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Epidermal growth factor receptor (EGFR) mutation testing is essential for individualized treatment using tyrosine kinase inhibitors. We evaluated two EGFR mutation tests, cobas v2 and PANAMutyper, for detection of EGFR activating mutations Ex19del, L858R, and T790M in tumor tissue and plasma from 244 non-small cell lung cancer (NSCLC) patients. The Kappa coefficient (95% CI) between the tests was 0.82 (0.74−0.92) in tumor samples (suggesting almost perfect agreement) and 0.69 (0.54−0.84) in plasma (suggesting substantial agreement). In plasma samples, both tests showed low to moderate sensitivity depending on disease stage but high diagnostic precision (86%−100%) in all disease stages (sensitivity: percentage of mutations in tumors that are also detected in plasma; precision: percentage of mutations in plasma which are also detected in tumors). Among the 244 patients, those previously diagnosed as T790M carriers who received osimertinib treatment showed dramatically better clinical outcomes than T790M carriers without osimertinib treatment. Taken together, our study supports interchangeable use of cobas v2 and PANAMutyper in tumor and plasma EGFR testing. Both tests have high diagnostic precision in plasma but are particularly valuable in late-stage disease. Our clinical data in T790M carriers strongly support the clinical benefits of osimertinib treatment guided by both EGFR mutation tests.

Simple Summary In this study, Sel-CapTM, a next-generation sequencing (NGS)-based genotyping platform, showed high sensitivity for detection of epidermal growth factor receptor (EGFR) gene mutations in plasma samples collected from 185 patients with non-small cell lung cancer (NSCLC). In the early-stage NSCLC, Sel-Cap liquid biopsy was able to detect more than half the EGFR mutations, which were detected in tumor tissue (sensitivity: 50% and 78% for Ex19del and L858R respectively, with tumor results as the references), while the conventional NGS could not detect any. Sel-Cap liquid biopsy was particularly sensitive for resistant mutation T790M (sensitivity: 88%). In addition, we conducted a retrospective study to monitor T790M using Sel-Cap in 34 patients who progressed on first-line tyrosine kinase inhibitors (EGFR-TKIs). The study suggested that the first appearance of T790M in plasma, ranging from at treatment baseline to over three years post-EGFR-TKI initiation, may be useful for prediction of disease progression (around 5 months in advance). Abstract Sel-CapTM, a digital enrichment next-generation sequencing (NGS)-based cancer panel, was assessed for detection of epidermal growth factor receptor (EGFR) gene mutations in plasma for non-small cell lung cancer (NSCLC), and for application in monitoring EGFR resistance mutation T790M in plasma following first-line EGFR-tyrosine kinase inhibitor (EGFR-TKI) treatment. Using Sel-Cap, we genotyped plasma samples collected from 185 patients for mutations Ex19del, L858R, and T790M, and compared results to those of PNAclampTM tumor biopsy (reference method, a peptide nucleic acid-mediated polymerase chain reaction clamping) and two other NGS liquid biopsies. Over two-thirds of activating mutations (Ex19del and L858R), previously confirmed by PNAclamp, were detected by Sel-Cap, which is 4–5 times more sensitive than NGS liquid biopsy. Sel-Cap showed particularly high sensitivity for T790M (88%) and for early-stage plasma samples. The relationship between initial T790M detection in plasma and progression-free survival (PFS) following first-line EGFR-TKIs was evaluated in 34 patients. Patients with T790M detected at treatment initiation (±3 months) had significantly shorter PFS than patients where T790M was first detected >3 months post treatment initiation (median PFS: 5.9 vs. 26.5 months; p < 0.0001). However, time from T790M detection to disease progression was not significantly different between the two groups (median around 5 months). In conclusion, Sel-Cap is a highly sensitive platform for EGFR mutations in plasma, and the timing of the first appearance of T790M in plasma, determined via highly sensitive liquid biopsies, may be useful for prediction of disease progression of NSCLC, around 5 months in advance.

To better understand the immune profile within the tumor microenvironment (TME) and the potential immune-evasion capacity that may arise from administration of immune checkpoint inhibitors, we investigated immunological phenotypes and their correlations with genomic alterations in order to provide predictive/prognostic implications in lung adenocarcinoma (LAC).Surgical sections with corresponding peripheral blood mononuclear cells were collected from 76 patients with pathologically confirmed Ia-IIIa. The histological contents expressing immune checkpoint molecules in the tumor nest and stromal area were imaged and quantified using a multi-spectral imaging system. Distribution of tumor mutation burden (TMB) and non-synonymous somatic mutations were analyzed in whole exome sequencing (WES) data.Total 76 patients consisted of EGFR-mutant (n=37) and EGFR-wild type (n=39) tumors. As is well known, the number of tumor-infiltrating cytotoxic T cells (174.7 ± 24.4 cells/mm2) and TMB (3.8 ± 0.4) in EGFR-mutant tumors was significantly lower than those of EGFR wild type tumors (394.2 ± 82.3 cells/mm2 and 6.7 ± 1.2) (p=0.0146 and p=0.0254, respectively), elucidating EGFR mutation-mediated immune evasion. The subjects carrying EGFR wild-type were classified into two subgroups by the density of tumor-infiltrating cytotoxic T cells, defined as the high TIL (n=20) and low TIL (n=19). There was great difference of mean density of cytotoxic T cells between low TIL group (129.6 ± 16.8 cells/mm2) and high TIL group (672.8±143.5 cells/mm2). Tumor-infiltrating T cells were granzyme B-positive, suggesting most cells were tumor-specific. Interestingly, no significant difference in TMB was found between the two subgroups (7.5 ± 1.9 in low TIL group vs. 5.8±1.3 in high TIL group; p=0.4642). Tumor proportion score (TPS) of PD-L1 was remarkably higher in high TIL group than low TIL group (29.7 ± 6.5% vs. 13.9 ± 2.8%, p=0.0008, respectively), but no significant differences of tumoral CD73 and IDO between the two groups (p=0.643 and p=0.923, respectively). Even there were no significant differences in counts of tumor-infiltrating macrophages in all types of macrophage, significantly higher counts of PD-L1+ M1-type macrophages (CD68+ CD163low) in the stromal area were discovered in the high TIL group compared to those of the low TIL group (396.2 ± 93.6 vs. 139.8 ± 30.1, p=0.0114, respectively), indicating that most of macrophages resided in the stromal area. Interestingly, the low TIL group exhibited higher counts of CD73+ M2-type macrophages (CD68+ CD163high) in the stromal area than the high TIL group (644.5 ± 110.9 vs. 387.9 ± 73.2, p=0.0639, respectively). Cell count ratio (M1/M2) of macrophages was significantly higher in the high TIL group (p=0.0042), suggesting that the TME with a smaller number of TILs tends to contain more M2-type macrophages. The tumor recurrence was more frequent in the low TIL group compared to the high TIL group with no statistical significance (p=0.127). In surgically resected lung adenocarcinoma patients, the subgroup with a low density of tumor-infiltrating cytotoxic T cells had a large number of CD73+ M2-type macrophages, which may contribute to poor prognosis. Acknowledgement: This study was sponsored by Ono Pharma Co., Ltd Citation Format: Jae-Hwan Kim, Tae-Min Kim, Sung-Gu Her, Kyoung-Ho Pyo, Jeong- Oh Kim, Beung-Chul Ahn, Byoung Chul Cho, Jin Hyoung Kang. Comprehensive analysis of immune phenotypes identifies CD73+ CD163high macrophages as a prognostic biomarker in lung adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4981.

The KRAS gain-of-function mutation confers intrinsic resistance to targeted anti-cancer drugs and cytotoxic chemotherapeutic agents, ultimately leading to treatment failure. KRAS mutation frequency in lung adenocarcinoma is ~15-30%. Novel therapeutic strategies should be developed to improve clinical outcomes in these cases. Deregulation of the p16/cyclin-dependent kinase (CDK) 4/retinoblastoma (Rb) pathway is frequently observed in various cancers and it represents an attractive therapeutic target. We compared the anti-tumor efficacy of genetically knocked-down CDK4 and a pharmacological inhibitor of CDK4/6, CINK4, in KRAS mutation-positive lung adenocarcinoma cells. We also investigated changes in anti-proliferative activity and downstream molecules with these treatments in combination with paclitaxel. CDK4 short interfering RNA (siRNA) significantly increased paclitaxel sensitivity in KRAS mutation-positive H23 cells. CINK4 demonstrated concentration- and time-dependent anti-proliferative activity in 5 adenocarcinoma lines. CINK4 induced G 1 arrest by downregulating the p16/cyclin D1/Rb pathway, resulting in apoptotic induction via increased expression of cleaved caspase3, cleaved PARP and Bax. Combined CINK4 and paclitaxel produced synergistic anti-proliferative activity and increased apoptosis through reduced cyclin D1 and Bcl-2 in KRAS mutation-positive cancer cells. These data suggest CDK4 is a promising target for development of anti-cancer drugs and CINK4 combined with paclitaxel may be an effective therapeutic strategy for enhancing anti-tumor efficacy in KRAS mutation-positive lung adenocarcinoma.

Patients with HPV-positive oropharyngeal cancer show better tumor response to radiation or chemotherapy than patients with HPV-negative cancer. HPV oncoprotein E6 binds and degrades a typically wild-type p53 protein product. However, HPV16 infection and p53 mutation infrequently coexist in a subset of HNSCCs. The purpose of this study was to investigate the mechanisms through which tumor biology and molecular genetic mechanisms change when two HPV-negative, p53-mutated oropharyngeal cell lines (YD8, non-disruptive p53 mutation; YD10B, disruptive p53 mutation) derived from patients with a history of heavy smoking are transfected with HPV E6 and E7 oncogenes in vitro. Transfection with HPV E6 and E7 oncogenes in YD8, reduced the abundance of proteins encoded by tumor suppressor genes, such as p-p53 and p-Rb. Cell proliferative activity was increased in the cells transfected with E6E7 compared to cells transfected with vector alone (P=0.09), whereas the invasiveness of E6E7-transfected cells was significantly reduced (P=0.02). cDNA microarray of the transfected cells with E6E7 showed significant changes in mRNA expression in several signaling pathways, including focal adhesion, JAK-STAT signaling pathway, cell cycle and p53 signaling pathway. Regarding the qPCR array for the p53 signaling pathway, the mRNA expression of STAT1 was remarkably upregulated by 6.47-fold (P

Background: Mechanisms of acquired resistance for EGFR tyrosine kinase inhibitor (TKI) in non-small cell lung cancer (NSCLC) has been studied broadly, and T790M mutation, bypass signaling, and phenotype change have been suggested. Questions remain in terms of the selection of sub-clones according to the sites during EGFR TKI therapy, especially in brain due to blood-brain barrier. Three metastatic NSCLC patients harboring sensitive EGFR mutation were treated with EGFT TKI with good response but became refractory and symptomatic brain oligo-metastasis occurred. Metastectomy was done and next-generation sequencing (NGS) was done with the brain tissue and blood. Methods: Fresh brain metastasis tissue was obtained during surgery and frozen in liquid nitrogen. Blood sampling was done within a week of the surgery and plasma was used for NGS. Amplicon sequencing (Thunderbolt) was performed. Library preparation and quality control sequencing amplicon libraries were prepared using ThunderBolts Cancer Panel, according to the manufacturer's instructions. The ThunderBolts Cancer Panel uses 230 amplicons to interrogate mutations and hotspots in 50 oncogenes, tumor suppressors, and drug resistance markers. Results: All 3 brain tissues had the same sensitive EGFR mutation (exon 19 deletion) as initial presentation, with PDGFRA, APC and TP53 mutations, while in the plasma, PDGFRA and APC mutations were identified in the 3 patients. From case 1, PDGFRA, APC, MLH1, and EGFR mutations were identified in the plasma, and additional PIK3CA, KDR, TP53, ERBB2, and GNA11 mutations were found in the brain. From case 2, PDGFRA, APC, EGFR, MET, PTEN, HRAS, and TP53 mutations were found in the plasma, and in the brain, PIK3CA, KDR, and ERBB2 mutations were identified in addition. From case 3, PDGFRA, APC, HRAS, and TP53 mutations in the plasma, and additional EGFR mutation in the brain. Conclusions: Our data identified several additional mutations in brain metastasis compared to the plasma from patients with acquired EGFR TKI resistance, other than T790M mutation. Every mutation in the blood sample was identified in the brain tissue, while additional mutations were found in the brain tissue. Citation Format: Hee Yeon Lee, Jeong-Oh Kim, Jin-Hyoung Kang. Comparison of next-generation sequencing results between brain metastasis and blood in NSCLC patients with acquired EGFR-TKI resistance other than T790M mutation [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A131.

Pemetrexed has demonstrated clinical activity in non-small cell lung cancer (NSCLC) as well as other solid tumors. It transports into the cells via reduced folate carrier (RFC) and is polyglutamated by folypolyglutamate synthetase (FPGS). Pemetrexed directly inhibits several folate-dependent enzymes such as thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT). We investigated the effects of genetic variations and the expression of RFC, FPGS, TS and DHFR enzymes on drug sensitivity to pemetrexed in NSCLC cells. Polymorphisms in RFC, FPGS, and DHFR were genotyped in four NSCLC cells - A549, PC14, HCC-1588, and H226. Real-time RT-PCR and Western blot was performed to evaluate mRNA transcripts and protein of these genes. The cytotoxicity of pemetrexed was measured by SRB assay. In PC14 and H226 cells, increased mRNA expressions of RFC and FPGS were associated with higher cytotoxicity to pemetrexed. 2R/2R genotype of TS and its increased mRNA expression were associated with drug resistance to pemetrexed in A549 cells, whereas 3R/3R genotype in TS with decreased mRNA expression was associated with higher sensitivity in H226 cells. After pemetrexed treatment, an inverse change of DHFR mRNA and protein expression was found. The strongest linkage disequilibrium (LD) was discovered between-1726C＞T and -1188A＞C SNP of DHFR gene. Our findings suggest the cytotoxic effect of pemetrexed may be associated with genetic polymorphisms and the expression level of genes involved in pemetrexed metabolisms in NSCLC cells.

Background: The KIF5B-RET rearrangement is detected with the frequency of 1 similar to 2 % in 'triple marker'-negative lung adenocarcinomas, i.e., EGFR, KRAS and EML4-ALK wild type. These mutational changes are known to be mutually exclusive, but the co-existence of ALK rearrangement with activating mutations of EGFR is rarely found. Methods: We examined the KIF5B-RET fusion gene in frozen tissues from 154 surgically resected lung tumors using RT-PCR with direct sequencing and the mutation status of EGFR and KRAS genes using PNA clamping. We tested KIF5B-RET translocation in Formalin Fixed Paraffin Embedded using fluorescence in situ hybridization. We also measured RET mRNA and protein expression by RT-PCR and immunohistochemistry, respectively. Results: The existence of KIF5B-RET fusion gene was identified in 9 patients. The mean age was 67.2 and M: F ratio 4: 5. Of 9 patients, 3 patients harbored wild type of EGFR and KRAS gene. However, KIF5B-RET fusion gene coincided with EGFR or KRAS mutation in 6 patients. These six pts were also positive for both RET break-apart probes (23.9 %) and KIF5B-RET fusion (44.4 %). However, there were no correlations between RET mRNA and protein expression in the KIF5B-RET-positive patients. The median disease free survival and overall survival were 23.9 months and 29.5 months, respectively. Conclusions: Taken together, our data suggest one-step screening platform for KIF5B-RET as well as EGFR, K-RAS, ALK oncogenic mutations be necessary for lung adenocarcinoma patients because EGFR or KRAS mutation are not infrequently found in KIF5B-RET-positive patients.

Purpose Matrix metalloproteinase (MMP) and tissue inhibitor of matrix metalloproteinase (TIMP) play an important role in tumor invasion and progression. The aim of this study was to evaluate the prognostic role of MMP-1 and TIMP-1 in non-small cell lung cancer (NSCLC). To find out a potential serum biomarker, tissue and serum levels were investigated together. Patients and methods For 85 surgically resected NSCLC patients who had pre-operative serum samplings, MMP-1 and TIMP-1 expression were investigated using immunohistochemistry in tumor tissue and ELISA in serum. Tumor cells and surrounding stromal cells were assessed separately. Results Higher expression of MMP-1 in tumor cells comparing to stromal cells was related to male gender (P = 0.006), ever smoker (P = 0.004), and pooly differentiated tumor (P = 0.043). For TIMP-1, adenocarcinoma showed higher tumor cell expression, while squamous cell carcinoma showed higher stromal expression (P = 0.007). Patients with high carcinoembryonic antigen (CEA) level, the presence of vascular invasion, recurrence or death showed higher serum MMP-1 level. There was no correlation between the tissue and serum levels of MMP-1 and TIMP-1. A tumor/stroma TIMP-1 intensity ratio ≥1 was strongly associated with early recurrence in multivariate analysis (hazard ratio = 280.55, 95% confidence intervals; 11.12–7080.45; P = 0.001). High serum MMP-1 (≥3,500 pg/ml) showed a trend for short overall survival (P = 0.080). When serum MMP-1 was combined with CEA level or presence of vascular invasion, its prognostic implication was statistically significant (P = 0.045 and P = 0.015, respectively). Conclusion The tumor/stroma TIMP-1 intensity ratio in tissue is useful to predict tumor recurrence. Serum MMP-1 level showed a possibility as a prognostic biomarker.

Background & Purpose In recent, KIF5B-RET gene rearrangement has been discovered as a driver oncogene in non-small cell lung cancers. This fusion gene was demonstrated to be able to induce tumorigenesis in vivo mouse model. Hereby, we investigated how KIF5B-RET fusion gene affect tumor differentiation and which transcription factors and signaling pathway are activated. Methods We analyzed cell proliferative activity in transformed HEK293T cells with KIF5B-RET fusion gene (K4) and empty vector (V5) using CCK8 assay. We used light microscopy and H&E staining to observe morphological changes of transformed cells. We evaluated protein and mRNA expressions of four EMT markers (E-cadherin, N-cadherin, Snail, and Twist) and two transcription factors (FOXA2, p-STAT5A) using transcription factor PCR array, Western blot, RT-PCR, immunofluorescence staining. Results K4 cells showed decreased cell proliferative activity compared with V5 cells. V5 cells had spreading pattern alike mesenchymal phenotype when their density was lower. However, their morphology changed to epithelial phenotype as they were overgrown with higher density. On the other hands, K4 cells exhibited the island growing pattern similar to epithelial phenotype in lower density of tumor cells, but changed to mesenchymal phenotype as the density of tumor cells gradually increased. On H&E staining, the cytoplasm of K4 cells was much smaller than that of V5 cells. We observed that E-cadherin mRNA was down-regulated, but N-cadherin and Twist were up-regulated in K4 cells. Based on the results of repeated screening assay, we finally selected two transcription factors, FOXA2 and STAT5A, which were significantly overexpressed in K4 cells. In K4 cells, phosphorylated-STAT5A was increased and FOXA2 was decreased compared to V5 cells. In addition, the expression of E-cadherin was reduced, but p-GSK3β, p-AKT, and p-ERK were markedly increased in K4 cells. The pattern of immunofluorescence staining of V5 and K4 cells were similar to the protein expressions of E-cadherin and p-STAT5A in Western blot analysis. Conclusion Taken together, FOXA2 and E-cadherin are down-regulated and p-STAT5A is up-regulated in HEK-293T cells stably expressing the KIF5B-RET. Our data suggest that KIF5B-RET fusion gene may induce EMT via the regulation of two transcription factors, FOXA2 and STAT5A. Citation Format: Min-Young Kim, Jung-Young Shin, Jeong-Oh Kim, Kyoung-Hwa Son, Jin Hyoung Kang. KIF5B-RET fusion gene may induce EMT via the regulation of two transcription factors, FOXA2 and STAT5A. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1687.

e20014Background: KIF5B-RET gene rearrangement occurs in about 1% of lung adenocarcinomas. In recent, the targeted agents to inhibit RET phosphorylation have been examined in several clinical studies. However, little was known about how this fusion gene drive lung cancer. Methods: We established transformed HEK293T cells with KIF5B-RET fusion gene using Lenti virus system. In transformed HEK293T cells, we identified transcription factors which are activated using qRT-PCR array and then, checked mRNA, protein, expressions of FOXA2. We performed immunohistochemistry to evaluate the expressions of FOXA2 protein in tumor tissues obtained from 38 lung adenocarcinoma patients who were screened by FISH, RT-PCR, and Nanostring analysis. Results: In the qRT-PCR array, the transcription factors of which the relative expression increased more than 1.5 in KIF5B-RET transformed HEK293T cells were MYF5, STAT5A, ESR1 and FOXA2. We confirmed the increased expressions of FOXA2 mRNA and protein in transformed HEK293T cells...

Introduction: The interaction between tumor cells and tumor micro-environment (TME) plays important role in tumor initiation, progression, treatment resistance, and prognosis in solid tumors including non-small cell lung cancer (NSCLC). The aim of this study is to evaluate the prognostic role of TME-related molecules in NSCLC patients who received surgical resection. To find out a possibility of a potential serum biomarker, protein expression in tissue from surgical specimen and pre-operative serum were investigated together. Methods: For 85 NSCLC patients who underwent surgical resection from August 2006 to September 2010 and had pre-operative serum samplings, we screened the serum levels of 60 TME-related molecules with angiogenesis array kit for 8 patients who had recurrences and 4 patients who did not. For matrix metalloproteinase (MMP)-1 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 in which significant difference was observed between recurrence and recurrence-free groups, protein expressions were investigated using ELISA in serum and immunohistochemistry in tumor tissue. Results: High serum MMP-1 was related to carcinoembryonic antigen (CEA) level≥5 at diagnosis (P = 0.034), higher frequency of the presence of vascular invasion (P = 0.041). High expression of MMP-1 in tumor cells (intensity score≥2) was related to tumor differentiation (P = 0.057), whereas high MMP-1 expression in stroma was significantly associated with lymph node metastasis and TNM stage (P = 0.021 and P = 0.022, respectively). In the case of TIMP-1, expression in tumor cells was higher for adenocarcinoma but higher in stromal cells for squamous cell carcinoma (P = 0.046). There was no correlation between the tissue and serum levels of MMP-1 and TIMP-1. High serum MMP-1 showed a trend for short overall survival (P = 0.080). When serum MMP-1 was combined with CEA level or presence of vascular invasion, the prognostic implication for overall survival was statistically significant. Neither tumor nor stromal positivity for MMP-1 and TIMP-1 was associated with survival outcome; however, a tumor/stroma TIMP-1 intensity ratio≥1 was strongly associated with early recurrence in multivariate analysis (HR = 13.1, P = 0.008). Conclusion: In surgically resected NSCLC patients, serum MMP-1 showed a possibility as a prognostic biomarker. In addition, the tumor/stroma TIMP-1 ratio (≥1) in tissue may be a useful to predict tumor recurrence. Citation Format: Ho Jung An, Jin-Hyoung Kang, Yoon-Jin Lee, Soon Uk Hong, Kyo Young Lee, Jeong-Oh Kim. The clinical implication of MMP-1 and TIMP-1 on prognosis in patients with non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1547. doi:10.1158/1538-7445.AM2015-1547

The KIF5B-RET gene is a novel fusion gene resulting from pericentric inversion between the kinesin family member 5B (KIF5B) and the rearranged during transfection (RET) gene, which recently isolated as an oncogene in non-small cell lung cancers. In the present study, we investigated the mechanisms associated with tumorigenic activity of KIF5B-RET fusion gene. We used a HEK-293T cell line stably expressing the KIF5B-RET fusion gene in pLOC lenti-viral vector. We investigated the tumor formation in a xenograft mouse models after subcutaneous injection and confirmed the expression of KIF5B-RET by PCR and western blot analysis. We also explored the mechanism of KIF5B-RET fusion gene in stable cell line. The KIF5B-RET stable cells showed upregulation of the cell growth, migration and colony formation. Furthermore, the KIF5B-RET stable cells induced the activation of BRAF, AKT and ERK compared with the parental cells. These results suggest that the KIF5B-RET fusion gene promotes the cell proliferation and tumorigenic activity though BRAF, AKT and ERK activation. These findings may also provide the possible that KIF5B-RET fusion gene as a ‘driver’ in some subgroups of lung cancers. Citation Format: Jung-Young Shin, Min-Young Kim, Kyoung-Hwa Son, Jeong-Oh Kim, Jin-Hyoung Kang. Tumorigenic activity of a novel KIF5B-RET fusion gene. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2710. doi:10.1158/1538-7445.AM2015-2710

e18528 Background: KIF5B-RET fusion genes are formed when part of the RET gene fuses with part of the KIF5B gene. This fusion gene induces the proliferation and differentiation of tumor cells by tr...

This study investigated the cytotoxic effect of zinc–citrate compound (CIZAR ® ) on choriocarcinoma cell lines. Primary cultured normal trophoblast cells (NPT), human tumorigenic poorly differentiated trophoblast cell line (HT), and choriocarcinoma cell line (BeWo) were exposed to different concentrations of CIZAR ® and cultured at different times. Cell viability was determined by CCK-8 assay. The effects on cell cycle progression, population distribution and apoptotic incidence were determined by flow cytometry. The appearance of apoptosis was confirmed by DNA laddering and DAPI staining. The quantitative analysis of telomerase was measured by TRAPeze ® telomerase detection kit. The molecular mechanism of CIZAR ® -induced apoptosis was examined with Western blot analysis and colorimetric caspase-3 activity assay. In in vitro condition, CIZAR ® had a selective cytotoxic effect on choriocarcinoma cell line in dose- and time-dependent patterns. Flow cytometric analysis, DNA laddering, and DAPI staining indicated that BeWo cells only have been induced apoptosis by CIZAR ® . Shortening of telomere was also observed only in BeWo cells. Results also displayed that CIZAR ® -induced apoptosis involves the up-regulation of p21 WAF1 and Bax protein and down-regulation of Bcl-2 which were accompanied by the activation of caspase-3. Taken together, our results suggest that CIZAR ® is an apoptotic inducer in malignant trophoblast cells (BeWo).

7537 Background: The KIF5B-RET rearrangement is detected with the frequency of 1~2% in ‘triple marker’-negative lung adenocarcinomas, i.e., EGFR, KRAS and EML4-ALK wild type. These mutational chang...

Direct sequencing remains the most widely used method for the detection of epidermal growth factor receptor (EGFR) mutations in lung cancer; however, its relatively low sensitivity limits its clinical use. The objective of this study was to investigate the sensitivity of detecting an epidermal growth factor receptor (EGFR) mutation from peptide nucleic acid-locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamp and Ion Torrent Personal Genome Machine (PGM) techniques compared to that by direct sequencing. Furthermore, the predictive efficacy of EGFR mutations detected by PNA-LNA PCR clamp was evaluated. EGFR mutational status was assessed by direct sequencing, PNA-LNA PCR clamp, and Ion Torrent PGM in 57 patients with non-small cell lung cancer (NSCLC). We evaluated the predictive efficacy of PNA-LNA PCR clamp on the EGFR-TKI treatment in 36 patients with advanced NSCLC retrospectively. Compared to direct sequencing (16/57, 28.1%), PNA-LNA PCR clamp (27/57, 47.4%) and Ion Torrent PGM (26/57, 45.6%) detected more EGFR mutations. EGFR mutant patients had significantly longer progressive free survival (14.31 vs. 21.61 months, P = 0.003) than that of EGFR wild patients when tested with PNA-LNA PCR clamp. However, no difference in response rate to EGFR TKIs (75.0% vs. 82.4%, P = 0.195) or overall survival (34.39 vs. 44.10 months, P = 0.422) was observed between the EGFR mutations by direct sequencing or PNA-LNA PCR clamp. Our results demonstrate firstly that patients with EGFR mutations were detected more frequently by PNA-LNA PCR clamp and Ion Torrent PGM than those by direct sequencing. EGFR mutations detected by PNA-LNA PCR clamp may be as a predicative factor for EGFR TKI response in patients with NSCLC.

Background High-risk type of human papillomavirus (HPV) has recently been found to be etiologically associated with 20 to 25% of SCCHN, particularly common in or pharyngeal tumors. We investigated the protein expression of PTEN/PI3K/AKT/mTOR signal pathway on HPV 16 +/− tonsillar squamous cell carcinomas. And, we analyzed microRNA expression profile in tongue cancer cell line transfected with HPV 16 E6/E7 oncogene. Method Tonsil cancer patients who had been treated at Seoul St. Mary's Hospital between 2000 and 2009 were reviewed. Tissue microarrays were prepared from formalin-fixed, paraffin-embedded tumor tissues of 65 surgical specimens, and stained with antibodies against PTEN, PIK3CA, phosphorylated AktSer473, and phosphorylated mTORSer2448. High-risk HPV infection status was determined by in situ hybridization. The protein expression of EGFR, PIK3CA, pAkt, mTOR, and PTEN was assessed by immunohistochemistry. MicroRNA expression was studied using microarrays (PANArray™ miRNA expression profiling kit) in two tongue cancer cell lines (YD8 and YD10B) transfected with HPV 16 E6/E7 oncogene. Results Of the 65 pts, 26 (40%) pts were HPV positive. Overall survival and relapse-free survival were significantly different between two groups (P = 0.05 and P = 0.036, respectively). PTEN expression was significantly higher in HPV-positive- than in HPV-negative patients (P = 0.02). PIK3CA, phosphorylated AktSer473, and phosphorylated mTORSer2448 expression was negatively correlated with HPV status, yet not significantly. HPV status (P = 0.03) and nodal stage (P = 0.05) were found to significantly affect overall survival, as determined by multivariate analysis. The expression profile of miRNAs was different in HPV 16 E6/E7 transformed cells compared with vector alone cells. In the final round, 8 of 158 miRNAs were selected to be most differently expressed miRNAs; let-7b, miR-1, miR-107, miR-122, miR-127-5p, miR-95, miR-23b and miR-370 (>1.2-fold). Conclusion We observed the higher expression of PTEN in HPV 16 positive tonsil tumor than HPV 16 negative tumor. Our data suggest that some microRNAs induced by E6/E7 oncogene might directly affect PTEN expression level independent of p53. We are underway on the experiments confirming that PTEN is regulated by which among these miRNAs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 185. doi:1538-7445.AM2012-185

Backgraound The eIF4B, a key molecule for initiation of protein translation, play an important role in cell growth, proliferation and survival. We investigated that eIF4B is associated with the molecular mechanism of de novo resistance against EGFR TKIs and its role on cell proliferation and survival in NSCLC cells. Method Among five NSCLC cell lines harboring EGFR and K-RAS wild type, PC14 cells showing the highest IC50, were selected for our experiment. In eIF4B silenced PC14 cells by shRNA, we evaluated cell proliferation by direct cell counts and SRB assay and cell cycle distribution using flow cytometry. The expression of signal molecules, transcription factors and apoptosis associated proteins was measured by Western blot. In vivo tumor model established by subcutaneous injection of eIF4B silenced PC14 cells, the retardation of tumor growth was measured. Results In PC14 cells transformed with eIF4B shRNA, we observed the protein expression of eIF4B was reduced (>70% at day 5). The proliferation of eIF4B silenced cells was greatly inhibited both in vitro and in vivo tumor model. Between eIF4B silenced cells and control cells, there was no significant difference in anti-proliferative activity of several target inhibitors (enzastaurin, gefitinib, LY294002, vandetanib) against EGFR-PI3K-AKT signaling molecules. The expression of proteins involved in cell survival (pEGFR, pAKT and mTOR), transcription factors (pNFkB and TS) and anti-apoptotic protein (bcl-2), was decreased and apoptosis associated proteins (phospho-p53 and active caspase-3) were increased. The G0/G1 cell fraction in eIF4B silenced cells was reduced (47.3% vs. 55.1% in control cells) and accumulation of apoptotic cells was observed (12.7% vs. 1.5% in control cells; shown as sub G1 population). Conclusion Our results demonstrated that eIF4B silencing resulted in a marked decrease of several anti-apoptotic and pro-proliferative proteins and eIF4B was required for cell proliferation and survival by regulating cell cycle and cell signaling molecules. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1291. doi:1538-7445.AM2012-1291

Background: Deregulation of the p16INK4A-cyclin D-cyclin-dependent kinases (CDK) 4/6-retinoblastoma (RB) pathway is frequently observed in various cancers and represents one of the attractive therapeutic targets. The gain of function mutation in KRAS gene confers intrinsic resistance to targeted anti-cancer drugs as well as cytotoxic chemotherapeutic agents leading to treatment failure. More effective treatment for adenocarcinomas harboring KRAS mutations should be developed to improve clinical outcomes. Purpose: CINK4, a chemical inhibitor of cyclin-dependent kinase 4, has demonstrated potent anti-proliferative activity in the presence of pRb. Herein, when combined with paclitaxel, we investigated that CINK4 may increase its anti-proliferative activity in the NSCLC cells harboring KRAS mutant. Methods: We measured the anti-proliferative activities of CINK4 in A549 (KRAS mutant), H358 (KRAS mutant), SK-LU-1 (KRAS mutant), H23 (KRAS mutant), PC14 (KRAS wild), and Calu-3 (KRAS wild) cell lines using SRB assay, cell cycle distribution quantified by flow cytometric analysis, and the expression of cell cycle regulators and apoptosis-related proteins was evaluated by Western blotting. Multiple drug effect was analyzed to study an interaction between two drugs. The nature of the interaction between two agents was calculated for the combination index (CI). Results: Concentration- and time- dependent anti-proliferative effects of CINK4 (0.1∼ 40 µM concentration range) and paclitaxel (0.1∼300 nM) alone were observed in six NSCLC cell lines, and were not significantly different between each cell line (48 h IC50: 10.4 ± 0.2 µM and 5.5 ± 0.9 nM, 10.1± 1.9 µM and 5.2 ± 0.9 nM, 6.6 ± 0.8 µM and 3.6 ± 1.0 nM, 8.6 ± 0.8 µM and >10 nM, 8.9 ± 0.5 µM and 3.5 ± 0.2 nM, and 8.4 ± 0.2 µM and > 10 nM, in A549, H358, PC14, and Calu-3, SKLU-1, and H23 cells, respectively). After 48 h CINK4 treatment alone, the G0/G1phase cell population increased with increment of subG1 faction in concentration-dependent manner. CINK4 reduced cyclinD1 and Rb phosphorylation and increase the expression of cleaved PARP in concentration-dependent manner in A549, H358, and PC14 cells. The CINK4 combined with paclitaxel in four KRAS mutant cell lines enhanced anti-proliferative activity of paclitaxel. The CI values of CINK4 and palitaxel in A549, H358, SKLU-1 and H23 were 0.51 ± 0.16, 0.63± 0.12, 0.60 ± 0.15, and 0.55 ± 0.15, respectively. Conclusions: Taken together, our data suggest that CINK4 alone has promising anti-tumor activity in NSCLC cells with or without KRAS mutation. In addition, CDK4/6 inhibitor in combined with paclitaxel demonstrated more enhanced anti-tumor activity in NSCLC cells harboring mutant KRAS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4896. doi:1538-7445.AM2012-4896

64 Background: Metastasis of cancer cells is associated with numerous activating molecules, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and tumor necrosis factor (TNF)-α. Excess VEGF production induces hematogenous metastasis in gastric cancer (GC) cells. To understand the anti-metastatic effect of bevacizumab, an anti-VEGF monoclonal antibody developed for selective inhibition of tumor angiogenesis, we investigated VEGF-induced expression of key adhesion molecules in human umbilical vein endothelial cells (HUVECs) and the inhibitory effect of bevacizumab on the adhesion of GC cells expressing high levels of VEGF. Methods: We measured the soluble VEGF (sVEGF) produced by 7 GC cells by ELISA. We evaluated intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin expression in HUVECs pretreated with SNU-1-conditioned medium (SNU-1 CM) or recombinant VEGF (rhVEGF) at different time points by western blotting. Results: We identified that SNU-1 cells secreted the highest sVEGF concentration in 7 GC cells. After rhVEGF pretreatment, ICAM-1 and E-selectin expression were highest at 4–6 h and 2 h, respectively, but VCAM-1 expression was unchanged. Bevacizumab cotreatment markedly decreased VCAM-1 and E-selectin expression to basal levels, whereas ICAM-1 expression was unaffected. The SNU-1 adherent cell percentage decreased following bevacizumab cotreatment of SNU-1 CM- or rhVEGF-pretreated HUVECs. Investigation of SNU-1-cell invasiveness through activated HUVECs revealed less than 10 invasive adherent cells, whereas no cells were found after bevacizumab cotreatment. Conclusions: Our preclinical data suggests that bevacizumab, might contribute to anti-metastasis by inhibiting SNU-1 cell adhesion to HUVECs by reducing E-selectin and VCAM-1 expression.

Background: Deregulation of the p16INK4A-cyclin D-cyclin-dependent kinases (CDK) 4/6-retinoblastoma (RB) pathway is a common paradigm in various solid tumors and represents one of the attractive targets in cancer treatment. KRAS mutation confers intrinsic resistance to cytotoxic chemotherapeutic agents, and to EGFR TKIs leading to treatment failure. An effective systemic treatment for adenocarcinomas harboring KRAS mutations are urgently needed to improve clinical outcomes. Purpose: CINK4, a chemical inhibitor of cyclin-dependent kinase 4, has demonstrated effective anti-proliferative activity in vivo in the presence of pRb. Herein, we investigated that CINK4 may increase its anti-proliferative activity in the NSCLC cells harboring KRAS mutation Methods: We measured the anti-proliferative activities of CINK4 in A549 (KRASG12S), H358 (KRASG12C), PC14 (KRASwild), and Calu-3 (KRASwild) cell lines using SRB assay, cell cycle distribution quantified by flow cytometry, and the expression of cell cycle regulators and apoptosis-related proteins was evaluated by Western blotting. Multiple drug effect was analyzed to study an interaction between two drugs. The nature of the interaction between two agents was calculated for the combination index (CI). Results: Concentration- and time-dependent anti-proliferative effects of CINK4 (0.1∼40 μM concentration range) and pacitaxel (0.1∼300 nM) alone were seen in four NSCLC cell lines, and were not significantly different between each cell line (48 h IC50: 10.4 ± 0.2 μM and 5.5 ± 0.9, 10.1± 1.9 μM and 5.2 ± 0.9, 6.6 ± 0.8 μM and 3.6 ± 1.0, and 8.4 ± 0.2 μM and > 10 nM, in A549, H358, PC14, and Calu-3, respectively). After 48 h CINK4 treatment alone, the G0/G1phase cell population increased with increment of subG1 faction in concentration-dependent manner. CINK4 reduced cyclinD1 and Rb phosphorylation and increase the expression of cleaved PARP in concentration-dependent manner in A549, H358, and PC14 cell lines. And CINK4 combined with paclitaxel in two KRAS mutated cell lines showed enhanced anti-proliferative activity of paclitaxel. The CI values of CINK4 and palitaxel combination in A549 and H358 were 0.51 ± 0.16 and 0.63± 0.12, respectively. Conclusions: CINK4 showed promising anti-tumor activity in NSCLC cell lines. NSCLC harboring KRAS mutation may have clinical benefit from CDK4/6 inhibitor in combined with chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A66.

Background As EBV-associated gastric cancer has unique features that are different from EBV (-) gastric cancer, EBV is considered to have a key role in gastric carcinogenesis. It has been reported that viral latent membrane protein 2A (LMP2A) in EBV-transformed tumor cells activates the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which provides a survival signal and chemo-resistance to cytotoxic anti-cancer drugs. This study was to evaluate anti-proliferative effect and cell cycle change when 5-FU and LY294002 (LY), a selective inhibitor of PI3K, were treated separately or combined with different schedules in EBV positive gastric cancer cell line, SNU-719. Methods After single treatment and sequential combination of 5-FU and LY, cytotoxic activity was measured by MTS assay. When 5-FU and LY were treated in single and sequential combinations, the expression of p-AKT, p-NFkB, p-p53 and bcl-2 was observed on different concentrations by Western blot analysis. We also investigated the effect on apoptosis and cell cycle distribution using flow cytometry. The LMP2A siRNA inhibition was done to confirm the reversal of decreased 5-FU activity and p-AKT. Results When 5-FU was sequentially combined with LY, the combination index (CI) value indicated synergistic anti-proliferative effect. The expression of p-AKT and p-NFκB was upregulated by 5-FU alone but sequential treatment of 5-FU and LY decreased the expression of both p-AKT and p-NFκB. When 5-FU was combined with LY, G0/G1 and sub G1 cell population (%) increased. When 5-FU was added to the cells transfected with LMP2A siRNA, its anti-proliferative effect increased and the expression of p-AKT decreased. In sequential combination of 5-FU and LY, the expression of p-p53 was increased and bcl-2 expression was diminished compared to 5-FU alone. Conclusion These data suggest that sequential combination of 5-FU and LY induce synergistic cytotoxicity and overcome intrinsic and acquired resistance of 5-FU via downregulation of activated p-AKT and mitochondria-dependent apoptosis in EBV gastric cancer cell line, SNU-719.

Purpose The aim of this study is to investigate the effect of genetic variations and the expression of the reduced folate carrier (RFC) and dihydrofolate reductase (DHFR) on the drug sensitivity to methotrexate (MTX) in different cancer cell lines. Materials and methods We examined the six human cancer cell lines (MCF-7, AGS, A549, NCI-H23, HCT-116 and Saos-2). The cytotoxicity of MTX was measured by sulforhodamine B (SRB) assay. The expressions of the DHFR and RFC were evaluated by real-time PCR and western blotting. Four single nucleotide polymorphisms (SNPs) of the DHFR and two SNPs of the RFC were genotyped. Results The IC₅₀s of MTX was in an extensively broad range from 6.05±0.81 nM to>1,000 nM in the cell lines. The Saos-2 (>1,000 nM) and MCF-7 (114.31±5.34 nM) cells were most resistant to MTX; in contrast, the AGS and HCT-116 cells were highly sensitive to MTX with an IC(50) of 6.05±0.81 nM and 13.56±3.76 nM, respectively. A reciprocal change of the RFC and DHFR mRNA expression was found between the MTX-sensitive AGS and MTX-resistant Saos-2 cells. There was no significant difference in the expression levels of RFC protein in both the AGS and Saos-2 cells, whereas DHFR protein was more increased in the MTX-resistant Saos-2 cells treated with MTX. The genotype of the MTX-sensitive AGS cells were mutant variants of the DHFR; in contrast, the Saos-2 cells had the wild-type of the DHFR. Conclusion In conclusion, this study showed that inverse change of the RFC and DHFR mRNA and protein expression was associated with RFC and DHFR polymorphisms and it is postulated that this phenomenon might play an important role in sensitivity of certain cancers to MTX.

11077 Background: The aromatase inhibitors cause bone loss by estrogen depletion. Zoledronic acid (ZA) can prevent bone mineral density (BMD) loss associated with the use of aromatase inhibitors. Accordingly interest has arisen in measuring surrogate markers of bone resorption to monitor the response of treatment of BMD loss in place of radiologic assessment. This study was designed to determine whether ZA would prevent bone loss that is known to occur with letrozole and identified surrogate markers of bone resorption in an animal model. Methods: In ovariectomized or sham-operated rat, we administrated ZA and letrozole to 5 different groups including: a sham operation control group (OC), a group in which an ovariectomy was performed followed by saline administration (OS), an ovariectomy with ZA treatment group (OZ), an ovariectomy with letrozole treatment group (OL) and an; ovariectomy with ZA and letrozole combined treatment group (OZL). The levels of serum osteocalcin, serum bone alkaline phosphatase (BALP), serum calcium and urine N-telopeptide (NTX) and BMD were estimated and compared at the same periods for each group. The distinct microscopic findings of proximal tibia at week sixteen were also compared. Results: Significantly intense increment of BDM in the OZ group and the OZL group and lower increment in the OL group were valued in correlation to the OS group, 31.3%, 35.0%, 10.5% and 13.0% respectively. Serum osteocalcin levels and urine calcium levels were close to each group. Serum calcium levels and BALP levels were relatively lowering in the OZ group and the OZL group than other group. Meaningfully lower levels of urine NTX were measured in the OZ group and the OZL group, 6.6% and 16.5% respectively. In the OL group levels of urine NTX were rise of 55.9%. Less cancellous bone loss and increase bone trabeculae were noted in the microscopic findings of tibia. Conclusions: The combination treatment with ZA and letrozole are effective in the inhibition of bone resorption and in the maintenance of BMD. Measurement of serum osteocalcin, urine NTX, and BMD, levels are recommended as surrogate markers for determining the response for the treatment of bone loss. No significant financial relationships to disclose.