Network Medicine in Pathobiology.

The past decade has witnessed exponential growth in the generation of high-throughput human data across almost all known dimensions of biological systems. The discipline of network medicine has rapidly evolved in parallel, providing an unbiased, comprehensive biological framework through which to interrogate and integrate systematically these large-scale, multi-omic data to enhance our understanding of disease mechanisms and to design drugs that reflect a deep knowledge of molecular pathobiology. In this review, we discuss the key principles of network medicine and the human disease network and explore the latest applications of network medicine in this multi-omic era. We also highlight the current conceptual and technological challenges, which serve as exciting opportunities by which to improve and expand the network-based applications beyond the artificial boundaries of the current state of human pathobiology.

Bacterial genome sequencing in the clinic: bioinformatic challenges and solutions.

The potential of bacterial whole-genome sequencing (WGS) to complement existing diagnostic infrastructures in clinical microbiology has been shown in proof-of-principle examples and extensively discussed. However, less attention has been drawn to bioinformatic challenges that are associated with the clinical adoption of WGS-based molecular diagnostics. This Perspective article discusses questions that are related to standard operating procedures, computational resource management, and data storage and integration in the context of recent developments in the sequencing and bioinformatics service markets.

Molecular Diagnostics of Vascular Tumors of the Skin.

In this article, the authors have reviewed all the recent news regarding how the discovery of some novel and recurrent molecular and genetic changes has modified the classification of some entities and have addressed to the description of new variants of vascular tumors. And even more important, the authors also reviewed on how these findings, in addition to gain insight into the tumoral biology, portend significant clinical consequences not only regarding to their diagnosis but also to their management and prognosis because some of these mutations are potential targets for treatment. The authors have also highlighted immunohistochemical markers can help us as a surrogate marker of those molecular alterations.

Aptamers as Valuable Molecular Tools in Neurosciences.

Aptamers are short nucleic acids that interact with a variety of targets with high affinity and specificity. They have been shown to inhibit biological functions of cognate target proteins, and they are identifiable by an in vitro selection process, also termed SELEX (Systematic Evolution of Ligands by EXponential enrichment). Being nucleic acids, aptamers can be synthesized chemically or enzymatically. The latter renders RNA aptamers compatible with the cell's own transcription machinery and, thus, expressable inside cells. The synthesis of aptamers by chemical approaches opens up the possibility of producing aptamers on a large scale and enables a straightforward access to introduce modifications in a site-specific manner (e.g., fluorophores or photo-labile groups). These characteristics make aptamers broadly applicable (e.g., as an analytical, diagnostic, or separation tool). In this TechSight, we provide a brief overview on aptamer technology and the potential of aptamers as valuable research tools in neurosciences.

Meeting the challenges of implementing rapid genomic testing in acute pediatric care.

PURPOSE: The purpose of the study was to implement and prospectively evaluate the outcomes of a rapid genomic diagnosis program at two pediatric tertiary centers. METHODS: Rapid singleton whole-exome sequencing (rWES) was performed in acutely unwell pediatric patients with suspected monogenic disorders. Laboratory and clinical barriers to implementation were addressed through continuous multidisciplinary review of process parameters. Diagnostic and clinical utility and cost-effectiveness of rWES were assessed. RESULTS: Of 40 enrolled patients, 21 (52.5%) received a diagnosis, with median time to report of 16 days (range 9-109 days). A result was provided during the first hospital admission in 28 of 36 inpatients (78%). Clinical management changed in 12 of the 21 diagnosed patients (57%), including the provision of lifesaving treatment, avoidance of invasive biopsies, and palliative care guidance. The cost per diagnosis was AU$13,388 (US$10,453). Additional cost savings from avoidance of planned tests and procedures and reduced length of stay are estimated to be around AU$543,178 (US$424,101). The clear relative advantage of rWES, joint clinical and laboratory leadership, and the creation of a multidisciplinary "rapid team" were key to successful implementation. CONCLUSION: Rapid genomic testing in acute pediatrics is not only feasible but also cost-effective, and has high diagnostic and clinical utility. It requires a whole-of-system approach for successful implementation.

Incorporating Advances in Molecular Pathology Into Brain Tumor Diagnostics.

Recent advances in molecular pathology have reshaped the practice of brain tumor diagnostics. The classification of gliomas has been restructured with the discovery of isocitrate dehydrogenase (IDH) 1/2 mutations in the vast majority of lower grade infiltrating gliomas and secondary glioblastomas (GBM), with IDH-mutant astrocytomas further characterized by TP53 and ATRX mutations. Whole-arm 1p/19q codeletion in conjunction with IDH mutations now define oligodendrogliomas, which are also enriched for CIC, FUBP1, PI3K, NOTCH1, and TERT-p mutations. IDH-wild-type (wt) infiltrating astrocytomas are mostly primary GBMs and are characterized by EGFR, PTEN, TP53, NF1, RB1, PDGFRA, and CDKN2A/B alterations, TERT-p mutations, and characteristic copy number alterations including gains of chromosome 7 and losses of 10. Other clinically and genetically distinct infiltrating astrocytomas include the aggressive H3K27M-mutant midline gliomas, and smaller subsets that occur in the setting of NF1 or have BRAF V600E mutations. Low-grade pediatric gliomas are both genetically and biologically distinct from their adult counterparts and often harbor a single driver event often involving BRAF, FGFR1, or MYB/MYBL1 genes. Large scale genomic and epigenomic analyses have identified distinct subgroups of ependymomas tightly linked to tumor location and clinical behavior. The diagnosis of embryonal neoplasms also integrates molecular testing: (I) 4 molecularly defined, biologically distinct subtypes of medulloblastomas are now recognized; (II) 3 histologic entities have now been reclassified under a diagnosis of "embryonal tumor with multilayered rosettes (ETMR), C19MC-altered"; and (III) atypical teratoid/rhabdoid tumors (AT/RT) now require SMARCB1 (INI1) or SMARCA4 (BRG1) alterations for their diagnosis. We discuss the practical use of contemporary biomarkers for an integrative diagnosis of central nervous system neoplasia.

The rapid evolution of molecular genetic diagnostics in neuromuscular diseases.

PURPOSE OF REVIEW: The development of massively parallel sequencing (MPS) has revolutionized molecular genetic diagnostics in monogenic disorders. The present review gives a brief overview of different MPS-based approaches used in clinical diagnostics of neuromuscular disorders (NMDs) and highlights their advantages and limitations. RECENT FINDINGS: MPS-based approaches like gene panel sequencing, (whole) exome sequencing, (whole) genome sequencing, and RNA sequencing have been used to identify the genetic cause in NMDs. Although gene panel sequencing has evolved as a standard test for heterogeneous diseases, it is still debated, mainly because of financial issues and unsolved problems of variant interpretation, whether genome sequencing (and to a lesser extent also exome sequencing) of single patients can already be regarded as routine diagnostics. However, it has been shown that the inclusion of parents and additional family members often leads to a substantial increase in the diagnostic yield in exome-wide/genome-wide MPS approaches. In addition, MPS-based RNA sequencing just enters the research and diagnostic scene. SUMMARY: Next-generation sequencing increasingly enables the detection of the genetic cause in highly heterogeneous diseases like NMDs in an efficient and affordable way. Gene panel sequencing and family-based exome sequencing have been proven as potent and cost-efficient diagnostic tools. Although clinical validation and interpretation of genome sequencing is still challenging, diagnostic RNA sequencing represents a promising tool to bypass some hurdles of diagnostics using genomic DNA.

Emerging Themes in Image Informatics and Molecular Analysis for Digital Pathology.

Pathology is essential for research in disease and development, as well as for clinical decision making. For more than 100 years, pathology practice has involved analyzing images of stained, thin tissue sections by a trained human using an optical microscope. Technological advances are now driving major changes in this paradigm toward digital pathology (DP). The digital transformation of pathology goes beyond recording, archiving, and retrieving images, providing new computational tools to inform better decision making for precision medicine. First, we discuss some emerging innovations in both computational image analytics and imaging instrumentation in DP. Second, we discuss molecular contrast in pathology. Molecular DP has traditionally been an extension of pathology with molecularly specific dyes. Label-free, spectroscopic images are rapidly emerging as another important information source, and we describe the benefits and potential of this evolution. Third, we describe multimodal DP, which is enabled by computational algorithms and combines the best characteristics of structural and molecular pathology. Finally, we provide examples of application areas in telepathology, education, and precision medicine. We conclude by discussing challenges and emerging opportunities in this area.

Rapid molecular diagnostics of severe primary immunodeficiency determined by using targeted next-generation sequencing.

BACKGROUND: Primary immunodeficiency diseases (PIDDs) are inherited disorders of the immune system. The most severe form, severe combined immunodeficiency (SCID), presents with profound deficiencies of T cells, B cells, or both at birth. If not treated promptly, affected patients usually do not live beyond infancy because of infections. Genetic heterogeneity of SCID frequently delays the diagnosis; a specific diagnosis is crucial for life-saving treatment and optimal management. OBJECTIVE: We developed a next-generation sequencing (NGS)-based multigene-targeted panel for SCID and other severe PIDDs requiring rapid therapeutic actions in a clinical laboratory setting. METHODS: The target gene capture/NGS assay provides an average read depth of approximately 1000×. The deep coverage facilitates simultaneous detection of single nucleotide variants and exonic copy number variants in one comprehensive assessment. Exons with insufficient coverage (<20× read depth) or high sequence homology (pseudogenes) are complemented by amplicon-based sequencing with specific primers to ensure 100% coverage of all targeted regions. RESULTS: Analysis of 20 patient samples with low T-cell receptor excision circle numbers on newborn screening or a positive family history or clinical suspicion of SCID or other severe PIDD identified deleterious mutations in 14 of them. Identified pathogenic variants included both single nucleotide variants and exonic copy number variants, such as hemizygous nonsense, frameshift, and missense changes in IL2RG; compound heterozygous changes in ATM, RAG1, and CIITA; homozygous changes in DCLRE1C and IL7R; and a heterozygous nonsense mutation in CHD7. CONCLUSION: High-throughput deep sequencing analysis with complete clinical validation greatly increases the diagnostic yield of severe primary immunodeficiency. Establishing a molecular diagnosis enables early immune reconstitution through prompt therapeutic intervention and guides management for improved long-term quality of life.

[Contemporary Historical Development and Analysis of In Vitro Diagnostics(IVD)].

By analyzing the contemporary key elements evolution of in vitro diagnostics (IVD), especially the rise in vitro diagnostic to the development of molecular diagnostics and precision medicine, from clinical laboratory to POCT, from basic research to the global regulation industry, this research is to expound the complex process and value of biomedical technology development and application.

A Clinician's perspective on clinical exome sequencing.

Clinical exome sequencing has clearly improved our ability as clinicians to identify the cause of a wide variety of disorders. Prior to exome sequencing, a majority of patients with apparent syndromes never received a specific molecular genetic diagnosis despite extensive diagnostic odysseys. Even for those receiving an answer to the question of what caused their disorder, the diagnostic odyssey often spanned years to decades. Determining the particular genetic cause in an individual patient can be challenging due to inherent phenotypic and genetic heterogeneity of disease, technical limitations of testing or both. Blended phenotypes, due to multiple monogenic disorders in the same patient, are true dilemmas for traditional genetic evaluations, but are increasingly being diagnosed through clinical exome sequencing. New sequencing technologies have increased the proportion of patients receiving molecular diagnoses, while significantly shortening the time scale, providing multiple benefits for the health-care team, patient and family.

Regulating whole exome sequencing as a diagnostic test.

In the last decade, there has been a flood of new technology in the sequencing arena. The onset of next-generation sequencing (NGS) technology has resulted in the vast increase in genetic diagnostic testing available to the ordering physician. Whole exome sequencing (WES) has become available as a diagnostic test performed in certified clinical laboratories. This has led to increased presence in the diagnostic marketplace, increased consumer awareness, and the question has been raised by various stakeholders to whether there is sufficient stringent regulation of WES and other NGS-based tests. We discuss the various WES services currently available in the marketplace, current regulation of WES as a laboratory developed test, the proposed FDA involvement in its oversight as well as the response of various laboratory groups that provide these diagnostic services. Overall, a rigorous process oversight and assessment of inter-lab reproducibility is strongly warranted for WES as it is used as a diagnostic test, but regulation should be mindful of the excessive administrative burden on academic and smaller diagnostic laboratories.

Training for cytotechnologists and cytopathologists in the developing world.

Currently there is a major challenge to train sufficient cytopathologists and cytotechnicians in developing countries that have poor medical and pathology infrastructure. Cytology requires well trained pathologists and laboratory staff but it needs only minimal laboratory resources. Cytology can provide the material for rapid, accurate and inexpensive diagnoses of infections such as tuberculosis and of benign and malignant palpable and impalpable lesions. Cytology can achieve this in the developing world by utilizing fine needle aspiration biopsy cytology, general fluids cytology and cervical cytology, in the same manner as is currently done in the developed world where cytology specimens are used to make cytomorphological diagnoses and increasingly to provide material for the full range of ancillary testing including molecular pathology. There are a number of ways to develop sustainable training in cytology in developing countries, especially in fine needle aspiration biopsy cytology, and these are presented and discussed.

SPR and SPR Imaging: Recent Trends in Developing Nanodevices for Detection and Real-Time Monitoring of Biomolecular Events.

In this paper we review the underlying principles of the surface plasmon resonance (SPR) technique, particularly emphasizing its advantages along with its limitations regarding the ability to discriminate between the specific binding response and the interfering effects from biological samples. While SPR sensors were developed almost three decades, SPR detection is not yet able to reduce the time-consuming steps of the analysis, and is hardly amenable for miniaturized, portable platforms required in point-of-care (POC) testing. Recent advances in near-field optics have emerged, resulting in the development of SPR imaging (SPRi) as a powerful optical, label-free monitoring tool for multiplexed detection and monitoring of biomolecular events. The microarrays design of the SPRi chips incorporating various metallic nanostructures make these optofluidic devices more suitable for diagnosis and near-patient testing than the traditional SPR sensors. The latest developments indicate SPRi detection as being the most promising surface plasmon-based technique fulfilling the demands for implementation in lab-on-a-chip (LOC) technologies.

The evolution of pre-analytic factors in Anatomic Pathology.

Anatomic Pathology has continuously evolved since launch by Virchow in Berlin. The era from 1990 to 2010 saw the rise of immunohistochemistry and its application for diagnosis, prognosis, and prediction of response to therapy. Currently the next wave of evolution is ongoing; molecular pathology, with emphasis on alterations to DNA, and expression of mRNA as biomarkers. The interrogation of biomolecules by specific probes is more demanding on specimens than the traditional application of histologic stains to tissue. This issue is juxtaposed to the fact that the majority of specimens are purely evaluated by histomorphology, for which current specimen practices are adequate. The capacity to identify a priori which cassette of tissue is appropriate for molecular analysis is difficult, if not impossible, the goal is to improve the quality of all pathology specimens in an economically viable model to enable advanced assay, when applicable.

[Trends and Challenges of the Statutory Regulation of Molecular Genetic Tests].

Advances in basic science have made it possible to characterize tumors at molecular levels and exploit the differences in the genetic makeup of tumors for personalized cancer treatment. Biomarker tests are essential to stratify patients with the same tumor histology for appropriate treatment. Such tests are developed together with drugs, involving mainly molecular genetic tests at present, and are called companion diagnostics (CDx). Under the universal health care system in Japan, molecular genetic tests are permitted in clinics, and the fees are reimbursed only when approved diagnostic reagents or kits are used. However, new tests are developed so fast that the regulation cannot keep up with the pace. To fill this gap, the framework of advanced medical technologies was introduced in 1984. In 2012, this framework was amended to classify medical technology using unapproved diagnostics or home-brew assays as advanced medical technologies A. In my talk at this symposium, trends and challenges of the statutory regulation of molecular genetic tests in Japan were discussed, followed by personal proposals to advance the clinical application of novel medical technologies in the field of personalized cancer treatment.

[Genetic Diagnosis and Molecular Therapies for Duchenne Muscular Dystrophy].

Duchenne muscular dystrophy (DMD) is the most common form of inherited muscle disease and is characterized by progressive muscle wasting, ultimately resulting in the death of patients in their twenties or thirties. DMD is characterized by a deficiency of the muscle dystrophin as a result of mutations in the dystrophin gene. Currently, no effective treatment for DMD is available. Promising molecular therapies which are mutation-specific have been developed. Transformation of an out-of-frame mRNA into an in-frame dystrophin message by inducing exon skipping is considered one of the approaches most likely to lead to success. We demonstrated that the intravenous administration of the antisense oligonucleotide against the splicing enhancer sequence results in exon skipping and production of the dystrophin protein in DMD case for the first time. After extensive studies, anti-sense oligonucleotides comprising different monomers have undergone clinical trials and provided favorable results, enabling improvements in ambulation of DMD patients. Induction of the read-through of nonsense mutations is expected to produce dystrophin in DMD patients with nonsense mutations, which are detected in 19% of DMD cases. The clinical effectiveness of gentamicin and PTC124 has been reported. We have demonstrated that arbekacin-mediated read-through can markedly ameliorate muscular dystrophy in vitro. We have already begun a clinical trial of nonsense mutation read-through therapy using arbekacin. Some of these drug candidates are planned to undergo submission for approval to regulatory agencies in the US and EU. We hope that these molecular therapies will contribute towards DMD treatment.

[Hopes and pitfalls of the molecular classification of breast cancer]. / Nadeje a úskalí molekulární klasifikace karcinomu prsu.

Traditional histopathological diagnosis of breast cancer has been extended in recent years through the results of additional methods. Today, the results of the detection of hormone receptors, HER-2/neu, and Ki67 antigen are thus an integral part of the histopathological diagnosis. A critical factor for the success of these tests is the fulfillment of pre-analytical phase conditions - i.e. optimal fixation, as well as taking into account the heterogeneous nature of the neoplastic population. In addition to the above-mentioned markers - which have become a routine practice in recent years, there are many efforts to include the molecular characteristics of tumors both in tumor classification as well as in the prediction of results of cancer treatment. Most of the work is based on the use of gene expression profiles. On the basis of the detection of increased or decreased expression of a large number of genes, it is possible to find a set of multiple genes correlating with the biological behavior of the tumor. Using this approach, four basic subgroups of breast cancer have been identified - luminal, basal-like, HER-2 enriched and normal gland-like. Over the course of time, the number of molecular categories has expanded - originally a homogenous group of luminal cancers has been subclassified into the luminal A, B and C. Also within basal-like carcinomas additional subgroups have been identified. However, the results of studies dealing with the analysis of gene expression profiles suggest that our understanding of the biology of breast cancer is far from being complete. The individual categories are defined differently in various publications and thus the comparison of the results of these studies is very difficult. Another approach for the molecular classification of breast cancer is the immunohistochemical detection of various proteins used as a surrogate marker instead of the detection of the mRNA of individual genes. The advantage of this approach is the possibility to use even archive material, as well as much lower costs. On the other hand, its main limitation is the inability of parallel detection of thousands of markers, unlike in genomic profiling. The results of molecular classification are, however, not fundamentally surprising. The fact that breast cancer tumor stem cells can differentiate towards myoepithelial (or basal) and luminal cells has been known for a long time. These two lines of differentiation are - among others - characterized by differential expression of cytoskeletal proteins as well as of other molecules. These findings have been confirmed by the results of molecular studies - either those based on gene expression profiling or immunohistochemical ones. Research results in gene expression profiling have relatively quickly translated into clinical practice. At present, several commercially available certified tests serve as a complementary source of information for decisions about clinical treatment.

[Lab-on-a-chip systems in the point-of-care diagnostics]. / Lab-on-a-chip rendszerek a betegágy melletti diagnosztikában.

The need in modern medicine for near-patient diagnostics being able to accelerate therapeutic decisions and possibly replacing laboratory measurements is significantly growing. Reliable and cost-effective bioanalytical measurement systems are required which - acting as a micro-laboratory - contain integrated biomolecular recognition, sensing, signal processing and complex microfluidic sample preparation modules. These micro- and nanofabricated Lab-on-a-chip systems open new perspectives in the diagnostic supply chain, since they are able even for quantitative, high-precision and immediate analysis of special disease specific molecular markers or their combinations from a single drop of sample. Accordingly, crucial requirements regarding the instruments and the analytical methods are the high selectivity, extremely low detection limit, short response time and integrability into the healthcare information networks. All these features can make the hierarchical examination chain shorten, and revolutionize laboratory diagnostics, evolving a brand new situation in therapeutic intervention.

[Hybrid imaging: clinical evidence, opportunities]. / Hibrid képalkotás: klinikai evidenciák, lehetoségek.

Nowadays the hybrid imaging technologies which combine the modern equipments of radiology and nuclear medicine play an important role in both the translational research process and clinical diagnostics. Among the routine diagnostic imaging procedures positron emission tomography and single photon emission computed tomography combined with computed tomography or magnetic resonance imaging currently belong to the most advanced techniques allowing that functional and morphological images can be superimposed on each other in the same position. The hybrid imaging equipments provide useful information about the pathological processes in the body due to their high sensibility and resolution. Furthermore, with the help of these imaging modalities we can get acquainted with the biochemical and pathobiochemical processes that are essential for understanding and treating diseases, or getting acquainted with the behaviour of a new drug candidate. With the help of the clinical and preclinical non-invasive in vivo molecular imaging systems the drug developing process can be shortened and its costs can be reduced.