Drug-induced DNA modification in buccal cells of cancer patients receiving carboplatin and cisplatin combination chemotherapy, as determined by an immunocytochemical method: interindividual variation and correlation with disease response.

Twenty-six patients with a variety of tumor types were treated according to a phase 1 experimental treatment protocol consisting of repetitive cycles of cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) (carboplatin, 200-480 mg/m2) at day 1 and cis-diamminedichloroplatinum(II) (cisplatin, 50-100 mg/m2) at day 3. Buccal cells were collected in one or two treatment cycles prior to carboplatin, 24 h after carboplatin, just prior to cisplatin, and approximately 24 h after cisplatin administration. Drug-induced DNA modification was visualized at the single cell level by anti-serum NKI-A59 and quantitated by microdensitometry. All (39 of 39) treatments with carboplatin, and almost all (33 of 35) treatments with cisplatin resulted in an increase in nuclear stain. Interindividual variation in drug-induced, adduct-specific nuclear stain amounted to a factor of 5-8 for carboplatin and 5-12 for cisplatin. This drug-induced increase was, however, not related to the dose of either carboplatin or cisplatin, suggesting that large interindividual differences in DNA adduct formation and/or repair obscured the effects of dose variation within the relatively small range used for the drugs (2.4 for carboplatin and 2.0 for cisplatin). This explanation was strengthened by the good reproducibility of the immunocytochemical assay and by the reasonable correlation between carboplatin-induced nuclear stain in cycles 1 and 2 (correlation coefficient, 0.69; P = 0.009). Mean carboplatin-induced nuclear stain was significantly higher in the first cycle than in the second cycle (P = 0.0001) but this difference was no longer significant when drug-induced nuclear stain was corrected for carboplatin dose. Differences in cisplatin-induced nuclear stain between cycle 1 and cycle 2 were small and not significant. Carboplatin-induced nuclear stain was significantly higher in the partial responders than in the nonresponders (P < 0.0001, two cycles combined); the level of statistical significance remained the same after dose correction. Cisplatin-induced nuclear stain did not differ significantly between partial responders and nonresponders; this result might, however, be confounded to some extent by remaining carboplatin-induced nuclear stain at the moment of cisplatin administration. It is concluded that determination of the extent of platinum-induced DNA modification might be helpful in predicting the tumor response in cancer patients.

Cytotoxic activity of human pulmonary alveolar macrophages.

The functions of human pulmonary alveolar macrophages (PAMs) have been relatively little studied compared with those of their circulating counterparts, blood monocytes. This study examined the ability of human PAMs to kill primary human tumor cell cultures and control normal fibroblasts in vitro. PAMs were derived by bronchial lavage from patients with lung cancer of various histological types and stages, patients with acute or chronic noncancerous pulmonary disorders, and subjects with a presumed illness who proved to be normal. After extensive washing, the PAMs were cocultured with [3H]proline-labeled tumor cells, principally lung cancers and melanomas, at various effector:target ratios for 60 hr. Cytotoxicity was measured by comparing radioactivity associated with the remaining adherent tumor cells cultured in the presence or absence of PAMs. Twenty-eight of 42 preparations of PAMs from 42 individuals were cytotoxic to one or more short-term primary tumor cultures. All 28 specimens from patients with lung cancer or chronic pulmonary disease were cytotoxic; all of the 14 PAM preparations lacking cytotoxicity were from individuals with acute pulmonary disorders or who were proved free of pulmonary disease. PAMs were cytotoxic even at effector:target ratios of 2.5:1 or 1.25:1. Fibroblasts were unaffected at any ratio. Sarcoidosis patients in remission had noncytotoxic PAMs, whereas the disease in relapse was characterized by cytotoxic PAMs. Serial study of 2 patients confirmed a loss of reactivity during remission. Smoking did not correlate with the presence or absence of spontaneous cytotoxicity and did not influence the degree of cytotoxicity in "reactors." Partially purified alpha-interferon enhanced the killing of cytotoxic PAMs in 10 of 21 instances but did not induce cytotoxicity in 9 tests on nonreactive PAMs. We conclude that human PAMs from patients with lung cancer or chronic pulmonary diseases, including active sarcoidosis, were cytotoxic to several recently explanted tumor cell cultures. PAMs from acute pulmonary dysfunctions and those from patients with inactive sarcoidosis were not spontaneously cytotoxic.

Correlation between cytotoxic and suppressor activities of human pulmonary alveolar macrophages.

We have reported previously that pulmonary alveolar macrophages (PAMs) from individuals with lung cancer and active chronic pulmonary diseases were cytotoxic to tumor cells in vitro, whereas PAMs from normal individuals or patients with acute pulmonary disorders were noncytotoxic. In the present study, we evaluated 20 PAM preparations for both suppressor and cytotoxic functions to determine if PAMs could function as suppressor cells and, if so, whether a correlation between the two functions exists. Cytotoxicity was assessed in a 60-hr cytotoxicity assay against [3H]proline-prelabeled human melanoma target cells. More than 20% cytotoxicity was considered to be significant. Suppressor activity was measured by determining whether admixing PAMs at various ratios with autologous or allogeneic mononuclear cells could suppress concanavalin A-induced blastogenesis by T-lymphocytes. At least 50% suppression was considered to be significant. Of the 20 specimens evaluated, 13 were cytotoxic and 5 of these exhibited suppressor activity. None of the 7 noncytotoxic PAM preparations had suppressor activity. Suppression was nonspecific and not HLA restricted, since autologous and allogeneic mononuclear cells were inhibited to a similar extent. Suppression was probably not due to prostaglandin production by the PAMs since assays were performed under optimal conditions and required extremely high concentrations of prostaglandins. A significant correlation between suppressor and cytotoxic activity was found. Suppression was observed only with PAM specimens that were also highly cytotoxic to tumors, but not all cytotoxic PAM specimens were suppressive. Whether these actions reflect different levels of activation of PAMs or are the properties of different macrophage subsets remains to be clarified.

Graft versus leukemia effect after transplantation with interleukin-2-activated bone marrow. Correlation with eradication of residual disease.

IL-2 has been used after autologous BMT (ABMT) with the aim of inducing graft versus leukemia (GVL) effect. Our studies in mice have shown that IL-2 therapy induces GVL effect when employed after BMT with bone marrow (BM) that has been activated with IL-2 in vitro (ABM). The present study was carried out to define the time of optimal GVL effect after BMT so that the immunomodulatory approaches could be concentrated at the time of maximum GVL effect. Our data show that GVL effect was induced if IL-2 was instituted immediately after BMT with ABM in mice with acute myeloid leukemia; institution of IL-2 1 week after BMT with ABM did not induce GVL effect. IL-2 therapy instituted immediately or 1 week after BMT with fresh bone marrow (FBM) did not induce any GVL effect. A significant increase in the NK activity was noticed whether IL-2 was instituted immediately or 1 week after BMT, either with FBM or with ABM. To evaluate the ability of IL-2 in the eradication of residual disease from the autograft and the host, BM with variable infiltration with leukemia was activated with IL-2 and used for BMT in leukemic mice. The GVL effect of BM with minimal leukemic infiltration (absence of morphologically demonstrable disease) was comparable to the GVL effect of normal BM. These findings suggest that: (a) maximum GVL effect after BMT with ABM is concentrated in the early post-transplant period possibly because of minimal residual disease during this time; (b) an increase in the NK activity induced by IL-2 therapy may not predict for an improved GVL effect; and (c) for optimum GVL effect, BM with minimal leukemic infiltration should be activated with IL-2 before BMT.

Correlation of meningioma hormone receptor status with hormone sensitivity in a tumor stem-cell assay.

Several investigators have detected progesterone receptors in a high percentage of meningioma specimens and have noted progesterone receptors to be more common than estrogen receptors in these specimens. However, a functional significance of such hormone receptor positivity in control of meningioma growth has not been described. This paper describes a paired test of the estrogen and progesterone receptor assay as the biochemical assay and of the human tumor stem-cell clonogenic assay (HTSCCA) as the functional assay in 17 meningioma specimens. Only one (6%) of the 17 specimens was estrogen receptor-positive, while 11 (69%) of 16 specimens were progesterone receptor-positive. The HTSCCA revealed that only two (15%) of 13 specimens were sensitive to estradiol while five (31%) of 16 specimens were sensitive to progesterone. Comparison of progesterone results for the 15 specimens on which both hormone receptor assay and HTSCCA were performed revealed correlation in a majority of cases; four specimens were positive for both assays and five specimens were negative for both assays. No specimen that was negative for progesterone receptors was sensitive to progesterone by HTSCCA. These results suggest that the hormone receptor and sensitivity pattern of meningiomas may differ from that of breast cancer, and that progesterone addition or ablation may be a reasonable therapeutic approach for meningiomas.

Dose-escalation study of carboplatin (day 1) and cisplatin (day 3): tolerance and relation to leukocyte and buccal cell platinum--DNA adducts.

Carboplatin and cisplatin have similar antitumor activities but different toxicities. Combining these two analogs may be expected to balance the toxicities and allow higher doses of platinum compounds to be administered with tolerable toxicity. To test this concept, a Phase I trial of carboplatin in combination with cisplatin was performed. Thirty-three eligible patients received carboplatin doses ranging from 200-480 mg/m2 on day 1 and cisplatin doses ranging from 50-100 mg/m2 on day 3 of the 28 day cycle. A 2-day interval ensured no interference in renal excretion of carboplatin by cisplatin. Myelosuppression was the dose limiting toxicity. With the usual full dose of carboplatin, 480 mg/m2, patients tolerated 50 mg/m2 of cisplatin, without apparent additional toxicity. At 100 mg/m2 of cisplatin, non-hematologic as well as hematologic toxicities frequently precluded administration of more than 300 mg/m2 of carboplatin. Platinum-DNA adduct quantitation was done in leukocytes and buccal cells during cycle 1 in most patients. The adduct-specific immunosignal in buccal cells was always increased after carboplatin and in all but one after cisplatin. The level of adducts in buccal cells increased with increasing doses of carboplatin and cisplatin. In leukocytes, measurable levels of adducts were formed after carboplatin with further contribution made by cisplatin but not obviously in a dose dependent fashion. We conclude from the toxicities observed, that combinations of carboplatin with cisplatin may have advantages over either drug alone in certain clinical situations; and that further study of platinum-DNA adducts may shed light on platinum dose-response relationships.

Immunological effects of recombinant interferon-alpha 2 in cancer patients.

Fifteen patients with various types of cancer, resistant to conventional therapy, were entered into a phase I trial of pure interferon-alpha 2 (IFN-alpha 2) produced by recombinant DNA technology. Groups of patients received either 3, 10, 30, or 50 x 10(6) units (U) of IFN-alpha 2 subcutaneously daily for 4 weeks and were closely followed for possible toxicity. At the higher doses, toxicity was encountered, which led to reduction in the frequency of administration. For immunological testing, peripheral blood mononuclear cells were obtained before treatment on day 1, on day 2, and during the 2nd, 3rd, and 7th weeks of the study. The cells were tested for natural killer (NK) activity, antibody-dependent cellular cytotoxicity (ADCC), monocyte-mediated antibody-dependent cellular cytotoxicity (MMADCC), and spontaneous monocyte-mediated cytotoxicity (SMC). ADCC was augmented at all doses in 9 of 15 patients, 6 of whom exhibited elevated levels by day 2. In direct contrast, MMADCC was decreased in 12 of 14 patients 7-20 days after beginning treatment. SMC was increased on day 2 in 1 of 2 patients given 3 x 10(6) U and in 3 of 4 patients given 10 x 10(6) U. SMC in the other patients given these doses was unchanged on day 2, with no decreases noted. In contrast, SMC was decreased in 2 of 4 patients given 30 x 10(6) U. SMC in the other patients given these doses was unchanged on day 2, with no decreases noted. In contrast, SMC was decreased in 2 of 4 patients given 30 x 10(6) U and in 2 of 3 patients given 50 x 10(6) U. NK cell activity was increased in 2 of 3 patients given 3 x 10(6) U, but was either unchanged or decreased in patients given higher doses. The only exception was an increase found in a patient given 50 x 10(6) U, whose renal cell carcinoma responded significantly to treatment. Data on NK and SMC from a phase II study of breast cancer treated daily with 3 x 10(6) U IFN-alpha 2 support our phase I observations. NK-cell activity was increased on day 2 in 3 of 8 patients, and SMC increased in 2 of 8 patients. As in the phase I study, no decreases in these functions occurred. In summary, for those responses that were dose dependent, such as NK and SMC, lower doses of recombinant IFN-alpha 2 (3 x 10(6) and possibly 10 x 10(6) U) may be more effective in increasing these antitumor activities than are higher doses.