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We studied 53 patients with bidimensionally measurable metastases of transitional cell cancer of the bladder who were treated with a planned regimen of 70 mg. per m. cisplatin intravenously on day 1, and 40 mg. per m. methotrexate intravenously on days 8 and 15 every 3 weeks. The toxicity of this regimen, with agranulocytosis and mucositis as the most important side effects, was so severe that only 17 per cent of the patients actually received the protocol regimen without modification. Six patients were ineligible and 47 were evaluable for toxicity, including 43 who were evaluable for response. The response to treatment was assessed after each second treatment cycle. A complete response was achieved in 10 patients (23 per cent) and a partial response was achieved in 10 (23 per cent). The median duration of response was 64 weeks for patients with a complete response and 23 weeks for those with a partial response, while the median duration of survival was 81 and 37 weeks, respectively. The aforementioned regimen with allowance of routine leucovorin rescue is tested as preoperative chemotherapy in patients with stages T3 to T4 nonmetastatic bladder cancer.

A retrospective evaluation of serial measurements of neuron-specific enolase (NSE) has been performed in 58 patients with small cell lung cancer (SCLC). All 58 patients received first-line chemotherapy and 11 patients received also second-line treatment after relapse. Samples were obtained every 3-4 weeks during treatment before each cycle of chemotherapy and every 6 or 12 weeks during follow-up. NSE values were depicted on semi-logarithmic paper. Fifty-one times a major response (complete or partial remission) was observed and 49 times the NSE level reached a plateau between 3.5-10 ng/ml. The NSE level did not discriminate between a complete or a partial remission. Seven times stable disease was obtained and the NSE level declined but remained above the normal plateau of 3.5-10 ng/ml. On 50 occasions progressive disease was found. In 3 cases progressive disease was due to a histologically-proven non-small cell lung cancer and NSE levels did not change. In only 5 out of the remaining 47 occasions NSE levels were normal at the time of relapse but rose later in 4. On 42 occasions of progressive SCLC an exponential rise of NSE was found, often within the range of 3.5-20 ng/ml. None of 6 patients, who are still incomplete remission for 1-5 years, showed a consistent rise of NSE. Serial measurements of serum NSE, can predict the occurrence of a major response, stable disease and progressive disease outside the brain with a very high accuracy and seem to be at least a useful addition to standard investigational methods to guide the treatment of SCLC.

Diamine oxidase (DAO) from tissues of mice, rats and humans showed different properties with respect to stability and kinetic parameters. DAO-activities in homogenates of rat or human tissues, but not of mouse tissues, rapidly decreased upon storage at -20 degrees C. The Km-value for putrescine was 90 microM in mouse kidney or intestine. In rats different Km-values were observed before (272 microM) and after freezing (102 microM). A similar effect was observed with DAO in human kidney (321 and 39 microM, respectively). Treatment of rats with heparin resulted in a depletion of intestinal DAO and the concomitant appearance of DAO in blood. The enzyme remaining in the intestine showed the lower Km-value.

During a retrospective analysis of the value of neuron specific enolase (NSE) in patients with small cell lung cancer (SCLC) it became apparent that at progressive disease (PD) NSE rose exponentially with a doubling time (NSE-Td) varying from 10 - 94 days. In this study the influence of the NSE-Td on the survival of 29 SCLC-patients has been investigated. A significant correlation between survival from the start of rise of NSE at PD and NSE-Td was observed. By extrapolating the exponential rise of NSE to the start of treatment a theoretical logarithmic value of NSE, called Yr, could be calculated. When the patients were grouped according to the Yr value greater than -1, between -1 and -4 and less than or equal to -4 a highly significant correlation between the survival from the start of treatment and NSE-Td was found in all 3 groups. These preliminary data suggest that by means of NSE-Td and Yr value the survival of an SCLC-patient from the time of rise of NSE and from the start of treatment may be predicted within certain limits.

Human lymphoid cell lines were studied as an experimental model for the spontaneous or induced occurrence of tuburloreticular structures (TRS). It was possible to induce TRS after culturing the EB-3 cell line with 20 mug/ml bromodeoxyuridine (BrUdR) during 96 h. Starvation, culturing at lower temperature (32 degrees) or inhibition of DNA synthesis did not give rise to the production of TRS. The response to BrUdR could be blocked with 60 mug/ml thymidine but not with 60 mug/ml deoxycytidine. The addition of 5 mug/ml cytarabine or the removal of BrUdR at different times resulted in inhibition of TRS induction, indicating that BrUdR had to be incorporated into DNA during at least 48 h. After incorporation, neither the presence of BrUdR nor DNA synthesis was necessary for the production of TRS. These experiments and the finding that in the cell line IHTC-33, which does not produce Epstein-Barr virus associated antigens, TRS were spontaneously present, exclude a correlation between TRS and these antigens. However, the induction of TRS by BrUdR may be related to the activation of another (latent) virus.

Five patients suffered from dysphagia due to breast carcinoma. Several aspects of diagnosis and therapy are discussed. It is recommended to start the therapy based on the clinical diagnosis and not to wait until the diagnosis is histologically confirmed.

Capping, independent of metabolic energy, of surface immunoglobulin (S-Ig) on 'Hairy cells' from patients with Hairy cell leukaemia (HCL) is described. As controls leukaemic cells from a patient with a prolymphocytic leukaemia (PLL) and blood lymphocytes from healthy individuals were used. The specificity of the energy-independent capping of the HCL-cells as compared to the controls was tested by incubation of the cells at 4 degrees C in the presence of 0.1 M sodium azide with different FITC-labelled ligands. In order to find an explanation for this phenomenon, the influence of cytochalasin B, colchicine and the combination of both drugs on capping of S-Ig and concanavalin (Con-A)-receptors at 37 degrees C was investigated. Furthermore the effect of Con A on S-Ig capping and vice versa was studied. The results show that only S-Ig on HCL cells could form caps at 4 degrees C in the presence of sodium-azide. Cytochalasin B alone induced a strong inhibition of Con A capping on all 3 cell types, whereas S-Ig capping was unaffected. Colchicine alone had practically no effect. Anti-Ig inhibited subsequent patch and cap formation with Con A on both HCL cells and PLL cells, whereas Con A caps and patches were redistributed by anti-Ig on PLL cells, but not on HCL cells. Conversely, Con A could link S-Ig to other receptors, leading to inhibition of S-Ig capping at 4 degrees C on HCL cells and to co-capping of S-Ig at 37 degrees C on both cell types. In addition Con A induced redistribution of S-Ig caps. The combination of co-capping of S-Ig by Con A, followed by redistribution of the caps by FITC-anti-Ig simulated inhibition of S-Ig capping by Con A on PLL cells. The major conclusions are: in some cases inhibition of capping may actually be caused by redistribution of caps; the energy-independent capping cannot be explained by free diffusion of S-Ig in the membrane through lack of any connexion with receptor-mobility regulating systems. It is proposed that the energy requirement of capping is needed to inactivate a specific mechanism,w which restrains receptor mobility and which is non-operative in HCL cells.

Cells from 32 adult patients with non-Hodgkin's lymphoma were studied with respect to surface markers and functional properties in short-term culture. Twenty-six lymphomas were of B-cell origin, including all nodular and diffuse lymphocytic lymphomas. Three tumors were of T-cell origin (one histiocytic lymphoma and two undifferentiated lymphomas). In the remaining three cases (histiocytic lymphomas) the immunological nature of the tumor cells could not be determined. All reactivity to mitogenic stimuli of cells from B-cell lymphomas was due to residual normal T cells. In follicular lymphocytic lymphomas more reactive T cells prevailed among the malignant B cells than in diffuse lymphocytic lymphomas. Heterogeneity among B-cell lymphomas was indicated by differences in intensity of fluorescence with anti-Ig reagents and in stimulatory capacity in mixed lymphocyte culture. T-cell lymphomas were characterized by high percentages of T cells together with impaired responses to stimuli. The results of immunological studies correlated well with the histological classifications of Rappaport, Lukes and Lennert.

A 61-year-old man underwent chemotherapy for a small-cell undifferentiated lung cancer. During this treatment the primary tumour showed complete remission, whereas a lesion in the contralateral lung increased in size. This second tumour was found on cytological examination to be a moderately differentiated adenocarcinoma which was resected. Histological examination of the resected lung segment provided confirmation that this was a second primary bronchogenic carcinoma. The treatment of synchronous primary bronchogenic carcinomas is reviewed.

In 37 patients with small cell lung cancer treatment with five cycles of cyclophosphamide, doxorubicin and etoposide (CDE), resulted in 23 complete (CR) and 14 partial responses (PR). Median response duration was 34 weeks. At relapse all patients were retreated with CDE. In 23 (62%) patients this gave a second response (6 CR, 17 PR). Factors influencing the occurrence of a second response were: 1. a CR after the first five cycles of CDE; 18 out of 23 CR patients responded again whereas only five of the 14 PR patients responded (P less than 0.01). 2. 15 out of 19 patients with a first response duration greater than 34 weeks reached a second response and in eight of the other 18 patients retreatment was successful (P less than 0.05). Reinduction at relapse, after short term chemotherapy and a treatment-free interval, with the induction regimen is an effective second line treatment in patients with an initial CR and a first response duration of greater than 34 weeks.

The incidence of mutations in cellular ras genes was determined in human seminoma, a germ cell tumor of the testis, with the aid of specific oligonucleotide probe hybridization. To eliminate the large number of nonneoplastic cells present in seminomas, aneuploid tumor cell nuclei were isolated from the tumor tissue by flow sorting. Mutations were detected in 40% of the seminomas at codons 12 or 61 of either the Ki-ras or the N-ras gene. No correlation was found with histopathological or clinical features. In some seminomas the mutant gene was present in only a fraction of the tumor cell population, suggesting tumor heterogeneity for ras gene mutations. Yet, flow cytometric measurement of nuclear DNA contents and histological examination of tumor tissue did not reveal two different tumor cell populations. We conclude from these observations that ras mutation is probably not the initial genetic event in the development of seminoma.

alpha-Difluoromethylornithine (DFMO) is a potent inhibitor of the synthesis of putrescine (pu) and spermidine (sd) in some benign and malignant tissues. Intracellular deprivation of pu and sd has been shown to induce an enhanced uptake of polyamine-analogs such as methyl-GAG (MGBG). The purpose of this study was to investigate the tolerance and the toxicity of the combination of DFMO and MGBG. Thirty-six patients received 4 X 2 g of DFMO/day orally and every 2 weeks 250-500 mg/m2 of MGBG as a 2-hr infusion, starting on day 14. Besides the well known acute and late side-effects of methyl-GAG, dose-limiting toxicity consisted also of thrombocytopenia, leucopenia, dyspnea, hemolysis and jaundice. The maximal tolerated dose of MGBG for one course was 350 mg/m2 and for repeated courses 250 mg/m2, due to cumulative toxicity. Furthermore, after 8 weeks of continuous administration of DFMO 70% of the patients had a severe hearing loss, which was reversible after a treatment delay of 4-6 weeks. Since the hearing loss prohibited the continuous use of DFMO, two different schedules of intermittent DFMO-administration together with two different infusion periods of MGBG have been investigated in 15 patients. In none of these patients did hearing loss occur. The schedule of continuous administration of 4 X 2 g of DFMO/day orally for 21 days and 250 mg/m2 of MGBG as a 24-hr infusion on days 7, 14 and 21, repeated on day 42, was tolerated best. In 28 evaluable patients two partial remissions were seen. Pretreatment with DFMO significantly enhanced the toxicity of MGBG and the combination of both drugs produced side-effects not seen with either drug alone.

In an attempt to define a biochemical marker of ornithine decarboxylase inhibition in humans, alpha-difluoromethylornithine hydrochloride (DFMO), an irreversible ornithine decarboxylase inhibitor, was infused i.v. in seven cancer patients over 10-day courses at doses of 10-90 g/day and 24-h urinary excretion of polyamines and decarboxylated-S-adenosylmethionine was determined before, during, and after treatment. DFMO produces marked increases in urinary decarboxylated-S-adenosylmethionine excretion, up to 84 times pretreatment values. This response appears to be time dependent, requiring several days to reach a maximum and lasting at least 4-5 days after stopping DFMO. In contrast, urinary excretion of the polyamines putrescine, cadaverine, spermidine, N1-monoacetylspermidine, N8-monoacetylspermidine, and spermine, were not consistently altered by DFMO. We conclude that urinary excretion of decarboxylated-S-adenosylmethionine represents a valid biochemical indicator of ornithine decarboxylase inhibition in humans, whereas urinary polyamines are of no value.

A rapid and simple method, originally described by Redmond and Tseng [J. Chromatogr., 170 (1979) 479] was applied to the analysis of di- and polyamines in cultured human tumour cells and human tumour xenografts. Optimization of the procedures and evaluation of the characteristic features of the assay are described. The (modified) procedure employs precolumn derivatization with benzoyl chloride, extraction of the derivatives by chloroform, separation by reversed-phase high-performance liquid chromatography under isocratic conditions and detection by ultraviolet absorbance measurement at 229 nm. The complete analysis was accomplished within 10 min per sample. The detection limit was ca. 1 pmol. The intra- and inter-assay coefficients of variation were 2.5-4.4% and 3.4-13.1%, respectively. The presence of well known inhibitors of polyamine biosynthesis, such as DL-alpha-difluoromethylornithine and methylglyoxal bis(guanylhydrazone), did not interfere with the assay, and disturbance by cyclohexylamine could be avoided by changing the polarity of the mobile phase. The method proved to be very suitable because it is rapid, simple, requires a minimum of sample pretreatment, and still provides sufficient sensitivity to quantitate polyamines in relatively small amounts of cells (10(5) cells) or tumour tissues (less than 1 mg), even after treatment with inhibitors of polyamine biosynthesis.

Total platinum kinetics were studied after the administration of two formulation products of cisplatin: the lyophilized form and a ready-to-use solution. Twelve patients received both preparations during two successive cycles in a randomized crossover study. Platinum concentrations in plasma and urine were measured by atomic absorption spectrometry. Data were analyzed by means of a mixed-effect analysis of variance. Areas under the concentration-time curves up to 96 h were increased (p = 0.026) and slopes of the elimination phase were decreased (p = 0.035) during cycle 2 when compared with cycle 1. However, no difference in these two parameters was observed when comparing the two formulations. Three-day urinary platinum excretion was not related to either the treatment cycle or the formulation used. Because of its convenience of use and reduced risk of aerosolization, the ready-to-use formulation seems preferable.

The pharmacokinetics of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of polyamine biosynthesis, were investigated in BALB/c (nude) mice after i.p. injection and after oral administration of radiolabeled drug. After i.p. injection the compound was rapidly cleared from the serum (t1/2 alpha = 14 min; t1/2 beta = 2.1 h) and from tissues such as muscle, liver and kidney (t1/2 alpha = 30-60 min; t1/2 beta = 2.1 h). DFMO concentrations were proportional to the administered dose (10-2000 mg/kg) in both serum and tissues. Oral administration of DFMO was carried out by dissolving the compound in drinking water at a concentration of 20 g/l. Studies on the distribution showed that DFMO did not accumulate preferentially in any particular tissue. An extremely wide variation in the dose actually achieved in different animals was observed; this ranged from 350 to 2800 mg/kg for a 14-h treatment period. A significant correlation (r = 0.83-0.92) between the dose of DFMO, calculated from the consumption of drinking water for each individual animal, and the DFMO concentrations in serum, muscle, spleen, liver and kidney was found. Similarly, it was shown that oral administration of DFMO during the daytime resulted in 10- to 15-fold lower levels than administration during the night. After discontinuation of treatment DFMO levels in serum and tissues decreased by 50% in approximately 6 h. From these results it is concluded that the optimal treatment schedule of mice with DFMO (or other drugs with similar pharmacodynamic properties) consists in a combination of oral administration via the drinking water and additional i.p. injection (during the daytime). Furthermore, the drug intake of the individual animals should be monitored to check whether the experimental requirements are actually fulfilled.

A radioimmunoassay for neuron specific enolase (NSE), a marker of neuroendocrine differentiation, has been evaluated in small cell lung cancer (SCLC). In untreated patients 25/38 (68%) with localized SCLC had raised blood levels of NSE (greater than 13 ng ml-1), in extensive disease 34/39 (87%) patients had raised NSE levels. In patients with non-small cell lung cancer (NSCLC) the serum levels were raised in 16/94 (17%). In extensive tumours of non-pulmonary origin NSE levels were increased in 24/116 (20%) patients. Longitudinal studies indicated a good correlation between the response to chemotherapy and fall of NSE levels. Tumour progression was accompanied by a rising NSE in 25/29 patients, with doubling times of 7-90 days. In patients with progression with a normal NSE the recurrence was a NSCLC. Cerebral metastases occurring as the only recurrence during clinical complete remission were not accompanied by a rise of NSE. Serum NSE levels provides a valuable monitor for SCLC during and after chemotherapy.