High incidence of migration inhibition reactivity to lung tumor-associated antigen by normal donors in close contact with lung cancer patients or materials.

A high incidence of indirect leukocyte migration inhibition reactivity of normal donors to a 3-M KCl extract from a fresh pleural effusion of a patient with a lung adenocarcinoma (designated 7661) was observed. When these normal donors were classified according to contact with lung cancer patients or materials, 22 of 32 (72%) normal donors in contact with lung cancer patients or materials were reactive with the 7661 extract as compared to only 3 of 76 (4%) who had no contact. Of normal donors involved in the direct care of lung cancer patients, 14 of 20 (70%) were positive, whereas only 2 of 10 (20%) hospital personnel who worked with noncancer patients were reactive. Among laboratory personnel who handled blood and tissue specimens from lung cancer patients, 8 of 11 (73%) were positive with the 7661 extract, whereas none of 5 laboratory workers who worked with cancer materials unrelated to lung cancer were positive. Also, none of 13 personnel working in laboratories adjacent to those where lung cancer tests were performed were reactive with 7661. None of the 16 blood bank donors and none of 11 secretarial and clerical staff who worked in biochemical laboratories were positive. Reactivity was no correlated with a smoking history. Thus development of reactivity appeared to require direct contact with lung cancer patients or materials. The results suggested a horizontal transmission of reactivity against an antigen associated with lung cancer.

Cellular microcytotoxicity in human tumor systems: analysis of results.

Using the tritiated-proline microcytotoxicity assay with cultured target cells, we tested a large series of melanoma, breast cancer, and bladder cancer patients for the presence of cell-mediated immunity. Specific, disease-related activity was infrequently observed, since the patients' lymphocytes exhibited selective activity against both disease-related and non-disease-related target cells. Most normal controls also demonstrated selective activity against these target cells. Neither the length of time the target cells had been cultured in vitro nor technical aspects of the assay, including the lymphocyte preparation methods, seemed to account for our results. We concluded that the experimental design of these tests may be the critical factor responsible for many of the disparate results being observed in different laboratories.

Lymphocytes forming rosettes with sheep erythrocytes in metastatic pleural effusions.

Percentages of lymphocytes forming rosettes with sheep erythrocytes at 29 degrees C were determined in 10 cancer patients with metastases to the pleural cavity. Compared with normal controls, the patients showed a decreased proportion of rosette-forming cells (RFC) in the peripheral blood. The same patients had elevated levels of RFC in their metastatic pleural effusions. However, 2 patients with benign diseases had normal levels of RFC in their peripheral blood and pleural transudates. These observations suggested that in cancer patients some T-cells might migrate from the peripheral blood and accumulate in sites of tumor infiltration such as the pleural cavity.

Cell-mediated immune responses of breast cancer patients to autologous tumor-associated antigens.

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.

Leukocyte migration inhibition by soluble extracts of MCF-7 tissue culture cell line derived from breast carcinoma.

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.

Leukocyte migration inhibition in patients with Ewing's sarcoma by 3-M potassium chloride extracts of fresh and tissue-cultured Ewing's sarcomas.

Leukocyte migration inhibition (LMI) assays were performed to detect cell-mediated immune reactions against tumor-associated antigens (TAA) of Ewing's sarcoma. With the use of crude antigen preparations obtained by 3M KCl extractions of fresh Ewing's sarcoma or of tissue culture cells derived from a pleural effusion of a Ewing's sarcoma patient, assays were performed with leukocytes from these patients, patients with other cancers, and normal donors. The results demonstrated approximately 60% or greater positive LMI reactivity in Ewing's sarcoma patients, as compared to less than 10% reactivity of normal donors, with the use of extracts of either fresh or tissue-cultured Ewing's sarcoma cells. A lower proportion of positive reactivity was observed in patients with breast and lung cancer. Further specificity tests indicated that a smaller proportion of patients with Ewing's sarcoma had LMI reactivity with KCl extracts of tissue-cultured cells derived from breast cancer of fresh lung cancer cells than did patients with the homologous disease. The results indicate that many patients with Ewing's sarcoma have cell-mediated immunity toward TAA on Ewing's sarcomas. Inasmuch as all the LMI assay were performed with allogeneic extracts, the data also suggested that different Ewing's sarcomas possess common antigens and that some breast and lung cancers may share some TAA with Ewing's sarcoma.

Immunologic relationship between breast carcinoma and benign breast disease as detected by the leukocyte migration inhibition assay.

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.

Lymphocyte blastogenesis induced by potassium chloride extracts of allogeneic breast carcinoma and lymphoid cells.

Lymphocytes from cancer patients and normal individuals demonstrated blastogenesis with allogeneic potassium chloride (3 M KCl) extracts of breast carcinoma cells. Normal individuals reacted with a greater frequency and stronger blastogenic responses to tumor extracts than did breast carcinoma patients; allogeneic extracts may have elicited recognition of normal alloantigens rather than tumor-associated antigens. Normal individuals also responded to 3 M KCl extracts of allogeneic pooled normal leukocytes, normal breast tissue, and other cancers, but did not react to extracts of autologous leukocytes.

Cell-mediated immunity to mouse mammary tumor virus antigens by patients with hyperplastic benign breast disease.

Peripheral blood leukocytes of patients with preoperative breast cancer, benign breast disease, and benign gynecologic disorders and normal healthy females were tested, as blind coded specimens, with murine mammary tumor virus (MuMTV) antigens in the direct and indirect leukocyte migration inhibition (LMI) assays. The incidence of reactivity by patients with breast cancer was low. (From 5 to 35% breast cancer patients reacted, depending on which group of control individuals they were compared to and what antigen was used.) Nonparametric analyses showed no differences between control groups (normal donors and patients with gynecologic disorders) and breast cancer patients with either assay. However, there was a significant difference between benign breast disease patients with hyperplasia and 1) benign breast disease patients without hyperplasia (P less than 0.03) and 2) patients with gynecologic disorders (P less than 0.04) in the direct assay when it was performed blindly with the gp52 antigen. Patients with hyperplasia (benign breast disease as well as breast cancer) had a higher incidence of enhanced migration in the indirect test than breast disease patients without hyperplasia. The enhanced migration to the MuMTV was correlated to enhanced migration to a 3-M KCI extract of the breast cancer cell line MCF-7 in simultaneous tests. Thus the LMI assays with MuMTV antigens do not appear valuable in breast cancer diagnosis, but they may help to identify a small group of benign breast disease patients whose breast pathology is thought to be associated with a high risk for developing breast cancer.

Indirect leukocyte migration inhibition in breast cancer and benign breast disease patients by mouse mammary tumor virus grown in feline kidney cells.

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.

Lack of correlation between natural killer activity and tumor growth control in nude mice with different immune defects.

To elucidate the in vivo role of natural killer (NK) cells, the growth of several murine and human tumors was studied in four variants of athymic, nude mice with different levels of NK activity. Beige-nude mice, homozygous for both the beige and the nude genes, had very low levels of NK activity, and their response to the B-cell mitogen, bacterial lipopolysaccharide, was lower than that of high-NK, adult NIH nude mice. Young and adult NIH nudes had different NK levels and showed different response in assays for K-cell, T-cell, and B-cell activity. The B-cell-defective NIH-II mice had slightly lower NK levels than adult NIH animals, but much lower response in the antibody-dependent cell-mediated cytotoxicity assay. No correlation was found between host NK activity and the s.c. growth of various human (LOX, CEM, K562) and murine (YAC-1) tumor cells. Low NK activity was not associated with increased lung colony formation in a metastasis model using i.v.-injected human (LOX) and murine (B16F10) melanoma cells. No relationship was found between host NK activity and the rate of elimination of i.v.-injected 5-iodo-2'-deoxyuridine-labeled LOX, B16F10, and YAC-1 cells from lungs, liver, or spleen. The results fail to support the view that NK cells exert significant direct effects on tumor cells in vivo.

Immunologically defined prognostic subgroups as predictors of response to BCG immunotherapy.

Fifty-two stage I and II patients with nonsmall cell lung cancer were randomized after resection to no further therapy, BCG, or BCG plus allogeneic tumor cells. Patients have been observed for 16-65 months (mean 39.5). When the two immunotherapy arms were combined and plotted against controls, trend analysis suggested (P = 0.088) an increase in disease-free interval (DFI) only for stage I patients. The one-way mixed lymphocyte culture (MLC) values were depressed in some patients prior to any immune stimulation. Immunotherapy significantly benefited DFI among patients with a depressed MLC.

Variations in the immune response to Herpesvirus saimiri in squirrel and rhesus monkeys.

Humoral and cell-mediated immunity (CMI) to herpesvirus saimiri (HVS), an oncogenic lymphotropic herpesvirus, was studied in squirrel and rhesus monkeys. Natural antibody to HVS was found in five of six squirrel monkeys but there was no evidence of specific CMI directed against HVS. Rhesus monkeys did not show natural antibody or CMI against HVS antigens. Immunization with HVS, however, produced both antibody and specific CMI in the rhesus monkeys, but no CMI developed in the squirrel monkeys. These findings are important in the development of animal models for the treatment of tumors associated with lymphotropic herpesviruses.

Cell-mediated immune responses--prognostic indicators of survival from breast cancer.

The prognostic significance of cell-mediated immunity measured by a single post-operative in vitro lympho-proliferation assay with an autologous tumor hypotonic membrane preparation (HMP) and in the mixed leukocyte culture (MLC) is shown in white and black breast cancer patients followed for as long as 14 years. Nine of 16 white patients and 6 of 11 black patients who did not respond to their tumor but who had a normal MLC died of systemic breast cancer as compared to 1 of 20 white and 2 of 5 black patients who responded to the HMP and/or had a depressed MLC. When high-risk clinical variables (nodal status, tumor size and cell differentiation) were combined with high-risk immunological risk factors in white patients, the prognostic values of both the immunological and clinical categories were enhanced. Seven of 9 concordant high-risk white patients died from systemic breast cancer. These results point to the necessity of immunological testing as an aid in clinical staging of breast cancer patients.

Relationship of human natural lymphocyte-mediated cytotoxicity to cytotoxicity of breast-cancer-derived target cells.

Mononuclear cells from 115 individuals were tested in a 4-h chromium release assay against two breast-cancer-derived cell lines, G11 and MCF-7, and a myeloid line, K-562, shown previously to be sensitive to natural cytotoxicity. These data were analyzed in a manner designed to detect hyperreactivity against the breast cell lines relative to the level of reactivity against K-562. A high proportion of breast cancer patients were found to be relatively hyperreactive against G11 (12/18 or 67%) and against MCF-7 (10/18 or 56%). Fibroadenoma patients were very similar to the normal females, with 0/11 hyperreactive to G11 and 1/11 (9%) to MCF-7. However, several normal males (7/17 or 41%) were hyperreactive to G11 but not to MCF-7 (2/17 or 12%). Colon cancer and lung cancer patients were also more hyperreactive to G11, 4/8 or 50% and 4/6 or 67%, respectively, than they were to MCF-7, 1/8 or 13% and 1/6 or 17%, respectively. Only fibrocystic patients resembled the breast cancer patients, with some but not as many individuals being hyperreactive to G11 (3/8 or 38%) and to MCF-7 (2/8 or 25%). With another group of individuals reproducibility of the method was demonstrated, with only 1/14 or 7% of normal females and 12/17 or 70% of breast cancer patients being hyperreactive to G11. Thus, natural cytotoxicity toward K-562 can be related to breast cancer-associated cytotoxicity toward MCF-7 in a way that distinguishes a majority of breast cancer patients specifically from other groups of individuals.