- Email: [email protected]
PD-004 Serum levels of vascular endothelial growth factor andepidermal growth factor receptor in advanced non-small cell lung cancer: Its correlation with clinical characteristics
$68
Poster Discussions t Basic science~Ceil and moiecular biology
in early stage non small cell lung cancers (NSCLC) and how this relates to tumor apoptosis Methods: High-density tissue microamays using tissues from 17"6 surgically rasected stage NSCLC were immunostalned with a phospho-tyrosine Stat3 antibody (pStat3) and phospho-tyrosine EGFR antibody (pEGFR) The score system included estimating the intensity of total cell pEGFR or nuclear pStat3 in a semi~uanfitative manner For triplicate samples, median values of the percentage of staining, the intensity, and a composite score (percentage multiplied by intensity) were derived Tumor apoptosis was evaluated by detecting apoptotlc cells and apoptotic bodies using In sltu labeling with an ApopTag Detection Kit. Results: We identified nuclear pStat3 expression in 54% of tumors. We found higher e~prossion of pStat3 in smaller tumors (p = 0.0002) and in patients with limited smoking history (p = 0.03). We found a bend towards higher pStat3 e~pross~on in adeneca~nomas compared with other histologies (p=0.13). pStat3 had no influence on either overall or disease-free survival following surgical resection Importantly. we found a very strong correlation between pEGFR expression and pStat3 expression in this patient cohort (Spearman correlation coefficient 052. p < 0 0 0 0 1 ) Finally. we identified a negative correlation between pStat3 and ApopTag (Spearman correlation coef 0 1g. p 0 01) consistent with less apoptosis in tumors expressing higher amounts of pStat3 Conclusions: Those results suggest that small NSCLC tumors have activated EGFR Stat3 signaling and may be dependent on oontinuous s~gnaling from this pathway for survival. Our finding that pEGFR and pStat3 is e~prossod in nearly 50% of patients suggests that agents targeting this pathway may have better effK~cy in earlier stage NSCLC compared with more advanced stage patients.
~Tserurn levels of vascular endothelial growth factor and epidermal growth factor receptor In advanced non-small cell lung cancer Its correlation with clinical ctlaracterlstlcs C Camps I . P, Sirera ~. R Bremnes 2. L Llobat ~. M Safont I . A Blasco ~. V. Alhorola ~. M. Taron 4. J. Sanchoz 5. R. Resell 4. ~Hospttal Genera/ Umvers~tano De Valencia, Valencia, Spain, 2 Unlverstty of Troms~, Norway;,
~Hospital Amau De Vflanova, Valencia, Spain, 4Institute Catala D'Oncotogia, Hosptal Germans Tnas t P~ol, Badalena, Spa]n, ~Untverslclad Autonoma De Madrid, Madrid, Spain Bad(ground: Vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) are angiogenic factors involved in tumour growth and metastasis. Serum levels of VLGF and EGFR were analyzed in advanced nor. small cell lung cancer (NSCLC) patients and correlated with clinical characteristics. Methods: 100 advanced NSCLC patients treated with gemcitabine+cisplatin were enrolled in this study. Blood was collected before chemotherapy and VEGF and EGFR levels wore cFJantlfied by FLISA Results: 91% were males. Mean ago was 61years (range. 35-82). 86 patients were ECOG PS ~ 1 and 14 PS 2 71% of patients were stage IV and 29% IIIB Histological types: 38.% sduamous coll. 3?'% adenocareinoma. ,5% anaplastic large call. 20% undifferentiated 7?" ,5% of patients had metastases out of the lung Response to therapy: 4% complete response (CR). 38% papal response (PR). 25% stable cisease (SD) and 30% progressive disease (PD) Serum levels of VEGF and EGFR were not different according to stage, histology and PS No significant differences were observed in response to therapy accorclng to VEGF and EGFR levels: in CR and PR patients, median EGFR level was 63.5 r'@'ml (range. 7.5-302) and median VEGF level was 364.4 pg/ml (range. 51-4015). while Ibr SD and PD patients, median EGFR level was 62.6 ng/ml (range. 5.3-227.8) and median VEGF level was 570.8 pg/ml (range. 143-1452). Patients with local metastases had lower EGFR levels (55.5 ng/ml [range. 7.5-85.7]) than patients with distant metastases (6&7ng/ml [range. 12.8-302]) (I3 < 0.05). EGFR and VEGF levels wore not preclctors of time to progression and survival Conclusions: In advanced NSCLC. EGFR levels were related to the presence of distant metastases, but there was no relationship between serum VEGF or EGFR levels with stage, histology, response, time to progression and mean survival
coil cycle arrest in H1299. a NSCLC cell line. Based on those data. it has been proposed that aCP-4 may be a tumor suppresser gone at 3p21 Another alternatively spliced form of aCP-4, termed aCP-4a, has also been described This form differs from aCP-4 in the pnmary sequence of the protein C-terminus and nothing is known about its function Interestingly. the eocpression ofaCP-4a is apparently normal in lung tumors Methods: In this study we have further characterized the funchonal implication of aCP-4 and aCP-4a in lung carcinogenesis For that purpose, by retroviral infection, we generated H1299 cells that induclbly express aCP 4 or aCP4a. Using these cells we have evaluated the effects of both vanants on (:ell growth. coil cycle, apoptosis and invasion. Besides. we have determined the subcollular localization of both vanants using GFP fusion protons and confecal microscopy. Result8: Induction of aCP4 expross~on reduced coil growth when measured by MTT assays, whereas the e~prossion of the splicing vanant aCP 4a did net affect it. In agreement with those results, cell cycle analysis by flow cytometry showed an induction of cells cycle arrest in G04G1 after aCP-4 overexprassion In the same conditions, aCP-4a cid not mocify the call cycle In addition, we did not observe any effect on apoptosis neither mediated by aCP-4 nor by aCP-4a In an in vitro model for call invasion, we found that the expression of aCP-4 decreased the invasion ability of H129g cells through a basement membrane, while expression ofthe alternative vabant aCP-4a did not modify this capacity Finally. subcallular localization of GFP-tagged proteins, as visualized by confocal microscopy, revealed a distinct distnbutlon of beth spliced variants. While. aCPAa was localized both in the nucleus and the cytoplasm. aCP 4 e~prossion was ras1~cted to the nucleolus. Conclusions: ]hose results are in agreement with the proposed role of aCP 4 as a tumor suppressor, suggesting an association of this RNA binding protein with lung carcinogenesis. Be=des. our data show functional clfferoncos between the two splicing forms, aCP 4 is involved in inhibition of cell proliferation and invasion, but aCP-4a does not show any of these activi'das These data prove the importance of the splicing process in the control of gone function in cancer In this case. a different compartmentalization of both vanants due to the changes in the pnmary sequence of the protein may be associated to the functional discrepancies
~
2-Met~oxyeetradlol (2-ME2, Panzem ®) and its new derivatives Inhibited lung cancer cells In vitro and prolonged survivals In atttymlc nude rats @ orthotoplc lung tumors In vlvo
D. Chan I . T LaVallee 2. Z Zhang ~. L Zhao ~. G Swartz 2. A Treston 2. W. Fooler 2, E Bunn Jr?. ~Umversrty of C_,o/ora¢o Cancer Center, Aurora,
Colorado, USA, 2EntreMed, tnc, Rockville, Maryland, USA Background: 2Methoxyeslzadiol (25ME2. Panzem®) is a natural metabolite of estradiol that offect?veiy induces spoptosis and inhibits proliferation in a wide range of cancer (:ell lines tn wtto. as well as showing anti angiogenic and anti tumor activities in preolinical models. The anti~rolifarativo activity of 2ME2 is independent of estrogen receptors, and has been found to ~srupt microtubulo sb-ucturas Ioa~ng to arrest in G 2 M stage. New drug formulations. Panzem nanecrystal colloidal dispersion (NCD) as well as new analogs of 2ME2 have been developed and show improved anti~roliferahve activities and pharmacokinetlc parameters as compared to onginal 2ME2 formulation Methods: We have used the MT]" assay to evaluate the growth inhibitory effects of 2ME2 and three near analogs ENMD-1198.. -1200. and -1237 against a panel of human small cell lung cancer cells (SCLC) and non-small cell lung cancer cells (NSCLC) Drug combinations were evaluated for synergistic effects using Cembieatlon Inde=-Isobologram In t~vo efficacy of 2ME2 and analogs was evaluated in athymic nude rats bearing orthotopically implanted H2122 adenocarclnomas in the loft lungs and survival curves were generated. Results: Average In ~ t o IC50 ~lV0 values are shown in the table below. which range from 0.21 to 1.10p.M for 2ME2; 0.028 to 0.22~.M for ENMD 1198; 0.058 to 0.43 ~.M for ENMD 1200 and 0.48 to 3.05 ~.M for ENMD 1237 respectively. Overall ENMD 1198 and ENMD 1200 are significantly more potent than 2NIE2 or ENMD 1237. There is net much clfforonca in growth inhibitory effects between adenecareinomas, squamous and large call by each of these agents SCLC cell line proliferation is more effectively inhibited than is NSCLC call line proliferation :21VIE2at concentrations ranging from 0 1 to 1 p.M strongly synergized the growth inhibitory effects of several therapau'dc agents in vitro
~05TRole of the putative tumor suppressor aCP-4 and Its altemaUvely spliced variant aCP-4a In In vlb'o lung cancer growth Z. Casta~5o. L. Montuonga. R. Pie. Dtvrstorl of Onco/ogy, CIMA, Ur~wrstty of Navarre, Pamp/ona, Spare
Background: aCP-4 is an RNA bincing protein coded by a gone mapped to 3p21. one of the most common altered regions in lung cancer In previous studios we have demonstrated that lung tumors show frequent lack of aCP 4 prote~n e~prossion assecmtod with aCP4 allele loss. This lack of aCP4 is associated with poody differentiated and highly proliferative tumors. Besides. a splicing vanant of aCP4 called MCG10 is able to induce apoptosis and
NSCLC(n 14) SCLC(n=6)
2ME21C~0 (F.M)
ENMD-1198 IC~0 (p.M)
ENMD-1200 IC~0 (F.M)
ENMD-1237 IC~ (F.M)
0768±01`53 0.476-J-0.088
0122±0023 0066±0027
0238.±0052 0.116d-0.025
1729±0307 0.9774-0.230
Rats (n 12Jgroup) were treated with saline control or compounds at 10 and 60 mgfkgiday by oral garage for 50 days Panzem NCD and ENMD-1200 effectively prolonged survival of rats in a doso~lopondont manner, with moan survival for control groqo at 27 days. 2ME2 10mgtkg at 39 days. 2ME2 60 mgtkg >50 days. and ENMD 1200 10mgfkg at 48 days. and ENMD 1200 60mgtkg >50 days respectively. ENMD 1198 and ENMD 1237 at 10 or 60mg/kg wore
Poster Discussions t Basic science~Ceil and moiecular biology
in early stage non small cell lung cancers (NSCLC) and how this relates to tumor apoptosis Methods: High-density tissue microamays using tissues from 17"6 surgically rasected stage NSCLC were immunostalned with a phospho-tyrosine Stat3 antibody (pStat3) and phospho-tyrosine EGFR antibody (pEGFR) The score system included estimating the intensity of total cell pEGFR or nuclear pStat3 in a semi~uanfitative manner For triplicate samples, median values of the percentage of staining, the intensity, and a composite score (percentage multiplied by intensity) were derived Tumor apoptosis was evaluated by detecting apoptotlc cells and apoptotic bodies using In sltu labeling with an ApopTag Detection Kit. Results: We identified nuclear pStat3 expression in 54% of tumors. We found higher e~prossion of pStat3 in smaller tumors (p = 0.0002) and in patients with limited smoking history (p = 0.03). We found a bend towards higher pStat3 e~pross~on in adeneca~nomas compared with other histologies (p=0.13). pStat3 had no influence on either overall or disease-free survival following surgical resection Importantly. we found a very strong correlation between pEGFR expression and pStat3 expression in this patient cohort (Spearman correlation coefficient 052. p < 0 0 0 0 1 ) Finally. we identified a negative correlation between pStat3 and ApopTag (Spearman correlation coef 0 1g. p 0 01) consistent with less apoptosis in tumors expressing higher amounts of pStat3 Conclusions: Those results suggest that small NSCLC tumors have activated EGFR Stat3 signaling and may be dependent on oontinuous s~gnaling from this pathway for survival. Our finding that pEGFR and pStat3 is e~prossod in nearly 50% of patients suggests that agents targeting this pathway may have better effK~cy in earlier stage NSCLC compared with more advanced stage patients.
~Tserurn levels of vascular endothelial growth factor and epidermal growth factor receptor In advanced non-small cell lung cancer Its correlation with clinical ctlaracterlstlcs C Camps I . P, Sirera ~. R Bremnes 2. L Llobat ~. M Safont I . A Blasco ~. V. Alhorola ~. M. Taron 4. J. Sanchoz 5. R. Resell 4. ~Hospttal Genera/ Umvers~tano De Valencia, Valencia, Spain, 2 Unlverstty of Troms~, Norway;,
~Hospital Amau De Vflanova, Valencia, Spain, 4Institute Catala D'Oncotogia, Hosptal Germans Tnas t P~ol, Badalena, Spa]n, ~Untverslclad Autonoma De Madrid, Madrid, Spain Bad(ground: Vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) are angiogenic factors involved in tumour growth and metastasis. Serum levels of VLGF and EGFR were analyzed in advanced nor. small cell lung cancer (NSCLC) patients and correlated with clinical characteristics. Methods: 100 advanced NSCLC patients treated with gemcitabine+cisplatin were enrolled in this study. Blood was collected before chemotherapy and VEGF and EGFR levels wore cFJantlfied by FLISA Results: 91% were males. Mean ago was 61years (range. 35-82). 86 patients were ECOG PS ~ 1 and 14 PS 2 71% of patients were stage IV and 29% IIIB Histological types: 38.% sduamous coll. 3?'% adenocareinoma. ,5% anaplastic large call. 20% undifferentiated 7?" ,5% of patients had metastases out of the lung Response to therapy: 4% complete response (CR). 38% papal response (PR). 25% stable cisease (SD) and 30% progressive disease (PD) Serum levels of VEGF and EGFR were not different according to stage, histology and PS No significant differences were observed in response to therapy accorclng to VEGF and EGFR levels: in CR and PR patients, median EGFR level was 63.5 r'@'ml (range. 7.5-302) and median VEGF level was 364.4 pg/ml (range. 51-4015). while Ibr SD and PD patients, median EGFR level was 62.6 ng/ml (range. 5.3-227.8) and median VEGF level was 570.8 pg/ml (range. 143-1452). Patients with local metastases had lower EGFR levels (55.5 ng/ml [range. 7.5-85.7]) than patients with distant metastases (6&7ng/ml [range. 12.8-302]) (I3 < 0.05). EGFR and VEGF levels wore not preclctors of time to progression and survival Conclusions: In advanced NSCLC. EGFR levels were related to the presence of distant metastases, but there was no relationship between serum VEGF or EGFR levels with stage, histology, response, time to progression and mean survival
coil cycle arrest in H1299. a NSCLC cell line. Based on those data. it has been proposed that aCP-4 may be a tumor suppresser gone at 3p21 Another alternatively spliced form of aCP-4, termed aCP-4a, has also been described This form differs from aCP-4 in the pnmary sequence of the protein C-terminus and nothing is known about its function Interestingly. the eocpression ofaCP-4a is apparently normal in lung tumors Methods: In this study we have further characterized the funchonal implication of aCP-4 and aCP-4a in lung carcinogenesis For that purpose, by retroviral infection, we generated H1299 cells that induclbly express aCP 4 or aCP4a. Using these cells we have evaluated the effects of both vanants on (:ell growth. coil cycle, apoptosis and invasion. Besides. we have determined the subcollular localization of both vanants using GFP fusion protons and confecal microscopy. Result8: Induction of aCP4 expross~on reduced coil growth when measured by MTT assays, whereas the e~prossion of the splicing vanant aCP 4a did net affect it. In agreement with those results, cell cycle analysis by flow cytometry showed an induction of cells cycle arrest in G04G1 after aCP-4 overexprassion In the same conditions, aCP-4a cid not mocify the call cycle In addition, we did not observe any effect on apoptosis neither mediated by aCP-4 nor by aCP-4a In an in vitro model for call invasion, we found that the expression of aCP-4 decreased the invasion ability of H129g cells through a basement membrane, while expression ofthe alternative vabant aCP-4a did not modify this capacity Finally. subcallular localization of GFP-tagged proteins, as visualized by confocal microscopy, revealed a distinct distnbutlon of beth spliced variants. While. aCPAa was localized both in the nucleus and the cytoplasm. aCP 4 e~prossion was ras1~cted to the nucleolus. Conclusions: ]hose results are in agreement with the proposed role of aCP 4 as a tumor suppressor, suggesting an association of this RNA binding protein with lung carcinogenesis. Be=des. our data show functional clfferoncos between the two splicing forms, aCP 4 is involved in inhibition of cell proliferation and invasion, but aCP-4a does not show any of these activi'das These data prove the importance of the splicing process in the control of gone function in cancer In this case. a different compartmentalization of both vanants due to the changes in the pnmary sequence of the protein may be associated to the functional discrepancies
~
2-Met~oxyeetradlol (2-ME2, Panzem ®) and its new derivatives Inhibited lung cancer cells In vitro and prolonged survivals In atttymlc nude rats @ orthotoplc lung tumors In vlvo
D. Chan I . T LaVallee 2. Z Zhang ~. L Zhao ~. G Swartz 2. A Treston 2. W. Fooler 2, E Bunn Jr?. ~Umversrty of C_,o/ora¢o Cancer Center, Aurora,
Colorado, USA, 2EntreMed, tnc, Rockville, Maryland, USA Background: 2Methoxyeslzadiol (25ME2. Panzem®) is a natural metabolite of estradiol that offect?veiy induces spoptosis and inhibits proliferation in a wide range of cancer (:ell lines tn wtto. as well as showing anti angiogenic and anti tumor activities in preolinical models. The anti~rolifarativo activity of 2ME2 is independent of estrogen receptors, and has been found to ~srupt microtubulo sb-ucturas Ioa~ng to arrest in G 2 M stage. New drug formulations. Panzem nanecrystal colloidal dispersion (NCD) as well as new analogs of 2ME2 have been developed and show improved anti~roliferahve activities and pharmacokinetlc parameters as compared to onginal 2ME2 formulation Methods: We have used the MT]" assay to evaluate the growth inhibitory effects of 2ME2 and three near analogs ENMD-1198.. -1200. and -1237 against a panel of human small cell lung cancer cells (SCLC) and non-small cell lung cancer cells (NSCLC) Drug combinations were evaluated for synergistic effects using Cembieatlon Inde=-Isobologram In t~vo efficacy of 2ME2 and analogs was evaluated in athymic nude rats bearing orthotopically implanted H2122 adenocarclnomas in the loft lungs and survival curves were generated. Results: Average In ~ t o IC50 ~lV0 values are shown in the table below. which range from 0.21 to 1.10p.M for 2ME2; 0.028 to 0.22~.M for ENMD 1198; 0.058 to 0.43 ~.M for ENMD 1200 and 0.48 to 3.05 ~.M for ENMD 1237 respectively. Overall ENMD 1198 and ENMD 1200 are significantly more potent than 2NIE2 or ENMD 1237. There is net much clfforonca in growth inhibitory effects between adenecareinomas, squamous and large call by each of these agents SCLC cell line proliferation is more effectively inhibited than is NSCLC call line proliferation :21VIE2at concentrations ranging from 0 1 to 1 p.M strongly synergized the growth inhibitory effects of several therapau'dc agents in vitro
~05TRole of the putative tumor suppressor aCP-4 and Its altemaUvely spliced variant aCP-4a In In vlb'o lung cancer growth Z. Casta~5o. L. Montuonga. R. Pie. Dtvrstorl of Onco/ogy, CIMA, Ur~wrstty of Navarre, Pamp/ona, Spare
Background: aCP-4 is an RNA bincing protein coded by a gone mapped to 3p21. one of the most common altered regions in lung cancer In previous studios we have demonstrated that lung tumors show frequent lack of aCP 4 prote~n e~prossion assecmtod with aCP4 allele loss. This lack of aCP4 is associated with poody differentiated and highly proliferative tumors. Besides. a splicing vanant of aCP4 called MCG10 is able to induce apoptosis and
NSCLC(n 14) SCLC(n=6)
2ME21C~0 (F.M)
ENMD-1198 IC~0 (p.M)
ENMD-1200 IC~0 (F.M)
ENMD-1237 IC~ (F.M)
0768±01`53 0.476-J-0.088
0122±0023 0066±0027
0238.±0052 0.116d-0.025
1729±0307 0.9774-0.230
Rats (n 12Jgroup) were treated with saline control or compounds at 10 and 60 mgfkgiday by oral garage for 50 days Panzem NCD and ENMD-1200 effectively prolonged survival of rats in a doso~lopondont manner, with moan survival for control groqo at 27 days. 2ME2 10mgtkg at 39 days. 2ME2 60 mgtkg >50 days. and ENMD 1200 10mgfkg at 48 days. and ENMD 1200 60mgtkg >50 days respectively. ENMD 1198 and ENMD 1237 at 10 or 60mg/kg wore