Reliability of tissue microarrays in detecting protein expression and gene amplification in breast cancer

Tissue microarrays allow high throughput molecular profiling of diagnostic or predictive markers in cancer specimens and rapid validation of novel potential candidates identified from genomic and proteomic analyses in a large number of tumor samples. To validate the use of tissue microarray technology for all the main biomarkers routinely used to decide breast cancer prognostication and postsurgical adjuvant therapy, we constructed a tissue microarray from 97 breast tumors, with a single 0.6 mm core per specimen. Inummostaining; of tissue microarray sections and conventional full sections of each tumor were performed using well-characterized prognostic markers (estrogen receptor ER, progesterone receptor PR and c-erbB2). The full section versus tissue microarray concordance for these stains was 97% for ER, 98% for PR, and 97% for c-erbB2, respectively, with a strong statistical association (kappa value more than 0.90). Fluorescence in situ hybridization analysis for HER-2/neu gene amplification from the single-core tissue microarray was technically successful in about 90% (87/97) of the cases, with a concordance of 95% compared with parallel analyses with the full sections. The correlation with other pathological parameters was not significantly different between full-section and array-based results. It is concluded that the constructed tissue microarray with a single core per specimen ensures full biological representativeness to identify the associations between biomarkers and clinicopathological parameters, with no significant associated sampling bias.

Evaluation of HER-2/neu oncogene status in breast tumors on tissue microarrays

The amplification and/or overexpression of the HER-2/neu oncogene and its encoded receptor protein are increasingly used for prognostication and prediction of therapeutic response to Herceptin in breast cancer. However, large-scale examination of archival tumor blocks by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) is prohibitively laborious and technically challenging. The tissue microarray (TMA) technique enables hundreds of tumors to be studied simultaneously in a single experiment. To evaluate the HER-2/neu status of a selection of the breast tumors in our tumor bank, we constructed a TMA from 97 breast tumors, with a single 0.6-mm core per specimen. HER-2/neu gene amplification by FISH was found in 20 of the 87 interpretable cases (23%): in 14 of 14 IHC 3+ cases (100%), 5 of 8 IHC 2+ cases (62.5%) and 1 of 65 IHC 0/1+ cases (1.5%). Three of the 67 cases with no evidence of HER-2/neu gene amplification by FISH were moderately positive (2+) by IHC. A close relationship was observed between these 2 assays as applied to the TMA (95.4% concordance: 95% CI, - 2.2% to 6.8%; P

Ultra-fast processing of gigapixel Tissue MicroArray images using high performance computing.

BACKGROUND:
tissue MicroArrays (TMAs) are a valuable platform for tissue based translational research and the discovery of tissue biomarkers. The digitised TMA slides or TMA Virtual Slides, are ultra-large digital images, and can contain several hundred samples. The processing of such slides is time-consuming, bottlenecking a potentially high throughput platform.
METHODS:
a High Performance Computing (HPC) platform for the rapid analysis of TMA virtual slides is presented in this study. Using an HP high performance cluster and a centralised dynamic load balancing approach, the simultaneous analysis of multiple tissue-cores were established. This was evaluated on Non-Small Cell Lung Cancer TMAs for complex analysis of tissue pattern and immunohistochemical positivity.
RESULTS:
the automated processing of a single TMA virtual slide containing 230 patient samples can be significantly speeded up by a factor of circa 22, bringing the analysis time to one minute. Over 90 TMAs could also be analysed simultaneously, speeding up multiplex biomarker experiments enormously.
CONCLUSIONS:
the methodologies developed in this paper provide for the first time a genuine high throughput analysis platform for TMA biomarker discovery that will significantly enhance the reliability and speed for biomarker research. This will have widespread implications in translational tissue based research.

The Role of Self-Help Efforts in the Reintegration of ‘Politically Motivated’ Former Prisoners: Implications from the Northern Irish experience

Self-help (or mutual aid) processes play a substantial role in the reintegration of stigmatized individuals, in particular, a substantial self-help movement has developed around addiction recovery. Prisoners and ex-prisoners have also established self-help groups around the world. This paper focuses in particular on the role of self-help principles and practices among “politically motivated” former prisoners from all sides of the Northern Irish conflict. The concept of self-help and its application to former prisoners are analysed theoretically, then applied to the Northern Irish case study through a series of interviews with ex-prisoners whose incarceration has been related to the conflict in Northern Ireland. We draw on the implications of this case study for wider issues of reintegration for politically motivated and ordinary prisoners.

PARP inhibition induces BAX/BAK-independent synthetic lethality of BRCA1-deficient non-small cell lung cancer

Evasion of apoptosis contributes to both tumourigenesis and drug resistance in non-small cell lung carcinoma (NSCLC). The pro-apoptotic BCL-2 family proteins BAX and BAK are critical regulators of mitochondrial apoptosis. New strategies for targeting NSCLC in a mitochondria-independent manner should bypass this common mechanism of apoptosis block. BRCA1 mutation frequency in lung cancer is low; however, decreased BRCA1 mRNA and protein expression levels have been reported in a significant proportion of lung adenocarcinomas. BRCA1 mutation/deficiency confers a defect in homologous recombination DNA repair that has been exploited by synthetic lethality through inhibition of PARP (PARPi) in breast and ovarian cells; however, it is not known whether this same synthetic lethal mechanism exists in NSCLC cells. Additionally, it is unknown whether the mitochondrial apoptotic pathway is required for BRCA1/PARPi-mediated synthetic lethality. Here we demonstrate that silencing of BRCA1 expression by RNA interference sensitizes NSCLC cells to PARP inhibition. Importantly, this sensitivity was not attenuated in cells harbouring mitochondrial apoptosis block induced by co-depletion of BAX and BAK. Furthermore, we demonstrate that BRCA1 inhibition cannot override platinum resistance, which is often mediated by loss of mitochondrial apoptosis signalling, but can still sensitize to PARP inhibition. Finally we demonstrate the existence of a BRCA1-deficient subgroup (11-19%) of NSCLC patients by analysing BRCA1 protein levels using immunohistochemistry in two independent primary NSCLC cohorts. Taken together, the existence of BRCA1-immunodeficient NSCLC suggests that this molecular subgroup could be effectively targeted by PARP inhibitors in the clinic and that PARP inhibitors could be used for the treatment of BRCA1-immunodeficient, platinum-resistant tumours. Copyright (C) 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The role of pea3 group transcription factors in esophageal squamous cell carcinoma.

The transcription factors Pea3, Erm, and Er81 can promote cancer initiation and progression in various types of solid tumors. However, their role in esophageal squamous cell carcinoma (ESCC) has not been elucidated. In this study, we found that the expression levels of Pea3 and Erm, but not that of Er81, were significantly higher in ESCC compared with nontumor esophageal epithelium. A high level of Pea3 expression was significantly correlated with a shorter overall survival in a cohort of 81 patients with ESCC and the subgroup with N1 stage tumor (Wilcoxon-Gehan test, P = 0.016 and P = 0.001, respectively). Pea3 was overexpressed in seven ESCC cell lines compared with two immortalized esophageal cell lines. Pea3 knockdown reduced cell proliferation and suppressed nonadherent growth, migration, and invasion in ESCC cells in vitro. In addition, Pea3 knockdown in ESCC cells resulted in a down-regulation of phospho-Akt and matrix metalloproteinase 13, whereas a significant positive correlation in the expression levels was observed between Pea3 and phospho-Akt (r = 0.281, P

Re-imagining DDR Ex-combatants, leadership and moral agency in conflict transformation

Drawing upon criminological studies in the field of prisoner rehabilitation, this essay explores the relevance of the Demobilisation, Disarmament and Reintegration (DDR) framework to the process of conflict transformation in Northern Ireland. In a similar fashion to the critique of 'passivity' offered by, for example, the 'strengths based' or 'good lives' approach to prisoner resettlement and reintegration more generally, the authors contend that the Northern Ireland peace process offers conspicuous examples of former prisoners and combatants as agents and indeed leaders in the process of conflict transformation. They draw out three broad styles of leadership which have emerged amongst ex-combatants over the course of the Northern Ireland transition from conflict-political, military and communal. They suggest that cumulatively such leadership speaks to the potential of ex-prisoners and ex-combatants as moral agents in conflict transformation around which peacemaking can be constructed rather than as obstacles which must be 'managed' out of existence.

Identification of MRP-8 (calgranulin A) as a major responsive protein in chronic periodontitis

The purpose of the study was to analyse how the protein composition of the inflammatory exudate associated with chronic periodontitis differed from the exudate in periodontal health. Gingival crevicular fluid (GCF) was collected from sites with chronic periodontal inflammation and from non-diseased sites in healthy control subjects. Microbore HPLC analysis revealed one major difference in GCF protein profiles between healthy controls and periodontitis patients. The protein enhanced in periodontitis patients was identified as migration inhibitory factor-related protein-8 (MRP-8) by a combination of N-terminal amino acid sequencing, mass spectrometry, and SDS-PAGE. Together, these data demonstrate, for the first time, the presence of monomeric MRP-8 in an inflammatory exudate. Whether monomeric MRP-8 is a unique feature of chronic periodontal inflammation is not yet clear, but the chemotactic properties of this peptide support a functional role for MRP-8 in periodontal inflammation. Copyright (C) 2000 John Wiley & Sons, Ltd.

Persistent Inflammation Subverts Thrombospondin-1–Induced Regulation of Retinal Angiogenesis and Is Driven by CCR2 Ligation

Neovascular retinal disease is a leading cause of blindness orchestrated by inflammatory responses. Although noninfectious uveoretinitis is mediated by CD4(+) T cells, in the persistent phase of disease, angiogenic responses are observed, along with degeneration of the retina. Full clinical manifestation relies on myeloid-derived cells, which are phenotypically distinct from, but potentially sharing common effector responses to age-related macular degeneration. To interrogate inflammation-mediated angiogenesis, we investigated experimental autoimmune uveoretinitis, an animal model for human uveitis. After the initial acute phase of severe inflammation, the retina sustains a persistent low-grade inflammation with tissue-infiltrating leukocytes for over 4 months. During this persistent phase, angiogenesis is observed as retinal neovascular membranes that arise from inflamed venules and postcapillary venules, increase in size as the disease progresses, and are associated with infiltrating arginase-1(+) macrophages. In the absence of thrombospondin-1, retinal neovascular membranes are markedly increased and are associated with arginase-1(-) CD68(+) macrophages, whereas deletion of the chemokine receptor CCR2 resulted in reduced retinal neovascular membranes in association with a predominant neutrophil infiltrate. CCR2 is important for macrophage recruitment to the retina in experimental autoimmune uveoretinitis and promotes chronicity in the form of a persistent angiogenesis response, which in turn is regulated by constitutive expression of angiogenic inhibitors like thrombospondin-1. This model offers a new platform to dissect the molecular and cellular pathology of inflammation-induced ocular angiogenesis.

Implications for Powering Biomarker Discovery Studies

This study examined variations in gene expression between FFPE blocks within tumors of individual patients. Microarray data were used to measure tumor heterogeneity within and between patients and disease states. Data were used to determine the number of samples needed to power biomarker discovery studies. Bias and variation in gene expression were assessed at the intrapatient and interpatient levels and between adenocarcinoma and squamous samples. A mixed-model analysis of variance was fitted to gene expression data and model signatures to assess the statistical significance of observed variations within and between samples and disease states. Sample size analysis, adjusted for sample heterogeneity, was used to determine the number of samples required to support biomarker discovery studies. Variation in gene expression was observed between blocks taken from a single patient. However, this variation was considerably less than differences between histological characteristics. This degree of block-to-block variation still permits biomarker discovery using either macrodissected tumors or whole FFPE sections, provided that intratumor heterogeneity is taken into account. Failure to consider intratumor heterogeneity may result in underpowered biomarker studies that may result in either the generation of longer gene signatures or the inability to identify a viable biomarker. Moreover, the results of this study indicate that a single biopsy sample is suitable for applying a biomarker in nonsmall-cell lung cancer. © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology.

Maximising trace soil evidence: An improved recovery method developed during investigation of a $26 million bank robbery

Obtaining as much particulate material as possible from questioned items is desirable in forensic science as this allows a range of analyses to be undertaken and the retention of material for others to check. A method of maximising particulate recovery is described using a kidnap case, where minimal staining on clothing (socks) remained as possible indications of where the victim had been held captive. Police intelligence led to a hostage scene that was sampled. Brushing of the socks recovered about 50 sand grains with some silt: ultrasonic agitation and centrifuging recovered over 300 grains of sand, silt and clay. These were visually compared to scene and control samples, allowing exclusion of 52 samples and the retention of one comparison sample as well as other possibles, saving time and money, but maximising sample quantity and quality. © 2011 Elsevier Ireland Ltd.