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Clinical Chemistry 45: 1768-1773, 1999;
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(Clinical Chemistry. 1999;45:1768-1773.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Time-resolved Immunofluorometric Assay of Trypsin-1 Complexed with {alpha}1-Antitrypsin in Serum: Increased Immunoreactivity in Patients with Biliary Tract Cancer

Johan Hedström1,a, Caj Haglund2, Esko Kemppainen2, Maarit Leinimaa1, Jari Leinonen1 and Ulf-Håkan Stenman1

1 Department of Clinical Chemistry and
2 Second Department of Surgery, University of Helsinki, FIN-00029 Helsinki, Finland.
a Address correspondence to this author at: Clinical Research Institute, Helsinki University Central Hospital Ltd, P.O. Box 105, FIN-00029 Helsinki, Finland. Fax 358-9-47174804; e-mail johan.hedstrom{at}huch.fi


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Increased serum concentrations of trypsin immunoreactivity occur in patients with biliary tract cancer. To characterize this trypsin, we developed a sensitive time-resolved immunofluorometric assay for trypsin-1 complexed with {alpha}1-antitrypsin (AAT) and studied the concentrations of this complex in sera from healthy individuals (n = 130) and patients with benign biliary disease (n = 32), biliary tract cancer (n = 17), pancreatic cancer (n = 27), and hepatocellular cancer (n = 12).

Methods: We used a trypsin-1-specific monoclonal antibody on the solid phase and a europium-labeled polyclonal antibody to AAT as tracer. The detection limit was 0.42 µg/L. The validity of the trypsin-1-AAT test for detection of biliary tract cancer was compared with trypsin-2-AAT and CA19-9.

Results: Increased concentrations of trypsin-1-AAT (>33 µg/L) were found in 76% of patients with biliary tract cancer, and the concentrations were significantly higher than in those with benign biliary disease (P <0.0001). The median concentration of trypsin-1-AAT in serum from patients with biliary tract cancer was 3.7-fold higher than in healthy controls, 2.6-fold higher than in patients with benign biliary tract disease, 1.7-fold higher than in patients with pancreatic cancer, and 2.0-fold higher than in patients with hepatocellular cancer.

Conclusions: Of the markers studied, trypsin-1-AAT had the largest area (0.83) under the receiver operating curve in differentiating biliary tract cancer from benign biliary tract disease. Our results suggest that trypsin-1-AAT is a new potential marker for biliary tract cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Trypsin (Mr 24 000) is produced by pancreatic acinar cells and is the main pancreatic protease responsible for the proteolysis of dietary proteins (1)(2). It consists of two major isoenzymes, trypsinogen-1 (cationic trypsinogen) and trypsinogen-2 (anionic trypsinogen) (3). Inadvertent intrapancreatic activation of trypsinogen is a key feature in pancreatitis; this leads to leakage of trypsin into circulation (4), where it is rapidly inactivated by the major trypsin inhibitors, {alpha}2-macroglobulin and {alpha}1-antitrypsin (AAT)1 (5).High serum concentrations of trypsin-2-AAT and trypsin-1-AAT, also called {alpha}1-proteinase inhibitor, have been demonstrated in acute pancreatitis (6)(7), and the concentrations on admission correlate with the severity of the disease (8)(9).

We previously showed that two trypsinogen isoenzymes are produced by ovarian tumors. These are similar to pancreatic trypsinogen-1 and -2 with respect to amino acid sequence, molecular weight, and immunoreactivity, but they differ from pancreatic trypsinogen with respect to catalytic properties and isoelectric point behavior. Hence, they were called tumor-associated trypsinogen-1 and -2 (TAT-1 and TAT-2, respectively) (10). Recently, TAT-2 and pancreatic trypsinogen were shown to have the same cDNA sequence (11).

Various cell lines derived from colon carcinomas, fibrosarcomas, pancreatic carcinomas, and leukemia cells secrete trypsinogen and mainly the TAT-2 isoenzyme (12). Very high concentrations of TAT-2 have been observed in cyst fluid of ovarian tumors; these concentrations correlate with the degree of malignancy (13). It has been suggested that tumor-associated trypsins play an essential role in cancer invasion and metastasis by degrading trypsin-sensitive extracellular matrix proteins (14). This is supported by the recent finding that trypsin-2 efficiently activates matrix metalloproteinase-2 and -9 (11), which degrade type 4 collagen and are thought to play a major role in tumor invasion (15). Pancreatic trypsinogen has been suggested to play a role in the invasiveness of pancreatic cancer (16).

Terada et al. (17) recently demonstrated by immunohistochemistry expression of trypsinogen in malignant biliary epithelial cells. Trypsinogen is also expressed by the epithelium of benign intrahepatic bile ducts and peribiliary glands (18), and in extrahepatic peribiliary glands (19). We previously observed high trypsinogen-2 and trypsin-2-AAT concentrations in serum of patients with malignant digestive tract diseases. Preferential increases were found in patients with biliary tract cancer, and the trypsinogen appeared to be derived from the biliary epithelium (20).

To determine the type of trypsinogen in the serum of patients with biliary tract cancer, we developed a new sensitive time-resolved immunofluorometric assay (IFMA) for trypsin-1 in complex with AAT and studied the concentrations of this complex in sera from healthy individuals and patients with benign biliary disease, biliary tract cancer, and pancreatic cancer. The validity of trypsin-1-AAT as a marker for biliary tract cancer was compared with those of trypsin-2-AAT, trypsinogen-1, and CA19-9.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
samples
Preoperative serum samples were obtained from 17 patients with biliary tract cancer, 27 with pancreatic cancer, 12 with hepatocellular carcinoma, and 32 with benign biliary tract disease from the Department of Surgery, Helsinki University Central Hospital, from June 1990 through February 1995. The diagnosis of cancer was verified by histology or cytology. The disease stage was determined on the basis of clinical examination, imaging methods, and surgical findings. Nine patients (20%) with malignant disease had a local tumor; all of the others had either locally spread or metastasized disease. The diagnosis of benign biliary disease was based on clinical and laboratory findings, ultrasonography, radiological examinations, or operative findings. Samples from patients with malignant disease were obtained before treatment. All samples from patients with benign biliary disease were drawn within 24 h of admission and before initiation of therapy. Serum samples from 130 blood donors (65 males) were obtained from the Finnish Red Cross Blood Bank. All serum samples were stored at -20 °C until assayed. Pancreatic juice (10 mL) was obtained by aspiration from the main pancreatic duct of patients undergoing endoscopic retrograde cholangiopancreatography and added to tubes containing 1 mL of 10 mmol/L benzamidine and stored at -20 °C until further preparation. The sampling procedures were approved by our institution's responsible committee.

reagents
A polyclonal antibody against AAT was obtained from Dakopatts. Production of the monoclonal antibodies against trypsinogen-1 and-2 has been described previously (21).

buffers and reagent solutions
The assay buffer in the IFMA was 50 mmol/L Tris-HCl, pH 7.7, with 150 mmol/L NaCl and 8 mmol of NaN3 [Tris-buffered saline (TBS)] containing 5 g/L bovine serum albumin and 0.15 g/L bovine globulin. The wash solution contained 150 mmol/L NaCl, 7.7 mmol/L NaN3, and 0.2 g/L Tween 20. Enhancement solution was obtained from Wallac Biochemical Laboratories.

gel chromatography
Serum samples (0.5 mL) were applied to a 1.6 x 80 cm Sephacryl S-200 column and eluted with TBS buffer at a flow rate of 20 mL/h. Fractions (1 mL) were collected, and the absorbance was monitored at 280 nm. Concentrated assay buffer (100 µL of 10-fold concentrate) was added to each fraction. Immunoreactivity of the fractions was determined by IFMA with a sample volume of 200 µL. The elution volumes of serum IgG (150 kDa) and albumin (69 kDa) were used for rough calibration of the column. Purification of the trypsin-1-AAT calibrator was performed by gel filtration using a 1 x 30 cm column of Superdex 200 HR 10/30 (Pharmacia) and TBS buffer for elution. The flow rate was 30 mL/h, and 500-µL fractions were collected. The tubes were prefilled with 50 µL of assay buffer containing aprotinin (1.0 mg/L) to prevent autolysis and nonspecific adsorption to the tubes. The column was calibrated with ovalbumin (43 kDa), soybean trypsin inhibitor (21 kDa), and aprotinin (6 kDa).

isolation of trypsinogen isoenzymes from pancreatic juice
Samples (10 mL) of pancreatic juice were diluted twofold with distilled water and applied (2 mL) to a Resource-Q ion exchange column (HR 5/5; Pharmacia) equilibrated with 50 mmol/L Tris-HCl buffer, pH 8.0 (buffer A). Buffer B was 50 mmol/L Tris-HCl buffer, pH 8.0, containing 1 mmol/L CaCl2, 0.5 mol/L NaCl, and 1 mL/L 2-propanol. The column was eluted with linear gradient consisting of 20 mL of each of buffers A and B, respectively. The flow rate was 1 mL/min, and the fraction volume was 1.5 mL. Immunoreactivity of the fractions was determined by IFMA as described below. The fractions containing each isoenzyme were pooled, and aprotinin was added (final concentration, 70 mg/L) to prevent autoactivation.

calibrators
Trypsin-1-AAT was prepared from pure human AAT (Athens Research and Technology) and trypsin-1, purified as described (21). Trypsinogen-1 was autoactivated by incubation at 37 °C for 2 h and then incubating for 16 h at 20 °C in TBS buffer, pH 7.4, with a sevenfold molar excess of AAT. The incubation mixture was separated by gel filtration, and the concentrations of trypsinogen-1 and trypsin-1-AAT in the fractions were estimated by a trypsinogen-1 IFMA. Incubation with AAT reduced the trypsinogen-1 immunoreactivity to 3% of that in the control incubated with aprotinin. On this basis, we assumed that 97% of trypsin had complexed to AAT. This preparation was used for calibration of the trypsin-1-AAT assay. Calibrators were prepared by diluting the complex with assay buffer to contain trypsin-1-AAT at concentrations of 0.1, 0.5, 1.0, 10, and 100 µg/L.

time-resolved ifma of trypsinogen-1 and trypsin-1-aat
Trypsinogen-1 was determined as described (21). For assay of trypsin-1-AAT, monoclonal antibody 3E8 to trypsin-1 (21) was coated onto microtitration wells, and the polyclonal rabbit antibody to AAT labeled with a europium chelate (22) was used as tracer. Sample and assay buffer (25 and 200 µL, respectively) were pipetted into the coated wells. After incubation for 1 h, the wells were emptied, washed twice with wash solution, using an automatic washer (DELFIA Platewash 1296-024; Wallac), and filled with 200 µL of assay buffer containing 200 ng of europium-labeled anti-AAT antibody. After further incubation for 1 h, the wells were emptied and washed four times. Enhancement solution (200 µL) was added to the wells, and after 5 min the fluorescence was measured with a 1234 DELFIA Research fluorometer (Wallac).

determination of ca19-9
The serum concentrations of CA19-9 were determined by a commercially available CA19-9 assay (Immuno-1; Bayer). A cutoff value of 37 kilounits/L was used (23).

recovery
Analytical recovery was measured by adding trypsin-1-AAT calibrator and trypsin-2-AAT-containing human serum into human sera samples containing 25 and 13 µg/L trypsin-1-AAT, respectively.

statistics
The detection limit of the assay was defined as the concentration corresponding to the fluorescence signal of assay buffer + 2 SD (calculated from 12 replicates). The reference range was determined on the basis of the central 95% reference interval, i.e., the 2.5 and 97.5 percentiles in sera from 130 blood donors.

The ability of various tests to differentiate between biliary tract cancer and benign biliary disease was estimated on the basis of sensitivity and specificity at various cutoff values. The validity of the tests was further evaluated by ROC curve analysis. A univariate z-score test (24) was used to estimate the significance of the difference between the areas under the ROC curves. The correlation between the values of trypsinogen-1 and trypsin-1-AAT and CA19-9 was calculated by the least-squares method. The significance of the differences between the values of various groups was calculated with the Mann–Whitney U-test. The ability of various tests to differentiate between malignant and benign biliary tract disease was estimated on the basis of sensitivity and specificity at various cutoff values. The specificities were compared using the McNemars test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Fractionation by gel chromatography indicated that the trypsin-1-AAT calibrator contained 4% free trypsinogen-1, which the IFMA for trypsin-1-AAT did not recognize (Fig. 1 ). The apparent cross-reaction of trypsin-1-AAT in the trypsinogen-1 IFMA was 3.3% on a molar basis (Fig. 1 ). The dose–response curve for the trypsin-1-AAT IFMA is shown in Fig. 2 . The detection limit was 0.42 µg/L, and the calibration curve was fairly linear to 100 µg/L. The within-run coefficient of variation (CV) for trypsin-1-AAT was 16%, 8%, and 7% at trypsin-1-AAT concentrations of 12, 46, and 69 µg/L, respectively. The between-run CVs were 7%, 15%, and 8%, at concentrations of 10, 43, and 71 µg/L, respectively. The analytical recovery of trypsin-1-AAT added at concentrations of 9.8–50 µg/L to human serum was 87–102%. The reference range for trypsin-1-AAT was 8.9–33 µg/L (median, 17 µg/L).



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  		Figure 1. Characterization by gel filtration of the trypsin-1-AAT calibrator.

The elution profile of trypsinogen-1 immunoreactivity is shown for comparison. The elution volumes of the IgG (150 kDa) and albumin (69 kDa) used to calibrate the column are indicated by arrows.



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  		Figure 2. Calibration curve for the IFMA for trypsin-1-AAT.

The background of ~750 cps has been subtracted.

Increased trypsinogen-1 (>50 µg/L) and trypsin-1-AAT (>33 µg/L) were found in 47% and 76%, respectively, of patients with biliary tract cancer (n = 17), and the concentrations of trypsin-1-AAT (median, 63 µg/L; range, 7–190 µg/L) were significantly higher than in those with benign biliary disease (n = 32; median 24 µg/L; range; 10–99 µg/L; P <0.0001; Fig. 3 ). The median concentration of trypsin-1-AAT in serum from patients with biliary tract cancer was 3.7-fold higher than in healthy controls, whereas it was 2.6-fold higher than in patients with benign biliary tract disease, 1.7-fold higher than in patients with pancreatic cancer, and 2.0-fold higher than in patients with hepatocellular cancer (Table 1 and Fig. 3 ). The median and range of trypsin-1-AAT at different stages of the malignancies studied are shown in Table 2 .



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  		Figure 3. Concentrations of trypsin-1-AAT in serum samples from 130 healthy subjects (HS), 32 patients with benign biliary tract disease (BD), 17 patients with biliary tract cancer (BC), 27 patients with pancreatic cancer (PC), and 14 patients with hepatocellular carcinoma (HC).


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  		Table 1. Concentrations of trypsin-1-AAT, trypsin-2-AAT, and CA19-9 in patients with malignant and benign diseases.


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  		Table 2. Median and range of trypsin-1-AAT concentrations for different stages of malignant disease.

The capacity of the different analytes to differentiate between malignant and benign biliary disease was evaluated by ROC analysis. The area under the ROC curve was 0.832 for trypsin-1-AAT, 0.761 for trypsin-2-AAT, 0.602 for trypsinogen-1, and 0.779 for CA19-9. Thus, the accuracy of trypsin-1-AAT was superior to those of the other determinations. Table 3 shows the sensitivity of the various tests to differentiate malignant biliary disease from benign biliary disease at clinically relevant specificity values.


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  		Table 3. Specificity and sensitivity of the various tests to differentiate malignant biliary disease from benign biliary disease.1


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In most patients, the diagnosis of biliary tract cancer is based on radiological examinations, and the disease is often already disseminated at diagnosis. Sometimes, however, it can be difficult to distinguish between benign and malignant structures of the bile ducts. In these patients, tumor markers might be of help. The most used markers today are CA19-9 and CEA. However, these markers are often increased not only in malignant but also in benign biliary obstruction (25).

In the present study, we have shown that the trypsin-1-AAT complex in serum is increased in ~75% of the patients with biliary cancer and that trypsin-1-AAT is slightly better than CA19-9 in differentiating between patients with cholangiocarcinomas and those with benign biliary tract disease, as evidenced by larger areas under the curves in the ROC plots. Increased trypsinogen-1 and trypsin-2-AAT occurred in about one-half of the cases. Thus, the increase in trypsin-1-AAT appears to be typical of hepatobiliary cancer, whereas trypsin-2-AAT is the best marker of acute pancreatitis (9).

The high trypsin-1-AAT concentrations in patients with cholangiocarcinoma may be explained by expression of trypsin-1 in malignant biliary epithelial cells, which has recently been demonstrated by immunohistochemistry (17). Expression of trypsin-1 has also been observed in the healthy epithelium of intrahepatic bile ducts and peribiliary glands (18) and in extrahepatic peribiliary glands (19). Hence, the increased serum concentrations seen in patients with biliary tract cancer are more likely the result of production of trypsin-1 by the tumor rather than by a nonspecific disturbance of pancreatic function. In a previous study, we showed that hepatic dysfunction, biliary obstruction, and pancreatic irritation did not appear to be the mechanisms causing increases of trypsinogen-2 and trypsin-2-AAT in biliary tract disease (20). We therefore believe that the same is true for trypsin-1-AAT.

The values of trypsinogen-1 correlated with those of trypsin-1-AAT (r = 0.6745; P = 0.001), but trypsin-1-AAT was more highly increases in cancer. This suggests that in biliary tract cancer, activated trypsin-1 leaks out into the extracellular fluid and into circulation, where it forms complexes with AAT and other protease inhibitors.

The serum concentrations of trypsin-1-AAT are also increased in patients with pancreatic disease, but measurement of trypsin-1-AAT has not been considered clinically useful in the diagnosis of pancreatitis because of a high frequency of increased values in other diseases, such as gastric ulcer and hepatobiliary obstruction (8)(26)(27). Because trypsin-1 is also expressed by healthy biliary epithelium, the increase of trypsin-1-AAT in benign hepatobiliary disease could be explained by leakage of trypsin-1 from benign biliary epithelium. We observed moderately increased concentrations of trypsin-1-AAT in benign biliary disease, but the increase in malignant disease was stronger. Thus, a differentiation between benign and malignant disease similar to that of CA19-9 could be obtained.

Recent studies have suggested that TAT plays an essential role in cancer invasion and metastasis by degrading trypsin-sensitive extracellular matrix proteins (14). Pancreatic trypsinogen has been suggested to play a role in tumor invasion of pancreatic cancer (16). Thus, the expression of trypsinogen in biliary tract cancer could be a factor contributing to the aggressive behavior of this tumor.

In conclusion, our study shows that the serum concentrations of trypsin-1-AAT are often increased in malignant biliary tract disease and that trypsin-1-AAT is a new potential marker for biliary tract cancer. However, our study was small, and larger prospective studies of patients with different stages of disease are needed to define the clinical value of this new marker.


   Acknowledgments
 
This work was supported by grants from the Finska Läkaresällskapet, the Albin Johansson Foundation, the Liv och Hälsa Foundation, the Research Foundation of Helsinki University Central Hospital, and the Finnish Academy of Sciences.


   Footnotes
 
1 Nonstandard abbreviations: AAT, {alpha}1-antitrypsin; TAT, tumor-associated trypsinogen; IFMA, immunofluorometric assay; and TBS, Tris-buffered saline.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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J. M. Andersen, J. Hedstrom, E. Kemppainen, P. Finne, and P. Puolakkainen
The Ratio of Trypsin-2-{{alpha}}1-Antitrypsin to Trypsinogen-1 Discriminates Biliary and Alcohol-induced Acute Pancreatitis
Clin. Chem., February 1, 2001; 47(2): 231 - 236.
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