Computer-assisted assessment of the Human Epidermal Growth Factor Receptor 2 immunohistochemical assay in imaged histologic sections using a membrane isolation algorithm and quantitative analysis of positive controls

<p>Abstract</p> <p>Background</p> <p>Breast cancers that overexpress the human epidermal growth factor receptor 2 (HER2) are eligible for effective biologically targeted therapies, such as trastuzumab. However, accurately determining HER2 overexpression, especially in immunohistochemically equivocal cases, remains a challenge. Manual analysis of HER2 expression is dependent on the assessment of membrane staining as well as comparisons with positive controls. In spite of the strides that have been made to standardize the assessment process, intra- and inter-observer discrepancies in scoring is not uncommon. In this manuscript we describe a pathologist assisted, computer-based continuous scoring approach for increasing the precision and reproducibility of assessing imaged breast tissue specimens.</p> <p>Methods</p> <p>Computer-assisted analysis on HER2 IHC is compared with manual scoring and fluorescence in situ hybridization results on a test set of 99 digitally imaged breast cancer cases enriched with equivocally scored (2+) cases. Image features are generated based on the staining profile of the positive control tissue and pixels delineated by a newly developed Membrane Isolation Algorithm. Evaluation of results was performed using Receiver Operator Characteristic (ROC) analysis.</p> <p>Results</p> <p>A computer-aided diagnostic approach has been developed using a membrane isolation algorithm and quantitative use of positive immunostaining controls. By incorporating internal positive controls into feature analysis a greater Area Under the Curve (AUC) in ROC analysis was achieved than feature analysis without positive controls. Evaluation of HER2 immunostaining that utilized membrane pixels, controls, and percent area stained showed significantly greater AUC than manual scoring, and significantly less false positive rate when used to evaluate immunohistochemically equivocal cases.</p> <p>Conclusion</p> <p>It has been shown that by incorporating both a membrane isolation algorithm and analysis of known positive controls a computer-assisted diagnostic algorithm was developed that can reproducibly score HER2 status in IHC stained clinical breast cancer specimens. For equivocal scoring cases, this approach performed better than standard manual evaluation as assessed by ROC analysis in our test samples. Finally, there exists potential for utilizing image-analysis techniques for improving HER2 scoring at the immunohistochemically equivocal range.</p

Rare, high-affinity anti-pathogen antibodies from human repertoires, discovered using microfluidics and molecular genomics

Affinity-matured, functional anti-pathogen antibodies are present at low frequencies in natural human repertoires. These antibodies are often excellent candidates for therapeutic monoclonal antibodies. However, mining natural human antibody repertoires is a challenge. In this study, we demonstrate a new method that uses microfluidics, yeast display, and deep sequencing to identify 247 natively paired anti-pathogen single-chain variable fragments (scFvs), which were initially as rare as 1 in 100,000 in the human repertoires. Influenza A vaccination increased the frequency of influenza A antigen-binding scFv within the peripheral B cell repertoire from <0.1% in non-vaccinated donors to 0.3-0.4% in vaccinated donors, whereas pneumococcus vaccination did not increase the frequency of antigen-binding scFv. However, the pneumococcus scFv binders from the vaccinated library had higher heavy and light chain Replacement/Silent mutation (R/S) ratios, a measure of affinity maturation, than the pneumococcus binders from the corresponding non-vaccinated library. Thus, pneumococcus vaccination may increase the frequency of affinity-matured antibodies in human repertoires. We synthesized 10 anti-influenza A and nine anti-pneumococcus full-length antibodies that were highly abundant among antigen-binding scFv. All 10 anti-influenza A antibodies bound the appropriate antigen at KD<10 nM and neutralized virus in cellular assays. All nine anti-pneumococcus full-length antibodies bound at least one polysaccharide serotype, and 71% of the anti-pneumococcus antibodies that we tested were functional in cell killing assays. Our approach has future application in a variety of fields, including the development of therapeutic antibodies for emerging viral diseases, autoimmune disorders, and cancer

Calcified multilocular thymic cyst associated with thymoma: a case report

<p>Abstract</p> <p>Introduction</p> <p>There are few case reports of thymoma with a thymic cyst. Such an association renders it difficult for any pathologist to differentiate from other neoplasms, such as a cystic thymoma.</p> <p>Case presentation</p> <p>A 50-year-old Berber woman from Morocco was admitted with a chronic cough of more than 10 years duration. Her medical history and physical examination were normal. Anterior chest radiography demonstrated a calcified opacity in her right anterior mediastinum. A chest-computed tomogram revealed a round cystic tumor, with significant calcification in her right anterior mediastinum. A surgical exploration was performed. The tumor seemed to be a well-encapsulated and totally calcified lesion, arising from the right lobe of her thymus. It was removed by partial resection of her thymus. Through histology, the calcified tumor exhibited some areas of multilocular fibrous-wall cysts. These cysts were partially lined by small cuboidal cells with severe chronic inflammation and an AB thymoma that arose from the wall of the cyst.</p> <p>Conclusion</p> <p>Greater attention should be given to multilocular thymic cysts, to exclude the possibility of neoplasm, especially when the cyst wall is thickened.</p

Revisiting the technical validation of tumour biomarker assays: how to open a Pandora's box

A tumour biomarker is a characteristic that is objectively measured and evaluated in tumour samples as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. The development of a biomarker contemplates distinct phases, including discovery by hypothesis-generating preclinical or exploratory studies, development and qualification of the assay for the identification of the biomarker in clinical samples, and validation of its clinical significance. Although guidelines for the development and validation of biomarkers are available, their implementation is challenging, owing to the diversity of biomarkers being developed. The term 'validation' undoubtedly has several meanings; however, in the context of biomarker research, a test may be considered valid if it is 'fit for purpose'. In the process of validation of a biomarker assay, a key point is the validation of the methodology. Here we discuss the challenges for the technical validation of immunohistochemical and gene expression assays to detect tumour biomarkers and provide suggestions of pragmatic solutions to address these challenges

3-D Ultrastructure of O. tauri: Electron Cryotomography of an Entire Eukaryotic Cell

The hallmark of eukaryotic cells is their segregation of key biological functions into discrete, membrane-bound organelles. Creating accurate models of their ultrastructural complexity has been difficult in part because of the limited resolution of light microscopy and the artifact-prone nature of conventional electron microscopy. Here we explored the potential of the emerging technology electron cryotomography to produce three-dimensional images of an entire eukaryotic cell in a near-native state. Ostreococcus tauri was chosen as the specimen because as a unicellular picoplankton with just one copy of each organelle, it is the smallest known eukaryote and was therefore likely to yield the highest resolution images. Whole cells were imaged at various stages of the cell cycle, yielding 3-D reconstructions of complete chloroplasts, mitochondria, endoplasmic reticula, Golgi bodies, peroxisomes, microtubules, and putative ribosome distributions in-situ. Surprisingly, the nucleus was seen to open long before mitosis, and while one microtubule (or two in some predivisional cells) was consistently present, no mitotic spindle was ever observed, prompting speculation that a single microtubule might be sufficient to segregate multiple chromosomes

Human α2β1HI CD133+VE epithelial prostate stem cells express low levels of active androgen receptor

Stem cells are thought to be the cell of origin in malignant transformation in many tissues, but their role in human prostate carcinogenesis continues to be debated. One of the conflicts with this model is that cancer stem cells have been described to lack androgen receptor (AR) expression, which is of established importance in prostate cancer initiation and progression. We re-examined the expression patterns of AR within adult prostate epithelial differentiation using an optimised sensitive and specific approach examining transcript, protein and AR regulated gene expression. Highly enriched populations were isolated consisting of stem (α(2)β(1)(HI) CD133(+VE)), transiently amplifying (α(2)β(1)(HI) CD133(-VE)) and terminally differentiated (α(2)β(1)(LOW) CD133(-VE)) cells. AR transcript and protein expression was confirmed in α(2)β(1)(HI) CD133(+VE) and CD133(-VE) progenitor cells. Flow cytometry confirmed that median (±SD) fraction of cells expressing AR were 77% (±6%) in α(2)β(1)(HI) CD133(+VE) stem cells and 68% (±12%) in α(2)β(1)(HI) CD133(-VE) transiently amplifying cells. However, 3-fold lower levels of total AR protein expression (peak and median immunofluorescence) were present in α(2)β(1)(HI) CD133(+VE) stem cells compared with differentiated cells. This finding was confirmed with dual immunostaining of prostate sections for AR and CD133, which again demonstrated low levels of AR within basal CD133(+VE) cells. Activity of the AR was confirmed in prostate progenitor cells by the expression of low levels of the AR regulated genes PSA, KLK2 and TMPRSS2. The confirmation of AR expression in prostate progenitor cells allows integration of the cancer stem cell theory with the established models of prostate cancer initiation based on a functional AR. Further study of specific AR functions in prostate stem and differentiated cells may highlight novel mechanisms of prostate homeostasis and insights into tumourigenesis

Visualization and quantitation of the expression of microRNAs and their target genes in neuroblastoma single cells using imaging cytometry

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) are regulatory molecules that play an important role in many physiological processes, including cell growth, differentiation, and apoptosis. In addition to modulating normal cellular functions, it has also been reported that miRNAs are involved in the development of many pathologies, including cardiovascular diseases, cancer, inflammation, and neurodegeneration. Methods for the sensitive detection and measurement of specific miRNAs and their cellular targets are essential for both basic research endeavours, as well as diagnostic efforts aimed at understanding the role of miRNAs in disease processes.</p> <p>Findings</p> <p>In this study, we describe a novel, imaging cytometry-based protocol that allows for simultaneous visualisation and quantification of miRNAs and their putative targets. We validated this methodology in a neuronal cell line by examining the relationship of the miRNA miR-124 and its known target, cyclin dependent kinase 6 (CDK6). We found that ectopic overexpression of miR-124 resulted in the downregulation of CDK6, decreased cellular proliferation, and induced cellular morphological changes.</p> <p>Conclusions</p> <p>This method is suitable for analysing the expression and cellular localisation of miRNAs and target proteins in small cell subsets within a heterogeneous cell suspension. We believe that our cytometry-based methodology will be easily adaptable to miRNA studies in many areas of biomedical research including neuroscience, stem cell biology, immunology, and oncology.</p

WNT signaling regulates self-renewal and differentiation of prostate cancer cells with stem cell characteristics

Prostate cancer cells with stem cell characteristics were identified in human prostate cancer cell lines by their ability to form from single cells self-renewing prostaspheres in non-adherent cultures. Prostaspheres exhibited heterogeneous expression of proliferation, differentiation and stem cell-associated makers CD44, ABCG2 and CD133. Treatment with WNT inhibitors reduced both prostasphere size and self-renewal. In contrast, addition of Wnt3a caused increased prostasphere size and self-renewal, which was associated with a significant increase in nuclear Β-catenin, keratin 18, CD133 and CD44 expression. As a high proportion of LNCaP and C4-2B cancer cells express androgen receptor we determined the effect of the androgen receptor antagonist bicalutamide. Androgen receptor inhibition reduced prostasphere size and expression of PSA, but did not inhibit prostasphere formation. These effects are consistent with the androgen-independent self-renewal of cells with stem cell characteristics and the androgen-dependent proliferation of transit amplifying cells. As the canonical WNT signaling effector Β-catenin can also associate with the androgen receptor, we propose a model for tumour propagation involving a balance between WNT and androgen receptor activity. That would affect the self-renewal of a cancer cell with stem cell characteristics and drive transit amplifying cell proliferation and differentiation. In conclusion, we provide evidence that WNT activity regulates the self-renewal of prostate cancer cells with stem cell characteristics independently of androgen receptor activity. Inhibition of WNT signaling therefore has the potential to reduce the self-renewal of prostate cancer cells with stem cell characteristics and improve the therapeutic outcome.Peer reviewe

A segmental radiological study of the spine and rib – cage in children with progressive Infantile Idiopathic Scoliosis

BACKGROUND: The role of rib cage in the development of progressive infantile idiopathic scoliosis (IIS) has not been studied previously. No report was found for rib growth in children with IIS. These findings caused us to undertake a segmental radiological study of the spine and rib-cage in children with progressive IIS. The aim of the present study is to present a new method for assessing the thoracic shape in scoliotics and in control subjects and to compare the findings between the two groups. MATERIALS AND METHODS: In the posteroanterior (PA) spinal radiographs of 24 patients with progressive IIS, with a mean age of 4.1 years old, the Thoracic Ratios (TRs) (segmental convex and concave TRs), the Cobb angle, the segmental vertebral rotation and vertebral tilt were measured. In 233 subjects, with a mean age of 5.1 years old, who were used as a control group, the segmental left and right TRs and the total width of the chest (left plus right TRs) were measured in PA chest radiographs. Statistical analysis included Mann-Whitney, Spearman correlation coefficient, multiple linear regression analysis and ANOVA. RESULTS: The comparison shows that the scoliotic thorax is significantly narrower than that of the controls at all spinal levels. The upper chest in IIS is funnel-shaped and the vertebral rotation at T4 early in management correlates significantly with the apical vertebral rotation at follow up. CONCLUSION: The IIS thorax is narrower than that of the controls, the upper chest is funnel-shaped and there is a predictive value of vertebral rotation at the upper limit of the thoracic curve of IIS, which reflects, impaired rib control of spinal rotation possibly due to neuromuscular factors, which contribute also to the funnel-shaped chest