Nicola J. Armstrong

Haemolysis during Sample Preparation Alters microRNA Content of Plasma

The presence of cell-free microRNAs (miRNAs) has been detected in a range of body fluids. The miRNA content of plasma/serum in particular has been proposed as a potential source of novel biomarkers for a number of diseases. Nevertheless, the quantification of miRNAs from plasma or serum is made difficult due to inefficient isolation and lack of consensus regarding the optimal reference miRNA. The effect of haemolysis on the quantification and normalisation of miRNAs in plasma has not been investigated in great detail. We found that levels of miR-16, a commonly used reference gene, showed little variation when measured in plasma samples from healthy volunteers or patients with malignant mesothelioma or coronary artery disease. Including samples with evidence of haemolysis led to variation in miR-16 levels and consequently decreased its ability to serve as a reference. The levels of miR-16 and miR-451, both present in significant levels in red blood cells, were proportional to the degree of haemolysis. Measurements of the level of these miRNAs in whole blood, plasma, red blood cells and peripheral blood mononuclear cells revealed that the miRNA content of red blood cells represents the major source of variation in miR-16 and miR-451 levels measured in plasma. Adding lysed red blood cells to non-haemolysed plasma allowed a cut-off level of free haemoglobin to be determined, below which miR-16 and miR-451 levels displayed little variation between individuals. In conclusion, increases in plasma miR-16 and miR-451 are caused by haemolysis. In the absence of haemolysis the levels of both miR-16 and miR-451 are sufficiently constant to serve as normalisers.

IL28B, HLA-C, and KIR variants additively predict response to therapy in chronic hepatitis C virus infection in a European Cohort: a cross-sectional study.

Vijayaprakash Suppiah and colleagues show that genotyping hepatitis C patients for the IL28B, HLA-C, and KIR genes improves the ability to predict whether or not patients will respond to antiviral treatment.

A comprehensive analysis of prognostic signatures reveals the high predictive capacity of the Proliferation, Immune response and RNA splicing modules in breast cancer

IntroductionSeveral gene expression signatures have been proposed and demonstrated to be predictive of outcome in breast cancer. In the present article we address the following issues: Do these signatures perform similarly? Are there (common) molecular processes reported by these signatures? Can better prognostic predictors be constructed based on these identified molecular processes?MethodsWe performed a comprehensive analysis of the performance of nine gene expression signatures on seven different breast cancer datasets. To better characterize the functional processes associated with these signatures, we enlarged each signature by including all probes with a significant correlation to at least one of the genes in the original signature. The enrichment of functional groups was assessed using four ontology databases.ResultsThe classification performance of the nine gene expression signatures is very similar in terms of assigning a sample to either a poor outcome group or a good outcome group. Nevertheless the concordance in classification at the sample level is low, with only 50% of the breast cancer samples classified in the same outcome group by all classifiers. The predictive accuracy decreases with the number of poor outcome assignments given to a sample. The best classification performance was obtained for the group of patients with only good outcome assignments. Enrichment analysis of the enlarged signatures revealed 11 functional modules with prognostic ability. The combination of the RNA-splicing and immune modules resulted in a classifier with high prognostic performance on an independent validation set.ConclusionsThe study revealed that the nine signatures perform similarly but exhibit a large degree of discordance in prognostic group assignment. Functional analyses indicate that proliferation is a common cellular process, but that other functional categories are also enriched and show independent prognostic ability. We provide new evidence of the potentially promising prognostic impact of immunity and RNA-splicing processes in breast cancer.

Calibration and discriminatory accuracy of prognosis calculation for breast cancer with the online Adjuvant! program: a hospital-based retrospective cohort study

BACKGROUND Adjuvant! is a web-based program that calculates individualised 10-year survival probabilities and predicted benefit of adjuvant systemic therapy. The Adjuvant! model has not been validated in any large European series. The aim of our study was to validate Adjuvant! in Dutch patients, investigating both its calibration and discriminatory accuracy. METHODS Patients who were at least partly treated at the Netherlands Cancer Institute for breast cancer between 1987 and 1998 were included if they met the following criteria: tumour size T1 (< or =2 cm), T2 (2-5 cm), or T3 (>5 cm), invasive breast carcinoma, with information about involvement of axillary lymph nodes available, no distant metastases, primary surgery, axillary staging, and radiotherapy according to national guidelines. Clinicopathological characteristics and adjuvant treatment data were retrieved from hospital records and medical registries and were entered into the Adjuvant! (version 8.0) batch processor with blinding to outcome. Endpoints were overall survival and the proportion of patients that did not die from breast cancer (breast-cancer-specific survival [BCSS]). FINDINGS 5380 patients were included with median follow-up of 11.7 years (range 0.03-21.8). The 10-year observed overall survival (69.0%) and BCSS (78.6%) and Adjuvant! predicted overall survival (69.1%) and BCSS (77.8%) were not statistically different (p=0.87 and p=0.18, respectively). Moreover, differences between predicted and observed outcomes were within 2% for most relevant clinicopathological subgroups. In patients younger than 40 years, Adjuvant! overestimated overall survival by 4.2% (p=0.04) and BCSS by 4.7% (p=0.01). The concordance index, which indicates discriminatory accuracy at the individual level, was 0.71 for BCSS in the entire cohort. INTERPRETATION Adjuvant! accurately predicted 10-year outcomes in this large-scale Dutch validation study and is of use for adjuvant treatment decision making, although the results may be less reliable in some subgroups.

One naive T cell, multiple fates in CD8+ T cell differentiation

The mechanism by which the immune system produces effector and memory T cells is largely unclear. To allow a large-scale assessment of the development of single naive T cells into different subsets, we have developed a technology that introduces unique genetic tags (barcodes) into naive T cells. By comparing the barcodes present in antigen-specific effector and memory T cell populations in systemic and local infection models, at different anatomical sites, and for TCR–pMHC interactions of different avidities, we demonstrate that under all conditions tested, individual naive T cells yield both effector and memory CD8+ T cell progeny. This indicates that effector and memory fate decisions are not determined by the nature of the priming antigen-presenting cell or the time of T cell priming. Instead, for both low and high avidity T cells, individual naive T cells have multiple fates and can differentiate into effector and memory T cell subsets.

Identification of candidate transcriptional modulators involved in successful regeneration after nerve injury

Successful regeneration of injured neurons requires a complex molecular response that involves the expression, modification and transport of a large number of proteins. The identity of neuronal proteins responsible for the initiation of regenerative neurite outgrowth is largely unknown. Dorsal root ganglion (DRG) neurons display robust and successful regeneration following lesion of their peripheral neurite, whereas outgrowth of central neurites is weak and does not lead to functional recovery. We have utilized this differential response to gain insight in the early transcriptional events associated with successful regeneration. Surprisingly, our study shows that peripheral and central nerve crushes elicit very distinct transcriptional activation, revealing a large set of novel genes that are differentially regulated within the first 24 h after the lesion. Here we show that Ankrd1, a gene known to act as a transcriptional modulator, is involved in neurite outgrowth of a DRG neuron‐derived cell line as well as in cultured adult DRG neurons. This gene, and others identified in this study, may be part of the transcriptional regulatory module that orchestrates the onset of successful regeneration.

Local Recurrence after Breast-Conserving Therapy in Relation to Gene Expression Patterns in a Large Series of Patients

Purpose: The majority of patients with early-stage breast cancer are treated with breast-conserving therapy (BCT). Several clinical risk factors are associated with local recurrence (LR) after BCT but are unable to explain all instances of LR after BCT. Here, gene expression microarrays are used to identify novel risk factors for LR after BCT. Experimental Design: Gene expression profiles of 56 primary invasive breast carcinomas from patients who developed a LR after BCT were compared with profiles of 109 tumors from patients who did not develop a LR after BCT. Both unsupervised and supervised methods of classification were used to separate patients into groups corresponding to disease outcome. In addition, for 15 patients, the gene expression profile in the recurrence was compared with that of the primary tumor. Results: The two main clusters found by hierarchical cluster analysis of all 165 primary invasive breast carcinomas revealed no association with LR. Predefined gene sets (molecular subtypes and “chromosomal instability” signature) are associated with LR (P = 0.0002 and 0.003, respectively). Significant analysis of microarrays revealed an association between LR and cell proliferation, not captured by histologic grading. Class prediction analysis constructed a gene classifier, which was successfully validated, cross-platform, on an independent data set of 161 patients (log-rank P = 0.041). In multivariate analysis, young age was the only independent predictor of LR. Conclusions: We have constructed and cross-platform validated a gene expression profile predictive for LR after BCT, which is characterized by genes involved in cell proliferation but not a surrogate for high histologic grade.

The multiple sclerosis whole blood mRNA transcriptome and genetic associations indicate dysregulation of specific T cell pathways in pathogenesis

Multiple sclerosis (MS) is an autoimmune disease with a genetic component, caused at least in part by aberrant lymphocyte activity. The whole blood mRNA transcriptome was measured for 99 untreated MS patients: 43 primary progressive MS, 20 secondary progressive MS, 36 relapsing remitting MS and 45 age-matched healthy controls. The ANZgene Multiple Sclerosis Genetics Consortium genotyped more than 300 000 SNPs for 115 of these samples. Transcription from genes on translational regulation, oxidative phosphorylation, immune synapse and antigen presentation pathways was markedly increased in all forms of MS. Expression of genes tagging T cells was also upregulated (P < 10(-12)) in MS. A T cell gene signature predicts disease state with a concordance index of 0.79 with age and gender as co-variables, but the signature is not associated with clinical course or disability. The ANZgene genome wide association screen identified two novel regions with genome wide significance: one encoding the T cell co-stimulatory molecule, CD40; the other a region on chromosome 12q13-14. The CD40 haplotype associated with increased MS susceptibility has decreased gene expression in MS (P < 0.0007). The second MS susceptibility region includes 17 genes on 12q13-14 in tight linkage disequilibrium. Of these, only 13 are expressed in leukocytes, and of these the expression of one, FAM119B, is much lower in the susceptibility haplotype (P < 10(-14)). Overall, these data indicate dysregulation of T cells can be detected in the whole blood of untreated MS patients, and supports targeting of activated T cells in therapy for all forms of MS.

Epigenetic-induced repression of microRNA-205 is associated with MED1 activation and a poorer prognosis in localized prostate cancer

Deregulation of microRNA (miRNA) expression can have a critical role in carcinogenesis. Here we show in prostate cancer that miRNA-205 (miR-205) transcription is commonly repressed and the MIR-205 locus is hypermethylated. LOC642587, the MIR-205 host gene of unknown function, is also concordantly inactivated. We show that miR-205 targets mediator 1 (MED1, also called TRAP220 and PPARBP) for transcriptional silencing in normal prostate cells, leading to reduction in MED1 mRNA levels, and in total and active phospho-MED1 protein. Overexpression of miR-205 in prostate cancer cells negatively affects cell viability, consistent with a tumor suppressor function. We found that hypermethylation of the MIR-205 locus was strongly related with a decrease in miR-205 expression and an increase in MED1 expression in primary tumor samples (n=14), when compared with matched normal prostate (n=7). An expanded patient cohort (tumor n=149, matched normal n=30) also showed significant MIR-205 DNA methylation in tumors compared with normal, and MIR-205 hypermethylation is significantly associated with biochemical recurrence (hazard ratio=2.005, 95% confidence interval (1.109, 3.625), P=0.02), in patients with low preoperative prostate specific antigen. In summary, these results suggest that miR-205 is an epigenetically regulated tumor suppressor that targets MED1 and may provide a potential biomarker in prostate cancer management.

Low Calretinin Expression and High Neutrophil-To- Lymphocyte Ratio Are Poor Prognostic Factors in Patients with Malignant Mesothelioma Undergoing Extrapleural Pneumonectomy

Introduction: Survival after extrapleural pneumonectomy (EPP) is variable in patients with malignant pleural mesothelioma (MPM), and there are no validated prognostic factors that could be used preoperatively. We investigated the calretinin and D2-40 expression and the neutrophil-to-lymphocyte ratio (NLR), an index of systemic inflammation as potential preoperative prognostic factors. Methods: Consecutive patients who underwent EPP were included in this retrospective study. Potential prognostic factors such as age, gender, histological subtype, baseline laboratory parameters including NLR, and immunohistochemical staining for calretinin and D2-40 were evaluated. Overall survival (OS) from the date of surgery was determined by the Kaplan-Meier method. The prognostic value of the variables was examined using Cox regression, and significant factors (p < 0.05) were entered into a multivariate model to determine their independent effect. Results: A total of 85 patients were included: median age 58 years; 80% men; 77% epithelial and 23% biphasic MPM. The median OS was 19.7 months. The following variables were predictive of longer OS: female gender (p = 0.02), epithelial subtype (p = 0.04), low NLR (p < 0.01), and high calretinin score (p < 0.001). In a multivariate analysis, only NLR ≥3 (hazard ratio 1.79; 95% confidence interval: 1.04–3.07; p = 0.04) and calretinin score ≤33 versus more than 67% (hazard ratio 4.72; 95% confidence interval: 1.97–11.32; p < 0.01) remained independent predictors. The addition of calretinin score increased the explained variation by 10.1%. Conclusions: Both low calretinin expression and high NLR were independently associated with poor prognosis in patients with MPM undergoing EPP, and the calretinin score seemed to improve the accuracy of the prognostic model.