Tumor Markers of Pancreatic Carcinoma

J. V. KLAVINS, MD, PHD

There is no well-defined specific tumor marker for carcinoma of the pancreas. Carcinoembryonic anti- gen (CEA) and alphafetal protein (AFP) are produced by some pancreatic cancer cells. A pancreatic oncofetal antigen (POA) cross-reacts with other malignant neoplasms and is not produced by all pan- creatic carcinomas. An ammonium sulfate fraction (0-25%) from an aqueous extract of pancreatic carcinoma tissues contains an antigen cross-reacting with extracts of other pancreatic carcinomas and various Carcinomas representing all three germinal layers. A heterologous antiserum to this antigen cross-reacts with sera from patients with pancreatic carcinomas in a dilution of 1:16,000. At lower dilu- tions this antiserum reacts with sera from patients with other carcinomas and with sera from some apparently healthy individuals.

Cancer 47:1597-1601, 1981.

MBRYoNAiJ G E N E products like carcinoembryonic E antigen (CEA), pancreatic oncofetal antigen (POA), and an antiserum recently produced in our laboratories can be used as markers associated with some pancreatic carcinomas. Presently there is no defined specific tumor marker for neoplasms of the pancreas.

Carcinoembryonic Antigen (CEA)

Determination of plasma CEA has contributed to the diagnosis of pancreatic carcinoma. Elevated plasma CEA levels were found in 85% of 27 patients with pancreatic carcinoma.” The CEA determination was superior to upper gastrointestional series, hypotonic duodenography, coeliac arteriography, percutaneous transhepatic cholangiography , and liver scanning. Elevated CEA levels were found in three patients whose biopsies showed no evidence of carcinoma. This test did not benefit the patients because, at the time of the testing, the carcinomas were far advanced.

Other studies have indicated that CEA, although

Presented at the International Meeting on Pancreatic Cancer, Louisiana State University School of Medicine, New Orleans, Louisiana. March 10- I t . 1980.

From the Departments of Pathology, Catholic Medical Center and Laboratories. Long Island Jewish-Hillside Medical Center, Queens Hospital Center Affiliation, New York, New York.

Professor of Pathology, State University of New York at Stony 9rook and Chairman of the Department of Pathology, Catholic Medical Center.

Supported by NCI Grant no. 3R26CA20128-03 through the dational Pancreatic Cancer Project.

Address f ix reprints: J. V . Klavins, MD, PhD, 88-25 153rd :treet, Suite ? J , Jamaica NY 11432.

Accepted for publication September 22, 1980.

greater than 2.5 ng/ml in most of the pancreatic cancer patients, also was present in patients with other cancers and noncancerous diseases.6 Serum CEA levels ranged from 0.6-36 ng/ml in cases of pancreatic carcinomas. Elevated levels above 2.6 ng/ml were found in 68% sera of patients with pancreatic cancer and 7% with chronic pancreatitis.29 With simultaneous serum CEA and beta 2-microglobulin measurements it was possible to differentiate chronic pancreatitis from ca~cinoma.~ Beta 2-microglobulin levels were not elevated in pa- tients with chronic pancreatitis.

Pancreatic juice of carcinoma patients contained significantly more CEA than that of patients with any other pancreatic disease.19 It is possible that elevated CEA values in pancreatic juice occur earlier than in serum. Among six patients with CEA about 2.5 ng/ml in duodenal drainage, only four had elevated plasma CEA.I7 Patients with pancreatic carcinoma had more than 30 ng/ml of CEA in pancreatic juice.25 However, from another study it appeared that endoscopic retro- grade cholangiopancreatography was a more reliable method to differentiate pancreatic carcinoma from chronic pancreatiti~.~ In both these diseases pancreatic juice CEA levels were greater than 110 ng/ml.

The measurement of CEA in duodenal fluid after secretin-cholecystokinin stimulation was not success- ful in differentiating pancreatic carcinoma from chronic p a n c r e a t i t i ~ . ~ ’ ~ ” ~ ’ ~ ~ A combined approach, using endo- scopic retrograde pancreatography with cytologic examination and CEA determination in pancreatic juice, was successful in achieving diagnostic accuracy in all 47 cases studied.’ Among these cases there were nine patients with pancreatic carcinoma, 16 with chronic pancreatitis, and 22 controls.

0008-543X/81/0315/1597 $0.75 0 American Cancer Society

1597

1598 CANCER March 15 S~lpplernent 1981 VOl. 47

A serial CEA determination in plasma of patients with pancreatic carcinoma serves as a monitoring procedure in cases with resectable tumor and elevated CEA levels. Its diagnostic value due to a wide range of variation is marginal. For example, in 34 patients with nonresectable tumors 21% had values of S ng/ml or less and in 57 patients with metastases 19% had such low levels of CEA.'?

Serum CEA-S, an isomeric form of CEA, may be a useful pancreatic carcinoma marker. It was elevated in 12 of 13 patients with pancreatic carcinoma.'*

There was a CEA-related compound in the ascitic fluid of a patient with pancreatic carcinoma.'o It con- tained IgM and a SS component reacting with an anti- serum to CEA; however, antiserum to the 5s protein did not react with CEA. This antigen may be a more specific pancreatic carcinoma-associated component than CEA.

Alpha Fetal Protein (AFP)

AFP has been associated with pancreatic car- cinoma.zH In comparison with CEA and pancreatic oncofetal antigen (POA), the AFP provided no ad- vantage in diagnosing pancreatic carcinoma. True posi- tive results in detecting 38 pancreatic carcinomas among 136 patients with suspected major intraab- dominal disease were obtained with AFP in 33%. True negative results that excluded pancreatic cancer were found in 71%. In this respect POA would be superior with a 60% positive and a 94% negative evaluation; corresponding percentages for CEA were 48 and 75%. From these studies it appears that AFP is less valuable than other tumor markers in evaluating patients with pancreatic carcinoma.

Pancreatic Oncofetal Antigen (POA)

An antiserum was raised in rabbits with fetal human pancreas. Using the immunodiffusion method the anti- serum reacted with three human pancreatic carcinoma tissue homogenates and with 36 out of 37 sera from patients with pancreatic carcin0ma.I This antiserum did not react with homogenates of normal adult pan- creas, fetal colon, fetal liver, clonic carcinoma, and hepatoma. It did not react with sera from patients with acute and chronic pancreatitis, obstructive jaundice, cirrhosis of the liver, hepatoma, and carcinomas of the colon or stomach. This oncofetal antigen was not immunologically related to AFP or CEA. It had a2- mobility, similar to lactoferrin, but was not immuno- logically related to lactoferrin.

Subsequently POA was purified and its molecular weight was estimated to be between 800,000 and 900.000 daltons.i This glycoprotein was unaffected by DNAse or RNAse, but the immunologic reactivity was

abolished by trypsin, papain, pronase, pepsin, or in- cubation with Con-A Sepharose. Using rocket im- munoelectrophoresis assay it was discovered that elevated serum levels of POA were found in predominantly pancreatic carcinoma patients as well as in bronchogenic carcinoma. carcinoma of large intestine, biliary carcinoma, and less frequently in some nonmalignant conditions and pregnancy.R It seemed more likely for patients with pancreatic cancer to have elevated serum POA values than patients with any other disease except biliary tract carcinomas. Using an enzyme immunoassay it was found2" that among 20 cancer patients 80% had abnormally high serum POA values.

The diagnostic sensitivity and specificity of the ultrasonography for patients with reported pancreatic carcinoma was improved by additional testing of CEA and POA.I5

Pancreatic Carcinoma Specific Components

There is some evidence that pancreatic carcinoma cells may contain some specific tumor markers. Using crude membrane preparation from pancreatic carci- noma, there was a significant inhibition of leukocyte adherence in the microassay.':3 Positive leukocyte adherence inhibition was observed in 18 of 19 pancreatic carcinoma patients. This inhibition was not evident of white blood cells in ten patients with acute pancreatitis, 28 healthy volunteers, ten patients with upper gastrointestinal malignancies,I3 and carcinomas of lung or colon.Li The white blood cells of the patients with pancreatic carcinoma were not affected by exposing them to crude membrane extracts of colonic carcinoma,I:' stomach carcinomas, or carcinomas of the lung."

There was a unique polycytidylic acid-specific serum ribonuclease isoenzyme in the serum of five patients with pancreatic carcinoma.22 On isoelectric focusing this isoenzyme had an Ip of 4.72.

From the author's laboratories a widely cross- reacting tumor-associated antigen in carcinoma of pancreas was reported. l 6

Materials and Methods

Human pancreatic carcinoma (4.5 g, obtained from an autopsy) was homogenized at 0-2 C in 75 ml of 10 mmol phosphate buffer at p H 7.5 containing 0.1 M NaCI, 0.6% eaminocaproic acid, and aprotinin (20 kallikrein inactivator U/ml). The homogenate was centrifuged at 4000~ for 20 minutes at 4 C . The supernatant was made to 25% saturation with am- monium sulfate and the precipitate was removed by centrifugation at l0,OOOg for 10 minutes at 4 C. Thepre- cipate was dialyzed against 100 volumes of homog-

N o h M A R K E R S OF PANCREAI- IC CARCINOMA . Kltii,iirs 1599

enizing buffer without NaCI, lyophilyzed, and used as an antigen CAPI. The other fractions (post 25% saturation with ammonium sulfate) contained no or very little of tumor-aswciated antigenic activity as compared with CAPI. They were not. therefore, studied further.

Antiserum was produced in rabbits by weekly subcutaneous injections of 0.5 mg of lyophilyzed CAPI with complete Freund's adjuvant for 14 weeks. Five milliliters of rabbit antiserum was mixed with equal volunie of pooled normal human serum (20 donors). 100 pl of heparin, and 5 mg (dry weight) each of normal pancreas, colonic mucosa, liver, kidney, and spleen to absorb antibodies against normal components. The mixture was incubated at 37 C for 2 hours and then kept at 4 C overnight. The mixture was centrifuged at 35,000g for 20 minutes at 4 C. The supernatant wits filtered through a glass wool column (10 x 0.1 cm). This clear, filtered, absorbed antiserum (uCAP1) was used for the studies.

lmmunoperoxidase PAP technique''' was used. Sec- tions cut from paraffin-embedded blocks of tissues were deparaffinized and rehydrated before staining. Goat anti- serum to rabbit IgG fraction (GARbGG) and rabbit anti- serum to horse radish peroxidase coupled to the enzyme (PAP) were obtained from commercial sources. Pre- immunization rabbit serum and phosphate-buffered d i n e at p H 7.6 (PBS) were used as controls. A saturated solution of 3-3' dimethoxyaminobenzidine (DAB) was prepared fresh by adding 75 mg (free base) to 100 ml of PBS. 'The solution was stirred con- tinuously for at least Z hours. filtered, and 0.1 ml of a freshly prepared solution of 3% H,Oy added. The re- hydrated tissue sections were treated sequentially with: ( I ) nonimmune rabbit serum in a dilution 1:5 to block nonspecific adherence of Fc portions of aCAPI; (2) aCAPI in a dilution of 1:500; (3) GARbGG in a dilution of 1:100; (4) PAP in a dilution of I : 1000: and (5) DAB-H,O,. In steps 1-4 the sections were covered with a few drops of antisera and the slides incubated in moist chambers at 37 C for 10 minutes. Before and after each step the slides were washed in PBS for 10 minutes with a total of three changes. After step 4 and washing in PBS, the slides were submerged in DAB-H'Q solution for 10 minutes, then washed, counterstained with methylene blue, de- hydrated in graded alcohol and xylene. and mounted wiht permount. Thus prepared sections were examined with a light microscope. Binding of aCAPI was indicated by a brown reaction product.

To test the possible relationship of CAP1 to CEA the following sequence was used: ( I ) nonimmune goat serum (1:5) to block nonspecific adherence of Fc portions of goat anti-CEA; (2) goat anti-CEA ( 1 : 100) to block the binding sites of CEA; ( 3 ) uCAP1 (1:500);

1 ' 4 ~ 1 t I . Reactivity of Malignant Neoplasms from Different Sites with uCAPl Using Immunoperoxidase Method

Site of malignant aCAPl aCAPl neoplasms reactive nonreactive Total

Rc\piratory 8 0 8 Gastrointestinal 29 1 30 Genitourinary 12 14 26 Breast 5 6 I I Basal cell carcinomas 0 2 2 Lymphoma 0 I I n) 1 . \ I 54 24 78

(4) GARbGG (l: l00); (5) Pap (1:IOOO); and 6. I)AB-H202. In our experience immunoperoxidase microscopy was more sensitive than complement fixation method.

For the microcomplement fixation method tissues obtained from autopsies and surgeries were homog- enized at 0-2 C in 10 V of 10 mmol phosphate buffer at pH 7.5 containing 0.1 M NaCI. 0.6% €-amino- caproic acid, aprotinin (20 kallikrein inactivator U/ml). The homogenate was centrifuged at 4000g for 20 minutes. The supernatant was lyophilyzed and stored. Before t h e test the lyophilyzed powder was redissolved in complement-fixation test buffer (Oxoid, Ltd., London, England). The use of this buffer yielded antigens frre of anticomplimentary activity." The tissue concentrations were from 0.02-0.2 ng/ml of dry weight.

Patients' sera, to be examined with microcomple- ment fixation method, were heated at 56 C for 30 minutes and diluted threefold with complement- fixation test buffer. Pooled normal human sera were treated identically. In case of residual anticomple- mentary activity the sera were mixed with 0.5 mg/ml of rabbit liver powder and kept at 4 C overnight. The mixture was centrifuged at 4 0 0 0 ~ for 10 minutes at 4 C. The supernatant serum was used for the studies.

Microcomplement fixation method'.' with a modifi- cation!' was used. This method was further modified in our laboratories by adding to all the wells 25 pl of complement fixation buffer containing 0.025 mg/ml of rabbit liver powder. This procedure eliminated anticomplementary activity. Absorbed aCAPI was heated at 56 C for 30 minutes and 25 pi of it was serially diluted in all raws (A-H) of the tray. Antigen ( 2 5 A) was added to each well. Complement was added so that its concentration in raws A and E was I :7, raws B and F was I : 15, raws C and G was 1:32, and raws D and H was 1:63. Raws A-D con- tained tumor antigens and raws E - H antigens from control tissues. The control tissues consisted of sera from healthy individuals and various nonneoplastic diseases. C'ornplenient titer was checked with each

1600 CANCER March 15 Supplement 1981 Vol. 47

TABLE 2 . Reactivity of Different Neoplasms with aCAPI by Using Immunoperoxidase Method

aCAPI aCAPI Type of tumor reactive nonreactive Total

Carcinomas Larynx Lung Tongue Esophagus Stomach Colon Pancreas Gall bladder

Endometrial carcinomas Adenomatous hyperplasias of

Carcinomas of cervix Mixed Mullerian tumor Leiom yosarcomas Leiom yomas Adenocarcinomas of ovary Endometroid carcinomas of

Embryonal carcinoma of ovary Undifferentiated carcinomas of

Carcinomas of prostata Carcinomas of urinary bladder Carcinoma of kidney Carcinomas of breast Cystosarcomas phylloides of

Fibroadenomas of breast Basal cell carcinomas Lymphoma

endometrium

ovary

ovary

breast

2 6 1 1 5

16 5 1 2

0 2 0 0 0 3

3 0

0 0 2 0 5

0 0 0 0

0 0 0 0 0 1 0 0 1

2 2 1 2 2 0

1 1

2 3 0 1 4

2 2 2 1

2 6 1 1 5

17 5 I 3

2 4 1 2 2 3

4 1

2 3 2 1 9

2 2 2 1

TABLE 3. Microcomplement Fixation Test of aCAPI with Various Tissues

Tissues

Carcinoma of pancreas* Normal pancreas Carcinoma of colon Normal colon Carcinoma of breast Normal breast Normal spleen Carcinoma of lung Normal lung Carcinoma of prostata Normal prostata Carcinoma of ovary Normal ovary Carcinoma of urinary bladder Normal bladder Carcinoma of nasopharynx Normal lymph nodes from nasopharynx Carcinoma of esophagus Normal esophagus

No. of tissues tested

8 4 8 8 6 6 2 2 3 2 2 2 2

No. of tissues reacted

8 0 8 0 6 6 0 2 0 2 0 2 0 2 0 1 0 I 0

* Reactions were positive when aCAPI was diluted 16,000-fold but the other tissues did not react at this dilution of the antiserum.

experiment separately. The trays were sealed with sealing tape and kept at 4 C for 18 hours. Then 100 A of hemolytic system (indicator) was added at room temperature for 1 hour and the results examined. Inhibition of hemolysis was considered a positive reaction.

Results

With the immunoperoxidase technique there was evidence of reactivity of an antigen with aCAPI in 54 of 78 malignant neoplasms examined (Table 1). This group of tumors constitutes derivatives of all three germinal layers. The antigen was present in the cyto- plasm of the tumor cells or in the lumen of the tumor glands. Blocking experiments using goat anti-CEA failed to abolish the specific staining of aCAPI. The reactivity of different kinds of neoplasms with aCAPI is indicated in Table 2. In a pancreatic carcinoma, there was an area of normal acinar tissue with a duct. Although the acinar tissues were CAPI- negative, the secretions in the duct reacted with aCAPI. From the nonneoplastic control tissues fetal colon of approximately 12 weeks gestation contained an antigen in the mucosal cells reacting with aCAPI. The nonneoplastic tissues adjacent to tumors did not react with the aCAPI. There was no reactivity when PBS or preimmunization serum was substituted for aCAPI.

Using microcomplement fixation method, the aCAPI reacted with tissue extracts from malignant neoplasms but not with the extracts of corresponding normal tissues (Table 3).

The reactivity of sera with aCAPI is summarized in Table 4. Among the sera of 42 healthy individuals there were 23 that reacted with aCAPI. There was no distinct pattern in different nonneoplastic diseases or in malignant neoplasms regarding the reactivity of sera with aCAPI. In studying the sera with microcom- plement fixation method absorption was not carried out with those sera from apparently healthy individuals that reacted with aCAPI. An exception was sera from all seven patients with pancreatic carcinoma. Only these sera reacted with aCAPI diluted 1: 16,000.

Discussion

From the immunoperoxidase microscopy studies it appears that CAPI is an antigen specifically present in the majority of the carcinomas. The reactivity of fetal colonic mucosa indicates that CAPI is an oncofetal antigen. It is not CEA. The blocking experiments failed to eliminate the reactivity with aCAPI.

No. 6 MARKERS OF PANCREATIC CARCINOMA . Klavins 1601

TAHLE 4. Reactivity of aCAPI with Sera ~ ~~

aCAPI aCAPI Type of serum reactive nonreactive Total

Patients with malignant neoplasms 74 I I 85

Patients with various diseases without malignant neoplasms 38 54 92

Healthy individuals 23 19 42

From the complement fixation studies it appears that the reactivity of sera from healthy individuals and patients with other nonneoplastic diseases with aCAPI is based on some component that is present only in serum and not in tissues. An alternate possibility is that a normal component present in human serum was incompletely absorbed.

Pancreatic carcinomas apparently contained a highly reactive component that is not present in other malignant neoplasms or in sera of patients with different diseases.

The composition of CAP1 is not yet defined. It probably is an antigen consisting of several sub- stances.

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