Abstracts/Lung Cancer II (1994) 123-150 131

genetic changes that precede the onset of invasive lung cancer, and to search for biological markers useful in screening multiple primary tumorsoftheupperaerodigeativetract, wehaveperformedacytogenetic and genetic study using normal bronchial epithelium and primary tumor specimens of 68 patients undergoing pulmonary resection for early stage lung cancer, and normal bronchial epithelium of 5 controls with metastatic sarcomas. Of the 68 lung cancer cases, 3 1 had a single tumor and 37 displayed multiple synchronous or metachronous tumors. Cytogenetic alterations were observed in 59% (23139) of the evaluable tumor specimens with complex rearranged karyotypes, particularly involving chromosomes 3 (70%). 17 (39%), 11(‘26%), 8,9,12 (22%), snd7(17%).Genealterationswerealsodete&dincludingoverexptession of epidermal growth factor receptor (EGFR) in 63 % (36157). HER21 NEU in 21% (12/56), and ~53 mutations in 50% (12124). The overall frequency of genetic changes (any type) in the tumors was 76 96 (52168). In the normal bronchial mucosa, we identified a rearranged karyotype in20 % oftheevaluablecases(13/63); particularly simple rearrangements involving chromosomes 3p (6 cases), 7 (6 cases), 17 (3 cases), 9, 11 (2 cases), 8 (1 case); as well as overexpression of EGFR in 39% (20151) and of HER2/NEU in 14% (7/51). The overall frequency of genetic changes (any type) in the normal epithelium was 46% (30165). The presence of a rearranged karyotype in the bronchial mucosa was associated with a rearranged karyotype in the tumor sample. Other istically significant correlations were found between histopathologic and clinical features and the occurrence of the different cytogenetic and genetic changes both in tumors and in the normal bronchial mucosa. No genetic abnormalities were found in the bronchial epithelium of the 5 controls.

Molecular epidemiology of lung cancer and the modulation of markers of chronic carcinogen exposure by chemopreventive agents Perera FP, Tang D, Grinberg-Funes RA, Blackwood MA, Dickey C, Blaner W et al. Columbia Univ. School of Public Hlth, Division of Environmental Sciences, 60 Haven Ave. New York, NY 10032. J Cell Biochem 1993;52 Suppl 17 F:ll9-128.

Chronic inhalation exposure to environmental carcinogens such as polycyclic aromatic hydrocarbons (PAHs), cigarette smoke, 4- aminobiphenyl (CABP), ethylene oxide, and styrene is associated with elevations in biomarkers such as DNA adducts, protein adducts, sister chromatid exchanges (SCEs), chromosomal aberrations, gene mutation, and/or oncogene activation. These biomarkers indicate an increased cancer risk for the exposed population, although quantitative estimates cannot be made with certainty. There is convincing epidemiological evidence that the antioxidant and free radical-scavenging vitamins C and E and A-carotene (B-C) protect against cancer of the lung and other epithelial tissues, with somewhat weaker evidence for retinol. Experimental studies demonstrate that these micronutrients are capable of blocking or reducing tumor formation caused by diverse carcinogens. A variety of mechanisms appear to be involved, including suppression of carcinogen activation, enhancement of carcinogen detoxification, induction of cellular differentiation, inhibition of mutagenesis, enhancementofimmunologicfunction, and/orreductionoftheformation of carcinogen-DNA adducts, SCEs, micronuclei, and other markers of genotoxic damage. Therefore, we have recently investigated the possible modifying effect of serum vitamins C and E, 8-C, and retinal on a number of such biomarkers in a case-control study of lung cancer, and in a cross-sectional study of heavy smokers. Preliminary results indicate an inhibitory effect of certain vitamins on DNA adduct formation. A significant number of human intervention trials are ongoing involving thesevitamins. Itappcarsthat biomarkerscanprovideusetid intermediate endpoints for assessment of both the mechanisms and the efficacy of chemopreventive agents.

Prognosticimpact of mutated K-ras genein surgically resected non-small cell lung cancer patients Rosell R, Li S, Skacel Z, Mate JL, Maestre J, Canela M et al. Depanment of Medical Oncology, Univ Hospital Germans Trias i Pujol, 08916 Badalona, Barcelona. Gncogene 1993;8:2407-12.

Mutated K-ras oncogenes have been detected in a third of lung adenocarcinomas, located usually in codon 12, its presence correlating negatively with survival. To further define the role of K-ras point mutations in non-small cell lung cancer, ne studied the presence of mutated K-ras genes in surgical specimens from 66 patients. Polymerase chain reaction was performed from sections of formalin-tixed paraffin- embedded tissue. We screened for point mutations in codons 12,13 and 61 of the K-r&s gene by dot blot hybridization analysis with mutation- specific oligonucleotide probes. Ras gene mutations were p-t in 13 of 66 carcinomas (20%), nine in codon 12 and four in codon 61. Three squamous cell carcinomas harbored two different point mutations in K- ras codon 12. Mutated K-ras genes were found more frequently in squamous cell carcinomas (eight of 38) than in adenocarcinoma (three of 22). Analysis of nucleotide sequence disclosed a multifarious mutation pattern of K-ras codon 12, where the most common conversion was from glycine (GGT) to valine (G’JT). K-ras point mutation positive subset had poorer survival, nine of the 13 patients died during the follow-up period as compared with 22 of 53 patients with no mutation in the K-ras gene (P = 0.01). The difference was also strikingly significant when stratified according to node status.

Abnormalities of retinoblastoma gene structure in human lung tumors Linardopoulos S, Gonosa ES, Spandidosa DA. Institute of Biological Research/, Biotechnology, National Helknic Res Foundation, 48 Vas Constantinou Avenue, Athenr 11635. Cancer Lett 1993;71:67:74.

The retinoblastoma (Rb) gene is associated with the pathogenesis of several typeaofhuman cancer, includmgretinoblastoma, osteosarcoma, soft tissue sarcomas, and lung, breast and bladder carcinomas. Loss of heteroxygosity is a common mode in allelic inactivation of Rb and other tumor-suppressor genes. We investigated DNA from 15 human lung tumors for loss of heteroxygosity of the Rb locus using a polymerase chain reaction (PCR) based restriction fragment length polymorphism assay. Of informative cases we found loss of heteroxygosity in 2 out of 3 squamous cell carcinomas and 1 out of 2 adenocarcinomas of the lung. We also found structural rearrangements in two out of fourteen Hind III digested lung tumors examined at the 5’ region of the human Rb gene using Southern blot hybridization analysis. Since these two tumors were classified as stage III it is possible that the alteration of Rb gene is involved in the progression of this type of cancer. Using specific primers for exons 15,16,21 and 22 of the Rb gene, we carried out amplification of these exons by polymeruse chain reaction. None of these tumors showed a deletion of exons 15, 16, 21 and 22.

Expression of carbohydrate antigens in human pulmonary adenocarcinoma Kawai T, Suzuki M, Kase K, Ozeki Y. Department of Pathology, Notional Defense Medical Colkge. 3-2 Namiki, Tokorozawa 359. Cancer 1993;72:1581-7.

Background. The altered and anomalous expression of carbohydrate antigens (CA) occur in patients with lung carcinomas. Methods. TO investigate the relationship between prognosis and immunohi&&~~ findings of CA in serum and pulmonary adenocarcinomas (PA), PA specimens surgically resected from 102 patients were studied with the use of A, R, H, Lewis&e)‘, Leb, Le(x), Le(y). sialyl L-9 (FH-7). sialyl Le(x-i) (SLX) (FH-6), sialosyl Tn (TKH-2), and CA 19-9. The Lewis blood group types were determined .by the dot blotting method on

132 Abstracts/Lung Cancer 11 (1994) 123-I50

patients’ serum using monoclonal anti-w, Leb, Le(x), and Le(y). Formalin-fixed paraffin-embedded PA sections were stained using the avidin-biotin-peroxidase complex methods. Results. There was no correlation between the patients’ postoperative survival and tissue expression of CA, although survival correlated with the clinical stages, histologic differentiation of PA, and serum SLX in Stage 4. The higher the positive rate of both Leb and Le(y). the better their differentiation in PA specimens. Regardless of blood group type, PA showed high positive reactions for FH6 (88%) and L&) (87%). The serum concentration of CA 19-9 correlated with immunohistochemical reactions of tumors (Spearman correlation coefficient = 0.33, P = 0.02). The expression of Le’ in Lewis (a-b+) phenotype patients and Type 2 CA, such as LeQ) and FH-6, is characteristic in PA. Conclusions. The findings suggest that CA tissue expression may not reflect postoperative survival length of patients with PA, but biological aberrant fucosylation or sialylation as well as morphologic alteration may occur in neoplastic cells.

Association of dissociation of a protease and its inhibitor on the surface of lung squamous eelI carcinoma cells Steven FS, Anees M, Myers J, Ha&ton P. Dept Biochemistry/ Molecular Biology, School of Biological Sciences, University of Manchester, Manchester Ml3 9PT. Anticancer Res 1993; 13: 1063-S.

Squamous cell carcinoma cells possess a cell surface protease, referred to asguanidiiobenzoatase(GB). GB isaplasminogen-activator- like enzyme which can be located by the fluorescent probe 9-amino acridiie in frozen sections. Fluorescence microscopy has been used to study the inhibition of this GB, the displacement of inhibitor from GB, thedisplacementofGBfmmthecellsurfacereceptorandthepreparation of both active GB and inhibitor, obtained from these frozen sections of tumour tissue.

Voltage-activated K+ conductance and cell proliferation in small-cell lung caner Pancrazio JJ, Tabbara IA, Kim YI. Department of Biomedical Engineering, Box 377, Univ Virginia Health Sciences Center, Charlottesville, VA 22908. Anticancer Rea 1993;13:1231-4.

Whole-cell patch-clamp measurements indicate that human small- cell lung cancer (SCLC) cells express voltage-dependent potassium channels, whose function is blocked by K’ channel antagonist 4- aminopyridine (4-AP). Exposure of the tumour cells to 4 mM 4-AP reduced the peak outward K’ current (evoked by a depolarization to +80 mV) from 2.05 f 0.24 nA (mean f SEM, n = 28 cells) to 0.98 f 0.12 nA (n = 27). Incubation of SCLC cells with 0.1, 4 and I6 mM 4-AP resulted in a concentration- and time-dependent reduction in the number of viable cells when compared with the control; over a period of 144 hours, the drug either significantly reduced the number of viable SCLC cells or caused an apparent cessation of neoplastic cell proliferation, whereas the untreated control cells demonstrated a more than l6-fold multiplication in the number of viable cells. The inhibitory effect on cell growth was also observed with an additional K’ channel antagonist, tetraethyl-ammonium. These data suggest that voltage-activated K’ channels expressed by SCLC cells play a role in neoplastic cell proliferation.

Activationof potassiumcbannets by hypoxia and reoxygenation in the human lung adenocarcinoma cell line AS@ Koong AC, Giaccia AJ, Hahn GM, Saad AH. Department of Radiation Oncology, Sranford University School of Med., Stmford, CA 94305 5468. J Cell Physiol 1993;156:341-7.

Active oxygen specie-s are generated in cells during pathophysiologic conditions such as inflammation and postischemic reperfusion. If

oxygen radical scavengers are added before repetiion, then the magnitude of injury is reduced. We investigated whether free radicals generated following exposure to hypoxia and reoxygenation activate voltagedependent K’ ion channels in tumor cells in vitro. Using the technique of whole cell voltage clamping, we recorded currents from two families of potassium (K+) channels that were activated following reoxygenation. One of these groups possessed the electrophysical charactetisticsofatetmethyla mmonium(TEA)-sf&tivedelayedrectitier channel and the other possessed characteristics of a Tea-insensitive slow inactivating channel. We present evidence which suggests that K* channels are activated following reoxygenation but not during the hypoxia phase. The K+ currents decayed with time following reoxygenation. The decay characteristics of theK* currents depended on the duration and level of hypoxia to which the cells were exposed. To determine whether activation of K+ channels by reoxygenation was initiated by free radicals, we pretreated cells with N-Acetyl L-Cysteine (NAC), a free radical scavenger, and found that this pretreatment abolished the currents induced by reoxygenation. We also present evidence that free radicals do not directly act on the channel itself, but activate a protein kinase which, in turn, activates the K’ channels. Taken together, these results indicate that one of the early responses to oxidative stress is the activation of K’ currents.

N-isopropyl-piodoamphetamine receptors in normal and cancerous tissue of the human lung Tanaka E, Mishima M, Kawakami K, Sakai N, Sugiura N, Taniguchi T et al. Depanment of Clinical Physiology, Chest Disease Research Institute, Kyoto University, 53 Shogoin Kawahara-cho, Sak@u, Kyoto 606. Eur J Nucl Med 1993;20:293-6.

N-Isopropyl-p-iodoamphetamine (IMP) receptors in normal human lung tissue were characterized using a radioligand biding assay with iodine-125 IMP as the ligand. Saturation binding studies revealed the presence of two binding sites with dissociation constant (K(d)) values of 53 f 2 and 4687 f 124 nM and maximum binding capacity (Brnax) values of 7 f 1 and 133 f 27 pmollmg protein (n = 5) respectively. The ICso values of various amines were as follows: IMP, 9 x IO’ M; propranolol, 5 x 10’ M; halopetidol, 6 x 1O’M; ketamine, 9 x l@’ M; dopamine, 1 x 10’ M. The IMP receptors of cancemus tissue obtained from human lung also had two bmdiig sites with K(d) values of 54 * 2 and 5277 f 652 nM and Bmax values of 7 f 1 and 103 f 21 pmoll mg protein (n = 3) respectively. There was no significant difference in binding parameters between normal and cancemus lung tissue. These results demonstrate the existence of IMP receptors and suggest that cancer doea not affect the nature of IMP receptors in human lung tissue.

Levels of complement regulatory molecules in lung cancer: Disappearance of the D17 epitope of CD55 in small-cell carcinoma Sakuma T, Kodama K, Hara T, Esbita Y, Shibata N, Matsumoto M et al. Depafiment of hnmunology, Center for Adult Diseases, l-3-3 Nakamichi, Hignshinari-ku. Osakxa537. JpnJ CancerRes 1993;84:753- 9.

The levelsofcomplement-regulatory molecules (complement receptor type one [CRI], decay-accelerating factor [DAF], membrane cofactor protein [MCP], and an inhibitor of membrane attack complex [CDSS]) in lung cancer cells were analyzed to investigate the relation between their expression and histological subtypes, and the possibility of homologous complement deposition on cancer cells. In 25 cell lines (10 adenocarcinoma, 3 large-cell carcinoma, 7 small-cell lung cancer (SCLC], and 5 squamous cell carcinoma), flow cytometric analysis revealed that MCP was expressed in all cell lines, whereas none of the cell lines was CRl-positive. CD59 was detected in all cells. The DAF