Immunological heterogeneity of serum carcinoembryonic antigen (CEA)

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CON~IUNICATION TO THE EDITOR I M M U N O L O G I C A L HETEROGENEITY OF SERUM CARCINOEMBRYONIC ANTIGEN (CEA)* RUDOLF

VRBA,÷ E L L I O T A L P E R T and K U R T J. I S S E L B A C H E R

Departments of Medicine. Harvard Medical School and the Gastrointestinal Laboratory of the Massachusetts General Hospital. Boston. MA 02114. U.S.A. lFirst receired 22 March 1975: in rerised form 9 June 1975)

Abstract--Sera specimens of some cancer patients with high CEA concentrations (more than 500 ng C E A m l by Roche CEA assay) were compared by radioimmunoassay tRIA) using t~vo different antiCEA antisera (Roche and Montreal). Both anti-CEA antisera (Roche and Montreal) recognized three different purified standard preparations of CEA by RIA. However. a large difference of more than two orders of magnitude was observed in the ability of the two antisera to detect serum CEA in these selected patients. This suggests that circulating CEA-like activity in some patients with carcinoma of the digestive tract is immunologically different from available CEA standards purified from human tumor extracts.

INTRODUCTION

(Thomson et al.. 19691 and the Roche Z-gel method for CEA assays. This quantitative difference, of more than two orders of magnitude, in the ability of two different antiCEA antisera to recognize a serum CEA. indicates that CEA found in some sera is immunologicalb different from some presently available CEA standards. A preliminary communication of these results was published IVrba et al.. 1975a). Typical experiments are described below.

Carcinoembryonic antigen (CEAI is a large complex glycoprotein demonstrated by Gold and his colleagues in fetal colon extracts and in adult human colon carcinoma (Gold & Freedman. 1965a, b). The initial demonstration of circulating CEA in the plasma of almost all patients with colon carcinoma by Thomson et al. (19691 suggested that the serologic measurement of this antigen could be a useful diagnostic test for this malignancy. Despite the frequency of small CEA elevations in a number of benign diseases. CEA assays are considered to be a helpful adjunct in the diagnosis of certain malignancies (Hansen et al.. 1974). Very high CEA levels (over 100 ng/ml) have been reported exclusively in the serum of patients with colonic or pancreatic cancer with wide-spread metastases (Dykes & King, 19721. Although marginal differences have been reported in the baseline values between laboratories due to variations in serum or plasma extraction technique and type of radioimmunoassay employed for CEA IFleisher et al.. 19731. clinical results have been fairly consistent (Dykes & King. 1972; Sorokin et al., 1972), In fact. Sorokin et al. (1972! reported a large series of estimations demonstrating an 85% correlation when comparing two different radioimmunoassays (RIM: the Farr technique described by Thomson et al. (19691 and the Z-gel method of Hansen et al. (197l, 1974). The latter method has been recently standardized and made available commercially for clinical use (Roche Procedure Manual for Carcinoembryonic Antigen Assay, 1974; The Medical Letter. 1974). The purpose of the present report is to describe large variation in measurable CEA concentrations in sera of certain patients with carcinoma of the digestive system when assayed by two different anti-CEA antisera. This variation was demonstrable when using both the Farr method

METHODS AND MATERIALS I. Sera o f cancer patients

Five serum specimens were kindly provided by Drs. Kupchik and Zamcheck, Boston. MA. selected on the basis of having over 500 ng/ml of CEA as determined by CEARoche assa~. All 5 patients had histologic-ally confirmed metastatic colonic carcinoma. These sera were tested individually with the 'direct method' using the commercially available Roche CEA assay kit and contained from 500 to 3300 ng, ml of CEA. These 5 sera were then pooled and the sera pool. when reassayed, contained 1263 ng CEA/ml (S.E.: +5°~o). 2. Anti-CEA antisera

In these studies the commercially available Roche goat anti-CEA antiserum was used. as well as goat anti-CEA antiserum kindly provided by Dr. P. Gold. Montreal. The latter antiserum was absorbed with 50 m g m l of dialyzed and lyophilized l'0 M perchloric acid extracts of normal human plasma, colon, liver and lungs. 3. Reference CEA Three preparations of purified CEA standards were used in these studies: (a) commercially available CEA-standard (Hoffman-La Roche. Belvidere, NJ); (b) a CEA preparation kindly provided by Dr. Phil Gold (Montreal); tc) First British Standard for Carcinoembryonic Antigen obtained from the British Medical Research Council (National Institute for Biological Standards and Control. Holly Hill, Hampstead, London).

* This work was supported in part by grants from the National Institutes of Health (CA-12389) and the American Cancer Society. + Supported by the Medical Research Council of Canada and the University of British Columbia. Vancou',cr. B.C.. Canada. 87

88

Communication to the Editor

4. CE,4 ~lssays Two types of assa',s were used in these experiments: {a) the Z-gel assay of Hansen et ~,l. 11974) using commercially available reagents (Roche Procedure Mamtal for C~lrcmocmhryonic .4nthJen Assayl and Ib) the Farr technique assa~ as described by Thomson et aI. (19691. la) Z-~.lel assay. Each tube contained 5 ml of ammonium acetate solution (pH 6.8. 0"01 M). Sera 11-32 ~zl) to be tested were diluted to 50 ul with normal goat serum. The standards sho~n in Fig. IA contained 10. 25. 50 and 100 ,ut 11-25. 3.125. 6.25 and 12.5 ng respectively) of Roche CEA-standard solutions and 50 #I of normal goat sera. Roche anti-CEA antiserum (25 ld) was added, the test tubes were incubated for 1. hr at 45:C and 25 .ul of t-'5ICEA (Roche) solution was subsequently added. After 1 hr of incubation. 5 mt of Z-gel tRoche) was added, the test tubes were centrifuged at I000 .q for 5 min and the sediment washed with 5 ml of ammonium acetate IpH 6.25. 0.1 M). After repeated centrifugation the radioactivity in the sediment was counted. In these assays essentially the procedure described in the Roche Procedure Mammal for Carcinoemhr)'onic Assay was followed as closely as possible. (b) Farr method. Goat anti-CEA antiserum 125 till. provided bv Dr. P. Gold. Montreal. was dissolved in 100 ml of 0"05 M borate buffer (pH 8"6) containing AB-serum (I :60 vv). Half a millilitre of this antibody solution was incubated ~ ith I0-I00 l~I of CEA-standard or with tested sera. The ~olume was made up to 600 ~1 with borate buffer (0.05 M, pH 8"6). This mixture was incubated at 37:C overnight. Roche t'5I-CEA solution (25 ml) was dissolved in 100 ml of the borate buffer, and 0.5 ml of this solution was added to each test tube. After 2 hr incubation. 11 ml of saturated ammonium sulphate was added, the test tubes were centrifuged and the radioactivity in the precipitate was counted (Thomson eral.. 19691. In these experiments the dilution of Gold's anti-CEA antiserum was 40.000 fold: selection of this concentration of the antiserum was based on antibod? titration against ~2sI-CEA (Roche) IVrba etal.. 1975,5). A concentration of antisera yielding about 30°0 binding of added t2sI-CEA was used in these radioimmunoassays.

RESULTS AND DISCUSSION

A typical standard curve using Roche CEA-standard and Roche reagents is shown in Fig. IA. Additions of increasing amounts of the cancer sera pool tip to 8 /.d resulted in an almost linear increase of inhibition in this assay. whereas identical amounts of pooled plasma from five healthy donors resulted in no significant inhibition (Fig. IB). The same serum pool was tested by the Farr technique

qThomson ~t uL. 1969) using __.oat anti-CEA antisera pro~ided b~ Dr. P. Gold. Montreal Isee Fig. 21. Roche CEAstandard t125 ng) or 5 ng of the Montreal CEA-standard could be clearly detected b~ this antisera using the Farr technique although the 2 standards reacted with different inhibition curves Wig. 2A). However. up to 32 ~LI of the studies pooled cancer serum, containing as much as 40 ng of CEA. gave negative results (Fig. 2BI. identical to aliquots of pooled sera from healthy donors !Fig. 2C). Similar negative results were obtained v, ith the Montreal anti-CEA antiserum when assaying up to S ul of another cancer serum shown to contain 144 ng Ior 18.000 ng. ml) of CEA when assa?ed with the Roche CEA-assa.~ kit. Prior perchtoric acid extraction of CEA from the serum or using the "direct method" (assaying whole seral had no significant effect upon the results by either Farr or Z-get method. The recovery of the Roche CEA-standard added to the cancer sera pool demonstrated that negative results in these sera when assayed by the Montreal anti-CEA antiserum were not caused by an', serum constituent interfering in these assays. The degree of inhibition by 3. I ng of Roche standard CEA was the same in absence of the tested sera (Fig. 2A) as well as in presence of up to 8 ,ul of normal control sera (Fig. 2CI or pooled cancer sera {Fig. 2B). The latter cancer sera contained 1263 ng CEA ml when assayed with Roche anti-CEA antisera (see Fig. I). In order to determine whether the observed differences were due to the assay methods or due to differences in the anti-CEA antisera, the assays were repeated using Roche anti-CEA antiserum in the Farr method of RIA under the exact same experimental conditions {Thomson ctaL. 1969). Figure 3A shows that the Roche anti-CEA antiserum did recognize as little as 2 ng of both Roche and Montreal CEA standards. The pooled cancer sera gave increasing inhibition with increasing amounts of sera by the Farr technique (Fig. 3B). similar to that previously demonstrated by the Z-gel method (cf. Fig. 1). It should be emphasized that both anti-CEA antisera used in this study reacted in RIA with Roche CEA standard. a purified CEA standard provided by Dr. Gold. as well as with the 1st British Standard for CEA. However. the two antisera used in these studies showed marked quantitative differences in their ability to detect circulating CEA by RIA in the pool of cancer sera. as well as in the other three sera (unpublished results) which were selected for having very high CEA levels {more than 500 ng ml) when assayed by the Roche CEA assa', kit. These results suggest that CEA-like immunoreactivity in these

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Fig. 1. Measurement of CEA in serum (Z-gel method) using Roche goat anti-CEA antiserum. (A) A CEA [Roche) standards; (B) O, pooled cancer sera; O, pooled sera from healthy donors.

Fig. 2. Estimation of CEA in serum (Farr technique) using Montreal goat anti-CEA antiserum. (A) A A standard curve with CEA (Roche); • • , standard curve with CEA provided by Dr. P. Gold (Montreal). IB) e - - - - - e , pooled sera from cancer patients: 4~---~---O, same sera with addition of 3.1 ng CEA. (C) O - - - - O . pooled sera from healthy donors; O O. same sera with addition of 3 i ng CEA (Roche/.

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Fig. 3. Estimation of CEA in sera (Farr method) using Roche anti-CEA antiserum, CEA standards from both Roche and Montreal were used simultaneously in these experiments. Each test tube contained 850 ,ul of borate buffer (0"05 M. pH 8-6) containing AB-serum (1:60 v/v) and either 10--100,td of Roche CEA standard soLution. 2-64#1 of Montreal's CEA standards, or I-81~1 of tested sera. The volume was adjusted in each case to 950 #1 with the above borate buffer. Twenty-five microlitres of Roche anti-CEA antiserum was added. This mixture was inct,bated at 37°C overnight. Thereafter. 25#1 of t-'sI-CEA (Roche) was added (approx 1-25 ng CEA activity). Further analytical procedure is described in the methods section (Farr method). (A) A. Standard curve with CEA (Roche): A. standard curve with CEA. Montreal. (B) e. Pooled sera from cancer patients; ©, pooled sera from healthy donors. selected sera may be due to antigenic determinants, distinctly different from the three CEA standards used in our studies. The relationship of these findings to the preliminary reports of a second ion sensitive CEA antigenic determinant (Hansen et al., 1971) is unclear. However. these data demonstrate that high levels of circulating CEA-like

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activity in serum of some patients with carcinoma of the digestive system reflect the presence of CEA. ~hich is immunolo~cally different from presently available CEA standard preparations purified from extracts of human tumors. It should be emphasized that the discrepancy in CEA assays when using Roche and Montreal anti-CEA was observed consistently in all eight cancer sera tested. selected solely because they contained more than 500 ng C E A m l when assayed with the commercially available Roche CEA assa? kit during routine clinical investigations. The quantity of the CEA isoantigen with determinants different from the CEA standards is very high in some patients with advanced carcinoma of the digestive system, REFERENCES

Dykes P. W. & King J. (1972) Gut 13. I000. Fleisher M.. Oettgen H. F.. Besenfelder E. & Schwartz M. K. (1973) Clin. Chem. 19. 1214. Gold P. & Freedman S. O. (1965a) J. exp. Med. 121. 439. Gold P. & Freedman S, O. (1965h) J. exp. Med. 122. 467. Hansen H, J.. Lance K. P. & Krupey J. (197i) J. Clin. Res. 19. 143 (abstract). Hansen H. J.. Snyder J. J.. Miller E,. Vandevoorde P. P., Miller O. M., Hines L. R. & Burns J. J. (19741 Human1 Patholoyy 5. 139. The Medical Letter (1974) 16, 94. Sorokin J. J.. Kupchik H. A., Zamcheck N. & Dhar P. A. (1972) hmmmol. Comm. 1, 11. Thomson D, M. P., Krupey J.. Freedman S. O. & Gold P. (1969) Proc. ~atn. Acad. Sci. U.S.A. 64. 161. Roche Procedure Manual for Careinoemhryonic ,4nti¢jen Assay. (1974) Hoffman-LaRoche. Nutley. N.J. Vrba R.. Alpert E. & Isselbacher K, J. 11975al Fedlt Prot'. 34, 551. Vrba R.. Alpert E., lsselbacher K. J. & Jeanloz R. W. (1975b) Immunochemistl"y (to be published}.