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We studied 107 specimens (38 tumors and 69 tumor cell lines) from 90 patients with small cell lung cancer to determine the characteristics and clinical situations of patients from whom tumor cell lines could be established and the myc family DNA copy number. The proportion of extensive stage small cell lung cancer patients from whom a tumor cell line could be established prior to the initiation of therapy increased during the 10 years of the study (P less than 0.001). Amplification of one of the myc family genes occurred in 3 of 40 (8%) of the untreated patient specimens compared to 19 of 67 (28%) of the treated patient specimens (P = 0.01). The myc family DNA amplification occurred in 17 of 54 (31%) of the specimens from patients treated with cyclophosphamide-based combinations and 2 of 13 (15%) of the specimens from patients treated with etoposide/cisplatin (P = 0.25). Both tumors and tumor cell lines were obtained from 17 patients with small cell lung cancer and the myc family DNA copy number was similar in 16 of the 17 patients. We conclude that: (a) myc family DNA amplification occurs more commonly in specimens from treated than untreated patients (b) there are no prominent differences in the frequency of amplification following treatment with different chemotherapy regimens; and (c) myc family DNA amplification is similar in tumors and tumor cell lines from the same patients.

An in vitro soft agar technique was used in an attempt to culture neuroblastoma cells from 71 bone marrow, 3 lymph node, and 2 solid tumor specimens from 18 patients with neuroblastoma. One-half of each specimen was sent for routine pathology studies and one-half was cultured in the soft agar system. Colonies appeared within 10 days in histologically positive bone marrows. Light microscopy, electron microscopy, catecholamine secretion, and karyology provided evidence that the colonies were composed of neuroblastoma cells. There were 38 instances in which histological study of the specimen demonstrated neuroblastoma cells. The soft agar system showed colony growth in 30 of these samples (79%). There were a total of 38 specimens that were histologically negative for neuroblastoma. Thirty of these 38 specimens showed no growth in the stem cell assay. Eight histologically negative specimens from 6 patients formed colonies in the soft agar system. Five of these 6 patients showed tumor histologically on prior or subsequent marrow examinations. In addition to a significant correlation between histological and soft agar culture results (p < 0.001), there exists a highly significant positive correlation between the number of colonies per plate and the histological status of the specimen (p < 0.005). Serial marrow samples were cultured on 7 patients. There appears to be an association between the number of colonies that develop in the plate and the clinical course and prognosis of the patient. Decreasing plating efficiencies (number of colonies per number of cells plated) correlated with tumor response. Increasing plating efficiencies indicated tumor relapse. A plating efficiency of greater than or equal to 0.1% portended a particularly poor prognosis. Neuroblastoma grows well in this soft agar culture system. This excellent growth provides a good model for both clinical and basic science studies of neuroblastoma.

Tumor specimens procured from 38 different small cell lung cancer patients were studied for DNA amplification of the myc family of protooncogenes (c-myc, N-myc, and L-myc). Six of the 38 specimens (16%) had 4-fold or greater myc family DNA amplification (N-myc in 4 and L-myc in 2). All 6 tumors with amplification came from patients who had received combination chemotherapy. The myc family gene copy number of the DNA prepared from 9 tumor cell lines established from these 38 patients was similar to the myc family gene copy number in the DNA prepared from fresh tumor specimens from these same patients. myc family DNA amplification is present in 16% of small cell lung cancer patients' tumors and the amplification pattern in the tumor cell lines is representative of the fresh tumors obtained from the same patients.

Hyperthermia has been shown to increase the cytotoxic effect of certain cancer chemotherapeutic agents in vitro and in vivo. It is not known whether the combination of hyperthermia plus these chemotherapeutic drugs will also increase the toxicity of the drugs. This study was undertaken to evaluate the toxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea combined with hyperthermia on normal mouse bone marrow in vitro. An increased effect of the combination was seen when the marrow was heated at 42 degrees in the presence of the drug for 15 min (p less than 0.025) and for 1 hr (p less than 0.005). Incubations for 2 hr produced a significant decrease of control growth, such that no increased effect of the combination of drug plus hyperthermia could be seen. In order for the increased effects to be seen, heating had to be done simultaneously in the presence of the drug. Clinicians using the combination of whole-body hyperthermia plus 1,3-bis(2-chloroethyl)-1-nitrosourea should be aware of possible increased marrow toxicity.

During a randomized trial of total parenteral nutrition (TPN) in patients with small cell lung cancer, we evaluated the short- and long-term effects of 4 weeks of TPN on nutritional assessment parameters. All 119 patients who were accrued to the study received the same chemotherapy and radiotherapy protocol which extended over a 1-year period: 57 patients received TPN; and 62 served as controls. At base line, patients with greater than 5% pretreatment weight loss had significantly lower levels of serum albumin, total iron-binding capacity, and creatinine/height index. TPN administration led to a significant increase in mean caloric intake and weight compared with controls (P less than 0.0001). In the short-term study, body fat, as measured by triceps skinfold thickness, was maintained, and there was a small increase in arm muscle circumference. Serum albumin and hematocrit decreased but promptly returned to pretreatment levels when TPN was stopped. There were no long-term differences in any of the nutritional assessment parameters between the two groups.

The applicability of a human tumor colony-forming assay to drug screening was investigated in terms of feasibility, validity, and potential for discovering new antitumor drugs. Feasibility was addressed in a pilot study during which basic methods, appropriate assay quality controls, and a standardized protocol for screening were developed. Considerable variability was noted in the availability and colony growth of different tumor types. The majority of the evaluable experiments utilized breast, colorectal, kidney, lung, melanoma, or ovarian tumors. For many tumor types, little evidence of growth was observed, or only rare specimens formed colonies. Colony-forming efficiencies ranged from 0.05 to 0.11% for the six most useful tumors listed above. A set of quality control measures was developed to address technical problems inherent in the assay. Testing of standard agents in the pilot study established that most of these agents could be detected as active. However, it also identified three assay limitations: compounds requiring systemic metabolic activation are inactive; medium constituents may block the activity of certain antimetabolites; and compounds without therapeutic efficacy may be positive in the assay. The assay categorized nontoxic clinically ineffective agents as true negatives with 97% accuracy. Of 79 compounds which were negative in the current National Cancer Institute prescreen (leukemia P388), 14 were active in the assay. Several demonstrated outstanding in vitro activity and are structurally unrelated to compounds already in development or in clinical trials. A subset of these active compounds were found to lack activity in a P388 in vitro colony-forming assay. This indication of differential cytotoxicity to human tumor cells makes this subset of compounds particularly interesting as antitumor drug leads. The demonstrated sensitivity to most standard agents, discrimination of nontoxic compounds, reproducibility of survival values within assays and between laboratories, and evidence of ability to identify active compounds which were negative in the in vivo prescreen suggest that the human tumor colony-forming assay may be a valuable tool for antitumor drug screening. However, because of technical limitations inherent in the current assay methodology, this must be confined to selected tumor types and limited to screening on a moderate scale.

Clinical tumor specimens and cultures of small cell lung cancer (SCLC) produce 10- to 100-fold higher quantities of the BB isoenzyme of creatine kinase (CK-BB) (EC 2.7.3.2) than did other types of lung cancer. Serum CK-BB levels were evaluated in 105 newly diagnosed, previously untreated patients with SCLC. All patients were thoroughly staged, including 42 patients with limited-stage and 63 patients with extensive-stage disease. Serum CK-BB was elevated (greater than 10 ng/ml) in 27 patients (26%) (range, 11 to 522 ng/ml; median, 40 ng/ml). Only 1 of 42 patients with limited disease had an elevated serum CK-BB, while 26 of 63 (41%) of patients with extensive disease did. When patients were subgrouped according to the number of metastatic sites detected in pretreatment staging, a significant association between the presence of an elevated serum CK-BB and the number of metastatic sites was observed (p less than 0.005). No association between the presence of metastatic disease in a specific site and an elevated serum CK-BB could be detected. After adjusting for the number of metastatic sites, survival among patients with a normal pretreatment CK-BB was significantly better than in patients with an elevated CK-BB (p = 0.014). Sequential serum CK-BB determinations in 33 patients revealed an excellent correlation between clinical response to therapy and serum CK-BB levels. Continuous SCLC cell lines established from 13 patients in this study all expressed high levels of CK-BB. These data suggest that serum CK-BB determinations may be of value in estimating the extent of tumor dissemination, assigning prognosis, and monitoring response to therapy in patients with SCLC.