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Cell-mediated immune (CMI) responses to tumor-associated antigens (TAA) in the early postoperative period were examined for correlations with disease recurrence and survival in a 13-year-prospective study of 77 stage 1 and 2 breast cancer patients treated with modified radical or radical mastectomy alone. Among the 21 patients who had positive lymphoproliferative tests using patients' peripheral blood mononuclear cells and autologous TAA of breast cancer cells, only one died from metastatic disease (5%). Among the 56 patients who had a negative test, 23 died from metastatic disease (41%). This difference is statistically significant (p = 0.002) Three other risk factors including tumor size, nodal status and cell differentiation patterns were also analyzed. When these three clinical-pathologic criteria were analyzed individually, none reliably predicted disease recurrence and survival. Nodal status was the most predictive clinical-pathologic risk factor, but was not significant (p = 0.089). The results of this study demonstrate the detection of CMI responses against autologous TAA by lymphoproliferative assays identifies a sub-set of stage 1 and 2 breast cancer patients who are at minimal risk of developing metastatic disease. This testing also identifies immunologically unreactive patients who are at risk for disease recurrence.

Estrogen (ER) and progesterone receptor (PgR) content of tumors were determined by both the dextran-coated charcoal (DCC) cytosol and immunocytochemical assays (ICA), and these hormone receptor results were compared to lymphocyte immunity against tumor antigen(s) for 52 breast carcinoma patients. Hormone receptor analysis by both methods demonstrated that 60% of the patients' tumors had ERs, while 44% were positive for PgRs. The ICA procedure was more sensitive than the cytosol technique for determining PgR content of the tumors. This increased sensitivity was not observed for ER by ICA. Patient age, tumor size, and nodal status were not related to the ER and PgR receptor status. A total of 21/52 (40%) of the patients had positive lymphocyte immunity against tumor antigen. This immunity was independent of patient age, tumor size, and nodal status. There was no significant relationship between lymphocytic immunity against tumor antigen and ER or PgR content of tumors, suggesting that patient lymphocyte immunity against tumor is independent of hormone receptor status. This is further evidence that lymphocyte immunity against tumor antigen status is an independent prognostic indicator that may be useful in the selection of a subset of node negative patients for adjuvant chemotherapy.

Fifty-two breast cancer patients were evaluated for levels of several molecules related to iron metabolism including determining their tumor tissue and serum ferritin levels, serum transferrin levels, and serum iron levels. In addition the patients' lymphocyte immunity against autologous tumor antigen was investigated. Forty percent (21 of 52) of the patients had lymphocyte immunity against tumor antigen. Iron metabolism molecules were expressed in abnormal quantities in some breast cancer patients: 27% (13 of 49) had elevated tumor tissue ferritin levels, 4% (2 of 49) had abnormally high serum ferritin, 10% (5 of 49) had abnormally low serum transferrin levels, and 43% (21 of 49) had depressed serum iron levels. None of these abnormalities in iron metabolism are associated with tumor immunity. These iron metabolism molecules may be indicative of rates of cell proliferation or may influence growth of breast cancer cells, but do not appear to influence host lymphocyte immunity against tumor associated antigens.

One hundred forty-two breast cancer patients were evaluated for three functional immunologic parameters: the ability of their lymphocytes to proliferate in response to general T-(phytohemagglutinin) and B-lymphocyte (pokeweed mitogen) stimulators and their ability to proliferate in response to specific autologous tumor antigens. Tests were performed on patient blood specimens collected approximately 2 hours prior to surgery or 2-4 weeks following chemotherapy. T-lymphocyte functional competence was impaired in 83/142 (58%) of the patients, while B-lymphocyte competence was impaired in 34/142 (24%) of these patients. A total of 21/52 (40%) of the patients had lymphocyte immunity against autologous tumor antigen. There were weak associations between the ability of patients' T- and B-lymphocytes to function normally, and their ability to respond to autologous tumor-antigen. There was no relationship of age, tumor burden (clinical or pathological tumor size), extension to skin and/or muscle, or metastasis to any of the three immunological parameters. A relationship (p = 0.0463) between T-lymphocyte competence and pathological nodal status was observed; individuals that were node positive for disease, tended to have impaired T-lymphocyte function. When evaluating T- and B-lymphocyte competence and lymphocytic immunity against tumor antigen in pre- and post-chemotherapy patients, an immunologic rebound (increase in immune parameters shortly after completion of chemotherapy) was observed in some patients. These results demonstrate the utility of measuring these immune parameters in breast cancer patients, their relevance to the natural biology of the disease, and the influence that chemotherapy may have on host immune function.

Lymphocyte proliferation (LP) assays were performed in microculture using the T-cell mitogens phytohemagglutinin (PHA) and concanavalin A (Con A); the T + B cell mitogens, pokeweed mitogen (PWM) and staphylococcal phage lysate (SPL); and a pool of allogeneic stimulating leukocytes in one-way mixed leukocyte cultures (MLC) in lung and breast cancer patients and normal individuals. The resultant data were expressed in three different ways: (1) as mean counts per minute (CPM) of tritiated thymidine incorporation; (2) as a stimulation index (SI) and (3) as a relative proliferation index (RPI). The RPI is defined as the ratio of net CPM (nCPM) in experimental cultures with stimulant (E) minus medium control cultures (C) of a test individual to the mean nCPM of three or more normal individuals examined in the same assay on the same day. These expressions were then compared for their ability to discriminate between LP responses in cancer patients and normal individuals. The RPI value and selected cut-off values gave the most sensitive measure for the determination of depressed proliferative responses. These analyses demonstrated that lung carcinoma patients were depressed to PHA (50%), MLC (47%), PWM (43%) and Con A (40%). To a lesser degree, breast carcinoma patients were also depressed to MLC (36%), PHA (31%), PWM (27%) and Con A (19%). Our data indicate that the use of the RPI in the analysis of LP response represents an improved method for detecting impaired response of lymphocytes to general mitogens and alloantigens which can consistently reveal immunosuppression in many cancer patients and may be useful for serial monitoring of individual patients.

Lymphocyte proliferation assays with autologous tumor material in mixed leukocyte-tumor interactions (MLTI) were employed to monitor tumor-associated cell-mediated immune responses of peripheral blood lymphocytes from patients with carcinoma of the breast. In addition, leukocyte migration inhibition (LMI) assays were employed to compare reactivity to autologous breast-tumor extracts versus allogeneic breast-tumor extracts. Positive lymphoproliferative responses to tumor-associated antigens (TAA) were observed in the MLTI assay with the use of either intact autologous tumor cells or crude extracts (in mug and ng quantities) in 12 of 34 (35%) breast cancer patients studied. Positive reactivity to tumor, but not to normal tissue of reactive patients, was observed in repeated assays. Finally, patients demonstrating positive MLTI responses to autologous tumor extracts likewise responded in LMI assays to these same autologus extracts as well as to allogeneic breast-tumor extracts, but not to non-breast-tumor extracts. Thus breast tumors appeared to possess common TAA among both male and female patients.

Patients with benign diseases of the breast reacted in a migration inhibition assay with extracts of breast cancer and benign breast lesions and a human breast cancer-derived cell line, MCF-7. The incidence of reactivity of the patients with benign breast diseases against these antigens was similar to that of breast cancer patients. In addition, patients with breast cancer reacted to some extracts of benign breast lesions. The reactivity occurred in patients with several different histopathologic types of breast lesions, but was not found in women with no detectable pathologic lesions.

Indirect migration inhibition assays were performed with normal and mammary tumor-bearing C3H/HeN mice and patients with breast disease to assess cellular immunity against three different mouse mammary tumor virus (MTV) preparations grown in feline kidney cell cultures and against a mouse-derived MTV preparation. MTV obtained after passage through feline kidney cells and the mouse-derived MTV were capable of eliciting macrophage migration inhibitory factor production by mouse spleen cells obtained from normal or mammary tumor-bearing C3H/HeN mice, thus demonstrating a similar degree of antigenicity of these preparations. In experiments with human breast cancer patients' leukocytes, leukocyte inhibitory factor (LIF) was produced by 32-50% of these patients in response to the mouse-derived MTV or to three different MTV preparations obtained after passage through feline kidney cells. A significant proportion (31-54%) of benign breast disease patients also reacted with both the mouse-derived and feline-derived MTV preparations. Patients with both malignant and benign breast disease, however, had a significantly different (P less than .05) pattern of reactivity to mouse- and feline-derived MTV preparations from that observed with normal donors. Finally, some LIF activity was also observed (but not statistically significant with the use of nonparametric analysis methods) when feline leukemia virus was used as antigen with these patients. The data suggest that both breast cancer and benign breast disease patients were reactive against antigens largely specific for MTV in the feline cells and, presumably, were not reactive against feline cellular components, although the second possibility cannot be completely ruled out.

The leukocyte migration inhibition assay was used to compare the antigenic reactivity of 3 M KCl extracts of human tumors. Many extracts demonstrated strong reactivity with patient leukocytes, whereas others demonstrated weak or no reactivity, Extracts prepared from primary tumors or local recurrent tumors were more antigenic than extracts from involved lymph nodes or pleural effusions. The least reactive preparations were extracts made from specimens of liver metastases obtained at autopsy. A large standard extract tested at a standard concentration was useful for the evaluation of antigenic reactivity of human tumor extracts. It served as a point of reference in simultaneous tests with one blood sample from each individual, thus eliminating the influence of patient variation on extract reactivity.

Studies were conducted to determine whether MCF-7, a tissue culture cell line derived from a pleural effusion of a patient with breast carcinoma, could be used as a source of tumor-associated antigen for direct leukocyte migration-inhibition (LMI) assays. Of 32 patients with breast carcinoma, 27 (84.4%) gave positive migration-inhibition results on their initial tests with a 25-mug protein/ml concentration of a 3 M KCl extract of MCF-7; 1 of 24 (4.5%) normal donors reacted with MCF-7. An intermediate incidence of reactivity (7/16) was observed with the extract when leukocytes of patients with melanoma, lung carcinoma, and Ewing's sarcoma were used. In further specificity studies, leukocytes of patients with breast carcinoma gave a lower incidence of LMl reactivity than did those of patients with Ewing's sarcoma and lung carcinoma with KCl extracts of the appropriate histologic type of tumor. The results indicated that the MCF-7 cells possessed a tumor-associated antigen to which many patients with breast carcinoma are sensitized.