Pathogenic Variants Associated with Epigenetic Control and the NOTCH Pathway Are Frequent in Classic Hodgkin Lymphoma.

Classic Hodgkin lymphoma (cHL) constitutes a B-cell neoplasm derived from germinal center lymphocytes. Despite high cure rates (80-90%) obtained with the current multiagent protocols, a significant proportion of cHL patients experience recurrences, characterized by a lower sensitivity to second-line treatments. The genomic background of chemorefractory cHL is still poorly understood, limiting personalized treatment strategies based on molecular features. In this study, using a targeted next-generation sequencing (NGS) panel specifically designed for cHL research, we compared chemosensitive and chemorefractory diagnostic tissue samples of cHL patients. Furthermore, we longitudinally examined paired diagnosis-relapsesamples of chemorefractory cHL in order to define patterns of dynamic evolution and clonal selection. Pathogenic variants in NOTCH1 and NOTCH2 genes frequently arise in cHL. Mutations in genes associated with epigenetic regulation (CREBBP and EP300) are particularly frequent in relapsed/refractory cHL. The appearance of novel clones characterized by mutations previously not identified at diagnosis is a common feature in cHL cases showing chemoresistance to frontline treatments. Our results expand current molecular and pathogenic knowledge of cHL and support the performance of molecular studies in cHL prior to the initiation of first-line therapies.

Differences in the Immune Response of the Nonmetastatic Axillary Lymph Nodes between Triple-Negative and Luminal A Breast Cancer Surrogate Subtypes.

Breast cancer (BC) comprises four immunohistochemical surrogate subtypes of which triple-negative breast cancer (TNBC) has the highest risk of mortality. Axillary lymph nodes (ALNs) are the regions where BC cells first establish before distant metastasis, and the presence of tumor cells in the ALN causes an immune tolerance profile that contrasts with that of the nonmetastatic ALN (ALN-). However, few studies have compared the immune components of the ALNs- in BC subtypes. The present study aimed to determine whether differences between immune populations in the primary tumor and ALNs- were associated with the luminal A or TNBC subtype. We evaluated a retrospective cohort of 144 patients using paraffin-embedded biopsies. The TNBC samples tended to have a higher histologic grade and proliferation index and had higher levels of immune markers compared with luminal A in primary tumors and ALNs-. Two methods for validating the multivariate analysis found that histologic grade, intratumoral S100 dendritic cells, and CD8 T lymphocytes and CD57 natural killer cells in the ALNs- were factors associated with TNBC, whereas CD83 dendritic cells in the ALNs- were associated with the luminal A subtype. In conclusion, we found that intratumoral regions and ALNs- of TNBC contained higher concentrations of markers related to immune tolerance than luminal A. This finding partially explains the worse prognosis of patients with TNBC.

The Immune Response in Nonmetastatic Axillary Lymph Nodes Is Associated with the Presence of Axillary Metastasis and Breast Cancer Patient Outcome.

Tumor cells can modify the immune response in primary tumors and in the axillary lymph nodes with metastasis (ALN+) in breast cancer (BC), influencing patient outcome. We investigated whether patterns of immune cells in the primary tumor and in the axillary lymph nodes without metastasis (ALN-) differed between patients diagnosed without ALN+ (diagnosed-ALN-) and with ALN+ (diagnosed-ALN+) and the implications for clinical outcome. Eleven immune markers were studied using immunohistochemistry, tissue microarray, and digital image analysis in 141 BC patient samples (75 diagnosed-ALN+ and 66 diagnosed-ALN-). Two logistic regression models were derived to identify the clinical, pathologic, and immunologic variables associated with the presence of ALN+ at diagnosis. There are immune patterns in the ALN- associated with the presence of ALN+ at diagnosis. The regression models revealed a small subgroup of diagnosed-ALN+ with ALN- immune patterns that were more similar to those of the ALN- of the diagnosed-ALN-. This small subgroup also showed similar clinical behavior to that of the diagnosed-ALN-. Another small subgroup of diagnosed-ALN- with ALN- immune patterns was found whose members were more similar to those of the ALN- of the diagnosed-ALN+. This small subgroup had similar clinical behavior to the diagnosed-ALN+. These data suggest that the immune response present in ALN- at diagnosis could influence the clinical outcome of BC patients.

Compositional and structural analysis of engineered stones and inorganic particles in silicotic nodules of exposed workers.

BACKGROUND: Engineered stone silicosis is an emerging disease in many countries worldwide produced by the inhalation of respirable dust of engineered stone. This silicosis has a high incidence among young workers, with a short latency period and greater aggressiveness than silicosis caused by natural materials. Although the silica content is very high and this is the key factor, it has been postulated that other constituents in engineered stones can influence the aggressiveness of the disease. Different samples of engineered stone countertops (fabricated by workers during the years prior to their diagnoses), as well as seven lung samples from exposed patients, were analyzed by multiple techniques. RESULTS: The different countertops were composed of SiO2 in percentages between 87.9 and 99.6%, with variable relationships of quartz and cristobalite depending on the sample. The most abundant metals were Al, Na, Fe, Ca and Ti. The most frequent volatile organic compounds were styrene, toluene and m-xylene, and among the polycyclic aromatic hydrocarbons, phenanthrene and naphthalene were detected in all samples. Patients were all males, between 26 and 46 years-old (average age: 36) at the moment of the diagnosis. They were exposed to the engineered stone an average time of 14 years. At diagnosis, only one patient had progressive massive fibrosis. After a follow-up period of 8 ± 3 years, four patients presented progressive massive fibrosis. Samples obtained from lung biopsies most frequently showed well or ill-defined nodules, composed of histiocytic cells and fibroblasts without central hyalinization. All tissue samples showed high proportion of Si and Al at the center of the nodules, becoming sparser at the periphery. Al to Si content ratios turned out to be higher than 1 in two of the studied cases. Correlation between Si and Al was very high (r = 0.93). CONCLUSION: Some of the volatile organic compounds, polycyclic aromatic hydrocarbons and metals detected in the studied countertop samples have been described as causative of lung inflammation and respiratory disease. Among inorganic constituents, aluminum has been a relevant component within the silicotic nodule, reaching atomic concentrations even higher than silicon in some cases. Such concentrations, both for silicon and aluminum showed a decreasing tendency from the center of the nodule towards its frontier.

Immune response profile of primary tumour, sentinel and non-sentinel axillary lymph nodes related to metastasis in breast cancer: an immunohistochemical point of view.

Approximately 1.67 million new cases of breast cancer (BC) are diagnosed annually, and patient survival significantly decreases when the disease metastasizes. The axillary lymph nodes (ALNs) are the main doorway for BC tumoral cell escape, through which cells can disseminate to distant organs. The immune response, which principally develops in the lymph nodes, is linked to cancer progression, and its efficacy at controlling tumoral growth is compromised during the disease. Immunohistochemistry (IHC) is one of the most widely used research techniques for studying the immune response. It allows the measurement of the expression of particular markers related to the immune populations. This review focuses on the role of the immune populations in the primary tumour in the locoregional metastasis of the ALN, and the relationship of the immune response in these regions to distant metastasis. We considered only studies of immune cells using IHC techniques. In particular, lymphocytes, macrophages and dendritic cells all play important roles in BC and have been extensively studied. Although further research is needed, there is much evidence of their role in the invasion of the ALN and distant organs. Their association with tumoral growth or protection has not yet been demonstrated decisively and is very likely to be determined by a combination of factors. Moreover, even though IHC is a widely used technique in cancer diagnosis and research, there is still room for improvement, since its quantification needs to be properly standardized.

A high-fat diet combined with food deprivation increases food seeking and the expression of candidate biomarkers of addiction.

A mouse model has been developed to study the effect of dietary fat combined with food deprivation periods on palatable food seeking and on the expression of three potential addiction biomarkers in the nucleus accumbens: fumarate hydratase (FH), ATP synthase subunit alpha (ATP5a1) and transketolase (TKT). Forty C57BL/6 J male mice, four-week old, were fed either with a high-fat (HF) diet or standard diet along the experiment. After 3 weeks of differential feeding, animals underwent a two-week training period of two daily sessions where visual cues were paired either to palatable food (chocolate cereals) or no food at all. This training was prolonged one more week with similar, one daily sessions preceded by 12 hours of food deprivation. A behavioural test was finally conducted where mice were confined for 30 minutes either in food unpaired compartments or in compartments previously paired with cereals, but now with empty food trays. Total activity during this behavioural test and serum corticosterone levels right after it were similar in all experimental groups. Mice tested in food-paired compartments showed a marked preference for the empty food tray that gradually disappeared in standard diet-fed individuals but persisted in HF-fed mice. HF-fed mice also overexpressed FH, ATP5a1 and TKT, which positively correlated with the persistence of preference for the empty food tray. It is suggested that HF diets combined with food deprivation may enhance food seeking behaviours while upregulating FH/ATP5a1/TKT, which are further envisaged as biomarkers of addiction.

International Clinical Guidelines for the Adoption of Digital Pathology: A Review of Technical Aspects.

Digital slides, also called whole-slide images, are being evaluated to replace conventional microscopy, and several guidelines have been published. This paper reviews technical specifications of digital pathology systems that have been included in the guidelines and position papers from the Canadian Association of Pathologists, the College of American Pathologists, the American Telemedicine Association, the Digital Pathology Association, the Food and Drug Administration, the Centers for Medicare and Medicaid Services, the Centers for Disease Control and Prevention, the Society of Toxicologic Pathology, the European Commission, the Spanish Society of Anatomic Pathology, The Royal College of Pathologists and The Royal College of Pathologists of Australasia. In conclusion, most technical aspects are well covered by these guidelines, although they offer limited information regarding image quality and compression, and file formats.

New Trends of Emerging Technologies in Digital Pathology.

The future paradigm of pathology will be digital. Instead of conventional microscopy, a pathologist will perform a diagnosis through interacting with images on computer screens and performing quantitative analysis. The fourth generation of virtual slide telepathology systems, so-called virtual microscopy and whole-slide imaging (WSI), has allowed for the storage and fast dissemination of image data in pathology and other biomedical areas. These novel digital imaging modalities encompass high-resolution scanning of tissue slides and derived technologies, including automatic digitization and computational processing of whole microscopic slides. Moreover, automated image analysis with WSI can extract specific diagnostic features of diseases and quantify individual components of these features to support diagnoses and provide informative clinical measures of disease. Therefore, the challenge is to apply information technology and image analysis methods to exploit the new and emerging digital pathology technologies effectively in order to process and model all the data and information contained in WSI. The final objective is to support the complex workflow from specimen receipt to anatomic pathology report transmission, that is, to improve diagnosis both in terms of pathologists' efficiency and with new information. This article reviews the main concerns about and novel methods of digital pathology discussed at the latest workshop in the field carried out within the European project AIDPATH (Academia and Industry Collaboration for Digital Pathology).

Clinical Neuropathology Views - 2/2016: Digital networking in European neuropathology: An initiative to facilitate truly interactive consultations.

Digital technology is progressively changing our vision of the practice of neuropathology. There are a number of facts that support the introduction of digital neuropathology. With the development of wholeslide imaging (WSI) systems the difficulties involved in implementing a neuropathology network have been solved. A relevant difficulty has been image standardization, but an open digital image communication protocol defined by the Digital Imaging and Communications in Medicine (DICOM) standard is already a reality. The neuropathology network should be established in Europe because it is the expected geographic context for relationships among European neuropathologists. There are several limitations in the implementation of a digital neuropathology consultancy network such as financial support, operational costs, legal issues, and technical assistance of clients. All of these items have been considered and should be solved before implementing the proposal. Finally, the authors conclude that a European digital neuropathology network should be created for patients' benefit.

Clinicopathological and Genomic Identification of Breast Cancers with No Impact on Mortality.

BACKGROUND: Implementing mammogram screening means that clinicians are seeing many breast cancers that will never develop metastases. The purpose of this study was to identify subgroups of breast cancer patients who did not present events related to long-term breast cancer mortality, taking into account diagnosis at breast screening, absence of palpability and axillary involvement, and genomic analysis with PAM50. PATIENTS AND METHODS: To identify them, a retrospective observational study was carried out selecting patients without any palpable tumor and without axillary involvement, and a genomic analysis was performed with PAM50. RESULTS: The probability of distant metastasis-free interval (DMFI) of 337 patients was 0.92 (95% CI, 0.90-0.93) at 20 years and 0.96 (95% CI, 0.92-1.00) in 95 patients (28%) with available PAM50 tests. In 22 (23.15%) luminal A tumors and in 9 (9.47%) luminal B tumors smaller than 1 cm, and in HER2 and basal type tumors, there were no metastatic events (20-year DMFI of 1.00). CONCLUSION: Patients with nonpalpable breast cancer found at screening with negative nodes are at very low risk. It is possible to identify subgroups without metastatic events by determining the intrinsic subtype and tumor size less than 1 cm. Therefore, de-escalation of treatment should be considered.

TMA vessel segmentation based on color and morphological features: application to angiogenesis research.

Given that angiogenesis and lymphangiogenesis are strongly related to prognosis in neoplastic and other pathologies and that many methods exist that provide different results, we aim to construct a morphometric tool allowing us to measure different aspects of the shape and size of vascular vessels in a complete and accurate way. The developed tool presented is based on vessel closing which is an essential property to properly characterize the size and the shape of vascular and lymphatic vessels. The method is fast and accurate improving existing tools for angiogenesis analysis. The tool also improves the accuracy of vascular density measurements, since the set of endothelial cells forming a vessel is considered as a single object.

Whole slide imaging of tumour microenvironment in classical Hodgkin's lymphoma: development of a clinical prediction model based on programmed death-ligand 1 and tumorous Reed-Sternberg cells.

AIMS: The prognostic impact of programmed death-ligand 1 (PD-L1) cells in classic Hodgkin lymphoma (cHL) tumour microenvironment remains undefined. METHODS: Model development via Transparent Reporting of a multivariable prediction model for Individual Prognosis Or Diagnosis guidelines were followed. PD-L1+ and CD30+ tumoral Reed-Sternberg cells were quantified through whole slide imaging and digital image analysis in 155 digital histopathological slides of cHL. Univariate and multivariate survival analyses were performed. The analyses were reproduced for patients with advanced stages (IIB, III and IV) using the Advanced-stage cHL International Prognostic Index. RESULTS: The PD-L1/CD30 ratio was statistically significantly associated with survival outcomes. Patients with a PD-L1/CD30 ratio above 47.1 presented a shorter overall survival (mean OS: 53.7 months; 95% CI: 28.7 to 78.7) in comparison with patients below this threshold (mean OS: 105.4 months; 95% CI: 89.6 to 121.3) (p=0.04). When adjusted for covariates, the PD-L1/CD30 ratio retained prognostic impact, both for the OS (HR: 1.005; 95% CI: 1.002 to 1.008; p=0.000) and the progression-free survival (HR: 3.442; 95% CI: 1.045 to 11.340; p=0.04) in a clinical and histopathological multivariate model including the male sex (HR: 3.551; 95% CI: 0.986 to 12.786; p=0.05), a percentage of tumoral cells ≥10.1% (HR: 1.044; 95% CI: 1.003 to 1.087; p=0.03) and high risk International Prognostic Score (≥3 points) (HR: 6.453; 95% CI: 1.970 to 21.134; p=0.002). CONCLUSIONS: The PD-L1/CD30 ratio identifies a group of cHL patients with an increased risk of treatment failure. Its clinical application can be performed as it constitutes an easy to implement pathological information in the diagnostic work-up of patients with cHL.

[Study of genetic variants in 169 non-small cell lung cancer patients]. / Estudio de variantes genéticas en 169 pacientes de cáncer de pulmón no microcítico.

INTRODUCTION: Lung cancer is the leading cause of cancer death in our country. Non-small cell lung cancer (NSCLC) represents the paradigm of personalized medicine. The main objective of this study is analysing the distribution of the most frequently described clinically significant variants in NSCLC, in our environment. MATERIAL AND METHODS: We studied the immunohistochemical expression of TTF1, p40 and PD-L1 and the genetic variants frequency using Next-Generation Sequencing (NGS) with a panel of 52 genes, in 174 NSCLC paraffin-embedded samples in 169 patients (111 men and 52 women) from the province of Cádiz. RESULTS: The immunohistochemical expression of TTF1, p40 and PD-L1 was positive in 87%, 0% and 46% in adenocarcinoma, and 0%, 100% and 41% in squamous cell carcinoma. In NGS, the most common single nucleotide variants (SNVs) were KRAS (36%), EGFR (14%), BRAF (10%), PIK3CA (8%), and MET (3%). The most frequent copy number variants (CNVs) were amplifications in NF1 (30%), EGFR (18%), CCND1 (9%), MYC (9%) and KRAS (7%). In women, SNV in EGFR are more frequent than in men (P<.0001). Adenocarcinoma is the most frequent histological type with SNV in KRAS (P=.007361) or in EGFR (P<.0001). Gene fusions were detected in 16 patients (9.47%), in 9 cases in the MET gene. CONCLUSIONS: We detected associations, not described so far, between immunohistochemical expression and specific gene variants, which could have an impact on the treatment of NSCLC patients.

Digital pathology in personalized cancer therapy.

The development of small molecule inhibitors of the growth factor receptors and discovery of somatic mutations of the thyrosine kinase domain resulted in new paradigms for the cancers therapy. Digital microscopy is an important tool for surgical pathologists. The achievements in the digital pathology field have modified the workflow of pathomorphology labs, enhanced the pathologists' role in the diagnostics and increased their contribution to the personalized targeted medicine. Digital image analysis is now available in a variety of platforms to improve quantification performance of diagnostic pathology. The authors describe the state of digital microscopy as it applies to the field of quantitative immunohistochemistry of biomarkers related to the clinical personalized targeted therapy of breast cancer, non-small lung cancer and colorectal cancer: HER-2, EGFR, KRAS and BRAF genes. The information is derived from the experience of the authors and review of the literature.

The COST Action IC0604 "Telepathology Network in Europe" (EURO-TELEPATH).

The COST Action IC0604 "Telepathology Network in Europe" (EURO-TELEPATH) is a European COST Action that has been running from 2007 to 2011. COST Actions are funded by the COST (European Cooperation in the field of Scientific and Technical Research) Agency, supported by the Seventh Framework Programme for Research and Technological Development (FP7), of the European Union. EURO-TELEPATH's main objectives were evaluating and validating the common technological framework and communication standards required to access, transmit and manage digital medical records by pathologists and other medical professionals in a networked environment. The project was organized in four working groups. orking Group 1 "Business modeling in pathology" has designed main pathology processes - Frozen Study, Formalin Fixed Specimen Study, Telepathology, Cytology, and Autopsy -using Business Process Modeling Notation (BPMN). orking Group 2 "Informatics standards in pathology" has been dedicated to promoting the development and application of informatics standards in pathology, collaborating with Integrating the Healthcare Enterprise (IHE), Digital Imaging and Communications in Medicine (DICOM), Health Level Seven (HL7), and other standardization bodies. Working Group 3 "Images: Analysis, Processing, Retrieval and Management" worked on the use of virtual or digital slides that are fostering the use of image processing and analysis in pathology not only for research purposes, but also in daily practice. Working Group 4 "Technology and Automation in Pathology" was focused on studying the adequacy of current existing technical solutions, including, e.g., the quality of images obtained by slide scanners, or the efficiency of image analysis applications. Major outcome of this action are the collaboration with international health informatics standardization bodies to foster the development of standards for digital pathology, offering a new approach for workflow analysis, based in business process modeling. Health terminology standardization research has become a topic of high interest. Future research work should focus on standardization of automatic image analysis and tissue microarrays imaging.

SNOMED CT in pathology.

Pathology information systems have been using SNOMED II for many years, and in most cases, they are in a migration process to SNOMED CT. COST Action IC0604 (EURO-TELEPATH) has considered terminology normalization one of its strategic objectives. This paper reviews the use of SNOMED CT in healthcare, with a special focus in pathology. Nowadays, SNOMED CT is mainly used for concept search and coding of clinical data. Some ontological errors found in SNOMED CT are described. The Integrating the Healthcare Enterprise (IHE) initiative has fostered the use of SNOMED CT, also in Pathology, as recommended in the Supplement Anatomic Pathology Structured Reports of the IHE Anatomic Pathology Technical Framework. Rule governing concept post-coordination is also described. Some recent initiatives are trying to define a SNOMED CT subset for Pathology. The Spanish Society of Pathology has defined a subset for specimens and procedures in Pathology. Regarding diagnosis coding, the morphological abnormality sub-hierarchy of SNOMED CT need to be significantly extended and improved to become useful for pathologists. A consensus is needed to encode pathology reports with the adequate hierarchies and concepts. This will make the implementation of pathology structured reports more feasible.

State of the art in pathology business process analysis, modeling, design and optimization.

For analyzing current workflows and processes, for improving them, for quality management and quality assurance, for integrating hardware and software components, but also for education, training and communication between different domains' experts, modeling business process in a pathology department is inevitable. The authors highlight three main processes in pathology: general diagnostic, cytology diagnostic, and autopsy. In this chapter, those processes are formally modeled and described in detail. Finally, specialized processes such as immunohistochemistry and frozen section have been considered.

State of the art and trends for digital pathology.

Anatomic pathology is a medical specialty where both information management systems and digital images systems paly a most important role. Digital pathology is a new concept that considers all uses of this information, including diagnosis, biomedical research and education. Virtual microscopy or whole slide imaging, resulting in digital slides, is an outreaching technology in anatomic pathology. Limiting factors in the expansion of virtual microscopy are formidable storage dimension, scanning speed, quality of image and cultural change. Anatomic pathology data and images should be an important part of the patient electronic health records as well as of clinical data warehouse, epidemiological or biomedical research databases, and platforms dedicated to translational medicine. Integrating anatomic pathology to the "healthcare enterprise" can only be achieved using existing and emerging medical informatics standards like Digital Imaging and Communications in Medicine (DICOM®1), Health Level Seven (HL7®), and Systematized Nomenclature of Medicine-Clinical Terms (SNOMED CT®), following the recommendations of Integrating the Healthcare Enterprise (IHE®). The consequences of the full digitalization of pathology departments are hard to foresee, but short term issues have arisen that imply interesting challenges for health care standards bodies.

Digital image analysis in breast cancer: an example of an automated methodology and the effects of image compression.

In the current practice of pathology, the evaluation of immunohistochemical (IHC) markers represents an essential tool. The manual quantification of these markers is still laborious and subjective, and the use of computerized systems for digital image (DI) analysis has not yet resolved the problems of nuclear aggregates (clusters). Furthermore, the volume of DI storage continues to be an important problem in computer-assisted pathology. In the present study we have developed an automated procedure to quantify IHC nuclear markers in DI with a high level of clusters. Furthermore the effects of JPEG compression in the image analysis were evaluated. The results indicated that there was an agreement with the results of both methods (automated vs. manual) in almost 90% of the analyzed images. On the other hand, automated count differences increase as the compression level increase, but only in images with a high number of stained nuclei (>nuclei/image) or with high area cluster (>25µm2). Some corrector factors were developed in order to correct this count differences. In conclusion, the proposed automated procedure is an objective, faster than manual counting and reproducible method that has more than 90% of similarity with manual count. Moreover, the results demonstrate that with correction factors, it is possible to carry out unbiased automated quantifications on IHC nuclear markers in compressed DIs.

Emerging trends: grid technology in pathology.

Grid technology has enabled clustering and access to, and interaction among, a wide variety of geographically distributed resources such as supercomputers, storage systems, data sources, instruments as well as special devices and services, realizing network-centric operations. Their main applications include large scale computational and data intensive problems in science and engineering. Grids are likely to have a deep impact on health related applications. Moreover, they seem to be suitable for tissue-based diagnosis. They offer a powerful tool to deal with current challenges in many biomedical domains involving complex anatomical and physiological modeling of structures from images or large image databases assembling and analysis. This chapter analyzes the general structures and functions of a Grid environment implemented for tissue-based diagnosis on digital images. Moreover, it presents a Grid middleware implemented by the authors for diagnostic pathology applications. The chapter is a review of the work done as part of the European COST project EUROTELEPATH.