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PD-016 Identification of predicative serum markers for chemotherapyresponse in non-small cell lung cancer by quantitative proteomics
Poster Discussions t Basic science/Ceil and rnoiecu/ar biology the relative level of ERCC1 or MAGE expression in 67 inoperable (stage Ilia IV) NSCLC palJents tTeated with taxanes (33 cases) or gemcitabine (34 cases) plus cisplabn every 3 weeks Re.=ult.=: In sputum. ERCC1 was detected in 74 6% and MAGE was in 40 2% of palJents By the median of ERCCI. the patients were olassified as high e0¢pression when above the median, or low expression when lower than the median Median overall survival (MST) was significantly longer in patients with high ERCC1 expression then those of low expression (84 weeks vs 44 weeks respectively. P = 0.017). In the taxen~based treatment group. MST was longer than gomcitabine group (79 weeks vs. 47 weeks, respectively. P = 0.03). In patients with MAGE~oslt]vo. the levels of ERCC1 wore significantly high (P = 0.003). In pat]ants with MAGE negative. MST was significantly longer in the tlgh ERCC1 group than the low ERCC1 group (103 weeks vs. 43 weeks. P = 0.008). but this finding was not found in patients with MAGE positive (62 weeks vs. 44 weeks. P = 0.348). Conduslon,=: These data suggest that ERCC1 e0¢pression in the sputum is a predictive factor for survival after chemotherapy in the inoperable NSCLC pabents
~ D ~ S ~ Reduction of PTEN protein and loss of epidermal growth factor receptor (EGFR) gone mutation In lung cancer wRh natural roe/stance to gefltlnlb (IRESSA) YKokubo 1'3.A Gamma ~.R Nero I . M Seike ~.K Kataoka I . K Matsuda ~. Y Minegishi ~. A Yoshimura ~. M Shibuya ~. S Kudoh ~ ~Four~ Department
of tntamal Medicine, t~ppon Madcal School, Tokyo, Japan, 2Respratoty Medicine, Tokyo Metropolitan Koma~me Hospital, Tokyo, Japan, 3Respiratory Medicine, Hakujtkal MemonaJ Hospital, Tokyo, Japan Background: Gofit]nib (IRESSA). an epidermal growth factor receptor (EGFR) tyrosino kinaso inhibiter, has ant]tumour activity in the advanced non small (:ell lung cancer (NSCLC) ,,;offing. However. in chemothorapy~aivo pat]ants with advanced NSCLC. the addit]on of gefit]nib to standard chemotherapy regimens failed to increase survival. These results suggest the need for improved pat]ant selection and cornbinat]en rat]onales for targeted therapies. Methods: We have identified subpopulations of an adenocarc~noma cell line that are naturally resistant to gefibrtb, and have analysed the cONA expression profiles, genomic status ofEGFR gene. and the ef~ct of geftinib on signalling pathways in these cell lines in order to identify k~/ mechanisms for naturallyacquired resistance to geftinib Reaulta: Gefitinib-resistant subpopulal~ons dernonst]"ated increased Akt phosphorylation (not inPibited by gefibnib), reduced PTEN protein espression, and loss of the EGFR gone mutal~on when compared with parental cell lines These differences in Akt and PTEN protein expression were not evident from the cONA array profiles. ¢onctuslons: These data suggests that (1) the EGFR gone mutation may be possibly lost in some cancer cells with ether addit]onal mechanisms Ibr activating Akt. (2) ReintToduet]on of PTEN or pharmacological downregulation oftbe const]tut]vo P I 3 K ~ ' t pathway activity may be an attractive therapeutic st]'atogy in cancers with gofitinib resistance. ~
Identification of prerllcatlve serum markers for chemotherapy response In non-small cell lung cancer by quantRatlve proteemlcs
K Lain. T Mek. T Peon. A Chan The Chinese UnivarsityofHong Kong,
Hong Kong Sar, China Background: A number of oncogenes or its gone products have bean known to have predical~ve value in tumor response to treatment or long-term prognosis Prcteomios may help to identify tissue or serum peptides/preteins that can predict chemotherapy responsiveness Method,=: The pro- and post-chemotherapy sere from 2,5 patients with partial response (PR) and 26 pal~ents with progressive disease (PD) in a completed phase II study on gemcltobine4~ased chemotherapy Ibr advanced non small cell lung cancer were profled with SELDI TOF PretomChip system (Cipbergen. Inc. USA) at 2 binding conditions with the use of CM 10 PretoinChip arrays. MS peeks that can different]ate between PR and PD pat]ants wore identified. Each of these peeks was compared between pro and post~hometherapy sore by WIcoxon Signed Reek Test. Results: Thirteen MS peaks that could differentiate between PR and PD wore identified (10 peaks with abundance in PD and 3 peaks with abundance in PR groups) 7 of 10 peeks in PD group have insignificant change between proand post-chemotherapy sere but 8 of these 10 peaks had significant increased post-chemotherapy level in PR group 3 of 10 peaks had significant increased post-chemotherapy level in both PR and PD groups 3 peaks in PR group were unchanged in post-chemotherapy Conclusion: By quantitative proteomics. 8 serum pepl~des may be related to chomo resistance. 3 peaks with increased post chemotherapy level in both PR and PD groups may be tumor specific. 5 poptidos with similar pro~ and post~hometheraby level in PD group but higher post chemotherapy level in PR group may be related to drug metabolism. These poptidos should be sequenced in the future study.
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$71
Detection of promoter byparme~ylaflon of RARII, RASSFIA, DAPK and P16/ INK4a genes In call-free DNA from serum and pleural liquid of patients wlt}l pleural effusion
B MassutJ~ S Benlloch 2. J Sanchez4~aya2. J Maf(~~. B E~aschwit~~. C. Fernandez 4. J. Ramirez ~. J. MartiCirlqulan I . S. Remora 4. J. Galbis ~.
1MeScal Oncotogy,, Genera/Hospital of AJicanfa, 2Research Unit, Genera/ Hospital ct A#cante, 31boracic Surge~ Genera/Hospital Of Al/cante, 4Pneumology, Genera/Hospital OrAl/canto, Ahcante, Spain: 5Medical Oncotogy, Germans Tnas I Pujot Hospital Badalona, Spatn Background: Aberrant gene promoter methylation is a major mechanism ~r tumor suppressor gene (TSG) silencing in all tumor types and affects their development and aggressiveness Elevated levels of CpG methylabon are considered to be one of the earliest and most frequent alterations in carcinogenesis Aberrant methylalJon of RARE. RASSE1A. DAPK and P16/ INK4a genes have bean shown to be involved in most tumors We designed this study with three obJoettves: 1) to detect promoter hypermethylation in call flee DNA from pleura/ liquid of patients with p/aural effusion; 2) to assess the concordance of gone promoter hyporroothylat]on in paired serum and pleural liquid samples of cancer patients with malignant pleural effusion; and 3) to invest]gate the role of hyperroothylat]on as a marker of pleura/ effusion malignancy. Methods: We evaluated ,53 consecutive pat]onts with pleura/ effusion and malignancy (24 lung. 11 digestive. 8 breast. 4 ovary. 6 others) at the General Hospital of Alicante. Spain HypermethylatJon of RARe. RASSF1A. DAPK. and P16/INK4a were assessed with methylalJon-specific PCR (MSP) after sodium bisulphite treatment Results: Convenbonal cytology of p/aural effusion samples revealed neoplasbc calls in 17 of 48 patients (35 b%) Aberrant methylatJon of at least one of the genes tested was detected in 24 of 53 patients (45 3%) in serum and in 31 of 53 pat]onts (58.6%) in pleural liquid. In pleura/ liquid. 41.,5% had no genes methylatod. 34% had only 1 gone methylated. 17% had 2 genes methylated. 5.7% had 3 genes mothylated, and only 1 (1.9%) had 4 genes mothylatod. In serum. 54.7% had no genes methylated. 26.4% had only 1 gone methylated. 15.1% had 2 genes mothylated, and oily 3.8% had 3 genes methylated. Concordance in the pattern of aberrant mothylation was found in paired serum and ploural liq,=d samples of 13 patients. In 11 patients, methylation was found only in pleural liquid and not in serum: in only 3 patients, methylatJon was found in serum and not in pleura/ liquid Aberrant metf~/lalJon of DAPK was found in 7 patients (13 2%) in serum and in 10 patients (18 9%) in pleura/ liquid P16/INK4a aberrant methylation was found in 10 patients (18 9%) in serum and in 20 palJents (37 7%) in p/aural liquid: RASSF1A aberrant methylatJon was found in g patients (17%) in serum and in 8 palJents (lb 1%) in pleural liquid Aberrant methylatJon of RAF~ was found in 10 patients (18 9%) in serum and in 11 patients (20.8%) in pleural liquid. The correlation coeffioent between serum and pleural liquid mothylated genes was r=0.6 Co <0.0ggl). Meclan survival t]me of patients with at least one gone methylatod in p/aural liquid was 12 months (95%C1: 5.95-63). while Ibr patients with no genes methylated. median survival has not yet been reached. Conclusions: Aberrant gone promoter methylat]on of PSG can be detected in pleural liquid. In ploural liquid. 58.6% of pat]onts had at least one TSG methylated while convenbonal cytology showed only 35 5% of pabents to have neoplastic cells Aberrant methylation was a negalJve prognostic marker Table 1 Companson of number of pabents with positive convenbonal cytology in pleural liquid and those with at least one hypermethylated gene in DNA from serum and pleural liquid
Positive conventional cytology At least one gone methylated in serum At least one gone mothylated in pleural liquid
•]
Number of patients
%
17/48 24/53 31/53
35.5 4b 3 58.6
Rsel-tlma quanUflcaflon of CK-19 mRNA-poslflva cells In peripheral blood of patients using with non-small cell lung cancer (NSCLC) using Real Time RT-PCR
G. Milaki I . D. Mevreudis I . S. Apostola~ 2, I. Souglakos ~. M. Perra~ 2, V Georgoulias ~ ~Univarsity Hosptal of Haraklion, Harskfion, Greece,
2LatJoratory tor Tumor Biology Umvers~ty of Crete, Herald/on, Greece Purpose: Was to develop a quentJtalJve real-time reverse transcdplJon~CR (RT~CR) for CK19-mRNA and evaluate its olinical potenlJal ~r the molecular detect]on of occult carcinoma cells in the peripheral blood of NSCLC pabents Patients and Method: The method is based on real-bme monitoring dudng PCR of fluorescently labeled specific hybridization probes Ibr CK19 mRNA. We analyzed blood samples from 6 patients with operable (stage IB IliA) postoperatively, and 48 patients with prevlcusly untreated locally advanced or metastatic disease (stage IV) before and after chemotherapy: All of the samples
~ D ~ S ~ Reduction of PTEN protein and loss of epidermal growth factor receptor (EGFR) gone mutation In lung cancer wRh natural roe/stance to gefltlnlb (IRESSA) YKokubo 1'3.A Gamma ~.R Nero I . M Seike ~.K Kataoka I . K Matsuda ~. Y Minegishi ~. A Yoshimura ~. M Shibuya ~. S Kudoh ~ ~Four~ Department
of tntamal Medicine, t~ppon Madcal School, Tokyo, Japan, 2Respratoty Medicine, Tokyo Metropolitan Koma~me Hospital, Tokyo, Japan, 3Respiratory Medicine, Hakujtkal MemonaJ Hospital, Tokyo, Japan Background: Gofit]nib (IRESSA). an epidermal growth factor receptor (EGFR) tyrosino kinaso inhibiter, has ant]tumour activity in the advanced non small (:ell lung cancer (NSCLC) ,,;offing. However. in chemothorapy~aivo pat]ants with advanced NSCLC. the addit]on of gefit]nib to standard chemotherapy regimens failed to increase survival. These results suggest the need for improved pat]ant selection and cornbinat]en rat]onales for targeted therapies. Methods: We have identified subpopulations of an adenocarc~noma cell line that are naturally resistant to gefibrtb, and have analysed the cONA expression profiles, genomic status ofEGFR gene. and the ef~ct of geftinib on signalling pathways in these cell lines in order to identify k~/ mechanisms for naturallyacquired resistance to geftinib Reaulta: Gefitinib-resistant subpopulal~ons dernonst]"ated increased Akt phosphorylation (not inPibited by gefibnib), reduced PTEN protein espression, and loss of the EGFR gone mutal~on when compared with parental cell lines These differences in Akt and PTEN protein expression were not evident from the cONA array profiles. ¢onctuslons: These data suggests that (1) the EGFR gone mutation may be possibly lost in some cancer cells with ether addit]onal mechanisms Ibr activating Akt. (2) ReintToduet]on of PTEN or pharmacological downregulation oftbe const]tut]vo P I 3 K ~ ' t pathway activity may be an attractive therapeutic st]'atogy in cancers with gofitinib resistance. ~
Identification of prerllcatlve serum markers for chemotherapy response In non-small cell lung cancer by quantRatlve proteemlcs
K Lain. T Mek. T Peon. A Chan The Chinese UnivarsityofHong Kong,
Hong Kong Sar, China Background: A number of oncogenes or its gone products have bean known to have predical~ve value in tumor response to treatment or long-term prognosis Prcteomios may help to identify tissue or serum peptides/preteins that can predict chemotherapy responsiveness Method,=: The pro- and post-chemotherapy sere from 2,5 patients with partial response (PR) and 26 pal~ents with progressive disease (PD) in a completed phase II study on gemcltobine4~ased chemotherapy Ibr advanced non small cell lung cancer were profled with SELDI TOF PretomChip system (Cipbergen. Inc. USA) at 2 binding conditions with the use of CM 10 PretoinChip arrays. MS peeks that can different]ate between PR and PD pat]ants wore identified. Each of these peeks was compared between pro and post~hometherapy sore by WIcoxon Signed Reek Test. Results: Thirteen MS peaks that could differentiate between PR and PD wore identified (10 peaks with abundance in PD and 3 peaks with abundance in PR groups) 7 of 10 peeks in PD group have insignificant change between proand post-chemotherapy sere but 8 of these 10 peaks had significant increased post-chemotherapy level in PR group 3 of 10 peaks had significant increased post-chemotherapy level in both PR and PD groups 3 peaks in PR group were unchanged in post-chemotherapy Conclusion: By quantitative proteomics. 8 serum pepl~des may be related to chomo resistance. 3 peaks with increased post chemotherapy level in both PR and PD groups may be tumor specific. 5 poptidos with similar pro~ and post~hometheraby level in PD group but higher post chemotherapy level in PR group may be related to drug metabolism. These poptidos should be sequenced in the future study.
[ ~ ]
$71
Detection of promoter byparme~ylaflon of RARII, RASSFIA, DAPK and P16/ INK4a genes In call-free DNA from serum and pleural liquid of patients wlt}l pleural effusion
B MassutJ~ S Benlloch 2. J Sanchez4~aya2. J Maf(~~. B E~aschwit~~. C. Fernandez 4. J. Ramirez ~. J. MartiCirlqulan I . S. Remora 4. J. Galbis ~.
1MeScal Oncotogy,, Genera/Hospital of AJicanfa, 2Research Unit, Genera/ Hospital ct A#cante, 31boracic Surge~ Genera/Hospital Of Al/cante, 4Pneumology, Genera/Hospital OrAl/canto, Ahcante, Spain: 5Medical Oncotogy, Germans Tnas I Pujot Hospital Badalona, Spatn Background: Aberrant gene promoter methylation is a major mechanism ~r tumor suppressor gene (TSG) silencing in all tumor types and affects their development and aggressiveness Elevated levels of CpG methylabon are considered to be one of the earliest and most frequent alterations in carcinogenesis Aberrant methylalJon of RARE. RASSE1A. DAPK and P16/ INK4a genes have bean shown to be involved in most tumors We designed this study with three obJoettves: 1) to detect promoter hypermethylation in call flee DNA from pleura/ liquid of patients with p/aural effusion; 2) to assess the concordance of gone promoter hyporroothylat]on in paired serum and pleural liquid samples of cancer patients with malignant pleural effusion; and 3) to invest]gate the role of hyperroothylat]on as a marker of pleura/ effusion malignancy. Methods: We evaluated ,53 consecutive pat]onts with pleura/ effusion and malignancy (24 lung. 11 digestive. 8 breast. 4 ovary. 6 others) at the General Hospital of Alicante. Spain HypermethylatJon of RARe. RASSF1A. DAPK. and P16/INK4a were assessed with methylalJon-specific PCR (MSP) after sodium bisulphite treatment Results: Convenbonal cytology of p/aural effusion samples revealed neoplasbc calls in 17 of 48 patients (35 b%) Aberrant methylatJon of at least one of the genes tested was detected in 24 of 53 patients (45 3%) in serum and in 31 of 53 pat]onts (58.6%) in pleural liquid. In pleura/ liquid. 41.,5% had no genes methylatod. 34% had only 1 gone methylated. 17% had 2 genes methylated. 5.7% had 3 genes mothylated, and only 1 (1.9%) had 4 genes mothylatod. In serum. 54.7% had no genes methylated. 26.4% had only 1 gone methylated. 15.1% had 2 genes mothylated, and oily 3.8% had 3 genes methylated. Concordance in the pattern of aberrant mothylation was found in paired serum and ploural liq,=d samples of 13 patients. In 11 patients, methylation was found only in pleural liquid and not in serum: in only 3 patients, methylatJon was found in serum and not in pleura/ liquid Aberrant metf~/lalJon of DAPK was found in 7 patients (13 2%) in serum and in 10 patients (18 9%) in pleura/ liquid P16/INK4a aberrant methylation was found in 10 patients (18 9%) in serum and in 20 palJents (37 7%) in p/aural liquid: RASSF1A aberrant methylatJon was found in g patients (17%) in serum and in 8 palJents (lb 1%) in pleural liquid Aberrant methylatJon of RAF~ was found in 10 patients (18 9%) in serum and in 11 patients (20.8%) in pleural liquid. The correlation coeffioent between serum and pleural liquid mothylated genes was r=0.6 Co <0.0ggl). Meclan survival t]me of patients with at least one gone methylatod in p/aural liquid was 12 months (95%C1: 5.95-63). while Ibr patients with no genes methylated. median survival has not yet been reached. Conclusions: Aberrant gone promoter methylat]on of PSG can be detected in pleural liquid. In ploural liquid. 58.6% of pat]onts had at least one TSG methylated while convenbonal cytology showed only 35 5% of pabents to have neoplastic cells Aberrant methylation was a negalJve prognostic marker Table 1 Companson of number of pabents with positive convenbonal cytology in pleural liquid and those with at least one hypermethylated gene in DNA from serum and pleural liquid
Positive conventional cytology At least one gone methylated in serum At least one gone mothylated in pleural liquid
•]
Number of patients
%
17/48 24/53 31/53
35.5 4b 3 58.6
Rsel-tlma quanUflcaflon of CK-19 mRNA-poslflva cells In peripheral blood of patients using with non-small cell lung cancer (NSCLC) using Real Time RT-PCR
G. Milaki I . D. Mevreudis I . S. Apostola~ 2, I. Souglakos ~. M. Perra~ 2, V Georgoulias ~ ~Univarsity Hosptal of Haraklion, Harskfion, Greece,
2LatJoratory tor Tumor Biology Umvers~ty of Crete, Herald/on, Greece Purpose: Was to develop a quentJtalJve real-time reverse transcdplJon~CR (RT~CR) for CK19-mRNA and evaluate its olinical potenlJal ~r the molecular detect]on of occult carcinoma cells in the peripheral blood of NSCLC pabents Patients and Method: The method is based on real-bme monitoring dudng PCR of fluorescently labeled specific hybridization probes Ibr CK19 mRNA. We analyzed blood samples from 6 patients with operable (stage IB IliA) postoperatively, and 48 patients with prevlcusly untreated locally advanced or metastatic disease (stage IV) before and after chemotherapy: All of the samples