Enhanced therapeutic efficacy of cisplatin by combination with diethyldithiocarbamate and hyperthermia in a mouse model.

A spontaneously metastasizing solid tumor model derived by transplanting the TA3Ha murine mammary carcinoma into the s.c. tail tissue of mice was used to develop a treatment strategy for enhancing the therapeutic efficacy of cisplatin (CDDP). This strategy was based on the findings that diethyldithiocarbamate (DDTC) reduces the toxicity of CDDP, and that localized hyperthermia (HT) augments the antitumor efficacy of CDDP. DDTC (500 mg/kg) reduced the CDDP-induced nephrotoxicity and gastrointestinal toxicity as well as increased the CDDP LD10 from 8 to 20 mg/kg in strain A mice. When CDDP and DDTC were used in multiple treatment schedules at 5-day intervals, DDTC protected the hosts but not the tumors against the toxicity of CDDP. HT administered locally to the tumor 1 h after the injection of CDDP (8 mg/kg) in 1 ml Hanks' balanced salt solution increased the antitumor effect but not the host toxicity. While administration of 8 mg/kg CDDP alone or with HT three times at 5-day intervals caused 100% host mortality, this dose of CDDP could be used with no mortality by combining it with DDTC. A combination of 8 mg/kg CDDP with DDTC (750 mg/kg) and HT (43 degree C for 60 min), administered three times at 5-day intervals, retarded the local tumor growth significantly compared to the untreated, CDDP plus DDTC plus HT control groups of mice. The frequency of lung metastasis in these groups on day 30 of tumor inoculation were 0, 90, 90, and 80%, respectively. The mean survival days of the mice treated with CDDP plus DDTC plus HT was 61 +/- 6 compared to 34 +/- 5 in the controls. The results presented here demonstrate that by combining CDDP with DDTC, high doses of CDDP can be safely administered. When localized HT is combined with high dose CDDP and DDTC, the tumor growth retardation and the host survival prolongation are significantly better than those obtained with the highest tolerable dose of CDDP alone or CDDP plus HT.

Combined effect of trans-diamminedichloroplatinum(II) and hyperthermia on murine and human tumor cells.

trans-Diamminedichloroplatinum(II), a paradigm of an inactive platinum compound, exhibited cytotoxic effect against HEP-2 human tumor cells, TA3Ha murine tumor cells, and freshly collected human ovarian carcinoma cells when combined with hyperthermia (43 degrees, 30 min). The heat treatment reduced the D0 of trans-platinum from 56 to 16.5 micrograms/ml in the HEP-2 system and from an undeterminable value at 37 degrees to 8.2 micrograms/ml in the TA3Ha system. Heat treatment before trans-platinum was more cytotoxic than that after trans-platinum in the TA3Ha system (P less than 0.001). TA3Ha cells treated in vitro with 40 micrograms/ml TDDP at 43 degrees failed to form tumors in mice upon subcutaneous implantation into the tails of mice. In contrast, these agents given singly did not alter the tumor-forming ability of TA3Ha cells. In vivo administration of trans-platinum after hyperthermia (43 degrees for 30 min) retarded the growth of TA3Ha tumors compared to either treatment alone. trans-Platinum did not form detectable DNA-interstrand cross-links in the HEP-2 cells treated at 37 degrees or 43 degrees. However, the DNA-protein cross-links were detectable under these conditions. The frequencies of DNA-protein cross-links were higher in the cells treated at 43 degrees than in those treated at 37 degrees, both immediately after and 12 h after the treatment with trans-platinum. Heat alone did not induce the formation of either DNA-interstrand or DNA-protein cross-links. Heat treatment did not appear to enhance the entry of trans-platinum into the cells.

Effect of localized hyperthermia on TA3Ha tumor transplanted subcutaneously in the tails of mice.

Localized hyperthermia (43 degrees) in single or multiple fractions was applied to mouse mammary adenocarcinoma TA3Ha implanted into the s.c. tail tissue of strain A mice. The effects of heat on the growth of local tumors, on the pattern of metastasis, and on the survival periods of the hosts were studied. Hyperthermia was administered by heating the tumor-bearing tails in a water bath. Multiple 30-min hyperthermia treatments at 5- or 7-day intervals controlled local tumor growth better than did a single 30-min treatment or multiple 30-min treatments at 3-day intervals or at intervals longer than 7 days. Heat treatments that produced cytostatic effects on tumors, sparing the normal tissue, had no effect on either the survival of the hosts or the extent of metastasis to the lungs and the lumbar lymph nodes. However, local treatments reduced the frequency of renal lymph node metastasis, indicating that concurrent metastases in different sites may exhibit differential heat sensitivities.

Growth and metastasis of human breast cancers in athymic nude mice.

To evaluate critically the merit of utilizing a wound model for growing human tumors, a series of increasingly difficult human tumor types were tested for growth at sites of trauma in athymic nude mice. In vitro tumor lines as well as fresh tumors from the breast, colon, rectum, lung, and a metastasis from an unknown primary were intraperitoneally injected into mice subjected to intra-abdominal organ injury. Successful xenografts were obtained from nine of 10 cell lines and 14 of 24 fresh tumors. The latter included five of six (83%) colon cancers, one lung tumor, metastatic tumor of unknown primary, three of four (75%) metastatic breast cancers and four of six (67%) estrogen receptor (ER)-negative breast primary tumors. Six ER-positive breast tumors tested failed to grow in mice without estrogen supplementation. Xenografts from two breast, two colon and the lung cancers formed spontaneous metastases and all xenografts tested were able to yield serial transplants in the surgical wound model. Histologically, all xenografts and their metastases were identical to their respective donor tumors. Transplantability in mice without exogenous estrogen supplementation was linked to the absence of estrogen and progesterone receptors in breast tumors. Transplantability of the cell lines was associated with the expression of cell surface receptors for fibronectin and hyaluronic acid. Receptors for other extracellular matrix components, namely, laminin, vitronectin, collagen, fibrinogen or von Willebrand factor were not associated with transplantability. These results demonstrate that a large proportion of human tumors, including the breast tumors, can be successfully xenografted into athymic mice by providing them with a healing wound environment, and that such xenografts grown at ectopic sites exhibit metastatic ability.

Inhibition of tumor implantation at sites of trauma by Arg-Gly-Asp containing proteins and peptides.

We report on the inhibition of wound implantation by TA3Ha mammary carcinoma cells by Arg-Gly-Asp containing proteins and peptides using a hepatic wedge resection model. Intravenously injected TA3Ha cells rarely form tumor in the liver of syngeneic mice, but after hepatic wedge resection, 45% (107/240) of the mice develop tumors in the hepatic wound. Hepatic wound implantation is significantly (P = 0.01) inhibited by pretreating the cells with whole mouse plasma, but not with fibrinogen-depleted plasma or serum. Tumor inhibition is also achieved by pretreatment of cells with fibrinogen (P = 0.05-0.0004), fibronectin (P = 0.007) and laminin, but not by albumin. The active domain appears to be the RGDS sequence since the deca- and tetrapeptides containing RGDS inhibit wound implantation (P less than 0.05). However, the tetrapeptide Arg-Gly-Glu-Ser has no such activity. None of these agents affects ascites tumor formation by the intraperitoneally injected cells, suggesting that anchorage independent growth of cells is not affected. We propose that proteins and peptides containing RGD occupy the binding sites and prevent the cells from interacting with cell adhesion proteins in healing wounds. Proteins and/or peptides containing RGD may be useful for preventing local recurrence in postsurgical cancer patients.

The role of fibronectin in tumor implantation at surgical sites.

Fibronectins are a family of glycoproteins with modular functional domains. They mediate cell-cell and cell-matrix interactions which are important in embryogenesis, wound healing, metastasis and other processes. We present data on the influence of fibronectin on wound implantation of a murine mammary carcinoma line, TA3Ha. Fibronectin used in these studies was derived from bovine plasma, human serum, human foreskin fibroblasts, and mouse embryo cultures. TA3Ha cells rarely form tumors in the liver of syngeneic mice when injected intravenously but after hepatic wedge resection, 45% (107/240) of the mice develop tumors in the hepatic wound. Wound implantation is markedly reduced when the cells are pre-exposed to 200 micrograms/ml bovine plasma fibronectin (13%, P = 0.007), human serum fibronectin (0%, P = 0.02), human cellular fibronectin (0%, P = 0.02), or mouse cellular fibronectin (0%, P = 0.04). Lung colonization is also reduced by these fibronectins. These effects are not due to a cytotoxic action of fibronectin, since intraperitoneally injected fibronectin-treated cells form ascites tumor as effectively as do control untreated cells. Local application of a solution containing 0.25 mg/ml mouse cellular fibronectin to the hepatic wound reduces the frequency of tumor implantation from 45% to 5% (1/21, P = 0.001). No tumor implantation inhibition is seen when only suspending medium or albumin in suspending medium is used. The mechanism by which topical application of fibronectin reduces hepatic wound implantation of tumor cells is unclear, but this finding raises an exciting possibility of preventing local recurrence of cancer.

The solitary pulmonary nodule in the patient with breast cancer.

A solitary pulmonary nodule appearing in a patient with breast cancer, either past or present, is most likely to be a second primary cancer originating in the lung rather than a metastasis from the breast cancer. Between 1970 and 1983 there were at this institution 1416 patients with breast cancer and 579 patients with bronchogenic cancer, 198 of whom were women. Among the patients with breast cancer, 42 (or 3% of all of the patients with breast cancer) had a solitary pulmonary nodule either at the time of presentation of their breast cancer or during the follow-up period. Fifty-two percent of the solitary pulmonary nodules proved to be a primary lung tumor, 5% proved to be benign lesions, and only 43% proved to be metastatic breast cancer. Patients with breast cancer with solitary pulmonary nodules should have a diagnostic workup appropriate for lung cancer. Since adenocarcinoma has become the most common lung cancer cell type, the usual diagnostic tests may not allow a firm differentiation between primary lung and secondary breast cancer. Therefore if malignancy is proved or suspected, thoracotomy with appropriate resection is the treatment of choice in most patients with breast cancer, even at the initial appearance of the breast cancer.

Thyroid carcinoma. Immunology, irradiation, and lymphocytic infiltration.

Patients undergoing thyroidectomies at Evanston (III) Hospital, during a six-month period had immunological studies performed preoperatively. No differential could be found between those with carcinoma or benign pathologic findings. T- and B-cell distribution and lymphocytic response to mitogens varied widely. Quantitative immunoglobulins showed slightly increased levels of IgG in patients with carcinoma and thyroiditis in comparison with those patients with adenomas. Antithyroglobulin antibodies were negative in all patients. Pathology slides from 107 patients with thyroid carcinoma between 1972 and 1978 at Evanston Hospital were reviewed for the presence of thyroiditis, either focal or diffuse. It was found that 50% of all carcinomas had either diffuse or focal thyroiditis. Diffuse thyroiditis was more common in patients with no history of irradiation and papillary carcinoma, and in younger age groups.

Improved detection of metastases to lymph nodes and estrogen receptor determination.

We present a new method for detection of micrometastases to axillary lymph nodes and estrogen receptor determination. Cellular suspensions from primary infiltrating ductal breast carcinoma or level I axillary lymph nodes of patients who underwent mastectomies were obtained, by loosely grinding fresh tumors or lymph nodes through a grid and then transferring the matrix to a slide using cytocentrifugation. Tumor samples were analyzed for estrogen receptor status using an immunocytochemical kit and compared with the dextran-coated charcoal method. Thirty-eight of 48 correlated (20 were estrogen positive, and 18 were estrogen negative). Seven of 46 were estrogen positive while results from the dextran-coated charcoal method were estrogen negative. One of 46 was estrogen negative, while the results from the dextran-coated charcoal method were estrogen positive. Lymph node slide preparations were stained to detect tumor cells using antikeratin monoclonal antibodies. Three of 8 node-negative patients were found to have micrometastases. Four of 15 node-positive patients had additional nodes with tumor. Our method combines the advantages of serial sectioning and immunohistochemical staining.

The morbidity of total thyroidectomy.

Of 245 total thyroidectomies done over a ten-year period, most were in patients who had previously received irradiation about the head and neck for benign conditions in infancy and childhood. There was no operative mortality, and only one patient had postoperative bleeding requiring reoperation. Of six patients with paralyzed recurrent laryngeal nerves in the immediate postoperative period, vocal cord function returned in all but one over one year. Forty-nine patients required at least one dose of calcium postoperatively, and 41 were taking calcium when discharged from the hospital. After one year, only two patients took calcium regularly; in both, lymph nodes were removed from both paratracheal grooves. In evaluating comparative statistics, increased morbidity of total thyroidectomy needs to be considered on a long-term basis rather than in the immediate postoperative period.

Thyroid disease following irradiation for benign conditions.

One hundred twenty-five patients with a history of prior irradiation to the head and neck region for benign disease underwent thyroidectomies between 1967 and 1976 at Evanston Hospital. One hundred twenty-four had a palpable abnormality. Forty-two had carcinoma, and nine of these had nodal metastases. Palpation was found to be more accurate than thyroid isotope scan in finding carcinoma within an abnormal gland. Some form of irradiation thyroiditis was found in one half of the resected specimens.

Head and neck neoplasia following irradiation for benign conditions.

A retrospective review of 143 patients with head or neck tumors who had received radiation therapy in the head and neck area for benign conditions during childhood or adolescence was conducted. This included an analysis of 1,080 patients from the Evanston Hospital Irradiated Thyroid Evaluation Clinic, which was established to define the relationship between irradiation and the subsequent development of thyroid neoplasia. The data support the following concepts of irradiation-induced neoplasia: (1) The thyroid, parathyroid, and salivary glands can develop benign and malignant changes after irradiation for benign conditions, with latent periods averaging about 30 years; (2) Once a glandular abnormality within the irradiated field appears, the risk of other glands in the field developing neoplastic changes is significantly increased.

Aspiration cytology of head and neck masses.

Fine needle aspiration biopsy and cytologic examination of the aspirate were performed on 203 masses involving glandular and nodal structures of the head and neck: thyroid gland (85); salivary glands (31); and lymph nodes (87). Overall cytologic-histologic correlation was 91 per cent, with a 10 per cent false-negative rate. The ease of the procedure coupled with the rapidity of obtaining a pathologic diagnosis allows a more intelligent therapeutic approach.

Irradiation-induced polyglandular neoplasia of the head and neck.

Eighteen patients are presented with twenty-one tumors of the head and neck, which include ten salivary gland tumors and eight parathyroid adenomas. Eight of the patients also had thyroid neoplasms. All patients had a history of prior irradiation to the head and neck. Seventy per cent of the salivary gland tumors and 37 per cent of the thyroid tumors were malignant. Recommendations are made for detection and treatment.

Preoperative physical assessment of thyroid glands in previously irradiated patients.

The Evanston Hospital maintains an Irradiated Thyroid Evaluation Clinic that has evaluated 695 patients since 1975. One hundred fourteen patients were retrospectively analyzed, and an attempt was made to correlate the preoperative physical examination with the pathologic specimen after thyroidectomy. There was no statistically significant difference between the incidence of carcinoma in glands containing a single nodule (23 per cent) and in multinodular glands. Postirradiation thyroiditis complicated the physical description of glands preoperatively. The categorization of physical findings served only to identify persistent thyroid abnormalities, which must be explored surgically. The overall incidence of carcinoma in the 114 available cases was 34 per cent, with nodal metastases in 18 per cent of the patients with carcinoma.

Diet intervention methods to reduce fat intake: nutrient and food group composition of self-selected low-fat diets.

A multicentered pilot study was conducted to test an intervention protocol designed to reduce fat intake to 15% of energy intake. Eligible subjects were postmenopausal women with stage II breast cancer whose baseline fat intake was more than 30% of energy intake. The low-fat diet intervention protocol consisted of bi-weekly individual counseling sessions with emphasis on substitution of lower-fat foods for high-fat foods and maintenance of nutritional adequacy. Nutrient intakes were calculated from 4-day food records collected at baseline and after 3 months of diet intervention. Mean daily fat intake for the 17 patients on the low-fat diet dropped significantly from 38.4 +/- 4.3% of energy intake at baseline to 22.8 +/- 7.8% at 3 months (p less than .001). A 25% reduction in mean energy intake, from 1,840 +/- 419 kcal at baseline to 1,365 +/- 291 kcal at 3 months, was accompanied by significant increases in protein and carbohydrate as percent of energy intake. A mean weight loss of 2.8 kg and a 7.7% reduction in serum cholesterol were observed; both changes were significant at the p less than .01 level. Absolute intakes of zinc and magnesium were significantly reduced. However, mean intake on the low-fat diet for 14 vitamins and minerals, including zinc and magnesium, exceeded two-thirds of the 1989 Recommended Dietary Allowances (RDAs). When expressed as nutrient density (i.e., amount of nutrient per 1,000 kcal), increases were observed for all micronutrients. These results support the hypothesis that a nutritionally adequate low-fat diet can be successfully implemented in a highly motivated, free-living population.

Immediate breast reconstruction: reducing the risks.

One-hundred and sixty-five consecutive immediate breast reconstructions in 157 patients were reviewed. Reconstructions were performed with tissue expanders (53 percent) or immediate gel prostheses (47 percent). Immediate reconstruction was associated with an 18 percent rate of implant loss. Certain risk factors were identified at the p less than 0.05 level using immediate gel implants: failure to achieve complete muscle coverage of the implant, smoking at the time of surgery, initial gel implants of 400 ml or more volume, and age. Expander loss was increased by detaching the pectoralis major (p less than 0.05) and probably by lack of complete muscle coverage in general. Chemotherapy, history of previous smoking, and clinical stage of the carcinoma did not seem to affect reconstructive success. Smoking and patient age should be considered during patient selection for immediate reconstruction. Muscle coverage of the prosthesis should always be attempted. Muscle coverage is mandatory in the smoker. Gel implants of 400 ml or more volume are to be avoided at the initial operation. This approach should enable all surgeons to achieve lower rates of implant loss.