnumbcr tested (positive ratio).
IO8
H.
H~&I
et al. I Cfin
Chbn. Acta 228 (NM)
101-112
Table 1 Comparison of the various tumor marker levels at each stage in patients with gastric cancer Stage
SeNm antigen kveIs g
I II III IV Total
uliiMml)a
16128 (57.1)b 3i5 (60.0) S/6 (83.3) IW13 (76.9) 34152(65.4)
CFJA (5 n@llI)
(20 Wmt)
AFP
CAl9-9 (37 wits/mI)
CA125 (SO units/ml)
I/28 (3.6) W5 (0) l/6 (16.7) 303 (23. I) 5J52 (9.6)
W20 (0) l/5 (20.0) W6 (0) WI2 (0) 1143(2.3)
3/2a (10.7) W5 (0) 4i6 (66.7) 6fl3 (46.2) 13152(25.0)
0119 (0) W2 (0) W5 (0) 3111 (27.3) 3127(8.1)
‘cut-off vahles. bNumben in parentheses: %.
2H6 levels were positive in 25 of 39 (64.1%)cases of C&9-9 negative serum and 2H6 levels were negative in 3 of 13 (23.1%)cases of CAM-9 positive serum. No significant correlation was recognized between 2H6 and CA199 levels in gastric cancer (Y = 0.054X + 27.76, r = 0.167, n = 52; X = 2H6, Y = CA199). Similarly, no correlation was seen between 2H6 antigen and CEA levels (Y = 0.001X + 3.20, r = 0.084, n = 52; X = 2H6, Y = CEA). Another analysis was done using combinations of 2H6, CA199, CEA, AFP and CAl25. Table 2 summa&es the percentages of patients with gastric -cancer(n = 52) whose serum samples contained either 2H6, CEA and/or CA199. Serum 2H6 (65.4%)alone was found in a higher percentage of patients with gastric cancer than either CEA (9.6%) or CA19-9 (25.0%) alone. Combining serum 2H6 measurement with CA19-9 increased the percentage of positive serum samples to 73.1%. However, no increase in the percentage of positive serum samples was observed when rn~~~t of 2H6 antigen was combined with CEA, or all three markers. 3.5. Serum 2H6 levels of patients with variom cancers before and after surgical treatment Serum 2H6 levels were measured in 11 patients with various cancers on one occa-
sion each before and after surgical treatment. No change in the serum 2H6 antigen Table 2 Summary of 2H6, CEA and CAl9-9 serum levels in patients with gastric cancer
Gastriccancer
2H6 CEA CA19-9 2H6 + CEA 2H6 + CA19-9 CEA +CAlP9 All markers ~Numbersin parentheses: %.
No. of oatients”
% of oatients*
34l52 5152 13152 34l52 38152 15152 38152
65.4 9.6 25.0 65.4 73.1 28.8 73.1
H. Muadaetd. ICiin. Chim.Aria 228(1994)Nl-If2
109
level was observed within 1 week tier surgical treatment. However, serum 2H6 antigen level tended to decrease slightly within 2-4 weeks, and it fell below 300 units/ml at 3 months. This indicated that the serum 2H6 level tended to decrease gradually from 4 weeks (Fig. 5). 4. DIscuwb The present study found that 2H6 antigen could be detected in sera from patients with various malignant diseases. Appro~a~ly, 60.6% of the sexa from patients with various carcinomas had elevated 2Ii6 antigen levels (> 300 undo). IP contrast, the false-positive rates in healthy donors and patients with benign disease were
2
432101 Pro Op..
Op..
3
4
s
3
7
Port Ope.
3
0
10 11 12
(W0.k~)
Fii 5. Swum kvels of 2H6ant&n beforeandafterthesurgical treatmentin patientswith gastriccancer.0, patientserumobtainedfroma pre-surgical treatment; l, packntacrumobtainedfrompostsurgical tleatmem.
110
H. Horada et rri./UittChim. Acta 228 (NH)
iOf-
relatively low, in the range of 1.2-8.3%. The false-positive rate in pregnant women was also very low in 1 of 28 (3.S”), indicating that 2H6 antigen dit&rs from oncof&talantigens like AFP (data not shown). The data indicated that it was possible to set a cut-off level of 300 aide to make a rough ~stin~on between mabgnant and benign disease. When the cut-off level is set at 400 units/ml, serum 2H6 antigen is clearly able to discriminate between carcinoma and benign disease. However, the positive rate of 2H6 antigen in patients with various carcinomas was decreased slightly, whereas none of the patients with benign diseases exceeded this level. Furthermore, it needs to be determined whether patients with gastric ulcer diseases who have inbox levels of 2H6 antigen in serum may constitute a high risk group. The results suggest that the assay of 2H6 antigen is useful for the serological diagnosis of carcinoma. Analysis of gastric cancer showed relatively high positive rates of serum 2H6 antigen. The positive rate of 2H6 antigen in patients with various stages of gastric cancer was stage dependent. In particular, appro~ately 60% of patients with early stage (I) gastric cancer showed positive serum levels of 2H6 antigen. Serum markers such as CEA and CA19-9 were slightly elevated in the early stages of gastric cancer. Similar results were obtained for AFP and CA125. These data indicate that serum 2H6 antigen is superior to CEA and CA19-9 in identifying the early stage of gastric cancer. In addition, no correlation was observed between the serum level of 2H6 antigen and the tumor markers tested (CA19-9, CEA, CA125 and AFP). It is unlikely that the mechanism by which 2H6 antigen increases in the sera of patients with carcinomas is related to that of CEA or CA19-9. CEA and CA19-9 are widely used as serum markers for the diagnosis of gastric cancer [ 191.Recently, TAG-72 was also reported to be a useful serum marker for the diagnosis of gastric cancer [7]. TAG-72 may be a possible serum marker for advanced stage (stages III and IV) gastric cancer. Goi et al. [20] and Bast et al. [21] reported that AFP-pr~u~g gastric carcinoma and gastrointestinal carcinoma could be slightly detected. Our results agree with reports that these tumor markers are difficult to detect in the early stage of gastric cancer. Thus, 286 may be a useful serum marker for the early diagnosis of gastric cancer, Recent findings have suggested a ~mplemen~ty among various tumor markers that may be exploited in the diagnosis of gas~ointestin~ carcinoma 171.Complementarity might increase sensitivity with respect to clinical diagnosis. The present data showed that elevated serum 2H6, CEA and CA19-9 levels in patients with gastric cancer appeared at rates of 65.4%, 9.6%, and 25,0%, respectively. Approximately, 35% of gastric cancer patients do not contain measurable serum levels of 2H6. Combining the me~~ment of serum 2H6 antigen with CA19-9 slightly increased the rates of detection of gastric carcinoma to 73.1% (stage I: 64.2% (18/28), stages III, IV: 89.5% (17/19)). It is suggested that the measurement of 2H6 antigen would be most useful in complementing CA19-9 for the serodiagnosis of gastric carcinoma. Additional studies with large numbers of samples are needed to investigate further whether the analysis of multiple serum tumor markers including CA72-4 may be ~v~~g~us in the batons of carcinomas, including gastric cancer. One of the important applications of any serum marker is its ability to be used
H. Harada et al. /Clin.
Chim. Acta 228 (1994) 101-112
Ill
in the msrnrremcnt of serum tumor antigen for predicting the clinical course of malignant disease. In the present study, serum samples obtained from 11 patients were assayed before and after surgical treatment on one occasion between 0 and 12weeks. Each patient had a serum level of 2H6 antigen in excess of 300 units/ml before surgery. The serum 2H6 antigen level tended to decrease slightly within 4 weeks after surgery and its level decreased significantly after 12 weeks. The serum CA125 level was significantly decreased at 24 h after surgical treatment (data not shown). Thus, the decrease in the 2H6 antigen level is different from that of CA125. Further study is needed to determine whether patients with clinically confirmed disease have elevated serum 2H6 levels at recurrence. When gastric cancer sections were stained with 2H6 antibody, the intercellular fluid was weakly positive or negative (data not shown). Elevation of the serum 2H6 antigen level in patients with carcinoma is not consistent with the results from tissue sections, suggesting that 2H6 antigen is not derived from carcinoma tissue. 2H6 antigen in sera may be a unique glycoprotein indicating the initiation of cancer. While additional studies with large population groups and different population bases are needed, these findings indicate that the serum 2H6 antigen level may be a useful marker for the clinical diagnosis of early stage with carcinoma, particularly gastric cancer. Referemces Muir C, Waterhouse T. Mack T, Powell J, Whelen S. Cancer incidence in five continents. Vol. V. JR Scientific Publication No. 88. IARC, Lyon, 1987. Kurihara M, Aoki K, Hisamichi S. Cancer mortality statistics in the world 1950-1985. The University of Nagoya Press, Nagoya, 1989. Hedin A, Carlsson L, Berghmd A, Hammarstrom S. A monoclonal enzyme immunoassay for serum CEA with incrca.sedspecificity for carcinoma. Proc Natl Acad Sci USA 1983;80:3470-3474. Koga T, Kano T, Souda K, Oka N, Inokichi K. The clinical usefulness of preoperative CEA determination in gastric cancer. Jpn J Surg 1987;17:342-347. Ritta Jr. RE, de1Villano BC, Go VL, Herberman RB, Klug TL, Zurawki Jr. VR. Initial clinical evaluation of an immunoradiometric assay CA19-9 using the NC1 serum bank. Int J Cancer 1984;33:339-345. 6 Herlyn M, Scars HF. Steplcwski Z. Koprowski H. Monoclonal antibody detection of a circulating tumor-associated antigen. I. Presence of antigen in scra of patients with colorcctal, pstric, and pancreatic carcinoma. J Clin Immunol 1982;2:135-140. Guadagni F, Roselli M, Amato T et al. CA72-4 measurement of tumor-associated glycoprotcin 72 (TAG72) as a serum marker in the management of gastric carcinoma. Cancer Res 1992;52:1222-1227. Byrne DJ, Browning M-CK. Cusieri A. CA-724 a new tumor marker for gastric cancer. Br J Surg 1990,77:1010-1013. Sikorska H, Shuster J, Gold P. Clinical applications of carcinoembryonic antigen. Cancer Detect Prev 1988;12:321-355, Sipponen P, Liidgren J. Sialylated Lewis’ determinant CA19-9 in benign and malignant gastric tissue. Acta Pathol Microbial Immunol Stand Select A 1986;94:305-311. Mach J-P, Jaeger P, Berholct M-M. Detection of recurrence of large-bowel carcinoma by radioimmunoasaay of circulating carcinocmbryonic antigen (CEA). Lancct 1974;7:535-540. Stein JA, Adler A, Goldfarb AJ, Czemobilsky B. Direct endolymphatic instillation (ELI) of BCGtumor cell mixture. A method for active immunization of cancer patients. Cancer Immunol Immunother 1978;5:31-39.
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hnmunity to tumor-asso&ed a@pns. Im13 Oldham RK, Herberman RR In: Ceil--ted rnudhgaob of cancer, Vol. 7, Part 5 New Yorkz Marcel Mtker, 1979$40-963. 14 Shinitzky M, Skomick Y. Haran-Chera N. RfYectivetumor immunization induced by cells of elevated membrane-lipid microviscosity. Proc Natl Acad Sci USA 1979;76:5313-5316. 15 Skomick YG, Rong OH, Sindelar WF et al. Active immunotherapy of human solid tumor with autologous cclls treated with choiesteryi hemisucoinate. Cancer 1986;58:650-654. 16 Harada H, Kubota T, Tsukada Y, Karasawa Y. Chracteriration of tumor-associated antigen (2H6 antigen) da&ii by monoclonal antibody 2H6. Clin Chim Acta 1994$CA 5864. 11 Skomick Y, Dmedale AR, Sindelar WF. Induction of delayed hypermmitivity reactions in cancer patients by cholesterol-hemituccinate-treated autologous tumor cells. J Natl Cancer Inst 1983;70:465-467. 18 Gueston JL. Temynek T, Arramas S. Use of avidin-biotin interaction in intmunoenaymatic techniques. J Histochem Cytodm 1979;27:1131-1139. 19 Goldenberg D, Neville AM, Cortes AC et al. Carcinoembryoaic antigen; its role as a marker in the measurement of cancer. A National Institutes of Health Consensus Development Conference. Ann Intern Med 1981;94407-409. 20 Ooi A, Nakanishi I, Sakamoto N et al. Alpha-f&protein (AFP)-producing carcinoma: is it hepatoid diierentiation? Cancer 1990;65:1741-1747. 21 Rast RC, Kiuf TL, John EST et al, A radioimmunoassay us& a monoclonal antibody to monitor the course of epithelial ovarian cancer. N Engl J Med 1983;309:883-887.
Clliea
Acts 228 (1994) 101-112
XF
Evahation of tumor-associated antigen (2H6 antigen) in detecting early stages of gastric cancer
AsaodwicbcoqmaI-liokcdionnuoosor beat assay(ELBA) wasdevelopedby usingmonoclonal antibody2H6 (2H6 MoAb). MoAb 2H6 could be usedto detect the 2H6 antigenin the em of severalauxcra, showingpositiveratesof 65.4%,66.7%.47.4%.SOB%,45.2%and 16.7%in gastriccancer.hepatoahlar carcinoma,cancersof thecolon,esophays, breastand m rerpsctively.On the other hand, the positive rates in benigndii or hepIthy donon were 3.4%,3.4%,4.2%,8.3%.6.9%,and 1.2%in myasthoia gmvis, polymyositis, gastritis,gastricuker, oth benigndireraa and normalhealthydonors,ra~pcctively.Fiftytwo patientawith gastriccancerw investigatedin detail.The positiveratesof serum2H6 antigen,CE& AFP, CAW9 and CA125 in patientawith gastriccancerwere 65.4%,9.W’ 2.3%,25.0%and 8.1% rapechly. Among thesetumormarkers,serum2H6 antigenkvels aloDewcrt aignifiitIy ekvatcd in patientswith earlystage(I andII) gastriccancer.Furthermore, combiningthe mewnxunt for merum 2H6 with CA19-9 increasedthe percentageof gastriccancerslightly.No correlationbctwcco2H6 antigenand theseother tumor markers was obecrved.The acrnmkv& of 2H6 antigenwere monitoredpost-sqically in 1I patients for12mekrondtbey~foundtodiminilhgradually.Theftndin~~suggertthatthemeasuremntof~2H6~ti~lrraybeau#fulmarlrerforan~y~~oflFastriccawer. Akinwiolkml: CEA, cudaoanbryonic antipn; CHS. choksterol hemisuccinatc; ELBA, mzymcdlinked immunoaodmnt anay; MoAb. monoclond antibody. lConerpading ruthof. OM9.8981/94&37.00 8 1994 Ekvk SSDI 0009-898 1(94)05865-P
Science B.V. All righta rwavcd
102
H. Harada et al. / Clin. Chim. Acta 228 (1994) 101-112
Keyword: Moneclonalantibody 2Hb; 2Hb antigen; Early gastriccancer;Tumotu mark !krological diagnosis
1. Intrluh&ion Gastric cancer is one of the most common cancers worldwide and in Japan it is still the leading type of cancer despite a declining trend [ 1,2J.The early stage of gastric cancer is often complicated and extremely difficult to diagnose. The develop ment of additional methods for diagnosis, is being influenced by the desire for efficient, non-invasive diagnostic procedures such as the identification of serum markers specifically associated with patients with gastric cancer. Carcinoembryonic antigen (CEA) and CA19-9 are widely used as serum markers for the diagnosis of gastric cancer. Analysis has shown that serum levels of CEA and CA19-9 are elevated in patients with gastric cancer [3-61. In a recent study, TAG-72 was found to be elevated in the serum of patients with primary gastric cancer [7,8].These serum markers are limited to use in the early diagnosis of gastric carcinoma [7,9- 111.Stein et al. [12] and Oldham and Herberman [13] reported that the immunogenicity of tumor cells is typically weak and has no clinically useful potential. Shinitxky and colleagues reported that augmentation of the immunogenicity of the tumor cells could be achieved by increasing their membrane microviscosity by the incorporation of cholesterol or its hydrophilic esters and that cholesterol bemisuccinate (CHS)treated tumor cells promoted the expression of latent tumor associated antigen [ 14,151. We hence attempted to produce a new monoclonal antibody which recognizes the early stage of cancer by immunixation with CHS-treated MKN-45 cells. Monoclonal antibody (MoAb) 2H6 was established by immunization with CHS-treated MKN-45 cells. The 2H6 antigen recognized by MoAb 2H6 was a glycoprotein consisting of several molecular species: 250,000, 75,000,68,009 and 50,000 V61. We investigated by testing with MoAb 2H6 whether various cancers in the early stage can be detected from serum, especiahy in serum from patients with gastric cancer. The present study evaluated the presperative serum levels of 2H6 antigen, CEA, CA19-9 and AFP, alone or in combination, in patients with cancer or benign disease. The findings suggest the potential utility of the 2H6 assay in detecting serum 2H6 antigen for the diagnosis of early stage gastric cancer. 2.MateriaIsatldmethods 2.1. Preparationof MoAb CHStreated MKN-45 cells were prepared by a modifmtion of the methods of Skomick et al. [17]. Establishment and purification of MoAb 2H6 were performed as described by Harada et al. [ 161. 2.2. 2H6 antigen preparation To prepare the standard antigen for sandwich enxymed-linked immunosorbent
assay (ELISA) a detergent extract of 1 x 10’ MKN-45 cells was collected. For the
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extraction of cell membrane proteins, ext~tion buffer intoning 50 mM Tris-HCl (pH 7.5), 0.15 M NaCl and 0.5% Nonidet P-40 (Nakarai Co, Lad., Tokyo) was used. The crude extracts were adjusted to a concentration of 1 mghnl in 50 mlb4Tris-NC1 containing 0.15 M NaCl and stored at -8OOCuntil assayed. 2H6 antigen-containing crude extracts were stable for at least 6 months. Crude extracts of 0.1 m&l represent 1,000units/ml. Crude extracts of 0.1 mg/ml corresponded to a ~n~ntration of a~ro~~tely 0.5 &ml of the 2H6 antigen purified from serum. 2.3. Sandwich ELISA Serum levels of 2H6 antigen were determined by a sandwich ELISA. 96Well plates (Nunc, 442402) coated with 10 &ml of 2H6 MoAb were incubated at 4OC overnight. The wells were then blocked with 50 n&i Tris-HCl (PH 7.5) containing 1% BSA, 0.15 M NaCl and 0,050/oTween 20, designated as the assay buffer, for 2 h at room temperature. One hundred microliters of standard antigen diluted 2-fold with assay buffer or patient’s serum diluted 5-fold with assay buffer were added to the wells and the plates were incubated for 3 h at room temperature. One hundred microliters of 2,000-fold diluted biotin-conjugated anti-2H6 MoAb were added and then the wells were incubated for 2 h at room tem~~t~. Biotinylation of 2H6 MoAb was performed by a modification of the method of Guesdon et al. [18]. One hundred microliters of 2,000-fold diluted peroxidase conjugated with streptavidin (Tago, 4025)were added and then the wells were incubated for 1.5 h at room temperature. Two hundred microliters of o-phenylenediamine solution were added and the wells were incubated for 20 min at room temperature. The enzyme reaction was stop ped with 50 &well of 5 N H#O., and absorbance at 492 nm was measured on a microplate reader (Dynatech, MR 5000). Serum 2H6 antigen levels (units/ml) in 5fold diluted samples were determined from a standard calibration curve. 2.4. CEA, CA19-9, CA125 and AFP radioimmunoassay Serum levels of CEA were determined with the CEA RIA BeadsfM Kit (Dainabot Co,, Tokyo, Japan). CA19-9 and CA125 serum levels were measured, respectively, with a CA19-9 and CA125 RIA Kit (Centocor, Malvern, PA). AFP serum levels were determined with alpha-Feto RIA BeadsTM(Dainabot Co., Tokyo, Japan). 2.5. Human ser4 Serum from human patients was kindly provided by Kanaxawa U~ve~ty (Cancer Res. Inst.). Serum samples from 83 healthy donors (50 males, 33 females; aged 21-56 years) and 353 patients (166 males; 187 females; aged 18-78 years) were examined. The panels of patients’ sera included 58 from patients with autoimmune diseases (myasthenia gravis, 29; polymyositis, 29), 95 from patients with benign gastric disease (gastritis 71; ulcer, 24) and 58 from patients with other benign diseases (duodenal ulcers, 9; chol~~holi~i~s, 8; chol~o~t~~s, 8; polyps of colon, 8; chronic hepatitis, 8; liver cirrhosis, 8; diabetes mellitus, 9). Sera obtained from malignancies included 71 from patients with gastric cancer, 9 from patients with hepatocellular carcinoma, 19 from patients with colon cancer, 5 from patients with w of the esophagus, 31 from patients with breast cancer and 7 from patients with cancer of the pancreas. Fifty-two of 72 with malignant gastric d&eases were
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pathologically staged: etage I (n = 28), stage II (n = 5), stage III (n = 6), stage IV (n = 13).Serum samples were obtained within 12 weeksprior to surgery.All sgdcs were stored at -40°C until assay. Assay performed was in a blind fashion. 3. Raladta 3.1. Standard curve of sandwich ELBA using 2H6 MoAb The extracts obtained from MKN-45 cells were serially diluted, and the levels of 2H6 antigen were determined by a sandwich ELISA using 2H6 MoAb. As shown in Fig. 1, the value of Am corresponded to the dilution rate of the extracts. The assay was detectable within the range of 8-500 units/ml. In&a-assay coefIicients of variation for the standard ranged from 3.5 to 8.4O/o, indicating s&kient reproducibility (data not shown). These results showed that 2H6 antigen levels in sera could be quantitatively measured by this sandwich ELISA. 3.2. Assay of sewn levels of 2H6 antigen in patients and normal controls The 2H6 levels in sera of the healthy donors, patients with carcinoma and those with various benign diseases were determined by sandwich ELISA. As shown in Fig. 2, only one of the test samples from the 83 healthy donors showed a serum level of
2
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2Ii6 lntlgon (U/ml) Fig. 1. Standard owva of undwbb
ELISA udqj 2H6 MoAb. The 2H6 antiSen lmd,
of toriallydihttad
2Ii6 antigen in excess.of 300 ~~~. The mean and standa& d~iation from normal heaIthy donors was 140.6 f 163.7 unit&l. From this result wt classified cases with 2H6 levels above 300 units/ml as 2H6 antigen positive. 2H6 antigen levels were elevated in 60.6% in the sera from patients with various cancers. Among patients diagnosed with carcinomas, 65.4%, 66.7?&,47,40/o,80.~~, 45.2% and 16.7% of the sera from patients with gastric cancer, hepatocellular carcinoma, cancers of the colon, esophagus, breast and pancreas had elevated serum 2H6 antigen levels (> 300 units/ml), respectively. In contrast, 5.2% of the sera from patients with various
l/b3 (1.2%)
34152 i (65.4%) I ,
Fig. 2. Senun Icvclsof 2H6 antigen in patientswith variouscancerdiseasesand healthy donors.The 2H6 antigenassay was perfomwdas describedin Materials and methods.The straightline denotes the cut-off k&s ofZH6 antigen(300 units/ml).Ca., c8aar, Upperordiite, number~~n~~ tested(positive ratio).
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bedpdi~hadeiev~2H62n~lewls.Amoagpotiantawith~ cIimams, 3.4%,3.4%,4.2%,8.3%and6.9?Aof the
sera from patients with myasthenia gravis, polymyositis, gastritis, gastric ulcer and gastrointestinal disease (pancreas, liver, colon) showed slightly positive results (Fig. 3). 3.3. Serum 2H6 levelsinpatientswithvariousstagesof gastriccancer The relationship between serum 2H6 levels and the clinical staging of 52 patients with gastric cancer were evaluated (Fig. 4). The positive rates summa&d according to stage were as follows: stage I, 57.1%; stage II, 60.0%; stage III, 83.3%; stage IV, 76.9%. In particular, relatively high serum 2H6 levels were found in patients diagnosed with early stage (I) gastric cancer. The positive rates observed in the advanced stages (III and IV) were higher than those observed in the relatively early stages (I and II).
1000
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200
900
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3.4. Comparison of various tumor markers in patients with gastric cancer The relationships between various tumor markers (2H6 antigen, CEA, CAl9-9,
AFP and CA123 and the clinical stage of the patients with gastric cancer were evaluated (Table 1). Positive serum 2H6 levels were found in a higher percentage of patients with various stages of gastric cancer than were those of CEA, CAl9-9, AFP, or CA125 In particular, serum 2H6 was elevated in the highest percentage of patients with early stap (I) gastric cancer. On the other hand, elevated serum CAl9-9, CEA and CA125 were observed predominantly in serum samples from patients with advanced stage (III and IV) gastric cancer. When the levels of CAl9-9 and 2H6 were analyzed in relation to gastric cancer,
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Qartric Cmcor 6trgo Fig. 4. Serum Ievclc of 2H6 antigen in patient8 with various stages of gastric cancer. The assay was perfornwd 91 de&bed in Fig. 1. The dashed line denotes the cut-off levels of the 2H6 antigen. Upper ordinate, number positivdnumbcr tested (positive ratio).
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Table 1 Comparison of the various tumor marker levels at each stage in patients with gastric cancer Stage
SeNm antigen kveIs g
I II III IV Total
uliiMml)a
16128 (57.1)b 3i5 (60.0) S/6 (83.3) IW13 (76.9) 34152(65.4)
CFJA (5 n@llI)
(20 Wmt)
AFP
CAl9-9 (37 wits/mI)
CA125 (SO units/ml)
I/28 (3.6) W5 (0) l/6 (16.7) 303 (23. I) 5J52 (9.6)
W20 (0) l/5 (20.0) W6 (0) WI2 (0) 1143(2.3)
3/2a (10.7) W5 (0) 4i6 (66.7) 6fl3 (46.2) 13152(25.0)
0119 (0) W2 (0) W5 (0) 3111 (27.3) 3127(8.1)
‘cut-off vahles. bNumben in parentheses: %.
2H6 levels were positive in 25 of 39 (64.1%)cases of C&9-9 negative serum and 2H6 levels were negative in 3 of 13 (23.1%)cases of CAM-9 positive serum. No significant correlation was recognized between 2H6 and CA199 levels in gastric cancer (Y = 0.054X + 27.76, r = 0.167, n = 52; X = 2H6, Y = CA199). Similarly, no correlation was seen between 2H6 antigen and CEA levels (Y = 0.001X + 3.20, r = 0.084, n = 52; X = 2H6, Y = CEA). Another analysis was done using combinations of 2H6, CA199, CEA, AFP and CAl25. Table 2 summa&es the percentages of patients with gastric -cancer(n = 52) whose serum samples contained either 2H6, CEA and/or CA199. Serum 2H6 (65.4%)alone was found in a higher percentage of patients with gastric cancer than either CEA (9.6%) or CA19-9 (25.0%) alone. Combining serum 2H6 measurement with CA19-9 increased the percentage of positive serum samples to 73.1%. However, no increase in the percentage of positive serum samples was observed when rn~~~t of 2H6 antigen was combined with CEA, or all three markers. 3.5. Serum 2H6 levels of patients with variom cancers before and after surgical treatment Serum 2H6 levels were measured in 11 patients with various cancers on one occa-
sion each before and after surgical treatment. No change in the serum 2H6 antigen Table 2 Summary of 2H6, CEA and CAl9-9 serum levels in patients with gastric cancer
Gastriccancer
2H6 CEA CA19-9 2H6 + CEA 2H6 + CA19-9 CEA +CAlP9 All markers ~Numbersin parentheses: %.
No. of oatients”
% of oatients*
34l52 5152 13152 34l52 38152 15152 38152
65.4 9.6 25.0 65.4 73.1 28.8 73.1
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level was observed within 1 week tier surgical treatment. However, serum 2H6 antigen level tended to decrease slightly within 2-4 weeks, and it fell below 300 units/ml at 3 months. This indicated that the serum 2H6 level tended to decrease gradually from 4 weeks (Fig. 5). 4. DIscuwb The present study found that 2H6 antigen could be detected in sera from patients with various malignant diseases. Appro~a~ly, 60.6% of the sexa from patients with various carcinomas had elevated 2Ii6 antigen levels (> 300 undo). IP contrast, the false-positive rates in healthy donors and patients with benign disease were
2
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Fii 5. Swum kvels of 2H6ant&n beforeandafterthesurgical treatmentin patientswith gastriccancer.0, patientserumobtainedfroma pre-surgical treatment; l, packntacrumobtainedfrompostsurgical tleatmem.
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relatively low, in the range of 1.2-8.3%. The false-positive rate in pregnant women was also very low in 1 of 28 (3.S”), indicating that 2H6 antigen dit&rs from oncof&talantigens like AFP (data not shown). The data indicated that it was possible to set a cut-off level of 300 aide to make a rough ~stin~on between mabgnant and benign disease. When the cut-off level is set at 400 units/ml, serum 2H6 antigen is clearly able to discriminate between carcinoma and benign disease. However, the positive rate of 2H6 antigen in patients with various carcinomas was decreased slightly, whereas none of the patients with benign diseases exceeded this level. Furthermore, it needs to be determined whether patients with gastric ulcer diseases who have inbox levels of 2H6 antigen in serum may constitute a high risk group. The results suggest that the assay of 2H6 antigen is useful for the serological diagnosis of carcinoma. Analysis of gastric cancer showed relatively high positive rates of serum 2H6 antigen. The positive rate of 2H6 antigen in patients with various stages of gastric cancer was stage dependent. In particular, appro~ately 60% of patients with early stage (I) gastric cancer showed positive serum levels of 2H6 antigen. Serum markers such as CEA and CA19-9 were slightly elevated in the early stages of gastric cancer. Similar results were obtained for AFP and CA125. These data indicate that serum 2H6 antigen is superior to CEA and CA19-9 in identifying the early stage of gastric cancer. In addition, no correlation was observed between the serum level of 2H6 antigen and the tumor markers tested (CA19-9, CEA, CA125 and AFP). It is unlikely that the mechanism by which 2H6 antigen increases in the sera of patients with carcinomas is related to that of CEA or CA19-9. CEA and CA19-9 are widely used as serum markers for the diagnosis of gastric cancer [ 191.Recently, TAG-72 was also reported to be a useful serum marker for the diagnosis of gastric cancer [7]. TAG-72 may be a possible serum marker for advanced stage (stages III and IV) gastric cancer. Goi et al. [20] and Bast et al. [21] reported that AFP-pr~u~g gastric carcinoma and gastrointestinal carcinoma could be slightly detected. Our results agree with reports that these tumor markers are difficult to detect in the early stage of gastric cancer. Thus, 286 may be a useful serum marker for the early diagnosis of gastric cancer, Recent findings have suggested a ~mplemen~ty among various tumor markers that may be exploited in the diagnosis of gas~ointestin~ carcinoma 171.Complementarity might increase sensitivity with respect to clinical diagnosis. The present data showed that elevated serum 2H6, CEA and CA19-9 levels in patients with gastric cancer appeared at rates of 65.4%, 9.6%, and 25,0%, respectively. Approximately, 35% of gastric cancer patients do not contain measurable serum levels of 2H6. Combining the me~~ment of serum 2H6 antigen with CA19-9 slightly increased the rates of detection of gastric carcinoma to 73.1% (stage I: 64.2% (18/28), stages III, IV: 89.5% (17/19)). It is suggested that the measurement of 2H6 antigen would be most useful in complementing CA19-9 for the serodiagnosis of gastric carcinoma. Additional studies with large numbers of samples are needed to investigate further whether the analysis of multiple serum tumor markers including CA72-4 may be ~v~~g~us in the batons of carcinomas, including gastric cancer. One of the important applications of any serum marker is its ability to be used
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in the msrnrremcnt of serum tumor antigen for predicting the clinical course of malignant disease. In the present study, serum samples obtained from 11 patients were assayed before and after surgical treatment on one occasion between 0 and 12weeks. Each patient had a serum level of 2H6 antigen in excess of 300 units/ml before surgery. The serum 2H6 antigen level tended to decrease slightly within 4 weeks after surgery and its level decreased significantly after 12 weeks. The serum CA125 level was significantly decreased at 24 h after surgical treatment (data not shown). Thus, the decrease in the 2H6 antigen level is different from that of CA125. Further study is needed to determine whether patients with clinically confirmed disease have elevated serum 2H6 levels at recurrence. When gastric cancer sections were stained with 2H6 antibody, the intercellular fluid was weakly positive or negative (data not shown). Elevation of the serum 2H6 antigen level in patients with carcinoma is not consistent with the results from tissue sections, suggesting that 2H6 antigen is not derived from carcinoma tissue. 2H6 antigen in sera may be a unique glycoprotein indicating the initiation of cancer. While additional studies with large population groups and different population bases are needed, these findings indicate that the serum 2H6 antigen level may be a useful marker for the clinical diagnosis of early stage with carcinoma, particularly gastric cancer. Referemces Muir C, Waterhouse T. Mack T, Powell J, Whelen S. Cancer incidence in five continents. Vol. V. JR Scientific Publication No. 88. IARC, Lyon, 1987. Kurihara M, Aoki K, Hisamichi S. Cancer mortality statistics in the world 1950-1985. The University of Nagoya Press, Nagoya, 1989. Hedin A, Carlsson L, Berghmd A, Hammarstrom S. A monoclonal enzyme immunoassay for serum CEA with incrca.sedspecificity for carcinoma. Proc Natl Acad Sci USA 1983;80:3470-3474. Koga T, Kano T, Souda K, Oka N, Inokichi K. The clinical usefulness of preoperative CEA determination in gastric cancer. Jpn J Surg 1987;17:342-347. Ritta Jr. RE, de1Villano BC, Go VL, Herberman RB, Klug TL, Zurawki Jr. VR. Initial clinical evaluation of an immunoradiometric assay CA19-9 using the NC1 serum bank. Int J Cancer 1984;33:339-345. 6 Herlyn M, Scars HF. Steplcwski Z. Koprowski H. Monoclonal antibody detection of a circulating tumor-associated antigen. I. Presence of antigen in scra of patients with colorcctal, pstric, and pancreatic carcinoma. J Clin Immunol 1982;2:135-140. Guadagni F, Roselli M, Amato T et al. CA72-4 measurement of tumor-associated glycoprotcin 72 (TAG72) as a serum marker in the management of gastric carcinoma. Cancer Res 1992;52:1222-1227. Byrne DJ, Browning M-CK. Cusieri A. CA-724 a new tumor marker for gastric cancer. Br J Surg 1990,77:1010-1013. Sikorska H, Shuster J, Gold P. Clinical applications of carcinoembryonic antigen. Cancer Detect Prev 1988;12:321-355, Sipponen P, Liidgren J. Sialylated Lewis’ determinant CA19-9 in benign and malignant gastric tissue. Acta Pathol Microbial Immunol Stand Select A 1986;94:305-311. Mach J-P, Jaeger P, Berholct M-M. Detection of recurrence of large-bowel carcinoma by radioimmunoasaay of circulating carcinocmbryonic antigen (CEA). Lancct 1974;7:535-540. Stein JA, Adler A, Goldfarb AJ, Czemobilsky B. Direct endolymphatic instillation (ELI) of BCGtumor cell mixture. A method for active immunization of cancer patients. Cancer Immunol Immunother 1978;5:31-39.
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hnmunity to tumor-asso&ed a@pns. Im13 Oldham RK, Herberman RR In: Ceil--ted rnudhgaob of cancer, Vol. 7, Part 5 New Yorkz Marcel Mtker, 1979$40-963. 14 Shinitzky M, Skomick Y. Haran-Chera N. RfYectivetumor immunization induced by cells of elevated membrane-lipid microviscosity. Proc Natl Acad Sci USA 1979;76:5313-5316. 15 Skomick YG, Rong OH, Sindelar WF et al. Active immunotherapy of human solid tumor with autologous cclls treated with choiesteryi hemisucoinate. Cancer 1986;58:650-654. 16 Harada H, Kubota T, Tsukada Y, Karasawa Y. Chracteriration of tumor-associated antigen (2H6 antigen) da&ii by monoclonal antibody 2H6. Clin Chim Acta 1994$CA 5864. 11 Skomick Y, Dmedale AR, Sindelar WF. Induction of delayed hypermmitivity reactions in cancer patients by cholesterol-hemituccinate-treated autologous tumor cells. J Natl Cancer Inst 1983;70:465-467. 18 Gueston JL. Temynek T, Arramas S. Use of avidin-biotin interaction in intmunoenaymatic techniques. J Histochem Cytodm 1979;27:1131-1139. 19 Goldenberg D, Neville AM, Cortes AC et al. Carcinoembryoaic antigen; its role as a marker in the measurement of cancer. A National Institutes of Health Consensus Development Conference. Ann Intern Med 1981;94407-409. 20 Ooi A, Nakanishi I, Sakamoto N et al. Alpha-f&protein (AFP)-producing carcinoma: is it hepatoid diierentiation? Cancer 1990;65:1741-1747. 21 Rast RC, Kiuf TL, John EST et al, A radioimmunoassay us& a monoclonal antibody to monitor the course of epithelial ovarian cancer. N Engl J Med 1983;309:883-887.