038

11

039

INTERPHASE CYTOGENETICS OF PRIMARY LUNG CANCER BY FLUORBSCENCE IN SITU HYBRIDIZATION (FISH) MKato, HMiyamoto, H.Hiroumi, K.Akie, H.Dosaka-Akita, YKawakami.

I.Kin~~$~~;~no,

Hokkaido University School of , Japan. ’ To investigate the relationship cytogenetic altnntial

and clinical finding of ptimgry lung cancer, direct interphase cytogenetic analysis was performed on nuclei of primary lung cancer using fluorescence in situ hybridization (FISH). F’rimary lung cancer specimens were obtained from surgical resection Q=33) or autopsy Q=lO), histologically diagnosed as adenccarcinoma (n=28), squamous cell carcinoma (n=12), large cell carcinoma (n=l) and small cell carcinoma (n=2). DNA p&es specific for chromosome 7,17, X and Y (Oncor , Inc., USA) were used to determine chromosome copy number in i&phase tumor cells.

Copy number distribution was varied greatly among specimens in every chromosome. The mean copy number was 3.23 for chromosome 7, 2.72 for chromaome 17, 1.94 for chromosome X in men, 3.0 for chromosome X in women and 1.13 for chromosome Y in men. The number of chromosome 17 tended to be decreased compared with chromosome 7 (p&06). In male case+ the number of chrumosome X was significantly increased axnpaEd wllh chfomasomeY (pcO.05). Furthermore, loss of chromosome Y was observed in 72% of male cases. Numerical abnormtity of chmmosom e was not correlated with histological types nor with clinicqxthological findings.

These findings suggest that primary lung cancer is a heterogenous tumor in cytogenetics, and that some of the specific cytogenetic changes observed may contribute to the development and progression of lung cancer.

040

Regulation by Pleural Cavity Macropbages and Peripheral Blood Monocyter to LAK Activity from Pleural Cavity Lymphocytes and Pcripherrl Blood Lymphocytes in Lung Cancer Patients

KcijiTakahashi, Saburo Sane’, Tad&i Uyama, Seiya Saito, Masayuki Sumitomo, Takeshi Ogura* and Yasumasa Monden

l?x Second Depanmcnt of Surgery and ??Tbc 3rd Department of Internal Medicine, School of Medicine, The University of Tokusbima.

The ability of pleural cavity lymphocytes (PLY) and periphcnl blood lymphocytes (BLY) to in&cc lymphokine(IL2)ativated killa (UK) aaivity and of pleural cavity mauophags @‘CM) and peripheral blood monocytcs (MO) to regulate the LAK in&aion wrc examined Purified PLY, BLY (>99%) and PCM, MO 090%) were separated by two step dsmntinuous gradient centtifugation. LAK adivity against Daudi ceils was measund by 4-hr %r relcasc assay.

Both non-stimulated PCM and LPS-stimulatcdPCM up-regulated L4K activity from BLY significantly @<0.05, P<O.OS). LAK activity in&ad from BLY up-regulated significantly by non-stimulated MO @<O.Ol) but not by stimulated Ma. Both non-stimulated PCM and LFS-stimulated PCM up-regulated LAK activity from PLY significantly (pcO.01, P<O.OS). WC activity indxed from PLY could not be upregulated by non-stimulated MO DOI LPS-stimulated MO.

These findngs in&ate the funaioos of PCM to regulate LAK activity from BLY and PLY is different from thorn of MO.

PCM MO non-stimulated LPS-stimulated non-stimulated LPS-stimulated

up-regulation of LAK from BLY +

I + +

up-regulation 0 l.AK from PLY + + +

+ : p-zO.05, versus L4K activity from lymphwytcs alone + + : pdO.01, versus L4Kactivity from lymphocytes alone

: not significant

HtGH FREOUENCY OF P53 fWTATtGNS IN ~tATE0 LUNGcWcEN,EulNoTm Ct4NONATE~TED LUNG CANCER. K. Kondo, H. Tsuzuki, M. Sass, S. Sakttama. Y. Kamarnura. A Umemoto, K. Takahashi, M. Sumttomo, T. Uyama, Y. Monden. The University of Tokushima, School of Medicine, Second Department of Surgery Chromate and tabacco smoktng increase the risk of lung cancer. To

investigate the assoctatton between these carcNogens and abnormality of cancer-associated genes, we examined mutattons of the p53 tumor suppressor gsne in lung cancers from 13 non-smokers, 40 smokers and 8 chromate workers. The potymerase chdn reaction-singts strand confamatton polymorphism (PCR-SSCP) anatysis and sequencing were applied to determine mutatkxe c4 exon 58 of p53 gene..

Mutations of p53 gene were found in only ons case(7.7%) of non-smokers, but in 24 cases(BO%) of smokers. The fquancy of p53 mutatkms in lung cancers was corretated with the amount of @arettesmoking as fottows:

Brinkman Index Mutations of p53 gene O<B.l.~500 3/7 (43%)

500<8.1.$1OQO S/l 5(60%) lOOO<B.I. W/18(67%)

Eight of 24 p53 mutations in smoker group were sequenced. These results revealed that most of base change ocurred at G-C paits(C+T; 3 cases, G +T; 3 cases).

P53 mutatkms in chromate workers were found in only one d eight cases, although seven of them were smoker(B.1.; o-1440, average 509 +424). The mutation case rweated an A-+Ttransverstcn in the first letter of codon 249, changing an Argtnine to a Ttyptophan. The present study ir&ates that p53 mutadons is assocMted wkh smoking

induced lung carcinogenests, but not chromate-induced lung rxrctnogenesis, and that the abnorrnattt of other cancer-assoctated genes attacked by the chromate may precede p53 mutations by smoking in chromate workers with smoking.

041

RASONCOGENEDETECTIONINBRONCHOALVEOL.4RLAVAGEPLUIB FROM PATIENTS WITB LUNG CANCER. NE Mills, CL Fishman, WN Ram, and DR Jacobson. Dqwtment of Medicine, Divisions of Hemstology, Oncology, and Pubnawy and Critical Care Medicine, New York University Medical Center, New York, NY, USA.

Activating point mutations in codon 12 of Le K-ras pmtwncogene have beat described in 40% of lung &ocarci~~omas, and less fnqueatly in other nw-srmll cell lung cancer histologies. Ras mutations have also been associated with poor prognosis in lung adatacarcinomn. These mutations may thus be. a clinically useful biomarker for lung cancer. We have developed a highly scasitive assay for activating ras mutations, and applied it to sp&mens obtained from patients umiergoing btrmchosqy for clinically sqr&d lung UDECI. ARer extmctim~ of DNA from bmnchc&eoIer Iwage. (&IL) fluid samples, K-ras sxon 1 was amplified by the pcdymemse chain teaction (PCR) using a mismstchd primer which introduced a new BstM cndonucleape restriction site into PCR products derived fmm normal, but not from mutant alleles. Multiple rounds of &tM dig&ion and further PCR were performed to entic41 for mutant alleles, with mut&iotts indicated by a digestion resistant band on agarose gel electm~hotis sRer ethidium bmmide staining. Samplra contain& a mutation were fur&r amplified using difforsnt mismatched primers designed to bmodwe into the PCR pmducts restriction sites depend& on the specific mutation pm@. lltese products were then digcstsd with the qpmpriate restriction enzymea to identify the mutant genotype. 25 of 31 BAL fluids analyzed were fmm patients dingwed witb lung -, and 7 wcm found to contain K-ms ccdon 12 mutations. No mutations were found in tbe samplea from the 6 @icuts with non-malignant dingnow. In 13 of the lung cdnccr patients, the BAL fluid was &ained during a ncwdiq~ostic bmnchoscopy. and the diagnosis of lung cancer required additional diagnostic procedures. In 3 case+ a K-ms codan 12 mutaion was detected in DNA from the nowdiagnostic BAL fluid; the same mutation wa found in tissue iiom each p&sat’s tumor. Diagnosis in these 3 patients required transthomcic needle aspiration in one case, medi&xwopy in one case, and 811 “topsy in one case, done 11 mat&s aftex the initial bmnchoscqy. Tissue samplea were also analyzed horn 10 patients with normal BAL fluid gcnotypcs, and none was found to carry B K-rar 12 mutation. We concbxde that this highly sensitive m&cd for detecting mutant ms genea is pote&ially useful clinically for the early diagnosis of lung cancer, as an adjunct to standard cytologic end pathologic analysis of samples from pulmonary diagnostic pmceduxs.