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EML4-ALK fusion was recently characterized as an “Addicted Oncogene” in a subset of human lung adenocarcinoma, and it plays an essential role in regulation of the tumor cell survival, growth and metastasis. Crizotinib is a potent and selective ATP competitive small molecule inhibitor of ALK and c-Met. It is currently in clinical trials for advanced non-small cell lung cancers positive for ALK fusion. This report summarizes the pre-clinical pharmacology studies for crizotinib in the EML4-Alk positive tumor models to assess the pharmacodynamic inhibition of EML4-ALK, antitumor efficacy, PKPD relationships and antitumor mechanism of action in vitro and in vivo. Crizotinib potently inhibited the catalytic activity of ALK kinase (Ki = 0.5 nM) and the autophosphorylation of cellular EML4-ALK V1, V2, V3a and V3b with IC50 values ranging from 26–74 nM. Crizotinib also inhibited cell proliferation and induced apoptosis in NCI-H3122 human NSCLC cells harboring EML4-ALK V1 fusion with IC50s of 63 nM and 110 nM respectively. In the EML4-ALK V3a/b positive NCI-H2228 human NSCLC cells, crizotinib completely inhibited ALK phosphorylation (IC50 = 74 nM) but only partial inhibited cell proliferation and survival. This observation is consistent with the notion that only a portion of H2228 cells are EML4-ALK positive by Exon Array analysis. Crizotinib demonstrated marked tumor growth inhibition and regression in H3122 xenograft model at well tolerated dose levels. The antitumor efficacy of crizotinib was dose dependent and demonstrated a strong correlation to pharmacodynamic inhibition of ALK phosphorylation in vivo. PKPD modeling was conducted to understand the relationships between crizotinib plasma concentration to ALK target inhibition (EC50 = 19 nM) and antitumor efficacy (EC50 = 23 nM). Collectively, the results from H3122 model indicated that significant inhibition of EML4-ALK during the entire treatment period was necessary to achieve robust antitumor efficacy. Additional in vivo studies with crizotinib demonstrated dose dependent inhibition of EML4-ALK mediated signal transduction (STAT3, AKT, Erk, PLCγ, c-Myc), tumor cell proliferation (Ki67) and induction of apoptosis (caspase-3). Crizotinib also dose dependently inhibited total EML4-ALK levels in H3122 tumors indicating an additional antitumor mechanism of action by crizotinib in these tumors. Furthermore, a dose dependent increase in phospho-EGFR levels was observed after 4 days of drug treatment in H3122 model, indicating a compensation mechanism of “oncogene switching” in tumor cell signaling, and a potential resistance mechanism that may compromise “patient” responses to crizotinib treatment in these tumors. In conclusion, crizotinib was shown to be a potent inhibitor of EML4-ALK. It demonstrated marked antitumor efficacy in EML4-ALK dependent human NSCLC tumors in vitro and in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-390. doi:10.1158/1538-7445.AM2011-LB-390

While the etiology and mechanism of Inflammatory Bowel Disease (IBD) is not fully understood, immunologic abnormalities play a role in these diseases. Monitoring of disease progression and response to therapeutics is typically invasive and lacking. Increased recruitment of lymphocytes to the gut has been demonstrated in IBD, leading to inflammation and ultimately to gut tissue damage. Lymphocyte circulation is tightly controlled by trafficking receptors (TRs) that act in combination to govern migration to the target organ. Plasmablasts (PBs) are immature circulating B cells that may reflect tissue localized plasma cells. We hypothesize that the PB blood levels and trafficking patterns can inform on the location of tissue damage and the clinical condition of the patients. Using multicolor flow cytometry we examined PB (IgA+/CD38high) and memory B cell (IgA+/CD19+) blood levels, and TRs expressed on these cells. We show that an increase in IgA+ PB blood levels reflect an aggravated clinical state, and a significantly greater amount of these cells in ulcerative colitis patients express colon associated TRs (CCR10+) as opposed to small intestine associated TRs (CCR9+). Additionally, these trafficking patterns are more evident on IgA+ PBs rather than memory B cells. This work in progress suggests that the analyses of the TR phenotype of circulating immature and mature B lymphocytes may provide valuable information for monitoring disease progression via a less invasive process.

To examine the incidence and prevalence of pediatric inflammatory bowel disease (IBD) during 1996-2006 in a community-based health-care delivery system.Members of Kaiser Permanente Northern California aged 0 to 17 years with IBD were identified by use of computerized medical information with confirmation obtained through review of the medical record.The average annual incidence of IBD per 100000 was 2.7 (95% confidence interval [CI], 2.3-3.1) for Crohn's disease (CD) and 3.2 (CI, 2.8-3.6) for ulcerative colitis (UC). During the 11-year study period, the annual incidence per 100000 increased from 2.2 to 4.3 for CD (P = .09) and from 1.8 to 4.9 for UC (P.001). The ratio of incident CD cases to incident UC cases was 0.9 in non-Hispanic whites, 1.6 in African Americans (P = .12), 0.3 in Hispanics (P.001) and 0.4 in Asians (P = .04). The average length of enrollment during the 11-year study period exceeded 8 years. The point prevalence on December 31, 2006, per 100000 was 12.0 for CD (CI, 9.6-14.4) and 19.5 (CI, 16.5-22.6) for UC.In this population the incidence of UC increased significantly by 2.7-fold and CD increased 2.0-fold without reaching statistical significance. Hispanic and Asian children had development of UC more often than CD, suggesting possible etiologic differences across racial and ethnic groups.

Emerging evidences suggest that cancer stem cells (CSC) may be critically responsible for tumor initiation, progression, metastasis, and drug resistance. It becomes important to ask whether anti-cancer agents are able to target the tumor-initiating subpopulation in relevant CSC models. In this study, we first established xenograft tumors in NOD/SCID mice from a colorectal cancer patient specimen and demonstrated that CD133/EpCAM-expressing CSC population was highly tumorigenic. We then sought to propagate the CSC population under a serum-free condition. In culture, the tumor cells formed non-adherent, 3-dimensional spheroids, a fraction of which retained expression of the CSC markers. When exposed to a serum-containing medium, tumor spheroid cells differentiated into epithelial-like adherent cells with an increase in cell proliferation rate. In comparison with the differentiated progeny, tumor spheroid cells exhibited resistance to the standard-of-care agent oxaliplatin and, in limiting dilution assays in mice, displayed substantially higher tumorigenic potential. In contrast to the tumors originated from the differentiated cells, tumor spheroid cell-derived tumors recapitulated not only the CSC frequency marked by CD133/EpCAM expression, but also the histological characters of the original tumor. Similarly, only were the fragments of spheroid cell-derived xenograft tumors capable of regenerating highly proliferative tumors in secondary transplantation. Thus, the tumor spheroid culture is indeed enriched of drug resistant, self-renewing, and tumor-initiating CSC populations. Mutation profiling of frequently mutated oncogenes using Sequenom OncoCarta™ panel identified a mutation in the kinase domain of PIK3CA (H1047R) in the cultured CSCs. This mutation has been reported present in a large number of colon cancer patients and likely functions as an oncogene (Samuels et al., Science 304:554; 2004). We further demonstrated that a dual mammalian target of rapamycin (mTOR)/phosphoinositide 3-kinase (PI3K) inhibitor (PF-04691502) exhibited a more potent effect on inhibition of in vitro proliferation of the mutated CSCs compared to the chemotoxic agent oxaliplatin. Collectively, our findings suggest that CSC models provide a novel avenue to drug sensitivity and efficacy studies. The well-characterized CSC model systems may assist in the development of more effective therapy against the subpopulation of tumors driven by the CSCs bearing specific mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4483.

[18F]FLT-PET imaging has been increasingly employed as a surrogate biomarker in early clinical trials to evaluate the mechanism of cancer drug candidates. In this report, we have assessed the preclinical application of [18F]FLT-PET imaging modality for several investigative agents that are currently under clinical development, including PF-03732010 (P-cadherin mAb), PD-0332991 (CDK4 inhibitor) and PF-03084014 (γ-secretase inhibitor). Docetaxel was also utilized as a standard comparator. Our goals included 1) building a preclinical understanding of the [18F]FLT tracer avidity in various xenograft models; 2) selecting models with appropriate tracer avidity and target molecular profiles for respective agents; 3) correlating suppression of [18F]FLT uptake with the changes of other pharmacodynamic endpoints and measures of antitumor efficacy; and 4) bridging preclinical and clinical [18F]FLT-PET imaging studies. Among the tested tumor models, [18F]FLT tracer avidity did not always correlate with the tumor growth rate. Affymetrix array and LC/MS analyses indicated that multiple factors including the expression levels of thymidine kinase 1 (TK1) and pyrimidine transporters, as well as the intrinsic thymidine level in tumor each contributed to the [18F]FLT tracer avidity. In the MDA-MB-435HAL-subrenal capsule model, administration of PF-03732010 resulted in a time- and dose-dependent inhibition of [18F]FLT tracer uptake, which corresponded with the modulation of β-catenin and Ki67 levels via IHC analysis. As β-catenin partners with P-cadherin for mediating the proliferation and invasiveness of tumor cells, this result suggests that the suppression in [18F]FLT uptake correspond with the target modulation. However, when we tested the [18F]FLT-PET imaging in the MDA-MB-231 model, treatment with an efficacious dose of PF-03084014 (GSI) exhibited an insignificant effect on the tracer uptake despite evidence in modulation of target genes. The GSI-induced impact on the tumor BrdU uptake was also minimal suggesting that the observed antitumor efficacy was not primarily mediated by modulation of cell cycle. In the Rb wild-type MDA-MB-231 and HCT116 tumor models, administration of an efficacious dose level of PD-0332991 (CDK4/6i) demonstrated a significant decline of [18F]FLT tracer uptake. Concurrent hypophosphorylation of RbSer780 and reduced BrdU uptake by IHC was also observed, indicating that [18F]FLT tracer uptake highly reflected the target modulation and predicted therapy. Therefore [18F]FLT-PET imaging modality presents a ideal proof-of-mechanism biomarker for PD-0332991. Collectively, these results indicate a potential applications for [18F]FLT-PET imaging in early clinical studies to demonstrate an impact on target endpoints depending on the mechanism of action of anticancer agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1758.

Determining the number of subpopulations from sample data is a major problem in cluster analysis. We assume in this study that the subpopulations correspond to modes of the population density function f. We then propose using test statistics based on the kth nearest neighbor clustering method to investigate the modality of f. A modified bootstrap procedure for estimating the sample significance levels of these statistics in the univariate case is described. The performance of this procedure in determining the number of subpopulations will be illustrated by generated and real data sets.

A hybrid method for clustering multivariate observations is proposed, which combines elements of the k-means and the single-linkage clustering techniques. One purpose of the proposed method is to discover the high-density clusters given a random sample of size N from some underlying population; a high-density cluster at level c in a population with density f is defined as a maximal connected set of points x with f(x) ≥ c. This clustering procedure is practicable for very large numbers of observations and is shown to be consistent, under certain regularity conditions, in one dimension.

The results of an exploratory study of patterns of purchase influence in the metal-working industry are reported. The data set used was a representative sample of 2151 purchase influencers in the metalworking industry. Respondents reported their involvement or noninvolvement in each of seven phases of the purchasing decision process. The authors show the presence of identifiable patterns of reported involvement in the organizational buying process and suggest an approach for analyzing those patterns.

Patients at Boston's Children's Hospital diagnosed as having cerebral palsy were filmed walking. These films were digitized and translated into measurements associated with leg motion. In this paper we use the gait measurements of 128 such patients to illustrate the the kth nearest neighbour clustering procedure results in a gait typology for patients with cerebral palsy. The procedure identifies four subpopulations from the sample data; the membership of a patient within this typology is mostly determined by the patient's motor control. The developed typology differs from the present diagnostic system which classifies a cerebral palsy patient as either quadriplegic, diaplegic or hemiplegic.

Excerpt To the editor: In their article on type A influenza prevention and control in nursing homes, Patriarca and colleagues (1) express cost-effectiveness as the ratio of the net health care cost...

O álcool é uma das substâncias psicoativas mais consumidas mundialmente e seu uso por mulheres em idade reprodutiva, em particular, tem representado grande preocupação por parte de especialistas e da sociedade em geral. Apesar dos efeitos adversos associados ao ato de ingerir bebidas alcoólicas durante a gestação ser bastante documentados e conhecidos, sabe-se que uma parcela de mulheres grávidas tem dificuldades em abandonar o hábito. O consumo excessivo de álcool durante a gravidez tem sido associado com a síndrome fetal pelo álcool (FAS), caracterizada por crianças com dificuldades comportamentais e de aprendizado. Entretanto, devido ao sentimento de culpa e medo de ações punitivas, mulheres raramente admitem terem utilizado álcool durante a gestação. Como resultado, uma série de marcadores biológicos tem sido estudada para se diagnosticar a exposição fetal ao etanol. Dentre os marcadores utilizados estão os ésteres etílicos de ácidos graxos (FAEE), que podem ser detectados em amostras de mecônio de recém-nascidos. No presente trabalho, um método analítico foi desenvolvido visando a detecção de oito FAEEs em amostras de mecônio e aplicada em amostras coletadas de recém-nascidos cujas mães admitiram ou não o uso de etanol durante a gestação. A microextração em fase sólida por Headspace (HS-SPME), uma técnica de preparação de amostras relativamente recente, foi utilizada para análise. Os FAEEs foram identificados e quantificados por cromatografia gasosa/espectrometria de massas (GC/MS), operado no modo de ionização química. Os correspondentes ésteres etílicos deuterados foram sintetizados e utilizados como padrão interno. Os limites de quantificação (LOQ) obtidos foram abaixo de 150 ng/g e limites de detecção (LOD) foram abaixo de 100ng/g para todos os analitos. O método mostrou boa linearidade na concentração estudada (LOQ-2000ng/g), com coeficiente (r2) melhor que 0.98. Os valores de precisão apresentaram coeficientes de variação menores que 15% para todos os FAEEs estudados. Quando o método foi aplicado em amostras de mecônio, foi possível detectar níveis de alguns FAEEs de recém-nascidos não suspeitos a exposição fetal ao etanol. Alcohol is the main psychoactive drug consumed worldwide and its increasing use by young women has been a great problem point out by specialists in the subject. Although the adverse effects associated to the habit of drinking alcoholic beverages during gestation being very much documented, it is known that a considerable number of pregnant women have difficulties to abandon the habit. Excessive alcohol use during pregnancy has been associated with Fetal Alcohol Syndrome (FAS) characterized by children with cognitive and behavioral disorders. However, because of denial, embarrassment and fear, maternal reports of gestational use of alcohol are often inaccurate. Consequently, a series of biomarkers have been studied to diagnose fetal exposure to alcohol. Recently, fatty acid ethyl esters (FAEE) have been studied as biomarkers found in meconium of neonates exposed in utero. In the present work, an analytical method was developed aiming the detection of eight FAEEs in meconium samples and applied to real specimens collected from newborns whose mothers admitted or not the use of alcohol during pregnancy. Headspace solid-phase microextraction (HS-SPME), a relatively recent sample preparation technique, was used for analysis. FAEEs were identified and quantified by gas chromatography/mass spectrometry (GC/MS), operated in chemical ionization mode. The corresponding deuterated ethyl esters were synthesized and used as internal standards. The lower limits of quantification (LOQ) obtained were below 150ng/g and limits of detection (LOD) were bellow 100ng/g for all analytes. The method showed good linearity in the range of concentration studied (LOQ-2000ng/g), with coefficient of linearity better than 0.98. The precision assay, given by the relative standard deviation (RSD) of the method was lower than 15% for all FAEEs studied. When the method was applied to real samples, it was possible to detect trace levels of some FAEEs from non-suspected ethanol exposed newborns.

As mortes por causas externas e o consumo de drogas são problemas crescentes nos grandes centros urbanos. No estudo foi investigado se estes fatores, também afrontam cidades de médio e pequeno porte da região Bragantina. Foram realizadas análises toxicológicas para verificar a exposição à cocaína, em amostras de cabelo e urina de indivíduos vítimas de morte violenta (n=42), submetidos à necropsia no Instituto Médico Legal de Bragança Paulista. Do total de indivíduos submetidos ao estudo, 14% apresentaram resultados positivos para cocaína. Foi possível verificar que 38% das vítimas de homicídios eram usuários de cocaína. O perfil sociodemográfico dos indivíduos vítimas de morte violenta foi: sexo masculino, branco, solteiro, faixa etária de 21-30 anos e empregados. No estudo foi constatada exposição à cocaína entre vítimas de mortes violentas ocorridas na Região Bragantina. The deaths for externaI causes and the consumption of drugs are increasing problems in the great urban centers. In the study it was investigated if these factors also confront medium and small cities of Bragantina region. Toxicological analyses had been carried through to verify the exposure to the cocaine, in samples of hair and urine of individuaIs victims of violent death (n=42), submitted to the autopsy in the Legal Medical Institute of Bragança Paulista. Of the total of individuaIs submitted to the study, 14% had presented positive results for cocaine. It was possible to verify that 38% of homicides victims were cocaine users. The sociodemographic profile of the individuaIs victims of violent death was: male sex, white, single, 21-30 years age group and employees. In the study, exposure to the cocaine between victims of violent deaths occurred in the Bragantina Region was evidenced.