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A triple-bridge, indirect, immunoperoxidase method for detecting and localizing carcinoembryonic antigen (CEA) in tissue sections is described. By this technique, a cell-surface localization of CEA in colonic carcinoma and ovarian mucinous cystadenocarcinoma cells could be visualized. In the case of the colonic cancer, both the tumor from the descending colon and a metastasis to the skin gave positive peroxidase reactions for CEA. This immunocytochemical method for demonstrating the presence of CEA functioned in both frozen, ethanol-fixed and formalin-fixed, paraffin-embedded tissues, thus making it applicable for use with tissue sections conventionally prepared for light microscopy.

The simultaneous distribution of monoclonal 131I-labeled anti-carcinoembryonic antigen (CEA) immunoglobulin (IgG) (NP-2) or 131I-labeled irrelevant myeloma IgG (Ag8) and [3H]thymidine was studied in hamsters bearing transplants of the GW-39 human colon carcinoma by qualitative double-tracer whole-body autoradiography. Autoradiography showed that large solid GW-39 tumors are characterized by heterogeneity of radioantibody retention and uneven [3H]thymidine accumulation, reflecting zonal variations in antibody reactivity and tumor cell proliferation, respectively. The autoradiographic images showed that both 131I-labeled-monoclonal antibody and control 131I-labeled IgG targeted nonproliferating tumor zones, suggesting a mechanism of nonspecific tumor uptake of radioantibodies in these areas. Absence of tumor center labeling with [3H]thymidine, associated with cellular necrosis, was confirmed by histology and microautoradiography in separate animal studies. In confirmation of earlier reports, 131I-labeled anti-CEA monoclonal antibody gave higher tumor-to-non-tumor labeling patterns than did control 131I-labeled IgG, at both 3 and 7 days following treatment. Immunohistochemical localization of CEA in GW-39 tumors with necrotic centers showed the presence of CEA in nonviable cells, but CEA antigen concentrations were diminished as compared to cells located in the tumor's periphery. The results indicate that double-tracer whole-body autoradiography is well suited for studying the kinetics of radioantibody localization in relation to regional tumor cell viability.

The site-specific conjugation of methotrexate, 4-amino-N10-methylpteroylglutamic acid, to a monoclonal anti-carcinoembryonic antigen (CEA) antibody, using an intermediate amino-dextran carrier system, resulted in a ratio of 30-50 molecules of MTX per molecule of immunoglobulin. The immunoreactivity of the conjugate was analyzed using flow cytometry or a competitive binding assay, which showed that the conjugate has significant retention of the antigen-binding activity. The pharmacokinetic behavior of the immunoconjugate in BALB/c mice and tumor localization in hamsters indicated that the conjugate remained in the circulation with higher concentration than free antibody, and could recognize the tumor as efficiently as the unconjugated antibody. The high degree of drug incorporation with retained immunoreactivity makes this method preferable to direct antibody conjugation.

The injection of an antiantibody (second antibody, SA) can enhance the clearance rate of a radiolabeled antitumor antibody (primary antibody, PA) from the blood. We have studied how the dose of the SA and the timing of the SA administration influence the rate of PA clearance and thereby improve tumor/nontumor ratios. Adult hamsters bearing the carcinoembryonic antigen-producing, GW-39 human colonic tumor xenograft were given injections of 131I-labeled, goat anti-carcinoembryonic antigen antibody, and after 6, 24, or 48 h, an injection of donkey anti-goat immunoglobulin was given at SA:PA ratios of 25, 50, 100, or 200:1. In comparison to a control group of animals that were only given 131I-PA, the administration of the SA improved tumor/blood ratios regardless of the SA:PA ratio or time the SA was given. The most important factor in optimizing this procedure was the timing of the SA injection. Significantly improved tumor/nontumor ratios were found when the SA was given between 24 and 48 h after the PA in comparison to 6 h. This was because maximum accretion of radiolabeled PA in the tumor was not achieved until 24 h. At SA:PA ratios of 25:1, only tumor/blood ratios were significantly improved in comparison to the control group. In addition, at SA:PA ratios of 25:1 and 50:1, tumor/spleen and tumor/kidney ratios were lower than the control group, whereas at higher SA:PA ratios, all tumor/nontumor ratios were significantly improved. These studies suggest that for this model, a ratio of SA:PA of 100:1 or higher given at 24 to 48 h after the PA is the best combination for maximizing tumor/nontumor ratios.

Quantitative whole-body autoradiography (WBAR) was used to study the biodistribution of goat anti-carcinoembryonic antigen and normal goat IgG, each labeled with 125I, in hamsters bearing the carcinoembryonic antigen-producing GW-39 human colonic carcinoma xenograft. Comparisons between computer-assisted videodensitometric profiles of WBARs and tissue radioactivity counts were made at 1, 3, and 7 days following administration of the radiolabeled IgGs. The results indicated that maximal tumor accretion of the radiolabeled antibody and normal IgG occurred within 1-3 days, with a marked selective accretion of antibody in the tumor being evidenced at 3-7 days because of clearance of normal IgG. Radioactivity derived from antibody IgG showed 6.5 to 118.7 times that found in other tissues, as measured by videodensitometry, whereas organ radioactivity counting revealed ratios of only 6.7 to 29.6. Specificity of tumor-cell accretion of the radiolabeled antibody was confirmed by microscopic autoradiography, showing intense labeling of the proliferating perimeters of GW-39 tumors. WBAR was found to have a resolution of 0.10 to 0.25 mm in 100-g hamsters, which appears to be greater than the resolving power of external body imaging by gamma camera scintigraphy. These studies suggest the use of WBAR and microautoradiography to complement external imaging methods for the analysis of antibody distribution and localization in cancer radioimmunodetection models.

Recombinant human interleukin 1 (IL-1) administered i.p. to tumor-bearing hamsters results in a dose-dependent 2- to 5-fold increase in total peripheral white blood cells. These levels remain elevated for 2 to 3 wk and then decline to base-line levels. Pretreatment of animals with similar doses of IL-1 prevents radioimmunotherapy-induced destruction of the radiosensitive hematopoietic system. Furthermore, recovery from radio-immunotherapy-induced myelosuppression is possible if animals are given IL-1 1 wk after radioimmunotherapy treatment. Thus, both protection and rescue from the hematopoietic damage associated with radioantibody treatment are feasible by pre- or posttreatment with IL-1.

Contingencies maintaining the bizarre speech of a 29-year-old woman with mild mental retardation and schizophrenia were analysed. Bizarre vocalizations occurred most frequently during demand conditions and least frequently during one-to-one interaction with attention contingent upon appropriate vocalizations. Treatment conditions derived from the assessment consisted of guided compliance and ignoring plus contingent attention. Treatment effects generalized to direct-care staff in day and residential settings. Analysis of the variables controlling bizarre speech facilitated development of interventions that were predominantly positive in nature, based on the specific and unique controlling behavior-environment interactions, and that were teachable to the direct-care staff.

Myelosuppression has been identified as the dose-limiting toxicity in radioimmunotherapy studies. Accurate bone marrow dosimetry is, therefore, necessary to evaluate bone marrow toxicity which may result from systemic cancer treatment with radiolabeled monoclonal antibodies. Dose to the red marrow was determined in 20 patient studies with 131I labeled anti-carcinoembryonic antigen, anti-alpha-feto-protein, or anti-human chorionic gonadotropin monoclonal antibody for diagnosis or treatment of diverse metastatic carcinomas, using a new technique involving sacral scintigraphy and a previously reported blood-based methodology. For the sacral technique, anterior and posterior gamma camera images of the pelvis were obtained at multiple times. Regions of interest were drawn around the sacrum in order to quantitate activity uptake as a function of time using the conjugate view counting method. Cumulated activity in red marrow was determined by curve integration and division by 0.099, since it has been estimated that 9.9% of the total red marrow is contained in the sacrum of the adult. Red marrow doses were then obtained by multiplying the cumulated activities by the appropriate S factor. These doses were compared to red marrow doses obtained from serial whole blood samples taken from these patients. Cumulated activity in the red marrow was determined from the blood with the assumption that the activity concentration in the blood and red marrow were equal. The mean red marrow dose per injected activity was 2.0 +/- 0.9 rad/mCi using the sacral data and 2.7 +/- 1.3 rad/mCi using the blood data (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Radiolabeled antibodies have been shown to have a therapeutic potential in tumor-bearing animal models. However, treatment with radiolabeled antibodies results in toxic effects to normal tissues, as monitored by losses in body weight and in peripheral wbcs. We have investigated the use of an anti-antibody, or second antibody (SA), as a means of reducing this toxicity. SA rapidly forms a complex with circulating radiolabeled antibody, causing an increase in the clearance rate of the radiolabeled antibody from the blood. Toxicity was significantly reduced in animals given the SA in comparison to the toxicity seen in animals given only the radiolabeled primary antibody (PA). The earlier the SA was administered, the lower was the toxicity. The therapeutic efficacy of radiolabeled antibody was not influenced by the administration of the SA given 48 hours after the PA. Thus, the controlled removal of circulating radiolabeled antibody by an anti-antibody can reduce the toxicity associated with radiolabeled antibody therapy without influencing the antitumor effect.

The advent of immunoperoxidase histochemistry techniques has allowed the more convenient visualization by light microscopy of antigenic cellular constituents. Application of the unlabeled and labeled antibody enzyme methods to the localization of carcinoembryonic antigen in conventionally prepared tissue specimens has revealed the feasibility of using immunoperoxidase techniques in routine diagnostic pathology. The methods involved are well defined and once antibody specificity and antigen stability are determined, they are adaptable to the identification of other immunological markers that may appear in malignant tissue specimens.

A comparison between two immunoperoxidase staining procedures, a triple-bridge and an indirect, antibody-conjugated method, was made to determine the relative sensitivity of each technique in the detection of carcinoembryonic antigen (CEA) in routine tissue sections. The enzyme-antibody conjugates for the indirect procedure were prepared according to the method of Nakane and Kawaoi (1974). The indirect, antibody-conjugated method, proved to be slightly more sensitive than the triple-bridge by two criteria. First, CEA could be localized using higher dilutions of the primary antiserum by the indirect technique, and secondly, tissues were shown to stain for CEA in specimens with lower tissue CEA levels by the indirect procedure than by the triple-bridge method. The preparation of enzyme-antibody conjugates is a relatively simple procedure and, in addition, the conjugates will remain stable when kept frozen or at 4 degrees C. Background staining due to non-specific interaction of the conjugate with the tissue can be eliminated easily by incubation with normal serum. These results indicate that the indirect, antibody-conjugated method can be used to enhance the staining of CEA in routine tissue sections.

Carcinoembryonic antigen (CEA) levels in plasma and the immunocytochemical detection of tumor CEA were studied in patients with endometrial adenocarcinoma treated at the University of Kentucky Medical Center from 1973 to 1976. The incidence of CEA elevation in a group of 60 patients varied directly with uterine size, histologic differentiation, and stage of disease. Immunoperoxidase staining for CEA was performed on 42 specimens, and four were positive, indicating a tissue concentration of CEA of at least 5 microng per gram. Following operation, CEA levels returned to normal within eight weeks in all but two patients, both of whom were later shown to have persistent or recurrent cancer. These data suggest that serial plasma CEA determinations may be useful in predicting occult recurrence of endometrial cancer in patients in whom the plasma or tumor initially contains elevated concentrations of this antigen.

A 3-layer immunoperoxidase technique was used to demonstrate carcinoembryonic antigen (CEA) in colonic polyps from patients with or without previous or concurrent malignancy. CEA was demonstrated in a higher percentage of the polyps received as fresh specimens that were rapidly frozen and fixed in ethanol, than in formalin-fixed, paraffin-embedded sections. Tissue CEA content of both colonic carcinomas and polyps was determined by radioimmunoassay, and it was found that benign colonic tumours had levels of tissue CEA comparable to colonic cancer, indicating that CEA concentration in a tumour does not reflect its grade of malignancy. In fact, in one case in which both colonic cancer and polyps were removed, the polyps had the higher quantities of tissue CEA. Further, tissue CEA concentration of a polyp was not dependent on its size or location. Studying the titres of circulating CEA in these patients revealed an elevation of plasma CEA in one-third of the patients with only colonic polyps, whilst the patients with cancer all had increased titres.