An integrative ChIP-chip and gene expression profiling to model SMAD regulatory modules

<p>Abstract</p> <p>Background</p> <p>The TGF-β/SMAD pathway is part of a broader signaling network in which crosstalk between pathways occurs. While the molecular mechanisms of TGF-β/SMAD signaling pathway have been studied in detail, the global networks downstream of SMAD remain largely unknown. The regulatory effect of SMAD complex likely depends on transcriptional modules, in which the SMAD binding elements and partner transcription factor binding sites (SMAD modules) are present in specific context.</p> <p>Results</p> <p>To address this question and develop a computational model for SMAD modules, we simultaneously performed chromatin immunoprecipitation followed by microarray analysis (ChIP-chip) and mRNA expression profiling to identify TGF-β/SMAD regulated and synchronously coexpressed gene sets in ovarian surface epithelium. Intersecting the ChIP-chip and gene expression data yielded 150 direct targets, of which 141 were grouped into 3 co-expressed gene sets (sustained up-regulated, transient up-regulated and down-regulated), based on their temporal changes in expression after TGF-β activation. We developed a data-mining method driven by the Random Forest algorithm to model SMAD transcriptional modules in the target sequences. The predicted SMAD modules contain SMAD binding element and up to 2 of 7 other transcription factor binding sites (E2F, P53, LEF1, ELK1, COUPTF, PAX4 and DR1).</p> <p>Conclusion</p> <p>Together, the computational results further the understanding of the interactions between SMAD and other transcription factors at specific target promoters, and provide the basis for more targeted experimental verification of the co-regulatory modules.</p

Differential Analysis of Ovarian and Endometrial Cancers Identifies a Methylator Phenotype

Despite improved outcomes in the past 30 years, less than half of all women diagnosed with epithelial ovarian cancer live five years beyond their diagnosis. Although typically treated as a single disease, epithelial ovarian cancer includes several distinct histological subtypes, such as papillary serous and endometrioid carcinomas. To address whether the morphological differences seen in these carcinomas represent distinct characteristics at the molecular level we analyzed DNA methylation patterns in 11 papillary serous tumors, 9 endometrioid ovarian tumors, 4 normal fallopian tube samples and 6 normal endometrial tissues, plus 8 normal fallopian tube and 4 serous samples from TCGA. For comparison within the endometrioid subtype we added 6 primary uterine endometrioid tumors and 5 endometrioid metastases from uterus to ovary. Data was obtained from 27,578 CpG dinucleotides occurring in or near promoter regions of 14,495 genes. We identified 36 locations with significant increases or decreases in methylation in comparisons of serous tumors and normal fallopian tube samples. Moreover, unsupervised clustering techniques applied to all samples showed three major profiles comprising mostly normal samples, serous tumors, and endometrioid tumors including ovarian, uterine and metastatic origins. The clustering analysis identified 60 differentially methylated sites between the serous group and the normal group. An unrelated set of 25 serous tumors validated the reproducibility of the methylation patterns. In contrast, >1,000 genes were differentially methylated between endometrioid tumors and normal samples. This finding is consistent with a generalized regulatory disruption caused by a methylator phenotype. Through DNA methylation analyses we have identified genes with known roles in ovarian carcinoma etiology, whereas pathway analyses provided biological insight to the role of novel genes. Our finding of differences between serous and endometrioid ovarian tumors indicates that intervention strategies could be developed to specifically address subtypes of epithelial ovarian cancer

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Incorporating Pharmacodynamic Considerations into Caffeine Therapeutic Drug Monitoring in Preterm Neonates

Background This study sought to assess the pharmacokinetic and pharmacodynamic relationships of caffeine citrate therapy in preterm neonates who had therapeutic drug monitoring (TDM) in the post-extubation period. Methods A retrospective observational study was conducted in preterm neonates who received caffeine citrate therapy for apnea of prematurity and had TDM done in the post-extubation period between January 2006 and October 2011. The relationships between pharmacodynamic effects (heart rate, respiratory rate, episodes of apnea, adverse events) and caffeine serum concentrations were explored. Results A total of 177 blood samples were obtained from 115 preterm neonates with a median (range) gestational age of 29 (24 – 33) weeks and birth weight of 1230 (607 – 2304) kg. Caffeine citrate therapy was initiated at a median (interquartile range) postnatal age of 1 (1 – 3) day and TDM was performed at a postnatal age of 15 (10 – 24) days. No direct correlations were found between respiratory rate or apneic episodes and caffeine serum concentrations; however, heart rate and caffeine serum concentrations were significantly correlated (p \u3c 0.05). Dosing regimen of 40/5 mg/kg q12h (loading dose/maintenance dose, time interval) led to similar endotracheal re-intubation rate but increased percentage of patients experiencing tachycardia compared to the standard regimen of 20/5 mg/kg q24h (44.7 % vs 10.2 %, p \u3c 0.001). Conclusion Based on this retrospective study, no correlation between episodes of apnea and caffeine serum concentrations was found in neonates who had TDM of caffeine citrate therapy in the post-extubation period, whereas a significant association between tachycardia and concentrations existed. Notwithstanding the absence of severe adverse reactions, TDM should be considered in critically ill neonates with unexplained adverse effects, such as tachycardia

Tyrosine Kinase in Pediatric Growth

The use of tyrosine kinase inhibitor (TKI) therapy has become more common in pediatric patients in the last few years. This trend is likely to continue as more TKIs are approved and the list of conditions for which TKIs have clinical utility expands. Imatinib (Gleevec™) is a tyrosine kinase inhibitor that is specifically indicated for Philadelphia positive chronic myelogenous leukemia (PHÆCML). Dasatinib (Sprycel™) and nilotinib (Tasigna™) are TKIs indicated for CML patients who are no longer benefitting from, or did not tolerate, other treatments including imatinib. OBJECTIVE:The objective of this study was to determine whether there is evidence of growth retardation as an adverse drug experience for TKIs. METHODS:The FDA Adverse Event Reporting System (FAERS) was reviewed for currently posted data from 4th quarter 2012 until 1st quarter 2014 for individuals ≤ 18 years of age. The most recent update of the FAERs data was in October of 2014. These are sponsor, patient and physician reported events. A search for approximate matches to the drug names using the generalized Levenshtein edit distance using the R and SAS™ 9.3 was used to search for patterns in the adverse experiences. These AEs were grouped by Preferred Term(PT), and the ranking of growth related AEs was conducted relative to other PTs. RESULTS:Of 574 self-reported adverse experiences reported for imatinib from 2012-2014, there were 12 (2.1%) cases of growth retardation. Growth retardation was the 5th most commonly occurring AE. Of 594 self-reported adverse experiences for dasatinib from 2012-2104, no cases of growth retardation occurred. Likewise, of 25 self-reported AEs for nilotinib, none were growth related. CONCLUSION:There appears to be some evidence of growth retardation in imatinib patients, and none for dasatinib patients. Not enough AEs have yet been reported for nilotinib to judge whether growth is also retarded in these patients

Tyrosine Kinase in Pediatric Growth

The use of tyrosine kinase inhibitor (TKI) therapy has become more common in pediatric patients in the last few years. This trend is likely to continue as more TKIs are approved and the list of conditions for which TKIs have clinical utility expands. Imatinib (Gleevec™) is a tyrosine kinase inhibitor that is specifically indicated for Philadelphia positive chronic myelogenous leukemia (PHÆCML). Dasatinib (Sprycel™) and nilotinib (Tasigna™) are TKIs indicated for CML patients who are no longer benefitting from, or did not tolerate, other treatments including imatinib. OBJECTIVE:The objective of this study was to determine whether there is evidence of growth retardation as an adverse drug experience for TKIs. METHODS:The FDA Adverse Event Reporting System (FAERS) was reviewed for currently posted data from 4th quarter 2012 until 1st quarter 2014 for individuals ≤ 18 years of age. The most recent update of the FAERs data was in October of 2014. These are sponsor, patient and physician reported events. A search for approximate matches to the drug names using the generalized Levenshtein edit distance using the R and SAS™ 9.3 was used to search for patterns in the adverse experiences. These AEs were grouped by Preferred Term(PT), and the ranking of growth related AEs was conducted relative to other PTs. RESULTS:Of 574 self-reported adverse experiences reported for imatinib from 2012-2014, there were 12 (2.1%) cases of growth retardation. Growth retardation was the 5th most commonly occurring AE. Of 594 self-reported adverse experiences for dasatinib from 2012-2104, no cases of growth retardation occurred. Likewise, of 25 self-reported AEs for nilotinib, none were growth related. CONCLUSION:There appears to be some evidence of growth retardation in imatinib patients, and none for dasatinib patients. Not enough AEs have yet been reported for nilotinib to judge whether growth is also retarded in these patients