superior antiproliferative effects mediated by interferon-α entrapped in liposomes against a newly...

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We have studied the short- and long-term effects of human recombi- nant tumor necrosis factor (TNF) and TNF/recombinant human inter- feron-gamma (IFN-gamma) mixtures on AS49 human lung carcinoma cells maintained in organotypic culture. Continuous treatments with 2 x 10; 2 x 10z, 2 x 101 and 2 x I@ U/ml TNF or with mixtures of TNF/ IFN-a at 2 x IO2 and IO3 U/ml, respectively, were administered. Nodule growth, cell proliferation and cell survival were studied. On the 2nd day of treatment with TNF, only the highest dose (2 x 104 U/ml) diminished cell proliferation significantly, as measured by tritiated thymidine uptake into DNA. On the 10th day, only the lowest TNF dose (2 x 10 U/ ml) Induced no significant growth inhibition. Necrosis and nodule disintegration were apparent in the 2 x 106 U/ml-treated noduleswhere DNAsynthesisdecreased. In thiscase,usingagarcloningassays,nocell survival could be observed. Similar results could be obtained with TNF at low concentration (2 x lo* U/ml) in combination with INF-gamma (lo3 U/ml), showing a synergistic effect on inhibition of cell prolifera- tion. In the long-term experiments, with the lower TNF doses, in situ evidence of regrowth was observed (outgrowing zones in the nodules) on about tbe40tb day of treatment, and nodule recovery was confirmed by the resumption of DNA synthesis measured on the 50th day of treatment. Noregrowth, however,occurred when theIFN-gammflNF combination was used, and the nodules disintegrated completely on the 35th day of treatment without evidence of any cellular survival. Superior antiproliferative effects mediated by interferon-a en- trapped in liposomes against a newly established human lung cancer cell line Shin DM, Fidler IJ, Bucana CD, Fan D, Hong WK, Killion JJ. Deparr- ment of Medical Oncology. University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. J Biol Re- sponse Modif 1990:9:35S-60. The purpose of this study was to characterize the antiproliferative activity of a recombinant interferon-a (IFN-a) against a newly estab- lished human adenocarcinoma cell line (DMS4C) and to determine whetherIFN-a enuappedinmultilamellarliposomes hadsuperioranti- tumor effects compared with free IFN-a. Treatment of DMS4C cells with 100 U/ml of free IFN-a resulted in 34% cytostasis. IFNa encap- sulated in phosphatidylcholine/phosphatidylserine multilamellar ves- icles produced growth inhibition of 67%, which was significantly greater than that produced by free IFNa or by control liposomes containing only medium combined with free IFN-a. Moreover, kinetic analysis revealed that to produce significant cytolysis, free IFN-a had to be incubated with target cells for at least 24 h, whereas IFN-a encapsulated into liposomes required only 30 min of exposure. Rationale for the use of chemotherapy in non-small cell lung cancer Miller TP. Cancer Center Clinics, Arizona Cancer Center. 151s N CampbellAve, Tucson, AZ85724. Semin 0nco11990;17:Supp17:1 l-3. The vast majority of patients have disseminated non-small cell lung cancer (NSCLC) at the time of diagnosis. Data from numerous studies clearly indicate that the disease is metastatic in asymptomatic patients whoappear to haveclinically resectable tumors. Adenocarcinomais the histologic subtype that is most frequently metastatic, and its incidence appears to be increasing in a manner relative to other tumors. Of all of tbe prognostic factors, performance status appears to be most directly related to response rates. In other words, the higher the performance status, the higher me response rates. Tumor burden has been found to have an effect not only on performance status, but also on response to chemotherapy. Therefore systemic chemotherapy is urged as adjuvant treatment early in the course of NSCLC when performance status is highest and tumor burden lowest. K-Ras oncogene activation as a prognostic marker in adenocar- cinema of the lung Slebos RJC, Kibbelaar RE, Dalesio 0 et al. Division of Experimental Therapy. Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam. New Engl J Med 1990;323:561-5. Background. The capability of activated oncogenes to induce malig- nant transformation of immortalized cells in vitro has suggested that they have a similar role in the pathogenesis of human tumors. We previously found that activation of the K-ras oncogene by a point mutation in codon 12 occurs in about one third of human lung adenocar- cinemas. Methods. We studied the clinical importance of this onco- gene-activation in 69 patients with lung adenoctucinoma in whom complete resection of the tumor was possible. The polymcrasc chain reaction was used to amplify ras-specific sequences of DNA isolated from frozen or paraffin-embedded tumor samples. Ras point mutations were subsequently detected and classified with the use of mutation- specific oligonucleotide probes. Results. Nineteen of the tumors har- bored a point mutation in codon 12 of the K-ras oncogene. There was no association between the K-ras point mutation and the age at diagnosis, sex, or presence of previous or concurrent neoplasms. Tumors positive for K-ras point mutations tended to be smaller and less differentiated than those without mutations. The K-ras codon- point mutation was a strong (and unfavorable) prognostic factor: 12 of the 19 patients with K-raspoint-mutation-positive tumorsdieddurmg the follow-upperiod, as compared with 16 of the 50 patients with no mutation in the K-ras oncogene (P = 0.002). This difference in prognosis was also reflected in the duration of disease-free survival (P = 0.038) and in the number of deaths due to cancer (P < 0.001). Conclusions. The presence of K-ras point mutations defines a subgroup of patients with lung adenocar cinema in whom the prognosis is very poor and disease-free survival is not usually long despite radical resection and a small tumor load. Rolycyclicaromatic hydrocarbon - DNA adducts in lung tissuefrom lung cancer patients Van Schoomn FJ, Hillebrand MJX, Van Leeuwen FE et al. Division of Chemical Carcinogen&s. Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam. Carcinogcnesis 1990,11:1677-81. In an attempt to probe for polycycbc aromatic hydrocarbon (PAH)- DNA adducts in human subjects resulting from smoking (or other chronic environmental exposure), lung tissue and lung tumours were obtained from patients hospitalized for lung cancer. DNA was Isolated from the tissue samples and examined both in an ELISA using a polyclonal antibody against (*)trans-7,8-dihydroxy-anti-9,10-epoxy- 7,8,9,10-tetrahydro benzo]a]pyr ene (BPDE) - DNA as well as by the nuclease PI-mediated modification of the 32P-post-labelling technique. The ELISA results showed BPDE DNA antigenicity in lung DNA from 6 out of 21 patients, and adduct levels ranged from 2 to 134 adducts per 108 nucleotides. For all 21 patients, the autoradiographs of chromato- grams of 32P-postlabelled digests of DNA from non-tumorous lung tissue showed a strong diagonal radioactive zone (DRZ). This DRZ was generally absent in tumorous tissue. DNA samples that were posttive in the ELISA contained a dominant spot within the DRZ that co-chroma- tographed with the major BPDE-DNA adduct (BPDE-dG). The quanti- ties of the BPDE-dG spots ranged from 2.1 to 42 adducts in IO9 nucleotides. These values were lower than the levels found in the ELISA but correlated well with the ELISA results (Kendall W = 0.97; P= 0.00). The levels of the DRZ adducts ranged from 1.9 to 34 adducts in 108 nucleotidcs. Correlations between smoking and DNA adduct levels were poor because of the small number of current smokers (n = 13). However, smokers of filter cigarettes had significantly lower DNA adduct levels compared with smokers of cigarettes without a filter (P = 0.02 by Fischer’s exact test). Restriction fragment length polymorphism analysis of the L-myc gene locus in a case-control study of lung cancer Tamai S, Sugimura H, Caporaso NE et al. Laboratory of Human Carcinogenesis, DCE. NCI, NIH, Bethesda, MD 20892. Int J Cancer 1990;46:41 l-5. The L-myc DNA-restriction fragment length polymorphism, re- vealed by EcoRI, has been studied in both a lung cancer case-control framework and a cohort of 40 nondiseased unrelated individuals. No association was found between the L-myc allelic frequencies and disease status, tumor stage or lung cancer histology. A strong associa- lion was, however, observed between the L-myc allelic frequencies and ethnic origin (black or white) of the subjects. Among American whites die allelic distribution at tbe L-myc proto-oncogene locus was almost identical to chat previously reported for Japanese subjects. Among the American black population there was a significantly higher frequency

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We have studied the short- and long-term effects of human recombi- nant tumor necrosis factor (TNF) and TNF/recombinant human inter- feron-gamma (IFN-gamma) mixtures on AS49 human lung carcinoma cells maintained in organotypic culture. Continuous treatments with 2 x 10; 2 x 10z, 2 x 101 and 2 x I@ U/ml TNF or with mixtures of TNF/ IFN-a at 2 x IO2 and IO3 U/ml, respectively, were administered. Nodule growth, cell proliferation and cell survival were studied. On the 2nd day of treatment with TNF, only the highest dose (2 x 104 U/ml) diminished cell proliferation significantly, as measured by tritiated thymidine uptake into DNA. On the 10th day, only the lowest TNF dose (2 x 10 U/ ml) Induced no significant growth inhibition. Necrosis and nodule disintegration were apparent in the 2 x 106 U/ml-treated noduleswhere DNAsynthesisdecreased. In thiscase,usingagarcloningassays,nocell survival could be observed. Similar results could be obtained with TNF at low concentration (2 x lo* U/ml) in combination with INF-gamma (lo3 U/ml), showing a synergistic effect on inhibition of cell prolifera- tion. In the long-term experiments, with the lower TNF doses, in situ evidence of regrowth was observed (outgrowing zones in the nodules) on about tbe40tb day of treatment, and nodule recovery was confirmed by the resumption of DNA synthesis measured on the 50th day of treatment. Noregrowth, however,occurred when theIFN-gammflNF combination was used, and the nodules disintegrated completely on the 35th day of treatment without evidence of any cellular survival.

Superior antiproliferative effects mediated by interferon-a en- trapped in liposomes against a newly established human lung cancer cell line Shin DM, Fidler IJ, Bucana CD, Fan D, Hong WK, Killion JJ. Deparr- ment of Medical Oncology. University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. J Biol Re- sponse Modif 1990:9:35S-60.

The purpose of this study was to characterize the antiproliferative activity of a recombinant interferon-a (IFN-a) against a newly estab- lished human adenocarcinoma cell line (DMS4C) and to determine whetherIFN-a enuappedinmultilamellarliposomes hadsuperioranti- tumor effects compared with free IFN-a. Treatment of DMS4C cells with 100 U/ml of free IFN-a resulted in 34% cytostasis. IFNa encap- sulated in phosphatidylcholine/phosphatidylserine multilamellar ves- icles produced growth inhibition of 67%, which was significantly greater than that produced by free IFNa or by control liposomes containing only medium combined with free IFN-a. Moreover, kinetic analysis revealed that to produce significant cytolysis, free IFN-a had to be incubated with target cells for at least 24 h, whereas IFN-a encapsulated into liposomes required only 30 min of exposure.

Rationale for the use of chemotherapy in non-small cell lung cancer Miller TP. Cancer Center Clinics, Arizona Cancer Center. 151s N CampbellAve, Tucson, AZ85724. Semin 0nco11990;17:Supp17:1 l-3.

The vast majority of patients have disseminated non-small cell lung cancer (NSCLC) at the time of diagnosis. Data from numerous studies clearly indicate that the disease is metastatic in asymptomatic patients whoappear to haveclinically resectable tumors. Adenocarcinomais the histologic subtype that is most frequently metastatic, and its incidence appears to be increasing in a manner relative to other tumors. Of all of tbe prognostic factors, performance status appears to be most directly related to response rates. In other words, the higher the performance status, the higher me response rates. Tumor burden has been found to have an effect not only on performance status, but also on response to chemotherapy. Therefore systemic chemotherapy is urged as adjuvant treatment early in the course of NSCLC when performance status is highest and tumor burden lowest.

K-Ras oncogene activation as a prognostic marker in adenocar- cinema of the lung Slebos RJC, Kibbelaar RE, Dalesio 0 et al. Division of Experimental Therapy. Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam. New Engl J Med 1990;323:561-5.

Background. The capability of activated oncogenes to induce malig- nant transformation of immortalized cells in vitro has suggested that they have a similar role in the pathogenesis of human tumors. We

previously found that activation of the K-ras oncogene by a point mutation in codon 12 occurs in about one third of human lung adenocar- cinemas. Methods. We studied the clinical importance of this onco- gene-activation in 69 patients with lung adenoctucinoma in whom complete resection of the tumor was possible. The polymcrasc chain reaction was used to amplify ras-specific sequences of DNA isolated from frozen or paraffin-embedded tumor samples. Ras point mutations were subsequently detected and classified with the use of mutation- specific oligonucleotide probes. Results. Nineteen of the tumors har- bored a point mutation in codon 12 of the K-ras oncogene. There was no association between the K-ras point mutation and the age at diagnosis, sex, or presence of previous or concurrent neoplasms. Tumors positive for K-ras point mutations tended to be smaller and less differentiated than those without mutations. The K-ras codon- point mutation was a strong (and unfavorable) prognostic factor: 12 of the 19 patients with K-raspoint-mutation-positive tumorsdieddurmg the follow-upperiod, as compared with 16 of the 50 patients with no mutation in the K-ras oncogene (P = 0.002). This difference in prognosis was also reflected in the duration of disease-free survival (P = 0.038) and in the number of deaths due to cancer (P < 0.001). Conclusions. The presence of K-ras point mutations defines a subgroup of patients with lung adenocar cinema in whom the prognosis is very poor and disease-free survival is not usually long despite radical resection and a small tumor load.

Rolycyclicaromatic hydrocarbon - DNA adducts in lung tissue from lung cancer patients Van Schoomn FJ, Hillebrand MJX, Van Leeuwen FE et al. Division of Chemical Carcinogen&s. Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam. Carcinogcnesis 1990,11:1677-81.

In an attempt to probe for polycycbc aromatic hydrocarbon (PAH)- DNA adducts in human subjects resulting from smoking (or other chronic environmental exposure), lung tissue and lung tumours were obtained from patients hospitalized for lung cancer. DNA was Isolated from the tissue samples and examined both in an ELISA using a polyclonal antibody against (*)trans-7,8-dihydroxy-anti-9,10-epoxy- 7,8,9,10-tetrahydro benzo]a]pyr ene (BPDE) - DNA as well as by the nuclease PI-mediated modification of the 32P-post-labelling technique. The ELISA results showed BPDE DNA antigenicity in lung DNA from 6 out of 21 patients, and adduct levels ranged from 2 to 134 adducts per 108 nucleotides. For all 21 patients, the autoradiographs of chromato- grams of 32P-postlabelled digests of DNA from non-tumorous lung tissue showed a strong diagonal radioactive zone (DRZ). This DRZ was generally absent in tumorous tissue. DNA samples that were posttive in the ELISA contained a dominant spot within the DRZ that co-chroma- tographed with the major BPDE-DNA adduct (BPDE-dG). The quanti- ties of the BPDE-dG spots ranged from 2.1 to 42 adducts in IO9 nucleotides. These values were lower than the levels found in the ELISA but correlated well with the ELISA results (Kendall W = 0.97; P= 0.00). The levels of the DRZ adducts ranged from 1.9 to 34 adducts in 108 nucleotidcs. Correlations between smoking and DNA adduct levels were poor because of the small number of current smokers (n = 13). However, smokers of filter cigarettes had significantly lower DNA adduct levels compared with smokers of cigarettes without a filter (P = 0.02 by Fischer’s exact test).

Restriction fragment length polymorphism analysis of the L-myc gene locus in a case-control study of lung cancer Tamai S, Sugimura H, Caporaso NE et al. Laboratory of Human Carcinogenesis, DCE. NCI, NIH, Bethesda, MD 20892. Int J Cancer 1990;46:41 l-5.

The L-myc DNA-restriction fragment length polymorphism, re- vealed by EcoRI, has been studied in both a lung cancer case-control framework and a cohort of 40 nondiseased unrelated individuals. No association was found between the L-myc allelic frequencies and disease status, tumor stage or lung cancer histology. A strong associa- lion was, however, observed between the L-myc allelic frequencies and ethnic origin (black or white) of the subjects. Among American whites die allelic distribution at tbe L-myc proto-oncogene locus was almost identical to chat previously reported for Japanese subjects. Among the American black population there was a significantly higher frequency