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Active-specific immunotherapy, which involves activation of host defenses toward antigenic factors distinct for each tumor, is a potentially useful antitumor strategy. Experimental studies have demonstrated the efficacy of autologous tumor cell vaccines supplemented with an adjuvant (Bacillus Calmette-Guerin, BCG) against intravenously injected tumor cells. In this study, we evaluated the vaccine's efficacy against a more clinically relevant model, spontaneously occurring metastases. Viable syngeneic hepatocarcinoma cells were injected intradermally into the left shoulder area of inbred guinea pigs. On Day 25, the primary tumor (1.5 cm in diameter) and the first draining node were excised. Animals were randomized into groups that received no further therapy or two, three, or four vaccinations with an autologous preparation of cryopreserved, irradiated tumor cells mixed with live BCG. Vaccinations were started 4 days after surgery and were given weekly on alternate sides of the shoulder and flank regions. Of those animals that received surgery alone, 5% survived, whereas of those that received two, three, or four vaccines, 35% ( P = 0.03), 45% ( P = 0.004), and 55% ( P = 0.002), respectively, were disease free at 200 days. That the effect was systemic and not due to BCG draining into the regionally involved lymph nodes was demonstrated in a repeat experiment in which all vaccinations were given in distant (contralateral) sites with equally effective results. Tumor cells in the lymph nodes beyond those excised were found in animals necropsied on the first day of therapy. Therefore, established distant lymph node metastases were controlled. Human tumors with tumor-specific antigens may respond similarly and should be studied.

It has been previously demonstrated that transplanted syngeneic tumors established in the skin of inbred (strain-2) guinea pigs regressed and regional lymph node metastases were eliminated after intralesional injection of viable Mycobacterium bovis (BCG). During the course of this reaction there is the development of tumor-specific immunity. This experimental model was further manipulated in order that it would more closely approximate a clinical reality. In the present study an evaluation was made of the effectiveness of the developing tumor-specific immunity in this BCG therapy model, to abrogate artifically induced distant tumor deposits and to assess the requirement for tumor-specific immunity in the local BCG-mediated tumor regression. During BCG-mediated regression of established intradermal tumor, the developing tumor-specific immunity inhibited the growth of artificially induced vascular metastases in animals receiving a 10(4) or 10(5) tumor cell dose. However, there is a direct causal relationship between the distant tumor burden and the escape of skin tumor and regional lymph node metastases from BCG-mediated regression. Thus, multiple tumor deposits as low as 10(4) cells are capable of competing for or preempting tumor-specific immune reactivity, which must be a requirement during some phase of the intralesional BCG-mediated therapy of established tumor and regional lymph node metastases. Thus, a significant therapeutic effect could be achieved in guinea pigs with established skin tumors and limited vascular metastases when the modality of therapy included BCG intralesional injection, followed 6 weeks later by surgery of the treated skin tumor and regional lymph node.

A vaccine of Bacillus Calmette-Guérin (BCG) admixed with tumor cells induced systemic immunity and had a therapeutic effect on subclinical, disseminated micrometastases. Inbred strain 2 guinea pigs given intravenous injections of either 10(4), 10(5) or 10(6) syngeneic L10 hepatocarcinoma cells were vaccinated after metastatic foci were established in the lung parenchyma. The studies demonstrate that under defined conditions of vaccine preparation and regimen, nontumorigenic preparations of BCG and tumor cells can cure the majority of animals of otherwise lethal visceral metastases. Histopathologically it was determined that immunization with these vaccines prevented the progressive growth of pulmonary micrometastatic foci approximately 0.1 mm in diameter. However, in this micrometastasis therapy model, the number of metastatic tumor foci is a major limitation in the efficacy of vaccine therapy. No protection against L10 tumor was achieved when antigenically distinct but syngeneic L1 hepatocarcinoma was used in the vaccine, suggesting that this is a tumor-specific immunotherapeutic procedure. This BCG-L10 tumor vaccine was also effective in curing guinea pigs of minimal disseminated tumor burden when administered after surgery of an established skin tumor and draining lymph node.

In a transplantable hepatocarcinoma (L10) model of inbred Sewall Wright strain 2 guinea pigs, established micrometastatic tumor foci could be eliminated from the viscera by specific tumor immunity induced by the systemic effect of a BCG-L10 tumor cell vaccine. The conditions of vaccine preparation and regimen were rigid, and a dose dependency for both BCG and tumor cells existed. Comparison of the influence of cryobiologic preservation procedures on the tumor cells to be used in the vaccine showed that cells frozen by an optimized cryobiologic procedure, improved for maintenance of viable cells, were more effective than cells frozen by a conventional glycerol method. The glycerol method resulted in a lower percentage of viable cells, and the effectiveness of the vaccine was greatly diminished. Thus some past failures of active specific immunotherapy could be associated with acute antigenic exposures resulting from suboptimal cryobiologic preservation of tumor cells in the vaccine preparation instead of prolonged or chronic tumor cell antigenic exposure resulting from optimal cryobiologic procedures.

We evaluated the use of Bacillus Calmette-Guérin admixed with tumor cells as a vaccine to induce systemic tumor immunity for therapy of subclinical (micrometastatic) disease. In several experiments inbred strain 2 guinea pigs were given i.v. injections of either 10(4), 10(5), or 10(6) syngeneic L10 hepatocarcinoma cells, and initial vaccinations were administered either 1 or 4 days after tumor inocluation. Variables in vaccine preparation, such as ratio of viable Bacillus Calmette-Guérin organisms to tumor cells, procedures for freezing the tumor cells, X-ray treatment of tumor cells, and vaccination regimen were evaluated. The studies demonstrated that under defined conditions nontumorigenic vaccines of Bacillus Calmette-Guérin and tumor cells can cure the majority of animals of otherwise lethal visceral micrometastases.

Intradermal vaccinations with Bacillus Calmette-Guérin (BCG) and viable but nontumorigenic syngeneic hepatocarcinoma cell vaccines were used successfully to treat micrometastatic disease experimentally induced in inbred guinea pigs at 100 times the minimal lethal dose. Several complications have been associated with the use of viable BCG organisms in the treatment of cancer patients and, in this animal model, intradermal administration of BCG uniformly results in severe ulceration and eschar formation at the injection sites leading to secondary microbial invasion and regional lymphadenopathy. We now report the use of isoniazid (Nydrazid) as part of an active specific immunization regimen. Incorporation of isoniazid into the immunization procedure for two weeks alleviates or reduces the side effects of BCG infection. Moreover, with proper consideration of BCG dosage, isoniazid does not impair the efficacy of the BCG plus tumor cell vaccines.

In inbred guinea pigs, administration of Mycobacterium bovis strain BCG by scarification at a site distant from an excised skin tumor, but in the regional lymph node drainage, was evaluated for its immunotherapeutic effect on the development of lymph node metastases. Scarification was performed after surgical excision of intradermally transplanted syngeneic (line-10) hepatocarcinoma at a time when microscopic foci of tumor cells were present in regional lymph nodes. Various strains of BCG were evaluated for their immunotherapeutic potential: fresh-frozen Phipps, Pasteur, and Tice; and lyophilized Pasteur, Tice, and Connaught. Scarification commenced 3 days after surgical removal of the tumor and continued once a week for 5 weeks. Only lymph nodes from fresh-frozen Phipps- and Pasteur-scarified animals were significantly smaller than those in the control groups. Differences in lymph node weight correlated histologically with less detectable metastases. This cytostatic effect was short lived; eventually, the metastatic tumor growth was not significantly different from that of control animals. No significant differences were observed in mean survival time: All animals died as a result of metastases 3 months after tumor inoculation. These results demonstrated that limited scarification with BCG of certain strains temporarily inhibits the growth and proliferation of metastases in regional lymph nodes after removal of the primary tumor.