BCG inhibition of murine leudemia: local suppression and systemic tumor immunity require different doses.

The quantitative relationship between bacillus Calmette-Guérin (BCG) and tumor cells which are optimal for suppressing the growth of tumor cells in BCG-tumor cell mixtures are detrimental to the development of a sustained, systemic tumor rejection immunity in the LSTRA murine leukemia.

Mammary gland hyperplastic alveolar noduligenesis in the LEW/Mai rat in relation to mitotic activity, estrous cycle status, and age.

Spontaneous mammary hyperplastic alveolar noduligenesis was studied in nulliparous female LEW/Mai rats in relation to age, estrous cycle status, and mammary gland mitotic activity. The morphometric composition of the mammary gland and the incidence of hyperplastic alveolar nodules (HAN) and dysplasias were determined by examination of stained wholemount preparations of one of the inguinal mammary glands. Mitotic activity in the contralateral mammary gland rose almost linearly from 30 to 108 days of age and then fell progressively thereafter, although mitotic activity remained significantly elevated compared to that in mammary glands of rats less than 100 days old. Mitotic activity was nonrandomly distributed in the mammary glands of young pubertal rats in contrast to that in glands of prepubertal rats and in rats in persistent estrus, in which it tended to be randomly distributed. HAN were present in some rats by 150 days of age and were frequent in the mammary glands of most rats older than 200 days. All animals with HAN were in persistent vaginal estrus and had significantly increased pituitary weights. This association suggested that hormone imbalance may be an important etiologic factor in spontaneous hyperplastic alveolar noduligenesis in the rat.

Suitability of rat mammary adenocarcinoma 13762 as a model for BCG immunotherapy.

We determined the optimal conditions for conducting experiments with the solid and ascites sublines of the 13762 rat mammary adenocarcinoma and examined the response of the tumor growth rate to BCG administered in admixture with tumor cells or separately at a remote site. Versene dissociation of the 13762 solid tumor produced better growth rates than did pronase-DNase, but the former decreased cell viability and yields. A dose of 10(6) or 10(5) tumor cells produced 100% growth by the sc and iv routes. Both sublines grew slower but produced metastases slightly sooner in the intradermal than in the sc site. The frequency of axillary lymph node metastases from the sc site increased as a function of the duration of the time interval between tumor implantation and surgical excision. Both solid and ascites tumors were weakly immunogenic. Administration of BCG in a split adjuvant protocol did not improve tumor immunity. Admixture of tumor cells with BCG suppressed tumor growth but when given at a remote site, BCG was ineffective. We concluded that the 13762 rat mammary adenocarcinoma is a useful system for BCG immunotherapy.

Evaluation of antitumor activity of Bordetella pertussis in two murine tumor models.

The antitumor activity of three preparations of killed Bordetella pertussis (Bp) (Eli Lilly crude and fluid pertussis vaccines and Parke-Davis pertussis vaccine) was studied in the B16 melanoma and CaD2 mammary adenocarcinoma models. In these tumor systems; Bp had weak and variable tumor inhibitory activity and did not augment tumor rejection immunity. The intratumor injection of Bp did not affect the growth of the B16 tumor but significantly inhibited the growth of the CaD2 tumor. However, the established tumor did not regress. Admixture of Bp with B16 cells before inoculation inhibited tumor growth and prolonged survival of inoculated mice. Admixture of Bp with CaD2 cells completely suppressed tumor cell growth in 60% of inoculated mice. Intratumor injection of CaD2 with Bp combined with surgery provided no protection against subsequent development of metastases.

Relationship between intradermal tumor suppression and tumor immunity.

Intradermal (id) injection of three tumor-immune stimulant mixtures (LSTRA-BCG, 13762A-BCG, CaD2-Corynebacterium parvum) was superior to the sc site for suppression of tumor growth: Suppression of LSTRA-BCG mixtures was even less efficient after an ip or iv injectiouppression at all four sites. In the LSTRA-BCG model, the id site was not uniquely favorable for either the afferent or efferent limb of the immune response; the other sites produced equally effectiveimmunization or rejection of tumor challenge. We concluded that local suppression of tumor cell-immune stimulant mixtures was frequently more effective in the skin than at other sites, that local tumor suppression did not depend primarily on tumor immunity, and that afferent and efferent tumor immunity were equally efficient by the four routes tested.

Inconsistent response of B16 melanoma to BCG immunotherapy.

We evaluated the potential of the B16 melanoma of mice as a model system for BCG immunotherapy of malignant melanoma. We studied a variety of treatment protocols: a) BCG given simultaneously but separately with a small number of B16 cells significantly inhibited tumor growth in only three of eight experiments. b) BCG injected directly into the tumor stimulated tumor growth in three of three experiments; the stimulation was at least partially attributable to the nutrient medium in which the BCG was suspended. c) The B16 tumor was weakly immunogenic and the addition of BCG to a tumor cell vaccine offered little improvement in subsequent resistance to tumor cell challenge: d) In a model of postsurgical residual tumor, metastatic to regional lymph nodes, BCG and tumor cell vaccination did not alter the development of nodal metastases. The B16 melanoma was not a useful model system for BCG immunotherapy, because the tumor inhibition was feeble, inconsistent, and not associated with augmented tumor immunity.

Antitumor activity of killed Corynebacterium parvum suspensions in a murine mammary adenocarcinoma CaD2) system.

We studied the antitumor activity of killed Corynebacterium parvum on the CaD2 mammary adenocarcinoma in DBA/2 mice. Intratumor treatment had little or no effect on subcutaneous tumor growth. Admixture of tumor cells with C. parvum before inoculation completely suppressed tumor growth. No tumor transplantation immunity was detected in mice inoculated with admixtures of C. parvum and tumor cells, but tumors were enhanced under certain circumstances. Growth of a tumor inoculated sc or iv was consistently retarded after iv or ip C. Parvum therapy, but tumors rarely regressed. Tumor transplantation immunity was detected in mice treated iv with C. parvum before surgical excision of established tumors. Certain adverse effects of iv or ip administration of C. parvum were also discussed.

Effects of dose and schedule of immune stimulant on efficacy of combination Corynebacterium parvum-cyclophosphamide treatment for a murine mammary adenocarcinoma.

Certain variables which might influence the outcome of combining cytotoxic drug and immune stimulant therapy were studied to optimize the effectiveness of Corynebacterium parvum combined with cyclophosphamide (CY) as treatment for a murine mammary adenocarcinoma (CaD2). Optimal effects of combined C. parvum-CY treatment in the CaD2 system were obtained when 443 to 1400 microgram of this immune stimulant per mouse were injected 2 to 3 days after CY chemotherapy and when combination treatment was continued on a weekly basis. The most critical factors contributing to the effectiveness of combination treatment in this system were the dose of C. parvum and the treatment frequency. The interval between chemotherapy and immune stimulant therapy was less critical to the outcome of combination treatment. Combination treatment given once or weekly significantly decreased tumor size in comparison to single or weekly CY treatment. A single treatment with CY and C. parvum significantly improved the survival over mice given a single CY treatment, but weekly CY and C. parvum treatment did not increase the survival over mice, given weekly chemotherapy.

Immunocytochemical evaluation of human prostatic carcinomas for carcinoembryonic antigen, nonspecific cross-reacting antigen, beta-chorionic gonadotrophin, and prostate-specific antigen.

The unlabeled antibody peroxidase-antiperoxidase technique was used to examine human malignant prostatic tissue (primary tumors) for the presence of prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), nonspecific cross-reacting antigen (NCA), and beta-chorionic gonadotrophin (HCG). The results were compared to those obtained with normal and hyperplastic prostate tissue (BPH). All specimens of neoplastic, hyperplastic, and normal prostate tissue showed immunostaining reactions for PSA. Immunostaining for PSA was relatively uniform among samples of normal and BPH tissue, but variations with respect to intensity of PSA immunostaining were noted among prostate tumors as well as between the neoplastic cells of individual tumors. Some areas of normal or hyperplastic prostatic epithelium within tumors showed stronger staining reactions for PSA than the tumor cells themselves. Using an antiserum which was able to detect both NCA and CEA, it was found that 16 of 38 tumors (42%) had positive immunostaining reactions. Of these, 15 were subsequently shown to contain only NCA immunoreactivity, and 1 tumor had both NCA and CEA immunoreactivity. NCA, but not CEA, immunoreactivity was identified in hyperplastic prostate tissue within tumor specimens and in BPH specimens. Neither antigen was detected in normal prostatic epithelium. Three of 38 tumors (8%) were found to contain neoplastic cells with HCG immunoreactivity. HCG immunoreactivity was not identified in BPH or normal prostatic tissue. Therefore, HCG and CEA immunoreactivity appear to be tumor-associated antigens in prostate cancer which are expressed with a low incidence. The results of the study identified prostate tumors with different patterns of immunocytochemical markers: 22 of 38 tumors (58%) contained only PSA immunoreactivity; 13 of 38 tumors (34%) contained PSA and NCA immunoreactivity; 2 of 38 tumors (5%) were positive for PSA, NCA, and HCG immunoreactivity; and 1 of 38 tumors (3%) contained PSA, NCA, HCG, and CEA immunoreactivity. Apart from PSA, which was present in all tumors, the markers studied here appeared to be more frequently expressed in well-differentiated tumors than in less-differentiated tumors. Our results suggest the possibility of subclassifying prostate tumors by means of immunocytochemistry.

Destruction of regional lymph node metastases of rat mammary adenocarcinoma 13762A by treatment with Corynebacterium parvum.

Intratumoral administration of Corynebacterium parvum to 13762A tumor-bearing rats on Day 7 of tumor growth, followed by primary tumor excision on Day 20, regularly cured about 40% of the animals and significantly prolonged survival in the remainder. Rats treated by surgery alone on either Day 7 or Day 20 died with metastases to axillary lymph nodes and lungs. Tumor was established in axillary lymph nodes by Day 7. Therefore, intratumoral injection of C. parvum on Day 7 destroyed metastases already established at this site. Growth of tumor in axillary nodes of rats treated but not cured by C. parvum was significantly slower than growth in untreated rats.

Corynebacterium parvum and cyclophosphamide as combination treatment for a murine mammary adenocarcinoma.

Weekly i.p. injections of killed Corynebacterium parvum and of cyclophosphamide (given on different days) strongly inhibited growth of a transplantable murine mammary adenocarcinoma. A significant portion (40 to 80%) of animals could be made tumor free by means of combined therapy. No tumor-free survivors were obtained with C. parvum alone, and tumor-free mice were obtained with cyclophosphamide alone only at the expense of a high incidence of deaths due to drug toxicity. No evidence of tumor rejection immunity was detected in the tumor-free survivors from the combined treatment protocols, suggesting that this therapeutic regimen is not associated with tumor rejection immunity.

Conditions for effective Bacillus Calmette-Guérin immunotherapy of postsurgical metastases of 13762A rat mammary adenocarcinoma.

We evaluated critical variables in Bacillus Calmette-Guérin (BCG) immunotherapy of residual 13762A rat mammary adenocarcinoma. BCG was given intratumorally on Day 7 of tumor growth and followed by primary tumor excisions on Day 20. Untreated animals died on about Day 40 with axillary nodal and pulmonary parenchymal metastases. BCG-treated animals experienced prolonged survival, and some were cured. The highest dose (5.0 X 10(7) colony-forming units) of BCG was more effective than the lowest (0.5 X 10(7) colony-forming units), but 1,500 micrograms Corynebacterium parvum were more effective than even the highest BCG dose. Previous sensitization to BCG did not improve the effects of BCG treatment. BCG treatment was effective when given on Day 7 and sometimes as late as Day 12 or 17, but C. parvum was ineffective if given after Day 7. Repeated injections of BCG or C. parvum were not more effective than single injections were. Rats cured of residual 13762A tumor by BCG treatment were strongly and specifically immune to rechallenge. We concluded that a high dose (5.0 X 10(7) colony-forming units) of BCG given early (7 days) was the most effective presurgical treatment of 13762A metastases. Repeated injections or host presensitization to BCG did not improve the benefits.

Immunotherapy of an established rat mammary adenocarcinoma (13762A) with intratumor injection of Corynebacterium parvum.

We studied the effects of intratumor injection of Corynebacterium parvum vaccine on the survival of 13762A tumor-bearing rats. Vaccine injection of established (7-day-old) tumors produced dose-related prolongation of survival and cured some animals. Although 30 to 40% of the vaccine-injected primary tumors regressed, recurrences and continued growth of metastases ultimately killed one-fourth of the regressors. Rats given 1500 microgram of C. parvum intratumorally at 7 days, with or without later primary tumor excision at 20 days were cured at a rate of 10 to 40%. Repeated injections improved the results (60%). C. parvum injections delayed until 12 and 17 days were ineffective. Cured rats were immune to rechallenge with 13762A tumor.

Comparative antitumor effects of Corynebacterium parvum, Bordetella pertussis, Bacillus Calmette-Guérin, and levamisole alone or in combination with cyclophosphamide in the CaD2 murine mammary adenocarcinoma system.

The antitumor efficacy of various immune stimulants [Corynebacterium parvum, Bordetella pertussis, and Bacillus Calmette-Guérin (BCG)] and levamisole alone or in conjunction with cyclophosphamide (CY) was studied in the CaD2 mammary adenocarcinoma system using schedules developed previously with C. parvum and CY. Weekly systemic treatment with C. parvum, B. pertussis, or BCG was effective in controlling tumor growth and had equivalent antitumor effects, but weekly treatment (or a single treatment) with levamisole was ineffective. Weekly treatment with B. pertussis was better than treatment given only once, but repeated treatment with C. parvum or BCG was not more effective than a single treatment with these agents. When administered as a single systemic injection, C. parvum was superior to B. pertussis in controlling tumor growth, but a single systemic injection of BCG was as effective as C. parvum. Systemic administration of immune stimulants had variable effects on survival, which were sometimes not correlated with effects on tumor size. Combined treatment with BCG and CY was significantly more effective than CY treatment alone in controlling tumor growth in most trials, as was combination treatment with C. parvum and CY. Combination treatment with B. pertussis and CY was not better in prolonging survival than CY alone. Levamisole rarely improved the antitumor effect of CY chemotherapy and had no effect on survival compared to the effect of CY alone.

Immunocytochemical evaluation of primary human esophageal carcinomas and their xenografts for keratin, beta-chorionic gonadotropin, placental lactogen, alpha-fetoprotein, carcinoembryonic antigen, and nonspecific cross-reacting antigen.

The unlabeled antibody peroxidase-antiperoxidase technique was used to examine esophageal neoplasms for the tumor markers beta-human chorionic gonadotropin, human placental lactogen (HPL), alpha-fetoprotein, carcinoembryonic antigen (CEA), and nonspecific cross-reacting antigen (NCA) before and after xenotransplantation to athymic nude mice. In addition, keratin was used as an epithelial cell marker. Immunoreactive beta-human chorionic gonadotropin was detected in four of seven primary tumors and in three of seven xenografts. Two of seven primary tumors contained HPL immunoreactive cells while four of seven tumor xenografts had HPL immunoreactivity. alpha-Fetoprotein was detected in two of seven primary tumors and in one of seven xenografts. NCA and CEA were detected in six of seven primary tumors and in all tumor xenografts. Five of seven primary neoplasms and six of seven tumor xenografts were found to contain both NCA and CEA, while one tumor and its xenografts displayed only NCA immunoreactivity. All seven primary carcinomas displayed keratin immunoreactivity, but only six of the seven xenograft tumors showed keratin positive cells. When a tumor marker was detected in a primary tumor, it was usually found in at least some of the xenografts arising from that tumor. However, marker loss did occur with repeated passage of tumors in some cases. On the other hand, markers were expressed in xenografts which were not present in the corresponding primary tumor. In three instances, HPL was detected in xenografts derived from HPL negative primary carcinomas. This was also true for CEA and NCA in one case. These results show that tumor markers are expressed to varying degrees by tumors growing as xenografts in nude mice. In primary tumors, HPL is associated with poorly differentiated squamous cell carcinomas and this marker was found to appear in HPL negative tumors as the tumor cells became less differentiated while growing as xenografts in nude mice.

Immunocytochemical evaluation of human esophageal neoplasms and preneoplastic lesions for beta-chorionic gonadotropin, placental lactogen, alpha-fetoprotein, carcinoembryonic antigen, and nonspecific cross-reacting antigen.

Human esophageal neoplasms were studied in comparison to normal, uninvolved, and preneoplastic human esophageal epithelium for the presence of human chorionic gonadotropin (HCG), human placental lactogen (HPL), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and nonspecific cross-reacting antigen (NCA) using the unlabeled antibody peroxidase-antiperoxidase technique. HCG immunoreactivity was identified in 10 of 33 squamous cell carcinomas (33%), in 1 of 6 adenocarcinomas (17%), and 1 of 6 preneoplastic esophageal lesions (17%); while 9 of 33 squamous cell carcinomas (33%) and 1 of 6 adenocarcinomas (17%) contained immunoreactive AFP. Immunoreactive HPL was detected in 6 of 33 squamous cell carcinomas (20%), but in none of the adenocarcinomas. Neither AFP nor HPL immunoreactivity was identified in the 6 hyperplastic lesions which were studied. When stained with an antiserum that was able to detect both CEA and NCA, 27 of 33 squamous cell tumors (82%) and 6 of 6 adenocarcinomas (100%) showed positive immunostaining reactions. Of these, 8 squamous cell carcinomas and 1 adenocarcinoma were subsequently shown to contain only NCA immunoreactivity, while 19 squamous cell carcinomas and 5 adenocarcinomas contained both NCA and CEA immunoreactivity. NCA immunoreactivity alone was identified in 3 of 6 preneoplastic lesions and NCA and CEA immunoreactivity in 1 of 6 preneoplastic lesions. None of the markers was detected in 8 specimens of normal esophageal epithelium which were studied as controls, nor in 6 specimens of uninvolved esophageal epithelium obtained from patients with esophageal cancer. Most tumors expressed 2 or 3 markers, and some tumors were identified which expressed up to 4 of the 5 markers investigated. Only 3 tumors failed to express any of the markers studied. No association was found between the degree of tumor differentiation and presence or absence of HCG immunoreactivity. However, HPL immunoreactivity was more common in poorly differentiated squamous cell carcinomas. In contrast, immunoreactive AFP was more common in well-differentiated squamous cell carcinomas than in other tumor types. Similarly, both CEA and NCA were more frequently expressed in well-differentiated squamous cell carcinomas, adenosquamous carcinomas, and adenocarcinomas than in less differentiated tumors. Our results suggest that HCG, HPL, AFP, CEA, and NCA are tumor-associated antigens in esophageal cancer. Therefore, they could be of value in screening tests for esophageal neoplasms and could be useful in subclassification of esophageal neoplasms.

Expression of carcinoembryonic antigen, T-antigen, and oncogene products as markers of neoplastic and preneoplastic colonic mucosa.

Several crypt abnormalities have been demonstrated in the mucosa of neoplastic and preneoplastic lesions of the large intestine. In addition, certain tumor markers are expressed in large intestinal carcinoma but not in normal mucosa. To determine whether any correlation exists between tumor marker expression and crypt abnormalities and at what stage markers are expressed, we studied specimens of large intestinal mucosa from 13 patients with preneoplastic conditions (adenomatous polyp, familial polyposis, Crohn's disease, and ulcerative colitis). The tumor markers examined include carcinoembryonic antigen (CEA), the ras gene products p21 and p21ser (mutated form), and beta-D-galactosyl-(1----3)-alpha-N-acetyl-D-galactosamine (gal--gal NAc, also known as T-antigen). Results were compared to those of five cases of adenocarcinoma of colon and three control cases of colonic mucosa obtained at immediate autopsy. All four markers were expressed in three of the five cases of adenocarcinoma, but none were expressed in the control cases. Variable expression of each marker was demonstrated in the dilated, distorted crypts of preneoplastic lesions. CEA and gal--gal NAc appeared to be expressed most frequently, suggesting that these are common markers or are expressed at an earlier stage in the neoplastic process than p21 or p21ser. Demonstration of such markers in preneoplastic conditions may be of use in determining the malignant potential and in monitoring these lesions.

Genotoxic effects of five polycyclic aromatic hydrocarbons in human and rat mammary epithelial cells.

Five polycyclic aromatic hydrocarbons (PAHs) of different carcinogenic activities were evaluated for their effects on DNA synthesis (3HTdR labeling index (L.I.] of rat and human mammary epithelial cells (MEC) and for their effects on chromosomes in MEC-mediated sister chromatid exchange (SCE) assays. When compared with DMSO-treated cells, exposures of rat MEC to the two most potent carcinogens (5 micrograms/ml for 24 hr), i.e., 7,12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (B[a]P), resulted in a 45-62% reduction in the L.I. of rat MEC. Another carcinogen, 20-methylcholanthrene (MCA), produced a 35-48% reduction in L.I., while the noncarcinogenic PAHs, 1,2-benzanthracene (BA) and benzo(e)pyrene (B[e]P), showed no effect. Similarly, exposures of human MEC to DMBA and B[a]P resulted in a 50-90% depression in L.I. while BA was significantly less effective (30% reduction). When co-cultivated with Chinese hamster V-79 cells in the presence of PAH, both rat and human MEC can activate and release the active metabolites to induce SCE in V-79 cells. In the rat MEC-mediated assay for all 5 PAHs, the frequencies of SCE per chromosome in DMBA-, B[a]P-, MCA-, BA-, B[e]P-, and DMSO (solvent control)-treated groups were 6, 3, 1.4, 0.7, 0.4, and 0.3, respectively. DMBA was most effective in increasing SCE, while B[e]P was ineffective. In the human MEC-mediated assay, B[a]P was more effective than DMBA in inducing SCE, and the frequencies of SCE per chromosome were 4.5 and 3.6 in B[a]P- and DMBA-treated groups, respectively. Comparing depression of L.I., SCE, and in vivo carcinogenicity for the 5 PAHs, SCE mediated by rat MEC is better correlated with carcinogenicity in rat than L.I. depression.