American Journal ofPathology, Vol. 137, No. 5, November 1990 Copyright © American Association ofPathologists
A,B Blood Group Antigens in Tissues of AB Heterozygotes Emphasis on Normal and Neoplastic Urothelium
Catherine Limas From the Department of Pathology, Department of Veterans Affairs Medical Center and the University of Minnesota School of Medicine, Minneapolis, Minnesota
The tissue distribution of the A and B blood group antigens was studied in 41 individuals with the heterozygous AB red blood cell (RBC) phenotype. A total of 134 biopsies from a variety of normal tissues (94from urothelium and 40from other tissues) were examined. In addition, changes in the expression of these antigens associated with neoplastic transformation were evaluated in 70 biopsies from transitional cell carcinomas of 19 AB heterozygous patients. There was heterogeneity in the distribution of tissue A and B antigens, depending on the cell type, as well as among cells of the same type. Ninety-one percent of AB heterozygotes expressed both A and B antigens in normal epithelial cells, with a mosaic distribution clearly apparent in 50% of these individuals. In 219% of these subjects, the A antigen was undetectable in the vascular endothelial cells in all biopsies from several organs. In most (79%) transitional cell neoplasms, only one of the two antigens was consistently expressed The results of this study may have implications for the clonal or specific gene deletion theories of neoplasia. They also demonstrate the existence of a subgroup ofAB individuals in whom the A antigen is absent specifically from the vascular endothelium. (AmJPathol 1990; 137:1157-1162)
Recent observations suggest that the expression of the blood group (BG) antigens A, B, and H in various tissues depends on a complex interaction between genetic background, embryologic origin, and cell differentiation.1`4 In addition, the study of these antigens has become relevant to cancer research because of the significance of cellsurface molecules in intercellular communication.5 7 This report focuses on the distribution of the A and B antigens in normal and neoplastic tissues from AB het-
erozygous individuals. Emphasis is placed on the expression of these antigens in the normal, hyperplastic, and neoplastic epithelium of the lower urinary tract. We considered the possibility that the pattern of expression of the A and B antigens in neoplastic growth from AB heterozygotes may be indicative of clonality or tumor cell heterogeneity. In addition, our observations in normal tissues document the existence of antigenic heterogeneity in various cell types, which suggests the presence of phenotypic subpopulations of AB heterozygotes.
Materials and Methods The tissues were derived from 41 patients (Table 1) whose red blood cells (RBCs) were agglutinable with both antiA and anti-B sera by the standard test tube method. The RBCs of 20 patients were also tested with the anti-A1 lectin of dolichos biflorus and with anti-Lea and -Leb sera. In eight of these patients, the biopsies contained only morphologically normal urothelium; in three, only neoplastic transitional epithelium; and in 16, both normal and neoplastic urothelial tissue. In 14 patients, the biopsies derived from nonurothelial tissues. In total, there were 94 biopsies from normal urothelium and 70 from transitional cell neoplasms obtained over a period of up to 11 years. In addition, 40 biopsies from various organs (prostate, 12; intestine, 9; oropharynx, 4; skin, 10; other, 5) were similarly studied. In all cases, sections from routinely processed tissue (fixed in 10% neutral buffered formalin and embedded in paraffin) were stained with hematoxylin and eosin for morphologic evaluation. Also, in all cases, sections from routinely processed tissues were used for immunohistochemical localization of the A and B antigens. In addition, 30 of the patients had fresh frozen tissue sections studied by the same methodology, as well as by the red cell adherence (RCA) test. Supported in part by a grant (CA 33239) from the National Cancer Institute, National Institutes of Health, Bethesda, Maryland. Accepted for publication June 29, 1990. Address reprint requests to Catherine Limas, MD, Department of Pathology, Department of Veterans Affairs Medical Center, One Veterans Dr., Minneapolis, MN 55417.
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Table 1. Material Examined Tissue examined
Number of patients
Normal urothelium only Transitional cell neoplasms only Normal and neoplastic urothelium Other normal tissues
8 3 16 14
Total
41
Monoclonal antibodies against synthetic oligosaccharides with A or B specificity were purchased from DAKO Co. (Santa Barbara, CA); biotinylated anti-mouse gamma M immunoglobulin (IgM), Ulex europeus lectin 1, dolichos biflorus lectin, and avidin-peroxidase reagent (Vectastain) from Vector Lab. Inc. (Burlingame, CA); polyclonal human anti-A, anti-B and anti-Lea, -Leb sera from Cooper Biomedical, Inc. (West Chester, PA). All other reagents were obtained from Sigma Chemical Co. (St. Louis, MO). The methodology for detecting blood group antigens (Bg-Ag) in tissue sections by immunohistochemistry with the biotin-avidin-peroxidase reaction and by the RCA test has been described in detail in our previous publications.89 Each biopsy was tested at least in duplicate for each antigen. Known A-positive B-positive controls were examined in parallel with the test tissues. The monoclonal anti-A antibody used for immunohistochemistry agglutinated both A1 and A2 RBCs up to a titer of 320, with somewhat weaker reactions for the A2 cells.
Results Normal Tissues The normal urothelium in 22 of 24 patients (91%) expressed both antigens, albeit to a variable extent. The Apositive areas were more extensive than B in 18 patients and about equal in four. Two patients had only A detectable in their normal urothelium. A distinctive mosaic distribution was demonstrable in 50% of the biopsies (Figure 1). We attempted to demonstrate more dramatically the mosaic expression of the two antigens by reacting the same sections sequentially with each of the monoclonal antibodies and developing the first (anti-B) reaction with alkaline phosphatase-avidin and the appropriate substrate (red) and the second (anti-A) with peroxidase-avidin and the corresponding substrate (brown). An example of this double staining is shown in Figure 2. The B antigen was detectable on endothelial cells in all tissues examined from all 41 patients. Apparently absent or extremely weak focal reactions with the anti-A were observed in the endothelium of all tissues from nine patients (21%). This apparent absence of A determinants
from the endothelium was consistently demonstrated in multiple biopsies from the same individual over several years and in biopsies from several organs, eg, skin, gastrointestinal, and genitourinary tract, which were obtained and tested at different times (Figure 3). To exclude the possibility that this phenomenon was induced by an adverse effect of the tissue processing on the endothelial blood group substances, fresh frozen sections also were examined. It was noted that endothelial reactivity for A in these nine patients was greatly diminished or absent, even in the fresh frozen, unfixed sections. Four of these patients had their RBCs tested with the antiA1 lectin of dolichos biflorus and all four gave negative reactions. The Lewis phenotype of the RBCs did not influence the endothelial reactions with anti-A, which indirectly implies that the secretor status of the individual had no bearing on this phenomenon. The reactions of the endothelium did not always correlate with those of the epithelial cells present in the same biopsy. As illustrated in Figure 4, the epithelium could be A-positive and B-negative, while the endothelium was Anegative and B-positive. This pattern was consistent in all biopsies obtained from various organs of the same individual through the years. The absence of A antigen from endothelial cells cannot be attributed to a peculiarity of the monoclonal antibody because similar results were obtained with the RCA test in which polyclonal anti-A serum was used. Also, the contrasting reactions between endothelium and epithelium (Figure 4) cannot be explained on the basis of the A2B phenotype. The reactions of endothelial cells with the Ulex europeus lectin were positive or weakly positive and did not correlate in any way with either the anti-A or anti-B reactions.
Urothelial Neoplasms The study of the 70 biopsies from the transitional cell neoplasms of 19 patients yielded the following results: In the neoplasms of 14 patients, only one of the two antigens was detectable, A in eight and B in six. One patient with an inverted transitional cell papilloma had both A and B antigens expressed in the tumor. The transitional cell carcinomas of three patients were negative for both A and B in all recurrences during a follow-up period of up to 5 years. A change in the detectable antigen from A to B was noted in the biopsies of the tumors from only one patient. This patient had at least two neoplasms: a grade 11 noninvasive transitional cell carcinoma involving the right wall of the bladder and a grade III carcinoma obstructing the left ureteral ostium. The first was A-positive and the latter B-positive. The non-neoplastic epithelium of this patient showed weak, patchy distribution of both antigens.
A,B Antigens in AB Heterozygotes 1159 AJP November 1990, Vol. 137, No. 5
Figure 1. Reactions of normal urothelium of an AB heterozygote for the BG antigens A(a) and B(b). Both antigens show apatchy distribution complementary to each other. This distribution results in an overall mosaic pattern. The vascular endothelium is bothA- and B-positive in this case. Routinely processed biopsjy stained with monoclonal antibodies as described in Methods (X40).
Figure 2. Normal urothelium from an AB heterozygote shows a mosaic at A-positive (brown) and B-positive (red) patches when stained for both A anid B antigens on1 the same section (for details refer to Methods) (X400).
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fj AE:.i_EK ..b Figure 3. Biopsy from the urinary bladder of a BGAB patient stained for A (a) and B (b). The epithelium is positive for both antigens but the endothelial cells are negative for A (X200).
All but one of the 14 patients with biopsies taken contemporaneously from both normal and neoplastic urothelium had both antigens detectable in the tumor-free mucosa, usually in a patchy distribution, regardless of the pattern of tumor cell reactivity. In one patient, only the A antigen was detectable in both normal and tumor cells in four sequential biopsies taken during a 7-year period. The reactions of the endothelium of the neovessels followed the same pattern as the patient's normal tissue vessels. Comparisons of the reactions on paraffin-processed tissue with those obtained on fresh frozen sections with the RCA method showed, in general, good correlation. There were quantitative differences between the results of the two procedures in the tumors of seven cases that had good positive reactions on the frozen and weak in the paraffinprocessed sections. In only one case was there a complete discrepancy, the tumor being A- and B-positive in the unfixed, frozen tissue and negative for both antigens in the paraffinized sections, while the blood vessels re-
Figure 4. Contrasting reactions of epithelial and endothelial cells for A and B antigens in sections from the pylorus of an AB heterozygote. a: A section incubated with monoclonal antiA shows positive epithelium and negative endothlium (X200). b: A section incubated with monoclonal anti-B s -ws negative epithelium and positive endothelium (X200).
mained positive. The RBC phenotype of this patient was A B, Lea-b+
Discussion The last several years have witnessed a renewed interest in the biochemistry and biology of the blood group (BG)associated antigens.3 Although the physiologic function of these antigens remains speculative, an increasing body of evidence underscores their role in the interactions of the cell with its milieu and suggests their involvement in a variety of disease processes.7 Thus knowledge of the biology of the BG-associated antigens A, B, and H has become relevant to several unresolved issues such as transplant rejection,10 malignant potential of neoplasms,6 and susceptibility to infections.1" We considered the possibility that the patterns of A and B antigen expression in the normal and neoplastic
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tissues of AB heterozygotes provide valuable insights into the expression of the genes of the BG biosynthetic enzymes and may be relevant to the tumor cell clonality or heterogeneity. The epithelium lining the human lower urinary tract is well suited to our study because it is morphologically and functionally rather uniform throughout its extent, so that the interpretation of the findings is not subject to sampling variability. Furthermore patients with urinary bladder lesions are often followed with endoscopy and biopsies from multiple sites, including neoplastic and morphologically uninvolved urothelium. To our knowledge, this is the first study designed to assess the detectability of A and B antigens in AB heterozygotes in multiple, sequential biopsies obtained over an extended period (up to 11 years) from both normal and neoplastic epithelium. The study was made more comprehensive by the availability of biopsies from several organs of patients with multiple medical problems, eg, prostate enlargement, skin lesions, gastrointestinal symptoms, etc. The heterogeneous distribution of the A and B antigens in normal tissues implies that the activities of the A- and B-specific glycosyltransferases vary in cells with similar histogenesis and differentiation. This variability sometimes results in an apparent mosaic pattern, as if small, randomly distributed cell colonies were derived from distinct clones of stem cells that express only one of the two antigens. Whether this is simply a quantitative phenomenon or involves specific gene expression or deletion can only be resolved by in situ hybridization with the DNA and RNA sequences corresponding to the relevant enzymes. The heterogeneous expression of the A and B antigens is not limited to patients with urothelial neoplasia, but is present in other organs of heterozygous individuals. For example, in the course of this study, we observed that other stratified as well as glandular epithelial structures displayed nonuniform reactivity. It has also been shown that the reactions of AB red blood cells with various antiA and anti-B sera are nonuniform, and one explanation offered invokes a competition of the A- and B-specific enzymes for substrate.12 The contrasting reactions in endothelial and epithelial cells in 21% of our cases highlight the cell-type-specific variability in the expression of each (A and B) antigen and suggest that the vascular endothelium of AB heterozygotes may have a different phenotype than the red blood cells. The diversity of the reactions may be strictly quantitative, but qualitative differences in the blood group substances in the endothelial cells of some individuals need also be considered. We tried to minimize the influence of methodologic factors, eg, inability to detect unusual variants of the A substance with monoclonal antibodies, interference by solvents and fixatives, by using various natural polyclonal antibodies and by modifying the procedure to avoid denaturation and extraction artifacts. We achieved
consistently reproducible results, even in biopsies obtained from various organs over long periods and tested independently by different methods. Therefore we are confident that the apparent absence of A determinants from the vascular endothelium is a stable, probably genetic, characteristic of some individuals with the AB RBC phenotype. This characteristic does not correlate with the Lewis phenotype and, therefore, is not likely to reflect the patient's secretor status. It did correlate with the negative agglutination reactions of the patient's RBCs with the Dolichos biflorus anti-A1 lectin. We have previously reported that the detectability of A antigen in the tissues of patients with A2 RBC phenotype is reduced compared with the A1 phenotype.13 However the marked discrepancy between endothelial and epithelial cell reactions noted in this series of AB heterozygotes is a new observation that cannot be directly explained on the basis of an A2B phenotype. This observation deserves detailed evaluation because of its implications for organ transplantation. For example, it may be possible to successfully transplant organs from BG AB donors to BG B recipients, provided that the absence of A antigens from the donor endothelial cells is verified, eg, in punch biopsies of skin. In this respect, it is interesting that kidneys from A2 donors have been successfully transplanted to BG 0 and B recipients.14 This study demonstrates that, as a rule, transitional cell neoplasms in BG AB heterozygotes express only one of the two antigens. Although this is not conclusive evidence, it does lend support to the theory of 'clonal' derivation of tumor cells. The frequency with which each of the two antigens was expressed in tumors was quite comparable, so that the presence or absence of either antigen seems to play no specific role in the pathogenesis of these tumors. Within a given tumor, the degree of reactivity for the expressed antigen may vary so that individual tumor cells show a spectrum of staining intensity from strongly positive to negative. This intratumor variability was more pronounced in paraffin-processed tissues. A preponderance of positive reactions at the basal and deep layers is frequently seen, but is due to the methodologic factors previously reported.13 We were impressed by the consistency of the antigenic makeup in recurrent tumors. It was not possible to trace accurately the anatomic origin of each recurrent lesion, so that, in several of our cases, we cannot firmly distinguish true recurrence of residual tumor from multicentricity. In the one discrepant case, the biopsies were obtained from clearly different sites and the tumors showed different morphology. Although an infrequent occurrence, this case suggests that the process of carcinogenesis can affect different clones of susceptible cells. Quantitative differences in the reactivity of cells within the same neoplasms are probably related to intratumor
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heterogeneity, which evolves after the initial stages of the transformation of a stem cell clone.15'16 There was only one phenotypically AB tumor that was morphologically distinct from all other cases (inverted papilloma) and did not recur. It is possible that this morphologic entity has a pathobiology distinct from the more common papillary transitional cell carcinomas. All the inverted papillomas we have studied so far are positive for the expected BG antigen and have shown no sign of progression. The pattern of A and B antigen expression in heterozygous individuals could serve as a means for distinguishing neoplastic from benign proliferative lesions. In this respect, it is significant that, in a given individual, both A and B antigens are usually detectable in non-neoplastic urothelium, albeit in a patchy distribution, while only one antigen A or B is demonstrable in truly neoplastic lesions. In summary, we have demonstrated that the distribution of A and B antigens in AB heterozygotes is heterogeneous and suggested that this phenomenon has relevance to organ transplantation and to the clonal derivation of urothelial neoplasms. We believe that this subject deserves detailed study in other normal tissues and their neoplastic processes.
References
5. Feizi T: Carbohydrate antigens in human cancer. Cancer Surv 1985, 4:246-269 6. Hakomori S: Aberrant glycosylation in cancer cell membranes as focused on glycolipids: Overview and perspectives. Cancer Res 1985, 45:2405-2414 7. Feizi T, Childs RA: Carbohydrate structures of glycoproteins and glycolipids as differentiation antigens, tumour-associated antigens and components of receptor systems. Trends Biochem Sci 1985,10:24-29 8. Limas C: Detection of urothelial Lewis antigens with monoclonal antibodies. Am J Pathol 1986, 125:515-523 9. Limas C, Lange P, Fraley EE, Vessella RL: ABH antigens in transitional cell tumors of the urinary bladder: Correlation with the clinical course. Cancer 1979, 44:2099-2107 10. Cook DJ, Graver B, Terasaki PI: ABO Incompatibility in cadaver donor kidney allografts. Transplant Proc 1987, 19:
4549-4552 11. Sheinfeld J, Schaeffer AJ, Cordon-Cardo C, Rogatko A, Fair WR: Association of the Lewis blood-group phenotype with recurrent urinary tract infections in women. N Engl J Med
1989, 320:773-777 12. Salmon C, Cartron JP, Rouger P: The Human Blood Groups. Chicago, Year Book Medical Publishers, 1984, p 114 13. Limas C, Lange P: ABH antigen detectability in normal and neoplastic urothelium. Influence of methodological factors. Cancer 1982, 49:2476-2484 14. Welsh KI, vanDam M, Koffman CG, Bewick ME, Rudge CJ, Taube DH, Clark AGB: Transplantation of Blood group A2 kidneys into 0 and B recipients: The effect of pretransplant anti-A titers on graft survival. Transplant Proc 1987,19:45654567 15. Nowell PC: The clonal evolution of tumor cell populations. Science 1976, 194:23-28 16. Nowell PC: Mechanisms of tumor progression. Cancer Res 1986, 46:2203-2207
1. Watkins WM: Glycosyltransferases: Early history, development and future prospects. Carbohydr Res 1986, 149:1-12 2. Oriol R, LePendue J, Mollicone R: Genetics of ABO, H, Lewis, X and related antigens. Vox Sang 1986, 51:161-171 3. Watkins WM: Biochemical genetics of blood group antigens: retrospect and prospect. Biochem Soc Trans 1987, 15:620-
Acknowledgment
624 4. Oriol R: ABH and related antigens. Biochem Soc Trans 1987, 15:596-599
The author thanks Dr. P. Lange of the Department of Urologic Surgery for his cooperation.
A,B Blood Group Antigens in Tissues of AB Heterozygotes Emphasis on Normal and Neoplastic Urothelium
Catherine Limas From the Department of Pathology, Department of Veterans Affairs Medical Center and the University of Minnesota School of Medicine, Minneapolis, Minnesota
The tissue distribution of the A and B blood group antigens was studied in 41 individuals with the heterozygous AB red blood cell (RBC) phenotype. A total of 134 biopsies from a variety of normal tissues (94from urothelium and 40from other tissues) were examined. In addition, changes in the expression of these antigens associated with neoplastic transformation were evaluated in 70 biopsies from transitional cell carcinomas of 19 AB heterozygous patients. There was heterogeneity in the distribution of tissue A and B antigens, depending on the cell type, as well as among cells of the same type. Ninety-one percent of AB heterozygotes expressed both A and B antigens in normal epithelial cells, with a mosaic distribution clearly apparent in 50% of these individuals. In 219% of these subjects, the A antigen was undetectable in the vascular endothelial cells in all biopsies from several organs. In most (79%) transitional cell neoplasms, only one of the two antigens was consistently expressed The results of this study may have implications for the clonal or specific gene deletion theories of neoplasia. They also demonstrate the existence of a subgroup ofAB individuals in whom the A antigen is absent specifically from the vascular endothelium. (AmJPathol 1990; 137:1157-1162)
Recent observations suggest that the expression of the blood group (BG) antigens A, B, and H in various tissues depends on a complex interaction between genetic background, embryologic origin, and cell differentiation.1`4 In addition, the study of these antigens has become relevant to cancer research because of the significance of cellsurface molecules in intercellular communication.5 7 This report focuses on the distribution of the A and B antigens in normal and neoplastic tissues from AB het-
erozygous individuals. Emphasis is placed on the expression of these antigens in the normal, hyperplastic, and neoplastic epithelium of the lower urinary tract. We considered the possibility that the pattern of expression of the A and B antigens in neoplastic growth from AB heterozygotes may be indicative of clonality or tumor cell heterogeneity. In addition, our observations in normal tissues document the existence of antigenic heterogeneity in various cell types, which suggests the presence of phenotypic subpopulations of AB heterozygotes.
Materials and Methods The tissues were derived from 41 patients (Table 1) whose red blood cells (RBCs) were agglutinable with both antiA and anti-B sera by the standard test tube method. The RBCs of 20 patients were also tested with the anti-A1 lectin of dolichos biflorus and with anti-Lea and -Leb sera. In eight of these patients, the biopsies contained only morphologically normal urothelium; in three, only neoplastic transitional epithelium; and in 16, both normal and neoplastic urothelial tissue. In 14 patients, the biopsies derived from nonurothelial tissues. In total, there were 94 biopsies from normal urothelium and 70 from transitional cell neoplasms obtained over a period of up to 11 years. In addition, 40 biopsies from various organs (prostate, 12; intestine, 9; oropharynx, 4; skin, 10; other, 5) were similarly studied. In all cases, sections from routinely processed tissue (fixed in 10% neutral buffered formalin and embedded in paraffin) were stained with hematoxylin and eosin for morphologic evaluation. Also, in all cases, sections from routinely processed tissues were used for immunohistochemical localization of the A and B antigens. In addition, 30 of the patients had fresh frozen tissue sections studied by the same methodology, as well as by the red cell adherence (RCA) test. Supported in part by a grant (CA 33239) from the National Cancer Institute, National Institutes of Health, Bethesda, Maryland. Accepted for publication June 29, 1990. Address reprint requests to Catherine Limas, MD, Department of Pathology, Department of Veterans Affairs Medical Center, One Veterans Dr., Minneapolis, MN 55417.
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Table 1. Material Examined Tissue examined
Number of patients
Normal urothelium only Transitional cell neoplasms only Normal and neoplastic urothelium Other normal tissues
8 3 16 14
Total
41
Monoclonal antibodies against synthetic oligosaccharides with A or B specificity were purchased from DAKO Co. (Santa Barbara, CA); biotinylated anti-mouse gamma M immunoglobulin (IgM), Ulex europeus lectin 1, dolichos biflorus lectin, and avidin-peroxidase reagent (Vectastain) from Vector Lab. Inc. (Burlingame, CA); polyclonal human anti-A, anti-B and anti-Lea, -Leb sera from Cooper Biomedical, Inc. (West Chester, PA). All other reagents were obtained from Sigma Chemical Co. (St. Louis, MO). The methodology for detecting blood group antigens (Bg-Ag) in tissue sections by immunohistochemistry with the biotin-avidin-peroxidase reaction and by the RCA test has been described in detail in our previous publications.89 Each biopsy was tested at least in duplicate for each antigen. Known A-positive B-positive controls were examined in parallel with the test tissues. The monoclonal anti-A antibody used for immunohistochemistry agglutinated both A1 and A2 RBCs up to a titer of 320, with somewhat weaker reactions for the A2 cells.
Results Normal Tissues The normal urothelium in 22 of 24 patients (91%) expressed both antigens, albeit to a variable extent. The Apositive areas were more extensive than B in 18 patients and about equal in four. Two patients had only A detectable in their normal urothelium. A distinctive mosaic distribution was demonstrable in 50% of the biopsies (Figure 1). We attempted to demonstrate more dramatically the mosaic expression of the two antigens by reacting the same sections sequentially with each of the monoclonal antibodies and developing the first (anti-B) reaction with alkaline phosphatase-avidin and the appropriate substrate (red) and the second (anti-A) with peroxidase-avidin and the corresponding substrate (brown). An example of this double staining is shown in Figure 2. The B antigen was detectable on endothelial cells in all tissues examined from all 41 patients. Apparently absent or extremely weak focal reactions with the anti-A were observed in the endothelium of all tissues from nine patients (21%). This apparent absence of A determinants
from the endothelium was consistently demonstrated in multiple biopsies from the same individual over several years and in biopsies from several organs, eg, skin, gastrointestinal, and genitourinary tract, which were obtained and tested at different times (Figure 3). To exclude the possibility that this phenomenon was induced by an adverse effect of the tissue processing on the endothelial blood group substances, fresh frozen sections also were examined. It was noted that endothelial reactivity for A in these nine patients was greatly diminished or absent, even in the fresh frozen, unfixed sections. Four of these patients had their RBCs tested with the antiA1 lectin of dolichos biflorus and all four gave negative reactions. The Lewis phenotype of the RBCs did not influence the endothelial reactions with anti-A, which indirectly implies that the secretor status of the individual had no bearing on this phenomenon. The reactions of the endothelium did not always correlate with those of the epithelial cells present in the same biopsy. As illustrated in Figure 4, the epithelium could be A-positive and B-negative, while the endothelium was Anegative and B-positive. This pattern was consistent in all biopsies obtained from various organs of the same individual through the years. The absence of A antigen from endothelial cells cannot be attributed to a peculiarity of the monoclonal antibody because similar results were obtained with the RCA test in which polyclonal anti-A serum was used. Also, the contrasting reactions between endothelium and epithelium (Figure 4) cannot be explained on the basis of the A2B phenotype. The reactions of endothelial cells with the Ulex europeus lectin were positive or weakly positive and did not correlate in any way with either the anti-A or anti-B reactions.
Urothelial Neoplasms The study of the 70 biopsies from the transitional cell neoplasms of 19 patients yielded the following results: In the neoplasms of 14 patients, only one of the two antigens was detectable, A in eight and B in six. One patient with an inverted transitional cell papilloma had both A and B antigens expressed in the tumor. The transitional cell carcinomas of three patients were negative for both A and B in all recurrences during a follow-up period of up to 5 years. A change in the detectable antigen from A to B was noted in the biopsies of the tumors from only one patient. This patient had at least two neoplasms: a grade 11 noninvasive transitional cell carcinoma involving the right wall of the bladder and a grade III carcinoma obstructing the left ureteral ostium. The first was A-positive and the latter B-positive. The non-neoplastic epithelium of this patient showed weak, patchy distribution of both antigens.
A,B Antigens in AB Heterozygotes 1159 AJP November 1990, Vol. 137, No. 5
Figure 1. Reactions of normal urothelium of an AB heterozygote for the BG antigens A(a) and B(b). Both antigens show apatchy distribution complementary to each other. This distribution results in an overall mosaic pattern. The vascular endothelium is bothA- and B-positive in this case. Routinely processed biopsjy stained with monoclonal antibodies as described in Methods (X40).
Figure 2. Normal urothelium from an AB heterozygote shows a mosaic at A-positive (brown) and B-positive (red) patches when stained for both A anid B antigens on1 the same section (for details refer to Methods) (X400).
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fj AE:.i_EK ..b Figure 3. Biopsy from the urinary bladder of a BGAB patient stained for A (a) and B (b). The epithelium is positive for both antigens but the endothelial cells are negative for A (X200).
All but one of the 14 patients with biopsies taken contemporaneously from both normal and neoplastic urothelium had both antigens detectable in the tumor-free mucosa, usually in a patchy distribution, regardless of the pattern of tumor cell reactivity. In one patient, only the A antigen was detectable in both normal and tumor cells in four sequential biopsies taken during a 7-year period. The reactions of the endothelium of the neovessels followed the same pattern as the patient's normal tissue vessels. Comparisons of the reactions on paraffin-processed tissue with those obtained on fresh frozen sections with the RCA method showed, in general, good correlation. There were quantitative differences between the results of the two procedures in the tumors of seven cases that had good positive reactions on the frozen and weak in the paraffinprocessed sections. In only one case was there a complete discrepancy, the tumor being A- and B-positive in the unfixed, frozen tissue and negative for both antigens in the paraffinized sections, while the blood vessels re-
Figure 4. Contrasting reactions of epithelial and endothelial cells for A and B antigens in sections from the pylorus of an AB heterozygote. a: A section incubated with monoclonal antiA shows positive epithelium and negative endothlium (X200). b: A section incubated with monoclonal anti-B s -ws negative epithelium and positive endothelium (X200).
mained positive. The RBC phenotype of this patient was A B, Lea-b+
Discussion The last several years have witnessed a renewed interest in the biochemistry and biology of the blood group (BG)associated antigens.3 Although the physiologic function of these antigens remains speculative, an increasing body of evidence underscores their role in the interactions of the cell with its milieu and suggests their involvement in a variety of disease processes.7 Thus knowledge of the biology of the BG-associated antigens A, B, and H has become relevant to several unresolved issues such as transplant rejection,10 malignant potential of neoplasms,6 and susceptibility to infections.1" We considered the possibility that the patterns of A and B antigen expression in the normal and neoplastic
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tissues of AB heterozygotes provide valuable insights into the expression of the genes of the BG biosynthetic enzymes and may be relevant to the tumor cell clonality or heterogeneity. The epithelium lining the human lower urinary tract is well suited to our study because it is morphologically and functionally rather uniform throughout its extent, so that the interpretation of the findings is not subject to sampling variability. Furthermore patients with urinary bladder lesions are often followed with endoscopy and biopsies from multiple sites, including neoplastic and morphologically uninvolved urothelium. To our knowledge, this is the first study designed to assess the detectability of A and B antigens in AB heterozygotes in multiple, sequential biopsies obtained over an extended period (up to 11 years) from both normal and neoplastic epithelium. The study was made more comprehensive by the availability of biopsies from several organs of patients with multiple medical problems, eg, prostate enlargement, skin lesions, gastrointestinal symptoms, etc. The heterogeneous distribution of the A and B antigens in normal tissues implies that the activities of the A- and B-specific glycosyltransferases vary in cells with similar histogenesis and differentiation. This variability sometimes results in an apparent mosaic pattern, as if small, randomly distributed cell colonies were derived from distinct clones of stem cells that express only one of the two antigens. Whether this is simply a quantitative phenomenon or involves specific gene expression or deletion can only be resolved by in situ hybridization with the DNA and RNA sequences corresponding to the relevant enzymes. The heterogeneous expression of the A and B antigens is not limited to patients with urothelial neoplasia, but is present in other organs of heterozygous individuals. For example, in the course of this study, we observed that other stratified as well as glandular epithelial structures displayed nonuniform reactivity. It has also been shown that the reactions of AB red blood cells with various antiA and anti-B sera are nonuniform, and one explanation offered invokes a competition of the A- and B-specific enzymes for substrate.12 The contrasting reactions in endothelial and epithelial cells in 21% of our cases highlight the cell-type-specific variability in the expression of each (A and B) antigen and suggest that the vascular endothelium of AB heterozygotes may have a different phenotype than the red blood cells. The diversity of the reactions may be strictly quantitative, but qualitative differences in the blood group substances in the endothelial cells of some individuals need also be considered. We tried to minimize the influence of methodologic factors, eg, inability to detect unusual variants of the A substance with monoclonal antibodies, interference by solvents and fixatives, by using various natural polyclonal antibodies and by modifying the procedure to avoid denaturation and extraction artifacts. We achieved
consistently reproducible results, even in biopsies obtained from various organs over long periods and tested independently by different methods. Therefore we are confident that the apparent absence of A determinants from the vascular endothelium is a stable, probably genetic, characteristic of some individuals with the AB RBC phenotype. This characteristic does not correlate with the Lewis phenotype and, therefore, is not likely to reflect the patient's secretor status. It did correlate with the negative agglutination reactions of the patient's RBCs with the Dolichos biflorus anti-A1 lectin. We have previously reported that the detectability of A antigen in the tissues of patients with A2 RBC phenotype is reduced compared with the A1 phenotype.13 However the marked discrepancy between endothelial and epithelial cell reactions noted in this series of AB heterozygotes is a new observation that cannot be directly explained on the basis of an A2B phenotype. This observation deserves detailed evaluation because of its implications for organ transplantation. For example, it may be possible to successfully transplant organs from BG AB donors to BG B recipients, provided that the absence of A antigens from the donor endothelial cells is verified, eg, in punch biopsies of skin. In this respect, it is interesting that kidneys from A2 donors have been successfully transplanted to BG 0 and B recipients.14 This study demonstrates that, as a rule, transitional cell neoplasms in BG AB heterozygotes express only one of the two antigens. Although this is not conclusive evidence, it does lend support to the theory of 'clonal' derivation of tumor cells. The frequency with which each of the two antigens was expressed in tumors was quite comparable, so that the presence or absence of either antigen seems to play no specific role in the pathogenesis of these tumors. Within a given tumor, the degree of reactivity for the expressed antigen may vary so that individual tumor cells show a spectrum of staining intensity from strongly positive to negative. This intratumor variability was more pronounced in paraffin-processed tissues. A preponderance of positive reactions at the basal and deep layers is frequently seen, but is due to the methodologic factors previously reported.13 We were impressed by the consistency of the antigenic makeup in recurrent tumors. It was not possible to trace accurately the anatomic origin of each recurrent lesion, so that, in several of our cases, we cannot firmly distinguish true recurrence of residual tumor from multicentricity. In the one discrepant case, the biopsies were obtained from clearly different sites and the tumors showed different morphology. Although an infrequent occurrence, this case suggests that the process of carcinogenesis can affect different clones of susceptible cells. Quantitative differences in the reactivity of cells within the same neoplasms are probably related to intratumor
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heterogeneity, which evolves after the initial stages of the transformation of a stem cell clone.15'16 There was only one phenotypically AB tumor that was morphologically distinct from all other cases (inverted papilloma) and did not recur. It is possible that this morphologic entity has a pathobiology distinct from the more common papillary transitional cell carcinomas. All the inverted papillomas we have studied so far are positive for the expected BG antigen and have shown no sign of progression. The pattern of A and B antigen expression in heterozygous individuals could serve as a means for distinguishing neoplastic from benign proliferative lesions. In this respect, it is significant that, in a given individual, both A and B antigens are usually detectable in non-neoplastic urothelium, albeit in a patchy distribution, while only one antigen A or B is demonstrable in truly neoplastic lesions. In summary, we have demonstrated that the distribution of A and B antigens in AB heterozygotes is heterogeneous and suggested that this phenomenon has relevance to organ transplantation and to the clonal derivation of urothelial neoplasms. We believe that this subject deserves detailed study in other normal tissues and their neoplastic processes.
References
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Acknowledgment
624 4. Oriol R: ABH and related antigens. Biochem Soc Trans 1987, 15:596-599
The author thanks Dr. P. Lange of the Department of Urologic Surgery for his cooperation.
