- Email: [email protected]
Molecular detection of genetic alterations in the plasma of non small cell lung cancer (NSCLC) patients
Biology digested by Eco RI, electrophoretically separated on an agarose gel, transferred on to Nylon membranes, and hybridised to a 1.8 Kb, Smal 5 Eco RI L-MYC radiolabelled probe. Data on S (6.6 Kb) and L (10 Kb) allele frequencies are shown in lower. S and L allele frequency in both groups were neady identical. However, the SS genotype was registered much more frequently in patients with metastasis (10/28; 36%) (p < 0.05) than in those with localized tumor (0/12). More over, overall frequency of S allele was significantly higher in lung cancer patients with node involvement (35•56; 63%) then in those with localized tumors (8•24; 33%) (p < 0.02). Finally, a significant correlation was found between S allele occurrence and distant metastases [MI: 19/28 (68%); MO: 26•58 (45%); p < 0.05]. Thus, S allele of L-MYC oncogene is associated with metatstatic lung cancer in patients from Moldova.
~-~
Isolation of full-length cDNAs of differentially expressed ESTs specific to adenocarcinoma of lung
Z. Xu, L. You, C. Xia, K. Eng, ×. Yuan, D.M. Jablons. School of
Medicine, Department of Surgery and Cancer Center, University of California, San Francisco, California, USA Lung cancer is the leading cause of cancer death in the United States and worldwide. Lack of suitable genetic markers for detecting lung cancer at early stage undermines the overall survival rate of lung cancer patients. We aim to develop novel markers that can be used for detecting molecular changes during early development of lung cancer. We have used differential mRNA display methods and high-throughput cDNA gene expression arrays to identify over expressed mRNAs in the form of expressed sequence tags (ESTs) from stage IA adenocarcinomas in the lung. A total of 30 ESTs were isolated, of which 27 were novel sequences. The magnitude of over expression of ESTs were 3 to 12-fold higher in stage IA adenocarcinoma than adjacent normal lung. Five over expressed ESTs were tested and confirmed using RNA northern blot analysis on a panel of lung cancer samples. To isolate full-length cDNA clones for the over expressed candidate ESTs, we have used a library-free system for direct cloning. Biotinylated primers designed from EST sequences were hybridized with anchored ligated first strand cDNAs. EST-specific first strand cDNAs were isolated using streptavidin coated magnetic bead capture and amplified using high-fidelity DNA polymerase. The amplified cDNAs were cloned into plasmid vector. We have isolated three full-length cDNA clones for the five over-expressed ESTs to date. The cDNA inserts were 2.5-3.8 kb in size, respectively and the complete cDNA inserts for the clones are now under sequencing. These ESTs and their full-length cDNA clones will be useful candidate gene markers for early diagnosis of lung cancer.
[•
GSTM1 and GSTT1 genotypes in Australians with non-small cell lung cancer
R. Gan, I. Yang, P. Zimmerman, K. Fong, A. Tunnicliffe, G. Rabnott, E. Duhig, B. Clarke. Prince Charles Hospital, Brisbane, Australia Despite the fact that the risk of lung cancer is dose-dependent on the number of cigarettes smoked, only 1 in 8-10 lifetime smokers develop lung cancer. Identifying genetic markers of individual susceptibility would Promote more efficient allocation of smoking cessation and case detection resources. Glutathione S-transferase (GST) gene products may help detoxity carcinogens in tobacco smoke such as epoxides of polycyclic aromatic hydrocarbons. Functional polymorphisms in enzyme activity between individuals can affect these processes. The GSTM1 locus is homozygously null or "deficient" in approximately 50% of the population. Some, but not all case control studies have linked the null genotype to an increased risk of lung cancer. The GSTT1 locus is homozygously null in -25% of the population and some small studies have associated this with an increased risk of lung cancer. To determine if the null genotypes of GSTM1 or T1 are linked to lung cancer risk in non-small cell lung cancer (NSCLC), we genotyped GSTM1 and GSTT1 with a PCR method in 108 Australian patients
201
with NSCLC in comparison to 54 healthy blood donors and 63 COPD patients without cancer. The null GSTM1 genotype was found in 591108 (54.6%) of NSCLC patients, 31/53 (58.5%) of healthy blood donors and 28154 (51.9%) of COPD patients. The null GSTT1 genotype was found in 141106 (13.2%) of NSCLC patients, 15/53 (28.3%) of healthy blood donors and 12163 (19%) of COPD patients. Our preliminary results suggest that GSTM1 null is not overrepresented in Australian lung cancer cases. However, a larger cohort should be studied to achieve sufficient statistical power to reject the null hypotheses. GSTT1 null genotype was underrepresented in the lung cancer cases. This could reflect between group differences in ethnicity particularly. GSTT1 has been observed to enhance mutagenicity of several of its substrates including haloalkanes, however, in vivo mechanisms of endogenous mutagenesis in which GST classes have been implicated are poorly defined biologically, and experimental clarification of the enzyme's role in human lung carcinogenicity is lacking.
~-~
Risk assesment of patients with non-small cell lung cancer by molecular and immunohistochemical analyses
K. Sugio, K. Yamazaki, S. Kase, M. Yamaguchi, K. Ondo, T. Okamoto, T. Koga, E Shoji, K. Sugimachi. Kyushu University, Fukuoka, Japan Several new findings regarding molecular oncogenesis and multistep carcinogenesis in non-small cell lung cancer (NSCLC) have helped to increase interest in this field of clinical research. The major purpose of research on biological factors is the identification of new prognostic markers that can indicate subsets of patients with a good or poor prognosis. We evaluated the genetic alteration or expression of 7 molecular markers in patients with NSCLC who underwent a complete resection, to determine the prognostic value of each marker. The molecular markers used in this study were p53 and K-ras to determine mutations, and cell cycle regulator (p27, p16), cell adhesion molecule (E-cadherin, beta-catenin), anti-angiogenic factor (Thrombospondin-l: TSP-1) to determine the immunohistochemical expression. Univariate analyses revealed mutations of p53, and abnormal expressions of p27, E-cadherin, beta-catenin, and TSP-1 which were all found to be unfavorable prognostic factors, in NSCLC. In addition, p53, p27, beta-catenin and TSP-1 were found to be prognostic factors in adenocarcinoma patients, while p27 was a prognostic factor for squamous cell carcinoma patients. In 273 patients, the 5 year survival rates were 82.9% in patients with no abnormal markers, 68.3% in those with 1 or 2 abnormal markers, 44.0% in those with 3 or 4 abnormal markers, and 0% in those with more than 5 abnormal markers (p < 0.0001). In patients with stage I disease, the 5 year survival rates were 92.1%, 87.3%, 63.1%, and 0%, respectively (p < 0.0001). These results suggested that biological analyses are useful for identifying subsets of patients who are at high risk of demonstrating a poor prognosis and therefore these patients are indicated to undergo adjuvant therapy in order to improve their survival.
•8-•
Molecular detection of genetic alterations in the plasma of non small cell lung cancer (NSCLC) patients
K. Takabe1, K. Tsuchiya 1, T. Fukuoka1, Y. Kodaira1, Y. ShinoharaI , S. Usui2, M. Inagaki2, N. Funakoshi2, K. Suzuki3, N. Inase4.
1Department of Internal Medicine; 2 Thoracic Surgery; 3pathology, Tsuchiura Kyodo Hospital, Ibaraki; 4Department of Respiratory Medicine, Tokyo Medical & Dental University, Tokyo, Japan In recent years, it has been possible to identity the same genetic alterations observed in tumor DNA in the plasma DNA of patients bearing diverse types of tumors including non small cell lung cancers (NSCLC). These genetic alterations include K-ras, N-ras, and p53 gene mutations, aberrant promoter region hypermethylation of tumor suppressor genes, and microsatellite alterations. To investigate the possible diagnostic value of these genetic alterations in the plasma of NSCLC patients, we analyzed the presence of microsatellite alterations and the p16 promoter region hypermethylation in both the plasma
Biology
202
and tumor DNA from seven NSCLC patients. The patients included 5 males and 2 females with a mean age of 68 years. Microsatellite alterations were analyzed using four microsatellite markers (D3S1228, D3S1286, D9S171, and IFNA). The p16 promoter region hypermethylation was examined using methylation-specific PCR (MSP) (Herman JG, et al., 1996). While 5 (71%) plasma samples showed microsatellite alterations in at least one loci, the same alterations were detected in only 2 tumor samples. The p16 promoter region hypermethylation was not detected in plasma DNA, but, 4 tumor samples showed hypermethylation. Further studies should be performed to enable the use of these genetic alterations in the plasma DNA for the diagnosis of NSCLC.
•6-•
~-~ Long time survival for patients with small cell lung cancer (SCLC)
Non-viral vectors for targeted gene delivery in small ceil lung cancer
K.L Abel, C. Albaek, K.S. Frederiksen, N. Abrahamsen, H.S. Poulsen.
Department of Radiation Biology, Finsen Center, National University Hospital, Copenhagen, Denmark
J. Kozielski, W. Dtubacz, K. Oklek, J. Kamifiski. DepL of
Pneumonology Silesian School of Medicine, Zabrze, Poland The one-year and two-year survival rates for the group of 244 patients with confirmed SCLC were analysed. One-year survival time was stated for 19.2% of patients who underwent treatment, while no oneyear survival was stated among the group of untreated patients. Twoyeas long survival time was established for 4.09% of treated patients. The extensiveness of disease influenced the time of survival (limited disease -8.86% of patients achieved two-years survival, compared to no patients with extensive disease) as well as patience's sex did (8.7% of women and 2.76% of men with two-years long survival rate). The 5-years long survival time was stated in case of 8 patients, it forms 2.5% of the whole group of patients hospitalised with SCLC diagnosis. The group consisted of 3 women and 3 men. All of them underwent chemotherapy, and in case of 4 patients, combined with radiotherapy. Two patients were operated on and further treated. Therapyapplied
No. of Women Men patients
Medium Disease Survival age exten(years) eion
Chemotherapy Chemotherapy+ Radiotherapy Surgery + Chemotherapy+ Radiotherapy Together Averages
2 2
1 1
1 1
53.7 50.7
Limited Limited
8.96 8.51
2
1
1
44.7
Limited
6.84
6
3 50%
3 50%
49.73
•
1) cancer growth in the inoculated site, 2) metastasis to lymph vessels of the perivascular or peribmnchiat space, 3) metastasis to mediastinal lymph nodes, was observed in 4 cell lines. UFT (17 mg/kg/day, 7 days) and CDDP (10 mg/kg) demonstrated the inhibitory effect of lymphogenous metastasis (UFT: 94%, CDDP: 92%) and the prolongation of life-span (mean survival time: UFT; 21.4 days ± 1.4 S.E. vs control; 15.8 + 1.2, CDDP; 25.2 ± 2.0 vs control; 17.2 ± 1.1, p < 0.01). Conclusion: We could recognize that our model was very useful for elucidating the mechanism of lymphogenous metastasis and for investigation of inhibitory effect of lymphogenous metastasis by anticancer drugs.
8.1
The incidence of lung cancer is increasing. Small cell lung cancer (SCLC) constitutes about 25% of all cases of lung cancer and is characterised by a high risk of metastasis mostly to liver, bone tissue and brain. The survival rate of patients with SCLC is poor, with a 5year survival of only 5-15%. This makes it of great interest to develop new therapeutical approaches and gene therapy will be a potential method. Our approach aims to specifically target the cancer ceils via receptor mediated endocytosis. Using a complex of cell-specific ligands coupled to a plasmid containing the therapeutical gene the cells can be specifically targeted. We used RT-PCR to elucidate the receptor status of our SCLC cell lines and a variety of normal human tissues. Investigating 11 receptors we found two to be very promising, as they were detected by RT-PCR in approximately 95% of the SCLC cell lines tested and in 24% and 27% respectively of normal tissue RNA. These two receptors are both 7TM G-protein coupled receptors. The identity, expression pattern and functionality of these receptors is currently being further elucidated. To increase the specificity and the security of our vector system we propose to use a bidimensional approach. To find genes higher expressed in SCLC cells than in normal lung tissue we are using Serial Analysis of Gene Expression (SAGE) to compare the transcription level in SCLC cells and a variety of normal tissue. Combining the promoter of one of the genes highly expressed in cancer cells with the above mentioned receptors we can specifically target the cancer cells and increase the security of our vector system. Earlier results suggested that 90% of our SCLC cell lines show mutations in the p53 cDNA. For this reason we suggest to include an apoptosis inducing gene in our vector to erase the cancer cells. The preliminary results in this study show promise in our effort to identify specific receptors for targeted gene delivery. An update will be presented.
Lymphogenous metastatic SClD mice model in human lung cancer cell line using orthotopic implantation
H. Ishikura, K. Kondo, T. Miyoshi, S. Sakiyama, H. Kinoshita, Y. Takahashi, H. Fujino, N. Tanida, K. Takaheshi, Y. Monden. 2nd
Friday, 15 S e p t e m b e r 2 0 0 0
Dept. of Surgery, School of Medicine, Tokushima University, Japan
POSTER SESSION
Background and Objectives: There are few animal models of metastasis of lung cancer to mediastinal lymph nodes which is similar to clinical course of human lung cancer. We established a new patientlike model of lymphogenous metastasis of lung cancer by orthotopic implantation using human non-small cell lung cancer cell lines. Methods: Male SCID mice (6 weeks of age) were anesthetized, and a 1-cm transverse incision was made on the left lateral thorax. A 30-gauge needle was inserted approximately 5 nun into the lung through the intercostal muscle. Tumor cells (2.0 x 104) with 4 ~g of Matrigel injected into the left lung. We examined the formation of lymphgenous mediastinal metastasis and time course of metastasis, and the inhibitory effect of metastasis by anticancer drugs. Result: Human lung cancer cell line (Ma44-3, Ma2, M a l 0 and Ma25) inoculated into the lung of SCID mice metastasized mediastinal lymph nodes after 2-8 weeks. The process of lymphogenous spread:
Biology ~-~
AnUoxidant activity in Bronchoalveolar Lavage Fluid from Patients with advanced NSCLC before and after chemotherapy with Mitomycin, Vindesin and Ifosfamid
B. Haering, S. Kampf, F. Herferth, R. Tischer-Neuhauss, U. Gatzemeier. Dep. of Thoracic oncology, Hospital Groflhansdorf,,
Center of Pneumology and Thoracic Surgery, 22927 Groflhansdorf,, Germany Introduction: Glutathione (GSH) is one of the key components of the lung antioxidant defenses. It was reported before that GSH in epithelial lining fluid (ELF) from patients with lung cancer was significantly
~-~
Isolation of full-length cDNAs of differentially expressed ESTs specific to adenocarcinoma of lung
Z. Xu, L. You, C. Xia, K. Eng, ×. Yuan, D.M. Jablons. School of
Medicine, Department of Surgery and Cancer Center, University of California, San Francisco, California, USA Lung cancer is the leading cause of cancer death in the United States and worldwide. Lack of suitable genetic markers for detecting lung cancer at early stage undermines the overall survival rate of lung cancer patients. We aim to develop novel markers that can be used for detecting molecular changes during early development of lung cancer. We have used differential mRNA display methods and high-throughput cDNA gene expression arrays to identify over expressed mRNAs in the form of expressed sequence tags (ESTs) from stage IA adenocarcinomas in the lung. A total of 30 ESTs were isolated, of which 27 were novel sequences. The magnitude of over expression of ESTs were 3 to 12-fold higher in stage IA adenocarcinoma than adjacent normal lung. Five over expressed ESTs were tested and confirmed using RNA northern blot analysis on a panel of lung cancer samples. To isolate full-length cDNA clones for the over expressed candidate ESTs, we have used a library-free system for direct cloning. Biotinylated primers designed from EST sequences were hybridized with anchored ligated first strand cDNAs. EST-specific first strand cDNAs were isolated using streptavidin coated magnetic bead capture and amplified using high-fidelity DNA polymerase. The amplified cDNAs were cloned into plasmid vector. We have isolated three full-length cDNA clones for the five over-expressed ESTs to date. The cDNA inserts were 2.5-3.8 kb in size, respectively and the complete cDNA inserts for the clones are now under sequencing. These ESTs and their full-length cDNA clones will be useful candidate gene markers for early diagnosis of lung cancer.
[•
GSTM1 and GSTT1 genotypes in Australians with non-small cell lung cancer
R. Gan, I. Yang, P. Zimmerman, K. Fong, A. Tunnicliffe, G. Rabnott, E. Duhig, B. Clarke. Prince Charles Hospital, Brisbane, Australia Despite the fact that the risk of lung cancer is dose-dependent on the number of cigarettes smoked, only 1 in 8-10 lifetime smokers develop lung cancer. Identifying genetic markers of individual susceptibility would Promote more efficient allocation of smoking cessation and case detection resources. Glutathione S-transferase (GST) gene products may help detoxity carcinogens in tobacco smoke such as epoxides of polycyclic aromatic hydrocarbons. Functional polymorphisms in enzyme activity between individuals can affect these processes. The GSTM1 locus is homozygously null or "deficient" in approximately 50% of the population. Some, but not all case control studies have linked the null genotype to an increased risk of lung cancer. The GSTT1 locus is homozygously null in -25% of the population and some small studies have associated this with an increased risk of lung cancer. To determine if the null genotypes of GSTM1 or T1 are linked to lung cancer risk in non-small cell lung cancer (NSCLC), we genotyped GSTM1 and GSTT1 with a PCR method in 108 Australian patients
201
with NSCLC in comparison to 54 healthy blood donors and 63 COPD patients without cancer. The null GSTM1 genotype was found in 591108 (54.6%) of NSCLC patients, 31/53 (58.5%) of healthy blood donors and 28154 (51.9%) of COPD patients. The null GSTT1 genotype was found in 141106 (13.2%) of NSCLC patients, 15/53 (28.3%) of healthy blood donors and 12163 (19%) of COPD patients. Our preliminary results suggest that GSTM1 null is not overrepresented in Australian lung cancer cases. However, a larger cohort should be studied to achieve sufficient statistical power to reject the null hypotheses. GSTT1 null genotype was underrepresented in the lung cancer cases. This could reflect between group differences in ethnicity particularly. GSTT1 has been observed to enhance mutagenicity of several of its substrates including haloalkanes, however, in vivo mechanisms of endogenous mutagenesis in which GST classes have been implicated are poorly defined biologically, and experimental clarification of the enzyme's role in human lung carcinogenicity is lacking.
~-~
Risk assesment of patients with non-small cell lung cancer by molecular and immunohistochemical analyses
K. Sugio, K. Yamazaki, S. Kase, M. Yamaguchi, K. Ondo, T. Okamoto, T. Koga, E Shoji, K. Sugimachi. Kyushu University, Fukuoka, Japan Several new findings regarding molecular oncogenesis and multistep carcinogenesis in non-small cell lung cancer (NSCLC) have helped to increase interest in this field of clinical research. The major purpose of research on biological factors is the identification of new prognostic markers that can indicate subsets of patients with a good or poor prognosis. We evaluated the genetic alteration or expression of 7 molecular markers in patients with NSCLC who underwent a complete resection, to determine the prognostic value of each marker. The molecular markers used in this study were p53 and K-ras to determine mutations, and cell cycle regulator (p27, p16), cell adhesion molecule (E-cadherin, beta-catenin), anti-angiogenic factor (Thrombospondin-l: TSP-1) to determine the immunohistochemical expression. Univariate analyses revealed mutations of p53, and abnormal expressions of p27, E-cadherin, beta-catenin, and TSP-1 which were all found to be unfavorable prognostic factors, in NSCLC. In addition, p53, p27, beta-catenin and TSP-1 were found to be prognostic factors in adenocarcinoma patients, while p27 was a prognostic factor for squamous cell carcinoma patients. In 273 patients, the 5 year survival rates were 82.9% in patients with no abnormal markers, 68.3% in those with 1 or 2 abnormal markers, 44.0% in those with 3 or 4 abnormal markers, and 0% in those with more than 5 abnormal markers (p < 0.0001). In patients with stage I disease, the 5 year survival rates were 92.1%, 87.3%, 63.1%, and 0%, respectively (p < 0.0001). These results suggested that biological analyses are useful for identifying subsets of patients who are at high risk of demonstrating a poor prognosis and therefore these patients are indicated to undergo adjuvant therapy in order to improve their survival.
•8-•
Molecular detection of genetic alterations in the plasma of non small cell lung cancer (NSCLC) patients
K. Takabe1, K. Tsuchiya 1, T. Fukuoka1, Y. Kodaira1, Y. ShinoharaI , S. Usui2, M. Inagaki2, N. Funakoshi2, K. Suzuki3, N. Inase4.
1Department of Internal Medicine; 2 Thoracic Surgery; 3pathology, Tsuchiura Kyodo Hospital, Ibaraki; 4Department of Respiratory Medicine, Tokyo Medical & Dental University, Tokyo, Japan In recent years, it has been possible to identity the same genetic alterations observed in tumor DNA in the plasma DNA of patients bearing diverse types of tumors including non small cell lung cancers (NSCLC). These genetic alterations include K-ras, N-ras, and p53 gene mutations, aberrant promoter region hypermethylation of tumor suppressor genes, and microsatellite alterations. To investigate the possible diagnostic value of these genetic alterations in the plasma of NSCLC patients, we analyzed the presence of microsatellite alterations and the p16 promoter region hypermethylation in both the plasma
Biology
202
and tumor DNA from seven NSCLC patients. The patients included 5 males and 2 females with a mean age of 68 years. Microsatellite alterations were analyzed using four microsatellite markers (D3S1228, D3S1286, D9S171, and IFNA). The p16 promoter region hypermethylation was examined using methylation-specific PCR (MSP) (Herman JG, et al., 1996). While 5 (71%) plasma samples showed microsatellite alterations in at least one loci, the same alterations were detected in only 2 tumor samples. The p16 promoter region hypermethylation was not detected in plasma DNA, but, 4 tumor samples showed hypermethylation. Further studies should be performed to enable the use of these genetic alterations in the plasma DNA for the diagnosis of NSCLC.
•6-•
~-~ Long time survival for patients with small cell lung cancer (SCLC)
Non-viral vectors for targeted gene delivery in small ceil lung cancer
K.L Abel, C. Albaek, K.S. Frederiksen, N. Abrahamsen, H.S. Poulsen.
Department of Radiation Biology, Finsen Center, National University Hospital, Copenhagen, Denmark
J. Kozielski, W. Dtubacz, K. Oklek, J. Kamifiski. DepL of
Pneumonology Silesian School of Medicine, Zabrze, Poland The one-year and two-year survival rates for the group of 244 patients with confirmed SCLC were analysed. One-year survival time was stated for 19.2% of patients who underwent treatment, while no oneyear survival was stated among the group of untreated patients. Twoyeas long survival time was established for 4.09% of treated patients. The extensiveness of disease influenced the time of survival (limited disease -8.86% of patients achieved two-years survival, compared to no patients with extensive disease) as well as patience's sex did (8.7% of women and 2.76% of men with two-years long survival rate). The 5-years long survival time was stated in case of 8 patients, it forms 2.5% of the whole group of patients hospitalised with SCLC diagnosis. The group consisted of 3 women and 3 men. All of them underwent chemotherapy, and in case of 4 patients, combined with radiotherapy. Two patients were operated on and further treated. Therapyapplied
No. of Women Men patients
Medium Disease Survival age exten(years) eion
Chemotherapy Chemotherapy+ Radiotherapy Surgery + Chemotherapy+ Radiotherapy Together Averages
2 2
1 1
1 1
53.7 50.7
Limited Limited
8.96 8.51
2
1
1
44.7
Limited
6.84
6
3 50%
3 50%
49.73
•
1) cancer growth in the inoculated site, 2) metastasis to lymph vessels of the perivascular or peribmnchiat space, 3) metastasis to mediastinal lymph nodes, was observed in 4 cell lines. UFT (17 mg/kg/day, 7 days) and CDDP (10 mg/kg) demonstrated the inhibitory effect of lymphogenous metastasis (UFT: 94%, CDDP: 92%) and the prolongation of life-span (mean survival time: UFT; 21.4 days ± 1.4 S.E. vs control; 15.8 + 1.2, CDDP; 25.2 ± 2.0 vs control; 17.2 ± 1.1, p < 0.01). Conclusion: We could recognize that our model was very useful for elucidating the mechanism of lymphogenous metastasis and for investigation of inhibitory effect of lymphogenous metastasis by anticancer drugs.
8.1
The incidence of lung cancer is increasing. Small cell lung cancer (SCLC) constitutes about 25% of all cases of lung cancer and is characterised by a high risk of metastasis mostly to liver, bone tissue and brain. The survival rate of patients with SCLC is poor, with a 5year survival of only 5-15%. This makes it of great interest to develop new therapeutical approaches and gene therapy will be a potential method. Our approach aims to specifically target the cancer ceils via receptor mediated endocytosis. Using a complex of cell-specific ligands coupled to a plasmid containing the therapeutical gene the cells can be specifically targeted. We used RT-PCR to elucidate the receptor status of our SCLC cell lines and a variety of normal human tissues. Investigating 11 receptors we found two to be very promising, as they were detected by RT-PCR in approximately 95% of the SCLC cell lines tested and in 24% and 27% respectively of normal tissue RNA. These two receptors are both 7TM G-protein coupled receptors. The identity, expression pattern and functionality of these receptors is currently being further elucidated. To increase the specificity and the security of our vector system we propose to use a bidimensional approach. To find genes higher expressed in SCLC cells than in normal lung tissue we are using Serial Analysis of Gene Expression (SAGE) to compare the transcription level in SCLC cells and a variety of normal tissue. Combining the promoter of one of the genes highly expressed in cancer cells with the above mentioned receptors we can specifically target the cancer cells and increase the security of our vector system. Earlier results suggested that 90% of our SCLC cell lines show mutations in the p53 cDNA. For this reason we suggest to include an apoptosis inducing gene in our vector to erase the cancer cells. The preliminary results in this study show promise in our effort to identify specific receptors for targeted gene delivery. An update will be presented.
Lymphogenous metastatic SClD mice model in human lung cancer cell line using orthotopic implantation
H. Ishikura, K. Kondo, T. Miyoshi, S. Sakiyama, H. Kinoshita, Y. Takahashi, H. Fujino, N. Tanida, K. Takaheshi, Y. Monden. 2nd
Friday, 15 S e p t e m b e r 2 0 0 0
Dept. of Surgery, School of Medicine, Tokushima University, Japan
POSTER SESSION
Background and Objectives: There are few animal models of metastasis of lung cancer to mediastinal lymph nodes which is similar to clinical course of human lung cancer. We established a new patientlike model of lymphogenous metastasis of lung cancer by orthotopic implantation using human non-small cell lung cancer cell lines. Methods: Male SCID mice (6 weeks of age) were anesthetized, and a 1-cm transverse incision was made on the left lateral thorax. A 30-gauge needle was inserted approximately 5 nun into the lung through the intercostal muscle. Tumor cells (2.0 x 104) with 4 ~g of Matrigel injected into the left lung. We examined the formation of lymphgenous mediastinal metastasis and time course of metastasis, and the inhibitory effect of metastasis by anticancer drugs. Result: Human lung cancer cell line (Ma44-3, Ma2, M a l 0 and Ma25) inoculated into the lung of SCID mice metastasized mediastinal lymph nodes after 2-8 weeks. The process of lymphogenous spread:
Biology ~-~
AnUoxidant activity in Bronchoalveolar Lavage Fluid from Patients with advanced NSCLC before and after chemotherapy with Mitomycin, Vindesin and Ifosfamid
B. Haering, S. Kampf, F. Herferth, R. Tischer-Neuhauss, U. Gatzemeier. Dep. of Thoracic oncology, Hospital Groflhansdorf,,
Center of Pneumology and Thoracic Surgery, 22927 Groflhansdorf,, Germany Introduction: Glutathione (GSH) is one of the key components of the lung antioxidant defenses. It was reported before that GSH in epithelial lining fluid (ELF) from patients with lung cancer was significantly