A TMA de-arraying method for high throughput biomarker discovery in tissue research

Background

Tissue MicroArrays (TMAs) represent a potential high-throughput platform for the analysis and discovery of tissue biomarkers. As TMA slides are produced manually and subject to processing and sectioning artefacts, the layout of TMA cores on the final slide and subsequent digital scan (TMA digital slide) is often disturbed making it difficult to associate cores with their original position in the planned TMA map. Additionally, the individual cores can be greatly altered and contain numerous irregularities such as missing cores, grid rotation and stretching. These factors demand the development of a robust method for de-arraying TMAs which identifies each TMA core, and assigns them to their appropriate coordinates on the constructed TMA slide.

Methodology

This study presents a robust TMA de-arraying method consisting of three functional phases: TMA core segmentation, gridding and mapping. The segmentation of TMA cores uses a set of morphological operations to identify each TMA core. Gridding then utilises a Delaunay Triangulation based method to find the row and column indices of each TMA core. Finally, mapping correlates each TMA core from a high resolution TMA whole slide image with its name within a TMAMap.

Conclusion

This study describes a genuine robust TMA de-arraying algorithm for the rapid identification of TMA cores from digital slides. The result of this de-arraying algorithm allows the easy partition of each TMA core for further processing. Based on a test group of 19 TMA slides (3129 cores), 99.84% of cores were segmented successfully, 99.81% of cores were gridded correctly and 99.96% of cores were mapped with their correct names via TMAMaps. The gridding of TMA cores were also extensively tested using a set of 113 pseudo slide (13,536 cores) with a variety of irregular grid layouts including missing cores, rotation and stretching. 100% of the cores were gridded correctly.     

Comparison of Attenuation of Striated Muscle between Postmortem and Antemortem Computed Tomography: Results of a Longitudinal Study

Objective

We evaluated the postmortem changes of striated muscle by comparing computed tomography (CT) images obtained postmortem and antemortem in the same patients.

Materials and Methods

We studied 33 consecutive patients who underwent antemortem CT, postmortem CT, and pathological autopsy in our tertiary care hospital between April 2009 and December 2010. Postmortem CT was performed within 20 h after death and was followed by pathological autopsy. Pathological autopsy confirmed the absence of muscular diseases such as amyotrophic lateral sclerosis, muscular dystrophy, myositis, and myasthenia, in all of the patients. The CT attenuation values of four cardiac muscle sites (anterior wall of the left ventricle, left ventricular free wall, posterior wall of the left ventricle, and the ventricular septum) and two skeletal muscle sites (the pectoralis major muscle and the erector spinae muscle) were compared between antemortem and postmortem CT using paired t test.

Results

Striated muscle had significantly greater attenuation on postmortem CT than on antemortem CT (P<0.001) in all six tissue sites. No significant association was found between postmortem change in the CT attenuation of striated muscle and gender, age, or elapsed time since death.

Conclusion

This is the first longitudinal study to show hyperattenuation of striated muscle on postmortem CT images compared with antemortem CT images in the same patients.     

The Relevance of External Quality Assessment for Molecular Testing for ALK Positive Non-Small Cell Lung Cancer: Results from Two Pilot Rounds Show Room for Optimization

Background and Purpose

Molecular profiling should be performed on all advanced non-small cell lung cancer with non-squamous histology to allow treatment selection. Currently, this should include EGFR mutation testing and testing for ALK rearrangements. ROS1 is another emerging target. ALK rearrangement status is a critical biomarker to predict response to tyrosine kinase inhibitors such as crizotinib. To promote high quality testing in non-small cell lung cancer, the European Society of Pathology has introduced an external quality assessment scheme. This article summarizes the results of the first two pilot rounds organized in 2012–2013.

Materials and Methods

Tissue microarray slides consisting of cell-lines and resection specimens were distributed with the request for routine ALK testing using IHC or FISH. Participation in ALK FISH testing included the interpretation of four digital FISH images.

Results

Data from 173 different laboratories was obtained. Results demonstrate decreased error rates in the second round for both ALK FISH and ALK IHC, although the error rates were still high and the need for external quality assessment in laboratories performing ALK testing is evident. Error rates obtained by FISH were lower than by IHC. The lowest error rates were observed for the interpretation of digital FISH images.

Conclusion

There was a large variety in FISH enumeration practices. Based on the results from this study, recommendations for the methodology, analysis, interpretation and result reporting were issued. External quality assessment is a crucial element to improve the quality of molecular testing.     

Enumeration of functional T-cell subsets by fluorescence-immunospot defines signatures of pathogen burden in tuberculosis

Background

IFN-γ and IL-2 cytokine-profiles define three functional T-cell subsets which may correlate with pathogen load in chronic intracellular infections. We therefore investigated the feasibility of the immunospot platform to rapidly enumerate T-cell subsets by single-cell IFN-γ/IL-2 cytokine-profiling and establish whether immunospot-based T-cell signatures distinguish different clinical stages of human tuberculosis infection.

Methods

We used fluorophore-labelled anti-IFN-γ and anti-IL-2 antibodies with digital overlay of spatially-mapped colour-filtered images to enumerate dual and single cytokine-secreting M. tuberculosis antigen-specific T-cells in tuberculosis patients and in latent tuberculosis infection (LTBI). We validated results against established measures of cytokine-secreting T-cells.

Results

Fluorescence-immunospot correlated closely with single-cytokine enzyme-linked-immunospot for IFN-γ-secreting T-cells and IL-2-secreting T-cells and flow-cytometry-based detection of dual IFN-γ/IL-2-secreting T-cells. The untreated tuberculosis signature was dominated by IFN-γ-only-secreting T-cells which shifted consistently in longitudinally-followed patients during treatment to a signature dominated by dual IFN-γ/IL-2-secreting T-cells in treated patients. The LTBI signature differed from active tuberculosis, with higher proportions of IL-2-only and IFN-γ/IL-2-secreting T-cells and lower proportions of IFN-γ-only-secreting T-cells.

Conclusions

Fluorescence-immunospot is a quantitative, accurate measure of functional T-cell subsets; identification of cytokine-signatures of pathogen burden, distinct clinical stages of M. tuberculosis infection and long-term immune containment suggests application for treatment monitoring and vaccine evaluation.     

Detection of setup uncertainties with 3D surface registration system for conformal radiotherapy of breast cancer

Aim

To investigate the clinical application of a technique for patient set-up verification in breast cancer radiotherapy based on a 3D surface image registration system.

Background

Accurate and reproducible patient set-up is a prerequisite to correctly deliver fractionated radiotherapy. Various approaches are available to verify and correct patient setup for 3D image acquisition in a radiation treatment room.

Materials and methods

The study analyzed the setup reproducibility of 15 patients affected by breast cancer and candidates for conformal radiotherapy by using the AlignRT system (VisionRT, London, UK). At the initial setup, electronic portal imaging device (EPID) images were compared with Digitally Reconstructed Radiographs (DRRs) and a reference three-dimensional (3D) surface image was obtained by AlignRT. Surface images were acquired prior to every subsequent setup procedure. The systematic and random errors along longitudinal and vertical directions were measured and compared for the two systems.

Results

The procedure for surface registration, image acquisition and comparison with the reference image took less than 1 min on average. The T test for systematic error showed no significant difference between the 2 verification systems along the longitudinal (p = 0.69) and vertical (p = 0.67) axes. The T-test for random error showed a significant difference between the 2 systems along the vertical axis (p = 0.05).

Conclusion

AlignRT is fast, simple, non-invasive and seems to be reliable in detecting patient setup errors. Our results suggest that it could be used to assess the setup reproducibility for breast cancer patients.     

An Automated System for Rapid Non-Destructive Enumeration of Growing Microbes

Background

The power and simplicity of visual colony counting have made it the mainstay of microbiological analysis for more than 130 years. A disadvantage of the method is the long time required to generate visible colonies from cells in a sample. New rapid testing technologies generally have failed to maintain one or more of the major advantages of culture-based methods.

Principal Findings

We present a new technology and platform that uses digital imaging of cellular autofluorescence to detect and enumerate growing microcolonies many generations before they become visible to the eye. The data presented demonstrate that the method preserves the viability of the microcolonies it detects, thus enabling generation of pure cultures for microbial identification. While visual colony counting detects Escherichia coli colonies containing about 5×10⁶ cells, the new imaging method detects E. coli microcolonies when they contain about 120 cells and microcolonies of the yeast Candida albicans when they contain only about 12 cells. We demonstrate that digital imaging of microcolony autofluorescence detects a broad spectrum of prokaryotic and eukaryotic microbes and present a model for predicting the time to detection for individual strains. Results from the analysis of environmental samples from pharmaceutical manufacturing plants containing a mixture of unidentified microbes demonstrate the method''s improved test turnaround times.

Conclusion

This work demonstrates a new technology and automated platform that substantially shortens test times while maintaining key advantages of the current methods.     

Area and Volumetric Density Estimation in Processed Full-Field Digital Mammograms for Risk Assessment of Breast Cancer

Introduction

Mammographic density, the white radiolucent part of a mammogram, is a marker of breast cancer risk and mammographic sensitivity. There are several means of measuring mammographic density, among which are area-based and volumetric-based approaches. Current volumetric methods use only unprocessed, raw mammograms, which is a problematic restriction since such raw mammograms are normally not stored. We describe fully automated methods for measuring both area and volumetric mammographic density from processed images.

Methods

The data set used in this study comprises raw and processed images of the same view from 1462 women. We developed two algorithms for processed images, an automated area-based approach (CASAM-Area) and a volumetric-based approach (CASAM-Vol). The latter method was based on training a random forest prediction model with image statistical features as predictors, against a volumetric measure, Volpara, for corresponding raw images. We contrast the three methods, CASAM-Area, CASAM-Vol and Volpara directly and in terms of association with breast cancer risk and a known genetic variant for mammographic density and breast cancer, rs10995190 in the gene ZNF365. Associations with breast cancer risk were evaluated using images from 47 breast cancer cases and 1011 control subjects. The genetic association analysis was based on 1011 control subjects.

Results

All three measures of mammographic density were associated with breast cancer risk and rs10995190 (p<0.025 for breast cancer risk and p<1×10⁻⁶ for rs10995190). After adjusting for one of the measures there remained little or no evidence of residual association with the remaining density measures (p>0.10 for risk, p>0.03 for rs10995190).

Conclusions

Our results show that it is possible to obtain reliable automated measures of volumetric and area mammographic density from processed digital images. Area and volumetric measures of density on processed digital images performed similar in terms of risk and genetic association.     

Counting pollen grains using readily available, free image processing and analysis software

Background and Aims

Although many methods exist for quantifying the number of pollen grains in a sample, there are few standard methods that are user-friendly, inexpensive and reliable. The present contribution describes a new method of counting pollen using readily available, free image processing and analysis software.

Methods

Pollen was collected from anthers of two species, Carduus acanthoides and C. nutans (Asteraceae), then illuminated on slides and digitally photographed through a stereomicroscope. Using ImageJ (NIH), these digital images were processed to remove noise and sharpen individual pollen grains, then analysed to obtain a reliable total count of the number of grains present in the image. A macro was developed to analyse multiple images together. To assess the accuracy and consistency of pollen counting by ImageJ analysis, counts were compared with those made by the human eye.

Key Results and Conclusions

Image analysis produced pollen counts in 60 s or less per image, considerably faster than counting with the human eye (5–68 min). In addition, counts produced with the ImageJ procedure were similar to those obtained by eye. Because count parameters are adjustable, this image analysis protocol may be used for many other plant species. Thus, the method provides a quick, inexpensive and reliable solution to counting pollen from digital images, not only reducing the chance of error but also substantially lowering labour requirements.     

An integrated approach for finding overlooked genes in Shigella

Background

The completion of numerous genome sequences introduced an era of whole-genome study. However, many genes are missed during genome annotation, including small RNAs (sRNAs) and small open reading frames (sORFs). In order to improve genome annotation, we aimed to identify novel sRNAs and sORFs in Shigella, the principal etiologic agents of bacillary dysentery.

Methodology/Principal Findings

We identified 64 sRNAs in Shigella, which were experimentally validated in other bacteria based on sequence conservation. We employed computer-based and tiling array-based methods to search for sRNAs, followed by RT-PCR and northern blots, to identify nine sRNAs in Shigella flexneri strain 301 (Sf301) and 256 regions containing possible sRNA genes. We found 29 candidate sORFs using bioinformatic prediction, array hybridization and RT-PCR verification. We experimentally validated 557 (57.9%) DOOR operon predictions in the chromosomes of Sf301 and 46 (76.7%) in virulence plasmid.We found 40 additional co-expressed gene pairs that were not predicted by DOOR.

Conclusions/Significance

We provide an updated and comprehensive annotation of the Shigella genome. Our study increased the expected numbers of sORFs and sRNAs, which will impact on future functional genomics and proteomics studies. Our method can be used for large scale reannotation of sRNAs and sORFs in any microbe with a known genome sequence.     

RiTE database: a resource database for genus-wide rice genomics and evolutionary biology

Background

Comparative evolutionary analysis of whole genomes requires not only accurate annotation of gene space, but also proper annotation of the repetitive fraction which is often the largest component of most if not all genomes larger than 50 kb in size.

Results

Here we present the Rice TE database (RiTE-db) - a genus-wide collection of transposable elements and repeated sequences across 11 diploid species of the genus Oryza and the closely-related out-group Leersia perrieri. The database consists of more than 170,000 entries divided into three main types: (i) a classified and curated set of publicly-available repeated sequences, (ii) a set of consensus assemblies of highly-repetitive sequences obtained from genome sequencing surveys of 12 species; and (iii) a set of full-length TEs, identified and extracted from 12 whole genome assemblies.

Conclusions

This is the first report of a repeat dataset that spans the majority of repeat variability within an entire genus, and one that includes complete elements as well as unassembled repeats. The database allows sequence browsing, downloading, and similarity searches. Because of the strategy adopted, the RiTE-db opens a new path to unprecedented direct comparative studies that span the entire nuclear repeat content of 15 million years of Oryza diversity.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1762-3) contains supplementary material, which is available to authorized users.     

Comparative genomics of 274 Vibrio cholerae genomes reveals mobile functions structuring three niche dimensions

Background

Vibrio cholerae is a globally dispersed pathogen that has evolved with humans for centuries, but also includes non-pathogenic environmental strains. Here, we identify the genomic variability underlying this remarkable persistence across the three major niche dimensions space, time, and habitat.

Results

Taking an innovative approach of genome-wide association applicable to microbial genomes (GWAS-M), we classify 274 complete V. cholerae genomes by niche, including 39 newly sequenced for this study with the Ion Torrent DNA-sequencing platform. Niche metadata were collected for each strain and analyzed together with comprehensive annotations of genetic and genomic attributes, including point mutations (single-nucleotide polymorphisms, SNPs), protein families, functions and prophages.

Conclusions

Our analysis revealed that genomic variations, in particular mobile functions including phages, prophages, transposable elements, and plasmids underlie the metadata structuring in each of the three niche dimensions. This underscores the role of phages and mobile elements as the most rapidly evolving elements in bacterial genomes, creating local endemicity (space), leading to temporal divergence (time), and allowing the invasion of new habitats. Together, we take a data-driven approach for comparative functional genomics that exploits high-volume genome sequencing and annotation, in conjunction with novel statistical and machine learning analyses to identify connections between genotype and phenotype on a genome-wide scale.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-654) contains supplementary material, which is available to authorized users.     

BEACON: automated tool for Bacterial GEnome Annotation ComparisON

Background

Genome annotation is one way of summarizing the existing knowledge about genomic characteristics of an organism. There has been an increased interest during the last several decades in computer-based structural and functional genome annotation. Many methods for this purpose have been developed for eukaryotes and prokaryotes. Our study focuses on comparison of functional annotations of prokaryotic genomes. To the best of our knowledge there is no fully automated system for detailed comparison of functional genome annotations generated by different annotation methods (AMs).

Results

The presence of many AMs and development of new ones introduce needs to: a/ compare different annotations for a single genome, and b/ generate annotation by combining individual ones. To address these issues we developed an Automated Tool for Bacterial GEnome Annotation ComparisON (BEACON) that benefits both AM developers and annotation analysers. BEACON provides detailed comparison of gene function annotations of prokaryotic genomes obtained by different AMs and generates extended annotations through combination of individual ones. For the illustration of BEACON’s utility, we provide a comparison analysis of multiple different annotations generated for four genomes and show on these examples that the extended annotation can increase the number of genes annotated by putative functions up to 27 %, while the number of genes without any function assignment is reduced.

Conclusions

We developed BEACON, a fast tool for an automated and a systematic comparison of different annotations of single genomes. The extended annotation assigns putative functions to many genes with unknown functions. BEACON is available under GNU General Public License version 3.0 and is accessible at: http://www.cbrc.kaust.edu.sa/BEACON/.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1826-4) contains supplementary material, which is available to authorized users.     

Comparison of the Accuracy of Voxel Based Registration and Surface Based Registration for 3D Assessment of Surgical Change following Orthognathic Surgery

Purpose

Superimposition of two dimensional preoperative and postoperative facial images, including radiographs and photographs, are used to evaluate the surgical changes after orthognathic surgery. Recently, three dimensional (3D) imaging has been introduced allowing more accurate analysis of surgical changes. Surface based registration and voxel based registration are commonly used methods for 3D superimposition. The aim of this study was to evaluate and compare the accuracy of the two methods.

Materials and methods

Pre-operative and 6 months post-operative cone beam CT scan (CBCT) images of 31 patients were randomly selected from the orthognathic patient database at the Dental Hospital and School, University of Glasgow, UK. Voxel based registration was performed on the DICOM images (Digital Imaging Communication in Medicine) using Maxilim software (Medicim-Medical Image Computing, Belgium). Surface based registration was performed on the soft and hard tissue 3D models using VRMesh (VirtualGrid, Bellevue City, WA). The accuracy of the superimposition was evaluated by measuring the mean value of the absolute distance between the two 3D image surfaces. The results were statistically analysed using a paired Student t-test, ANOVA with post-hoc Duncan test, a one sample t-test and Pearson correlation coefficient test.

Results

The results showed no significant statistical difference between the two superimposition methods (p<0.05). However surface based registration showed a high variability in the mean distances between the corresponding surfaces compared to voxel based registration, especially for soft tissue. Within each method there was a significant difference between superimposition of the soft and hard tissue models.

Conclusions

There were no significant statistical differences between the two registration methods and it was unlikely to have any clinical significance. Voxel based registration was associated with less variability. Registering on the soft tissue in isolation from the hard tissue may not be a true reflection of the surgical change.     

The grapevine gene nomenclature system

Background

Grapevine (Vitis vinifera L.) is one of the most important fruit crops in the world and serves as a valuable model for fruit development in woody species. A major breakthrough in grapevine genomics was achieved in 2007 with the sequencing of the Vitis vinifera cv. PN40024 genome. Subsequently, data on structural and functional characterization of grape genes accumulated exponentially. To better exploit the results obtained by the international community, we think that a coordinated nomenclature for gene naming in species with sequenced genomes is essential. It will pave the way for the accumulation of functional data that will enable effective scientific discussion and discovery. The exploitation of data that were generated independently of the genome release is hampered by their heterogeneous nature and by often incompatible and decentralized storage. Classically, large amounts of data describing gene functions are only available in printed articles and therefore remain hardly accessible for automatic text mining. On the other hand, high throughput “Omics” data are typically stored in public repositories, but should be arranged in compendia to better contribute to the annotation and functional characterization of the genes.

Results

With the objective of providing a high quality and highly accessible annotation of grapevine genes, the International Grapevine Genome Project (IGGP) commissioned an international Super-Nomenclature Committee for Grape Gene Annotation (sNCGGa) to coordinate the effort of experts to annotate the grapevine genes. The goal of the committee is to provide a standard nomenclature for locus identifiers and to define conventions for a gene naming system in this paper.

Conclusions

Learning from similar initiatives in other plant species such as Arabidopsis, rice and tomato, a versatile nomenclature system has been developed in anticipation of future genomic developments and annotation issues. The sNCGGa’s first outreach to the grape community has been focused on implementing recommended guidelines for the expert annotators by: (i) providing a common annotation platform that enables community-based gene curation, (ii) developing a gene nomenclature scheme reflecting the biological features of gene products that is consistent with that used in other organisms in order to facilitate comparative analyses.