142 Abstracts/Lung Cancer I5 (19%) 139-157

Hopkins Uniwrsiry, School of Medicine, 424 North Bond Street, Baltimore, MD 21231. J Cell Physiol 19%;166:43-8.

In an effort to study the mechanism underlying the observed phenotype-specific response of human lung cancer cell lines to a polyamine analogtie, N’,N’r-bis(ethyl)spermine(BESpm), we have isolated a BESpm resistant cell line from the BESpm-sensitive large cell lung carcinoma line NC1 H157. The mutant line exhibits identical growth rates in the presence or absence of the analogue. However, the overall growth of mutant cells reaches stationary phase earlier than that of the parental cells. In contrast to the parental cells, where a superinduction of spermidinelspermine N’-acetyltransferase (SSAT) is associated with BESpm toxicity, treatment of this resistant line with BESpm did not induce SSAT mRNA or enzyme activity. BESpm treatment was not effective in depleting the intracellular polyamine pools and very low intracellular BESpm levels were detected. This BESpm resistance is not mediated by multidrug resistance (MDR) protein, since these cells maintain their sensitivity to the antineoplastic agent adriamycin. Treatment of these cells with methylglyoxal bis(guanyIhydrazone) (MCBG), an AdoMetDC inhibitor which enters cell using polyamine transport system, shows no inhibition of cell growth. Our data suggest that these mutant cells are deficient in polyamine transport. Consistent with this hypothesis, exogenous polyamines did not prevent difluoromethylomithine (DFMO) induced growth inhibition in the mutant cells.

Glucocorticoid receptors and growth inhibitory effects of dexamethasone in human lung cancer ceil lines Hotinann J, Kaiser U, Maasberg M, Havernann K. Depanmenr of Internal Medicine, Division of Hematology and Oncology, Philipps- University, Baldingerstrasse, D-35033 Marburg. Eur J Cancer Part A Gen Top 1995;31:2053-8.

Expression of glucocorticoid receptors (GR) and growth effects of dexamethasone and the antiglucocorticoid RU486 were investigated in six cell lines originating from small cell lung cancer (SCLC) and 13 cell lines from non-small cell lung cancer (NSCLC; four adenocarcinoma, four squamous cell carcinoma, four large cell carcinoma and one mesothelioma). AI1 cell lines contained specific and saturable binding sites for the synthetic glucocorticoid dexamethasone, as determined by whole cell assays and by cytosolic receptor assays. The presence of GRs in the carcinoma cells was confirmed by immunocytochemistry. In NSCLC cell lines, GRs were present in large amounts (37-638 fmollmg cytosolic protein). In SCLC cell lines, GRs were also detectable but in considerably lower concentrations. Growth inhibitory effects of dexamethasone were seen in the cultures of two squamous cell carcinoma lines (EPLC32M 1 and NCI-H 157), one adenocarcinoma line (A-549), one large cell carcinoma cell line (LCLC-97TM 1) and the cell line of a meaothelioma(MSTO-21 IH). Allcell linesresponsivetodexamethasone had high GR concentrations (164 fmollmg cytosolic protein). The antiglucocorticoid RU486 was virtually inactive when administered alonebutwasabletoblockthegrowth-inhibitoryeffectofdexamethasone. The results indicate that glucocorticoids may inhibit the progression of individual non-small cell lung carcinoma.

Sequential molecular genetic changes in lung cancer development Chung GTY, Sundaresan V, Hasleton P, Rudd R, Taylor R, Rabbitts PH. MRC Clin Oncol Radiorlrerapeuric Unit, MRC Centre, HiIk Rood, Cambridge CB2 2QH. Oncogene 1995; 11:2591-S.

Epithelial tumours develop through a sequence of preinvasive lesions of increasing disarray driven by underlying somatic genetic changes. We have studied the occurrence of the two most common somatic genetic changes associated with lung cancer in a series of

premalignant bronchial lesions representing different stages in lung tumorigenesis. We present evidence that allele loss on chromosome 3 precedes damage to the ~53 gene. Damage to chromosome 3 itself appears to be sequential in that the pattern of allele loss seen in dysplasia is often much more discrete than in invasive tumours. This implies that preneoplastic lesions may be a useful source of material for deletion mapping studies aimed at localising the position of tumour suppressor genes. We illustrate this by the comparison of an interstitial deletion described in this study with a homozygous deletion we have described previously, which has resulted in a better definition of the localisation of a tumour suppressor gene believed to be involved in lung cancer development.

Putrescine accumulation in human pulmonary tumours Hoet PHM, Dinsdale D, Verbeken EK, Demedts M, Nemery B. Laborarorium voor Pneumologie. Herestraat 49, B-3ooO Leuven. Br J Cancer 1996;73:96-100.

Type II pneumocytes and Clara cells, both epithelial cells that possess an active uptake system for polyaminea, have been identified as possible precursor cells of atleast some types of lung turnours. In this study we have investigated whether human pulmonary tumours exhibit putrescine uptake. Lung slices from both tumoral tissue and non- tumoral tissue, obtained from patients undergoing surgery for lung cancer, were incubated with radiolabelled putrescine at both 37°C and 4°C. The accumulation of putrescine was evaluated by its apparent kinetic parameters, in the presence or absence of cystamine, and by autoradiography. The investigated tumoral tissue (six squamous carcinomas and live adenocarcinomas) did not show accumulation of putrescine above that attributable to simple diffusion, except for one adenocarcinoma. In this specimen autoradiography showed that the accumulation was not specifically associated with any particular cell type, but that practically every cell accumulated putrescine. We conclude that human pulmonary tumours do not accumulate polyamines in a manner similar to normal pulmonary epithelial cells.

Ras oncoproteins in human plasma from lung cancer patients and healthy controls Anderson D, Hughes JA, Cebulska-Wasilewska A, Nixankowska E, Graca B. BIBRA Inrernnrional. Woodmansterne Road, Carshalton, SurreySM54DS. MutatResFundamMol MechMutagen 1996;349: l21- 6.

In order to explore the significance of ras oncoproteins in plasma in the carcinogenic process, we have examined samples from 40 Polish human lung cancer patients prior to treatment. They were compared with 35 healthy donors and have been screened using a direct analysis of the plasma. Proteins were separated by gel electrophoresis, transferred to a nitrocellulose membrane by Western blotting and detected by chemiluminescence, using monoclonal ras antibody as the primary antibody. Elevated increasesin rasoncoproteins weredetermined where an increase was considered to be greater than 2 standard deviations above the mean negative control values. The results showed that in 45 % of cancer patients ras oncoprotein levels were statistically significantly increased (P < 0.001, pooled two-sample t-test untransformed, and non-parametric Mann-Whitney test) in the plasma by comparison with 6% in the-controls. Thiswouldsuggest thatanincmaseinrasoncoproteins in plasma could be a possible prognostic marker or biomarker for lung cancer.

Allelic deletion mapping on chromosome 5 in human lung carcinomas WieIand I, Bohm M, Arden KC, Ammermuller T, Bogatz S, Viars CS,

Abstracts/Lung Cancer 15 (19%) 139-157 143

Rajewsky MF. Insz. Cell Biology - Cancer Research, University of

Essetr Medical School, Virchowstr. 173, D-4S122 Essen. Oncogene 1996;12:97-102.

We analysed allelic deletions on chromosome 5 in microdissected human non-small cell lung cancers. Thirty-four primary squamous cell carcinomas, 15 primary adenocarcinomas and five regional lymph node metastases were investigated for loss of heterozygosity (LOH) in chromosomal region 5p15-q21. The sites analysed included the APC tutnorsnppressorgeneat 5q21, fivepolymorphic rnicrosatellitemarkers and the putative tumor suppressor locus del-27, that was assigned to chromosomal region 5~13-12 by fluorescence in situ hybridization (FISH) analysis. Allelic deletions encompassed larger genomic regions more often in squatnous cell carcinomas than in adenocarcinomas. The del-27 amd APC regions were identified as two distinct regions with the highest LOH frequencies within 5plSq21. In squamous cell carcinomas LOH frequencies were 73 % at the del-27 and 70 96 at the APC locus. In adenocarcinotnas LOH at the del-27 and APC loci occurred in 38 % of the informative cases. Allelic deletion of the APC gene and at the del- 27 locus was also detected in the tnetastases. The results suggest involvement of at least two tumor suppressor genes on chromosome 5 in lung tnmorigenesis.

Radioloealization of squamous lung carcinoma with ‘% labeled epidermal growth factor Cuartero-Plaza A, Martinez-Miralles E, Rosell R, Vadell-Nadal C, Farre M, Real FX. Depanament d’lmmunoiogia, Carrer de1 Dr. Aiguader, 80, E-3 Barcelona. Clin Cant Res 1996;2: 13-20.

Overexpression of epidertnal growth factor receptor (EGFr) in squamous carcinomas has been demonstrated extensively. Preliminary clinical studies have shown that radiolabeled anti-EGFr monoclonal antibodies can localize. to these tnmors. The aims of this study were to determine the tolerance, phartnacokinetics, and radiolocalization properties of “II-labeled EGF in patients (n = 9) with advanced squamonslnngcancer. Patients’vitalsignsandsymptotnsweremonitored regularly for 3 days. Daily scintigrams and biological samples for pharmacokinetic analysis were obtained for 3-4 days, 99’Tc-labeled human serum albumin was administered to patients with positive tumor scans. Six patients had positive tumor scans, and live of them had received 1.0 trig EGF. In all of these cases, tumors were visualized the same day of the infusion, although best tumor-background contrast was obtained at 50-74 h. There were no false-positive images. Whole-body radioactivity retention rose significantly with increasing EGF doses; most labeled EGF was eliminated by urinary excretion. Tumor:normal tissue uptake ratios increased during the course of the study. All patients presented self-limited, dose-related gastrointestinal adverse effects. In conclusion, recombinant “‘I-labeled EGF administered iv. can localize to squamous lung cancer efficiently, can be administered safely to patients, and has more advantageous pha-okinetic properties than monoclonal antibodies. Further studies are warranted to detertnine more accurately the potential of EGF and EGF-related peptides in the imaging and/or therapy of EGFr-overexpressing human cancers.

Activity of anti-erbE2 recombinant toxin OLX-209 on lung cancer cell lines in the absence of erbB-2 gene amplification Kasprzyk PG, Sullivan TL. Hunt JD. Gubish CT, Scoppa CA, Oelkuct M et al. lhe Lombardi Cancer Center, Georgetown University Medical School, Ihe Research Building. 3970Reservoir Road, Washington. DC 2oW7. Clin Cant Res 1996;2:75-80

The recombinant oncotoxin OLX-209 [e23(Fv)PE38KDEL] has been developed to target cancers with erbB-2 expression and is nearing a clinical trial. Important in clinical planning is the selection of patients on the basis of tumor expression of erbB-2. ErbB-2 gene amplification

occurs in cancers of the breast, stomach, and ovary. Patients with these diseases and evident overexpression are candidates for OLX-209 therapy. In lung cancer, overexpression of erbB-2 is also frequent, but in most cases, it is not caused by gene amplification. This study demonstrates that OLX-209 has activity on lung cancer cells with varying levels of erbB-2 expression in the presence and absence of gene amplification. In vitro sensitivity of cell lines to OLX-209 is related to erbB-2 expression level. Normal bronchial epithelial cells were not sensitive, Effective treatment of lung cancer cell lines growing as xenografts in nude mice was shown with Calu-3 (a lung adenocarcinoma line with high levels of pltJS(erbB-2) caused by gene amplitication) and three other lung adenocarcinomas (A549, NCI-H1466, and 2OlT) with lower levels of pl85(erbB-2) andnogeneamplification. TheZOlTcell linewasisolated recently from a lung tnmor with erbB-2 expression in the original hunor. The results of this study indicate that patients with erbB-2-positive, non- small cell lung cancer should be included in clinical trials of OLX-209.

~53 protein in non-small cell lung cancer as quantitated by enzyme-linked immunosorbent assay: Relation to prognosis Pappot H, Francis D, Btunner N, Grondahl-Hansen J, Osterlind K. 7be Finsen Laboratory, Strandboulevarden 49, DK-2100 Copenhagen. Clin Cant Res 1996;2: 155-60.

The prognostic value of p53 protein in tumor extracts as measured by ELISA was studied retrospectively in 228 non-small cell lung cancer (NSCLC) patients. The assay measuresboth wild-typeand mutated ~53. The specimens on which this study was performed have been used earlier to analyze the prognostic impact of components of the plasminogen activation system, which enabled an analysis of relationships behveen these components and p53 protein. The median of the p53 protein values in the 228 patients was 0.10 (range, o-0.70) ngltng protein. Survival analysis comparing patients with p53 levels below versus above the median showed no significant difference (P = 0.67). When analyzing the histological types, adenocarcinoma (n = 106). squamous cell carcinoma (n = 84), and large cell carcinoma of the lung (n = 38) separately, similarly, no significant differences in survival between patients having low versus high tumor p53 levels were found. When comparing levels of p53 protein in the three histological types, a significant difference (P < 0.0001) was found, with adenocarcinomas having the lowest levels. There was a weak positive correlation (r = 0.22) between ~53 protein and plasminogen activator inhibitor type 1 (PAI-1). Multivariate analysis proved no impact of p53 on survival; tumor size, PAI-1, and lymph node involvement were theonly variables with significant influence on survival. These data indicate that p53 protein quantitated with a sandwich ELISA in tumor extracts from NSCLC hasno prognosticvalue, but theobserved statistically significant difference of p53 protein content between histological subgroups may be related to differences in etiology and biology in different NSCLC subtypes. In addition, the weak association found behveen p53 protein andtheindependentptognosticmarkerPAI-1 conldsuggestyetnndefined interactions in lung cancer.

Prognostic significance of CCNDl (cyclin Dl) overexpression in primary resected non-small-cell lung cancer Betticher DC, Heighway J, Ha&ton P.S. Alterntatt I-II, Ryder WDJ, Cemy T et al. CRC Departrnenr of Cancer Generics. Paterson Institute Cancer Research, Christie Hospiral (NHS) Trust, Wilmslow Road, Manchester M20 9BX. Br J Cancer 1996;73:294-300.

AmplilicationoftheCCDNl geneencodingcyclinD1 wasexamined by Southern blotting and multiplex polymerase chain reaction (PCR) and occurred in 8 of53 patients (15 %) with primary resected non-small- cell lung cancer (NSCLC). These tumours and 17 additional tumours with a normal gene copy number showed overexpression of cyclin Dl