Cushing syndrome: Old and new genes.

Cushing syndrome (CS) describes the signs and symptoms caused by exogenous or endogenous hypercortisolemia. Endogenous CS is caused by either ACTH-dependent sources (pituitary or ectopic) or ACTH-independent (adrenal) hypercortisolemia. Several genes are currently known to contribute to the pathogenesis of CS. Germline gene defects, such as MEN1, AIP, PRKAR1A and others, often present in patients with pituitary or adrenal involvement as part of a genetic syndrome. Somatic defects in genes, such as USP8, TP53, and others, are also involved in the development of pituitary or adrenal tumors in a large percentage of patients with CS, and give insight in pathways involved in pituitary or adrenal tumorigenesis.

An overview of 20 years of genetic studies in pheochromocytoma and paraganglioma.

Paragangliomas and pheochromocytomas (PPGL) are rare neuroendocrine tumours characterized by a strong genetic determinism. Over the past 20 years, evolution of PPGL genetics has revealed that around 40% of PPGL are genetically determined, secondary to a germline mutation in one of more than twenty susceptibility genes reported so far. More than half of the mutations occur in one of the SDHx genes (SDHA, SDHB, SDHC, SDHD, SDHAF2), which encode the different subunits and assembly protein of a mitochondrial enzyme, succinate dehydrogenase. These susceptibility genes predispose to early forms (VHL, RET, SDHD, EPAS1, DLST), syndromic (RET, VHL, EPAS1, NF1, FH), multiple (SDHD, TMEM127, MAX, DLST, MDH2, GOT2) or malignant (SDHB, FH, SLC25A11) PPGL. The discovery of a germline mutation in one of these genes changes the patient's follow-up and allows genetic screening of affected families and the presymptomatic follow-up of relatives carrying a mutation.

Harnessing the full potential of reproductive genetics and epigenetics for male infertility in the era of "big data".

The complexity of male reproductive impairment has hampered characterization of the underlying genetic causes of male infertility. However, in the last 20 years, more powerful and affordable tools to interrogate the genetic and epigenetic determinants of male infertility have accelerated the number of new discoveries in the characterization of male infertility. With this explosion of new data, integration in a systems-based approach-including complete phenotypic information-to male infertility is imperative. We briefly review the current understanding of genetic and epigenetic causes of male infertility and how findings may be translated into a practical component for the diagnosis and treatment of male infertility.

Regulatory perspectives on next-generation sequencing and complementary diagnostics in Japan.

INTRODUCTION: The 'one biomarker/one drug' scenario is unsustainable because cancer is a complex disorder that involves a number of molecular defects. In the past decade, major technological advances have lowered the overall cost and increased the efficiency of next-generation sequencing (NGS). AREAS COVERED: We review recent regulations on NGS and complementary diagnostics in Japan, mainly focusing on high-quality studies that utilized these new diagnostic modalities and were published within the last 5 years. We highlight significant changes in regulation, and explain the direction of efforts to translate the results of NGS and complementary diagnostics into clinical practice. EXPERT OPINION: NGS holds a number of advantages over conventional companion and complementary diagnostics that enable simultaneous analyzes of multiple cancer genes to detect actionable mutations. Parallel technological developments and regulatory changes have led to the rapid adoption of NGS into clinical practice. NGS-based genomic data have been leveraged to better understand the characteristics of a disease that affects its patient's response to a given therapy. As NGS-based tests become more widespread, however, Japanese authorities will face significant challenges particularly with respect to the complexity of genomic data, which will have to be managed if NGS is to benefit patients.

Evolving Significance of Tumor-Normal Sequencing in Cancer Care.

Molecular tests assist at various stages of cancer patient management, including providing diagnosis, predicting prognosis, identifying therapeutic targets, and determining hereditary cancer risk. The current testing paradigm involves germline testing in a subset of patients determined to be at high risk for having a hereditary cancer syndrome, and tumor-only sequencing for treatment decisions in advanced cancer patients. A major limitation of tumor-only sequencing is its inability to distinguish germline versus somatic mutations. Tumor-normal sequencing has emerged as a comprehensive analysis for both hereditary cancer predisposition and somatic profiling. Here, we review recent studies involving tumor-normal sequencing, discuss its benefits in clinical care, challenges for its implementation, and novel insights it has provided regarding tumor biology and germline contribution to cancer.

Ovarian cancer and the evolution of subtype classifications using transcriptional profiling†.

Ovarian cancer is a complex disease with multiple subtypes, each having distinct histopathologies and variable responses to treatment. This review highlights the technological milestones and the studies that have applied them to change our definitions of ovarian cancer. Over the past 50 years, technologies such as microarrays and next-generation sequencing have led to the discovery of molecular alterations that define each of the ovarian cancer subtypes and has enabled further subclassification of the most common subtype, high-grade serous ovarian cancer (HGSOC). Improvements in mutational profiling have provided valuable insight, such as the ubiquity of TP53 mutations in HGSOC tumors. However, the information derived from these technological advances has also revealed the immense heterogeneity of this disease, from variation between patients to compositional differences within single masses. In looking forward, the emerging technologies for single-cell and spatially resolved transcriptomics will allow us to better understand the cellular composition and structure of tumors and how these contribute to the molecular subtypes. Attempts to incorporate the complexities ovarian cancer has resulted in increasing sophistication of model systems, and the increased precision in molecular profiling of ovarian cancers has already led to the introduction of inhibitors of poly (ADP-ribose) polymerases as a new class of treatments for ovarian cancer with DNA repair deficiencies. Future endeavors to define increasingly accurate classification strategies for ovarian cancer subtypes will allow for confident prediction of disease progression and provide important insight into potentially targetable molecular mechanisms specific to each subtype.

Molecular genetics of Conn adenomas in the era of exome analysis.

Aldosterone-producing adenomas (APA) are a major cause of primary aldosteronism (PA), the most common form of secondary hypertension. Exome analysis of APA has allowed the identification of recurrent somatic mutations in KCNJ5, CACNA1D, ATP1A1, and ATP2B3 in more than 50 % of sporadic cases. These gain of function mutations in ion channels and pumps lead to increased and autonomous aldosterone production. In addition, somatic CTNNB1 mutations have also been identified in APA. The CTNNB1 mutations were also identified in cortisol-producing adenomas and adrenal cancer, but their role in APA development and the mechanisms specifying the hormonal production or the malignant phenotype remain unknown. The role of the somatic mutations in the regulation of aldosterone production is well understood, while the impact of these mutations on cell proliferation remains to be established. Furthermore, the sequence of events leading to APA formation is currently the focus of many studies. There is evidence for a two-hit model where the somatic mutations are second hits occurring in a previously remodeled adrenal cortex. On the other hand, the APA-driver mutations were also identified in aldosterone-producing cell clusters (APCC) in normal adrenals, suggesting that these structures may represent precursors for APA development. As PA due to APA can be cured by surgical removal of the affected adrenal gland, the identification of the underlying genetic abnormalities by novel biomarkers could improve diagnostic and therapeutic approaches of the disease. In this context, recent data on steroid profiling in peripheral venous samples of APA patients and on new drugs capable of inhibiting mutated potassium channels provide promising preliminary data with potential for translation into clinical care.

Molecular analysis in liquid biopsies for diagnostics of primary central nervous system lymphoma: Review of literature and future opportunities.

Primary central nervous system lymphoma (PCNSL) is an aggressive lymphoma with a poor prognosis, for which accurate and timely diagnosis is of utmost importance. Unfortunately, diagnosis of PCNSL can be challenging and a brain biopsy (gold standard for diagnosis) is an invasive procedure with the risk of major complications. Thus, there is an urgent need for an alternative strategy to diagnose and monitor these lymphomas. Currently, liquid biopsies from cerebrospinal fluid (CSF) are used for cytomorphologic and flow cytometric analysis. Recently, new biomarkers such as genetic mutations and interleukins have been identified in these liquid biopsies, further expanding the diagnostic armamentarium. In this review we present an overview of genetic aberrations (>70) reported in this unique lymphoma. Of these genes, we have selected those that are reported in ≥3 studies. Half of the selected genes are implicated in the NFκB pathway (CARD11, CD79B, MYD88, TBL1XR1 and TNFAIP3), while the other half are not related to this pathway (CDKN2A, ETV6, PIM1, PRDM1 and TOX). Although this underlines the crucial role of the NFκB pathway in PCNSL, CD79B and MYD88 are at present the only genes mentioned in liquid biopsy analysis. Finally, a stepwise approach is proposed for minimally invasive liquid biopsy analysis and work-up of PCNSL, incorporating molecular analysis. Prioritization and refinements of this approach can be constructed based upon multidisciplinary collaboration as well as novel scientific insights.

Genetic alterations crossing the borders of distinct hematopoetic lineages and solid tumors: Diagnostic challenges in the era of high-throughput sequencing in hemato-oncology.

Owing to the introduction of next-generation sequencing (NGS) new challenges for diagnostic algorithms and the interpretation of the results for therapeutic decision making in hemato-oncology have arisen. Recurrent somatic mutations crossing the borders between different hematological entities and solid neoplasms have been detected. In analogy to mutant TP53, the same mutation type may occur in myeloid, B- or T-lymphatic malignancies or solid neoplasms. At the same time, a certain mutation can show different prognostic outcomes in different entities and co-existence of certain mutations may change the prognostic relevance. These insights may spark the investigation of targeted therapies with the same substances across different disease entities. This review article summarizes mutations that can emerge in different hematologic and solid malignancies and summarizes other obstacles in the era of modern molecular diagnostics, such as the phenomenon of "clonal hematopoiesis of indeterminate potential" being difficult to interpret in the individual patient.

Molecular Diagnostics in Pathology: Time for a Next-Generation Pathologist?

CONTEXT: - Comprehensive molecular investigations of mainstream carcinogenic processes have led to the use of effective molecular targeted agents in most cases of solid tumors in clinical settings. OBJECTIVE: - To update readers regarding the evolving role of the pathologist in the therapeutic decision-making process and the introduction of next-generation technologies into pathology practice. DATA SOURCES: - Current literature on the topic, primarily sourced from the PubMed (National Center for Biotechnology Information, Bethesda, Maryland) database, were reviewed. CONCLUSIONS: - Adequate evaluation of cytologic-based and tissue-based predictive diagnostic biomarkers largely depends on both proper pathologic characterization and customized processing of biospecimens. Moreover, increased requests for molecular testing have paralleled the recent, sharp decrease in tumor material to be analyzed-material that currently comprises cytology specimens or, at minimum, small biopsies in most cases of metastatic/advanced disease. Traditional diagnostic pathology has been completely revolutionized by the introduction of next-generation technologies, which provide multigene, targeted mutational profiling, even in the most complex of clinical cases. Combining traditional and molecular knowledge, pathologists integrate the morphological, clinical, and molecular dimensions of a disease, leading to a proper diagnosis and, therefore, the most-appropriate tailored therapy.

Beyond screening for chromosomal abnormalities: Advances in non-invasive diagnosis of single gene disorders and fetal exome sequencing.

Emerging genomic technologies, largely based around next generation sequencing (NGS), are offering new promise for safer prenatal genetic diagnosis. These innovative approaches will improve screening for fetal aneuploidy, allow definitive non-invasive prenatal diagnosis (NIPD) of single gene disorders at an early gestational stage without the need for invasive testing, and improve our ability to detect monogenic disorders as the aetiology of fetal abnormalities. This presents clinicians and scientists with novel challenges as well as opportunities. In addition, the transformation of prenatal genetic testing arising from the introduction of whole genome, exome and targeted NGS produces unprecedented volumes of data requiring complex analysis and interpretation. Now translating these technologies to the clinic has become the goal of clinical genomics, transforming modern healthcare and personalized medicine. The achievement of this goal requires the most progressive technological tools for rapid high-throughput data generation at an affordable cost. Furthermore, as larger proportions of patients with genetic disease are identified we must be ready to offer appropriate genetic counselling to families and potential parents. In addition, the identification of novel treatment targets will continue to be explored, which is likely to introduce ethical considerations, particularly if genome editing techniques are included in these targeted treatments and transferred into mainstream personalized healthcare. Here we review the impact of NGS technology to analyse cell-free DNA (cfDNA) in maternal plasma to deliver NIPD for monogenic disorders and allow more comprehensive investigation of the abnormal fetus through the use of exome sequencing.

Screening for fetal chromosomal and subchromosomal disorders.

Screening for fetal chromosomal disorders has evolved greatly over the last four decades. Initially, only maternal age-related risks of aneuploidy were provided to patients. This was followed by screening with maternal serum analytes and ultrasound markers, followed by the introduction and rapid uptake of maternal plasma cell-free DNA-based screening. Studies continue to demonstrate that cfDNA screening for common aneuploidies has impressive detection rates with low false-positive rates. The technology continues to push the boundaries of prenatal screening as it is now possible to screen for less common aneuploidies and subchromosomal disorders. The optimal method for incorporating cfDNA screening into existing programs continues to be debated. It is important that obstetricians understand the biological foundations and limitations of this technology and provide patients with up-to-date information regarding cfDNA screening.

Detection of RAS mutations in circulating tumor cells: applications in colorectal cancer and prospects.

The somatic mutations in the RAS genes (KRAS and NRAS) are widely associated with non-response to immunotherapies targeting the epidermal growth factor receptor in metastatic colorectal cancer. The detection of these mutations is carried out from tissue biopsies and become mandatory to prescribe these treatments. Nethertheless, this analysis is not possible in about 25% of cases and the development of alternative methods is therefore required. Among them, the search for mutations directly in the blood of patients are promising approaches. Circulating tumor cells (CTCs) represent a particularly relevant direct target. These cells, some of them have inducing-metastasis capabilities, have been able to detach themselves from the primary tumor, then migrate and finally enter into the bloodstream. In this sense, they are particularly resistant to physical-chemical and immunological constraints used by the organism to prevent their dissemination. Consequently, they represent a particularly valuable source of information on the most aggressive tumor cells. As a corollary, these cells are very rare requiring particularly highly performant technologies to be detected. In this presentation, we focus mainly on the molecular methods used to detect these mutated RAS cells by analyzing the performance of a solution based on a filtration device followed by detection with digital PCR. Finally, we will discuss the biological significance of these cells before highlighting prospects in colorectal cancer but also in other cancers.

Review of Clinical Next-Generation Sequencing.

CONTEXT: - Next-generation sequencing (NGS) is a technology being used by many laboratories to test for inherited disorders and tumor mutations. This technology is new for many practicing pathologists, who may not be familiar with the uses, methodology, and limitations of NGS. OBJECTIVE: - To familiarize pathologists with several aspects of NGS, including current and expanding uses; methodology including wet bench aspects, bioinformatics, and interpretation; validation and proficiency; limitations; and issues related to the integration of NGS data into patient care. DATA SOURCES: - The review is based on peer-reviewed literature and personal experience using NGS in a clinical setting at a major academic center. CONCLUSIONS: - The clinical applications of NGS will increase as the technology, bioinformatics, and resources evolve to address the limitations and improve quality of results. The challenge for clinical laboratories is to ensure testing is clinically relevant, cost-effective, and can be integrated into clinical care.

Colorectal cancer molecular profiling: from IHC to NGS in search of optimal algorithm.

Advances in defining the mutational landscape of colorectal cancer (CRC) over the past decades have revolutionized the molecular understanding and clinical testing algorithms for this disease. Mutation testing is standard of care for the work-up of CRCs. This review focuses on the current indications and strategies for molecular testing in CRC and discusses the potential changes in CRC testing approach associated with the emerging clinical application of genomic-based technologies.

[Next-generation DNA sequencing in clinical diagnostics]. / Le séquençage d'ADN à haut débit en pratique clinique.

The advent of next generation sequencing (NGS) technologies is so scale-changing that it modifies molecular diagnostics indications and induces laboratories to rethink their diagnostic strategies, until now based on the Sanger sequencing routine. Several high-throughput approaches are available from the sequencing of a gene panel, to an exome, or even a genome. In all cases, a tremendous amount of data is generated, which has to be filtered, interpreted and analyzed using powerful bioinformatics tools. In parallel, ethical considerations are raised to avoid the potential drifts of these powerful approaches. In all medical fields, and particularly in pediatrics, this new strategy offers better efficacy and faster mutation identification, allowing better support and care for patients and their families and even improving genetic counseling. In the present paper, we discuss the different NGS-based approaches and strategies as well as the issues involved in these new technologies applied to molecular diagnosis of rare diseases. Altogether, rare diseases affect more than 3 million people in France and are responsible for about one-third of childhood deaths.

Lab-on-a-chip technologies for genodermatoses: Recent progress and future perspectives.

In recent years, molecular biology has proven to be a great asset in our understanding of mechanisms in genodermatoses. However, bench to bedside translation research lags far behind. Advances in lab-on-a-chip technologies enabled programmable, reconfigurable, and scalable manipulation of a variety of laboratory procedures. Sample preparation, microfluidic reactions, and continuous monitoring systems can be integrated on a small chip. These advantages have attracted attention in various fields of clinical application including diagnosis of inherited skin diseases. This review lists an overview of the underlying genes and mutations and describes prospective application of lab-on-a-chip technologies as solutions to challenges for point-of-care genodematoses diagnosis.

[Research Advancement on EGFR Mutation Detection of Cell-free DNA and Tumor Cell in Peripheral Blood of Patients with Non-small Cell Lung Cancer].

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. Epideral growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are the most important treatments currently for advanced NSCLC patients harboring activating EGFR gene mutations, and achieve significant clinical efficacy. T790M mutation occurs in half of NSCLC patents with acquired EGFR-TKI resistance. Screening for EGFR gene mutations in histological and/or circulating tumor cell or DNA samples of NSCLC patients can identify patients who would have a response to EGFR-TKIs or acquire resistance during the treatment. Quantitative analysis of plasma EGFR mutation is of great importance not only in early diagnosis of tumors, but also in curative effect evaluation and for follow-up. However, a strict requirement is proposed on the detection technique because of low DNA content and the fragmentation of the genes. To date, many methods have been applied to detect cfDNA EGFR mutations, including sequencing, real-time PCR (RT-PCR), amplification refractory mutation system (ARMS), mutant-enriched PCR (ME-PCR), denaturing high-performance liquid chromatography (DHPLC), digital PCR, and droplet digital PCR (ddPCR). However, of all the methods above, ddPCR has the highest sensitivity, allows high throughput operation. In conclusion, the ddPCR has a lot of future promise in clinical gene diagnosis.