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Supplementary Figure S1; Supplementary Tables S1-S3.

Supplementary Data from Prediction of Recurrence-Free Survival in Postoperative Non–Small Cell Lung Cancer Patients by Using an Integrated Model of Clinical Information and Gene Expression

Purpose: This study was performed to discover prognostic genomic markers associated with postoperative outcome of stage I to III non–small cell lung cancer (NSCLC) that are reproducible between geographically distant and demographically distinct patient populations.Experimental Design: American patients (n = 27) were stratified on the basis of recurrence and microarray profiling of their tumors was performed to derive a training set of 44 genes. A larger Korean patient validation cohort (n = 138) was also stratified by recurrence and screened for these genes. Four reproducible genes were identified and used to construct genomic and clinicogenomic Cox models for both cohorts.Results: Four genomic markers, DBN1 (drebrin 1), CACNB3 (calcium channel beta 3), FLAD1 (PP591; flavin adenine dinucleotide synthetase), and CCND2 (cyclin D2), exhibited highly significant differential expression in recurrent tumors in the training set (P < 0.001). In the validation set, DBN1, FLAD1 (PP591), and CACNB3 were significant by Cox univariate analysis (P ≤ 0.035), whereas only DBN1 was significant by multivariate analysis. Genomic and clinicogenomic models for recurrence-free survival (RFS) were equally effective for risk stratification of stage I to II or I to III patients (all models P < 0.0001). For stage I to II or I to III patients, 5-year RFS of the low- and high-risk patients was approximately 70% versus 30% for both models. The genomic model for overall survival of stage I to III patients was improved by addition of pT and pN stage (P < 0.0013 vs. 0.010).Conclusion: A 4-gene prognostic model incorporating the multivariate marker DBN1 exhibits potential clinical utility for risk stratification of stage I to III NSCLC patients. Clin Cancer Res; 17(9); 2934–46. ©2011 AACR.

Supplementary Data from EML4-ALK Fusion Gene and Efficacy of an ALK Kinase Inhibitor in Lung Cancer

Purpose: One of the main challenges of lung cancer research is identifying patients at high risk for recurrence after surgical resection. Simple, accurate, and reproducible methods of evaluating individual risks of recurrence are needed.Experimental Design: Based on a combined analysis of time-to-recurrence data, censoring information, and microarray data from a set of 138 patients, we selected statistically significant genes thought to be predictive of disease recurrence. The number of genes was further reduced by eliminating those whose expression levels were not reproducible by real-time quantitative PCR. Within these variables, a recurrence prediction model was constructed using Cox proportional hazard regression and validated via two independent cohorts (n = 56 and n = 59).Results: After performing a log-rank test of the microarray data and successively selecting genes based on real-time quantitative PCR analysis, the most significant 18 genes had P values of CALB1, MMP7, SLC1A7, GSTA1, CCL19, and IFI44). Two pathologic variables, pStage and cellular differentiation, were developed. Validation by two independent cohorts confirmed that the proposed model is significantly accurate (P = 0.0314 and 0.0305, respectively). The predicted median recurrence-free survival times for each patient correlated well with the actual data.Conclusions: We have developed an accurate, technically simple, and reproducible method for predicting individual recurrence risks. This model would potentially be useful in developing customized strategies for managing lung cancer.

Supplementary Tables 1-8 from Clinical Validity of the Lung Cancer Biomarkers Identified by Bioinformatics Analysis of Public Expression Data

Identification of molecular markers often leads to important clinical applications such as early diagnosis, prognosis, and drug targeting. Lung cancer, the leading cause of cancer-related deaths, still lacks reliable molecular markers. We have combined the bioinformatics analysis of the public gene expression data and clinical validation to identify biomarker genes for non–small-cell lung cancer. The serial analysis of gene expression and the expressed sequence tag data were meta-analyzed to produce a list of the differentially expressed genes in lung cancer. Through careful inspection of the predicted genes, we selected 20 genes for experimental validation using semiquantitative reverse transcriptase-PCR. The microdissected clinical specimens used in the study consisted of three groups: lung tissues from benign diseases and the paired (cancer and pathologic normal) tissues from non–small-cell lung cancer patients. After extensive statistical analyses, seven genes (CBLC, CYP24A1, ALDH3A1, AKR1B10, S100P, PLUNC, and LOC147166) were identified as potential diagnostic markers. Quantitative real-time PCR was carried out to additionally assess the value of the seven identified genes leading to the confirmation of at least two genes (CBLC and CYP24A1) as highly probable novel biomarkers. The gene properties of the identified markers, especially their relationship to lung cancer and cell signaling pathway regulation, further suggest their potential value as drug targets as well. [Cancer Res 2007;67(15):7431–8]

Background Treatment of human lung squamous cell carcinoma (LUSC) using current targeted therapies is limited because of their diverse somatic mutations without any specific dominant driver mutations. These mutational diversities preventing the use of common targeted therapies or the combination of available therapeutic modalities would require a preclinical animal model of this tumor to acquire improved clinical responses. Patient-derived xenograft (PDX) models have been recognized as a potentially useful preclinical model for personalized precision medicine. However, whether the use of LUSC PDX models would be appropriate enough for clinical application is still controversial. Methods In the process of developing PDX models from Korean patients with LUSC, the authors investigated the factors influencing the successful initial engraftment of tumors in NOD scid gamma mice and the retainability of the pathological and genomic characteristics of the parental patient tumors in PDX tumors. Conclusions The authors have developed 62 LUSC PDX models that retained the pathological and genomic features of parental patient tumors, which could be used in preclinical and co-clinical studies. Trial registration Tumor samples were obtained from 139 patients with LUSC between November 2014 and January 2019. All the patients provided signed informed consents. This study was approved by the institutional review board (IRB) of Samsung Medical Center (2018-03-110)

The roles of miRNAs in lung cancer have not yet been explored systematically at the genome scale despite their important regulatory functions. Here, we report an integrative analysis of miRNA and mRNA sequencing data for matched tumor–normal samples from 109 Korean female patients with non‐small‐cell lung adenocarcinoma (LUAD). We produced miRNA sequencing (miRNA‐Seq) and RNA‐Seq data for 48 patients and RNA‐Seq data for 61 additional patients. Subsequent differential expression analysis with stringent criteria yielded 44 miRNAs and 2322 genes. Integrative gene set analysis of the differentially expressed miRNAs and genes using miRNA–target information revealed several regulatory processes related to the cell cycle that were targeted by tumor suppressor miRNAs (TSmiR). We performed colony formation assays in A549 and NCI‐H460 cell lines to test the tumor‐suppressive activity of downregulated miRNAs in cancer and identified 7 novel TSmiRs (miR‐144‐5p, miR‐218‐1‐3p, miR‐223‐3p, miR‐27a‐5p, miR‐30a‐3p, miR‐30c‐2‐3p, miR‐338‐5p). Two miRNAs, miR‐30a‐3p and miR‐30c‐2‐3p, showed differential survival characteristics in the Tumor Cancer Genome Atlas (TCGA) LUAD patient cohort indicating their prognostic value. Finally, we identified a network cluster of miRNAs and target genes that could be responsible for cell cycle regulation. Our study not only provides a dataset of miRNA as well as mRNA sequencing from the matched tumor–normal samples, but also reports several novel TSmiRs that could potentially be developed into prognostic biomarkers or therapeutic RNA drugs.

In this study, we report a novel function of FCN3 (Ficolin 3), a secreted lectin capable of activating the complement pathway, as a tumor suppressor of lung adenocarcinoma (LUAD). First, the expression of FCN3 was strongly down-regulated in cancer tissues compared to matched normal lung tissues, and down-regulation of FCN3 was shown to be significantly correlated with increased mortality among LUAD patients. Interestingly, while ectopic expression of FCN3 led to cell cycle arrest and apoptosis in A549 and H23 cells derived from LUAD, the secreted form of the protein had no effect on the cells. Rather, we found evidence indicating that activation of the unfolded protein response from endoplasmic reticulum (ER) stress is induced by ectopic expression of FCN3. Consistently, inhibition of ER stress response led to enhanced survival of the LUAD cells. Of note, the fibrinogen domain, which is not secreted, turned out to be both necessary and sufficient for induction of apoptosis when localized to ER, consistent with our proposed mechanism. Collectively, our data indicate that FCN3 is a tumor suppressor gene functioning through induction of ER stress.

Summary Exome and transcriptome analyses of clinically homogeneous early-stage never-smoker female patients with lung adenocarcinoma were performed to understand tumor-T cell interactions and immune escape points. Using our novel gene panels of eight functional categories in the cancer-immunity cycle, three distinct subgroups were identified in this immune checkpoint blockade-refractory cohort by defective gene expression in two major domains, i.e., type I interferon production/signaling pathway and antigen-presenting machinery. Our approach could play a critical role in understanding immune evasion mechanisms, developing a method for effective selection of rare immune checkpoint blockade responders, and finding new treatment strategies., Graphical Abstract, Highlights • GMM-based clustering of cancer-immune gene expression helps to find LUAD classifiers • Classifiers are found in the earlier-phased cancer-immunity cycle • Classifiers include ubiquitination, Ag-presenting machinery, and type I IFN signaling • Tumor-intrinsic classifiers strongly influence the outcome of cancer immunity, Immunology; Cancer Systems Biology; Genomics

In this study, we describe a novel function of TNNC1 (Troponin C1, Slow Skeletal and Cardiac Type), a component of actin-bound troponin, as a tumor suppressor of lung adenocarcinoma (LUAD). First, the expression of TNNC1 was strongly down-regulated in cancer tissues compared to matched normal lung tissues, and down-regulation of TNNC1 was shown to be strongly correlated with increased mortality among LUAD patients. Interestingly, TNNC1 expression was enhanced by suppression of KRAS, and ectopic expression of TNNC1 in turn inhibited KRAS G12D-mediated anchorage independent growth of NIH3T3 cells. Consistently, activation of KRAS pathway in LUAD patients was shown to be strongly correlated with down-regulation of TNNC1. In addition, ectopic expression of TNNC1 inhibited colony formation of multiple LUAD cell lines and induced DNA damage, cell cycle arrest and ultimately apoptosis. We further examined potential correlations between expression levels of TNNC1 and various clinical parameters and found that low-level expression is significantly associated with invasiveness of the tumor. Indeed, RNA interference-mediated down-regulation of TNNC1 led to significant enhancement of invasiveness in vitro. Collectively, our data indicate that TNNC1 has a novel function as a tumor suppressor and is targeted for down-regulation by KRAS pathway during the carcinogenesis of LUAD.

Liquid biopsy isolation of circulating tumor cells (CTCs) allows the genomic analysis of CTCs, which is useful in the determination of personalized cancer therapy. In the present study, CTCs from patients with breast cancer were enriched and successfully analyzed using cancer gene panel analysis. Blood samples from 11 patients with breast cancer were collected and CTCs enriched for using size-based filtration. The enriched CTCs were analyzed using immunofluorescence staining with antibodies directed against epithelial cell adhesion molecule (EpCAM) and cluster of differentiation 45. The genomic DNA of CTCs was extracted, amplified and 50 genes screened for mutations using the Ion AmpliSeq™ Cancer Hotspot Panel v2. EpCAM staining detected CTCs in 10/11 patients and the average CTC count was 3.9 in 5 ml blood. The average purity of enriched CTCs was 14.2±29.4% and the average amount of amplified DNA was 28.6±11.9 µg. Catalogue Of Somatic Mutations In Cancer mutations were detected in the CTCs and included IDH2, TP53, NRAS, IDH1, PDGFRA, HRAS, STK11, EGFR, PTEN, MLH1, PIK3CA, CDKN2A, KIT and SMARCB1. In conclusion, a novel size-based filtration approach for the isolation of CTCs was evaluated and successfully applied for the genomic analysis of CTCs from patients with breast cancer.

Additional file 3: Table S3. Clinical characteristics between XALD and successful PDX engraftment.

Additional file 1: Table S1. The clinical chracteristics of LUSC patients.

Additional file 6: Figure S1. Rate of successful engraftment along the LUSC PDX passages.

Additional file 4: Table S4. The freqency of somatic mutations in LUSC patients and PDX models.

Additional file 5: Table S5. Somatic mutations and Differently expression genes (DEGs) in LUSC patients and PDX models.

SAdditional file 2: Table S2. Primary antibodies used in the study.

Area-selective atomic layer deposition has attracted considerable interest as a means for enabling versatile fabrication of selectively formed thin films in both vertical and lateral direction in extremely downscaled 3D semiconductor devices. Herein, we report a methodology for an enhanced selectivity ALD process with a discrete feeding method (DFM), capable of improving the chemisorption density of small alkylating agents onto desired substrates. Using the DFM strategy of (N, N-diethylamino)trimethylsilane as an aminosilane inhibitor to render surface alkyl groups, we explored its efficacy with regard to improving its blocking capability against subsequent Ru ALD on various substrates such as Si, SiO2, and SiN substrates to provide H-, OH-, and NHx-terminated surface groups, respectively. As a result, a denser chemisorption layer could be selectively obtained on OH-terminated SiO2 and NH-terminated SiN but not on H-terminated Si, which in turn led to significant growth retardation on both SiO2 and SiN during Ru ALD. Adding a process sequence composed of intermittent Ru etching followed by DFM retreatment on both SiO2 and SiN allowed for selective removal of Ru moieties and restoration of inhibitory alkyl groups. In this way, we finally achieved enhanced deposition selectivity with a combination of sequential DFM and Ru ALD-etch supercycles.

Many phthalic acid esters (PAEs), including DMP, DEP, DBP, BBP, and DEHP, as well as DEHA are widely used as plasticizers in plastics. An analytical method was developed and used to analyze these compounds at 41 industrial facilities. The coefficient of determination (R) for each constructed curve was higher than 0.98. The method detection limit (MDL) values were 0.4–0.7 μg/L for PAEs and 0.6 μg/L for DEHA. In addition, the recovery rate was shown to be 77.0–92.3%, while the relative standard deviation was shown to be in the range of 5.8-10.5%. DMP (n = 3), DEP (n = 2), DBP (n = 2), BBP (n = 2), and DEHA (n = 3) were detected in the range of 2.2-11.1% in the influent. DEHP was a predominant compound and was detected at > MDL in both the influent (n = 16, 35.6%) and the effluent (n = 4, 10.0%) at a high removal efficiency (92–100%). The highest levels of residue in industrial wastewater influent were 137.4 μg/L of DEHP at plastic products manufacturing facility, 12.5 μg/L of DEHA at a chemical manufacturing facility, and 14.0 μg/L of DEP at an electronics facility. The highest concentration of effluent was 12.5 μg/L of DEHP at a chemical manufacturing facility, which indicated that the effluent was below the allowable concentration (800 μg/L). Therefore, the levels of PAEs and DEHA that are discharged into nearby streams could not influence the health of the ecosystem. 요 약: 본 연구에서는 플라스틱의 가소제로 널리 사용되고 있는 프탈산에스테르(DMP, DEP, DBP, BBP, ★ Corresponding author Phone : +82-(0)32-560-7686 Fax : +82-(0)32-560-7905 E-mail : boystone@korea.kr This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons. org/licenses/ by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 58 Hyesung Kim et al. Analytical Science & Technology DEHP) 5종과 디에틸헥실아디페이트(DEHA)의 수질 중 분석법을 확립하고, 41 개 지점의 산업폐수를 분 석하였다. 검정곡선은 분석대상성분 모두 결정계수 0.98이상으로 수질오염공정시험기준을 만족하는 직선 성을 나타내었으며, 방법검출한계(MDL)는 프탈산에스테르가 0.4~0.7 μg/L, 디에틸헥실아디페이트가 0.6 μg/L를 수준이었다. 그리고 회수율은 77.0~92.3 %였으며, 상대표준편차는 5.8~10.5 % 범위였다. 41개 지 점의 산업폐수 중 유입수 45점과 방류수 40점을 분석한 결과, 유입수에서 DMP (n=5), DEP (n=2), DBP (n=1), BBP (n=2), DEHA (n=3)의 검출율은 2.2~11.1 %였다. DEHP는 유입수 (n=16)에서 35.6 %, 방류수 (n=4)에서 10.0 %을 나타내 92~100 %의 높은 제거효율을 보였다. 산업폐수 업종별 유입수 중 최고 잔류 수준은 플라스틱제품제조업 137.4 μg/L (DEHP), 기타화학제품 제조업 12.5 μg/L(DEHA), 전기업 14.0 μg/ L(DEP)이었다. 방류수 중 최고농도는 기초화학물질 제조업 2.1 μg/L(DEHP)이었다. 전 지점에서의 DEHP 는 수질오염물질의 배출허용기준(특례지역 800 μg/L)을 초과하지 않았다. 따라서 인근 하천에 흐르는 산 업폐수 중 프탈산에스테르(Phthalic Acid Esters)와 디에틸헥실아디페이트가 수생태계환경에 미치는 영향 이 크지 않을 것이다.

Sciatica can occur due to a spinal lesion, intrapelvic tumor, diabetic neuropathy, and rarely piriformis syndrome. The causes of piriformis syndrome vary by a space-occupying lesion. A ganglionic cyst can occur in various lesions in the body but seldom around the hip joint. In addition, sciatica due to a ganglionic cyst around the hip joint has been reported in one patient in Korea who underwent surgical treatment. We experienced two cases of sciatica from a piriformis ganglionic cyst and we report the clinical characterics and progress after non-operative treatment by ultrasonography-guided aspiration. The two cases were diagnosed by magnetic resonance imaging and were treated by ultrasonography-guided aspiration. We followed the patients for more than 6 months. The symptoms of piriformis syndrome from the ganglion improved following aspiration and this conservative treatment is a treatment method that can be used without extensive incision or cyst excision. Level of evidence Level IV historical case.

Background To identify predictive markers for responders in lapatinib-treated patients and to demonstrate molecular changes during lapatinib treatment via cell-free genomics. Patients and methods We prospectively evaluated the efficacy of combining lapatinib with capecitabine and oxaliplatin as first line neoadjuvant therapy in patients with previously untreated, HER2-overexpressing advanced gastric cancer. A parallel biomarker study was conducted by simultaneously performing immunohistochemistry and next-generation sequencing (NGS) with tumor and blood samples. Results Complete response was confirmed in 7/32 patients (21.8%), 2 of whom received radical surgery with pathologic-confirmed complete response. Fifteen partial responses (46.8%) were observed, resulting in a 68.6% overall response rate. NGS of the 16 tumor specimens demonstrated that the most common co-occurring copy number alteration was CCNE1 amplification, which was present in 40% of HER2+ tumors. The relationship between CCNE1 amplification and lack of response to HER2-targeted therapy trended toward statistical significance (66.7% of non-responders versus 22.2% of responders harbored CCNE1 amplification; P = 0.08). Patients with high level ERBB2 amplification by NGS were more likely to respond to therapy, compared with patients with low level ERBB2 amplification (P = 0.02). Analysis of cfDNA showed that detectable ERBB2 copy number amplification in plasma was predictive to the response (100%, response rate) and changes in plasma-detected genomic alterations were associated with lapatinib sensitivity and/or resistance. The follow-up cfDNA genomics at disease progression demonstrated that there are emergences of other genomic aberrations such as MYC, EGFR, FGFR2 and MET amplifications. Conclusions The present study showed that HER2+ GC patients respond differently according to concomitant genomic aberrations beyond ERBB2, high ERBB2 amplification by NGS or cfDNA can be a positive predictor for patient selection, and tumor genomic alterations change significantly during targeted agent therapy.

Organic electronics devices could have many advantages such as flexibility, simple process and low fabrication cost. Minimizing damage to organic layers during sputtering is important for the improvement of organic optoelectronic performance and lifetime. In order to achieve improved device performance, degradation of organic layers, which could be occurring during manufacturing, should be suppressed. Among the methods available for the fabrication of transparent electrodes, sputtering deposition possess many advantage over others, such as simple apparatus and procedure, high deposition rate, and wide deposition area. [1] Because of its unique structure, the use of a facing targets sputtering (FTS) system could effectively reduce damage during sputtering. [2] In this study, we prepared transparent, organic, GAZO thin films deposited onto Alq3 using an FTS system for the purpose of investigating the damage caused to organic layer by sputter deposition. To measure the damage sustained by organic materials d...

Objectives Lung adenocarcinoma (LUAD) is a common subtype of non-small cell lung cancer prevalent in Asia. There is a dearth of understanding regarding the transcriptome landscape of LUAD without primary known driver mutations. In this study, LUAD samples without well-known driver mutations occurring in EGFR, KRAS, ALK, ROS1 or RET (quintuple-negative) were used for transcriptome study with a focus on long noncoding RNAs (lncRNAs), alternative splicing and gene fusions. Materials and methods 24 pairs of LUAD and adjacent normal samples and 13 tumor-only samples derived from 37 quintuple-negative patients were used. Differentially expressed lncRNA transcripts were detected by paired t-test and were validated by qPCR. Functions of lncRNAs were predicted by co-expressed mRNAs. Aberrant splicing events in LUAD were identified using MISO. In addition, gene fusions were screened by SOAPfuse. Results and conclusion In total, 90 and 153 up- or down-regulated lncRNA transcripts were detected in LUAD samples in comparison with the adjacent normal samples. The most significantly differentially expressed lncRNA transcript was ENST00000598996.1 (FENDRR) down-regulated in LUAD. By lncRNA-mRNA co-expression analysis, functions of 14 lncRNAs were predicted. The predicted functions included vasculature development, immune response, cell cycle and respiratory gaseous exchange. Furthermore, six co-expressed pairs of lncRNAs and their nearby protein coding genes were identified as associated with lung development. This study also identified two highly recurrent (22 in 24) differential exon skipping events occurring in MYH14 and ESYT2 with exon including isoforms of both genes up-regulated in isoform percentage in LUAD samples. On the other hand, two out of 24 LUAD samples possessed the driver mutation exon 14 skipping of MET. The transcriptional alterations of LUAD samples without well-known driver mutations identified in the study can be used as references for future research. The translational values of these transcriptional changes are also worthy of further investigation.

Background Circulating tumor DNA (ctDNA) analysis has been successfully applied to therapy selection and treatment monitoring in advanced cancer patients. However, it is not yet established whether ctDNA can be used clinically for early cancer detection or recurrence prediction in early stage lung cancer patients. Methods We analyzed pre-operative plasma samples from 55 early stage NSCLC patients (stages I-IIIA) using next-generation sequencing assay incorporating somatic and epigenomic analysis, and a bioinformatic classifier to filter non-tumor derived variants. Table: 111P . Cell type Stage Somatic mutation Epigenetic pattern Total number Recurrence+, n (%) Site of recurrence Adenocarcinoma stage 1 ctDNA- methylation- 9 1 (11) Lung n = 17 methylation+ 6 2 (33.3) Stump, bone ctDNA+ 2 1 (50) lung stage 2 ctDNA- methylation- 0 0 (0) n = 2 methylation+ 0 0(0) ctDNA+ 1 1 (100) multiple stage 3 ctDNA- methylation- 0 0 (0) n = 4 methylation+ 2 0 (0) ctDNA+ 2 2 (100) brain, multiple Sqaumous cell carcinoma stage1 ctDNA- methylation- 0 0 (0) n = 7 methylation+ 3 0 (0) ctDNA+ 4 1 (25) multiple stage2 ctDNA- methylation- 0 0 (0) n = 9 methylation+ 0 0 (0) ctDNA+ 9 2 (22.2) multiple, lung stage3 ctDNA- methylation- 0 0 (0) n = 4 methylation+ 1 0 (0) ctDNA+ 3 1 (33.3) Mediastinal LNs Results Somatic mutation analysis alone detected ctDNA in 42% (23/55) of patients, whereas combined mutational and epigenomic analysis detected ctDNA in 67% (37/55). ctDNA detection rate varied by pathological subtypes; using combined approach, ctDNA was detected in all squamous cell carcinoma patients, while only 55% (12/22) in adenocarcinoma (ADC) (p=0.006). Within the ADC subgroup, ctDNA detection rates using the combined approach were dependent on disease stage: 47% (8/17) in stage I, 100% (2/2) in stage II, and 100% (2/2) in stage IIIA. Importantly, within 2 years of follow-up, pre-operative ctDNA status was correlated with tumor recurrence after resection; among 17 stage I ADC patients, three of eight (38%) ctDNA-positive cases showed recurrence, while only one of nine (11%) ctDNA-negative cased did (p=0.29). Interestingly, patients with somatic mutation in their ctDNA have shown higher recurrence rate. Conclusion Utilizing a plasma-only sequencing assay incorporating somatic genomic and epigenomic analysis, ctDNA detection rate in early stage lung cancer (stage I-III) can far outperform the detection rate of somatic sequence variant detection alone. And, the presence of pre-operative ctDNA in patients with early stage lung adenocarcinoma may identify those who are more likely to have disease recurrence. Legal entity responsible for the study: Guradant Health, Inc. Funding Guardant Health, Redwood City, CA, USA. Disclosure I. Kim: Full / Part-time employment, Officer / Board of Directors: Guardant Health. M. Shultz: Officer / Board of Directors: Guardant Health. A. Jaimovich: Officer / Board of Directors: Guardant Health. J. Odegaard: Officer / Board of Directors: Guardant health, Inc. S. Olsen: Officer / Board of Directors: Guardant Health, Inc. A. Talasaz: Officer / Board of Directors: Guardant health. J. Kim: Research grant / Funding (self): Guardant health, Inc. All other authors have declared no conflicts of interest.

e14557 Background: Circulating tumor DNA (ctDNA) analysis has been successfully applied to therapy selection and treatment monitoring in advanced cancer patients. However, it is not yet established whether ctDNA can be used clinically for early cancer detection or predicting tumor recurrence in early stage lung cancer patients. Methods: We analyzed pre-operative plasma samples from 55 early stage NSCLC patients (stages I-IIIA) using next-generation sequencing to detect somatic mutations and differential epigenomics patterns, including methylation signatures. Results: Using somatic mutation analysis alone, ctDNA was detected in 42% (23/55) of patients, whereas combined mutational and epigenomic analysis detected ctDNA in 71%. ctDNA detection rate also varied markedly between lung squamous cell carcinoma (SCC) and adenocarcinoma (ADC);using combined analysis of somatic mutations and epigenomic patterns, ctDNA was detected in all SCC patients, while only 55% of ADC (12/22) were ctDNA-positive (p= 0.006). Within the ADC subgroup, ctDNA detection rates using the combined approach were dependent on disease stage: 47% (8/17) in stage I, 100% (2/2) in stage II, and 100% (2/2) in stage IIIA. Importantly, pre-operative ctDNA status was correlated with tumor recurrence post-resection; three of eight (38%) ctDNA-positive stage I ADC patients recurred within 2 years of resection, while only one of nine (11%) ctDNA-negative stage I ADC patients recurred (p= 0.29). Conclusions: Taken together, we show that the combination of somatic mutation detection and epigenomic analysis outperforms each individual biomarker in the detection of ctDNA in early stage lung cancer. Importantly, we also demonstrate that pre-operative ctDNA detection may identify a high-risk population of early stage lung cancer patients that may benefit from (neo)adjuvant therapy.

The sixty nine chemicals that are closely linked to chemical accident are designated as the substances requiring preparation for accidents and managed for public health under the Toxic Chemicals Control Act. In this study, storage stability of allyl chloride (AC) and carbon disulfide (CD), which are highly inflammable and volatile in tedlar bags, was studied for gaseous chemicals sampling. Storage stability was studied considering storage temperature (2 oC, 25 oC), chemical concentration (low conc. ppm, high conc. ppm) and storage time (0, 48, 96, and 144 hr). Also, the stability of bags containing one type of chemical substance and the bags containing a mixture of chemicals was compared against each other. As a result, two chemicals showed decreasing storage stability based on storage time. Also two chemicals presented statistical significance of concentration and mixing type. 요 약: 본 연구는 유해화학물질관리법에 지정되어 있는 사고대비물질 중 휘발성과 화재위험성 등급이 높은 알릴클로라이드(allyl chloride, AC)와 이황화탄소(carbon disulfide, CD)에 대하여 테들라 백에서의 보존 특성을 연구하였다. 테들라 백에서의 시료 보존성을 연구하기 위하여 시료 보존성에 영향을 줄 수 있는 시료보관 온도(2 oC, 25 oC), 물질 농도(저농도, 고농도), 보관 시간(0 시간, 48 시간, 96시간, 144시 간), 단일물질과 혼합물질 등의 영향인자를 비교하여 연구하였다. 테들라 백 내에서의 시료 보존율은 시 간 경과에 따라 감소하였고 각 영향인자에 대한 T-검정결과 농도와 혼합형태에 따라 유의한 차이를 보 였다. 본 연구결과는 테들라 백을 사용하여 시료 채취 시 대상물질에 대한 정량적 분석정보를 제공하는 데 도움이 될 것으로 판단된다.

Background: Inflammation is a key regulatory process in breast cancer progression and severity. Several studies have demonstrated that prolonged exposure of breast tumor cells to inflammatory cytokines leads to epithelial-mesenchymal transitions (EMT), which is the principle mechanism involved in metastasis and tumor invasion. Disruption of the signaling pathways involved in EMT may therefore provide an effective treatment strategy for currently difficult to treat or untreatable cancers such as TNBC. The interleukin (IL)-1 plays a pivotal role on breast cancer proliferation, invasion and/or inflammation. Here, we investigated the correlation of MMP-3 and IL-8 on IL-1b-induced cell migration and invasion as well as the inhibitory effect of zerumbone on IL-1β-induced MMP-3 and IL-8. Methods: Triple negative breast cancer cells (Hs578T and MDA-MB231) were cultured DMEM with 10% FBS and 1% antibiotics. The levels of IL-8 and MMP-3 mRNA were analyzed by real-time PCR. The levels of secreted IL-8 and MMP-3 protein expression were analyzed by ELISA and western blot analysis, respectively. Cell viabilities by drug were analyzed by MTT assay. Cell invasion and migration was detected by Boyden chamber assay. Results: The level of IL-8 and MMP-3 mRNA and protein expression significantly increased by IL-1β treatment in both Hs578T cells and MDA-MB231 cells. In addition, IL-1β-induced cell migration and invasion also increased in Hs578T and MDA-MB231 cells. On the other hand, the levels of basal and IL-1β-induced IL-8 and MMP-3 expression were decreased by zerumbone. Conclusion: IL-1β induces the migration and invasion of breast cancer cells through IL-8 and MMP-3 expression. These effects are significantly suppressed by zerumbone. Therefore, we suggest that zerumbone may act as a useful therapeutic agent for treatment of triple negative breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-19.

Background: Endocrine therapy has dramatic improvements of prognosis in patients with hormonal receptor positive breast cancer. However, long-term follow up shows that endocrine resistance is still major clinical concern. Although the value of estrogen receptor (ER) level can predict response to endocrine treatment in hormonal receptor positive breast cancers, few markers are available that can predict response to endocrine treatment. We attempted to identify molecular markers, especially progesterone receptor (PR), associated with endocrine failure in breast cancer. Methods: We reviewed the medical records of 2513 breast cancer patients who underwent breast surgery and endocrine therapy at Samsung Medical Center between March 2007 and July 2011. Patients were compared according to ER and PR expression and to assess the clinical and biological features of ER+positive/PR-negative breast cancers to understand how PR might be a useful marker of these activities. ER and PR expression accessed using Allred score, the ‘positive” was defined that total score is larger than 2. Results PR negative tumors were found more frequently in postmenopausal women (post- 11.2% vs. pre- 2.5%, P < 0.001) and PR negative tumors were significantly associated with higher grade, higher level of Ki 67 and more expression of EGFR and CK5/6. In Kaplan-Meier analysis and multivariate analysis, PR negativity was significantly poorer prognostic factor of DFS (HR 3.1, 95% CI 1.2-8.2, P = 0.021) and OS (HR 89.8, 95% CI 3.4-2352.3, P = 0.007). Stratified by the menopausal status, there was not shown significant difference of survival between the PR+ and PR- tumor in postmenopausal women (DFS, HR 2.1, 95% CI 0.4-10.8, P = 0.381; OS, HR 10.6, 95% CI 0.6-171.4, P = 0.096). However, PR negative tumors showed an association with poor outcome in premenopausal women (DFS, HR 7.2, 95% CI 2.5-20.6, P Conclusion: In HR positive breast cancers, PR negative tumors were found more frequently in elderly, postmenopausal women. However, PR negative tumors are shown more aggressive and poorer survival than PR positive tumors despite of endocrine therapy, especially in premenopausal women. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-17.

An increasing number of chromosomal aberrations is being identified in solid tumors providing novel biomarkers for various types of cancer and new insights into the mechanisms of carcinogenesis. We applied next generation sequencing technique to analyze the transcriptome of the non-small cell lung carcinoma (NSCLC) cell line H2228 and discovered a fusion transcript composed of multiple exons of ALK (anaplastic lymphoma receptor tyrosine kinase) and PTPN3 (protein tyrosine phosphatase, nonreceptor Type 3). Detailed analysis of the genomic structure revealed that a portion of genomic region encompassing Exons 10 and 11 of ALK has been translocated into the intronic region between Exons 2 and 3 of PTPN3. The key net result appears to be the null mutation of one allele of PTPN3, a gene with tumor suppressor activity. Consistently, ectopic expression of PTPN3 in NSCLC cell lines led to inhibition of colony formation. Our study confirms the utility of next generation sequencing as a tool for the discovery of somatic mutations and has led to the identification of a novel mutation in NSCLC that may be of diagnostic, prognostic, and therapeutic importance.

Purpose: The purpose of this study is to determine the long-term significance of internal mammary lymph node (IMLN) detected by lymphoscintigraphy. Background IMLN metastasis is an important prognostic indicator in breast cancer. However, the necessity of internal mammary sentinel lymph node (IMSLN) biopsy for accurate staging, for choosing adjuvant treatment, and as a prognostic indicator, has remained controversial. Methods: From January 2001 until December 2006, 525 female breast cancer patients underwent radical surgery after preoperative lymphatic scintigraphy. We retrospectively analyzed the follow-up results, recurrences and deaths of all patients. Results: There was no significant difference in clinicopathologic characteristics between the axilla and the IMLN group. The median follow-up period was 118.8 (range, 7–122) months in the axilla group and 107.7 (range, 14–108) months in the IMLN group. During the median follow-up period, the breast cancer-related death rate in the axilla group was 3.6%, which was not significantly different from that of the IMLN group (1.3%) (P = 0.484). The five-year survival rates (5YRS) did not differ between the two groups (P = 0.306). The overall recurrence rate and the locoregional (LR) recurrence rate also did not differ between the two groups (P = 0.835 and P = 0.582, respectively. The recurrence rate of IMLN (both ipsilateral and contralateral) metastasis was very low, accounting for 0.5% in the axilla group and 1.3% in the IMLN group (P = 0.416). Conclusion: The long-term follow up results showed that there was no significant difference in both overall outcome and regional recurrence between the two groups. Therefore, the necessity of identification of nodal basins outside the axilla or the necessity of IMLN sentinel biopsy should be reconsidered. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-07-19.

Purpose: This study was performed to discover prognostic genomic markers associated with postoperative outcome of stage I to III non–small cell lung cancer (NSCLC) that are reproducible between geographically distant and demographically distinct patient populations. Experimental Design: American patients (n = 27) were stratified on the basis of recurrence and microarray profiling of their tumors was performed to derive a training set of 44 genes. A larger Korean patient validation cohort (n = 138) was also stratified by recurrence and screened for these genes. Four reproducible genes were identified and used to construct genomic and clinicogenomic Cox models for both cohorts. Results: Four genomic markers, DBN1 (drebrin 1), CACNB3 (calcium channel beta 3), FLAD1 (PP591; flavin adenine dinucleotide synthetase), and CCND2 (cyclin D2), exhibited highly significant differential expression in recurrent tumors in the training set (P < 0.001). In the validation set, DBN1, FLAD1 (PP591), and CACNB3 were significant by Cox univariate analysis (P ≤ 0.035), whereas only DBN1 was significant by multivariate analysis. Genomic and clinicogenomic models for recurrence-free survival (RFS) were equally effective for risk stratification of stage I to II or I to III patients (all models P < 0.0001). For stage I to II or I to III patients, 5-year RFS of the low- and high-risk patients was approximately 70% versus 30% for both models. The genomic model for overall survival of stage I to III patients was improved by addition of pT and pN stage (P < 0.0013 vs. 0.010). Conclusion: A 4-gene prognostic model incorporating the multivariate marker DBN1 exhibits potential clinical utility for risk stratification of stage I to III NSCLC patients. Clin Cancer Res; 17(9); 2934–46. ©2011 AACR.

This prospective study explored the relationship between expression of AKR1B10 mRNA and various clinical parameters in non-small-cell lung cancer (NSCLC) in terms of its validation as a marker for NSCLC. Tumour tissue samples were collected from 229 patients with NSCLC. Tissue samples from adjacent non-malignant lung tissue (> 5 cm from the tumour) of 89 of these patients and samples from 20 patients with benign lung disease were used as controls. Quantitative reverse transcription–polymerase chain reaction showed significantly higher levels of AKR1B10 mRNA expression in NSCLC tumour tissue than in adjacent non-malignant lung tissue and benign lung tissue. Statistically significant factors for AKR1B10 mRNA over-expression were found to be male gender, smoking, squamous cell carcinoma and moderate or poor cell differentiation. It is concluded that AKR1B10 seems to have potential as a prognostic marker for NSCLC and warrants further investigation.