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Local immunotherapy is a form of cancer treatment where exogenous antigen is introduced into the area of the tumor. Under favorable circumstances, the perfused tumor regresses, systemic tumor-specific transplantation immunity is augmented, and distant microscopic metastases regress. Successful local immunotherapy requires an immunologically competent host, small tumor burden, and tumor located usually in the skin. A wide variety of biologic agents are capable of promoting local immunotherapy. BCG has been most widely studied. The antitumor activity of two different preparations of the Tice substrain of BCG were compared. No significant differences in antitumor activity were found. Alternative approaches to intralesional injection were sought. Intradermal injection of BCG adjacent to dermal tumors, prior to surgery, led to eradication of axillary metastases and to the development of tumor-specific transplantation immunity.

Guinea pigs with established intradermal tumors and microscopic lymph node metastases were treated by intralesional injection of graded doses of living BCG or BCG CW. The lowest dose of living BCG used produced a significant cure rate and no grossly evident toxicity. An intermediate and the highest dose of living BCG used cured some animals but others lost weight and a significant number died as a result of the treatment. Histologic examination of animals with significant weight loss showed fatty degeneration of the liver, granulomatous hepatitis and histiocytic infiltration of the spleen. None of the doses of BCG CW used was toxic and they were at least as effective as living BCG in intralesional treatment. In some experiments in which treatment was delayed it was found that the extent of disease required to render treatment ineffective was about the same for living BCG and for BCG CW.

We developed a reliable method for reconstituting thymectomized, lethally irradiated guinea pigs. Injection of 2.5 − 10 × 107 syngeneic bone marrow cells into adult thymectomized, lethally irradiated guinea pigs produced survival of 46–100% of treated animals. Gentamycin sulfate (5 mg/kg of body weight) for 10 days was required for optimal results. Acidified drinking water (pH 2.5) appeared to be required for optimal results. Thymectomized, lethally irradiated, bone marrow reconstituted (‘B’) guinea pigs had impaired ability to develop delayed cutaneous hypersensitivity to mycobacterial antigens and cutaneous basophil hypersensitivity to keyhole limpet hemocyanin; proliferative responses to phytohemmagglutinin were impaired.

The malignant disease produced in guinea pigs by intradermal inoculation of line-10 was allowed to progress to stage II, at which time the dermal tumor and the first draining lymph node were grossly evident. At that stage, the external appearance of the next draining lymph node was normal, but it contained tumor cells. Limited surgery consisting of excision of the dermal tumor and first draining lymph node was not curative; palpable metastases developed in the second and other draining lymph nodes, and at autopsy, some animals were found to have gross, visible lung metastases. Immunization of guinea pigs with a mixture of irradiated syngeneic tumor cells plus mycobacterial cell walls in an oil-in-water emulsion eradicated tumor cells remaining in lymph nodes after limited surgery for stage II experimental cancer and prevented progression of the disease to stage III. Tumor intravenously implanted in the lungs of animals after limited surgery for stage II disease was also eliminated by immunization.

CLINICAL evaluation of BCG for the treatment of cancer is under way in at least 50 centers throughout the world. Use of BCG in such cases has been encouraged by work with animal tumors indicating that BCG treatment can modify the action of carcinogens, prevent growth of tumor transplants and eliminate established tumors. In some reports BCG has not been effective or has even enhanced tumor growth. Sufficient data are now available to identify at least some of the factors that contribute to the success or failure of BCG immunotherapy. BCG was originally isolated by Calmette and Guerin at the . . .

Strain-2 male guinea pigs with established intradermal (id) tumors and microscopic regional lymph node metastases were treated by systemic transfer of syngeneic peritoneal exudate (PE) cells from tumor-immune guinea pigs. This treatment produced complete regressions of established id tumor nodules (10-11 mm in diameter) and prevented the growth of lymph node metastases in 32 (80%) of the 40 treated animals. All untreated animals died with progressive id and lymphatic tumor growth. Lymph node tumor metastases that remained after id tumor excision were also suppressed by immune cell transfer. PE cells from guinea pigs immune to an antigenically distinct tumor line (line-1), BCG, or PE cells from nonimmune guinea pigs failed to produce tumor regression or prolongation of survival time. PE cells from allogeneic guinea pigs and from sheep immune to line-10 failed to transfer tumor immunity to strain-2 guinea pigs. The effectiveness of therapy was reduced by increasing the tumor burden or decreasing the number of transferred lymphoid cells. This study demonstrated that systemic transfer of cells from syngeneic immune donors could effectively eliminate tumors as well as early metastases.

Guinea pigs with established intradermal tumors and microscopic axillary lymph node metastases were treated with a combination of surgery and BCG. The tumors were excised and BCG was given in attempts to eliminate residual malignant disease. Injection of BCG into established intradermal tumors 7 days before local excision successfully eradicated microscopic axillary lymph node metastases and cured significant numbers of animals. Injection of BCG into dermal tumors 20 minutes or 1 day before excision prolonged survival but did not cure a significant number of animals. Injection of BCG into the skin adjacent to the dermal tumor 7 days before local excision eradicated microscopic axillary lymph node metastases. However, such injection 1 day before local excision did not eradicate metastases. BCG administered by intravenous, intra-arterial, or intranodal injection did not eliminate reidual malignant disease. Several factors were evaluated as possible correlates of successful immunotherapy. The development of tuberculin hypersensitivity, the magnitude of regional adenopathy, and the number of BCG organisms in axillary nodes were not useful correlates. Histologically, the presence of tumor cells, multiple focal granuloma, or histiocytosis in axillary nodes faiiled to correlate with results of therapy. The development of tumor-specific transplantation immunity provided the best correlate of successful immunotherapy.

Oral tumors with associated cervical lymph node metastases developed after injection of tumor cells into buccal pads of inbred guinea pigs. Intralesional injection of living BCG or BCG cell walls (CW) caused regression of established tumors, prevented the development of cervical lymph node metastases and led to the development of host resistance to the growth of subsequent tumor transplant.

Birt-Hogg-Dube syndrome (BHD), an inherited autosomal genodermatosis characterized by benign tumors of the hair follicle, has been associated with renal neoplasia, lung cysts, and spontaneous pneumothorax. To identify the BHD locus, we recruited families with cutaneous lesions and associated phenotypic features of the BHD syndrome. We performed a genomewide scan in one large kindred with BHD and, by linkage analysis, localized the gene locus to the pericentromeric region of chromosome 17p, with a LOD score of 4.98 at D17S740 (recombination fraction 0). Two-point linkage analysis of eight additional families with BHD produced a maximum LOD score of 16.06 at D17S2196. Haplotype analysis identified critical recombinants and defined the minimal region of nonrecombination as being within a

In this Focus (Cancer Cell 6, pp. 223–228), the legends for Figure 1, Figure 2 were reversed. They are reprinted below with the proper captions. Additionally, Figure 1Figure 1 should be cited at the end of the second-to-last sentence of the section “Studies of dominantly inherited, epithelial forms of renal carcinoma,” the citation at the end of the last paragraph of the section “VHL gene pathway: Opportunity for disease-specific approaches to therapy” should refer to Figure 2Figure 2, not Figure 1Figure 1, and the citation to Figure 2Figure 2 at the end of the first paragraph of the section “Sporadic kidney cancer” should be disregarded.Figure 1Kidney cancer is not a single disease, it is made up of a number of different types of cancer that occur in the kidneyThese different types of kidney cancer are characterized by different histologies, have different clinical courses, respond differently to therapy, and are associated with alteration of different genes. Modified from Linehan et al. (2003).View Large Image | View Hi-Res Image | Download PowerPoint SlideFigure 2Molecular targeting of the VHL pathway in clear cell renal carcinomaMutation of the VHL gene in clear cell kidney cancer results in increased accumulation of HIF and the resulting increase in transcription of downstream targets such as VEGFR, EGFR, and TGFα (A). Mutation of the VHL gene in the α domain (shown here) inhibits binding to elongin C and formation of the VHL complex (Stebbins et al., 1999). Mutation in other parts of the gene, such as the β domain, prevents binding to and ubiquitin mediated degradation of HIF (Ohh et al., 2000). Potential disease-specific therapeutic approaches include agents which block the function of HIF (B), VEGFR, or EGFR (C). Modified from Linehan et al. (2002, 2003).View Large Image | View Hi-Res Image | Download PowerPoint Slide

A freeze dried preparation of BCG with a low ratio of living to dead organisms (LV) was compared to a frozen liquid preparation with high viability (HV) for its ability to eradicate established dermal tumors and microscopic lymph node metastases in guinea pigs. The cure rate achieved by the intralesional injection of LV-BCG did not differ significantly from that of HV-bcg when similar numbers of viable organisms were injected.

Successful treatment of a solid tumor was accomplished by repeated intradermal injection of living tumor cells.

In small cell lung carcinoma, one of the short arms of chromosome 3 is typically lost. To investigate chromosome 3 in extrapulmonary small cell carcinoma, we used DNA probes that detect restriction-fragment-length polymorphisms at loci on 3p. These probes were used to study DNA extracted from tumors and normal tissues and/or tumor cell lines from five patients with extrapulmonary small cell cancer. Tumor DNA from four of the five patients with extrapulmonary small cell cancer retained heterozygosity at loci on 3p. Cytogenetic studies of the tumor cell lines established from these four patients showed retention of both short arms of chromosome 3. We conclude that the loss of genetic material from 3p observed in small cell lung cancer is not typical in extrapulmonary small cell cancer.

Previous reports showed that the loss of DNA sequences on the short arm of chromosome 3 (3p) is consistently found in sporadic renal cell carcinomas. To evaluate the significance of this genetic change, we looked for the loss of 3p alleles in hereditary renal cell carcinomas and other tumors from patients with von Hippel-Lindau disease. Specific loss of alleles from chromosome 3p was detected with polymorphic DNA markers in 11 renal cell carcinomas, one pheochromocytoma, two spinal hemangioblastomas and one cerebellar hemangioblastoma from von Hippel-Lindau patients. Multiple renal cell carcinomas in individuals with von Hippel-Lindau disease showed loss of the same chromosome 3p alleles, which demonstrated that the same chromosome was deleted in each tumor. Analysis of haplotypes indicated that the loss of chromosome 3p alleles was from the chromosome bearing the balancing, wild-type allele of the VHL gene. These results are consistent with the concept that the VHL gene is a recessive oncogene. Renal cell carcinoma, pheochromocytoma, and spinal and cerebellar hemangioblastomas develop in predisposed family members when somatic mutational events lead to loss of chromosome 3p sequences bearing the wild-type allele of the VHL gene.

The growth in the colon wall of a diethylnitrosamine-induced hepatocarcinoma was suppressed by BCG. Injection of BCG into established colon-wall tumors caused tumor regression. Successful therapy required a limited tumor burden. Guinea pigs in which growth of the neoplasm was suppressed at the site of BCG infection developed systemic tumor transplantation immunity.

Oil-in-water emulsions containing either mycobacterial cell walls (CW) or killed whole cells attached to the oil droplets were prepared by ultrasonication and by a modified grinding procedure. The immunotherapeutic potency of the ultrasonically prepared vaccines was at least as great as that of similar vaccines prepared by emulsification in a tissue grinder. Among the advantages of ultrasonication over grinding for the preparation of mycobacterial cell and cell wall vaccines are simplicity and the ease with which sterility may be maintained. Both the ultrasonic and modified grinding methods are less time-consuming than published procedures for preparing mycobacterial vaccines.

Growth of a guinea pig hepatoma was suppressed when tumor cells were mixed with viable Listeria monocytogenes (LM) before intradermal (id) injection into syngeneic recipients. Heat-killed LM were less effective than viable organisms in suppressing tumor growth. A vaccine containing oil droplets and LM cell walls lacked antitumor activity. Intratumor injection of viable LM on the 7th day after id injection of tumor cells prolonged survival of guinea pigs that did not succumb to LM infection. After intratumor injection of 0.6 times 10-8-1.0 times 10-8 LM, 5 of 22 guinea pigs died from acute infection (23 percent). In the 17 survivors, 3 tumors regressed completely (18 percent). Animals surviving injections of LM and tumor cells were immune to a second challenge with tumor cells. Immunization ofguinea pigs with an intravenous injection of LM decreased the mortality from intratumor injection of LM, but the intratumor injection of LM failed to cure a significant fraction of LM-immune animals bearing 7-day hepatoma transplants. BCG was more effective than LM in producing tumor regression. Synergism between LM and BCG was not observed, and simultaneous intratumor injection of BCG and LM was no more effective than intratumor injection of BCG alone in the treatment of 12-day tumor transplants.

Intraperitoneal administration of pyran copolymer (pyran-2-succinic anhydride-4,5-dicarboxytetrahydro-6-methylanhydride polymer) protected C3H/HeN male mice against tumor development after intradermal challenge with cells of the transplantable, methylcholanthrene-induced, syngeneic fibrosarcoma 1038. Peritoneal cells from pyran-treated but not from normal animals suppressed tumor development in local passive-transfer experiments. Adherent peritoneal cells from pyran-treated mice were cytotoxic in vitro to several syngeneic murine tumor cell lines including 1038.

Growth of a murine fibrosarcoma was suppressed when tumor cells were mixed with viable Listeria monocytogenes (LM) before intradermal injection into nonimmune syngeneic recepients. Immunization of recipients, by intravenous injection of LM 11 days before transplantation of LM-tumor cell mixtures, eliminated the mortality associated with large doses of LM but did not alter the antitumor activity of the microorganisms. Simultaneous injection of LM and tumor cells at separate sites failed to affect tumor growth, which suggested that contact between LM and tumor cells was required for tumor suppression. Tumor-specific immunity was not observed; mice surviving injection of LM and tumor cells did not resist a second tumor-cell challenge. At least 100 times more heat-killed LM was required to produce the antitumor effect of viable organisms. The ability of heat-killed LM to suppress tumor growth was abolished by treatment of recipients with rabbit antiserum to mouse thymocytes, which was consistent with a requirement for a host response to the LM. Regression of established fibrosarcoma transplants was produced by the intratumor injection of viable LM 5 days after injection of tumor cells. Intratumor injection of BCG at this interval was not effective. The incidence of tumor regression was not increased by multiple intratumor injections of LM, by intratumor injection of a combination of LM and BCG, or by preimmunization with LM prior to the intratumor injection of the same organism.

We used immunologic selection to remove contaminating leukocytes from primary renal cell carcinomas and to improve detection of chromosome 3p allele loss. Leukocytes were removed from disaggregated renal cell carcinomas by a double-antibody, magnetic bead separation technique. Before immunologic selection, the preparations contained 26% +/- 15% (SD) tumor cells (n = 7); after immunologic selection, the preparations contained 76% +/- 12% tumor cells. The recovery of tumor cells in the purified preparations was about 10%. Detection of allele deletion was facilitated by immunologic selection. This method may be useful in allele deletion analysis of other human solid tumors that are contaminated with host leukocytes and, with appropriate modification, may be applied to tumors that are contaminated with other cell types.

Living BCG, killed Mycobacterium tuberculosis cells, or BCG cell walls (CW) augmented the immunogenicity of lyophilized syngeneic ascites hepatoma (line 10) of strain-2 guinea pigs. Effective vaccine contained living BCG and lyophilized line-10 cells, or mycobacterial cells or CW attached to oil droplets and lyophilized line-10 cells. Protection against the challenge tumor was evident 14 or 21 days after one administration of either vaccine.

Fetal guinea pig cells were transformed by treatment with four different chemical carcinogens including nitroso compounds and polycyclic hydrocarbons. As a consequence of this treatment, oncogenes capable of transforming NIH/3T3 cells became activated in each of five independently established clonal guinea pig cell lines. Molecular characterization of representative NIH/3T3 transformants revealed that the same oncogene was present in each of the cell lines tested. Moreover, detection of this transforming gene paralleled the acquisition of tumorigenic properties by these neoplastic cells.

Metastases to the lungs of guinea pigs occurred at high frequency as a consequence of intradermal implantation of tumor cells derived from the syngeneic hepatocellular carcinoma line-10. Surgery had a major influence on the proportion of guinea pigs found to have pulmonary metastases at necropsy. Without surgery all guinea pigs died with extensive lymph node metastases; macroscopic pulmonary metastases were present in a minority of the animals. Animals treated by excision of dermal tumors survived longer than untreated animals, and macroscopic pulmonary metastases were present in the majority of the animals. Animals treated by excision of dermal tumor and regional lymph nodes were rendered tumor-free. The data suggest that lymph node metastases were the most likely source of the tumor cells that spread to the lungs in animals from whom the dermal tumor transplant had been removed.

The effectiveness of each of two mycobacterial components and a synthetic analog of one of them in the eradication of pulmonary deposits of intravenously injected syngeneic fibrosarcoma 1023 in C3H mice was studied. BCG cell walls (BCG CW), trehalose 6,6′-dimycolate (TDM) or 6,6′-di-0-2-tetradecyl, 3-hydroxyoctadecanoyl-α,α-trehalose (C76), a synthetic analog of TDM, was administered in emulsified form by three different routes: intraperitoneal, intradermal, or intravenous, 24 h after intravenous injection of 1023 tumor cells. The most effective form of therapy was TDM given by the intraperitoneal route; about 50% of treated animals were cured. Higher doses of BCG CW or C76 also led to a significant number of cures. Each agent caused a significant prolongation of survival time of the treated mice at two or more of the dosages tested; however, their routes of optimal activity varied.

The mechanism by which tumor allografts escape host immunologic attack was investigated. B16-BL6 cells (the bladder 6 subline of the B16 melanoma) (H-2b) were transfected with a gene (Dd) encoding an allogeneic class I major histocompatibility complex antigen. Clones that expressed Dd antigen were injected into the footpads of nonimmune syngeneic mice, syngeneic immune mice, and nude mice. Under conditions of immunologic selection a clone that contained multiple copies of the transfected gene formed variants that lacked the transfected gene. Primary tumors and pulmonary metastases of immunized mice and pulmonary metastases of nonimmunized mice had lost the Dd gene and, in most cases, all of the associated plasmid. In contrast, in immunodeficient nude mice, primary tumors and pulmonary metastases retained the Dd gene and the associated plasmid. Deletion of genes encoding cell surface antigens may be one of the mechanisms by which allogeneic tumors escape immunologic attack.

Inbred Sewall Wright strain 2 male guinea pigs with established intradermal tumors and microscopic lymph node metastases were treated either by local excision, cryosurgery, or intralesional injection of BCG. Cryosurgery and local excision were effective in eliminating growth of the primary tumor but failed to prevent growth of lymph node metastases. In contrast, intralesional injection of BCG caused regression of primary tumors and prevented growth of lymph node metastases.

Injection of living Mycobacterium bovis (strain BCG) into established intradermal tumors caused tumor regression and prevented the development of metastases.

Tumor immunity to a transplantable diethylnitrosamine-induced hepatoma in inbred guinea pigs has been found to be immunologically specific and cell mediated. We have investigated this cellular immunity using a quantitative, reproducible, and simple assay based on the ability of leucocytes to inhibit the incorporation of tritiated thymidine (TdR 3 H) by tumor cells. Tumor cell suspensions were obtained from the ascites form or tissue culture monolayers of the hepatoma. Cells from the peritoneal exudate of immunized guinea pigs inhibited tritiated thymidine uptake by tumor target cells to a significantly greater degree than cells from the peritoneal exudate of unimmunized animals. Immune lymph node, peripheral blood, and spleen leucocytes were not inhibitory. The assay was sufficiently sensitive to detect relatively weak tumor immunity. The in vitro inhibition was correlated directly with the presence of delayed hypersensitivity and/or inhibition of tumor cell growth in local passive transfer studies. Irradiation of peritoneal exudate cells (3000 R) blocked their inhibitory effects on tumor cells. Fractionation of the peritoneal exudate cells by centrifugation in zones of bovine serum albumin of different density also revealed the lymphocytes to be responsible for the specific inhibitory effects whereas macrophages inhibited in an immunologically nonspecific fashion.