Combined cytotoxicity effect of hyperthermia and anthracycline antibiotics on human tumor cells.

The effects of temperature on the anthracycline antibiotics-induced cell kill of DND-1A human malignant melanoma (MM) and DND-39A Burkitt's lymphoma (BL) cell lines were studied by means of a clonogenic assay. The two cell lines differed in sensitivity when exposed to heat: The MM cells were unaffected by hyperthermia (42 degrees C), whereas BL cells were sensitive to this temperature. With the MM cells, hyperthermia potentiated the cytotoxic effects of doxorubicin (ADM), daunorubicin, mitoxantrone (DHAD), and quelamycin but did not enhance that of aclacinomycin (ACM). Conversely, the exposure of cells to the anthracycline compounds at 0 degree C resulted in almost complete disappearance of cell kill effects except with ACM; ACM retained substantial cell kill effects even at the given low temperature. For BL cells, ADM- or DHAD-induced cell lethality was also potentiated by hyperthermia; ACM produced only additive cell kill. At 0 degree C, ACM's effects virtually disappeared. These data indicate that human tumor cell lines have a substantial variety in heat sensitivity and that not every anthracycline antitumor agent is potentiated by temperature. ACM's thermoresponse is unique among anthracycline antibiotics studied. Additionally, it was shown that normothermic cell kill by ADM was not affected by hyperthermic preheating; however, preheating of appropriate duration produced important influence on subsequent hyperthermic ADM-induced cell kill.

Establishment of methotrexate-resistant human acute lymphoblastic leukemia cells in culture and effects of folate antagonists.

A human acute lymphoblastic T-cell line, MOLT-3, was fed with Roswell Park Memorial Institute Medium 1640-10% fetal bovine serum-antibiotics, containing increasing concentrations of methotrexate (MTX). After 10 months of feeding, the cells became resistant to 10(-7) M MTX; resistance was not reversed when the cells were placed in the original MTX-free medium. At 10(-7) M MTX, the concentration which produced complete inhibition of the parent MOLT-3 cell growth, the resistant cells were not inhibited at all. On a 50% inhibitory concentration basis, the resistant cells were approximately 30-fold more resistant to MTX. The parent MOLT-3 and the resistant line had the same doubling time of approximately 36 hr. There were no differences in light microscopic morphology. MOLT-3 produced loose colonies on 0.5% agar enriched with fetal bovine serum, whereas the colonies of the resistant line were tightly packed. The development of resistance was accompanied by a 4- to 5-fold decrease in [3H]MTX transport (MOLT-3/MTXt). Kinetic analysis of MTX uptake showed that the resistant subline did not have an altered Km for MTX (6.6 microM) but had a decreased Vmax of about 20% of the parent cell line. These data suggest that either the number of folate transport sites or the turnover rate of these sites has been reduced in the MTX-resistant cell line. Dihydrofolate reductase was only minimally elevated in the resistant cell line. The MTX-resistant cell line was cross-resistant to dichloromethotrexate. The sensitivity of the resistant line to the substituted 2,4-diaminoquinazoline and pyrimidine compounds, 2,4-diamino-5-methyl-6-[(3',4',5'-trimethoxyanilino) methyl] quinazoline (JB-11) and 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine, increased more than 3-fold. While leucovorin equally reversed the MTX effects on the parent and resistant cells, leucovorin reversal of 2,4-diamino-5-methyl-6-[(3',4',5'-trimethoxyanilino) methyl] quinazoline and 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine effects was limited only to the parent cell line. 2,4-diamino-5-methyl-6-[(3',4',5'-trimethoxyanilino) methyl] quinazoline or 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine plus leucovorin might prove to be unique in treating patients with acute lymphoblastic leukemia when the leukemic cells develop transport resistance to MTX.

Evolution of methotrexate resistance of human acute lymphoblastic leukemia cells in vitro.

A human acute lymphoblastic T-cell line, MOLT-3, was fed with Roswell Park Memorial Institute Medium 1640 supplemented with 10% fetal bovine serum and antibiotics which contained increasing concentrations of methotrexate (MTX). The development of drug resistance was associated initially with progressive decrease in MTX transport. When the cells became 200-fold resistant, a rise in the dihydrofolate reductase was noted which was short-lived in the absence of the drug. A 10,000-fold increase in MTX resistance was accompanied, in addition to further decrease in MTX transport, by a 10-fold increase in the dihydrofolate reductase activity. While the purely transport-related resistant cell lines had a collateral sensitivity to lipid-soluble antifols, the sublines which had both transport- and enzyme-related MTX resistance contained a subpopulation highly resistant to these antifols. Chromosome analysis of the subline with increased dihydrofolate reductase activity showed an expanded abnormally banded region in chromosome 5.

Placebo-controlled double-blind comparative study on the preventive efficacy of mesna against ifosfamide-induced urinary disorders.

In order to evaluate the preventive efficacy, safety and usefulness of mesna (Sodium 2-mercaptoethane sulfonate) against ifosfamide-induced urinary disorders, a placebo-controlled double-blind comparative study was performed. Ifosfamide was administered by intravenous drip infusion at a daily dose of 2 g/m2 for 5 consecutive days, and mesna was intravenously administered at 20% of the ifosfamide dose, three times daily for 5 consecutive days. The results obtained are as follows. (a) Of 101 accrued patients, 91 patients were evaluated consisting of 45 for the mesna group and 46 for the placebo group. There was no intergroup difference in the number of the evaluated cases and patient characteristics. (b) Micturition pain and feeling of residual urine graded as moderate or severe were not observed for the mesna group, but were observed for the placebo group with incidences of 19.6% (9/46) for micturition pain and 15.2% (7/46) for feeling of residual urine; the intergroup differences in the appearance of these urinary symptoms were statistically significant (P = 0.0003 for micturition pain; P = 0.0009 for feeling of residual urine). The incidence of hematuria graded as moderate or severe was 6.7% (3/45) in the mesna group, which was significantly lower than the 32.6% (15/46) in the placebo group (P = 0.0008). (c) No side-effect attributable to mesna was observed. (d) A judgment of "useful" was obtained in 80.0% (36/45) of the patients treated with mesna, which was significantly higher than the 34.8% (16/46) of the patients treated with placebo (P = near 0). On the basis of the above results, we conclude that the preventive efficacy, safety and usefulness of mesna against ifosfamide-induced urinary disorders have been well demonstrated in this study.

Primary pulmonary non-Hodgkin's lymphoma in a Japanese renal transplant recipient.

Pulmonary non-Hodgkin's lymphoma (NHL) developed in a 31-year-old renal transplant recipient 3 years after transplantation. Chest roentgenogram showed rapidly progressive multiple nodules in both lungs. The pleural fluid obtained by needle aspiration contained many atypical cells with surface B cell antigen and abnormal karyotype. The patient died of pulmonary edema shortly after combination chemotherapy was begun without regression of the tumors. Autopsy revealed diffuse large cell NHL confined to both lungs. Immunological examination and DNA analysis of the tumor showed monoclonal B cell NHL.

Brief induction chemotherapy followed by involved-field irradiation for the treatment of localized intermediate- and high-grade non-Hodgkin's lymphoma.

In an attempt to assess the effects and toxicity of brief induction chemotherapy plus involved-field irradiation for localized intermediate- and high-grade non-Hodgkin's lymphoma, we conducted a single-arm prospective trial between May 1987 and July 1991. Patients received four cycles of a five-drug chemotherapy regimen consisting of cyclophosphamide, doxorubicin, vincristine, prednisolone, and bleomycin (CHOP-Bleo) followed by involved-field irradiation of 30 Gy. Of 32 patients consecutively enrolled, 30 patients had measurable and/or evaluable disease, while two had no evaluable disease, as their major tumors had been surgically removed. All the patients were in complete remission at the conclusion of all treatment. Four patients have relapsed so far and one died of the disease at 17 months. With a median follow-up time of 33 months (range, 8 to 58), the actuarial disease-free survival rate was 82% and the overall survival rate was 96%. The toxicity was generally mild and all the patients received the planned treatment course. This treatment modality appeared to be safe and effective for localized intermediate- and high-grade non-Hodgkin's lymphoma.

Alternating chemotherapy of CHOP-Bleo and POEM-Bleo for diffuse large-cell lymphoma: a single-institutional study with a long-term follow-up.

Diffuse large-cell lymphoma (DLCL) is a neoplasm that is curable with chemotherapy in an appreciable percentage of patients. However, not all patients are cured and the best drug combination and optimal dose intensity have not yet been established. In an attempt to improve complete response rate and survival with minimal toxicity, we devised an alternating combination chemotherapy consisting of CHOP-Bleo (cyclophosphamide, doxorubicin, vincristine, prednisolone, and bleomycin) and POEM-Bleo (prednisolone, vincristine, etoposide, mitoxantrone, and bleomycin). Between March 1986 and October 1990, 30 newly-diagnosed patients with advanced DLCL were treated with the regimen. Of these 30 patients, 14 (47%) were 61 years of age or more, 15 (50%) had stage IV disease, 14 (47%) presented with constitutional symptoms, and 7 (23%) had T-cell lymphoma. After the completion of therapy, 23 (77%) achieved a complete response and 6 (20%) had a partial response. The actuarial relapse-free survival at 5 years is 52% and the overall survival projected to 5 years is 47%. Toxicity was generally mild and well tolerated. Although this alternating regimen had substantial activity as front-line chemotherapy for advanced DLCL, we conclude that the observed response rate and survival do not essentially differ from those achieved with conventional regimens and further clinical trials are thus not warranted.

Cisplatin-resistant human small cell lung cancer cell line shows collateral sensitivity to vinca alkaloids.

A cisplatin-resistant cell line, SBC-3/CDDP, was established from a human small-cell lung cancer cell line, SBC-3. The SBC-3/CDDP cells were 13.1-fold more resistant to cisplatin than the parent SBC-3 cells. We investigated the cellular changes of this cell line with regard to the development of resistance to cisplatin. The SBC-3/CDDP cells showed various characteristics as follows: a) increased intracellular glutathione and glutathione S-transferase content b) decreased intracellular accumulation of cisplatin, c) increased topoisomerase I activity and the same topoisomerase II activity as the parent SBC-3 cells, and 4) strong cross-resistance to the platinum analogues and mitomycin C, moderate cross-resistance to 7-ethyl-10-hydroxy-camptothecin (SN-38), 4-hydroperoxy cyclophosphamide, etoposide, Adriamycin and methotrexate, and collateral sensitivity to vinca alkaloids and 5-fluorouracil. From these observations, the SBC-3/CDDP cells could be useful as a well characterized cisplatin-resistant cell line, and the resistance pattem in this cell line will give us much information for eradication of cisplatin-resistant tumor cells.

Successful treatment of non-Hodgkin's lymphoma in a patient with common variable immunodeficiency.

A case of common variable immunodeficiency (CVID) who developed non-Hodgkin's lymphoma (NHL) of the rectum is reported. A 22-year-old male student in whom CVID was diagnosed at 7 years of age was referred to our department for the treatment of rectal NHL. The patient had stage IE disease confined to the rectum after clinical diagnostic procedures. He was initially treated with radiation therapy alone, but a relapse soon occurred in the paraaortic lymph nodes. He was successfully treated with CHOP-Bleo chemotherapy and supplementation with immunoglobulin preparations. He has since remained free of NHL and infectious complications for over 30 months despite his persistent immunodeficiency.

OK-432 induces production of neutrophil chemotactic factors in malignant pleural effusion.

We investigated the changes in cellular components and neutrophil chemotactic factors in pleural fluid from 19 lung cancer patients who received intrapleural injection of OK-432 to treat malignant pleurisy. Not only neutrophil chemotactic activity (NCA) but also neutrophil count and percentage were increased significantly at 6 hours after OK-432 injection. The neutrophil count was significantly correlated with NCA level. The levels of C5a and IL-8 in pleural fluid were increased significantly after OK-432 injection. The increased IL-8 level was associated with a increase of both NCA and neutrophil count. OK-432 treatment also induced a marked increase of IL-1 beta and IL-6 in pleural fluid. Thus, intrapleural injection of OK-432 induced production of neutrophil chemotactic factors (IL-8 and C5a) and cytokines (IL-1 beta and IL-6), which eventually attracted neutrophils into the pleural space. These observations suggest that neutrophil migration mediated by these factors and cytokines may contribute to the sclerosing effects of OK-432 treatment.

Long-term results of combination chemotherapy with or without irradiation in small cell lung cancer: a 5- to 11-year follow-up.

Between April 1981 and December 1987, 148 patients with newly diagnosed small cell lung cancer (SCLC) were treated using combination chemotherapy with or without thoracic irradiation and prophylactic cranial irradiation (PCI) in a series of cooperative therapeutic trials. With a minimum follow-up of 4.7 years, 13 (9%) patients survived and were free of SCLC. These included 11 (15%) of 76 patients with limited disease and two (3%) of 72 patients with extensive disease. Three died without any evidence of SCLC (one each from second leukemia, non-small cell lung cancer, and unrelated disease). The remaining 10 (7%) patients are currently alive and free of SCLC beyond 4.7 years. Since late relapse beyond 5 years is a very rare event, these patients may have been cured. However, late toxicity of PCI must be kept in mind. Three among the 10 patients have suffered from neuropsychologic symptoms of varying degrees in severity. Although the long-term survival rate is a benchmark in the treatment of SCLC, modifications of therapy that may potentially avoid such toxicities should be considered hereafter.

Neural cell adhesion molecule expression and clinical features in small cell lung cancer: a semi-quantitative immunohistochemical approach using an immunogold-silver staining method.

The neural cell adhesion molecule (NCAM) is a family of cell surface sialoglycoproteins mediating homotypic and heterotypic cell-cell adhesion. In tumors, NCAM is supposed to be involved with the malignant features characterized by invasive growth and metastasis. In the present study, we evaluated the correlation between NCAM expression of tumors obtained from small cell lung cancer (SCLC) patients and the clinical outcome. NCAM expression was determined semi-quantitatively by an immunogold-silver staining method using the SCLC cluster 1 monoclonal antibody NCC-LU-243. Of 20 SCLC patients studied, six patients with tumors with high NCAM expression had a poor response to chemotherapy, and a short disease-free (p = 0.011) and overall (p = 0.003) survival as compared with 14 patients having tumors with low NCAM expression. These findings indicate that the therapeutic outcome of SCLC may be partly predicted by determining the NCAM expression of the tumor.

Establishment of an adriamycin-resistant subline of human small cell lung cancer showing multifactorial mechanisms of resistance.

A subline highly resistant to Adriamycin (SBC-3/ADM100) was isolated in vitro from the human small cell lung cancer cell line, SBC-3, by culturing in progressively higher concentrations of Adriamycin. The SBC-3/ADM100 cells were 106-fold more resistant to the drug than the parent cells in an inhibitory concentration of 50% determined by the MTT assay. The population-doubling time was much longer in SBC-3/ADM100 than in the parent cells. Northern blot hybridization revealed marked overexpression of the MDR1 mRNA in the resistant cells. P-glycoprotein overexpression and a decrease in intracellular accumulation of Adriamycin were demonstrated in SBC-3/ADM100, indicating that outward drug transport was the major mechanism of resistance in this subline. Additionally, a significant elevation of the intracellular glutathione content coupled with the glutathione S-transferase (GST) pi level and a decrease in DNA topoisomerase II (Topo II) activity were noted in this resistant subline. These results indicate that the mechanism of resistance to Adriamycin is multifactorial; involving altered growth characteristics, an enhanced outward transport, enhanced drug detoxification process, and decreased target enzyme activity. The resistant subline will serve as a useful tool in the search for ways to overcome drug resistance.

MDR1 gene expression and treatment outcome in small cell lung cancer: MDR1 gene expression as an independent prognostic factor.

We report a preliminary study to determine whether MDR1 gene expression level in small cell lung cancer (SCLC) tumors is a useful predictor of tumor response to chemotherapy and patient survival in association with myc amplification in the tumor. We analyzed 18 patients with SCLC receiving adriamycin and etoposide combination chemotherapy between August 1989 and November 1991; 16 males and 2 females, median age of 68 years, and 7 with limited disease and 11 with extensive disease. MDR1 mRNA expression level and myc family gene amplification were simultaneously determined by polymerase chain reaction using transbronchial biopsy specimens which were obtained at diagnosis. Patients with tumors expressing low MDR1 mRNA responded more favorably to chemotherapy than those with tumors expressing high MDRI mRNA, however, the difference in tumor response was statistically not significant (84.6% versus 40%). The overall survival was significantly shorter in the latter than in the former (7.2 months versus 11.7 months; p = 0.023). The survival of the 4 patients with tumor showing myc family gene amplification was almost identical to that of patients with tumors showing no amplification of the gene (8.2 months versus 8.8 months; p = 0.73). Multivariate Cox's regression analysis supports the notion that MDR1 may be a useful independent prognostic factor.

Comparison of antitumor activity of new anthracycline analogues, ME2303, KRN8602, and SM5887 using human lung cancer cell lines.

In an attempt to predict the clinical activity of newly developed anthracycline analogues, ME2303, KRN8602, and SM5887 in the treatment of lung cancer, we compared antitumor activity of these drugs with that of adriamycin, using six human lung cancer cell lines and two drug-resistant human lung cancer sublines. Taking the pharmacokinetic data into consideration, we evaluated the relative antitumor activity: the ratio of area under the concentration-time curve of each drug to the 50% inhibitory concentration of the drug. Regarding this ratio, ME2303 was more potent than adriamycin, SM5887, and KRN8602. Cross-resistance of the new analogues to adriamycin was investigated using an adriamycin-resistant small cell lung cancer subline, SBC-3/ADM100 and an etoposide-resistant subline, SBC-3/ETP. SBC-3/ADM100 being 106-fold more resistant to adriamycin than the parent SBC-3 showed less resistance to the analogues: 1.80-fold to KRN8602, 3.80-fold to SM5887, and 8.60-fold to ME2303. SBC-3/ETP which was 52.1-fold more resistant to etoposide and 39.5-fold more resistant to adriamycin were also less resistant to the new analogues: 3.27-fold to KRN8602, 9.07-fold to SM5887, and 17.3-fold to ME2303. In conclusion, ME2303 was found to be the most potent agent among drugs tested for the treatment of lung cancer, and KRN8602 can be expected to be beneficial for the treatment of drug-resistant small cell lung cancer.

Establishment and characterization of an etoposide-resistant human small cell lung cancer cell line.

An etoposide-resistant subline, SBC-3/ETP, from a human small cell lung cancer cell line, SBC-3, was developed by continuous exposure to increasing concentrations of etoposide in culture. The SBC-3/ETP was 52.1-fold more resistant to etoposide than the parent cell line. The SBC-3/ETP was highly cross-resistant to teniposide, adriamycin, vinca alkaloids, 4-hydroperoxycyclophosphamide, CPT-11 and mitomycin C, and marginally cross-resistant to cisplatin, while the subline showed a collateral sensitivity to bleomycin. Topoisomerase I activity in the SBC-3/ETP was reduced to an extent of one half and topoisomerase II activity to an extent of one eighth in comparison with those of the SBC-3. Intracellular accumulation of [3H]-etoposide in the SBC-3/ETP was significantly lower in comparison to the SBC-3. An overexpression of MDR1 mRNA, and the presence of its product, P-glycoprotein, were detected in the SBC-3/ETP by Northern blotting and flowcytometry using a monoclonal antibody of the protein, MRK16. These results indicate that a decreased activity of topoisomerase II is the major factor for the development of etoposide resistance, and that an overexpression of the MDR1 gene is responsible, in part, for the development of resistance to the drug and some structurally unrelated compounds such as adriamycin and vinca alkaloids.

A case of non-Hodgkin's lymphoma associated with hypercalcemia.

A patient with a diffuse, small cleaved cell, non-Hodgkin's lymphoma associated with marked hypecalcemia was described. Antibody to the adult T-cell leukemia-lymphoma virus was absent. Although bone marrow was infiltrated by lymphoma cells, destructive or lytic bone lesions could not be detected. The serum level of immunoreactive parathyroid hormone C-terminal (PTH-C) was normal. The serum level of 1, 25-dihydroxyvitamin D was lower than normal. This case suggests that other humoral substances produced by lymphoma cells may be responsible for hypercalcemia.

Alveolar lymphocyte proliferation induced by Propionibacterium acnes in sarcoidosis patients.

The proliferation of lymphocytes induced by Propionibacterium acnes (P. acnes) was measured by the in vitro incorporation of 3H-thymidine. The mean response rate of alveolar lymphocytes obtained by bronchoalveolar lavage was 2.23 +/- 0.89 in nine untreated sarcoidosis patients, 0.85 +/- 0.17 in five sarcoidosis patients given corticosteroids and 0.78 +/- 0.29 in 11 controls. The proliferation was significantly enhanced in the untreated patients compared to both the treated patients (p less than 0.01) and controls (p less than 0.001), but there was no significant difference in response rates between the treated patients and controls. The response rate of alveolar lymphocytes was significantly higher in four active patients (3.05 +/- 0.61) than in four inactive patients (1.77 +/- 0.44) (p less than 0.05) and in the controls (p less than 0.001). In sarcoidosis patients, the response rates showed a good correlation with activities of serum lysozyme (r = 0.695, p less than 0.01), and with percentages of lymphocytes in bronchoalveolar lavage fluid (r = 0.591, p less than 0.05). There was a low correlation between angiotensin-converting enzyme activities and the response rates (r = 0.508, p less than 0.1). Neither peripheral blood lymphocytes in sarcoidosis patients nor in controls showed any response to P. acnes, but alveolar lymphocytes of the untreated active sarcoidosis patients were sensitive to P. acnes. The lymphocytes activated by P. acnes may play a central role in the induction of alveolitis in sarcoidosis patients.

Anticancer drug sensitivity by human tumor clonogenic assay.

The anticancer drug sensitivity of human cancers was tested by the human tumor clonogenic assay (HTCA). Of 152 human cancer specimens tested, 63 (41%) formed more than 30 tumor cell colonies in control plates and could be used to evaluate the drug sensitivity of tumor cells. In 42 (93%) of 45 clinical trials in 24 patients, a parallel correlation was observed between the in vitro anticancer drug sensitivity measured by the HTCA and the clinical response of tumors to anticancer drugs. These results suggest that the HTCA is a good technique for the in vitro test of the anticancer drug sensitivity of human cancers.

Cytological and histological correlation of primary lung cancer: a preliminary study of 106 cases with resectable tumors.

In order to increase the accuracy of diagnosis in lung cancer, analysis concerning cytological and histological correlation was attempted. The present study consists of 106 patients, who were seen during the past approximately five years and underwent radical surgery to remove tumors completely; mere biopsy specimens were excluded. These patients were 63 years old on the average, 78 males and 28 females, 29 cases of the hilar type (H) and 77 of the peripheral type (P), and 27 and 76 cases of the clinical stage I in H and P, respectively. Histologically, there were 53 adenocarcinomas (Ad), 38 squamous cell carcinomas (Sq), 4 adenosquamous cell carcinomas (Ad + Sq), 5 large cell carcinomas (LCC), and 6 small cell carcinomas (SCC); among them, 3 Ad and 21 Sq in H, and 50 Ad and 17 Sq in P. The overall positive percentages were 65.5 (H) and 26.0 (P) by combination of spontaneous, airsol-induced and Saccomanno's methods, against 96.6 (H) and 72.8 (P) with inclusion of brushing method. 94.8% of Sq in H and 66.7% of Ad and 70.6% of Sq in P were positive by the brushing. A comparative study of these four methods, performed at least once on the same patient, also confirmed the superiority of brushing. Cyto- and histological agreement was 21/21 (100%) for Sq in H, whereas 30/34 (88.2%) for Ad and 13/15 (86.7%) for Sq in P. In conclusion, cyto- and histological findings in H and P corresponded well, and as far as cytology of peripheral type is concerned, a combined method, especially with brushing, is strongly recommended.