Distribution of individual components of basement membrane in human colon polyps and adenocarcinomas as revealed by monoclonal antibodies.

Double-label immunofluorescence was used to monitor basement-membrane composition and integrity in 22 human colon polyps, 36 adenocarcinomas and 2 metastases. Cryostat sections were stained with polyclonal anti-laminin anti-serum combined with monoclonal antibodies (MAbs) to all major basement-membrane components (laminin, entactin/nidogen, collagen type IV and large heparan sulfate proteoglycan), as well as to keratin 8. In all adenocarcinomas, including mucinous, basement membranes were altered more at the invasive front than in the parenchyma. The degree of this alteration was inversely correlated with the level of tumor differentiation. An uncoordinated loss of basement membrane components (dissociation of markers), previously described by us in rat colon adenocarcinomas, was also found in human tumors. In the great majority of adenocarcinomas a pronounced stromal reaction was seen. It was manifested by the presence of fibrillar deposits of basement-membrane components, mainly of collagen type IV and/or heparan sulfate proteoglycan. This reaction was never observed in polyps and may be derived from myofibroblasts reported to accumulate in colon cancer stroma. The combined use of antibodies to basement-membrane components and to a specific keratin may constitute an adequate immunohistochemical test for the presence of invasion, and may be useful in the histologic analysis of polyps, especially in dubious cases.     

Immunohistological observation of blood group-related antigens in lung adenocarcinomas using monoclonal antibodies

The immunohistological distribution of blood group (BG)-related antigens including A, B, H type 2, and sialylated Lex in lung adenocarcinomas was examined using monoclonal antibodies. BG-A, B, and H type 2 compatible with the ABO status in tumor cells were expressed in 60% of the cases. Accumulation of H type 2, associated with loss of BG-A and B, was observed in tumor cells of patients with BG status other than 0. Tumor-associated antigens, Lex and sialylated Lex were detected in 36.0% and 72.0%, respectively. Modification of carbohydrate antigens in cancer may be associated with incomplete synthesis; accumulation of precursor antigen; and activated sialylation.     

Differentiation patterns of testicular germ-cell tumours as revealed by a panel of monoclonal antibodies

A panel of monoclonal antibodies against different keratin polypeptides, epithelial glycoproteins, placental alkaline phosphatase and collagen type IV was used to evaluate immunohistochemically the expression of the target antigens in 30 different human testicular germ-cell tumours of various types. Antikeratin antibodies detecting markers of different routes of epithelial differentiation revealed remarkable similarity of differential expression of various keratins in epithelial structures of teratomas and combined tumours as compared with normal human epithelial tissues. A considerable proportion of embryonal carcinoma cells stained positively for keratins 8, 18 and 19, while a minor subpopulation of tumour cells in embryonal carcinomas, some seminomas and many atypical intratubular cells expressed keratins 8 and 18 but usually lacked keratin 19. Antibody RICEO-MFG-06.3, specific for epithelial glycoproteins, gave negative results with seminomas as opposed to positivity in all but two nonseminomatous tumours. All but two neoplasms showed positivity for placental alkaline phosphatase, thus supporting its reliability as a marker of germ-cell tumours. It is concluded that the monoclonal antibody RICEO-MFG-06.3 and especially the keratin-19-specific antibodies BA16 and BA17 can be helpful in distinguishing embryonal carcinoma from seminoma and, together with antibodies to other keratins, in the study of the origin and histogenesis of testicular germ-cell tumours.     

Heterogeneity of keratin expression in mouse mammary hyperplastic alveolar nodules and adenocarcinomas

The keratins and other cytoskeletal proteins expressed by normal, preneoplastic, and malignant mammary tissues in BALB/c mice and by cells in primary cultures established from these tissues were analyzed and compared. The preneoplastic lesions were hyperplastic alveolar nodules (HAN) derived originally from mice treated by hormonal stimulation (D2), exposed to a chemical carcinogen (C4), or spontaneously expressing mouse mammary tumor virus (CV2) and maintained by serial transplantation. All tumors were mammary adenocarcinomas which developed as primary neoplasms from the HAN outgrowth lines. Cytoskeletal extracts were prepared from the tissues and cultured cells and subjected to two-dimensional polyacrylamide gel electrophoresis. Comparison of the major polypeptides in the normal and abnormal tissue extracts revealed considerable similarities in the cytoskeletal profiles. Three basic and seven acidic polypeptides ranging in molecular weight from 40,000 to 90,000 were regularly identified. However, notable differences were also found. A Mr 55,000 keratin (IEF 55) was prominent in one HAN, the D2, and all tumor tissues but not in normal gland. Likewise, a Mr 46,000 polypeptide (IEF 46), which has been tentatively identified previously as a keratin, was absent in normal epithelium but present in all abnormal tissues except the C4 and CV2 HAN. A Mr 58,000 polypeptide (NEPHGE 58) was not detected in normal gland or the C4 lesions but was found in all other abnormal tissues. The overall pattern of polypeptides in cytoskeletal extracts from normal and abnormal mammary cells in primary culture resembled that of the corresponding tissue but also had important differences. In all cell cultures, IEF 46 and IEF 55 were major species, while the larger and more basic components were markedly reduced. A Mr 56,000 polypeptide (NEPHGE 56) was detected only in C4 HAN and C4 and CV2 tumor cells. Trace or small, variable amounts of a Mr 57,000 basic keratin (NEPHGE 57) were present in normal and D2 tissues and cultured cells. NEPHGE 57 was dramatically increased in C4 and CV2 tissues and cultured cells and may be related to expression of squamous metaplasia and keratinization which are characteristic of these lesions. Although production of IEF 46 and IEF 55 may be associated with neoplastic progression of mammary epithelium, particularly in vivo, the association is not exclusive since normal cells express these polypeptides when grown in primary culture. In addition, correlations between altered keratin expression and the mode of induction of the mammary lesions were not obvious.     

Heterogeneous expression of cell-surface antigens in normal epithelia and their tumours, revealed by monoclonal antibodies

Most monoclonal antibodies that have been raised to human epithelial tumours bind to only some of the cells in a tumour, showing that tumour cells are very heterogeneous in their expression of antigens. Normal epithelia show the same heterogeneity of antigen expression, as also do cell lines and clones of epithelial cells in culture. It is not related to the mitotic cell cycle. Many, probably most of the antigenic determinants to which the antibodies bind are carbohydrate structures. It is not clear whether variations in antigen expression reflect variations in the differentiated state of the cells or merely variations in the carbohydrate structures on otherwise identical cells, nor is ir clear whether antibodies could be made that bind to all tumour cells by avoiding antibodies to carbohydrate structures. The normal and apparently reversible nature of this heterogeneity of antigen expression conflicts with conventional views that heterogeneity among cells of a tumour is due to permanent genetic change. The heterogeneity within normal clones suggests that cloning is not an adequate way to study heterogeneity in tumour cells. The implications of heterogeneous expression of antigens within tumours for therapeutic and diagnostic application of antibodies are discussed.     

Heterogeneity of cell surface antigen expression of human small cell lung cancer detected by monoclonal antibodies

Using immunohistochemistry, radiobinding, and indirect immunofluorescence assays, seven distinct cell surface antigens, detected by monoclonal antibodies, were analyzed for the degree of homogeneity or heterogeneity of antigen expression on a panel of human small cell lung cancers. The panel included 7 tumors taken directly from patients, 21 established cell lines (9 of which were derived from different metastatic sites of 3 patients), and 33 clonal derivatives of 3 lines. With all assays, considerable heterogeneity of antigen expression between tumors from different patients was observed. In both fresh tumors and in cell lines, as well as in cell lines established from different metastatic sites in an individual patient, we observed intratumor heterogeneity finding antigen positive and negative cells and variation in antigenic density, by immunohistochemistry and indirect immunofluorescence assays. Antigenic expression was not cell cycle dependent. In addition, when cell lines or patient samples expressing antigen positive and antigen negative tumor cells were cloned, heterogeneity of antigenic expression was still present in the clonal lines. This suggests that either the expression of the antigen was not heritable and/or the ability to regenerate antigenic heterogeneity is an intrinsic property of the tumor cells. The heterogeneity of antigen expression on lung cancer cells has significant implications for the use of these and other monoclonal antibodies in the study and therapy of lung cancer.     

Human monoclonal antibodies against cytokeratin 18 generated from patients with gastric cancer

By co-culturing regional lymph node B-cells and HAT-sensitive mutant cells obtained from RPMI-1788 cells, no less than 20,000 Epstein-Barr (EB)-transformed colonies were obtained from 32 patients with gastric cancer. From B-cell cultures generating antibodies reactive with gastric cancer tissues as well as cultured gastric cancer cells, two EB-transformed cell clones termed C418-59 and C1218-39 were isolated. Both of them produced human IgM-class antibodies, termed Mab418-59 and Mab1218-39, respectively. Both antibodies reacted with an antigen with a molecular weight of 45 kd existing in gastric cancer MKN-45, MKN-1, and Kato-III cells, and also with all of 4 adenocarcinomas of the stomach in paraffin sections. The antigen recognized by both antibodies was identified as a kind of cytoskeletal protein, cytokeratin 18. In this study, it was confirmed that B-cell clones generating autoantibodies against cytokeratin 18 were present in some patients with gastric cancer.     

Heterogeneity of gastrin-containing G-cells and its expression in gastric adenocarcinomas and endocrine tumors

The heterogeneity of gastrin-containing G cells present in human gastric mucosa has been examined immunohistochemically. Calcitonin gene-related peptide (CGRP), calcitonin and human chorionic gonadotropin (hCG)-immunoreactivity were detected in about 500, 20 and 10 cells pro 1,000 G cells, respectively, these findings supporting the "one cell, multi-hormone theory". Gastrin, calcitonin immunoreactive tumor cells were demonstrated in 13%, 3% of the antral adenocarcinomas and 17% and 10% of antral endocrine tumors, but they were not found in fundic adenocarcinomas and endocrine tumors. Cell hybridization between the tumor cell and the G-cell might be a possible mechanism for the occurrence of gastric and calcitonin in the gastric tumors. HCG-immunoreactive tumor cells were detected in 27% of antral adenocarcinomas, and in 24% of the fundic adenocarcinomas, and the production of hCG by gastric tumor cells might be based on the gene expression during carcinogenesis, regardless of the tumor localization.     

Complementation of anti-CEA and anti-TAG-72 monoclonal antibodies in reactivity to human gastric adenocarcinomas

Monoclonal antibody (MAb) B72.3 and MAb COL-4 are reactive with the high-molecular-weight (Mr greater than 10(6] tumor-associated glycoprotein (TAG)-72, and the Mr 180,000 carcino-embryonic antigen (CEA), respectively. Antibody competition radioimmunoassays (RIAs) using 125I-MAb B72.3 or 125I-COL-4 have demonstrated that each MAb also recognizes a distinct antigenic determinant. Solid-phase RIAs using MAbs B72.3 and COL-4, however, demonstrated similar reactivity for each MAb with gastric carcinomas versus normal gastric mucosa. Tissue sections from all of 17 gastric adenocarcinomas also reacted with both MAb B72.3 and MAb COL-4 when immunoperoxidase techniques were used. Double-staining techniques using both MAbs on the same section were performed on formalin-fixed, paraffin-embedded gastric tissue sections using the combination of avidin-biotin peroxidase complex and avidin-biotin alkaline phosphatase complex immunohistochemical methods. The double-staining technique revealed that some carcinoma cells react with MAb B72.3, some react with MAb COL-4, and others react with both MAbs. This technique has demonstrated that more carcinoma cells can be detected by both MAbs as compared to the number of stomach carcinoma cells shown to be reactive with either one or the other MAb. These studies thus define the rationale for the use of combinations of MAbs which recognize different tumor-associated antigens as immunological adjuncts for detection and perhaps therapy of gastric carcinoma.     

Proteoglycans detected by monoclonal antibodies in adenoid cystic carcinoma of salivary glands

Thirty-two cases of adenoid cystic carcinoma (ACC) of the salivary glands were examined immunohistochemically by monoclonal antibodies to proteoglycans (PG) such as chondroitin 6 sulfate (C6SPG), chondrotin 4 sulfate (C4SPG), dermatan sulfate (DSPG), heparan sulfate (HSPG) and keratan sulfate (KSPG) in conjunction with specific enzymatic digestion. The cribriform structure of ACC consisted of basaloid, myoepithelium-derived, and luminal tumor cells. The myoepithelial tumor cells were positive for PG, whereas luminal tumor cells were unstained. Occasional pseudocysts also gave positive staining for PG. Tubular structures consisting of modified myoepithelial cells indicated a high intensity reaction for C6SPG and C4SPG, and a slight one for DSPG, HSPG, and KSPG. Immunodepositions in solid and cluster structures were comparatively light in terms of PG. Basement membrane in ACC stained strongly for C4SPG, slightly for C6SPG, and very slightly for DSPG, HSPG, and KSPG. In ACC, immunohistochemical staining of PG was regularly positive in myoepithelium-derived tumor cells, but was irregular in other types of tumor cells.     

Multidrug resistance gene 1 expression in salivary gland adenocarcinomas and oral squamous-cell carcinomas

In combined chemotherapy for head-and-neck cancer (HNC), salivary gland-cell adenocarcinoma (SGA) shows insufficient clinical outcome, and it has been suggested that the sensitivity and/or the mechanism of resistance to anti-cancer drugs are different between SGA and oral squamous-cell carcinoma (SCC). The aim of our study was to clarify whether P-glycoprotein (P-gp) expression is associated with multidrug resistance (MDR) in HNC and the difference in the process of its development between SGA and SCC. In immunohistochemical analysis, P-gp expression was found in the ductal cells of salivary glands but not in oral mucosal epithelium. In cancer tissues, a few SCC cells in 12 of 37 and most cells in all SGAs expressed P-gp. The intensive P-gp expression was significantly found in SGA compared with SCC. In an in vivo chemotherapeutic model using tumor-bearing nude mice, P-gp expression in counterparts was observed in only a few cells of the HSY line, while no P-gp expression was observed in Hepd cells. However, P-gp expression was developed in both HSY and Hepd cell lines after vincristine (VCR) treatment. RT-PCR showed that the mean ratios of mdr1 mRNA expression levels in HSY clones were 3.7-fold higher than those in Hepd clones after VCR treatment, while each cell line exhibited both induction and activated production of P-gp. These results suggest that P-gp-related MDR in SGA is an inherent phenotype caused by both high levels of P-gp induction and activated P-gp production during VCR treatment, while that in SCC is an acquired phenotype chiefly caused by induction of P-gp.     

Differential expression of carcinoembryonic antigen in early gastric adenocarcinomas versus benign gastric lesions defined by monoclonal antibodies reactive with restricted antigen epitopes

Murine monoclonal antibodies (MAbs) reactive with distinct epitopes on carcinoembryonic antigen (CEA) have been analyzed systematically by radioimmunoassays, Western blotting, and immunohistochemical assays to define CEA expression in adenocarcinomas, benign lesions, and normal tissues of the stomach. Each of four COL-MAbs (COL-1, COL-4, COL-6, and COL-12) reacted preferentially with cell extracts of adenocarcinomas versus those of normal mucosae in solid-phase radioimmunoassays. Using Western blotting analyses MAbs COL-1, COL-4, COL-6, and COL-12 detected only the Mr 180,000 molecule characteristic of CEA in adenocarcinoma of the stomach; no reactivity was observed in an extract of normal gastric mucosa. Antibody competition radioimmunoassays were then carried out to define relations among COL-MAbs using 125I-radiolabeled MAbs, and nonradiolabeled MAbs as competitors. A spectrum of formalin-fixed, paraffin-embedded normal, benign, and malignant tissue sections of the stomach were examined for immunoreactivities with COL-MAbs using immunohistochemical assays to define whether the COL-MAbs were able to detect CEA expression in early foci of gastric carcinomas. All of the COL-MAbs generally demonstrated selective reactivities to adenocarcinomas (n = 40) versus benign lesions (n = 15) and normal mucosae (n = 6) of the stomach. From 72 to 100% of adenocarcinomas at early stage (n = 18) were reactive with the COL-MAbs, suggesting that these MAbs might serve as immunohistochemical diagnostic tools to detect early foci of gastric carcinoma. The data reported here indicate that the COL-MAbs can potentially be utilized as radioimmunological and immunohistochemical adjuncts to differentiate early adenocarcinomas from normal mucosae or benign lesions of the stomach on the basis of differential CEA expression.     

Heterogeneity among the non-Hodgkin's lymphomas. Implications for autologous bone marrow transplantation with in vitro purging using monoclonal antibodies

To investigate the possible implications of heterogeneity among the non-Hodgkin's lymphomas for bone marrow purging using complement-fixing monoclonal antibodies to lymphoma-associated antigens, a panel of large cell lymphoma cell lines of diverse phenotypes was treated with monoclonal antibodies DLC-48 and LN-1. An association was demonstrated between the percentage of suppression of colony formation by the cell line and both the percentage of cells staining with the antibody and the intensity of its binding. Flow cytometric analysis of cells surviving treatment with antibody and complement demonstrated that the population that escaped lysis showed weak immunofluorescent staining. Similarly, 40% of the clones derived from cells surviving treatment with antibody and complement stained weakly compared with the parent cell line. For a given fluorescence intensity, cells differed in their susceptibility to treatment. Some cells with moderate to strong staining survived incubation with antibody and complement. In five cases, treatment of bone marrow contaminated with malignant lymphoma cells resulted in complete eradication of even cells with weak staining. In two cases, a population of cells that stained dimly survived treatment with either antibody. DNA-content analysis showed that the cell cycle distribution of cells surviving treatment with DLC-48 or LN-1 and complement was similar to that of cells treated with control antibody 46-1G7 and complement. Phenotypic heterogeneity may hinder efforts to purge malignant lymphoma cells from human bone marrow with complement-fixing monoclonal antibody reagents. Relative resistance to complement-mediated lysis may underlie differences in the susceptibility of cells to treatment and also limit the effectiveness of this technique.     

Six new monoclonal antibodies to serous, mucinous, and poorly differentiated ovarian adenocarcinomas

Six monoclonal antibodies directed against ovarian adenocarcinoma were generated by use of 100,000 x g supernatants of Triton-X-100 solubilized extracts of ovarian serous adenocarcinoma as the antigen source. Immunoperoxidase preparation of frozen-sections and routinely processed paraffin section specimens revealed a highly restricted reactivity of these antibodies when tested with adult (n = 2) and fetal (n = 3) tissue types. Coreactivities were occasionally observed with epithelia of the kidney, mammary gland, and pancreas. One monoclonal antibody, Ki-OC I-6-2, cross-reacted only with epididymal epithelia. No coreaction occurred with normal tissue of the ovary, Fallopian tube, or uterus. All antibodies were additionally tested on 74 cases of nonovarian malignancies, 15 cases of ovarian metastases of nonovarian carcinomas, and 114 specimens of ovarian neoplasms other than carcinomas. Ki-OC I-6-2 had no cross-reactivity with these tumors except for one case of renal cell carcinoma. This monoclonal antibody recognized serous, mucinous, and poorly differentiated adenocarcinoma cell types. None of the six antibodies reacted with clear cell or endometrioid carcinoma. All were found to be of the IgG-1 subclass. The tumor antigen to which Ki-OC I-6-2 immunoreacted was estimated to have a molecular weight of 80 kilodaltons (KD).     

Immunohistochemical localization of glycosaminoglycans with the use of monoclonal antibodies in salivary pleomorphic adenomas

Immunohistochemical identification of glycosaminoglycans (GG) in salivary pleomorphic adenomas (63 cases) was made to evaluate chondrogenesis in modified myoepithelial cell regions. Monoclonal antibodies (MoAbs) to GGs were used in conjunction with specific enzyme digestion, and chondroitin 4S proteoglycan (C4SPG), chondroitin 6S PG(C6SPG), dermatan sulfate PG(DSPG), heparan sulfate PG(HSPG) and keratan sulfate PG(KSPC) were identified. Modified myoepithelial cells showing fibrillar and plasmatoid shapes contained KSPG (68%), DSPG (32%) and CSPG(C6SPG:22%, C4SPG:33%). Foci of chondroidally changed cells stained intensely for KSPG (53%), and less so for CSPG(C6SPG:22%, C4SPG:33%), and DSPG (19%). Perinuclear matrix in chondroidal tissue reacted most strongly. Almost all types of modified myoepithelial cells, or outer layer tumor cells of tubulo-ductal structures, produced or synthesized CSPG, DSPG, and HSPG. Certain cells located in hyalinous and myxomatous tissues may undergo chondroidal metaplasia, and clusters of such cells may produce GGs and PGs in the perinuclear zone similar to those GGs in matrix synthesized in chondroidal tissue. GG-synthesizing cells might continuously produce KSPG until the cartilage matrix is completed.     

Characterization of monoclonal antibodies B1 and B3 that react with mucinous adenocarcinomas.

B1 and B3 are two newly isolated monoclonal antibodies that react uniformly with the surface of many mucinous carcinomas of the colon, stomach, and ovary but with a limited number of normal tissues, among which are glands of the stomach, epithelia of the trachea and bladder, differentiated epithelium of the esophagus, and small bowel mucin. They also react uniformly with many human tumor cell lines, including MCF7, MDA-MB-468, and HTB20 (breast), A431 (epidermoid), HT29 (colon), HTB33 (cervical), and DU145 (prostate). Immunoprecipitation experiments indicate that B1 and B3 react with epitopes present on a large number of glycoproteins, ranging in molecular weight from greater than 200,000 to less than 40,000. Using a panel of 37 different carbohydrate residues attached to albumin to form neoglycoproteins, it was found that B1 reacts with Ley and H-type 2 and B3 reacts with Ley, di-Lex, and tri-Lex antigens. Thus, each antibody reacts with a distinct portion of a carbohydrate residue. Because of the limited reactivity of these antibodies with normal tissues, they merit evaluation in the treatment of cancer.     

Radiotherapy and tumor immunity--analysis using monoclonal antibodies

In order to better understand the immunologic effects of irradiation, blood levels of lymphocyte subsets were sequentially monitored in 37 patients before and during irradiation treatment for lung cancer. Tumor infiltrating lymphocytes induced by radiotherapy were analysed by the avidin-biotin-horseradish peroxidase method. 49 cases of head and neck cancer were examined. In some cases, remarkable infiltration of lymphocytes was observed surrounding cancer cells during radiotherapy. This infiltration was mainly composed of anti-Leu-3a + 3b positive lymphocytes, and HLA-DR positive cancer cells were remarkably observed.