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The internet has become an important component of our daily lives. The most common internet service is web surfing. Many individuals use their browser to do things like online shopping, bill payment, cell phone recharge, and banking transactions. Customers confront different security dangers, such as cyber crime, as a result of the widespread usage of this service. Cyber phishing is a type of web threat that entices users to connect with a false website. The main goal of this research paper is to prevent the user's sensitive information. The proposed model is developed in three steps in step1 we choose a dataset to train on, and then test classifiers on the dataset. In step2 we have applied the three classifiers finding phishing detection accuracy and finally step3 after completing all of the predictions, we discovered that XGBoost outperformed AdaBoost and Gradient boosting machine learning algorithms.

One of the specialties of microspheres is that they are controlled systems of drug delivery designed for sustained release, to reduce the ordinary dose frequency and easy patient compliance. Controlled drug delivery of microspheres to enhance bioavailability, stability, efficiency, and to reduce the toxicity of drug into the body. The preparation is done for targeted local site delivery with a predetermined rate. Microspheres are a significant part of a novel drug delivery system and have solid spherical particles showing free-flowing properties and the particle size range 1-1000μm. Microspheres are matrix-type systems in which the drug is entrapped, dissolve, suspended. Generally solid or liquid drug entrap, dissolve in a matrix system prepared by several types of microspheres such as Bioadhesive microspheres, Magnetic microspheres, floating microspheres, polymeric microspheres, radioactive microspheres. And the following techniques are used in the preparation of various types of microspheres such as spray drying technique, single emulsion technique, Double emulsion technique, solvent evaporation, phase separation coacervation technique. The review of microspheres follows the introduction, characteristics, Advantages, limitations, types of microspheres, method of preparation, evaluation, and application of microspheres. Keywords: Bio-adhesive microspheres, controlled drug Delivery, entrap dissolve matrix, enhance the bioavailability, floating microspheres.

Study of relationships/interactions among students and teachers in structured learning environments have been a keen area of interests among educational analysts since decades. These relationships have been found to have direct as well as indirect impacts on the performance of students through varied interdisciplinary researches. The study presented in this paper is a novel application of Social Network Analytics to deeply understand and quantify direct or indirect relational impacts on undergrad level student's scholastic execution in India. Undergrad Computer Science students from a class of Kalindi College are surveyed to pull-together information on varied relational parameters of student-student and student-teacher relationships for generating varied networks and deep analytical analysis using specialized graph (network) analytic software `Gephi'. The quantitative results generated with the help of `Gephi' software successfully signaled significant relational aspects which have direct impact on students's performance. The generated results hence can be utilized in various ways to target specific issues beforehand with the sole aim to improve student's performance. The presented study displays a scope for substantial future research and can help in leading to fresh perspectives in handling relationships data for decision making and reframing educational policies for betterment.

Background: KRAS-mt CRC has constitutively activated RAF-MEK-ERK pathways and resistance to anti-EGFR therapies. In pre-clinical models, we found that cells with inflammatory subtype tumors were sensitive to the MEK-inhibitor, MEK162, plus N, regardless of KRAS mutations. However, both KRAS-mt and -wt stem-like CRC cells were resistant to this combination. In C-07 and C-08 clinical studies, patients (pts) with stem-like subtype tumors were resistant to chemotherapy and had a very poor prognosis. We hypothesized that dual-HER2 targeting may provide more robust ERBB inhibition than N alone. We therefore tested Tz combined with Tm+N using PDX organoids (PDXOs) derived from KRAS-mt CRC tumors. PDOs have emerged as powerful preclinical models to predict clinical outcomes. The goal of this study was to identify more efficacious drug combinations for KRAS-mt tumors and stem-like subtype tumors using PDXOs. Methods: PDXO’s were generated using recently described methods from Hans Clevers' laboratory. Three KRAS-mt PDX tissues from Champions Oncology were used to generate four PDXOs for drug testing. The CTG-0406 PDX model was used to develop PDXOs from two different mice. Results: All three KRAS-mt PDXOs: CTG-0406 (inflammatory), CTG-1170 (inflammatory), and CTG-0079 (stem-like), were resistant to Tm, N, and Tz, as single agents. N+Tz was effective in inhibiting cell viability in all four models; however, more significant viability inhibition occurred when Tm was added to N+Tz in three of the PDXOs including the stem-like model. In all PDXOs, Tm+N+Tz inhibited 67-76% viability. Conclusion: We demonstrate that KRAS-mt PDXOs were inhibited to a greater extent with Tm+N+Tz compared to any of these drugs alone. PDXOs provide a rapid and cost-effective preclinical platform for screening of unique drug combinations for sensitivity or resistance. Table 1.% PDXO cell viabilityDrugsCTG-0406 (1) (KRAS mt) InflammatoryCTG-0406 (2) (KRAS mt) InflammatoryCTG-1170 (KRAS mt) InflammatoryCTG-0079 (KRAS mt) Stem-LikeTm (10nM)96%116%95%94%N (125nM)109%97%96%92%Tz (20 µg/ml)92%119%97%93%Tm (10nM) + N (125nM)64%59%31%56%Tm (10nM) + Tz (20 µg/ml)74%75%41%70%N (125nM) + Tz (20 µg/ml)34%70%23%43%Tm (10nM) + N (125nM)+ Tz (20 µg/ml)26%33%24%31% Support: NSABP Foundation, Inc. Citation Format: Rekha Pal, Ashok Srinivasan, Peter C. Lucas, Carmen J. Allegra, Angela M. Davies, Alshad S. Lalani, Samuel A. Jacobs, Katherine L. Pogue-Geile. KRAS-mutant (mt) colorectal cancer (CRC) organoid models generated from patient-derived xenografts (PDX) show response to combination of trametinib (Tm), neratinib (N), and trastuzumab (Tz) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1923.

Diabetic retinopathy is a one of the increasing medical situation occurs due to fluctuating insulin level in the blood that leads to loss of vision. It is an ophthalmic disease which is mainly occurs due to the generation of the new abnormal blood vessels. Diabetic retinopathy with exudates are causing main health problem that leads to loss of sight. Patient suffering from diabetes are advised to undergo continual retinal test by reason of diabetic retinopathy. As the population is quite large as compared to healthcare system available, tests should be optimised and identification of the disease is complex and time consuming task. In this paper, clustering technique is used among the various data mining techniques, clustering is the good approach to handle the complex task. Experiment is conducted to identify the best clustering technique which can easily identify the various impacting factors of DR in less complex way. The experimental results reflect that the performance of K-means is better than other clustering techniques. This analysis will help the medical practitioner to identify best algorithm for disease detection and provide preventive measures in advance.

Background: KRAS MT CRC tumors demonstrate constitutively activated RAF-MEK-ERK signaling pathways and resistance to anti-EGFR therapies. In preclinical studies using KRAS MT CRC cell lines, resistance to MEK inhibitors (MEKi) led to induction of ERBB3. Using kinome-centered synthetic lethality screen, suppression of ERBB3 receptor tyrosine kinase is strongly synergistic with MEKi in CRC cell lines. We previously showed that KRAS MT cell lines of intrinsic inflammatory subtype were sensitive to MEKi and N, but stem-like subtype cell lines were resistant regardless of KRAS MT status. In this study, we evaluated treatment with T + N in 5 PDX models (Champions Oncology). PDX models were characterized by genomic signature for intrinsic subtypes, and molecularly profiled for KRAS, BRAF, PI3K, NRAS, and MSI status. Methods: An initial study was performed for dose tolerability. Efficacy studies were undertaken with 10 mice in each of 4 cohorts: vehicle, N 40 mg/kg po qd , T 1.0 mg/kg po qd, and N 40 mg/kg + T 1.0 mg/kg. Doses were given until the model reached endpoint. Mean tumor volumes (MTV) of treatment groups were compared to control group to determine % tumor growth inhibition (TGI). Tissue specimens were obtained from vehicle and all treatment arms for WES, RNA seq, and phosphoprotein analysis. Results: Model NoSubtypeMT profile% TGI (T,N, T+N)*p-value v controlCTG-0117InflammatoryKRAS WT/MSS43% v 49% v 76%0.215 v 0.17 v 0.009CTG-0382InflammatoryKRAS MT/MSS62% v 40% v 80%0.009 v 0.11 v 0.001CTG-0406InflammatoryKRAS MT/MSS43% v 46% v 67%0.001 v 0.0006 v 0.0001CTG-0069Stem-likeKRAS MT/MSS39% v 32% v 61%0.12 v 0.31 v 0.015CTG-0079Stem-likeKRAS MT/MSS10% v 22% v 42%0.91 v 0.51 v 0.068 *TGI was determined by calculating % TGI (100% x [1-(final MTV − initial MTV of a treated group) / (final MTV – initial MTV of the control group)]). Conclusions: T + N was well tolerated as evaluated by body weight and demonstrated TGI in all 3 inflammatory models and in 1 of two stem-like CRC PDX models. Importantly, these tumors were KRAS MT and MSS suggesting that T + N may be a promising targeted therapy for related CRC pts. WES, RNA seq, and phosphoprotein analyses from vehicle and treatment arms will be performed. This analysis may define potential biomarkers related to sensitivity or resistance to T + N and inform additional targeted therapies. Support: Champions Oncology; Puma Biotechnology Citation Format: Rekha Pal, Nan Song, Ying Wang, Ashok Srinivasan, Peter C. Lucas, Carmen J. Allegra, Angela M. Davies, Alshad S. Lalani, Samuel A. Jacobs, Katherine L. Pogue-Geile. Inflammatory and stem-like colorectal cancer (CRC) subtypes identified in patient-derived xenograft (PDX) models show tumor growth inhibition (TGI) by the combination of trametinib (T) and neratinib (N) irrespective of KRAS mutation (MT) status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-087.

As big data computing is in emergence, therefore handling of large amount of data becoming a problematic day by day. Analyzing, visualizing, and managing this massive amount of data causing remarkable issues and challenges which are not even resolve through traditional technologies. Therefore it requires innovative technologies or methods to resolve issues and challenges. The main aim of this paper is to explore the effectiveness of combination of big data with soft computing to overcome the issues and challenges more efficiently and in less time. Here emphasis is given on three main soft computing techniques namely neural network, fuzzy logic and genetic for minimizing big data related problems. Research questions are also formed for this purpose in the present paper from 2004–2015 as primary study that will help the researchers in future in their related study.

Background: Infection with hepatitis E virus (HEV) is a major cause of acute viral hepatitis in several developing countries. Although usually self-limiting and benign, the disease is particularly severe among pregnant women, with mortality rates reaching 15–20%. Methods: Immune parameters among pregnant women with acute hepatitis E (P-HEV) were investigated and compared with those in non-pregnant patients with hepatitis E (N-HEV), and healthy pregnant (PC) and non-pregnant (NPC) women. Results: Peripheral blood mononuclear cells (PBMC) from P-HEV patients had lower lymphocyte proliferation response to phytohemagglutinin (PHA) than those in the PC and NPC groups. A positive lymphocyte proliferation response to HEV antigen (HEVAg), a mixture of eight peptides derived from HEV proteins, was observed in 7/19 (37%) P-HEV patients, 3/9 (33%) N-HEV patients and only 2/21 (10%) PC and 2/14 (14%) NPC subjects; the stimulation indices in the P-HEV group were similar to the N-HEV group and higher than the PC group. Measurement of cytokine production by PBMC in response to PHA and HEVAg showed a reduction in production of T-helper 1 (Th1) cytokines and an increase in that of Th2 cytokines in the P-HEV group. Cytokine mRNA levels showed similar changes. Conclusion: These results show the existence of a Th2 bias in pregnant women with acute hepatitis E. The role of this Th2 bias in the greater severity of hepatitis E among pregnant women needs further investigation.

The genetic diagnosis of Gaucher disease by molecular methods is complicated by the existence of a highly homologous transcribed pseudogene (96% identity) that is found in close proximity to the true gene on chromosome 1q21. In addition, the pseudogene sequence can mimic disease-causing mutations in the true gene. Selective polymerase chain reaction (PCR) amplification of the true gene can be accomplished in extracted DNA from fresh-frozen samples by designing oligonucleotide primers to hybridize to defined regions that are not present in the pseudogene. This standard molecular approach, which entails amplification of relatively long segments of intact DNA, is not feasible in archival, paraffin-embedded, solid-tissue specimens in which the negative effects of chemical fixation result in DNA strand scission and breakdown of nucleic acid. A novel approach, specifically created for use with archival, fixative-treated tissue specimens, was developed for detection and characterization of common mutations of Gaucher disease. Three separate robust PCR reactions were formulated, 2 for selective amplification of portions of only the true gene exons 2 and 9, with a third reaction targeting exon 10, wherein both the true and pseudogene were coamplified. In the latter, DNA sequencing was used to determine the presence of true and pseudogene allele content in addition to identification of base sequence alterations. This method, requiring a single, 4-microm-thick histologic section, was successfully applied to archival paraffin block tissue specimens that had been in storage for up to 75 years. It was capable of accurately genotyping common Gaucher disease mutations as well as discovering a novel mutation and genetic polymorphism. We recommend our approach when only fixative-treated tis sue is available for molecular genotyping.

BACKGROUND Bile duct brushing is the procedure of choice for the assessment of neoplasia of the biliary and pancreatic ducts. Conventional cytopathologic evaluation has been reported to have high specificity but relatively low sensitivity. Although a number of molecular studies regarding biliary tract tissue specimens have been performed, to the authors' knowledge their precise applicability to cytopathology specimens has not been critically analyzed. METHODS Bile duct brushing specimens with the cytopathologic diagnosis of “suspicious” or “positive for malignant cells” along with corresponding surgical pathology specimens demonstrating adenocarcinoma were searched for in the files of UPMC-Presbyterian Hospital for the years 1990–1996. Tumor cells from representative cytopathology and histology slides were microdissected and analyzed for loss of heterozygosity (LOH) in a panel of microsatellite markers. The results obtained from cytopathologic and surgical pathology specimens were compared. RESULTS Eight paired surgical and cytopathology cases of adenocarcinoma involving the biliary tract were identified. The fractional allelic loss (FAL) for the surgical specimens (FAL-S) ranged from 12.5–71.4% and the FAL for the cytopathology specimens (FAL-C) ranged from 25–71.4%. However, when evaluating the actual loci of LOH, the concordance rate of the surgical and cytopathology specimens ranged from 71.4–100% (mean, 88.6%). Only 3 of the 8 cases (37.5%) were found to have identical matching of the LOH loci. CONCLUSIONS Although the overall concordance rate of LOH in biliary cytology and surgical specimens by molecular analysis is relatively high, the issue of molecular tumoral heterogeneity must be considered if clinical decisions are to be based exclusively on cytopathologic analysis. Cancer (Cancer Cytopathol) 2003;99:379–84. © 2003 American Cancer Society.

Using a rat model of chronic central leptin infusion in which neuropeptide Y neurons develop leptin resistance, we examined whether leptin signal transduction mechanism in the hypothalamus is altered during central leptin infusion. Adult male rats were infused chronically into the lateral cerebroventricle with leptin (160 ng/h) or vehicle via Alzet pumps for 16 d. In the leptin-infused group, the initial decrease in food intake was followed by a recovery to their preleptin levels by d 16, although food intake remained significantly lower than in artificial cerebrospinal fluid controls; and body weight gradually decreased reaching a nadir at d 11 and remained stabilized at lower level thereafter. Phosphorylated leptin receptor and phosphorylated signal transducer and activator of transcription-3 (p-STAT3) remained elevated in association with a sustained elevation in DNA-binding activity of STAT3 in the hypothalamus throughout 16-d period of leptin infusion. However, phosphorylated Janus kinase-2 was increased during the early part of leptin infusion but remained unaltered on d 16. Although hypothalamic suppressors of cytokine signaling-3 (SOCS3) mRNA levels were increased throughout leptin infusion, SOCS3 protein levels were increased only on d 16. This study demonstrates a sustained elevation in hypothalamic leptin receptor signaling through Janus kinase-STAT pathway despite an increased expression of SOCS3 during chronic central leptin infusion. We propose that an alteration in leptin signaling in the hypothalamus through pathways other than STAT3 and/or a defect in downstream of STAT3 signaling may be involved in food intake recovery seen after an initial decrease during chronic central leptin infusion.

Background: FC-7 is a phase Ib study hypothesizing that dual ERBB blockade (neratinib and cetuximab) could overcome an acquired resistance mechanism to anti-EGFR tx in KRAS/NRAS/BRAF/PI3KCA wild type (wt) mCRC patients (pts). Previously we demonstrated a 36% rate of HER2 amp in post- anti-EGFR biopsies (bxs). Assay of plasma cfDNA is minimally invasive and may provide comprehensive genomic profiling. We tested the accuracy of a cfDNA assay for HER2 amp, comparing results from matched bxs to evaluate molecular changes in HER2 copy number. Methods: 23 pts with mCRC resistant to anti-EGFR tx were enrolled and treated with cetuximab and neratinib. 17 paired bxs (6 post-tx bxs had insufficient tumor) were assessed for HER2 amp using chromogenic in situ hybridization (CISH) (Dako PharmDxTM). HER2 was considered amplified when HER2/CEP17 ratio was ≥2. cfDNA was extracted from plasma of 11 pts after 1st line anti-EGFR tx but before addition of cetuximab and neratinib. 5-30 ng of DNA was sequenced across 512 exons in 54 genes with HiSeq (Illumina). Copy number variants (CNV) were quantified using a customized pipeline (Guardant 360). One plasma sample failed to yield sufficient cfDNA. 10 pts had comparison of cfDNA with pre- or post- anti-EGFR bx. Results: 4 of 9 plasma samples (1 non evaluable) had HER2 amp, all concordant with pre- or post- anti-EGFR tx tissue. 3 pts converted to HER2 amp after anti-EGFR tx (pts 3, 5, 7), and for 2 of these conversions (pts 5, 7), the amplification was evident in cfDNA. PtPre- anti-EGFR CISH RatioPost- anti-EGFR CISH RatioPost- anti-EGFR cfDNA CNVConcordance of cfDNA with pre- or post-anti-EGFR Tissue Samples1Neg (1.0)Neg (1.3)Non evaluableNA2Pos (2.5)Neg (1.5)Pos (2.66)Pre-Tx3Neg (1.0)Pos (3.7)NegPre-Tx4Neg (1.0)Neg (1.8)NegBoth5Neg (1.2)Pos (2.7)Pos (2.48)Post-Tx6Pos (>10)QNSPos (21.86)Pre-Tx7Neg (1.1)Pos (2.0)Pos (2.20)Post-Tx8Neg (1.3)Neg (1.1)NegBoth9Neg (1.0)Neg (1.3)NegBoth10Neg (1.0)Neg (1.5)NegBoth Conclusion: In this sample set, HER2 amp detected in plasma-derived cfDNA from mCRC pts resistant to anti-EGFR tx is comparable to amp assessed in primary or metastatic tissue. Given advantages of liquid bx relative to repeated tissue bxs, measurement of HER2 amp in cfDNA may be of utility in monitoring pts for resistance to anti-EGFR tx. This will need to be confirmed in a trial incorporating both plasma and tissue samples. Support: Puma Biotechnology, Inc. Citation Format: S. Rim Kim, Ashok Srinivasan, Carmen J. Allegra, Samuel Jacobs, Rebecca J. Nagy, Richard B. Lanman, AmirAli Talasaz, Patrick Gavin, Rekha Pal, Peter C. Lucas, Kay Pogue-Geile. NSABP FC-7 correlative study: HER2 amplification (amp) in circulating cell-free DNA (cfDNA) in metastatic colorectal cancer (mCRC) resistant to anti-EGFR therapy (tx) [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 5684. doi:10.1158/1538-7445.AM2017-5684

Estrogen receptors (ER) were studied in rat bone cytosol using immunoprecipitation, and Western blot technique. Ligand specificity of bone ER was studied using various known modulators of ER. Competitive experiments were performed under exchange conditions in bone tissue obtained from one day old rats. ER alpha and beta subtypes were identified using immunoblotting experiments compared with that of ovarian and uterine tissues. In competitive binding assay, maximum inhibition in specific 3H-E2 binding was shown by E2 followed by tamoxifen and diethylstilbestrol. 7-Hydroxycentchroman and 85/287 also inhibited specific 3H-E2 binding but were less potent as compared to tamoxifen and diethylstilbestrol. However, 85/287 was less effective (81%) as compared to 7-hydroxycentchroman. Polyacrylamide gel electrophoresis of cytosol and Western blot analysis revealed the presence of 55 kD and 66 kD ER immunoreactive bands corresponding to alpha and beta subtypes, respectively, in bone as well as in uterus. Interestingly, the concentration of 55 kD ER was 3-fold higher than that of 66 kD ER. Ovarian cytosol revealed the presence of a 55 kD band only in Western blot analysis. These studies suggest the action of estrogens/ER modulators on osteoblasts which contain a limited number of classical alpha as well as beta sub types of ER that are known to be structurally different in their hormone-binding domains.

l,2-Bis(4-substituted phenyl)-2-methyl ethanone (2,4-dlnitrophenyl)hydrazones and 1-naphthyl-1-(4-substituted phenyl)-methanone (2,4-dinitrophenyl)hydrazones have been synthesized and evaluated for their anti-implantation, uterotrophic, antiuterotrophic, anticancer and antimicrobial activities. Diphenolic hy-drazone (compound 6) showed maximum uterotrophic inhibition of 70 %, whereas compound 20 exhibited cytotoxicity in the range of 50–70 % against MCF-7 and ZR-75-1 human malignant breast cell lines.

The immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide yield high response rates in patients with multiple myeloma, but the use of IMiDs in multiple myeloma is associated with neutropenia and increased risk for venous thromboembolism (VTE) by mechanisms that are unknown. We show that IMiDs down-regulate PU.1, a key transcription factor involved in granulocyte differentiation in vitro and in patients treated with lenalidomide. Loss of PU.1 results in transient maturation arrest with medullary accumulation of immature myeloid precursors and subsequent neutropenia. Accumulation of promyelocytes leads to high levels of the platelet aggregation agonist, cathepsin G stored in the azurophilic granules of promyelocytes. High levels of cathepsin G subsequently may increase the risk of VTE. To our knowledge, this is the first report investigating the underlying mechanism of IMiD-induced neutropenia and increased risk of VTE in multiple myeloma.

CCAAT/enhancer-binding protein β (C/EBPβ), also known as nuclear factor–interleukin-6 (NF-IL6), is a transcription factor that plays an important role in the regulation of growth and differentiation of myeloid and lymphoid cells. Mice deficient in C/EBPβ show impaired generation of B lymphocytes. We show that C/EBPβ regulates transcription factors critical for proliferation and survival in multiple myeloma. Multiple myeloma cell lines and primary multiple myeloma cells strongly expressed C/EBPβ, whereas normal B cells and plasma cells had little or no detectable levels of C/EBPβ. Silencing of C/EBPβ led to down-regulation of transcription factors such as IRF4, XBP1, and BLIMP1 accompanied by a strong inhibition of proliferation. Further, silencing of C/EBPβ led to a complete down-regulation of antiapoptotic B-cell lymphoma 2 (BCL2) expression. In chromatin immunoprecipitation assays, C/EBPβ directly bound to the promoter region of IRF4, BLIMP1, and BCL2. Our data indicate that C/EBPβ is involved in the regulatory network of transcription factors that are critical for plasma cell differentiation and survival. Targeting C/EBPβ may provide a novel therapeutic strategy in the treatment of multiple myeloma.

Background: Improvement of colorectal cancer (CRC) treatment depends on finding new effective agents and associated biomarkers that can predict patient response to drugs. Recent cell culture experiments show that a combination of MEK and pan ERBB inhibitors may be efficacious in treating CRC (Sun et al 2014). The purpose of this study was to determine whether a cell line's response to targeted agents would correlate with its mutation profile and/or gene expression subtype. Methods: K-RAS mutation status of 7 cell lines were identified and classified into stem-like or inflammatory subtypes using gene expression data from the cancer cell line encyclopedia and an algorithm described by Sadanandam et al. We tested 5 stem-like cell lines (SW480, SW620, HCT116, C2BBE1, HS675T) and 2 inflammatory cell lines (NCI-H747, SW837) for response to inhibitors of the RAS/RAF and PI3K pathways. MEK-162 and neratinib were tested as single agents and in combination, using cell viability and clonogenic assays. Results: Cell lines varied in response to the drugs when used as single agents. All cell lines were resistant to the EGFR inhibitor (Gefitinib) at concentrations ≥5 μM. Two pan ERBB inhibitors (neratinib and afatinib) showed IC50 values at or >4 μM and 2 MEK inhibitors (Selumetinib and MEK162) had IC50 values at or >1μM. The inflammatory cell lines, SW837 and NCI-H747, had greater sensitivity to the MEK inhibitors (IC50 value 1μM) while the KRAS mutant stem-like cell lines were relatively more resistant (SW480, SW620) with IC50 values ≥4-5 μM. Stem-like cell lines with WT-KRAS (C2BBE1, HS675T) were even more resistant; IC50s were not achieved at concentrations of 5 μM. Similar results were obtained in clonogenic growth assay. The MEK and pan ERBB inhibitor combinations were more effective at decreasing cell viability and inducing apoptosis in inflammatory cell lines than in stem-like cell lines. This combination effectively inhibited 60-70% of cell viability in inflammatory cell lines at a dose of 0.5 μM and 0.062 μM for neratinib and MEK-162 respectively. The lethality of this combination was the same even if the doses were reversed (0.5 μM MEK-162 and 0.062 μM neratinib). In contrast, we observed that this combination was not nearly as effective against stem-like cell lines irrespective of KRAS mutation status. Profiling of the phosphorylation status of proteins in the RAF-MEK-ERK signaling pathway after exposure to the MEK-162 plus neratinib combination showed a substantial inhibition of ERK phosphorylation in inflammatory cell lines but not in 4 of the 5 stem-like cell lines. Conclusion: This study demonstrates that colon cancer cell lines classified as the inflammatory subtype are sensitive to the combination of MEK-162 plus neratinib at concentrations at which cell lines of the stem-like subtype are resistant. The PA DOH disclaims responsibility for analysis, interpretations, or conclusions. Citation Format: Rekha Pal, Nan Song, Ashok Srinivasan, Samuel A. Jacobs, Soonmyung Paik, Katherine L. Pogue-Geile. Inflammatory and stem-like colorectal cell lines show differential response to MEK-162 and neratinib in combination. [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 706. doi:10.1158/1538-7445.AM2015-706

Infection with hepatitis E virus (HEV) is a major cause of acute viral hepatitis in several developing countries. Although usually self-limiting and benign, the disease is particularly severe among pregnant women, with mortality rates reaching 15-20%.Immune parameters among pregnant women with acute hepatitis E (P-HEV) were investigated and compared with those in non-pregnant patients with hepatitis E (N-HEV), and healthy pregnant (PC) and non-pregnant (NPC) women.Peripheral blood mononuclear cells (PBMC) from P-HEV patients had lower lymphocyte proliferation response to phytohemagglutinin (PHA) than those in the PC and NPC groups. A positive lymphocyte proliferation response to HEV antigen (HEVAg), a mixture of eight peptides derived from HEV proteins, was observed in 7/19 (37%) P-HEV patients, 3/9 (33%) N-HEV patients and only 2/21 (10%) PC and 2/14 (14%) NPC subjects; the stimulation indices in the P-HEV group were similar to the N-HEV group and higher than the PC group. Measurement of cytokine production by PBMC in response to PHA and HEVAg showed a reduction in production of T-helper 1 (Th1) cytokines and an increase in that of Th2 cytokines in the P-HEV group. Cytokine mRNA levels showed similar changes.These results show the existence of a Th2 bias in pregnant women with acute hepatitis E. The role of this Th2 bias in the greater severity of hepatitis E among pregnant women needs further investigation.

Brush cytology of biliary strictures to diagnose pancreaticobiliary malignancy suffers from poor sensitivity.To improve the diagnostic yield of pancreaticobiliary brush cytology through analysis of tumour suppressor gene linked microsatellite marker loss of heterozygosity (LOH) and k-ras codon 12 mutation detection.Twenty six patients with biliary strictures underwent endoscopic retrograde cholangiography with brush cytology. A panel of 12 polymorphic microsatellite markers linked to six tumour suppressor genes was developed. Genomic DNA from cell clusters acquired from brush cytology specimens and microdissected surgical malignant and normal tissue underwent polymerase chain amplification reaction (PCR). PCR products were compared for LOH and k-ras codon 12 mutations.Seventeen patients were confirmed to have pancreaticobiliary adenocarcinoma. Nine patients had benign strictures (eight proven surgically, one by follow up). Cytomorphological interpretation was positive for malignancy (n = 8), indeterminate (n = 10), and negative for malignancy (n = 8). Selected malignant appearing cytological cell clusters and microdissected histological samples from cancer showed abundant LOH characteristic of malignancy while brushings from nine cases without cancer carried no LOH (p0.001). LOH and k-ras mutations profile of the cytological specimens was almost always concordant with the tissue samples. Presence of k-ras mutation predicted malignancy of pancreatic origin (p0.001).LOH and k-ras codon 12 mutation analysis of PCR amplified DNA from biliary brush cytology discriminates reactive from malignant cells, with 100% sensitivity, specificity, and accuracy. Minor variations in LOH in brushings and in different sites within the same tumour likely reflect intratumoral mutational heterogeneity during clonal expansion of pre- and neoplastic lineages.

We undertook epidemiologic and laboratory studies during an epidemic of acute hepatitis in Sindri town, in District Dhanbad, Bihar in 1998.A sample survey covering 201 randomly selected houses in the town was conducted during the epidemic, and records of patients admitted to the only large hospital in this town were reviewed. We also tested serum and stool specimens from some of the affected persons for hepatitis E virus (HEV) RNA and IgM anti-HEV antibodies.Of the 1088 persons residing in the surveyed houses, 82 (7.54%) had developed acute hepatitis during the outbreak. Attack rate was higher among male residents than among female residents (71/604 vs. 11/484; 11.75% vs. 2.27%; relative risk [RR] 5.17 [95% confidence interval 2.77-9.65]; p10(-6)) and was the highest in the 10-29 year age group. Hospital admission data showed similar age and gender distribution. Disease occurrence had no relation with source of drinking water (handpump 7.56% vs. municipal tap 7.53%; p=ns), or with habit of boiling (RR 1.10 [0.61-1.98]; p=ns) or filtering (RR 0.59 [0.33-1.06]; p=ns) water before drinking. Jaundice occurred more frequently among persons who had traveled outside Sindri town during the last two months than among those who had not (26.4% vs. 4.7%; RR 5.67 [3.81-8.43]; p10(-6)); this risk persisted after correction for age (Mantel-Haenszel weighted OR 6.74 [4.12-11.01]; p10(-6)). Men traveled more frequently than women and were more often affected. In multivariate analysis, travel and male gender were the only two independent risk factors. Data from a hospital in a neighboring large city, Dhanbad, suggested that there was an outbreak of hepatitis in that city too at the same time. Seventy-three of the 1088 study subjects had history of jaundice in the past; disease attack rate among these persons (9.6%) was similar to that among those without such history (7.5%; RR 1.31 [0.49-2.98]; p=ns). Of the 13 sera tested, 10 were positive for IgM anti-HEV. HEV RNA was detected in 9 of the 12 stool specimens and 10 of the 13 sera tested.The hepatitis epidemic in Sindri was caused by HEV and had several features resembling those of previous HEV epidemics. However, the occurrence of hepatitis E showed a strong relationship with history of travel, a finding not hitherto described.

Introduction: The role of microRNAs (miRNAs) in the pathology of medulloblastoma (MB), the most common malignant brain tumor in children, is being elucidated at present. miRNA expression profiling studies have revealed a number of miRNAs implicated in medulloblastoma initiation and progression by controlling the expression of various genes responsible for cell proliferation or cell death. The effect of miRNA-10b on MB growth and proliferation through transcriptional induction of Bcl-2, an anti-apoptotic cellular proto-oncogene and tumor promoter, was explored in this study. Methods: Assays were carried out using six established MB cell lines and 13 primary MB samples. A screening cancer-specific miRNA array identified miRNA-10b as highly expressed in MB compared to normal cerebellum. Real-time PCR showed that miRNA-10b is significantly up-regulated (42-100 fold) compared to the normal cerebellum in all MB cell lines. Two MB cell lines (DAOY and UW228) were transiently transfected with control miRNA, miRNA-10b inhibitor, and miRNA-10b mimic for 48 to 72 hrs. The transfected cells were subjected to RT-PCR, MTT, apoptosis, clonogenic assay, and western blot analysis. Results: Transfection of miRNA-10b inhibitor induced a significant down-regulation of miRNA-10b expression in the MB cell lines, while miRNA-10b mimic induced miRNA-10b expression as measured by RT-PCR. Down-regulation of miRNA-10b expression resulted in a significant down-regulation of mRNA and protein levels of Bcl-2 and Mcl-1, decreased proliferation (p ≤ 0.001) and induced apoptosis (p ≤ 0.001) as compared to control. Inhibition of miRNA-10B further blocked the colony-forming capability of MB cells (p ≤ 0.001). miRNA-10b mimic transfection exerted an opposite effect. Down-regulation of miRNA-10b resulted in abrogation of phosphorylation of Bcl-2 at Ser70, a site previously reported to be critical in inhibiting apoptosis. Analyses of miRNA-10b levels in 13 primary MB samples revealed that the 2 patients with high levels of miRNA-10b (more than 200-fold increase versus control) had multiple recurrences (4.5) and died within 5 years of diagnosis, compared with the 11 patients with low levels of miRNA-10b who had a mean of 1.2 recurrences and 40% long-term survival. Conclusions: In conclusion, our data indicate that miRNA-10b may act as an oncomir in MB tumorigenesis, and reveal a previously unreported mechanism with Bcl-2 as an important mediator of miRNA-10b activity in MB cell survival. Targeting of miRNA-10b may provide a novel therapeutic approach in the treatment of MB. Citation Format: Rekha Pal, Stephanie Greene. miRNA-10b promotes medulloblastoma proliferation and survival via Bcl-2. [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 3082. doi:10.1158/1538-7445.AM2013-3082

Using male Sprague-Dawley rats implanted with third intracerebroventricular (ICV) cannulae, we found that cilostamide, a phosphodiesterase 3 (PDE3) inhibitor, (i) reversed the established effects of leptin on food intake and body weight, (ii) blocked, at the hypothalamic level, the leptin-induced tyrosine phosphorylation of signal transducer and activator of transcription 3 (Stat3) and (iii) blocked the DNA binding of p-Stat3. Additionally, ICV administration of leptin increased hypothalamic phosphatidylinositol 3-kinase (PI3K) and PDE3B activities and decreased cyclic AMP (cAMP) concentration. These results indicate that a PI3K-PDE3B-cAMP pathway interacting with the Janus kinase 2 (Jak2)-Stat3 pathway constitutes a critical component of leptin signaling in the hypothalamus.

Abstract 135 Background: Lenalidomide and pomalidomide are IMiD® immunomodulatory compounds that have been shown to be highly active in the treatment of multiple myeloma (MM). IMiD compounds exert their anti-tumor effects via acting on costimulatory proteins of T cells and NK cells, augmenting both the adaptive and innate immune system. But the mechanisms by which IMiD compounds directly inhibit MM cell proliferation are still unclear. Here we focused on the direct effects of IMiD compounds alone on MM cells. Results and Methods: We found that IMiDs, at concentrations as low as 0.01 μ M, induce significant inhibition of DNA synthesis in MM cells as shown by thymidine uptake. Since our previous work demonstrated that C/EBPβ is an important transcription factor which controls the growth and proliferation of myeloma cells, we analyzed the effects of IMiD compounds on C/EBPβ. We found that both pomalidomide and lenalidomide significantly decreased the protein level of C/EBPβ LAP-isoform in MM cell lines and primary MM cells. IMiD compound-induced suppression of C/EBPβ protein expression led to impaired transcription of the downstream IRF4, and subsequently to downregulation of BLIMP1 and XBP1, which are all critical for MM survival. To confirm our findings in vivo, we analyzed IRF4 expression by double labeling (IRF4+/CD138+) immunohistochemical staining of bone marrow biopsy samples of 23 myeloma patients prior to therapy and during therapy with lenalidomide. During lenalidomide therapy, the bone marrow MM cells showed a significantly weaker staining intensity for IRF4 in comparison to prior therapy. This was quantified by a significant (p Conclusions: Our studies, for the first time, provide evidence that IMiD compounds inhibit MM cell proliferation and survival by affecting the translation of C/EBPβ and subsequently multiple downstream transcription factors including IRF4, BLIMP1 and XBP1. Due to the critical role of C/EBPβ in mediating effects of IMiD compounds in MM, it might be a target to overcome drug resistance to IMiD compounds. The fact that pomalidomide can overcome resistance to lenalidomide in MM requires still further evaluation. Disclosures: Schafer: Celgene Corporation: Employment, Equity Ownership. Mapara:Gentium: Equity Ownership. Lentzsch:Celgene Corp: Research Funding.

Abstract 1844 Poster Board I-870 Background: Lenalidomide is an immunomodulatory drug that has been shown to be highly active in the treatment of multiple myeloma (MM). In combination with dexamethasone, remission rates of up to 61% are achieved in newly diagnosed MM patients. Nevertheless, the mechanisms of how immunomodulatory compounds induce high remission rates in MM remains elusive. Methods and Results: Here we investigate the underlying anti-MM mechanism of lenalidomide. Lenalidomide significantly (p Disclosures: Schafer: Celgene Corporation: Employment. Lentzsch:Celgene: Consultancy, Speakers Bureau; Pfizer: Consultancy.

Previous clinical trials have shown that immunomodulatory derivatives of thalidomde (IMiDs) including lenalidomide or pomalidomide induce high response rates in patients with relapsed or refractory multiple myeloma. However, the most dose limiting adverse effect of IMiDs is severe neutropenia. This is in contrast to our findings that IMiDs promote a shift in hematopoietic progenitor lineage commitment toward myeloid precursors with a subsequent maturation stop. In colony forming assays using human CD34+ hematopoietic precursors treated with IMiDs, we observed a highly significant increase in myeloid progenitor formation at the expense of erythroid colonies. Treatment of hematopoietic progenitors with IMiDs showed an upregulation of CD33, a marker for immature myeloid cells. Further, in vitro treatment with IMiDs resulted in downregulation of the transcription factor PU.1. PU.1 gene knock-down models show increased granulopoiesis with impaired neutrophil maturation, resulting in an accumulation of promyelocytes. In accordance with that, Cathepsin G as a promyelocytic marker was highly upregulated under treatment with IMiDs measured by high-density oligonucleotide microarray, quantitative RT-PCR, western blot and ELISA, confirming the maturation arrest of neutrophil granulocytes. To evaluate medullary changes in granulopoiesis in human subjects treated with lenalidomide, we evaluated sequential bone marrows from 6 patients comparing bone marrow features prior to treatment, during treatment, and at the time of grade 4 neutropenia. Marrows at the time of neutropenia showed an accumulation of myeloid precursors (myelocytes, metamyelocytes) supporting impaired myeloid maturation. In 5 out of 6 patients the myeloid:erythroid (M:E) ratio increased with an M:E median of 1.95 at pretreatment and 9.2 at the time of the neutrophil nadir. At the same time bone marrow cellularity was unchanged. In summary we show that IMiDs downregulate PU.1, a key transcription factor involved in granulocyte differentiation. The loss of PU.1 results in medullary accumulation of immature myeloid precursors, with corresponding neutropenia in the peripheral blood. Further high amounts of azurophilic granules of promyelocytes subsequently increase Cathepsin G and neutrophil elastase 2 resulting in enhanced platelet aggregation and a possible higher risk for venous thromboembolism. Our findings show that IMiDs do not induce neutropenia by marrow suppression but rather by transient block of maturation, which might be overcome by application of G-CSF.

Lenalidomide induces impressive response rates in patients with multiple myeloma. Unfortunately, one of the major side effects of this therapy is the increased risk of thromboembolic events (TEE) up to 17%. The reason for a higher risk of TEE associated with Lenalidomide is not known, but it has been found that aspirin is very effective in preventing TEEs induced by thalidomide and lenalidomide (Rajkumar, 2005). Cathepsin G is a serine proteinase present in the azurophilic granules of polymorphonuclear neutrophils (PMNs) and is a strong platelet aggregation agonist with potency similar to thrombin. In the current study we investigated the effect of the immunomodulatory derivatives of thalidomide (IMiDs) including Actimid and lenalidomide on cathepsin G in vitro and in vivo. To investigate the effect of IMiDs in vitro CD34+ hematopoietic progenitors were cultured in the presence of IL-3, IL-6 and SCF supporting the development of granulocytes and treated with Actimid or DMSO (control). RNA and protein were extracted for the analysis of cathepsin G. A significant up-regulation of cathepsin G (7.7 fold) was detected in oligonucleotide gene array analysis after 3 days of treatment with Actimid as compared to control group. These results were confirmed by RT-PCR, which showed a 3.7 and 7.6 fold mRNA-increase on day 6 and day 10 of culture compared to control. Next we analyzed the cathepsin G levels of patients (n=10) treated with lenalidomide before treatment, on day 15 of each cycle (1–4) as well as 1, 2, and 3 month after stop of lenalidomide treatment. We observed a continuous significant increase of the mRNA levels of PMNs over the course of treatment (baseline: 1 fold, cycle 2: 1.7 fold, cycle 3: 4.8 fold, cycle 4: 20.7 fold). These data were confirmed by measuring cathepsin G levels in patient serum by ELISA. Cathepsin G significantly increased from a baseline mean of 53 ng/ml to 77.5 ng/ml (cycle 2), 129.2 ng/ml (cycle 3) and 145.5 ng/ml (cycle 4). After stop of the lenalidomide treatment we observed a time dependent normalization to baseline levels of the cathepsin G with 61 ng/mL (1 month post treatment), 42.5 ng/mL (2 months post treatment), and 49.2 ng/mL (3 months post treatment). Our data show that IMiDs up-regulate the potent platelet aggregation activator cathepsin G in hematopoietic cells and thereby might contribute to the development of TEE in patients receiving IMiD treatment. Based on our data TEE prophylaxis should be continued for at least 1 more month after stop of therapy. For the first time our results provide an explanation why aspirin is effective in preventing TEE in patients receiving IMiDs. Further studies are needed to determine whether cathepsin G helps to predict TEE. Inhibition of cathepsin G might be a potential therapeutic target for preventing the hypercoaguable state induced by IMiD treatment.

The development and maturation of plasma cells is dictated by multiple interacting transcription factors (TFs). C/EBPb (NF-IL6) is a TF regulated by IL-6 and has profound effects on the regulation of growth, survival and differentiation of B-cells. Mice deficient in C/EBPb show impaired generation of B lymphocytes suggesting that C/EBPb plays an important role in B lymphopoiesis. In this study we delineated the effect of C/EBPb on transcription factors critical for myeloma cell proliferation by over-expressing and inhibiting C/EBPb in myeloma cells. Multiple myeloma (MM) cell lines MM.1S, RPMI-8226 and H929 were transiently transfected with GFP, C/EBPb (pcNF-IL6), and truncated C/EBPb with a deletion of the internal spII-spII fragment [pcmNF-IL6(Dspl)] by using Bio-Rad Gene Pulser Xcell, followed by G418 selection. A pool of transfected cells was selected and subjected to thymidine incorporation, flow cytometry and western blot analysis. We found that transfection of a truncated form of C/EBPb induced a down-regulation of C/EBPb in MM cell lines (MM.1S, RPMI-8226 and H929) as measured by western blot. Down-regulation of C/EBPβ significantly inhibited proliferation and induced apoptosis of MM cell lines analyzed by annexin V-FITC/PI staining. This was accompanied by a complete down-regulation of the anti-apoptotic protein BCL-2. Further, inhibition of C/EBPb completely decreased IRF-4 expression. In contrast, over-expression of C/EBPb increased protein levels of IRF-4 suggesting that IRF-4 is under control of C/EBPb. IRF-4, which was over-expressed in all our tested MM cells lines, is an essential TF for the generation of plasma cells by regulating TFs like Blimp-1 and PAX-5, which are critical for plasma cell differentiation. Our studies showed that down-regulation of IRF-4 resulted in a complete abrogation of Blimp-1 and PAX-5 suggesting that the expression of these factors is C/EBPb/IRF-4 dependent. In conclusion, our data indicate that C/EBPb is an important key regulator for survival and growth of MM cells. We show for the first time that C/EBPb is a critical regulator upstream of IRF-4. Down-regulation of the C/EBPb and consequently IRF-4 results in complete disruption of the network of TFs necessary for MM growth and survival. Targeting C/EBPb may provide a novel therapeutic approach in the treatment of MM.

l,2-Bis(4-substituted phenyl)-2-methyl ethanone (2,4-dlnitrophenyl)hydrazones and 1-naphthyl-1-(4-substituted phenyl)-methanone (2,4-dinitrophenyl)hydrazones have been synthesized and evaluated for their anti-implantation, uterotrophic, antiuterotrophic, anticancer and antimicrobial activities. Diphenolic hy-drazone (compound 6) showed maximum uterotrophic inhibition of 70 %, whereas compound 20 exhibited cytotoxicity in the range of 50–70 % against MCF-7 and ZR-75-1 human malignant breast cell lines.