Tumor-specific cell-mediated immunity in household contacts of cancer patients..

Patients with osteogenic sarcoma (and related tumors), hypernephroma, and breast carcinoma, and their household contacts were tested for tumor-specific cell-mediated immunity against these tumors with the use of a short-term chromium-51 release assay. This assay, reproducible over many months and well-correlated with the clinical course of the patients, was used to demonstrate that household contacts of patients with osteogenic sarcoma and breast carcinoma have specific immunity against the tumor type with which they have been in contact. In both types of tumors, the range of cytotoxicity values produced by lymphocytes from the household contacts was significantly higher than that of the normal population. The incidence of immunity was much higher in household contacts of patients with breast carcinoma than in those of patients with osteogenic sarcoma. Immunity was found with equal frequency in men and women, as well as in genetically and nongenetically related household contacts (guardians, adopted children, spouses). Immunity against hypernephroma was not demonstrated in either patients with hypernephroma or their household contacts.

Osteogenic sarcoma. Immunologic parameters before and during immunotherapy with tumor-specific transfer factor.

18 patients with osteogenic sarcoma were followed by serial measurements in vitro of tumor-specific cell-mediated cytotoxicity and of "active" and total rosette-forming T-cells. 13 of these patients have had or are currently receiving injections of osteogenic sarcoma-specific dialyzable transfer factor derived from healthy donors. In three patients with very small lesions, cytotoxicity was high before amputation and decreased within 2 mo after removal of tumor. Cytotoxicity was low at time of diagnosis in all patients with large tumor masses. The cytotoxicity of the patients' lymphocytes increased after administration of tumor-specific transfer factor in all patients so treated. Patients receiving nonspecific transfer factor showed evidence of declining cell-mediated cytotoxicity. Tumor-specific transfer factor may produce an increase in cell-mediated cytotoxicity to the tumor in patients with osteogenic sarcoma. This possibility is suggested by the pain and edema that occurred in the area of the tumor in patients who had metastatic disease when therapy was started and by lymphocytic infiltrates in the tumor, as well as by the increase in cell-mediated cytotoxicity and the increase in percentage of active rosette-forming cells from subnormal to normal. Serial measurements of cell-mediated cytotoxicity are helpful in monitoring the efficacy of transfer factor and other modes of therapy in these patients, and these measurements are the best available criteria for selection of donors of tumor-specific transfer factor.

Internal antigens accessible in breast cancer: implications for tumor targeting.

Proponents of monoclonal antibody (MAb)-mediated cancer therapy often assume that a major limitation in clinical application of MAbs is their lack of absolute specificity for malignant cells. In addition, the presence of surface target antigens is thought to be essential. These requirements may be more stringent than necessary for the clinical usefulness of MAbs. We have demonstrated selective localization of a MAb to keratin polypeptides in malignant breast epithelium under conditions of passive infusion of antibody in fresh surgical specimens of breast carcinoma. Although these proteins are normal intracellular constituents of epithelial cells throughout the body, localization of antikeratin antibodies only within the tumor population is most probably associated with the presence of cells permeable to macromolecules. This permeable tumor cell fraction could be recruited for targeting neighboring impermeable tumor cells with radioisotopes or other antitumor agents conjugated to antibodies directed against intracellular antigens.

Expression of basal and luminal epithelium-specific keratins in normal, benign, and malignant breast tissue.

We characterized the subclass-specific keratins in the epithelium of normal, benign, and malignant breast tissue. Monoclonal antibody 34BE12 stained luminal as well as basal epithelium in normal and benign specimens and all tumor cells in malignant specimens. Antibody 312CS-1 reacted only with basal cells, and antibody LE61 reacted only with luminal cells in the normal and benign specimens. In 34 of 36 breast carcinomas examined, the basal and luminal cell-specific antibodies showed complementary patterns of reactivity, while in the remaining 2 specimens, neither antibody was reactive. The findings reported in this study demonstrate that expression of subclass-specific keratins is mutually exclusive not only in normal and benign mammary specimens but also in breast carcinoma. These findings suggest a role for epithelial subclass-specific antibodies in the histogenetic and prognostic subclassifications of breast carcinoma.

Response to doxorubicin of cultured normal and cancerous human mammary epithelial cells.

Epithelial cells were isolated and cultured from a number of human mammary specimens of both cancerous and noncancerous origin. Doxorubicin (Dx) sensitivity was measured at second passage with the use of a highly efficient clonogenic assay. For 23 different tumor specimens derived from patients without previous chemotherapy, the drug concentrations required to kill 50% of the cells varied approximately 35-fold. In contrast, for 11 tumor specimens from patients who relapsed after regimens containing Dx, the drug concentration for 50% survival varied only fivefold and the dose-response curves for these specimens clustered at the more resistant end of the spectrum. A wide range of sensitivities was also observed among 13 noncancerous mammary specimens; however, tumor tissue and noncancerous tissue from the same donor were similar. When cultures were subjected to drug incubation periods of 1 and 4 hours, dose-response curves were superimposable when plotted as a function of drug concentration multiplied by time.

Immortalization in culture: occurrence at a late stage in the progression of breast cancer.

The properties in culture of 3 breast cancer effusion metastases, obtained over approximately 2 years from the same patient, were examined. Despite repeated attempts with cryopreserved cells, only the last specimen reproducibly exhibited immortality in culture; the first 2 specimens grew initially but failed to develop into cell lines. Each specimen was unique in morphology and growth properties, although karyotypic markers indicated a common origin. Aberrations of chromosomes 1 and 11 marked these near-diploid cells, and further structural alterations of chromosome 11 accompanied the transition of biological properties observed in the third specimen.

Two syngeneic cell lines from human breast tissue: the aneuploid mammary epithelial (Hs578T) and the diploid myoepithelial (Hs578Bst) cell lines.

We characterized two human cell lines (Hs578T and Hs578Bst), which provide several unique features that should be useful in the study of breast disease. Hs578T, derived from a carcinosarcoma, is epithelial in origin. Hs578Bst, established from normal tissue peripheral to the tumor, is myoepithelial in origin. This is the first report of companion cell lines, one malignant and one normal, established from the same organ.

Nuclear ultrastructure of epithelial cell lines derived from human carcinomas and nonmalignant tissues.

The nuclear ultrastructure of sixteen human epithelial cell lines has been characterized in detail by transmission electron microscopy. The cell lines were derived from normal tissues, nonmalignant tissues of cancerous organs, primary carcinomas, and metastatic carcinomas. Every cell section on a grid containing a clearly defined nucleus and nucleolus was scored blindly utilizing a checklist of markers. The goal of these studies was to determine whether any ultrastructural markers consistently distinguished the different stages of malignant progression represented among the lines. Nuclear bodies and perichromatin granules were found in all lines derived from cancer and were not observed in any nonmalignant lines. Nuclear envelope dilation was seen in all lines derived from cancerours organs as well as from malignant tissues but not in any lines derived from normal tissue. Margination of chromatin, irregularity of nuclear outline, redistribution of nucleolar components, and margination were expressed slightly by the normal lines, to variable degrees by the lines derived from cancerous organs, and to a much greater extent by all lines derived from malignant tissues. No differences were found between lines derived from primary carcinomas and those derived from metastatic specimens. There were no ultrastructural differences comparing subconfluent and confluent cells or cells at different passage levels. In addition, the nuclear ultrastructure of a malignant line in culture was similar to that of a tumor induced by those cells in an immunosuppressed mouse.

Quantitative immunofluorescence studies of the tumor antigen-bearing cell in giant cell tumor of bone and osteogenic sarcoma.

Tumor-associated antigen was found by reacting sera from two patients with giant cell tumor of bone with cells derived from their tumors, using autologous serum as intermediate reactant and fluorescein-conjugated goat anti-human IgG as final reactant. Approximately 40% of the plump, spindle-shaped cells that formed the background stroma of these tumors possessed the antigen; however, it was not present on giant cells. Fluorescence was much greater than that on similarly stained cells from 4 osteogenic sarcomas, suggesting that the antigenic density on cells from giant cell tumor was greater than that on cells from osteogenic sarcoma. Antibodies in sera from giant cell tumor patients and osteogenic sarcoma patients showed specific cross-reactivity. Stromal cells of giant cell tumors were established in culture and retained tumor-associated antigen, whereas giant cells failed to divide and detached from the flask within two weeks. Intensity of fluorescence (antigenic density) decreased with progressive passage levels, but a larger percentage of cells showed fluorescence. At the tenth passage, all cells bore tumor-associated antigen. Cultured cells that were injected s.c. into mice formed progressively growing nodules, the cells of which were morphologically indistinguishable from stromal cells of the original tumor; all cells retained tumor-associated antigen, but antigenic density had decreased to about one-seventh of the value found originally. No giant cells were present in the nodules.

Clonal proliferation of cultured nonmalignant and malignant human breast epithelia.

We have developed a method for clonal growth of human mammary epithelial cells of both nonmalignant and malignant origin. Plating efficiencies of 1 to 50% were obtained by seeding second-passage mammary epithelial cells on fibroblast feeder layers in an enriched medium composed of various hormones and growth factors, as well as conditioned media from three specific human cell lines. Single mammary epithelial cells seeded sparsely onto the fibroblasts underwent at least eight population doublings to form large, readily visible colonies. Optimal colony formation required both feeder cells and the enriched medium. Epithelial colonies containing at least 16 cells were visible 5 days postseeding, and these colonies continued to grow progressively. Plating efficiency and colony size were similar on ultraviolet-irradiated or nonirradiated fibroblasts. The number of colonies formed was proportional to the number of epithelial cells plated. The colonies were identified as epithelial by the presence of human mammary epithelial antigens.

Preliminary correlations of clinical outcome with in vitro chemosensitivity of second passage human breast cancer cells.

These studies describe the clinical correlations of 63 in vitro chemosensitivity assays on breast cancer cells after short-term monolayer culture. Forty-five of the assays were single agent correlations. Based on cut-off values determined empirically, the test accurately predicted resistance for 36 of 41 patients (88%) who did not respond to the drug. It also predicted sensitivity with a high degree of accuracy: 21 of 22 patients (95%) who responded to the drug tested had a sensitive assay. In five cases, two biopsies were evaluated from the same patient. Whenever assays were performed before and after treatment with a given drug, tumor cells from the second biopsy were more resistant in vitro if the patient failed on therapy. If the patient did not fail, but stopped therapy for other reasons, or if there was no intervening therapy with the tested drug, the two biopsies remained similar in drug sensitivity. These results suggest that in vitro chemosensitivity assays which accurately predict both sensitivity and resistance can be obtained with breast cancer cells after short-term culture and that further prospective trials are warranted.

The biology of breast cancer at the cellular level.

Two properties seem fundamental to cancer; heterogeneity and progression (Foulds (1975) Academic Press, New York; Heppner et al. (1979) Commentaries on Research in Breast Disease, Vol. 1 (Bulbrook, R. and Taylor, D.J., eds.), pp. 177-191, Plenum Press, New York). Relatively little is understood about the premalignant stages of human breast disease in vivo. When the disease manifests as invasive carcinoma, its behavior exhibits great diversity, sometimes metastasizing rapidly, while in other cases 10-30 years pass before metastases proliferate. Here we review various aspects of breast cancer in vivo and consider how they predict properties of breast cancer found in culture. All of the experiments are consistent with the hypothesis proposed by Nowell (1976) Science 194, 23-28, that a fundamental aspect of malignancy is an increased genetic instability and that many of the cells within tumors are nonviable results of genetic instability. We suggest that most of the viable cells within primary breast carcinomas are diploid and are not yet capable of aspects of metastatic spread. What these cells have attained is an increased propensity for genetic instability which enables them to generate randomly aneuploid but frequently lethal genetic configurations. Occasionally one of these altered genomes is associated with the ability to proliferate at a metastatic site. This hypothesis implies that metastases from various patients may have arisen by divergent pathways and may also be divergent in many other aspects of their physiology, unrelated to malignancy. Such extreme heterogeneity may hamper attempts to understand fundamental aspects of malignancy. Hence we suggest that the less anaplastic and less divergent diploid cells within the primary carcinomas might be an important resource to gain insights into the critical alterations that are responsible for initiating frankly malignant behavior.

Physiological performance and work capacity of Sudanese cane cutters with Schistosoma mansoni infection.

Physiological tests of work performance and measurement of field productivity were made in 194 Sudanese cane cutters in order to study the effect of Schistosoma mansoni infection. The cane cutters were selected from two age ranges (16-24 and 25-45 years) and subdivided into three clinical groups: not infected, infected with, and infected without clinical signs of hepatosplenomegaly. Men infected with Schistosoma haemotobium, malaria (blood film), or with hemoglobin levels less than 10 g/100 ml were excluded. There was a statistically significant (P less than 0.002) higher mean hemoglobin concentration in those not infected but the mean difference was less than 1 g/100 ml. Submaximal responses to exercise on a stationary bicycle ergometer, oxygen intake, ventilation, tidal volume, cardiac frequency and estimated maximal aerobic power output calculated both in absolute terms and relative to lean body mass and leg volume were similar in the six groups of cane cutters. No significant differences were found in physique, body composition or in thermoregulatory function tests. The cane cutters were found to have little natural acclimatization to heat in terms of sweating capacity when compared with a group of fully acclimatized Sudanese soldiers. The mean productivity (mean daily weight of cane cut per man) was significantly correlated with the individual's estimated maximum aerobic capacity determined in the laboratory, but not with the degree of S. mansoni infection. The noninfected group was less "efficient" (mean productivity:oxygen intake) during cutting than the infected groups but a larger proportion of the noninfected were in their first season of cutting. There was a positive correlation between the number of seasons' cutting experience and the individual's age, degree of infection and mean productivity. Cane cutters studied in this investigation were a relatively fit, active population from whom the more seriously ill were excluded. These results do not, therefore necessarily reflect the effects of S. mansoni on physiological work capacity and productivity of more static populations in areas of high endemicity.

Growth of diploid cells from breast cancers.

Cell cultures were derived from normal and cancerous breast tissues and from metastases by methods that selected for relatively adherent epithelial aggregates. Karyotypic analyses of first or second passage cultures yielded predominantly normal diploid cells. Nonclonal aberrations were more common in tumor-derived than in normal cultures. Three of the cultures that originated from metastases were characterized by abnormal clones. These results support observations based on DNA content, which indicate that a considerable fraction of breast cancers are composed predominantly of diploid cells. They differ greatly from chromosomal findings in long-term cultures of tumor effusions and thus emphasize the karyotypic diversity that can be found in tumors from a single tissue of origin--the breast.

Use of an efficient method for culturing human mammary epithelial cells to study adriamycin sensitivity.

Techniques are described for isolating, cryopreserving, and culturing human mammary epithelial cells of both normal and malignant origin. The cells can be grown either in mass culture or as a clonal assay suitable for quantitating drug sensitivity. With this clonal assay plating efficiencies of 6%-41% were routinely obtained. We examined the response to adriamycin of five different primary carcinoma cultures from patients without prior drug therapy. We were able to detect heterogeneity in response to adriamycin among the breast carcinoma cultures as well as heterogeneity among subpopulations within a single carcinoma. The differences in adriamycin sensitivity were unrelated to growth rates in culture.

Metabolism of progesterone in the pregnant sheep near term: identification of 3 beta-hydroxy-5 alpha-pregnan-20-one 3-sulfate as a major metabolite.

Two pregnant ewes near term were given a single injection of progesterone-4-14C via the left jugular vein, and serial blood samples were taken from the right jugular vein at 5 min intervals over a period of 40 min. Radioactive steroids in the plasma were separated into unconjugated and conjugated fractions which were further isolated and analysed by established methods. The injected hormone was rapidly metabolized with a half-life of approximately 10 min and metabolic clearance rate about 3.5 liters min. The bulk of the metabolites was found in the sulfate fraction from which a major metabolite was identified as 3 beta-hydroxy-5 alpha-pregnan-20-one. From the unconjugated fraction, 20 alpha-hydroxy-pregn-4-en-3-one, a known minor metabolite was also isolated. No radioactive estrogens were found. It is concluded that a major portion of circulating progesterone in the pregnant ewe near term is cleared by 5 alpha-reduction of ring A, followed by sulfo-conjugation.

Testosterone of estradiol treatment of heifers or freemartins to detect estrus in confined dairy cattle.

Dairy heifers and freemartins were injected with either testosterone or estradiol and then used to detect estrus in mature dairy cows maintained indoors year-round on slatted-concrete floors. During a 15-mo period, 1,026 occurrences of estrus were confirmed from 339 cows. Observations by the herdsmen detected 92% of all occurrences of estrus compared to the heifers (39%) and freemartins (23%). There was great variation among animals in their ability to detect estrus. Conception rates were not affected by the method of detection of estrus.

Effect of two superovulation treatments on subsequent fertility in the confined dairy cow.

During a 13-month period, 64 lactating dairy cows of 2 genetic lines, Holstein and crossbred, housed indoors year-round were subjected to 2 superovulations and embryo collections within 112 days post partum. Half of the follicle stimulating hormone (FSH) treatments were given in a descending dosage regimen (Treatment A; 6.5 mg, 5.5 mg, 4.5 mg, and 3.5 mg, twice a day, total 40 mg) over 4 days; the remaining half of the treatments were administered in a constant dosage regimen (Treatment B) of 5 mg twice a day over 4 days. There were no significant differences due to treatment in the number of cows stimulated (more than 2 corpora lutea) nor in the number of ova/embryos collected. However, embryos were obtained from more cows (P<0.05) when treated with the descending dosage regimen. More cows (P<0.05) were stimulated by the superovulatory treatment during the first period than during the second period regardless of the regiment used, treatment A or B. More embryos (P<0.05) were obtained from the Holstein line than from the crossbred line. Fifty-two cows were inseminated at least once after the second embryo collection. Overall, 41 cows (79%) became pregnant after the second collection, requiring up to 4 services. These results suggest that the reproduction of dairy cattle housed indoors year-round is not adversely affected by 2 superovulation treatments and embryo collections within 112 days post partum. The question as to whether the administration of FSH is more efficacious in a descending dosage regimen or a constant dosage regimen was not resolved.

Effect of dose and time of injection of prostaglandin F(2alpha) in cycling ewes.

A study was done to evaluate the efficacy of graded doses of prostaglandin F(2alpha) (PGF(2alpha)) to induce regression of the corpus luteum and hence estrus, in cycling ewes when given on various days of the estrous cycle. One hundred cycling cross-bred ewes were observed twice daily (08:00 and 20:00 h) for marking by raddled vasectomized rams. After estrus was confirmed in marked ewes by assay of plasma progesterone concentration, the ewes were treated in pairs with 0, 5, 10, 15 or 20 mg PGF(2alpha) on day 2, 3, 4, 7, 8, 9, 12, 13, 14 or 15 of an estrous cycle and then exposed to a raddled ram of known libido and fertility. Plasma progesterone levels were determined on the day of, and on the day following PGF(2alpha)-treatment to monitor luteal function. Ewes marked between one and five days after treatment and having a decrease in plasma progesterone were considered to have responded to the treatment. The percentages of ewes responding were 10, 35, 60, 70 and 95 to doses of 0, 5, 10, 15 and 20 mg PGF(2alpha) respectively. Differences due to dose were significant (P < 0.01) with the two higher doses being more effective. There were differences due to the day of injection, with treatments on days 2 and 3 being less effective.

Effect of PMSG on the reproductive performance of totally confined ewes bred at synchronized estrus.

The reproductive performance (fertility, prolificacy and fecundity of totally confined sheep in a controlled breeding program at the Animal Research Centre (ARC) was analyzed to evaluate the effect of pregnant mares' serum gonadotrophin (PMSG) on ewes synchronized for estrus with fluorogestone acetate (FGA). PMSG did not significantly enhance reproductive performance. Fertility for ewes receiving 500 IU PMSG was 57% and for ewes not receiving PMSG it was 47%. Corresponding values for prolificacy were 2.4 and 2.2, and for fecundity, 144 and 103%. The reproductive performance was not affected by any of the other factors (flock, strain, lighting regime, ram age or reproductive status of the ewe) indigenous to the controlled breeding program for sheep housed in total confinement.