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32P PIGMENT EPITHELIUM-DERIVED FACTOR ENHANCES TUMOR RESPONSE TO RADIATION BY NORMALIZING VASCULATURE IN A XENOGRAFTED MODEL OF LUNG CANCER
TRANSLATIONAL RESEARCH cell carcinoma. After we had found differentially expressed genes, we conducted immunohistochemistry (IHC) to validate the results in a different NSCLC group. Results: In RNA-Seq analysis, the total counted reads were 25.41±8.90 million and 24.70±4.70 million in NSCLC tissues and normal lung tissues, respectively (mean ± SD). Among the genes expressed in both tissues, 335 were up-regulated whereas 728 were down-regulated in lung cancer tissues (fold changes 2 and p-value <0.001). Among up-regulated genes, we selected four genes that have not been reported previously in lung cancer, namely CBX3, GJB2, CRABP2 and DSP. We carried out IHC for four genes in another set of NSCLC tissues (n = 154). Total positive rates of CBX3, GJB2, CRABP2 and DSP were 90.3% (139 cases), 22.7% (35 cases), 72.1% (111 cases), and 17.5% (27 cases), respectively. Conclusion: The identified genes, such as CBX3 and CRABP2, showed distinctly increased expression in NSCLC tissues over matched normal lung tissues. So they may be promising candidate genes for diagnostic markers and therapeutic targets of NSCLC. Disclosure: All authors have declared no conflicts of interest. 30P DIFFERENTIAL MICRORNA EXPRESSION AND TARGET GENES BETWEEN LUNG CANCER AND NORMAL TISSUE H. Lee1 , S. Song1 , K. Lim1 , S. Han1 , W.J. Kim1 , Y. Oh2 , J.S. Lee2 1 Internal Medicine, Kangwon National University, Kangwon National University Hospital, Chuncheon, Korea, 2 Department of Pulmonary and Critical Care Medicine, and Clinical Research Center for Chronic Obstructive Airway Diseases, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea Rationale: MicroRNAs (miRNAs) are single-stranded RNA species that constitute a class of noncoding RNAs emerging as key regulators of gene expression at the post-transcriptional level. Aberrant miRNA expression has been implicated in the pathogenesis of several human diseases including malignancies. In the present study, we analyzed differential miRNA expression profiling between lung tissue and adjacent normal lung tissue to define pathogenesis of non-small cell lung cancer (NSCLC). Methods: Lung tissues were obtained, both lung cancer tissue and normal tissue, from 19 NSCLC patients. RNA isolated from these samples was processed with RNA Seq using HiSeq 2000. Gene expression measurements were calculated using Cufflinks software. Differentially expressed miRNAs and mRNAs were chosen using t-test. We identified putative target genes using miRBase Targets. We selected target pairs which showed negative correlation among significantly expressed miRNA and putative target mRNAs. Results: We identified 182miRNAs that were significantly different between lung tissues from the two groups (1.5-fold change and p < 0.05). We identified putative negative regulatory 47 miRNA-mRNA target pairs. Of these 47 miRNAs, 19 miRNAs were upregulated and 458 mRNAs were downregulated. And 28 miRNAs were downregulated and 457mRNAs were upregulated. Increased expression of miR-210, miR-429, and miR-615 3p and decreased expression of miR-874 were identified. MiR-210 showed negative correlation with RASAL2 (RAS protein activator like 2) and miR654 5p showed significant negative correlation with WISP2 (WNT1 inducible signaling pathway protein 2). Conclusions: There were significantly differentially expressed miRNAs and mRNAs between lung cancer and normal tissue. Further investigating miRNA and their target genes will be warranted to enhance our understanding of NSCLC. Disclosure: All authors have declared no conflicts of interest.
S13 31P ALK ASSESSMENT WITH FISH, IHC AND AQUA IN GREEK NSCLC PATIENTS V. Kotoula1 , P. Kosmidis1 , M. Bobos1 , M. Vassilakopoulou2 , E. Tsolaki1 , S. Chrysafi1 , A. Psyrri1 , G. Fountzilas1 1 Data Office, Hellenic Cooperative Oncology Group, Athens, Greece, 2 Department of Pathology, Yale University, School of Medicine, New Haven, CT, United States of America Recent recommendations for calling NSCLC ALK positive with the FDA approved FISH test (cut-off: 50% FISH-positive cells) yield a broad range of equivocal cases needing reevaluation (recommended final cut-off: 15%). Based on these FISH results NSCLC patients are selected to receive crizotinib. We retrospectively evaluated diagnostic tissue material from 128 NSCLC patients (age 38 78 years [median 62]; 71% current smokers; 46% adenocarcinomas [AC]) for ALK gene rearrangements with diagnostic FISH (informative n = 125); and, for ALK protein expression with immunohistochemistry (IHC, clone D5F3, n = 89) and with automated quantitative analysis (AQUA, clone D5F3, n = 83) (all methods informative, n = 75). With the 50% FISH cut-off, only 1 AC was ALK positive (66% positive cells) while 32 cases were equivocal. Reevaluation of the latter with the 15% cutoff yielded 4 additional ALK positive tumors (2 AC and 2 squamous cell carcinomas [SCC], with 25%, 43%, 16% and 16% positive cells, respectively). All 5 FISH ALK positive tumors were EGFR/KRAS/BRAF mutation negative. FISH positive AC (n = 3) demonstrated strong cytoplasmic staining in all (100%) neoplastic cells with ALK IHC, while the 2 SCC exhibited 15% weak nuclear staining. ALK IHC patterns of weak or moderate, cytoplasmic or nuclear staining in 10 40% of neoplastic cells were observed in additional 18 tumors (20%); 5 of these were equivocal with FISH, while ALK gene copy number was not associated with any IHC pattern. AQUA was positive in FISH/100%-IHC positive AC only (2 available tumors). All other tumors (n = 81), including those with various IHC patterns, equivocal FISH, and the FISH positive SCC were AQUA negative. Based on all above results, only the 3 AC were considered ALK positive (6% of AC; 2.4% of the NSCLC tested). Patients were women, age 68 78 years, 1 smoker. One patient (25% FISH-positive cells) received crizotinib, had a partial response, and remains progression-free after 5 months. In the present small series, our results highlight the need for further standardization of diagnostic ALK testing. If further validated, the 100%-strong-cytoplasmic IHC pattern may be sufficient and cost-effective for identifying NSCLC patients to be treated with crizotinib, since it seems to reflect functional ALK gene fusions. Disclosure: All authors have declared no conflicts of interest. 32P PIGMENT EPITHELIUM-DERIVED FACTOR ENHANCES TUMOR RESPONSE TO RADIATION BY NORMALIZING VASCULATURE IN A XENOGRAFTED MODEL OF LUNG CANCER Z. Xu1 , F. Peng2 , Z. Yu1 , Y. Zuo1 , Y. Chen3 , J. Wang3 , X. Hu3 , Q. Zhou2 , Y. Bao2 , M. Chen3 1 Department of Oncology, Affiliated Hospital of Guangdong Medical College, Zhanjiang, China, 2 Department of Radiation Oncology, Sun Yat-Sen University Cancer Center, State Key Laboratory of Oncology in South China, Guangzhou, China, 3 Department of Radiation Oncology, Zhejiang Cancer Hospital, Hangzhou, China Background: Pigment epithelium-derived factor (PEDF), a 50-kDa secreted glycoprotein, is a member of serpin superfamily encoded by gene SERPINF1 located on chromosome 17p13. Over the past decade, a host of studies have shown that PEDF could inhibit the angiogenesis and growth of several types of cancers, including lung cancer. However, the effects of PEDF on tumor vascular normalization remain unknown. The purpose of the present study is to explore the effects of PEDF on tumor vascular normalization and the therapeutical potential of PEDF in combination with radiation and the underlying mechanism on lung cancer.
S14 Methods: The LLC xenografts were treated by PEDF and the morphologic changes of tumor vasculature and hypoxic fraction were detected in mice bearing LLC xenografts. The combined effects of PEDF and radiation on LLC xenografts growth were investigrated and the potential mechanisms were explored. Results: We observed that blood vessels in PEDF-treated xenografts were less irregular, less tortuous, more uniform in caliber in contrast to those of the control group in Lewis lung cancer xenografts. Meanwhile, the thickness of basement membrane was remarkably reduced and pericyte coverage was significantly increased by PEDF, which is considered to be a key property of vascular normalization. We also found that tumor hypoxic fraction decreased on the 3rd-7th day after PEDF treatment as measured by pimonidazole staining, which indicated that intratumoral hypoxia was improved during the 3rd-7th day after PEDF treatment. These results together show that PEDF can create a vascular normalization window from the 3rd to the 7th day after treatment in Lewis lung cancer xenografts. Furthermore, a significant tumor growth delay effect was shown when radiaiton was delivered during the “normalization vasculature window” created by PEDF. We also found that PEDF might enhance the tumor growth delay effect of radiation by induction of cancer cell apoptosis. Conclusion: Our findings provide the basis for the scheduling of PEDF in combination with radiation to improve anti-tumor efficacy on lung cancer, which is of translational importance. In addition, these findings imply the potential role of PEDF as an adjuvant agent for lung cancer treatment. Disclosure: All authors have declared no conflicts of interest.
33P GENE EXPRESSION PROFILE OF CLASSICAL (CD14++CD16 ) MONOCYTES IN PATIENTS WITH NON-SMALL CELL LUNG CANCER IS ALTERED BY CHEMOTHERAPY F. McCarthy, C. Ghirelli, R. Zollinger, M. Phillips, J. Candido, R. Trehy, R. Roshani, J. Steele, T. Hagemann Centre for Cancer and Inflammation, Barts Cancer Institute, London, United Kingdom Human monocytes are important members of the innate immune system and play a vital role in immunity and inflammation. Monocytes are considered a heterogenous population with three populations identified depending on cell surface CD14 and CD16 expression: Classical (CD14++ CD16 ), intermediate (CD14+CD16 ) and non-classical (CD14 CD16++). These populations have varied and opposing functions and have been postulated to play both anti and pro-inflammatory roles in a variety of diseases including atherosclerosis and sarcoidosis. However, the prevalent monocyte populations in cancer have not as yet been identified. We aim to define the prevalent monocyte populations in non-small cell lung cancer prior to initiation of treatment as well as immediately post chemotherapy. We also aim to further characterise the monocyte populations using flow cytometry and Affymetrix technology. Methods: Blood was obtained from 24 newly diagnosed patients with advanced non-small cell lung cancer. Blood was taken immediately prior to and one hour post administration of their first cycle of Pemetrexed and Cisplatin chemotherapy. Monocyte subpopulations were sorted using flow cytometry. Gene expression profiling was performed using Affymetrix Human 133a 2.0 array. Results: The classical (CD14++CD16 ) monocyte population is significantly increased in non-small cell lung cancer patients by the administration of chemotherapy (p < 0.01). The intermediate (CD14+CD16+) and non-classical (CD14 CD16++) populations are unchanged. Analysis of the gene expression profile of the classical monocyte subset identified 70 up-regulated and 14 down-regulated genes in cancer patients post chemotherapy when compared to pretreatment samples (p < 0.05, fold change >2).
TRANSLATIONAL RESEARCH Conclusions: By identifying the prevalent monocyte subsets as well as their function in cancer, there is the potential to target detrimental effects and promote their beneficial functions. Monocytes subsets may be used as a possible therapeutic target. Fully distinguishing the changes induced in monocyte number and function by chemotherapy may allow us to target harmful monocyte subsets as an adjunct to standard treatment in order to yield an improved response to treatment. Disclosure: All authors have declared no conflicts of interest. 34P BRONCHIAL-PULMONARY CARCINOMA: PET SCAN SUVS AND MITOCHONDRIAL DNA COPY NUMBER CORRELATE WITH HISTOLOGICAL SUB-TYPING L. Carvalho1 , J.A. Dias1 , A. Alarc˜ ao1 , M.R. Silva1 , A.F. Ladeirinha1 , acio2 , M.J. Santos3 , M.J. D’Aguiar1 , T. Ferreira1 , C. Bonif´ M. Grazina2 1 Institute of Anatomical Pathology, Faculty of Medicine, University of Coimbra, Coimbra, Portugal, 2 CNC Center for Neuroscience and Cell Biology, University of Coimbra, Biochemistry, Faculty of Medicine, University of Coimbra, Coimbra, Portugal, 3 CNC Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal Introduction: Bronchial-pulmonary carcinoma continues to show poor survival as near 70% of cases are diagnosed in non-surgical stages. Epidermoid carcinomas comprising common histological criteria have better prognosis than the other types while adenocarcinomas still represent a number of heterogeneous subtypes, complicated further by polymorphic carcinomas that have obscure biology. PET SUV Max 18 F-fluordeoxyglucose (FDG) and tumoural mitochondrial DNA (mtDNA) copy number were used to validate their value as clinical laboratory parameters to correlate with histological and clinical diagnosis. Material and Methods: Tumor samples of surgical specimens of 8 epidermoid carcinomas, 5 TTF1+ adenocarcinomas, 4 pleomorphic carcinomas and 4 large cell neuroendocrine carcinomas combined with adenocarcinomas (2) and with epidermoid carcinomas (2) were submitted to DNA extraction by the Maxwell® 16 FFPE Tissue LEV DNA Purification Kit; mtDNA copy number was determined by real-time PCR assay and correlated with PET Max-18-FDG. Results: A significant difference (p = 0.006) was found between epidermoid carcinomas and TTF1+ adenocarcinomas 18-FDG uptake. Pleomorphic carcinomas and combined LCNEC showed 18-FDG uptake in between the former histological types; mtDNA copy number and SUVmax were correlated using linear regression, and statistically significant direct proportionality was found (p = 0.0059, r2 = 0.2485). Discussion: These results reinforce the role of tumoural classification for “personalized” treatment based in biological characteristics as SUVmax and mtDNA copy number correlated. These characteristics should be considered when biopsy tissue is the unique representation of advanced pulmonary carcinomas. The recent PET scans interpretation comment of G. Weiss and R. Korn (JTO Dec 2012) sustains the biological approach here presented. Disclosure: All authors have declared no conflicts of interest.
S13 31P ALK ASSESSMENT WITH FISH, IHC AND AQUA IN GREEK NSCLC PATIENTS V. Kotoula1 , P. Kosmidis1 , M. Bobos1 , M. Vassilakopoulou2 , E. Tsolaki1 , S. Chrysafi1 , A. Psyrri1 , G. Fountzilas1 1 Data Office, Hellenic Cooperative Oncology Group, Athens, Greece, 2 Department of Pathology, Yale University, School of Medicine, New Haven, CT, United States of America Recent recommendations for calling NSCLC ALK positive with the FDA approved FISH test (cut-off: 50% FISH-positive cells) yield a broad range of equivocal cases needing reevaluation (recommended final cut-off: 15%). Based on these FISH results NSCLC patients are selected to receive crizotinib. We retrospectively evaluated diagnostic tissue material from 128 NSCLC patients (age 38 78 years [median 62]; 71% current smokers; 46% adenocarcinomas [AC]) for ALK gene rearrangements with diagnostic FISH (informative n = 125); and, for ALK protein expression with immunohistochemistry (IHC, clone D5F3, n = 89) and with automated quantitative analysis (AQUA, clone D5F3, n = 83) (all methods informative, n = 75). With the 50% FISH cut-off, only 1 AC was ALK positive (66% positive cells) while 32 cases were equivocal. Reevaluation of the latter with the 15% cutoff yielded 4 additional ALK positive tumors (2 AC and 2 squamous cell carcinomas [SCC], with 25%, 43%, 16% and 16% positive cells, respectively). All 5 FISH ALK positive tumors were EGFR/KRAS/BRAF mutation negative. FISH positive AC (n = 3) demonstrated strong cytoplasmic staining in all (100%) neoplastic cells with ALK IHC, while the 2 SCC exhibited 15% weak nuclear staining. ALK IHC patterns of weak or moderate, cytoplasmic or nuclear staining in 10 40% of neoplastic cells were observed in additional 18 tumors (20%); 5 of these were equivocal with FISH, while ALK gene copy number was not associated with any IHC pattern. AQUA was positive in FISH/100%-IHC positive AC only (2 available tumors). All other tumors (n = 81), including those with various IHC patterns, equivocal FISH, and the FISH positive SCC were AQUA negative. Based on all above results, only the 3 AC were considered ALK positive (6% of AC; 2.4% of the NSCLC tested). Patients were women, age 68 78 years, 1 smoker. One patient (25% FISH-positive cells) received crizotinib, had a partial response, and remains progression-free after 5 months. In the present small series, our results highlight the need for further standardization of diagnostic ALK testing. If further validated, the 100%-strong-cytoplasmic IHC pattern may be sufficient and cost-effective for identifying NSCLC patients to be treated with crizotinib, since it seems to reflect functional ALK gene fusions. Disclosure: All authors have declared no conflicts of interest. 32P PIGMENT EPITHELIUM-DERIVED FACTOR ENHANCES TUMOR RESPONSE TO RADIATION BY NORMALIZING VASCULATURE IN A XENOGRAFTED MODEL OF LUNG CANCER Z. Xu1 , F. Peng2 , Z. Yu1 , Y. Zuo1 , Y. Chen3 , J. Wang3 , X. Hu3 , Q. Zhou2 , Y. Bao2 , M. Chen3 1 Department of Oncology, Affiliated Hospital of Guangdong Medical College, Zhanjiang, China, 2 Department of Radiation Oncology, Sun Yat-Sen University Cancer Center, State Key Laboratory of Oncology in South China, Guangzhou, China, 3 Department of Radiation Oncology, Zhejiang Cancer Hospital, Hangzhou, China Background: Pigment epithelium-derived factor (PEDF), a 50-kDa secreted glycoprotein, is a member of serpin superfamily encoded by gene SERPINF1 located on chromosome 17p13. Over the past decade, a host of studies have shown that PEDF could inhibit the angiogenesis and growth of several types of cancers, including lung cancer. However, the effects of PEDF on tumor vascular normalization remain unknown. The purpose of the present study is to explore the effects of PEDF on tumor vascular normalization and the therapeutical potential of PEDF in combination with radiation and the underlying mechanism on lung cancer.
S14 Methods: The LLC xenografts were treated by PEDF and the morphologic changes of tumor vasculature and hypoxic fraction were detected in mice bearing LLC xenografts. The combined effects of PEDF and radiation on LLC xenografts growth were investigrated and the potential mechanisms were explored. Results: We observed that blood vessels in PEDF-treated xenografts were less irregular, less tortuous, more uniform in caliber in contrast to those of the control group in Lewis lung cancer xenografts. Meanwhile, the thickness of basement membrane was remarkably reduced and pericyte coverage was significantly increased by PEDF, which is considered to be a key property of vascular normalization. We also found that tumor hypoxic fraction decreased on the 3rd-7th day after PEDF treatment as measured by pimonidazole staining, which indicated that intratumoral hypoxia was improved during the 3rd-7th day after PEDF treatment. These results together show that PEDF can create a vascular normalization window from the 3rd to the 7th day after treatment in Lewis lung cancer xenografts. Furthermore, a significant tumor growth delay effect was shown when radiaiton was delivered during the “normalization vasculature window” created by PEDF. We also found that PEDF might enhance the tumor growth delay effect of radiation by induction of cancer cell apoptosis. Conclusion: Our findings provide the basis for the scheduling of PEDF in combination with radiation to improve anti-tumor efficacy on lung cancer, which is of translational importance. In addition, these findings imply the potential role of PEDF as an adjuvant agent for lung cancer treatment. Disclosure: All authors have declared no conflicts of interest.
33P GENE EXPRESSION PROFILE OF CLASSICAL (CD14++CD16 ) MONOCYTES IN PATIENTS WITH NON-SMALL CELL LUNG CANCER IS ALTERED BY CHEMOTHERAPY F. McCarthy, C. Ghirelli, R. Zollinger, M. Phillips, J. Candido, R. Trehy, R. Roshani, J. Steele, T. Hagemann Centre for Cancer and Inflammation, Barts Cancer Institute, London, United Kingdom Human monocytes are important members of the innate immune system and play a vital role in immunity and inflammation. Monocytes are considered a heterogenous population with three populations identified depending on cell surface CD14 and CD16 expression: Classical (CD14++ CD16 ), intermediate (CD14+CD16 ) and non-classical (CD14 CD16++). These populations have varied and opposing functions and have been postulated to play both anti and pro-inflammatory roles in a variety of diseases including atherosclerosis and sarcoidosis. However, the prevalent monocyte populations in cancer have not as yet been identified. We aim to define the prevalent monocyte populations in non-small cell lung cancer prior to initiation of treatment as well as immediately post chemotherapy. We also aim to further characterise the monocyte populations using flow cytometry and Affymetrix technology. Methods: Blood was obtained from 24 newly diagnosed patients with advanced non-small cell lung cancer. Blood was taken immediately prior to and one hour post administration of their first cycle of Pemetrexed and Cisplatin chemotherapy. Monocyte subpopulations were sorted using flow cytometry. Gene expression profiling was performed using Affymetrix Human 133a 2.0 array. Results: The classical (CD14++CD16 ) monocyte population is significantly increased in non-small cell lung cancer patients by the administration of chemotherapy (p < 0.01). The intermediate (CD14+CD16+) and non-classical (CD14 CD16++) populations are unchanged. Analysis of the gene expression profile of the classical monocyte subset identified 70 up-regulated and 14 down-regulated genes in cancer patients post chemotherapy when compared to pretreatment samples (p < 0.05, fold change >2).
TRANSLATIONAL RESEARCH Conclusions: By identifying the prevalent monocyte subsets as well as their function in cancer, there is the potential to target detrimental effects and promote their beneficial functions. Monocytes subsets may be used as a possible therapeutic target. Fully distinguishing the changes induced in monocyte number and function by chemotherapy may allow us to target harmful monocyte subsets as an adjunct to standard treatment in order to yield an improved response to treatment. Disclosure: All authors have declared no conflicts of interest. 34P BRONCHIAL-PULMONARY CARCINOMA: PET SCAN SUVS AND MITOCHONDRIAL DNA COPY NUMBER CORRELATE WITH HISTOLOGICAL SUB-TYPING L. Carvalho1 , J.A. Dias1 , A. Alarc˜ ao1 , M.R. Silva1 , A.F. Ladeirinha1 , acio2 , M.J. Santos3 , M.J. D’Aguiar1 , T. Ferreira1 , C. Bonif´ M. Grazina2 1 Institute of Anatomical Pathology, Faculty of Medicine, University of Coimbra, Coimbra, Portugal, 2 CNC Center for Neuroscience and Cell Biology, University of Coimbra, Biochemistry, Faculty of Medicine, University of Coimbra, Coimbra, Portugal, 3 CNC Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal Introduction: Bronchial-pulmonary carcinoma continues to show poor survival as near 70% of cases are diagnosed in non-surgical stages. Epidermoid carcinomas comprising common histological criteria have better prognosis than the other types while adenocarcinomas still represent a number of heterogeneous subtypes, complicated further by polymorphic carcinomas that have obscure biology. PET SUV Max 18 F-fluordeoxyglucose (FDG) and tumoural mitochondrial DNA (mtDNA) copy number were used to validate their value as clinical laboratory parameters to correlate with histological and clinical diagnosis. Material and Methods: Tumor samples of surgical specimens of 8 epidermoid carcinomas, 5 TTF1+ adenocarcinomas, 4 pleomorphic carcinomas and 4 large cell neuroendocrine carcinomas combined with adenocarcinomas (2) and with epidermoid carcinomas (2) were submitted to DNA extraction by the Maxwell® 16 FFPE Tissue LEV DNA Purification Kit; mtDNA copy number was determined by real-time PCR assay and correlated with PET Max-18-FDG. Results: A significant difference (p = 0.006) was found between epidermoid carcinomas and TTF1+ adenocarcinomas 18-FDG uptake. Pleomorphic carcinomas and combined LCNEC showed 18-FDG uptake in between the former histological types; mtDNA copy number and SUVmax were correlated using linear regression, and statistically significant direct proportionality was found (p = 0.0059, r2 = 0.2485). Discussion: These results reinforce the role of tumoural classification for “personalized” treatment based in biological characteristics as SUVmax and mtDNA copy number correlated. These characteristics should be considered when biopsy tissue is the unique representation of advanced pulmonary carcinomas. The recent PET scans interpretation comment of G. Weiss and R. Korn (JTO Dec 2012) sustains the biological approach here presented. Disclosure: All authors have declared no conflicts of interest.