382 Abstracts/Lung Cancer 14 (19%) 377-408

of loss of human DNA in these propagated tumors in nude mice have yet to be delineated.

Shared cytogenetic abnormalities in lung tumours and corresponding peripheral blood lymphocytes Dave BJ. Hopwood VL, Spitz MR, Pathak S. Cellular Generics Laboratory, Box 181, M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Int J Oncol 1995;1:1297-305.

We analyzed chromosomal alterations in primary lung tumours and peripheral blood lymphocytes(PBLs) from 10 lungcancerpatients(nine with non-small cell lung carcinoma and one with small cell lung carcinoma) to determine whether there were shared chromosomal changes in the normal and diseased tissue of these cases. This study revealed that each paired sample had at least three chromosomes and two chromosomal regions in common for structural rearrangements. The chromosomes most frequently found structurally altered in paired analysis were 1 and 3 (60% each), 5 (SO%), 6,7, and 9 (40% each), and 14 (30%). Chromosome region 3~13-3~21 was structurally rearranged in both the normal and tumour tissues of three patients. chromosome 3 was structurally rearranged in all ten turnours. The chromosome arms most commonly affected in the tumours were 3p (nine times), 9p and 5q (eight times each). lp and 7q (six times each), 1Oq and 1 lq (five times each), 14q and 6q (four times each). The most frequently affected chromosomal regions in these tumours were (in decreasing order) 9p23- p24,3p21-3p13,5qll, lp34,7q22, and 1 lq13. Frequent polysomy of 7 and 12 and loss of D-group chromosomes were also observed in the tumours analyzed. Comparing the changes found only in tumours with those found in both PBLs and tumours was helpful in shedding some light on the probable sequence of genetic events leading to lung cancer. This investigation also offered compelling evidence that genomic instability at the chromosomal level in PBLs corresponds with the genetic changes observed in tumours inchcating that PBL analysis can help identify the early chromosomal changes in lung cancer. PBL chromosomal analysis thus has a promising future in thegenetic analysis of lung cancers.

Alterations of CDKN2 (~16) in non-small cell lung cancer De Vos S, Miller CW, Takeuchi S, Gombart AF, Cho SK, Koeffler HP. Cedars-Sinai Medical Center, Davis-B&. 5034, UCLA School of Medicine, 8700 Beverly Blvd.. Los Angeles, CA 90048. Genes Chromosomes Cancer 1995; 14: 164-70.

The cyclindependent kinase inhibitor known as ~16 (CDK41, CDKN2, INK4A, MTSl) hasbeenproposedasatumor suppressorgene on chromosome segment 9p21. We have evaluated CDKNZ alterations in 34 non-small cell lung cancers (NSCLCs) with matched normal tissue controls and in 9 NSCLC cell lines by Southern blotting, single-strand conformationpolymorphism(SSCP)withthepolymerasechain reaction, anddirect sequencing. Inaddition, lossofheterozygosityatchromosome segment 9p21, with the use of the microsatellite marker D9S171, was studied in these samples. Whereas CDKN2 was either deleted or mutated in NSCLC cell linesat a high frequency (6/9,67%), alterations were much less frequent (7/34, 21%) in primary tumor samples. Only one sample contained a point mutation in exon I of CDKN2. In addition, two samples had homozygous deletions of CDKNZ in exon I; one had a homozygous and three a hemizygous deletion of exon 2. Possibly normal tissue contaminating our tumor samples may have masked homozygous deletions in these cases. Four patient samples had LOH in the region of CDKN2 on chromosome segment 9p21; two of these samples had potentially inactivating alterations of CDKNZ; one sample had a mutation of CDKNZ, and the other had a homozygous deletion of exon 1. In summary, inactivation of CDKN2 is implicated in the development of about 20% of NSCLC, but the possibility of another tumor suppressor gene on chromosome segment 9p2 1 important in lung cancer cannot be eliminated.

Immunoreactive prostate-specific antigen in lung tumors Levesque M, Yu H, D’Costa M, Tadross L, Diamandis EP. Laboratory Medicine. Department of Pathology, Mount Sinai Hospital, 600 UniversityAwwe, Toronto, Onr. M5GlX5. JClinLab Anal 1995;9:375- 9.

Prostate-specific antigen (PSA) is a glycoprotein produced by the epithelial ceils of the prostate. PSA is currently used clinically to diagnose and monitor prostate carcinoma. In previous work we have demonstrated that 30 46 of breast tumors and, more rarely other tumors, contain significant amounts of PSA. PSA appears to be a favorable prognostic indicator in breast cancer here, using a sensitive assay, we demonstrated for the tirst timethat lung adenocarcinomasand squamous cell.carcinomas also contain PSA. PSA in lung tumor extracts was present mainly in its 33 KDa form (free PSA), at levels measurable by commercial methods. The presence of PSA was associated more closely with male patients and adenocarcinomas. The physiological role of PSA in lung tissue and the prognostic significance of PSA in lung cancer remain to be determined. These and our previous data as well as reports by other groups support the view that PSA is a ubiquitous biochemical marker of steroid hormone action.

Evaluation of proliferation parameters in in vivo bromodeoxyuridine labelled lung cancers Tinnemans MMFJ, Lenders M-HJH, Ten Velde GPM, Wagenaar SS, Blijham GH, Ramaekers FCS et al. Dept Molecular Cell Biology Genetics, University of Limburg, PO Box 616. 62W MD, Maastricht. Virchows Arch 1995;427:295-301.

In a series of 44 bronchial biopsies from patients suspected of having endobronchial lung carcinoma, the validity of proliferating cell nuclear antigen (PCNA) and Ki67 antigen as proliferative indicators was evaluated in ethanol fixed, paraffm embedded tissue. The percentages of cells positive for these markers were compared to the in viva bromodeoxyuridine (BrdU) labelling index. A good correlation was found between PCNA immunoreactivity and BrdU labelling index, while Ki67-antigen expression showed a significant relation with BrdU labelling index and with PCNA expression. All three parameters showed a trend towards similar values for the individual cases. Based on the fact that Ki67 antigen is expressed in all cycling cells, whereas replicon-associated PCNA and BrdU only reflect the S-phase fraction, the differences between Ki67-antigen scores on the one hand and BrdU and PCNA scores on the other were smaller than expected. In order to determine the degree of concordance between immunohistochemically and flow cytometrically detected proliferation variables, BrdU incorporation was measured using both methods in duplicate bronchial specimens. Discrepancies in labelling indices were observed predominantly in DNA diploid samples, with consistently lower values in the flow cytometrically analysed specimens. In tumour specimens with an aneuploid DNA content, flow cytometric determination of proliferative activity yielded results similar to those obtained by tissue section examination. We conclude that the scores for PCNA and Ki67 antigen, immunohistochemitally detected in ethanol fixed, paraffin embedded tissue reflect functional proliferative activity.

Evidence of cuinulative gene losses with progression of premalignant epithelial lesions to carcinoma of the bronchus Thiberville L, Payne P, Vielkinds J, LeRiche J, Horsman D, Nouvet G et al. Unite 295, INSERM, Faculte Medecine/Pharmacie de Rouen, 76803 Rouen Cede-x. Cancer Res 1995;55:5133-9.

Humanbronchialcarcinomaisthoughttodevelopthroughprogressive stages from basal cell hyperplasia to squamous metaplasia, dysplasia, carcinoma in situ, and finally invasive cancer. In this study, we used tissuemicrodissection toexaminelossofheterozygosityofchromosomes

Abstracts/Lung Cancer 14 (19%) 377-408 383

3~21, 5q21, and 9~21 at each stage of the epithelial progression to Background: Transforming growth factor-alpha (TGF-a) may play invasive cancers. Forty-eight premalignant/malignant bronchial sites important roles in carcinogenesis, tumor growth, and angiogenesis. were biopsied from 13 patients (including 9 subjects without cancer) Transforming growth factor-beta (TGF-8) are known to be involved in using fluorescence bronchoscopy. Eighteen sites with moderate/severe cell-cycle control and regeneration. TGFa positively acts on growth dysplasia in 6 patients were subjected to bronchoscopic and molecular control of many epithelial cells in contrast to the negative role of TGF- follow-up during a 3-month to 2-year period. Seven separate cases of 8. Method: To evaluate the possible role of TGFa and TGF-O in human advanced non-small cell bronchial cancers were also analyzed. From the primary lung cancers, the expression of TGFa and TGF-R were baseline biopsies, the prevalence of 3p and 9p deletions increased immmunohistochemically investigated in tissue sections from forty significantly from no deletion in the hyperplasialmetaplasia samples (n seven cases with lung cancers and ten cases with non-cancerous lung = 9) to 37 and 31% of the informative cases, respectively, in the tissues. Recombinant cloned monoclonal antibody of TGF-a and dysplasia samples (n = 29). to 100 and 83%, respectively, for the neutralizing antibody of TGF-a were employed as primary antibodies carcinomas in situ (n = 6), and 100% in the invasive cancers (n = 11). after dewaxing the formalin-fixed, paraffin&d tissue sections. Results: Chromosome 5q deletion was significantly more frequent in invasive TGFa was expressed in the cytoplasms of tumor cells in thirty five cancers (70% of the informative cases) as compared to carcinoma in situ cases of forty seven (74.5 %) primary lung cancers, whereas the control (40%), dysplasias (33%), and hyperplasialmetaplasia samples (11%). expressed in two of ten bronchial epithelial cells. The expression of The number of chromosome alterations also increased significantly TGF-a was disclosed in four cases of eleven (36.4%) small cell from the lowest to the highest grade lesions, showing evidence of carcinomas and thirty one cases of thirty six (86.1 W) non-small cell accumulation of genetic damage from one group to another. The carcinomas of the lung. Expressions of TGF-13 was discernible in molecular follow-up analysis showed that the same genomic alteration bronchial epithelium in eight of ten non-cancerous lung tissues. The can persist in a given dysplastic bronchial area for several months or expression of TGF-8 was noted in the cytoplasms of tumor cells in eight years, and that the persistence or the regression of the molecular cases of forty seven (17.0%) primary lung cancers. The expression of abnormality is well correlated with the evolution of the disease on TGF-I3 disclosed in two cases of eleven (18.2%) small cell carcinomas follow-up. Our results suggest that molecular analysis of bronchial and six cases of thirty six (I 6.7 46) non-small cell carcinomas of the lung. biopsies obtained by fluorescence bronchoscopy may be a very useful Conclusion: These findings suggest that up-regulation of TGFa and means to study the natural history of preinvasive bronchial lesions and down-regulation of TGF-R are involved during development and the outcome of interventions, such as with chemopreventive treatment. growth of primary lung cancers.

Pituitary adenylatecyclaseactivating peptidereceptors regulate the growth of non-small cell lung cancer cells Zia F, Fagarasan M, Bitar K, Coy DH, Pisegna JR, Wank SA et al. Biomarkers/Prevetuion Res. Branch, National Cancer fnstitute, Buitiing C. 9610 Medical Center Drive, Rockvitte. MD 2OS50. Cancer Res 1995;55:4886-91.

We have identified pituitary adenylate cyclase activating peptide (PACAP) receptors on small cell lung cancer cell line NCI-N417 in a previous study. In this study, the role of PACAP in the growth and signal transduction of non-small cell lung cancer cells was investigated. Northern blot analysis with a full-length human PACAP receptor cDNA probe revealed a major 7.5-kb hybridizing transcript when total RNA extracted from NCI-H838 cells was used. PACAP bound with high affinity (K(d) = 1 nM) to a single class of sites-(B(max) = 14,OOUcell) when NCI-H838 cells were used. Specific lz51- labeled PACAP binding was inhibited with high affinity by PACAP- and PACAP- 38, with moderateaffinity by PACAP(6-38), and with low affinity byvasoactive intestinal polypeptide, PACAP(28-38), and PACAP( 16-38). PACAP- 27 elevated CAMP in a dose-dependent manner, and the increase in CAMP caused by PACAP was reversed by PACAP(6-38). PACAP-27, but not vasaactive intestinal polypeptide, elevated cytosolic Ca2’ in individual NCI-H838 cells. PACAP- 27 stimulated arachidonic acid release, and the increase caused by PACAP was reversed by PACAP(6- 38). PACAP- stimulated colony formation in NCI-H838 cells, whereas the PACAP antagonist PACAP(638) reducedcolony formation in the absence or presence of exogenous PACAP-27. In nude mice bearing NCI-H838 xenografts, PACAP(6-38) slowed tumor growth significantly. Thesedata suggest that biologically active type 1 PACAP receptors are present on human non- small cell lung cancer cells, which exhibit dual signal transduction pathways and regulatecell proliferation.

Mitogenic effect of serotonin in human small cell lung carcinoma cells via both S-HT(lA) and 5HT(lD) receptors Cattaneo MG, Fesce R, Vicentini LM. Department of Medical Pharmacology, University ofhfilan, Via Vanvirelli 32, 20129 Milan. Eur J Pharmacol Mel Pharmacol Sect 1995;29 1:209-l I.

We have recently shown that the mitogenic effect of serotonin (5- hydroxytryptamine, 5-HT) on human small cell lung carcinoma (SCLC) cells is at least partly due to stimulation of a 5-HT(lD) receptor type. We now report that the 5-HT( 1A) receptor agonist R( +)-8-hydroxy-2- (di-n-propylamino)tetralin (8-OH-DPAT) is also capableof stimulating [‘Hlthymidine incorporation into SCLC GLC-8 cells, although with lower efficacy than 5-HT. The simultaneous administration of maximal doses of 8-OH-DPAT and the 5-HT( 1 D) receptor agonist sumatriptan reproduced the maximal [‘Hlthymidine incorporation observed with 5- HT alone. The 5-HT( IA) receptor antagonists spiperone and SDZ 216- 525 completely abolished the effect of II-OH-DPAT (IC, 30 nM for both drugs) behaving as pure antagonists. Accordingly, the two drugs partially inhibited the mitogenic effect of 5-HT. These data indicate that the mitogenic effect of5-HT in SCLC cells involves both S-HT( 1 A) and 5-HT( 1 D) receptor types.

Pathology Differential diagnosis of thymiccarcinoma and lung carcinoma with the use of antibodies to cytokeratins Fukai I, Masaoka A, Hashimoto T, Yamakawa Y, Niwa H, Kiriyama M et al. Second Deparrmenr of Surgery, Nagoya City Univ. Med. School, Miruho-ku, Nagoya. JThoracCardiovascSurg 1995; 110: 1670- 5.

Expression of transforming growth factor-aand transforming growth factor-t3 in human primary lung cancer Lew WJ, Shin DH, Park SS, Lee DH, Lee JD, Lee JH. Deparrment of Internal Medicine, Hanyang University, College of Medicine, Seoul. Tuberc Respir DIS. 1995;42:492-501.

There are few specific pathologic findings that can be relied on to distinguish primary thymic carcinomas from lung carcinomas with mediastinal extension or showing metastasis to the anterior mediastinum. The immunohistochemical reactivity on frozen sections of thymic carcinomas and lung carcinomas, which are histologically similar to each other, was examined with the use of monoclonal antibodies to