American Journal ofPathology, Vol. 136, No. 3, March 1990 Copyright© American Association ofPathologists
Expression of Human Tumor-associated Antigens in Pancreatic Cancer Induced in Syrian Hamsters Yoshiyuki Takiyama,* Hiroshi Egami,* and Parviz M. Pour*t From the Eppley Institute* and the Department of Pathology and Microbiology,t University ofNebraska Medical Center, Omaha, Nebraska
Our previous studies have shown that pancreatic cancer induced in Syrian hamsters by N-nitrosobis(2-oxopropyl)amine (BOP) shows remarkable similarities with the human disease in morphologic and biologic characteristics. Moreover, both human and hamster pancreatic cancer share expression of some tumor-associated antigens, such as those with blood group specificities, including A, B, H, Leb, Le7, and Ley. By examining other antigens commonly expressed in human pancreatic cancer, we have found that monoclonal antibodies COI 7IA (recognizing 17-lA antigen), OC 125 (recognizing CA 125 antigen), B72.3 (recognizing TAG72 antigen) and DU-PAN-2 react with inducedpancreatic cancer in a pattern similar to that seen in human pancreatic cancer. Remarkably, although the epitopes of the antigens recognized by these three antibodies are different, many tumor cells were reactive with all these antibodies. However, in contrast to the human cancer, none of these antigens were expressed in the normal hamster pancreatic tissue, exceptfor 17-JA. However, all of these antigens were expressed in some hamster tissues showing the same cellular localization as pancreatic cancer cells and corresponded, to a great extent, with findings in human tissue. Expression of these antigens was diminished in vitro (cell culture) but was regained in vivo (homologous transplantation). The results emphasize the usefulness of this experimental model for studying some aspects oftissue antigenicity, particularly as it relates to pancreatic cancer. (Am JPathol 1990, 136: 707-
715)
Monoclonal antibodies (MAbs) detecting tumor-associated antigens (TAAs) have been used for immunodi-
agnosis and immunotherapy. A number of these TAAs were closely related to blood group substances. The epitope of CA19-9 is a sialylated Leal and that of TAG-72 is believed to be a sialosyl-Tn.2 In previous studies, we
reported remarkable similarities between human pancreatic cancer and that induced in Syrian hamsters by Nnitrosobis(2-oxopropyl)amine (BOP) in morphologic, biologic, and biochemical characteristics.34 Furthermore, as in humans,5 BOP-induced hamster pancreatic cancer expresses blood group antigens.6 To further examine the similarities of BOP-induced pancreatic cancer to the human disease, we investigated the expression of some antigens known to be common TAAs in human pancreatic cancer, ie, CA19-9, DU-PAN-2, CA125, TAG-72,7e and 17-1 A9 in BOP-induced pancreatic neoplasms, as well as in the normal tissues of hamsters.
Materials and Methods
Animals Eight-week-old outbred Syrian golden hamsters (Eppley Colony) with initial body weights of about 100 g were housed in plastic cages (Macrolon) on granular cellulose bedding (Bed-O-Cobs, Anderson Cob Co., Maumee, OH) in groups of five. They were kept under standard laboratory conditions (temperature 22±3 C; relative humidity 40±5%; 12-hour light/i 2-hour dark cycle), and given Wayne pelleted diet (Allied Mills, Chicago, IL) and water ad libitum.
Normal Hamster Tissues Hamsters at the age of 8 weeks and 30 weeks, 10 hamsters each, were killed and all tissues were fixed in Bouin's Supported by NCI Laboratory Cancer Research Center Support Grant CA36727 and ACS Special Institutional Grant SIG- 16. Accepted for publication November 6, 1989. Address reprint requests to Dr. Parviz M. Pour, the Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, NE 68105-1065.
707
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solution and processed for histology by conventional methods.
esthesia. After 3 weeks, the hamsters were sacrificed and their pancreases were processed for histology and immunohistochemistry.
Primary Pancreatic Cancer Human Pancreatic Cancer Tissue N-nitrosobis(2-oxopropyl) amine (BOP) was synthesized in our laboratory as described,10 dissolved in physiologic saline before use, and injected subcutaneously (10 mg/ kg body weight) into 18 male hamsters weekly for 4 weeks. At the 30th week, hamsters were necropsied and their pancreases were fixed in Bouin's solution and processed for histology.
Cancer Cell Line (PC-1) Establishment and characteristics of pancreatic cancer cell line (PC-1) from a BOP-induced primary tumor have been reported.11 We have shown that this cell line retains the ability to produce blood group-related substances and can be propagated in vivo by their injection into the homologous pancreas. For histologic examination, the culture cells were removed from the bottom of the culture flask with a rubber policeman and then fixed in absolute alcohol for 20 minutes. After washing with PBS, a small volume of 1% agarose (Sigma Chemical Co., St. Louis, MO) was added to the pellet at 370C. The agar plug was dehydrated and processed for histology.
Intrapancreatic Transplanted PC-1 Cells A total of 1 X 107 PC-1 cells from the 30th passage were resuspended in physiologic saline and injected by a 26guage syringe into the splenic lobe of nine recipient male hamsters via abdominal incision under pentobarbital an-
Pancreatic cancer tissues from our previous study5 were used as control material and were similarly processed for immunohistochemistry.
Histology Paraffin-embedded tissues were cut into serial sections, each 4 um thick. One of these sections was stained with hematoxylin and eosin, and others were processed by immunoperoxidase procedures as described below.
Monoclonal Antibodies CO1 9-9 and CO1 7-1A were provided by Dr. Z. Steplewski (Wistar Institute, Philadelphia, PA) and OC125 by Centocor Inc. (Malvern, PA). Productions and characterizations of these antibodies have been reported.12-16 DU-PAN-2 was a gift of Dr. R. Metzger (Duke University, Durham, NC) and B72.3 was provided by Dr. J. Schlom (NIH,
Bethesda, MD). Productions and characterizations of DUPAN-2 and B72.3 have been reported.17-21 The immunoglobulin isotype of CO1 9-9, OC1 25, and B72.3 was IgG1, that of CO1 7-1 A was IgG2a, and that of DU-PAN-2 was IgM. As control materials, MOPC-21 (Litton Bionetics, Charleston, SC) was used as IgGl, MAb UPC-10 (Litton Bionetics, Charleston, SC) as IgG2a,22 and the culture supernatant of P3x63Ag8 murine myeloma cell line as IgM.
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Table 1. Immunoreactivity ofHuman Tumor-AssociatedAntigens in Normal Pancreatic Cells and in Pancreatic Cancer Cells
Primary
Transplantable pancreatic
Antibody
Acinar cells
Islet cells
Duct cells
cancer cells
PC-1 cells
C019-9
* -
_
-
_ -
_
_
-/+
-
-/+
++
++
+
++
++
-
-
-
++ ++
++
++ +++ +++
DUPAN-Il C017-1A OC 125 B72.3 *
+
cancercell
See Materials and Methods.
Immunoperoxidase Procedure Vectastain ABC kit (Vector Laboratories, Burlingame, CA) was used as described earlier.6-8,23 Control slides were examined as follows: (i) tissues were incubated with PBS instead of primary antibodies; (ii) tissues were incubated with UPC-10, MOPC-21, or culture supernatant of P3x63Ag8 murine myeloma cell line instead of primary MAbs.
Scoring The reactivity of tumor cells with each MAb was given in arbitrary units: 3+ when more than 50% of the cells were reactive, 2+ when 10% to 50% of the cells were reactive, 1 + when less than 10% of the cells were reactive, and when no cells were reactive. Cellular localization of each antigen was classified as cytoplasmic granular, Golgi, or glycocalyx pattern. Cytoplasmic granular staining was defined as presence of fine granular material dispersed through the cytoplasm. If the stained coarse granules were confined to the projected region of the Golgi apparatus, Golgi pattern of reactivity was used. Staining of the luminal surface of the cells was termed glycocalyx pattern of staining.
Results
Histology Pseudoductular formations and ductular adenomas were found in almost all hamsters 30 weeks after the last BOP injection. Ductular carcinomas in situ were found in all hamsters and single or multiple ductal/ductular adenocarcinomas (all of the well-differentiated type) in 16 of 18 hamsters. Serial morphologic changes in treated animals in comparison with human pancreatic cancer have been previously reported,34 as have been the pattern of PC-i cells and their growth characteristic in the homologous pancreas.11
Histologically, the primary and transplanted tumors showed identical patterns (Figure 1). Implantation of cancer cells into pancreatic tissue of nine hamsters resulted in 100% tumor take within 3 weeks, with invasion of surrounding normal pancreatic tissue and occasional metastases into the regional lymphnodes.
Immunohistochemistry Control slides of tissues showed no staining with MAbs UPC-10, MOPC-21, and culture supernatant of P3x63Ag8 murine myeloma cell line.
Primary Pancreatic Cancer Contrary to the findings in human material, C019-9 was not immunoreactive with any normal, premalignant, or malignant pancreatic cells. However, as with human pancreatic cancer, CO1 7-1 A, OC 125, B72.3, and DU-PAN-2 were immunoreactive with cancer cells of all 20 tumors (all of the well-differentiated type) examined. The percentages of the reactive cells were 30%, 50%, 30%, and 5%, respectively (Table 1), and this was consistent in all tumors, except for DU-PAN-2, in which reactivity was weak and inconsistent. The reactivity of B72.3 and DU-PAN-2 showed a decreasing trend in relation to the size of tumors. In tumors of 10 mm, the reactivity of B72.3 was 5% to 10% and of DU-PAN-2 reactivity was 0% to 5%. On the other hand, in all hyperplastic, dysplastic lesions and carcinomas in situ, a larger number of cells were reactive with all C017-1A, OC 125, and B72.3. The localization of the reactive material was in all cases, including human tumors, cytoplasmic and in granular form (Table 2, Figures 2 to 4). Goblet cell-like tumor cells in hamsters showed a strong diffuse cytoplasmic staining with B72.3, as did the luminal contents of malignant glands (Figure 4). In the normal pancreatic tissue only C017-1 A showed positive reactivity with acinar cells in granular cytoplasmic form.
Takiyama, Egami, and Pour
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AJP March 1990, Vol. 136, No. 3
.a
Figure 2. Reactivity of CO1 7-lA with primarypancreatic cancer(a), with PC- I cells (itnset in a), and with human pancreatic cancer cells(b). The staininig is in granular form and supranuclear (ABC method. X200; intset, X 240).
PC-1 Cells
of the cell membranes that was not seen in the primary tumor cells.
Approximately 30% of cells reacted with C017-1A and OC125, less than 10% of them reacted with B72.3, and none reacted with DU-PAN-2 (Table 1). Compared with primary pancreatic cancer cells, the reactivity of B72.3 cells in PC-1 cells was less (+ vs. ++). Although the staining pattern was mostly cytoplasmic granular, as in primary tumors (Table 2), some cells showed an intense staining Table 2. The Staining Patterns of Tumor-Associated Antigen in Pancreatic Cancer Cells
Cytoplasmic Golgi pattern C019-9 DUPAN-II C017-1 A OC 125 B72.3 *
granular pattern
Glycocalyx pattern
Luminal contents
+ +
_
_
+*
+*
-
-
-
Occasional findings.
+ +
Intrapancreatic Transplanted Tumor The patterns of immunostaining of intrapancreatic cancer cells were similar to those seen in the primary tumors (Table 2). However, the reactivity of these tumor cells with OC125 and B72.3 was greater than in the primary tumor cells (Table 1). Also in these hamsters, the reactivity of B72.3 and DU-PAN-2 was dependent on the size of tumors and decreased with the increasing size. Normal pancreatic acinar cells surrounding the tumors showed a cytoplasmic fine granular staining pattern with C017-1 A, as seen in the pancreas of normal hamsters. Metastatic cells in the regional lymphnodes were stained more intensely with OC125 than were the tumor cells within the pancreas (Figure 3).
TAAs in Hamster Pancreatic Cancer Cells 711 AJPMarch 1990, Vol. 136, No. 3
Figure 3. Reactivity of OC125 with hamster (a, b) and human (c) pancreatic cancer cells. Note a granular staining pattern in
all cancer cells. The intensity of the staining is greater in the metastatic canscer cells into the regional lymphonode (b X200) (ABC method. Inset, stainingpattern ofPCI cells with OC125, X240).
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Figure 4. Reactivity of B72.3 with hamster (a, b) and human (c) pancreatic cancer cells in a fine granular (a, c) or diffuse cytoplasmic pattern (b) (ABC method, X200. inset, in PC-1 cells, there are cytoplasmic granular and membrane staining, X240).
TAAs in Hamster Pancreatic Cancer Cells 713 AJPMarch1990, Vol. 136, No. 3 Table 3. Reactivity ofHamster Tissues with MonoclonalAntibodies* C019-9 DU-PAN-11 Tissue Olfactory mucosa Thymus Trachea Larynx Lung Heart
Spleen Thyroid Parathyroid Saliva Esophagus Fore-stomach
Glandular-stomach Duodenum Upper Lower
Jejejunum Ileum Cecum Colon Rectum Liver Gall Bladder Pancreas Kidney Paraurethral gland Urethra Prostata Testis Vagina Fallopian tube Uterus Ovary
-
-
-
-
-
--
C017-1A
OC 125
+
B72.3 +
-
+
+ +
+
+
+ + + + + + +
-
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Means no staining, + means positive staining. t Positive staining reported in human tissues. Not all the tissues listed here have been examined in humans.'51'6'1927 f Superficial layer of the squamous cell epithelium. § Goblet cells. 1" Collecting tubules. ¶ Proximal tubules. # A few follicular cells. *
Normal Hamster Tissues Reactivities of hamster tissues (positive or negative) with the MAbs are summarized in Table 3. There were no differences in the reactivities of the MAbs with the tissues of hamsters of different ages (8 weeks vs. 30 weeks).
Discussion CA1 9-9, CA1 25, DU-PAN-2, and B72.3 have been shown to be expressed in many human pancreatic cancer cells, and C01 7-1A and B72.3 are being used for immunotherapy24'25 and radioimmunoscintigraphy,26 respectively. However, the use of these MAbs for detailed studies was limited due to the lack of expression of some of these antigens, such as 17-1A and TAG-72 (recognized by B72.3) in human cancer cell lines,27 and the absence of a suitable experimental model. The present study demonstrates that pancreatic cancers induced in Syrian ham-
sters provide a unique tool for such studies because these tumors share expression of TAAs known to be commonly expressed in human pancreatic cancer cells. Despite these similarities, there are also some dissimilarities in the antigenicity of tumor cells in humans and in hamsters. This includes lack of CAl 9-9 antigens in the hamster cancer cells. In humans, CAl 9-9 is detectable in normal human pancreas in minute concentration, but is expressed in about 80% of pancreatic cancer tissues.7 However, this antigen was missing in both the normal and malignant pancreatic cells in hamsters. Possible reasons for the absence of this antigen in hamsters have been addressed (Takiyama et al, manuscript submitted for publication) and could be related to the lack of Le' substance in hamster tissues. DU-PAN-2, occurring in approximately 90% of human pancreatic cancer and, occasionally also in normal human tissues,7 was also missing in the normal pancreatic tissues in hamsters but was present in a few pancreatic cancer cells in vivo and in the goblet cells of the upper but not lower intestinal mucosa of this species.
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The distribution patterns of this antigen in hamster tissue confirms the view that the antibody recognizes some epitope of mucin substances,17'18 and that the nature of mucin differs in different segments of the hamster intestinal tract. C017-1 A, which detects a 37-kd protein,28 has been found by us9 and others28 in almost all normal and malignant human pancreatic tissues. As in humans, this antigen is produced in large amounts by normal hamster tissues, particularly in the gastrointestinal tract, including the pancreas. The localization of the antigen in both human and hamster pancreatic cancer tissue was similar. The determinant of MAb OC125 is thought to be a highmolecular weight glycoprotein complex14 and possibly a peptide.15 In hamsters this antigen was missing in the normal pancreas but was present in more than 50% of pancreatic cancer cells, as well as in the normal gastrointestinal and reproductive tissues. The pattems of the reactivity and the cellular localization were, again, similar to those seen in humans (Figure 3). Because the expression of this antigen in human tissues was found by us to be closely related to blood group A antigen (Pour et al, manuscript submitted for publication), and A antigen had been shown to be expressed by pancreatic cancer cells in hamsters,45 its expression in hamster tissues was expected. The reactivity and cellular localization of MAb B72.3 in pancreatic cancer tissues in hamsters and in humans was also similar (Figure 4). However, compared with the findings in the normal human tissues,19'0 this antigen had a broad spectrum of distribution in hamster tissues but was missing in the normal pancreas. The remarkably high concentration of the antigen TAG-72, a proposed sialosylTn,2 in the hamster salivary gland is consistent with the findings in cows.2 Interestingly, the immunoreactivity of OC125 and B72.3 was strikingly similar in both normal and cancer tissues in hamsters. The possibility that these two MAbs recognize the same antigen is remote because the epitopes of CAl 25 and TAG-72 were shown to be different and the antibodies are not cross reactive.-9 Furthermore, the reactivity of B72.3 (and of DU-PAN-2) decreased with the increasing size of the tumors, a finding that was not true for other MAbs. Whether hamster cells can coproduce these two antigens remains to be seen. Expression of these two antigens in PC-1 cells was found in vitro (cultured cells) and in vivo. However, in vitro expression of TAG-72 was decreased (as in the cells of large tumors), whereas in transplants of PC-1 cells within the pancreas the reactivity with OC125 and B72.3 was increased as compared with the primary cancer cells. Furthermore, in contrast to the cancer cells of the primary and intrapancreatic transplants, a reactivity of all three antibodies was also seen with the membrane of PC-1 cells. These findings indicate that the site and quantity of antigen is related
to the environment of tumor cells and, in the case of B72.3, also to the differentiation. Interestingly, the expression of B72.3 was retained in PC-1 cells, whereas it is lost in human cancer cell lines.27 Nevertheless, the present study demonstrates that hamsters share with humans the expression of some antigens in their normal tissues and pancreatic cancer cells and highlight the usefulness of this model for studying antigenicity of normal and malignant pancreatic tissues.
References 1. Magnani JL, Nilsson B, Brockhaus M, Zopf D, Steplewski Z, Koprowski H, Ginsburg V: A monoclonal antibody-defined antigen associated with gastrointestinal cancer is a ganglioside containing sialylated lacto-N-fucopentaose 11. J Biol
Chem 1982,257:14365-14369 2. Kjeldsen T, Clausen H, Hirohashi S, Ogawa T, lijima H, Hakomori S: Preparation and characterization of monoclonal antibodies directed to the tumor associated 0-linked sialosyl2-6 a-N-acetyl-galactosaminyl (Sialosyl-Tn) epitope. Cancer Res 1988, 48:2214-2220 3. Pour P, Mohr U, Cardesa A, Althoff J, Kruger FW: Pancreatic neoplasms in an animal model: Morphological, biological and comparative studies. Cancer 1975,36:379-389 4. Pour PM, Runge RG, Birt D, Gingel R, Lawson T; Nagel D, Wallcave L, Salmasi SZ: Current knowledge of pancreatic carcinogenesis in the hamster and its relevance to the human disease. Cancer 1981, 47:1573-1587 5. Pour PM, Tempero MA, Takasaki H, Uchida E, Takiyama Y, Burnett DA, Steplewski Z: Expression of blood group-related antigens ABH, Lewis A, Lewis B, Lewis X, Lewis Y, and CA 19-9 in pancreatic cancer cells in comparison with the patient's blood group type. Cancer Res 1988,48:5422-5426 6. Pour PM, Uchida E, Burnett DA, Steplewski Z: Blood-group antigen expression during pancreatic cancer induction in hamsters. Int J Pancreatol 1986,1:327-340 7. Takasaki H, Uchida E, Tempero MA, Burnett DA, Metzgar RS, Pour PM: Correlative study on expression of CA19-9 and DU-PAN-2 in tumor tissue and in serum of pancreatic cancer patients. Cancer Res 1988, 48:1435-1438 8. Takasaki H, Tempero MA, Uchida E, Buchler M, Ness MJ, Burnett DA, Metzgar RS, Colcher D, Schlom J, Pour PM: Comparative studies on the expression of tumor-associated glycoprotein (TAG-72), CAl 9-9 and DU-PAN-2 in normal, benign and malignant pancreatic tissue. Int J Cancer 1988, 42: 681-686 9. Takiyama Y, Tempero MA, Takasaki H, Onda M, Tsuchiya R, Buchler M, Ness M, Colcher D, Schlom J, Pour PM: Reactivity of CO1 7-A and B72.3 in benign and malignant pancreatic diseases. Hum Pathol 1989 20:832-838 10. Pour P, Althoff J, Kruger FW, Mohr U: A potent pancreatic carcinogen in Syrian hamsters: N-nitrosobis(2-oxopropyl) amine. J Natl Cancer Inst 1977, 58:1449-1453 11. Egami H, Takiyama Y, Cano M, Houser WH, Pour PM: Establishment of hamster pancreatic carcinoma cell line (PC-1) producing human blood group-related antigens. Carcinogenesis 1989,10:861-869
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12. Herlyn M, Steplewski Z, Herlyn D, Koprowski H: Colo-rectal carcinoma-specific antigen: Detection by means of monoclonal antibodies. Proc Natl Acad Sci USA 1979, 76:14381442 13. Herlyn D, Koprowski H: IgG2a monoclonal antibodies inhibit human tumor growth through interaction with effector cells. Proc Natl Acad Sci USA 1982, 79:4761-4765 14. Bast RC Jr, Feeney M, Lazarus H, Nadler LM, Colvin RB, Knapp RC: Reactivity of a monoclonal antibody with human ovarian carcinoma. J Clin Invest 1981, 68:1331-1337 15. Kabawat SE, Bast RC Jr, Bhan AK, Welch WR, Knapp RC, Colvin RB: Tissue distribution of a coelomic epitheilium-related antigen recognized by the monoclonal antibody OC125. Int J Gynecol Pathol 1983, 2:275-285 16. Davis HM, Zurawski VR Jr, Bast RC Jr, Klug TL: Characterization of the CA125 antigen with human epithelial ovarian carcinomas. Cancer Res 1986, 46:6143-6148 17. Metzger RS, Gaillard MT, Levine SJ, Tuck FL, Bossen EH, Borowitz MJ: Antigens of human pancreatic adenocarcinoma cells defined by murine monoclonal antibodies. Cancer Res 1982,42:601-608 18. Metzgar RS, Rodriguez N, Finn OJ, Lan MS, Daasch VN, Fernsten PD, Meyers WC, Sindelar WF, Sandler RS, Seigler HF: Detection of a pancreatic cancer-associated antigen (DU-PAN-2 antigen) in serum and ascites of patients with adenocarcinoma. Proc Natl Acad USA 1984, 81:5242-5246 19. Colcher D, Horan Hand P, Nuti M, Schlom J: A spectrum of monoclonal antibodies reactive with human mammary tumor cells. Proc Natl Acad Sci USA 1981, 78:3199-3203 20. Thor A, Ohuchi N, Szpak CA, Johnston WW, Schlom J: Distribution of oncofetal antigen tumor-associated glycoprotein-72 defined by monoclonal antibody B72.3. Cancer Res 1986,46:3118-3124 21. Klug TL, Sattler MA, Colcher D, Schlom J: Monoclonal antibody immunoradiometric assay for an antigenic determinant
22.
23.
24.
25.
26.
27.
28.
29.
(CA 72) on a novel pancarcinoma antigen (TAG-72). Int J Cancer 1986, 38:661-669 Potter M: Immunoglobulin-producing tumors and myeloma proteins of mice. Physiol Rev 1972, 52:632-684 Hsu SM, Raine L, Fanger H: Use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase technique: A comparison between ABC and unlabeled antibody (PAP) procedure. J Histochem Cytochem 1981, 29:577-580 Sears HF, Herlyn D, Steplewski Z, Koprowski H: Phase II clinical trial of a murine monoclonal antibody cytotoxic for gastrointestinal adenocarcinoma. Cancer Res 1985, 45:59105913 Tempero MA, Pour PM, Uchida E, Herlyn D, Steplewski Z: Monoclonal antibody C01 7-1A and leukopheresis in immunotherapy of pancreatic cancer. Hybridoma 1986, 5:s133s138 Colcher D, Esteban JM, Carrasquillo JA, Sugarbaker P, Reynolds JC, Bryant G, Larson SM, Schlom J: Quantitative analyses of selective radiolabeled monoclonal antibody localization in metastatic lesions of colorectal cancer patients. Cancer Res 1987,47:1185-1189 Horan Hand P, Colcher D, Salomon D, Ridge J, Noguchi P, Schlom J: Influence of spatial configuration of carcinoma cell populations on the expression of a tumor associated glycoprotein. Cancer Res 1985, 45:833-840 Gottlinger HG, Funke I, Johnson JP, Gokel JM, Riethmuller G: The epithelial cell surface antigen 1 7-1 A, a target for antibody-mediated tumor therapy: Its biochemical nature, tissue distribution and recognition by different monoclonal antibodies. Int J Cancer 1986, 38:47-53 Lan MS, Bast RC Jr, Colmaghi Ml, Knapp RC, Colcher D, Schlom J, Metzgar RS: Co-expression of human cancer-associated epitopes on mucin molecules. Int J Cancer 1987, 39:68-72
Expression of Human Tumor-associated Antigens in Pancreatic Cancer Induced in Syrian Hamsters Yoshiyuki Takiyama,* Hiroshi Egami,* and Parviz M. Pour*t From the Eppley Institute* and the Department of Pathology and Microbiology,t University ofNebraska Medical Center, Omaha, Nebraska
Our previous studies have shown that pancreatic cancer induced in Syrian hamsters by N-nitrosobis(2-oxopropyl)amine (BOP) shows remarkable similarities with the human disease in morphologic and biologic characteristics. Moreover, both human and hamster pancreatic cancer share expression of some tumor-associated antigens, such as those with blood group specificities, including A, B, H, Leb, Le7, and Ley. By examining other antigens commonly expressed in human pancreatic cancer, we have found that monoclonal antibodies COI 7IA (recognizing 17-lA antigen), OC 125 (recognizing CA 125 antigen), B72.3 (recognizing TAG72 antigen) and DU-PAN-2 react with inducedpancreatic cancer in a pattern similar to that seen in human pancreatic cancer. Remarkably, although the epitopes of the antigens recognized by these three antibodies are different, many tumor cells were reactive with all these antibodies. However, in contrast to the human cancer, none of these antigens were expressed in the normal hamster pancreatic tissue, exceptfor 17-JA. However, all of these antigens were expressed in some hamster tissues showing the same cellular localization as pancreatic cancer cells and corresponded, to a great extent, with findings in human tissue. Expression of these antigens was diminished in vitro (cell culture) but was regained in vivo (homologous transplantation). The results emphasize the usefulness of this experimental model for studying some aspects oftissue antigenicity, particularly as it relates to pancreatic cancer. (Am JPathol 1990, 136: 707-
715)
Monoclonal antibodies (MAbs) detecting tumor-associated antigens (TAAs) have been used for immunodi-
agnosis and immunotherapy. A number of these TAAs were closely related to blood group substances. The epitope of CA19-9 is a sialylated Leal and that of TAG-72 is believed to be a sialosyl-Tn.2 In previous studies, we
reported remarkable similarities between human pancreatic cancer and that induced in Syrian hamsters by Nnitrosobis(2-oxopropyl)amine (BOP) in morphologic, biologic, and biochemical characteristics.34 Furthermore, as in humans,5 BOP-induced hamster pancreatic cancer expresses blood group antigens.6 To further examine the similarities of BOP-induced pancreatic cancer to the human disease, we investigated the expression of some antigens known to be common TAAs in human pancreatic cancer, ie, CA19-9, DU-PAN-2, CA125, TAG-72,7e and 17-1 A9 in BOP-induced pancreatic neoplasms, as well as in the normal tissues of hamsters.
Materials and Methods
Animals Eight-week-old outbred Syrian golden hamsters (Eppley Colony) with initial body weights of about 100 g were housed in plastic cages (Macrolon) on granular cellulose bedding (Bed-O-Cobs, Anderson Cob Co., Maumee, OH) in groups of five. They were kept under standard laboratory conditions (temperature 22±3 C; relative humidity 40±5%; 12-hour light/i 2-hour dark cycle), and given Wayne pelleted diet (Allied Mills, Chicago, IL) and water ad libitum.
Normal Hamster Tissues Hamsters at the age of 8 weeks and 30 weeks, 10 hamsters each, were killed and all tissues were fixed in Bouin's Supported by NCI Laboratory Cancer Research Center Support Grant CA36727 and ACS Special Institutional Grant SIG- 16. Accepted for publication November 6, 1989. Address reprint requests to Dr. Parviz M. Pour, the Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, NE 68105-1065.
707
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Takiyama, Egami, and Pour
AJPMarch 1990, Vol. 136, No. 3
-a,
-.
.
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.
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.-
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.
.
-
.
.
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Figure 1. Histologic appearance of cancer cells produced by intrapancreatic injection ofPC- I cells (X 100, H&E staining).
solution and processed for histology by conventional methods.
esthesia. After 3 weeks, the hamsters were sacrificed and their pancreases were processed for histology and immunohistochemistry.
Primary Pancreatic Cancer Human Pancreatic Cancer Tissue N-nitrosobis(2-oxopropyl) amine (BOP) was synthesized in our laboratory as described,10 dissolved in physiologic saline before use, and injected subcutaneously (10 mg/ kg body weight) into 18 male hamsters weekly for 4 weeks. At the 30th week, hamsters were necropsied and their pancreases were fixed in Bouin's solution and processed for histology.
Cancer Cell Line (PC-1) Establishment and characteristics of pancreatic cancer cell line (PC-1) from a BOP-induced primary tumor have been reported.11 We have shown that this cell line retains the ability to produce blood group-related substances and can be propagated in vivo by their injection into the homologous pancreas. For histologic examination, the culture cells were removed from the bottom of the culture flask with a rubber policeman and then fixed in absolute alcohol for 20 minutes. After washing with PBS, a small volume of 1% agarose (Sigma Chemical Co., St. Louis, MO) was added to the pellet at 370C. The agar plug was dehydrated and processed for histology.
Intrapancreatic Transplanted PC-1 Cells A total of 1 X 107 PC-1 cells from the 30th passage were resuspended in physiologic saline and injected by a 26guage syringe into the splenic lobe of nine recipient male hamsters via abdominal incision under pentobarbital an-
Pancreatic cancer tissues from our previous study5 were used as control material and were similarly processed for immunohistochemistry.
Histology Paraffin-embedded tissues were cut into serial sections, each 4 um thick. One of these sections was stained with hematoxylin and eosin, and others were processed by immunoperoxidase procedures as described below.
Monoclonal Antibodies CO1 9-9 and CO1 7-1A were provided by Dr. Z. Steplewski (Wistar Institute, Philadelphia, PA) and OC125 by Centocor Inc. (Malvern, PA). Productions and characterizations of these antibodies have been reported.12-16 DU-PAN-2 was a gift of Dr. R. Metzger (Duke University, Durham, NC) and B72.3 was provided by Dr. J. Schlom (NIH,
Bethesda, MD). Productions and characterizations of DUPAN-2 and B72.3 have been reported.17-21 The immunoglobulin isotype of CO1 9-9, OC1 25, and B72.3 was IgG1, that of CO1 7-1 A was IgG2a, and that of DU-PAN-2 was IgM. As control materials, MOPC-21 (Litton Bionetics, Charleston, SC) was used as IgGl, MAb UPC-10 (Litton Bionetics, Charleston, SC) as IgG2a,22 and the culture supernatant of P3x63Ag8 murine myeloma cell line as IgM.
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Table 1. Immunoreactivity ofHuman Tumor-AssociatedAntigens in Normal Pancreatic Cells and in Pancreatic Cancer Cells
Primary
Transplantable pancreatic
Antibody
Acinar cells
Islet cells
Duct cells
cancer cells
PC-1 cells
C019-9
* -
_
-
_ -
_
_
-/+
-
-/+
++
++
+
++
++
-
-
-
++ ++
++
++ +++ +++
DUPAN-Il C017-1A OC 125 B72.3 *
+
cancercell
See Materials and Methods.
Immunoperoxidase Procedure Vectastain ABC kit (Vector Laboratories, Burlingame, CA) was used as described earlier.6-8,23 Control slides were examined as follows: (i) tissues were incubated with PBS instead of primary antibodies; (ii) tissues were incubated with UPC-10, MOPC-21, or culture supernatant of P3x63Ag8 murine myeloma cell line instead of primary MAbs.
Scoring The reactivity of tumor cells with each MAb was given in arbitrary units: 3+ when more than 50% of the cells were reactive, 2+ when 10% to 50% of the cells were reactive, 1 + when less than 10% of the cells were reactive, and when no cells were reactive. Cellular localization of each antigen was classified as cytoplasmic granular, Golgi, or glycocalyx pattern. Cytoplasmic granular staining was defined as presence of fine granular material dispersed through the cytoplasm. If the stained coarse granules were confined to the projected region of the Golgi apparatus, Golgi pattern of reactivity was used. Staining of the luminal surface of the cells was termed glycocalyx pattern of staining.
Results
Histology Pseudoductular formations and ductular adenomas were found in almost all hamsters 30 weeks after the last BOP injection. Ductular carcinomas in situ were found in all hamsters and single or multiple ductal/ductular adenocarcinomas (all of the well-differentiated type) in 16 of 18 hamsters. Serial morphologic changes in treated animals in comparison with human pancreatic cancer have been previously reported,34 as have been the pattern of PC-i cells and their growth characteristic in the homologous pancreas.11
Histologically, the primary and transplanted tumors showed identical patterns (Figure 1). Implantation of cancer cells into pancreatic tissue of nine hamsters resulted in 100% tumor take within 3 weeks, with invasion of surrounding normal pancreatic tissue and occasional metastases into the regional lymphnodes.
Immunohistochemistry Control slides of tissues showed no staining with MAbs UPC-10, MOPC-21, and culture supernatant of P3x63Ag8 murine myeloma cell line.
Primary Pancreatic Cancer Contrary to the findings in human material, C019-9 was not immunoreactive with any normal, premalignant, or malignant pancreatic cells. However, as with human pancreatic cancer, CO1 7-1 A, OC 125, B72.3, and DU-PAN-2 were immunoreactive with cancer cells of all 20 tumors (all of the well-differentiated type) examined. The percentages of the reactive cells were 30%, 50%, 30%, and 5%, respectively (Table 1), and this was consistent in all tumors, except for DU-PAN-2, in which reactivity was weak and inconsistent. The reactivity of B72.3 and DU-PAN-2 showed a decreasing trend in relation to the size of tumors. In tumors of 10 mm, the reactivity of B72.3 was 5% to 10% and of DU-PAN-2 reactivity was 0% to 5%. On the other hand, in all hyperplastic, dysplastic lesions and carcinomas in situ, a larger number of cells were reactive with all C017-1A, OC 125, and B72.3. The localization of the reactive material was in all cases, including human tumors, cytoplasmic and in granular form (Table 2, Figures 2 to 4). Goblet cell-like tumor cells in hamsters showed a strong diffuse cytoplasmic staining with B72.3, as did the luminal contents of malignant glands (Figure 4). In the normal pancreatic tissue only C017-1 A showed positive reactivity with acinar cells in granular cytoplasmic form.
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.a
Figure 2. Reactivity of CO1 7-lA with primarypancreatic cancer(a), with PC- I cells (itnset in a), and with human pancreatic cancer cells(b). The staininig is in granular form and supranuclear (ABC method. X200; intset, X 240).
PC-1 Cells
of the cell membranes that was not seen in the primary tumor cells.
Approximately 30% of cells reacted with C017-1A and OC125, less than 10% of them reacted with B72.3, and none reacted with DU-PAN-2 (Table 1). Compared with primary pancreatic cancer cells, the reactivity of B72.3 cells in PC-1 cells was less (+ vs. ++). Although the staining pattern was mostly cytoplasmic granular, as in primary tumors (Table 2), some cells showed an intense staining Table 2. The Staining Patterns of Tumor-Associated Antigen in Pancreatic Cancer Cells
Cytoplasmic Golgi pattern C019-9 DUPAN-II C017-1 A OC 125 B72.3 *
granular pattern
Glycocalyx pattern
Luminal contents
+ +
_
_
+*
+*
-
-
-
Occasional findings.
+ +
Intrapancreatic Transplanted Tumor The patterns of immunostaining of intrapancreatic cancer cells were similar to those seen in the primary tumors (Table 2). However, the reactivity of these tumor cells with OC125 and B72.3 was greater than in the primary tumor cells (Table 1). Also in these hamsters, the reactivity of B72.3 and DU-PAN-2 was dependent on the size of tumors and decreased with the increasing size. Normal pancreatic acinar cells surrounding the tumors showed a cytoplasmic fine granular staining pattern with C017-1 A, as seen in the pancreas of normal hamsters. Metastatic cells in the regional lymphnodes were stained more intensely with OC125 than were the tumor cells within the pancreas (Figure 3).
TAAs in Hamster Pancreatic Cancer Cells 711 AJPMarch 1990, Vol. 136, No. 3
Figure 3. Reactivity of OC125 with hamster (a, b) and human (c) pancreatic cancer cells. Note a granular staining pattern in
all cancer cells. The intensity of the staining is greater in the metastatic canscer cells into the regional lymphonode (b X200) (ABC method. Inset, stainingpattern ofPCI cells with OC125, X240).
.
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*.
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Takiyama, Egami, and Pour
AJPMarch 1990, VoL 136, No. 3
Figure 4. Reactivity of B72.3 with hamster (a, b) and human (c) pancreatic cancer cells in a fine granular (a, c) or diffuse cytoplasmic pattern (b) (ABC method, X200. inset, in PC-1 cells, there are cytoplasmic granular and membrane staining, X240).
TAAs in Hamster Pancreatic Cancer Cells 713 AJPMarch1990, Vol. 136, No. 3 Table 3. Reactivity ofHamster Tissues with MonoclonalAntibodies* C019-9 DU-PAN-11 Tissue Olfactory mucosa Thymus Trachea Larynx Lung Heart
Spleen Thyroid Parathyroid Saliva Esophagus Fore-stomach
Glandular-stomach Duodenum Upper Lower
Jejejunum Ileum Cecum Colon Rectum Liver Gall Bladder Pancreas Kidney Paraurethral gland Urethra Prostata Testis Vagina Fallopian tube Uterus Ovary
-
-
-
-
-
--
C017-1A
OC 125
+
B72.3 +
-
+
+ +
+
+
+ + + + + + +
-
+t
-
--+
+t
-
-
-
+
_
-
+
+
+t
-
-
+t
-
-
-t
-
-
+t
-
_ --
+
-
-
-t
+ + + + + + + -
-
-t +11
+t -t
-
-
-
-
-
+ +
+ +
+ +
+
+
+t +t
+t
-
--t
-
-
-
-
-
-
-
_
_
-
-t +
+
+#
Means no staining, + means positive staining. t Positive staining reported in human tissues. Not all the tissues listed here have been examined in humans.'51'6'1927 f Superficial layer of the squamous cell epithelium. § Goblet cells. 1" Collecting tubules. ¶ Proximal tubules. # A few follicular cells. *
Normal Hamster Tissues Reactivities of hamster tissues (positive or negative) with the MAbs are summarized in Table 3. There were no differences in the reactivities of the MAbs with the tissues of hamsters of different ages (8 weeks vs. 30 weeks).
Discussion CA1 9-9, CA1 25, DU-PAN-2, and B72.3 have been shown to be expressed in many human pancreatic cancer cells, and C01 7-1A and B72.3 are being used for immunotherapy24'25 and radioimmunoscintigraphy,26 respectively. However, the use of these MAbs for detailed studies was limited due to the lack of expression of some of these antigens, such as 17-1A and TAG-72 (recognized by B72.3) in human cancer cell lines,27 and the absence of a suitable experimental model. The present study demonstrates that pancreatic cancers induced in Syrian ham-
sters provide a unique tool for such studies because these tumors share expression of TAAs known to be commonly expressed in human pancreatic cancer cells. Despite these similarities, there are also some dissimilarities in the antigenicity of tumor cells in humans and in hamsters. This includes lack of CAl 9-9 antigens in the hamster cancer cells. In humans, CAl 9-9 is detectable in normal human pancreas in minute concentration, but is expressed in about 80% of pancreatic cancer tissues.7 However, this antigen was missing in both the normal and malignant pancreatic cells in hamsters. Possible reasons for the absence of this antigen in hamsters have been addressed (Takiyama et al, manuscript submitted for publication) and could be related to the lack of Le' substance in hamster tissues. DU-PAN-2, occurring in approximately 90% of human pancreatic cancer and, occasionally also in normal human tissues,7 was also missing in the normal pancreatic tissues in hamsters but was present in a few pancreatic cancer cells in vivo and in the goblet cells of the upper but not lower intestinal mucosa of this species.
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The distribution patterns of this antigen in hamster tissue confirms the view that the antibody recognizes some epitope of mucin substances,17'18 and that the nature of mucin differs in different segments of the hamster intestinal tract. C017-1 A, which detects a 37-kd protein,28 has been found by us9 and others28 in almost all normal and malignant human pancreatic tissues. As in humans, this antigen is produced in large amounts by normal hamster tissues, particularly in the gastrointestinal tract, including the pancreas. The localization of the antigen in both human and hamster pancreatic cancer tissue was similar. The determinant of MAb OC125 is thought to be a highmolecular weight glycoprotein complex14 and possibly a peptide.15 In hamsters this antigen was missing in the normal pancreas but was present in more than 50% of pancreatic cancer cells, as well as in the normal gastrointestinal and reproductive tissues. The pattems of the reactivity and the cellular localization were, again, similar to those seen in humans (Figure 3). Because the expression of this antigen in human tissues was found by us to be closely related to blood group A antigen (Pour et al, manuscript submitted for publication), and A antigen had been shown to be expressed by pancreatic cancer cells in hamsters,45 its expression in hamster tissues was expected. The reactivity and cellular localization of MAb B72.3 in pancreatic cancer tissues in hamsters and in humans was also similar (Figure 4). However, compared with the findings in the normal human tissues,19'0 this antigen had a broad spectrum of distribution in hamster tissues but was missing in the normal pancreas. The remarkably high concentration of the antigen TAG-72, a proposed sialosylTn,2 in the hamster salivary gland is consistent with the findings in cows.2 Interestingly, the immunoreactivity of OC125 and B72.3 was strikingly similar in both normal and cancer tissues in hamsters. The possibility that these two MAbs recognize the same antigen is remote because the epitopes of CAl 25 and TAG-72 were shown to be different and the antibodies are not cross reactive.-9 Furthermore, the reactivity of B72.3 (and of DU-PAN-2) decreased with the increasing size of the tumors, a finding that was not true for other MAbs. Whether hamster cells can coproduce these two antigens remains to be seen. Expression of these two antigens in PC-1 cells was found in vitro (cultured cells) and in vivo. However, in vitro expression of TAG-72 was decreased (as in the cells of large tumors), whereas in transplants of PC-1 cells within the pancreas the reactivity with OC125 and B72.3 was increased as compared with the primary cancer cells. Furthermore, in contrast to the cancer cells of the primary and intrapancreatic transplants, a reactivity of all three antibodies was also seen with the membrane of PC-1 cells. These findings indicate that the site and quantity of antigen is related
to the environment of tumor cells and, in the case of B72.3, also to the differentiation. Interestingly, the expression of B72.3 was retained in PC-1 cells, whereas it is lost in human cancer cell lines.27 Nevertheless, the present study demonstrates that hamsters share with humans the expression of some antigens in their normal tissues and pancreatic cancer cells and highlight the usefulness of this model for studying antigenicity of normal and malignant pancreatic tissues.
References 1. Magnani JL, Nilsson B, Brockhaus M, Zopf D, Steplewski Z, Koprowski H, Ginsburg V: A monoclonal antibody-defined antigen associated with gastrointestinal cancer is a ganglioside containing sialylated lacto-N-fucopentaose 11. J Biol
Chem 1982,257:14365-14369 2. Kjeldsen T, Clausen H, Hirohashi S, Ogawa T, lijima H, Hakomori S: Preparation and characterization of monoclonal antibodies directed to the tumor associated 0-linked sialosyl2-6 a-N-acetyl-galactosaminyl (Sialosyl-Tn) epitope. Cancer Res 1988, 48:2214-2220 3. Pour P, Mohr U, Cardesa A, Althoff J, Kruger FW: Pancreatic neoplasms in an animal model: Morphological, biological and comparative studies. Cancer 1975,36:379-389 4. Pour PM, Runge RG, Birt D, Gingel R, Lawson T; Nagel D, Wallcave L, Salmasi SZ: Current knowledge of pancreatic carcinogenesis in the hamster and its relevance to the human disease. Cancer 1981, 47:1573-1587 5. Pour PM, Tempero MA, Takasaki H, Uchida E, Takiyama Y, Burnett DA, Steplewski Z: Expression of blood group-related antigens ABH, Lewis A, Lewis B, Lewis X, Lewis Y, and CA 19-9 in pancreatic cancer cells in comparison with the patient's blood group type. Cancer Res 1988,48:5422-5426 6. Pour PM, Uchida E, Burnett DA, Steplewski Z: Blood-group antigen expression during pancreatic cancer induction in hamsters. Int J Pancreatol 1986,1:327-340 7. Takasaki H, Uchida E, Tempero MA, Burnett DA, Metzgar RS, Pour PM: Correlative study on expression of CA19-9 and DU-PAN-2 in tumor tissue and in serum of pancreatic cancer patients. Cancer Res 1988, 48:1435-1438 8. Takasaki H, Tempero MA, Uchida E, Buchler M, Ness MJ, Burnett DA, Metzgar RS, Colcher D, Schlom J, Pour PM: Comparative studies on the expression of tumor-associated glycoprotein (TAG-72), CAl 9-9 and DU-PAN-2 in normal, benign and malignant pancreatic tissue. Int J Cancer 1988, 42: 681-686 9. Takiyama Y, Tempero MA, Takasaki H, Onda M, Tsuchiya R, Buchler M, Ness M, Colcher D, Schlom J, Pour PM: Reactivity of CO1 7-A and B72.3 in benign and malignant pancreatic diseases. Hum Pathol 1989 20:832-838 10. Pour P, Althoff J, Kruger FW, Mohr U: A potent pancreatic carcinogen in Syrian hamsters: N-nitrosobis(2-oxopropyl) amine. J Natl Cancer Inst 1977, 58:1449-1453 11. Egami H, Takiyama Y, Cano M, Houser WH, Pour PM: Establishment of hamster pancreatic carcinoma cell line (PC-1) producing human blood group-related antigens. Carcinogenesis 1989,10:861-869
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12. Herlyn M, Steplewski Z, Herlyn D, Koprowski H: Colo-rectal carcinoma-specific antigen: Detection by means of monoclonal antibodies. Proc Natl Acad Sci USA 1979, 76:14381442 13. Herlyn D, Koprowski H: IgG2a monoclonal antibodies inhibit human tumor growth through interaction with effector cells. Proc Natl Acad Sci USA 1982, 79:4761-4765 14. Bast RC Jr, Feeney M, Lazarus H, Nadler LM, Colvin RB, Knapp RC: Reactivity of a monoclonal antibody with human ovarian carcinoma. J Clin Invest 1981, 68:1331-1337 15. Kabawat SE, Bast RC Jr, Bhan AK, Welch WR, Knapp RC, Colvin RB: Tissue distribution of a coelomic epitheilium-related antigen recognized by the monoclonal antibody OC125. Int J Gynecol Pathol 1983, 2:275-285 16. Davis HM, Zurawski VR Jr, Bast RC Jr, Klug TL: Characterization of the CA125 antigen with human epithelial ovarian carcinomas. Cancer Res 1986, 46:6143-6148 17. Metzger RS, Gaillard MT, Levine SJ, Tuck FL, Bossen EH, Borowitz MJ: Antigens of human pancreatic adenocarcinoma cells defined by murine monoclonal antibodies. Cancer Res 1982,42:601-608 18. Metzgar RS, Rodriguez N, Finn OJ, Lan MS, Daasch VN, Fernsten PD, Meyers WC, Sindelar WF, Sandler RS, Seigler HF: Detection of a pancreatic cancer-associated antigen (DU-PAN-2 antigen) in serum and ascites of patients with adenocarcinoma. Proc Natl Acad USA 1984, 81:5242-5246 19. Colcher D, Horan Hand P, Nuti M, Schlom J: A spectrum of monoclonal antibodies reactive with human mammary tumor cells. Proc Natl Acad Sci USA 1981, 78:3199-3203 20. Thor A, Ohuchi N, Szpak CA, Johnston WW, Schlom J: Distribution of oncofetal antigen tumor-associated glycoprotein-72 defined by monoclonal antibody B72.3. Cancer Res 1986,46:3118-3124 21. Klug TL, Sattler MA, Colcher D, Schlom J: Monoclonal antibody immunoradiometric assay for an antigenic determinant
22.
23.
24.
25.
26.
27.
28.
29.
(CA 72) on a novel pancarcinoma antigen (TAG-72). Int J Cancer 1986, 38:661-669 Potter M: Immunoglobulin-producing tumors and myeloma proteins of mice. Physiol Rev 1972, 52:632-684 Hsu SM, Raine L, Fanger H: Use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase technique: A comparison between ABC and unlabeled antibody (PAP) procedure. J Histochem Cytochem 1981, 29:577-580 Sears HF, Herlyn D, Steplewski Z, Koprowski H: Phase II clinical trial of a murine monoclonal antibody cytotoxic for gastrointestinal adenocarcinoma. Cancer Res 1985, 45:59105913 Tempero MA, Pour PM, Uchida E, Herlyn D, Steplewski Z: Monoclonal antibody C01 7-1A and leukopheresis in immunotherapy of pancreatic cancer. Hybridoma 1986, 5:s133s138 Colcher D, Esteban JM, Carrasquillo JA, Sugarbaker P, Reynolds JC, Bryant G, Larson SM, Schlom J: Quantitative analyses of selective radiolabeled monoclonal antibody localization in metastatic lesions of colorectal cancer patients. Cancer Res 1987,47:1185-1189 Horan Hand P, Colcher D, Salomon D, Ridge J, Noguchi P, Schlom J: Influence of spatial configuration of carcinoma cell populations on the expression of a tumor associated glycoprotein. Cancer Res 1985, 45:833-840 Gottlinger HG, Funke I, Johnson JP, Gokel JM, Riethmuller G: The epithelial cell surface antigen 1 7-1 A, a target for antibody-mediated tumor therapy: Its biochemical nature, tissue distribution and recognition by different monoclonal antibodies. Int J Cancer 1986, 38:47-53 Lan MS, Bast RC Jr, Colmaghi Ml, Knapp RC, Colcher D, Schlom J, Metzgar RS: Co-expression of human cancer-associated epitopes on mucin molecules. Int J Cancer 1987, 39:68-72
