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The CDK4/6 inhibitor palbociclib reduces tumor growth by decreasing retinoblastoma (RB) protein phosphorylation and inducing cell cycle arrest at the G1/S phase transition. Palbociclib in combination with anti-hormonal therapy brings significant benefit to breast cancer patients. In this study, novel combination approaches and underlying molecular/cellular mechanisms for palbociclib were explored in squamous cell lung cancer (SqCLC), the second most common subtype of non-small cell lung cancer. While approximate 20% lung patients benefit from immunotherapy, most SqCLC patients who receive platinum-doublet chemotherapy as first-line treatment, which often includes a taxane, are still in need of more effective combination therapies. Our results demonstrated enhanced cytotoxicity and anti-tumor effect with palbociclib plus taxanes at clinically achievable doses in multiple SqCLC models with diverse cancer genetic backgrounds. Comprehensive gene expression analysis revealed a sustained disruption of pRB-E2F signaling by combination that was accompanied with enhanced regulation of pleiotropic biological effects. These included several novel mechanisms such as abrogation of G2/M and mitotic spindle assembly checkpoints, as well as impaired induction of hypoxia-inducible factor 1 alpha (HIF-1α). The decrease in HIF-1α modulated a couple key angiogenic and anti-angiogenic factors, resulting in an enhanced anti-angiogenic effect. This preclinical work suggests a new therapeutic opportunity for palbociclib in lung and other cancers currently treated with taxane based chemotherapy as standard of care.

To elucidate the effects of neoadjuvant chemotherapy (NAC), we conduct whole transcriptome profiling coupled with histopathology analyses of a longitudinal breast cancer cohort of 146 patients including 110 pairs of serial tumor biopsies collected before treatment, after the first cycle of treatment and at the time of surgery. Here, we show that cytotoxic chemotherapies induce dynamic changes in the tumor immune microenvironment that vary by subtype and pathologic response. Just one cycle of treatment induces an immune stimulatory microenvironment harboring more tumor infiltrating lymphocytes (TILs) and up-regulation of inflammatory signatures predictive of response to anti-PD1 therapies while residual tumors are immune suppressed at end-of-treatment compared to the baseline. Increases in TILs and CD8+ T cell proportions in response to NAC are independently associated with pathologic complete response. Further, on-treatment immune response is more predictive of treatment outcome than immune features in paired baseline samples although these are strongly correlated., Neoadjuvant chemotherapy is a therapeutic option for the treatment of breast cancer. Here, the authors characterize changes in the gene expression profiles and immune microenvironment in serial breast cancer biopsies taken before, during and after neoadjuvant chemotherapy.

Macroautophagy (autophagy) is an essential cellular catabolic process required for survival under conditions of starvation. The role of autophagy in cancer is complex, context-dependent and at times contradictory, as it has been shown to inhibit, promote or be dispensable for tumor progression. In this study, we evaluated the contribution of the immune system to the reliance of tumors on autophagy by depleting autophagy-related 7 (ATG7) in murine tumor cells and grafting into immunocompetent versus immunodeficient hosts. Although loss of ATG7 did not affect tumor growth in vitro or in immunodeficient mice, our studies revealed that cancer cell reliance on autophagy was influenced by anti-tumor immune responses, including those mediated by CD8+ T cells. Furthermore, we provide insights into possible mechanisms by which autophagy disruption can enhance anti-tumor immune responses and suggest that autophagy disruption may further benefit patients with immunoreactive tumors.

Chronic hypoxia is a common inducer of end-stage cardiovascular disease. In cells under hypoxia, the hypoxia-inducible factor-1 (HIF-1) plays a vital role in regulating downstream target genes. However, the mechanism of hypoxia in cardiomyocytes is still unclear. In this study, we aimed to identify novel downstream epigenetic targets of HIF-1α in cardiomyocytes under hypoxia. H9c2 cells were exposed to hypoxia condition, and quantitative real-time PCR analysis was performed to evaluate the expression of miR-20b-5p. The results indicated that the expression of miR-20b-5p was down-regulated in H9c2 cells under low oxygen condition. Meanwhile, HIF-1α overexpression further down-regulated the miR-20b-5p expression in H9c2 cells transfected with HIF-1α plasmids. In addition, Annexin-V-FITC/PI flow cytometry analysis suggested that overexpression of miR-20b-5p attenuated cell apoptosis under hypoxia condition in H9c2 cells. Western blot analysis showed that the hypoxia apparently increased Bax and cleaved-caspase-3, but decreased Bcl-2 expression in H9c2 cells, indicating that hypoxia-induced NF-κB signaling pathway activation is mediated by miR-20b-5p. Hypoxia-induced H9c2 cell apoptosis was reduced after HIF-1α knockdown as shown by the flow cytometry analysis. In conclusion, we identified that miR-20b-5p plays an important role in mediating cardiomyocytes apoptosis under hypoxia, which is mediated by the HIF-1/NF-κB signaling pathway.

In this phase I study (NCT01307267), we evaluated safety, pharmacokinetics, clinical activity, and pharmacodynamics of treatment with utomilumab plus rituximab in patients with relapsed/refractory follicular lymphoma (FL) and other CD20Primary objectives were to assess treatment safety and tolerability for estimating the MTD, using a modified time-to-event continual reassessment method, and selecting the recommended phase II dose (RP2D).Sixty-seven patients received utomilumab (0.03-10.0 mg/kg every 4 weeks) and rituximab (375 mg/mUtomilumab in combination with rituximab demonstrated clinical activity and a favorable safety profile in patients with CD20

Background: Percutaneous coronary intervention with the new generation drug eluting stents (DES) is 1 among the revascularization procedures required to treat patients with coronary artery disease (CAD). Since late stent thrombosis and silent myocardial infarction are highly associated with type 2 diabetes mellitus (T2DM), an analysis comparing the newer generation DES in this specific subgroup of patients would be scientifically relevant. In this analysis, we aimed to systematically compare the cardiovascular outcomes observed with the ultrathin bioresorbable polymer sirolimus eluting stents (SES) versus thin, durable polymer everolimus eluting stents (EES) following percutaneous coronary intervention in patients with T2DM. Methods: Through online databases, relevant studies comparing ultrathin bioresorbable polymer SES versus the durable polymer EES were carefully searched. The cardiovascular outcomes were assessed during a follow-up time period of 1 year and more than 1 year (1–5 years) respectively. This meta-analysis was carried out by the latest version of the RevMan software. Following analysis, the results were represented by odds ratios (OR) with 95% confidence intervals (CI). Results: A total number of 1967 patients with T2DM were included in this analysis. During a 1 year follow-up time period, target lesion failure (TLF) (OR: 0.59, 95% CI: 0.34–1.02; P = .06, target vessel revascularization (TVR) (OR: 0.97, 95% CI: 0.55–1.70; P = .91) and target lesion revascularization (TLR) (OR: 0.91, 95% CI: 0.44–1.87; P = .79) were similarly observed with ultrathin bioresorbable polymer SES versus the thin, durable polymer EES in these patients with T2DM. Other cardiovascular outcomes including myocardial infarction (MI), major adverse cardiac events, all-cause mortality (OR: 0.72, 95% CI: 0.37–1.40; P = .34), cardiac death and stent thrombosis (OR: 0.85, 95% CI: 0.45–1.62; P = .63) were also similarly observed with these 2 types of new stents. During a follow-up time period above 1 year (1–5 years), still no significant difference was observed in TLF, TVR, TLR, major adverse cardiac events, MI, all-cause mortality, cardiac death and stent thrombosis (OR: 0.62, 95% CI: 0.33–1.16; P = .14). Conclusions: The ultrathin bioresorbable polymer SES were similar to the durable polymer EES in these patients with T2DM. These 2 types of new generation stents were comparable in terms of cardiovascular outcomes. Hence, they might be recommended in patients with T2DM. Upcoming trials should be able to confirm this hypothesis.

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.

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.

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.

// 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.

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.

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.

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.

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.

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

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.

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.

This study determined gender and ethnic differences in physical work capacity (PWC170) of 8–16-year-old American youth as they progress from elementary to high school. A multicohort group of 2,540 youth participated: 50.4% girls (21.4% African Americans, and 78.6% Caucasians). PWCI70 was predicted from cycle ergometer testing six times over a 7-year period. The absolute PWC 170 of girls stabilized in early adolescence, while boys increased with each trial. The PWC170 relative to body weight of girls decreased steadily, whereas the boys remained stable. African Americans had greater absolute PWC170 values than Caucasians (p =. 0001). The relative PWC 170 was lower for African American girls than Caucasian girls (p = . 0001), but there were no ethnic differences for boys (p > .05). Although correlations and grouping suggested moderate tracking, girls with high relative PWC170 tended to migrate to lower levels as they aged, where as high-performing boys maintained their PWC170 as they aged.

PURPOSE To determine the effects of age, gender, and ethnicity on the predicted aerobic power of youth as they age from 8 to 16 yr. METHODS The sample was a multicohort group of 2540 African Americans (N = 543) and Caucasians (N = 1997), 1279 (50.4%) girls and 1261 (49.6%) boys. Heights, weights, and sum of skin folds (triceps + subscapular) were measured. Aerobic power ((p)VO(2max)), expressed in relative (mL x kg(-1) x min(-1)) or absolute (L x min(-1)) terms, was predicted from a three-stage cycle ergometry test. RESULTS Quadratic mixed-model analysis indicated that boys had higher relative and absolute (p)VO(2max) than the girls (P = 0.0004). The African American subjects had a higher absolute (p)VO(2max) (L x min(-1)) than the Caucasians, but their relative (p)VO(2max) was lower than the Caucasians (P = 0.031). Finally, age had a significant effect on (p)VO(2max) (P = 0.0001). The absolute (p)VO(2max) of the girls increased 9%.yr(-1) until age 14; but their relative (p)VO(2max) declined approximately 1.2 mL x kg(-1) x min(-1) x yr(-1). The absolute (p)VO(2max) for the boys increased yearly by approximately 14% from ages 8 to 16 yr, but the relative (p)VO(2max) of the African American males was stable from ages 8 to 11, then dropped, and stabilized again between ages 12 and 16 yr. The relative (p)VO(2max) of the Caucasian boys declined from ages 8 to 10, but then increased slightly from age 12 to 16 yr. In addition, the interactions of age and gender, and age squared and gender were significant (P < 0.02). CONCLUSION These data indicate that although absolute (p)VO(2max) (L x min(-1)) increases from age 8 to 16 yr, relative (p)VO(2max) (mL x kg(-1) x min(-1)) declines. The decline appears to be related to increased fat mass. Similarly, the higher relative (p)VO(2max) (mL x kg(-1) x min(-1)) of the Caucasian youth compared with the African American youth was related to lower weights and skin folds of the Caucasian youth.

Purpose: This study was a longitudinal examination of the change in both the activities done and the intensity of those activities from childhood to adolescence. Methods: Common activities were assessed by questionnaire initially on 656 subjects from 21 elementary schools; 50.5% were female, 83.4% were Caucasian, 20.6% African-American, and 6.0% were other races. Results: Girls more often reported sedentary activities overall. Weighted least squares analyses showed boys consistently reported more vigorous activities than girls (P < 0.0008). African-American girls reported fewer vigorous activities than Caucasian or other race girls (P = 0.027). Sedentary activities were more frequently reported with increasing age (X 2 P < 0.001). The youngest African-American and Caucasian boys reported similar activity patterns. However, boys from other races reported more intense activities until sixth and seventh grades when African-American boys began reporting more sedentary activities than Caucasians or other races (P = 0.004). During sixth-eighth grades, Generalized Estimating Equations (GEE) models show that girls with more advanced pubertal status reported more sedentary activities than girls who were less developed (P < 0.0001). For high school girls, race was a marginally significant predictor (P = 0.05) of activity status. Neither race nor pubertal status were significant factors in activities chosen by middle school boys. However, for male high school students, Caucasians were more likely than African-Americans to report vigorous activities (P = 0.005). Conclusions: Variation in activities by race within gender suggests that establishing activity patterns in youth may be race-specific as well as gender-specific and must be accounted for in designing physical activity interventions. Also, pubertal maturation is a factor in activity choices in middle school girls.

Smokeless tobacco has seen a resurgence of popularity among adolescents despite its association with oral cancer and altered cardiovascular function. This study examined age, gender, ethnicity, self-esteem, physical activity, parental smoking, and socioeconomic status as predictors of smokeless tobacco use among middle school children. Subjects included 1,211 youth (White (64%), Black (24%), Hispanic (6%), and Other (6%); age 12.2) participating in the Cardiovascular Health in Children and Youth (CHIC II) study. All data were collected by questionnaire. Factors related to ever using smokeless tobacco included older age (p < .001), being male (p < .001), lower self-esteem (p < .001), and having parents who currently (p = .02) or formerly (p = .05) smoked. Hispanics reported a higher current usage rate than other ethnic groups (p < .001). White youth in the lowest socioeconomic status were most likely to be experimental users (p = .007), while those in the high socioeconomic status were more likely to be current users (p = .006). Physical activity was not associated with smokeless tobacco use.