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Background/aim LY2835219 (LY), a novel CDK4/6 inhibitor, prevents cell proliferation through G1 arrest. Docetaxel (DTX) and paclitaxel (PTX) are cytotoxic drugs targeting tubulin-mediated apoptotic cell death via G2/M arrest. We evaluated the antitumor effects of DTX/PTX and LY individually and in combination in lung adenocarcinoma cells with or without KRAS mutations and xenograft mice harboring KRAS mutations. Materials and methods We investigated in vitro/in vivo changes in signaling molecules and analyzed cell proliferation, cycle, and apoptosis via flow cytometry and western blotting. Results LY cytotoxicity was dose-dependent and varied with KRAS mutation status. DTX→LY showed synergistic cytotoxicity regardless of KRAS mutation. Furthermore, the synergistic effect of PTX→LY was significantly greater than that of PTX+LY. DTX→LY remarkably reduced the number of G0/G1 cells and increased the number of G2/M arrested cells, resulting in an increase in apoptosis and subG1 cells. Conclusion DTX→LY has synergistic antitumor effect in lung cancer cells and xenograft mice regardless of KRAS mutation.

Introduction: Non-small cell lung cancer (NSCLC) patients with activating EGFR mutations (mEGFR) well respond to EGFR TKIs, but the majority of patients eventually acquire drug resistance, leading to treatment failure. Recently, intracellular potential autophagy in addition to primary genetic alterations implicates the acquired drug resistance to targeted drugs in various carcinomas such as melanoma, pancreas, and breast cancer. We aimed to identify autophagy flux-related signal molecules which may be closely linked to acquired resistance in mEGFR NSCLC. Methods: We constructed the TMA composed of 579 surgically resected tumor tissues from lung adenocarcinoma patients and evaluated protein expression of autophagy flux-related signal molecules including CAGE, p-Beclin1, LC3B, p-AMPK, ULK1, p62, and ATG5 using immunohistochemistry (IHC). We established H1975OSI (166-folds) and PC9ERL (3,650-folds) resistant sub cell lines through the prolonged exposure of Osimertinib and Erlotinib to H1975 (L858R/T790M) and PC9 (Del19) cells, respectively. We also explored genes that induce autophagy flux activity using mRNA seq and qRT-PCR. Results: H-score of CAGE ( 95.6±7.9, 70±5.5), p-AMPK (73.4±7.8, 32±5.6) and p-Beclin1 (44.4±16.1, 19.7±5.5), ULK1( 57.4±4.9, 41.9±4.9) were significantly higher in mEGFR, compared to those of wild type EGFR NSCLC (P Conclusion: Taken together, our results strongly support the evidence that autophagy flux-related signal molecules as well as known genetic alterations are closely connected to the acquired resistance during period of EGFR TKI treatment in mEGFR NSCLC patients. Now, we are undergoing additional RNA profiling and function study of identified novel autophagy-related genes to examine a possibility as potential target to overcome EGFR TKI resistance. Citation Format: Jeong-Oh Kim, Jung-Young Shin, Min-Young Kim, Mi-Ran Lee, Seoree Kim, Hankyu Lee, Doyong Jeon, Seung Ryel Baek, Kim Joo Il, Kyoungjune Lee, Youn Soo Lee, Jin-Hyoung Kang. The identification of novel autophagy flux-related molecules closely linked to the acquired resistance to EGFR TKIs in NSCLC patients harboring EGFR mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2232.

Introduction: There are similar and different properties between the epidermal growth factor receptor mutations (mEGFR), which are classified into common, uncommon, and compound mutation subtypes depending on their location and pattern on the EGFR gene. We investigated the RWD and RNA expression profile of non-common mutations (uncommon and compound mutations) in the mEGFR+ NSCLC patients. Method: We analyzed the NGS data extracted from the Foundation Medicine (FMI) SRC. And, we collected the medical information for stage I-IIIA mEGFR+ NSCLC patients receiving curative surgical resection. In Böehringer Ingelheim (BI) open data access (ODA), we analyzed treatment results in stage IV mEGFR+ NSCLC patients treated with EGFR TKI. We explored the RNA expression profile by High-Plex Digital Spatial Profiling. A total of 18,676 DEG libraries were constructed, and 419 significance genes with > 2-fold changes, 3-log 2 normalized read counts, and p-value = 0.05 (up and down) were identified. Result: In FMI-SRC cohort, 10,059 mEGFR+ cases of total 78,656 cases consist of common, 6,994 (69.5%), uncommon, 1,966 (19.6%), and compound, 1,099 (10.9%). In hospital cohort, 941 mEGFR+ cases consist of common, 860 (91.4%), uncommon, 60 (7.4%), and compound, 21 (2.2%). Median relapse-free survival (mRFS) in non-common mutations (31.4M and 33.7M) was shorter than common mutations (40.1M, p=0.877). CNS recurrence in the non-common mutations (40.0% and 35.3%) tended to be higher than that of common mutations (27.4%, p=0.361). In BI-ODA, median time on treatment (mTOT) in 163 uncommon mutations (10.0M) and 139 compound mutations (11.5M) were shorter than historical data of common mutations (17.3M). Spatial RNA-seq data of the ratio of deconvolution individual cells showed that genes involved in inflammatory and immune response were relatively lower in non-common mutations. Gene ontology string analysis revealed RNA expression of genes in compound mutations was functionally associated with chemokine production (23.4%), electron transfer activity (14.9%) and T cell selection (8.5%), and, in uncommon mutations, was associated with regulation of oligopeptide transport (26.1%), hypermethylation of CpG island (21.7%), and CD4+ CD25+ regulatory T cell lineage commitment (13.0%), compared to common mutations. Based on the GSEA, relatively highly clustered genes in compound mutations were the protein modification process, G-protein coupled receptor, regulation of cytosolic calcium ion concentration, and IL-5 production pathways, and lower ES levels in oxidative phosphorylation and RNA processing pathways, when compared to common mutations. Conclusion: Taken together, our results suggest that uncommon and compound mEGFR subtypes exhibiting shorter mRFS and mTOT, and distinguishing RNA expression profile of functional signaling pathways be not same disease entity as common mEGFR subtype Citation Format: Seoree Kim, Yeoun Eun Sung, Joo Ri Kim, Chan Kwon Jung, Jeong-Oh Kim, Jeong-yeon Shin, Guk Jin Lee, Sang hoon Chun, Young Ho Lee, Jin Hyoung Kang. Real world data and multi-genomic analysis of non-common mutations in the EGFR mutation-positive non-small cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4391.

Immune checkpoint inhibitors (ICIs) offer improved survival for patients with advanced malignant melanomas. However, only a subset of these patients exhibit an objective response rate of 10-40 % with ICIs. We aimed to ascertain the effects of RNA signatures and the spatial distribution of immune cells on the treatment outcomes of patients with malignant melanomas undergoing ICI therapy. Clinical data were retrospectively collected from ICI-treated patients with malignant melanoma; RNA expression profiles were examined via next-generation sequencing, whereas the composition, density, and spatial distribution of immune cells were determined via multiplex immunohistochemistry. Patients with poor and good responses to ICIs showed significant differences in mRNA expression profiles. Different spatial distributions of T-cells, macrophages, and NK cells as well as RNA signatures of immune-related genes were found to be closely related to therapeutic outcomes in ICI-treated patients with malignant melanomas. The spatial distributions of PD-1+ T-cells and activated M1 macrophages showed a significant correlation with favorable responses to ICIs. Our findings highlight the clinical relevance of the spatial proximity of immune cell subsets in the treatment outcomes of metastatic malignant melanoma.

Purpose Isoform 2 of tight junction protein claudin-18 (CLDN18.2) is a potential target for gastric cancer treatment. A treatment targeting CLDN18.2 has shown promising results in gastric cancer. We investigated the clinical significance of CLDN18.2 and other cell-adherens junction molecules (Rho GTPase-activating protein [RhoGAP] and E-cadherin) in metastatic diffuse-type gastric cancer (mDGC). Materials and Methods We evaluated CLDN18.2, RhoGAP, and E-cadherin expression using two-plex immunofluorescence and quantitative data analysis of H-scores of 77 consecutive mDGC patients who received first-line platinum-based chemotherapy between March 2015 and February 2017. Results CLDN18.2 and E-cadherin expression was significantly lower in patients with peritoneal metastasis (PM) than those without PM at the time of diagnosis (P=0.010 and 0.013, respectively), whereas it was significantly higher in patients who never developed PM from diagnosis to death than in those who did (P=0.001 and 0.003, respectively). Meanwhile, CLDN18.2 and E-cadherin expression levels were significantly higher in patients with bone metastasis than in those without bone metastasis (P=0.010 and 0.001, respectively). Moreover, we identified a positive correlation between the expression of CLDN18.2 and E-cadherin (P

Background Immune checkpoint blockades (ICBs) are characterized by a durable clinical response and better tolerability in patients with a variety of advanced solid tumors. However, we not infrequently encounter patients with hyperprogressive disease (HPD) exhibiting paradoxically accelerated tumor growth with poor clinical outcomes. This study aimed to investigate implications of clinical factors and immune cell composition on different tumor responses to immunotherapy in patients with non-small cell lung cancer (NSCLC). Methods This study evaluated 231 NSCLC patients receiving ICBs between January 2014 and May 2018. HPD was defined as a > 2-fold tumor growth kinetics ratio during ICB therapy and time-to-treatment failure of ≤2 months. We analyzed clinical data, imaging studies, periodic serologic indexes, and immune cell compositions in tumors and stromata using multiplex immunohistochemistry. Results Of 231 NSCLC patients, PR/CR and SD were observed in 50 (21.6%) and 79 (34.2%) patients, respectively and 26 (11.3%) patients met the criteria for HPD. Median overall survival in poor response groups (HPD and non-HPD PD) was extremely shorter than disease-controlled group (SD and PR/CR) (5.5 and 6.1 months vs. 16.2 and 18.3 months, respectively, P = 0.000). In multivariate analysis, HPD were significantly associated with heavy smoker (p = 0.0072), PD-L1 expression ≤1% (p = 0.0355), and number of metastatic site ≥3 (p = 0.0297). Among the serologic indexes including NLR, PLR, CAR, and LDH, only CAR had constantly significant correlations with HPD at the beginning of prior treatment and immunotherapy, and at the 1st tumor assessment. The number of CD4+ effector T cells and CD8+ cytotoxic T cells, and CD8+/PD-1+ tumor-infiltrating lymphocytes (TIL) tended to be smaller, especially in stromata of HPD group. More M2-type macrophages expressing CD14, CD68 and CD163 in the stromal area and markedly fewer CD56+ NK cells in the intratumoral area were observed in HPD group. Conclusions Our study suggests that not only clinical factors including heavy smoker, very low PD-L1 expression, multiple metastasis, and CAR index, but also fewer CD8+/PD-1+ TIL and more M2 macrophages in the tumor microenvironment are significantly associated with the occurrence of HPD in the patients with advanced/metastatic NSCLC receiving immunotherapy.

Additional file 6: Supplementary Table S2. Serologic inflammatory markers associated with HPD by univariate and multivariate analyses (n = 155)† (DOCX 16 kb)

Additional file 5: Supplementary Table S1. Clinical and pathologic characteristics (N = 231)

Introduction: The immune checkpoint inhibitors (ICIs) against CTLA-4 and PD-1/PD-L1, have improved survival with long-term durable response for advanced malignant melanomas. However, despite this success, only a minority of them respond to ICIs, with 10-40% objective response rate. The clinical experience with ICIs has identified several potential biomarkers associated with treatment efficacy, including tumor mutational burden, tumor-infiltrating lymphocytes, PD-L1 expression, and intestinal microbiota, but they still have not been useful in clinical practice. Our study aimed to investigate the influences of RNA gene signature and spatial distribution of immune cells on the treatment outcomes of malignant melanoma patients receiving ICIs. Method Clinical: data were retrospectively collected malignant melanoma patients who had been treated with ICIs between Jan. 2016 and Dec. 2018 at Seoul St. Mary’s Hospitals. Among them, 23 cases with sufficient tumor tissue to examine RNA expression profile by NGS and the composition, and spatial distance of immune cells by multiplex immunofluorescent staining. Results: The cell densities of T cells, cytotoxic T cells, Treg cells, CD8+Treg cells, and exhausted T cells in the good response group tended to be higher, which was clearer in the peritumoral (PT) area than the intratumoral (IT) area. Meanwhile, the cell densities of macrophage and activated M1 were significantly higher, which was more pronounced in the IT area than the PT area. For the good response group, M1/M2 ratio in the IT area was significantly higher compared to that of poor response group (p=0.045). The distribution (%) of cancer cells present at a close distance from T cells in the PT area was relatively higher in the good responders compared to that of the poor responders. In most cases, the distribution of cancer cells (%) present at a close distance from M1, M2, and NK cells tended to be higher in the IT area than PT area. For the cases with higher distribution of cancer cells at a close distance from M1 in the IT area, OS was significantly extended (p=0.036). For the cases with higher distribution of cancer cells at a close distance from NK cells in the IT area, PFS was significantly prolonged (p=0.057), but OS was not (p=0.324). The gene sets with relatively higher RNA expression in the good response group, were as follows; hematopoietic cell dedifferentiation gene, T-cell related adaptive immunity gene, interferon-related cytokine gene, and macrophage, NK, cell, T-cell innate immunity gene set. In the good response group, innate immune response (TLR7, PTPRC, CCL5), INFγ and JAK signaling molecules (CD8A, STAT1, CD86, IRF3, CXCL10, CXCL9, OAS1, IL10R α, IRF1, IFNAR2, CXCR3, PYHIN1, KIRK1, IFIH1) showed more than 2-folds increase of RNA expression. Meanwhile, in the poor response group, inflammatory immune response (CTSV, HMGB3, KIT, CDC4EP4, TRIM28), extracellular immune response (SPON1, SRPX, SOX10, SNAI2, LING01, LAMC3), fibrosis response genes (TGFβ2, TGFβ1) was increased. Conclusion: Taken together, our results suggest that the distinguishing spatial distribution of T-cells, macrophages and NK cells and RNA signature of immune-related genes have close associations with therapeutic outcomes in malignant melanoma patients treated with ICIs. Citation Format: Seoree Kim, Su In Jo, Sang-Yeob Kim, Dong-Jun Bae, Jeong-Oh Kim, Jeong-yeon Shin, Joo-re Kim, Cookjin Lee, Sang hoon Chun, Gyeongsin Park, Jin Hyoung Kang. The RNA signature of immune-related genes and distinct spatial distribution of immune cells are closely associated with the treatment outcomes in the malignant melanoma patients receiving immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB139.

BackgroundHypoxic tumors are known to be highly resistant to radiotherapy and cause poor prognosis in non-small cell lung cancer (NSCLC) patients. CKD-516, a novel vascular disrupting agent (VDA), mainly affects blood vessels in the central area of the tumor and blocks tubulin polymerization, thereby destroying the aberrant tumor vasculature with a rapid decrease in blood, resulting in rapid tumor cell death. Therefore, we evaluated the anti-tumor efficacy of CKD-516 in combination with irradiation (IR) and examined tumor necrosis, delayed tumor growth, and expression of proteins involved in hypoxia and angiogenesis in this study.MethodsA xenograft mouse model of lung squamous cell carcinoma was established, and the tumor was exposed to IR 5 days per week. CKD-516 was administered with two treatment schedules (day 1 or days 1 and 5) 1 h after IR. After treatment, tumor tissues were stained with hematoxylin and eosin, and pimonidazole. HIF-1α, Glut-1, VEGF, CD31, and Ki-67 expression levels were evaluated using immunohistochemical staining.ResultsShort-term treatment with IR alone and CKD-516 + IR (d1) significantly reduced tumor volume (p = 0.006 andp = 0.048, respectively). Treatment with CKD-516 + IR (d1 and d1, 5) resulted in a marked reduction in the number of blood vessels (p p = 0.02) and decreased HIF-1α, Glut-1, VEGF, and Ki-67 expression. Long-term administration of CKD-516 + IR reduced tumor volume and delayed tumor growth. This combination also greatly reduced the number of blood vessels (p = 0.0006) and significantly enhanced tumor necrosis (p = 0.004). CKD-516 + IR significantly increased HIF-1α expression (p = 0.0047), but significantly reduced VEGF expression (p = 0.0046).ConclusionsTaken together, our data show that when used in combination, CKD-516 and IR can significantly enhance anti-tumor efficacy compared to monotherapy in lung cancer xenograft mice.

Background Immune checkpoint blockers (ICBs) are characterized by a durable clinical response and better tolerability in patients with a variety of advanced solid tumors. However, we not infrequently encounter patients with hyperprogressive disease (HPD) exhibiting paradoxically accelerated tumor growth with poor outcomes. This study aimed to determine differences in factors among patients with non-small cell lung cancer (NSCLC) displaying different tumor responses to immunotherapy. Methods This study evaluated 231 NSCLC patients receiving ICBs between January 2014 and May 2018. HPD was defined as a > 2-fold tumor growth kinetics ratio during ICB therapy and time-to-treatment failure of ≤ 2 months. We analyzed clinical data, imaging studies, periodic serologic indexes, and immune cell compositions in tumors and stromata using multiplex immunohistochemistry. Results Twenty-six patients (11.3%) met the HPD criteria. HPD was more frequent in patients with oncogenic driver mutations, ≥ 3 metastatic sites, ≥ 4 prior systemic treatments, and low PD-L1 expression (

Background Isoform 2 of the tight junction protein claudin-18 (CLDN18.2) is a potential target of gastric cancer treatment. A treatment targeting CLDN18.2 has shown promising results in gastric cancer. We investigated the clinical significance of CLDN18.2 and other cell-adherens junction molecules (Rho GTPase-activating protein [RhoGAP] and E-cadherin) in metastatic diffuse-type gastric cancer (mDGC).Methods We evaluated CLDN18.2, RhoGAP, and E-cadherin expression by performing two-plex immunofluorescence and quantitative data analysis of H-scores of 77 consecutive mDGC patients who received first-line, platinum-based chemotherapy between March 2015 and February 2017.Results CLDN18.2 and E-cadherin expression was significantly reduced in patients with peritoneal metastasis (PM) compared with those without PM at the time of diagnosis (P=0.010 and 0.013, respectively), while it was significantly higher in patients who never exhibited PM from diagnosis to death than in those who did (p=0.001 and 0.003, respectively). Meanwhile, CLDN18.2 and E-cadherin were expressed at significantly higher levels in patients with bone metastasis than in those without it (p=0.010 and 0.001, respectively). We identified a positive correlation between the expression of CLDN18.2 and E-cadherin (p < 0.001), RhoGAP and CLDN18.2 (p=0.004), and RhoGAP and E-cadherin (p=0.001). CDLN18.2, RhoGAP, and E-cadherin expression was not associated with chemotherapy response and survival.Conclusions CLDN18.2 expression was reduced in PM but significantly intact in bone metastasis. Furthermore, a positive correlation was identified between the expression of CLDN18.2 and other adherens junction molecules, which has clinical implications for mDGC and PM pathogenesis.

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.

Additional file 2. Toxicity of low- or high-dose IR in BALB/c nude mice. (A) The shielding device was made of 4 mm thick lead. The anesthetized mice were fixed in a 50 mL tube and irradiated with a lead shield. (B) Tumor growth and (C) body weight according to radiation dose. (D) Tumor growth during long-term treatment with 4 Gy of IR.

Additional file 1. Short-term and long-term drug administration schedules.

Additional file 3. IHC staining with HIF-1α, VEGF, Glut-1, and Ki-67 antibodies in all five treatment groups (72 h after the end of treatment). Mice were divided into five groups according to the dosing and treatment schedule: vehicle (PBS), IR alone (4 Gy/day), CKD-516 alone (3 mg/kg), and CKD-516 (3 mg/kg, day 1 or days 1 and 5) combined with IR.Scale bar: 200 μm. Magnification: × 100.

The effects of chamber pressure ( ${P}_{\text {C}}$ ) on the structural and chemical properties of an indium gallium tin oxide (IGTO) channel layer were examined. Smoother and denser IGTO films were obtained with decreasing ${P}_{\text {C}}$ , which were explained based on the enhanced kinetic energy of sputtered particles due to fewer collisions with the plasma atmosphere. The resulting IGTO thin-film transistor exhibited a high mobility of 35.0 cm2/Vs, subthreshold gate swing of 0.17 V/decade, threshold voltage ( ${V}_{\text {TH}}$ ) of −0.45 V, and ${I}_{\mathrm {\scriptscriptstyle {ON/OFF}}}$ ratio >108, even at a low annealing temperature of 150 °C.

Background: To evaluate the anti-tumor efficacy of CKD-516 in combined with irradiation (IR) and examine tumor necrosis, delayed tumor growth, and expression of molecules involved in hypoxia and angiogenesis. Methods : A xenograft mouse model of lung cancer was established. The tumor was exposed to irradiation (IR) for 5 days per week. CKD-516 was administered with ​​two treatment schedules (day 1 or days 1 and 5) at one hour after IR. After the administration, tumor tissues were stained with hematoxylin and eosin and pimonidazole. HIF1 , Glut-1, VEGF, CD31 and Ki-67 expression were evaluated by Immunohistochemical staining. Results: With short-term administration, IR and CKD-516+IR (d1) significantly reduced tumor size ( p = 0.0062 and p = 0.0051, respectively). CKD-516+IR groups were remarkably reduced blood vessels ( p < 0.005). In particular, CKD-516+IR (d1) resulted in the most extensive tumor necrosis, which was significantly increased with large hypoxic area ( p = 0.02) and decreased HIF1 , Glut-1, VEGF, and Ki-67 expressions. Long-term administration of CKD-516+IR reduced tumor size and delayed tumor growth. This combination also greatly reduced the number of blood vessels ( p = 0.0006) and significantly enhanced tumor necrosis ( p = 0.004). CKD-516+IR notably increased HIF1 expression ( p = 0.0047), but significantly diminished VEGF expression ( p = 0.0046). Conclusion: Taken together, our results demonstrate that CKD-516 in combination with IR can significantly enhance the anti-tumor efficacy compared to CKD-516 or IR alone in lung cancer xenograft mice. Keywords: Irradiation, Tumor necrosis, Hypoxia, Squamous cell carcinoma of lung, Xenograft mice

Background Atypical hemolytic uremic syndrome (aHUS) involves dysregulation of the complement system, but whether this also occurs in thrombotic thrombocytopenic purpura (TTP) remains unclear. Although these conditions are difficult to differentiate clinically, TTP can be distinguished by low (

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.

Background: c-MET is co-expressed with EGFR in approximately 70% of EGFR mutant-positive tumors. In addition, c-Met amplification was found in 10-20% of lung cancer patients with acquired tolerance to EGFR TKI, and c-Met overexpression was found in 14-69% of patients receiving EGFR TKI. CKD-702, a double antibody, is designed to simultaneously block both EGFR and c-MET expression. Purpose: We evaluated the anti-tumor efficacy of CKD-702 with PDX model derived from lung adenocarcinoma patients. Methods: We constructed the PDX model by transplanting surgical tissue from lung adenocarcinoma patient into NOD/SCID mice after the approval of research protocol in Seoul St. Mary's Hospital IRB. We confirmed the co-existence of activating EGFR mutation and c-MET amplification with PNAclamp and FISH methods. We also checked overexpression of EGFR and c-MET protein by immunohistochemistry. Results: The characteristics of tumor tissue transplanted for PDX model were: 1) Korean, lung cancer with EGFR TKI chemotherapy failure confirmed c-MET amplification (> 5 copies) and overexpression (IHC score 3+), 19del Positive EGFR mutation, 2) European lung adenocarcinoma, c-MET amplification (> 2.5 copy) and overexpression (IHC score 3+) were confirmed, and L858R EGFR mutation positive tumor tumor confirmed. The anti-cancer efficacy of CKD-702 was reduced by more than 80% compared to the tumor size of the control group regardless of the concentration. In the CKD-702 20mg/kg group, there was no significant difference in the change in the overall tumor size until the time of disappearing or ending during drug administration. The %TGI was 93.6 and 97.5 according to the concentration of CKD-702, and the % TGD was 118.5 and 311.8, respectively. CKD-702 has a significantly higher tumor growth inhibitory effect regardless of the concentration, but the tumor growth retardation effect is 2.6 times different depending on the concentration. % TGI of CKD-702 20mg / kg group was similar to Hu8c4 group or Hu8c4 + Vectibix group. However, % TGD was 1.4 times higher than Hu8c4 group efficacy and 3.1 times higher than Hu8c4 + Vectibix group% TGD. Even after the completion of drug administration, tumor still remained at 47 days and 67 days in the CKD-702 10 mg/kg and 20 mg/kg groups, respectively. Although c-MET protein expression decreased during the administration of CKD-702, it was confirmed that c-MET protein expression increased again in tumor tissue at the time of tumor growth. Although the tumor size was rather large (605.6 ~ 816.2mm3), CKD-702 showed twice the tumor growth inhibition effect (% TGI: 32.8 vs. 68.1) and the tumor growth delay effect was 4.4 times higher depending on the drug concentration (% TGI: 2.8 vs 12.3). Conclusion: Our data showed that CKD-702 have effective anti-tumor activity in EGFR TKI-resistant PDX model harboring both activating EGFR mutation and c-MET overexpression. Citation Format: Jung-Young Shin, Min-Young Kim, Mi-Ran Lee, Jeong-Oh Kim, Eun-Ju Jeon, Ji Hye Choi, Soon-Ki Hong, Kyu-Jin Park, Jin Hyoung Kang. Anti-tumor efficacy of CKD-702 in EGFR TKI-resistant patient-derived xenograft model [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 3046.

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.

Purpose Dose-limiting toxicities of docetaxel are widely considered to be neutropenia, anemia, skin toxicity, and nausea. One of the factors that limit the use of docetaxel is its unpredictability of inter-individual variation in toxicity. Materials and methods In order to identify the genetic factors that affect the risk of docetaxel-induced toxicities, we recruited patients who received docetaxel chemotherapy. We genotyped 92 patients with single-nucleotide polymorphisms (SNPs) in 5 genes: CYP3A4 (CYP3A4(*)1B, CYP3A4(*)18, and CYP3A4(*)3), CYP3A5 (CYP3A5(*)2 and CYP3A5(*)3), ABCB1 (C1236T, G2677G/T, and C3435T), SLCO1B3 (rs11045585), and ABCC2 (rs12762549). Results Out of 92 patients, 70 had grade 3 or 4 neutropenia; 4 had grade 1 or 2; and 18 had no toxicity (76.1%, 4.3%, and 19.6%, respectively). The findings of the SNP analysis showed that patients with TT genotype of ABCB1 3435C>T polymorphism showed significantly higher risk of neutropenia and anemia (p=0.029 and p=0.044, respectively). There were significant associations between docetaxel-induced leucopenia and 2677G/T of ABCB1 and rs12762549 of ABCC2 (p=0.025 and p=0.028, respectively). In a multivariate analysis, we observed that patients carrying 2677G>T in ABCB1might be associated with higher risk of chemo-resistance when treated with docetaxel (odds ratio [OR], 6.48; confidence interval, 1.92 to 21.94; p=0.003). In a subgroup analysis of non-small cell lung cancer patients, a significant association of tumor response with G2677T/A (OR, 4.54) in ABCB1 and SLCO1B3 (OR, 9.44) was observed. Conclusion Our data suggest that ABCB1 (2677G/T) and SLCO1B3 (rs11055585) might be major genetic predictors of docetaxel-related toxicities in patients receiving docetaxel chemotherapy.

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

Objectives: To establish a system which provides the NTIS (National Science and Technology Information Service)-owned information in a LOD (Linked Open Data) format for the purpose of opening national R&D information to realize ‘Government 3.0’. Methods/Statistical Analysis: The NTIS ontology has been designed using the CERIF (Common European Research Information Format) ontology which efficiently manages research information and BIBO (Bibliographic Ontology) targeted to describe literature data in a library with the RDF (Resource Description Framework). To decide a scope to provide the NTIS information in LOD format and convert it into triple data, R2RML is used. In addition, Pubby is adopted for an interface between the server and application while a triple store uses OntoBase2.0. Findings: Among a total of 389 national R&D information items provided by the NTIS, those except for personal information, secret and national defense and management items were set to be provided in LOD format. After analyzing 30 tables, the matters to be referred to during modeling were derived. In addition, ontology was designed by deriving item-concept or inter-concept relationships. To convert NTIS information into RDF, R2RML was used and the extracted triple data are stored in a triplestore. Users are able to get access to LOD data, using a SPARQL endpoint. Improvements/Applications: It is able to provide a lot of related information through connection with diverse LODs after opening national R&D information in LOD format. Then, this information would be available as base data in science and technology.

Epilepsy is the most prevalent chronic neurological disorder and can be controlled by antiepileptic drugs (AEDs) in up to 70% of patients. We performed an association study between adverse drug reactions and the genetic polymorphisms of CYP2C9, CYP2C19, ABCB1, and SCN1A. The clinical data of 83 epilepsy patients who had received AEDs containing carbamazepine (CBZ) were collected. We extracted genomic DNA from peripheral blood and then genotyped CYP2C9 (CYP2C9*2, CYP2C9*3), CYP2C19 (CYP2C19*2, CYP2C19*3), ABCB1 (C3435T), and SCN1A (IVS5N+5 G>A) using direct sequencing. The allele frequencies of CYP2C9*3, CYP2C19*2, CYP2C19*3, ABCB1 (3435C>T), and SCN1A (IVS5N+5 G>A) were 0.93, 0.72, 0.91, 0.61, and 0.55, respectively. Statistically significant differences were indicated from the data obtained. Patients with SCN1A genotype CC or CT were compared with patients with SCN1A genotype TT while using more than 500mg of carbamazepine. We have associated functional polymorphisms with the dose used in regular clinical practice for Korean epilepsy patients who had received antiepileptic drugs (AEDs) containing carbamazepine. For AEDs, we found that one of the SCN1A genotypes is associated with a 500 mg dose. There was no association found with CNS ADR caused by AEDs.

Background/Objectives: There has been a necessity of a new system to protect and share user information with cooperative research institutes and manage subjects for managing and providing national R&D information.Methods/Statistical analysis: The National Science & Technology Information Service (NTIS) used the SSO API to share user information with cooperative research institutes safely. The API included minimum information only to prevent personal information such as user ID and authorization code from being leaked and observed related laws. For the authorization and management of user information targeted to open R&D information, moreover, 2-stage authentication has been established, using the certificate authentication system.Findings: Since information is collected and provided in diverse manners by multiple institutes, user information has been scattered, and there has been a risk of the leak of personal information. With the centralized collection and management of user information, however, the protection of personal information and observance of laws have become more convenient. Furthermore, the information is provided to the authorized managers only through the construction of a security system and utilization of access control system, and security has been secured. To make users utilize information properly at access to raw data, authorization procedures were strengthened, keeping national R&D information and users more reliable.Improvements/Applications: Through this framework, secure information-sharing & management systems were applied, improving the safety of information management.

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.

The local oxidation on Si substrate has been studied by atomic force microscope lithography. Heights of protruded patterns were changed during the lithographic process with thin films of 2-amino-6-methoxybenzothiazole-azo (MBT-A) and 2-amino-6-methoxybenzothiazol-azo-Ni ([MBT-A]2Ni2+) on Si substrates. The current-value in a tip–sample junction was investigated by using scanning tunneling spectroscopy with a contact mode atomic force microscope (AFM), and it was confirmed that a change of current-values depends on applied voltages. The difference of potential barrier between [MBT-A]2Ni2+ and MBT-A was also confirmed by using UV–vis spectrophotometry and ultraviolet photoelectron spectroscopy. The tunneling current value of a [MBT-A]2Ni2+ film was larger than that of MBT-A film and the difference from threshold voltages was also observed.

Background & Purpose LY2835219(LY), a novel CDK4/6 inhibitor, inhibits phosphorylation of RB and E2F activation, thereby arresting the cell cycle in the G1 phase and suppressing the cell proliferation and cell division. Docetaxel(DTX) is a cytotoxic anti-cancer drug, which inhibits mitotic cell division, thus induces G2/M arrest and apoptosis of various cancer cells. In present study, we evaluated the activities of CDK4/6 inhibitor alone or combined with docetaxel on the anti-proliferation, cell cycle and apoptosis in lung cancer cell lines harboring KRAS mutations. Methods We measured the anti-proliferative activities of LY or DTX single and their combinations (DTX+LY 72h and DTX 24h->LY 48h) on cell proliferation in A549(G12S) and H727(G12V) cells using CCK-8 asssy. We evaluated the expression of CDK2, CDK4, cPARP and caspase-3 by Western blot. The cell cycle distribution and apoptosis in subG1 phase were analyzed by flow cytometry. Results The IC50 values of the LY and DTX alone were 0.4 ± 0.2 uM and 0.9 ± 0.2 nM in A549 cells and 2.0 ± 0.7 uM and 3.1 ± 0.3 nM in H727 cells, respectively. The CI (Combination index) of the DTX->LY sequential and the DTX+LY simultaneous treatments were 0.8 and 0.9 (CILY showed decreased cell viability by 60% at very low concentration of DTX (3.0e−5 nM), but DTX+LY showed significantly decreased cell viability by 40% at higher concentration of DTX (IC50 values can not be measured). In A549 cells, after DTX treatment for 72h, the cell population in G2/M phase increased by 54.5% relative to control (24.6%). With LY alone, the fraction of A549 cells in G0/G1 phase increased compared to control (78.4% vs. 49.5%). In case of DTX+LY combination, the G2/M fraction of A549 cells significantly increased by 33% vs 24.6% in a dose-dependent manner. Meanwhile, in DTX->LY treatment, cell fractions in G2/M were increased (44.5% vs 24.6%), especially those in G0/G1 phases were remarkably reduced (14.7% vs 49.5%). In addition, the subG1 fraction was accumulated in response to individual drug treatments. In case of DTX single treatment, the subG1 fractions of both A549 and H727 cells increased in a dose-dependent manner (0.7% vs. 17.4% and 2.5% vs. 28.1%, respectively). We were not able to observe the change of subG1 fraction in DTX+LY combination treatment in A549 cells. However, in DTX->LY sequential treatment, the subG1 fraction increased in a dose-dependent manner by relative to control (23.3% vs 10.9%). The expression of c-PARP and caspase-3 was increased by the DTX alone and two different combination schedule and CDK2 and CDK4 expressions were decreased by LY alone and DTX->LY sequential treatment. Conclusions Taken together, our results suggest that DTX->LY sequential treatment has enhanced antitumor efficacy with synergistic interaction in lung cancer cell lines harboring KRAS mutations. Citation Format: Kyoung Hwa Son, Jung-Young Shin, Jeong-Oh Kim, JinHyoung Kang. Synergistic effect of combined cdk4/6 inhibitor with docetaxel in lung cancer cell lines harboring KRAS mutations. [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 2832.

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.

Warfarin is a commonly prescribed anticoagulant drug for the prevention of thromboembolic disorders. We investigated the contribution of genetic variations of four genes and clinical factors to warfarin dose requirement and provided a warfarin-dosing algorithm based on genetic and clinical variables in Korean patients. We recruited 564 Korean patients on stable anticoagulation. Single nucleotide polymorphisms (SNPs) for the VKORC1, CYP2C9, CYP4F2 and GGCX were analyzed. Using multiple regression analysis, we developed a model to predict the warfarin requirement. The SNPs of VKORC1, CYP2C9, CYP4F2 and GGCX showed significant correlation with warfarin dose. Patients with the 3730AA genotype received significantly higher doses of warfarin than those with the 3730GG (P=0.0001). For CYP2C9, the highest maintenance dose was observed in the patients with wild-type genotype compared with the variant allele carriers (P

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