Exact P-value Research Articles

Osteopontin as a Prognostic Indicator in Grading of Ovarian Epithelial Tumors

ABSTRACT Aim To determine if osteopontin expression by immunohistochemistry (IHC) can correlate with histological type and grade of epithelial ovarian cancer. Materials and methods The present work has been carried out at department of pathology, of a reputed medical college in Belgaum district of Karnataka state. The study is carried out for a period of 18 months for data collection, evaluation of the marker and data analysis. All consecutive specimens of ovarian tumors are included in the study. The study included 55 epithelial ovarian tumors and these are analyzed histopathologically. In all the cases, osteopontin expression has been studied with IHC on paraffin slides. Results This study included 41 (74.5%) benign tumors and 14 (25.5%) malignant tumors. High grade (grade III) was common in serous carcinomas than in other histological types. Fisher's exact test p-value < 0.001 was significant for expression of osteopontin and all the malignant ovarian epithelial tumors expressed osteopontin by IHC procedure on paraffin tissue sections. All high-grade epithelial ovarian tumors showed increased expression of osteopontin of +++ and low-grade tumors expressed +/++osteopontin. Conclusion The osteopontin expression was seen in all the malignant epithelial ovarian cancers and the osteopontin expression was significantly higher in high-grade epithelial ovarian cancers than low-grade epithelial ovarian cancers. How to cite this article Pilli GS, Patil PV, Karadesai SG. Osteopontin as a Prognostic Indicator in Grading of Ovarian Epithelial Tumors. J South Asian Feder Obst Gynae 2015;7(2): 61-63.

Doctoral Theses from Nursing Postgraduate Programs in Brazil and their Association with the Millennium Development Goals.

OBJECTIVES: The Millennium Development Goals are centered around combatting poverty and other social evils all over the world. Thus, this study seeks to identify the Millennium Development Goals as an object of study in theses from Postgraduate Nursing Programs in Brazil scoring 5 (national excellence) and 6 or 7 (international excellence), and evaluate the association between the score for the program and achieving the Millennium Development Goals. METHOD: Exploratory descriptive document research. Data were collected from the Notes on Indicators/Coordination for Higher Education Personnel Improvement for the 15 Postgraduate Nursing Courses scoring between 5 and 7 in the three-year-period of 2010/2012. RESULTS: of the 8 Millennium Development Objectives, 6 were dealt with in the theses. There was an association (Fisher's exact test p-value=0.0059) between the distribution of the theses and the program scores in relation to the Millennium Development Objectives (p-valor=0.0347) CONCLUSION: the doctoral theses were slightly related to the Millennium Development Objectives, covering the population's economic development, health conditions and quality of life. It is recommended that Postgraduate Programs in Nursing pay closer attention to the Millennium Development Objectives..

Genome-wide association study on susceptibility genes associated with yang-deficiency constitution: A small sample case-control study

To explore susceptibility genes associated with yang-deficiency constitution using single nucleotide polymorphism (SNP) genotyping. Based on an epidemiological survey, 30 volunteers with yang-deficiency constitution and 30 volunteers with a balanced constitution were included according to the Classification and Determination Standards of Constitutions in Traditional Chinese Medicine. Peripheral blood was collected and DNA was extracted from white blood cells. A genome-wide association study (GWAS) was conducted by SNP 6.0 genotyping at the Beijing CapitalBio Corporation Ltd. A minimum association P-value (Fisher's exact value) of less than 10(-4) in the allele, genotype, dominant, and recessive models served as the standard for significant association of SNP with yang-deficiency constitution. Among the four genetic models, a total of 42 SNPs were significantly associated with yang-deficiency constitution (Fisher's exact P-values P<10(-4)). These SNPs were adjacent to more than 20 genes, including RGS6, mGluR5, GAPDHL19, and IKZF1. Yang-deficiency constitution exhibits the characteristics of polygenic inheritance. This pilot study suggests that the polymorphisms in RGS6, mGluR5, GAPDHL19, and IKZF1 are associated with changes in cyclic adenosine monophosphate and cyclic guanosine monophosphate levels, memory, metabolic energy status, and immune function, respectively in people with yang-deficiency constitution.

Age and Population-Based Early Mortality Rate (EMR) in Acute Myeloid Leukemia (AML) Versus the EMR Reported By SWOG

Background: In their seminal paper SWOG described the expected EMR for patients by age enrolled on clinical trials in AML (Appelbaum et al Blood 2006; 107: 3481-5). We sought to contrast these to population based outcomes.Methods: Using case listing session of SEER 18 (1973-2010) we identified patients (pt) 18 years of age and older diagnosed (dx) from 1990-2005 with AML (Site recode ICD-O-3/WHO 2008 Acute Myeloid Leukemia which includes ICD-0-3 9861/3, 9873/3, 9920/3, 9910/3, 9840/3, 9891/3, 9867/3, and 9895/3 and includes all FAB and WHO subtypes except M3 ). This was done in order to match the accrual periods of the SWOG studies. We then examined the EMR of pt with non-M3 AML and compared them to those reported by SWOG. EMR in SEER was defined as death within 0 to 1 month from dx; EMR in the SWOG studies was defined as death within 30 days of initiation of induction chemotherapy. Analyses were conducted with SEER*Stat 8.1.2, Microsoft Excel 2007 and GraphPad Prism 6. All p-values were 2-sided.Results: 26,272 pt were identified within SEER and 955 pt were in the SWOG EMR cohort. 54% of patients were male in SEER and 55% were male in SWOG; 85% of patients were white in SEER and 89% were white in SWOG. The EMR was 38.6% (10,130) in the SEER cohort; EMR was 12.2% (116) in the SWOG cohort (chi-squared with Yates correction p<0.001). Age distribution and mortality by age are presented in the table below. In general the SEER patients were older and experienced a two-fold greater EMR than SWOG patients in each age subgroup.Conclusion: EMR in pt with non-M3 AML is much higher across all studied age groups in population based data than reported in SWOG studies. TableSEERSWOGEMR Fisher’s exact p-value% of ptEMR% of ptEMR<562616383P<0.00156-6516282511P<0.00166-7526412820P<0.001>753260832P<0.001 DisclosuresNo relevant conflicts of interest to declare.

Novel Chromatin Modifying Gene Alterations and Significant Survival Association of ATM and P53 in Mantle Cell Lymphoma

Background: The survival of mantle cell lymphoma (MCL) has improved (median OS now >5 years) due to the use of dose-intensive strategies in younger patients (pts) achieving a deeper and earlier response (including molecular CR) and the application of novel therapies in the relapsed/refractory setting, where patients are typically chemoresistant. However, given the median age at onset in the mid to late 60’s, and the remaining variability in outcomes, additional strategies are needed. A number of prognostic factors have been reported in MCL including MIPI, Ki67 and blastoid variants among others, the latter being frequently associated with del 17p or p53 mutations and a particularly poor outcome. Recent studies have begun to describe the genomic landscape of MCL by whole-genome, whole-exome and targeted sequencing. However, comprehensive evaluation of genomic alterations relevant for MCL pathogenesis and that could be potentially informative for patient’s prognosis and patient’s stratification remains to be elucidated.Methods: We studied 33 FFPE or cryopreserved blood MCL specimens by performing integrated DNA and RNA sequencing (N=18) or DNA sequencing alone (N=15). All samples were obtained at diagnosis before patients received Rituximab-HyperCVAD as frontline therapy. In this MCL cohort, treated at our institution, the median age was 59 y (range 35-75), all were stage IV, median Ki-67 was 30 % (range 5-80%), MIPI was low risk (27%), intermediate risk (30%), high risk (42%), and there were 4 blastoid variants (2 with del 17p by FISH or cytogenetics). Genomic DNA and total RNA were extracted and captured using custom bait-sets targeting all exons of 405 cancer-related genes by DNA-Seq, and 265 frequently rearranged genes by RNA-Seq (FoundationOne Heme). Captured libraries were sequenced to high depth in a CLIA-certified, CAP-accredited, NYS-approved clinical laboratory, with an average depth of 400X for DNA and >8M unique pairs for RNA.Results: In total, we identified 125 genomic alterations with an average of 3.8 per sample, including 45 base substitutions/indels, 33 truncations, 1 gene amplification, 6 gene deletions, and 40 rearrangements (Figure 1). IGH/CCND1 t(11,14) translocation was detected in 32/33 samples, and CCND1 over-expression was found in all translocation positive cases by RNA-seq, when available. Consistent with published reports, ATM and TP53 were among the most frequently altered genes, found in 45% and 18% of the samples, respectively. Mutations of TP53 and ATM were correlated with the short overall survival (OS) in our cohort. The median OS was not reached in TP53wt pts compared with 5.3 months in TP53mut pts, and not reached in pts with wild-type TP53 and ATM versus 10.4 months in either TP53mut or ATMmut pts (Fisher’s exact test p-value=0.01, Figure 2).Gene alterations of chromatin remodeling pathway were identified in 18 out of 33 samples, including SMARCB1 (4), SMARCA4 (2), ARID1A (3), ARID2 (1), PBRM1 (1), CHD2 (1), WHSC1 (5), MLL2 (2), SETD2 (1), EZH2 (1), and ASXL1 (1). Alterations in SMARCB1 (also known as SNF5 and INI1) and ARID1A, members of the SWI/SNF chromatin-remodeling complex, have not been reported in MCL previously. Other frequently altered genes included NOTCH1/2 (12%), and the cell cycle genes CDKN2A/B (9%). GAs associated with approved agents or clinical trials of targeted therapies were identified in 67% of the samples. The most common genes with potential therapeutic implications were ATM (PARP inhibitors), CDKN2A and CCND1 (CDK4/6 inhibitors) and NOTCH1 (Notch1 inhibitors).Summary:1. We identified relevant genomic alterations in MCL patients from baseline peripheral blood and/or tissue (with no significant difference in source of materials) in all samples tested.2. We discovered novel recurrent mutations in chromatin remodeling genes SMARCB1 and ARID1A in MCL, and demonstrated that 55% of tumors carried alterations in epigenetic regulation overall.3. In our frontline cohort of MCL patients treated with R-HyperCVAD, we identified the prognostic value of DNA damage response genes ATM and TP53 on outcome. These findings highlight the possibilities for improved prognosis and novel therapeutic strategies based on comprehensive genomic analysis of these pathways [Display omitted] [Display omitted] DisclosuresWang:Foundation Medicine, Inc.: Employment, Equity Ownership. Nahas:Foundation Medicine, Inc.: Employment, Equity Ownership. Yelensky:Foundation Medicine, Inc.: Employment, Equity Ownership. Otto:Foundation Medicine, Inc.: Employment, Equity Ownership. Lipson:Foundation Medicine, Inc.: Employment, Equity Ownership. He:Foundation Medicine, Inc.: Employment, Equity Ownership. Ross:Foundation Medicine, Inc.: Employment, Equity Ownership. Stephens:Foundation Medicine, Inc.: Employment, Research Funding. Miller:Foundation Medicine, Inc.: Employment, Equity Ownership.

Genomic Alterations of Histone Modification Genes Are Significantly Less Common in Non-Hodgkin Lymphomas of Adolescents and Young Adults Compared to Older Patients

Background: Non-Hodgkin lymphoma (NHL) is a large group of hematolymphoid malignant neoplasms that can occur at any age. Genomic alterations of histone modifying genes and the NF-kB signaling pathway are characteristic of NHLs. Histone deacetylase and methyltransferase inhibitors including the EZH2 inhibitors E7438, GSK126 and El1 have shown anti-tumor activity in NHLs (PMID: 24217204). Cancers arising in adolescent and young adults (age 15-39, AYA) often exhibit different clinical and biological features than older adults. However, there is very limited information on age-related genomic profiles of NHLs to facilitate clinical investigation of targeted therapies in the AYA population.Methods: Nucleic acid was extracted from 149 cases of NHLs including diffuse large B-cell lymphoma (DLBCL, 45%), chronic lymphocytic leukemia (CLL, 18%), follicular lymphoma (FL, 9%), mantle cell lymphoma (MCL, 7%), NK/T-cell lymphoma (NKTCL, 5%), anaplastic large cell lymphoma (ALCL, 3%), cutaneous T-cell lymphoma (CTCL, 3%), B-cell lymphoma (3%), Burkitt lymphoma (BL, 2%), T-cell large granular lymphocytic leukemia (T-LGL, 2%) and lymphoblastic lymphoma as well as not otherwise specified NHL (3% in total), and captured using custom baits targeting all exons up to 405 cancer-related genes and 265 frequently rearranged genes (FoundationOne HemeTM). All captured libraries were sequenced to high uniform depth in a CLIA-certified, CAP-accredited laboratory with an average depth of >500x for DNA and >6M unique pairs for RNA. There were 29 AYA and 120 older adult (>39 years) NHL cases in this study. DLBCL was the most common diagnosis across groups in 34% of AYAs and 47% of older adults, respectively.Results: The most frequent genomic alterations (GAs) in AYAs were IGH rearrangement (31%), TP53 (28%), MYC (21%), CIITA (16%), DDX3X (16%), ALK (14%), CDKN2A (10%), and TNFAIP3 (10%). Older adults often harbored IGH rearrangements (33%), TP53 (26%), CDKN2A (24%), MLL2 (21%), CREBBP (15%), TNFAIP3 (14%), and MYD88(13%) alterations.Only 1 MLL2 mutation was identified in our AYA samples (3%). In contrast, MLL2 mutations were found in 25/120 (21%) of adult NHLs, including 30% of DLBCLs and 58% of FLs (two tailed Fisher’s exact test p-value=0.03). Ninety-seven percent of the MLL2 mutations were reading frame disruptions or truncations, supporting the concept of MLL2 functioning as a tumor suppressor in NHLs. Patients whose tumors harbored mutant MLL2 frequently contained 2 mutations simultaneously (35%). No additional alteration of histone modification genes was found in AYAs. But 39% of adult NHLs carried alterations of those genes (ASXL1, CREBBP, EP300, EZH2, KDM5A, MLL2, MLL3, SETD2, and WHSC1) across different subtypes, including 92% of FL cases, 46% of DLBCLs, 50% of ALCLs, 33% of CTCLs, 33% of NKTCLs, 27% of MCLs, and 12% of CLLs. The difference in the alteration frequency of histone modification genes between AYA (1/29) and older adult (47/120) is significant (p=0.0001). This observation was also seen when excluding the FL cases (1/27 versus 36/108, p=0.001).Conclusions: The unique genomic features of AYA NHLs and guarded clinical outcome strongly favor that AYA NHLs exhibit distinct molecular characteristics compared with older adults. Using comprehensive genomic sequencing (FoundationOne Heme), we discovered that the alterations of histone modifying genes are extremely rare in our AYA samples. Further study of the unique genomic profile of AYA NHLs to expand our knowledge of their biology and discover potential new therapeutic targets for AYA patients appears warranted. DisclosuresWang:Foundation Medicine, Inc. : Employment, Equity Ownership. Morosini:Foundation Medicine, Inc. : Employment, Equity Ownership. Yelensky:Foundation Medicine, Inc. : Employment, Equity Ownership. Palma:Foundation Medicine, Inc.: Employment, Equity Ownership. Johnson:Foundation Medicine, Inc. : Employment, Equity Ownership. Lipson:Foundation Medicine, Inc. : Employment, Equity Ownership. Chmielecki:Foundation Medicine, Inc. : Employment, Equity Ownership. Ali:Foundation Medicine, Inc.: Employment, Equity Ownership. Ross:Foundation Medicine, Inc. : Employment, Equity Ownership. Stephens:Foundation Medicine, Inc. : Employment, Equity Ownership. Miller:Foundation Medicine, Inc. : Employment, Equity Ownership.

Analysis of pattern overlaps and exact computation of P-values of pattern occurrences numbers: case of Hidden Markov Models.

BackgroundFinding new functional fragments in biological sequences is a challenging problem. Methods addressing this problem commonly search for clusters of pattern occurrences that are statistically significant. A measure of statistical significance is the P-value of a number of pattern occurrences, i.e. the probability to find at least S occurrences of words from a pattern in a random text of length N generated according to a given probability model. All words of the pattern are supposed to be of same length.ResultsWe present a novel algorithm SufPref that computes an exact P-value for Hidden Markov models (HMM). The algorithm is based on recursive equations on text sets related to pattern occurrences; the equations can be used for any probability model. The algorithm inductively traverses a specific data structure, an overlap graph. The nodes of the graph are associated with the overlaps of words from . The edges are associated to the prefix and suffix relations between overlaps. An originality of our data structure is that pattern need not be explicitly represented in nodes or leaves. The algorithm relies on the Cartesian product of the overlap graph and the graph of HMM states; this approach is analogous to the automaton approach from JBCB 4: 553-569. The gain in size of SufPref data structure leads to significant improvements in space and time complexity compared to existent algorithms. The algorithm SufPref was implemented as a C++ program; the program can be used both as Web-server and a stand alone program for Linux and Windows. The program interface admits special formats to describe probability models of various types (HMM, Bernoulli, Markov); a pattern can be described with a list of words, a PSSM, a degenerate pattern or a word and a number of mismatches. It is available at http://server2.lpm.org.ru/bio/online/sf/. The program was applied to compare sensitivity and specificity of methods for TFBS prediction based on P-values computed for Bernoulli models, Markov models of orders one and two and HMMs. The experiments show that the methods have approximately the same qualities.Electronic supplementary materialThe online version of this article (doi:10.1186/s13015-014-0025-1) contains supplementary material, which is available to authorized users.

A fast algorithm for determining bounds and accurate approximate p-values of the rank product statistic for replicate experiments.

BackgroundThe rank product method is a powerful statistical technique for identifying differentially expressed molecules in replicated experiments. A critical issue in molecule selection is accurate calculation of the p-value of the rank product statistic to adequately address multiple testing. Both exact calculation and permutation and gamma approximations have been proposed to determine molecule-level significance. These current approaches have serious drawbacks as they are either computationally burdensome or provide inaccurate estimates in the tail of the p-value distribution.ResultsWe derive strict lower and upper bounds to the exact p-value along with an accurate approximation that can be used to assess the significance of the rank product statistic in a computationally fast manner. The bounds and the proposed approximation are shown to provide far better accuracy over existing approximate methods in determining tail probabilities, with the slightly conservative upper bound protecting against false positives. We illustrate the proposed method in the context of a recently published analysis on transcriptomic profiling performed in blood.ConclusionsWe provide a method to determine upper bounds and accurate approximate p-values of the rank product statistic. The proposed algorithm provides an order of magnitude increase in throughput as compared with current approaches and offers the opportunity to explore new application domains with even larger multiple testing issue. The R code is published in one of the Additional files and is available at http://www.ru.nl/publish/pages/726696/rankprodbounds.zip.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-014-0367-1) contains supplementary material, which is available to authorized users.

Comparison of noninferiority margins reported in protocols and publications showed incomplete and inconsistent reporting

ObjectivesTo compare noninferiority margins defined in study protocols and trial registry records with margins reported in subsequent publications. Study Design and SettingComparison of protocols of noninferiority trials submitted 2001 to 2005 to ethics committees in Switzerland and The Netherlands with corresponding publications and registry records. We searched MEDLINE via PubMed, the Cochrane Controlled Trials Register (Cochrane Library issue 01/2012), and Google Scholar in September 2013 to identify published reports, and the International Clinical Trials Registry Platform of the World Health Organization in March 2013 to identify registry records. Two readers recorded the noninferiority margin and other data using a standardized data-abstraction form. ResultsThe margin was identical in study protocol and publication in 43 (80%) of 54 pairs of study protocols and articles. In the remaining pairs, reporting was inconsistent (five pairs, 9%), or the noninferiority margin was either not reported in the publication (five pairs, 9%) or not defined in the study protocol (one pair). The confidence interval or the exact P-value required to judge whether the result was compatible with noninferior, inferior, or superior efficacy was reported in 43 (80%) publications. Complete and consistent reporting of both noninferiority margin and confidence interval (or exact P-value) was present in 39 (72%) protocol-publication pairs. Twenty-nine trials (54%) were registered in trial registries, but only one registry record included the noninferiority margin. ConclusionThe reporting of noninferiority margins was incomplete and inconsistent with study protocols in a substantial proportion of published trials, and margins were rarely reported in trial registries.

Lack of Association of the CD247 SNP rs2056626 with Systemic Sclerosis in Han Chinese.

Systemic sclerosis (SSc) is a complex disease involving multiple genetic factors. A recent genome-wide association study (GWAS) indicated that CD247 was strongly associated with SSc, which was subsequently confirmed in a SSc cohort of European population. However, genetic heterogeneity in different ethnic populations may significantly impact the complex trait of SSc. The studies herein aimed to examine whether the SSc-associated SNP rs2056626 of CD247 identified in Caucasian is also associated with Han Chinese SSc. A Han Chinese cohort consisting of 387 SSc patients and 523 healthy controls were examined in the studies. TaqMan assays were performed to examine the SNP. Exact p-values were obtained (Fisher’s test) from 2x2 tables of allele counts and disease status. The results showed that there was no association between rs2056626 of CD247 and SSc or any SSc subtypes of Han Chinese. The negative results are important in understanding genetics of SSc in different ethnic populations, which further suggest complex nature of genetics of SSc.

Quantifying Fecal Tumor Necrosis Factor-α (TNF-α) in Active IBD Compared to Other Forms of Enterocolitis: A Prospective Study

Introduction: The ability to utilize inflammatory markers in inflammatory bowl disease (IBD) for diagnosis, disease activity and prognosis is an active area of ongoing research. Markers such as neutrophil gelatinase-associated lipocalin, calprotectin, interleukin-8 and TNF-α have been studied in the stool samples of IBD patients. Identifying a more sensitive and specific marker to detect and monitor progression of disease could provide a less invasive and more feasible tool in management of patient care. Specifically, TNF-α in stool samples has been shown to be elevated in acute flares of IBD compared to other causes of diarrhea. To date, there have been limited studies conducted in adults comparing levels of TNF-α in stool samples from patients with acute IBD flares verses other causes of enterocolitis. Studies in children have revealed statistically significant differences in stool TNF-α levels in patients with IBD compared to infectious causes. Aim: To analyze levels of TNF-α and calprotectin in stool specimens between two cohorts of patients to assess the accuracy of these cytokine markers in differentiating between IBD and other inflammatory conditions of the GI tract. Methods: Subjects fulfilling the diagnostic criteria for ulcerative colitis (UC) and Crohn’s disease who were symptomatic and others with symptomatic enterocolitis were recruited from both the outpatient GI clinic and in-patient hospital. Subjects were identified if diagnosed as having symptomatic colitis or flare of IBD identified by stool cultures, signs and symptoms, or endoscopy/histology reports revealing gastrointestinal inflammation. After obtaining informed consent, subjects were enrolled in the study and stool samples were collected and stored in -80 C freezer. Samples were then processed and ran to determine levels of TNF-α and calprotectin via ELISA. Results: Seventy-four stool samples were collected from adults comprising of 22 CD, 19 UC and 33 other colitides including C. dificile, Salmonella, Shigella, diverticular colitis, ischemic colitis, microscopic colitis and indeterminate colitis. TNF-α levels were found in only 6 fecal samples; 3 CD with mean 0.63 pg/mL, 2 UC with mean 0.78 pg/mL, and 1 other colitides with mean 0.21 pg/mL (Fisher’s exact test p-value: 0.36). In comparison, calprotectin levels were found in 21 CD with mean 661.13 ug/g, 17 UC with mean 855.26 ug/g, and other colitides with mean 177.15 ug/g (chi-square p-value: <0.0001). Upon further analysis, the only significant difference in calprotectin levels amongst the three variables was when comparing IBD (UC, CD or both) with other colitides (chi-square p-value: <0.0001). Conclusion: The study reaffirms fecal calprotectin as the preferred inflammatory marker in IBD compared to TNF-α in adults. This research was supported by an industry grant from UCB.

Abstract 859: Regulation of cell cycle and NAD biosynthesis II pathways are associated with differential response to preoperative radiotherapy in locally advanced breast carcinoma

Abstract Human breast carcinomas have heterogeneous pathologies, molecular profiles and different responses to therapy. For non-inflammatory locally-advanced breast cancer patients (LABC), preoperative radiotherapy (pRT) is currently indicated after neoadjuvant chemoherapy (CHT), if there is no response to CHT. However, there are no predictive markers of response to pRT in routine clinical practice. Furthermore, understanding of the molecular mechanisms associated to radiotherapy response could open new avenues for overcoming radio-resistance in these patients. In this retrospective study, we have analyzed genes associated with different response to pRT in a neo-adjuvant setting in LABC patients treated with preoperative RT with a total dose of 45 Gy in 15 fractions every second day to the breast and regional lymph nodes, without previous or concomitant systemic therapy. Radical mastectomy was performed 6 weeks after pRT, and adjuvant systemic therapy was administered as per protocol. Response to pRT was classified as pathological complete response (pCR) if there is no evidence of tumor in the breast after mastectomy, partial response (PR) if regression of tumor was &amp;gt;30%, and no response if regression was &amp;lt;30%. We have analyzed global gene expression profiles by Agilent 8x60K microarray platform in a set of 14 samples of formalin-fixed paraffin-embedded (FFPE) initial LABC biopsies. Unsupervised clustering analysis (Pearson correlation, un-centered metrics) over top 20% most variable genes revealed clear differences in the gene expression profile between radio-resistant and radio-sensitive tumors, despite the small sample size. We have found 165 genes significantly differentially expressed (FDR &amp;lt; 0.10) between responders and non-responders to pRT. To explain the biological processes and signaling pathways of radioresistance, a gene set enrichment using Ingenuity Pathways Analysis was applied. Top scoring molecular functions associated with this gene list included regulation of cellular development, cellular growth and proliferation and cell cycle. Not surprisingly, the most significantly enriched canonical pathways (Fisher's exact p-value&amp;lt;0.05) within the gene list of significantly differentially expressed genes between RT resistant and RT sensitive tumors were cell cycle control of chromosomal replication and pathways related to glucose metabolism and NAD biosynthesis II route. These results set basis for further understanding of molecular response to radiotherapy and highlight genes potentially useful as predictive markers of response to preoperative radiation treatment. Further confirmatory research of potentially RT sensitive/resistant genes and pathways is warranted. Citation Format: Miljana Tanic, Ana Krivokuca, Jasmina Mladenovic, Snezana Susnjar, Sinisa Radulovic, Radmila Jankovic. Regulation of cell cycle and NAD biosynthesis II pathways are associated with differential response to preoperative radiotherapy in locally advanced breast carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 859. doi:10.1158/1538-7445.AM2014-859

Abstract 1414: Race-associated variation in the airway transcriptome response to cigarette smoke

Abstract INTRODUCTION: Tobacco-induced lung cancer is the leading cause of cancer death. Lung cancer incidence is 15% higher in African Americans (AFA) than Caucasians (CAU) even after taking into account variations in smoking behavior and other environmental variables, and the reasons for this difference are unclear. As 90% of patients with lung cancer have a smoking history, we hypothesize that race-related differences in the response to tobacco smoke might contribute to the elevated lung cancer risk observed in AFA. To begin to test this hypothesis, we have used gene expression profiling to identify differences in the biologic response of the bronchial airway epithelium of AFA and CAU to tobacco smoke. METHODS: Cytologically normal bronchial epithelial cells obtained during bronchoscopy from current (C) and former (F) smokers with and without lung cancer were hybridized to Affymetrix Human Gene 1.0 ST microarrays (n=885). After quality control, n=810 samples (from n=710 subjects) were selected for subsequent analysis. RMA-normalized gene expression levels were batch-corrected using ComBat. Genes whose expression is altered by smoking differently in the airway epithelium of AFA (n=155; nC=76, nF=79) as compared to CAU (n=655; nC=283, nF=372), were identified via ANOVA using a linear model including terms for self-reported race, smoking status, and the interaction between race and smoking status, as well as other covariates including gender, cancer status, COPD status, age, cumulative cigarette smoke exposure, and RIN. Smoking-associated race-dependent pathways were identified using Enrichr. An independent dataset of cytologically normal bronchial epithelial cells hybridized to the Affymetrix HGU133A microarrays (nCAU=154, nAFA=58) was analyzed as detailed above and relationships between the two datasets were established using GSEA for the genes associated with race, and the interaction between race and smoking status. RESULTS: We identified 361 genes that displayed smoking-associated race-dependent patterns of expression (FDR&amp;lt;0.05) and were linked to multiple biological processes, pathways, and phenotypes, including DNA damage response, membrane organization, endoplasmic reticulum stress, and abnormal lung morphology and respiration. In addition, 2771 genes were found to be associated with race (FDR&amp;lt;0.01) and enriched in pathways related to metabolism of xenobiotics, integrin cell surface interactions, and response to chemical stimulus. (Enrichr; Fisher exact test p-value&amp;lt;0.05). Both sets of genes were significantly concordantly enriched in the independent dataset by GSEA (FDR&amp;lt;0.05). CONCLUSIONS: These findings suggest that race influences the airway epithelial response to cigarette smoke at the level of individual genes and pathways. Our findings also suggest that gene expression profiling of the airway epithelium might be useful to understand the molecular basis of the elevated risk of lung cancer among African Americans. Citation Format: Anna Tassinari, Duncan Whitney, Kate Porta, Marc Lenburg, Avrum Spira, Jennifer Beane. Race-associated variation in the airway transcriptome response to cigarette smoke. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1414. doi:10.1158/1538-7445.AM2014-1414

Abstract LB-315: Comprehensive genomic characterization of cutaneous melanoma

Abstract Melanoma has emerged as the latest success story for both immuno- and genomics-guided targeted therapy following the advent of both monoclonal antibodies directed toward the inhibitory immune receptors CTLA-4, PD-1, and PD-L1, as well as BRAF inhibitors targeting mutant BRAF found in over 50% of melanoma patients. To characterize the genomic alterations in cutaneous melanoma, The Cancer Genome Atlas Project (TCGA) analyzed exome sequence, DNA structural variations (copy number and translocations), promoter methylation, mRNA, miRNA, and protein expression in primary and metastatic melanoma samples originating from non-glabrous skin in up to 336 samples. Interestingly, clustering analysis of RNA-seq data demonstrated 3 expression module subgroups of melanomas, categorized by elevated lymphocytic, keratin, and melanocyte differentiation markers. Consistent with previous findings, the high lymphocytic expression subgroup was found to possess a better overall survival. Mutation analysis from exome sequencing identified a broad spectrum of samples possessing a signature of UV mutagenesis dominated by C &amp;gt; T transitions at dipyrimidines. We observed a mean somatic mutation rate of 17 mutations per megabase (Mb), almost doubling the highest mutation rate reported previously for a cancer type analyzed by TCGA. The InVEx algorithm identified 13 significantly mutated genes (P. Bonferroni &amp;lt;0.05, Q &amp;lt;0.1), which included: known melanoma oncogenes and tumor suppressors (BRAF, NRAS, CDKN2A, TP53, and PTEN); genes previously implicated in melanoma, but found here for the first time to be significantly mutated (including the RAS-GAP NF1, IDH1 and RB1); recently implicated melanoma genes possessing either characteristic hotspot or loss of function mutations (RAC1, MAP2K1, PPP6C, and ARID2); and finally, a gene not previously implicated in melanoma, the RNA helicase DDX3X. Over 80% of samples possessed at least one mutation in the significantly mutated genes BRAF, NRAS, or NF1 involved in the MAPK pathway. Importantly, hotspot mutations in BRAF were found to be anti-correlated with both NRAS (Fisher's exact test p-value = 6.461e-16) and NF1 mutations (p-value 1.888e-06). Given this finding, we propose melanoma can be categorized into four genetic subgroups distinguished by MAPK driver mutations: BRAF, NRAS, NF1, and triple-wild type melanomas. Integrative analysis with additional platforms revealed enrichment of copy number gains in KIT, PDGFRA, KDR (VEGFR2), CCND1,CDK4, MDM2, and TERT in the triple wild-type melanomas. Integration of reverse phase protein array data also revealed differentially downstream signaling of the MAPK pathway in these four genetic subgroups. In summary, insights provided by this analysis have shed light on the different genetic mechanisms leading to cutaneous melanoma. Citation Format: Ian R. Watson, Chang-Jiun Wu, Jeffrey E. Gershenwald, Lynda Chin, On Behalf of the Melanoma Analysis Working Group and The Cancer Genome Analysis Research Network. Comprehensive genomic characterization of cutaneous melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-315. doi:10.1158/1538-7445.AM2014-LB-315

Interleukin-4 receptor alpha overexpression in human bladder cancer correlates with the pathological grade and stage of the disease.

Previously, we have demonstrated that interleukin-4 receptor α (IL-4Rα) is overexpressed on a variety of human cancers and can serve as target for IL-4 immunotoxin comprised of IL-4 and a mutated Pseudomonas exotoxin. However, its expression and association with grade and clinical stage of bladder cancer has not been studied. IL-4Rα expression was examined in human bladder cancer cell lines, mouse xenografts, and biopsy specimens at mRNA and protein levels by real-time RT-PCR and IHC/ISH techniques. We also examined the effect of IL-4 on proliferation and invasion of bladder carcinoma cell lines. For tissue microarray (TMA) results, we analyzed the precision data using exact binomial proportion with exact two-sided P-values. We used Cochran–Armitage Statistics with exact two-sided P-values to examine the trend analysis of IL-4Rα over grade or stage of the bladder cancer specimens. The influence of age and gender covariates was also analyzed using multiple logistic regression models. IL-4Rα is overexpressed in five bladder cancer cell lines, while normal bladder and human umbilical vein cell lines (HUVEC) expressed at low levels. Two other chains of IL-4 receptor complex, IL-2RγC and IL-13Rα1, were absent or weakly expressed. IL-4 modestly inhibited the cell proliferation, but enhanced cell invasion of bladder cancer cell lines in a concentration-dependent manner. Bladder cancer xenografts in immunodeficient mice also maintained IL-4Rα overexpression in vivo. Analysis of tumor biopsy specimens in TMAs revealed significantly higher IL-4Rα immunostaining (≥2+) in Grade 2 (85%) and Grade 3 (97%) compared to Grade 1 tumors (0%) (P ≤ 0.0001). Similarly, 9% stage I tumors were positive for IL-4Rα (≥2+) compared to 84% stage II (P ≤ 0.0001) and 100% stages III–IV tumors (P ≤ 0.0001). IL-13Rα1 was also expressed in tumor tissues but at low levels and it did not show any correlation with the grade and stage of disease. However, the IL-2RγC was not expressed. Ten normal bladder specimens demonstrated ≤1+ staining for IL-4Rα and IL-13Rα1 and no staining for IL-2RγC. These results demonstrate that IL-4Rα is overexpressed in human bladder cancer, which correlates with advanced grade and stage of the disease. Thus, IL-4Rα may be a bladder tumor-associated protein and a prognostic biomarker.

P-Value for two-Sample Linear Rank Tests Using Permutation Simulations and the Normal Approximation Method

Test statistics from the class of two-sample linear rank tests are commonly used to compare a treatment group with a control group. Two independent random samples of sizes m and n are drawn from two populations. As a result, N = m + n observations in total are obtained. The aim is to test the null hypothesis of identical distributions. The alternative hypothesis is that the populations are of the same form but with a different measure of central tendency. This article examines mid p-values from the null permutation distributions of tests based on the class of two-sample linear rank statistics. The results obtained indicate that normal approximation-based computations are very close to the permutation simulations, and they provide p-values that are close to the exact mid p-values for all practical purposes.

Computing Exact p-values for a Cross-correlation Shotgun Proteomics Score Function

The core of every protein mass spectrometry analysis pipeline is a function that assesses the quality of a match between an observed spectrum and a candidate peptide. We describe a procedure for computing exact p-values for the oldest and still widely used score function, SEQUEST XCorr. The procedure uses dynamic programming to enumerate efficiently the full distribution of scores for all possible peptides whose masses are close to that of the spectrum precursor mass. Ranking identified spectra by p-value rather than XCorr significantly reduces variance because of spectrum-specific effects on the score. In combination with the Percolator postprocessor, the XCorr p-value yields more spectrum and peptide identifications at a fixed false discovery rate than Mascot, X!Tandem, Comet, and MS-GF+ across a variety of data sets.

Calculation of exact p-values when SNPs are tested using multiple genetic models.

BackgroundSeveral methods have been proposed to account for multiple comparisons in genetic association studies. However, investigators typically test each of the SNPs using multiple genetic models. Association testing using the Cochran-Armitage test for trend assuming an additive, dominant, or recessive genetic model, is commonly performed. Thus, each SNP is tested three times. Some investigators report the smallest p-value obtained from the three tests corresponding to the three genetic models, but such an approach inherently leads to inflated type 1 errors. Because of the small number of tests (three) and high correlation (functional dependence) among these tests, the procedures available for accounting for multiple tests are either too conservative or fail to meet the underlying assumptions (e.g., asymptotic multivariate normality or independence among the tests).ResultsWe propose a method to calculate the exact p-value for each SNP using different genetic models. We performed simulations, which demonstrated the control of type 1 error and power gains using the proposed approach. We applied the proposed method to compute p-value for a polymorphism eNOS -786T>C which was shown to be associated with breast cancer risk.ConclusionsOur findings indicate that the proposed method should be used to maximize power and control type 1 errors when analyzing genetic data using additive, dominant, and recessive models.

OP0258-HPR Self-Reported Disease Characteristics do not Explain Why Younger Women with SPA Are Less Physically Active than Older Women with the Disease

BackgroundExercise is a commonly used treatment for patients with spondyloarthritis (SpA) but younger women reach WHOs recommended level of physical activity (PA) to a less extent than peers in the...

Comparison of Bier's block with Lidocaine versus Lidocaine plus Verapamil

Background: General anaesthesia and regional anaesthesia both are used for upper extremity surgeries. Bier’s Blockusing local anaesthetic alone or in combination with other adjuvants provides effective analgesia intraoperatively as wellas postoperatively with fewer complications as compared to general anaesthesia.Objective: This study was designed to compare effectiveness, haemodynamic alterations and total duration of analgesiawith Lidocaine versus Lidocaine plus Verapamil in Bier’s Block.Methods: This is a prospective randomised double blind trial conducted in Kathmandu Medical College TeachingHospital from February 2012 to December 2012 after approval from the ethical committee and informed consent fromthe participants. Total 40 adult patients of age 20 to 50 years, weight 50 to 70 kg, of both gender, belonging to AmericanSociety of Anaesthesiologists Physical Status I and II undergoing elective distal upper extrimity surgery lasting at one toone and half hours with Bier’s Block were included in this study. They were randomly divided into two groups of twentyeach to receive either 40 ml of 0.5% Lidocaine alone (Group A) or 40 ml of 0.5% Lidocaine plus 2.5 mg Verapamil (GroupB). The two groups were compared in terms of onset and recovery from sensory and motor blockade, tourniquet paintolerance time, duration of analgesia, alteration of haemodynamics and major side effects. Data analysis was done byMicrosoft Offi ce Excel 2007 [Polystat, Microsoft Offi ce Excel worksheet.XLS] using student’s two tailed t test. Categoricalparamatres were tested by Fisher Exact test and p-value of &lt;0.05 was considered statistically signifi cant.Result: Onset of sensory blockade was faster in group B (Mean± SD: 3.07± 0.25 minutes) as compared to group A (Mean±SD: 5.59 ± 0.41 minutes). The onset of motor block in group B was 10.4 ± 0.77 minutes versus 13.17 ± 1.45 minutes ingroup A. Recovery of sensory block in group B occurred at 29.95 ± 6.96 minutes versus 11.45 ± 2.16 minutes in groupA. Similarly recovery of motor block in group B occurred in 13.6 ± 1.79 minutes versus 7.65 ± 1.04 minutes in group A.Tolerance of fi rst tourniquet pain in group B was long 41.15 ± 3.82 minutes versus 22.00 ± 2.9) minutes in group A. Secondtourniquet pain tolerance time was again longer (48.25 ± 3.96 minutes) in group B versus 28.05 ± 4.84 minutes in groupA. Total duration of analgesia was more in group B (207.25 ± 21.1 minutes) versus 32.2 ± 5.78 minutes in group A. Totalconsumption of analgesic (Tramadol) in 24 hours in group B was lesser than group A (47.5 ± 38.0 mg versus 112.5 ± 35.8mg). All these differences were signifi cant statistically (p value &lt;0.05). Both of the groups showed stable haemodynamicparameters intraoperatively as well as postoperatively without any signifi cant adverse effects.Conclusion: Addition of Verapamil to Lidocaine was more effective than Lidocaine alone in Bier’s block.DOI: http://dx.doi.org/10.3126/jkmc.v2i1.10513Journal of Kathmandu Medical College, Vol. 2, No. 1, Issue 3, Jan.-Mar., 2013, Page: 12-17