Development of an Immunohistochemical Assay to Detect the Ataxia-Telangiectasia Mutated (ATM) Protein in Gastric Carcinoma.

Ataxia-telangiectasia mutated (ATM), a key activator of DNA damage response mechanisms, represents a potential biomarker for targeted gastric carcinoma therapies. A phase II study (Study 39; NCT01063517) designed to investigate the combination olaparib plus paclitaxel in patients with recurrent or metastatic gastric cancer did not meet its primary endpoint of progression-free survival; however, an improvement in the secondary endpoint of overall survival was recorded with a greater overall survival benefit noted in patients with ATM-negative tumors. An ATM immunohistochemical (IHC) diagnostic assay was developed to identify patients who may respond favorably to targeted therapies and deployed in the confirmatory phase III GOLD trial (NCT01924533). The VENTANA ATM (Y170) assay was developed for investigational use in formalin-fixed, paraffin-embedded gastric carcinoma samples using an anti-ATM rabbit monoclonal antibody (clone Y170) and was optimized with OptiView DAB IHC Detection Kit on a BenchMark ULTRA instrument. The assay was deployed in studies assessing sensitivity, specificity, robustness, precision, and determining optimal ATM staining cutoff to define ATM-deficiency (ATM-low). The ATM (Y170) assay met all predefined product development acceptance criteria. Multiple parameters were characterized, including repeatability, reproducibility, analytical sensitivity, specificity, robustness, and product stability. The scoring algorithm was defined; gastric carcinoma samples were considered ATM-negative or ATM-positive when <25% or ≥25%, respectively, of tumor cell nuclei expressed ATM at any IHC stain intensity and nuclei of immune and/or endothelial cells expressed ATM at a moderate stain intensity (internal positive control). Results highlight reproducibility of the assay, supporting suitability for investigational use for evaluation of gastric carcinoma samples using tumor cell staining cutoff of <25% to define ATM-deficiency. Using this ATM assay, phase III GOLD trial (NCT01924533) clinical trial did not meet its primary endpoint, only suggesting, but not demonstrating, that assessment of ATM levels by IHC could possibly be useful in assessing the degree of benefit that may be achieved by adding olaparib to paxitaxel when treating gastric carcinoma. The utility of ATM (Y170) assay as a companion diagnostic requires further clinical validation.

Relevance of deep learning to facilitate the diagnosis of HER2 status in breast cancer.

Tissue biomarker scoring by pathologists is central to defining the appropriate therapy for patients with cancer. Yet, inter-pathologist variability in the interpretation of ambiguous cases can affect diagnostic accuracy. Modern artificial intelligence methods such as deep learning have the potential to supplement pathologist expertise to ensure constant diagnostic accuracy. We developed a computational approach based on deep learning that automatically scores HER2, a biomarker that defines patient eligibility for anti-HER2 targeted therapies in breast cancer. In a cohort of 71 breast tumour resection samples, automated scoring showed a concordance of 83% with a pathologist. The twelve discordant cases were then independently reviewed, leading to a modification of diagnosis from initial pathologist assessment for eight cases. Diagnostic discordance was found to be largely caused by perceptual differences in assessing HER2 expression due to high HER2 staining heterogeneity. This study provides evidence that deep learning aided diagnosis can facilitate clinical decision making in breast cancer by identifying cases at high risk of misdiagnosis.

N-3 long-chain polyunsaturated fatty acids inhibit smooth muscle cell migration by modulating urokinase plasminogen activator receptor through MEK/ERK-dependent and -independent mechanisms.

Smooth muscle cell (SMC) migration is a major and complex feature of atherosclerosis and restenosis. N-3 long-chain polyunsaturated fatty acids (LCPUFAs) affect SMC migration; however, the mechanisms involved are unclear. This study investigated the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the MEK/ERK pathway and urokinase plasminogen activator receptor (uPAR) in relation to SMC migration. Transwell migration assays revealed that both EPA and DHA decreased cell migration. Western blotting and real-time reverse transcription polymerase chain reaction showed that n-3 LCPUFAs decreased uPAR expression, but not urokinase plasminogen activator (uPA) expression, without changing plasmin and uPA activity. DHA also inhibited the activation of the MEK/ERK signaling pathway, whereas EPA switched the SMC phenotype from synthetic to contractile. siRNA technology targeting uPAR expression showed that decreased uPAR led to a significant decrease in migration, demonstrating the role of uPAR on SMC migration. We also showed that MEK/ERK pathway activation was involved in the regulation of uPAR gene expression in SMCs. Our results suggest that n-3 LCPUFAs decrease SMC migration through the inhibition of uPAR expression, with DHA affecting its expression via the modulation of MEK/ERK signaling pathway, while EPA induces a change in SMC phenotype. This could represent another means by which to explain how n-3 LCPUFAs exert their preventive properties against atherosclerosis.

Determinants of meat quality: tenderness.

Meat quality is a term used to describe a range of attributes of meat. Consumer research suggests that tenderness is a very important element of eating quality and that variations in tenderness affect the decision to repurchase. The present paper highlights recent information on the factors that affect tenderness. While the precise aetiology is not fully understood, a number of factors have been shown to affect tenderness. Of these factors, postmortem factors, particularly temperature, sarcomere length and proteolysis, which affect the conversion of muscle to meat, appear most important. However, it is now becoming clear that variation in other factors such as the muscle fibre type composition and the buffering capacity of the muscle together with the breed and nutritional status of the animals may also contribute to the observed variation in meat tenderness.

Increased muscle proteolysis after local trauma mainly reflects macrophage-associated lysosomal proteolysis.

Rat gastrocnemius showed increased protein degradation (+75-115%) at 48 h after traumatic injury. Injured muscle showed increased cathepsin B activity (+327%) and mRNA encoding cathepsin B (+670%), cathepsin L (+298%), cathepsin H (+159%), and cathepsin C (+268%). In in situ hybridization, cathepsin B mRNA localized to the mononuclear cell infiltrate in injured muscle, and only background levels of hybridization were observed either over muscle cells in injured tissue or in uninjured muscle. Immunogold/electron microscopy showed specific staining for cathepsin B only in lysosome-like structures in cells of the mononuclear cell infiltrate in injured muscle. Muscle cells were uniformly negative in the immunocytochemistry. Matrix metalloproteinase-9 (granulocyte-macrophage gelatinase) mRNA and activity were not present in uninjured muscle but were expressed after trauma. There was no activation of the ATP-ubiquitin-proteasome-dependent proteolytic pathway in injured muscle, by contrast to diverse forms of muscle wasting where the activity of this system and the expression of genes encoding ubiquitin and proteasome elements rise. These results suggest that proteolytic systems of the muscle cells remain unstimulated after local injury and that lysosomal enzymes of the inflammatory infiltrated cells are likely to be the major participant in protein catabolism associated with local trauma.