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Rapid Analysis of Carcinoembryonic in Gallbladder Bile

Antigen

Levels

Identification ofPatients at High Risk of Colorectal Liver Metastasis TIMOTHY J. YEATMAN, M.D.,* ARTHUR K. KIMURA, PH.D.,tt EDWARD M. COPELAND III, M.D.,* and KIRBY 1. BLAND, M.D.* From the Departments of Surgery* and Pathology,t University of Florida, College of Medicine, Gainesville, Florida

Recently it was found that immunoanalysis of carcinoembryonic antigen (CEA) levels in gallbladder bile may be a sensitive method to detect colorectal liver metastases in humans. Methods used in the past for the detection of CEA in various body fluids were cumbersome and time consuming, requiring acid extraction, extensive dialysis, and column purification. Single-step, solid-phase radioimmunoassays, designed specifically for serum CEA analysis, were developed commercially to replace these methods. Parameters and methodology necessary to adapt these kits for use with gallbladder bile are presented here. A combination of pretreatment procedures for bile, before radioimmunoassay, permit rapid, reproducible, and accurate measurement of CEA levels in gallbladder bile.

C

ARCINOEMBYRIONIC ANTIGEN (CEA) is a 200,000 molecular weight (MW) glycoprotein that is ex-

creted by certain embryonic and adult tissues in addition to adenocarcinomata of the digestive organs.' Extensive studies of patients bearing primary and metastatic colorectal neoplasms have determined that its primary use is in the detection of local and metastatic cancer recurrence after initial resection of the primary tumor, through periodic postoperative analysis of CEA in serum or plasma.2 Recently the notion that fluids bathing tumors in metastatic sites might contain higher levels of CEA than those found in the blood pool led us to analyze CEA levels in gallbladder bile from patients bearing colorectal liver metastases.3 From these investigations, it was determined that CEA levels in gallbladder bile were strikingly higher than those in serum. Furthermore linear regression analysis of tumor volume versus gallbladder bile CEA levels in pa-

tients with liver metastases predicted that tumors as small as 1 cm3 would produce easily measurable gallbladder bile CEA levels as high as 41 ng/mL. This data suggested that measuring biliary CEA levels in patients with primary colorectal lesions might permit detection of small, occult colorectal liver metastases earlier than is now possible through conventional methods (computed tomographic liver scanning, ultrasound, intraoperative exploration). Previously used radioimmunoassays4 were cumbersome and required lengthy dialysis; commercially available radioimmunoassays and enzyme-linked assays were designed only for serum CEA analysis. In addition we have found that the use of these assays required, at minimum, a 1:40 dilution of bile before analysis to dilute out an unknown interfering substance(s). This dilution process increased the lowest level of detectable CEA from 1 ± 0.9 ng/mL to 40 ± 0.9 ng/mL and limited the predictive, clinical use of the assay. We designed a new, clinically applicable method for analysis of CEA levels in bile samples, which may be completed in approximately 6 hours. Using perchloric acid to extract CEA from other interfering biliary components (i.e., bilirubin, proteins), followed by neutralization, dilution, and ultrafiltration, we were able to assay rapidly and accurately for CEA levels ranging from 5 to more than 40 ng/mL with a solid-phase, two-site ra-

dioimmunoassay.

t Deceased. This work was supported in part by grants from the Southern Medical Association, Florida Chapter of the American Cancer Society, Research Development Award (Division of Sponsored Research, University of Florida), and National Institutes of Health grant CA 43051, DHHS. Address reprint requests to Kirby I. Bland, M.D., Department of Surgery, Box J-286, JHMHC, University of Florida, College of Medicine, Gainesville, FL 32610. Accepted for publication March 23, 1990.

Materials and Methods

Specimens Gallbladder bile for analysis was obtained from patients without biliary pathology (controls) and from patients bearing colorectal liver metastases. All samples were ob-

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Ann. Surg. * February 1991

YEATMAN AND OTHERS

taned in accordance with the policies of the Institutional Review Board of the University of Florida, Gainesville, Florida. Reagents and Equipment

Carcinoembryonic antigen solid-phase, two-site radioimmunoassay test kits were provided by Hybritech, Inc. (San Diego, CA). These kits contained human serum standardized samples with known levels of CEA from 0 to 100 ng/mL to be used as standards in analysis. Also included in the kit were murine monoclonal anti-CEA antibody-coated plastic beads and 1251-labeled murine monoclonal IgG tracer anti-CEA antibodies (directed against an antigenic site distinct from that detected by the solid-phase antibody). Centricon 30,000 MW cutoff ultrafiltration microconcentrators were purchased from Amicon, Inc. (Danvers, MA). Dialysis was performed using a 28-well microdialyzing system (Bethesda Research Laboratories, Gaithersburg, MD). Gamma counting was performed using an automated gamma counter (LKB/ Wallac, Turku, Finland). A microfuge (Beckman-B, Beckman Instruments, Palo Alto, CA) was used to remove precipitates from perchloric acid extracts and a fixed-angle rotor (Beckman JA-20) was used for ultrafiltration. CEA Analysis by Solid-phase Radioimmunoassay The methodology used in the 4-hour radioimmunoassay test by Hybritech for CEA analysis previously was described in detail and has a sensitivity of 1.0 ± 0.9 ng/ ml.5 Briefly, following single-step incubation (at 37 C) of samples (100 ,L), 125I-labeled tracer antibody (100 AL), and antibody-coated beads, a standard curve was constructed from linear regression analysis of values from CEA serum standards and used to determine experimental values.

Specimens for Assay Standardization Serum standard reagents (provided in the Hybritech kit) were used to add known amounts of exogenous CEA to gallbladder bile obtained from normal patients (without endogenous CEA) and from patients bearing colorectal liver metastases (with endogenous CEA). For each admixed sample (to which exogenous CEA was added), in addition to known serum standard samples used as controls, an expected CEA level was compared with levels measured by radioimmunoassay before or after sample preparation.

Sample Preparation Before CEA Analysis Method 1 (2 hours). Serum and admixed gallbladder bile standard samples (100 AL) were analyzed after a 2hour pretreatment procedure. This procedure involved

extraction with 0.5 mL of 1.2 mol/L (molar) perchloric acid for 30 minutes at room temperature and centrifugation at 9000g for 10 minutes to remove any precipitate. Supernatants were collected and neutralized with 0.5 mL of 1.0 mol/L TRIS in 0.15 mol/L phosphate-buffered NaCl (PBS), pH = 10.5. Samples then were brought up to 2.5 mL final volume with 0. 15 mol/L PBS. These samples were concentrated by ultrafiltration using 30,000 MW cutoff microconcentrators (Amicon) for 30 minutes to 1 hour at 5000g (using a fixed-angle rotor at 32°). Retentate (40 ± 5 ,L) once again was resuspended to 2 mL with 0.15 mol/L PBS and concentrated through ultrafiltration for 30 to 60 minutes. Centrifugation times varied with the amount of biliary pigment soluble in perchloric acid; longer periods of centrifugation were required for samples with larger concentrations of soluble pigment. Final retentate (40 ± 5 uL) was measured and mixed with approximately 60 ML CEA-free serum standard to yield 100 ,ML total sample volume for CEA radioimmunoassay. Method 2 (72 hours). Serum and composite gallbladder bile standard samples (100 uL) were analyzed after extraction with 100 ML of 1.2 mol/L perchloric acid (30 minutes at room temperature), centrifugation to remove any precipitate at 9000g for 1O minutes, and supernatant collection. The supernatants were dialyzed (10,000 MW cutoff dialysis membranes) against four changes ofdistilled water ( 16 L total) for 72 hours and CEA levels were measured directly by radioimmunoassay. Results

Standard serum samples (containing 100 ng/mL CEA), when diluted with CEA-free serum, produced a linear dilution curve with the 'expected' CEA recovered at each dilution. When CEA was admixed with normal bile (undetectable endogenous CEA content), however, radioimmunoassay did not accurately yield the expected amount of CEA until the admixed bile sample was diluted approximately 40 times (Fig. 1). This limitation severely reduced the sensitivity of CEA determinations in bile such that levels less than 40 ng/mL could not be detected accurately by this method (i.e., a 1:40 dilution of bile containing less than 40 ng/mL would reduce the measurable CEA content to an undetectable level of less than 1 ng/mL). To remove the substance(s) interfering with the direct assay of bile, we used perchloric acid to maintain CEA solubility while precipitating out proteins and pigments (method 1). After this extraction, samples were neutralized with TRIS, concentrated and dialyzed simultaneously by ultrafiltration, and tested in solid-phase radioimmunoassay (6 hours total time for method). Expected levels of CEA in the serum and bile samples were compared with measured CEA levels (Fig. 2). Levels of exogenously added

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CEA LEVELS IN GALLBLADDER BILE

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200

FIG. 1. Both standard serum samples (containing 100 ng/ mL endogenous CEA) and normal bile samples (undetectable CEA) admixed 1:1 with standard serum samples (containing 200 ng/mL CEA) were analyzed directly by radioimmunoassay after dilution with CEA-free serum. Percentage of expected CEA was determined by comparing predicted with measured CEA levels at various dilutions. Accurate measurement of CEA levels in gallbladder bile, unlike serum, required 40-fold dilution (dilution factor includes the 1:1 dilution incurred during admixing) to remove inhibitory substance(s).

Srum Contaking 100 ng/ml CEA 0 Normal Blh Contabking 100 ng/ml exogenous CEA 150 'u

0 0 0

0

x

XU 100 owc

E} --''

0

0 0

50

0 1:1

1:20

1:10

1:30

1:40

1:50

1:60

1:70

Dilution CEA as low as 5 ng/mL were measured accurately and reproducibly from serum, normal bile, and 'metastatic bile' (known to contain 32.4 ng/mL endogenous CEA). Expected levels of CEA for metastatic bile were determined by adding the known value of endogenous CEA,

measured by 1:40 dilution of bile, to the amount of exogenous CEA admixed with the bile before analysis. Pearson correlation coefficients were all very high (0.997) and were associated with low root mean square (RMS) errors

(< 2.3).

Metastatic Bile

Normal Ble

Normal Serum

(32.4 ng/mI CEA) 100 E

E

E

C

c

-

(Y.

50 X

61

6

0

so

50 Added CEA (ng/ml)

r.m.s.

error-2.3

Added CEA (ng/ml)

r.m.s. error-

1.4

50 Added CEA (ng/ml)

r.m.s. error-

104

1.6

FIG. 2. Specimens were pretreated by perchloric acid extraction without dialysis (method 1) before analysis. Measurement of CEA concentrations in normal serum (endogenous CEA, 0 ng/mL), normal bile (endogenous CEA, 0 ng/mL), and 'metastatic' bile obtained from a patient with documented colorectal liver metastasis (endogenous CEA, 32.4 ng/mL) after the addition of various amounts of exogenous CEA demonstrates that expected CEA levels (based on the known amounts of CEA added to each sample) correlate closely with measured CEA levels using this method.

Ann. Surg. . February 1991

YEATMAN AND OTHERS

116

Normal Bile

Normal Serum 100

FiG. 3. Specimens were pretreated by perchloric acid extraction with 72 hours of dialysis (method 2) before analysis. Measurement of CEA concentrations in normal serum (endogenous CEA, 0 ng/mL) and normal bile after the addition of various amounts of exogenous CEA demonstrates high correlation between expected CEA levels and measured CEA levels using this method.

E CP c

E CP c _

_

0

50 0

L)

L)

0

0 0

100

50 Added CEA (ng/ml)

0

r.m.s. error=

0

100

50

Added CEA (ng/ml)

1.5

r.m.s. error

=0.8

The results of the 6-hour assay using ultrafiltration for concentration and dialysis (method 1) were paired to those obtained from the 72-hour assay (method 2), which required extensive dialysis and often generated errors in estimation of subsequent sample volume (Fig. 3). From this data it is clear that similar results are obtained for most samples using either method 1 or 2; however, for samples containing higher levels of CEA (30 and 50 ng/ mL) added to bile, dialysis does not appear to remove completely all the inhibitory factors because CEA levels significantly lower than expected were recovered. While the sensitivity and reproducibility of the Hybritech assay is known for serum CEA analysis, these values have not been established yet for gallbladder bile. To determine the precision of this CEA radioimmunoassay for use with bile specimens, we first examined the variation of the assay for determining CEA levels in the same replicate groups (Table 1). Four normal (undetectable CEA content) bile samples were admixed with known amounts of CEA provided in the Hybritech kit, pretreated by method 1, and aliquoted into multiple groups or replicates (3 to 5 aliquots per sample). These aliquots then were

assayed for CEA content. The data in Table 1 demonstrate that, for each of the four CEA levels (12.3 to 60.0 ng/ mL), the variability of measured CEA levels within replicates is quite low, with standard deviations ranging from 0.4 to 3.9 and coefficients of variation ranging from 3.4% to 7.5%. Having established the reproducibility of the assay for the detection of CEA within the same pretreated/aliquoted specimen, we examined the variability of results obtained first by individually processing (pretreating) multiple aliquots of the same bile sample (endogenous CEA level known to be 12.5 ng/mL) and then measuring CEA content (Table 2). In other words, we measured the variation inherent in the pretreatment procedures for the bile. Once again variability among replicates was low (standard deviations ranged from 0.5 to 1.8) and the mean CEA levels detected within each separate, pretreated aliquot (samples to 3) were almost identical (1 1.6 to 12.9 ng/mL).

TABLE 1. Precision of Bile Assay (Method 1): Within Assay Variation

TABLE 2. Precision of Bile Assay (Method 1): Within Sample Variation

Discussion The sensitivity of gallbladder bile CEA analysis for detecting colorectal liver metastases has been shown to be

Sample

1

2

3

4

Sample

1

2

3

Number of replicates Mean CEA level (ng/mL) Standard deviation Coefficient of variation

3 12.3 0.4 3.4%

5 16.7

5 33.1 1.3 3.9%

5 60.0 3.9 6.4%

Number of replicates Mean CEA level (ng/mL) Standard deviation Coefficient of variation

3 11.6 0.5 4.6%

3 12.3 0.4 3.4%

3 12.9

CEA, carcinoembryonic antigen.

1.3 7.5%

CEA, carcinoembryonic antigen.

1.8 13.8%

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CEA LEVELS IN GALLBLADDER BILE

TABLE 3. Analysis of Serum and Gallbladder Bile CEA Levels in Control Patients and in Patients with Known Colorectal Liver Metastases CEA Levels (ng/mL)* Group

n

Serum

Bile

Normal controls Liver metastases

5 16

0.8 ± 0.5 58.1 ± 23.8t

0.2 ± 0.1 1574.0 ± 380.2t

* Values represent mean carcinoembryonic antigen (CEA) levels ± standard error of the mean. t p = 0.0009.

superior to serum CEA analysis.3 Control patients with normal livers had low serum and bile CEA levels, whereas patients with colorectal liver metastases had CEA levels in bile markedly greater (up to 259 times greater) than serum (Table 3). With knowledge of the potential for biliary CEA analysis to detect colorectal liver metastases and to identify patients with occult hepatic metastases, a rapid, reliable assay was needed for clinical application. Initial attempts to measure CEA levels in gallbladder bile demonstrated that an inhibitor(s) was present in all bile samples (n = 45) tested.3 For this reason available assay test kits (designed for serum) are not suitable for the detection of CEA levels less than 40 ng/mL in bile because a 1:40 dilution is required before analysis to remove the inhibitor(s). We developed a new quantitative assay for detecting CEA in bile that represents a significant improvement over previous methodologies designed for serum. Per-

117

chloric acid extraction of bile, along with neutralization and ultrafiltration, were used to separate CEA from interfering biliary substances. Solid-phase radioimmunoassay then was used to measure directly CEA levels. With this new methodology, CEA analysis of gallbladder bile is now possible, without end-sample dilution or loss, in approximately 6 hours. At levels of detection as low as 5.0 ± 0.9 ng/mL, we hope to detect occult colorectal liver lesions producing low levels of CEA in the bile (O to 40 ng/mL) that now escape routine methods ofdetection. Prospective clinical trials are underway using this new method for CEA analysis of bile to evaluate fully its potential for early detection of colorectal liver metastases.

Acknowledgments The authors thank Carolyn Hansen for performing statistical analyses.

References 1. Gold P, Freedman SO. Demonstration of tumor-specific antigens in human colonic carcinomata by immunological tolerance and absorption techniques. J Exp Med 1965; 121:439-462. 2. Steele G, Ellenberg S, Ramming K, et al. CEA monitoring among patients in multi-institutional adjuvant G.I. therapy protocols. Ann Surg 1982; 196(2):162. 3. Yeatman TJ, Bland KI, Hollenbeck JI, et al. Relationship between colorectal liver metastases and CEA levels in gallbladder bile. Ann Surg 1989; 210(4):505-512. 4. Hansen HJ, Snyder JJ, Miller E, et al. Carcinoembryonic antigen (CEA) assay. Human Path 1974; 5(2):139. 5. Hybritech, Inc. Monograph. Tandem-R CEA Immunoradiometric Assay: for the quantitative measurement of carcinoembryonic antigen (CEA) in serum; 1985, pp 1-7.