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O-020 Global genome damage assessment and lung cancer outcome
$10
Oral Sessions/Basic science/Ceil and molecular biology
and preclct divergent clinical courses. In future studies, we will cornblne these analyses with data from comparat]ve genomic hybddizat]on studies of the same specimens to boild a comprehensive genomewvide assessment of lung adenocarcinomas These stucles may provide insight into the tumor biology. drug responsiveness, and clinical behavior of lung adenocarcinomas
I0•18•
Mlcroarray profiles and prognosis of completely resected non-small cell lung cancer (NSCLC) patients
Netherlands Cancer Institute, Amsterdam, Nethefands Background: Micrcarray prefding studes in other tumor types have shown the value of these techniques in predicting prognos~s. A more accurate pregnesticabon of patients with NSCLC may improve the selection of patients for adjuvant therapy. We have undertaken a cooperat]ve effort to ident] fy profiles that predict prognosis in NSCLC. Methods: Data from more than 700 NSCLC patients (from 5 academic inst]tut]ons), who underwent surgical treatment and from whom frozen tumor tissue was available were entered in a common database Two series of pat]ants were selected: One with eady metastatic spread shortly after complete resect]on of pat]ants with a "favorable" "I3NM and another group st]ll alive and free of cancer more than ,5 years after surgery The isolat]on of RNA. labeling of complementary RNA (ClRNA) bybridizat]on of labeled cRNA to 44.000-gene arrays, and expression rat]os were all performed as previously described (van 't Veer LJ et al Nature 2002:41,5:,539 548) Results: Will be clsctosed at the meet]ng. Conclusions: Will be available at the time of the meet]ng.
Molecular Biology IV: Immunology and prognostic factors Wednesday, 6 July 2005
•0•9•
17:00-19:00
Modulation of the classical pathway or complement Increases the suscepUblllty of lung cancer cells to complement-mediated lysis
D /~ona. L Montuenga. R Pie Division ~ Oncology, CIMAqJniversity
or Navarra, Pamp/ona, Spain Background: Many molecular targeted therapeutic strategies against cancer are based on moneclonal ant]bodies to tumor associated ant]gens Among other mechanisms, monodonal ant]bodies can initiate complement-dependent call lysis However. malignant calls are usually resistant to complement through the enhanced e0¢pression of complement control proteins In previous studies we have shown that non small cell lung cancer (NSCLC) cells produce and bind factor H. an inhibitor of complement ac0vation, as a mechanism to escape the activation of the altemat]ve pathway of complement. Methods: In this study, we examine the role of factor H and CD59. a coil membrane complement inilbltor, in the ragulat]on of the classical pathway of complement. First. we generated polyolonal ant]sere to NSCLC cell lines A549 and H1264 by immunizing rabbits with these cell axe'acts. Flow cytometz-y studes revealed that both cell lines reacted with their respective ant]serum Single coil suspensions of both cell lines wore first treated v~th their corresponcing rabbit antiserum and. in a second step. with normal human serum To evaluate the activation of complement, we examined by flow cytometTy the deposition of C3 (a k~/ element in the complement cascade) Generat]on of anapbylatoxin C,sa dunng complement activation was quant]fied by ELISA We also analyzed complement-mediated lysis by a calcoin-AM cytotoxicity assay Results: Our study revealed deposition of C3 fragments and C,sa release on both cancer coil lines but a limited cell lye,s (around 10%). These results are in agreement with previous studies in which a high resistance to complement mediated lysis in NSCLC cells had been described. However. when factor H activity was blocked by neutralizing ant]bodies (MoAb OX 24). deposit]on of C3 fragments and C5a release from normal human serum was more efficient. and the suscopt]bll~ to complement mediated lye,s increased moderately. On the other hand. when CD59 actrv~y was blocked by neulzalizlng ant]bodies (MoAb Y'13-153 1). calls were more susceptible to complement-mediated lysis. enabling lysis of up to 50% of H1264 calls and 70% ofA549 cells A combined treatroent with anti-factor H and ant]~D,sg antibocles was much more effec~ve in increasing cell susceptibility to lysis, with up to 90% of the calls lysed by complement Finally. in order to evaluate the in ~#vorole of factor H. we knocked down factor H expression on A549 cells using shRNA silencing of the gene tn v/tro growth of these cells was not af~cted when compared to cells transacted with an s~RNA targeted to a scramble sequence. However. grow~ of xenograft tumors in athymic mice was reduced to 50%. Conclusions: We have shown that lung cancer coils are able to prevent significant complement act]vat]on upon thedr coil membrane. Furthermore. we demonstrate that lack of activation can be prevented by combined
factor H and CD59 inilblt]on. Our results suggest that complementmediated immunotherapy based on modulat]on of complement acOvation is an attradive strategy for lung cancer tTeatment ~ ]
Global ganome damage assessment and lung cancer outcome
Z Zheng. A Cantor. G Bepler M~l~Jt Cancer Canter, Tampa, Florida, USA
Background: Genome damage is a hallmark of human cancer. Efforts at assessing the impact of global genome damage on tumor phenotype and patients' outcome have focused on measurements of the relabve DNA content in tumor cells compared to normal cells (DNA irides) and the assessment of allele loss (loss of heterezygoslty. LOF0 at single or multiple selected Icol that are thought to harbor genes important in cancer biology. Methods: We have adapted a global, high-resolLrdon genetyping method for determination of global and unbiased genome damage and generated a global genome damage score (GGDS). which is a measure of the extent of damaged DNA in individual tumors The score is a cont]nuous variable from zero to one where zero indicates absence of any damage and one indicates complete
damage Results: In surgically resected human non-small-cell lung cancers, the GGDS ranged from 0.003 to 0.204 indicating that between 0.3-20.4% of the geneme is damaged. Patients with high scores (>0.049) had a significantly worse outcome and their tumors espressed DNA damage repair genes at lower levels than those v~th low scores (median overall su~val time 38.3 months vs. 94.0 months, p = 0.007). The expression of genes involved in DNA damage repair was inversely correlated with GGDS. Conclusions: Our study of global high~-esolut]on genome damage analys~s in non-small-call lung cancer convincingly demonstrates a stat]st]cally significant and clinically meaningful associat]on with the in Wvo tumor phenotype This implies that the clinical behavior of tumors with low GGDS is relat]vely benign while tumors with high GGDS are aggressive resulting in early death of pat]ants Since GGDS determinat]on is a robust and reliable technology, it can easily he integrated into clinical decisions on cancer care For instance, adjuvant treatroent of epithelial malignancies benefits only a minority of patients while t~ic4ty is substantial. GGDS may prove useful in selecting patients at high nsk for tumor associated mortality for adjuvant therapeutic interventions. [ 0 - - 0 ~ Prognostic and predictive role of alteration6 of the PS3-bax-b¢12 pathway of apoptosls In the IALT 0nternatlonal Adjuvant Lung Cancer Trlal) E Brambilla ~ A Dunant 2. M Filiplts 3. S Lantuejoul ~. M Tarayre 2. L. DavidBoudet I . R. Pirker 2. J. Sorta4. J. Pignen 2. ]. Le Chevalier'¢.
1INSERM U5?'8. Department ot Pathology, Grenoble Hospttal, France, Grenoble, France, 2Biostatistics Unit, tnstJtut GustaveJTtoussy, Villejuff, France: 3Laboratory ot Moleca~ar Research and Drug Resistance, Department of lntama] Medicine t, Vienna, Austt?a, 4Department of Medicine, tnstitut Gustave-Roussy, Villejuff, France Background: IALT was the largest clinical thai ever performed (1867 patients) to assess the benefit of clsplat]n based adjuvant chemotherapy in operable non small cell lung ca~noma (NSCLC). A significant survival advantage in the chemotherapy group was demonstrated (p = 0.03). To idenhfy subgroups of patients who would benefit most of chemotherapy, we studed clfferent blomarkers and their relationship with chemotherapy effect on survival Coredict]ve analysis) Cisplat]n chemosensit]vlty is dependent on tumor cell suscept]bllity to apoptosis The P`534)ax-bcl2 pathway represents a cht]cal functional network of mltochondrial-dependent apoptosis influencing cytotoxic drug tTiggered apoptosis Methods: Quality control assessment and pathology ravie.~r was carded out on 867 collected blocks and 783 representative NSCLC cases were retained for an immunehistochomical analysis Ventana immunostainer, standardized rethaving and the same batches of ant]bodies were used. Staining avaluat]on was performed blindy by the same pathologists (LB. SL) for distnbut]on of pos~]ve cells (0 to 100%) and intens~y of stanlng (0 to 3). A final score was achieved on each section by the product of percentage of labeled cells and intensity of staining (range from 0 to 300). P53. bax and bcl2 wore considered as pesltlve for a score >20. Pregnest]c and predictive analysis was based on a C ~ model adjusted on ilstopathological type. stage, pathological N statu~. WHO performance status, age. sex. type of surgery, and canter Due to mult]ple blomarkers test]rig, only p-values belew 0 01 were considered significant Results: Due to the absence of relevant internal controls. 48. immunostainings were une0¢ploltable In tumor calls. P,53 immunostaining was posit]ve in ,55% of all cases. 57% of squamous cell carcinoma (SCC). 44% of adenocarcinema (ADC). and 74% of others Pb3 posit]ve espression did not correlate with overall survival but did correlate with histological type (p < 0 0001) and vascular invasion Co < 0.0003) in a Iogist]c model. Bax was positive in 70% of all cases. 70% of SCC. 66% of ADC. and 74% of others. Bax rcact]vlty vaned with (:enters Co < 0.0001) but net with any of the pathological variables. Bci2 was pesltrve in 17% of all cases, and its pos~t]v~ vaned v~th histological type (p <0.0009): 19% ofSCC. 10% of ADC. 32% of large coll. and 7% ofsarcomatoid ca~noma.
Oral Sessions/Basic science/Ceil and molecular biology
and preclct divergent clinical courses. In future studies, we will cornblne these analyses with data from comparat]ve genomic hybddizat]on studies of the same specimens to boild a comprehensive genomewvide assessment of lung adenocarcinomas These stucles may provide insight into the tumor biology. drug responsiveness, and clinical behavior of lung adenocarcinomas
I0•18•
Mlcroarray profiles and prognosis of completely resected non-small cell lung cancer (NSCLC) patients
Netherlands Cancer Institute, Amsterdam, Nethefands Background: Micrcarray prefding studes in other tumor types have shown the value of these techniques in predicting prognos~s. A more accurate pregnesticabon of patients with NSCLC may improve the selection of patients for adjuvant therapy. We have undertaken a cooperat]ve effort to ident] fy profiles that predict prognosis in NSCLC. Methods: Data from more than 700 NSCLC patients (from 5 academic inst]tut]ons), who underwent surgical treatment and from whom frozen tumor tissue was available were entered in a common database Two series of pat]ants were selected: One with eady metastatic spread shortly after complete resect]on of pat]ants with a "favorable" "I3NM and another group st]ll alive and free of cancer more than ,5 years after surgery The isolat]on of RNA. labeling of complementary RNA (ClRNA) bybridizat]on of labeled cRNA to 44.000-gene arrays, and expression rat]os were all performed as previously described (van 't Veer LJ et al Nature 2002:41,5:,539 548) Results: Will be clsctosed at the meet]ng. Conclusions: Will be available at the time of the meet]ng.
Molecular Biology IV: Immunology and prognostic factors Wednesday, 6 July 2005
•0•9•
17:00-19:00
Modulation of the classical pathway or complement Increases the suscepUblllty of lung cancer cells to complement-mediated lysis
D /~ona. L Montuenga. R Pie Division ~ Oncology, CIMAqJniversity
or Navarra, Pamp/ona, Spain Background: Many molecular targeted therapeutic strategies against cancer are based on moneclonal ant]bodies to tumor associated ant]gens Among other mechanisms, monodonal ant]bodies can initiate complement-dependent call lysis However. malignant calls are usually resistant to complement through the enhanced e0¢pression of complement control proteins In previous studies we have shown that non small cell lung cancer (NSCLC) cells produce and bind factor H. an inhibitor of complement ac0vation, as a mechanism to escape the activation of the altemat]ve pathway of complement. Methods: In this study, we examine the role of factor H and CD59. a coil membrane complement inilbltor, in the ragulat]on of the classical pathway of complement. First. we generated polyolonal ant]sere to NSCLC cell lines A549 and H1264 by immunizing rabbits with these cell axe'acts. Flow cytometz-y studes revealed that both cell lines reacted with their respective ant]serum Single coil suspensions of both cell lines wore first treated v~th their corresponcing rabbit antiserum and. in a second step. with normal human serum To evaluate the activation of complement, we examined by flow cytometTy the deposition of C3 (a k~/ element in the complement cascade) Generat]on of anapbylatoxin C,sa dunng complement activation was quant]fied by ELISA We also analyzed complement-mediated lysis by a calcoin-AM cytotoxicity assay Results: Our study revealed deposition of C3 fragments and C,sa release on both cancer coil lines but a limited cell lye,s (around 10%). These results are in agreement with previous studies in which a high resistance to complement mediated lysis in NSCLC cells had been described. However. when factor H activity was blocked by neutralizing ant]bodies (MoAb OX 24). deposit]on of C3 fragments and C5a release from normal human serum was more efficient. and the suscopt]bll~ to complement mediated lye,s increased moderately. On the other hand. when CD59 actrv~y was blocked by neulzalizlng ant]bodies (MoAb Y'13-153 1). calls were more susceptible to complement-mediated lysis. enabling lysis of up to 50% of H1264 calls and 70% ofA549 cells A combined treatroent with anti-factor H and ant]~D,sg antibocles was much more effec~ve in increasing cell susceptibility to lysis, with up to 90% of the calls lysed by complement Finally. in order to evaluate the in ~#vorole of factor H. we knocked down factor H expression on A549 cells using shRNA silencing of the gene tn v/tro growth of these cells was not af~cted when compared to cells transacted with an s~RNA targeted to a scramble sequence. However. grow~ of xenograft tumors in athymic mice was reduced to 50%. Conclusions: We have shown that lung cancer coils are able to prevent significant complement act]vat]on upon thedr coil membrane. Furthermore. we demonstrate that lack of activation can be prevented by combined
factor H and CD59 inilblt]on. Our results suggest that complementmediated immunotherapy based on modulat]on of complement acOvation is an attradive strategy for lung cancer tTeatment ~ ]
Global ganome damage assessment and lung cancer outcome
Z Zheng. A Cantor. G Bepler M~l~Jt Cancer Canter, Tampa, Florida, USA
Background: Genome damage is a hallmark of human cancer. Efforts at assessing the impact of global genome damage on tumor phenotype and patients' outcome have focused on measurements of the relabve DNA content in tumor cells compared to normal cells (DNA irides) and the assessment of allele loss (loss of heterezygoslty. LOF0 at single or multiple selected Icol that are thought to harbor genes important in cancer biology. Methods: We have adapted a global, high-resolLrdon genetyping method for determination of global and unbiased genome damage and generated a global genome damage score (GGDS). which is a measure of the extent of damaged DNA in individual tumors The score is a cont]nuous variable from zero to one where zero indicates absence of any damage and one indicates complete
damage Results: In surgically resected human non-small-cell lung cancers, the GGDS ranged from 0.003 to 0.204 indicating that between 0.3-20.4% of the geneme is damaged. Patients with high scores (>0.049) had a significantly worse outcome and their tumors espressed DNA damage repair genes at lower levels than those v~th low scores (median overall su~val time 38.3 months vs. 94.0 months, p = 0.007). The expression of genes involved in DNA damage repair was inversely correlated with GGDS. Conclusions: Our study of global high~-esolut]on genome damage analys~s in non-small-call lung cancer convincingly demonstrates a stat]st]cally significant and clinically meaningful associat]on with the in Wvo tumor phenotype This implies that the clinical behavior of tumors with low GGDS is relat]vely benign while tumors with high GGDS are aggressive resulting in early death of pat]ants Since GGDS determinat]on is a robust and reliable technology, it can easily he integrated into clinical decisions on cancer care For instance, adjuvant treatroent of epithelial malignancies benefits only a minority of patients while t~ic4ty is substantial. GGDS may prove useful in selecting patients at high nsk for tumor associated mortality for adjuvant therapeutic interventions. [ 0 - - 0 ~ Prognostic and predictive role of alteration6 of the PS3-bax-b¢12 pathway of apoptosls In the IALT 0nternatlonal Adjuvant Lung Cancer Trlal) E Brambilla ~ A Dunant 2. M Filiplts 3. S Lantuejoul ~. M Tarayre 2. L. DavidBoudet I . R. Pirker 2. J. Sorta4. J. Pignen 2. ]. Le Chevalier'¢.
1INSERM U5?'8. Department ot Pathology, Grenoble Hospttal, France, Grenoble, France, 2Biostatistics Unit, tnstJtut GustaveJTtoussy, Villejuff, France: 3Laboratory ot Moleca~ar Research and Drug Resistance, Department of lntama] Medicine t, Vienna, Austt?a, 4Department of Medicine, tnstitut Gustave-Roussy, Villejuff, France Background: IALT was the largest clinical thai ever performed (1867 patients) to assess the benefit of clsplat]n based adjuvant chemotherapy in operable non small cell lung ca~noma (NSCLC). A significant survival advantage in the chemotherapy group was demonstrated (p = 0.03). To idenhfy subgroups of patients who would benefit most of chemotherapy, we studed clfferent blomarkers and their relationship with chemotherapy effect on survival Coredict]ve analysis) Cisplat]n chemosensit]vlty is dependent on tumor cell suscept]bllity to apoptosis The P`534)ax-bcl2 pathway represents a cht]cal functional network of mltochondrial-dependent apoptosis influencing cytotoxic drug tTiggered apoptosis Methods: Quality control assessment and pathology ravie.~r was carded out on 867 collected blocks and 783 representative NSCLC cases were retained for an immunehistochomical analysis Ventana immunostainer, standardized rethaving and the same batches of ant]bodies were used. Staining avaluat]on was performed blindy by the same pathologists (LB. SL) for distnbut]on of pos~]ve cells (0 to 100%) and intens~y of stanlng (0 to 3). A final score was achieved on each section by the product of percentage of labeled cells and intensity of staining (range from 0 to 300). P53. bax and bcl2 wore considered as pesltlve for a score >20. Pregnest]c and predictive analysis was based on a C ~ model adjusted on ilstopathological type. stage, pathological N statu~. WHO performance status, age. sex. type of surgery, and canter Due to mult]ple blomarkers test]rig, only p-values belew 0 01 were considered significant Results: Due to the absence of relevant internal controls. 48. immunostainings were une0¢ploltable In tumor calls. P,53 immunostaining was posit]ve in ,55% of all cases. 57% of squamous cell carcinoma (SCC). 44% of adenocarcinema (ADC). and 74% of others Pb3 posit]ve espression did not correlate with overall survival but did correlate with histological type (p < 0 0001) and vascular invasion Co < 0.0003) in a Iogist]c model. Bax was positive in 70% of all cases. 70% of SCC. 66% of ADC. and 74% of others. Bax rcact]vlty vaned with (:enters Co < 0.0001) but net with any of the pathological variables. Bci2 was pesltrve in 17% of all cases, and its pos~t]v~ vaned v~th histological type (p <0.0009): 19% ofSCC. 10% of ADC. 32% of large coll. and 7% ofsarcomatoid ca~noma.