Mediation of immunity to tumor isografts in mice by heterologous ribonucleic acid.

The growth of tumor isografts in inbred mice is inhibited by intra-peritoneal injections of syngeneic spleen incubated, in vitro, with ribonucleic acid extracted from guinea pigs immunized with the same mouse tumor. This inhibition is partially tumor-specific. Treatment with ribonuclease abolishes the response.

Mediation of cytotoxic immune responses against human tumor-associated antigens by xenogeneic immune RNA.

Xenogeneic immune RNA (I-RNA), extracted from the lymphoid organs of sheep or guinea pigs immunized with human tumor cells, mediated in vitro cytotoxic immune responses that were directed specifically against tumor-associated antigens of human tumor target cells. Normal human peripheral blood lymphocytes from healthy donors became markedly more cytotoxic for human tumor target cells after being incubated with I-RNA extracted from the lymphoid organs of animals that had been immunized with that particular tumor. Gastric carcinoma, malignant melanoma, and carcinoma of the breast were studied. Lymphocytes incubated with RNA from animals immunized with only complete Freund's adjuvant evidenced no increased cytotoxic activity. RNA extracted from the lymphoid organs of animals immunized with normal skin fibroblasts that were autologous to the immunizing tumor, when incubated with normal allogeneic lymphocytes, also mediated cytotoxic immune reactions against tumor target cells. These immune responses probably were directed principally against normal transplantation antigens. However, when lymphocytes that were autologous to the immunizing tumor and/or the tumor target cells were incubated with RNA from animals immunized with autologous normal fibroblasts, cytotoxicity did not increase. Only I-RNA extracted from donor animals specifically immunized with tumor cells mediated cytotoxic antitumor immune responses when incubated with autologous lymphocytes.

Mediation of cytotoxic immune responses against human tumor-associated antigens by allogeneic immune RNA.

Allogeneic immune RNA (I-RNA), extracted from the peripheral blood lymphocytes of patients putatively cured of cancer, mediated cytotoxic immune reactions that apparently were directed specifically against human tumor-associated antigens. I-RNA was extracted from the peripheral blood lymphocytes of patients with various types of cancer. Patients selected had not been previously sensitized to HL-A or other normal transplantation antigens or to blood group antigens. Normal human peripheral blood lymphocytes were incubated with these allogeneic I-RNA preparations and tested for cytotoxicity against human target cells in vitro. Allogeneic I-RNA mediated cytotoxic immune reactions only against tumor target cells of the same histologic type as the I-RNA donor. I-RNA's extracted from peripheral blood lymphocytes of melanoma patients mediated cytotoxic immune reactions only against melanoma cells. Similarly, only I-RNA's extracted from the lymphocytes of patients with colon cancer mediated cytotoxic immune reactions against colon carcinoma cells, and only I-RNA's from the lymphocytes of breast cancer patients mediated immune reactions against breast cancer target cells. Allogeneic I-RNA extracted from peripheral blood lymphocytes of cancer patients possibly mediated specific cytotoxic immune reactions that were directed against common tumor-associated antigens shared by human tumors of similar histologic type.

Specificity of antitumor immune reactions mediated by xenogeneic immune RNA.

Evidence that xenogeneic immune RNA (I-RNA) mediated specific cytotoxic immune responses against human tumor-associated antigens was obtained from in vitro studies in two autologous melanoma systems. In these systems, malignant melanoma target cells, matching normal fibroblast target cells, lymphocyte effector cells, and melanoma and normal skin tissue used to immunize RNA donor animals were derived from the same autochthonous hosts. When incubated with autologous lymphocytes, I-RNA extracted from the lymphoid organs of donor animals immunized with melanoma tissue mediated immune reactions against autologous melanoma target cells in vitro. I-RNA from animals immunized with normal skin tissue from autochthonous hosts did not increase the cytotoxicity of autologous lymphocytes for autologous melanoma cells. Using autologous fibroblasts as target cells, we detected no increase in cytotoxicity when autologous lymphocytes were incubated with RNA from animals immunized either with melanoma tissue or normal skin tissue from the autochthonous host. By contrast, when allogeneic lymphocytes were used as effector cells, RNA extracted from animals immunized either with melanoma tissue or normal skin mediated cytotoxic immune reactions against melanoma target cells and normal fibroblast target cells derived from the same patient.

Production of antisera with specificity for malignant melanoma and human fetal skin.

Complement-dependent cytotoxic antibodies to common cell surface antigens of cultured melanoma cells were produced in guinea pigs. At appropriate dilution, melanoma antisera were cytotoxic only to melanoma target cells. Following absorption with pooled lymphoid cells, additional absorption with melanoma cells but not absorption with fibroblasts or carcinoma cells was found to remove all cytotoxic activity from melanoma antisera. Absorption with human fetal skin cells but not with autologous fetal visceral cells was found to remove cytotoxicity from melanoma antisera. Tissue type-specific antigens may be shared by human malignant melanomas and fetal skin of black racial origin (at 16 to 18 weeks of gestation). The methods may be useful in the production of xenogeneic antisera with "operational monospecificity" for common melanoma-specific antigens. Sera from 47 patients with malignant melanoma failed to evidence specific cytotoxicity for melanoma target cells.

Immunogenicity of chemically induced murine colon cancers.

The antigenicity and immunogenicity of three colorectal carcinomas induced in BALB/c mice by 1,2-dimethylhydrazine or N-methyl-N-nitrosourethan were studied. All tumors were readily transplantable. Two of these tumors metastasized when transplants reached sufficient size. All tumors were found to be immunogenic in the strain of origin, and all tumors were shown to contain unique tumor-specific transplantation antigens in cross-protection experiments. The use of these tumors as an animal model for studies of adjuvant immunotherapy and chemoimmunotherapy is suggested.

A new micromethod for the detection of HL-A antigens on cultured human tumor cells.

A rapid microcytotoxicity assay for the detection of HL-A antigens on tissue culture cells derived from human solid tumors is described. Tumor cells were prelabeled with 125Iododeoxyuridine. Isotopically labeled tumor cells were reacted with up to 37 highly selected HL-A antisera and diluted rabbit complement. Results of the HL-A typing of nine human tumor cell lines are reported. Three melanoma cell lines showed individually distinct HL-A profiles at the first HL-A locus which agreed with the antigenic pattern of the tumor donor's autologous lymphocytes. Less reactivity was noted with HL-A antisera defining second locus specificities on the three melanoma cell lines, whereas some other cell lines showed more HL-A reactions than required to present a "full house". This method obviates the necessity for visually enumerating residual tumor target cells.

Mediation of immune responses to tumor antigens in vitro by immune RNA.

It was shown that normal nonimmune C3H mouse spleen cells became specifically cytotoxic to chemically-induced syngeneic C3H tumor cells by incubation with xenogeneic I-RNA extracted from the lymphoid organs of specifically immunized guinea pigs. This response was specific for the tumor used to immunize the I-RNA donor. In a totally syngeneic system, we showed that syngeneic I-RNA extracted from the spleens of tumor-bearing rats mediated cytotoxic immune reactions which were directed specifically against the tumor-associated antigens of syngeneic rat tumor target cells. Active antitumor I-RNA synthesis in the lymphoid organs of I-RNA donor animals reached a maximum between days 14 and 21, depending on the route of administration and the nature of the immunizing tumor. Active I-RNA preparations were insensitive to treatment with deoxyribonuclease or pronase, but were inactivated by ribonuclease treatment; thereby indicating that the active moiety was one or more species of RNA. The active fractions of the I-RNA preparations had sedimentation values in sucrose density gradients of 12-16S, and comprised only a small fraction of the total RNA present in the lymphoid cells. Active antitumor I-RNA appeared to be localized in the cytoplasm of sensitized lymphoid cells, rather than in the nucleus. Lymphocytes from normal human donors as well as from cancer patients, when incubated with xenogeneic or allogeneic I-RNA, became specifically cytotoxic for human tumor cells in vitro. Crossreactivity among tumors of the same histologic type was observed, but not crossreactivity with tumors of other histologic types. Xenogeneic I-RNA extracted from the lymphoid organs of donor animals immunized either iwth tumor cells or normal tissues, following incubation with normal allogeneic lymphocytes, mediated cytotoxic immune reactions which were directed both against tumor-associated antigens and normal transplantation antigens. However, when autologous lymphocytes were used as effector cells, only immune reactions directed against tumor-associated antigens were observed. Allogeneic I-RNA extracted from peripheral blood lymphocytes of human cancer patients mediated specific cytotoxic immune reactions that were directed against common tumor-associated antigens shared by human tumors of similar histologic type. I-RNA's directed against "self" normal cell surface antigens appear to be recognized as self by lymphocytes, and immune responses against these self antigens are not elicited. On the other hand, I-RNA's directed against "nonself" tumor-associated antigens induce lymphocytes to effect specific antitumor immune responses. Our data are consistent with the hypothesis that I-RNA is an information-containing ribonucleic acid molecule capable of mediating immune reactions in vitro which are specific for the tumor-associated antigens of the tumor used to immunize the I-RNA donor.

Colonic epithelial impedance analysis in a murine model of large-bowel cancer.

Death rates from large-bowel cancer have remained essentially unchanged over the past 40 years because the diagnosis is made late, after the tumor has spread to other sites. This study was undertaken to examine whether alterations in mucosal electrical capacitance precede the development of gross malignancy, since this parameter may reflect functional or structural changes in the colonocyte plasma membrane, which is of importance in the regulation of cell growth. Distal colonic mucosal capacitance was decreased at low frequencies after only four weeks of treatment with the carcinogen dimethylhydrazine in C1 mice. Alterations in electrical capacitance may be a useful marker in identifying patients with a propensity to develop large-bowel cancer.

Changing concepts in establishing the diagnosis of breast masses.

Experience with 297 consecutive biopsies of breast masses for 235 benign lesions and 62 carcinomas over a thirty month period has been reviewed. The correct preoperative clinical diagnosis was made in 91% of cases. Of patients with carcinoma, 66% were suspected clinically, and 88% of those clinically suspected were confirmed by needle biopsy alone. The mammographic diagnosis was correct in 89% of cases with 6% false-negatives. The clinical and mammographic diagnosis differed in 27 patients, with the clinical diagnosis being correct in 85%. No patient thought to have cancer on both clinical and mammographic grounds had a benign lesion. These data indicate that the approach to establishing a tissue diagnosis in women with breast masses can be simple and inexpensive. Local anesthesia can almost always be employed, and the diagnosis of breast cancer can usually be confirmed with certainty by needle biopsy alone.

Local delayed cutaneous hypersensitivity reactions in breast cancer patients with and without removal of axillary lymph nodes.

Fifty-five patients were sensitized to dinitrochlorobenzene (DNCB). Two weeks later they were challenged. The minimal concentration yielding 2+ reactivity and one dilution above and below were then applied to the anterior chest wall on both the operated and nonoperated sides. Using multinomial chi-square statistical analysis, we found that the operated and nonoperated sides evidenced equal reactivity. Futhermore, the absence of axillary lymph nodes did not diminish the reactivity in the operated area. These data support the contention that maintenance of local cellular immunity, as assessed by DNCB skin test reactivity, is systemic and counters the argument that regional lymphadenectomy impairs local and/or systemic cellular immunity.

Immunotherapy of cancer with "immune" RNA. A preliminary report.

A phase I clinical trial of immunotherapy with "Immune" RNA was undertaken fifteen months ago. Twenty-six cancer patients were treated with RNA extracted from the lymphoid organs of sheep immunized with either autologous tumor cells or allogeneic tumor cells of the same histologic type. Eighteen patients had gross disease and eight had minimum residual disease. RNA was administered weekly, intradermally, at doses up to 9 mg/week without any significant local or systemic toxicity. Four patients improved, thirteen achieved stability of disease or possible improvement, seven were treatment failures, and two are indeterminate. Lymphocyte-mediated cytotoxicity to allogeneic tumor target cells of the same histologic type were monitored in eleven patients. In seven patients, cytotoxicity increased after "Immune" RNA therapy; no change was observed in three patients; a decrease was noted in one patient. There appeared to be a possible correlation between cytotoxicity assessed in vitro and clinical response. There is some evidence that these responses may be specific for the particular tumor used to immunize the RNA donor.

Breast cancer treatment--current status. 2. Segmental mastectomy, alternative to total breast excision?

Loss of a breast to cancer can have a profound psychologic effect on a woman. Within the last few years, research in breast cancer has centered on trying to find procedures that are less mutilating than but just as effective as the conventional surgical procedures discussed in part 1 (page 126). In this second part by Dr Pilch, recent and ongoing studies of a newer promising method known as segmental mastectomy are reviewed. How simple excision with irradiation is being used in early disease is described in part 3 (page 151). Part 4 (page 161) considers adjuvant therapies in early and late disease.

Breast cancer treatment--current status. 1. Mastectomy, standard surgical approach.

For about three quarters of a century, radical mastectomy was considered standard initial treatment for invasive breast cancer. Within the past 15 years, modified radical mastectomy (total mastectomy with axillary dissection) has replaced radical mastectomy as standard treatment, even though superiority of the former has not been scientifically proven, with the exception of a single study. During the same period, total mastectomy with postoperative irradiation of axillary lymph nodes came into vogue, and debate centered on whether irradiation of nodes was as efficacious as surgical extirpation. Results of several prospective studies indicated that when used prophylactically, neither method enhanced survival but both prevented subsequent nodal disease, and when used therapeutically, both methods controlled cancer growth equally well. Today, modified radical mastectomy is still standard treatment for patients with operable breast cancer, since axillary dissection is now routinely performed for staging purposes, thereby being preferable to irradiation of nodes.