Tumor antigen detection in human carcinoma

A search for antigenic substances distinctive for human adenocarcinoma has been made. Tumors have been investigated by examining the specificities of antisera produced in rabbits to certain mucinous materials. Fluids were collected from benign, human, pseudomucinous cyst-adenomas of the ovary. These cystic fluids were used for prolonged antigenic stimulation in New Zealand White female rabbits. The antisera from these rabbits were reacted by the gel double diffusion technique with saline extracts of normal and cancerous tissues of humans. The antisera, after absorption with human serum, produced a characteristic precipitation band when reacted with extracts of colonic adenocarcinoma tissue and not with extracts of normal colonic tissue from the same patient. Other extracts from adenocarcinomas of lung, breast, kidney, ovary, and stomach did not produce the characteristic precipitation band when reacted with the rabbit antisera. It appears that cancer of the colon produces an antigen common to mucinous cystadenoma fluid of the ovary. It is postulated that this antigenic substance is an altered mucin. Evidence indicated that this tumor-specific substance is present in all cancerous colonic tissues and is identical in all patients with cancer of the colon. It is suggested that materials present in adenocarcinoma of the colon may be distinctive without being unique and that this possibility offers a future diagnostic technique to effect earlier detection of adenocarcinoma of the colon in humans..

TUMOR ANTIGEN DETECTION IN HUMAN CARCINOl\1A by Myron Wayne \ientz A dissertation submitted to the faculty of the University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Hicrobio1ogy University of Utah June 1972 1�is Disscrtatj.on for the Doctor of Philosophy Dcgr,)c by Myron Wayne Wentz has been approved Septem1"lcr' 19"71 ;;.;.�:�.i.�.("�. .;: ,;7?L� �:.j;.. � (.. _.l/;;. :.:.:�_. _.. ...:_l..-'--'---_ - __ D8c'LD, Grflc7�(1te :/ :=;chool ACKNOWLEDGE~~NTS The author wishes to express his sincere appreciation to Dr. Stanley Marcus and Dr. Crichton McNeil for their guidance and encouragement during the course of this work. Grateful acknowledgement is made to Dr. Thomas F. Dougherty, Dr. Louis P. Gebhardt, Dr. Douglas W. Hill, and Dr. Bill B. Wiley for their helpful suggestions and criticisms in the preparation of this thesis. Appreciation is also extended to my wife Jacquelyn for her support and patience throughout this endeavor. She is thanked for typing this thesis. This research was supported by National Cancer Institute Grant CA 05965 from the National Institutes of Health. TABLE OF CONTENTS Page ABSTRACT ...................... INTRODUCTION ix • • • • • • • • • • • • • • • • • • • • I LITERATURE REVIE\v • • • • • • • • • • • • • • • • • • 3 Blood Group Mucopolysaccharide • • • • • • • • • • 3 Association of Blood Group Substances and Cancer 3 0 • • • • • • • • • • • • • • • • Secretion of Blood Group Substances Tissue Mucopolysaccharides • • • • • • • • • Colon ~~copolysaccharide Content • • Cyst Fluid Nucopo1ysaccharide Content • • ·. ·.. ·.. ·.. 0 • • 5 7 8 10 Mucopolysaccharide Content of Other Adenocarcinomas ••••• • • 12 Cellular Growth Involvement 12 0 • • • • • • • • • • • • • • • 0 • Cancer Cell Surface Properties • • • • • • • Serum f\;Iucoids and Glycoproteins ~!\..i\J'D NETHODS • • Human Tissues ..• • ·........ Human Cancer Antigens and Their Detection MATERIALS • • 0 • • 14 15 20 • • • • • • • • • • • 32 • • • • • • • • • • • • • • • • • • 32 ·..... 32 Antisera Preparation • • • • • • • • • • • • • • • 35 Cyst Fluids • • • • • • • • • • • Immunization of Animals • • • • • • • • • • • 35 • • • • • • • • • • • • • 37 • • • • • • • • • • • • • • • 38 Bleeding of Animals Antisera Absorption Absorption by Absorbent Incorporation in Agar iv 38 Page Dialysis Chamber Method Beaker Method • • • 0 • • • • • • • • • • • • • • • • • • • • • • • 39 39 Preparation of Tissue Test Antigen • • • • • • • • 40 Immunology Assay • • • • • • • • • • • • • • • 40 Preparation of Ouchterlony Double Diffusion Agar Plates • • • • • • • • • 41 Reaction Measurement • • • • • • • • • • • • • 42 RESULTS. • • • • • • • • • • • • • • • • • • • • • • 44 0 Preliminary Findings • • • • • • • • • o • • • • • • • • • • • • • 44 Reactions of Prepared Antisera • • • • • • • • • • 45 First Immunization Series o 00. • • • • • • • Second Immunization Series • • • • • • • • • • 51 · . .0. . • • . 53 Third Immunization Series Fourth Immunization Series • • • • • • • • • Fifth Immunization Series o • 0 • Sixth Immunization Series • • • 00. Seventh Immunization Series Ninth Immunization Series Tenth Immunization Series • • • • • • 60 • • • • 61 68 • • • • • • • 70 • • • • • • • • • • 81 • o • • • • • 0 • • • • Eleventh Immunization Series • • • • • • • • • DISCUSSION Sffi.1MARY • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • LITERATURE CITED 55 • • • • • • • • • Eighth Immunization Series • VITA 45 o 90 91 93 • • • • • • • 105 • • • • • • • • • • • • • • • • • • 106 • • • • • • • • • • • • • • • • • • • • • • • • 118 v LIST OF TABLES AND FIGURES Page Tables ... ... 1. Description of Malignant Tissues Used • • 2. Cyst Fluids Used for Immunization • • • • 3. Comparison of Titers of Antisera H-13, H-14, H-17, and H-23 • • • • • • • • • • • • • • • • 2. 3. 4. 5. 6. 7. 8. 36 72 Page Figures 1. 33 Ouchterlony reaction of antiserum U-14 with saline-diluted cyst fluid in 1 per cent agar 46 Ouchterlony reaction of antisera U-14 and U-lS with normal rabbit sera, normal human sera, and cyst fluid in human serum-agar • • • • • • 46 Ouchterlony reaction of antiserum U-14 with extracts of normal and cancerous colonic tissues, saliva, and cyst fluid in 1 per cent agar • • • 48 Ouchterlony reaction of antiserum U-14 with extracts of normal and cancerous colonic tissues, saliva, and cyst fluid in human serum-agar •• 48 Ouchterlony reaction of antiserum U-14 with cyst fluid and extracts of normal and cancerous colonic tissues in 1 per cent agar • • • • • • 49 Ouchterlony reaction of antiserum U-14 with cyst fluid and extracts of normal and cancerous colonic tissues in human serum-agar • • • • •• 49 Ouchterlony reaction of antiserum U-14 with extracts of normal and cancerous colonic tissues and cyst fluid in human serum-agar • • • • • • 50 Ouchterlony reaction of antiserum U-15 with extracts of normal and cancerous colonic tissues and cancerous breast tissue, and cyst fluid in human serum-agar • • • • • • • • • • • • • •• 50 vi Page Figures 9. 10. 11. 12. 13. 14. 15. 16 0 17. 18. 19. Ouchterlony reaction of antisera U-14 and W-17 with cyst fluid and cancerous colonic tissue extracts and of antisera \i-14, \v-15, and \i-16 with cancerous colonic tissue extracts in human serum-agar • • • • • • • • • • • • • 57 Ouchterlony reaction of antiserum W-17 with extracts of normal and cancerous colonic tissues and cyst fluid in human serum-agar • • • • • • 57 Ouchterlony reaction of antiserum W-14 with cancerous colonic tissue extracts and gamma globulin of serum in 1 per cent agar •• • • • 59 Ouchterlony reaction of antisera R-l through R-5 with cyst fluid in human serum-agar • • • • Ouchterlony reaction of antiserum R_2 x with extracts of normal and cancerous colonic tissues in human serum-agar • • • • • • • • • • • • Ouchterlony reaction of antiserum R_2x with extracts of normal and cancerous stomach tissues and cancerous colonic tissue in human serumagar • • • • • • • • • • • • • • • • • • • Ouchterlony reaction of antiserum R_2 x and antiserum R_2x concentrated with 10 per cent PVP and adsorbed with red blood cells to extracts of cancerous colonic tissue in human serum-agar • • • • • • • • • • • • • • • • • • 63 65 67 69 Ouchterlony reaction of antiserum II-14 with extracts of normal and cancerous colonic tissues in human serum-agar ••••••••••••• 74 Ouchterlony reaction of antiserum II-17 with extracts of normal and cancerous colonic tissues and cancerous lung and ovarian tissues in human serum-agar • • • • • • • • • • • • • • • 74 Ouchterlony reaction of antiserum H-17 with extracts of cancerous colonic tissue and normal and cancerous stomach tissue in human serumagar • • • • • • • • • • • • • • • • • • • • • 76 Ouchterlony reaction of antisera H-14 and H-17 with extracts of normal and cancerous colonic tissues in human serum-agar • • • • • • • • • • 77 vii Page Figures 20. 21. 22. 23. 24. 25. 26. 27. 28. Ouchterlony reaction of antisera H-14 and H-17 with extracts of normal and cancerous colonic tissues in human serum-agar • • • • • • • • • • 77 Ouchterlony reaction of antisera H-14 and H-17 with extracts of normal and cancerous colonic tissues in human serum-agar with 15 per cent M normal colon extract incorporated • • • • • • 79 Ouchterlony reaction of antisera H-14 and H-17 with extracts of normal and cancerous colonic tissues in human serum-agar with 10 per cent A normal colon extract incorporated • • • • • • 79 Ouchterlony reaction of antiserum H-17 with cyst fluid and extracts of normal and cancerous colonic t issues in human serum-agar 'vi th 10 per cent 0 normal colonic extract incorporated 80 Ouchterlony reaction of antiserum H-17 'vi th cyst fluid and extracts of normal and cancerous colonic tissues in human serum-agar with 10 per cent 0 cancerous colonic extract incorporated. 80 Ouchterlony reaction of antiserum 2-31 with extracts of normal and cancerous colonic tissues in human serum-agar • • • • • • • • • • • •• 83 Ouchterlony reaction of antiserum 2-35 with extracts of normal and cancerous colonic tissues in human serum agar • • • • • • • • • • • • • • 85 Ouchterlony reaction of pooled antiserum from rabbits 2-31, Z-33, and ~-35 with cyst fluid and extracts of normal and cancerous colonic tissue in human serum-agar • • • • • • • • • • 89 Ouchterlony reaction of pooled antiserum from rabbits Z-31, 2-33, and Z-35 and antiserum H-17 with extracts of normal and cancerous colonic tissue in human serum-agar • • • • • • • • • • 89 viii ABSTRACT A search for antigenic substances,distinctive for human adenocarcinoma has been made. Tumors have been investigated by examining the specificities of antisera produced in rabbits to certain mucinous materials. Fluids were collected from benign, human, pseudomucinous cystadenomas of the ovary. These cystic fluids were used for prolonged antigenic stimulation in New Zealand Ivhite female rabbits. The antisera from these rabbits were reacted by the gel double diffusion technique with saline extracts of normal and cancerous tissues of humans. The antisera, after absorption with human serum, produced a characteristic precipitation band when reacted with extracts of colonic adenocarcinoma tissue and not with extracts of normal colonic tissue from the same patient. Other extracts from adenocarcinomas of lung, breast, kidney, ovary, and stomach did not produce the characteristic precipitation band when reacted with the rabbit antisera. It appears that cancer of the colon produces an antigen common to mucinous cystadenoma fluid of the ovary. It is postulated that this antigenic substance is an altered mucin. Evidence indicated that this tumor- specific substance is present in all cancerous colonic tissues and is identical in all patients with cancer of the colon. It is suggested that materials present in adenocarcinoma of the colon may be distinctive without being unique and that this possibility offers a future diagnostic technique to effect earlier detection of adenocarcinoma of the colon in humans. x INTRODUCTION The search for human tumor-specific antigens and their antibodies has centered primarily on finding a diagnostic method for detection of cancer and secondarily on finding a possible cure through active or passive immunization. Detection of tumor antigens in humans has most often been explored by injection of tumor extracts into animals followed by reaction of the animals' sera with human materials. Although the use of the prepared antisera, after removal of antibodies to normal tissue components by absorption, has been reported to be sucessful in detecting cancer-specific antigens in man, widespread doubt still exists concerning these reports. Skepticism rises from the technical problems involved in each research effort. In the work reported in this thesis, a new hypothesis has been examined; that is, that fluid obtained from certain benign cystic tumors contains tumor-specific antigenic material that yields distinctive antibody in rabbits. Such rabbit antibody has been employed sero- logically to examine extracts of mucin-producing tumors. The pathological changes of adenocarcinomas of colon, lung, stomach, breast, and ovary, are characterized by an elevated secretion and accumulation of mucinous substances in these tissues. It is hypothesized that these mucinous materials in mucin-producing tumors may 2 show qualitative as well as quantitative differences from the normal. The study of such substances has been ham- pered by difficulty in collecting sufficient amounts for meaningful studies (Jeanloz, 1966). The use of cyst fluid presents a possible means for alleviating this problem. While ovarian cyst formation is not common, a single cyst can supply ample amounts of mucinous material for study. The data to be reported are derived from an investigation into mucinous substances produced during the cancer process. The antigenicity of certain mucinous materials and their antigenic realtionship to tumor tissues have been ex~lored to contribute to the knowledge concerning the pathogenesis of neoplastic disease. LITERATURE REVIE\i Blood Group Mucopolysaccharides Active interest currently exists in the search for distinctive antigenic materials in autochthonous tumors. Literature relative to blood group substances or mucopolysaccharides and their possible relationship to antigens related to cancer is reviewed here. Association of Blood Group Substances and Cancer Aird, Bentall, and Roberts (1953) reported a statistically significant association between the distribution of ABO blood groups and cancer of the stomach. Cancer of the stomach was significantly more common in patients of blood group A than in patients of other blood types. A year later McConnell, Clarke, and Downton (1954) in studying the blood groups of 777 cases of carcinoma of the lung found no significant differences in the ABO blood group distribution of these cases and blood donor controls. They did, however, find an excess of group A at the expense of group a in patients with "oat-cell" tumors, a histologically differentiated carcinoma confined to the epithelium of the lungg They also found a statistically significant association of Rh-positive people and lung cancer. The above reports kindled much interest in the association of ABO blood groups and incidence of 4 neoplastic disease. As a result, additional epidemiologic evidence that gastric cancer occurred more frequently in people of blood group A has been collected by many investigators (Kpster, Sindrup, and Steele, 1955; Buckwalter et al., 1956; Zographov, 1962; and Desai and Creger, 1962). Clarke (1961), in gathering reports from 15 medical centers, concluded that persons of group A ,,,ere about 20 per cent more likely to develop gastric cancer than those of blood group O. Patients of blood group A were found also to have a higher frequency of benign and malignant tumors of the salivary glands (Cameron, 1958 and Osborne and De George, 1963) and of the ovary (Osborne and De George, 1963)0 A significantly higher proportion of cancers of the pancreas and the esophagus occurred among group A patients (Clarke, 1961). Fadhli and Dominguez (1963) reported a statistically significant high occurrence of blood group A in 123 white patients with multiple primary cancers. This finding has been refuted by Tsukada et ale (1964) and Holley, Taswell, -- and Moertel (1966). Roberts (1957) and Clarke (1961) re- ported no significant association of the ABO blood groups and cancer of the breast, colon, and rectum. Jouvenceaux et ale (1964) presented interesting evidence indicating the occurrence of unusual blood group reactivity in patients with cancero In cases reported by 5 these workers, red blood cells from cancer patients of blood group A were partially agglutinated \V'i th high titered anti-B typing serum. This was interpreted to mean that patients of blood type A acquired a ufeeble" B blood group antigen during the disease. This phenomenon oc- curred in highest frequency in patients with cancers of the colon and rectum. pseudo-B antigen. The "feeble" B antigen was termed Normal anti-B antibody was found in the sera of these patients. The pseudo-B antigen was not found in the saliva of patients who were secretors. Secretion of Blood Group Substances Secretor status, as determined by salivary secretion of blood group substances, and its relationship to gastric cancer have been analyzed by Desai and Creger (1962, 1964). They found that gastric cancer patients secreted significantly more H substance and possibly more A substance than controls. r-lcNeil et al. (1965), in studying the frequencies of secretors (Se) and non-secretors (sese) of blood group substances, noted a significant correlation between Se status and bronchogenic carcinoma. They found no cor- relation of secretor status with other high-incidence tumors, such as of colon and breast. McNeil and co-workers (1964) also provided the interesting suggestion that nossession of the Se gene 6 favored metastases of cancer. Conversely, sese status was reflected in localization of and improved survival from all kinds of malignancy. Speculation as to the importance of secretor status in the pathogenesis of cancer has been offered. Desai and Creger (1964), in regard to gastric cancer, suggested that virus could penetrate a gastric epithelial cell containing A substance more easily than one containing H substance or no blood group substance at all. hypothesized that the n Zographov (1962) antigen could exert some pro- tective effect against carcinogens. Osborne and De George (1963) suggested that different potentials for atypical cell differentiation could be associated with the different ABO blood group genotypes o l\lcNeil et ale (1965), in noting the increased incidence of bronchogenic and gastric carcinoma in Se patients, have proposed two possible eA~lanations. Since both mucosal tissues involved were potent secretors of blood group substances, it was suggested that water-soluble blood group substances could trattract or alter carcinogens, favouring neoplasia"; (McNeil et al., 1965) or the reduction in Lewis substance which was associated with Se status could result in a diminution of some protective effect afforded by this substance. 7 Tissue Mucopolysaccharides The biological significance of changes in the content of polysaccharide substances in tissues is difficult to define due to the difficulties involved in quantitatively extracting these substances from tissues (Antonopoulos et al., 1964). Likewise, histochemical methods, while showing the presence and location of mucopolysaccharides, are not able to distinguish the various mucopolysaccharides quantitatively. Even more perplexing in reviewing the literature on the involvement of mucinous sUbstances in disease is the confusion created by the lack of a standardized nomenclature for these substances. "l\lucin is a term of convenience rather than precision (Franks, O'Shea, and Thomson, 1964). The term mucin as used in this thesis applies to a group of substances that have certain chemical and physical properties (e.g., sliminess, soluble in alkaline water, precipitated by acetic acid and alcohol) and stain with the standard mucin stains (mucicarmine, mucihematin). These mucinous sub- stances (mucosubstances) can be divided into three groups (Stacey and Barker, 1962)--mucoproteins (polysaccharideprotein complexes predominantly protein), mucopolysaccharides (polysaccharide-protein complexes predominantly carbohydrate), and mucolipid (polysaccharide-fat). A pre- fix before the word mucin usually denotes what the mucin contains in high abundance. For example, fucomucin and 8 sialomucin refer to mucin which contain high amounts of fucose and sialic acid, respectively. The term mucoid can be equated with mucoprotein and neutral mucopolysaccharide. Glycoproteins are distinguished from muco- proteins in that they (glycoproteins) contain relatively lesser amounts of carbohydrate, less than 4 per cent. As will be seen later in a review of cancer antigens, detailed chemical information concerning these mucosubstances was rarely supplied by researchers. Therefore, the precise chemical nature of some of the mucosubstances to be discussed can only be speculated. Colon Mucopolysaccharide Content Sky-Peck, Lundgren, and Bornstein (1966) have quantitatively studied the chemical composition of mucopolysaccharides of human colon. Tissues of whole colons ob- tained at autopsy were defatted and hydrolyzed with papain. The mucopolysaccharides were extracted by forma- tion of a complex with cetylpyridinium chloride (CPC). The complex was extracted with ~1gC12 solution, freed of CPC by the addition of excess NaSCN, and further separated by column chromatography. Examination of the mucopoly- saccharides obtained revealed that hyaluronic acid comprised about 70 per cent of the acid mucopolysaccharides. The remaining acidic mucopolysaccharides consisted of heparin monosulfate, chondroitin sulfates, heparin, and 9 keratosulfate in decreasing amounts. Of the chondroitin sulfates, chondroitin sulfate-A was in greatest concentration in human colonic tissues followed by chondroitin sulfate-C and chondroitin sulfate-B. HaKkinen, Hartiala, and Terho (1965) reported chondroitin sulfate-B to be the predominant chondroitin in the gastric wall of the dog. A substantial amount of neutral mucopolysaccharide material \\Tas also found in human colon (Sky-Peck et a1., 1966). Glycoproteins containing sialic acid, hexose, and large amounts of hexosamine were extracted. The content of one such glycopeptide (high percentage of sialic acid and presence of galactose, fucose, galctosamine, and glucosamine) was similar to the content of submaxillary sialomucins, blood group substances, gastric mucins, and other mucoproteins found in the gastrointestinal tract (Kent, 1962). Goldman and Ning (1968) reported that a reduction of sulfomucin was detected in colonic carcinomas. The colonic tissue contained much polysaccharide material not precipitible with CPC (Sky-Peck et al., 1966). All such fractions contained galactose, mannose, sialic acid, and fucose. The amino sugar, if present, was either glucosamine or galactosamine. Werner (1953) and Whitehouse and Bostrom (1961) reported the existence of two principal glycoproteins in the intestine, one containing fucose and the other containing sialic acid. \ierner identified these two mucins as blood 10 group substances and sialomucin. The evidence of Sky-Peck et ale (1966) also indicated that at least two glycoproteins were present. However, they always contained both fucose and sialic acid. Dobrogorski and Braunstein (1963) studied the use of histochemical staining techniques in the differential diagnosis of neoplasms. The periodic acid-Schiff (PAS) staining technique was found to be useful in demonstrating the presence of neutral and acidic mucopolysaccharides in adenocarcinomas of the alimentary tract. Cyst Fluid Nucopolysaccharide Content The fact that cystic ovarian tumors are capable of secreting mucinous sUbstances has been well established. Hammarsten in 1882 discovered that ovarian cyst fluid mUCin, unlike other body mucins, was not precipitated with acetic acid (Hammarsten, 1882). He thus used the term "pseudomucinous ll to note this difference. This term, as in "pseudomucinous cystadenoma", is still used to describe these tumors. Histochemical studies, however, do not reveal any fundamental differences between ovarian pseudomucin and other body mucins (Zilliacus, 1952; Fisher, 1954; Kiekhofer, Holmen, and Peckham, 1962; \vynn, 1962; and Johnson and Helwig, 1963). ~lucopolysaccharides common to various body secretions have been demonstrated in 11 ovarian cyst mucins: fucomucins (~1organ, 1963), blood group mucopolysaccharides (Morgan and Van Heyningen, 1944; and Szulman, 1962), hyaluronic acid (Jensen, 1954), and sialomucins (Spicer and Warren, 1960). Garcia-Bunuel and Monis (1964) were intrigued as to why a mucin-secreting tumor was able to arise from an organ (ovary) essentially devoid of mucin-secreting epithelium. They contributed much to the general knowledge of mucin secretion by studying the mucins in 49 ovarian neoplasms and 14 ovarian and juxtaovarian structures of non-neoplastic nature. All of the neoplasms and non- neoplastic structures of coelomic derivation secreted mucin. As e~~ected, mucin was most predominant in mucin- ous cystadenomas and cystadenocarcinomas. In the benign tumor mucin was contained in cytoplasmic granules of all epithelial cells. In the malignant tumor mucin accumu- lated in vacuoles in the cytoplasm of most, but not all, epithelial cells. Strongly acidic mucins containing sialic acids predominated in these mucinous tumors. Serous cystadenomas and cystadenocarcinomas also secreted mucins although to a much lesser extent. Unlike mucinous cystadenomas, mucin in serous cystadenomas was diffusely distributed in the cytoplasm and not found in granules. In serous cystadenocarcinomas mucin contained in vacuoles was present in only a few epithelial cells. Weakly acidic mucins predominated in these benign and malignant serous 12 tumors, but small amounts of strongly acidic and neutral mucins were also present. Thus, it has been shown that ovarian tumor cells undergo certain pathological changes relative to cellular mucin content and secretion. Mucopolysaccharide Content of Other Adenocarcinomas ~'lucopolysaccharides appeared to be involved in the pathological changes of other tumor tissues. Franks et ale -- (1964) reported that prostatic carcinoma cells secreted sialic acid-containing mucin. of the mucin were sulfated. The sialic acid residues Cancer cells of colloidal prostates also produced this material but to a greater extent. This mucin was rarely present in the non- neoplastic gland. Greater than normal amounts of acidic mucopolysaccharides were seen in stained smears of cancer tissues of the breast (~lajewski, Kubiak, and Szulc, 1963). Spicer et ale (1962) have sho\V'n that the histochemical properties of these acidic mucopolysaccharides were attributable to enzymatically digestible sialomucin Q Greater than normal amounts of mucinous substances, belonging to the group of nonsulfated sialomucins, occurred in bladder carcinomas (Hukill and Vidone, 1965). Cellular Growth Involvement Lippman (1965) has presented a detailed review and has built an excellent case for the important involvement of mucopolysaccharides in the initiation and control of 13 cell division. His work suggested that the absence of certain mucopolysaccharides was associated with unblocked cell division cycles in normal physiological situations, in tissue culture, in embryonic growth, and in tumor growth. The restoration of acidic polysaccharides to the cell surface blocked cell division. During the course of a tissue cell's lifetime and during different physiological conditions, the cell exhibits qualitative and quantitative differences in its mucopolysaccharide coat. As a result, these differences determined whether the cell would remain blocked at the interphase stage after all interphase conditions were completed, or whether it would spontaneously enter the prophase stage. Cellular growth at this stage could be checked either by the cell's own production of new mucopolysaccharides or by the mucopolysaccharides provided by mast cellso 1iithout this mucopolysaccharide block, cells exhibited active proliferation. Lippman implied that polysaccharide molecules acted as ion exchangers at the cell's surface. These molecules may have acted as surface blocking and triggering agents for prophase events. There was evidence that calcium com- plexed to mucopolysaccharides or other surface polysaccharides could act as a mitotic block, and that altering or removing the mucopolysaccharide-calcium complex resulted in release of the block. While other sites of blocking of cell division were possible, it was concluded that 14 normal blocking of cell division occurred either during interphase or by prevention of prophase. Lippman postu- lated that genetic, metabolic, viral, carcinogenic, immunological, and injury theories of cancer could have a common focal point--regulation of the cell surface complex. Cancer Cell Surface Properties In neoplastic cells unblocked cell division was coincident with reduced cell adhesiveness--that force which resists mechanical separation of attached pairs of cells (Lippman, 1965). Reduced adhesiveness was credited to be the primary factor in the ability of cells to metastasize (Coman, 1944). Investigations on adhesive- ness and on stickiness of tumor cells have shown an interesting paradox. Coman (1961) and Kojima and Sakai (1964) have shown that cell stickiness--the tendency of cells to cling to a foreign substrate--was inversely related to cell adhesiveness. Tumor cells of ascites hepa- toma of the rat were feund to be extremely sticky but poorly adhesive. The reverse was true of normal cells. Gasic and Gasic (1962) reported that intravenous injection of neuraminidase before injection of mouse ascitic tumor cells into homologous hosts significantly reduced metastases. There was much evidence to confirm that removal of sialic acid from the mucopolysaccharide structure reduced cell stickiness. Kojima and Sakai (1964) have 15 shown that in an acid medium cell stickiness was significantly decreased, while in an alkaline medium it was increased. Neoplastic transformation resulted in a greater negative surface charge of cells (Abercrombie and Ambrose, 1962). This difference in surface charge \"las reflected in changes in electrophoretic mobility of tumor cells. Wallach and De Perez Esandi (1964) have shown, by treatment of cells with sialidases, that the content of sialic acid was responsible for the increase in electrophoretic mobility of cancer cells. Reduction in the sialic acid component of the cell membrane during cancer has been implicated by Eylar et ale (1962) as being responsible for the retardation in elec- trophoretic mobility of the red blood cell. A factor present in the serum of persons with malignant disease appeared to alter the surface electrical charge of the red blood cell. This slowing factor has been found in the alpha 1 fraction of serum globulins distinguishing it from a slowing factor seen in infectious mononucleosis patients; the latter was found in the a1pha w0 fraction of serum globulins. Serum ~;ucoids and Glycoproteins Winzler and Smyth (1948) indicated that a class of mucoproteins, perchloric acid soluble and phosphotungstic acid insoluble, was present in greater than normal amounts 16 in plasma of cancer patients. Shetlar et ale (1949, 1950) reported high amounts of polysaccharide associated with serum proteins in patients with cancer. Greenspan (1955) found an increase of serum mucoproteins in 80 per cent of the cases of patients with leukemia, carcinomas, and tumors of the lymph glands. The highest values \vere observed among the cancer patients with multiple metastases. ~Ioschides and co-workers (1958) also reported the muco- protein fraction and total hexosamine of serum \-Jere quantitatively increased in diseases of a neoplastic nature. Significant variations of the protein-bound and mucoprotein-bound hexoses were observed. Numerous other re- ports of high mucoprotein levels in serum of cancer patients have been reviewed by Biserni (1961). From early reports, ho\vever, it became evident that neoplastic disease could not be diagnosed merely by the observation of a general serum mucoprotein elevation. According to Greenspan (1954), serum mucoprotein levels were affected by several tissue proliferative and/or degenerative processes such as inflammatory, neoplastic, or traumatic disease. Hepatic and endocrine functions also affected mucoprotein levels. Saifer and Weintraub (1961) reported that rheumatoid arthritic patients and cancer patients had statistically significant differences from a normal control group of patients in protein-bound fucose and in the ratio of protein-hound fucose to total protein. 17 Much controversy has arisen concerning the source of mucoproteins in serum 0 Seibert et ale (1947) and Catchpole (1950) proposed that the mucoproteins were products of the depolymerization of basic connective tissue sUbstance. This theory has been discounted since in- creased amounts of hexuronic acids, essential connective tissue components, have not been found in serum concomitantly with high mucoprotein levels. (1949) and ~~si1 Shetlar et ale (1960) suggested that increased cellular proliferation was responsible for high mucoprotein levels. Other workers (Herner, 1949;' Greenspan et al., 1951, 1952; Macbeth and Bekesi, 1964; and Schultze, 1963) cited the liver as somehow being responsible for the elevated levels of serum mucoproteins. Less than normal levels of serum mucoproteins have been found in diseases of the liver, such as hepatitis and cirrhosis. Other findings have con- firmed that there is a significantly elevated concentration of protein-hound carbohydrates in the developing tumor itself as \VeIl as in the tissue adjacent to the tumor (Barker et al., 1959 and Tombs, Burston, and Mac1agan, 1962). Macbeth and Bekesi (1962, 1964) reported greater than normal levels of protein-bound galactose and mannose in plasma of persons with cancer. Data from 161 cancer cases suggested that the fucose of neutral glycoprotein may be elevated in concentration exclusively in malignant 18 disease. Significant elevation in the level of protein- bound sialic acids in serum lias observed in all forms of malignant disease except localized carcinoma of the breast. Polarographic wave studies (Yamaguchi, Sasai, and Kokei, 1963) and electrophoretic analyses (Parrella, Di Perna, and Carratu, 1964) have also suggested possible qualitative differences existing in serum mucoproteins in patients with cancer. Other investigations into qualitative differences of serum glycoproteins in patients with cancer \vere conducted by nakomori, Kawauchi, and Ishimoda (19Gl). They observed increases in branching, molecular weight, and ratio of amino-sugar to non-amino-sugar in serum glycoproteins of patients with cancer. These changes were not observed in the serum glycoproteins of patients with inflammatory disease. Harshman and Bryant (1964) induced high serum levels of "mucoids" (mucoproteins) in rats by implants of \{alker 256 tumor tissue. The elevated levels of mucoproteins re- turned to normal after surgical extirpation of the tumor. The levels remained high if substantial metastasis had occurred. Ion-exchange chromatographic studies indicated heterogeneity of the serum mucoproteins in tumor-bearing rats and normal rats. In humans, data indicated that fol- lowing surgery a high serum level of mucoproteins was associated with persistent or recurrent malignant disease 19 whereas a low serum level of mucoproteins following surgery correlated with transient or prolonged remission (Harshman et al., 1967). The work of Bacchus, Kennedy, and Blackwell (1967) stressed the importance of seromucoid investigation in understanding neoplastic disease. Human sera were sepa- rated into eight fractions by ion-exchange chromatography. The protein, hexose, and hexosamine contents and hexosamine/protein ratios of various fractions differed in sera from normal subjects and from patients with cancer. Disc electrophoretic and immunoelectrophoretic studies of these fractions further demonstrated qualitative differences in these seromucoid substanceso The seromucoid sUbstances in patients with neoplastic disease have been shown to have an electrophoretic mobility in the alpha globulin range--predominantly alpha 1 (Ferri, Cassermelli, and DeCourt, 1962). Numerous investigators have shown an association between an increase in the alpha globulin fraction of serum and the presence of cancer (Winzler, 1953; Campbell, Kernot, and Roitt, 1959; Fahey and I3oggs, 1960; Biserte et al., 1961; and Graham, 1963). Tombs, James, and Maclagan (1961) demonstrated that an alpha globulin fraction of serum in cancer patients increased up to ten times the normal value. Sera from patients with colonic cancer studied in our laboratory have also shown marked increases in the alpha fraction. 20 Human Cancer Antigens and Their Detection Witebsky, working in Heidelberg, was among the first to claim the demonstration of a cancer-distinctive antigen. In his well controlled experiments, Witebsky (1929, 1930) prepared rabbit antisera against boiled human can- cer tissue. These antisera reacted in a complement fixa- tion test with alcoholic extracts of tumors but not with extracts of normal tissues. These antisera, however, also reacted with blood group substances from red blood cells. As a result, \fitebsky controlled for blood group reactivity by using tissues from patients with the same blood type for his test and for his immunizing antigens. Thus, the first reported antiserum to cancer-distinctive antigens cannot be considered cancer-specific since it also reacted with blood group SUbstances. gen of Witebsky was considered to be lipid. The tumor antiThis conclu- sion was presumably based on the use of alcoholic tracts as test antigens. In the same year that \1itebsky reported his findings, Hirszfeld, Halber, and Laskowski (1929) published an extensive and detailed report on the serological specificity of lipids in malignant tissue. Their work confirmed Witebsky's finding (existence of a cancer-specific lipid antigen), eliminated the possibility of involvement of Forssman antigen, and presented several other findings pertaining to the lipid antigens. They reported that 21 several lipid antigens existed. A few lipid hapten anti- gens serologically distinctive for cancer were shared by a limited group of neoplasms. A lipid antigen existed in normal stomach tissues and a variety of carcinoma tissues of cancer patients. Slightly anaplastic tissues were the best source of serologically distinctive cancer lipids. The ratio of useable antisera to those prepared was low. (Out of 87 antisera prepared in rabbits, 11 were found to contain cancer-specific antibodies. Eight of the 11 sera were considered useable as reagents.) Trawinsl\:i (1937) immunized rabbi ts with extracts of normal and cancerous tissues. The macerated tissues had been extracted in sterile saline at 4 C for 8 days. Anti- sera to extracts of normal stomach, liver, or uterus cross reacted with each other. was used. The precipitin ring test These antisera did not react with extracts of cancerous tissues. Antisera to extracts of cancerous tissues reacted rather specifically with the homologous antigens. None reacted with normal tissue extracts. Although the data appeared to be convincing, the experiments did not control for genetic differences of individuals and, most importantly, blood group substances. r.1ann and Helker (1940) prepared rabbit antisera against saline extracts of gastric carcinoma tissue preparations. The antisera reacted well with the homol- ogous antigen and did not react with normal tissue 22 extracts from stomach or other organs. The antisera were not reacted with other malignant tissues. The antisera also reacted with the sera of a certain percentage (57 per cent) of patients with carcinoma of the stomach but not with sera of normal persons. This suggested that the cancer-specific antigen is detectable in the serum of patients with cancer of the stomach. The rabbits were injected intramuscularly with the antigen absorbed on aluminum hydroxide. The experimental design \vas based on the following findingo In rabbits injected with tissue antigens, precipitins to human serum components were not detectable after a period of a month after immunization whereas the precipitins to tissue remained in relatively high titers (l1ektoen and ~ifelker, 1935) 0 In rabbi ts immu- nized \vi th antigens which ,,,ere absorbed on aluminum hydroxide, persistance of precipitins ultimately corresponded to the quantity and differential retention times of the antigens in the aluminum hydroxide (Hektoen and l{elker, 1934). As a result of this immunization procedure, precipitins to normal serum components were absent by the sixth month after immunization. The precipitins to cancer tissue per- sisted for more than 2 months after the disappearance of the precipitins to human serum components. Graham and Graham (1955) sholved that 12 of 48 cancer patients possessed antibodies to antigens prepared from their own tumors. The sera of these patients when reacted 23 with the water-extracted tumor antigens had complementfixation titers of 1:16 to 1:128. The antigens and anti- bodies, however, could not be considered specific since normal tissues, prepared by the same procedure, were not tested. Kobayashi (1956) isolated and purified a tumorspecific antigen and a normal tissue antigen from human gastric tissue. He showed these two antigens to be immunologically different. He controlled for blood group substance reactivity, and by using numerous solvent e:'~­ tractions he characterized these antigens as lipids, probably phospholipids. Bjorklund (1956) pooled tissues from several hundred human carcinomas so that individual differences between the tumors would be diluted and any common antigens present would not be diluted. Hyperimmune sera were made by injecting the pooled antigen into horses. The sera proved to have a cytotoxic effect on various kinds of freshly established cell cultures of human cancerous tissues. There was no effect on cultures of 14 different types of normal human tissues (Bjorklund and Bjorklund, 1957) • Significant contributions to human cancer immunology were made by Zilberbeginning in 1957. He showed the presence of specific antigens in. many human carcinomas as well as in spontaneous and induced animal tumors 24 (Zilber, 1957). The tumor-specific antigens were detected using the gel-precipitation technique (Zilber, 1962) and the sensitization-desensitization-anaphylaxis (SDA) method (Zilber, 1958). In the SDA method guinea pigs were sensi- tized to extracts of human tumors, desensitized to extracts from the corresponding normal human tissues and then challenged with the sensitizing antigen--measuring the degree of analphylactic response. The now well known cytolipin H was discovered by Rapport et al. (1959) using very extensive extraction and purification procedures. The isolation of this cancer- distinctive substance from human epidermoid carcinoma has stressed the importance of immunochemistry in cancer research. Cytolipin H was undoubtedly the first cancer- specific substance to be artificially synthesized (Shapiro and Rachaman, 1964). The antigen, while being character- ized as a lipid, contained lactose. The lactose was an important component and the formula of cytolipin H was written as a ceramide lactoside. Graf and Rapport (1960), in attempting to distinguish cytolipin II from the \lassermann cardiolipin and Forssman cardiolipin antigen~ dis- covered a new lipid hapten from gastrointestinal carcinoma which they termed cytolipin G. The cytolipin G antigen was separated from cytolipin H by chromatography of gastrointestinal tumor lipids on silicic acid. The lipid antigens (described above) contained 25 carbohydrates and would 'better be termed glycolipids. The lipid hapten isolated from human adenocarcinoma tissue by Hakomori and co-workers (1967) was termed a glycolipid since it was shown to contain fucose, glucose, galactose, and glucosamine. They have also reclassified the tumor antigens of Witebsky and Rapport as glycolipids. The glycolipid isolated from tumor tissue by Hakomori et alo (1967) had weak II and moderate Le\ofis blood substance activity. After mild acid hydrolysis the glycolipid hydrolysate was precipitated by antiserum prepared to Type XIV pneumococcal polysaccharide. Antisera to the glycolipid agglutinated type A erythrocytes more strongly than erythrocytes of type B, An, or O. Data on the similarities of the glycolipid with blood group A substance suggested that this glycolipid was perhaps ident cal with that of ~1itebsky's. The structural similarity of group A substance and tumor glycolipid was referable to the carbohydrate moiety. Hakomori et al. (1967) suggested that tumor glycolipid resulted from an aberration in synthesis of glycolipids possessing blood group activity. The evidence of Hakomori et al. (1967) of the similarity between tumor glycolipid and blood group substance shed some light on immunological competence (or defectiveness) in respect to cancer. They suggested that in aberrant synthesis of glycolipid in tumors, blood group 26 precursor or precursor-like substances accumulated at the surface of the tumor cells. Since tolerance to these substances has been established during embryologic development, the host may fail to recognize the tumor cell as foreign and thus may fail to reject it (Hakomori et al., 1967). The relationship between blood group A substance and tumor glycolipid could eA~lain the increased incidence of stomach carcinoma in patients of blood type A. Thus, it may be more difficult for the immunological system of a type A patient to recognize tumor cells coated \vi th the glycolipid because of its similarity to the host's own blood group sUbstance. The relationship, if any, of the above phenomenon to changes in the blood types of leukemia patients (van der Hart, van der Veer, and van Loghem, 1962; Gold et al., 1959; and Hoogstraten, Hosenfield, and Wassermann, 1961) is not known. Treacy, Geiger, and Goss (1967) reported excessively high concentrations of soluble, anti-A or anti-B inhihiting substances in sera of 12 cancer patients. The ~ases con- sisted of patients with ovarian cysts and adenocarcinomas of the stomach, colon, and pancreas. Sufficient soluble substance was present in these sera to completely neutralize the antiserum used for blood group determination. It seems likely that these soluble substances originated in the ovarian cysts and tissues of· the adenocarcinomas. Specific antigens in human gastric cancer tissue 27 and other human tumors have been reported by Tee, lvang, and Watkins (1964); McKenna, Sanderson, and Blakemore (1964); and Itakura (1963). Zilber (1962) immunized rabbits to alkaline (pH 8.3 to 8.5), aqueous extracts of cancerous stomach tissue as well as to normal tissues from a variety of organs. Antibodies to normal tissue antigens were absorbed from the prepared antiserum to tumor tissue by adding extracts of normal tissues (including stomach) to the serum. The antisera to normal tissues were absorbed with extracts from other normal tissues. The resulting precipitates were removed by centrifugation. The excess of absorbing antigens was re- moved by either preparative agar electrophoresis or by ion-exchange chromatography on DEAE-cellulose. These antisera were then reacted with tissue extracts using the agar-gel diffusion technique. Of the three antisera prepared against cancerous stomach tissue, two formed a precipitation band when reacted with the cancerous stomach antigen. The third one formed two bands. None of the antisera contained antibodies against antigens of normal stomach tissue. The purified antisera to carcinoma ex- tracts cross-reacted extensively with extracts from other types of cancers, but when absorptions were carried out with extracts of the other cancerous tissues, crossreactivity was eliminated. The absorption left antibody that still reacted with stomach carcinoma extracts. The 28 cancerous gastric tissue antigen was purified by column electrophoresis through a sucrose density gradient. The immunological activity was found in two fractions which were shown to migrate in the zone of slowly migrating proteins defined by beta- and gamma-globulins. Avdeyev, Bashkayev, and Rogalsky (1967) have since reported that the antitumor sera obtained from Zilber's laboratory also reacted with certain antigens of normal colonic mucosa. The above reports, relative to gastric tumors, have implicated gastric tumors as producing additional antigenic substances not common to their normal tissue counterpart. In addition, gastric tumors have been re- ported to undergo antigenic simplification (Rapp et al., 1965; and Nairn et al., 1962b). That is to say, sub- stances present in normal tissue were lost as the tissue underwent neoplastic transformation. Kosyakov and Korosteleva (1963) showed that specific antigenic substances appeared in the cells of human malignant tumors. These substances were absent in normal tis- sues of the same individual. The specific antigenic properties of the incipient cancer were also found in its metastases. No single antigen specific for all human tumors was shown to exist. There were, however, anti genically similar tumors. Antigens specific for human tumors \vere not affected by high temperature (100 C) and 29 were not destroyed by proteolytic enzymes (Kosyakov and Korosteleva, 1967). Normal organ-specific antigens were unstable at 100 C and were destroyed by proteolytic enzymes. Based on these characteristics they concluded that the antigenic determinants of the tumor-specific substances were either lipid or carbohydrate. Gold and Freedman (1965a), in preparing rabbit antiserum to tissues of human cancerous colons, demonstrated that all human colonic cancers contained identical tumorspecific antigens. These antigens were shown to be pres- ent in all adenocarcinomas arising from entodermally derived epithelium of the digestive system, such as rectum, duodenum, stomach, esophagus, and pancreas. These anti- gens were absent from normal alimentary tract tissue and from all other adult normal, diseased, or cancerous human tissues (Gold and Freedman, 1965b). They found, however, that these tumor-specific antigens of the digestive tract were contained in normal human fetal gut, liver, and pancreas until the end of the second trimester of gestation. These antigens, which were protein-polysaccharide in nature, were therefore named carcinoembryonic antigens of the human digestive system. Although it is not possible to document all research on the serological search for cancer-specific substances, all serological approaches reviewed appeared to consist of five components: immunizing anti~en, prepared antibody, 30 test antigen, detection method, and control of crossreactions. The immunizing antigen usually consisted of some form of tumor tissue. ~jaterials used have been whole tumors, viable cell cultures, treated tumor cells, tumor extracts, and fractions of tumor cells. The test antigen has usually corresponded to the immunizing antigen. The prepared antibody has been most often derived from rabbits. However, other animals, such as horse and guinea pig, have been used. The mouse has been the most used animal for preparation of iso- and auto-antibody. De- tection methods for cancer-specific antigens have varied greatly among researchers. The antigenic test systems used have been complement fixation, cytotoxicity, in vivo protection, anaphalaxis, agar-gel precipitation, immunofluorescence, and localized radioantibody reaction. Techniques for the control of cross-reactions have been the most obscure and problematic area in the search for cancer-specific antigens. In addition, the accepted essential requirements among researchers for establishing specificity of serological reactions have changed with time. Various techniques used have been absorption of antisera, comparative reaction of other antisera or other test antigens, purification of test antigens, employment of heat and solvent stable antigens, desensitization to cross-reacting antigens, use of pooled antigens for immunization, and usc of natural tolerance. 31 The research to be reported here made use of cyst fluid from human cystadenomas of the ovary as the immunizing antigen. The test antigens used were saline extracts of normal and cancerous tissues. The use of an immunizing antigen so unrelated to the test antigen is unique in cancer research. MATERIALS AND METHODS Human Tissues Tissues, malignant and normal, were obtained from surgery and at autopsy. Cancerous tissue was excised from the central portion of the tumor. In the case of tumors of the digestive tract, care was taken to excise only the malignant tissue, avoiding areas in which there was deeper wall infiltration. The term tumor tissue will hereafter refer to malignant tissue. Normal tissue of the same organ, if available, was also obtained. Normal digestive tract tissues were removed from a site at least 10 cm from the pathological area. The normal mucosa tissues of the stomach and colon were stripped of all underlying muscle tissue. extracted. The tissues were immediately frozen or Tissues used as specific examples are de- scribed in Table 1. Cyst Fluids Fluids from benign, pseudomucinous cystadenomas of the human ovary obtained from surgery were aspirated aseptically into sterile, evacuated blood donor bottles. The fluids were checked for sterility by bacteriological testing. The amounts of fluid necessary for immunization were stored at 4 C; the remainder were frozen. Occasion- ally, the cyst fluids required homogenization or dilution with saline in order to render them sufficiently fluid TABLE 1 DESCRIPTION OF MALIGNANT TISSUES USED* Designation Type of Pathological Tissue Blood Type of Patient A cancer colon Adenocarcinoma of colon B cancer colon Adenocarcinoma of colon o Br cancer colon Adenocarcinoma of colon o negative Bu cancer colon Adenocarcinoma of colon o positive C cancer breast Adenocarcinoma of breast E cancer colon Adenocarcinoma of colon B positive En cancer stomach Adenocarcinoma of stomach All negative II cancer lung Bronchogenic carcinoma o positive L cancer colon Adenocarcinoma of colon o positive M cancer colon Adenocarcinoma of colon A positive 1'-1a cancer colon Adenocarcinoma of colon A positive 1'-lc cancer colon Adenocarcinoma of colon A positive Me cancer stomach Adenocarcinoma of stomach A positive (Continued on nexf 11age) positive** Ijl VI TABLE 1 (continued) Designation Type of Pathological Tissue Blood Type of Patient ° cancer colon Adenocarcinoma of colon AB negative P cancer colon Adenocarcinoma of colon A negative Pi cancer colon Adenocarcinoma of colon A positive Pr cancer colon Adenocarcinoma of colon o positive S cancer ovary Cystadenocarcinoma of ovary o positive T cancer colon Adenocarcinoma of colon o positive *Normal organ tissues from the same patients were also used. **Type 0, RhO(D) positive. ~ ~ 35 for injection. A list of the cyst fluids used is pre- sented in Table 2. stain~d The materials of the cyst fluids intensely with periodic acid-Schiff stain (for polysaccharides) and faintly with amido-Schwarz stain (for proteins) after paper electrophoresis (Holy Cross Hospital Laboratory, Salt Lake City). Antisera Preparation New Zealand White, virgin female rabbits were used for all immunizations. Animal weights, while generally uniform in an individual immunization series, varied with the different series as will be indicated. Animal condi- tion throughout immunization was determined by appearance and normal weight gain. A few animals displaying poor health were discarded. The individually caged animals were immunized using separate sterile syringes. Intraperitoneal (IP), intra- muscular (1M), and intradermal (ID) injection routes were used in this research. used for I~1 A site on the upper hind leg was and ID injections. Adjuvants used were Freund's complete adjuvant (Hyland Laboratories, Costa Mesa, California) and sodium alginate with calcium chloride (CaC1 2 )o Na alginate and CaC1 2 solutions were prepared in 4 per cent and 0.33 per cent concentrations, respectively. Procedures of immunization varied with each immunization series. Specific details concerning the 36 TABLE 2 CYST FLUIDS USED FOR INNUNIZATION Designation Blood Type of Patient W cyst fluid A negative* R cyst fluid A positive 0 cyst fluid 0 positive H cyst fluid A positive Z cyst fluid 0 negative S cyst fluid 0 positive L cyst fluid A negative *Type A , RhO(D) negative 37 immunization of the animals will be reported in the results of each series. Bleeding of Animals All animals to be immunized were weighed and a preimmunization blood sample prior to injection was taken. Approximately 20 ml of blood were collected by intracardial (IC) bleeding. Blood samples were permitted to clot for 1 to 2 hours at room temperature. "rimmed" and stored overnight at 4 C. The clots were The sera were separated, preserved with 0.1 per cent sodium azide, and frozen. All preimmunization sera were reacted with the test antigens used in this work by the Ouchterlony double diffusion test. All preimmunization sera failed to react in a demonstrable way with the test antigens. During the immunization series trial bleedings were made. Reactivity of the sera was evaluated by screening with one or more of the test antigens. The procedures for further immunization were based on the results of the screening. The bleeding schedule varied with each immuni- zation series and will be described with the results of each series. At the conclusion of antigen injection, the animals were usually rested a week and then bled one to four times at weekly intervals. were exsanguinated. Animals producing potent antisera The use of terms such as potent, 38 strong, suitable, or satisfactory, relative to describing antisera will hereafter refer to the relative ability of the antisera to react demonstrably with the test antigens. Also, the use of the terms strong, weak, or faint in describing precipitation reactions will refer to the comparative intensity of the precipitation bands. Antisera were divided into 3 ml aliquots; one aliquot for immediate use was stored at 4 C and the remainder were frozen. Antisera Absorption Absorption ~ Absorbent Incorporation in Agar On the basis of results to be reported, examination of antisera from the first immunization series revealed that the cyst fluids contained serum components. The inability to distinguish between precipitation bands created by antibodies against normal serum components and tissue antigens was an obvious problem. Therefore, effort was directed toward finding a simple but reliable method for removing antibodies to normal human tissue and serum components from the rabbit sera. A method was devised whereby absorbing antigens such as human sera, cyst fluid, and homogenates of malignant and normal tissues were added to the cooling agar immediately before pouring the agar into plates. Differing volumes of these absorbing re- agents were added at the expense of equal volumes of buffered saline. Concentrations of absorbing antigens in 39 the agar were dependent upon the strength of the antisera and the designed experimental effect and thus were varied. Dialysis Chamber ~1ethod An absorbent medium containing 0.5 per cent agar, 30 per cent human sera, and 10 per cent saline extract of colonic tissue was prepared. Mixtures using cancerous colonic tissue and mixtures using normal colonic tissue were prepared. The absorbents were added to the melted agar as it cooled. These mixtures in volumes of 0.5 ml were pipetted onto supporting filters, 1.2~ average pore diameter, (Gelman Instrument Co., Ann Arbor, Michigan) which were designed to fit TechniLab E 1 Diffusion Chambers (TechniLab Instruments, Inco, Pequannock, New Jersey) 0 The agar was allowed to set for 2 hours. The filters were then placed in the diffusion chambers and the apparatus were assembled. One side of each chamber was designated as sample side and the other side as collection side. One ml of the antisera to be absorbed was placed in the sample side. placed in the other sideo One ml of saline was The chambers with their ports unsealed were allowed to stand at 5 C for 24 hours. The contents of each side were then withdrawn and retained for testing. Beaker Method The absorbent mixtures used in this method were 40 prepared in the same manner as in the Dialysis Chamber Method described above. The melted mixture in volumes of 2 ml was poured into 30 ml beakers and was allowed to harden. One ml of the antisera to be absorbed was layered over the agar. The beakers were then rotated for 3 hours at a setting of 50 on a Thomas Rotator (Arthur H. Thomas Co., Philadelphia, Pennsylvania). The sera were removed and retained for testing. Preparation of Tissue Test Antigens Extracts of normal tissues and of histologically proven malignant tissues were prepared. Treatment con- sisted of homogenization of the tissues using either a semimicro or micro (depending upon tissue volume) Eberbach attachment (Eberbach Corp., Ann Arbor, Michigan) for A volume of normal physiological the Waring Blender. saline solution equal in weight to double the weight of the sample was added. The homogenates were centrifuged at 4500 X g for 30 minutes at 4-5 C. The lipid material forming a pellicle was removed with a spatula. The super- natant fluid was then poured off the saline-insoluble tissue brei and filtered through \,hatman #1 filter paper. The extracts were divided into volumes of 2 to 3 ml and stored in the frozen state. Immunological Assay Precipitation antibody was detected by immuno- 41 diffusion in gel. The plate method ad modum Ouchterlony (1958) of double diffusion in two dimensions was used. Agar was employed as the stabilizing gel for the reactants. Preparation of Ouchterlony Double Diffusion Agar Plates Two main types of agar preparations were used; these were 1 per cent agar and 1 per cent agar containing pooled human serum. Various modifications of the human serum- agar were used in this research and are described under Antisera Absorption. The agar solution was prepared by placing 1 g of Difco Noble agar in 100 ml of 0.15 M phosphate (Sorenson) buffered saline (pH 7.3) which contained 0.1 per cent sodium azide. The agar was autoclaved for 10 minutes and poured into separate, scratch-free plastic Petri plates in 16 ml volumes. Human serum-agar consisted of 1 per cent agar with 30 per cent human serum (pooled sera of greater than 100 blood donors from the American Red Cross Regional Blood Center, Salt Lake City). One gram of agar was autoclaved in 70 ml of buffered saline. The agar solution was cooled to 70 C and 30 ml of pooled human serum, prewarmed to 40 C, were added. The agar was gently swirled and poured as described above. After solidification of the agar the plates were placed in a moist chamber at room temperature and allowed to stand overnight. Human serum-agar was used 42 in all tests except when noted otherwise. A Feinberg cutter (Consolidated Laboratories, Inc., Chicago Heights, Illinois) was initially used to punch the diffusion wells in the agar plates. The use of this cutter produced a "six-shooter" pattern--a central well surrounded by six peripheral wells (Ouchterlony, 1968). Antisera were inoculated into the larger center well. However, during the first immunization series it was realized that a more flexible procedure was needed for well-cutting. In order to vary patterns (the number and spacing of holes) and to permit the use of antisera in larger periphery wells, wells were punched with a #4 cork bore of 8 mm diameter and a #3 cork bore of 6 mm diameter. In most cases wells designed for the placement of antisera were punched with the larger #4 cork bore. Distances between the wells were varied depending unon the strength of the reactants. The distances ranged from 13 to 20 mm. Agar from the wells was removed and the extracts and antisera were added to their respective wells with a Pasteur pipette until visually level with the surface of the agar. Reaction Measurement The Ouchterlony plates were incubated at room temperature in a moist chamber and observed daily for a period of 2 weeks. The formation of antigen-antibody complexes was observed by the appearance of precipitation 43 bands in the agar. The bands were more easily seen using a specially constructed viewing box which permitted light to be obliquely transmitted through the bottom of the Petri plate. RESULTS Preliminary Findings Preliminary experiments showed that normal sera from humans and rabbits did not react with ovarian cyst fluid as determined by the Ouchterlony double diffusion test. The cyst fluid was also reacted with sera of patients with adenocarcinomas of the colon, kidney, pancreas, liver, breast, and stomach. No precipitation bands were observed. Sera from cancer patients were also reacted with negative results. Pooled sera from blood donors did not react with any of the above antigens. Commercially prepared antihuman globulin (Sigma Chemicals, St. Louis, Missouri) was reacted with antigens of cyst fluid and saline extracted tissue from ovarian cysts of two patients, one with blood type A and the other with blood type O. A faint, diffuse band was produced with the ovarian cyst antigens from the type A patient. Two bands were produced by reaction of the antihuman globulin with the ovarian cyst antigens from the type 0 patient. These bands were not produced when 30 per cent human serum was incorporated in the agar. Thus, cystic antigen preparations contained serum globulins and antibody produced in rabbits to these globulins could be absorbed by use of human serum-agar. 45 Reactions of Prepared Antisera First Immunization Series Two rabbits, labeled U-14 and U-15, were immunized by IP injection with mucinous ovarian cyst fluid from an unknown patient. Rabbit U-14 was initially injected with 5 ml of the cyst fluid and observed for 4 days. no apparent effect of the injection. There was Both rabbits were then injected IP with 1 ml followed by 2 ml injections twice a week for 6 weeks. The rabbits were bled by car- diac puncture 1 week after the last injection. A slightly diffuse precipitation band was produced when antisera U-14 and U-15 were reacted with various dilutions of cyst fluid antigen from patient W (W cyst fluid) as seen in Figure 1. The supply of cyst fluid from the unknown patient was exhausted during immunization and was not available for subsequent testing. A 1:5 and 1:10 dilution of the W cyst fluid antigen exhibited a strong reaction. The band was also produced in agar containing 30 per cent human serum. A faint second band developed when antiserum U-14 was reacted with the undiluted cyst fluid antigen (Figure 2). This second band was seen only when the cyst fluid was placed in the larger 8 mm well. The use of human serum-agar did not prevent the formation of the second band. Antisera U-14 and U-15 were then reacted in 1 per 46 Antiserum U-14 0 ~ 0 W cyst fluid (1:5) (1:20) 0 0 W cyst fluid (1:10) Ii cyst fluid (1:40) W cyst fluid Figure 1. Ouchterlony reaction of antiserum U-14 with -' saline-diluted cyst fluid in 1 per cent agar. W cyst fluid Normal rabbit sera 0 Antiserum U-15 Figure 2. ~ 0 0 0 Normal human sera Antiserum U-14 Ouchterlony reaction of antisera U-14 and U-15 \Y'i th normal rabbit sera, normal human sera, and cyst fluid in human serum-agar. 47 cent agar with saline extracts of tissues, as well as with salivas and blood group substances. One or two bands were produced with saliva from various patients. No precipi- tation bands were exhibited by reaction with 1 per cent solutions of type A and of type B blood group substances (Sharpe and Dohme, West Point, Pennsylvania). Reaction with extracts of malignant tissues of the breast, lung, liver, ovary, and spleen either did not produce any precipitation bands or produced one or two bands. None of the above bands, however, appeared when human serum-agar was used. The precipitation reactions shown in Figure 3 were the first indications of a specific reaction of antisera to cyst fluid with tumor tissue extracts. The figure diagrammatically illustrates the reaction of antiserum U-14 with W cyst fluid, saliva from patients Nand G, and extracts of normal and cancerous colonic tissue from patient E. Precipitation reactions occurred with all these antigens. The use of human serum-agar eliminated all bands except the band with cyst fluid and one of the bands with tumor tissue (Figure 4). This pattern held true when testing extracts of colonic tissues from other patients (Figures 5 and 6). The precipitation band to colonic tumor was also produced with antiserum U-15 (Figures 7 and 8). Surprisingly, the reactions of the antisera with the W cyst fluid and tumor tissue were 48 Center well--Antiserum U-14 N saliva W cyst fluid (1:5) G saliva Boiled W cyst fluid (1:5) E normal colon E cancer colon Figure 3. Ouchterlony reaction of antiserum U-14 with extracts of normal and cancerous colonic tissues, saliva, and cyst fluid in 1 per cent agar. Center well--Antiserum U-14 N saliva G saliva E normal colon 0 0 0\0 /0 0 0 W cyst fluid (1:5) Boiled W cyst fluid (1:5) E cancer colon Figure 4. Ouchterlony reaction of antiserum U-14 with extracts of normal and cancerous colonic tissues, saliva, and cyst fluid in human serum-agar. 49 Center well--Antiserum U-14 W cyst fluid (1:1) o E cancer colon W cyst fluid (1:5) T cancer colon E normal colon T normal colon Figure 5. Ouchterlony reaction of antiserum U-l4 with cyst fluid and extracts of normal and cancerous colonic tissues in 1 per cent agar. Center well--Antiserum U-14 W cyst fluid (1:1) o E cancer colon 0 _,-S---- T cancer colon 0 ow o cyst fluid (1:5) E normal colon o T normal colon Figure 6. Ouchterlony reaction of antiserum U-14 with cyst fluid and extracts of normal and cancerous colonic tissues in human serum-agar. 50 Center well--Antiserum U-14 W cyst fluid T normal colon 0 o --..oa...- o E normal colon 0 o o T cancer colon E cancer colon W cyst fluid Figure 7. Ouchtcrlony reaction of antiserum U-14 with extracts of normal and cancerous colonic tisse.es and cyst fluid in human serum-agar. Center well--Antiserum U-l5 C cancer breast 0 Btl Crulcer colon tv cyst fluid ~~\O o 0 E cancer colon P normal colon 0 p cancer colon Figure 8. Ouchterlony reaction of antiserum U-15 with extracts of normal and cancerous colonic tissues and cancerous breast tissue, and cyst fluid in human serum-agar. 51 reactions of non-identity. Antiserum U-14 was reacted in human serum-agar with sera from cancer patients having high concentrations of alpha 2 serum globulins. No precipitation bands were produced. Second Immunization Series Three rabbits, numbered W-1, W-2, and W-3, were immunized with a mucinous cyst fluid from a pseudomucinous cystadenoma of the ovary from patient W. A fourth rabbit was injected with 10 per cent sterile polyvinyl pyrrolidone (Nutritional Biochemicals Company, Cleveland, Ohio). The materials were injected IP in 2 ml amounts twice weekly for 5 weeks. All rabbits were bled 1 week after the last injection. These antisera were reacted with the cyst fluid antigen from patient W. Antisera \{-l and W-2 developed a faint precipitation band with the homologous cyst fluid antigen after 3 days of incubation. The band faded away with continued incubation. Antiserum W-3 and the antiserum from the rabbit immunized with PVP failed to elicit any reaction with the cyst fluid. Three days after the first bleeding, immunization of all rabbits was resumed for 3 more weeks using the above procedure. Rabbit W-l died after the last injection. serum was not collected. Its The remaining rabbits were allowed to rest 1 week before the final bleeding. Antisera 52 from the final bleedings of rabbits W-2 and W-3 produced many bands with cyst fluid and colonic tissue extracts when tested in 1 per cent agar. None of these bands were produced when the experiments were repeated using human serum-agar. The anti-PVP serum was negative to all re- actants in both types of agar. At this point a technique for concentration of antisera was employed. In reacting antihuman globulin (AHG) with human sera, it was found that concentrating MIG 5:1 with Carbowax 20-M (Union Carbide Chemicals Co., New York, New York) greatly intensified the precipitation bands. A similar concentration (7:1) of antisera W-2 and W-3 was of no value in enhancing existing or revealing new precipitation bandso All sera from this immunization series were discarded. Third Immunization Series This series was designed mainly to explore the effectiveness of different routes of immunization. The W cyst fluid was again used. ~v-5 Eight rabbits, numbered through W-12, were injected with this antigen. W-5 throu~l pounds 0 Rabbits W-IO were young animals weighing 3.5 to 4 Rabbits W-ll and W-12 were older with weights of 5 and 7.5 pounds, respectivelyo The rabbits were injected IP with 2 ml of the cyst fluid twice weekly for 3 weeks. Rabbits W-5 and W-6 were rested 1 week and trial bled 53 while immunization of the remaining rabbits continued with two more 1 ml injections. Hereafter, antisera were always tested in human serum-agar unless indicated otheniise. Antisera W-5 and W-6 produced a faint band when reacted with the W cyst fluid antigen. No bands were produced with tissue extracts. Immunization of all rabbits was continued with 1 ml IP injections twice weekly for 3 more weeks. All rabbits were bled 2 weeks after the last injections. Two faint diffuse bands were produced by the reaction of antisera W-5, \y-6, and W-9 with the \~ cyst fluid. Antisera \v-7, W-B, \v-lO, and \i-ll produced one band and antiserum \.J-12 did not react with the cyst fluid. No bands were produced by reaction of any of the antisera with tissue extracts. Immunization of all rabbits was resumed the following week using a different route; 1M injections of W cyst fluid with Na alginate and CaC1 2 solutions were given twice weekly for 2 weeks. These injections consisted of 0.25 ml of W cyst fluid, 0.25 ml of Na alginate solution, and 0.5 ml of CaC1 2 solution. Rabbit W-6 died after the last injection. Two more blood samples were taken from the remaining animals 11 and 19 days after the last antigen injections. Rabbit W-9 died after the third bleeding and its serum (negative against tissue extracts) was discarded. Antisera W-5, W-B, li-lO, and \v-ll from these bleedings produced a strong band when reacted with the W 54 cyst fluid antigen while the sera from rabbits \v-7 and W-12 produced none. All antisera except for W-8 and W-IO failed to react with any extracts of tissues tested. Antisera \,-8 and \v-lO each produced a band with colonic tumor antigen; however, the bands were very weak. Rabbi ts \,-8 and \v-lO were bled for the fourth time 2 weeks after the third bleeding. In order to enhance antibody production they were given two more 1M injections (4 days apart) of a solution of 1 ml W cyst fluid, 1 ml Na alginate, and 2 ml CaCI 2 • The rabbits were bled a fifth and a sixth time, 10 and 25 days after the last injections. In comparing the gel reactions of the fourth and fifth bleedings there did not appear to be any strengthening in intensity of the band to colonic tumor antigen. How- ever, the reaction bands which required 7 days to appear with the sera from the fourth bleeding appeared in 4 days with the sera from the fifth bleeding. The antibody was not demonstrable in the sera from the sixth bleeding which was collected 25 days after the last challenge. As noted above, antisera from the third bleeding of rabbits W-5 and W-7 failed to react with colonic tumor extracts. Immunization of these rabbits was resumed 2 weeks after the third bleeding with an 1M injection of a solution of 1 ml of freshly collected cyst fluid from patient 0 (0 cyst fluid) and 1 ml of Freund's adjuvant. This injection \\"as repeated after 40 days and was followed by 55 three more semiweekly injections. These animals were bled 11 days after the last injection. Rabbit W-5 refused food and water the week prior to bleeding. it suffered convulsions and died. After the bleeding Precipitation bands appeared when antisera W-5 and \v-7 from this bleeding were reacted with extracts of colonic tumors. Antiserum \i-7 exhibited a fairly strong band while the band produced by antiserum \1-5 was very faint. The precipitation band which antiserum W-5 had originally produced against W cyst fluid was not discernible. Antiserum W-7 which did not exhibit a band with the W cyst fluid after the second and third bleedings now produced a weak band when reacted with that antigen. It thus appeared that in this rabbit the immunization with 0 cyst fluid resulted in a reinforcement of antibody response to the \v cyst fluid. Rabbi t \v-7 was bled a fifth time I week after the fourth bleeding. The antibody response to the tumor antigens as well as to the W cyst fluid was weaker in the antiserum from this bleeding. Rabbi ts lv-II and \.)'-12 were not further immunized but were bled for the fourth time 4 weeks after the third bleeding. These antisera still failed to react with the tissue extracts. Fourth Immunization Series Another immunization. series utilizing the remaining 56 W cyst fluid was begun immediately after the start of the preceding series. Rabbits W-14 through \v-17, weighing 4 to 4.5 pounds, received progressively increasing 1M doses of a solution of \1 cyst fluid, Na alginate, and CaCll) ... • The injections given twice weekly consisted of a corresponding volume of CaC1 2 solution to combined, equal volumes of cyst fluid and Na alginate solution. The first injection contained 0.2 ml of cyst fluid, 0.2 ml of Na alginate, and 0.4 ml CaC1 2 for a total volume of 0.8 mI. This total volume was progressively increased 0.4 ml each injection until a total injection volume of 4 ml (11 injections) was reached. All animals were bled 10 and 25 days after the last injections. All antisera from the first bleeding produced one strong and two weaker bands when reacted wi tIl the \y cyst fluid. Testing the antisera with tissue extracts showed that antisera \1-14 and \v-17 produced a precl.pi tation band \vi th colonic tumor antigen (Figure 9). No bands were seen after reaction of the anti- sera with normal colonic tissue extracts (Figure 10) or with extracts of other normal or cancerous tissues, such as breast, liver, spleen, and lung. The bands produced by reaction of the W antisera with colonic tumor extracts appeared to be "in identity" with the bands produced by antiserum U-14 (from the first immunization series) with colonic tumor extracts (Figure 9). It appeared from Figures 9 and 10 that the band to 57 Center well--P cancer colon \v cyst fluid Antiserum U-14 \V-17 Antiserum \Y'-14 Antiserum \v-16 Antiserum \V-15 Figure 9. Ouchterlony reaction of antisera U-14 and \v-17 with cyst fluid and cancerous colonic tissue extracts and of antisera W-14, W-15, and W-16 with cancerous colonic tissue extracts in human serum-agar. Center well--Antiserum W-17 Bu cancer colon Bu normal colon B cancer colon o o ' \ 0 O~ 0\ o 0 \v cyst fluid Mc cancer colon B normal colon Figure 10. Ouchterlony reaction of antiserum li-17 with extracts of normal and cancerous colonic tissues and cyst fluid in human serum-agar. 58 colonic tumor extracts was not "in identity" with any of the three bands to cyst fluid. The use of 1 per cent agar instead of human serumagar revealed that the W antisera contained antibodies to two antigens common to both colonic tumor tissue and human serum. It can be seen in Figure 11 that the components of human serum found in the extracts of tumor tissue were not contained in the gamma globulin fraction. Comparison of the sera from the first two bleedings showed that the strength of the reactions of sera from the second bleeding was considerably reduced. Of the three bands to cyst fluid produced by reactions of sera from the first bleeding, only the prominent band was exhibited by sera from the second bleeding. Also, the bands to extracts of colonic tumor tissue produced by antisera li-14 and H-17 were not discernible wi th sera from the second bleeding. Immediately following the second bleeding, ID injections of 0.2 ml of cyst fluid were given twice weekly for 2 weeks. The animals were bled 10, 20, and 35 days after the last ID injections. Results of the third bleeding showed the reactions of the antisera to extracts of colonic tumor to be moderately enhanced over those of the second bleeding. However, the reactions were not as strong as those of the sera from the first bleeding. cipitation reactions of the antisera to the cyst fluid Pre- 59 Antiserum \tl-14 Gamma globulin (l: 2)0 P cancer 0./ 0 COlon~;O Gamma globulin (1:10) P cancer colon Gamma globulin (1:5) Figure 11. Ouchterlony reaction of antiserum W-14 with cancerous colonic tissue extracts and gamma globulin of serum in 1 per cent agar. 60 remained weak in the sera of the third bleeding. In the fourth bleeding the antisera's reactions to both colonic tissues and cyst fluid were comparable or slightly stronger than those in the third blood sample. Antisera from the fifth bleeding failed to react with the cyst fluid or colonic tissue extracts. Immediately following the fifth bleeding two more 4 rul 1M injections of cyst fluid were given 5 days apart. Reactions from sera collected 10 days after the two booster injections were comparable to those of the sera from the fifth bleeding. Fifth Immunization Series Results of the preceding immunization series suggested that intramuscular injection showed promise of being an effective route of immunization. Therefore, the 1M route of injection was further explored using a newly collected cyst fluid. Four rabbits were immunized with mucinous ovarian cyst fluid from patient O. The 0 cyst fluid was very viscous and required dilution with sterile saline (1:3) to render it suitable for injection. rabbits, labeled 0-20 through 0-23, were injected The I~I with o cyst fluid, Na alginate, and CaC1 2 in a ratio of 1:1:2. The initial injection consisted of a solution of 0.2 ml of o cyst fluid, 0.2 ml of Na alginate, and 0.4 ml of CaC1 2 for a total volume of 0.8 mI. This total volume was 61 progressively increased by 0.4 ml on each semiweekly injection until a total volume of 2.4 ml (five injections) was reached. This series was followed 4 days later with a 4 ml injection (1 ml:l ml:2 rol). The rabbits were bled 10 days after the final antigen injection. Rabbits 0-20 and 0-21 suffered convulsions after the bleeding. 0-23 had convulsions before the bleeding. survived. Rabbit All animals The sera from the four animals did not contain antibody to 0 cyst fluid and colonic tissue. Immunization was resumed 25 days after this bleeding repeating the immunization program used in this series except for the omission of the final 4 ml injection. The animals were bled a final time 8 days after the last injection. Antibody response of the sera to 0 cyst fluid and colonic tissue was still negative and all collections were discarded. Sixth Immunization Series Five rabbits, weighing 3.5 to 4.5 pounds, were immunized with mucinous cyst fluid of a pseudomucinous cystadenoma of the ovary from patient R (R cyst fluid) using 1M and IP routes of injection. The rabbits, labeled R-l through R-5, were ini tially in.jected IP \vi th 5 ml of the R cyst fluid. This injection was followed with three successive IP injections of 2 ml given at 3 day intervals. Immunization was continued with 1M injections of a 62 solution of 1 ml of cyst fluid, 1 ml of Na alginate, and 2 ml of CaC1 2 • A total of six 11\1 injections were given semiweekly. Lesions developed at the site of 1M injection of rabbits R-4 and R-5 after the second injection. Be- cause of the severity of the lesion of rabbit R-5, the animal was exsanguinated 3 days after the fourth 1M injection. The remaining four rabbits were bled 1, 2, and 3 weeks after the last injection. All rabbit sera from the first bleeding reacted with the homologous antigen of R cyst fluid. bands were produced (Figure 12). Many distinct Four bands were produced by reaction of antiserum R-2 with the cyst fluid. The re- maining sera exhibited three bands with the cyst fluid. The reactions of the antisera to tissue extracts were many and varied. All the antisera produced a band when reacted with extracts of malignant colonic tissue. However, all but one (antiserum R-2) also reacted with extracts of normal colonic tissues. Antiserum R-l produced two bands while antisera R-3, R-4, and R-5 each produced one band when reacted with extracts of normal colonic tissues. Examination of sera from the second bleeding revealed diminished reactivity in all sera except R-2 which showed enhanced reactivity. Antiserum R-2 from the second bleeding produced a weak second band to cancerous colonic tissues and a weak band to normal colonic tissues. Serum from the third bleeding of rabbit R-2 compared 63 Center well--R cyst fluid Antiserum R-l o Antiserum R-2 Antiserum R-3 Figure 12. O~, (0 ~j o 0 Antiserum R-5 Antiserum R-4 Ouchterlony reaction of antisera R-l through R-5 with cyst fluid in human serum-agar. 64 to that of the first bleeding in that it produced only one band when reacted with cancerous colonic tissues. Reactions of the sera from the third bleeding of the other rabbits were weaker than those of the second bleeding. The sera from the three blood samples of rabbit R-2 were pooled in the ratio of 1:1:2, respectively, and designated as antiserum R_2 x • In testing the pooled reagent, none of the reactions of the sera from the individual bleedings was masked. The prominent precipitation band to malignant colonic tissue of all three bleedings appeared as well as the fainter second precipitation band from the second bleedin~. The weak reaction to normal colonic tis- sue was also exhibited by this pooled antiserum. The re- actions of antiserum R_2x with colonic tissue arc seen in Figure 13. The weak reactions (faint precipitation bands) to extracts of normal and cancerous colonic tissue were reactions of identity. This was the first evidence of an antigen common to colonic tissues, normal or cancerous. Antiserum R_2 x (unlike sera from previous series) reacted with numerous extracts of other tissues--liver, kidney, breast, testes, lung, and thyroid. was usually seen as a weak and diffuse band. The reaction While the band appeared more frequently with the extracts of cancerous tissues than with their normal counterparts, the band, nonetheless, appeared to be in common with the band 65 Center well--Antiserum R_2 x A cancer colon o O/~~\ A normal colon I' Mc cancer colon 0 \ 0 O\~/O o 1\1 normal colon }\1 cancer colon Mc normal colon Figure 13. Ouchterlony reaction of antiserum R_2x with extracts of normal and cancerous colonic tissues in human serum-agar. 66 to normal colonic tissue. The reactions of this anti- serum with stomach tissue extracts, however, presented a different pattern. The antiserum reacted strongly with extracts of stomach tissue (Figure 14). The tissue extracts from normal stomach generally exhibited one more band when reacted with the antiserum than did the tissue extracts from cancerous stomach. In no case did the bands to stomach tissue appear to be in common with the band specific for colonic tumor tissue (intense outermost band in Figure 14). The tissue antigen common to both normal and malignant colonic tissue is demonstrated in Figure 14 as the weak band closest to the center well. To strengthen the intensity of the precipitation band common to extracts of normal and cancerous colonic tissue, the antiserum was concentrated using various procedures. In the first method antibody globulins were pre- cipitated with half-saturated ammonium sulfate. The pre- cipitate was resuspended with a small amount of saline and dialyzed overnight against saline. This procedure did not intensify the reactions of the antiserrun or reveal any new bands. The precipitins to extracts of colonic tissue even appeared to be diminished. Adsorption onto membrane and dilution by uptake of water during dialysis were the probable causes of the failure of this method to enhance reactivity of the antiserum. Concentration of the anti- serum by dialysis against 10 per cent PVP was then tried. 67 Center well--Antiserum R_2 x P cancer colon 1 o Me cancer stomach 0 ~(o 0 He normal stomach En cancer stomach 0 \' ~r 0 En normal stomach o P cancer colon Figure 14. Ouchterlony reaction of antiserum R_2x with extracts of normal and cancerous stomach tissues and cancerous colonic tissue in human serum-agar. 68 The bands to colonic tissue extracts were significantly enhanced. The precipitation band common to normal and cancerous colonic extracts, the outer ring in Figure 15, was notably strengthened (compare the reaction at well 1 with reactions at wells 2 and 3). band specific for colonic tumor. The inner ring was the Adsorption of the anti- serum with types 0, AI' and B red blood cells had no effect on the intensity of the precipitation bands to colonic tissues (Figure 15--wells 4 and 5). Seventh Immunization Series Based on the favorable results of the sixth series, the R cyst fluid was again used in immunizing five rabbits. The rabbits, labeled R-6 through R-IO, weighed 4 to 5.5 pounds. The procedure of IP injections used in the sixth series was repeated in this series with the exception that an additional IP injection of 2 ml R cyst fluid was given. (Rabbit R-9 suffered convulsions and died after the initial 5 ml injection.) This was followed by six 1M injections also given according to the procedure used in the sixth series. The animals were bled 1 week after the last injections. Rabbits R-7 and R-8 developed deep necrotic lesions at the site of 1M injection. A prominant band was produced by reaction of antisera from the first bleeding of rabbits R-7 and R-8 with extracts of malignant colonic tissues. Antiserum R-IO 69 Center well--P cancer colon Antiserum R_2x Adsorbed antiserum R_2x (O+A 1 ) 0 Concentrated ~dsorbed antiserum R-2 (A 1+B) o fn\\ 0 ~ 0 0 Concentrated x antiserum R_2 (2:1) Concentrated antiserum R_2x (3:1) (2:1) Figure 15. Ouchterlony reaction of antiserum R_2x and antiserum R_2x concentrated with 10 per cent PVP and adsorbed with red blood cells to extracts of cancerous colonic tissue in human serum-agar. 70 produced a weak band with these extracts. These bands, however, were common to those of normal colonic tissues. Therefore, these antisera did not contain precipitins specific for colonic tumor tissue. Antiserum R-6 did not react with extracts of colonic tissues. Rabbits R-7 and R-8 were bled a second time 1 \Veek after the first bleeding. Reactions of the antisera from this bleeding were weaker than those of the first bleeding. All sera were discarded. Eighth Immunization Series Seventeen rabbits, weighing 3.5 to 4.75 pounds, were immunized with cyst fluid from a mixed, pseudomucinous and serous cystadenoma of the ovary from patient H (H cyst fluid). An aliquot of 130 ml of the cyst fluid was heated in a water bath at 56 C for 8 hours. Four rabbits, H-24 through H-27, were immunized with the heated cyst fluid. Rabbits H-ll through H-23 were immunized with the unheated cyst fluid. a week for 7 weeks o IP injections were given twice The first injection consisted of 5 ml and the second of 1 mI. The remaining 6 injections were given in 2 ml amounts. Rabbit H-15 developed an abscess at the site of the initial injection and \Vas discarded. All other rabbits were bled 1 week after the last injections. These antisera were screened by reaction with extracts of colonic tumor tissues. Antisera H-13, II-14, 71 and H-17 produced two strong precipitation bands when reacted with colonic tumor tissue extracts. One strong band and a second weaker band were produced by antiserum H-23. Antiserum H-27 produced one weak band. Based on these results, rabbits H-13, H-14, H-l?, H-23, and H-27 were bled a second time 1 week after the first bleeding. Rabbit H-2? was found dead in his cage the day after the second bleeding. The antisera from the second bleeding were also screened by reaction with extracts of colonic tumor tissues. Of the five sera collected, antisera H-14 and H-17 produced the strongest reactions. Antisera H-13 and H-23 reacted weakly and antiserum H-2? failed to produce a precipitation band by reaction with the tissue extracts. Four days after the second bleeding, the four remaining animals (H-13, H-14, H-l?, and H-23) were exsanguinated. A comparison of the strength of the reactions of antisera from the three bleedings can be seen in Table 3. The antisera were titered as the greatest dilution of antisera before loss of resolution of any of the bands when reacted with colonic tumor tissue. Antisera H-13 and H-23 had a dilution titer of 1:4 and 1:3 respectively on the first bleeding. This was reduced to 1:2 on the second bleeding and less than 1:2 on the third bleeding. Antisera H-14 and II-17 had a dilution titer of 1:5 on the first bleeding. The titer was reduced to 1:3 on the 72 TABLE 3 COMPARISON OF TITERS OF ANTISERA H-13, H-14, H-17, AND H-23 Bleeding Antiserum dilution* First 1:5 14,17 1:4 13 1:3 23 1:2 < 1:2 Second Third 14,17 14,17 13,23 *Highest dilution showing reaction with tumor extract. 73 second bleeding and 1:2 on the third. The above four antisera produced numerous precipitation bands when reacted with the H ovarian cyst fluid in 1 per cent agar. However, only one dense precipitation band appeared with the use of human serum-agar. This dense band appeared to be composed of two bands formed closely together. Reaction of the antisera with colonic tissue extracts is seen in Figure 16. Two bands, one of which was "in identity" with the normal colonic tissue band, were produced by reaction with tumor tissue extracts. pattern held true for all colonic tissues tested. This Anti- sera H-14 and H-17 had the strongest reactions and were used in all succeeding experimentso Each antiserum was composed of equal parts of the sera from the first and second bleedings. The precipitation band (Figure 16) common to all extracts of colonic tissues will be referred to hereafter as the normal tissue band. The precipitation band specific for extracts of cancerous colonic tissues will be referred to as the cancer-specific band. In one instance (Figure 17) the normal tissue band was separated into two bands o reproducible. The phenomenon was not Either inappropriate incorporation of human serum in the agar or dissolution and reprecipitation of the antigen-antibody complex was the probable cause. 74 Center well--Antiserum II-14 Br cancer colon o 0/ ~-rl L normal colon 0 /0 ~ o\~j0 L cancer colon o Figure 16. \ Br normal colon 0 cancer colon o normal colon Ouchterlony reaction of antiserum H-14 with extracts of normal and cancerous colonic tissues in human serum-agar. Center well--Antiserum II-17 Br cancer colon S cancer ovary 0 Br normal colon II cancer lung 0 Pi cancer colon Pi normal colon Figure 17. Ouchterlony reaction of antiserum H-17 with extracts of normal and cancerous colonic tissues and cancerous lung and ovarian tissues in human serum-agar. 75 The antiserum also reacted with other carcinomatous tissues (Figure 17). One or two bands were produced by reaction of the H antiserum with normal and cancerous tissue extracts of stomach (Figure 18), lung, kidney, ovary, and breast. None of these bands appeared to be specific for the particular tissues tested. In no case did any of these tissues exhibit a band in common with the colonic cancer-specific band. Reactions of antisera H-14 and H-17 with extracts of normal and cancerous colonic tissue were investigated using a four well pattern of the Ouchterlony assay. In Figure 19 it appeared that the reactions of ider..tity, produced by the two antisera reacting with each tissue extract, constituted normal tissue bands. An interesting effect was pro- duced by interchanging the different sized wells (or volumes of the reactants). When the antisera were put into 6 mm wells and the colonic tissue extracts into 8 mm \vells, the result in Figure 20 was produced. Consequently, the normal tissue bands produced by each antisera reacting with both extracts constituted reactions of identity_ Absorption experiments were performed to further establish the specificity of the antisera for cancerous colonic tissue. Absorption of antibody to normal tissue was accomplished by adding extract of normal colonic tissue to the agar as described earlier o Incorporation of this tissue extract in the agar at a concentration of 76 Center well--Antiserum H-17 P cancer colon o Me normal stomach o ~\ 0 / 0 \: o '~" Me cancer stomach I <=----./' o 0 En cancer stomach En normal stomach P cancer colon Figure 18. Ouchterlony reaction of antiserum H-17 with extracts of cancerous colonic tissue and normal and cancerous stomach tissue in human serum-agar. 77 Antiserum H-14 A cancer colon A normal colon Antiserum H-17 Figure 19. Ouchterlony reaction of antisera H-14 and B-17 with extracts of normal and cancerous colonic tissues in human serum-agar. Antiserum H-l4 Br cancer colon Br normal colon Antiserum H-17 Figure 20. Ouchterlony reaction of antisera H-14 and H-17 with extracts of normal and cancerous colonic tissues in human serum-agar. 78 15 per cent (Figure 21) prevented the formation of the Vshaped normal tissue bands seen in Figure 19. Varying the concentrations of extracts of normal colonic tissues in the agar revealed that the normal tissue band was stronger with the cancerous than with the normal tissue extracts. For example, in Figure 22 extract of normal colonic tissue incorporated into the agar in a concentration of 10 per cent prevented the formation of the normal tissue band to normal colonic tissue but not to cancerous colonic tissue. The concentration of normal colonic tissue extract necessary for absorption was also dependent upon the arrangement of the reactants. For example, incorporation of normal colonic tissue extract in a 10 per cent concentration was insufficient in preventing the formation of the normal tissue band to normal colon in Figure 23. If the well arrangement in Figure 23 was used, a slightly higher concentration of the absorbing antigen was necessary to produce the absorption effects seen in Figures 21 and 22. The absorption experiments were continued by incorporation of colonic tumor tissue extract into the agar. A concentration of 5 per cent colonic tumor extract in the agar prevented the formation of the cancer-specific bands seen in earlier figures (e.g. Figure 16). mal tissue band remained. The nor- Absorption with 10 per cent colonic tumor tissue removed both the normal tissue and the cancer-specific bands (Figure 24). None of the bands 79 Antiserum H-14 o M cancer colon O~ 0 r.1 normal colon 10 Antiserum H-17 Figure 21. Ouchterlony reaction of antisera H-14 and H-17 with extracts of normal and cancerous colonic tissues in human serum-agar with 15 per cent M normal colon extract incorporated. Antiserum H-14 A cancer colon A normal colon Antiserum H-17 Figure 22. Ouchterlony reaction of antisera H-14 and H-17 with extracts of normal and cancerous colonic tissues in human serum-agar with 10 per cent A normal colon extract incorporated. 80 Center well--Antiserum H-17 Ma cancer colon o ~> o normal colon O? 0 \) 0 v ~ 0 ~;O Ma normal colon ~1' ~v:6:;::/ o cancer colon Figure 23. H cyst fluid Ouchterlony reaction of antiserum B-17 with cyst fluid and extracts of normal and cancerous colonic tissues in human serum-agar with 10 per cent 0 normal colonic extract incorporated. Center well--Antiserum H-17 Ma cancer colon o o normal colon o o cancer colon Figure 24. o 0 45f o /;:'0 1\la normal colon H cyst fluid Ouchterlony reaction of antiserum H-17 with cyst fluid and extracts of normal and cancerous colonic tissues in human serum-agar with 10 per cent 0 cancerous colonic extract incorporated. 81 to colonic extracts were affected by incorporation of 0.1 per cent A or B blood g~oup substance into the agar. It should be noted that absorption of antibody to tissue did not require the use of a tissue-absorbing antigen corresponding to the patient whose tissue extract was being reacted. The formation of a patient's normal tissue band could be prevented by absorption with any normal colonic tissue extract. Likewise, the formation of the cancer-specific band could be prevented by absorption with any cancerous colonic extract. Attention is called also to the reaction of the antiserum \Vi th the 23 and 24)0 homologo~ls cyst fluid antigen (Figures For some unknown reason, tissue extract in- corporated in the agar enhanced resolution of the original diffuse band into many bands. Ninth Immunization Series A very mucinous fluid from a pseudomucinous cystadenoma of the ovary from patient Z (Z cyst fluid) was blended aseptically in a Waring Blender for 1 minute. Before the immunization series was started, a test for toxicity of the cyst fluid was performed by injecting 3 ml of the fluid IV into a rabbit. action. The test animal showed no apparent re- Ten rabbits, labeled Z-28 through Z-37 and weigh- ing 5 to 6 pounds, were injected with the cyst fluid. rabbits were given 14 injections of 1 ml of cyst fluid. The 82 The injections were given IP on a Tuesday/Friday schedule for 7 weeks. jection. Rabbit Z-28 died 1 day after the eighth in- Rabbit Z-29 developed an abdominal abscess fol- lowing the eighth injection. given to this rabbit. ated. Two more injections were It was rested 1 week and exsanguin- Blood samples were taken from the remaining eight rabbits 1 week after the fourteenth injection. A second blood sample was taken a week later. Sera from the first and second bleedings were reacted with extracts of colonic tumor tissues in the usual manner. Antisera from both bleedings of rabbits Z-30, Z-36, and 2-37 failed to react and these collections were discarded. A single band was produced by antisera from rabbits 2-29, Z-32, Z-33, and Z-34. The single band of antiserum 2-33 was stronger and more diffuse than those of the other antisera exhibiting single bands. The antisera from rab- bits Z-31 and 2-35 produced two bands when reacted with extracts of colonic tumor tissue. Based on these results, rabbits 2-31, 2-33, and Z-35 were selected to be exsanguinated 3 days after the second bleeding. Antisera from all three bleedings of rabbit Z-31 produced intense precipitation bands when reacted with colonic tissue extracts. Serum from the first bleeding, however, produced the strongest reactions. The antiserum produced the pattern seen in Figure 25 when reacted in the arrangement of alternating normal and cancerous colonic 83 Center well--Antiserum Z-31 Br cancer colon L normal colon Rr normal colon L cancer colon Pr normal colon Pr normal colon Figure 25. Ouchterlony reaction of antiserum Z-31 with extracts of normal and cancerous colonic tissues in human serum-agar. 84 tissue extracts. Serum from the first bleeding of rabbit Z-33 also produced the best reacting antiserum. The reactivity of the sera from the second and third bleedings was considerably weaker. The cancer-specific band produced by antiserum Z-33 was much stronger than the normal tissue band. The cancer-specific band appeared in 1 to 2 days whereas the normal tissue band required 4 to 6 days to appear. Antiserum Z-33 produced the same reaction pattern seen in Figure 25. The reactivity of ant~serum Z-35 to colonic tissue extracts was greater than that of the above two antisera. Suitable antisera were obtained from all three bleedings. The reaction pattern of antiserum Z-35 (Figure 26), ho\,,ever, differed from that seen in Figure 25. The cancer- specific band appeared to be on or outside the normal tissue band. A faint, third band to extracts of colonic tumor tissues also appeared. However, by dilution of antiserum Z-35 with antiserum Z-3l (equal parts) or by agar absorption with a subabsorptive amount of cancerous colonic tissue (10 per cent), the pattern in Figure 25 could be produced. The third band to colonic tumor tissue could therefore be the continuation of the normal tissue band, which had been interrupted by the cancer-specific band. Thus, it appeared that ~he precipitation complex formed by the unusually strong cancer-specific precipitin 85 Center well--Antiserum Z-35 Br cancer colon L normal colon Br normal colon L cancer colon Pr cancer colon Pr normal colon Figure 26. Ouchterlony reaction of antiserum 2-35 with extracts of normal and cancerous colonic tissues in human serum-agar. 86 altered the mobility of the antibody to normal colonic tissue and subsequently its precipitation site. Absorption experiments using colonic tissue extracts as absorbants were carried out as before. The precipitin to extracts of normal colonic tissues could be removed by absorption with 10 to 15 per cent extract of normal colonic tissue. Cancerous colonic tissue extract in a 10 to 15 per cent concentration absorbed the antibody to cancerous tissue as well as the normal tissue precipitin. Although the cancer-specific band was much stronger than the normal tissue band, the cancer-specific precipitin was absorbed sooner than the normal tissue precipitin. In the preceding immunization series absorption with increasing amounts of colonic tumor tissue extract removed the precipitin to normal tissue sooner than the precipitin to cancerous colonic tissue. \~hile the method of absorption in agar was success- ful and used throughout this research, other methods of absorption of the rabbit antisera were investigated. Two of these methods were dialysis chamber method and beaker method. The dialysis chamber method was developed as described in Methods. The absorbing material was incor- porated into agar supported on a filter membrane. The antiserum to be absorbed was placed in the sample side of the chamber, collected from the collection side, and reacted in the usual manner in 1 per cent agar plates. 87 Incorporation of human serum in the agar membrane was effective in removing the precipitins to serum components. Incorporation of normal colonic tissue extract in a 10 per cent concentration likewise removed the antibody to normal colonic tissue. Incorporation of 10 per cent extract of colonic tumor tissue in the agar membrane did not absorb all antibody to colonic tumor tissue. A beaker method for absorption as described in Methods was also used. Precipitins to serum components were not completely absorbed by agar containing 30 per cent human serum. The extracts of cancerous and normal colonic tissues in the concentrations used were effective in absorbing the precipitins directed to normal tissue. The cancer-specific antibody was not completely removed from the antisera by absorption with the colonic tumor tissue extract at the concentration used. The above two methods could perhaps be effective absorptive techniques if appropriate concentrations of absorbing materials were used. Removal of the cancer- specific precipitin from the antisera required a greater than 10 per cent incorpor["tion of extract of colonic tumor tissue in the agar. However, at these higher concentra- tions the agar medium did not retain the absorbing antigen. Ant~gen migrated from the agar into the antiserum creating a condition of "antigen in excess" in the antiserum. result, absorbed antisera were able to react with As a 88 unabsorbed antisera. This precluded the use of absorbed and unabsorbed antisera in the s:une reaction plate. There- fore, the use of these methods was not pursued. Experimentation with the Z antisera ,,,as continued by preparing a pooled reagent. Sera from the first bleedings of rabbits 2-31 and Z-33 plus the sera from all three bleedings of rabbit 2-35 were pooled. Reacting this anti- serum with colonic tissue extracts revealed the antiserum assumed the characteristics of antiserum Z-35. Z cyst fluid produced many bands when reacted with the pooled Z antiserum. Two of the bands to cyst fluid were "in iden- tity" with the normal tissue and cancer-specific bands (Figure 27). Therefore, the antigens in the Z cyst fluid common to colonic tissues were not destroyed by storage. (It was previously thought that failure of the immunizing cyst fluid to exhibit bands in common with colonic tissue extracts, when reacted with the anti-cyst fluid antisera, was due to antigen deterioration during storage of the cyst fluid.) Z cyst fluid incorporated into the agar at a 10 per cent concentration prevented the formation of all the bands to colonic tissues. A comparison of antiserum H-17 and the pooled Z ant serum is seen in Figure 28. The cancer-specific bands produced by reaction of the two antisera with colonic tumor tissue extracts were Ifin identity". The normal tis- sue bands produced by reactions of extracts of malignant 89 Z cyst fluid o o OG:§-O ~\ cancer colon o normal colon Pooled antiserum Figure 27. Ouchterlony reaction of pooled antiserum from rabbits Z-31, Z-33, and Z-35 with cyst fluid and extracts of normal and cancerous colonic tissue in human serum-agar. Ma cancer colon Pooled antiserum Antiserum H-17 Ma normal colon Figure 28. Ouchterlony reaction of pooled antiserum from rabbits Z-31, Z-33, and Z-35 and antiserum B-17 with extracts of normal and cancerous colonic tissue in human serum-agar. 90 and normal colonic tissues with the pooled antiserum were also "in identity". The cancer-specific band of antiserum H-17 located closest to the antiserum well interrupted the normal tissue bands common to cancerous and normal colonic tissues. Tenth Immunization Series Mucinous cyst fluid from patient 8 (8 cyst fluid) was collected and aseptically blended with an equal volume of sterile saline. The fluid was stored at 4 to 6 C for 48 hours to allow the foam to dissipate. The fluid was tested for toxicity by injecting 3 ml IV into a rabbit. No reaction occurred. Fourteen rabbits, numbered 8-38 through S-51 and weighing 4 to 5.5 pounds, received 14 IP injections of 1 ml on a l\londay/Thursday schedule. Thirty ml of blood were collected from each animal 1 week after the fourteenth injection. All antisera from this bleeding produced normal tissue bands when reacted with malignant and normal colonic tissue extracts. Six of the fourteen antisera appeared to produce cancer-specific bands when reacted with extracts of malignant colonic tissue. The bands, however, were too close to the normal tissue bands for easy distinction. Also, the precipitation complexes seemed to dissociate readily and moved in and out of each other. It was concluded that antiserum to S cyst fluid did not 91 lend itself well to aSEay by the testing methods used in this research. Eleventh Immunization Series Thirteen rabbits, labeled L-I through L-13, were injected with a serous and only slightly mucinous cyst fluid from patient L (L cyst fluid). The test for tox- icity of the fluid by injecting 3 ml IV into a test animal was negative. Fourteen I ml injections were given IP on a Tuesday/Friday schedule. A trial bleeding from the ear was performed I week after the fourteenth injection. The sera were screened by reaction with the L cyst fluid. Rabbits failing to produce suitable reacting anti- sera were discarded. Rabbit L-9 developed an ear infection midway through the immunization series and its antiserum was not tested. Antisera from rabbits L-3, L-4, L-5, and L-IO produced a single band when reacted with the cyst fluid. Antiserum L-7 developed three strong, precipita- tion bands while antiserum L-14 produced a heavy, diffuse band which appeared to be composed of two band;:;. These six rabbits were exsanguinated I week after the trial bleeding. Antisera L-7 and L-14 reacted strongly with other cyst fluids. The two antisera did not react as strongly with colonic tissue extracts as they did with cyst fluids. 92 Antiserum L-7 produced a fairly strong normal tissue band with colonic tissue extracts but only a suggestion of a cancer-specific band. Antiserum L-14 also produced a normal tissue band with extracts of colonic tissues. A very faint and diffuse cancer-specific band was occasionally produced with extracts of colonic tumors. The sera were considered unsatisfactory and were discarded. DISCUSSION The most significant result of the described research indicates that an antiserum which produces a specific reaction with extracts of human adenocarcinoma of the colon can be prepared in rabbits. This antibody response in rabbits can be evoked by injection of mucinous fluid from benign, human cystadenomas of the ovary. It would appear that a tumor-specific substance found in extracts of colonic tumor tissues is also contained in some ovarian cyst fluids. The presence of the cancer- specific antibody in rabbit ·antisera prepared against cyst fluid can be demonstrated by reaction in agar-gel of the sera with colonic tumor extracts. The antisera to U cyst fluid in the first series and W cyst fluid in the third and fourth series produced a single precipitation band when reacted with extracts of cancerous colonic tissues in human serum-agar. These antisera did not react with ex- tracts of normal colonic tissues. The antisera to R, H, Z, and S cyst fluids in the sixth, eighth, ninth, and tenth series, respectively, did produce a precipitation band to extracts of normal colonic tissues. Two bands were exhibited by these antisera when reacted with colonic tumor extracts. One of these bands was identical to the band produced to normal colonic tissues. In preliminary experiments ovarian cyst fluid was reacted with normal human serum and sera from patients 94 with cancer. fluid. These sera failed to react with the cyst Thus, it appeared that the colonic tumor antigen (common to ovarian cyst fluid) did not evoke an immunological response with the formation of detectable antibody in patients with cancer of the colon. This then would appear to be a case wherein the immunological defense mechanisms were not brought into play to curb the neoplastic disease process. This tumor-specific substance, however, was antigenic and did evoke an antibody response in rabbits. \ihile differences \Iere observed in antibody response in the different immunization series, it did not appear that immunization procedures and routes were the critical determinants for good antibody production. Biological variation of the individual rabbits used for immunization seemed to be the most important factor in the variation in reactivity of the antisera. Not all rabbits within the same series and under the same testing conditions produced sera possessing the tumor-specific antibody. Another important determinant in antibody response was the nature and condition of the cyst fluid used for immunization. Some cyst fluids appeared to be better immunizing antigens than others. Generally, the more mucinous fluids were the better antigens. \vhile cyst fluids contain relatively little protein, serous ovarian fluids contain more protein than mucinous ovarian fluids 95 (Kiekhofer et al., 1962). It is possible that the higher concentration of mucopolysaccharides in mucinous fluids may have contributed to the greater effectiveness of these fluids in inducing the antibody response to the tumorspecific substance. The data also suggest that storage reduced the antigenicity of some of the cyst fluids. For example, in the sixth immunization series the antisera from all five rabbits produced a normal tissue band when reacted with colonic tissue extracts. In addition, one of these anti- sera produced a cancer-specific band. The immunization procedures were repeated 6 weeks later using the same cyst fluid (seventh immunization series). Of the antisera col- lected, none produced a cancer-specific band. Three pro- duced a band to normal colonic tissue--one band, however, was very faint. tissue extracts. One antiserum did not react with colonic The inability of the reacting antisera to produce reactions of identity with the cyst fluid and colonic tissue extracts was also interpreted to mean that storage (of the test antigen) resulted in deterioration of the antigenic components in cyst fluid. Storage, however, did not seem to have an adverse effect on the Z cyst fluid. The antisera to the Z cyst fluid reacted strongly with the cyst fluid antigen and produced reactions of identity with those to colonic carcinoma extracts. In the first immunization series it was also observed 96 that boiling the cyst fluid test antigen for 15 minutes reduced the intensity of the precipitation band formed by reaction of the cyst fluid with the prepared antisera. However, the formation of the band was not completely prevented by the heat treatment. Therefore, while the anti- gen in cyst fluid could be considered thermostable, some of its antibody-complexing determinants were not. Antibody response was greatest in sera collected within 1 week of the last antigen injection. The antibody did not persist and was rapidly lost \vi th the discontinuation of immunization. Booster injections did not appear to be of value in maintaining antibody titers. As investigators prepared antisera to normal and malignant tissues, it soon became apparent that these antisera necessarily contained antibodies directed to serum proteins (Mann and Welker, 1940). Even with fine grinding or homogenizing and thorough washing, these tissue antigen preparations still contained sufficient blood proteins to elicit antibodies in animals. The production of antibodies to normal serum components lvas not averted by the use of cyst fluid as the immunizing antigen. However, absorption of the antibodies directed to human serum was simply but effectively accomplished by incorporation of pooled human serum (at a concentration of 30 per cent) into the agar (McNeil et al., 1969). No advantage over this method was found with the use of the 97 dialysis chamber or beaker methods of absorption other than the absence of "inhibition halos" around the antisera wells. These latter methods were laborious and not as effective in absorbing as the method using human serumagar. Moreover, absorbed antisera reacted with unabsorbed antisera (if placed in adjacent reactant wells) due to the presence of excess absorbing antigen in the absorbed sera. Five of the eight cyst fluids studied stimulated antibody formation to extracts of normal colonic tissues. In all cases, antisera possessing the normal tissue precipitin also produced the normal tissue band when reacted with cancerous colonic tissues. Nairn et ale (1962a) described a colonic antigen detected by rabbit antisera against microsomal fractions of cells of normal human colon. The antigen was present in small amounts in cells of gastric mucosa, but was absent from gastric and colonic carcinoma tissues. The antigen was concentrated in the luminal cytoplasm of colon cells and appeared to be acidic mucopolysaccharide. The antigenic activity of the sub- stance was evidently associated with a differentiated state of the intestinal epithelial cell and was lost on malignant change. in human colon. Lord (1962) described a similar antigen Several other reports of colonic antigens have evolved from stUdies of ulcerative colitis and were reviewed by Dumonde (1966). All these antigens appeared to be polysaccharide-containing substances. Broberger 98 and Perlmann (1963) detected a colonic antigen in 20-weekold human fetal colon, as well as in colonic carcinoma. They indicated that the alimentary antigens were related to epithelial mucins. The antigen of normal colonic tissues reported in this thesis was not organ-specific. It was found in tissues other than colon. Gold and Freedman (1965a) demonstrated cancerspecific antigens in colonic carcinomas by hemagglutination using erythrocytes coated with tumor extracts, by fluorescent antibody methods, by passive cutaneous anaphylaxis, and by immunodiffusion methods. In their studies antisera against extracts of pooled human colonic carcinomas were prepared in rabbits. The antisera were rendered tumor-specific by either absorption of the antisera or induction of immunological tolerance to normal colonic tissue. Absorption of sera was performed by repeatedly absorbing the sera with pooled human plasma and extracts of normal colonic tissues. Isoantigenic differences were controlled by the use of normal and tumor tissues from the same patient. Under these condi- tions, it was shown that at least two distinct tumor antigens in tumor extracts could be detected by immunodiffusion and hemagglutination methods. Furthermore, immunofluorescence studies demonstrated intense staining of tumor tissue with antisera produced in rabbits tolerant to normal colonic tissue. These antisera produced only 99 slight reactions with adjacent normal tissue. The findings of Gold and Freedman strongly suggested that these colonic carcinoma antigens were tumor-specific and not merely present in higher concentrations in cancerous tissue than in normal tissue. Their evidence indicated that tumor-specific antigens were identical in all human colonic tumors. Kronman (cited in Klein, 1968) also prepared rabbit antisera against normal and cancerous colonic tissue. Reaction of the antisera using the Ouchterlony technique indicated that the rabbits produced many antibodies to colonic tissues. All antibodies, in the antiserum to normal colon, were absorbed with either normal or cancerous tissue. The antiserum to colonic carcinoma tissue produced one precipitation band with colonic carcinoma tissue after absorption with normal colonic t sue. All antibodies to colonic tissue were absorbed from both antisera by use of cancerous colonic tissue. This absorption was accomplished with any of the cancerous colonic tissues irrespective of which patientfs colonic tissue was used for immunization. The findings presented in this thesis also revealed that colonic tumors were antigenically different from normal colonic tissues. Possible isoantigenic differences were controlled by use of normal and malignant tissues from the same patient. However, in this research cyst 100 fluid rather than extracts of colonic tumors was used as the immunizing antigen. Only one distinct tumor-specific band was detected in this research whereas a minimum of two cancer-specific precipitins was found by Gold and Freedman (1965a). The use of an immunizing antigen heterologous to the test antigen in the research presented in this thesis could account for the different number of precipitins. The antisera of Gold and Freedman (1965a) contained numerous antibodies to normal tissue. The anti- sera against cyst fluid contained no more than one. How- ever, antisera from both of these studies required absorption by human sera. All cancerous colonic tissues ex- hibited the cancer-specific band when reacted with the antisera against cyst fluid. Absorption of the tumor- specific antibody from these antisera was effected by colonic tumor tissue from any patient. Therefore, the re- sults of this research were in complete agreement with the conclusion drawn by Gold and Freedman (1965a) and Kronman (cited in Klein, 1968) that the same tumorspecific antigen is contained in all human colonic tumors. Later findings (Gold and Freedman, 1965b) indicated that these colonic tumor-specific antigens \vere also present in all adenocarcinomas of the entodermally-derived epithelium of the gastrointestinal tract. These antigens were reported to be absent from all other adult normal, diseased, or cancerous human tissueo The antisera 101 prepared against cyst fluid in this research did not detect the tumor-specific antigen in any adenocarcinoma tissues tested other than those of the colon. However, Gold (cited in Klein, 1968) indicated that the concentration of tumor-specific substances varied according to the site of the tumor in the gastrointestinal tract. Low concentrations were found in tumors situated in the upper tract. Antigen concentration was quite low at the level of the stomach and was highest in tumors of the bowel and rectum. It was noted throughout this research that the antisera against cyst fluid displayed a high degree of reactivity with extracts of stomach tissues. Specific reference is made to the R cyst fluid in the sixth immunization series. Although there was a high degree of re- activity of the antisera with gastric tissues, no antigen common to the colonic tumor antigen was discernible. Gold (cited in Klein, 1968) reported that detection of the tumor-specific antigen in gastric tumors by the Ouchterlony reaction required the use of concentrated extracts of gastric cancer tissue. None of the tissue extracts tested in the research reported in this thesis were concentrated. Therefore, the results of this research did not necessarily contradict the evidence for the presence of tumor-specific antigens in other tumors of the digestive tract. The cancer-specific antigen found in this research did not appear to be related to the lipid or glycolipid 102 gastrointestinal antigens described earlier by numerous workers. Such antigens were invariably associated with blood group substances. Despite the similarity between blood group substances and mucins, blood group substances were not responsible for the normal tissue or colonic tumor-specific precipitin. Absorption with blood group substances did not affect the cancer-specific bands produced by the antisera to cyst fluid. Likewise, there was no correlation of the blood types of patients whose tissues were used and of patients whose cyst fluids were used. Preliminary characterization of the colonic carcinoma antigens by Gold and Freedman (1965a) suggested that the important antigenic determinants of the tumorspecific substances were polysaccharide in nature. The production of the same antibodies (if they were the same) in rabbits by injection of mucinous (polysaccharide-rich and relatively thermostable) cyst fluid would seem to support their hypothesis. Fluorescent antibody studies indicated that the carbohydrate determinants for tumor specificity were located at the cell surface (Gold, Gold, and Freedman, 1968). Avdeyev et al. (1967) have apparently detected a similar (or identical) colonic antigen with rabbit antisera prepared against saline extracts of rectum and sigmoid carcinomas. The antisera absorbed with normal 103 colonic tissue reacted with extracts of malignant colonic tissues. The antisera also reacted with some cancers of the stomach and esophagus. This antigen, however, was not detected in tissues of human embryos 6 to 10 weeks old. This was in opposition to the findings of Gold and Freedman (1965a). They found that the tumor-specific antigens of the digestive system were present in differentiating gut, liver, and pancreas of the human fetus at 2 to 6 months of gestation. It may be that the 6 to 10 week old embryos did not yet contain enough of the substance to be detected. Avdeyev et ale (1967) also re- vealed that the tumor-specific antigen was detected in 17 out of 25 patients with polyps of the colon. It is pos- sible, therefore, that this antigen could be found in colonic tissues before the appearance of morphological symptoms of malignancy. Characterization of this antigen indicated that its immunological activity was not destroyed by proteinases or by boiling. The antigenic material did not stain with stains specific for DNA or lipid. stained, however, by the PAS reagent. It was It can be concluded, therefore, that the colonic tumor antigen was polysaccharide in nature. The preponderance of PAS staining material in adenocarcinoma tissues as reported by other workers was also observed in adenocarcinoma tissues used in this research. Histologic examination of colonic tissues revealed that 104 mucosal cells produced a mucinous material which assumed a finely punctate PAS staining in the cellular cytoplasm. In contrast, cells making up carcinoma of the colon produced coarser PAS staining material which was apically located. This mucoid material accumulated noticeably in the lumens of the glandular epithelial tissue. It would seem likely that these mucinous materials could diffuse into the blood stream and account in some part for elevated levels of serum mucoproteins and glycoproteins reported by numerous investigators. This material could also account for the material in cancer patients (described by Treacy et al., 1967) which mimics blood group substance. It would also seem possible that mucinous substances from tumors of the colon and ovary could be of similar etiology as \vell as composi tion. Results of this research support the hypothesis that mucinous secretions of cancerous colonic tissue and ovarian cystadenoma tissue possess a common antigenic component. SUNl'-1ARY Study of the antigenicity of human mucin-producing tumors has revealed an antigenic relationship between mucinous ovarian cyst fluid and colonic adenocarcinoma tissue. Immunization of rabbits with mucinous fluid from benign, human cystadenoma of the ovary produced an antiserum which reacted specifically with extracts of human colonic cancer tissue, as determined by the Ouchterlony double diffusion reaction. The antiserum did not produce the characteristic precipitation band when reacted with other adenocarcinoma extracts or with extracts of normal colonic mucosa from the same patient. 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