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Prior to registering and marketing any new pharmaceutical, (agro)chemical or food ingredient product manufacturers must, by law, generate data to ensure human safety. Safety testing requirements vary depending on sector, but generally repeat-dose testing in animals form the basis for human health risk assessments. Dose level selection is an important consideration when designing such studies, to ensure that exposure levels that lead to relevant hazards are identified. Advice on dose level selection is provided in test guidelines and allied guidance documents, but it is not well harmonised, particularly for selection of the highest dose tested. This paper further builds on concepts developed in a technical report by the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) which recommends pragmatic approaches to dose selection considering regulatory requirements, animal welfare and state of the art scientific approaches. Industry sectors have differing degrees of freedom to operate regarding dose level selection, depending on the purpose of the studies and the regulatory requirements/legislation, and this is reflected in the overall recommended approaches. An understanding of systemic exposure should be utilised where possible (e.g., through toxicokinetic approaches) and used together with apical endpoints from existing toxicity studies to guide more appropriate dose level selection. The highest dose should be limited to a reasonable level, causing minimal but evident toxicity to the test animals without significantly compromising their well-being. As the science of predictive human exposure further develops and matures, this will provide exciting and novel opportunities for more human-relevant approaches to dose level selection.

Recently, many attempts have been made to use injectable materials in the subcutaneous fat layer anywhere in the body, including the breast and face, for cosmetic purposes. A 56-year-old woman presented with multiple palpable lumps without tenderness or skin color changes on the anterior and lateral chest and the abdominal walls. Magnetic resonance imaging showed fluid-like collections without surrounding soft tissue inflammatory changes in the chest wall, abdominal wall, and deeper within the abdomen. The lesions penetrated the peritoneum and were observed adjacent to the liver dome. Ultrasonography also showed hypoechogenicity suggestive of fluid collection in the left axilla and trunk. The differential diagnosis based on radiologic findings included parasite manifestation, non-specific inflammatory conditions, and chronic granulomatous infections such as tuberculosis or non-tuberculous mycobacterial infections. However, these conditions are usually accompanied by changes in the adjacent subcutaneous fat layers, but our patient did not show any other abnormalities in the adjacent soft tissue. After biopsy and aspiration analysis, the patient was found to have a history of filler injection for breast augmentation approximately 17 years prior. It is often difficult to make a differential diagnosis without detailed knowledge of the patient's medical history. Here we describe a rare case of distant migration of the filler to the axilla, chest wall, abdominal wall, and peritoneum following breast augmentation with filler injection. Knowledge of the radiologic characteristics and migration patterns of gel fillers and their related complications is useful for making an accurate diagnosis.

This study aims to identify the acoustic and perceptual properties that contribute to identifying the meaning of Korean sentences that are ambiguous between wh-question and yes-no question readings. While in most cases the Accentual Phrase (AP) tonal pattern (Jun 1993) differs between the two question readings, there are cases where the two readings are predicted to have the same AP tonal pattern. However, our experimental results indicate that even in those cases a typical AP tonal contrast between the two question interpretations, i.e. the presence vs. absence of the tone in the syllable that immediately follows the wh-word, is observed in production and plays a meaningful role in perception. The results suggest that there is a production and processing strategy to utilize a consistent contrast in accentual phrasing between the two types of questions for disambiguation.

The transmembrane 4 L six family member 5 (TM4SF5) is aberrantly expressed in hepatocellular and colorectal cancers, and has been implicated in tumor progression, suggesting that it could serve as a novel therapeutic target. Previously, we screened a murine antibody phage-display library to generate a novel monoclonal antibody, Ab27, that is specific to the extracellular loop 2 of TM4SF5. In this study, we evaluated the effects of chimeric Ab27 using cancer cells expressing endogenous TM4SF5 or stably overexpressing TM4SF5 in vivo and in vitro. Monotherapy with Ab27 significantly decreased tumor growth in liver and colon cancer xenograft models, including a sorafenib-resistant model, and decreased the phosphorylation of focal adhesion kinase (FAK), p27Kip1, and signal transducer and activator of transcription 3 (STAT3). No general Ab27 toxicity was observed in vivo. Combination treatment with Ab27 and sorafenib or doxorubicin exerted higher antitumor activity than monotherapy. In addition, we humanized the Ab27 sequence by the complementarity-determining region (CDR) grafting method. The humanized antibody Ab27-hz9 had reduced immunogenicity but exhibited target recognition and antitumor activity comparable with those of Ab27. Both Ab27 and Ab27-hz9 efficiently targeted tumor cells expressing TM4SF5 in vivo. These observations strongly support the further development of Ab27-hz9 as a novel therapeutic agent against liver and colorectal cancers.

Amniotic products are potent immunomodulators used clinically to repair tissue injury. Little information exists regarding the potential of cell-free human amniotic fluid (hAF) to treat cardiovascular disease. Herein, we sought to determine the influence and efficacy of acellular hAF on myocardial ischemia-reperfusion injury. Processed hAF was obtained from volunteer donors at the time of elective caesarean section and manufactured using proprietary methods. Left anterior descending coronary artery ligation was performed on rats for 60 min. Thirty minutes after release and reperfusion, either saline or hAF was injected intramyocardially. Serial echocardiography revealed that compared with saline-injected rats, hAF animals maintained their ejection fraction and did not adversely remodel through the 4-wk period. This preserved ventricular function correlated with decreased infarct size, less fibrosis, and reduced expression of cytokines and infiltrating inflammatory cells. Comparative arrays of different donor hAF lots confirmed the presence of a wide array of immunomodulatory and host-defense proteins. The observed functional cardioprotection was furthermore evident when given intravenously and across multiple hAF donors. In conclusion, our data demonstrate, for the first time, the cardioprotective effect of acellular hAF on myocardial injury. These observations spanned across diverse donors and likely result from the mixture of a plethora of naturally produced cytokines, chemokines, and immune-modulating proteins rather than a single, defined mechanistic culprit. The ubiquitous availability of hAF as a cell-free solution further suggests its potential for widespread adoption as a therapy for myocardial ischemia-reperfusion injury.

Purpose The objective is to develop a real-time Mitochondrial ATP contents and Response Against Inhibiting and Stimulating Substrates (MitoRAISE) assay and assess its potential to evaluate the mitochondrial oxidative phosphorylation function. Methods MitoRAISE measures the total ATP contents, ATP synthesis capacity, and the response to inhibitory substrates. The measurements were all quantified with an ATP standard curve and validated with in vitro testing. MitoRAISE was then applied using peripheral blood mononuclear cells (PBMCs) from 35 healthy volunteers and 20 breast cancer patients. Result Results from isolated mitochondria, cells with different permeabilization percentages, and cells with damaged mitochondria demonstrated the ability of capturing ATP signals specifically from the mitochondria. Breast cancer PBMCs exhibited a significant increase in glutamic acid- and malic acid-induced mitochondrial ATP synthesis capacity yet a significant decrease in mitochondrial DNA copy number (mtDNA CN) compared to healthy PBMCs. Furthermore, breast cancer PBMCs mostly showed negative correlation between mtDNA CN and parameters from MitoRAISE but healthy individuals showed positive correlation. Conclusion We developed a quick and easy method to detect real-time mitochondrial activity in live cells. Monitoring mitochondrial ATP synthesis capacity and sensitivity to inhibitory substrates could aid in assessing the functional status of mitochondrial oxidative phosphorylation.

A Gram-negative, non-motile, and reddish-orange-colored bacterium, designated CAU 1643T, was isolated from a mudflat collected in Ganghwa Island, Republic of Korea. The bacterium was found to grow optimally at 30°C, pH 9.0–9.5, and with 0–1% (w/v) NaCl. The highest 16S rRNA gene sequence similarities to the bacterium were Algoriphagus kandeliae XY-J91T (97.9%), Algoriphagus aquimaris F21T (97.1%), Algoriphagus formosus XAY3209T (97.0%) and Algoriphagus marincola DSM 16067T (96.2%). The DNA G + C content of the type strain was 40.35 mol%. The average nucleotide identity and digital DNA–DNA hybridisation values between strain CAU 1643T and the reference strains were below the threshold value for species demarcation. The predominant cellular fatty acids were iso-C15:0, iso-C17:0 3-OH and Summed Feature 9. The major respiratory quinone was menaquinone-7. The genome showed three putative biosynthetic gene clusters (BGCs) that are responsible for different secondary metabolites. Moreover, CAU 1643T contains 72 genes that encode carbohydrate-active enzymes. Based on phenotypic, phylogenetic, and chemotaxonomic evidence, strain CAU 1643T represents novel species in the genus Algoriphagus, for which the name Algoriphagus limi sp. nov. is proposed. The type strain is CAU 1643T ( = KCTC 92080T, = MCCC 1K07150T).

Background COVID-19 has affected innumerable aspects of life, including education, economy, and religion. Economic problems and inequality are associated with poor mental health in adolescents. This study aimed to identify the relationship between economic damage to families due to COVID-19 and various mental health problems in Korean adolescents and to evaluate the risk factors of mental health. Methods In total, 54,948 Korean adolescent students from 398 middle and 395 high schools were surveyed between August and November 2020. Complex sample logistic regression was performed to calculate odds ratios (ORs) and 95% confidence intervals (CI) for depression and suicidal ideation, respectively. A generalized linear model analysis was used to examine the association between mental health (unhappiness, loneliness, and stress) and the economic impact of COVID-19. Analyses were adjusted for age, gender, school grade, perceived academic achievement, perceived family economic status, and economic support. Results The ORs of depression (OR = 1.77, 95% CI:1.57–2.00), suicidal ideation (OR = 2.14, 95% CI:1.84–2.50), unhappiness (OR = 1.51 95% CI 1.42–1.60) and lonely (OR = 1.38 95% CI 1.27–1.49) for the low level of perceived family economic status was higher compared to middle level. Adolescents who experienced economic deterioration in their households as COVID-19 showed a higher risk of depression (OR = 1.42, 95% CI:1.35–1.49), suicide ideation (OR = 1.36, 95% CI:1.28–1.44), unhappiness (OR = 2.23 95% CI 2.19–2.27), lonely (OR = 1.20 95% CI 1.17–1.22), and stress (OR = 1.14 95% CI 1.12–1.16) than those who did not. Conclusions The findings revealed an association between the decline in household economic status due to COVID-19 and mental health problems, such as stress, loneliness, suicidal ideation, depression, and unhappiness.

Rice cultivars with floury endosperm provide a useful raw material for producing dry-milled rice flour, helping to enhance the processed rice food industry. To expand the genetic resources for breeding floury endosperm rice cultivars, we developed Samkwang(SA)-flo3 (SK-flo3), a floury endosperm mutant line derived from the chemical mutagenesis of Samkwang, an elite Korean japonica rice cultivar. Compared with Samkwang, SK-flo3 showed significantly lower grain hardness, which is suitable for producing dry-milled flour without the soaking and drying processes required in regular wet rice milling. The dry-milled flour of SK-flo3 exhibited excellent physicochemical properties with less damaged starch and finer flour particles relative to Samkwang. Genetic analyses revealed a G-to-A point mutation in exon 7 of cyOsPPDKB, substituting glycine with aspartic acid as a causative mutation for the floury endosperm of SK-flo3. We named this allele flo4-6 and developed a molecular marker to efficiently transfer it to commercial rice cultivars. Our results provide useful genetic resources and information for developing specialty rice cultivars for high-quality rice flour production with reduced milling costs.

This study presents ultra-low frequency (ULF) Pc5 discrete spectrum simultaneously observed in the magnetosphere and high- to low-latitude ionospheres near noon hours (~10-14 MLT) during the recovery phase of geomagnetic storm on November 4, 2021. During the recovery phase, magnetospheric toroidal mode oscillations (GOES-16 Bn) appeared according to solar wind dynamic pressure enhancements after GOES Bp and Be (poloidal mode) oscillations precede during high solar wind speeds. When Bn oscillates, the ionospheric line-of-sight (LOS) velocity and echo power oscillate at the same discrete frequencies of 1.7 and 2.2 mHz (9.7 and 7.5 min), observed by Super Dual Auroral Radar Network (SuperDARN) at Saskatoon (eastward LOS). The period of negative LOS velocity (away from the radar) for 7.5 min or 9.7 min corresponds to echo power increase. This signifies that both the ionospheric density and poleward convection velocity increase are driven by the periodic forcing of the convection electric field and energetic electron precipitation. The same frequency pulsations have also been observed in the geomagnetic field (H-component) and Global Positioning System (GPS) total electron content (TEC) from high- to low-latitude ionosphere. The oscillation frequency of the H-component is consistently preserved at 1.7 mHz (9.7 min) down to low latitudes. The Pc5 oscillations at high to low latitudes can be attributed to toroidal mode Alfven waves and the compressional mode propagating across magnetic field lines as well as the fast magnetosonic waveguide mode at work by the solar wind dynamic pressure enhancements at high solar wind speeds.

Background The assessment of pharmacy students’ readiness to begin the education of an advanced pharmacy practice experience (APPE) in clinical pharmacy settings continues to gain increasing attention. This study aimed to develop an objective structured clinical examination (OSCE) in the core domains acquired through an introductory pharmacy practice experience (IPPE), for evaluating its appropriateness as a tool of assessing clinical pharmacist competency for APPEs in Korean pharmacy students throughout a pilot study. Methods OSCE’s core competency domains and case scenarios were developed through a literature review, ideation by researchers, and external experts’ consensus by a Delphi method. A prospective single-arm pilot test was conducted to implement the OSCE for Korean pharmacy students who completed a 60-h course of in-class simulation IPPE. Their competencies were assessed by four assessors in each OSCE station with a pass-fail grading system accompanied by a scoring rubric. Results OSCE competency areas including patient counseling, provision of drug information, over-the-counter (OTC) counseling, and pharmaceutical care services were developed with four interactive and one non-interactive cases. Twenty pharmacy students participated in the OSCE pilot test, and their competencies were evaluated by 20 assessors. The performance rate was the lowest in the area of patient counseling for a respiratory inhaler (32.1%) and the highest (79.7%) in OTC counseling for constipation. The students had an average performance rate of 60.4% in their communication skills. Most participants agreed on the appropriateness, necessity, and effectiveness of the OSCE in evaluating pharmacy students’ clinical performance and communication skills. Conclusions The OSCE model can be used to assess pharmacy students’ readiness for off-campus clinical pharmacy practice experience. Our pilot study suggests the necessity of conducting an OSCE domain-based adjustment of difficulty levels, and strengthening simulation-based IPPE education.

Supplementary Data from The Role of MET Activation in Determining the Sensitivity to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors

The development of resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) seems almost inevitable, even in patients with lung cancer that initially respond well to EGFR-TKIs. MET amplification was recently found to be a mechanism of escape from the anticancer effect of EGFR inhibitors. In the present study, we investigated the means whereby MET affects sensitivity to EGFR-TKIs in PC-9 cells. Gefitinib- or erlotinib-resistant sublines were established by exposing the parental PC-9 cell line to chronic, repeated treatments with these drugs. These resistant sublines showed more than 100-fold more resistance to gefitinib and erlotinib and acquired cross-resistance to other EGFR-TKIs. The T790M EGFR mutation was found by pyrosequencing, and this seemed to be the cause of drug resistance. Resistant cells also showed MET activation, although gene amplification was not detected. Furthermore, the induction of MET activity was not found to be associated with sensitivity to EGFR-TKIs. Interestingly, increased passage number without exposure to gefitinib or erlotinib caused MET activation, but this did not affect sensitivity to EGFR-TKIs. In addition, hepatocyte growth factor was found to block the ability of EGFR-TKIs to inhibit MET activation. However, sustained MET activation by hepatocyte growth factor did not modulate the cellular effects of gefitinib or erlotinib. Rather, activated MET enhanced migration and invasion abilities. Summarizing, MET activation may be acquired during cancer cell proliferation and enhances migratory and invasive abilities without affecting cellular sensitivity to EGFR-TKIs. Accordingly, the present study suggests that MET activation caused by factors other than MET gene amplification is not a suitable surrogate marker of resistance to EGFR-TKIs. (Mol Cancer Res 2009;7(10):1736–43)

Supplementary Information

Purpose: Activation of YAP1, a novel oncogene in the Hippo pathway, has been observed in many cancers, including colorectal cancer. We investigated whether activation of YAP1 is significantly associated with prognosis or treatment outcomes in colorectal cancer.Experimental Design: A gene expression signature reflecting YAP1 activation was identified in colorectal cancer cells, and patients with colorectal cancer were stratified into two groups according to this signature: activated YAP1 colorectal cancer (AYCC) or inactivated YAP1 colorectal cancer (IYCC). Stratified patients in five test cohorts were evaluated to determine the effect of the signature on colorectal cancer prognosis and response to cetuximab treatment.Results: The activated YAP1 signature was associated with poor prognosis for colorectal cancer in four independent patient cohorts with stage I–III disease (total n = 1,028). In a multivariate analysis, the impact of the YAP1 signature on disease-free survival was independent of other clinical variables [hazard ratio (HR), 1.63; 95% confidence interval (CI), 1.25–2.13; P < 0.001]. In patients with stage IV colorectal cancer and wild-type KRAS, IYCC patients had a better disease control rate and progression-free survival (PFS) after cetuximab monotherapy than did AYCC patients; however, in patients with KRAS mutations, PFS duration after cetuximab monotherapy was not different between IYCC and AYCC patients. In multivariate analysis, the effect of YAP1 activation on PFS was independent of KRAS mutation status and other clinical variables (HR, 1.82; 95% CI, 1.05–3.16; P = 0.03).Conclusions: Activation of YAP1 is highly associated with poor prognosis for colorectal cancer and may be useful in identifying patients with metastatic colorectal cancer resistant to cetuximab. Clin Cancer Res; 21(2); 357–64. ©2014 AACR.

Supplementary Figures S1-S5. Supplementary Figure S1. Expression of YAP1 in NCI-H716 CRC cells. Supplementary Figure S2. YAP1-specific gene expression signature used in our study of YAP1 activation in CRC patients. Supplementary Figure S3. Gene set enrichment analysis (GSEA) of gene expression data from two cancer cells with activated YAP1. Supplementary Figure S4. Prognostic impact of the YAP1 signature for CRC according to disease stages. Supplementary Figure S5. PFS according to KRAS mutation status (Cohort 5)

Recurrence risk score and its clinical significance.

Peroxiredoxins (PRDX) are a family of thiol-dependent peroxidases. Among the six mammalian members of this family, PRDX6 is the only protein that additionally exhibits phospholipase A2 (PLA2) activity. The physiologic role of this interesting PRDX6 feature is largely unknown at present. In this study, we show that PRDX6 increases the metastatic potential of lung cancer cells. Functional analyses of the enzymatic activities of PRDX6, using specific pharmacologic inhibitors and mutagenesis studies, reveal that both peroxidase and PLA2 activities are required for metastasis. Specifically, peroxidase activity facilitates the growth of cancer cells, and PLA2 activity promotes invasiveness. Further investigation of the latter event discloses that PLA2 activity promotes accumulation of arachidonic acid, which, in turn, induces the invasive pathway involving p38 kinase, phosphoinositide 3-kinase, Akt, and urokinase-type plasminogen activator. This study is the first to define the functions of the enzymatic activities of PRDX6 in metastasis and to show the involvement of arachidonic acid in PRDX6 action in intact cells. These novel findings provide a significant step toward elucidating the role of PRDX6 in cancer and the mechanism of its action. Mol Cancer Ther; 9(4); 825–32. ©2010 AACR.

Combination therapy of ALK and β3 inhibitors shows promising results in vivo and in ALK-rearranged NSCLC

Purpose:Although first-line crizotinib treatment leads to clinical benefit in ROS1+ lung cancer, high prevalence of crizotinib-resistant ROS1-G2032R (ROS1G2032R) mutation and progression in the central nervous system (CNS) represents a therapeutic challenge. Here, we investigated the antitumor activity of repotrectinib, a novel next-generation ROS1/TRK/ALK-tyrosine kinase inhibitor (TKI) in ROS1+ patient-derived preclinical models.Experimental Design:Antitumor activity of repotrectinib was evaluated in ROS1+ patient-derived preclinical models including treatment-naïve and ROS1G2032R models and was further demonstrated in patients enrolled in an on-going phase I/II clinical trial (NCT03093116). Intracranial antitumor activity of repotrectinib was evaluated in a brain-metastasis mouse model.Results:Repotrectinib potently inhibited in vitro and in vivo tumor growth and ROS1 downstream signal in treatment-naïve YU1078 compared with clinically available crizotinib, ceritinib, and entrectinib. Despite comparable tumor regression between repotrectinib and lorlatinib in YU1078-derived xenograft model, repotrectinib markedly delayed the onset of tumor recurrence following drug withdrawal. Moreover, repotrectinib induced profound antitumor activity in the CNS with efficient blood–brain barrier penetrating properties. Notably, repotrectinib showed selective and potent in vitro and in vivo activity against ROS1G2032R. These findings were supported by systemic and intracranial activity of repotrectinib observed in patients enrolled in the on-going clinical trial.Conclusions:Repotrectinib is a novel next-generation ROS1-TKI with improved potency and selectivity against treatment-naïve and ROS1G2032R with efficient CNS penetration. Our findings suggest that repotrectinib can be effective both as first-line and after progression to prior ROS1-TKI.

Supplementary table 1

Distribution of candidate genes in biological process categories of the Gene Ontology database.

Methods

Treatment information of patients who received crizotinib

Purpose: This study was aimed at developing and validating a quantitative multigene assay for predicting tumor recurrence after gastric cancer surgery.Experimental Design: Gene expression data were generated from tumor tissues of patients who underwent surgery for gastric cancer (n = 267, training cohort). Genes whose expression was significantly associated with activation of YAP1 (a frequently activated oncogene in gastrointestinal cancer), 5-year recurrence-free survival, and 5-year overall survival were first identified as candidates for prognostic genes (156 genes, P < 0.001). We developed the recurrence risk score (RRS) by using quantitative RT-PCR to identify genes whose expression levels were significantly associated with YAP1 activation and patient survival in the training cohort.Results: We based the RRS assay on 6 genes, IGFBP4, SFRP4, SPOCK1, SULF1, THBS, and GADD45B, whose expression levels were significantly associated with YAP1 activation and prognosis in the training cohort. The RRS assay was further validated in an independent cohort of 317 patients. In multivariate analysis, the RRS was an independent predictor of recurrence [HR, 1.6; 95% confidence interval (CI), 1.02–2.4; P = 0.03]. In patients with stage II disease, the RRS had an HR of 2.9 (95% CI, 1.1–7.9; P = 0.03) and was the only significant independent predictor of recurrence.Conclusions: The RRS assay was a valid predictor of recurrence in the two cohorts of patients with gastric cancer. Independent prospective studies to assess the clinical utility of this assay are warranted. Clin Cancer Res; 22(24); 6228–35. ©2016 AACR.

The precise characteristics that distinguish normal and oncogenic RAS signaling remain obscure. Here, we show that oncogenic RAS and BRAF induce perinuclear relocalization of several RAS pathway proteins, including the kinases CK2 and p-ERK1/2 and the signaling scaffold KSR1. This spatial reorganization requires endocytosis, the kinase activities of MEK-ERK and CK2, and the presence of KSR1. CK2α colocalizes with KSR1 and Rab11, a marker of recycling endosomes, whereas p-ERK associates predominantly with a distinct KSR1-positive endosomal population. Notably, these perinuclear signaling complexes (PSC) are present in tumor cell lines, mouse lung tumors, and mouse embryonic fibroblasts undergoing RAS-induced senescence. PSCs are also transiently induced by growth factors (GF) in nontransformed cells with delayed kinetics (4–6 hours), establishing a novel late phase of GF signaling that appears to be constitutively activated in tumor cells. PSCs provide an essential platform for RAS-induced phosphorylation and activation of the prosenescence transcription factor C/EBPβ in primary MEFs undergoing senescence. Conversely, in tumor cells, C/EBPβ activation is suppressed by 3′UTR-mediated localization of Cebpb transcripts to a peripheral cytoplasmic domain distinct from the PSC region. Collectively, our findings indicate that sustained PSC formation is a critical feature of oncogenic RAS/BRAF signaling in cancer cells that controls signal transmission to downstream targets by regulating selective access of effector kinases to substrates such as C/EBPβ.Significance: In addressing the long-standing question of the difference between normal and oncogenic RAS pathway signaling, this study shows that oncogenic RAS specifically triggers constitutive endocytosis-dependent movement of effector kinases to a perinuclear region, thereby creating connections to unique downstream targets such as the core prosenescence and the inflammatory regulatory transcription factor C/EBPβ. Cancer Res; 78(4); 891–908. ©2017 AACR.

Purpose: Although many genomic alterations have been observed in lung cancer, their clinicopathologic significance has not been thoroughly investigated. This study screened the genomic aberrations across the whole genome of non–small cell lung cancer cells with high-resolution and investigated their clinicopathologic implications.Experimental Design: One-megabase resolution array comparative genomic hybridization was applied to 29 squamous cell carcinomas and 21 adenocarcinomas of the lung. Tumor and normal tissues were microdissected and the extracted DNA was used directly for hybridization without genomic amplification. The recurrent genomic alterations were analyzed for their association with the clinicopathologic features of lung cancer.Results: Overall, 36 amplicons, 3 homozygous deletions, and 17 minimally altered regions common to many lung cancers were identified. Among them, genomic changes on 13q21, 1p32, Xq, and Yp were found to be significantly associated with clinical features such as age, stage, and disease recurrence. Kaplan-Meier survival analysis revealed that genomic changes on 10p, 16q, 9p, 13q, 6p21, and 19q13 were associated with poor survival. Multivariate analysis showed that alterations on 6p21, 7p, 9q, and 9p remained as independent predictors of poor outcome. In addition, significant correlations were observed for three pairs of minimally altered regions (19q13 and 6p21, 19p13 and 19q13, and 8p12 and 8q11), which indicated their possible collaborative roles.Conclusions: These results show that our approach is robust for high-resolution mapping of genomic alterations. The novel genomic alterations identified in this study, along with their clinicopathologic implications, would be useful to elucidate the molecular mechanisms of lung cancer and to identify reliable biomarkers for clinical application.

Purpose: Anaplastic lymphoma kinase (ALK)-positive cancers are sensitive to small-molecule ALK kinase inhibitors, but most cases experience failure following treatment. Hence, additional drug targets and combination therapeutic treatments are needed. We investigated gene expression that is regulated by the expression of ALK and explored its roles in cancer progression and therapeutic implication.Experimental Design: We screened ALK-rearranged non–small cell lung cancer (NSCLC) cases using immunohistochemistry and fluorescence in situ hybridization and then conducted multiplex gene expression analysis. We also performed a clinicopathologic analysis to validate the findings. Additional cellular experiments, including inhibition and migration assays, and in vivo lung cancer model studies were performed.Results: Among patients with ALK-rearranged NSCLC, integrin β3 (ITGB3) was one of the overexpressed genes in comparison with that in ALK-negative NSCLC (P = 0.0003). ALK and integrin β3 expression were positively correlated, and we discovered that high integrin β3 mRNA expression was associated with metastasis and more advanced tumor stages (P < 0.005; P < 0.05). Furthermore, we found that inhibition of both ALK and integrin β3 led to increased drug sensitivity in vitro and in vivo (both P < 0.05).Conclusions: We discovered a positive correlation between ALK and integrin β3 expression levels in ALK-rearranged NSCLC. Our findings suggest that high integrin β3 expression in ALK-rearranged NSCLC is associated with tumor progression and a worse prognosis. This finding demonstrates the prognostic value of integrin β3 and provides a rationale for combination treatment with ALK and integrin β3 inhibitors in patients with ALK-rearranged NSCLC. Clin Cancer Res; 24(17); 4162–74. ©2018 AACR.

Supplementary Tables S1-S4 from Genome-Wide Screening of Genomic Alterations and Their Clinicopathologic Implications in Non–Small Cell Lung Cancers