Search

An unbiased scanning methodology using ultra high-performance liquid chromatography coupled with high-resolution mass spectrometry was used to bank data and plasma samples for comparing the data generated at different dates. This method was applied to bank the data generated earlier in animal samples and then to compare the exposure to metabolites in animal versus human for safety assessment. With neither authentic standards nor prior knowledge of the identities and structures of metabolites, full scans for precursor ions and all ion fragments (AIF) were employed with a generic gradient LC method to analyze plasma samples at positive and negative polarity, respectively. In a total of 22 tested drugs and metabolites, 21 analytes were detected using this unbiased scanning method except that naproxen was not detected due to low sensitivity at negative polarity and interference at positive polarity; and 4'- or 5-hydroxy diclofenac was not separated by a generic UPLC method. Statistical analysis of the peak area ratios of the analytes versus the internal standard in five repetitive analyses over approximately 1 year demonstrated that the analysis variation was significantly different from sample instability. The confidence limits for comparing the exposure using peak area ratio of metabolites in animal plasma versus human plasma measured over approximately 1 year apart were comparable to the analysis undertaken side by side on the same days. These statistical analysis results showed it was feasible to compare data generated at different dates with neither authentic standards nor prior knowledge of the analytes.

Background Ultra-deep next-generation sequencing of circulating tumor DNA (ctDNA) holds great promise as a tool for the early detection of cancer and for monitoring disease progression and therapeutic responses. However, the low abundance of ctDNA in the bloodstream coupled with technical errors introduced during library construction and sequencing complicates mutation detection. Results To achieve high accuracy of variant calling via better distinguishing low-frequency ctDNA mutations from background errors, we introduce TNER (Tri-Nucleotide Error Reducer), a novel background error suppression method that provides a robust estimation of background noise to reduce sequencing errors. The results on both simulated data and real data from healthy subjects demonstrate that the proposed algorithm consistently outperforms a current, state-of-the-art, position-specific error polishing model, particularly when the sample size of healthy subjects is small. Conclusions TNER significantly enhances the specificity of downstream ctDNA mutation detection without sacrificing sensitivity. The tool is publicly available at https://github.com/ctDNA/TNER. Electronic supplementary material The online version of this article (10.1186/s12859-018-2428-3) contains supplementary material, which is available to authorized users.

Aim This study aimed at exploring changes in YAP expression and their effect on periodontitis (PD) combined with traumatic occlusion (TO). Materials and methods BALB/cJ mice were used to establish a PD model by local administration of Porphyromonas gingivalis (P.g, ATCC 33277) and a TO model by occlusal elevation (OE) using composite resin bonding on the bilateral maxillary molar. The mouse fibroblast cell line (L929) and pre-osteoblast cell line (MC3T3-E1) were subjected to cyclic tensile/compressive stress and inflammatory stimuli (lipopolysaccharide from Escherichia coli) to verify in vivo results. Results Severe bone resorption was observed by microCT scanning in OE with P.g group, when compared to OE only and P.g only groups. Mechanical stress caused by OE activated the Hippo-YAP pathway in periodontal tissues and upregulated the expression of JNK/AP-1. OE with P.g further promoted the expression of YAP and JNK/AP1, leading to the upregulation of the JNK/AP-1 related inflammatory cytokines TNF-α and IL6. Similar results were obtained when osteoblasts were subjected to mechanical stress in vitro. Conclusions Our study demonstrated that periodontitis with TO caused severe inflammation-induced bone resorption. Activation of YAP and upregulation of JNK/AP-1 induced by TO potentially aggravated the symptoms of PD.

SummaryWe report the preclinical evaluation of PF-06463922, a potent and brain-penetrant ALK/ROS1 inhibitor. Compared with other clinically available ALK inhibitors, PF-06463922 displayed superior potency against all known clinically acquired ALK mutations, including the highly resistant G1202R mutant. Furthermore, PF-06463922 treatment led to regression of EML4-ALK-driven brain metastases, leading to prolonged mouse survival, in a superior manner. Finally, PF-06463922 demonstrated high selectivity and safety margins in a variety of preclinical studies. These results suggest that PF-06463922 will be highly effective for the treatment of patients with ALK-driven lung cancers, including those who relapsed on clinically available ALK inhibitors because of secondary ALK kinase domain mutations and/or brain metastases.

To investigate the plasma levels of amyloid beta (Aβ) and select inflammatory mediators in patients with various stages of AMD compared to that of age-matched controls, and discern a relationship to disease severity. Plasma samples were obtained from AMD subjects at various stages of disease—early (drusen only), geographic atrophy (GA), neovascular AMD (CNV)—and from controls of similar age without AMD. Samples were analyzed using a commercially available ELISA kit (sixteen cytokines) or LC/MS/MS (Aβ isotypes). Descriptive statistics were compiled on all analytes. Analysis of covariance (ANCOVA) was conducted to compare each analyte across AMD groups while adjusting for sex and age of the patients, and in comparison to the control group. Receiver operating characteristics plots were generated for the strongest predictor variables. Levels of alternative spliced CC3 proteins were significantly different between controls and CNV groups (p

The fetal oncogene 5T4 is a cell surface protein, with overexpression observed in a variety of cancers as compared to normal adult tissue. The ability to select patients with tumors that express high levels of 5T4 may enrich a clinical trial cohort with patients most likely to respond to 5T4 targeted therapy. To that end, we developed assays to measure 5T4 in both tumors and in circulating tumor cells (CTCs). We identified the presence of 5T4 in both adenocarcinoma and squamous cell carcinoma of lung, in all clinical stages and grades of disease. CTCs were identified in peripheral blood from the majority of patients with NSCLC, and 5T4 was detectable in most samples. Although 5T4 was present in both CTCs and tumors in most patients, there was no concordance between relative amount in either sample type. Clinical response rates of patients treated with the therapies directed against 5T4 in early stage clinical trials, as determined by these assays, may provide important insights into the biology of 5T4 in tumors and the mechanisms of action of 5T4-targeting therapy.

Objectives Amongst the fourth generation of PHAs is bio-plasticpoly3-hydroxybutyrate4-hydroxybutyrate (P34HB); it is thus appropriate to perform novel research on its uses and applications. The main objective of this study was to determine whether electrospun P34HB fibres would accommodate viability, growth and differentiation of mouse adipose-derived stem cells (mASCs). Materials and methods In the present study, we looked at P34HB in two forms, electrospun P34HB fibres and P34HB film. Morphology of electrospun P34HB fibres and P34HB film were characterized using scanning electron microscopy, fluorescence microscopy and confocal laser scanning microscopy, after cell seeding. Cell adhesion, proliferation and cytotoxicity tests were conducted on both by MTT and CCK-8 assays, respectively. After being cultured with osteogenic induction, expression of adipogenic genes Runx2, OPN and OCN, were examined by real-time PCR. Results By scanning electron microscopy, light microscopy and confocal laser scanning microscopy, we observed that the mASCs grew well associated with the P34HB materials. After MTT and CCK-8 assay, we concluded that P34HB would, indeed, be a material suitable for further cell adhesion and proliferation studies. More importantly, we found that the P34HB matrices promoted expression of Runx2, OPN and OCN with osteogenic induction. Conclusions In this investigation, we can confirm that the electrospun P34HB fibres accommodated survival, proliferation and differentiation of mASCs, and we have been able to draw the conclusion that fibre scaffolds produced by the electrospinning process are promising for application of bone tissue engineering.

Approximately 7% of non-small cell lung carcinomas (NSCLCs) harbor oncogenic fusions involving ALK, ROS1, and RET. Although tumors harboring ALK fusions are highly sensitive to crizotinib, emerging preclinical and clinical data demonstrate that patients with ROS1 or RET fusions may also benefit from inhibitors targeting these kinases. Using a transcript-based method, we designed a combination of 3' overexpression and fusion-specific detection strategies to detect ALK, ROS1 and RET fusion transcripts in NSCLC tumors. We validated the assay in 295 NSCLC specimens and showed that the assay is highly sensitive and specific. ALK results were 100% concordant with fluorescence in situ hybridization (FISH) (n = 52) and 97.8% concordant with IHC (n = 179) [sensitivity, 96.8% (95% CI 91.0%-98.9%); specificity, 98.8% (95% CI 93.6%-99.8%)]. For ROS1 and RET, we also observed 100% concordance with FISH (n = 46 and n = 15, respectively). We identified seven ROS1 and 14 RET fusion-positive tumors and confirmed the fusion status by RT-PCR and FISH. One RET fusion involved a novel partner, cutlike homeobox 1 gene (CUX1), yielding an in-frame CUX1-RET fusion. ROS1 and RET fusions were significantly enriched in tumors without KRAS/EGFR/ALK alterations. ALK/ROS1/RET/EGFR/KRAS alterations were mutually exclusive. As a single-tube assay, this test shows promise as a more practical and cost-effective screening modality for detecting rare but targetable fusions in NSCLC.

Current methods to quantify T-cell clonal expansion only account for variance due to random sampling from a highly diverse repertoire space. We propose a beta-binomial model to incorporate time-dependent variance into the assessment of differentially abundant T-cell clones, identified by unique T Cell Receptor (TCR) β-chain rearrangements, and show that this model improves specificity for detecting clinically relevant clonal expansion. Using blood samples from ten healthy donors, we modeled the variance of T-cell clones within each subject over time and calibrated the dispersion parameters of the beta distribution to fit this variance. As a validation, we compared pre- versus post-treatment blood samples from urothelial cancer patients treated with atezolizumab, where clonal expansion (quantified by the earlier binomial model) was previously reported to correlate with benefit. The beta-binomial model significantly reduced the false-positive rate for detecting differentially abundant clones over time compared to the earlier binomial method. In the urothelial cancer cohort, the beta-binomial model enriched for tumor infiltrating lymphocytes among the clones detected as expanding in the peripheral blood in response to therapy compared to the binomial model and improved the overall correlation with clinical benefit. Incorporating time-dependent variance into the statistical framework for measuring differentially abundant T-cell clones improves the model's specificity for T-cells that correlate more strongly with the disease and treatment setting of-interest. Reducing background-level clonal expansion, therefore, improves the quality of clonal expansion as a biomarker for assessing the T cell immune response and correlations with clinical measures.

Background: CDK4/6 inhibition combined with endocrine therapy (ET) is now the standard of care for advanced hormone receptor (HR) positive breast cancer patients (pts). Previous ctDNA analysis of paired pre-treatment (pre-T) and end of treatment (EoT) plasma samples from the PALOMA-3 trial of the CDK4/6 inhibitor P with/without F demonstrated that acquired PIK3CA, ESR1, RB1 and rare growth factor receptor mutations (mut) likely contribute to resistance (Turner et al, ASCO 2018, Abstract 1001). We now report results from an extended ctDNA analysis of paired PALOMA-3 plasma samples using hybridization capture-based, next-generation sequencing (NGS) assay covering 87 breast cancer, HR signaling, and cell cycle-related genes. Methods: Pre-/post-menopausal pts whose disease progressed after prior ET (N=521) were randomized 2:1 to receive F 500 mg + P (125 mg/d, 3 wk on/1 wk off) or placebo. Cell-free DNA extracted from paired plasma samples (n=194) was analyzed with a custom 87-gene NGS assay, to identify single nucleotide variants and copy number amplification (CNA), with molecular barcoding, high NGS coverage (10,000-20,000 reads per nucleotide position), and background error correction. Differences in ctDNA mut frequencies detected in P+F compared to F alone were assessed and their association to clinical outcome estimated. Results: Blinded assay qualification and droplet digital PCR validation suggested a mut detection frequency cutoff at 0.3%. A high coefficient of correlation with previously presented data was observed for ESR1 and PIK3CA variants (r=0.94 and 0.97, respectively), with acquisition of PIK3CA and ESR1 mutations at EoT in P+F and F groups. Gene level mut analysis of EoT plasma revealed no significant difference between P+F vs F alone (Table), although there was an increase in RB1 mutations in P+F consistent with previous data. Other acquired muts at EoT (SMAD4, NOTCH2, and CDKN1B) were observed at low frequencies. Muts in CDK4 and CDK6 were rarely observed (< 1% of pts), regardless of treatment arm. Detected Variants, Frequency, n (%) P+FF GenePre-TEoTPre-TEoTPre-T pvalEoT pvalPIK3CA47(37)51(40)19(28)22(33)0.2660.352ESR136(28)45(35)19(28)24(36)11TP5330(24)33(26)23(34)25(37)0.1280.137RB12(2)9(7)2(3)2(3)NA0.336PTEN5(4)7(6)3(4)3(4)11AKT17(6)7(6)2(3)2(3)0.7210.721 Conclusions: Our results corroborate the previously reported results demonstrating that genomic profiles of treatment resistant cancer are similar between P + F and F therapy. Expanded analysis of cell-cycle genes identified no new recurrently mutated genes, and confirmed that RB1 mutations are selected at low frequency after P+F treatment. Alterations in cell cycle genes may not be a common mechanism of resistance to CDK4/6 inhibitors in HR+ HER2- advanced breast cancer. Sponsor: Pfizer Citation Format: O'Leary B, Lira ME, Huang S, Deng S, Xie T, Kinong JK, Hardwick J, Rejto P, Liu Y, Huang X, Huang Bartlett C, André F, Loibl S, DeMichele A, Cristofanilli M, Turner NC. Longitudinal ctDNA sequencing using an expanded genomic panel in the PALOMA3 trial of palbociclib plus fulvestrant [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD2-02.

// Seung Tae Kim 1, * , Maruja Lira 2, * , Shibing Deng 2 , Sujin Lee 1 , Young Suk Park 1 , Ho Yeong Lim 1 , Won Ki Kang 1 , Mao Mao 3 , Jin Seok Heo 4 , Wooil Kwon 4 , Kee‑Taek Jang 5 , Jeeyun Lee 1, 6 , Joon Oh Park 1, 6 1 Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 2 Precision Medicine, Oncology Research Unit, Pfizer, Inc, San Diego, CA, USA 3 WuXi AppTec, Shanghai, China 4 Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 5 Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 6 Innovative Cancer Medicine Institute, Samsung Medical Center, Seoul, Korea * These authors have contributed equally to this work Correspondence to: Joon Oh Park, e-mail: oncopark@skku.edu Jeeyun Lee, e-mail: jyunlee@skku.edu Keywords: cell free DNA (cfDNA), PIK3CA mutation, droplet digital PCR (ddPCR) Received: July 13, 2015 Accepted: October 05, 2015 Published: October 16, 2015 ABSTRACT PIK3CA mutation is considered a good candidate for targeted therapies in cancers, especially biliary tract cancer (BTC). We evaluated the utility of cell free DNA (cfDNA) from serum by using droplet digital PCR (ddPCR) as an alternative source for PIK3CA mutation analysis. To identify matching archival tumour specimens from serum samples of advanced BTC patients, mutation detection using ddPCR with Bio-Rad’s PrimePCR mutation and wild type assays were performed for PIK3CA p.E542K, p.E545K, and p.H1047R. Thirty-eight patients with metastatic BTC were enrolled. Only one (BTC 29T) sample ( n = 38) was positive for PIK3CA p.E542K and another (BTC 27T) for p.H1047R mutation; none was positive for PIK3CA p.E545K. Matched serum sample (BTC 29P) was positive for PIK3CA p.E542K with 28 mutant copies detected, corresponding to 48 copies/ml of serum and an allelic prevalence of 0.3%. Another matched serum sample (BTC 27P) was positive for PIK3CA p.H1047R with 10 mutant copies detected, i.e. 18 copies/ml and an allelic frequency of 0.2%. High correlation was noted in the PIK3CA mutation status between tumour gDNA and serum cfDNA. Low-level PIK3CA mutations were detectable in the serum indicating the utility of cfDNA as a DNA source to detect cancer-derived mutations in metastatic biliary cancers.

Background Progression of hepatocellular carcinoma (HCC) often leads to vascular invasion and intrahepatic metastasis, which correlate with recurrence after surgical treatment and poor prognosis. The molecular prognostic model that could be applied to the HCC patient population in general is needed for effectively predicting disease-free survival (DFS).

Breast cancers (BC) in younger, premenopausal patients (YBC) tend to be more aggressive with worse prognosis, higher chance of relapse and poorer response to endocrine therapies compared to breast cancers in older patients. The proportion of YBC (age ≤ 40) among BC in East Asia is estimated to be 16-32%, significantly higher than the 7% reported in Western countries. In addition, approximately half of the Asian BC patients were premenopausal compared to 15-30% in the West. To characterize the molecular bases of Asian YBC, we have performed whole-exome sequencing (WES) and whole-transcriptome sequencing (WTS) on tumor and matched normal samples from 168 Korean BC patients consisting of 106 YBC cases (age ≤ 40) and 62 OBC cases (age > 40). We then performed comparison analyses with the TCGA BC cohort consisting of 1,116 tumors from primarily Caucasian patients, also grouped by age into YBC (age ≤ 40), IBC (40 < age ≤ 60) and OBC (age > 60). We performed logistic regression analyses to identify differentially expressed (DE) genes and pathways among age-based cohorts while controlling for the confounding effects of molecular subtype, tumor purity and stage. Within the Asian cohort, we found that estrogen response, endocrine therapy resistance, and various metabolism pathways are up-regulated in YBCs while cell cycle, proliferation and inflammatory pathways are up-regulated in OBCs. To separately examine molecular signatures from tumor, stroma and normal compartments, we used non-negative matrix factorization (NMF) analyses to virtually dissect bulk tumor expression data and identified 14 factors including 3 factors associated with normal tissues, 1 factor associated with stroma and 1 factor associated with tumor infiltrating leukocytes (TILs). By examining the correlation between pathway gene expression and NMF factors, we inferred that DE pathways such as fatty acid metabolism, bile acid biosynthesis, and epithelial-to-mesenchymal transition (EMT) were mainly active in stromal and normal tissue compartments. The TIL factor was significantly enriched in Asian BCs relative to Caucasian BCs with the highest TIL factor weight observed in Asian OBCs. Using gene expression signatures representing distinct types of TILs, we classified the combined cohort into three subtypes of varying TIL activities. Consistent with results from the NMF analysis, the TIL-high subtype is also significantly enriched in Asian BCs relative to Caucasian BCs. To our knowledge, this is the first large-scale multi-omics study of Asian breast cancer. Comparative analyses of multi-omics profiles from Asian and primarily Caucasian BC cohorts identified distinguishing molecular signatures associated with Asian BCs. Further, many signatures appeared to be specific to non-tumor compartments within bulk tumor, indicating that young Asian BCs may harbor distinctive tumor microenvironment. Citation Format: Yeon Hee Park, Ying Ding, Soo-Hyeon Lee, Hae Hyun Jung, Woosung Chung, Soonweng Cho, Jin-Ho Kim, Shibing Deng, Yoon-la Choi, Julio Fernandez, Se Kyung Lee, Seok Won Kim, Jeong Eon Lee, Ji-Yeon Kim, Jin Seok Ahn, Young-Hyuck Im, Seok Jin Nam, Woong-Yang Park, Zhengyan Kan. Comparative analyses of multi-omics profiles reveal distinctive molecular signatures of young Asian breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3370. doi:10.1158/1538-7445.AM2017-3370

Liquid biopsies have the potential to revolutionize the way physicians select personalized anti-cancer therapies, monitor patient responses to treatment, and characterize acquired resistance to cancer drugs. New tests that use a simple peripheral blood draw offer snapshots of a patient‘s total tumor DNA mutation profile and are attractive because of their minimally-invasive modality and because they integrate information from both primary and metastatic disease. Currently, most plasma cell-free DNA (cfDNA) mutation detection tests used in clinical research detect known hotspot mutations in a limited number of genes. Technologies that interrogate multi-gene panels in cfDNA are advancing, but commercially-available options suitable for clinical use are limited, come at a high cost, and are not customizable. We designed and developed a customized, next generation sequencing-based, liquid biopsy assay capable of detecting somatic mutations in 87 breast cancer genes involved in cell cycle and estrogen receptor signaling. Targeted regions (147 Kb) were enriched using hybrid capture resulting in an average capture specificity and uniformity of 65.93% and 96.38%, respectively. When tested on cfDNA from healthy donors (n=14), we demonstrated a level of specificity >99.99%. Analytical sensitivity of 0.1% was established on HapMap and reference mutant cell line DNA. Using a pool of HapMap genomic DNA (n=10), 96% (48/50) of SNPs with expected allele frequency of 0.1% were detected. In reference mutant cell line DNA with 1% or 0.1% mutant allele frequencies, we were able to reliably detect all mutations present at 1% and mutations at 0.1% in 50% of the cases. Assay validation on plasma cfDNA with matching tumor from ER+, HER2- breast cancer patients will be presented. In conclusion, we developed a highly sensitive and specific liquid biopsy assay to interrogate 87 breast cancer-related genes. The high level of specificity and sensitivity makes the test ideal not only for detecting known cancer gene hotspot mutations but also for detection of novel gene mutations that may arise during treatment as a result of acquired drug resistance. Citation Format: Maruja E. Lira, Tao Xie, Shibing Deng, Jennifer Kinong, Jingjin Gao, Zhou Zhu, Nathan Lee, Paul Rejto, Jadwiga Bienkowska, James Hardwick, Kai Wang, Stephen Huang. Liquid biopsy testing allows highly-sensitive detection of plasma cfDNA mutations in 87 breast cancer-related genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2749. doi:10.1158/1538-7445.AM2017-2749

Norepinephrine reuptake inhibitors (NRIs) acutely increase norepinephrine (NE) levels, but therapeutic antidepressant activity is only observed after weeks of treatment because central NE levels progressively increase during continued drug exposure. Similarly, while NRIs acutely increase blood pressure (BP) and heart rate (HR) due to enhanced sympathetic neurotransmission, chronic treatment changes the responsiveness of the central noradrenergic system and suppresses these effects via autonomic regulation. To better understand the relationship between NE increases and cardiovascular safety, we investigated acute and chronic effects of the NRI reboxetine on central NE release and on BP and HR and electrical alternans, a measure of arrhythmia liability, in guinea pigs. NE release was assessed by microdialysis in medial prefrontal cortex (mPFC) and hypothalamic paraventricular nucleus (PVN); BP and HR were measured by telemetry. Animals were treated for 28 days with 15 mg/kg/day of reboxetine or vehicle via an osmotic minipump and then challenged with acute intravenous doses of reboxetine. Animals chronically treated with reboxetine had 2-fold higher extracellular basal NE levels in mPFC and PVN compared to basal levels after chronic vehicle treatment BP was significantly increased after the first day of treatment, and gradually returned to vehicle levels by day 21. These data indicate that chronic NRI treatment may lead to an increase in central NE levels and a concomitant reduction in BP based on exposure-response curves compared to vehicle treatment, suggesting a larger separation between preclinical estimates of efficacy vs. safety compared to acute NRI treatment. (c) 2012 Elsevier Inc. All rights reserved.

Schizophrenia is characterized by affective, cognitive, neuromorphological, and molecular abnormalities that may have a neurodevelopmental origin. MicroRNAs (miRNAs) are small noncoding RNA sequences critical to neurodevelopment and adult neuronal processes by coordinating the activity of multiple genes within biological networks. We examined the expression of 854 miRNAs in prefrontal cortical tissue from 100 control, schizophrenic, and bipolar subjects. The cyclic AMP-responsive element binding- and NMDA-regulated microRNA miR-132 was significantly down-regulated in both the schizophrenic discovery cohort and a second, independent set of schizophrenic subjects. Analysis of miR-132 target gene expression in schizophrenia gene-expression microarrays identified 26 genes up-regulated in schizophrenia subjects. Consistent with NMDA-mediated hypofunction observed in schizophrenic subjects, administration of an NMDA antagonist to adult mice results in miR-132 down-regulation in the prefrontal cortex. Furthermore, miR-132 expression in the murine prefrontal cortex exhibits significant developmental regulation and overlaps with critical neurodevelopmental processes during adolescence. Adult prefrontal expression of miR-132 can be down-regulated by pharmacologic inhibition of NMDA receptor signaling during a brief postnatal period. Several key genes, including DNMT3A , GATA2 , and DPYSL3 , are regulated by miR-132 and exhibited altered expression either during normal neurodevelopment or in tissue from adult schizophrenic subjects. Our data suggest miR-132 dysregulation and subsequent abnormal expression of miR-132 target genes contribute to the neurodevelopmental and neuromorphological pathologies present in schizophrenia.

The genotoxicity testing battery is highly sensitive for detection of chemical carcinogens. However, it features a low specificity and provides only limited mechanistic information required for risk assessment of positive findings. This is especially important in case of positive findings in the in vitro chromosome damage assays, because chromosome damage may be also induced secondarily to cell death. An increasing body of evidence indicates that toxicogenomic analysis of cellular stress responses provides an insight into mechanisms of action of genotoxicants. To evaluate the utility of such a toxicogenomic analysis we evaluated gene expression profiles of TK6 cells treated with four model genotoxic agents using a targeted high density real-time PCR approach in a multilaboratory project coordinated by the Health and Environmental Sciences Institute Committee on the Application of Genomics in Mechanism-based Risk Assessment. We show that this gene profiling technology produced reproducible data across laboratories allowing us to conclude that expression analysis of a relevant gene set is capable of distinguishing compounds that cause DNA adducts or double strand breaks from those that interfere with mitotic spindle function or that cause chromosome damage as a consequence of cytotoxicity. Furthermore, our data suggest that the gene expression profiles at early time points are most likely to provide information relevant to mechanisms of genotoxic damage and that larger gene expression arrays will likely provide richer information for differentiating molecular mechanisms of action of genotoxicants. Although more compounds need to be tested to identify a robust molecular signature, this study confirms the potential of toxicogenomic analysis for investigation of genotoxic mechanisms.

Our group has been using the ECVAM Embryonic Stem Cell assay to predict developmental toxicity. In order to improve the separation of non-teratogens from weak teratogens, we have employed measures of gene expression, and different statistical methods from those originally used to develop the test. These approaches have fundamentally not improved the discrimination of 'weaks' from 'nons'. A realization that a very low value for cytotoxicity IC50 would drive a final result for the test in ways that were inappropriate for pharmaceuticals has led us to re-examine the cytotoxicity component. Our current efforts are focused on other, perhaps more sensitive, measures of cytotoxicity, combined with gene expression changes in mouse stem cells in an attempt to correctly identify weak teratogens and non-teratogens.

Breast cancers (BC) in younger, premenopausal patients (YBC) tend to be more aggressive with worse prognosis, higher chance of relapse and poorer response to endocrine therapies compared to breast cancers in older patients (OBC). The proportion of YBC (age ? 40) among BC in East Asia is estimated to be 16-32%, significantly higher than the 7% reported in Western countries. Genomic and molecular characterizations have deepened our understanding of breast cancer biology in areas ranging from intrinsic subtypes to treatment responses, however, the molecular bases of Asian YBC remains poorly characterized. We have performed whole-exome sequencing (WES), whole-transcriptome sequencing (WTS) and high coverage targeted sequencing on tumor and matched normal samples from 133 Korean BC patients consisting of 74 YBC cases (age ? 40). We further performed immunohistochemistry (IHC) analyses to characterize tumor-infiltrating lymphocytes (TILs) in 46 tumors using four markers (CD45, CD4, CD8 and CD163). We found that BRCA1/2 germline deleterious mutations are enriched in YBC and the ER+/HER2- subtype, indicating that Asian ER+ YBC has a significant germline contribution. MutSig analysis4 identified ARID1A as a significantly mutated gene, implicating chromatin modeling as a cancer driver in Asian BC. Differential expression analyses suggested that Asian YBC differ in energy metabolism and are more active in protein synthesis than OBC tumors, whereas OBC is more proliferative than YBC. Using gene expression signatures representing distinct immune cell types and immunohistochemistry, we classified our cohort into four subtypes of varying TIL activities: high, medium, low and quiet. The majority of immunogenic cases with high TIL levels lie in ER+ or HER2+ subtypes although higher proportion is seen in TNBC. Moreover, YBC tumors appear to harbor lower levels of TIL activities than OBC, suggesting that younger patients may be less likely to benefit from immunomodulatory therapies than older patients. To our knowledge, this is the first large-scale multi-omics study of Asian breast cancer and would significantly contribute to the compendium of molecular data available for young, premenopausal breast cancer. While the major landmarks in the molecular and immune landscape of Asian BC look similar to that of the predominantly Caucasian BC cohorts, we have identified a number of distinguishing characteristics pointing to distinctive oncogenic mechanisms underlying Asian BC. Citation Format: Yeon Hee Park, Ying Ding, Soonweng Cho, Soo-Hyeon Lee, Hae Hyun Jung, Woosung Chung, Jinho Kim, Woong-Yang Park, Eric Powell, Pamela Vizcarra, Shibing Deng, Se Kyung Lee, Seok Won Kim, Jeong Eon Lee, Ji-Yeon Kim, Jin Seok Ahn, Young-Hyuck Im, Seok Jin Nam, Zhengyan Kan. Multi-omics and immuno-oncology profiling of an Asian breast cancer cohort enriched in young and premenopausal patients. [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 LB-325.

One of the main challenges in immuno-oncology is to quantify different types of immune cells in the tumor microenvironment, as such data can greatly facilitate our understanding of the mechanism of action of, and response to cancer immunotherapies. Traditionally immune profiling is performed by immunohistochemistry or flow cytometry experiments using surface markers specific to each immune cell type. However, both approaches suffer from practical limitations such as reagent availability and real world specimen conditions. Recently several computational deconvolution approaches using expression profiles of the bulk tumor tissue samples have been developed. This alternative approach utilizes existing database of purified immune cell gene signatures and relies on a rigorous mathematical framework of signal deconvolution. One of such algorithms, called CIBERSORT (Newman et al. 2015), has been demonstrated to perform robustly in deconvoluting the relative fractions of 22 human leukocyte subsets in solid tumor tissue samples, and benchmarked favorably against FACS analysis. In this work, we performed immune phenotyping by the CIBERSORT-based expression deconvolution on a cohort of 100 gastric cancer (GC) patients. We found that the fractions of activated CD4 memory T cells, rather than CD8+ T cell, are most significantly correlated with tumor neo-antigen load, whereas the latter is most significantly associated with the expression of a “Cytolytic Activity” metagene and an interferon gamma signature. There is stronger pre-existing host immune response against the MSI and EBV subtypes of GCs mediated by either cytotoxic T cells or Natural Killer cells. Lastly, we found a high M2/M1 tumor associated Macrophages (TAMs) ratio is strongly associated with poor GC prognosis, corroborating recent reports on the role of a TAM and stromal response in gastric cancer angiogenesis. Taken together, we demonstrated in this work that critical information about the tumor infiltrating immune cells can be gleaned from the computational deconvolution of bulk tumor gene expression profiling data. The comprehensiveness and cost-effectiveness of this approach can complement other immune profiling techniques in characterizing a wide variety of tumor specimens under various treatment conditions. We foresee its application in characterizing pharmacodynamics, understanding immunological mechanism of action and monitoring treatment response and disease progression for cancer immunotherapies and their combinations in both pre-clinical and clinical settings. Citation Format: Kai Wang, Hoicheong Siu, Shibing Deng, Jadwiga R. Bienkowska, Suet Yi Leung. Characterizing the immunogenicity of gastric cancer by transcriptomic expression based immune phenotyping. [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 4143.

Consistency and coherence of gene expression data across multiple sites depends on several factors such as platform (oligo, cDNA, etc.), environmental conditions at each laboratory, and data quality. The Hepatotoxicity Working Group of the International Life Sciences Institute Health and Environmental Sciences Institute consortium on the application of genomics to mechanism-based risk assessment is investigating these factors by comparing high-density gene expression data sets generated on two sets of RNA from methapyrilene (MP) experiments conducted at Abbott Laboratories and Boehringer-Ingelheim Pharmaceuticals, Inc. using a single platform (Affymetrix Rat Genome U34A GeneChip) at seven different sites. This article focuses on the evaluation of data quality and statistical models that facilitate the comparison of such data sets at the probe level. We present methods for exploring and quantitatively assessing differences in the data, with the principal goal being the generation of lists of site-insensitive genes responsive to low and high doses of MP. A combination of numerical and graphical techniques reveals important patterns and partitions of variability in the data, including the magnitude of the site effects. Although the site effects are significantly large in the analysis results, they appear to be primarily additive and therefore can be adjusted in the statistical calculations in a way that does not bias conclusions regarding treatment differences.

Characteristic patterns of gene expression measured by DNA microarrays have been used to classify tumors into clinically relevant subgroups. In this study, we have refined the previously defined subtypes of breast tumors that could be distinguished by their distinct patterns of gene expression. A total of 115 malignant breast tumors were analyzed by hierarchical clustering based on patterns of expression of 534 “intrinsic” genes and shown to subdivide into one basal-like, one ERBB2 -overexpressing, two luminal-like, and one normal breast tissue-like subgroup. The genes used for classification were selected based on their similar expression levels between pairs of consecutive samples taken from the same tumor separated by 15 weeks of neoadjuvant treatment. Similar cluster analyses of two published, independent data sets representing different patient cohorts from different laboratories, uncovered some of the same breast cancer subtypes. In the one data set that included information on time to development of distant metastasis, subtypes were associated with significant differences in this clinical feature. By including a group of tumors from BRCA1 carriers in the analysis, we found that this genotype predisposes to the basal tumor subtype. Our results strongly support the idea that many of these breast tumor subtypes represent biologically distinct disease entities.

This study evaluated factors that contribute to the increased energy cost of locomotion in youth. The subjects were 321 8-18-year-old youth, similar dispersed by age and sex. Oxygen uptake (VO2) was measured during rest (REE), running at 8 km · h−1 and cycling at 16 km · h−1, using a COSMED K4b2 metabolic system. Developmental stage was obtained via questionnaire. Stature, body mass, and skinfolds (triceps & subscapular) were measured. Both sexes had similar absolute VO2 (mL · min−1) at rest (p = 0.065) and running (p = 0.084), but the males had a higher VO2 during cycling (p = 0.046). There were no sex differences in relative VO2 (mL · kg−1 · min−1) at rest (p = 0.083); however, the males had a higher VO2 than the females during cycling and running (p £ 0.002). Multiple regression, tested for collinearity, found that absolute VO2 during cycling and running was mostly related to fat-free mass (p = 0.0001). Similar analyses for relative VO2 (mL · kg−1 · min−1) during cycling found that fat-free mass, sex, and skinfolds were significant contributors (p ‡ 0.003). During running the relative VO2 was related to skinfolds, fat-free mass, and resting energy expenditure (p < 0.05). Neither age nor developmental stage was a significant contributor. The results indicate that the VO2 of locomotion is most closely associated with fat-free mass. Thus, to compare youth of varying age or pubertal developmental status, fat-free mass should be taken into consideration.

Purpose: To determine the effect of increasing the aerobic component of the school's physical activity program and improving the knowledge about weight control and blood pressure on the blood pressure and body fat of early adolescents. Methods: The subjects were 1140 youth aged 11 to 14 years (630 females, 510 males; 64% white, 24.4% African-American, and 11.6% "other"), who were randomly assigned by school into four treatment groups: exercise only, education only, exercise and education combined, and control group. Heights, weights, and skinfold thicknesses were measured, and body mass index (BMI) was computed (kg/m 2 ). Blood pressure was obtained in duplicate using a random-zero mercury sphygmomanometer. Maximal oxygen uptake was predicted from a submaximal cycle ergometer test. Data were analyzed using analysis of covariance statistics, adjusting for gender, ethnicity, age, socioeconomic status, and initial baseline characteristics. Results: Systolic and diastolic blood pressures increased more in the control group than in the intervention groups ( p = .001). The BMI did not change significantly ( p = .709), but the sum of skinfolds increased less in subjects in the exercise intervention groups than the education only or control groups ( p = .0001). The small increase in p VO 2 max of the combined exercise and education group was significantly greater than the education only group ( p = .0001). Conclusion: An exercise program for youth can have a positive effect on blood pressure independent of body weight loss.

Background: African American women living in the Southeast experience a higher mortality due to cardiovascular (CV) disease than their White counterparts. It is unclear if this vulnerability to CV disease is due to race, socioeconomic status, or health behaviors. Objectives: To examine the disparities in cardiovascular health between Southern rural, African American and White women to determine if a CV Risk-Index differed by race, education, or income levels and if differences persisted when controlling for body mass index (BMI). Methods: Subjects were 1,110 women (27% African American, 73% White) residing in rural North Carolina. Data were collected by mailed questionnaire and analyzed using analysis of variance (ANOVA) and analysis of covariance (ANCOVA). Results: African American women had significantly lower education and lower income than Whites, higher BMI, and a much greater prevalence of hypertension, angina, and diabetes. In a three-way ANOVA including race, income, and education, education and race were significant predictors of the CV Risk Index, but when adjusted for BMI race was no longer significant (p = .3039); the only significant predictors were BMI and educational level. Discussion: Women with the least education had the highest CV Risk-Index, regardless of race. These findings suggest the need to focus risk reduction interventions on all Southern rural women with limited education, not only African American women. This supports the current literature that suggests race should be viewed as a risk marker rather than a risk factor.

Gastric cancer is a heterogeneous disease with diverse molecular and histological subtypes. We performed whole-genome sequencing in 100 tumor-normal pairs, along with DNA copy number, gene expression and methylation profiling, for integrative genomic analysis. We found subtype-specific genetic and epigenetic perturbations and unique mutational signatures. We identified previously known (TP53, ARID1A and CDH1) and new (MUC6, CTNNA2, GLI3, RNF43 and others) significantly mutated driver genes. Specifically, we found RHOA mutations in 14.3% of diffuse-type tumors but not in intestinal-type tumors (P < 0.001). The mutations clustered in recurrent hotspots affecting functional domains and caused defective RHOA signaling, promoting escape from anoikis in organoid cultures. The top perturbed pathways in gastric cancer included adherens junction and focal adhesion, in which RHOA and other mutated genes we identified participate as key players. These findings illustrate a multidimensional and comprehensive genomic landscape that highlights the molecular complexity of gastric cancer and provides a road map to facilitate genome-guided personalized therapy.

Serum β-nerve growth factor (NGF) concentrations were determined in pregnant female, nonpregnant female, and male cynomolgus monkeys using a highly selective and sensitive immunoaffinity liquid chromatography-tandem mass spectrometry assay. NGF was significantly higher in pregnant monkeys than in nonpregnant female and male monkeys. NGF increased over pregnancy (mean NGF±SD: 541±448, 1590±520, and 3560±1430 pg/ml during the first, second, and third trimesters, respectively). These data will aid in further understanding the role of NGF during pregnancy.

Handling, including blood collection, has often been discouraged in molting penguins because it is considered an additional stress imposed on birds already experiencing major physiologic stress associated with molting. To evaluate the degree of physiologic stress posed by molting, we compared the hematologic and plasma biochemical values and hormone levels of molting and nonmolting African penguins, Spheniscus demersus. Five male and 5 female penguins randomly chosen were given complete physical examinations, were weighed, and blood samples were taken at 7 time points before, during, and after the molt. Data were analyzed by linear mixed-model analysis of variance. Throughout the study, behavior and appetite remained normal. Catecholamine levels were highly variable within and among subjects, whereas mean corticosterone levels were significantly different between baseline, molt, and postmolt values. Significant differences from baseline values were observed in many of the hematologic analytes; however, only decreases in hematocrit and red blood cell count values were considered clinically significant. Anemia due to experimentally induced blood loss as a possible cause of the significant hematologic changes was ruled out based on results of a follow-up control study during the nonmolt season, which showed no significant changes in hematocrit level or total red blood cell counts when using similar sampling protocols, which indicates that these changes were associated with molt.

Colorectal cancer (CRC) patients have poor prognosis after formation of distant metastasis. Understanding the molecular mechanisms by which genetic changes facilitate metastasis is critical for the development of targeted therapeutic strategies aimed at controlling disease progression while minimizing toxic side effects. A comprehensive portrait of somatic alterations in CRC and the changes between primary and metastatic tumors has yet to be developed. We performed whole genome sequencing of two primary CRC tumors and their matched liver metastases. By comparing to matched germline DNA, we catalogued somatic alterations at multiple scales, including single nucleotide variations, small insertions and deletions, copy number aberrations and structural variations in both the primary and matched metastasis. We found that the majority of these somatic alterations are present in both sites. Despite the overall similarity, several de novo alterations in the metastases were predicted to be deleterious, in genes including FBXW7, DCLK1 and FAT2, which might contribute to the initiation and progression of distant metastasis. Through careful examination of the mutation prevalence among tumor cells at each site, we also proposed distinct clonal evolution patterns between primary and metastatic tumors in the two cases. These results suggest that somatic alterations may play an important role in driving the development of colorectal cancer metastasis and present challenges and opportunities when considering the choice of treatment.

Defects in the leptin gene or the leptin receptor may be a genetic cause of obesity, but little is known about the familial associations of leptin and obesity. This study compared plasma leptin and measures of obesity in a sample of 248 subjects (124 mother-offspring pairs); 34% were African American and 66% were white. Youth were aged 12 to 16 years. Plasma leptin and body mass index (BMI) were higher in mothers than in their offspring and, among the offspring, higher in girls than boys, even after correcting for BMI or body fat. Racial differences in leptin were present in both mothers and youth when adjusting for percentage body fat but disappeared when adjusting for BMI. In univariate analyses of the associations between mothers and offspring, BMI was associated with leptin in all groups but was most strongly associated in white pairs and in mother-son pairs. In multiple regression analyses, when adjusting for BMI, significant predictors of leptin level for the boys and girls together were gender, BMI, and pubertal status of the offspring; in girls only BMI was significant (R2= 0.72), and in boys the significant predictors were their BMI (R2= 0.66) followed by their pubertal status (R2= 0.06) and the leptin level of their mothers (R2= 0.02). When adjusting for body fat, the predictors were the offspring’s percentage body fat (R2= 0.67) and mother’s leptin (R2= 0.03), with similar results in gender-specific analyses. The authors conclude that leptin levels of youth are most closely associated with their degree of obesity or body fat; mother’s leptin and, for boys only, pubertal status also play a small role. Although the small association between maternal leptin on leptin in their offspring could be due to either heredity or shared environment, the results of this study suggest that individual obesity and environmental factors are important predictors of leptin levels in children.

Tobacco is the leading cause of preventable death in the United States, and its use is increasing in adolescents. To determine the interventions needed to prevent the initiation of smoking, it is important to know the factors related to tobacco use by adolescents. In this study the following factors related to cigarette use were examined: age, gender, ethnicity, self-esteem, physical activity, parental smoking, and socioeconomic status. Participants were 1,207 youth completing a written survey for the Cardiovascular Health in Children and Youth Study (CHIC II). Participants ranged in age from 10 to 15 years, with a mean age of 12.2 years; 64.2% were White, 24.0% Black, 5.8% Hispanic, and 6.0% other races. White and Hispanic youth and youth of other races had significantly higher rates of smoking than did Black youth. Significant risk factors for smoking were: higher grade in school, White race, and for girls only, lower self-esteem. In White youth those in the lowest socioeconomic status were most likely to be current and experimental smokers. Smoking was as common in girls as in boys at these ages. Multivariate analysis showed that neither physical activity nor parental smoking were significant predictors of smoking behaviors. These results suggest that smoking prevention programs for adolescents should specifically target White and Hispanic youth and those from families with low socioeconomic status. In addition, these interventions should include ways to increase self-esteem in girls.

Overcoming resistance to targeted kinase inhibitors is a major clinical challenge in oncology. For 1st and 2nd generation ALK inhibitors acquired resistance due to ALK kinase domain mutations and/or pharmacological drug resistance are major causes for disease relapse. Here, we report the preclinical evaluation of PF-06463922, a potent and brain penetrant ALK/ROS1 inhibitor with sub to low nanomolar cell potency against ALK fusions and all known clinically-acquired resistant mutations. PF-06463922 exhibited marked cytoreductive activity in tumor xenografts driven by various ALK mutants. Furthermore, PF-06463922 led to significant regression of EML4-ALK driven lung cancer brain metastasis and prolonged mouse survival. Compared to other clinically available ALK inhibitors, PF-06463922 is unique in its superior potency against a broad spectrum of acquired ALK mutations, including the highly resistant G1202R mutant and its robust antitumor activity in the brain. Furthermore, PF-06463922 demonstrated remarkable selectivity and safety margins in a variety of preclinical studies. These results suggest that PF-06463922 may be highly effective for the treatment of patients with ALK-driven lung cancers, including those who relapsed on clinically available ALK inhibitors due to ALK secondary mutations and/or brain metastases. Citation Format: Luc Friboulet, Helen Zou, David P. Kodack, Lars D. Engstrom, Qiuhua Li, Melissa West, Ruth W. Tang, Hui Wang, Konstantinos Tsaparikos, Jinwei Wang, Sergei Timofeevski, Dac M. Dinh, Hieu Lam, Justine L. Lam, Shinji Yamazaki, Wenyue Hu, Bhushankumar Patel, Divya Bezwada, Sidra Mahmood, Eugene Lifshits, Timothy Affolter, Patrick B. Lappin, Hovhannes Gukasyan, Nathan Lee, Shibing Deng, Rakesh K. Jain, Ted W. Johnson, Alice T. Shaw, Valeria R. Fantin, Tod Smeal. PF-06463922, a novel next generation ALK/ROS1 inhibitor, overcomes resistance to 1st and 2nd generation ALK inhibitors in pre-clinical models. [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 130. doi:10.1158/1538-7445.AM2015-130

The fetal oncogene 5T4 is a cell surface protein, with over-expression observed in a variety of cancers as compared to normal adult tissue. Recent studies have shown that expression of 5T4 appears to be associated with the undifferentiated state and the epithelial-mesenchymal transition (EMT), and thus has been associated with a more invasive phenotype. We have developed assays to measure the expression of the fetal oncogene, 5T4, in both the primary tumor compartment and in the circulating tumor cell compartment. These assays were then used to investigate 5T4 expression in a small cohort of patients with NSCLC. We obtained matched primary tumor and blood samples, with the blood being obtained prior to resection of the primary tumor. The expression of 5T4 was found to be robust and measurable in both the primary and circulating tumor compartments. We observed expression of 5T4 in both adenocarcinoma and squamous cell carcinoma, in all stages and grades of tumor, with no specific correlation between expression and stage, grade or pathology. We further observed robust enumeration of CTCs in NSCLC samples. The expression of 5T4 was heterogeneous in the CTC compartment with no correlation to grade, stage or pathology. Finally, we observed no concordance between 5T4 expression in the primary tumor and the circulating tumor cell compartment. We discuss the current utility of target expression in predicting response to targeted therapy in the context of antibody based therapy, and the role that CTCs may have in the clinic. Citation Format: Steven R. Pirie-Shepherd, Shibing Deng, Jonathon Golas, Pamela Vizcarra, Eric Tucker, Dena Marrinuci, Hans-Peter Gerber, Eric L. Powell. The expression of fetal oncogene 5T4 in CTCs obtained from NSCLC patients is discordant with the expression measured in the primary tumor. [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 564. doi:10.1158/1538-7445.AM2015-564

Previous studies on the influence of a rural/urban setting on the prevalence of cardiovascular disease risk factors in children have not sufficiently controlled for socioeconomic status, race, gender, and perhaps, may not have included a representative sample of rural and urban children. This study compared the cardiovascular disease risk factors and rate of obesity of children living in rural and urban settings. It also determined the magnitude of the effect of the rural/urban setting on cardiovascular disease risk factors and obesity when controlling for race, socioeconomic status, and gender. The subjects were 2,113 third- and fourth-grade children; 962 from an urban setting and 1,151 from a rural setting. Height, weight, skinfolds, resting blood pressure, and total cholesterol levels were measured. Aerobic power (pV02max) was estimated from cycle ergomerty. Physical activity and smoking history were obtained from a questionnaire. Clustering analyses using adjustment for sample error indicated that total cholesterol, blood pressure, smoking, and physical activity levels of rural and urban children were not different (P>0.10); however, body mass index and sum of skinfolds was greater for rural youth (P

Low-resolution mid-UV spectra of M32 and 47 Tuc have been extracted from the IUE archival database, along with spectra of 41 F and G dwarfs with well-determined atmospheric parameters and integrated spectra of 24 Galactic globular clusters. We have used five mid-UV spectral indices defined by Fanelli et al. to constrain the stellar content of M32 and 47 Tuc and to make a comparative study between the two stellar systems. In the case of 47 Tuc, the bulk of the mid-UV light is shown to come from the main-sequence turnoff stars, with much smaller (but significant) contributions coming from red horizontal-branch stars, red giants, and A stars (presumably, blue stragglers). In contrast, M32 is shown to have no significant contribution from a red horizontal-branch population, has a more metal-rich main-sequence turnoff, and has a significantly larger hot star contribution than is inferred to be present in 47 Tuc. These inferences are consistent with conclusions obtained from integrated light studies of M32 and 47 Tuc in the blue.

Purpose: To describe smoking initiation, and to investigate factors that predict the early initiation of smoking in schoolchildren using a longitudinal approach. Methods: A prospective study of smoking habits of children from the third and fourth grades through the eighth and ninth grades. The initial study population was 1970; 79.8% were white and 20.2% were African-American. Children were classified as "nonsmokers," "experimental smokers," or "current smokers" at five time points over 6 years. Multivariate regression models examined relationships of demographic and developmental factors with smoking initiation. Results: Experimental smoking increased from 4% at Grades 3–4 to 42% at Grades 8–9, and current smoking prevalence rose from 0.4% to 9% over the same period. The mean age of initiation of smoking was 12.3 years. Smoking initiation (experimental smoking) was significantly different by racial group, socioeconomic status (SES), and pubertal development. White children and those of low SES were more likely to be experimental smokers, and also started earlier than African-American children and children of high SES. Once they started, white children advanced more rapidly to become current smokers. Boys had a higher prevalence of experimental smoking than girls at all time points. Children in rural areas were more likely than urban children to start smoking after age 12 years. Children who were at a higher pubertal stage than their peers were also more likely to experiment with smoking. Conclusions: Race, SES, and pubertal stage are important predictors of initiation of smoking in schoolchildren. This study indicates a need for smoking prevention classes in elementary and middle school, especially in areas with large numbers of white and low-SES youth. Also, smoking cessation programs, as well as smoking prevention classes, would be useful for middle school and high school students.

Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide and has no effective treatment, yet the molecular basis of hepatocarcinogenesis remains largely unknown. Here we report findings from a whole-genome sequencing (WGS) study of 88 matched HCC tumor/normal pairs, 81 of which are Hepatitis B virus (HBV) positive, seeking to identify genetically altered genes and pathways implicated in HBV-associated HCC. We find beta-catenin to be the most frequently mutated oncogene (15.9%) and TP53 the most frequently mutated tumor suppressor (35.2%). The Wnt/beta-catenin and JAK/STAT pathways, altered in 62.5% and 45.5% of cases, respectively, are likely to act as two major oncogenic drivers in HCC. This study also identifies several prevalent and potentially actionable mutations, including activating mutations of Janus kinase 1 (JAK1), in 9.1% of patients and provides a path toward therapeutic intervention of the disease.

In the 2012 Food and Drug Administration (FDA) draft guidance on drug-drug interactions (DDIs), a new molecular entity that inhibits Pglycoprotein (P-gp) may need a clinical DDI study with a P-gp substrate such as digoxin when themaximumconcentration of inhibitor at steady state divided by IC50 ([I1]/IC50) is0.1 or concentration of inhibitor based on highest approved dose dissolved in 250 ml divide by IC50 ([I2]/IC 50) is10. In this article, refined criteria are presented, determined by receiver operating characteristic analysis, using IC50 values generated by 23 laboratories. P-gp probe substrates were digoxin for polarized cell-lines and N-methyl quinidine or vinblastine for P-gp overexpressed vesicles. Inhibition of probe substrate transport was evaluated using 15 known P-gp inhibitors. Importantly, the criteria derived in this article take into account variability in IC50 values. Moreover, they are statistically derived based on the highest degree of accuracy in predicting true positive and true negative digoxin DDI results. The refined criteria of [I1]/IC50 0.03 and [I2]/IC50 45 and FDA criteria were applied to a test set of 101 in vitro-in vivo digoxin DDI pairs collated from the literature. The number of false negatives (none predicted but DDI observed) were similar, 10 and 12%, whereas the number of false positives (DDI predicted but not observed) substantially decreased from 51 to 40%, relative to the FDA criteria. On the basis of estimated overall variability in IC50 values, a theoretical 95%confidence interval calculation was developed for single laboratory IC 50 values, translating into a range of [I1]/IC50 and [I2]/IC50 values. The extent by which this range falls above the criteria is a measure of risk associated with the decision, attributable to variability in IC50 values. © 2013 by The American Society for Pharmacology.

Let $G_w$ be a weighted graph. The number of the positive, negative and zero eigenvalues in the spectrum of $G_w$ are called positive inertia index, negative inertia index and nullity of $G_w$, and denoted by $i_{+}(G_w)$, $i_{-}(G_w)$, $i_{0}(G_w)$, respectively. In this paper, sharp lower bound on the positive (resp. negative) inertia index of weighted bicyclic graphs of order $n$ with pendant vertices is obtained. Moreover, all the weighted bicyclic graphs of order $n$ with at most two positive, two negative and at least $n-4$ zero eigenvalues are identified, respectively., Comment: 12 pages, 5 figures, 2 tables. arXiv admin note: text overlap with arXiv:1307.0059 by other authors

Aberrant activation of the Hedgehog (Hh) signaling pathway has been implicated in a variety of cancers and a small molecule inhibitor of Smoothened (SMO), Vismodegib, has been approved for the treatment of basal cell carcinoma, a disease frequently driven by Hh pathway signaling due to pathway mutations. SMO dependent Hh signaling has also been implicated in models of myeloid leukemia, primarily CML, where genetic loss of SMO or pharmacological inhibition limits disease progression in part through targeting the leukemic stem cell (LSC). Outside of BCR-ABL driven leukemia the role of Hh signaling and impact of SMO inhibition on disease progression and the LSC remains unclear. To explore the role of Hedgehog pathway signaling and interrogate responses to SMO inhibition in AML, a panel of primary AML patient-derived models was utilized to examine responses to PF-04449913, an oral small molecule SMO inhibitor currently in early phase clinical trials targeting myeloid malignancies. AML patient samples were characterized for Hh pathway expression levels and screened for responses to PF-04449913. Ex vivo treatment of AML bone marrow cells showed PF-04449913 was capable of inhibiting Hh pathway activity, reducing expression of key LSC regulators and decreasing populations of cells expressing LSC markers. Use of AML xenotransplant models to assess in vivo responses to PF-04449913 as single agent and in combination with Cytarabine have shown potential for combination efficacy of the two agents in select models suggesting patient selection strategies may be critical for SMO inhibitor-based therapies in AML. Citation Format: Amy Jackson-Fisher, Pamela Whalen, Mark Elliott, Melissa McMahon, Enhong Chen, Xianxian Zheng, Mark Ozeck, Donghui Huang, Paul Lira, Joseph Lee, Cathy Zhang, Justine Lam, Mary Spilker, Shibing Deng, Patrick Lappin, Penny Venne, Cynthia Heinlein, Annelie Schairer, Karen McLachlan, Todd VanArsdale. Interrogating hedgehog pathway and smoothened inhibition by PF-04449913 in patient-derived acute myeloid leukemia models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1958. doi:10.1158/1538-7445.AM2014-1958

Cancer is a genetic disease with frequent somatic DNA alterations. Studying recurrent copy number aberrations (CNAs) in human cancers would enable the elucidation of disease mechanisms and the prioritization of candidate oncogenic drivers with causal roles in oncogenesis. We have comprehensively and systematically characterized CNAs and the accompanying gene expression changes in tumors and matched nontumor liver tissues from 286 hepatocellular carcinoma (HCC) patients. Our analysis identified 29 recurrently amplified and 22 recurrently deleted regions with a high level of copy number changes. These regions harbor established oncogenes and tumor suppressors, including CCND1 (cyclin D1), MET (hepatocyte growth factor receptor), CDKN2A (cyclin-dependent kinase inhibitor 2A) and CDKN2B (cyclin-dependent kinase inhibitor 2B), as well as many other genes not previously reported to be involved in liver carcinogenesis. Pathway analysis of cis-acting genes in the amplification and deletion peaks implicates alterations of core cancer pathways, including cell-cycle, p53 signaling, phosphoinositide 3-kinase signaling, mitogen-activated protein kinase signaling, Wnt signaling, and transforming growth factor beta signaling, in a large proportion of HCC patients. We further credentialed two candidate driver genes (BCL9 and MTDH) from the recurrent focal amplification peaks and showed that they play a significant role in HCC growth and survival. Conclusion: We have demonstrated that characterizing the CNA landscape in HCC will facilitate the understanding of disease mechanisms and the identification of oncogenic drivers that may serve as potential therapeutic targets for the treatment of this devastating disease. (Hepatology 2013;58:706–717)

The recently approved ALK kinase inhibitor crizotinib has demonstrated successful treatment of metastatic and late stage ALK fusion positive non-small cell lung cancer (NSCLC). However, the median duration of clinical benefit is ~10–11months due to the emergence of multiple and simultaneous resistance mechanisms in these tumors. Mutations in the ALK kinase domain confer resistance to crizotinib in about one-third of these patients. We developed a multiplex deep sequencing method using semiconductor sequencing technology to quickly detect resistance mutations within the ALK kinase domain from tumor biopsies. By applying a base-pair specific error-weighted mutation calling algorithm (BASCA) that we developed for this assay, genomic DNA analysis from thirteen relapsed patients revealed three known crizotinib resistance mutations, C1156Y, L1196M and G1269A. Our assay demonstrates robust and sensitive detection of ALK kinase mutations in NSCLC tumor samples and aids in the elucidation of resistance mechanisms pertinent to the clinical setting.

Anaplastic lymphoma kinase gene (ALK) fusions have been identified in approximately 5% of non-small-cell lung carcinomas (NSCLCs) and define a distinct subpopulation of patients with lung cancer who are highly responsive to ALK kinase inhibitors, such as crizotinib. Because of this profound therapeutic implication, the latest National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology recommend upfront ALK screening for all patients with NSCLC. The Food and Drug Administration–approved companion diagnostic test (ie, fluorescence in situ hybridization) for identification of ALK-positive patients, however, is complex and has considerable limitations in terms of cost and throughput, making it difficult to screen many patients. To explore alternative screening modalities for detecting ALK fusions, we designed a combination of two transcript-based assays to detect for presence or absence of ALK fusions using NanoString's nCounter technology. By using this combined gene expression and ALK fusion detection strategy, we developed a multiplexed assay with a quantitative scoring modality that is highly sensitive, reproducible, and capable of detecting low-abundant ALK fusion transcripts, even in samples with a low tumor cell content. In 66 archival NSCLC samples, our results were highly concordant to prior results obtained by fluorescence in situ hybridization and IHC. Our assay offers a cost-effective, easy-to-perform, high-throughput, and FFPE-compatible screening alternative for detection of ALK fusions.

MCMURRAY, ROBERT G., JOANNE S. HARRELL,SHIBING DENG, CHYRISE B. BRADLEY, LORI M.COX, AND SHRIKANT I. BANGDIWALA. The influenceof physical activity, socioeconomic status, and ethnicity onthe weight status of adolescents. Obes Res. 2000;8:130–139.Objective: This study examined the effects of physicalactivity, television viewing, video game play, socioeco-nomic status (SES), and ethnicity on body massindex (BMI).Research Methods and Procedures: The sample was 2389adolescents, 10 to 16 years of age (12.7 6 1.0 years); 1240(52%) females and 1149 (48%) males; 77% white and 23%African American; from rural (77%) and urban (23%) set-tings. BMI and skinfolds were directly assessed. All otherdata were obtained from questionnaires.Results: Watching television on non-school days was re-lated to being overweight (p , 0.005). However, when BMIanalyses were adjusted for ethnicity and SES, there were nosignificant effects of television viewing on BMI (p .0.061). Increased hours of video game play enhanced therisk of being overweight for both genders when analyseswere adjusted for ethnicity and SES (p , 0.019). In males,participation in as little as one high-intensity physical ac-tivity 3 to 5 days a week decreased the ethnic- and SES-adjusted relative risk of being overweight (RR 5 0.646; CI:0.427 to 0.977). For females, the ethnic- and SES-adjustedrelative risk for being overweight was not significantlyaltered by physical activity. The logistic analyses furtherindicated the influence of low SES and African Americanethnicity overshadowed any direct effect of televisionor videos.Discussion: Because weight status of male adolescents ap-pears to be more related to exercise habits than to televisionor video game habits, increased participation in high-inten-sity exercise appears to be important. For females, neithervideos nor exercise habits appear to be related to risk ofbeing overweight. However, ethnicity and SES may beimportant factors that can influence body weight status,while television viewing may be of some importance. Thus,programs to reduce obesity in female adolescent shouldfocus their efforts in lower SES communities.Key words: television, video games, body mass index,adolescents

Recent regulatory guidance suggests that metabolites identified in human plasma should be present at equal or greater levels in one of the animal species used in safety assessments. In this report, a high-performance liquid chromatography-tandem mass spectrometry method is described whereby quantitative comparisons of exposures to metabolites between species can be obtained in the absence of authentic standards of the metabolites, calibration curves, and other attributes of standard bioanalytical methods. This novel method was tested using six drug-metabolite combinations. Plasma samples from animals are mixed with control plasma from humans and vice versa to remove possible differential effects of matrices. Through multiple ion monitoring-triggered enhanced product ion (EPI) scans, all metabolites were qualitatively confirmed, and daughter ions were selected for the most sensitive mass transitions to trigger EPI scans. Direct comparisons of metabolites in animal versus human plasma were achieved by calculating the peak area ratios of the metabolites versus an internal standard. Linearity of instrument responses was established by serial dilution. A statistical analysis demonstrated that experimentally measured ratios of the parent and metabolites in rat versus human correlated well with the nominal ratios of concentrations using linear regression with an average slope of 0.99 ± 0.08 (r = 0.994 ± 0.005). This analysis showed that if the experimentally determined ratio of mass spectrometer responses is ≥ 2.0, then the actual exposure ratio is unity or greater (p < 0.01). This method offers time- and resource-sparing advantages to ascertaining metabolite exposure comparisons between humans and laboratory animal species. A strategy for application of this approach within standard drug development processes is described.

Prolongation of the electrocardiogram QT interval by some, but not all drugs, has been associated with increased incidence of sudden cardiac death. Current preclinical regulatory assays cannot discriminate the arrhythmia liability of these drugs. Consequently, many new medications that prolong the QT interval are not developed despite their potential therapeutic benefit. Alternans (action potential duration alternations) is a measure of cardiac instability in humans and animals associated with the onset of ventricular fibrillation. Due to potential arrhythmia risk from observed QT prolongation, alternans was assessed in the anesthetized guinea pig after azithromycin or chloroquine alone and after combination treatment at clinically relevant concentrations proposed for the management of malaria. Chloroquine alone, but not azithromycin, caused a profound increase in action potential duration but with only minimal effects on alternans (approximately 10 ms). Azithromycin alone and in combination with chloroquine showed no increase in alternans beyond vehicle baseline responses indicating no additional arrhythmia liability.

Cancer is a genetic disease with frequent somatic alterations in DNA. Study of recurrent copy number aberrations (CNAs) in human cancers would enable the elucidation of disease mechanisms and the identification of key oncogenic drivers with causal roles in oncogenesis. We have comprehensively and systematically characterized CNAs and accompanied gene expression changes in the tumors and their matched non-tumor liver tissues from 286 hepatocellular carcinoma (HCC) patients. Our analysis identified 29 recurrently amplified regions and 22 deleted regions with a high level of copy number changes, harboring established oncogenes and tumor suppressors, including CCND1, MET, CDKN2A and CDKN2B, as well as many other genes not previously reported to be involved in liver carcinogenesis. Pathway analysis of cis-acting genes in the amplification and deletion peaks suggests the alterations of core cancer pathways, including cell cycle, p53, PI3K, MAPK, Wnt and TGFβ signaling, in large proportions of HCC patients. We further credentialed two candidate driver genes, BCL9 and MTDH, from the recurrent focal amplification peaks and showed that they play a significant role in HCC growth and survival. In summary, we have demonstrated that characterizing the CNA landscape in HCC will facilitate the understanding of disease mechanisms and the identification of oncogenic drivers that may serve as potential therapeutic targets for the treatment of this devastating disease. Citation Format: Kai Wang, Ho Yeong Lim, Stephanie Shi, Jeeyun Lee, Shibing Deng, Tao Xie, Zhou Zhu, Yuli Wang, David Pocalyko, Wei J. Yang, Paul A. Rejto, Mao Mao, Cheol-Keun Park, Jiangchun Xu. Genomic landscape of copy number aberrations enables the identification of oncogenic drivers in hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2002. doi:10.1158/1538-7445.AM2013-2002

Oncogenic fusions involving ALK, ROS1 and RET define a small subpopulation (∼7%) of non-small cell lung carcinomas (NSCLC). Tumors harboring ALK and ROS1 rearrangements are highly sensitive to crizotinib whereas RET inhibitors may be of potential benefit for the treatment for RET-fusion positive tumors1. Current methods for detection of ALK, ROS1, and RET rearrangements involve fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) or RT-PCR, each test offering its own advantages and disadvantages, and making it difficult for screening large numbers of patient samples for all three targetable fusions. To explore screening modalities for the simultaneous detection of ALK, ROS1 and RET fusions from a single analyte, we designed a multiplexed, transcript-based assay to detect for presence or absence of fusions. Utilizing a combined 3’ over-expression and fusion-specific detection strategy, we developed a single-tube multiplex assay with a quantitative scoring modality that is highly sensitive, reproducible and capable of detecting low abundant ALK, ROS1 and RET fusion transcripts. We successfully validated the assay in 273 NSCLC specimens. For ALK, our results were highly concordant (97.4%) to prior results obtained by IHC (n=189). We confirmed ROS1 (n=7) and RET (n=11) fusion-positive tumors by RT-PCR plus sequencing. There is a significant enrichment for ROS1 and RET fusions in triple negative NSCLC (i.e. KRAS/EGFR/ALK wildtype) (Table 1). ALK, ROS1, RET, KRAS and EGFR mutations are mutually exclusive in this study. Our assay offers an easy to perform, high-throughput, and FFPE-compatible screening method for detection of ALK, ROS1 and RET fusions. Table 1. Incidence of ALK, ROS1 and RET fusions cohorts clinical/molecular feature # cases ALK+ ROS1+ RET+ Set 1 ALK IHC+, Kras/EGFR wt 95 91 0 0 Set 2 ALK IHC−, Kras/EGFR wt 94 1 5 11 Set 3 never smokers 84 10 2 1 Citation Format: Maruja Lira, Yoon-La Choi, Shibing Deng, Donghui Huang, Mark Ozeck, Joungho Han, Ji Yun Jeong, Keunchil Park, Jhingook Kim, Myung-Ju Ahn, Mao Mao. A single-tube, multiplexed, transcript-based assay to detect ALK, ROS1 and RET fusions in lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3493. doi:10.1158/1538-7445.AM2013-3493