Fikri Icli

The combination of cytotoxic treatment modalities including oncolytic viral gene therapies and immunotherapy usually yields a synergistic effect. In the current study, we constructed a bicistronic adenoviral vector, Ad-CD-GMCSF, carrying the cytosine deaminase (CD) and granulocyte macrophage-colony stimulating factor (GM-CSF) transcription units driven by a CMV promoter and tested the in vitro efficacy of the vector in tumor cell lines and a syngeneic mouse model of colon cancer. The tumor cells infected with Ad-CD-GMCSF vector were found to produce a substantial amount of GM-CSF tumor cell lines. Accordingly, the vector carrying CD and GM-CSF transcription units together induced a potent anti-tumor immunity with a significantly increased number of tumor-specific T cells, and tumor-specific T cell cytotoxicity (p<0.001). The tumor growth rate of Ad-CD-GMCSF treated mice was significantly lower when compared to the control and an adenoviral vector carrying only CD transcription unit (Ad-CD) (P<0.05). Likewise, the median overall survival of the Ad-CD-GMCSF vector group was significantly higher than that of the control and Ad-CD groups (34.0±12.8 days vs. 14.0±0.5 and 23.0±2.8, respectively, P<0.001). In conclusion, along with its cytotoxic effect, the high immunostimulatory effect of the bicistronic Ad-CD-GMCSF has excellent potential in the treatment of cancers.

Aim: There are inconsistent results about the effects of vitamin D level on breast cancer prognosis. We aimed to investigate the effect of vitamin D levels on the prognosis of resectable breast cancer in a patient group with highly different clothing styles. Patients & methods: A total of 186 breast cancer patients were enrolled in the study. Results: Vitamin D level was sufficient, insufficient and deficient in 17.2, 52.2 and 30.6% of patients, respectively. There was a significant relationship between clothing style and serum 25 (OH) D levels. We could not establish any relation between vitamin D level and tumor characteristics or survival. Conclusion: Vitamin D supplementation can be more important than diagnostic serum vitamin D level on prognosis of breast cancer.

E23013 Background: The efficacy of the metronomic use of conventional chemotherapeutic drugs in the clinic has been linked to their anti-angiogenic activity. However, the dose level for the metronomic use of the drugs with regard to anti-angiogenic activity is not known. In the current study, we aimed to study the effects of very low doses of 5-florouracil (5-FU), irinotecan and oxaliplatin on the synthesis of pro-angiogenic factors in tumor cells. Methods: We first determined the IC50 doses of 5-FU, irinotecan, and oxaliplatin in human tumor cell lines (HT-29: colon cancer cells, MCF-7: breast cancer cells and LNCaP: prostatic carcinoma cells). Then we applied the drugs to the assigned cells at the dose levels of IC50, -1log, -2log and -3log of IC50. The effects of the drugs on both gene expression and protein secretion levels of pro-angiogenic genes including vascular endothelial growth factor A (VEGF-A), basic-fibroblast growth factor (bFGF), matrix metalloproteinase-9 (MMP-9), platelet-derived growth factor-beta (PDGF-B), interleukin-8 (IL-8) and factor secretion by real-time PCR and sandwich ELISA. Results: The IC50 levels of 5-FU, irinotecan, and oxaliplatin in tumor cells were found as shown in the Table. We found that -1logIC50 dose level of 5-FU efficiently suppressed the pro-angiogenic genes both at the gene expression and protein secretion levels. However, the -2logIC50 and -3logIC50 dose levels of both irinotecan and oxaliplatin significantly inhibited gene expression and protein secretion. Conclusions: In conclusion, our results suggest that irinotecan and oxaliplatin could be used as anti-angiogenic agents at the very low doses. The -1logIC50 dose for 5-FU and the -2log and -3logIC50 doses of irinotecan and oxaliplatin could be further tested in animal models and clinical trials. Table. The IC50 dose levels of the drugs in tumor cell lines. [Table: see text]

E23014 Background: Angiogenesis is one of the key processes leading tumor progression and metastasis. Conventional chemotherapeutic drugs are usually cytotoxic to tumor cells at the therapeutic doses. The lowered doses of those drugs have been shown to induce apoptosis in endothelial cells and implemented as metronomic schedule in the clinic. We aimed to study the effects of very low doses of chemotherapeutic drugs on endothelial cell functions and gene and protein expression of pro-and anti-angiogenic factors. Methods: We first determined the IC50 doses of 5-fluorouracil, irinotecan, and oxaliplatin in a human umbilical endothelial cell line (HUVEC). Then we’ve tested the effects of the IC50, -1log, -2log and -3log of IC50 doses on endothelial cell migration and tube formation. We’ve also studied the effects of the assigned doses on expression levels of the genes VEGF-A, VEGFR2, PDGF-B, angiopoietin-2 (Ang-2), endothelin-1 (End-1), thrombospondin-1 (Tbs-1), iNOS, eNOS by real-time PCR, protein secretion levels of VEGF-A, PDGF-BB, Ang-2, End-1 and Tbs-1 by ELISA and nitrate/nitrite production by a colorimetric assay. Results: The IC50 dose levels of 5-FU, irinotecan, and oxaliplatin were 9,2 μM, 9,8 μM and 11,8 μM, respectively. We found that all the -1log, -2log and -3log of IC50 doses of the drugs tested in the study significantly decreased the migration capacity of the HUVEC cells. However, no significant changes were observed in tube formation of endothelial cells. Likewise, the expression levels of pro-angiogenic genes including Ang-2, iNOS, eNOS, PDGF-B and VEGF-A were significantly decreased at the same doses of the drugs. Accordingly, the levels of Tbs-1 were significantly increased at all 3 dose levels of the drugs. The most efficacious doses of the drugs to decrease NO levels in HUVEC cells were -1logIC50 for 5-FU, -2logIC50 for irinotecan and -3logIC50 for oxaliplatin. Conclusions: In conclusion, our results suggest that 5-FU, irinotecan, and oxaliplatin could decrease the pro-angiogenic and increase anti-angiogenic factor production in endothelial cells at the doses much lower than the usual metronomic schedules and warrant further research in animal models and clinical trials as an anti-angiogenic treatment modality.

E15103 Background: The tumor-associated macrophage (TAM) is one of the major components of tumor microenvironment. Neuroendocrine differentiation (NED) of the tumor has been linked to poor prognosis. We aimed to investigate the role of TAM infiltration and NED in tumor tissue on the efficacy of bevacizumab plus chemotherapy in patients with advanced colorectal cancer. Methods: A total of 123 consecutive patients with advanced colorectal cancer treated with bevacizumab plus either irinotecan or oxaliplatin-based combination chemotherapy were included in the study. Along with clinicopathological parameters including age, tumor location (right versus left colon), pretreatment CEA level, KRAS mutation status, tumor grade, histologic subtype (mucinous vs non-mucinous), the presence of NED and the level of TAM infiltration were studied as covariates for survival analysis. The patients with at least 2% or more staining with either synaptophysin or chromogranin A were regarded as NED positive. The +2 or less staining with anti-CD68 was regarded as low for TAM infiltration and +3 or +4 staining as high. The overall (OS) and progression-free survival (PFS) times were calculated from the start of bevacizumab. Results: Thirty-five percent of the patients were given bevacizumab in the first line setting, the others mainly at the second line. The chemo backbone was FOLFIRI in 75% of the patients. The median OS was 15,1 and PFS 7,1 months. The ratio of NED negative and TAM infiltration low patients were 73,9% and 13,8 %, respectively. The univariate analysis yielded only the NED and higher TAM infiltration as the significant factors for PFS. However, only NED was significant for PFS in the multivariate analysis (HR:0.542). The left-sided tumors and the low levels of TAM infiltration were the favorable factors for OS in the univariate analysis. However, the TAM level was the only independent factor predicting the OS (26,7±8,8 vs 14,1±1,7 months, respectively; HR: 0,301). Conclusions: Our results suggest that the TAM infiltration in the tumor tissue could be used as a predictive marker of efficacy of bevacizumab plus combination chemotherapy in patients with advanced colorectal cancer.

Backgrounds: The serum leptin level (SLL) has been shown to increase in patients with nonsmall cell lung cancer (NSCLC). However, available data regarding the relation between SLL and tumor subtypes, survival, cachexia, and tumor respectability in NSCLC are still under debate. The aim of this study is to evaluate SLL in NSCLC patients with and without cachexia. Materials and Methods: A total of 71 patients with early stage NSCLC were enrolled in this prospective study. SLL was measured by enzyme-linked immunosorbent assay. The relationship between SLL and clinicopathological factors including histopathological subtypes, weight loss, overall survival, and tumor resectability were evaluated. Results: Of the 71 patients, 57 (81%) were male with a mean age of 63.3 ± 8.2 years. The rates of histological subtypes of NSCLC were as follows: Squamous cell carcinoma 60.5%, adenocarcinoma 32%, and others 7.2%. Mean SLL was 12.9 ± 38.4 pmol/mL. There was no distinctive difference between SLL, weight loss, and survival. However, when stratifying the groups according to the lung cancer histological subtypes, mean SLL was significantly higher in patients with adenocarcinoma than those with squamous cell subtype (26.9 ± 6.2 pmol/mL vs. 5.1 ± 9.1 pmol/mL, P = 0.004). Conclusions: SLL might be beneficial as a useful biomarker in preclinical setting of NSCLC to guide detecting the lung cancer subtypes as well as monitoring the patients.

Background/aim: The role of angiogenic factors in gastric cancer is not clear. We aimed to assess the role of vascular endothelial growth factor A (VEGFA), angiopoietin 1 (Ang-1), and placental growth factor (PlGF) in the prognosis of patients with advanced gastric cancer. Materials and methods: Thirty consecutive patients treated with a modified DCF (docetaxel, cisplatin, and fluorouracil) regimen were included in the study. The plasma VEGFA, Ang-1, and PlGF levels of the patients before treatment and following two cycles of chemotherapy were measured and evaluated as prognostic factors. Results: Poor performance status and lower Ang-1 levels were correlated with poor overall survival (OS). No significant correlation between VEGFA or PlGF and OS was found. An angiogenesis prognostic index (API) based on the levels of VEGFA, Ang-1, and PlGF was found to be highly correlated with OS. Performance status and API were found as independent prognostic factors for OS. Furthermore, a decrease in VEGFA by 25% from the pretreatment level was also found as a prognostic factor for OS independent of response to DCF regimen. Conclusion: Our results support the use of the new API including VEGFA, Ang-1, and PlGF levels in patients with advanced gastric cancer as a predictor of survival.

E14573 Background: Insufficient targeting of the therapeutic genes to tumor cells is one of the major reasons of failure in cancer gene therapy. Mesenchymal stem cells (MSC) can easily home to the tumor sites when administered intravenously because of the chronic inflammatory process induced by the tumor cells. Methods: In the current study, we constructed baculoviral vectors (BVs) carrying cytosine deaminase (CD) (BV-CD) or green fluorescence protein genes and tested the transduction efficiency of the vectors and in vivo efficacy of the bone marrow derived MSC (BM-MSC) loaded with BV-CD vector in a colon cancer model in BALB/c mice. Three groups of mice with 6 mice in each having approximately 50 mm³ of tumor nodules established by subcutaneous injection of CRL2638 colon cancer cells were used. The control group was given iv PBS, the second group iv injection of 1×10⁵ BM-MSCs and the third group iv injection of 1×10⁵ BM-MSCs loaded with 100 MOI of BV-CD. All the mice were given ip 500 mg/kg 5-FC in 0.5 ml of volume for 10 days. Results: While the transduction efficiency of the recombinant baculoviral vectors was almost 100% in Sf-9 insect cells at the dose of 100 MOI, it was approximately 70% in mouse tumor cells and BM-MSCs of the BALB/c mice. The BV-CD treatment caused a significant in vitro cytotoxicity when used with 5-fluorocytosine in various tumor cells. No toxicity was observed when the vector used alone in mammalian cells. The recombinant vector loaded BM-MSCs caused a significant delay in tumor growth (p < 0.01) and increased the median survival time of the tumor bearing mice approximately 40% when compared to either controls or unloaded MSCs (p < 0.05). Conclusions: Our results show that the recombinant BV-CD vector could be used either alone or loaded into BM-MSCs, in the treatment of colorectal cancer.