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1.  Cervicovaginal cytology in patients undergoing pelvic radiotherapy using the Focalpoint system: results from the RODEO study 
Diagnostic Pathology  2015;10:1.
Background
Evaluate the performance of the Focalpoint system in identifying and classifying cervical cytology alterations from samples collected from patients treated with Radiotherapy (RT).
Methods
The reproducibility of manual and automated screening by cytotechnologists using the BD Focalpoint GS Imaging System was examined. Samples were collected from May 2010 to August 2011.
Results
A total of 378 treated with RT and 8,967 cytology samples from patients without previous RT, were evaluated. The kappa values for cytological diagnoses read manually and automated in cases without previous RT were as follows: < ASC-H vs. ≥ ASC-H = 0.71; < LSIL vs. ≥ LSIL = 0.66; and < HSIL vs. ≥ HSIL = 0.67. The kappa for cytological diagnoses in post-RT women have showed: < ASC-H vs. ≥ ASC-H = 0.71; < LSIL vs. ≥ LSIL = 0.65; < HSIL vs. ≥ HSIL = 0.57.
Conclusions
There was no significant difference among the kappa values we found. Post-RT cytology showed small diagnostic agreement between manual and automated examination.
Virtual Slides
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_231
doi:10.1186/s13000-014-0231-7
PMCID: PMC4304134  PMID: 25591792
Pap test; Radiotherapy; Automation; Focalpoint; SurePath
2.  Decreased expression of microRNA-126 is associated with poor prognosis in patients with cervical cancer 
Diagnostic Pathology  2014;9(1):220.
Background
MicroRNA-126(miR-126) has been shown to be frequently down-regulated in a variety of malignancies and act as a potential tumor suppressor. However, its correlations with the clinicopathological characters of cervical cancer remain unclear.
Methods
TaqMan quantitative RT-PCR was used to determine the expression level of miR-126 in tissue samples. The associations of miR-126 expression with clinicopathologic variables were analyzed. Kaplan-Meier survival analysis was performed to analyze the association of miR-126 expression with overall survival (OS) of patients. Univariate and multivariate Cox regression analyses were performed.
Results
miR-126 expression level in human cervical cancer tissues was significantly lower than that in adjacent nontumorous tissues (mean ± SD: 0.59 ± 0.44 vs. 1.00 ± 0.51, P < 0.0001). Decreased miR-126 expression in cervical cancer was found to be significantly associated with lymphatic invasion (P = 0.002), distant metastasis (P < 0.001), FIGO stage (P = 0.009), and histological grade (P = 0.005). Kaplan-Meier analysis showed that patients with lower levels of miR-126 had significantly poorer survival than those with higher expression of this miRNA in patients, with a 5-year OS of 45.7% and 70.9%, respectively (P = 0.002). Multivariate analysis revealed that miR-126 expression (HR = 3.97, 95% CI: 2.01-20.22; P = 0.003) was independently associated with the OS.
Conclusion
Our data suggests the potential of miR-126 as a prognostic biomarker for cervical cancer.
Virtual Slides
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_220
doi:10.1186/s13000-014-0220-x
PMCID: PMC4299682  PMID: 25551621
miRNA; miR-126; Cervical cancer; Quantitative RT-PCR; Prognosis
3.  Magnetic resonance image tissue classification using an automatic method 
Diagnostic Pathology  2014;9(1):207.
Background
Brain segmentation in magnetic resonance images (MRI) is an important stage in clinical studies for different issues such as diagnosis, analysis, 3-D visualizations for treatment and surgical planning. MR Image segmentation remains a challenging problem in spite of different existing artifacts such as noise, bias field, partial volume effects and complexity of the images. Some of the automatic brain segmentation techniques are complex and some of them are not sufficiently accurate for certain applications. The goal of this paper is proposing an algorithm that is more accurate and less complex).
Methods
In this paper we present a simple and more accurate automated technique for brain segmentation into White Matter, Gray Matter and Cerebrospinal fluid (CSF) in three-dimensional MR images. The algorithm’s three steps are histogram based segmentation, feature extraction and final classification using SVM. The integrated algorithm has more accurate results than what can be obtained with its individual components. To produce much more efficient segmentation method our framework captures different types of features in each step that are of special importance for MRI, i.e., distributions of tissue intensities, textural features, and relationship with neighboring voxels or spatial features.
Results
Our method has been validated on real images and simulated data, with desirable performance in the presence of noise and intensity inhomogeneities.
Conclusions
The experimental results demonstrate that our proposed method is a simple and accurate technique to define brain tissues with high reproducibility in comparison with other techniques.
Virtual Slides
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_207
doi:10.1186/s13000-014-0207-7
PMCID: PMC4300026  PMID: 25540017
Statistical segmentation; Magnetic resonance imaging; Image segmentation; Histogram-based segmentation method; SVMs; Brain tissue classification
4.  Breast cancer stromal elastosis is associated with mammography screening detection, low Ki67 expression and favourable prognosis in a population-based study 
Diagnostic Pathology  2014;9(1):230.
Background
Mammography screen-detected breast cancers have a better prognosis than predicted from established prognostic markers. A search for additional features that are characteristic for these tumours and their prognosis is needed to reduce overtreatment, a recognized challenge in breast cancer patient management today. Here, we have investigated the occurrence and importance of tumour elastosis.
Methods
We performed a population based retrospective study of breast cancers detected in the Norwegian Breast Cancer Screening Programme in Vestfold County during 2004–2009. In total, 197 invasive screen-detected cancers and 75 interval cancers in patients aged 50–69 years were compared with regard to standard clinico-pathological parameters and tumour shape, as well as ER, PR, HER2 and Ki67 expression. In particular, the presence of elastotic material in tumours was graded on a 4-tiered scale (score 0–3).
Results
Screen-detected cancers had a significantly higher content of stromal elastosis than interval cancers (p < 0.001). High content of elastosis (score 3) correlated strongly with stellate tumour shape, low histological grade, and ER+/HER2- status. Further, high elastosis score was significantly associated with lower Ki67 expression. In survival analyses, cases with high elastosis demonstrated increased recurrence free (p = 0.03) and disease-specific survival (p = 0.11) compared to cases with low elastosis.
Conclusion
There is a strong correlation between the presence of tumour elastosis, stellate tumour shape and mammography detection of breast cancers. To our knowledge, this is the first time elastosis has been studied in relation to breast cancer detection method. Presence of elastosis is associated with low tumour cell proliferation (Ki67) and a good prognosis.
Virtual Slides
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_230
doi:10.1186/s13000-014-0230-8
PMCID: PMC4300053  PMID: 25522915
Breast cancer; Mammography screening; Elastosis; Ki67
5.  The Eastern Québec Telepathology Network: a three-year experience of clinical diagnostic services 
Diagnostic Pathology  2014;9(Suppl 1):S1.
Background
The Eastern Quebec Telepathology Network (called Réseau de Télépathologie de l'Est du Québec in French) was created to provide uniform diagnostic telepathology services in a huge territory with low population density. We report our first 3-year experience.
Methods
The network was funded equally by the Québec ministry of Health and Canada Health Infoway, a federal telehealth funding agency. The coverage includes intraoperative consultations (IOC), expert opinions, urgent analyses and supervision of macroscopic description. The deployment of the equipment and software started in 2010 and clinical activities began in January 2011. This network comprises 24 hospitals providing oncologic surgery, of which 7 have no pathology laboratory and 4 have a pathology laboratory but no pathologist. The real-time gross evaluation during IOC was performed using a macroscopy station and the sample selection was performed distantly by a technician, a pathology assistant or the surgeon under on-site pathologist supervision. Slides were scanned into whole-slide images (WSI).
Results
As per March 2014, 7,440 slides had been scanned for primary/urgent diagnosis; 1,329 for IOC cases and 2,308 for expert opinions. A 98% concordance rate was found for IOC compared to paraffin material and the average turnaround time was 20 minutes. Expert opinion reports were signed out within 24 hours in 68% of cases and within 72 hours in 85%. A recent multi-method evaluation study of the Network demonstrated that, thanks to telepathology: 1. interruption of IOC service was prevented in hospitals with no pathologist on site; 2. two-stage surgeries and patients transfers were prevented according to surgeons and pathologists; 3. retention and recruitment of surgeons in remote hospitals were facilitated; and 4. professional isolation among pathologists working alone was reduced. This study also demonstrated that wider adoption of telepathology would require technological improvement and that the sustainability of the network requires better coordination and the development of a supra-regional pathology organisation.
Conclusion
The Eastern Quebec Telepathology Network allowed the maintenance of rapid and high quality pathology services in more than 20 sites disseminated on a huge territory. A second phase is underway to expand telepathology to other regions across the province.
doi:10.1186/1746-1596-9-S1-S1
PMCID: PMC4305967  PMID: 25564940
6.  Digital immunohistochemistry platform for the staining variation monitoring based on integration of image and statistical analyses with laboratory information system 
Diagnostic Pathology  2014;9(Suppl 1):S10.
Background
Digital immunohistochemistry (IHC) is one of the most promising applications brought by new generation image analysis (IA). While conventional IHC staining quality is monitored by semi-quantitative visual evaluation of tissue controls, IA may require more sensitive measurement. We designed an automated system to digitally monitor IHC multi-tissue controls, based on SQL-level integration of laboratory information system with image and statistical analysis tools.
Methods
Consecutive sections of TMA containing 10 cores of breast cancer tissue were used as tissue controls in routine Ki67 IHC testing. Ventana slide label barcode ID was sent to the LIS to register the serial section sequence. The slides were stained and scanned (Aperio ScanScope XT), IA was performed by the Aperio/Leica Colocalization and Genie Classifier/Nuclear algorithms. SQL-based integration ensured automated statistical analysis of the IA data by the SAS Enterprise Guide project. Factor analysis and plot visualizations were performed to explore slide-to-slide variation of the Ki67 IHC staining results in the control tissue.
Results
Slide-to-slide intra-core IHC staining analysis revealed rather significant variation of the variables reflecting the sample size, while Brown and Blue Intensity were relatively stable. To further investigate this variation, the IA results from the 10 cores were aggregated to minimize tissue-related variance. Factor analysis revealed association between the variables reflecting the sample size detected by IA and Blue Intensity. Since the main feature to be extracted from the tissue controls was staining intensity, we further explored the variation of the intensity variables in the individual cores. MeanBrownBlue Intensity ((Brown+Blue)/2) and DiffBrownBlue Intensity (Brown-Blue) were introduced to better contrast the absolute intensity and the colour balance variation in each core; relevant factor scores were extracted. Finally, tissue-related factors of IHC staining variance were explored in the individual tissue cores.
Conclusions
Our solution enabled to monitor staining of IHC multi-tissue controls by the means of IA, followed by automated statistical analysis, integrated into the laboratory workflow. We found that, even in consecutive serial tissue sections, tissue-related factors affected the IHC IA results; meanwhile, less intense blue counterstain was associated with less amount of tissue, detected by the IA tools.
doi:10.1186/1746-1596-9-S1-S10
PMCID: PMC4305968  PMID: 25565007
7.  Exploring the spatial dimension of estrogen and progesterone signaling: detection of nuclear labeling in lobular epithelial cells in normal mammary glands adjacent to breast cancer 
Diagnostic Pathology  2014;9(Suppl 1):S11.
Background
Comprehensive spatial assessment of hormone receptor immunohistochemistry staining in digital whole slide images of breast cancer requires accurate detection of positive nuclei within biologically relevant regions of interest. Herein, we propose a combination of automated region labeling at low resolution and subsequent detailed tissue evaluation of subcellular structures in lobular structures adjacent to breast cancer, as a proof of concept for the approach to analyze estrogen and progesterone receptor expression in the spatial context of surrounding tissue.
Methods
Routinely processed paraffin sections of hormone receptor-negative ductal invasive breast cancer were stained for estrogen and progesterone receptor by immunohistochemistry. Digital whole slides were analyzed using commercially available image analysis software for advanced object-based analysis, applying textural, relational, and geometrical features. Mammary gland lobules were targeted as regions of interest for analysis at subcellular level in relation to their distance from coherent tumor as neighboring relevant tissue compartment. Lobule detection quality was evaluated visually by a pathologist.
Results
After rule set optimization in an estrogen receptor-stained training set, independent test sets (progesterone and estrogen receptor) showed acceptable detection quality in 33% of cases. Presence of disrupted lobular structures, either by brisk inflammatory infiltrate, or diffuse tumor infiltration, was common in cases with lower detection accuracy. Hormone receptor detection tended towards higher percentage of positively stained nuclei in lobules distant from the tumor border as compared to areas adjacent to the tumor. After adaptations of image analysis, corresponding evaluations were also feasible in hormone receptor positive breast cancer, with some limitations of automated separation of mammary epithelial cells from hormone receptor-positive tumor cells.
Conclusions
As a proof of concept for object-oriented detection of steroid hormone receptors in their spatial context, we show that lobular structures can be classified based on texture-based image features, unless brisk inflammatory infiltration disrupts the normal morphological structure of the tubular gland epithelium. We consider this approach as prototypic for detection and spatial analysis of nuclear markers in defined regions of interest. We conclude that advanced image analysis at this level of complexity requires adaptation to the individual tumor phenotypes and morphological characteristics of the tumor environment.
doi:10.1186/1746-1596-9-S1-S11
PMCID: PMC4305969  PMID: 25565114
8.  iPathology cockpit diagnostic station: validation according to College of American Pathologists Pathology and Laboratory Quality Center recommendation at the Hospital Trust and University of Verona 
Diagnostic Pathology  2014;9(Suppl 1):S12.
Background
Validation of digital whole slide images is crucial to ensure that diagnostic performance is at least equivalent to that of glass slides and light microscopy. The College of American Pathologists Pathology and Laboratory Quality Center recently developed recommendations for internal digital pathology system validation. Following these guidelines we sought to validate the performance of a digital approach for routine diagnosis by using an iPad and digital control widescreen-assisted workstation through a pilot study.
Methods
From January 2014, 61 histopathological slides were scanned by ScanScope Digital Slides Scanner (Aperio, Vista, CA). Two independent pathologists performed diagnosis on virtual slides in front of a widescreen by using two computer devices (ImageScope viewing software) located to different Health Institutions (AOUI Verona) connected by local network and a remote image server using an iPad tablet (Aperio, Vista, CA), after uploading the Citrix receiver for iPad. Quality indicators related to image characters and work-flow of the e-health cockpit enterprise system were scored based on subjective (high vs poor) perception. The images were re-evaluated two weeks apart.
Results
The whole glass slides encountered 10 liver: hepatocarcinoma, 10 renal carcinoma, 10 gastric carcinoma and 10 prostate biopsies: adenocarcinoma, 5 excisional skin biopsies: melanoma, 5 lymph-nodes: lymphoma. 6 immuno- and 5 special stains were available for intra- and internet remote viewing. Scan times averaged two minutes and 54 seconds per slide (standard deviation 2 minutes 34 seconds). Megabytes ranged from 256 to 680 (mean 390) per slide storage. Reliance on glass slide, image quality (resolution and color fidelity), slide navigation time, simultaneous viewers in geographically remote locations were considered of high performance score. Side by side comparisons between diagnosis performed on tissue glass slides versus widescreen were excellent showing an almost perfect concordance (0.81, kappa index).
Conclusions
We validated our institutional digital pathology system for routine diagnostic facing with whole slide images in a cockpit enterprise digital system or iPad tablet. Computer widescreens are better for diagnosing scanned glass slide that iPad. For urgent requests, iPad may be used. Legal aspects have to be soon faced with to permit the clinical use of this technology in a manner that does not compromise patient care.
doi:10.1186/1746-1596-9-S1-S12
PMCID: PMC4305970  PMID: 25565219
9.  The influence of the microscope lamp filament colour temperature on the process of digital images of histological slides acquisition standardization 
Diagnostic Pathology  2014;9(Suppl 1):S13.
Background
The aim of this study is to compare the digital images of the tissue biopsy captured with optical microscope using bright field technique under various light conditions. The range of colour's variation in immunohistochemically stained with 3,3'-Diaminobenzidine and Haematoxylin tissue samples is immense and coming from various sources. One of them is inadequate setting of camera's white balance to microscope's light colour temperature. Although this type of error can be easily handled during the stage of image acquisition, it can be eliminated with use of colour adjustment algorithms. The examination of the dependence of colour variation from microscope's light temperature and settings of the camera is done as an introductory research to the process of automatic colour standardization.
Methods
Six fields of view with empty space among the tissue samples have been selected for analysis. Each field of view has been acquired 225 times with various microscope light temperature and camera white balance settings. The fourteen randomly chosen images have been corrected and compared, with the reference image, by the following methods: Mean Square Error, Structural SIMilarity and visual assessment of viewer.
Results
For two types of backgrounds and two types of objects, the statistical image descriptors: range, median, mean and its standard deviation of chromaticity on a and b channels from CIELab colour space, and luminance L, and local colour variability for objects' specific area have been calculated. The results have been averaged for 6 images acquired in the same light conditions and camera settings for each sample.
Conclusions
The analysis of the results leads to the following conclusions: (1) the images collected with white balance setting adjusted to light colour temperature clusters in certain area of chromatic space, (2) the process of white balance correction for images collected with white balance camera settings not matched to the light temperature moves image descriptors into proper chromatic space but simultaneously the value of luminance changes. So the process of the image unification in a sense of colour fidelity can be solved in separate introductory stage before the automatic image analysis.
doi:10.1186/1746-1596-9-S1-S13
PMCID: PMC4305971  PMID: 25565329
10.  A nationwide telepathology consultation and quality control program in China: implementation and result analysis 
Diagnostic Pathology  2014;9(Suppl 1):S2.
Background
Telepathology may play an important role in pathology consultation and quality control for cancer diagnosis in China, as the country has the largest population of cancer patients worldwide. In 2011, the Pathology Quality Control Center of China and Ministry of Health developed and implemented a nationwide telepathology consultation and quality control program for cancer diagnosis in China. We here report the results of the two-year implementation and experiences.
Methods
the program built an Internet based telepathology platform to connect participating hospitals and expert consultants. The hardware and software used for the platform were validated in previous validation studies in China. The program had three regional centers consisting of Peking Union Medical College, Huasi Medical College of Sichuan and 2nd affiliated hospital of Zhejiang University. It also had 20 provincial consultation centers based in the provincial referral hospitals. 80 provincial or national pathologists served as expert consultants for the program, providing telepathology consultation for cancer diagnosis for more than 60 participating hospitals.
Results
from 2011 to July 2013, 16,247 pathology cases were submitted to the platform for consultation. Among them, 84% were due to diagnostic difficulty and 16% were due to request by patients. The preliminary diagnosis provided by submitting pathologists were in agreement with expert opinion in 59.8% of cases but was in disagreement with expert opinion in 24.2% of cases. 16.0% of cases were not provided with preliminary diagnosis. The distribution of pathology cases by system or organ were: digestive system, 17.3%; gynecologic system, 16.7%; head and neck, 15.7%; bone and soft tissue, 10.4%; lung and mediastinum, 8.6%; breast, 7.6%; urinary system, 7.5%; hematopathology, 6.4%; skin, 5.2%; neuropathology, 2.5% and cytopathology, 1.3%. Expert consultants also provided assessment of quality of slide preparation and staining, online lectures and guidance for pathology quality control.
Conclusion
our results of two years' implementation indicated that telepathology could solve the problem of uneven distribution of pathology resources and provide a solution for countrywide pathology quality control in China. Telepathology could play an important role in improving pathology diagnosis in China.
doi:10.1186/1746-1596-9-S1-S2
PMCID: PMC4305972  PMID: 25565398
11.  Towards better digital pathology workflows: programming libraries for high-speed sharpness assessment of Whole Slide Images 
Diagnostic Pathology  2014;9(Suppl 1):S3.
Background
Since microscopic slides can now be automatically digitized and integrated in the clinical workflow, quality assessment of Whole Slide Images (WSI) has become a crucial issue. We present a no-reference quality assessment method that has been thoroughly tested since 2010 and is under implementation in multiple sites, both public university-hospitals and private entities. It is part of the FlexMIm R&D project which aims to improve the global workflow of digital pathology. For these uses, we have developed two programming libraries, in Java and Python, which can be integrated in various types of WSI acquisition systems, viewers and image analysis tools.
Methods
Development and testing have been carried out on a MacBook Pro i7 and on a bi-Xeon 2.7GHz server. Libraries implementing the blur assessment method have been developed in Java, Python, PHP5 and MySQL5. For web applications, JavaScript, Ajax, JSON and Sockets were also used, as well as the Google Maps API. Aperio SVS files were converted into the Google Maps format using VIPS and Openslide libraries.
Results
We designed the Java library as a Service Provider Interface (SPI), extendable by third parties. Analysis is computed in real-time (3 billion pixels per minute). Tests were made on 5000 single images, 200 NDPI WSI, 100 Aperio SVS WSI converted to the Google Maps format.
Conclusions
Applications based on our method and libraries can be used upstream, as calibration and quality control tool for the WSI acquisition systems, or as tools to reacquire tiles while the WSI is being scanned. They can also be used downstream to reacquire the complete slides that are below the quality threshold for surgical pathology analysis. WSI may also be displayed in a smarter way by sending and displaying the regions of highest quality before other regions. Such quality assessment scores could be integrated as WSI's metadata shared in clinical, research or teaching contexts, for a more efficient medical informatics workflow.
doi:10.1186/1746-1596-9-S1-S3
PMCID: PMC4305973  PMID: 25565494
12.  A reference model based interface terminology for generic observations in Anatomic Pathology Structured Reports 
Diagnostic Pathology  2014;9(Suppl 1):S4.
Background
Current terminology systems for structured reporting in pathology are more or less focused on tumor pathology. They have not been compiled in a systematic approach, therefore they gather terms of very different granularity. Generic models for terminology development could help in establishing reference terminologies for all fields of anatomic pathology.
The core principle of those models is the ontological structure of native speaking terminology. By analyzing the PathLex interface a generic terminology model will be derived.
Methods
For each element template of PathLex its possible generic nature and its value set was analyzed, looking for the uniqueness or multiplicity of the values in the value sets.
The generic terms were mapped to SNOMED-CT terms using "ArtDecor".
Results
The 488 PathLex element templates for Anatomic Pathology (AP) observations can be reduced to 53 generic templates, leaving out only 17 templates very specific for organ and/or disease. Among those 53 templates 28 are describing UICC-TNM staging, ICD-O-classification, and grading. Further 15 templates describe the results from marker investigations. Almost all of the terms, used in those templates could be mapped to SNOMED CT.
All of the generic elements have their "organ specific" counterparts by assigning them to one of 20 organs and invasive or noninvasive cancer, respectively. Studying the structure of generic and specific terms it becomes obvious that any AP observation
- occurs always in a context
- consists of three basic elements (target of observation, property of observation, additional qualifiers, added by value sets for coded data).
Conclusions
If a machine-readable terminology is aimed to preserve all the information of native speaking, then two principal solutions exist:
- ystematic consideration of all the aspects mentioned above in each single term
- ocusing on the generic elements of terms and combining this with the structure of communication, reflecting the non-obvious elements of the terminology.
The fastest way for establishing an interface terminology is the first approach, which lists all of the terms needed for e.g. a checklist in a comprehensive manner (precoordination).
However, if the list of terms and problems increases, or new requirements have to be met, considerable difficulties may arise in keeping the terminology consistent and complete.
The second, postcoordination approach offers some advantages. It does not have limitations in the organ- or disease specificity, and it keeps the number of terms limited, making them more easily to survey.
doi:10.1186/1746-1596-9-S1-S4
PMCID: PMC4305974  PMID: 25565606
13.  Online teaching of inflammatory skin pathology by a French-speaking International University Network 
Diagnostic Pathology  2014;9(Suppl 1):S5.
Introduction
Developments in technology, web-based teaching and whole slide imaging have broadened the teaching horizon in anatomic pathology. Creating online learning material including many types of media such as radiologic images, whole slides, videos, clinical and macroscopic photographs, is now accessible to most universities. Unfortunately, a major limiting factor to maintain and update the learning material is the amount of resources needed. In this perspective, a French-national university network was initiated in 2011 to build joint online teaching modules consisting of clinical cases and tests. The network has since expanded internationally to Québec, Switzerland and Ivory Coast.
Method
One of the first steps of the project was to build a learning module on inflammatory skin pathology for interns and residents in pathology and dermatology. A pathology resident from Québec spent 6 weeks in France and Switzerland to develop the contents and build the module on an e-learning Moodle platform under the supervision of two dermatopathologists. The learning module contains text, interactive clinical cases, tests with feedback, virtual slides, images and clinical photographs. For that module, the virtual slides are decentralized in 2 universities (Bordeaux and Paris 7). Each university is responsible of its own slide scanning, image storage and online display with virtual slide viewers.
Results
The module on inflammatory skin pathology includes more than 50 web pages with French original content, tests and clinical cases, links to over 45 virtual images and more than 50 microscopic and clinical photographs. The whole learning module is being revised by four dermatopathologists and two senior pathologists. It will be accessible to interns and residents in the spring of 2014. The experience and knowledge gained from that work will be transferred to the next international resident whose work will be aimed at creating lung and breast pathology learning modules.
Conclusion
The challenges of sustaining a project of this scope are numerous. The technical aspect of whole-slide imaging and storage needs to be developed by each university or group. The content needs to be regularly updated and its accuracy reviewed by experts in each individual domain. The learning modules also need to be promoted within the academic community to ensure maximal benefit for trainees. A collateral benefit of the project was the establishment of international partnerships between French-speaking universities and pathologists with the common goal of promoting pathology education through the use of multi-media technology including whole slide imaging.
doi:10.1186/1746-1596-9-S1-S5
PMCID: PMC4305975  PMID: 25564778
14.  Preliminary results from a crowdsourcing experiment in immunohistochemistry 
Diagnostic Pathology  2014;9(Suppl 1):S6.
Background
Crowdsourcing, i.e., the outsourcing of tasks typically performed by a few experts to a large crowd as an open call, has been shown to be reasonably effective in many cases, like Wikipedia, the Chess match of Kasparov against the world in 1999, and several others. The aim of the present paper is to describe the setup of an experimentation of crowdsourcing techniques applied to the quantification of immunohistochemistry.
Methods
Fourteen Images from MIB1-stained breast specimens were first manually counted by a pathologist, then submitted to a crowdsourcing platform through a specifically developed application. 10 positivity evaluations for each image have been collected and summarized using their median. The positivity values have been then compared to the gold standard provided by the pathologist by means of Spearman correlation.
Results
Contributors were in total 28, and evaluated 4.64 images each on average. Spearman correlation between gold and crowdsourced positivity percentages is 0.946 (p < 0.001).
Conclusions
Aim of the experiment was to understand how to use crowdsourcing for an image analysis task that is currently time-consuming when done by human experts. Crowdsourced work can be used in various ways, in particular statistically agregating data to reduce identification errors. However, in this preliminary experimentation we just considered the most basic indicator, that is the median positivity percentage, which provided overall good results. This method might be more aimed to research than routine: when a large number of images are in need of ad-hoc evaluation, crowdsourcing may represent a quick answer to the need.
doi:10.1186/1746-1596-9-S1-S6
PMCID: PMC4305976  PMID: 25565010
15.  Automated image analysis in the study of lymphocyte subpopulation in eosinophilic oesophagitis 
Diagnostic Pathology  2014;9(Suppl 1):S7.
Background
Eosinophilic oesophagitis (EoE) is characterized by the presence of eosinophils in oesophageal mucosa. Other inflammatory cells, mainly lymphocytes, dendritic cells, and mast cells may also play an important role in this disease. The aim of this study is to compare the inflammatory pattern of the mucosa between EoE and gastro-oesophageal reflux disease (GERD), using automatic image analysis in digital slides, and to assess treatment response after elimination diet and food challenge test.
Methods
From 2010 to 2013, 35 oesophageal biopsies from EoE and GERD patients were randomly selected. In six EoE biopsies, patients had been treated with selective food exclusion diet. Immunohistochemical study with CD3, CD20, CD4, and CD8 for lymphocyte populations, CD1a for dendritic cells, and CD117/c-kit for mast cells was performed. Slides were scanned using Leica Aperio Scanscope XT with 40× magnification. Immunohistochemical expression was quantified in 245 immunohistochemistry digital slides with Leica Aperio positive pixel count algorithm using two different approaches: whole slide analysis versus selection of a 2 mm2 hot spot area.
Results
Average eosinophil cell count was significantly higher (p < 0.001) in the first biopsy of EoE patients before treatment (30.75 eosinophils per high power field - HPF) than in GERD patients (0.85 eosinophils/HPF) or in EoE patients after treatment with elimination diet (1.60 eosinophils/HPF). In the immunohistochemical study, manual count and automatic image analysis showed a significant increase in the number of CD3 and CD8 cells in EoE patients, compared with GERD patients. However, the increase of CD117/c-kit was only statistically significant when manual counting procedures were used. CD20 positive cell count also showed a non-statistically significant tendency to reduce after elimination diet treatment.
Manual eosinophil count correlated much better with CD3 and CD8 count using hot spot approach than with a whole slide approach.
Conclusions
Positive pixel count algorithm can be a useful tool to quantify the immunohistochemical expression of inflammatory cells in the diagnosis and follow up of eosinophilic oesophagitis.
doi:10.1186/1746-1596-9-S1-S7
PMCID: PMC4305977  PMID: 25565117
16.  Digital immunohistochemistry wizard: image analysis-assisted stereology tool to produce reference data set for calibration and quality control 
Diagnostic Pathology  2014;9(Suppl 1):S8.
Background
Digital image analysis (DIA) enables better reproducibility of immunohistochemistry (IHC) studies. Nevertheless, accuracy of the DIA methods needs to be ensured, demanding production of reference data sets. We have reported on methodology to calibrate DIA for Ki67 IHC in breast cancer tissue based on reference data obtained by stereology grid count. To produce the reference data more efficiently, we propose digital IHC wizard generating initial cell marks to be verified by experts.
Methods
Digital images of proliferation marker Ki67 IHC from 158 patients (one tissue microarray spot per patient) with an invasive ductal carcinoma of the breast were used. Manual data (mD) were obtained by marking Ki67-positive and negative tumour cells, using a stereological method for 2D object enumeration. DIA was used as an initial step in stereology grid count to generate the digital data (dD) marks by Aperio Genie and Nuclear algorithms. The dD were collected into XML files from the DIA markup images and overlaid on the original spots along with the stereology grid. The expert correction of the dD marks resulted in corrected data (cD). The percentages of Ki67 positive tumour cells per spot in the mD, dD, and cD sets were compared by single linear regression analysis. Efficiency of cD production was estimated based on manual editing effort.
Results
The percentage of Ki67-positive tumor cells was in very good agreement in the mD, dD, and cD sets: regression of cD from dD (R2=0.92) reflects the impact of the expert editing the dD as well as accuracy of the DIA used; regression of the cD from the mD (R2=0.94) represents the consistency of the DIA-assisted ground truth (cD) with the manual procedure. Nevertheless, the accuracy of detection of individual tumour cells was much lower: in average, 18 and 219 marks per spot were edited due to the Genie and Nuclear algorithm errors, respectively. The DIA-assisted cD production in our experiment saved approximately 2/3 of manual marking.
Conclusions
Digital IHC wizard enabled DIA-assisted stereology to produce reference data in a consistent and efficient way. It can provide quality control measure for appraising accuracy of the DIA steps.
doi:10.1186/1746-1596-9-S1-S8
PMCID: PMC4305978  PMID: 25565221
17.  Building of a composite virtual slide from contiguous tissue samples 
Diagnostic Pathology  2014;9(Suppl 1):S9.
Background
Currently available microscope slide scanners produce whole slide images at various resolutions from histological sections. Nevertheless, acquisition area and so visualization of large tissue samples are limited by the standardized size of glass slides, used daily in pathology departments. The proposed solution has been developed to build composite virtual slides from images of large tumor fragments.
Materials and methods
Images of HES or immunostained histological sections of carefully labeled fragments from a representative slice of breast carcinoma were acquired with a digital slide scanner at a magnification of 20×. The tiling program involves three steps: the straightening of tissue fragment images using polynomial interpolation method, and the building and assembling of strips of contiguous tissue sample whole slide images in × and y directions. The final image is saved in a pyramidal BigTiff file format. The program has been tested on several tumor slices. A correlation quality control has been done on five images artificially cut.
Results
Sixty tumor slices from twenty surgical specimens, cut into two to twenty six pieces, were reconstructed. A median of 98.71% is obtained by computing the correlation coefficients between native and reconstructed images for quality control.
Conclusions
The proposed method is efficient and able to adapt itself to daily work conditions of classical pathology laboratories.
doi:10.1186/1746-1596-9-S1-S9
PMCID: PMC4305979  PMID: 25565295
18.  Association of apolipoprotein A5 genetic polymorphisms with steroid-induced osteonecrosis of femoral head in a Chinese Han population 
Diagnostic Pathology  2014;9(1):229.
Background
Previous studies suggested that apolipoprotein A5 (ApoA5) genetic polymorphisms (SNPs) may result in lipid metabolism disorders. Therefore, genetic polymorphisms in ApoA5 may be associated with the occurrence of osteonecrosis of femoral head (ONFH).
Methods
We designed a case–control study including 223 patients of osteonecrosis and 201 age- and sex-matched control subjects to analyze the association between ApoA5 polymorphisms and susceptibility of steroid-induced ONFH. We utilized polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to genotype two SNPs (rs662799 and rs3135506) in ApoA5 gene.
Results
We found both rs662799 and rs3135506 were associated with the risk of ONFH in codominant, dominant, and recessive model, respectively. Haplotype analyses suggested that T-C haplotype was associated with decreased risk of ONFH, whereas the haplotype C-C was significantly associated with an increased risk of ONFH.
Conclusion
Our study suggested that ApoA5 genetic polymorphisms were associated with susceptibility to ONFH in Chinese population. However, our results need further investigation with large sample size and various populations.
Virtual Slides
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_229
doi:10.1186/s13000-014-0229-1
PMCID: PMC4282735  PMID: 25515090
Apolipoprotein A5; Steroid-induced osteonecrosis; Gene polymorphism
19.  Difference in distribution profiles between CD163+ tumor-associated macrophages and S100+ dendritic cells in thymic epithelial tumors 
Diagnostic Pathology  2014;9(1):215.
Background
In a number of human malignancies, tumor-associated macrophages (TAMs) are closely involved in tumor progression. On the other hand, dendritic cells (DCs) that infiltrate tumor tissues are involved in tumor suppression. However, there have been very few reports on the distribution profiles of TAMs and DCs in thymic epithelial tumors. We examined the difference in the distribution profiles between TAMs and DCs in thymoma and thymic carcinoma.
Methods
We examined 69 samples of surgically resected thymic epithelial tumors, namely, 16 thymic carcinomas and 53 thymomas, in which we immunohistochemically evaluated the presence of TAMs using CD68 and CD163 as markers and DCs using S100 as the marker in tumor tissue samples in comparison with normal thymic tissues.
Results
The percentage of samples with a large number of CD68+ TAMs was not significantly different between thymic carcinoma and thymoma (7/16 versus 16/53, p = 0.904). However, the percentage of sample with a large number of CD163+ TAMs was significantly higher in thymic carcinoma than in thymoma (15/16 versus 34/53, p = 0.024). In contrast, the percentage of samples with a large number of S100+ DCs was significantly lower in thymic carcinoma than in thymoma (2/16 versus 23/53, p = 0.021).
Conclusions
To the best of our knowledge, we are the first to show a high percentage of CD163+ TAMs and a low percentage of S100+ DCs in thymic carcinoma samples, and our findings may provide an idea for future targeted therapeutic strategies for thymic carcinoma using antibodies that inhibit monocyte differentiation to TAMs, thereby skewing TAMs differentiation toward DCs.
Virtual Slides
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_215
doi:10.1186/s13000-014-0215-7
PMCID: PMC4302590  PMID: 25499804
Thymic carcinoma; Thymoma; Tumor-associated macrophages; CD163; Dendritic cells; S100
20.  Identification of a novel gene fusion (BMX-ARHGAP) in gastric cardia adenocarcinoma 
Diagnostic Pathology  2014;9(1):218.
Background
Gastric cardia adenocarcinoma (GCA) is one of the major causes of cancer related mortality worldwide. We aim to provide new understanding in the pathogenesis of GCA through investigations on gene expression alterations.
Methods
We preformed RNA-Seq for one pair of GCA and matched non-tumor tissues. Differentially expressed genes (DEGs) and fusion genes were acquired. PCR and gel analysis in additional 14 pairs of samples were performed to validate the chimeric transcripts.
Results
1590 up-regulated and 709 down-regulated genes were detected. Functional analysis revealed that these DEGs were significantly overrepresented in gene ontology items of cell cycle, tumor invasion and proliferation. Moreover, we firstly discovered 3 fusion genes in GCA, including BMX-ARHGAP, LRP5- LITAF and CBX3-C15orf57. The chimeric transcript BMX-ARHGAP was validated and recurrently occurred in 4/15 independent tumor tissues.
Conclusions
Our results may provide new understanding of GCA and biomarkers for further therapeutic studies.
Virtual Slides
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_218
doi:10.1186/s13000-014-0218-4
PMCID: PMC4282731  PMID: 25499959
Gastric cardia adenocarcinoma (GCA); RNA-Seq; Gene fusion
21.  p16INK4a expression in retinoblastoma: a marker of differentiation grade 
Diagnostic Pathology  2014;9(1):180.
Background
The tumor suppressor protein p16INK4a has been extensively studied in many tumors with very different results, ranging from its loss to its clear overexpression, which may be associated with degree of tumor differentiation and prognosis. However, its expression remains unclear in human retinoblastoma (RB), a common malignant tumor of retina in childhood. The aim of this study was to explore the expression pattern of p16INK4a in RB, and the correlation between p16INK4a expression and histopathological features of RB.
Methods
Sixty-five cases of RB were retrospectively analyzed. Paraffin-embedded blocks were retrieved from the archives of ocular pathology department at Zhongshan Ophthalmic Center of Sun Yat-sen University, China. Serial sections were cut and subjected to hematoxylin and eosin staining. Immunohistochemical staining was further done with antibodies p16INK4a, CRX and Ki67. The correlation of p16 INK4a expression with CRX and Ki67 and clinicopathological features of RB were analyzed.
Results
RB tumor histologically consists of various differentiation components including undifferentiated (UD) cells, Homer-Wright rosettes (HWR) or Flexner-Winterstein rosettes (FWR) and fleurettes characteristic of photoreceptor differentiation or Retinocytoma (RC). p16INK4a expression was negative in both fleurette region and the residual retinal tissue adjacent to the tumor, weakly to moderately positive in FWR, strongly positive in both HWR and UD region. However, CRX had the reverse expression patterns in comparison with p16INK4a. It was strongly positive in photoreceptor cells within the residual retina and fleurettes, but weakly to moderately positive in UD area. Together with Ki67 staining, high p16INK4a expression was associated with poor histological differentiation of RB tumors, which had higher risk features with the optic nerve invasion and uveal invasion.
Conclusions
p16INK4a expression increased with the decreasing level of cell differentiation of RBs. RB tumors extensively expressing p16INK4a tended to have higher risk features with poor prognosis. This study suggested that p16INK4a would be a valuable molecular marker of RB to distinguish its histological phenotypes and to serve as a predictor of its prognosis.
Virtual Slides
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_180
doi:10.1186/s13000-014-0180-1
PMCID: PMC4300043  PMID: 25499675
Retinoblastoma; Differentiation; p16INK4a; Immunohistochemistry
22.  IgG4-related disease of the paratestis in a patient with Wells syndrome: a case report 
Diagnostic Pathology  2014;9(1):225.
Background
We report a case of a 33-year-old man who presented with immunoglobulin (Ig)G4-related disease (IgG4-RD) forming a pseudotumor in the left paratesticular region during oral administration of corticosteroid for Wells syndrome, which involves cellulitis with eosinophilia.
Case presentation
The patient was introduced to our institution from a private hospital with a 3-month history of asymptomatic left scrotal mass. A 5-cm diameter nodule was palpable in the left scrotum. Tumor lesion in the left paratestis involving the epididymis and spermatic cord was observed on computed tomography and magnetic resonance imaging. Blood testing showed no abnormalities other than a minimal increase in C-reactive protein levels. Urine examination likewise revealed no significant findings. Left radical orchidectomy was performed under a diagnosis of left paratesticular neoplasm suspected as malignant tumor. The tumor was pathologically identified as IgG4-RD of the left paratestis involving the epididymis and spermatic cord.
Conclusions
We present a first description of IgG4-RD in a patient with Wells syndrome and the ninth case of IgG4-RD in a scrotal organ, and discuss this very rare entity with reference to the literature.
Virtual Slides
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_225
doi:10.1186/s13000-014-0225-5
PMCID: PMC4265405  PMID: 25487870
IgG4-related disease; Paratestis; Pseudotumor; Wells syndrome
23.  Mucinous tubular and spindle cell carcinoma and solid variant papillary renal cell carcinoma: a clinicopathologic comparative analysis of four cases with similar molecular genetics datum 
Diagnostic Pathology  2014;9(1):194.
Mucinous tubular and spindle cell carcinoma (MTSC) was first recognized as a specific entity in the World Health Organization 2004 classification. The “classic” tumor presentation includes an extracellular blue-gray mucinous/myxoid matrix accompanying the typical tubular and spindle cell epithelial components. Tubules are lined by cuboidal to columnar cells with bland nuclei, central small to medium sized nucleoli, and few to no mitoses. By expanding the histologic spectrum, a number of studies highlighted the distinction between MTSC and solid variant of papillary renal cell carcinoma (sPRCC), although controversy still exists. Here, we evaluated two cases of MTSC and compared two cases of sPRCC by light microscopy, special staining, immunohistochemical staining and fluorescence in situ hybridization (FISH). We found that morphologic and immunophenotyping features showed more overlap between MTSC and sPRCC. In addition, gains of chromosomes 7 and 17 and loss of Y, which are characteristic of PRCC, were observed in two cases of sPRCC and one case of MTSC, suggesting that MTSC is similar to sPRCC or may be a subtype of PRCC.
Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_194
doi:10.1186/s13000-014-0194-8
PMCID: PMC4262063  PMID: 25476569
Carcinoma; Renal cell; Mucinous tubular and spindle cell carcinoma; Fluorescence in situ hybridization; Immunohistochemistry
24.  Ductal carcinoma in situ of the breast: correlation between histopathological features and age of patients 
Diagnostic Pathology  2014;9(1):227.
Background
The histopathological subtype, nuclear grade and presence or absence of comedonecrosis are established as critical elements in the reporting of ductal carcinoma in situ (DCIS) of the breast. The aims of this study were to determine the frequencies of morphological subtypes of DCIS, nuclear grade and comedonecrosis; to compare the age of patients with the histopathological characteristics of DCIS, and to assess the agreement of grade between in situ and invasive components in DCIS cases that were associated with invasive carcinoma.
Methods
We evaluated a series of 403 cases of DCIS, pure or associated with invasive mammary carcinoma, consecutively identified from the histopathology files of the Breast Pathology Laboratory, Federal University of Minas Gerais, Brazil, from 2003 to 2008.
Results
DCIS displayed a single growth pattern in most cases (55.1%) and the solid subtype was the most common morphology (42.2% of the total). High-grade DCIS was identified in 293/403 cases (72.7%) and comedonecrosis was present in 222/403 cases (55%). Among DCIS with a single architectural pattern, high grade was more common in the solid subtype (151/168 cases, 89.9%; p < 0.001). Only 32% of tumours with a cribriform pattern had high nuclear grade. Comedonecrosis was more common in the solid morphology than in the cribriform, papillary and micropapillary subtypes (p < 0.001). Patients with high-grade DCIS were younger in relation to patients with low-grade DCIS (p = 0.027) and patients with tumours with comedonecrosis were also younger in comparison to patients with tumours without comedonecrosis (p = 0.003). Fair agreement was observed between in situ and invasive components with regard to grade (weighted kappa = 0.23).
Conclusions
The high nuclear grade and the presence of comedonecrosis were identified more frequently in younger patients and more often correlated with the solid pattern of DCIS.
Virtual Slides
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_227
doi:10.1186/s13000-014-0227-3
PMCID: PMC4260240  PMID: 25471940
Ductal carcinoma in situ; Histological pattern; Morphology; Breast cancer
25.  Upregulation of microRNA-106b is associated with poor prognosis in hepatocellular carcinoma 
Diagnostic Pathology  2014;9(1):226.
Background
MicroRNA-106b (miR-106b) is a member of the miR-106b ~ 25 cluster. It has been reported that miR-106b acts as an oncogene and is upregulated in many human cancers. However, the prognostic value of miR-106b in hepatocellular carcinoma (HCC) remains unclear. The aim of this study was to investigate the clinical significance of miR-106b expression in HCC.
Methods
We determined the expression level of miR-106b in 104 cases of paired HCC and adjacent non-tumor tissues by quantitative real-time PCR (qRT-PCR). The correlation between miR-106b expression and prognosis of HCC was studied by univariate and multivariate analysis. Multivariate analysis of the prognostic factors was performed with Cox proportional hazards model.
Results
MiR-106b expression was significantly upregulated in as high as 76.0% of HCC tissues, compared with their non-tumor counterparts (P < 0.001). High miR-106b expression was significantly associated with large tumor size (P = 0.019) and vascular invasion (P = 0.016). Kaplan-Meier analysis showed that patients with high miR-106b expression had a worse overall survival than patients with low miR-106b expression (log-rank P = 0.004). The multivariate Cox regression analysis indicated that miR-106b expression was an independent prognostic factor for overall survival (HR, 2.002; 95% CI, 1.130-6.977; P = 0.027).
Conclusion
Our data indicated that miR-106b expression was significantly upregulated in HCC and could serve as a potential unfavorable prognostic biomarker.
Virtual Slides
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_226
doi:10.1186/s13000-014-0226-4
PMCID: PMC4261545  PMID: 25466449
Hepatocellular carcinoma; miR-106b; Prognosis

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