Marie C. South

RefPlus: an R package extending the RMA Algorithm

UNLABELLED RMA has become a widely used methodology to pre-process Affymetrix gene expression microarrays. A limitation of RMA is that the calculated probeset intensities change when a set of microarrays is re-pre-processed after the inclusion of additional microarrays into the analysis set. Here we report the availability of the RefPlus package containing functions to perform the Extrapolation Strategy and Extrapolation Averaging algorithms which address these issues. AVAILABILITY The software is implemented in the R language and can be downloaded from the Bioconductor project website (http://www.bioconductor.org). SUPPLEMENTARY INFORMATION Further details of the workings and evaluation of these functions are given in the documentation available on the Bioconductor website.

Identification of biomarkers in human head and neck tumor cell lines that predict for in vitro sensitivity to gefitinib.

Potential biomarkers were identified for in vitro sensitivity to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib in head and neck cancer. Gefitinib sensitivity was determined in cell lines, followed by transcript profiling coupled with a novel pathway analysis approach. Eleven cell lines were highly sensitive to gefitinib (inhibitor concentration required to give 50% growth inhibition [GI50] < 1 μM), three had intermediate sensitivity (GI50 1–7 μM), and six were resistant (GI50 > 7 μM); an exploratory principal component analysis revealed a separation between the genomic profiles of sensitive and resistant cell lines. Subsequently, a hypothesis‐driven analysis of Affymetrix data (Affymetrix, Inc., Santa Clara, CA, USA) revealed higher mRNA levels for E‐cadherin (CDH1); transforming growth factor, alpha (TGF‐α); amphiregulin (AREG); FLJ22662; EGFR; p21‐activated kinase 6 (PAK6); glutathione S‐transferase Pi (GSTP1); and ATP‐binding cassette, subfamily C, member 5 (ABCC5) in sensitive versus resistant cell lines. A hypothesis‐free analysis identified 46 gene transcripts that were strongly differentiated, seven of which had a known association with EGFR and head and neck cancer (human EGF receptor 3 [HER3], TGF‐α, CDH1, EGFR, keratin 16 [KRT16], fibroblast growth factor 2 [FGF2], and cortactin [CTTN]). Polymerase chain reaction (PCR) and enzyme‐linked immunoabsorbant assay analysis confirmed Affymetrix data, and EGFR gene mutation, amplification, and genomic gain correlated strongly with gefitinib sensitivity. We identified biomarkers that predict for in vitro responsiveness to gefitinib, seven of which have known association with EGFR and head and neck cancer. These in vitro predictive biomarkers may have potential utility in the clinic and warrant further investigation.

Can you trust your animal study data

persists, thus increasing the risk of poor decisions and contributing to the attrition of early-stage drug projects4–7. We therefore conclude that we have an ethical and scientific obligation to improve the quality of preclinical animal studies, ensuring that where they are used, they include as few animals as possible and are run in a way that provides reproducible and valid information. We believe that a systematic approach to reviewing animal study protocols against key statistical principles supports this goal, providing a robust challenge to study design, analysis and interpretation, thereby helping to ensure the internal validity of the studies and increasing confidence in resulting decisions.

Proteomic Biomarkers for Acute Interstitial Lung Disease in Gefitinib-Treated Japanese Lung Cancer Patients

Interstitial lung disease (ILD) events have been reported in Japanese non-small-cell lung cancer (NSCLC) patients receiving EGFR tyrosine kinase inhibitors. We investigated proteomic biomarkers for mechanistic insights and improved prediction of ILD. Blood plasma was collected from 43 gefitinib-treated NSCLC patients developing acute ILD (confirmed by blinded diagnostic review) and 123 randomly selected controls in a nested case-control study within a pharmacoepidemiological cohort study in Japan. We generated ∼7 million tandem mass spectrometry (MS/MS) measurements with extensive quality control and validation, producing one of the largest proteomic lung cancer datasets to date, incorporating rigorous study design, phenotype definition, and evaluation of sample processing. After alignment, scaling, and measurement batch adjustment, we identified 41 peptide peaks representing 29 proteins best predicting ILD. Multivariate peptide, protein, and pathway modeling achieved ILD prediction comparable to previously identified clinical variables; combining the two provided some improvement. The acute phase response pathway was strongly represented (17 of 29 proteins, p = 1.0×10−25), suggesting a key role with potential utility as a marker for increased risk of acute ILD events. Validation by Western blotting showed correlation for identified proteins, confirming that robust results can be generated from an MS/MS platform implementing strict quality control.

Claudin-2 Expression Levels in Ulcerative Colitis: Development and Validation of an In-Situ Hybridisation Assay for Therapeutic Studies.

Ulcerative colitis is a chronic inflammatory disease affecting the colon and is characterized by epithelial damage and barrier dysfunction. Upregulation of the tight junction protein claudin-2 by cytokines is hypothesized to contribute to the dysregulation of the epithelial barrier. New therapeutic agents which block the action of cytokines are being investigated in patients with ulcerative colitis. In order to understand the potential of these therapies, it is important to have reliable assays that can assess downstream endpoints that reflect drug mechanism of action. The aim of the current study was therefore to establish & validate an assay to reproducibly assess the expression and distribution of claudin-2 in human colon biopsy samples. Initially, the potential to measure claudin-2 protein by immunohistochemistry (IHC) was investigated. To identify suitable reagents to develop an IHC assay, pre-established criteria were used to screen five commercial antibodies by Western blotting, immunofluorescence and immunohistochemistry on claudin-2 positive and negative cells and healthy and ulcerative colitis colon tissue. Despite some of these antibodies specifically detecting claudin-2 using some of these techniques, none of the antibodies showed the expected specific staining pattern in formalin fixed human colon samples. As an alternative method to detect claudin-2 expression and distribution in formalin fixed biopsy sections, an in situ hybridization assay was developed. This assay underwent a novel tiered approach of validation to establish that it was fit-for-purpose, and suitable for clinical deployment. In addition, to understand the possible relationship of claudin-2 in the context of disease severity, expression was compared to the Geboes score. Overall, the microscopical Geboes score correlated with the claudin-2 biomarker score for samples that retained crypt morphology; samples with the highest Geboes score were not specifically distinguished, probably due to crypt destruction. In summary, we have applied a strategy for identifying target-specific antibodies in formalin fixed biopsy samples and highlighted that (published) antibodies may not correctly identify the intended antigen in tissues fixed using this method. Furthermore, we have developed and, for the first time, validated an in situ hybridization assay for detection of claudin-2 mRNA, suitable for use as a supportative method in clinical trials. Using our validated assay, we have demonstrated that increased claudin-2 expression correlates with the severity of ulcerative colitis, where crypt destruction is not seen.

Incorporation of capillary microsampling into whole body plethysmography and modified Irwin safety pharmacology studies in rats

UNLABELLED Limited toxicokinetic data is generated during modified Irwin screen and whole body plethysmography safety pharmacology studies, due to disturbance of primary endpoints by standard blood sampling methods. We evaluated if incorporation of microsampling would impact on data quality, as providing toxicokinetic data from main test animals could reduce the need for satellite animals. METHODS A modified Irwin screen was performed, testing oral clonidine (0, 0.03, 0.1, or 0.3 mg/kg). One cohort of rats per dose level underwent standard blood sampling (post-4 h Irwin assessment), and another cohort underwent microsampling after the 0.5, 1, 2, 4 and 24 h Irwin assessments. The respiratory effects of oral theophylline (0 or 10 mg/kg) and oral baclofen (0 or 5 mg/kg), were tested using whole body plethysmography. Groups of animals underwent standard blood sampling (at end of recording at 4 h post-dose) or microsampling at either 0.5 h or 1 h intervals (4 or 8 microsamples, respectively). RESULTS Microsampling did not impact on the quality of the data generated. The expected effects of clonidine on behavioural parameters, and of theophylline and baclofen on changes in ventilatory parameters were observed at a similar magnitude and duration independent of sampling method. DISCUSSION The incorporation of multiple capillary microsamples into the modified Irwin or respiratory study did not adversely affect the primary endpoints. The capillary microsampling method is a refinement in blood sampling technique which can easily be adapted into safety pharmacology studies to generate pharmacokinetic profiles within the same animal as the primary data, thus enhancing scientific robustness and reducing the satellite animals required.

Nonclinical Safety Assessment: An Introduction for Statisticians

This chapter provides an overview of the nonclinical drug safety testing process, and the statistical challenges associated with work in this area. Whilst other chapters in this book focus on specific types of designs and analyses which you may encounter during nonclinical drug development, we provide here the context for a statistician working in nonclinical safety assessment, and seek to prepare them for some of the practical issues and decisions they are likely to face. We will describe the scope and framework for the studies run within a nonclinical safety assessment programme, and recommend when and how a statistician needs to engage to add value. We will look generally at design and analysis considerations for safety studies which, whilst not unique to this context, are particularly pertinent to this area of work. Finally we will highlight some practical considerations and industry trends. If this chapter provides you with insight that complements rather than replicates what is taught in conventional statistics courses, and inspires you to believe that statistical work in this area can be both valuable and rewarding, we will have achieved our goal.